CYTOTOXICITY OF Se-LABELED ANTIBODIES AND SELENOFOLATE AGAINST TRIPLE NEGATIVE BREAST CELL LINES MDA-MB-231 AND MDA-MB-468

By

Soni Khandelwal B.Sc., B.Ed., M.Sc.

A Dissertation in

NUTRITIONAL SCIENCES

Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Approved

Dr. Julian E. Spallholz Co-Chair of Committee

Dr. Lauren S. Gollahon Co-Chair of Committee

Dr. Nikhil Dhurandhar

Dr. Mallory L. Boylan

Dr. Ruth Serra-Moreno

Dr. Mark Sheridan Dean of the Graduate School

May, 2018

Copyright 2018, Soni Khandelwal

Texas Tech University, Soni Khandelwal, May 2018

ACKNOWLEGDEMENTS

There have been many people who have walked alongside me during the last four years. They have guided me, placed opportunities in front of me and showed me the doors that might be beneficial to open. I am truly grateful to each and every one of them.

I would like to thank my research co-advisors, Dr. Julian E. Spallholz and Dr. Lauren S. Gollahon for their enduring support, patience, guidance and advice through this project. They have been a constant source of motivation which has helped me to build positive attitude during these years. Special thanks to Dr. Boylan for believing in my abilities and introducing me to Dr. Spallholz. Exchanging conversations with Dr. Boylan have helped me to remain engrossed on my path.

Today, if I am able complete this research project it is because of Dr. Nikhil Dhurandhar, who helped us arrange funds for the research. Dr. Dhurandhar always helped me whenever I needed one and made sure the research continued to progress towards a finished dissertation.

I would like to thank Dr. Serra-Moreno for valuable insights and experiences on Western Blot, which incented me to widen my research from various perspectives. Whenever I had question, I knew Dr. Serra-Moreno would have answers to those questions.

Besides my committee members, I appreciate Dr. Gilbert Kirsh for synthesizing Selenofolate and providing me with the compound during the entire study period.

I would like to express my sincere gratitude to the College of Human Sciences and Department of Nutritional Sciences for providing the financial support of this study. I would also like to extend my gratitude to the Faculty and Staff of Department of Nutritional Sciences and Biological Sciences. My appreciation is extended to the doctoral and undergraduate students Caroline, Sergio, Drake, Noshin, Maliha, Zahid, Shao-Hua, Samudani, Sanjoy, Sagor, Belinda, Stacy, Maria and Roberto for their help, friendship and stimulation discussion. I would like to thank Shane Scoggin for his suggestions for

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Texas Tech University, Soni Khandelwal, May 2018 alternatives while conducting my experiments. I would also extend my gratitude to my friends- Anuradha, Arwa, Nadeeja, Pooja, Priyanka, Sneha, Sriporna, Swati and Shwetha and would like to thank them for their unconditional support throughout the study.

I am most grateful to my family in India and my husband Mr. Rahul Khandelwal who has played a key role in shaping my career and helped me achieve my goals with patience and focus.

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TABLE OF CONTENTS

ACKNOWLEGDEMENTS ...... ii ABSTRACT ...... viii LIST OF TABLES ...... ix LIST OF FIGURES ...... x LIST OF ABBREVIATIONS ...... xxviii I. INTRODUCTION ...... 1 GENERAL CHARACTERISTICS OF CANCER ...... 1 BREAST CANCER ...... 2 ETIOLOGY AND RISK FACTORS ...... 2 CLASSIFICATION OF BREAST ...... 4 STAGES OF BREAST CANCER ...... 6 TREATMENT FOR BREAST CANCER ...... 8 TRIPLE NEGATIVE BREAST CANCER ...... 11 SELENIUM: ESSENTIAL AND TOXIC MICRONUTRIENT ...... 12 MAJOR FUNCTIONS OF SELENIUM IN THE HUMAN BODY ...... 13 DISEASES ASSOCIATED WITH SELENIUM DEFICIENCY ...... 15 SELENIUM TOXICITY ...... 16 MECHANISM OF TOXICITY OF SELENIUM ...... 17 SELENIUM IN CANCER PREVENTION ...... 20 ...... 22 PROPOSAL AND GOALS ...... 24 HYPOTHESIS ...... 25 SPECIFIC AIMS ...... 25 II. CYTOTOXICITY OF , SELENOFOLATE AND SELENITE AGAINST TRIPLE NEGATIVE BREAST LINES MDA-MB-231 AND MDA-MB- 468 ...... 26 ABSTRACT ...... 26 KEYWORDS ...... 27 iv

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INTRODUCTION ...... 27 MATERIALS AND METHODS ...... 29 Materials ...... 29 Synthesis of 1-propanol selenocyanate ...... 29 Synthesis of Selenofolate ...... 30 Cell Culture ...... 31 Detection of Superoxide In Vitro: Selenofolate ...... 32 Detection of Intracellular Reactive Oxygen Species Accumulation In Situ: Superoxide Generation Assay ...... 32 Optimization of Cell Density for Treatment ...... 32 Cell Treatments ...... 33 Visual Assessments of Cellular Morphology ...... 34 Cell Viability Measured by Trypan Blue Exclusion ...... 34 Experimental Cell Cultures ...... 35 MTT Assay ...... 35 MitoTracker® Red and Annexin V Staining ...... 36 Western Blotting ...... 37 STATISTICAL ANALYSES ...... 38 RESULTS AND DISCUSSION ...... 41 III. CYTOTOXICITY OF AVASTIN®, SELENOAVASTIN AND SELENITE AGAINST TRIPLE NEGATIVE BREAST CANCER CELL LINES MDA-MB-231 AND MDA-MB- 468 ...... 71 ABSTRACT ...... 71 KEYWORDS ...... 72 INTRODUCTION ...... 72 MATERIALS AND METHODS ...... 74 Materials ...... 74 Monoclonal antibodies ...... 75 Synthesis of Bolton-Hunter Seleno ester ...... 75 Conjugation of Redox Se to Avastin® ...... 77

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Analysis of Selenoavastin ...... 78 Detection of Superoxide In Vitro by Selenoavastin ...... 78 Detection of Intracellular Reactive Oxygen Species Accumulation In Situ: Superoxide Generation Assay ...... 79 BCA Assay for Protein Determination of Avastin® and Selenoavastin ...... 79 Western Blotting Under Denaturing Conditions...... 80 Western Blotting Under Non-Denaturing Conditions ...... 80 Cell Culture ...... 81 Optimization of Cell Density for Treatment ...... 81 Cell Treatments ...... 81 Visual Assessments of Cellular Morphology ...... 82 Cell Viability Measured by Trypan Blue Exclusion ...... 82 Experimental Cell Cultures ...... 82 Measuring Cell Viability Using MTT Assay ...... 83 MitoTracker® Red and Annexin V Staining ...... 84 Western Blotting ...... 85 STATISTICAL ANALYSES ...... 86 RESULTS AND DISCUSSION ...... 89 IV. CYTOTOXICITY OF HERCEPTIN®, SELENOHERCEPTIN AND SELENITE AGAINST TRIPLE NEGATIVE BREAST CANCER CELL LINES MDA-MB-231 AND MDA-MB-468 ...... 125 ABSTRACT ...... 125 KEYWORDS ...... 126 INTRODUCTION ...... 126 MATERIALS AND METHODS ...... 128 Materials ...... 128 Monoclonal antibodies ...... 129 Synthesis Bolton-Hunter of Seleno ester ...... 130 Conjugation of Redox Se to Herceptin® ...... 130 Analysis of Selenoherceptin ...... 132

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Detection of Superoxide In Vitro By Selenoherceptin ...... 132 Detection of Intracellular Reactive Oxygen Species Accumulation In Situ: Superoxide Generation Assay ...... 133 BCA assay for Protein Determination of Herceptin® and Selenoherceptin ...... 133 Western Blotting Under Denaturing Conditions...... 134 Western Blotting Under Non-Denaturing Conditions ...... 134 Cell Culture ...... 135 Optimization of Cell Density for Treatment ...... 135 Cell Treatments ...... 135 Visual Assessments of Cellular Toxicity ...... 136 Cell Viability Measured by Trypan Blue Exclusion ...... 136 Cell Experimental Cultures ...... 136 Measuring Cell Viability Using the MTT Assay ...... 137 MitoTracker® Red and Annexin V Staining ...... 138 Western Blotting ...... 139 STATISTICAL ANALYSES ...... 140 RESULTS AND DISCUSSION ...... 143 V. IMPLICATIONS AND LIMITATION ...... 181 VI. CONCLUSIONS ...... 183 REFERENCES ...... 188 APPENDICIES A. QUALITY CONTROL AND MICRO-PHOTOGRAPHS OF DIFFERENT TREATMENTS ...... 201 B. CELL DIAMETER FROM DIFFERENT TREATMENTS...... 223

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ABSTRACT

Breast cancer (BC) is the second leading cause of death among women. Triple negative breast cancer (TNBC) accounts for 10-15% of all breast cancer. As the TNBC name suggest they lack hormone receptors-estrogen and progesterone receptors and amplification of HER-2. These receptors are considered essential for “targeted therapies” of BC. Thus, systemic is the only TNBC drug treatment option for these patients. In this study, Folate or two clinical monoclonal antibodies; Herceptin® or Avastin® were covalently labeled with redox selenium and used to treat two TNBC cell lines (MDA-MB-231 and MDA-MB-468 cells) and HME50-5E cells in culture. All the three cell lines were treated with vehicle control, Selenite, increasing concentrations of Selenofolate, Selenoherceptin or Selenoavastin and were compared with equal concentrations of Folate or native antibodies. Superoxide generation was detected in vitro using Chemiluminescence assay and in situ by Dihydroethidium (DHE) assay. Morphological changes in cells were observed under phase contrast microscopy in all selenium treated cells. Cell counts, and viability were analyzed by Trypan Blue exclusion and MTT assay. Annexin V was used to detect apoptosis pathway and western blot to observe protein expression levels. Selenofolate, Selenoavastin and Selenoherceptin generated superoxide in vitro and in situ in TNBC cells. Selenofolate and Se- immunoconjugates were observed to be much more cytotoxic over Se dose and time to both TNBC cell lines in comparison to vehicle control cells and cells treated with native antibodies alone. Selenofolate and Se-immunoconjugates induced apoptosis in TNBC cell lines but not in HME50-5E cells and protein bands were detected at their respective molecular weight. This is the first report of selenium Antibody-Drug Conjugates (ADCs) being demonstrated to be cytotoxic to these TNBC cell lines, suggesting a potential strategy to design more effective treatments of TNBCs resistant to chemotherapy.

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LIST OF TABLES

Table I-1: Human Triple Negative Breast Cancer Cells and the Immortalized Mammary Epithelial 50-5E Cell Line. Adapted from [48] ...... 12 Table I-2: Functions of Selenoproteins in Human Body...... 13 Table I-3: Effects of Different Forms of Selenium on Cancer...... 21 Table II-1: Conversion of Selenite and Selenofolate from µM Se to µg Se ...... 34 Table II-2: Statistical analyses of Folate and Selenofolate treatments against MDA-MB-231 Cells ...... 39 Table II-3: Statistical analyses of Folate and Selenofolate treatments against MDA-MB-468 Cells ...... 40 Table II-4: Statistical analyses of Folate and Selenofolate treatments against HME 50-5E Cells 41 Table III-1: Statistical Analyses of Avastin® and Selenoavastin treatments against MDA-MB-231 Cells...... 87 Table III-2: Statistical Analyses of Avastin® and Selenoavastin treatments against MDA-MB-468 Cells...... 88 Table III-3: Statistical Analyses of Avastin® and Selenoavastin treatments against HME50-5E Cells...... 88 Table III-4: Selenium and Protein Concentration of Avastin® and Selenoavastin...... 90 Table IV-1: Statistical Analyses of Herceptin® and Selenoherceptin treatments in MDA-MB-231 Cells...... 141 Table IV-2: Statistical Analyses of Herceptin® and Selenoherceptin treatments in MDA-MB-468 Cells...... 142 Table IV-3: Statistical Analyses of Herceptin® and Selenoherceptin treatments in HME50-5E Cells...... 143 Table IV-4: Selenium and Protein Concentration of Herceptin® and Selenoherceptin...... 144 Table B-1: MDA-MB-231 Cell Diameter (microns). Data is expressed as mean (n=3)...... 223 Table B-2: MDA-MB-231 Cell Circularity (microns). Data is expressed as mean (n=3)...... 223 Table B-3: MDA-MB-468 Cell Diameter (microns). Data is expressed as mean (n=3)...... 224 Table B-4: MDA-MB-468 Cell Circularity (microns). Data is expressed as mean (n=3)...... 224

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LIST OF FIGURES

Figure I-1: Timeline of Selenium ...... 13 Figure I-2: Redox Cycling Between Selenium Compounds and GSH...... 18 Figure II-1: Structure of Folate, Molecular Weight: 441 g/mol ...... 30 Figure II-2: Structure of Selenofolate; Molecular weight: 573 g/mol ...... 31 Figure II-3: Photographs of Folate (A) and Selenofolate (B) ...... 42 Figure II-4: Photographs of Folate (B) and Selenofolate (A) dissolved in 1X PBS. Sample pH was 7.0-7.5...... 42 Figure II-5: Time Dependent Superoxide Generation as Measured by Lucigenin Chemiluminescence (CL) for Blank, Folate and Selenofolate. Control Blank, Folate and Selenofolate CL was measured over 5 minutes with 30 second integrations...... 44 Figure II-6: Total Superoxide Generation as Measured by Lucigenin Chemiluminescence (CL) with different concentrations of Glutathione (GSH). Chemiluminescence (CL) for Control Blank, Folate and Selenofolate measured over 5 minutes...... 44 Figure II-7: Photomicrographs of Intracellular Superoxide Generation by DHE Red Florescence from MDA-MB-231 Cells after 30 minutes of Blank, Control, Selenite (20 µM), Folate (100 µM) and Selenofolate (100 µM) Treatments. Scale bar=200 µm...... 45 Figure II-8: Photomicrographs of Intracellular Superoxide Generation by DHE Red Florescence from MDA-MB-468 Cells after 30 minutes of Blank, Control, Selenite (20 µM), Folate (100 µM) and Selenofolate (100 µM) Treatments. Scale bar=100 µm...... 45 Figure II-9: Photomicrographs of Intracellular Superoxide Generation by DHE Red Florescence from HME50-5E Cells after 30 minutes of Blank, Control, Selenite (20 µM), Folate (100 µM) and Selenofolate (100 µM) Treatments. Scale bar=200 µm...... 45 Figure II-10: Morphological Changes of Control, Selenite (4 µM), Folate (50 µM) and Selenofolate (50 µM) Treatments in MDA-MB-231 Cells. Treatment of MDA-MB-231 Cells with Selenite and Selenofolate morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-231 cells. Cells were visualized under 20X magnification...... 47 Figure II-11: Morphological Changes of Control, Selenite (4 µM), Folate (50 µM) and Selenofolate (50 µM) Treatments in MDA-MB-468 Cells. Treatment of MDA-MB-468 Cells with Selenite and Selenofolate morphological changes indicative of membrane disruption with

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decreased cell viability. Representative fields of view of MDA-MB-468 cells. Cells were visualized under 20X magnification...... 48 Figure II-12: Morphological Changes of Control, Selenite (4 µM), Folate (50 µM) and Selenofolate (50 µM) Treatments in HME50-5E Cells. Treatment of HME50-5E cells with Selenite, Folate and Selenofolate did not induce severe morphological change comparable to those seen in TNBC cells. Representative fields of view of HME50-5E cells. Cells were visualized under 20X magnification...... 49 Figure II-13: Cytotoxicity of Control, Selenite, Folate and Selenofolate in Dose and Time Dependent Treatments against MDA-MB-231 cells. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding. Control cells were incubated for 7 days with Selenite (4 µM, 10 µM, 20 µM and 40 µM), Folate and Selenofolate at concentrations ranging from 5 µM to 100 µM. Viable cells were counted at time periods (0-7 days) by Trypan Blue exclusion as described in Methods. The cell growth data are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table II-2...... 51 Figure II-14: Cytotoxicity of Control, Selenite, Folate and Selenofolate in Dose and Time Dependent Treatments against MDA-MB-468 cells. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding. Control cells were incubated for 7 days with Selenite (4 µM, 10 µM, 20 µM and 40 µM), Folate and Selenofolate at concentrations ranging from 5 µM to 100 µM. Viable cells were counted at time periods (0-7 days) by Trypan Blue exclusion as described in Methods. The cell growth data are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table II-3...... 52 Figure II-15: Cytotoxicity of Control, Selenite, Folate and Selenofolate in Dose and Time Dependent Treatments against HME50-5E cells. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding. Control cells were incubated for 7 days with Selenite (4 µM, 10 µM, 20 µM and 40 µM), Folate and Selenofolate at concentrations ranging from 50 µM to 100 µM. Viable cells were counted at time periods (0-7 days) by Trypan Blue exclusion as described in Methods. The cell growth data are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table II-4...... 53

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Figure II-16: Metabolic activity of Control, Selenite, Folate and Selenofolate treated MDA-MB- 231 cells as determined by the MTT Assay. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding with Selenite (4 µM, 10 µM, 20 µM), Folate and Selenofolate at concentrations ranging from 1 µM to 100 µM over 6 days by MTT as described in Methods. The cell viabilities are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table II-2...... 55 Figure II-17: Metabolic activity of Control, Selenite, Folate and Selenofolate treated MDA-MB- 468 cells as determined by the MTT Assay. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding with Selenite (4 µM, 10 µM, 20 µM), Folate and Selenofolate at concentrations ranging from 1 µM to 100 µM over 6 days by MTT as described in Methods. The cell viabilities are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table II-3...... 56 Figure II-18: Metabolic activity of Control, Selenite, Folate and Selenofolate treated HME50-5E cells as determined by the MTT Assay. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding with Selenite (4 µM, 10 µM, 20 µM), Folate and Selenofolate at concentrations ranging from 1 µM to 100 µM over 6 days by MTT as described in Methods. The cell viabilities are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table II-4...... 57 Figure II-19: IC50 for MDA-MB-231 Cells with Selenofolate...... 58 Figure II-20: IC50 for MDA-MB-468 Cells with Selenofolate...... 59 Figure II-21: Folate, Selenofolate and Selenite as Se Treatments induced apoptosis in MDA-MB- 231 Cells. MDA-MB-231 cells were stained with Annexin V/PI and subjected to flow cytometric analysis. The four quadrants represent- living cells (Lower Left; LL: Annexin V- PI--), early apoptotic (Lower Right; LR: Annexin V+PI-), late apoptotic (Upper Right; UR: Annexin+PI+) or necrotic (Upper Left; UL: Annexin V-PI+) stages...... 62 Figure II-22: Representative of four quadrants for MDA-MB-231 cells were treated with Folate, Selenofolate and Selenite. Data is expressed as Mean (n=3) of the total percent of MDA-MB- 231 cells...... 63

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Figure II-23: Percentage level of MDA-MB-231 apoptotic cells when the cells were treated with Folate, Selenofolate and Selenite as Se. Data is expressed as Mean ± SE (n=3). Treatments were compared using two sample t test. Treatments were statistically significant if p ≤ 0.05 and summarized in Table II-2...... 63 Figure II-24: Folate, Selenofolate and Selenite as Se Treatments induced apoptosis in MDA-MB- 468 Cells. MDA-MB-468 cells were stained with Annexin V/Mitotracker Red and subjected to flow cytometric analysis. The four quadrants represent- living cells (Upper Left; UL: Annexin V- Mitotracker Red+), early apoptotic (Upper Right; UR: Annexin+ Mitotracker Red +), late apoptotic (Lower Right; LR: Annexin V+ Mitotracker Red-) or necrotic (Lower Left; LL: Annexin V- Mitotracker Red-) stages...... 64 Figure II-25: Representative of four quadrants for MDA-MB-468 cells were treated with Folate, Selenofolate and Selenite. Data is expressed as Mean (n=3) of the total percent of MDA-MB- 468 cells...... 65 Figure II-26: Percentage of MDA-MB-468 apoptotic cells when the cells were treated with Folate, Selenofolate and Selenite as Se. Data is expressed as Mean ± SE (n=3). Treatments were compared using two sample t test. Treatments were statistically significant, if p ≤ 0.05 and summarized in Table II-3...... 65 Figure II-27: Folate, Selenofolate and Selenite as Se Treatments did not induced apoptosis in HME50-5E Cells. HME50-5E cells were stained with Annexin V/ Mitotracker Red and subjected to flow cytometric analysis. The four quadrants represent- living cells (Upper Left; UL: Annexin V- Mitotracker Red+), early apoptotic (Upper Right; UR: Annexin+ Mitotracker Red +), late apoptotic (Lower Right; LR: Annexin V+ Mitotracker Red-) or necrotic (Lower Left; LL: Annexin V- Mitotracker Red-) stages...... 66 Figure II-28: Representative of four quadrants for HME50-5E cells were treated with Folate, Selenofolate and Selenite. Data is expressed as Mean (n=3) of the total percent of HME50-5E cells...... 67 Figure II-29: Percentage level of HME50-5E apoptotic cells when the cells were treated with Folate, Selenofolate and Selenite as Se. Data is expressed as Mean ± SE (n=3)...... 67 Figure II-30: Western blot analysis of the Folate Receptor Alpha in MDA-MB-231 Control Cells and cells treated with Selenite Folate, Selenofolate. Total cell lysates were subjected to SDS- PAGE followed by Western blotting. Membranes were probed with the anti-folate receptor alpha (FRA) or anti β-actin antibodies followed by peroxidase conjugated rabbit anti-mouse xiii

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antibodies and visualization was performed by the ECL detection system. Lane 1: Control, Lane 2: Selenite 4 µM treatment, Lane 3: Folate 50 µM treatment, Lane 4: Folate 75 µM treatment, Lane 5: Folate 100 µM treatment, Lane 6: Selenofolate 50 µM treatment, Lane 7: Selenofolate 75 µM treatment, Lane 8: Selenofolate 100 µM treatment...... 68 Figure II-31: Western blot analysis of the Folate Receptor Alpha in MDA-MB-468 Control Cells and cells treated with Selenite Folate, Selenofolate. Total cell lysates were subjected to SDS- PAGE followed by Western blotting. Membranes were probed with the anti-folate receptor alpha (FRA) or anti β-actin antibodies followed by peroxidase conjugated rabbit anti-mouse antibodies and visualization was performed by the ECL detection system. Lane 1: Control, Lane 2: Selenite 4 µM treatment, Lane 3: Folate 50 µM treatment, Lane 4: Folate 75 µM treatment, Lane 5: Folate 100 µM treatment, Lane 6: Selenofolate 50 µM treatment, Lane 7: Selenofolate 75 µM treatment, Lane 8: Selenofolate 100 µM treatment...... 68 Figure II-32: Western blot analysis of the Folate Receptor Alpha in MDA-MB-231 Control Cells and cells treated with Selenite Folate, Selenofolate. Total cell lysates were subjected to SDS- PAGE followed by Western blotting. Membranes were probed with the anti-folate receptor alpha (FRA) or anti β-actin antibodies followed by peroxidase conjugated rabbit anti-mouse antibodies and visualization was performed by the ECL detection system...... 69 Figure III-1: The Procedure for Conjugation of Seleno-Ester to the Lysine Residues of Monoclonal Antibodies...... 76 Figure III-2: Selenocyanate Propionic Ester of N-hydroxysuccinimide (Bolton-Hunter Seleno- Ester) ...... 76 Figure III-3: Saturated Solution of Se-Ester Dissolved In Tetrahydrofuran...... 78 Figure III-4: Selenoavastin and Avastin...... 90 Figure III-5: Total Superoxide Generation as Measured by Lucigenin Chemiluminescence (CL) with Glutathione (GSH). Chemiluminescence (CL) for Control Blank, Avastin® and Selenoavastin measured over 12.5 minutes. Data is expressed as Mean ± SE. CL for Avastin® and Selenoavastin was compared with CL Blank. CL for Selenoavastin was statistically significant (p≤0.001). 100µL of Selenoavastin contains 8.1µg of Se...... 91 Figure III-6: Time Dependent Superoxide Generation as Measured by Lucigenin Chemiluminescence (CL) for Blank, Avastin® and Selenoavastin. Control Blank Avastin® and Selenoavastin CL was measured over 12.5 minutes with 30 second integrations. 100µL of Selenoavastin contains 8.1µg of Se...... 92 xiv

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Figure III-7: Photomicrographs of Intracellular Superoxide Generation by DHE Red Florescence from MDA-MB-468 Cells after 30 minutes of Blank, Control, Selenite (10 µg), Avastin® (153.7 µg protein and Selenoavastin (10 µg Se and 153.7 µg protein) Treatments. Scale bar=100 µm...... 93 Figure III-8: Photomicrographs of Intracellular Superoxide Generation by DHE Red Florescence from MDA-MB-231 Cells after 30 minutes of Blank, Control, Selenite (10 µg), Avastin® (153.7 µg protein) and Selenoavastin (10 µg Se and 153.7 µg protein) Treatments. Scale bar=200 µm...... 93 Figure III-9: Photomicrographs of Intracellular Superoxide Generation by DHE Red Florescence from HME50-5E Cells after 30 minutes of Blank, Control, Selenite (10 µg), Avastin® (153.7 µg protein) and Selenoavastin (10 µg Se and 153.7 µg protein) Treatments. Scale bar=200 µm...... 93 Figure III-10: SDS-PAGE of Native and Se-conjugated mAbs under reducing conditions followed by Coomassie Blue R-250 staining. The photograph was taken under Coomaisse Blue filter. Lane 1: Kadcyla® 20 μg; Lane 2: Marker; Lane 3: Avastin® 5 µg; Lane 4: Avastin® 10 µg; Lane 5: Avastin® 20 µg; Lane 6: Selenoavastin 5 µg; Lane 7: Selenoavastin 10 µg; Lane 8: Selenoavastin 20 µg; Lane 9: Herceptin® 5 µg; Lane 10: Herceptin® 10 µg; Lane 11: Herceptin® 20 µg, Lane 12: Selenoherceptin 10 µg; Lane 13: Selenoherceptin 20 µg; Lane 14: Gamma globulin 20 µg ...... 95 Figure III-11: mAbs migration on 4-20% Tris-Glycine PAGE gel under non-reducing conditions followed by Coomaisse Blue R-250 stain. The photograph was taken under Coomassie Blue filter. Lane 1: Molecular Marker; Lane 2: Purified Human IgG 10 μg; Lane 3: Bovine Gamma Globulin 5 μg; Lane 4: Bovine Gamma Globulin 10 μg; Lane 5: Kadcyla® 5 μg; Lane 6: Kadcyla® 10 μg; Lane 7: Avastin® 5 μg; Lane 8: Avastin® 10 μg; Lane 9: Selenoavastin 5 μg; Lane 10: Selenoavastin 10 μg; Lane 11: Herceptin® 5 μg; Lane 12: Herceptin® 10 μg; Lane 13: Selenoherceptin 5 μg; Lane 14: Selenoherceptin 10 μg...... 96 Figure III-12: Morphological Changes of Control, Selenite, Avastin® and Selenoavastin Treatments in MDA-MB-231 Cells. Treatment of MDA-MB-231 Cells with Selenite and Selenoavastin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-231 cells. Cells were visualized under 20X magnification...... 98

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Figure III-13: Morphological Changes of Control, Selenite, Avastin® and Selenoavastin Treatments in MDA-MB-468 Cells. Treatment of MDA-MB-468 Cells with Selenite and Selenoavastin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-468 cells. Cells were visualized under 20X magnification...... 99 Figure III-14: Morphological Changes of Control, Selenite, Avastin® and Selenoavastin Treatments in HME50-5E Cells. Treatment of HME50-5E cells with Selenite, Avastin® and Selenoavastin did not induce severe morphological change comparable to those seen in TNBC cells. Representative fields of view of HME50-5E cells. Cells were visualized under 20X magnification...... 100 Figure III-15: Cytotoxicity of Control, Selenite, Avastin® and Selenoavastin in Dose and Time Dependent Treatments against MDA-MB-231 cells. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding. Control cells were incubated for 7 days with Selenite, Avastin® and Selenoavastin at different concentrations. Viable cells were counted at time periods (0-7 days) by Trypan Blue exclusion as described in Methods. The cell growth data are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table III-1...... 102 Figure III-16: Cytotoxicity of Control, Selenite, Avastin® and Selenoavastin in Dose and Time Dependent Treatments against MDA-MB-468 cells. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding. Control cells were incubated for 7 days with Selenite, Avastin® and Selenoavastin at different concentrations. Viable cells were counted at time periods (0-7 days) by Trypan Blue exclusion as described in Methods. The cell growth data are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table III-2...... 103 Figure III-17: Cytotoxicity of Control, Selenite, Avastin® and Selenoavastin in Dose and Time Dependent Treatments against HME50-5E cells. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding. Control cells were incubated for 7 days with Selenite, Avastin® and Selenoavastin at different concentrations. Viable cells were counted at time periods (0-7 days) by Trypan Blue exclusion as described in Methods. The cell growth data are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table III-3...... 104

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Figure III-18: Metabolic activity of Control, Selenite, Avastin® and Selenoavastin treated MDA- MB-231 cells as determined by the MTT Assay. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding with Selenite, Avastin® and Selenoavastin at different concentrations over 6 days by MTT as described in Methods. The cell viabilities are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table III-1...... 106 Figure III-19: Metabolic activity of Control, Selenite, Avastin® and Selenoavastin treated MDA- MB-468 cells as determined by the MTT Assay. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding with Selenite, Avastin® and Selenoavastin at different concentrations over 6 days by MTT as described in Methods. The cell viabilities are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table III-2...... 107 Figure III-20: Metabolic activity of Control, Selenite, Avastin® and Selenoavastin treated HME50-5E cells as determined by the MTT Assay. 40,000 cells were seeded in 48 well- plates and were treated on day 5 post seeding with Selenite, Avastin® and Selenoavastin at different concentrations over 6 days by MTT as described in Methods. The cell viabilities are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table III-3...... 108 Figure III-21: IC50 for MDA-MB-231 Cells with Selenoavastin...... 109 Figure III-22: IC50 for MDA-MB-468 Cells with Selenoavastin...... 110 Figure III-23: Avastin®, Selenoavastin and Selenite as Se Treatments induced apoptosis in MDA-MB-231 Cells. MDA-MB-231 cells were stained with Annexin V/PI and subjected to flow cytometric analysis. The four quadrants represent-living cells (Lower Left; LL: Annexin V-PI-), early apoptotic (Lower Right; LR: Annexin V+PI-), late apoptotic (Upper Right; UR: Annexin+PI+) or necrotic (Upper Left; UL: Annexin V-PI+) stages...... 113 Figure III-24: Representative of four quadrants when MDA-MB-231 cells were treated with Avastin®, Selenoavastin and Selenite as Se. Data is expressed as Mean (n=3)...... 114 Figure III-25: Percentage level of MDA-MB-231 apoptotic cells when the cells were treated with Avastin®, Selenoavastin and Selenite as Se. Data is expressed as Mean ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table III-1...... 115

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Figure III-26: Avastin®, Selenoavastin and Selenite as Se Treatments induced apoptosis in MDA-MB-468 cells. MDA-MB-468 cells were stained with Annexin V/ Mitotracker Red and subjected to flow cytometric analysis. The four quadrants represent- living cells (Upper Left; UL: Annexin V- Mitotracker Red+), early apoptotic (Upper Right; UR: Annexin+ Mitotracker Red +), late apoptotic (Lower Right; LR: Annexin V+ Mitotracker Red-) or necrotic (Lower Left; LL: Annexin V- Mitotracker Red-) stages...... 116 Figure III-27: Representative of four quadrants when MDA-MB-468 cells were treated with Avastin®, Selenoavastin and Selenite as Se. Data is expressed as Mean (n=3)...... 117 Figure III-28: Percentage level of MDA-MB-468 apoptotic cells when the cells were treated with Avastin®, Selenoavastin and Selenite as Se. Data is expressed as Mean ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant p ≤ 0.05 are summarized in Table III-2...... 118 Figure III-29: Avastin®, Selenoavastin and Selenite as Se Treatments did not induced apoptosis in HME50-5E cells. HME50-5E cells were stained with Annexin V/ Mitotracker Red and subjected to flow cytometric analysis. The four quadrants represent- living cells (Upper Left; UL: Annexin V- Mitotracker Red+), early apoptotic (Upper Right; UR: Annexin+ Mitotracker Red +), late apoptotic (Lower Right; LR: Annexin V+ Mitotracker Red-) or necrotic (Lower Left; LL: Annexin V- Mitotracker Red-) stages...... 119 Figure III-30: Representative of four quadrants when HME50-5E cells were treated with Avastin®, Selenoavastin and Selenite as Se. Data is expressed as Mean (n=3)...... 120 Figure III-31: Percentage level of MDA- apoptotic cells when the cells were treated with Avastin®, Selenoavastin and Selenite as Se. Data is expressed as Mean ± SE (n=3)...... 121 Figure III-32: Western blot analysis of the expression level of VEGF in MDA-MB-231 Cells treated with Avastin®, Selenoavastin and Selenite as Se. Total cell lysates were subjected to SDS-PAGE followed by Western blotting. Membranes were probed with the anti-VEGF or anti β-actin antibodies followed by peroxidase conjugated rabbit anti-mouse antibodies and visualization was performed by the ECL detection system. Lanes 1 and 2: Control, Lanes 3 and 4: Selenite (2µg Se) treatments, Lanes 5 and 6: Avastin® (30.7µg protein) treatment, Lanes 7 and 8: Selenoavastin (2µg Se, 30.7 µg protein) treatments...... 122 Figure III-33: Western blot analysis of the expression level of VEGF in MDA-MB-468 Cells treated with Avastin®, Selenoavastin and Selenite as Se. Total cell lysates were subjected to SDS-PAGE followed by Western blotting. Membranes were probed with the anti-VEGF or xviii

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anti β-actin antibodies followed by peroxidase conjugated rabbit anti-mouse antibodies and visualization was performed by the ECL detection system. Lanes 1 and 2: Control, Lanes 3 and 4: Selenite (2µg Se) treatments, Lanes 5 and 6: Avastin® (30.7µg protein) treatment, Lanes 7 and 8: Selenoavastin (2µg Se, 30.7 µg protein) treatments...... 123 Figure III-34: Western blot analysis of the expression level of VEGF in HME50-5E Cells treated with Avastin®, Selenoavastin and Selenite as Se. Total cell lysates were subjected to SDS- PAGE followed by Western blotting. Membranes were probed with the anti-VEGF or anti β- actin antibodies followed by peroxidase conjugated rabbit anti-mouse antibodies and visualization was performed by the ECL detection system. Lane 1: MDA-MB-468 as VEGF positive loading control, Lanes 2 and 3: Control, Lanes 4 and 5: Selenite (2µg Se) treatments, Lanes 6 and 7: Avastin® (30.7µg protein) treatment, Lanes 8 and 9: Selenoavastin (2µg Se, 30.7 µg protein) treatments...... 123 Figure IV-1: The Procedure for Conjugation of Seleno-Ester to the Lysine Residues of Herceptin®...... 130 Figure IV-2: Selenocyanate Propionic Ester of N-hydroxysuccinimide (Bolton-Hunter Seleno- Ester)...... 131 Figure IV-3: Saturated Solution Of Se-Ester Dissolved In Tetrahydrofuran...... 132 Figure IV-4: Selenoherceptin and Herceptin®...... 144 Figure IV-5: Total Superoxide Generation as Measured by Lucigenin Chemiluminescence (CL) with Glutathione (GSH). Chemiluminescence (CL) for Control Blank, Herceptin® and Selenoherceptin measured over 12.5 minutes. Data is expressed as Mean ± SE. CL for Herceptin® and Selenoherceptin was compared with CL Blank. CL for Selenoherceptin was statistically significant (p≤0.001). 100µL of Selenoherceptin contains 8.8µg of Se...... 146 Figure IV-6: Time Dependent Superoxide Generation as Measured by Lucigenin Chemiluminescence (CL) for Blank, Avastin® and Selenoavastin. Control Blank, Herceptin® and Selenoherceptin CL was measured over 12.5 minutes with 30 second integrations. 100µL of Selenoherceptin contains 8.8µg of Se...... 146 Figure IV-7: Photomicrographs of Intracellular Superoxide Generation by DHE Red Florescence from MDA-MB-231 Cells after 30 minutes of Blank, Control, Selenite (10 µg as Se), Herceptin® (131.1 µg protein) and Selenoherceptin (10 µg Se and 131.1 µg protein) Treatments. Scale bar=200 µm...... 147

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Figure IV-8: Photomicrographs of Intracellular Superoxide Generation by DHE Red Florescence from MDA-MB-468 Cells after 30 minutes of Blank, Control, Selenite (10 µg as Se), Herceptin® (131.1 µg protein) and Selenoherceptin (10 µg Se and 131.1 µg protein) Treatments. Scale bar=100 µm...... 147 Figure IV-9: Photomicrographs of Intracellular Superoxide Generation by DHE Red Florescence from HME50-5E Cells after 30 minutes of Blank, Control, Selenite (10 µg as Se), Herceptin® (131.1 µg protein) and Selenoherceptin (10 µg Se and 131.1 µg protein) Treatments. Scale bar=200 µm...... 147 Figure IV-10: SDS-PAGE of Native Herceptin® and Selenoherceptin mAbs under denaturing or reducing conditions followed by Coomassie Blue R-250 staining. The photograph was taken under Coomassie Blue. Lane 1: Kadcyla 20 μg; Lane 2: Marker; Lane 3: Avastin® 5 µg; Lane 4: Avastin® 10 µg; Lane 5: Avastin® 20 µg; Lane 6: Selenoavastin 5 µg; Lane 7: Selenoavastin 10 µg; Lane 8: Selenoavastin 20 µg; Lane 9: Herceptin® 5 µg; Lane 10: Herceptin® 10 µg; Lane 11: Herceptin® 20 µg, Lane 12: Selenoherceptin 10 µg; Lane 13: Selenoherceptin 20 µg; Lane 14: Gamma globulin 20 µg...... 149 Figure IV-11: mAbs migration on 3-20% Tris-Glycine PAGE gel under non-reducing conditions followed by Coomaisse Blue R-250 stain. The photograph was taken under Commaisse blue filter. Lane 1: Molecular Marker; Lane 2: Purified Human IgG 10 μg; Lane 3: Bovine Gamma Globulin 5 μg; Lane 4: Bovine Gamma Globulin 10 μg; ; Lane 5: Kadcyla® 5 μg; Lane 6: Kadcyla® 10 μg; Lane 7: Avastin® 5 μg; Lane 8: Avastin® 10 μg; Lane 9: Selenoavastin 5 μg; Lane 10: Selenoavastin 10 μg; Lane 11: Herceptin® 5 μg; Lane 12: Herceptin® 10 μg; Lane 13: Selenoherceptin 5 μg; Lane 14: Selenoherceptin 10 μg...... 150 Figure IV-12: Herceptin® migration on 3-20% Tris-Glycine PAGE gel under non-reducing conditions followed by Coomaisse Blue R-250 stain. The photograph was taken under Commaisse Blue filter. Lane 3: Herceptin® 5 μg; Lane 4: Herceptin® 10 μg...... 151 Figure IV-13: Morphological Changes of Control, Selenite, Herceptin® and Selenoherceptin Treatments in MDA-MB-231 Cells. Treatment of MDA-MB-231 Cells with Selenite and Selenoherceptin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-231 cells. Cells were visualized under 20X magnification...... 153 Figure IV-14: Morphological Changes of Control, Selenite, Herceptin® and Selenoherceptin Treatments in MDA-MB-468 Cells. Treatment of MDA-MB-468 Cells with Selenite and

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Selenoherceptin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-468 cells. Cells were visualized under 20X magnification...... 154 Figure IV-15: Morphological Changes of Control, Selenite, Herceptin® and Selenoherceptin Treatments in HME50-5E Cells. Treatment of HME50-5E cells with Selenite, Herceptin® and Selenoherceptin did not induce severe morphological change comparable to those seen in TNBC cells. Representative fields of view of HME50-5E cells. Cells were visualized under 20X magnification...... 155 Figure IV-16: Cytotoxicity of Control, Selenite, Herceptin®, Selenoherceptin in Dose and Time Dependent Treatments against MDA-MB-231 cells. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding. Control cells were incubated for 7 days with Selenite, Avastin® and Selenoavastin at different concentrations. Viable cells were counted at time periods (0-7 days) by Trypan Blue exclusion as described in Methods. The cell growth data are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table IV-1...... 157 Figure IV-17: Cytotoxicity of Control, Selenite, Herceptin®, Selenoherceptin in Dose and Time Dependent Treatments against MDA-MB-468 cells. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding. Control cells were incubated for 7 days with Selenite, Avastin® and Selenoavastin at different concentrations. Viable cells were counted at time periods (0-7 days) by Trypan Blue exclusion as described in Methods. The cell growth data are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table IV-2...... 158 Figure IV-18: Cytotoxicity of Control, Selenite, Herceptin®, Selenoherceptin in Dose and Time Dependent Treatments against HME50-5E cells. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding. Control cells were incubated for 7 days with Selenite, Avastin® and Selenoavastin at different concentrations. Viable cells were counted at time periods (0-7 days) by Trypan Blue exclusion as described in Methods. The cell growth data are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table IV-3...... 159 Figure IV-19: Metabolic activity of Control, Selenite, Herceptin®, Selenoherceptin treated MDA- MB-231 cells as determined by the MTT Assay. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding with Selenite, Avastin® and Selenoavastin at different

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concentrations over 6 days by MTT as described in Methods. The cell viabilities are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table IV-1...... 161 Figure IV-20: Metabolic activity of Control, Selenite, Herceptin®, Selenoherceptin treated MDA- MB-468 cells as determined by the MTT Assay. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding with Selenite, Avastin® and Selenoavastin at different concentrations over 6 days by MTT as described in Methods. The cell viabilities are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table IV-2...... 162 Figure IV-21: Metabolic activity of Control, Selenite, Herceptin®, Selenoherceptin treated HME50-5E cells as determined by the MTT Assay. 40,000 cells were seeded in 48 well- plates and were treated on day 5 post seeding with Selenite, Avastin® and Selenoavastin at different concentrations over 6 days by MTT as described in Methods. The cell viabilities are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table IV-3...... 163 Figure IV-22: IC50 for MDA-MB-231 Cells with Selenoherceptin...... 164 Figure IV-23: IC50 for MDA-MB-468 Cells with Selenoherceptin...... 165 Figure IV-24: Herceptin®, Selenoherceptin and Selenite as Se Treatments induced apoptosis in MDA-MB-231 cells. MDA-MB-231 cells were stained with Annexin V/PI and subjected to flow cytometric analysis. The four quadrants represent-living cells (Lower Left; LL: Annexin V-PI-), early apoptotic (Lower Right; LR: Annexin V+PI-), late apoptotic (Upper Right; UR: Annexin+PI+) or necrotic (Upper Left; UL: Annexin V-PI+) stages...... 168 Figure IV-25: Representative of four quadrants when MDA-MB-231 cells were treated with Herceptin®, Selenoherceptin and Selenite as Se. Data is expressed as Mean (n=3) of the total percent MDA-MB-231 cells...... 169 Figure IV-26: Percentage level of apoptotic cells when MDA-MB-231 cells were treated with Herceptin®, Selenoherceptin and Selenite as Se. Data is expressed as Mean ± SE (n=3). Treatments that were statistically significant p ≤ 0.05 and summarized in Table IV-1...... 170 Figure IV-27: Herceptin®, Selenoherceptin and Selenite as Se Treatments induced apoptosis in MDA-MB-468 cells. MDA-MB-468 cells were stained with Annexin V/ Mitotracker Red and subjected to flow cytometric analysis. The four quadrants represent-living cells (Upper Left; UL: Annexin V- Mitotracker Red+), early apoptotic (Upper Right; UR: Annexin+ Mitotracker xxii

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Red +), late apoptotic (Lower Right; LR: Annexin V+ Mitotracker Red-) or necrotic (Lower Left; LL: Annexin V- Mitotracker Red-) stages...... 171 Figure IV-28: Representative of four quadrants when MDA-MB-468 cells were treated with Herceptin®, Selenoherceptin and Selenite as Se. Data is expressed as Mean (n=3) of the total percent of MDA-MB-468 cells...... 172 Figure IV-29: Percentage level of apoptotic cells when MDA-MB-468 cells were treated with Herceptin®, Selenoherceptin and Selenite as Se. Data is expressed as Mean ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant p ≤ 0.05 and summarized in Table IV-2...... 173 Figure IV-30: Herceptin®, Selenoherceptin and Selenite as Se Treatments did not induced apoptosis in HME50-5E cells. HME50-5E cells were stained with Annexin V/ Mitotracker Red and subjected to flow cytometric analysis. The four quadrants represent- living cells (Upper Left; UL: Annexin V- Mitotracker Red+), early apoptotic (Upper Right; UR: Annexin+ Mitotracker Red +), late apoptotic (Lower Right; LR: Annexin V+ Mitotracker Red-) or necrotic (Lower Left; LL: Annexin V- Mitotracker Red-) stages...... 174 Figure IV-31: Representative of four quadrants when HME 50-5E cells were treated with Herceptin®, Selenoherceptin and Selenite as Se. Data is expressed as Mean (n=3) of the total percent of HME50-5E cells...... 175 Figure IV-32: Percentage level of HME50-5E apoptotic cells when the cells were treated with Herceptin®, Selenoherceptin and Selenite as Se. Data is expressed as Mean ± SE (n=3). Treatments were compared using two sample t test...... 176 Figure IV-33: Western blot analysis of the expression level of HER2 in MDA-MB-231 Cells treated with Herceptin®, Selenoherceptin and Selenite as Se. Total cell lysates were subjected to SDS-PAGE followed by Western blotting. Membranes were probed with the anti-HER2 or anti β-actin antibodies followed by peroxidase conjugated rabbit anti-mouse antibodies and visualization was performed by the ECL detection system. Lane 1: BT474 as HER2 positive loading control, Lanes 2 and 3: Control, Lanes 4 and 5: Selenite (2µg as Se) treatments, Lanes 6 and 7: Herceptin® (26.22µg as protein) treatment, Lanes 8 and 9: Selenoherceptin (2µg as Se, 26.22µg as protein) treatments...... 177 Figure IV-34: Western blot analysis of the expression level of HER2 in MDA-MB-468 Cells treated with Herceptin®, Selenoherceptin and Selenite as Se. Total cell lysates were subjected to SDS-PAGE followed by Western blotting. Membranes were probed with the anti-HER2 or xxiii

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anti β-actin antibodies followed by peroxidase conjugated rabbit anti-mouse antibodies and visualization was performed by the ECL detection system. Lane 1: BT474 as HER2 positive loading control, Lanes 2 and 3: Control, Lanes 4 and 5: Selenite (2µg as Se) treatments, Lanes 6 and 7: Herceptin® (26.22µg as protein) treatment, Lanes 8 and 9: Selenoherceptin (2µg as Se, 26.22µg as protein) treatments...... 178 Figure IV-35: Western blot analysis of the expression level of HER2 in HME 50-5E Cells treated with Herceptin®, Selenoherceptin and Selenite as Se. Total cell lysates were subjected to SDS-PAGE followed by Western blotting. Membranes were probed with the anti-HER or anti β-actin antibodies followed by peroxidase conjugated rabbit anti-mouse antibodies and visualization was performed by the ECL detection system. Lane 1: BT474 as HER2 positive loading control, Lane 2: Molecular weight markers, Lanes 3 and 4: Control, Lanes 5 and 6: Selenite (2µg as Se) treatments, Lanes 7 and 8: Herceptin® (26.22µg as protein) treatment, Lanes 9 and 10: Selenoherceptin (2µg as Se, 26.22µg as protein) treatments...... 178 Figure IV-36: SDS-PAGE of HME50-5E Total Protein Cell Lysate from Control, Herceptin®, Selenoherceptin and Selenite as Se Treatments. Total cell lysates were subjected to SDS- PAGE followed by Coomassie Blue R-250 stain. Lane 1: HCC1419 as HER2 positive loading control, Lane 2: Molecular markers, Lanes 3 and 4: Control, Lanes 5 and 6: Selenite (2µg as Se) treatments, Lanes 7 and 8: Herceptin® (26.22µg as protein) treatment, Lanes 9 and 10: Selenoherceptin (2µg as Se, 26.22µg as protein) treatments...... 179 Figure VI-1: Summarized diagrammatic representation of the study...... 187 Figure A-1: Folate did not auto-fluoresce under UV light spectrum. Folate was run through the Attune™ NxT Flow Cytometer (ThermoFisher Scientific, Carlsbad, CA) as a quality control to detect if there was any kind of fluorescence emitted by Folate, because this would if present effect fluorescent assays...... 201 Figure A-2: Selenoavastin exhibited elemental fluorescence under UV light spectrum. Selenoavastin was run through the Attune™ NxT Flow Cytometer (ThermoFisher Scientific, Carlsbad, CA) as a quality control to detect if there was any kind of fluorescence emitted by Selenoavastin, because this would if present effect fluorescent assays...... 202 Figure A-3: Morphological Changes of Control, Selenite (10 µM), Folate (75 µM) and Selenofolate (75 µM) Treatments in MDA-MB-231 Cells. Treatment of MDA-MB-231 Cells with Selenite and Selenofolate morphological changes indicative of membrane disruption with

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decreased cell viability. Representative fields of view of MDA-MB-231 cells. Cells were visualized under 20X magnification...... 203 Figure A-4: Morphological Changes of Control, Selenite (20 µM), Folate (100 µM) and Selenofolate (100 µM) Treatments in MDA-MB-231 Cells. Treatment of MDA-MB-231 Cells with Selenite and Selenofolate morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-231 cells. Cells were visualized under 20X magnification...... 204 Figure A-5: Morphological Changes of Control, Selenite (10 µM), Folate (75 µM) and Selenofolate (75 µM) Treatments in MDA-MB-468 Cells. Treatment of MDA-MB-468 Cells with Selenite and Selenofolate morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-468 cells. Cells were visualized under 20X magnification...... 205 Figure A-6: Morphological Changes of Control, Selenite (20 µM), Folate (100 µM) and Selenofolate (100 µM) Treatments in MDA-MB-468 Cells. Treatment of MDA-MB-468 Cells with Selenite and Selenofolate morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-468 cells. Cells were visualized under 20X magnification...... 206 Figure A-7: Morphological Changes of Control, Selenite (10 µM), Folate (75 µM) and Selenofolate (75 µM) Treatments in HME50-5E Cells. Treatment of HME50-5E cells with Selenite, Folate and Selenofolate did not induce severe morphological change comparable to those seen in TNBC cells. Representative fields of view of HME50-5E cells. Cells were visualized under 20X magnification...... 207 Figure A-8: Morphological Changes of Control, Selenite (20 µM), Folate (100 µM) and Selenofolate (100 µM) Treatments in HME50-5E Cells. Treatment of HME50-5E cells with Selenite, Folate and Selenofolate did not induce severe morphological change comparable to those seen in TNBC cells. Representative fields of view of HME50-5E cells. Cells were visualized under 20X magnification...... 208 Figure A-9: Morphological Changes of Control, Selenite (5 µg as Se), Avastin® and Selenoavastin Treatments in MDA-MB-231 Cells. Treatment of MDA-MB-231 Cells with Selenite and Selenoavastin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-231 cells. Cells were visualized under 20X magnification...... 209

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Figure A-10: Morphological Changes of Control, Selenite (5 µg as Se), Avastin® and Selenoavastin Treatments in MDA-MB-468 Cells. Treatment of MDA-MB-468 Cells with Selenite and Selenoavastin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-468 cells. Cells were visualized under 20X magnification...... 210 Figure A-11: Morphological Changes of Control, Selenite (5 µg as Se), Avastin® and Selenoavastin Treatments in HME50-5E Cells. Representative fields of view of HME50-5E cells. Cells were visualized under 20X magnification...... 211 Figure A-12: Morphological Changes of Control, Selenite (10 µg as Se), Avastin® and Selenoavastin Treatments in MDA-MB-231 Cells. Treatment of MDA-MB-231 Cells with Selenite and Selenoavastin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-231 cells. Cells were visualized under 20X magnification...... 212 Figure A-13: Morphological Changes of Control, Selenite (10 µg as Se), Avastin® and Selenoavastin Treatments in MDA-MB-468 Cells. Treatment of MDA-MB-468 Cells with Selenite and Selenoavastin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-468 cells. Cells were visualized under 20X magnification...... 213 Figure A-14: Morphological Changes of Control, Selenite (5 µg as Se), Avastin® and Selenoavastin Treatments in HME50-5E Cells. Representative fields of view of HME50-5E cells. Cells were visualized under 20X magnification...... 214 Figure A-15: Morphological Changes of Control, Selenite, Herceptin® and Selenoherceptin Treatments in MDA-MB-231 Cells. Treatment of MDA-MB-231 Cells with Selenite and Selenoherceptin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-231 cells. Cells were visualized under 20X magnification...... 215 Figure A-16: Morphological Changes of Control, Selenite, Herceptin® and Selenoherceptin Treatments in MDA-MB-468 Cells. Treatment of MDA-MB-468 Cells with Selenite and Selenoherceptin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-468 cells. Cells were visualized under 20X magnification...... 216

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Figure A-17: Morphological Changes of Control, Selenite, Herceptin® and Selenoherceptin Treatments in HME50-5E Cells. Representative fields of view of HME50-5E cells. Cells were visualized under 20X magnification...... 217 Figure A-18: Morphological Changes of Control, Selenite, Herceptin® and Selenoherceptin Treatments in MDA-MB-231 Cells. Treatment of MDA-MB-231 Cells with Selenite and Selenoherceptin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-231 cells. Cells were visualized under 20X magnification...... 218 Figure A-19: Morphological Changes of Control, Selenite, Herceptin® and Selenoherceptin Treatments in MDA-MB-468 Cells. Treatment of MDA-MB-468 Cells with Selenite and Selenoherceptin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-468 cells. Cells were visualized under 20X magnification...... 219 Figure A-20: Morphological Changes of Control, Selenite, Herceptin® and Selenoherceptin Treatments in HME50-5E Cells. Representative fields of view of HME50-5E cells. Cells were visualized under 20X magnification...... 220 Figure A-21: Standard Curve prepared from a serial dilution of Bovine Gamma Globulin used for protein determination of Avastin®, Selenoavastin, Herceptin® and Selenoherceptin...... 221 Figure A-22: Time dependent superoxide generation as measured by lucigenin chemiluminescence (CL) for blank, sodium selenite...... 221 Figure A-23: HME50-5E MTT Plate Photo on Day 3 Post Treatment showing the insoluble formazan salt...... 222

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LIST OF ABBREVIATIONS

ANOVA Analysis of Variance

BV Bevacizumab/Avastin/Native BV

BCA Bicinchoninic Acid

BT-474 Her2+ Breast Cancer Cell line

CL Chemiluminescence

CNS Central Nervous System

DMSO Dimethyl sulfoxide

DHE Dihydroethidium

DCC N, N’-dicyclohexylcarbodimide

EGF Epidermal Growth Factor

EGFR Epidermal Growth Factor Receptor

FDA US Food and Drug Administration

FR Folate Receptor

G1 Growth Phase

GPx Glutathione Peroxidase

GSH Glutathione

GSSeSG Selenodiglutathione

HME50-5E Human Mammary Epithelial 50-5E

Her2/neu/ Erb B2 Human Epidermal Growth Factor Receptor 2

IC50 50% Inhibitory Concentration

ICP-MS Inductively Coupled Plasma-Mass Spectrophotometry

IHC Immunohistochemistry mAbs Monoclonal Antibodies xxviii

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MAPK Mitogen Activated Protein Kinase

MWCO Molecular Weight Cut-Off

MDA-MB-231 Triple Negative Breast Cancer Cell line

MDA-MB-468 Triple Negative Breast Cancer Cell line

MIT Massachusetts Institute of Technology

NIH National Institutes of Health

NO Nitric Oxide

- O2 Superoxide

PI3K Phosphatidylinositol-4,5-bisphosphate 3-kinase

RAS Rat Sarcoma

RAF Rapidly Accelerated Fibrosarcoma

RAS-RAF-MAPK Intracellular Signaling Pathway

ROCH2CH2SeCN N-Hydroxysuccinimide Ester of 3-Selenocyanopropanoic Acid

RDI Recommended Dietary Intake

RDA Recommended Dietary Allowance

ROS Reactive Oxygen Species

RSe- Selenide Anion

Se Selenium

SeCys Selenocysteine

SEM Scanning Electron Microscope

Se-TZ Seleno-Trastuzumab; Immunoconjugate

Se-BV Seleno-Bevacizumab; Immunoconjugate

SOD Superoxide Dismutase

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TB Trypan Blue

THF Tetrahydrofuran

TZ Trastuzumab/Herceptin/Native TZ

VEGF Vascular Endothelial Growth Factor

VEGFR Vascular Endothelial Growth Factor Receptor

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CHAPTER I

INTRODUCTION

GENERAL CHARACTERISTICS OF CANCER

Cancer is widely accepted as a major public health problem [1]. It is the leading cause of death worldwide [2] and the second leading cause of death after cardiovascular disease in the United States [1]. An estimated 1,735,350 new cases and 609,640 deaths from cancer are expected in the United States across both genders in 2018 [3]. According to the World Cancer Research Fund International/American Institute for Cancer Research (WCRF/AICR) and National Cancer Institute (NCI), cancers of the lung and colorectal are common cancers observed in both in men and women, in addition to cancers of prostrate and breast, respectively.

Tumorigenesis is a series of several processes within the cell of origin and is initiated by different signals from the microenvironment. Alterations or a genetic defect in somatic cells leads to inactivation of tumor suppressor genes and activation of oncogenes. Tumor cells that adapt can bypass cell death processes through constitutive cell proliferation signaling pathways, which help them to attain immortality and sometimes induce angiogenesis and metastasis [4]. Cancer cells also possesses the capacity to alter their metabolism, avoid immunological destruction, and support tumor- related inflammation, genome instability and mutations [5]. Some tumor cells can spread to other parts of the body through the blood and lymphatic systems successfully metastasizing.

There are different types of cancer. Carcinomas are cancers that arise in the tissue or in skin that line or cover internal organs (epithelium). Sarcomas are cancers that begin in cartilage, bone, fat, blood vessels, muscle, or other connective or supportive tissues. Leukemias are cancers found in blood-forming tissue, like the bone marrow. These cause considerable numbers of anomalistic white blood cells to be formed and disrupt normal blood function. Cancers that initiate in the immune cells/tissues are called

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Texas Tech University, Soni Khandelwal, May 2018 lymphomas and multiple myelomas. Central nervous system cancers are found in the tissues of spinal cord and brain (National Cancer Institute).

BREAST CANCER

Breast cancer is the second leading cause of cancer-related death among women in United States [6]. Estimates of new breast cancer cases and related deaths for 2018 are predicted to be about 268,670 with about 41,400 deaths in the United States [3]. The statistics also show a significantly higher number of invasive cancers as compared to in situ cancer in women of all ages, although the overall trends show an increased incidence of in situ cancer as well. Paradoxically, there has been a reduction in the overall breast cancer mortality rates. This is most likely due to improved diagnostic techniques that have enabled early detection such as mammography [7]; magnetic resonance imaging [8]; detection of CA 15-3 (cancer antigen 15-3) [9, 10], HSP90A and PAI-1 (plasminogen activator inhibitor-1) [10].

ETIOLOGY AND RISK FACTORS

There are quite a few risk factors that influence individuals with an increased risk of breast cancer [11, 12]. The recognized major risk factors are as follows:

1. Gender – Females are 100 times more prone to breast cancer as compared to males [13].

2. Age – The risk for breast cancer increases with age for all ethnic groups [7].

3. Ethnicity - Incidence is higher in non-Hispanic white women as compared to African American women of all ages [1]. In contrast to other ethnic groups, African American women have a higher risk of mortality due to breast cancer [14].

4. Family History/Genetics – There are two types of genetic mutations - acquired and germline [15]. Acquired mutations are caused due to tobacco, ultraviolet (UV) radiation, viruses, and age. Cells with acquired mutations may lead to sporadic cancers. In contrast,

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Texas Tech University, Soni Khandelwal, May 2018 germ line mutations are passed from parents to the child. Germline mutations also lead to inherited cancer and account for 5% to 10% of all the cancers [15, 16].

A family history of breast cancer also contributes to an increased risk of breast cancer [17]. The major causes of breast cancer are mutations in tumor suppressor genes like p53, PTEN, BRCA1 and BRCA2; or activation of proto-oncogenes like ras, myc [18]. Overall 20-40% of hereditary breast cancer cases are known to be due to mutations in BRCA1 [15]. Sometimes mutations in DNA repair genes may also lead to breast cancer [19]. However, the causes for the remaining breast cancers show no discernible pattern. Confounding this is the genetic predisposition that patients with ovarian and uterine cancers are more prone to breast cancer [20].

5. Endocrine – Several endocrine factors are known to increase the risk of breast cancer. Abnormalities in menstrual cycles, early on set menarche or late on set natural menopause are correlated to breast cancer [15, 21, 22].

6. Exogenous Hormonal – Hormone replacement therapy (HRT) with estrogen and/ or progestin appears to increase the risk of breast cancer in postmenopausal women [23].

7. Diet and Lifestyle –Diet and Lifestyle play an important role in the etiology of breast cancer. An association between dietary fat and breast cancer is seen in postmenopausal women [24]. Alcohol intake also increases the risk of breast cancer [25]. Studies have shown a significant correlation between breast cancer occurrence and obesity in post- menopausal women [26].

8. Circadian Rhythm – Studies suggest an association between increased incidence of breast cancer and disruption of circadian rhythm [27]. It was observed that disturbance of light and dark cycles can disturb melatonin homeostasis [28]. The risk of breast cancer was increased by 48% in women working on night shifts [29]. This suggests an important role of the circadian rhythm optimal exposure to light and dark cycle in reducing the risk of breast cancer.

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9. Parity status – As per some studies, age of first birth and the parity status of a female are associated with incidence of breast cancer [30]. Some studies have shown a 30% increased risk of breast cancer due to null parity [31] but no direct evidence exists that can correlate breast cancer incidence with parity status [32]. However, a strong correlation between reduced risk of breast cancer and breast feeding has been observed [33].

10. Others – Several other factors are also known to increase the risk of breast cancer. For example, infections with oncogenic viruses like MMTV [34], radiation exposure [35], physical inactivity [36], cigarette smoking [37], past history of Hodgkin’s disease, other cancers like ovarian and uterine cancer are associated with breast cancer occurrence [36].

CLASSIFICATION OF BREAST CANCERS

Breast cancer is a heterogeneous disease with some sub-types. Breast cancer can be classified based on histology, tumor size and stage or molecular characteristics of the tumor. The following information was collected from National Cancer Institute and Susan G. Komen Foundation website.

A. Histological classification: Structurally, breast tissue consists of 8-10 lobes and each lobe consists of several lobules. The lobes and lobules are interconnected by ducts that carry milk. Based on the normal histology, breast cancer can be classified into four major types:

I. Lobular carcinoma in situ (LCIS) - LCIS is a pre-cancerous lesion and an indicator for the development of breast cancer. It is defined by the presence of abnormal cells in the lobules of breast tissue that are still encapsulated within the basement membrane.

II. Ductal carcinoma in situ (DCIS) – DCIS is defined as the presence of abnormal cells in the milk ducts that are still encapsulated within the basement membrane.

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III. Invasive lobular carcinoma (ILC) - ILC is the most common form of invasive breast cancer after ductal carcinoma, accounting for 5% to 10% of all invasive breast cancers. ILC originates in the lobules but becomes progressively malignant and eventually invasive.

IV. Invasive ductal carcinoma (IDC) – IDC or infiltrating ductal carcinoma originates in the milk ducts, becoming progressively more malignant invading neighboring tissues. IDC represents about 80 % of all diagnosed breast cancer cases.

B. Molecular classification: Based on the gene expression pattern of cancer cells, a phenotypic classification for breast cancer has been proposed. Gene expression analysis for breast cancer is called PAM50 [38]. It is commercially available but whether it presents an advantage over conventional testing of hormone receptors and HER2 status toward drug design is not known. The following information on molecular classes of breast cancer was collected from National Cancer Institute and Susan G. Komen website. I. Luminal A and Luminal B Tumor Types: The Luminal tumors are closer to normal cells of the breast ducts and glands and are estrogen receptor (ER) positive. Luminal cancers are further classified as type A and type B. Luminal type A is a low-grade cancer and contributes to 30-70% of total breast cancer cases. Considered low grade tumors, these tumors progress very slowly and have better prognosis. Luminal type B cancers represent 10-20% of breast cancer cases, are generally of higher grade and grow faster than Luminal A cancers.

II. HER2: This classification was included due to a high expression of Human Epidermal Growth Factor Receptor 2 (HER2) in a subset of patients. HER2 overexpression is associated with poor prognosis and reduced hormonal therapeutic outcomes, but these cancers are more responsive to chemotherapy due to increased proliferation. The HER2 positive tumors being more aggressive can be treated with targeted therapies like Trastuzumab (Herceptin®), Pertuzumab and Lapatinib (Tykerb). However, these Her2 tumor cells frequently develop 5

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resistance to therapy. For example, JIMT-1 cells are Trastuzumab resistant. The progression free survival advantage of Trastuzumab is lost after the drug treatment is stopped.

III. Basal type: These are usually triple-negative (TN) breast cancer cells, as they lack estrogen receptors, progesterone receptors as well as the HER2 receptor. These TN breast cancers contribute to 15-20% of all breast cancer cases. The genetic patterns of these cancers are similar to cells in the deeper basal layers of the breast ducts and glands. Basal like and TN are mostly BRCA-1 related mutations. These tumors are highly aggressive with a poor prognosis. These tumors are difficult to treat by hormonal or targeted therapies due to the absence of estrogen/progesterone and HER2 receptors. Lacking the three receptors, chemotherapy is mainly used to treat basal cancer types.

STAGES OF BREAST CANCER

Breast cancer can be divided into four major stages based on the extent of progression and advancement of the disease. Cancer can spread via tissues, blood or lymphatic systems. Generally, an invasive tumor spreads to the neighboring tissues spontaneously but it needs conduits like blood vessels or lymphatic vessels to spread to other parts of the body.

Stage 0 – Carcinoma in situ (ductal or lobular). Stage I - Tumor is usually very small and have started to invade. This stage is subdivided into stage IA and IB. Stage IA – Tumor is less than 2 cm and no lymph nodes are involved. Stage IB – No tumor in breast or less than 2 cm and cell cluster ranging from more than 0.2 mm but less than 2 mm present in lymph nodes. Stage II – Tumor is small with metastasis to axillary lymph nodes. This stage is divided into two sub-stages IIA and IIB. Stage IIA – No tumor in breast but cell cluster greater than 2 mm present in 1-3 axillary lymph nodes or lymph nodes near sternum (breast bone) OR tumor is about 2 cm and

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Texas Tech University, Soni Khandelwal, May 2018 spread to axillary lymph nodes OR tumor ranges from 2-5 cm but not spread to lymph nodes. Stage IIB - Tumor size ranges from 2-5 cm and cell clusters (0.2-2 mm) present in lymph nodes OR tumor size ranges from 2-5 cm and spread to 1-3 axillary lymph nodes or lymph nodes near sternum OR tumor is larger than 5 cm but not spread. Stage III – Tumor spread to distant lymph nodes or even to the skin of the breast. This is subdivided into IIIA, IIIB and IIIC.

Stage IIIA - No or small tumor in breast and cell clusters in 4 to 9 axillary lymph nodes or in the lymph nodes near the sternum OR tumor is larger than 5 cm and cell clusters (0.2 – 2 mm) are found in the lymph nodes OR tumor is larger than 5 cm and cell clusters are found in 1 to 3 axillary lymph nodes or lymph nodes near the breast bone.

Stage IIIB - Tumor may be of any size and has spread to the chest wall (near collar bone clavicle) and/or skin of the breast and caused swelling or an ulcer and may have spread to 9 axillary lymph nodes OR may have spread to lymph nodes near the breast bone sternum. Stage IIIC – No tumor in breast or a tumor of any size and may have spread to the chest wall and/or the skin of the breast and the cancer has spread to 10 or more axillary lymph nodes OR the cancer has spread to lymph nodes above or below the clavicle OR the cancer has spread to axillary lymph nodes or to lymph nodes near the sternum.

Stage IV - Cancer has spread to other organs of the body, most often to the bones, lungs, liver, or brain.

TNM staging of breast cancer: The most common system used to describe the stages of breast cancer is the TNM system given by the American Joint Committee on Cancer (AJCC). T = tumor size (0-4) N = Lymph nodes affected (0-3) M = Metastasis (0 or 1)

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This classification grouped with stages is used to grade the breast cancer spread and size. This classification helps in designing the optimal therapeutic options for breast cancer patients.

TREATMENT FOR BREAST CANCER

Cancer staging is useful for planning and deciding a best therapeutic regimen for the patient. The treatments can be either local or systemic. The two main goals of treatment are to eliminate, shrink or limit the growth of existing tumors and secondly to prevent the relapse. To achieve this, the most common approach is used for treating breast cancer patients is combinatorial. Some therapeutic options are accessible depending on the stage of cancer.

1. Surgery – Surgery is done to remove the tumor. It includes breast conserving surgery and mastectomy. Options for surgical procedure include a sentinel lymph node biopsy and an axillary (armpit) lymph node dissection. Surgery can remove about 70-80% patients with stage I cancer and about 50% patients with stage II cancer [39, 40]. 2. Radiation - Radiation therapy is performed using a high-energy X-ray beam or other type of radiation to destroy cancer cells or to prevent their growth. Radiation therapy can be external or internal. External radiation therapy uses a radiation source placed outside the body, whereas, internal radiation therapy uses a sealed radioactive isotope placed directly into or near the tumor. Radiation therapy may be used to treat breast cancer at almost every stage and it is often effective to preventing the relapse and spread of the cancer [39]. 3. Chemotherapy – Chemotherapy is the most commonly used therapy for cancer. In this treatment small molecules are used to suppress cancer growth and progression. Chemotherapeutic drugs can be divided into several classes based on their mechanism of action and their chemical structure [40]. a. Alkylating agents - Alkylating agents act directly upon and damage DNA by attaching an alkyl group (CnH2n+1) to the n7 nitrogen of guanine. These agents are not selective for any phase. Due to their DNA damaging effects, their long-term use can cause

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Texas Tech University, Soni Khandelwal, May 2018 severe bone marrow toxicity, which can eventually lead to acute leukemia [41]. There are five sub-classes of alkylating agents: if. Nitrogen mustards: e.g. ,

ii. : e.g. , (BCNU) and

iii. Alkyl sulfonates: e.g.

iv. Triazines: e.g. (DTIC) and

v. Ethyleneimines: e.g. and (hexamethylmelamine) b. The Platinum Drugs (, , and oxalaplatin). These agents also cause DNA damage by crosslinking DNA due to platinum binding, leading to DNA damage [41]. c. - These agents interrupt the synthesis of nucleic acids required for cell growth and proliferation. These agents are cell cycle specific and act on the S phase of cell division, during which the nucleic acids are synthesized. Examples of antimetabolites include 5-, hydroxyurea, [41]. d. Anti-tumor Antibiotics interfere with enzymes required for DNA replication. These drugs work in all phases of the cell cycle. Examples include , , and . However, use of anthracyclines are associated with dose-dependent cardiotoxicity. Other Anti-tumor Antibiotics – Non- antibiotics include actinomycin-D, , mitomycin-C and [41]. e. Topoisomerase Inhibitors – Topoisomerases are enzymes required for DNA replication during cell division. Hence, inhibition of topoisomerases causes suppression of cell proliferation. Examples include (topoisomerase I inhibitior), (topoisomerase II inhibitor). However, the side effects of these drugs are associated with an increased risk of leukemia [41].

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Texas Tech University, Soni Khandelwal, May 2018 f. Mitotic Inhibitors – These are plant-derived compounds and inhibit cell mitosis. Mitotic inhibitors are more effective in M phase. However, they can have some effects in all the phases of cell cycle. Examples include (, ), (), vinca alkaloids (, , ) and estramustine. Major potential toxicity associated with mitotic inhibitors is peripheral nerve damage [41]. 4. Hormone Therapy – Breast cancer is a hormonal cancer and tumor growth is most dependent on estrogen hormone levels in many cases. Hence, inhibition of hormone synthesis or blocking of their cell division function can suppress tumor growth. Different therapeutic strategies have been utilized to exploit this hormonal dependency [42]. a. Disruption of Ovarian Function – This is done by Oophorectomy or by radiation. Ovarian function can be suppressed by gonadotropic releasing hormone agonists such as luteinizing releasing hormone (LHRH), which inhibits the stimulation of the ovaries by the pituitary gland to release estrogen. Examples are goserelin and leuprolide [43]. b. Inhibition of Estrogen Synthesis - Aromatase is a major enzyme that is required for the synthesis of estrogen. Inhibition of Aromatase is used to block estrogen synthesis. In postmenopausal women, estrogen is synthesized only by peripheral tissues whereas in premenopausal women, the ovaries produce the majority of circulating estrogen. Aromatase inhibitors (AI) are primarily useful in postmenopausal women [44]. In premenopausal women, ovaries produce estrogen in such relative large quantity that AIs cannot inhibit it completely. Examples of this class of anticancer drugs include anastrozole, letrozole (temporary effect) and exemestane (permanent effect). Temporary effect refers to the reversible binding of the drug to the Aromatase by competitive inhibition. This inhibits the conversion of androgens to estrogens in peripheral tissues. However, exemestane is an irreversible Aromatase inhibitor, structurally related to the natural substrate androstenedione. It mimics the substrate for the Aromatase, and an intermediate is formed that binds irreversibly to the active site of the enzyme leading to permanent non-competitive inactivation. This effect is also known as "suicide inhibition" [45, 46].

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Texas Tech University, Soni Khandelwal, May 2018 c. Inhibition of Estrogen Activity: Estrogen binds to its cell receptors to exert its effects on cell division. Thus inhibition of the interaction between estrogen and its receptors can help block cell division induced by estrogens. These agents are called selective estrogen receptor modulators (SERMs). Examples include tamoxifen, raloxifene and toremifene. These drugs can also have a partial estrogen mimicking effect due to binding to estrogen receptors. However, SERMs can also act as estrogen agonists or antagonists in an organ specific manner. For example, tamoxifen acts as an antagonist in breast tissue but as an agonist in uterus and bone. In addition to SERMs, there are additional anti-estrogen drugs like fulvestrant. that have only an antagonistic action [47].

5. Biological Therapy – Cancer treatment with biologic vaccines or large molecules, such as monoclonal antibodies are a relatively new form of cancer therapy. Most of these therapies potentiate the body’s immune system or mimic the immune system. Recently, vaccines have been introduced into the cancer therapy regimen. In 2010, the FDA approved the first vaccine; Provenge® to treat advanced prostate cancer. The search for other cancer vaccines is ongoing.

TRIPLE NEGATIVE BREAST CANCER

Breast cancers are classified based on molecular markers. These molecular markers are the estrogen, progesterone hormone receptors and amplification of the HER- 2/Neu portion of the EGF family of receptors. [48]. Of all breast cancers, 10-15% are of the TNBC genotype.[49]. By definition, triple negative breast cancers (TNBC) lack amplification of all three molecular markers. As a result, they lack therapeutic targets and are associated with poor prognoses [49]. However, there are other molecular markers such as VEGF [50], EGFR [51], Src [52] and mTOR [53].

Breast cancer cell lines are important for contributing to in vitro studies as they can be cultured easily and information about their proteome, transcriptome, genome and epigenome can be evaluated. There are about 27 TNBC lines. MDA-MB-231 was established in 1970’s. Currently, MDA-MB-231 is widely studied to identify gene and

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Texas Tech University, Soni Khandelwal, May 2018 pathway involved in metastasis. In the present study, MDA-MB-231, MDA-MB-468 and HME50-5E cell lines were used. MDA-MB-231, MDA-MB-468 and HME50-5E are adherent epithelial cells. MDA-MB-231 cells grow with stellate colony-disorganized nuclei and elongated cell body. Stellate projections are associated with more invasive phenotype [54, 55]. In contrast, MDA-MB-468 grows with a grape-like cluster morphology and is less aggressive [55]. HME50-5E have cuboidal cobblestone-like appearance [56]. Other details of MDA-MB-231, MDA-MB-468 and HME50-5E [56, 57] are summarized in Table I-1.

Table I-1: Human Triple Negative Breast Cancer Cells and the Immortalized Mammary Epithelial 50-5E Cell Line. Adapted from [48]

Ethnicity Molecular p53 BRCA1 PI3K Classification Cell Line Pathology Grade Age (years) MDA- Adenocarcinoma Poorly 51 Caucasian Basal B Mutation Wild Wild MB-231 differentiated type type MDA- Adenocarcinoma Poorly 51 Black Basal A Mutation Wild PTEN MB-468 differentiated type homo deletion HME50- Li-Fraumeni 31 Hispanic 5E Syndrome (LFS)

SELENIUM: ESSENTIAL AND TOXIC MICRONUTRIENT

In the Periodic Table, Selenium occurs in Period 4 Group 16 and is a non-metal element but having metallic properties it is also classed as a metalloid. Selenium has the atomic number 34 and an atomic mass of 79. Initially, selenium was only considered to be a toxicant and most of the early research on Se was targeted towards addressing this toxicity. Se is known to cause poisoning of horses and cattle grazing on seleniferious plants. By the 1950s Se was identified as a micronutrient and the last of the micronutrients to be established as dietary essential. Other major discoveries related to Selenium [58-64] are depicted in Figure I-1.

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Figure I-1: Timeline of Selenium

MAJOR FUNCTIONS OF SELENIUM IN THE HUMAN BODY

Selenium is incorporated as selenocysteine (Sec) in 25 selenoproteins [65, 66].

Some selenoproteins exist as anti-oxidant enzymes and are of great importance to human

health. The Table I-2 lists all the selenoproteins and their known function [67-70].

Table I-2: Functions of Selenoproteins in Human Body.

Selenoprotein Occurrence and Function Glutathione peroxidases (4 Usually a tetramer and 4 Sec residues. GPx1 is found in liver and kidney; GPx2 in isoforms: GP×1, GP×2, gastrointestinal epithelium; GPx3 synthesized by kidney and secreted into plasma, GP×3, GP×4) GPx4 has 3 isoforms (cytosolic, nuclear and mitochondrial).

Antioxidant enzymes, removes hydrogen peroxide, and lipid and phospholipid hydroperoxides.

GPx4 protects developing sperm cells from oxidative damage and later polymerizes into the structural protein required for stability/motility of mature sperm. Iodothyronine deiodinases (3 DI1 and DI3 are found in plasma membrane whereas DI2 is found in endoplasmic isoforms: DI1, DI2 and DI3) reticulum (ER). DI1 is found in liver and kidney, DI2 is most abundant in thyroid,

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heart, skeletal muscle, brown adipose tissue, and the central nervous system, DI3 is found in fetal tissue and placenta.

DI1 produces most of the circulating T3; DI2 produces peripheral T3; DI3 prevents overexposure of T3 to fetus. Thioredoxin reductases (3 TRx1 is present in the cytosol, TRx2 in mitochondria and TRx3 in testis. TRx1 isoforms: TRx1, TRx2 and regulates gene expression by redox control of binding of transcription factors to TRx3) DNA, maintenance of intracellular redox state. TRx2 helps to maintain cell viability and proliferation. Methionine-R-Sulfoxide Present in liver and kidney and reduces oxidized methionine. Reductase

Selenophosphate synthetase, SPS2 Required for biosynthesis of selenophosphate, the precursor of selenocysteine, and therefore for selenoprotein synthesis. Selenoprotein P (Sepp1) Produced in liver and is secreted into plasma. Human Sepp1 has 10 Sec residues (one in N terminal and 9 in C terminal) and rats have 7-8 Sec residues. It has molecular weight of 60 kDa and has 4 isoforms. Sepp1 delivers selenium to organs where apolipoprotein E receptor or megalin is expressed. It is the best characterized selenoprotein and has a stoichiometry of 7.5 gms atoms of selenium per mole as Sec [71]. Selenoprotein W (SelW) Found in muscle. It is the smallest selenoprotein of 6 kDa. Needed for muscle function and was the first selenoprotein to be associated with muscular disorders. Selenoprotein V (SelV) Function is unknown. Selenoprotein T (SelT) Present in kidney, heart, brain, and thymus. Role in regulating calcium equilibrium. Selenoprotein M (SelM) It is found in brain and plays a role in calcium regulation in the ER. Selenoprotein 15 kDa Takes part in reorganizing disulfide bonds or reduction of incorrectly formed disulfide bonds in misfolded glycoproteins bound to UDP-glucose. Selenoprotein O and I (SelO SelI is found in the ER. Functions are not known. and SelI) Selenoprotein H (SelH) Found in the nucleus. It is involved in glutathione synthesis and detoxification reaction. Selenoprotein S (SelS) It interacts with Derlin, a membrane integral protein of the ER. Selenoprotein K (SelK) Found in the ER. It is believed to have a function in calcium efflux in immune cells. Selenoprotein N (SelN) Found in the ER membrane of muscle. Deficiency is associated with muscular dystrophy.

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DISEASES ASSOCIATED WITH SELENIUM DEFICIENCY

Three diseases have been reported to be associated with severe selenium deficiency, based on their occurrence in areas with selenium poor soils and their reversal upon selenium (Se) supplementation. It should be noted that Se may be a cofactor in these diseases, with other factors contributing to their incidence or severity.

• Keshan disease, was first described as a juvenile cardiomyopathy in 1930s in the

Chinese medical literature [72]. Women and children were susceptible to the

development of Keshan disease, which had a high mortality rate. The average

intake of Se in Keshan disease endemic areas was as low as 10 µg/d. Autopsy

revealed cellular edema, mitochondrial swelling and overlapping striations of

fibrotic tissue, indicating multiple bouts of localized necrosis. Heart tissues from

Keshan disease victims contained Coxsackie viruses. Studies in selenium-

deficient mice demonstrated that Coxsackie virus B4 infection induced severe

heart pathology as compared to Se-adequate mice in the development of Keshan

disease [73].

• Kashin-Beck disease is a chronic, endemic osteo-arthropathy, accompanied by

joint necrosis [74]. This syndrome affects individuals in regions of Tibet, China,

and North Korea. While individuals with this disease show skeletal pathology,

they are not reported to develop dysfunction of other organs or tissues. A

polymorphism in the Gpx1 gene is considered to be a genetic risk factor for

Kashin-Beck disease. Average serum Se concentrations among residents of the

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Kashin–Beck prevalent areas was <11 ng/ml in contrast to 60–105 ng/ml without

mineral deficiency [75].

• Myxedematous endemic cretinism, is caused by thyroid atrophy and results in

mental retardation. Myxedematous endemic cretinism is found in central Africa

[76, 77].

SELENIUM TOXICITY

Selenosis, a condition defined by blood selenium levels > 100 µg/dL, may result in symptoms including gastrointestinal upset, hair loss, white blotchy nails, garlicy breath odor, fatigue, irritability, and mild nerve damage [78].

Chronic selenium toxicity in horses was likely described by Marco Polo, while traveling the “Silk Road” in China [79]. These horses nibbled poisonous plants and the resulting selenium toxicity was characterized by malformations of the hooves and hair loss. Alkali disease and blind staggers in livestock have been documented in areas with seleniferious shales, Se-accumulating plants, and alkaline soils. One recent incident involved 21 polo ponies [80]. These animals were injected with a vitamin-mineral supplement dose containing selenium levels at higher amounts than needed. The Biodyl formula normally called for 500 µg of sodium selenite per mL of formulary. However the amount of selenium added, contained 5000 µg of sodium selenite per mL in the Biodyl formulation. The lethal oral dose of sodium selenite to horses is 3300 µg or 3.3 mg/kg per body weight. This treatment resulted in higher liver selenium concentrations than normal and the horses began to die shortly before the United States Polo Open in 2009 due to acute selenium toxicity leading to extensive hemolysis [81]. Experimental chronic

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Texas Tech University, Soni Khandelwal, May 2018 selenium toxicity in laboratory animals has been shown to affect the major organs including the liver, spleen, kidneys, heart and pancreas [82]. High Se levels in waterways were found to cause congenital disorders in wetland birds and fish [83].

• Selenosis in humans is a rare event outside of accidental industrial exposures. An

episode of human selenium poisoning that occurred due to a manufacturing error

of a dietary supplement, resulted in a product that contained 27,300 μg of Se (182

times the amount of selenium as declared on its label) [84, 85]. The toxic

symptoms included fatigue, irritability and peripheral neuropathy in those taking

the excessive amounts. Significant adverse effects occurred within a few days to

weeks after consumption and included effects on hair, nails, and liver [84]. Motor

weakness, tingling in limbs, diarrhea, convulsions, and paralysis are some other

symptoms caused by Se intoxication. Extreme cases of selenosis can be lethal due

to cirrhosis of liver [86]. In order to prevent the risk of potential selenosis in

humans, the Institute of Medicine of the National Academy of Sciences has set

tolerable upper intake levels (TUL) for selenium at 400 µg/day [87], the Lowest

Observed Adverse Effect Levels (LOAEL) of 910 µg Se/day [88] and the No

Observed Adverse Effect Levels (NOAEL) of 200 µg Se/day [89, 90].

MECHANISM OF TOXICITY OF SELENIUM

Selenium is toxic as compounds of selenium that form selenides, selenopersulfides, or isoselenocyanates oxidize reduced thiols (RSH) generating

- superoxide (O2 ). Selenium is present in the catalytic site of GPx.

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In the presence of thiols (RSH) selenite, diselenides generate a selenide or selenolate anion (RSe-). For example, the reaction of selenite with glutathione initially generates a selenotrisulfide (RSSeSR). Upon further reduction an unstable reduced selenopersulfide is formed; (RSSeH), ionization at physiological pH forms the selenopersulfide anion, RSSe-. This selenide catalyst oxidizes additional thiols present cycling electrons through the selenium anion from thiols to oxygen forming superoxide

- (O2 ), as originally proposed by Seko (equation I-1) for inorganic selenite [91] and by

Chaudière for organic diselenides (Figure I-2) [92].

Figure I-2: Redox Cycling Between Selenium Compounds and GSH.

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(I-1)

Glutathione (GSH) and other thiols reduce diselenides (RSeSeR) to selenenylsufides (RSe-SG) and forms the RSe- a catalytic species. The one electron

- - transfer from RSe to oxygen (O2) yields superoxide (O2 ) and a selenyl radical (RSe*).

The superoxide is converted to hydrogen peroxide (H2O2) by superoxide dismutase. The hydrogen peroxide is reduced to water (this is the antioxidant property of GPx) forming a selenolate (RSeOH), which is associated with decay of selenyl radical to diselenides. This happens at low concentrations of selenium. At high concentrations, RSe- oxidizes GSH to

GSSG, which in turn depletes the intracellular GSH concentration and the cell is subjected to oxidative stress, the basis of selenium toxicity. Crampsie et al. [93] showed that selenocyanates and isoselenocyanates also continuously redox cycle and generate superoxide. In the present study, an organic selenocyanate has been attached to Folate,

Herceptin® and Avastin®, forming redox active Se-immunoconjugates used to target and induce cell death triple negative breast cancer (TNBC) cell growth.

Irregular redox reactions in cancer cells are well documented at different stages of cancer. Both the enzymatic and non-enzymatic redox systems are modulated in cancer cells, providing growth and survival advantages necessary for the progression of the disease. As reviewed elsewhere [94], the enzymatic defense systems comprise the expression of superoxide dismutase, catalase, glutathione peroxidase (GPx), 19

Texas Tech University, Soni Khandelwal, May 2018 peroxiredoxins and selenoproteins with antioxidant functionalities. These enzymes are increased in many cancer types and decreased in other types of cancer [95]. Such contrasting results indicate a complex and varied functional regulation of antioxidant enzymes in different tumor types. In the cytoplasm, the homeostatic ratio of (reduced)

GSH to (oxidized) GSSG may be as high as 100:1 In contrast, in the endoplasmic reticulum, the ratio is lower at approximately 3:1 [96]. Increased biosynthesis of glutathione (GSH) is often seen in many tumor cell lines. Increased GSH levels not only protect the cancer cells from reactive oxygen species (ROS)-induced oxidative stress but also plays a role in drug resistance. To overcome this resistance, several cytostatic drugs have been designed to target intracellular GSH homeostasis and antioxidant enzymes

[97].

SELENIUM IN CANCER PREVENTION

A study in 1996 reported a decrease in the rate of lung, colorectal and prostate cancers in the Se supplemented group of older Americans [98]. In contrast to these results, a more recent study on Se supplementation failed to show a reduction in the risk of prostate cancer among healthy men [99] most likely because varied forms of Se were used, in which selenomethionine was compared to a Se-enriched yeast [98]. Additionally, negative results may be due to Se status and genetic differences of individuals taking part in the trial [100-102]. However, most animal studies have demonstrated that both organic and inorganic forms of Se compounds can decrease tumor size at doses higher than minimum dietary concentrations. Selenium has been shown to specifically hinder cell growth and to induce programmed cell death in cancer cell culture models. These

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Texas Tech University, Soni Khandelwal, May 2018 outcomes have triggered an in-depth interest in the mechanism by which Se can negatively impact cancer progression. There are numerous Se studies that have concentrated on the role that dietary minerals play in cancer prevention while less attention has been given to the toxic effects of selenium that may serve as a means to treat cancer [103]. Table I-3 lists relevant studies that show the importance of selenium as an anti-cancer agent.

Table I-3: Effects of Different Forms of Selenium on Cancer.

Study Study Design Selenium Form and Dose Population Outcome Weisberger et Case series (N=4) Selenocysteine average dose Acute Decrease in leukocyte al. [104] 100 mg/day, ranging from 50- leukemia, particle concentration 200 mg/day for 10-57 days Chronic counts. Reversible myeloid adverse effects like leukemia nausea, vomiting, anorexia, and finger nail destruction. No organ changes related to selenium toxicity was reported. Kasseroller Randomized 1000 µg of sodium selenite/ Secondary Selenium prevented [105] controlled (N=60) day in first week, 1300 µg/day lymphedema erysipelas. Adverse for weeks 2 and 3, then 200- post head and effects not recorded. 300 µg/day for 3 months. neck surgery Cumulative dose selenium ~17 mg Yu et al. [106] Randomized Selenized yeast as 200 µg Hepatitis Inhibition of liver placebo- Se/day surface cancer controlled trial antigen (N= 226) carriers Yu et al. [107] Population based Selenite in table salt; 15 mg of General Inhibition of intervention sodium selenite/kg of table salt population preneoplastic liver lesions

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Lee et al. In vitro study α-keto-ϒ-methylselenobutyrate LNCaP, PC-3, Cell growth inhibition. [108] and β-methylselenobutyrate DU-145 (prostate cancer)

Pinto et al., HT29, HCT Altered HDAC activity [109] 116 (colon and histone acetylation cancer) status in colon cancer cells. Gowda et al. In vivo study Bis(5-phenylcarbamoylpentyl) Melanoma Tumor inhibition [110] diselenide (SelSA-1) and 5- xenograft phenylcarbamoylpentyl model selenocyanide (SelSA-2) Gowda et al. Selenocoxib-1-GSH Melanoma Tumor inhibition [111] xenograft model Clark et al. Randomized, Selenized yeast as 200 µg Patients with No selenium toxicity [98], double blind, Se/day prior skin reported. Primary end Witherington placebo- cancer point: no effect on basal et al., [112] controlled trial cell or squamous cell Nutrition carcinoma. Prevention of Secondary end point: Cancer (NPC) Inhibition of lung, (N=1312) colon and prostate cancer.

TARGETED THERAPY

Targeted therapy for breast as well as other cancers is directed towards overly expressed cellular receptors as described for HER2, EGFR and the Folate receptor-alpha FRA. Monoclonal antibodies whose antigens are the targeting receptors target cancer cells blocking growth signals (e.g. Erbitux®; Herceptin®), inhibiting angiogenesis (e.g. Avastin®), delivering radioactive isotopes to cancer cells (e.g. Zevalin), and delivering chemotherapeutic drugs to cancer cells (e.g. Kadcyla®).

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Recently, folate receptors have been considered to be a therapeutic target for treating cancer [113-116]. Farletuzumab (Morphotek), a humanized FR targeted mAb showed effective preclinical results in xenograft models of and was under Phase 3 clinical trial in platinum-sensitive ovarian cancer patients. Unfortunately, the efficacy data of Farletuzumab were conflicting and did not meet the primary end point of progression free survival [117]. Vintafolide (Endocyte Inc), a folate cytotoxic drug conjugate was under Phase 3 clinical trial in platinum-resistant ovarian cancer patients. The trial was terminated early because pre-specified criteria (progression-free survival) for continuation of the study was unmet [118]. Vintafolide, a small molecule drug conjugate, targets the folate receptor and was under development of treatment of TNBC in a Phase II clinical trial. This trial is now withdrawn [119]. Since TNBC do not adequately benefit from conventional therapies and tumor associated Folate receptor- alpha seems to be a therapeutic target for breast cancer patients; in this study we discuss an in vitro treatment approach other than monoclonal antibodies for FRA.

Bevacizumab (Avastin®) is a 149 kDa, a humanized monoclonal immunoglobulin (IgG1) antibody [120] which blocks the vascular endothelial growth factor (VEGF) [121]. Avastin® (manufactured by Genentech/Roche) targets the VEGF protein, which is synthesized by tumor cells to induce angiogenesis. Bevacizumab attaches to the VEGF, which blocks the signals for new blood vessels formation. This monoclonal antibody is approved for use along with chemotherapy to treat kidney cancer, colorectal cancer, glioblastoma, lung cancer, cervical and ovarian cancer [120] and is being studied for use against other cancers, such as breast cancer.

The first example of a successfully targeted monoclonal mAb therapy is Trastuzumab (TZ, Herceptin®), first approved for clinical use by the FDA in 1998 [122]. TZ is a 148 kDa humanized monoclonal immunoglobulin (IgG1) antibody [123] developed to target the HER2 receptor that is over expressed in 25 to 30% of breast cancer patients [124, 125]. The mechanism of action of Trastuzumab is to bind to the extracellular domain of the HER2 receptor to prevent homo-dimerization or heterodimerization [126] and arrest the cell proliferation that the overexpressed HER2 23

Texas Tech University, Soni Khandelwal, May 2018 drives [127]. Trastuzumab checks the cell cycle via preventing phosphatidylinositol 3- kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways cascades [126, 128].

PROPOSAL AND GOALS

This present research study, stems from prior work in the laboratory which has focused on developing Se-labeled redox toxic compounds of existing clinical mAbs. This study then evaluated the cytotoxic effect of redox Se attached to Folate forming Selenofolate was employed to treat the TNBC cell lines MDA-MB-468, MDA-MB-231 as well as the HME50-5E mammary epithelial cell line. Additionally, Se covalently attached to the monoclonal antibodies; Avastin®, Herceptin®, against two triple negative breast cancer cells (TNBC) MDA-MB-231, MDA-MB-468 and an immortalized human epithelial mammary cell line HME50-5E. Additionally, Se attached to Folic acid forming Selenofolate was also employed to treat the same TNBC cell lines MDA-MB-468, MDA- MB-231 as well as the HME50-5E mammary epithelial cell line ex vivo.

The Se-conjugated anti-cancer antibodies, Herceptin® (Figure IV-4 in CHAPTER IV) and Avastin® (Figure III-4 in CHAPTER III) and the Se-conjugated vitamers Selenofolate (Figure II-3 and Figure II-4 in CHAPTER II) were tested in the present proposal in a dose and time dependent fashion to evaluate cancer and control cell death (apoptosis), inhibition of cell growth (metabolism), formation of reactive oxygen species (ROS) and peroxide radicals in a Se-concentration and time-dependent manner against the known toxicity of sodium selenite. The ultimate goal of all research from this laboratory is to improve the therapeutics of current clinically used monoclonal antibodies and targeting the Folate Receptor-alpha. The TNBC cells tested in the present studies using the redox selenium conjugates represent a difficult genotype of breast cancer to clinically treat. The experimental results speak for themselves.

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HYPOTHESIS

Redox selenium covalently labeled to Folate, Avastin® and Herceptin® will be more effective in preventing the cell proliferation of MDA-MB-231 and MDA-MB-468 cells in vitro than Folate, Avastin® and Herceptin® alone.

SPECIFIC AIMS

1. To covalently modify Folate and Monoclonal Antibodies-Avastin® and Herceptin® with redox Se. 2. To examine that Selenofolate, Selenoavastin and Selenoherceptin redox cycle generating superoxide using the Lucigenin Chemiluminescence assay in vitro and intracellular superoxide generation using Dihydroethidium (DHE) Assay in situ. 3. To determine the Cytotoxic and Therapeutic activity of Selenofolate, Selenoavastin and Selenoherceptin against two Triple Negative Breast Cancer Cell lines-MDA-MB-231, MDA-MB-468 and the normal mammary epithelial cell line HME50-5E.

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CHAPTER II

CYTOTOXICITY OF FOLATE, SELENOFOLATE AND SELENITE AGAINST

TRIPLE NEGATIVE BREAST CANCER CELL LINES MDA-MB-231 AND

MDA-MB-468

ABSTRACT

Previous studies have demonstrated that selenium compounds through their pro- .- oxidative activity by generating superoxide (O2 ) against cancer cells in vitro. Currently, there are no efficacious treatment options for people with triple negative breast cancer cells (TNBC). However, the association between overexpression of the folate receptor alpha (FRA) on TNBC and other cancer cells, has led to the possibility that TNBC might be treated by targeting the FRA with covalent folate drug conjugates. The present study attached the redox active compound, 1-propanol selenocyanate [HO(CH2)2SeCN] to folic .- acid by a DCC reaction forming Selenofolate. Superoxide (O2 ) generation by Selenofolate was assessed by an in vitro chemiluminescence assay and by the DHE in vivo assay. The cytotoxicity of Selenofolate was then compared to Folate, and selenite in a time and dose dependent manner against the TNBC cell lines MDA-MB-231, MDA- MB-468 and an immortalized mammary epithelial cell line, HME50-5E. Cells were treated with either 50 µM, 75 µM or 100 µM of Selenofolate or Folate on day 5 post- cellular inoculation. Selenite treatment was at 10 µM. Selenofolate treatment resulted in inhibition of cell proliferation as evaluated by Trypan Blue exclusion, MTT, and Annexin V assays. Folate receptor alpha (FRA) protein expression was assessed by Western blotting. The experimental results show that redox active Selenofolate and selenite, but not Folate uptake into FRA overexpressing TNBC cells is cytotoxic in vitro and suggests a possible clinical option for treating Triple Negative Breast Cancer.

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KEYWORDS

Selenium, Triple Negative Breast Cancer Cell lines, MDA-MB-231 cells, MDA-MB-468 cells, HME50-5E cells, Selenofolate, Sodium Selenite, Folate, Chemiluminescence, AnnexinV, Reactive Oxygen Species, Dihydroethidium

INTRODUCTION

Folate, folic acid or vitamin B9, plays an important role in the synthesis of purines and pyrimidines and other cellular methylation reactions including DNA production and repair [129]. Folate from foods (like spinach, Brussel sprouts, broccoli, asparagus, turnip greens, mushrooms, legumes and fortified foods) is absorbed in the intestines and released as 5-methyl-tetrahydrofolate (5-MTHF) into the bloodstream, taken up by cells of various tissues [130]. The four folate receptor isoforms (folate receptor alpha, FRA; beta, FRB, gamma, FRG; and delta, FRD) belong to a class of proteins that helps take up folic acid into cells, maintains folic acid homeostasis and may have effects on cellular proliferation [114, 131]. FRA, a 38 kDa single chain glycosylphosphatidylinositol (GPI)- anchored cell surface cysteine-rich glycoprotein [132]. FRA binds 5-MTHF, allowing the vitamin to enter into the cell [133]. The FOLR1 gene located on chromosome 11q13.3- q14.1, being responsible for the synthesis of FRA protein [134]. FRA is predominantly expressed on the apical surface of a subset of polarized epithelial cells [135, 136] including lung, parotid, thyroid, kidney, and breast cancers with a high affinity for folic acid and reduced [115, 137-139]. In normal breast cells, FRA expression is limited to the luminal borders of secretory cells, consistent with the secretion of FRA into breast milk [140]. FRA is produced in large amounts in the choroid plexus (a region in brain which is responsible for releasing cerebrospinal fluid) helping in transportation of folate from the bloodstream to brain cells [141]. In brain, folate is essential for the synthesis of neurotransmitters and myelin, which play a major role in transmission of nerve impulses in the nervous system. In kidney and normal intestinal cells, FRA helps in the absorption and re-absorption of folate from the luminal ‘exterior’ cavities to the absorbed ‘interior’ cellular environment [142, 143]. FRA and FRB are expressed in

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Texas Tech University, Soni Khandelwal, May 2018 placenta tissue which helps in folate transportation during different stages of fetal growth and development [132].

FRA was first discovered by Coney et al. in 1991 [144] in IGROV-1, a human ovarian carcinoma cell line. FRA is upregulated in ovarian, endometrial, colorectal, kidney, lung and breast carcinoma. According to Parker et al. [116] there is low or negligible expression of FRA levels in primary breast carcinoma, though Kelley et al. [145] confirmed higher expression among estrogen (ER-) negative primary breast cancers. In 2014, Zhang et al. [146] reported similar results that triple negative breast cancers express FRA. Furthermore, FRA over expression is associated with poor clinical outcomes. The limited distribution of FRA in normal tissues, and over expression of FRA in several cancers makes it a potential target for therapeutic purposes. The FRA has been targeted in ovarian cancer with 99mTc-etarfolatide, covalent folate florescent tags [147, 148] to enhance metastasized and ovarian surgical cancer treatment with a folate-drug conjugate of vinblastine; Vintafolide. The folate fluorescent tag helps to improve surgical outcome while Vintafolide failed its Phase 3 clinical trial [149].

For some time, folate receptors have been considered to be a therapeutic target for treating cancer [113-116]. Vintafolide (Endocyte Inc) having showed promising preclinical activity in TNBC xenograft models [150], it was under experimental treatment of TNBC in a Phase II clinical trial. This trial is now withdrawn [119]. Since TNBC does not adequately benefit from conventional therapies and tumor associated FRA seems to be a therapeutic target for many cancers overexpressing the FRA, Selenofolate seemed a potential small molecule for targeting cancer. Folic acid is composed of 3 fused components, a pteridine ring, para-amino benzoic acid and glutamic acid. Folate anchors to its receptor, FRα with the pterin head group buried inside the folate receptor [151].

Due to this interaction, 1-propanol selenocyanate [OH(CH2)2SeCN] was covalently attached to the carboxylic acid of the glutamate moiety of folic acid. The conjugate, .- Selenofolate, is observed in vitro to generate superoxide (O2 ) and because it produces oxidative stress it would be expected to be cytotoxic to cancers overexpressing FRA, including the TNBC lines MDA-MB-231 and MDA-MB-468. 28

Texas Tech University, Soni Khandelwal, May 2018

MATERIALS AND METHODS

Materials

DMEM high glucose (Catalog# 11965-092), Fetal Bovine Serum (Catalog# 10082-147), 1% Penicillin-Streptomycin (Catalog# 15-140-122), were purchased from Life technologies, Gibco. pH test strips (Catalog# 8882-1) were purchased from Ricca Chemical Company. PVDF membranes (Catalog# 1620177), Non-Fat Milk Protein (Catalog# 1706404), 3-20% Tris-Glycine Polyacrylamide Gel (Catalog# 456-1096) and 2X native PAGE sample buffer (Catalog# 161-0738) were purchased from BIO-RAD. Mammary Epithelial Cell Media (Catalog# 50-306-176) was purchased from PromoCell. Trypsin (Catalog# 30-2101) was purchased from ATCC. Rabbit Anti-Mouse IgG H&L HRP (Catalog# ab6728) was purchased from Abcam. 0.22 µM filter (Catalog# SLGV033RS), Anti-β-Actin Antibody (Catalog# MAB1501) and Accutase (Catalog# SCR005) from EMD Millipore. Corning® cell culture 75 cm2 (Catalog# CLS430641) and 25 cm2 (Catalog# CLS430639) vented cap flasks, Corning® Costar® TC-Treated Multiple Well Plates (Catalog# CLS3527), Corning® Costar® TC-Treated Multiple Well Plates (Catalog# CLS3548), Sodium selenite (Catalog# 214485), MTT (Catalog# M2128), Superoxide Dismutase (Catalog# S7571), Bovine Erythrocytes (Catalog# C1345-1G), 2′,7′-Dichlorofluorescin diacetate (Catalog# D6883), Dihydroethidium (Catalog# D7008) were purchased from Sigma-Aldrich. RIPA lysis buffer (Catalog# 89900), Anti-Folate Receptor Alpha antibody (Catalog# MA5-23917), SuperSignal™ West Femto Maximum Sensitivity Substrate (Catalog# 34095), Gamma Globulin (Catalog# 23212), BCA asay kit (Catalog# 23225), MitoTracker™ Red CMXRos (Catalog# M7512), Annexin V-FITC Apoptosis Detection Kit (Catalog# BMS500FI-300) were purchased from ThermoFisher Scientific.

Synthesis of 1-propanol selenocyanate

0.1 mol of 3-chloropropanol [Cl(CH2)3OH] and 0.1 mol of KSeCN were dissolved in 50 mL of acetonitrile. This mixture was refluxed for 4 hours as shown in Equation II-1. Acetonitrile was then evaporated under vacuum and the residue was taken 29

Texas Tech University, Soni Khandelwal, May 2018 up into water and extracted with diethyl ether (2X 50mL). The organic phase was washed once with water, dried with MgSO4, and evaporated. The propan-1-ol 3-selenocyanate obtained is used without further purification.

(II-1)

Synthesis of Selenofolate

Selenofolate (Figure II-2) was synthesized using one mmol (441 mg) of folate (Figure II-1) dissolved in 250 mL DMSO and 2 mL of pyridine and 1 mM DCC. The mixture was stirred for 30 minutes at room temperature. One mmol of the alcohol

[(NCSe(CH2)2OH] 1-propanol selenocyanate (140 mg) was then added and the mixture was stirred for 72 hours at room temperature.

Afterwards, the reaction mixture is poured into 500 mL of a mixture of diethyl ether and acetone (3:1) and stirred until a precipitate was formed. The solid precipitate was filtered and wash with ether and acetone. (It is possible that the solid has to be boiled in acetone to eliminate all DMSO). The yield of the red Selenofolate was about 60% and used without further purification.

Figure II-1: Structure of Folate, Molecular Weight: 441 g/mol

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Figure II-2: Structure of Selenofolate; Molecular weight: 573 g/mol Cell Culture

MDA-MB-231 and MDA-MB-468 cells were cultured in 75 cm2 tissue culture flasks and maintained in high glucose Dulbecco's Modified Eagle's Media (DMEM) supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin and cells were incubated for 2 to 3 days at 37°C under humid conditions in 5% CO2 incubator (ThermoFischer Scientific, Carlsbad, CA). The growth media was changed twice weekly. Cells were grown to 75–85% confluence then washed with 1X phosphate buffer saline (PBS, pH 7.4), trypsinized with 5 mL of 0.25% (v) trypsin-0.0.3% EDTA, diluted with fresh media, and counted using a Beckman Coulter ViCell analyzer (Beckman Coulter Inc., Model VI-CELL SGL).

HME50-5E cells were also cultured in 25 cm2 tissue culture flask and maintained in Mammary Epithelial Cell Media and were incubated for 7 to 8 days at 37°C under humid conditions in 5% CO2 incubator. The media was changed every other day. Cells were grown to 60-70% confluence then washed with ice cold 1X phosphate buffer saline (PBS), trypsinized with 5 mL of Accutase, diluted with fresh media, and counted using a Beckman Coulter ViCell analyzer (Beckman Coulter Inc., Model VI-CELL SGL).

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Detection of Superoxide In Vitro: Selenofolate

Lucigenin (Bis-N-Methylacridinium Nitrate) was used to detect superoxide in a chemiluminescent assay consisting of phosphate buffer and reduced GSH. The amount of superoxide generated is measured by the chemiluminescent produced light using a Turner TD-20e Luminometer (Turner Designs Inc., Mountain View, CA), connected to a circulating water bath. The source of electrons for superoxide is GSH upon oxidation and the reaction is catalyzed by some but not all Se compounds, including Selenofolate. Chemiluminescence measurement of Folate and Selenofolate was taken by adding different volumes (0 µL, 20 µL, 40 µL, 60 µL, 80 µL, 100 µL) of Folate (5 mg/mL) and Selenofolate (5 mg/mL) to 500 µL of the chemiluminescent cocktail at 37°C with integrations of 30 seconds over 5 minutes, N=10.

Detection of Intracellular Reactive Oxygen Species Accumulation In Situ: Superoxide Generation Assay

MDA-MB-231, MDA-MB-468 and HME50-5E cells were seeded at a density of 2X105 cells/well in 24 well flat bottom plate. After 48 hours, DMEM high glucose media without phenol red supplemented with FBS was added. 50 units/well of superoxide dismutase (SOD) from bovine erythrocytes and 100 units/well of catalase from bovine liver in media were added to all wells. All cells-control, Selenite (20 µM), Folate (100 µM) and Selenofolate (100 µM) and were appropriately treated for 30 minutes. DHE was added at a final concentration of 10 µM in each well. Cells were visually assessed using a fluorescence microscope EVOS FL Auto Cell Imaging System (ThermoFisher Scientific, Carlsbad, CA) or Cytation 3 plate reader (BioTek, Winooski, VT, USA).

Optimization of Cell Density for Treatment

Log growth cell curves in 48 well plate was performed by seeding different densities of cells, 5,000; 10,000; 20,000; 40,000 and 100,000 cells/well and cell growth was plotted against time for 7 days. This was done to determine and assure a logarithmic growth of cells throughout the experimental time period. 32

Texas Tech University, Soni Khandelwal, May 2018

Cell Treatments

All experiments were performed in a cell culture hood in an aseptic environment. Exponentially growing cells were harvested from flasks, counted by the Trypan Blue exclusion, and plated into 48 well plates at the predetermined optimal seeding density of 40,000 cells (MDA-MB-231, MDA-MB-468, HME50-5E) per well. Cells were allowed to grow for 5 days post seeding prior to day 0 of treatment. Media was changed on day 3 post seeding and on day 5 treatments were begun with the addition of fresh culture media.

Folate was used as a control for Selenofolate. Sodium selenite (2.2 mg of sodium selenite containing 1 mg of Se) was used as a positive control for cell-death. Control cells were treated with 1X PBS. Folate, Selenofolate or selenite was added to wells in final concentrations to MDA-MB-231, MDA-MB-468 cells and an immortalized human mammary epithelial cells, HME50-5E. Folate (1 µM, 5 µM, 25 µM, 50 µM, 75 µM, 100 µM), Selenofolate (1 µM, 5 µM, 25 µM, 50 µM, 75 µM, 100 µM) and selenite (4 µM, 10 µM, 20 µM and 40 µM) were tested in triplicate in 48 well plates and analyzed on days 0,1, 2,3,4,5,6 and 7 days for cytotoxicity and cell viability. For conversion of Selenite and Selenofolate from µM to µg of Se, please refer Table II-1.

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Table II-1: Conversion of Selenite and Selenofolate from µM Se to µg Se

Name of the Compound µM Se µg Se

Selenite 4 µM 0.04 µM 2 µg

Selenite 10 µM 0.1 µM 5 µg

Selenite 20 µM 0.2 µM 10 µg

Selenite 40 µM 0.4 µM 20 µg

Selenofolate 1 µM 0.14 µM 0.08 µg (80 ng)

Selenofolate 5 µM 0.7 µM 0.4 µg

Selenofolate 25 µM 3.5 µM 2 µg

Selenofolate 50 µM 7 µM 4 µg

Selenofolate 75 µM 10.5 µM 6 µg

Selenofolate 100 µM 14 µM 8 µg

Visual Assessments of Cellular Morphology

A phase-contrast microscope, EVOS XL Core (Life Technologies, Carlsbad, CA) was used for photographing cell morphological changes due to Folate, Selenofolate and Selenite treatments.

Cell Viability Measured by Trypan Blue Exclusion

Cell viability from control and experimental treated cells was determined using a Beckman Coulter Vi-Cell Viability Analyzer (Beckman Coulter, Inc. Model VI-CELL SGL) with viability based on Trypan Blue cell exclusion.

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Experimental Cell Cultures

TNBC and the immortalized breast epithelial cells; 40,000 cells/well were seeded in 48 well flat-bottom plate, cell volumes were adjusted with media and cells were allowed to grow for 5 days prior to treatments. Media was changed on day 3 post seeding and just before treatment. Cells were treated with 5 µM, 25 µM, 50 µM, 75 µM and 100 µM of Folate and Selenofolate for 7 days in triplicate. Cells were treated with selenite 2 µg, 5 µg, 10 µg and 20 µg as Se.

On days 0 through 7 of treatments; 500 µl of media (containing treatments) from each well was collected in a coulter cup. The cells were rinsed with 500 µl of 1X PBS (kept in incubator for 5 minutes) and this 1X PBS was collected in coulter-cups. 200 µl of 0.025% trypsin-EDTA was added to harvest adherent cells. After 5 minutes of incubation, trypsin was added to the coulter-cups containing media and 1X PBS. Wells were observed to make sure all adherent cells were collected under the microscope. Cells were analyzed with the Beckman Coulter Vi-Cell Viability Analyzer for this experiment with the following settings and criteria:

Cell type: BrCa cells Minimum diameter (microns): 12 Maximum diameter (microns): 50 Cell brightness (%): 85 Cell sharpness: 100 Viable cell spot brightness (%): 65 Viable cell spot area (%): 5

MTT Assay

TNBC and the immortalized breast epithelial cells; 40,000 cells/well were seeded into 48 well flat-bottom plates, volumes were adjusted with media, and cells were allowed to grow for 5 days prior to treatment. Cells were treated (on day 5 post seeding) 35

Texas Tech University, Soni Khandelwal, May 2018 with 0 µM, 5 µM, 25 µM, 50 µM, 75 µM and 100 µM of Folate or Selenofolate for 6 days. Blanks without cells contained complete growth media (Phenol red free DMEM high glucose + 10% FBS). MTT dissolved in phenol red-free media, 5 mg/mL was passed and sterilized through a 0.22 µM filter inside the cell culture hood. The tube containing the MTT solution was wrapped with foil paper and used immediately. To each well 10% (v/v) i.e., 50 µl MTT (5 mg/mL) was added and incubated at 37oC for 3 hours. Following incubation with MTT, formazan solubilization solution [acidified isopropanol (0.1N HCl) with 10% Triton X-100] equal to the original volume of media was added in each well. Dissolved Formazan in each well was determined by a Cytation 3 plate reader (BioTek, Winooski, VT, USA) at 570 nm absorption with baseline subtraction of 690 nm absorption to assess cell viability. The control cells were considered to be 100% viable. Cell viability percentage was calculated using following equation II-2.

퐴푏푠표푟푏푎푛푐푒 표푓 퐹표푙푎푡푒 표푟 푆푒푙푒푛표푓표푙푎푡푒−푡푟푒푎푡푒푑 푐푒푙푙푠 퐶푒푙푙 푣푖푎푏푖푙푖푡푦 (%) = 푋 100 (II-2) 퐴푏푠표푟푏푎푛푐푒 표푓 푐표푛푡푟표푙 푐푒푙푙푠

Cell viability data was then used to calculate the 50% cell inhibition concentration; 50% (IC50).

MitoTracker® Red and Annexin V Staining

Into 48 well flat-bottom plates 40,000 cells/well were seeded were allowed to grow for 5 days prior to treatment. Media was changed on day 3 post seeding and volume was adjusted before treatment. Cells were treated (on day 5 post seeding) with selenite (2 µg Se/well); 50 µM, 75 µM and 100 µM of Folate and Selenofolate and incubated for 2 and 3 days of treatment. Additionally, the control cells were untreated. Another set of control cells for staining were prepared - unstained control was untreated+unstained. For necrosis control (double-negative), cells were treated with 50 µl of 0.01% Triton X-100 for 30 minutes prior to staining or 200 µl of H2O2 for 24 hours prior to staining. For single staining controls, appropriate wells were treated with the dyes separately (Annexin V – 488 were treated with apoptosis inducer i.e., 5 µl of 1 mM Sutent; MitoTracker® Red-untreated cells). For double staining controls (double-positive), appropriate wells

36

Texas Tech University, Soni Khandelwal, May 2018 were treated with both dyes of the same volume. These controls were set aside in 2 mL Eppendorf tubes. Each treatment was repeated in triplicate.

A 10 mM stock solution of MitoTracker® Red dye was prepared by adding 9.4 μL DMSO to a vial of MitoTracker® Red dye. A 10 μM working solution of the MitoTracker® Red dye was prepared by pipetting 1 μL of 10 mM MitoTracker® Red stock solution into 1,000 μL of DMEM high glucose phenol red free cell media. 2 μL of 10 μM MitoTracker® Red working solution was added to each well (500 µl) and stained for 30 minutes at 37°C in an atmosphere of 5% CO2. After 30 minutes of incubation, the media was collected in a sterile 2 mL Eppendorf tube. The cells were washed with 500 µl of 1X PBS and collected in the appropriate Eppendorf tube. Cells were trypsinized using 200 µl of 0.025% trypsin-EDTA and centrifuged. Cells were resuspended in 100 µl of 1X Annexin-binding buffer. This suspension was transferred into 96 well flat-bottom plates. 4 µl of Annexin V dye was added to each well. The 96-well plate was wrapped in aluminum foil paper. The cells were incubated for 15 minutes at 37°C in an atmosphere of 5% CO2. After the incubation, 100 µl of 1X Annexin-binding buffer was added. The plate was placed immediately on ice. The stained cells were analyzed by flow cytometry on the Attune™ NxT Flow Cytometer (ThermoFisher Scientific, Carlsbad, CA). The laser lines were set to BL1 (200 nm) and YL1 (336 nm). Voltage for Forward Scatter was set to 120 nm and Side Scatter to 290 nm.

Western Blotting

One million MDA-MB-231, MDA-MB-468 and HME50-5E cells were allowed to attach to T-25 flasks overnight. The following day cells were treated with 50 µM, 75 µM and 100 µM of Folate or Selenofolate and incubated for 3 days. Control cells were treated with 1X PBS and incubated in the media with using same time, 3 days.

On day 3 of treatment, cultured cells fluid was collected in a 15 mL centrifuge tube and cells were washed with ice cold 1X PBS in the same tube. The tube was centrifuged at 4000 rpm for 4-5 minutes and the media was aspirated. 200 µl of RIPA lysis buffer was added to the tube and everything was transferred to T25 flask. T25 flasks 37

Texas Tech University, Soni Khandelwal, May 2018 were kept at -80oC for one day. This was done to get a better protein yield when the flask was thawed. Cancer cell lysates were collected upon thawed. To collect HME50-5E cell lysates, flasks were broken with a hammer and cells were scrapped and kept on ice for 5 minutes. HME50-5E lysates were passed through a 20 G needle and kept on ice for 5 minutes. All cell lysates were kept on an inverter for 15 minutes at 4°C and centrifuged at 12,000 rpm for 15 minutes at 4°C. Total protein concentration in the cleared lysate was determined by the BCA assay according to manufacturer’s instructions. Fifty µg of total protein was separated on 8% denaturing polyacrylamide gels and transferred to PVDF membranes by electroblotting. Membranes were blocked for 1 hour with Phosphate- buffered saline containing 0.05% Tween-20 (PBST) and 5% non-fat milk protein. They were incubated overnight with an anti-folate receptor alpha antibody diluted 2 µg/mL in PBST or anti-β-actin antibody diluted 1:1000 in PBST containing 1% non-fat milk protein. The next day, the membrane was washed 3 times for 45 minutes in PBST, incubated for 1 hour with horse-radish peroxidase conjugated with rabbit anti-mouse IgG diluted 1:10000 in PBST, and washed once in PBST for 15 minutes. Bound antibody complexes were visualized using SuperSignal™ West Femto Maximum Sensitivity Substrate.

STATISTICAL ANALYSES

All experimental assays were conducted in triplicate and the data are representative of three independent experiments. The results are expressed as the mean ± standard error (SE). Statistical analyses were performed using MATLAB (2017A) two sample t test across treatments. Differences were considered significant at p≤0.05. IC50 was calculated using a two-parameter sigmoidal model. The results of the comparative statistical analyses are summarized in Table II-2, Table II-3 and Table II-4.

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Table II-2: Statistical analyses of Folate and Selenofolate treatments against MDA-MB-231 Cells

Experiment Treatments p value Day 3 Trypan Blue Folate 75 µM vs Selenofolate 75 µM 0.005 (Figure II-13) Folate 100 µM vs Selenofolate at 100 µM 0.001 Day 4 Trypan Blue Control vs Selenofolate 75 µM 0.015 (Figure II-13) Control vs Selenofolate 100 µM 0.003 Folate 25 µM vs Selenofolate 25 µM 0.009 Folate 50 µM vs Selenofolate 50 µM 0.005 Folate 75 µM vs Selenofolate 75 µM 0.001 Folate 100 µM vs Selenofolate 100 µM 0.001 Day 5 Trypan Blue Control vs Selenofolate 75 µM 0.006 (Figure II-13) Control vs Selenofolate 100 µM 0.001 Folate 75 µM vs Selenofolate 75 µM 0.001 Folate 100 µM vs Selenofolate 100 µM 0.001 Day 3 MTT Control vs Selenofolate 25 µM 0.002 (Figure II-16) Control vs Selenofolate 50 µM 0.025 Control vs Selenofolate 75 µM 0.008 Control vs Selenofolate 100 µM 0.002 Folate 75 µM vs Selenofolate 75 µM 0.002 Folate 100 µM vs Selenofolate 100 µM 0.003 Day 4 MTT Control vs Selenofolate 50 µM 0.004 (Figure II-16) Control vs Selenofolate 75 µM 0.002 Control vs Selenofolate 100 µM 0.001 Folate 75 µM vs Selenofolate 75 µM 0.001 Folate 100 µM vs Selenofolate 100 µM 0.001 Annenix V Folate 75 µM vs Se-Folate 75 µM (late apoptosis) 0.002 (Figure II-23) Folate 100 µM vs Se-Folate 100 µM (late apoptosis) 0.008 Control vs Se-Folate 75 µM (late apoptosis) 0.004 Control vs Se-Folate 100 µM (late apoptosis) 0.006

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Table II-3: Statistical analyses of Folate and Selenofolate treatments against MDA-MB-468 Cells

Experiment Treatments p value Day 3 Trypan Blue Folate 100 µM vs Selenofolate 100 µM 0.015 (Figure II-14)

Day 4 Trypan Blue Folate 50 µM vs Selenofolate 50 µM 0.008

(Figure II-14) Folate 75 µM vs Selenofolate 75 µM 0.006

Folate 100 µM vs Selenofolate 100 µM 0.004

Day 5 Trypan Blue Control vs Selenofolate 75 µM 0.020

(Figure II-14) Control vs Selenofolate 100 µM 0.035

Folate 50 µM vs Selenofolate 50 µM 0.041

Folate 75 µM vs Selenofolate 75 µM 0.001

Folate 100 µM vs Selenofolate 100 µM 0.001

Day 3 MTT Folate 75 µM vs Selenofolate 75 µM 0.024 (Figure II-17)

Day 4 MTT Control vs Selenofolate 100 µM 0.022 (Figure II-17)

Annexin V Folate 50 µM vs Se-Folate 50 µM (early apoptosis) 0.001

(Figure II-26) Folate 75 µM vs Se-Folate 75 µM (early apoptosis) 0.001

Folate 100 µM vs Se-Folate 100 µM (early apoptosis) 0.002

Control vs Se-Folate 50 µM (early apoptosis) 0.001

Control vs Se-Folate 75 µM (early apoptosis) 0.002

Control vs Se-Folate 100 µM (early apoptosis) 0.004

Folate 50 µM vs Se-Folate 50 µM (late apoptosis) 0.004

Folate 75 µM vs Se-Folate 75 µM (late apoptosis) 0.001

Folate 100 µM vs Se-Folate 100 µM (late apoptosis) 0.001

Control vs Se-Folate 50 µM (late apoptosis) 0.003

Control vs Se-Folate 75 µM (late apoptosis) 0.001

Control vs Se-Folate 100 µM (late apoptosis) 0.001

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Table II-4: Statistical analyses of Folate and Selenofolate treatments against HME 50-5E Cells

Experiment Treatments p value Day 3 Trypan Blue Folate 50 µM vs Selenofolate 50 µM 0.004 (Figure II-15) Folate 75 µM vs Selenofolate 75 µM 0.002 Day 4 Trypan Blue Folate 50 µM vs Selenofolate 50 µM 0.002 (Figure II-15) Folate 75 µM vs Selenofolate 75 µM 0.001 Folate 100 µM vs Selenofolate 100 µM 0.001 Day 4 MTT Folate 100 µM vs Selenofolate 100 µM 0.010 (Figure II-18) Day 5 MTT Folate 50 µM vs Selenofolate 50 µM 0.007 (Figure II-18) Folate 75 µM vs Selenofolate 75 µM 0.001 Folate 100 µM vs Selenofolate 100 µM 0.001

RESULTS AND DISCUSSION

Progress in molecular biology has resulted in the identification and greater understanding of molecular markers that may have prognostic and predictive value for breast cancer patients. The FRA is now being recognized as a potential therapeutic target in cancer [152] therapy as it has been used for the delivery of fluorescent molecules for ovarian surgery [147]; for the development of Vintafolide, a Vinblastine conjugates [153]; and is the crown jewel receptor target for Endocyte, Inc. Studies have shown that FRA is expressed in stage IV metastatic TNBC tumor [140]. Moreover, it has been demonstrated that patients with overexpression of the FRA receptor protein have poorer clinical prognosis [154]. With the ongoing development of targeted therapy, the goal of the therapeutic strategy of chemotherapeutic agents is to selectively inhibit cell proliferation in cancer cells, thereby reducing the toxicity towards normal cells. Over expression of folate receptors have been reported in many types of cancer [152] and in this present study we conjugated covalent couple redox Se with folic acid and exploited the FRA receptor-mediated endocytosis as a delivery system to the TNBC cells lines MDA-MB-231, MDA-MB-468 [113].

Successful redox Se conjugation with 1-propanol selenocyanate generated a newly synthesized Selenofolate (Molecular weight: 573 g/mol) with 14 % Se

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Texas Tech University, Soni Khandelwal, May 2018 concentration. Additionally, a color shift from yellow to red was also observed due to Se attachment (Figure II-3). Both Folate and Selenofolate were stored in glass scintillation vials and away from light exposure. Folate and Selenofolate remained clear for the duration of the study with no precipitates or particulates detected by eye. No change in color or turbidity was observed. Neither was there a change in the pH of the Folate or Selenofolate as shown in Figure II-4 throughout the study period.

Figure II-3: Photographs of Folate (A) and Selenofolate (B)

Figure II-4: Photographs of Folate (B) and Selenofolate (A) dissolved in 1X PBS. Sample pH was 7.0-7.5.

Selenofolate and Folate were analyzed by lucigenin superoxide chemiluminescence detection a Turner TD-20e Luminometer. Figure II-6 and Figure II-5 demonstrates the experimental CL results. Over a 5-minute CL counting period (Figure 42

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II-5), the reaction between Selenofolate and glutathione (GSH) was seen to be dependent upon the concentration of GSH. In these reactions, lucigenin was reduced by the superoxide anion generated and chemiluminescence was observed. These experiments confirmed the generation of superoxides from the oxidation of GSH by selenite as shown by Seko et al., [91] and extended the observation to Selenofolate but not CL cocktail or Folate. After determining the chemiluminescence activity of Folate and Selenofolate at different GSH concentrations, experimental steps were taken to determine if Folate and Selenofolate would generate superoxides in situ. The photographs taken on an EVOS FL Auto Cell Imaging System in Figure II-7 and Figure II-8 show the intracellular production of superoxide by Selenofolate and selenite, but not Folate, analyzed by DHE assay in MDA-MB-231 and MDA-MB-468 cells. Similar results have been reported from prior studies in the laboratory that have compared the cytotoxicity of selenite, selenate, selenocystine and selenomethionine towards human mammary tumor cell line HTB123/DU4475 in vitro [155]. In contrast to the TNBC cell line, Selenofolate did not induce such severe superoxide production in HME50-5E cells (Figure II-9). This difference in TNBC cells and the HME50-5E cells resistance to redox selenium suggests a biochemical variance that can provide a basis for more selective killing of cancer cells with lesser toxicity to normal cells. The results are consistent with cancer cells being known to overexpress FRA relative to normal cells, kidney cells but the one exception.

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Figure II-6: Total Superoxide Generation as Measured by Lucigenin Chemiluminescence (CL) with different concentrations of Glutathione (GSH). Chemiluminescence (CL) for Control Blank, Folate and Selenofolate measured over 5 minutes.

Figure II-5: Time Dependent Superoxide Generation as Measured by Lucigenin Chemiluminescence (CL) for Blank, Folate and Selenofolate. Control Blank, Folate and Selenofolate CL was measured over 5 minutes with 30 second integrations.

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Figure II-7: Photomicrographs of Intracellular Superoxide Generation by DHE Red Florescence from MDA-MB-231 Cells after 30 minutes of Blank, Control, Selenite (20 µM), Folate (100 µM) and Selenofolate (100 µM) Treatments. Scale bar=200 µm.

Figure II-8: Photomicrographs of Intracellular Superoxide Generation by DHE Red Florescence from MDA-MB-468 Cells after 30 minutes of Blank, Control, Selenite (20 µM), Folate (100 µM) and Selenofolate (100 µM) Treatments. Scale bar=100 µm.

Figure II-9: Photomicrographs of Intracellular Superoxide Generation by DHE Red Florescence from HME50-5E Cells after 30 minutes of Blank, Control, Selenite (20 µM), Folate (100 µM) and Selenofolate (100 µM) Treatments. Scale bar=200 µm.

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To visualize morphological toxicity MDA-MB-231, MDA-MB-468 and HME50- 5E cells were treated with Selenofolate, Folate and selenite as Se. Images were taken under a light microscope at 20X magnification. Images were taken under a light microscope at 20X magnification and results are shown in Figure II-10 (MDA-MB-231), Figure II-11 (MDA-MB-468), Figure II-12 (HME50-5E). Selenofolate and selenite treated cells show gross cell morphological changes, with both cell swelling and shrinkage, and cell membrane disruption ultimately detachment from the cell culture substratum. MDA-MB-231, MDA-MB-468 and HME50-5E cells are all adherent epithelial cells. With adherent cells, detachment from the substratum as well as morphological change is indicative of apoptosis [156]. Additionally, the morphological change (from the grape-like structure in MDA-MB-468 cells and the stellate-elongated structure in MDA-MB-231 cells to the extensive cytoplasmic vacuolization) became more remarkable with increased treatment exposure. Similar morphological change in cancer cells treated with other Se compounds have been reported in other in vitro studies [157]. From a therapeutic point of view, the clinical relevance of this observation is that Selenofolate can be effective in suppressing tumor growth if targeting and endocytosis is optimized. Selenite also caused morphological change in cell anatomy but since it is a non-targeting treatment, it demonstrated general toxicity. Interestingly, Folate treatment alone, showed morphological changes from cuboidal, cobblestone-like to fibroblast-like structure in HME50-5E cells (Figure II-12). Additional photomicrographs of morphological changes due to Folate and Selenofolate treatments are included in APPENDIX A (Figure A-3, Figure A-4, Figure A-5, Figure A-6, Figure A-7 and Figure A-8).

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Figure II-10: Morphological Changes of Control, Selenite (4 µM), Folate (50 µM) and Selenofolate (50 µM) Treatments in MDA-MB-231 Cells. Treatment of MDA-MB-231 Cells with Selenite and Selenofolate morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-231 cells. Cells were visualized under 20X magnification.

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Figure II-11: Morphological Changes of Control, Selenite (4 µM), Folate (50 µM) and Selenofolate (50 µM) Treatments in MDA-MB-468 Cells. Treatment of MDA-MB-468 Cells with Selenite and Selenofolate morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-468 cells. Cells were visualized under 20X magnification.

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Figure II-12: Morphological Changes of Control, Selenite (4 µM), Folate (50 µM) and Selenofolate (50 µM) Treatments in HME50-5E Cells. Treatment of HME50-5E cells with Selenite, Folate and Selenofolate did not induce severe morphological change comparable to those seen in TNBC cells. Representative fields of view of HME50-5E cells. Cells were visualized under 20X magnification.

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Following optimization of cell growth of the MDA-MB-231, MDA-MB-468 and the HME50-5E cells, the cells were treated with Folate, Selenofolate or selenite. The dose response results of control cell growth and the three treatments are shown in Figure II-13, Figure II-14 and Figure II-15 as measured for cell viability by the Trypan Blue exclusion. These three experimental treatments show that selenite and Selenofolate exhibit a general loss of viable cells over Se dose and time as measured against control and folate treated cells. Selenofolate 100 µM had cytotoxic effects on MDA-MB-231, MDA-MB-468 and HME50-5E cells. Overall treatment of Selenofolate at 5 µM and 25 µM dose did not cause significant reduction in MDA-MB-231, MDA-MB-468 cell viability. This may be due to selenium reverting to its nutritional role in support of Glutathione peroxidase and other selenium proteins. Two, five, ten and twenty µg of Se (as selenite) per well, was cytotoxic to all three cell types tested. Sodium selenite an inorganic chemical form of selenium is toxic and inhibits cell proliferation by apoptosis not only in human and mouse breast cancer cell lines but also in human colon carcinoma cells, human ovarian cancer, human prostate cancer cells in vitro [158].

Trypan Blue exclusion experiments were not conducted with HME50-5E at 5 µM and 25 µM Selenofolate. In part, successful analysis was difficult due to the strong adherence properties of the HME50-5E cells, causing experimental bias. Initially 200 µl of 0.025% trypsin-EDTA was added and incubated for 10-15 minutes but when viewed under a microscope not all the cells completely detached. So, another 200 µl of 0.025% trypsin-EDTA was added to cells to try to collect the whole population. Despite incubating with double the amount (400 µl) of trypsin, a substantial number of cells remained. This inability to adequately remove all HME50-5E cells from the plate resulted in a high percentage loss of cells inadequate representation for the Trypan Blue assay.

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Figure II-13: Cytotoxicity of Control, Selenite, Folate and Selenofolate in Dose and Time Dependent Treatments against MDA-MB-231 cells. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding. Control cells were incubated for 7 days with Selenite (4 µM, 10 µM, 20 µM and 40 µM), Folate and Selenofolate at concentrations ranging from 5 µM to 100 µM. Viable cells were counted at time periods (0-7 days) by Trypan Blue exclusion as described in Methods. The cell growth data are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table II-2

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Figure II-14: Cytotoxicity of Control, Selenite, Folate and Selenofolate in Dose and Time Dependent Treatments against MDA-MB-468 cells. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding. Control cells were incubated for 7 days with Selenite (4 µM, 10 µM, 20 µM and 40 µM), Folate and Selenofolate at concentrations ranging from 5 µM to 100 µM. Viable cells were counted at time periods (0-7 days) by Trypan Blue exclusion as described in Methods. The cell growth data are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table II-3.

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Figure II-15: Cytotoxicity of Control, Selenite, Folate and Selenofolate in Dose and Time Dependent Treatments against HME50-5E cells. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding. Control cells were incubated for 7 days with Selenite (4 µM, 10 µM, 20 µM and 40 µM), Folate and Selenofolate at concentrations ranging from 50 µM to 100 µM. Viable cells were counted at time periods (0-7 days) by Trypan Blue exclusion as described in Methods. The cell growth data are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table II-4.

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To better understand the mechanism of cell death demonstrated by the Trypan Blue exclusion results, an alternative cytotoxic assessment assay of cell viability, i.e., the MTT assay (3-[4, 5-dimethylthioazol-2-yl]-2-5-diphenyltetrazolium bromide) was performed, where the tetrazolium salt ring is cleaved by viable cells forming the insoluble Formazan. This method would also help address the question of cell viability in the HME50-5E cells when treated with Selenofolate. The results for the three cell lines tested are illustrated in Figure II-16, Figure II-17 and Figure II-18. Percent viability for MDA- MB-231 cells treated with 100 μM Selenofolate was 59%. This percentage increased to 88% at 50 μM Selenofolate (Figure II-16). Interestingly, MDA-MB-468 cells were more resistant as viability ranged from 97-102% at these same treatments (Figure II-17). Se as selenite was toxic to cells at all concentrations (Figure II-16 and Figure II-17). Folate treatments alone showed high cell viability for all doses and time periods, as folate is non-toxic at these concentrations and serves as a nutrient for cancer cells.

MTT incubation in the HME50-5E cells yielded an interesting result. When the MTT solubilization media for Formazan was added to HME50-5E cells, the Formazan salt could not be dissolved completely because of the adherence properties of the cells (Figure A-23). Also, the intensity of Formazan color was so dark that plate reader displayed ‘OVERFLOW’ for results on day 1 and day 3 of treatments indicating that the metabolic activity was so high, it saturated the limits of detection for the instrumentation. This observation coupled with the confluent, adherent populations, suggested that the vast majority of the population was metabolically healthy following Se treatments. Control HME50-5E cell viability was therefore set at 100% viability for days 1 and 3 treatments. Moreover, no significant differences were observed on day 3 for Folate or Selenofolate treatments. So we made HME50-5E cell viability as 100% for days 1 and 3 treatments. Hence no significant differences were observed on day 3 folate, Selenofolate treatments. Ip [157] mentioned that normal cells, early transformed cells and late stage preneoplastic cells may respond differently to selenium intervention with respect to molecular pathways involving cell cycle proteins and apoptotic proteins. The photomicrographs reflect this result (shown in Figure II-18) with no morphological changes evident and loss of viable cells in the Trypan Blue assay.

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Figure II-16: Metabolic activity of Control, Selenite, Folate and Selenofolate treated MDA-MB-231 cells as determined by the MTT Assay. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding with Selenite (4 µM, 10 µM, 20 µM), Folate and Selenofolate at concentrations ranging from 1 µM to 100 µM over 6 days by MTT as described in Methods. The cell viabilities are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table II-2.

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Figure II-17: Metabolic activity of Control, Selenite, Folate and Selenofolate treated MDA-MB-468 cells as determined by the MTT Assay. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding with Selenite (4 µM, 10 µM, 20 µM), Folate and Selenofolate at concentrations ranging from 1 µM to 100 µM over 6 days by MTT as described in Methods. The cell viabilities are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table II-3.

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Figure II-18: Metabolic activity of Control, Selenite, Folate and Selenofolate treated HME50-5E cells as determined by the MTT Assay. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding with Selenite (4 µM, 10 µM, 20 µM), Folate and Selenofolate at concentrations ranging from 1 µM to 100 µM over 6 days by MTT as described in Methods. The cell viabilities are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table II-4.

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Dose dependent calculations were used to determine the IC50s of Selenofolate on at 72 hours of post treatment are shown in Figure II-19 for MDA-MB-231 cells, Figure

II-20 for MDA-MB-468 cells. Selenofolate showed an IC50 of 58.68 µM in MDA-MB- 231 cells and 18.21 µM in MDA-MB-468 cells. Since cell viability for HME50-5E cells was assumed to be 100% as describe above, IC50 of HME50-5E was not able to be calculated as treatment values did not approach close enough to 50% to provide an accurate estimate.

Figure II-19: IC50 for MDA-MB-231 Cells with Selenofolate.

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Figure II-20: IC50 for MDA-MB-468 Cells with Selenofolate.

Together the Trypan Blue exclusion and MTT experiments show that Selenofolate is able to significantly inhibit cell proliferation, and cell viability in the TNBC cell lines; MDA-MB-231, MDA-MB-468. This is consistent with the ability of both selenite and Selenofolate to generate superoxide in both the in vitro CL assay as shown in Figure II-6 and Figure II-5 and as detected by the intracellular increase in the red fluorescence of the intracellular DHE in Figure II-7, Figure II-8 and Figure II-9. These experimental results also confirm the optical assessment of cell morphology as photographed and shown in Figure II-10, Figure II-11 and Figure II-12.

Previous studies have established that selenium induces apoptosis by the intrinsic (mitochondrial-mediated) pathway [159-163]. Furthermore, apoptosis or programmed cell death is associated with morphological, biochemical and molecular changes occurring in the cell [164]. In order to confirm that Selenofolate treatments also induce apoptosis, an Annexin V assay was performed. Both Selenofolate and Selenite treated

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Texas Tech University, Soni Khandelwal, May 2018 cells showed prominent morphological changes, characteristic of apoptosis as evidenced by photomicrographs (Figure II-10 and Figure II-11). Due to different sensitivities identified at day 3 for all cell lines tested in both Trypan Blue and MTT assays, cells were incubated with three different concentrations of Selenofolate to determine apoptosis would be induced. Flow data (Figure II-21, Figure II-22, Figure II-23, Figure II-24, Figure II-25 and Figure II-26) of Selenofolate treatments showed MDA-MB-468 cells exhibited more changes than MDA-MB-231 cells. A majority of the MDA-MB-231 cell population was in the lower left (LL) quadrant, corresponding to living cells (Figure II-21). A substantial proportion of MDA-MB-468 cells in the upper right (UR) and lower right (LR) quadrant (as shown in Figure II-24), correspond to early and late apoptosis respectively. The morphological phenotypes (Figure II-10 and Figure II-11) of these apoptotic cells showed a strongly correlated with flow analysis. In contrast, considerable proportions of HME50-5E cells were found in the upper left (UL) quadrant (as shown in Figure II-27), corresponding to living cells. The total percentages of cell are shown in Figure II-22, Figure II-25 and Figure II-28. The percentages of cells undergoing early and late apoptosis were measured by flow cytometry and results are shown in Figure II-23, Figure II-26 and Figure II-29.

Due to its apoptotic activity, Sutent (Pfizer) is used as a positive control for induction of apoptosis in TNBC [165] and HME50-5E cells. Interestingly, Sutent demonstrated even greater potency in killing the normal cells. To our knowledge, this is the first report of the toxicity of Sutent on normal cells. This suggests important consequences for clinical success of Sutent with respect to side effects. Further study is needed to investigate this observation. Control and Folate treated cells also showed apoptosis (Figure II-23, Figure II-26 and Figure II-29). One possibility for this phenomenon is that the rapidly dividing cell became overconfluent in the limited well space, causing cell death. Increased cell viability and cell proliferation was observed in Folate alone treatments in both Trypan Blue exclusion (Figure II-13, Figure II-14 and Figure II-15) and MTT (Figure II-16, Figure II-17 and Figure II-18) assays. To address the appearance of a population of apoptotic cells in the vehicle control, prior studies of candidate anticancer therapies also demonstrated [162, 166] this phenomenon in their control groups.

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A loss of mitochondrial membrane potential (ΔψM) indicates a loss of cell viability due to proton pumps across the inner membrane during the process of electron transport and oxidative phosphorylation driving the conversion of ADP to ATP [167]. Therefore, MDA-MB-468 cells and HME50-5E cells were stained with MitoTracker Red CMXRos (instead of Propidium Iodide), which stains mitochondria in live cells and its accumulation is dependent upon the ΔψM. In this study the ΔψM was measured by flow cytometer and the results demonstrated a Selenofolate dose-dependent decrease and a concurrent dose-dependent increase in the percent apoptotic MDA-MB-468 cells. In contrast, there was no change in the ΔψM (UL quadrant) of control vs Selenofolate treated HME50-5E cells (Figure II-27), indicating to living cells. This validates the intense Formazan color observed from MTT assay assessing cellular metabolic activity (Figure II-18). Hence, HME50-5E cells do not undergo apoptosis following Selenofolate treatment. These observations again suggest the probable role of selenium-induced oxidative stress/glutathione triggered apoptosis associated with both increased generation of superoxide and a decrease in mitochondrial GSH levels as well as ΔψM. Studies have reported that modulation of mitochondrial functions regulating apoptosis is one of the most affected pathways of Se compounds in cancer therapy [168]. Selenite also caused necrosis in MDA-MB-468 cells and apoptosis in MDA-MB-231 cell, while few events were detected in HME50-5E cells. Selenite is known to induce necrosis in other breast cancer cells [157].

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Figure II-21: Folate, Selenofolate and Selenite as Se Treatments induced apoptosis in MDA-MB-231 Cells. MDA-MB-231 cells were stained with Annexin V/PI and subjected to flow cytometric analysis. The four quadrants represent- living cells (Lower Left; LL: Annexin V-PI--), early apoptotic (Lower Right; LR: Annexin V+PI-), late apoptotic (Upper Right; UR: Annexin+PI+) or necrotic (Upper Left; UL: Annexin V- PI+) stages.

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Figure II-22: Representative of four quadrants for MDA-MB-231 cells were treated with Folate, Selenofolate and Selenite. Data is expressed as Mean (n=3) of the total percent of MDA-MB-231 cells.

Figure II-23: Percentage level of MDA-MB-231 apoptotic cells when the cells were treated with Folate, Selenofolate and Selenite as Se. Data is expressed as Mean ± SE (n=3). Treatments were compared using two sample t test. Treatments were statistically significant if p ≤ 0.05 and summarized in Table II-2.

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Figure II-24: Folate, Selenofolate and Selenite as Se Treatments induced apoptosis in MDA-MB-468 Cells. MDA-MB-468 cells were stained with Annexin V/Mitotracker Red and subjected to flow cytometric analysis. The four quadrants represent- living cells (Upper Left; UL: Annexin V- Mitotracker Red+), early apoptotic (Upper Right; UR: Annexin+ Mitotracker Red +), late apoptotic (Lower Right; LR: Annexin V+ Mitotracker Red-) or necrotic (Lower Left; LL: Annexin V- Mitotracker Red-) stages.

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Figure II-25: Representative of four quadrants for MDA-MB-468 cells were treated with Folate, Selenofolate and Selenite. Data is expressed as Mean (n=3) of the total percent of MDA-MB-468 cells.

Figure II-26: Percentage of MDA-MB-468 apoptotic cells when the cells were treated with Folate, Selenofolate and Selenite as Se. Data is expressed as Mean ± SE (n=3). Treatments were compared using two sample t test. Treatments were statistically significant, if p ≤ 0.05 and summarized in Table II-3.

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Figure II-27: Folate, Selenofolate and Selenite as Se Treatments did not induced apoptosis in HME50-5E Cells. HME50-5E cells were stained with Annexin V/ Mitotracker Red and subjected to flow cytometric analysis. The four quadrants represent- living cells (Upper Left; UL: Annexin V- Mitotracker Red+), early apoptotic (Upper Right; UR: Annexin+ Mitotracker Red +), late apoptotic (Lower Right; LR: Annexin V+ Mitotracker Red-) or necrotic (Lower Left; LL: Annexin V- Mitotracker Red-) stages.

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Figure II-28: Representative of four quadrants for HME50-5E cells were treated with Folate, Selenofolate and Selenite. Data is expressed as Mean (n=3) of the total percent of HME50-5E cells.

Figure II-29: Percentage level of HME50-5E apoptotic cells when the cells were treated with Folate, Selenofolate and Selenite as Se. Data is expressed as Mean ± SE (n=3).

Western blotting was used to detect the protein expression levels of Folate Receptor Alpha on the surface of MDA-MB-231, MDA-MB-468 and HME50-5E cells (Figure II-30, Figure II-31 and Figure II-32). A band for the FRA protein was detected at ~38 kDa and β-actin ~ 42 kDa in control cells, Folate and Selenofolate treatments. Selenite show cleaved expression for both FRA and β-actin. Selenite toxicity is shown in Figure II-10, Figure II-11 and Figure II-12. One possibility for cleaved FRA expression

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in selenite treatment is its non-specific toxicity. Cleavage of β-actin by selenite treatment was found in all three cell lines. These may have some biological and therapeutic implications like cell-cell interactions, cell migration (metastasis) and proliferation [169]. This interesting observation requires study and was outside the scope of this current project. Furthermore, the safety and efficacy of Sodium selenite was evaluated in Phase I clinical trial. The results from this trial show that no major toxicity was reported when 10.2 mg/m2 of sodium selenite was administered in cancer patients [170].

Figure II-30: Western blot analysis of the Folate Receptor Alpha in MDA-MB-231 Control Cells and cells treated with Selenite Folate, Selenofolate. Total cell lysates were subjected to SDS-PAGE followed by Western blotting. Membranes were probed with the anti-folate receptor alpha (FRA) or anti β-actin antibodies followed by peroxidase conjugated rabbit anti- mouse antibodies and visualization was performed by the ECL detection system. Lane 1: Control, Lane 2: Selenite 4 µM treatment, Lane 3: Folate 50 µM treatment, Lane 4: Folate 75 µM treatment, Lane 5: Folate 100 µM treatment, Lane 6: Selenofolate 50 µM treatment, Lane 7: Selenofolate 75 µM treatment, Lane 8: Selenofolate 100 µM treatment.

Figure II-31: Western blot analysis of the Folate Receptor Alpha in MDA-MB-468 Control Cells and cells treated with Selenite Folate, Selenofolate. Total cell lysates were subjected to SDS-PAGE followed by Western blotting. Membranes were probed with the anti-folate receptor alpha (FRA) or anti β-actin antibodies followed by peroxidase conjugated rabbit anti-mouse antibodies and visualization was performed by the ECL detection system. Lane 1: Control, Lane 2: Selenite 4 µM treatment, Lane 3: Folate 50 µM treatment, Lane 4: Folate 75 µM treatment, Lane 5: Folate 100 µM treatment, Lane 6: Selenofolate 50 µM treatment, Lane 7: Selenofolate 75 µM treatment, Lane 8: Selenofolate 100 µM treatment.

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(I)

(I)

(II)

(II)

(III)

(III)

Figure II-32: Western blot analysis of the Folate Receptor Alpha in MDA-MB-231 Control Cells and cells treated with Selenite Folate, Selenofolate. Total cell lysates were subjected to SDS-PAGE followed by Western blotting. Membranes were probed with the anti-folate receptor alpha (FRA) or anti β-actin antibodies followed by peroxidase conjugated rabbit anti-mouse antibodies and visualization was performed by the ECL detection system.

(I) Lane 1: Control, Lane 2: Selenite 4 µM treatment, Lane 3: Folate 50 µM treatment, Lane 4: Selenofolate 50 µM treatment.

(II) Lane 1: Control, Lane 2: Selenite treatment, Lane 3: Folate 75 µM treatment, Lane 4: Selenofolate 75 µM treatment.

(III) Lane 1: Control, Lane 2: Selenite treatment, Lane 3: Folate 100 µM treatment, Lane 4: Selenofolate 100 µM treatment.

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In summary, the FRA is upregulated and over expressed on many cancer cell lines. This FRA is thus a potential major target for the clinical treatment of many cancers with folate or the folate specific mAb not yet readily available or in experimental treatment. The results from this research definitively demonstrated the cytotoxic effects of selenocompounds that form selenides and generate superoxide on different malignant cancer cell lines. Furthermore, the effects between these different TNBC cells was very similar. The metabolic and apoptotic experimental results substantiate the loss of cell viability most likely from induction of programmed cell death through the intrinsic apoptotic pathway. Furthermore, the selenium redox chemistry targeting of GSH and other thiols, the cysteines of the mitochondrial membrane and other proteins, possibly actin.

A major finding from this study was the significantly lower cytotoxicity of the Selenofolate on HME50-5E cells. This also correlated with the quantitative expression of the FRA on the three cell lines, the IC50 values calculated, the generation of intracellular superoxide, and observed changes in morphology of the cells. This data corroborates the report that folate isotopes were focused in effect only to cancer and normal kidney cells, that folate-fluorescein conjugates can be internalized by ovarian cancers [147, 148] . Another important advantage of this strategy is more obvious when considering the failure of Vintafolide (folate-Vinblastine conjugates) in a Phase 3 clinical trial [149]. Application of a small, redox conjugate that does not require disassociation from folate, opens the possibility that Selenofolate may be a significant treatment modality for many cancers frequently detected and diagnosed at Stage IV progression, as evidenced by its effectiveness in TNBC cell demonstrated from this research.

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CHAPTER III

CYTOTOXICITY OF AVASTIN®, SELENOAVASTIN AND SELENITE

AGAINST TRIPLE NEGATIVE BREAST CANCER CELL LINES MDA-MB-231

AND MDA-MB-468

ABSTRACT

A humanized monoclonal IgG1, vascular endothelial growth factor (VEGF) specific antibody Avastin® was conjugated with the N-hydroxysuccinamide ester of 3- selenocyanopropionic acid forming a Selenoavastin immunoconjugate. The selenium - containing immunoconjugate, Selenoavastin was shown to produce superoxide (O2 ) anion in the presence of glutathione using lucigenin chemiluminescence. With cells in vitro, Selenoavastin has exhibited intracellular superoxide generation exhibited by the DHE assay. The cytotoxicity of Selenoavastin was then compared to Avastin, and selenite in a time and dose dependent manner against the TNBC cell lines MDA-MB- 231, MDA-MB-468 and an immortalized mammary epithelial cell line, HME50-5E. Cells were treated with either 2 µg, 5 µg, 10 µg or 20 µg of Se as Selenoavastin or an equivalent concentration of Avastin on day 3 post-cellular inoculation. In all experiments sodium selenite was used as a positive control for cell death at comparative Se concentrations of 2, 5, 10 and 20 µg as Se. Selenoavastin was shown to arrest the MDA- MB-231, MDA-MB-468 and HME50-5E cells in vitro as measured by Trypan Blue exclusion, MTT and Annexin V assays. VEGF protein expression was assessed by immunoblotting using Western blot. These results show that selenium toxicity can be targeted by Avastin and provide a reasonable expectation that antibodies by attachment of Selenium may be useful in pharmaceutical applications for treatment of triple negative breast cancer.

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KEYWORDS

Selenium, Triple Negative Breast Cancer Cell lines, MDA-MB-231 cells, MDA-MB-468 cells, HME50-5E cells, Selenoavastin, Sodium Selenite, Avastin®, Chemiluminescence, AnnexinV, Reactive Oxygen Species, Dihydroethidium

INTRODUCTION

In 1971, Judah Folkman hypothesized that tumor angiogenesis is a pre-requisite for the growth of tumors [171]. In 1983, the vascular endothelial growth factor (VEGF) was discovered to be was secreted by tumors to promote angiogenesis [172]. Angiogenesis is the formation of new blood vessels from pre-existing ones to provide oxygen and nutrients to the cells. VEGFs are produced and secreted by several normal cell types including smooth muscle, luteal and adrenal cortex cells [173]. But in cancer, VEGF facilitates the cancer cell’s survival and metastasis.

VEGF appears as a protein of molecular mass of about 45 kDa under non- reducing conditions and 23 kDa under reducing conditions by SDS-PAGE. In 1989, it was purified from bovine pituitary follicular cells by Ferrara and Henzel [174]. VEGF, originally known as the vascular permeability factor, is a homo dimeric, heparin binding, disulfide-bonded glycoprotein. Different isoforms of VEGF include; VEGF-A, VEGF-B, VEGF-C and VEGF-D [175-178]. The VEGF-A gene is located on chromosome 6p.21.3 [179]. VEGF-A, VEGF-B, VEGF-C and VEGF-D have three tyrosine kinase receptors: VEGFR-1 (Flt-1) plays an important role in hematopoiesis in the growth and development of cells; VEGFR-2 (KDR/Flk-1) is important for vascular endothelial cell development and VEGFR-3 (Flt4) is important for lymphatic endothelial cell development or lymph angiogenesis [180]. VEGFRs are involved in signaling pathways like MAPK/ERK and Akt. In humans, VEGF-A165 is widely expressed as a 46 kDa homodimer and consists of 23 kDa subunits. VEGF-A is produced by macrophages, vascular smooth muscle cells and tumor cells [181]. H1F1-α regulates expression of VEGF-A, which is released in response to hypoxia (oxygen stress) [120]. VEGFR is over expressed in cancers; ovarian, colorectal, cervical, kidney and breast cancer cells. For breast cancer, over expression of VEGFR is seen in 60% of women at the time of first diagnosis [182]. Due to the importance of VEGFs and VEGFRs for tumor angiogenesis,

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Folkman proposed the idea of anti-angiogenesis treatment, which lead to the development of Bevacizumab, (Avastin®) a humanized monoclonal antibody that binds to VEGF-A and suppresses angiogenesis [121].

Bevacizumab (Avastin®) is 149 kDa humanized monoclonal immunoglobulin (IgG1) antibody [120]. It blocks the vascular endothelial growth factor (VEGF) and inhibits formation of new blood vessels [121]. Avastin® (manufactured by Genentech/Roche) targets the VEGF protein, which is synthesized by tumor cells to induce angiogenesis. It is approved for use along with chemotherapy to treat kidney cancer, colorectal cancer, glioblastoma, lung cancer, cervical and ovarian cancer [120]. Efficacy of Avastin® was investigated in several clinical trials, of which three are well- known- the E2100 study [183], the Avastin & Docetaxel (AVADO) trial [184] and the Regimens in Bevacizumab for Breast Oncology (RiBBOn)-1 trial [185]. The results from the E2100 trial showed that the risk of progression in first line TNBC patients reduced by 51% when Avastin® was administered in combination with paclitaxel [183]. On the contrary combination of Avastin® and Docetaxel in the AVADO trial showed 47% decrease in disease progression [184]. Results from RiBBOn-1 did not show any clear benefit from combination of Avastin and chemotherapy regimens [185]. The results from other two trials are inconsistent [186, 187]. Avastin® in combination with paclitaxel (Taxol) was approved by the FDA for untreated metastatic breast cancer in February 2008 [188] but in June 2011, the FDA revoked the 2008 approval of the mAb as it was not helping breast cancer patients to live longer and the size of the tumors were not reduced and had it posed serious side effects [189, 190]. Investigators reported at the 2013 San Antonio Breast Cancer Symposium that carboplatin (an angiogenesis inhibitor) along with Avastin shrank tumors in triple negative breast cancer patients [191]. Currently, there is no evidence if Avastin will either help women with breast cancer live longer or improve quality of life.

In this study, the labeling of Avastin® (Bevacizumab) with an essential dietary nutrient, selenium, in redox active form removes some of the obstacles mentioned above. As previously shown by Goswami [192] for Transferrins and Bapat [193] for Herceptin® conjugation of proteins and mAbs, directed selenium cytotoxicity is possible using

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MATERIALS AND METHODS

Materials

DMEM high glucose (Catalog# 11965-092), Fetal Bovine Serum (Catalog# 10082-147), 1% Penicillin-Streptomycin (Catalog# 15-140-122), were purchased from Life technologies, Gibco. pH test strips (Catalog# 8882-1) were purchased from Ricca Chemical Company. PVDF membranes (Catalog# 1620177), Non-Fat Milk Protein (Catalog# 1706404), 3-20% Tris-Glycine Polyacrylamide Gel (Catalog# 456-1096) and 2X native PAGE sample buffer (Catalog# 161-0738) were purchased from BIO-RAD. Mammary Epithelial Cell Media (Catalog# 50-306-176) was purchased from PromoCell. Trypsin (Catalog# 30-2101) was purchased from ATCC. Rabbit Anti-Mouse IgG H&L HRP (Catalog# ab6728) was purchased from Abcam. 0.22 µM filter (Catalog# SLGV033RS), Anti-β-Actin Antibody (Catalog# MAB1501) and Accutase (Catalog# SCR005) from EMD Millipore. Corning® cell culture 75 cm2 (Catalog# CLS430641) and 25 cm2 (Catalog# CLS430639) vented cap flasks, Corning® Costar® TC-Treated Multiple Well Plates (Catalog# CLS3527), Corning® Costar® TC-Treated Multiple Well Plates (Catalog# CLS3548), Sodium selenite (Catalog# 214485), MTT (Catalog# M2128), Superoxide Dismutase (Catalog# S7571), Bovine Erythrocytes (Catalog# C1345-1G), 2′,7′-Dichlorofluorescin diacetate (Catalog# D6883), Dihydroethidium (Catalog# D7008), Tetrahydrofuran (Catalog# 34865) were purchased from Sigma-

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Aldrich. RIPA lysis buffer (Catalog# 89900), VEGF (JH121) monoclonal antibody (Catalog# MA5-13182), SuperSignal™ West Femto Maximum Sensitivity Substrate (Catalog# 34095), Gamma Globulin (Catalog# 23212), BCA asay kit (Catalog# 23225), MitoTracker™ Red CMXRos (Catalog# M7512), Annexin V-FITC Apoptosis Detection Kit (Catalog# BMS500FI-300) were purchased from ThermoFisher Scientific. Dialysis tube (Catalog# 132542) was purchased from Spectra/Por membrane. Bovine Serum Albumin (Catalog# BP1600-100) was purchased from Fisher Scientific. Protein ladders of size of 10-245 kDa was purchased from (GeneTex, Catalog# GTX50875).

Monoclonal antibodies

Bevacizumab (Avastin®) was obtained through the collaborative efforts of TTUHSC Cancer Center Clinic with Dr. Everado Cobos, Director of the Southwest Cancer Center of TTUHSC. Selenoavastin was synthesized in this laboratory using a selenium modified Bolton-Hunter reagent (Se ester; ROCH2CH2SeCN). The covalent attachment of a selenide Se moiety to proteins and antibodies via the Se-Bolton-Hunter reagent is only a recent development [194]. The Bolton-Hunter reagent was originally developed in the 1970’s for radioimmunoassays using radioactive iodine. A Se-modified Bolton-Hunter reagent was synthesized using this technique and was subsequently used to attach catalytic redox Se to Avastin®.

Synthesis of Bolton-Hunter Seleno ester

The N-hydroxysuccinamide ester of 3-selenocyanopropionic acid (a modified Se Bolton-Hunter Reagent; Selenoester) (Eburon Organics N.V.; Belgium) was synthesized from N-hydroxysuccinamide and 3-selenocyanopropionic acid by an N, N’- dicyclohexylcarbodimide (DCC) reaction and was used as received without further preparation as shown in Figure III-1. The final Se ester product has a dark red color (refer Figure III-2).

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Figure III-1: The Procedure for Conjugation of Seleno-Ester to the Lysine Residues of Monoclonal Antibodies.

Figure III-2: Selenocyanate Propionic Ester of N- hydroxysuccinimide (Bolton-Hunter Seleno-Ester)

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Conjugation of Redox Se to Avastin®

Avastin® (Genentech, Catalog# 10133725) was diluted to a concentration of 3 mg protein/mL from a stock solution of 25 mg/mL with 0.05 M sodium borate buffer (pH 8.5) at 4°C. The Se-ester was dissolved in tetrahydrofuran at a concentration of 20 mg/mL (refer Figure III-3) and 2.5 ml (20 mg/mL) of seleno-ester in THF was added into 10 ml of dilute Avastin® with the glass vial kept on ice and packed cold water. The reaction was carried out for 72 hours at 4°C. Dialysis tubing in cold 0.05 M phosphate buffer (pH 7.4) was soaked for 24 hours at 4°C. After incubation for 72 hours, Avastin® from the glass vial was transferred to dialysis tubing (MWCO 50,000) and was placed in the refrigerator for another 72 hours in dialysis buffer. The dialysis buffer (0.05 M phosphate buffer, pH 7.4) was changed 3-4 times the first day and then twice daily thereafter. For dialysis 10 ml of the antibody in 2 L of dialysis buffer was used. After 72 hours, Avastin® and Selenoavastin containing dialysis tubing in PBS buffer was taken out and rinsed with sterile water, and the antibodies were drawn from the dialysis tubing with a 16-gauge needle and syringe and was filtered into a scintillation vial using 0.22 μM sterile filter. Avastin® and Selenoavastin were stored at 4°C. The scintillation vial containing antibodies were kept in containers and covered with aluminum foil paper to avoid light. Exposure to increasing temperature was avoided by keeping the mAbs on ice during their handling and transport. Selenoavastin was run through the Attune™ NxT Flow Cytometer (ThermoFisher Scientific, Carlsbad, CA) as a quality control to detect if there was any kind of fluorescence emitted by Selenoavastin, because this would if present effect fluorescent assays.

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Figure III-3: Saturated Solution of Se-Ester Dissolved In Tetrahydrofuran.

Analysis of Selenoavastin

Inductively Coupled Plasma Mass Spectrometry or ICP-MS is an analytical technique used for elemental Se determinations. Selenoavastin was sent to TraceAnalysis, Inc. (Lubbock, TX) for selenium analysis was performed.

Detection of Superoxide In Vitro by Selenoavastin

Lucigenin (Bis-N-Methylacridinium Nitrate) was used to detect superoxide in a chemiluminescent assay consisting of phosphate buffer and reduced GSH. The amount of superoxide generated is measured by the chemiluminescent produced light using a Turner TD-20e Luminometer (Turner Designs Inc., Mountain View, CA), connected to a circulating water bath. The source of electrons for superoxide is GSH catalyzed by some but not all Se compounds, including Selenoavastin. Chemiluminescence (CL) measurements of Avastin® and Selenoavastin were performed in triplicates by adding

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100 µL of Avastin® and Selenoavastin to 500 µL of the chemiluminescent cocktail at 37°C with integrations of 30 seconds over 12.5 minutes, N=25.

Detection of Intracellular Reactive Oxygen Species Accumulation In Situ: Superoxide Generation Assay

MDA-MB-231, MDA-MB-468 and HME50-5E cells were seeded at a density of 2X105 cells/well in 24 well flat bottom plates. After 48 hours, DMEM high glucose media without phenol red supplemented with FBS was added. 50 units/well of superoxide dismutase (SOD) from bovine erythrocytes and 100 units/well of catalase from bovine liver in media were added to all wells. All cells-control, Selenite (10 µg), Avastin® (153.7 µg protein) and Selenoavastin (10 µg Se and 153.7 µg protein) and were appropriately treated for 30 minutes. DHE was added at a final concentration of 10 µM in each well. Cells were visually assessed using EVOS FL Auto Cell Imaging System (ThermoFisher Scientific, Carlsbad, CA) or Cytation 3 plate reader (Biotek, Winooski, VT).

BCA Assay for Protein Determination of Avastin® and Selenoavastin

Total protein content of Avastin® and Selenoavastin were determined using bicinchoninic acid (BCA) assay kit. 10 µL of Avastin® and Selenoavastin were diluted in 90 µL of phosphate-buffered saline before addition of 2 mL of working reagent. After addition of working reagent, the samples were incubated in cuvette at 37°C in water bath for 30 minutes. One empty 96-well plate was kept in the incubator. After 30 minutes of incubation both-the cuvettes and 96-well plate was cooled down to room temperature. 225 μL of the colored solution from cuvette was added to each of 3 wells in 96-well plate. Absorbance was read using Cytation 3 plate reader (BioTek, Winooski, VT, USA) at 562 nm absorption. Protein concentrations were determined from a standard curve prepared from a serial dilution of bovine gamma globulin and purified human IgG. Protein concentrations were also determined using nanodrop, Cytation 3 plate reader (BioTek, Winooski, VT, USA).

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Western Blotting Under Denaturing Conditions

Native unconjugated Avastin® and Se-conjugated Avastin® were placed on ice. Protein ladders of 10-245 kDa were used to determine the unknown protein molecular weights. 20 µg of bovine gamma globulin and purified human IgG were loaded as controls. Concentrations of 5 µg, 10 µg and 20 µg of protein from each antibody- Avastin® and Selenoavastin in 6X Laemmli buffer was mixed and boiled for 5 minutes at 95°C. The mAbs were briefly centrifuged for 1 minute at 11,000 rpm. The total volume of the samples was loaded into a BIO-RAD miniprotean 14 well comb (1.5 mm) 8% polyacrylamide gel and was used for electrophoresis. Initially, the gel was run at 50 V for 5 minutes and then the voltage was increased to 90 V for 90 minutes. The SDS-PAGE gel was stained with Coomassie Blue R-250 for 15 minutes and then destained using Coomassie blue destaining solution. Photographs were taken under Coomassie filter using LI-COR (Model: 2800, S/N OFC- 0786, LI-COR, Inc., Lincoln, NE, USA).

Western Blotting Under Non-Denaturing Conditions

Avastin® and Selenoavastin were placed on ice. Protein ladders 10-245 kDa were used to verify the molecular weight. 20 µg of bovine gamma globulin and purified human IgG were also used as loading control. Five and ten micrograms of protein from Avastin® and Selenoavastin in 12 µL, was mixed with 6 µL of 2X native PAGE sample buffer. Samples were run on a gradient; 3-20% Tris-Glycine Polyacrylamide Gel. Initially, the gel was run at 50 V for 5 minutes and then the voltage was increased to 160 V to finish for 1 hour. The gradient gel was stained with Coomassie Blue R-250 stain for 15 minutes and destained with Coomassie Blue destaining solution for 20 minutes, then transferred to water. Photographs were taken under Coomassie filter using LI-COR (Model: 2800, S/N OFC- 0786, LI-COR, Inc., Lincoln, NE, USA). Samples were not boiled, the gel, sample buffer and the running buffer did not contain sodium-dodecyl sulfate.

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Cell Culture

MDA-MB-231 and MDA-MB-468 cells were cultured in 75 cm2 tissue culture flasks and maintained in high glucose Dulbecco's Modified Eagle Medium (DMEM) media supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin. The cells were incubated for 2 to 3 days at 37°C under humid conditions in 5% CO2 incubator (ThermoFisher Scientific, Carlsbad, CA). The growth media was changed twice weekly. Cells were grown to 75–85% confluence then washed with 1X phosphate buffer saline (PBS), trypsinized with 5 mL of 0.25% (v) trypsin-0.0.3% EDTA, diluted with fresh media, and counted using a Beckman Coulter ViCell (Beckman Coulter Inc., Model VI- CELL SGL).

HME50-5E cells were also cultured in 25 cm2 tissue culture flask and maintained in Mammary Epithelial Cell Media and were incubated for 7 to 8 days at 37°C under humid conditions in 5% CO2 incubator. The media was changed every other day. Cells were grown to 60-70% confluence then washed with ice cold 1X phosphate buffer saline (PBS), trypsinized with 5 mL of Accutase, diluted with fresh media, and counted using a Beckman Coulter ViCell (Beckman Coulter, Inc. Model VI-CELL SGL).

Optimization of Cell Density for Treatment

Log growth cell curves in 48 well plate was performed by seeding different densities of cells, 5,000; 10,000; 20,000; 40,000 and 100,000 cells/well and cell growth was plotted against time for 7 days. This was done to determine and assure a logarithmic growth of cells throughout the experimental time period.

Cell Treatments

All experiments were performed in a cell culture hood in an aseptic environment. Exponentially growing cells were harvested from flasks, counted by Trypan Blue exclusion, and plated into 48 well plates at the predetermined optimal seeding density of 40,000 cells (MDA-MB-231, MDA-MB-468, HME50-5E) per well. Cells were allowed to grow for 5 days post seeding prior to day 0 of treatment. Media was changed on day 3 post seeding and treatments were begun with the addition of fresh culture media.

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Avastin® was used as the experimental control for Selenoavastin. Sodium selenite (2.2 mg of sodium selenite containing 1 mg of Se) was used as a positive control for cell-death. Control Cells were treated with 1X PBS. Avastin®, Selenoavastin or selenite was added to wells in increasing concentrations to MDA-MB-231, MDA-MB- 468 cells and an immortalized human mammary epithelial cells, HME50-5E. Avastin® (30.7 µg protein; 76.7 µg protein; 153.7 µg protein; 307.5 µg protein), Selenoavastin (2 µg Se and 30.7 µg protein; 5 µg Se and 76.7 µg protein;10 µg Se and 153.7 µg protein; 20 µg Se and 307.5 µg protein) or selenite (2 µg, 5 µg, 10 µg, 20 µg as Se) were tested in triplicate in 48 well plates and analyzed on days 0,1,2,3,4,5,6 and 7 for cytotoxicity and cell viability.

Visual Assessments of Cellular Morphology

A phase-contrast microscope, EVOS XL Core (Life Technologies, Carlsbad, CA) was used for photographing cell morphological changes due to Selenite, Avastin® and Selenoavastin treatments.

Cell Viability Measured by Trypan Blue Exclusion

Cell viability from control and experimental treated cells was determined using a Beckman Coulter Vi-Cell Viability Analyzer (Beckman Coulter, Inc. Model VI-CELL SGL) with viability based on Trypan Blue cell exclusion.

Experimental Cell Cultures

TNBC and the immortalized breast epithelial cells; 40,000 cells/well were seeded in 48 well flat-bottom plates, cell volumes were adjusted with media and cells were allowed to grow for 5 days prior to treatments. Media was changed on day 3 post seeding and just before treatment. Cells were treated with Selenoavastin as 2 µg Se and 30.7 µg protein, 5 µg Se and 76.7 µg protein, 10 µg Se and 153.7 µg protein, 20 µg Se and 307.5 µg protein in triplicate for 7 days. Cells were treated with selenite 2, 5, 10 and 20 µg as Se. Additionally, cells were concentration and time dependent treated in comparison to native parent Avastin® (30.7 µg protein; 76.7 µg protein; 153.7 µg protein; 307.5 µg protein).

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On days 0 through 7 of treatments; 500 µl of media (containing treatments) from each well was collected in a coulter cup. The cells were rinsed with 500 µl of 1X PBS (kept in incubator for 5 minutes) and this 1X PBS was collected in coulter-cups. 200 µl of 0.025% trypsin-EDTA was added to harvest adherent cells. After 5 minutes of incubation, trypsin was added to the coulter-cups containing media and 1X PBS. Wells were observed to make sure all adherent cells were collected under the microscope. Cells were analyzed with the Beckman Coulter Vi-Cell Viability Analyzer for this experiment with the following settings and criteria:

Cell type: BrCa cells Minimum diameter (microns): 12 Maximum diameter (microns): 50 Cell brightness (%): 85 Cell sharpness: 100 Viable cell spot brightness (%): 65 Viable cell spot area (%): 5

Measuring Cell Viability Using MTT Assay

TNBC and the immortalized breast epithelial cells; 40,000 cells/well were seeded into 48 well flat-bottom plate, volumes were adjusted with media, and cells were allowed to grow for 5 days prior to treatment. Cells were treated (on day 5 post seeding) with three different doses of Selenoavastin 2 µg Se and 30.7 µg protein, 5 µg Se and 76.7 µg protein, 10 µg Se and 153.7 µg protein in triplicates for 6 days. Cells were treated with equivalent protein Avastin® treatments and selenite (2, 5, and 10 µg as Se). Blanks without cells contained complete growth media (Phenol red free DMEM high glucose + 10% FBS). MTT was dissolved in phenol red-free media, 5 mg/mL was passed and sterilized through a 0.22 µM filter inside the cell culture hood. The tube containing the MTT solution was wrapped with foil paper and used immediately. To each well 10% (v/v) i.e., 50 µl MTT (5 mg/mL) was added and incubated at 37oC for 3 hours. Following incubation with MTT, Formazan solubilization solution [acidified isopropanol (0.1N

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HCl) with 10% Triton X-100] equal to the original volume of media (500 µl) was added in each well. Dissolved Formazan in each well was determined by a Cytation 3 plate reader (BioTek, Winooski, VT, USA) at 570 nm absorption with baseline subtraction of 690 nm absorption to assess cell viability. The control cells were considered to be 100% viable. Cell viability percentage was calculated using following equation III-1.

퐴푏푠표푟푏푎푛푐푒 표푓 퐴푣푎푠푡푖푛 표푟 푆푒푙푒푛표푎푣푎푠푡푖푛 − 푡푟푒푎푡푒푑 푐푒푙푙푠 퐶푒푙푙 푣푖푎푏푖푙푖푡푦 = 푋 100 (III-1) 퐴푏푠표푟푏푎푛푐푒 표푓 푐표푛푡푟표푙 푐푒푙푙푠

Cell viability data was then used to calculate the 50% cell inhibition concentration; 50%

(IC50).

MitoTracker® Red and Annexin V Staining

Into 48 well flat-bottom plates 40,000 cells/well were seeded were allowed to grow for 5 days prior to treatment. Media was changed on day 3 post seeding and volume was adjusted before treatment. Cells were treated (on day 5 post seeding) with Selenoavastin 2 µg Se and 30.7 µg protein; equivalent protein concentration of unconjugated, native Avastin® and selenite (2 µg Se/well). Additionally, the control cells were untreated. Another set of control cells for staining were prepared- unstained control was untreated+unstained. For necrosis control (double-negative), cells were treated with

50 µl of 0.01% Triton X-100 for 30 minutes prior to staining or 200 µl of H2O2 for 24 hours prior to staining. For single staining controls, appropriate wells were treated with the dyes separately (Annexin V – 488 were treated with apoptosis inducer i.e., 5 µl of 1 mM Sutent; MitoTracker® Red-untreated cells). For double staining controls (double- positive), appropriate wells were treated with both dyes of the same volume. These controls were set aside in 2 mL Eppendorf tubes. Each treatment was repeated in triplicate.

A 10 mM stock solution of MitoTracker® Red dye was prepared by adding 9.4 μL DMSO to a vial of MitoTracker® Red dye. At a 10 μM working solution the MitoTracker® Red dye was prepared by pipetting 1 μL of 10 mM MitoTracker® Red

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Texas Tech University, Soni Khandelwal, May 2018 stock solution into 1,000 μL of DMEM high glucose phenol red free cell media. 2 μL of 10 μM MitoTracker® Red working solution was added to each well (500 µl) and stained for 30 minutes at 37°C in an atmosphere of 5% CO2. After 30 minutes of incubation, the media was collected in a sterile 2 mL Eppendorf tube. The cells were washed with 500 µl of 1X PBS and collected in the appropriate Eppendorf tube. Cells were trypsinized using 200 µl of 0.025% trypsin-EDTA and centrifuged. Cells were resuspended in 100 µl of 1X Annexin-binding buffer. This suspension was transferred into 96 well flat-bottom plates. 4 µl of Annexin V dye was added to each well. The 96-well plate was wrapped in aluminum foil paper. The cells were incubated for 15 minutes at 37°C in an atmosphere of 5% CO2. After the incubation, 100 µl of 1X Annexin-binding buffer was added. The plate was placed immediately on ice. The stained cells were analyzed by flow cytometry on the Attune™ NxT Flow Cytometer (ThermoFisher Scientific, Carlsbad, CA). The laser lines were set to BL1 (200 nm) and YL1 (336 nm). Voltage for Forward Scatter was set to 120 and Side Scatter to 290.

Western Blotting

One million MDA-MB-231, MDA-MB-468 and HME50-5E cells were allowed to attach to T-25 flasks overnight. The following day cells were treated with Selenoavastin as 2 µg of Se and 30.75 µg protein and incubated for 3 days. Control cells were treated with 1X PBS under the same conditions and with the same points.

On day 3 of treatment, cultured cells fluid was collected in a 15 mL centrifuge tube and cells were washed with ice cold 1X PBS and collected in the same tube. The tube was centrifuged at 4000 rpm for 4-5 minutes and the media was aspirated with a pasteur pipette. 200 µl of RIPA lysis was added to the tube and everything was transferred to T25 flask. T25 flasks were kept at -80oC for one day. This was done to get a better cancer cell protein lysate yield when the flask was thawed. To collect the HME50-5E cell lysates, flasks were broken with a hammer and cells were scrapped and kept on ice for 5 minutes. HME50-5E lysates were passed through a 20 G needle and kept on ice for 5 minutes. All cell lysates were kept on an inverter for 15 minutes at 4°C and centrifuged at 12,000 rpm for 15 minutes at 4°C. Total protein concentration in the

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Texas Tech University, Soni Khandelwal, May 2018 cleared lysate was determined by the bicinchoninic acid (BCA) assay according to manufacturer’s instructions. Forty µg of total protein was separated on 8% denaturing polyacrylamide gels and transferred to PVDF membranes by electroblotting. Membranes were blocked for 1 hour with Tris-buffered saline containing 0.05% Tween-20 (TBST) and 5% bovine serum albumin fatty acid free. They were incubated overnight with VEGF (JH121) monoclonal antibody diluted 1:100 in TBST or anti-β-actin antibodies diluted 1:1000 in TBST containing 1% bovine serum albumin. The next day, the membrane was washed 3 times for 45 minutes in TBST, incubated for 1 hour with horse-radish peroxidase conjugated with rabbit anti-mouse IgG diluted 1:10000 in TBST, and washed once in PBST for 15 minutes. Bound antibody complexes were visualized using SuperSignal™ West Femto Maximum Sensitivity Substrate.

STATISTICAL ANALYSES

All experimental assays were conducted in triplicate and the data are representative of three independent experiments. The results are expressed as the mean ± standard error (SE). Statistical analyses were performed using the MATLAB ver 9.2 (Release R2017a) two sample t test for across treatments. Differences were considered significant at p≤0.05. IC50 was calculated using a two-parameter sigmoidal model. The results of statistical analyses are summarized in Table III-1, Table III-2 and Table III-3.

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Table III-1: Statistical Analyses of Avastin® and Selenoavastin treatments against MDA-MB- 231 Cells.

Experiment Treatments p value Day Trypan Blue Avastin® as 76.75 µg protein vs Selenoavastin as 5 µg Se and 76.7 µg protein 0.009 3 (Figure III-15) Avastin® as 153.75 µg protein vs Selenoavastin as 10 µg Se and 153.7 µg 0.001 protein Avastin® as 307.5 µg protein vs Selenoavastin as 20 µg Se and 307.5 µg protein 0.001 Day Trypan Blue Control vs Selenoavastin as 2 µg Se and 30.7 µg protein 0.003 4 (Figure III-15) Control vs Selenoavastin as 5 µg Se and 76.7 µg protein 0.001 Avastin® as 30.7 µg protein vs Selenoavastin as 2 µg Se and 30.7 µg protein 0.002 Day MTT Control vs Selenoavastin as 2 µg Se and 30.7 µg protein 0.005 3 (Figure III-18) Control vs Selenoavastin as 5 µg Se and 76.7 µg protein 0.002 Control vs Selenoavastin as 10 µg Se and 153.7 µg protein 0.0020 Avastin® as 30.7 µg protein vs Selenoavastin as 2 µg Se and 30.7 µg protein 0.019 Avastin® as 76.75 µg protein vs Selenoavastin as 5 µg Se and 76.7 µg protein 0.003 Day MTT Control vs Selenoavastin as 2 µg Se and 30.7 µg protein and 0.001 4 (Figure III-18) Control vs Selenoavastin as 5 µg Se and 76.7 µg protein 0.001 Control vs Selenoavastin as 10 µg Se and 153.7 µg protein 0.001 Avastin® as 30.7 µg protein vs Selenoavastin as 2 µg Se and 30.7 µg protein 0.001 Annexin V Avastin® as 30.7 µg protein vs Selenoavastin as 2 µg Se and 30.7 µg protein 0.001 (Figure III-25) (late apoptosis)

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Table III-2: Statistical Analyses of Avastin® and Selenoavastin treatments against MDA-MB- 468 Cells.

Experiment Treatments p value Day Trypan Blue Control vs Selenoavastin as 5 µg Se and 76.7 µg protein 0.033 3 (Figure Control vs Selenoavastin as 10 µg Se and 153.7 µg protein 0.001 III-16) Control vs Selenoavastin as 20 µg Se and 307.5 µg protein 0.001 Avastin® as 76.75 µg protein vs Selenoavastin as 5 µg Se and 76.7 µg protein 0.002 Avastin® as 153.75 µg protein vs Selenoavastin as 10 µg Se and 153.7 µg protein 0.001 Avastin® as 307.5 µg protein vs Selenoavastin as 20 µg Se and 307.5 µg protein 0.001 Day Trypan Blue Control vs Selenoavastin as 5 µg Se and 76.7 µg protein 0.003 4 (Figure Control vs Selenoavastin as 10 µg Se and 153.7 µg protein 0.001 III-16) Avastin® as 30.7 µg protein vs Selenoavastin as 2 µg Se and 30.7 µg protein 0.009 Day MTT Control vs Selenoavastin as 5 µg Se and 76.7 µg protein 0.008 3 (Figure Control vs Selenoavastin as 10 µg Se and 153.7 µg protein 0.001 III-19) Avastin® as 76.75 µg protein vs Selenoavastin as 5 µg Se and 76.7 µg protein 0.005 Avastin® as 153.75 µg protein vs Selenoavastin as 10 µg Se and 153.7 µg protein 0.002 Day MTT Control vs Selenoavastin as 2 µg Se and 30.7 µg protein 0.024 4 (Figure Control vs Selenoavastin as 5 µg Se and 76.7 µg protein 0.001 III-19) Control vs Selenoavastin as 10 µg Se and 153.7 µg protein 0.001 Avastin® as 76.7 µg protein vs Selenoavastin as 5 µg Se and 76.7 µg protein 0.020 Annexin V Avastin® as 30.7 µg protein vs Selenoavastin as 2 µg Se and 30.7 µg protein (early 0.001 (Figure apoptosis) III-28) Avastin® as 30.7 µg protein vs Selenoavastin as 2 µg Se and 30.7 µg protein (late 0.001 apoptosis)

Table III-3: Statistical Analyses of Avastin® and Selenoavastin treatments against HME50- 5E Cells.

Experiment Treatments p value Day 3 Trypan Blue Control vs Selenoavastin as 5 µg Se and 76.7 µg protein 0.047 (Figure III-17) Avastin® as 30.7 µg protein vs Selenoavastin as 2 µg Se and 30.7 µg protein 0.001 Avastin as 76.75 µg protein vs Selenoavastin as 5 µg Se and 76.7 µg protein 0.001 Avastin® as 153.75 µg protein vs Selenoavastin as 10 µg Se and 153.7 µg protein 0.046 Day 4 Trypan Blue Control vs Selenoavastin as 2 µg Se and 30.7 µg protein 0.001 (Figure III-17) Avastin® as 30.7 µg protein vs Selenoavastin as 2 µg Se and 30.7 µg protein 0.001 Avastin® as 76.75 µg protein vs Selenoavastin as 5 µg Se and 76.7 µg protein 0.001 Day 4 MTT Control vs Selenoavastin as 2 µg Se and 30.7 µg protein 0.001 (Figure III-20) Avastin® as 30.7 µg protein vs Selenoavastin as 2 µg Se and 30.7 µg protein 0.014 Avastin® as 76.75 µg protein vs Selenoavastin as 5 µg Se and 76.7 µg protein 0.001

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RESULTS AND DISCUSSION

Avastin® is a derivative of a murine VEGF monoclonal antibody,A4.6.1 the first approved anti-angiogenesis treatment for human cancer [195]. It is of ~93% human and ~7% murine protein sequences [195]. The efficacy of Avastin® is well documented in inhibiting tumor angiogenesis in various mouse and human tumor models [196]. Additionally, in a pre-clinical study, Avastin® neutralized all the isoforms of VEGF leading to tumor growth inhibition [197]. Clinical trials/studies have shown that the combination of Avastin® with standard chemotherapy produces far better response rates than does Avastin® alone. As a model, we chose Avastin® conjugated with redox Se with the rationale that Avastin® may target a subset of carcinoma cells that tend to be highly sensitive to the toxic activity of Se.

Successful redox Se conjugation to Avastin® resulted in pale red colored final solution (Figure III-4). To estimate Se concentration, Selenoavastin was analyzed by inductively coupled plasma mass spectrometry (ICP-MS; Trace Analysis, Inc., Lubbock, TX). The Selenoavastin Se concentration was 32.12 µg Se/mg protein (shown in Table III-1, Table III-2 and Table III-3) after 72 hours of conjugation time and another 72 hours of exhaustive dialysis. Se concentrations of native Avastin® was <0.007 µg Se/mg protein. Table III-4 shows the protein concentration of both native Avastin® and Selenoavastin determined using bicinchoninic acid (BCA) assay and bovine gamma globulin used as the standard reference protein. Similar protein concentrations were determined using nanodrop and BCA assay where bovine serum albumin was used as the standard protein for reference (data not shown).

Avastin® and Selenoavastin were stored in glass scintillation vials away from light exposure. Both Avastin® and Selenoavastin remained clear for the duration of the study with no precipitates or particulates detected by eye. No change in color or turbidity was observed. Neither was there a change in the pH of the antibodies tested throughout the study period as shown in Figure III-4.

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Figure III-4: Selenoavastin and Avastin.

Table III-4: Selenium and Protein Concentration of Avastin® and Selenoavastin.

Avastin® Selenoavastin

81.3 Selenium Concentration (mg/L) <0.0200 Protein Concentration (mg/mL) 2.73 2.53

Selenium Concentration/ mg of protein (µg/mg) 0.007 32.12

Avastin® and Selenoavastin were analyzed by lucigenin superoxide chemiluminescence (CL) detection Turner TD-20e Luminometer. Figure III-5 and Figure III-6 demonstrates the experimental CL results. In these reactions, lucigenin is reduced by the superoxide anion generated and chemiluminescence was observed for Avastin® and Selenoavastin. Over a 12.5-minutes CL counting period (Figure III-6), the reaction between Selenoavastin and glutathione (GSH) was seen to be generally higher than Avastin® at the same GSH concentration. These experiments confirmed the generation of superoxide from the oxidation of GSH by selenite as shown by Seko et al., [91] and extended the observation to Selenoavastin but not CL cocktail or Avastin®. After determining the chemiluminescence activity of Avastin® and Selenoavastin, experimental steps were taken to determine if Avastin® and Selenoavastin would generate superoxide in situ. The photographs taken on an EVOS FL Auto Cell Imaging

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System in Figure III-7, Figure III-8 and Figure III-9 show the intracellular production of superoxide by Selenoavastin and selenite but not Avastin® as analyzed by DHE in MDA-MB-231, MDA-MB-468 and HME50-5E cells. Similar results have been reported from prior studies in the laboratory that have compared the cytotoxicity of selenite, selenate, selenocystine and selenomethionine towards human mammary tumor cell line HTB123/DU4475 in vitro [155]. In contrast to the TNBC cell line, Selenoavastin did not induce such severe superoxide production in HME50-5E cells (Figure III-9). This difference in TNBC cells and the HME50-5E cells resistance to redox selenium suggests a biochemical variance that can provide a basis for more selective killing of cancer cells with lesser toxicity to normal cells. These results collectively suggest the ability of Selenoavastin to generate superoxide by the oxidation of glutathione and other thiols within TNBC cells.

Figure III-5: Total Superoxide Generation as Measured by Lucigenin Chemiluminescence (CL) with Glutathione (GSH). Chemiluminescence (CL) for Control Blank, Avastin® and Selenoavastin measured over 12.5 minutes. Data is expressed as Mean ± SE. CL for Avastin® and Selenoavastin was compared with CL Blank. CL for Selenoavastin was statistically significant (p≤0.001). 100µL of Selenoavastin contains 8.1µg of Se.

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Figure III-6: Time Dependent Superoxide Generation as Measured by Lucigenin Chemiluminescence (CL) for Blank, Avastin® and Selenoavastin. Control Blank Avastin® and Selenoavastin CL was measured over 12.5 minutes with 30 second integrations. 100µL of Selenoavastin contains 8.1µg of Se.

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Figure III-8: Photomicrographs of Intracellular Superoxide Generation by DHE Red Florescence from MDA-MB-231 Cells after 30 minutes of Blank, Control, Selenite (10 µg), Avastin® (153.7 µg protein) and Selenoavastin (10 µg Se and 153.7 µg protein) Treatments. Scale bar=200 µm.

Figure III-7: Photomicrographs of Intracellular Superoxide Generation by DHE Red Florescence from MDA-MB-468 Cells after 30 minutes of Blank, Control, Selenite (10 µg), Avastin® (153.7 µg protein and Selenoavastin (10 µg Se and 153.7 µg protein) Treatments. Scale bar=100 µm.

Figure III-9: Photomicrographs of Intracellular Superoxide Generation by DHE Red Florescence from HME50-5E Cells after 30 minutes of Blank, Control, Selenite (10 µg), Avastin® (153.7 µg protein) and Selenoavastin (10 µg Se and 153.7 µg protein) Treatments. Scale bar=200 µm.

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To assess what changes may have been made to the Avastin® by the conjugation of the redox Se, a polyacrylamide gel electrophoresis of labeled and native Avastin® was conducted showing little change in the electrophoretic pattern of labeled Avastin® (Lanes 6, 7, 8 in Figure III-10) as compared to native Avastin® (Lanes 3, 4, 5 in Figure III-10). Subsequent separation of unconjugated Avastin® and Selenoavastin on SDS-PAGE was done as shown in Figure III-10 to evaluate the homogeneity of the heavy and light chains of native Avastin® and Selenoavastin (5 µg, 10 µg and 20 µg protein/lane). Using Coomassie Blue stain, the high molecular weight band of above 245 kDa was observed suggestive of new Se construct of Selenoavastin. Protein bands also migrated to 180 kDa, 135 kDa 75kDa, 48 kDa and with a lower molecular weight band of approximately 25 kDa (Figure III-2), corresponding to the heavy and light chains of Avastin®, an IgG1 molecule. Kadcyla® (Lane 1), Bovine gamma globulin (Lane 14) and Avastin® (Lanes 3, 4 and 5) formed a doublet at all concentrations between molecular masses of 245 and 180 kDa. Formation of the doublet on SDS-PAGE was due to incomplete reduction of IgG1 molecules into heavy and light chains. However, migration of unreduced IgG1 as a doublet is a consistent phenomenon [198].

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Figure III-10: SDS-PAGE of Native and Se-conjugated mAbs under reducing conditions followed by Coomassie Blue R-250 staining. The photograph was taken under Coomaisse Blue filter. Lane 1: Kadcyla® 20 μg; Lane 2: Marker; Lane 3: Avastin® 5 µg; Lane 4: Avastin® 10 µg; Lane 5: Avastin® 20 µg; Lane 6: Selenoavastin 5 µg; Lane 7: Selenoavastin 10 µg; Lane 8: Selenoavastin 20 µg; Lane 9: Herceptin® 5 µg; Lane 10: Herceptin® 10 µg; Lane 11: Herceptin® 20 µg, Lane 12: Selenoherceptin 10 µg; Lane 13: Selenoherceptin 20 µg; Lane 14: Gamma globulin 20 µg

Native PAGE protein migration depends on the charge and conformational state of the protein and it is considered as an excellent tool to assess protein stability. To assess protein stability, at least two different amounts of Avastin® were loaded on 4-20% Tris- Glycine PAGE and were run under non-reducing conditions as shown in Figure III-11. Unreduced native mAbs- Kadcyla® (Lanes 5 and 6) and Avastin® (Lanes 7 and 8) show identical band intensities and mobilities. A trailing band was seen for Selenoavastin (Lanes 9 and 10). The trailing band of Selenoavastin was similar to the trailing band of Bovine gamma globulin (Lanes 3 and 4). Both Selenoavastin (Lanes 9 and 10) and Selenoherceptin (Lanes 13 and 14) show identical band mobilities. An identical mobility would mean an identical molecular weight and charge [199]. No smaller products representing protein degradation or larger products representing protein aggregation were detectable on native gels. Hence the conjugated and unconjugated mAbs were uncontaminated. The photograph of native Tris-Glycine PAGE gel of Avastin® and Selenoavastin (Figure III-11) show that the band pattern was qualitatively independent of

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Texas Tech University, Soni Khandelwal, May 2018 the sample volume loaded across all the different sample concentrations. This was true for both native Tris-Glycine PAGE (Figure III-11) and SDS-PAGE (Figure III-10). This is in line with one study which determined the prolonged storage of Avastin® [200].

Figure III-11: mAbs migration on 4-20% Tris-Glycine PAGE gel under non-reducing conditions followed by Coomaisse Blue R-250 stain. The photograph was taken under Coomassie Blue filter. Lane 1: Molecular Marker; Lane 2: Purified Human IgG 10 μg; Lane 3: Bovine Gamma Globulin 5 μg; Lane 4: Bovine Gamma Globulin 10 μg; Lane 5: Kadcyla® 5 μg; Lane 6: Kadcyla® 10 μg; Lane 7: Avastin® 5 μg; Lane 8: Avastin® 10 μg; Lane 9: Selenoavastin 5 μg; Lane 10: Selenoavastin 10 μg; Lane 11: Herceptin® 5 μg; Lane 12: Herceptin® 10 μg; Lane 13: Selenoherceptin 5 μg; Lane 14: Selenoherceptin 10 μg.

Bovine gamma globulin is considered as a control for running mAbs because gamma globulins are good reference for protein quantitation of immunoglobulins. Kadcyla®, another monoclonal antibody as another control because of its similar framework. The structure of Kadcyla® consists of Herceptin®, linker and a cytotoxic

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Texas Tech University, Soni Khandelwal, May 2018 agent Emtansine [201]; which is identical to Selenoavastin, linker and Se. Additionally, the main function of Kadcyla® delivers the toxin specifically to tumor cells; which is like Selenoavastin.

To visualize morphological effects to toxicity, MDA-MB-231, MDA-MB-468 and HME50-5E cells were treated with equal amounts of selenium as of Selenoavastin, selenite and Avastin®. Images were taken under a light microscope at 20X magnification and results shown in Figure III-12 (MDA-MB-231), Figure III-13 (MDA-MB-468), Figure III-14 (HME50-5E). Selenoavastin and selenite treated cells show gross cell morphological changes, cell membrane disruption, growth inhibition, with both cell swelling and shrinkage, and ultimately detachment from the cell culture substratum. MDA-MB-231, MDA-MB-468 and HME50-5E cells are all adherent epithelial cells. With adherent cells, detachment from the substratum as well as morphological change is indicative of apoptosis [156]. Additionally, the morphological change (from the grape- like structure in MDA-MB-468 cells and the stellate-elongated structure in MDA-MB- 231 cells to the extensive cytoplasmic vacuolization) became more remarkable with increased treatment exposure. Similar morphological change in cancer cells treated with other redox active Se compounds have been reported in other in vitro studies [157]. From a therapeutic point of view, the clinical relevance of this observation suggests that Selenoavastin may be effective in suppressing tumor growth than Avastin®. Selenite also caused morphological change in cell anatomy but since it is a non-targeting treatment, it demonstrated general toxicity. Additional photomicrographs of morphological changes due to Selenite, Avastin® and Selenoavastin are included in APPENDIX A (Figure A-9, Figure A-10, Figure A-11, Figure A-12, Figure A-13 and Figure A-14).

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Figure III-12: Morphological Changes of Control, Selenite, Avastin® and Selenoavastin Treatments in MDA-MB-231 Cells. Treatment of MDA-MB-231 Cells with Selenite and Selenoavastin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-231 cells. Cells were visualized under 20X magnification.

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Figure III-13: Morphological Changes of Control, Selenite, Avastin® and Selenoavastin Treatments in MDA-MB-468 Cells. Treatment of MDA-MB-468 Cells with Selenite and Selenoavastin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-468 cells. Cells were visualized under 20X magnification.

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Figure III-14: Morphological Changes of Control, Selenite, Avastin® and Selenoavastin Treatments in HME50-5E Cells. Treatment of HME50-5E cells with Selenite, Avastin® and Selenoavastin did not induce severe morphological change comparable to those seen in TNBC cells. Representative fields of view of HME50-5E cells. Cells were visualized under 20X magnification.

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Following optimization of cell growth of the MDA-MB-231, MDA-MB-468 and the HME50-5E cells, the cells were treated with Avastin®, Selenoavastin or selenite. The dose response results of control cell growth and the three treatments are shown in Figure III-15, Figure III-16 and Figure III-17 as measured for cell viability by the Trypan Blue exclusion. These three experimental treatments show that selenite and Selenoavastin exhibit a general loss of viable cells over Se dose and time as measured against control and Avastin® treated cells. Selenoavastin as 20 µg Se and 307.5 µg protein showed highest cytotoxicity in MDA-MB-231 and MDA-MB-468 cells but lowest treatment concentration Selenoavastin as 2 µg Se and 30.7 µg protein was also cytotoxic. This is evident from Figure III-12, Figure III-13 and Figure III-14. Two, five, ten and twenty µg of Se (as selenite) per well, was cytotoxic to all three cell types tested. Sodium selenite an inorganic chemical form of selenium is toxic, inhibits cell proliferation by apoptosis not only in human and mouse breast cancer cell lines but also in human colon carcinoma cells, human ovarian cancer cells, and human prostate cancer cells in vitro [158]. MDA- MB-231, MDA-MB-468 control cells receiving native unconjugated Avastin® alone at the highest protein concentrations tested all show high cell viability at all time periods.

Trypan Blue exclusion experiments were not conducted with HME50-5E at Selenoavastin as 20 µg Se and 307.5 µg protein treatment. In part, successful analysis was difficult due to the strong adherence properties of the HME50-5E cells, causing experimental bias. Initially 200 µl of 0.025% trypsin-EDTA was added and incubated for 10-15 minutes but when viewed under a microscope not all the cells completely detached. So, another 200 µl of 0.025% trypsin-EDTA was added to cells to try to collect the whole population. Despite incubating with double the amount (400 µl) of trypsin, a substantial number of cells remained. This inability to adequately remove all HME50-5E cells from the plate resulted in a high percentage loss of cells inadequate representation for the Trypan Blue assay.

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Figure III-15: Cytotoxicity of Control, Selenite, Avastin® and Selenoavastin in Dose and Time Dependent Treatments against MDA-MB-231 cells. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding. Control cells were incubated for 7 days with Selenite, Avastin® and Selenoavastin at different concentrations. Viable cells were counted at time periods (0-7 days) by Trypan Blue exclusion as described in Methods. The cell growth data are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table III-1.

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Figure III-16: Cytotoxicity of Control, Selenite, Avastin® and Selenoavastin in Dose and Time Dependent Treatments against MDA-MB-468 cells. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding. Control cells were incubated for 7 days with Selenite, Avastin® and Selenoavastin at different concentrations. Viable cells were counted at time periods (0-7 days) by Trypan Blue exclusion as described in Methods. The cell growth data are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table III-2.

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Figure III-17: Cytotoxicity of Control, Selenite, Avastin® and Selenoavastin in Dose and Time Dependent Treatments against HME50-5E cells. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding. Control cells were incubated for 7 days with Selenite, Avastin® and Selenoavastin at different concentrations. Viable cells were counted at time periods (0-7 days) by Trypan Blue exclusion as described in Methods. The cell growth data are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table III-3.

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To better understand the mechanism of cell death demonstrated by the Trypan Blue exclusion results, an alternative cytotoxic assessment assay of cell viability, i.e., the MTT assay (3-[4, 5-dimethylthioazol-2-yl]-2-5-diphenyltetrazolium bromide) was performed, where the tetrazolium salt ring is cleaved by viable cells forming the insoluble Formazan. This method would also help address the question of cell viability in the HME50-5E cells when treated with Selenoavastin. The results for the three cell lines tested is illustrated in Figure III-18, Figure III-19 and Figure III-20. The results demonstrate that Selenoavastin and Selenite treatments are effective in repressing cell proliferation in these cell lines. Percent viability for MDA-MB-231 cells treated with Selenoavastin as 10 µg Se,153.7 µg protein was 64% cell viability whereas Selenoavastin as 2 µg Se,30.7 µg protein treatment was 77% on day 3 post treatment. Interestingly, MDA-MB-468 cells were more resistant as viability was 104% at Selenoavastin as 2 µg Se,30.7 µg protein treatment. MDA-MB-468 percent viability was 35% at Selenoavastin as 10 µg Se,153.7 µg protein treatment. Thus, the results from the MTT assay show that Selenoavastin dose-dependently and significantly reduced cell viability.

MTT incubation in the HME50-5E cells yielded an interesting result. When the MTT solubilization media was added to the HME50-5E cells, the Formazan salt could not be dissolved completely because of the adherence tendency of the cells. Also, the concentration of Formazan was so dark that plate reader displayed ‘OVERFLOW’ for results on days 1 and 3 of treatments indicating that the metabolic activity was so high, it saturated the limits of detection for the instrumentation. This observation coupled with the confluent, adherent populations, suggested that the vast majority of the population was metabolically healthy following Se treatments. Control HME50-5E cell viability was therefore set at 100% viability for days 1 and 3 treatments. Moreover, no significant differences at low concentration of Avastin® and Selenoavastin treatments were observed on day 3. Ip [157] mentioned that normal cells, early transformed cells and late stage preneoplastic cells may respond differently to selenium intervention with respect to molecular pathways involving cell cycle proteins and apoptotic proteins. The photomicrographs reflect this result (shown in Figure III-14) with no morphological changes evident and loss of viable cells in the Trypan Blue assay.

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Figure III-18: Metabolic activity of Control, Selenite, Avastin® and Selenoavastin treated MDA-MB-231 cells as determined by the MTT Assay. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding with Selenite, Avastin® and Selenoavastin at different concentrations over 6 days by MTT as described in Methods. The cell viabilities are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table III-1.

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Figure III-19: Metabolic activity of Control, Selenite, Avastin® and Selenoavastin treated MDA-MB-468 cells as determined by the MTT Assay. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding with Selenite, Avastin® and Selenoavastin at different concentrations over 6 days by MTT as described in Methods. The cell viabilities are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table III-2.

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Figure III-20: Metabolic activity of Control, Selenite, Avastin® and Selenoavastin treated HME50-5E cells as determined by the MTT Assay. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding with Selenite, Avastin® and Selenoavastin at different concentrations over 6 days by MTT as described in Methods. The cell viabilities are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table III-3.

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Dose dependent calculations were used to determine the IC50s of Selenoavastin on day 3 of post treatments as shown in Figure III-21 for MDA-MB-231 cell viability, and

Figure III-22 for MDA-MB-468 cell viability. Selenoavastin showed an IC50 of 1.91 µg Se for MDA-MB-231 cells and 1.47 µg Se for MDA-MB-468 cells on day 3 of post treatment. Since cell viability for HME50-5E cells was assumed to be 100% as described above, IC50 of HME50-5E was not able to be calculated as treatment values did not approach close enough to 50% to provide an accurate estimate.

Figure III-21: IC50 for MDA-MB-231 Cells with Selenoavastin.

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Figure III-22: IC50 for MDA-MB-468 Cells with Selenoavastin.

The experimental study results uniformly and collectively indicate that Selenoavastin can significantly inhibit cell proliferation and cell viability in the TNBC cell lines MDA-MB-231 and MDA-MB-468, but not the immortalized normal HME50- 5E epithelial cells. The cytotoxic data is consistent with the ability of both selenite and Selenoavastin but not Avastin® to generate superoxide in both the in vitro CL assay (Figure III-5 and Figure III-6) and as detected by the intracellular increase in the fluorescence of the intracellular DHE (Figure III-7, Figure III-8 and Figure III-9). The experimental results also appear to be confirmed by the optical assessment of altered cell morphology as photographed and shown in Figure III-12, Figure III-13 and Figure III-14.

Previous studies have established that selenium induces apoptosis by the intrinsic (mitochondrial-mediated) pathway [159-163]. Furthermore, apoptosis or programmed cell death is associated with morphological, biochemical and molecular changes occurring in the cell [164]. In order to confirm that Selenoavastin treatments also induce apoptosis, an Annexin V assay was performed. Both Selenoavastin and also selenite

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Texas Tech University, Soni Khandelwal, May 2018 treated cells showed prominent morphological changes, characteristic of apoptosis as evidenced by photomicrographs (Figure III-7, Figure III-8 and Figure III-9). Due to different sensitivities identified at day 3 for all cell lines tested in both Trypan Blue and MTT assays, cells were incubated with a low dose of Selenoavastin to determine apoptosis would be induced. Flow cytometry data of 2 µg Se as Selenoavastin treatments showed MDA-MB-468 cells exhibited more changes than MDA-MB-231 cells. A majority of the MDA-MB-231 cell population were in lower left (LL) quadrant, corresponding to living cells (Figure III-23). A substantial proportion of the MDA-MB- 468 cells in the upper right (UR) and lower right (LR) quadrants (as shown in Figure III-26), corresponding to early and late apoptosis respectively. The morphological phenotypes (Figure III-12, Figure III-13 and Figure III-14) of these apoptotic cells strongly correlated with flow analyses reported here. In contrast to the cancer cells, significant percentages of the HME50-5E cells were found in the upper left (UL) quadrant (as shown in Figure III-29), corresponding to living cells. The total percentages of cells are shown in Figure III-24, Figure III-27 and Figure III-30. The percentages of cells undergoing early and late apoptosis were measured by flow cytometry and results are shown in Figure III-25, Figure III-28 and Figure III-31.

Due to its apoptotic activity, Sutent (Pfizer) is used as a positive control for induction of apoptosis in TNBC [165] and HME50-5E cells. Interestingly, Sutent demonstrated even greater potency in killing the normal cells. To our knowledge, this is the first report of the toxicity of Sutent on normal cells. This suggests important consequences for clinical success of Sutent with respect to side effects. Further study is needed to investigate this observation. Control cells also showed apoptosis (Figure III-25, Figure III-28 and Figure III-31). One possibility for this phenomenon is that the rapidly dividing cells became over confluent in the limited well space, causing cell death. However, this was not observed in Trypan Blue exclusion (Figure III-15, Figure III-16 and Figure III-17) and MTT (Figure III-18, Figure III-19 and Figure III-20) assays. To address the appearance of a population of apoptotic cells in the vehicle control, prior studies of candidate anticancer therapies also demonstrated [162, 166] this phenomenon in their control groups.

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A loss of mitochondrial membrane potential (ΔψM) indicates the loss of cell viability as the electrical potential reflects the pumping of protons across the inner mitochondrial membrane during the processes of electron transport and oxidative phosphorylation that drives the conversion of ADP to ATP [167]. Therefore, MDA-MB- 468 and HME50-5E cells were stained with MitoTracker Red CMXRos (instead of Propidium Iodide), which stains mitochondria in live cells and its accumulation is dependent upon its ΔψM. In this study, the ΔψM was measured by flow cytometer and the results demonstrated that a dose of 2 µg Se as Selenoavastin caused a decrease in the ΔψM potential and increase in the percent apoptotic MDA-MB-468 cells (Figure III-26). In contrast, there was no change in the ΔψM (UL quadrant) of control vs Selenoavastin treated HME50-5E cells (Figure III-29), corresponding to full cell viability. This validates the intense Formazan color observed from the MTT assay assessing cellular metabolic activity (Figure III-20). Hence, HME50-5E cells do not undergo apoptosis following Selenoavastin treatment as determined by the cells ΔψM. These observations again suggest the probable role of selenium-induced oxidative stress/glutathione triggers apoptosis associated with both increased generation of superoxide and a decrease in level of the mitochondrial membrane potential. Studies have reported that modulation of mitochondrial functions to regulate apoptosis is one of the most affected pathways of Se compounds in cancer therapy [168]. Selenite also caused necrosis in MDA-MB-468 cells and apoptosis in MDA-MB-231 cells, and while few events were detected in HME50-5E cells, yet selenite is known to also induce necrosis in other breast cancer cells [157].

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Figure III-23: Avastin®, Selenoavastin and Selenite as Se Treatments induced apoptosis in MDA-MB-231 Cells. MDA-MB-231 cells were stained with Annexin V/PI and subjected to flow cytometric analysis. The four quadrants represent-living cells (Lower Left; LL: Annexin V-PI- ), early apoptotic (Lower Right; LR: Annexin V+PI-), late apoptotic (Upper Right; UR: Annexin+PI+) or necrotic (Upper Left; UL: Annexin V-PI+) stages.

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Figure III-24: Representative of four quadrants when MDA-MB-231 cells were treated with Avastin®, Selenoavastin and Selenite as Se. Data is expressed as Mean (n=3).

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Figure III-25: Percentage level of MDA-MB-231 apoptotic cells when the cells were treated with Avastin®, Selenoavastin and Selenite as Se. Data is expressed as Mean ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table III-1.

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Figure III-26: Avastin®, Selenoavastin and Selenite as Se Treatments induced apoptosis in MDA-MB-468 cells. MDA-MB-468 cells were stained with Annexin V/ Mitotracker Red and subjected to flow cytometric analysis. The four quadrants represent- living cells (Upper Left; UL: Annexin V- Mitotracker Red+), early apoptotic (Upper Right; UR: Annexin+ Mitotracker Red +), late apoptotic (Lower Right; LR: Annexin V+ Mitotracker Red-) or necrotic (Lower Left; LL: Annexin V- Mitotracker Red-) stages.

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Figure III-27: Representative of four quadrants when MDA-MB-468 cells were treated with Avastin®, Selenoavastin and Selenite as Se. Data is expressed as Mean (n=3).

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Figure III-28: Percentage level of MDA-MB-468 apoptotic cells when the cells were treated with Avastin®, Selenoavastin and Selenite as Se. Data is expressed as Mean ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant p ≤ 0.05 are summarized in Table III-2.

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Figure III-29: Avastin®, Selenoavastin and Selenite as Se Treatments did not induced apoptosis in HME50-5E cells. HME50-5E cells were stained with Annexin V/ Mitotracker Red and subjected to flow cytometric analysis. The four quadrants represent- living cells (Upper Left; UL: Annexin V- Mitotracker Red+), early apoptotic (Upper Right; UR: Annexin+ Mitotracker Red +), late apoptotic (Lower Right; LR: Annexin V+ Mitotracker Red-) or necrotic (Lower Left; LL: Annexin V- Mitotracker Red-) stages.

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Figure III-30: Representative of four quadrants when HME50-5E cells were treated with Avastin®, Selenoavastin and Selenite as Se. Data is expressed as Mean (n=3).

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Figure III-31: Percentage level of MDA- apoptotic cells when the cells were treated with Avastin®, Selenoavastin and Selenite as Se. Data is expressed as Mean ± SE (n=3).

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Western blotting was used to detect the protein expression levels of VEGF in MDA-MB-231, MDA-MB-468 and HME50-5E cells for Avastin®, Selenoavastin or selenite treatments (Figure III-32, Figure III-33 and Figure III-34). A band for the VEGF protein was detected at ~23 kDa and β-actin ~42 kDa in control cells, Avastin®, Selenoavastin. Cleaved VEGF expression was found in MDA-MB-231 and HME50-5E cells following selenite treatment. One possibility for cleaved VEGF expression in selenite treatment is its non-specific toxicity. Selenite toxicity is evident from Figure III-12, Figure III-13 and Figure III-14. However, cleaved β-actin expression was detected only after selenite (as 2 µg Se) treatment in all three cell lines and Selenoavastin treatment in HME50-5E cells. These may have some biological and therapeutic implications like cell-cell interactions, cell migration (metastasis) and proliferation [169]. This interesting observation requires study and was outside the scope of this current project. Furthermore, the safety and efficacy of Sodium selenite was evaluated in Phase I clinical trial. The results from this trial show that no major toxicity was reported when 10.2 mg/m2 of sodium selenite was administered in patients with terminal cancer [170].

Figure III-32: Western blot analysis of the expression level of VEGF in MDA-MB-231 Cells treated with Avastin®, Selenoavastin and Selenite as Se. Total cell lysates were subjected to SDS-PAGE followed by Western blotting. Membranes were probed with the anti-VEGF or anti β-actin antibodies followed by peroxidase conjugated rabbit anti-mouse antibodies and visualization was performed by the ECL detection system. Lanes 1 and 2: Control, Lanes 3 and 4: Selenite (2µg Se) treatments, Lanes 5 and 6: Avastin® (30.7µg protein) treatment, Lanes 7 and 8: Selenoavastin (2µg Se, 30.7 µg protein) treatments.

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Figure III-33: Western blot analysis of the expression level of VEGF in MDA-MB-468 Cells treated with Avastin®, Selenoavastin and Selenite as Se. Total cell lysates were subjected to SDS-PAGE followed by Western blotting. Membranes were probed with the anti-VEGF or anti β-actin antibodies followed by peroxidase conjugated rabbit anti-mouse antibodies and visualization was performed by the ECL detection system. Lanes 1 and 2: Control, Lanes 3 and 4: Selenite (2µg Se) treatments, Lanes 5 and 6: Avastin® (30.7µg protein) treatment, Lanes 7 and 8: Selenoavastin (2µg Se, 30.7 µg protein) treatments.

Figure III-34: Western blot analysis of the expression level of VEGF in HME50-5E Cells treated with Avastin®, Selenoavastin and Selenite as Se. Total cell lysates were subjected to SDS-PAGE followed by Western blotting. Membranes were probed with the anti-VEGF or anti β-actin antibodies followed by peroxidase conjugated rabbit anti-mouse antibodies and visualization was performed by the ECL detection system. Lane 1: MDA-MB-468 as VEGF positive loading control, Lanes 2 and 3: Control, Lanes 4 and 5: Selenite (2µg Se) treatments, Lanes 6 and 7: Avastin® (30.7µg protein) treatment, Lanes 8 and 9: Selenoavastin (2µg Se, 30.7 µg protein) treatments.

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This study demonstrates that TNBC cells, MDA-MB-231 and MDA-MB-468 are inhibited from proliferating and are induced to undergo programmed cell death by the cytotoxic effects of selenocompounds that form selenides generating superoxide by oxidizing thiols, in different cultured cancer cells. The Trypan Blue exclusion, MTT and Annexin V experimental results substantiates the loss of cell viability most likely from induction of apoptosis, possibly some necrosis, with the selenium redox chemistry targeting GSH and other thiols, the cysteines membranes and the mitochondrial membrane. The data also correlated with the expression of the VEGF on the three cell lines, the IC50 values calculated for the two TNBC cell lines being susceptible to low levels of redox selenium. The HME50-5E cells were found difficult to test because of their extreme adherence. The generation of intracellular superoxide and photographic change in morphology of the HME50-5E cells from selenium treatments TNBC cells suggest these more “normal” epithelial cells are not as sensitive to selenium toxicity.

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CHAPTER IV

CYTOTOXICITY OF HERCEPTIN®, SELENOHERCEPTIN AND SELENITE

AGAINST TRIPLE NEGATIVE BREAST CANCER CELL LINES MDA-MB-231

AND MDA-MB-468

ABSTRACT

A humanized monoclonal IgG1, human epidermal growth factor receptor 2 (HER2) specific antibody Herceptin® was conjugated with the N-hydroxysuccinamide ester of 3-selenocyanopropionic acid forming a Selenoherceptin immunoconjugate. The selenium immunoconjugate, Selenoherceptin was shown to produce superoxide anion - (O2 ) in the presence of glutathione using lucigenin chemiluminescence. With cells in vitro, Selenoherceptin exhibited intracellular superoxide generation as determined by the DHE assay. The cytotoxicity of Selenoherceptin was compared to the cytotoxicity of Herceptin® and selenite in a time and dose dependent manner against the triple negative breast cancer (TNBC) cell lines MDA-MB-231, MDA-MB-468 and an immortalized mammary epithelial cell line, HME50-5E. Cells were treated with either 2, 5, 10 or 20 µg Se as selenite. An equivalent concentration of Herceptin® protein was compared to Selenoherceptin. All cells were treated on day 3 post-seeding and Selenoherceptin was shown to impair cell growth of the MDA-MB-231, MDA-MB-468 and HME50-5E cells in vitro as measured by Trypan Blue exclusion, MTT and Annexin V assays. HER2 protein expression was assessed by immunoblotting using Western blots. The results show that selenium toxicity may be targeted by Herceptin® and provide a reasonable expectation that antibodies with covalently attached redox selenium may be useful in clinical applications for treatment of Her/neu expressed breast cancer.

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KEYWORDS

Selenium, Triple Negative Breast Cancer Cell lines, MDA-MB-231 cells, MDA-MB-468 cells, HME50-5E cells, Selenoherceptin, Sodium Selenite, Herceptin®, Chemiluminescence, AnnexinV, Reactive Oxygen Species, Dihydroethidium

INTRODUCTION

In 1978, Stanley Cohen and co-workers discovered the Epidermal Growth Factor Receptor 1 (EGFR, ErB-1, Her1) [202]. Between 1982-1984, a group of scientists discovered the neu gene also known as Her-2/neu (Erb B-2) or P185 [203, 204] located on chromosome 17q12 [205]. Her2 is a proto-oncogene that encodes a 185 kDa trans- membrane tyrosine kinase receptor glycoprotein, belonging to the family of epidermal growth factor receptors (EGFR) [206]. HER2 proteins are receptors are found on both normal and breast cancer cells. HER2 activation occurs through homo or hetero- dimerization with other EGFR family members EGFR1 (HER1), HER3 and HER4 resulting in auto phosphorylation of tyrosine residues within the cytoplasmic domain of the receptors. This activates signal transduction pathways like the MAP-kinase pathway [206]. Normally ~20,000 HER2 receptors are present on a normal cell and they promote cell division of healthy breast cells. HER2+ breast cancer cells can have up to 2 million HER2 receptors/cell. Over expression of the HER2 gene leads to over production of the HER2 protein and uncontrolled cancer cell growth, resulting in excess cell signaling and an invasive cancer [207, 208].

Over expression of Her2 is also seen in ovarian, uterine, endometrial, gastric and lung cancer. The Her2 receptor protein is upregulated in about 25-30% of all breast cancer patients and is linked with a poor 5-year survival prognosis [124, 125, 209]. Furthermore, duration to cancer relapse is also as these tumors have a tendency to be more aggressive than HER2 negative cells [210]. These reasons make HER2 an important therapeutic target in breast cancer and have led to the development of anti- HER2 therapies like Herceptin® (Trastuzumab –a monoclonal antibody).

Herceptin® (Trastuzumab) manufactured by Genentech (Roche) was the first monoclonal antibody (mAb) to find its way into the clinic to treat breast cancer; initially

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Texas Tech University, Soni Khandelwal, May 2018 approved for clinical use by the FDA in 1998 [122]. Herceptin® is a 148 kDa humanized monoclonal immunoglobulin (IgG1) antibody [123] has developed to target the HER2 receptor, one of the 4 members of the ErbB family of tyrosine kinases, occurring in 15-30% of all breast cancers as well as ovarian, lung, uterine, gastric and salivary duct cancers [124, 125]. Breast cancer prognosis with over expressed Her2/neu receptors is not welcomed news. HER2/neu breast cancer treatment with monoclonal antibodies is at least a two- step process if metastases occurs. Herceptin® is the first treatment option for Her2/neu positive breast cancer and if relapse and metastasis occurs in patients, treatment can be started with Kadcyla® [211]. Kadcyla® (T-DM1) is Herceptin® (Trastuzumab) with attached Emtansine, one of the very first fast-tracked antibody-drug conjugate (ADCs) by the US FDA. Like Herceptin®, Kadcyla® binds to the Her2/neu receptor and with about 30% of the ADC entering the cancer cells, and the Emtansine being expected and perhaps necessary to be released from the ADC by hydrolytic enzymes, attaches itself at the Rhizoxin binding site inducing cancer cell death. In a clinical trial of T-DM1 vs two chemotherapeutic drugs [212] disease-free survival was ~ 3-4 months with T-DM1 vs the chemotherapeutic drug arm, and approximately 96% of all patient deaths in both arms of the clinical trial was attributable to disease progression. Herceptin® (Trastuzumab) nor Kadcyla® (T-DM1) would be expected to have therapeutic value against Triple Negative Breast cancers (TNBC), as by definition TNBC cancers either do not overly express or the HER2/neu receptor target is absent from TNBC cells, along with the two other therapeutic targeted receptors; estrogen and progesterone [213]. Breast cancers or women susceptible and at high risk for breast cancer may be treated with Tamoxifen to block the estrogen binding to cancer cells inducing cell division, or inhibitors of Aromatase [45]. Aromatase is an enzyme that converts testosterone in women to the estrogen; estradiol and estrogen synthesis inhibition often leads to cancer cell death [46]. While more than 70 ADCs may be in various stages of clinical trials, and ADC has been shown to be widely effective across patients with similar cancers by treating a targeted cancer, although that is precisely the goal, to delivery specifically a mAb to which a radioisotope or toxin is covalently attached. None of the presently approved

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ADCs are without serious side effects beyond their targeted treatment [214], as most drug conjugates may have to be released intracellularly for them to be effective and then those that upon escape beyond treatment are otherwise toxic and most treated by Phase I and II liver enzymes for excretion [215]. The labeling of Herceptin® (Trastuzumab) and Avastin® (Bevacizumab, as presented in Chapter II) with an essential dietary nutrient, selenium, in redox active form removes some of the obstacles to ADC conjugation and metabolism noted above. As previously shown by Goswami [192] for Transferrins and Bapat [193] for Herceptin® conjugation of proteins and mAbs, directed selenium cytotoxicity is possible using decades old chemistry of Bolton-Hunter modification with redox selenium. Attachment is a one-step process, does not require any linker, and does not require that the selenium be released from the protein or mab to induce cytotoxicity. In this section, Chapter IV, the data presented below shows and suggests that Selenoherceptin is cytotoxic to the TNBC cell lines MDA-MB-231 and MDA-MB-468 whereas Herceptin® is not at all cytotoxic. Similar data presented in Chapter III also shows that Selenoavastin is likewise cytotoxic to the same cells lines whereas Avastin® is not. The same theme applies to Selenofolate and Folate as presented in Chapter II, when redox selenium is covalently attached to a presumed targeting molecule, it is made more cytotoxic than the parent targeting native parent targeting molecule.

MATERIALS AND METHODS

Materials

DMEM high glucose (Catalog# 11965-092), Fetal Bovine Serum (Catalog# 10082-147), 1% Penicillin-Streptomycin (Catalog# 15-140-122), were purchased from Life technologies, Gibco. pH test strips (Catalog# 8882-1) were purchased from Ricca Chemical Company. PVDF membranes (Catalog# 1620177), Non-Fat Milk Protein (Catalog# 1706404), 3-20% Tris-Glycine Polyacrylamide Gel (Catalog# 456-1096) and 2X native PAGE sample buffer (Catalog# 161-0738) were purchased from BIO-RAD. Mammary Epithelial Cell Media (Catalog# 50-306-176) was purchased from PromoCell. Trypsin (Catalog# 30-2101) was purchased from ATCC. Rabbit Anti-Mouse IgG H&L

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HRP (Catalog# ab6728) was purchased from Abcam. 0.22 µM filter (Catalog# SLGV033RS), Anti-β-Actin Antibody (Catalog# MAB1501) and Accutase (Catalog# SCR005) from EMD Millipore. Corning® cell culture 75 cm2 (Catalog# CLS430641) and 25 cm2 (Catalog# CLS430639) vented cap flasks, Corning® Costar® TC-Treated Multiple Well Plates (Catalog# CLS3527), Corning® Costar® TC-Treated Multiple Well Plates (Catalog# CLS3548), Sodium selenite (Catalog# 214485), MTT (Catalog# M2128), Superoxide Dismutase (Catalog# S7571), Bovine Erythrocytes (Catalog# C1345-1G), 2′,7′-Dichlorofluorescin diacetate (Catalog# D6883), Dihydroethidium (Catalog# D7008), Tetrahydrofuran (Catalog# 34865) were purchased from Sigma- Aldrich. RIPA lysis buffer (Catalog# 89900), SuperSignal™ West Femto Maximum Sensitivity Substrate (Catalog# 34095), Gamma Globulin (Catalog# 23212), BCA asay kit (Catalog# 23225), MitoTracker™ Red CMXRos (Catalog# M7512), Annexin V-FITC Apoptosis Detection Kit (Catalog# BMS500FI-300) were purchased from ThermoFisher Scientific. Dialysis tube (Catalog# 132542) was purchased from Spectra/Por membrane. Bovine Serum Albumin (Catalog# BP9704100) was purchased from Fisher Scientific. HER2/ErbB2 (44E7) mouse mAb (Catalog# 2248S) was purchased from Cell Signaling Technology. Protein ladders of size10-245 kDa was purchased from (GeneTex, Catalog# GTX50875).

Monoclonal antibodies

Trastuzumab (Herceptin®) was obtained through the collaborative efforts of TTUHSC Cancer Center Clinic with Dr. Everado Cobos, Director of the Southwest Cancer Center of TTUHSC. Selenoherceptin was synthesized in this laboratory using a selenium modified Bolton-Hunter reagent (Se ester; ROCH2CH2SeCN). The covalent attachment of a selenide Se moiety to proteins and antibodies via the Se-Bolton-Hunter reagent is only a recent development [194]. The Bolton-Hunter reagent was originally developed in the 1970’s for radioimmunoassays using radioactive iodine. A Se-modified Bolton-Hunter reagent was synthesized using this technique and was subsequently used to attach catalytic redox Se to Herceptin®.

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Synthesis Bolton-Hunter of Seleno ester

The N-hydroxysuccinamide ester of 3-selenocyanopropionic acid (a modified Se Bolton-Hunter Reagent; Selenoester) (Eburon Organics N.V.; Belgium) was synthesized from N-hydroxysuccinamide and 3-selenocyanopropionic acid by an N,N’- dicyclohexylcarbodimide (DCC) reaction and was used as received without further preparation as shown in Figure IV-1. The final Se ester product has a dark red color (refer Figure IV-2).

N-hydroxysuccinamide and 3- selenocyanopropionic acid

DCC reaction

N-hydroxysuccinamide ester of 3-selenocyanopropionic acid

Dissolved in THF and added to mAbs

Seleno-ester, R-O-CH2-CH2-Se-C≡N attaches to lysine residues of mAbs

Figure IV-1: The Procedure for Conjugation of Seleno-Ester to the Lysine Residues of Herceptin®.

Conjugation of Redox Se to Herceptin®

Herceptin® (Genentech) was diluted to a concentration of 3 mg protein/mL from a stock solution of 21 mg/mL with 0.05 M sodium borate buffer (pH 8.5) at 4°C. The Se- ester was dissolved in tetrahydrofuran at a concentration of 20 mg/mL (refer Figure IV-3) and 2.5 mL (20 mg/mL) of selenoester dissolved in THF was added into 10 ml of dilute Herceptin® (3 mg/mL) with the glass vial kept on ice and packed cold water at for 72 hours 4°C. Dialysis tubing stored in cold 0.05 M phosphate buffer (pH 7.4) was soaked

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Texas Tech University, Soni Khandelwal, May 2018 for 24 hours at 4°C. After incubation for 72 hours, Herceptin® from the glass vial was transferred to dialysis tubing (MWCO 50,000) and was placed in the refrigerator for another 72 hours in dialysis buffer. The dialysis buffer (0.05 M phosphate buffer, pH 7.4) was changed 3-4 times the first day and then twice every day. For dialysis 10 ml of the antibody 2 L of dialysis buffer was used. After 72 hours, dialysis tubing from PBS buffer was taken out and rinsed with water and the antibody were drawn very slowly from the dialysis tubing with a 16 gauge needle and syringe. This was filtered into a scintillation vial using a 0.22 μM syringe filter. Herceptin® and Selenoherceptin were stored in 4°C. The scintillation vial containing antibodies were kept in containers that was covered with aluminum foil paper to avoid light. . Exposure to increasing temperature was avoided by keeping the mAbs on ice during their handling and transport. The pH of Herceptin® and Selenoherceptin was tested using a pH Test Strip 4.5-10. Selenoherceptin was run through the Attune™ NxT Flow Cytometer (ThermoFisher Scientific, Carlsbad, CA) as a quality control to detect if there was any kind of fluorescence emitted by Selenoherceptin, because this would if present effect fluorescent assays.

Figure IV-2: Selenocyanate Propionic Ester of N-hydroxysuccinimide (Bolton-Hunter Seleno-Ester).

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Figure IV-3: Saturated Solution Of Se-Ester Dissolved In Tetrahydrofuran.

Analysis of Selenoherceptin

Inductively Coupled Plasma Mass Spectrometry or ICP-MS is an analytical technique used for elemental Se determinations. Selenoherceptin was sent to TraceAnalysis, Inc. (Lubbock, TX) for selenium analysis was performed.

Detection of Superoxide In Vitro By Selenoherceptin

Lucigenin (Bis-N-Methylacridinium Nitrate) was used to detect superoxide in a chemiluminescent assay consisting of phosphate buffer and reduced GSH. The amount of superoxide generated is measured by the chemiluminescent produced light using a Turner TD-20e Luminometer (Turner Designs Inc., Mountain View, CA), connected to a circulating water bath. The source of electrons for superoxide is GSH catalyzed by

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Selenoavastin, but not all Se compounds. Chemiluminescence measurements of Herceptin® and Selenoherceptin was performed in triplicates by adding 100 µL volumes of Herceptin® and Selenoherceptin to 500 µL of the chemiluminescent cocktail at 37°C with integrations of 30 seconds over 12.5 minutes, n=25.

Detection of Intracellular Reactive Oxygen Species Accumulation In Situ: Superoxide Generation Assay

MDA-MB-231, MDA-MB-468 and HME50-5E cells were seeded at a density of 2X105 cells/well in 24 well flat bottom plate. After 48 hours, DMEM high glucose media without phenol red supplemented with FBS was added. 50 units/well of superoxide dismutase (SOD) from bovine erythrocytes and 100 units/well of catalase from bovine liver in media were added to all wells. All cells-control, Selenite (10 µg as Se), Herceptin® (131.1 µg protein) and Selenoherceptin (10 µg Se and 131.1 µg protein) were appropriately treated for 30 minutes. DHE was added at a final concentration of 10 µM in each well. Cells were visually assessed using EVOS FL Auto Cell Imaging System (ThermoFisher Scientific, Carlsbad, CA) or Cytation 3 plate reader (BioTek, Winooski, VT, USA).

BCA assay for Protein Determination of Herceptin® and Selenoherceptin

Total protein content of Herceptin® and Selenoherceptin were determined using bicinchoninic acid (BCA) assay kit. 10 µL of Herceptin® and Selenoherceptin were diluted in 90 µL of phosphate-buffered saline before addition of 2 mL of working reagent. After addition of working reagent, the samples were incubated in cuvette at 37°C in water bath for 30 minutes. One empty 96-well plate was kept in the incubator. After 30 minutes of incubation both-the cuvettes and the 96-well plate was cooled down to room temperature. 225 μL of the solution from cuvette was added to each of 3 wells in 96-well plate. Absorbance was read using Cytation 3 plate reader (BioTek, Winooski, VT, USA) at 562 nm absorption. Protein concentrations were determined from a standard curve prepared from a serial dilution of bovine gamma globulin and purified human IgG. Protein concentrations were also determined using nanodrop, Cytation 3 plate reader (BioTek, Winooski, VT, USA).

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Western Blotting Under Denaturing Conditions

Native unconjugated Herceptin® and Selenoherceptin were placed on ice. Protein ladders of 10-245 kDa were used to determine the unknown protein molecular weights. 20 µg of bovine gamma globulin and purified human IgG were loaded as controls. Concentrations of 5 µg, 10 µg and 20 µg of protein from each antibody, Herceptin® and Selenoherceptin in 6X Laemmli buffer was mixed and samples were boiled for 5 minutes at 95°C. The mAbs were briefly centrifuged for 1 minute at 11,000 rpm. The total volume of the samples was loaded into a BIO-RAD miniprotean 14 well comb (1.5 mm) 8% Polyacrylamide Gel and was used for electrophoresis. Initially, the gel was run at 50 V for 5 minutes and then the voltage was increased to 90 V for 90 minutes. The SDS-PAGE gel was stained with Coomassie Blue R-250 for 15 minutes and then destained using Coomassie blue destaining solution. Photographs were taken under a Coomassie filter using LI-COR (Model: 2800, S/N OFC- 0786, LI-COR, Inc., Lincoln, NE, USA).

Western Blotting Under Non-Denaturing Conditions

Herceptin® and Selenoherceptin were placed on ice. Protein ladders of 10-245 kDa were used to verify the molecular weight of the mAbs. 20 µg of bovine gamma globulin and purified human IgG were also used as loading control. Five and ten micrograms of protein from Herceptin and Selenoherceptin in a 12 µL, was mixed with 6 µL of 2X native PAGE sample buffer. Samples were run on a gradient; 3-20% Tris- Glycine Polyacrylamide Gel. Initially, the gel was run at 50 V for 5 minutes and then the voltage was increased to 160 V for 1 hour. The gradient gel was stained with Coomassie Blue R-250 stain for 15 minutes and destained with Coomassie Blue destaining solution for 20 minutes and then transferred to distilled water. Photographs were taken under Coomassie filter using LI-COR (Model: 2800, S/N OFC- 0786, LI-COR, Inc., Lincoln, NE, USA). Samples were not boiled, the gel, the native sample buffer and the running buffer did not contain sodium-dodecyl sulfate.

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Cell Culture

MDA-MB-231 and MDA-MB-468 cells were cultured in 75 cm2 tissue culture flasks and maintained in high glucose Dulbecco's Modified Eagle's Media (DMEM) media supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin. The cells were incubated for 2 to 3 days at 37°C under humid conditions in 5% CO2 incubator (ThermoFisher Scientific, Carlsbad, CA). The growth media was changed twice weekly. Cells were grown to 75–85% confluence then washed with 1X phosphate buffer saline (PBS), trypsinized with 5 mL of 0.25% (v) trypsin-0.0.3% EDTA, diluted with fresh media, and counted using a Beckman Coulter ViCell (Beckman Coulter, Inc. Model VI- CELL SGL).

HME50-5E cells were also cultured in 25 cm2 tissue culture flask and maintained in Mammary Epithelial Cell Media and were incubated for 7 to 8 days at 37°C under humid conditions in 5% CO2 incubator. The media was changed every other day. Cells were grown to 60-70% confluence then washed with ice cold 1X phosphate buffer saline (PBS), trypsinized with 5 mL of Accutase, diluted with fresh media, and counted using a Beckman Coulter ViCell (Beckman Coulter, Inc. Model VI-CELL SGL).

Optimization of Cell Density for Treatment

Log growth cell curves in 48 well plate was performed by seeding different densities of cells, 5,000; 10,000; 20,000; 40,000 and 100,000 cells/well and cell growth was plotted against time for 7 days. This was done to determine and assure a logarithmic growth of cells throughout the experimental time period.

Cell Treatments

All experiments were performed in cell culture hood in an aseptic environment. Exponentially growing cells were harvested, counted by the Trypan Blue exclusion, and plated onto 48 well plates at an optimal seeding density of 40,000 cells (MDA-MB-231, MDA-MB-468, HME50-5E) per well. Cells were allowed to grow for 5 days post seeding prior to day 0 of treatment. Media was changed on day 3 post cell seeding and

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Texas Tech University, Soni Khandelwal, May 2018 treatments (on day 5 post seeding) were done with the addition of fresh culture media and drugs.

Herceptin® was used as the experimental control for Selenoherceptin. Sodium selenite (45% Se) was used as a positive control for cell-death and was added to wells with MDA-MB-231, MDA-MB-468 cells and HME50-5E cells in final concentrations. Control cells were treated with 1X PBS. Herceptin®, Selenoherceptin or selenite were tested in triplicate in 48 well plates and analyzed at 0,1,2,3,4,5,6 and 7 for cytotoxicity and cell viability.

Visual Assessments of Cellular Toxicity

A phase-contrast microscope, EVOS XL Core (Life Technologies, Carlsbad, CA) was used for photographing cell morphological changes due to Selenite, Herceptin® and Selenoherceptin treatments.

Cell Viability Measured by Trypan Blue Exclusion

Cell viability from control and experimental treated cells was determined using a Beckman Coulter Vi-Cell Viability Analyzer (Beckman Coulter, Inc. Model VI-CELL SGL) with viability based on Trypan Blue cell exclusion.

Cell Experimental Cultures

TNBC and the immortalized breast epithelial cells; 40,000 cells/well were seeded in 48 well flat-bottom plates, cell volumes were adjusted with media and cells were allowed to grow for 5 days prior to treatment. Media was changed on day 3 post seeding and just before treatments. Cells were treated with four doses of Selenoherceptin 2 µg Se and 26.2 µg protein; 5 µg Se and 65.5 µg protein; 10 µg Se and 131.1 µg protein; 20 µg Se and 262.2 µg protein of Selenoherceptin for 7 days in triplicate. Cells were treated with Herceptin® (26.22 µg, 65.5 µg, 131.1 µg and 262.2 µg protein) and selenite (2, 5, 10 and 20 µg as Se).

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On days 0 through 7 of treatments; 500 µl of media (containing treatments) from each well was collected in a coulter cup. The cells were rinsed with 500 µl of 1X PBS (kept in incubator for 5 minutes) and was collected in coulter-cups. 200 µl of 0.025% trypsin- EDTA was added to harvest the adherent cells. After 5 minutes of incubation, trypsin was added to the coulter-cups containing media and 1x PBS. Wells were observed to make sure all adherent cells were collected under the microscope to make sure all adherent cells remaining after treatments were collected. Cells were analyzed with the Beckman Coulter Vi-Cell Viability Analyzer for this experiment with the following settings and criteria:

Cell type: BrCa cells Minimum diameter (microns): 12 Maximum diameter (microns): 50 Cell brightness (%): 85 Cell sharpness: 100 Viable cell spot brightness (%): 65 Viable cell spot area (%): 5

Measuring Cell Viability Using the MTT Assay

TNBC and the breast epithelial cells; 40,000 cells/well were seeded into 48 well flat-bottom plates, the volumes were adjusted with media, and cells were allowed to grow for 5 days prior to treatment. Cells were treated (on day 5 post seeding) with 2 µg Se and 26.2 µg protein, 5 µg Se and 65.5 µg protein, 10 µg Se and 131.1 µg protein of Selenoherceptin for 6 days. Cells were treated with selenite (2, 5, and 10 µg as Se) and unconjugated native Herceptin®. Each treatment was repeated in triplicates. Blanks without cells contained complete growth media (Phenol red free DMEM high glucose + 10% FBS). MTT dissolved in phenol red-free media, 5 mg/mL was passed and sterilized through a 0.22 µM filter inside the cell culture hood. The tube containing the MTT solution was wrapped with aluminum foil paper and used immediately. To each well 10% (v/v) i.e., 50 µl MTT (5 mg/mL) was added and incubated for 3 hours at 37oC. Following incubation with MTT, the Formazan solubilization solution [acidified isopropanol (0.1N

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HCl) with 10% Triton X-100] equal to the original volume of media was added in each well. Dissolved Formazan in each well was determined by a Cytation 3 plate reader (BioTek, Winooski, VT, USA) at 570 nm absorption with baseline subtraction of 690 nm absorption to assess cell viability. The control cells were considered to be 100% viable. Cell viability percentage was calculated using following equation IV-1

퐴푏푠표푟푏푎푛푐푒 표푓 퐻푒푟푐푒푝푡𝑖푛 표푟 푆푒푙푒푛표ℎ푒푟푐푒푝푡𝑖푛−푡푟푒푎푡푒푑 푐푒푙푙푠 Cell viability (%) = 푋 100 (IV-1) 퐴푏푠표푟푏푎푛푐푒 표푓 푐표푛푡푟표푙 푐푒푙푙푠

Cell viability data was then used to calculate the 50% cell inhibition concentration; 50% (IC50).

MitoTracker® Red and Annexin V Staining

Into 48 well flat-bottom plates 40,000 cells/well were seeded were allowed to grow for 5 days prior to treatment. Media was changed on day 3 post seeding and volume was adjusted before treatment. Cells were treated (on day 5 post seeding) with 2 µg Se 26.22 µg protein of Selenoherceptin for 3 days. Cells were also treated with selenite (2 µg Se/well) and unconjugated native Herceptin®. Additionally, the control cells were 1X PBS treated. Another set of control cells for staining were prepared- unstained control was untreated+unstained. For necrosis control (double-negative), cells were treated with

50 µl of 0.01% Triton X-100 for 30 minutes prior to first staining or 200 µl of H2O2 for 24 hours prior to first staining. For single staining controls, appropriate wells were treated with the dyes separately (Annexin V – 488 were treated with apoptosis inducer i.e., 5 µl of 1 mM Sutent; MitoTracker® Red – untreated cells). For double staining controls (double-positive), appropriate wells were treated with both dyes of the same volume. These controls were set aside in 2 mL Eppendorf tubes. Each treatment was repeated in triplicate.

A 10 mM stock solution of MitoTracker® Red dye was prepared by adding 9.4 μL DMSO to a vial of MitoTracker® Red dye. A 10 μM working solution of the MitoTracker® Red dye was prepared by pipetting 1 μL of 10 mM MitoTracker® Red stock solution into 1,000 μL of DMEM high glucose phenol red free cell media. 2 μL of 10 μM MitoTracker® Red working solution was added to each well (500 µl) and stained

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for 30 minutes at 37°C in an atmosphere of 5% CO2. After 30 minutes of incubation, the media was collected in a sterile 2 mL Eppendorf tube. The cells were washed with 500 µl of 1X PBS and collected in the appropriate Eppendorf tube. Cells were trypsinized using 200 µl of 0.025% trypsin-EDTA and centrifuged. Cells were resuspended in 100 µl of 1X Annexin-binding buffer. This suspension was transferred into 96 well flat-bottom plates. 4 µl of Annexin V dye was added to each well. The 96-well plate was wrapped in aluminum foil paper. The cells were incubated for 15 minutes at 37°C in an atmosphere of 5% CO2. After the incubation, 100 µl of 1X Annexin-binding buffer was added. The plate was placed immediately on ice. The stained cells were analyzed by flow cytometry on the Attune™ NxT Flow Cytometer (ThermoFisher Scientific, Carlsbad, CA). The laser lines were set to BL1 (200 nm) and YL1 (336 nm). Voltage for Forward Scatter was set to 120 and Side Scatter to 290.

Western Blotting

One million MDA-MB-231, MDA-MB-468 and HME50-5E cells were allowed to attach to T-25 flasks overnight. The following day cells were treated with 2 µg as Se- 26.22 as protein µg of Selenoherceptin and incubated for 3 days. Control cells were treated with 1X PBS using same experimental time.

On day 3 of treatment, cultured cells fluid was collected in a 15 mL centrifuge tube and cells were washed with ice cold 1X PBS and collected in the same tube. The tube was centrifuged at 4000 rpm for 4-5 minutes and the media was aspirated using a Pasteur pipette. 200 µl of RIPA lysis buffer was added to the tube and everything was transferred to the T25 flasks. The T25 flasks were kept at -80oC as it is for one day. Cancer protein cell lysates yield was better protein when the flask was thawed. To collect the HME50-5E cell lysates, the flasks were broken with a hammer and cells were scrapped and kept on ice for 5 minutes. The HME50-5E lysates were passed through a 20 G needle and kept on ice for 5 minutes. All cell lysates were kept on an inverter for 15 minutes at 4°C and centrifuged at 12,000 rpm for 15 minutes at 4°C. Total protein concentration in the cleared lysate was determined by the bicinchoninic acid (BCA) assay according to manufacturer’s instructions. BT-474 cell lysate was used as a positive

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Texas Tech University, Soni Khandelwal, May 2018 loading control for HER2. Forty µg of total protein was separated on 8% denaturing polyacrylamide gels and transferred to PVDF membranes by electroblotting. Membranes were blocked for 1 hour with Tris-buffered saline containing 0.05% Tween-20 (TBST) and 5% non-fat milk protein. They were incubated overnight with HER2/ErbB2 (44E7) mouse mAb diluted 1:1000 in TBST or anti-β-actin antibody diluted 1:1000 in TBST containing 1% non-fat milk protein. The next day, the membrane was washed 3 times for 45 minutes in TBST, incubated for 1 hour with horse-radish peroxidase conjugated with rabbit anti-mouse IgG diluted 1:10000 in PBST, and washed once in TBST for 15 minutes. Bound antibody complexes were visualized using SuperSignal™ West Femto Maximum Sensitivity Substrate.

Forty microgram of total protein cell lysates of HME50-5E control, Herceptin®, Selenoherceptin and Selenite treated were separated on 8% SDS-PAGE gel. The gel was stained with Coomassie Blue R-250. HCC1419 cell lysate was used as a positive loading control for HER2.

STATISTICAL ANALYSES

All experimental assays were conducted in triplicate and the data are representative of three independent experiments. The results are expressed as the mean ± standard error (SE). Statistical analyses were performed using the MATLAB ver 9.2 (Release R2017a) two sample t test for across treatments. Differences were considered significant at p≤0.05. IC50 was calculated using a two-parameter sigmoidal model. The results of statistical analyses are summarized in Table IV-1, Table IV-2 and Table IV-3.

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Table IV-1: Statistical Analyses of Herceptin® and Selenoherceptin treatments in MDA-MB- 231 Cells.

Experiment Treatments p value Day Trypan Blue Herceptin® as 26.22 µg protein vs Selenoherceptin 2 µg Se and 26.22 µg 0.005 3 (Figure IV-16) protein Herceptin® as 65.55 µg protein vs Selenoherceptin 5 µg Se and 65.5 µg 0.003 protein Herceptin® as 131.1 µg protein vs Selenoherceptin 10 µg Se and 131.1 µg 0.001 protein Day Trypan Blue Control vs Selenoherceptin 2 µg Se and 26.2 µg protein 0.004 4 (Figure IV-16) Control vs Selenoherceptin 5 µg Se and 65.5 µg protein 0.001 Herceptin® as 26.22 µg protein vs Selenoherceptin 2 µg Se and 26.22 µg 0.001 protein Herceptin® as 65.55 µg protein vs Selenoherceptin 5 µg Se and 65.5 µg 0.001 protein Herceptin® as 131.1 µg protein vs Selenoherceptin 10 µg Se and 131.1 µg 0.001 protein Day MTT Control vs Selenoherceptin 2 µg Se and 26.2 µg protein 0.002 3 (Figure IV-19) Control vs Selenoherceptin 5 µg Se and 65.5 µg protein 0.001 Herceptin® as 26.22 µg protein vs Selenoherceptin 2 µg Se and 26.22 µg 0.012 protein Herceptin® as 65.55 µg protein vs Selenoherceptin 5 µg Se and 65.5 µg 0.001 protein Herceptin® as 131.1 µg protein vs Selenoherceptin 10 µg Se and 131.1 µg 0.001 protein Day MTT Control vs Selenoherceptin 2 µg Se and 26.2 µg protein 0.001 4 (Figure IV-19) Control vs Selenoherceptin 5 µg Se and 65.5 µg as protein 0.006 Herceptin® as 26.22 µg protein vs Selenoherceptin 2 µg Se and 26.22 µg 0.001 protein Herceptin® as 65.55 µg protein vs Selenoherceptin 5 µg Se and 65.5 µg 0.002 protein Herceptin® as 131.1 µg protein vs Selenoherceptin 10 µg Se and 131.1 µg 0.001 protein Annexin V (Figure Herceptin® as 26.22 µg protein vs Selenoherceptin 2 µg Se and 26.22 µg 0.004 IV-26) protein (late apoptosis)

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Table IV-2: Statistical Analyses of Herceptin® and Selenoherceptin treatments in MDA-MB- 468 Cells.

Experiment Treatments p value Day Trypan Blue Control vs Selenoherceptin 5 µg as Se and 65.5 µg as protein 0.008 3 (Figure Control vs Selenoherceptin 10 µg Se and 131.1 µg protein 0.001 IV-17) Herceptin® as 26.22 µg protein vs Selenoherceptin 2 µg Se and 26.22 µg protein 0.005 Herceptin® as 65.55 µg protein vs Selenoherceptin 5 µg Se and 65.5 µg protein 0.001 Herceptin® as 131.1 µg protein vs Selenoherceptin 10 µg Se and 131.1 µg protein 0.001 Herceptin® as 262.2 µg protein vs Selenoherceptin as 20 µg Se and 262.2 µg 0.001 protein Day Trypan Blue Control vs Selenoherceptin 2 µg Se and 26.2 µg protein 0.017 4 (Figure Control vs Selenoherceptin 5 µg Se and 65.5 µg protein 0.001 IV-17) Herceptin® as 26.22 µg protein vs Selenoherceptin 2 µg Se and 26.22 µg protein 0.002 Herceptin® as 65.55 µg protein vs Selenoherceptin 5 µg Se and 65.5 µg protein 0.001 Herceptin® as 131.1 µg protein vs Selenoherceptin 10 µg Se and 131.1 µg protein 0.001 Day MTT Control vs Selenoherceptin 2 µg Se and 26.2 µg protein 0.007 3 (Figure Herceptin® as 65.55 µg protein vs Selenoherceptin 5 µg Se and 65.5 µg protein 0.003 IV-20) Herceptin® as 131.1 µg protein vs Selenoherceptin 10 µg Se and 131.1 µg protein 0.002 Day MTT Control vs Selenoherceptin 2 µg Se and 26.2 µg protein 0.005 4 (Figure Control vs Selenoherceptin 5 µg Se and 65.5 µg protein 0.001 IV-20) Herceptin® as 26.22 µg protein vs Selenoherceptin 2 µg Se and 26.22 µg protein 0.015 Herceptin® as 65.55 µg protein vs Selenoherceptin 5 µg Se and 65.5 µg protein 0.001 Herceptin® as 131.1 µg protein vs Selenoherceptin 10 µg Se and 131.1 µg protein 0.007 Annexin V Herceptin® as 26.22 µg protein vs Selenoherceptin 2 µg Se and 26.22 µg protein 0.003 (Figure (early apoptosis) IV-29) Herceptin® as 26.22 µg protein vs Selenoherceptin 2 µg Se and 26.22 µg protein 0.004 (late apoptosis)

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Table IV-3: Statistical Analyses of Herceptin® and Selenoherceptin treatments in HME50-5E Cells.

Experiment Treatments p value Day 3 Trypan Blue Herceptin® as 26.22 µg protein vs Selenoherceptin 2 µg Se and 26.22 µg protein 0.002 (Figure IV-18) Herceptin® as 65.55 µg protein vs Selenoherceptin 5 µg Se and 65.5 µg protein 0.001 Herceptin® as 131.1 µg protein vs Selenoherceptin 10 µg Se and 131.1 µg protein 0.001 Day 4 Trypan Blue Control vs Selenoherceptin 2 µg Se and 26.2 µg protein 0.010 (Figure IV-18) Herceptin® as 26.22 µg protein vs Selenoherceptin 2 µg Se and 26.22 µg protein 0.008 Herceptin® as 65.55 µg protein vs Selenoherceptin 5 µg Se and 65.5 µg protein 0.001 Herceptin® as 131.1 µg protein vs Selenoherceptin 10 µg Se and 131.1 µg protein 0.001 Day 4 MTT Control vs Selenoherceptin 2 µg Se and 26.2 µg protein 0.001 (Figure IV-21) Control vs Selenoherceptin 5 µg Se and 65.5 µg protein 0.001 Herceptin® as 26.22 µg protein vs Selenoherceptin 2 µg Se and 26.22 µg protein 0.003 Herceptin® as 65.55 µg protein vs Selenoherceptin 5 µg Se and 65.5 µg protein 0.001 Herceptin® as 131.1 µg protein vs Selenoherceptin 10 µg Se and 131.1 µg protein 0.002

RESULTS AND DISCUSSION

Following redox Se conjugation with the selenoester at pH 8.5 and exhaustive dialysis with PBS (pH 7.4) the amount of Se-conjugated to Herceptin® were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) (Trace Analysis, Inc., Lubbock, TX,). The Selenoherceptin Se concentrations was 38 µg Se/mg protein after 72 hours of conjugation time (as shown in Figure IV-4). Se concentrations of native Herceptin® was <0.007 µg Se/mg protein. Table III-4 shows the protein concentration of both native Herceptin® and Selenoherceptin were determined using BCA assay and bovine gamma globulin as a standard protein for reference. Similar protein concentration was determined using nanodrop and BCA assay where bovine serum albumin was used as a standard protein for reference (data not shown).

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Figure IV-4: Selenoherceptin and Herceptin®.

Table IV-4: Selenium and Protein Concentration of Herceptin® and Selenoherceptin.

Herceptin® Selenoherceptin Selenium Concentration (mg/L) <0.0200 88 Protein Concentration (mg/mL) 2.82 2.32 Selenium Concentration/ Protein concentration (µg/mg) <0.007 38

Both Herceptin® and Selenoherceptin were stored in glass scintillation vials and steps were taken to avoid light exposure. Herceptin® and Selenoherceptin remained clear for the duration of the study with no precipitates or particulates detected with the naked eye. No change in color or turbidity was observed. Neither was there a change in the pH of Herceptin® and Selenoherceptin as shown in Figure IV-4 throughout the study period.

Herceptin® and Selenoherceptin were analyzed by lucigenin superoxide chemiluminescence using a Turner TD-20e Luminometer. Figure IV-5 and Figure IV-6 demonstrates the experimental CL results. Over a 12.5-minutes CL counting period (Figure IV-6) the reaction between Selenoherceptin and glutathione (GSH) shows superoxide generation in Selenoherceptin vs Herceptin®. In these reactions, lucigenin is reduced by the superoxide anion and chemiluminescence was observed in Selenoherceptin. These experiments using lucigenin-chemiluminescence confirmed the generation of superoxide by the oxidation of GSH by selenite as shown by Seko et al.,

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[91] and extended the observation to Selenoherceptin but not CL cocktail or Herceptin®. After determining the chemiluminescence activity of Herceptin® and Selenoherceptin, the experimental steps were taken to determine Herceptin® and Selenoherceptin would generate superoxide in situ. The photographs taken on an EVOS FL Auto Cell Imaging System in Figure IV-7 and Figure IV-8 show the production of superoxide by Selenoherceptin and selenite, but not Herceptin®, analyzed by DHE assay in MDA-MB- 231 and MDA-MB-468 cells. Similar results have been reported from prior studies in the laboratory that have compared the cytotoxicity of selenite, selenate, selenocystine and selenomethionine towards the human mammary tumor cell line HTB123/DU4475 in vitro [155]. In contrast to the TNBC cell line, Selenoherceptin did not induce such severe superoxide production in HME50-5E cells (Figure IV-9), probably because of their natural antioxidant systems to more readily maintain their redox balance. This difference in TNBC cells and the HME50-5E cells resistance to redox selenium suggests a biochemical variance that can provide a basis for more selective killing of cancer cells with lesser toxicity to normal cells. These results collectively suggest ability of Selenoherceptin to generate superoxide by the oxidation of glutathione (GSH) and other thiols within TNBC cells.

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Figure IV-5: Total Superoxide Generation as Measured by Lucigenin Chemiluminescence (CL) with Glutathione (GSH). Chemiluminescence (CL) for Control Blank, Herceptin® and Selenoherceptin measured over 12.5 minutes. Data is expressed as Mean ± SE. CL for Herceptin® and Selenoherceptin was compared with CL Blank. CL for Selenoherceptin was statistically significant (p≤0.001). 100µL of Selenoherceptin contains 8.8µg of Se.

Figure IV-6: Time Dependent Superoxide Generation as Measured by Lucigenin Chemiluminescence (CL) for Blank, Avastin® and Selenoavastin. Control Blank, Herceptin® and Selenoherceptin CL was measured over 12.5 minutes with 30 second integrations. 100µL of Selenoherceptin contains 8.8µg of Se.

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Figure IV-7: Photomicrographs of Intracellular Superoxide Generation by DHE Red Florescence from MDA-MB-231 Cells after 30 minutes of Blank, Control, Selenite (10 µg as Se), Herceptin® (131.1 µg protein) and Selenoherceptin (10 µg Se and 131.1 µg protein) Treatments. Scale bar=200 µm.

Figure IV-8: Photomicrographs of Intracellular Superoxide Generation by DHE Red Florescence from MDA-MB-468 Cells after 30 minutes of Blank, Control, Selenite (10 µg as Se), Herceptin® (131.1 µg protein) and Selenoherceptin (10 µg Se and 131.1 µg protein) Treatments. Scale bar=100 µm.

Figure IV-9: Photomicrographs of Intracellular Superoxide Generation by DHE Red Florescence from HME50-5E Cells after 30 minutes of Blank, Control, Selenite (10 µg as Se), Herceptin® (131.1 µg protein) and Selenoherceptin (10 µg Se and 131.1 µg protein) Treatments. Scale bar=200 µm.

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To assess what changes may have been made to the Herceptin® by the conjugation of the redox Se; a polyacrylamide gel electrophoresis of Se-labeled and native Herceptin® was conducted and showed little change in the electrophoretic pattern of Se-labeled Herceptin® as compared to native parent Herceptin®. Subsequent separation of unconjugated Herceptin® (Lanes 9, 10, 11) and Selenoherceptin (Lanes 12, 13) on SDS-PAGE was performed as shown in Figure IV-10 to evaluate the homogeneity of the heavy and light chains of native Herceptin® and Selenoherceptin (5 µg, 10 µg and 20 µg protein/lane). Using Coomassie Blue stain, high molecular weight bands of above 245 kDa were observed in Selenoherceptin lanes 12 and 13 suggestive of new Se constructs. Similar results from SDS-electrophoretogram was observed when diphtheria toxin was attached to Herceptin [216]. Protein bands in lanes 12 and 13 in Figure IV-10, also migrated to 180 kDa, 135 kDa 75 kDa, 48 kDa and a lower molecular weight band of approximately 25 kDa was detected (Figure IV-10), corresponding to the heavy and light chains of Herceptin® IgG1 molecule. Kadcyla® (Lane 1), Bovine gamma globulin (Lane 14) and Herceptin® all formed a doublet at all concentrations between molecular masses of 245 and 180 kDa. Formation of doublet on SDS-PAGE was due to incomplete reduction of the IgG1 molecule into heavy and light chains. However, migration of unreduced IgG1 was observed consistently as a doublet [198].

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Figure IV-10: SDS-PAGE of Native Herceptin® and Selenoherceptin mAbs under denaturing or reducing conditions followed by Coomassie Blue R-250 staining. The photograph was taken under Coomassie Blue. Lane 1: Kadcyla 20 μg; Lane 2: Marker; Lane 3: Avastin® 5 µg; Lane 4: Avastin® 10 µg; Lane 5: Avastin® 20 µg; Lane 6: Selenoavastin 5 µg; Lane 7: Selenoavastin 10 µg; Lane 8: Selenoavastin 20 µg; Lane 9: Herceptin® 5 µg; Lane 10: Herceptin® 10 µg; Lane 11: Herceptin® 20 µg, Lane 12: Selenoherceptin 10 µg; Lane 13: Selenoherceptin 20 µg; Lane 14: Gamma globulin 20 µg.

Native PAGE protein migration depends on the charged and conformational state of protein. It is considered as an excellent tool to assess protein stability. To assess protein stability, at least two different amounts of Herceptin® were loaded on 4-20% Tris-Glycine PAGE and were run under non-reducing conditions. A trailing band was seen for Selenoherceptin (Lanes 13 and 14) and it was similar to the trailing band of bovine gamma globulin (Lanes 3 and 4) as shown in Figure IV-11. The photograph of native Tris-Glycine PAGE gel (Figure IV-11) of Selenoherceptin shows that the band pattern was independent of the sample volume loaded across all the different samples. This was true for both native Tris-Glycine PAGE and SDS-PAGE (Figure IV-10). This is in line with one study which determined the prolonged storage of Avastin® [200]. Both the Se-conjugated mAbs, Selenoavastin (Lanes 9 and 10) and Selenoherceptin (Lanes 13 and 14) show identical band mobilities. An identical mobility would mean an identical molecular weight and charge [199]. No smaller products representing protein degradation

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or larger products representing protein aggregation were detectable on native gels. Surprisingly, no migration was seen for Herceptin® in lanes 11 and 12 of Figure IV-11.

Figure IV-11: mAbs migration on 3-20% Tris-Glycine PAGE gel under non-reducing conditions followed by Coomaisse Blue R-250 stain. The photograph was taken under Commaisse blue filter. Lane 1: Molecular Marker; Lane 2: Purified Human IgG 10 μg; Lane 3: Bovine Gamma Globulin 5 μg; Lane 4: Bovine Gamma Globulin 10 μg; ; Lane 5: Kadcyla® 5 μg; Lane 6: Kadcyla® 10 μg; Lane 7: Avastin® 5 μg; Lane 8: Avastin® 10 μg; Lane 9: Selenoavastin 5 μg; Lane 10: Selenoavastin 10 μg; Lane 11: Herceptin® 5 μg; Lane 12: Herceptin® 10 μg; Lane 13: Selenoherceptin 5 μg; Lane 14: Selenoherceptin 10 μg.

Bovine gamma globulin is considered as a control for running mAbs because gamma globulins are good reference for protein quantitation of immunoglobulins. Kadcyla®, another monoclonal antibody as another control because of its similar framework. The structure of Kadcyla® consists of Herceptin®, linker and a cytotoxic agent Emtansine [201]; which is identical to Selenoherceptin, linker and Se. Additionally, the main function of Kadcyla® delivers the toxin specifically to tumor cells; which is like Selenoherceptin.

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In another gel two concentrations of Herceptin® and a protein ladder was loaded on a 4-20% Tris-Glycine PAGE and were run under non-reducing conditions. This time the power supply was flipped, from positive to negative to negative to positive, red to black and black wire to red outlet. This time Herceptin® migrated in its lane (Lanes 3 and 4) as shown in Figure IV-12. Unreduced native mAb- Kadcyla® lanes 5 and 6 in Figure IV-11 and Herceptin® in Figure IV-12 again show identical band mobilities.

Figure IV-12: Herceptin® migration on 3-20% Tris-Glycine PAGE gel under non-reducing conditions followed by Coomaisse Blue R-250 stain. The photograph was taken under Commaisse Blue filter. Lane 3: Herceptin® 5 μg; Lane 4: Herceptin® 10 μg.

To visualize morphological effects of toxicity, MDA-MB-231, MDA-MB-468 and HME50-5E cells were treated with selenite, Herceptin® and Selenoherceptin. Images were taken under a light microscope at 20X magnification and results shown in Figure IV-13 (MDA-MB-231), Figure IV-14 (MDA-MB-468), and Figure IV-15 (HME50-5E). Selenoherceptin and selenite treated cells show growth inhibition, gross cell morphological changes, cell membrane disruption, both cell swelling and shrinkage, and ultimately detachment from the cell culture substratum. MDA-MB-231, MDA-MB-468

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Texas Tech University, Soni Khandelwal, May 2018 and HME50-5E cells are all adherent epithelial cells. With adherent cells, detachment from the substratum and morphological changes is indicative of apoptosis [156]. Additionally, the morphological change (from grape-like structure in MDA-MB-468 cells and stellate-elongated structure in MDA-MB-231cells to extensive cytoplasmic vacuolization) became more remarkable with increased treatment exposure. Similar morphological change in cancer cells treated with other redox active Se compounds have been reported in other in vitro studies [157]. From a therapeutic point of view, the clinical relevance of this observation suggests that Selenoherceptin may be effective in suppressing tumor growth than Herceptin®. Selenite also caused morphological change in cell anatomy but since it is a non-targeting treatment, it demonstrated general toxicity. Additional photomicrographs of morphological changes due to Selenite, Herceptin® and Selenoherceptin treatments are included in APPENDIX A (Figure A-15, Figure A-16, Figure A-17, Figure A-18, Figure A-19 and Figure A-20).

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Figure IV-13: Morphological Changes of Control, Selenite, Herceptin® and Selenoherceptin Treatments in MDA-MB-231 Cells. Treatment of MDA-MB-231 Cells with Selenite and Selenoherceptin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-231 cells. Cells were visualized under 20X magnification.

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Figure IV-14: Morphological Changes of Control, Selenite, Herceptin® and Selenoherceptin Treatments in MDA-MB-468 Cells. Treatment of MDA-MB-468 Cells with Selenite and Selenoherceptin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-468 cells. Cells were visualized under 20X magnification.

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Figure IV-15: Morphological Changes of Control, Selenite, Herceptin® and Selenoherceptin Treatments in HME50-5E Cells. Treatment of HME50-5E cells with Selenite, Herceptin® and Selenoherceptin did not induce severe morphological change comparable to those seen in TNBC cells. Representative fields of view of HME50-5E cells. Cells were visualized under 20X magnification.

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Following optimization of cell growth of MDA-MB-231, MDA-MB-468 and the HME50-5E cells, all cells were treated with selenite, Herceptin® and Selenoherceptin. The dose response results of control cell growth and the three treatments are shown in Figure IV-16, Figure IV-17 and Figure IV-18 as measured for cell viability Trypan Blue exclusion. These three experimental treatments show that selenite and Selenoherceptin exhibit a general loss of viable cells over Se dose and time, as measured against control and Herceptin® treated cells. A low concentration treatment of Selenoherceptin as 2 µg Se and 26.2 µg protein treatments resulted in significant Se cell dose killing effects in MDA-MB-231 and MDA-MB-468 cells. Two, five, ten and twenty µg of Se (as selenite) per well, was cytotoxic to all three cell types tested. Sodium selenite used here as a positive control of toxicity is known to have inhibitory growth effects and induce apoptosis not only in human and mouse breast cancer cell line but also in human colon carcinoma cells, human ovarian cancer, human prostate cancer cells in vitro [158]. MDA- MB-231, MDA-MB-468 control cells receiving native unconjugated Herceptin® alone at the highest protein concentrations tested all show high cell viability at all time periods.

Trypan Blue exclusion experiments were not conducted with HME50-5E at 20 µg Se 262.2 µg protein as Selenoherceptin treatment. In part, successful analysis was difficult due to the strong adherence properties of the HME50-5E cells, causing experimental bias. Initially 200 µl of 0.025% trypsin-EDTA was added and incubated for 10-15 minutes but when viewed under a microscope not all the cells completely detached. So, another 200 µl of 0.025% trypsin-EDTA was added to cells to try to collect the whole population. Despite incubating with double the amount (400 µl) of trypsin, a substantial number of cells remained. This inability to adequately remove all HME50-5E cells from the plate resulted in a high percentage loss of cells inadequate representation for the Trypan Blue assay.

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Figure IV-16: Cytotoxicity of Control, Selenite, Herceptin®, Selenoherceptin in Dose and Time Dependent Treatments against MDA-MB-231 cells. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding. Control cells were incubated for 7 days with Selenite, Avastin® and Selenoavastin at different concentrations. Viable cells were counted at time periods (0-7 days) by Trypan Blue exclusion as described in Methods. The cell growth data are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table IV-1.

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Figure IV-17: Cytotoxicity of Control, Selenite, Herceptin®, Selenoherceptin in Dose and Time Dependent Treatments against MDA-MB-468 cells. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding. Control cells were incubated for 7 days with Selenite, Avastin® and Selenoavastin at different concentrations. Viable cells were counted at time periods (0-7 days) by Trypan Blue exclusion as described in Methods. The cell growth data are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table IV-2.

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Figure IV-18: Cytotoxicity of Control, Selenite, Herceptin®, Selenoherceptin in Dose and Time Dependent Treatments against HME50-5E cells. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding. Control cells were incubated for 7 days with Selenite, Avastin® and Selenoavastin at different concentrations. Viable cells were counted at time periods (0-7 days) by Trypan Blue exclusion as described in Methods. The cell growth data are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table IV-3.

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To better understand the mechanism of cell death demonstrated by the Trypan Blue exclusion results, an alternative cytotoxic assessment assay of cell viability, i.e., the MTT assay (3-[4, 5-dimethylthioazol-2-yl]-2-5-diphenyltetrazolium bromide) was performed, where the tetrazolium salt ring is cleaved by viable cells forming the insoluble Formazan. This method would also help address the question of cell viability in the HME50-5E cells when treated with Selenoherceptin. The results for the three cell lines tested is illustrated in Figure IV-19, Figure IV-20 and Figure IV-21. Percent viability for MDA-MB-231 cells treated with Selenoherceptin as 10 µg Se and 131.1 µg protein was 57%. This percentage increased to 71% at 2 µg Se, 26.22 µg protein of Selenoherceptin in MDA-MB-231 cells on day 3 post treatment (Figure IV-19). At these same treatments, percent viability was 22% and 81% in MDA-MB-468 cell. The results from MTT assay show that Selenoherceptin dose-dependently and significantly reduced cell viability. Se as selenite was toxic to cells at all concentrations (Figure IV-19 and Figure IV-20). Herceptin® treatments alone showed high cell viability for all doses and time periods. MTT incubation in the HME50-5E cells yielded an interesting result. When the MTT solubilization media for Formazan was added to HME50-5E cells, the Formazan salt could not be dissolved completely because of the adherence properties of the cells. Also, the intensity of formazan color was so dark that plate reader displayed ‘OVERFLOW’ for results on day 1 and day 3 of treatments indicating that the metabolic activity was so high, it saturated the limits of detection for the instrumentation. This observation coupled with the confluent, adherent populations, suggested that the vast majority of the population was metabolically healthy following Se treatments. Control HME50-5E cell viability was therefore set at 100% viability for days 1 and 3 treatment. Moreover, no significant differences were observed on day 3 Herceptin® or Selenoherceptin treatments. Ip [157] mentioned that normal cells, early transformed cells and late stage preneoplastic cells may respond differently to selenium intervention with respect to molecular pathways involving cell cycle proteins and apoptotic proteins [155]. The photomicrographs reflect this result (shown in Figure IV-15) with no morphological changes evident nor viable loss of cells.

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Figure IV-19: Metabolic activity of Control, Selenite, Herceptin®, Selenoherceptin treated MDA-MB-231 cells as determined by the MTT Assay. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding with Selenite, Avastin® and Selenoavastin at different concentrations over 6 days by MTT as described in Methods. The cell viabilities are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table IV-1.

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Figure IV-20: Metabolic activity of Control, Selenite, Herceptin®, Selenoherceptin treated MDA-MB-468 cells as determined by the MTT Assay. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding with Selenite, Avastin® and Selenoavastin at different concentrations over 6 days by MTT as described in Methods. The cell viabilities are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table IV-2.

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Figure IV-21: Metabolic activity of Control, Selenite, Herceptin®, Selenoherceptin treated HME50-5E cells as determined by the MTT Assay. 40,000 cells were seeded in 48 well-plates and were treated on day 5 post seeding with Selenite, Avastin® and Selenoavastin at different concentrations over 6 days by MTT as described in Methods. The cell viabilities are Means ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant, p≤ 0.05 are summarized in Table IV-3.

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Dose dependent calculations were used to determine IC50s of Selenoherceptin on day 3 of post treatments as shown in Figure IV-22 for MDA-MB-231 cell viability and

Figure IV-23 for MDA-MB-468 cell viability. Selenoherceptin showed an IC50 of 2.07 µg Se in MDA-MB-231 cells and 1.71 µg Se in MDA-MB-468 cells on day 3 of post treatment. Since cell viability for HME50-5E cells was assumed to be 100% as described above, IC50 of HME50-5E was not able to be calculated as treatment values did not approach close enough to 50% to provide an accurate estimate.

Figure IV-22: IC50 for MDA-MB-231 Cells with Selenoherceptin.

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Figure IV-23: IC50 for MDA-MB-468 Cells with Selenoherceptin.

The experimental results uniformly and collectively indicate that Selenoherceptin can significantly inhibit cell proliferation and cell viability in the TNBC cell lines; MDA- MB-231 and MDA-MB-468 but not the immortalized normal HME50-5E epithelial. The cytotoxic data is consistent with the ability of both selenite and Selenoherceptin but not Herceptin® to generate superoxide in both the in vitro CL assay (Figure IV-5 and Figure IV-6) and as detected by the intracellular increase in the red fluorescence of the intracellular DHE (Figure IV-7, Figure IV-8 and Figure IV-9). The experimental results also appear to be confirmed by the optical assessment of altered cell morphology as photographed and shown in Figure IV-13, Figure IV-14 and Figure IV-15.

Previous studies have established that selenium induces apoptosis by the intrinsic (mitochondrial-mediated) pathway [159-163]. Furthermore, apoptosis or programmed cell death is associated with morphological, biochemical and molecular changes occurring in the cell [164]. In order to confirm that Selenoherceptin treatments also induce apoptosis, an Annexin V was performed. Both Selenoherceptin and also selenite treated cells showed prominent morphological changes, a characteristic of apoptosis as

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Texas Tech University, Soni Khandelwal, May 2018 evidenced by microscopic studies (Figure IV-13 and Figure IV-14). Due to different sensitivities identified at day 3 for all cell lines tested in both Trypan Blue and MTT assays, cells were incubated with three different concentrations of Selenofolate to determine apoptosis would be induced. Flow cytometry data shows that a dosage of Selenoherceptin as 2 µg Se and 26.2 µg protein treated MDA-MB-468 cells exhibited more changes than MDA-MB-231 cells. A majority of the MDA-MB-231 cell population were in lower left (LL) quadrant, corresponding to living cells (Figure IV-24). A substantial proportion of the MDA-MB-468 cells in the upper right (UR) and lower right (LR) quadrants (as shown in Figure IV-27), corresponding to early and late apoptosis respectively. The morphological phenotypes (Figure IV-13 and Figure IV-14) of these apoptotic cells strongly correlated with flow analysis. In contrast to the TNBC cells, significant percentages of HME50-5E cells were found in the upper left (UL) quadrant (as shown in Figure IV-30), corresponding to living cells. The total percentages and distribution of cells are shown in Figure IV-25, Figure IV-28 and Figure IV-31. The percentages of cells undergoing early and late apoptosis were measured by flow cytometry and results are shown in Figure IV-26, Figure IV-29 and Figure IV-32. The percentages of cell undergoing apoptosis were measured by flow cytometry as shown in Figure IV-24, Figure IV-25 and Figure IV-26.

Due to its apoptotic activity, Sutent (Pfizer) is used as a positive control for induction of apoptosis in TNBC [165] and HME50-5E cells. Interestingly, Sutent demonstrated even greater potency in killing the normal cells (Figure IV-30). To our knowledge, this is the first report of the toxicity of Sutent on normal cells. This suggests important consequences for clinical success of Sutent with respect to side effects. Further study is needed to investigate this observation. Control cells also showed apoptosis, one speculation for this phenomenon is that the cells divided rapidly and the over-growth of the cells in the limited well space might cause the cell death. To address the appearance of a population of apoptotic cells in the vehicle control, prior studies of candidate anticancer therapies also demonstrated [162, 166] this phenomenon in their control groups. However, this was not observed in Trypan Blue exclusion (Figure IV-16, Figure IV-17 and Figure IV-18) and MTT (Figure IV-19, Figure IV-20 and Figure IV-21) assays.

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A loss of mitochondrial membrane potential (ΔψM) indicates a loss of cell viability due to proton pumps across the inner membrane during the process of electron transport and oxidative phosphorylation driving the conversion of ADP to ATP [167]. Therefore, MDA-MB-468 and HME50-5E cells were stained with MitoTracker Red CMXRos (instead of Propidium Iodide), which stains mitochondria in live cells and its accumulation is dependent upon its ΔψM. In this study, the ΔψM was measured by the flow cytometry and the results demonstrated that a dose of 2 µg Se and 26.2 µg protein as Selenoherceptin caused a decrease in the ΔψM and increase in the percent apoptotic MDA-MB-468 cells. In contrast, there was no change in the ΔψM (UL quadrant) of control vs Selenoherceptin treated HME50-5E cells (Figure IV-30), corresponding to full cell viability. This data agrees with the intense Formazan color developed from MTT assays of the HME50-5E cellular metabolic activity (Figure IV-21). Hence, HME50-5E cells do not undergo apoptosis following Selenoherceptin treatment on day 3. These observations again suggest the probable role of selenium-induced oxidative stress/glutathione oxidation that triggers apoptosis associated with both increased generation of superoxide and a decreases in level of the mitochondrial membrane potential, ΔψM. Studies have reported that modulation of mitochondrial functions to regulate apoptosis is one of the most affected pathways of Se compounds in cancer therapy [160]. Selenite also caused necrosis in MDA-MB-468 cells and apoptosis in MDA-MB-231 cell and few events were detected in HME50-5E cells, yet selenite is known to also induce necrosis in other breast cancer cells [155].

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Figure IV-24: Herceptin®, Selenoherceptin and Selenite as Se Treatments induced apoptosis in MDA-MB-231 cells. MDA-MB-231 cells were stained with Annexin V/PI and subjected to flow cytometric analysis. The four quadrants represent-living cells (Lower Left; LL: Annexin V-PI-), early apoptotic (Lower Right; LR: Annexin V+PI-), late apoptotic (Upper Right; UR: Annexin+PI+) or necrotic (Upper Left; UL: Annexin V-PI+) stages.

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Figure IV-25: Representative of four quadrants when MDA-MB-231 cells were treated with Herceptin®, Selenoherceptin and Selenite as Se. Data is expressed as Mean (n=3) of the total percent MDA-MB-231 cells.

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Figure IV-26: Percentage level of apoptotic cells when MDA-MB-231 cells were treated with Herceptin®, Selenoherceptin and Selenite as Se. Data is expressed as Mean ± SE (n=3). Treatments that were statistically significant p ≤ 0.05 and summarized in Table IV-1.

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Figure IV-27: Herceptin®, Selenoherceptin and Selenite as Se Treatments induced apoptosis in MDA-MB-468 cells. MDA-MB-468 cells were stained with Annexin V/ Mitotracker Red and subjected to flow cytometric analysis. The four quadrants represent-living cells (Upper Left; UL: Annexin V- Mitotracker Red+), early apoptotic (Upper Right; UR: Annexin+ Mitotracker Red +), late apoptotic (Lower Right; LR: Annexin V+ Mitotracker Red-) or necrotic (Lower Left; LL: Annexin V- Mitotracker Red-) stages.

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Figure IV-28: Representative of four quadrants when MDA-MB-468 cells were treated with Herceptin®, Selenoherceptin and Selenite as Se. Data is expressed as Mean (n=3) of the total percent of MDA-MB-468 cells.

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Figure IV-29: Percentage level of apoptotic cells when MDA-MB-468 cells were treated with Herceptin®, Selenoherceptin and Selenite as Se. Data is expressed as Mean ± SE (n=3). Treatments were compared using two sample t test. Treatments that were statistically significant p ≤ 0.05 and summarized in Table IV-2.

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Figure IV-30: Herceptin®, Selenoherceptin and Selenite as Se Treatments did not induced apoptosis in HME50-5E cells. HME50-5E cells were stained with Annexin V/ Mitotracker Red and subjected to flow cytometric analysis. The four quadrants represent- living cells (Upper Left; UL: Annexin V- Mitotracker Red+), early apoptotic (Upper Right; UR: Annexin+ Mitotracker Red +), late apoptotic (Lower Right; LR: Annexin V+ Mitotracker Red-) or necrotic (Lower Left; LL: Annexin V- Mitotracker Red-) stages.

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Figure IV-31: Representative of four quadrants when HME 50-5E cells were treated with Herceptin®, Selenoherceptin and Selenite as Se. Data is expressed as Mean (n=3) of the total percent of HME50-5E cells.

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Figure IV-32: Percentage level of HME50-5E apoptotic cells when the cells were treated with Herceptin®, Selenoherceptin and Selenite as Se. Data is expressed as Mean ± SE (n=3). Treatments were compared using two sample t test.

Western blotting was used to detect the protein expression levels of HER2 in MDA-MB-231, MDA-MB-468 and HME50-5E cells. BT-474 cell lysate was used as a positive loading control for immunoblotting and HER2 expression ~185 kDa was detected. . Following experimental treatments of cells, expression of HER2 in TNBC and HME50-5E cell lines were not detected (Figure IV-33, Figure IV-34 and Figure IV-35). According to ATCC website, MDA-MB-468 expresses 1 X 106 epidermal growth factor receptor (EGFR)/cell. Herceptin® targets HER2 and since HER2 belongs to EGFR family, probably MDA-MB-468 was sensitive to Selenoherceptin treatment. Expression of β-actin was detected ~42 kDa and cleaved β-actin expression was detected only after selenite treatment in all three cell lines. This was evident from Figure IV-13, Figure IV-14 and Figure IV-15 which shows Selenite toxicity. Cleavage of β-actin by selenite treatment was found in all three cell lines. These may have some biological and therapeutic implications like cell-cell interactions, cell migration (metastasis) and

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proliferation [169]. This interesting observation requires study and was outside the scope of this current project. Furthermore, the safety and efficacy of Sodium selenite was evaluated in Phase I clinical trial. The results from this trial show that no major toxicity was reported when 10.2 mg/m2 of sodium selenite was administered in cancer patients [170].

Figure IV-33: Western blot analysis of the expression level of HER2 in MDA-MB-231 Cells treated with Herceptin®, Selenoherceptin and Selenite as Se. Total cell lysates were subjected to SDS-PAGE followed by Western blotting. Membranes were probed with the anti-HER2 or anti β-actin antibodies followed by peroxidase conjugated rabbit anti-mouse antibodies and visualization was performed by the ECL detection system. Lane 1: BT474 as HER2 positive loading control, Lanes 2 and 3: Control, Lanes 4 and 5: Selenite (2µg as Se) treatments, Lanes 6 and 7: Herceptin® (26.22µg as protein) treatment, Lanes 8 and 9: Selenoherceptin (2µg as Se, 26.22µg as protein) treatments.

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Figure IV-34: Western blot analysis of the expression level of HER2 in MDA-MB-468 Cells treated with Herceptin®, Selenoherceptin and Selenite as Se. Total cell lysates were subjected to SDS-PAGE followed by Western blotting. Membranes were probed with the anti-HER2 or anti β-actin antibodies followed by peroxidase conjugated rabbit anti-mouse antibodies and visualization was performed by the ECL detection system. Lane 1: BT474 as HER2 positive loading control, Lanes 2 and 3: Control, Lanes 4 and 5: Selenite (2µg as Se) treatments, Lanes 6 and 7: Herceptin® (26.22µg as protein) treatment, Lanes 8 and 9: Selenoherceptin (2µg as Se, 26.22µg as protein) treatments.

Figure IV-35: Western blot analysis of the expression level of HER2 in HME 50-5E Cells treated with Herceptin®, Selenoherceptin and Selenite as Se. Total cell lysates were subjected to SDS-PAGE followed by Western blotting. Membranes were probed with the anti-HER or anti β-actin antibodies followed by peroxidase conjugated rabbit anti-mouse antibodies and visualization was performed by the ECL detection system. Lane 1: BT474 as HER2 positive loading control, Lane 2: Molecular weight markers, Lanes 3 and 4: Control, Lanes 5 and 6: Selenite (2µg as Se) treatments, Lanes 7 and 8: Herceptin® (26.22µg as protein) treatment, Lanes 9 and 10: Selenoherceptin (2µg as Se, 26.22µg as protein) treatments.

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Additionally, Herceptin®, Selenoherceptin and Selenite treated HME50-5E cell lysates protein were separated on 8% SDS-PAGE gel and stained with Coomaisse Blue R-250 dye (Figure IV-36) to see total number of proteins expressed after treatment. Low molecular intense bands ~35 kDa were detected in Selenite and Selenoherceptin treatment. This result suggest an identical posttranslational mechanism due to presence of Se. Similar banding pattern was seen in HCC1419 (loading HER2 positive control), control and Herceptin® treated cells- bands above 75 kDa and below 48 kDa.

Figure IV-36: SDS-PAGE of HME50-5E Total Protein Cell Lysate from Control, Herceptin®, Selenoherceptin and Selenite as Se Treatments. Total cell lysates were subjected to SDS-PAGE followed by Coomassie Blue R-250 stain. Lane 1: HCC1419 as HER2 positive loading control, Lane 2: Molecular markers, Lanes 3 and 4: Control, Lanes 5 and 6: Selenite (2µg as Se) treatments, Lanes 7 and 8: Herceptin® (26.22µg as protein) treatment, Lanes 9 and 10: Selenoherceptin (2µg as Se, 26.22µg as protein) treatments.

This study demonstrates that TNBC cells, MDA-MB-231 and MDA-MB-468 are inhibited from proliferating and are induced to undergo programmed cell death by the cytotoxic effects of selenocompounds that form selenides generating superoxide by

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CHAPTER V

IMPLICATIONS AND LIMITATION

The present study demonstrates that Selenofolate, seleno-immunoglobulins Selenoavastin and Selenoherceptin reduced MDA-MB-231 and MDA-MB-468 cell viability and proliferation in vitro as a function of dose and time. The decreased cell viability and proliferation in these cell lines by Selenofolate and the seleno- immunoglobulins were primarily apoptotic induced by the intracellular generation of superoxide. These conclusions are drawn from the assessment of cell morphology, cell viability subjected to Trypan Blue staining, MTT assay, visual assessment of reactive oxygen species using DHE, detection of apoptosis by flow cytometry and identification of the FRA and HER2+, EGFR proteins on these TNBC cell lines using western blots.

The potential toxicity of Selenofolate to other than targeted cancer cells in an in vivo trial remains the major concern as in any other chemotherapy. Although folate receptors are expressed at very low levels in normal cells, Weitman et al. [136] reported that only the normal renal epithelium cells express quite a considerable amount of folate receptor. Because of the small molecular weight of Selenofolate (MW = 573 g/mol), this conjugate may be easily reabsorbed into the proximal tubules of the kidney following excretion by the kidneys. Thus, Selenofolate may accumulate in the kidney cells due to FRA expression in the apical membrane of the proximal tubules and in turn result in kidney damage. However, Leamon and Reddy [217] found that a FR-drug conjugate, EC72, was effective as a chemotherapeutic agent in mice. EC72 caused no harm to normal tissues, including the FR-positive cells in the kidneys. Furthermore, results from folate conjugate-based have not yet reported any renal toxicity [218] ] and so optimism remains that Selenofolate might be a widely employed therapeutic drug without toxicity to any normal cells in the light of the failure of the Phase 3 Ovarian trial of Vintafolide.

These studies further suggested a general technique which can be applicable to other proteins, other mAbs and compounds with primary amines where Bolton-Hunter Chemistry can be applied. The development of target specific “magic bullets” in the form of immunoconjugates is widely being accepted and developed for the treatment of

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The study also demonstrates many potential therapeutic advantages of using Selenofolate over other folate-conjugates to treat Ovarian and other cancers which over- express the FRA. Selenofolate is of smaller molecular weight and may better penetrate tumors and probably can be more rapidly cleared from body. As the conjugate carries a nutrient escaping selenium levels will not be toxic to other cells. Selenium toxicity depends totally on delivery of sufficient amounts to induce oxidative stress in excess of cancer cell’s capacity to resist ROS. The immunoglobulins, Selenoavastin and Selenoherceptin are toxic to two TNBC cells lines, MDA-MB-231 and MDA-MB-468, whereas Avastin® and Herceptin® exhibit no toxicity. Like Selenofolate the attachment of redox selenium to these antibodies is a one-step process without the need of linkers nor any conjugation of “cytotoxic drugs” which may have to be precisely uncoupled to be effective against the cancer cells. In the selenium attachment, the effective concentration of the end selenium adduct to the immunoglobulin is controlled only by time and dose. Development time, cost and effectiveness in controlling cancer cells growth with selenoadducts is possible across a broad spectrum of small molecules, proteins, antibodies and antibiotics.

Additional studies of Selenofolate, Selenoavastin, Selenoherceptin and combined fluorescent or gold labeling to see Selenofolate, Selenoavastin, Selenoherceptin binding the FRA, VEGF and HER2 respectively in MDA-MB-231, MDA-MB-468 and HME50- 5E cells could be extremely rewarding but was outside the scope of this current project.

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CHAPTER VI

CONCLUSIONS

In summary, these in vitro studies demonstrate that Selenofolate and the selenium immunoconjugates, Selenoavastin and Selenoherceptin decrease cell viability and proliferation of Triple Negative Breast Cancer (TNBC) cells, MDA-MB-231 and MDA- MB-468 cell lines, in a Se dose and time dependent manner. All selenium compounds, modified Folate and immunoconjugates with redox selenium exhibited the generation of .- superoxide (O2 ) as determined by CL in vitro and in intracellular DHE assays. This ROS activity was verified by cell morphology changes documented photographically over time. The Trypan Blue exclusion and MTT assays at varied Selenofolate and Seleno- immunoglobulin concentrations demonstrated significant loss of cell viability. Decreased cell viability was mostly due to the intrinsic (mitochondrial) apoptosis pathway in both MDA-MB-468 and MDA-MB-231 cells, as determined by the MTT assay and flow cytometry. The latter MDA-MB-231 cells were not as responsive to the selenium treatments by dose and over time. Immortalized but otherwise normal epithelial breast cells, HME50-5E cells, were way less sensitive to the comparable selenium treatments and not highly responsive to selenite treatments.

Triple Negative Breast Cancer Cells are reported not to possess the HER2+, progesterone and estrogen receptors. In this study with the TNBC cell lines, MDA-MB- 468 and MDA-MB-231, these receptors were not detected by western blot assays. The TNBC cells also revealed by western blot assays, the presence of the Folate Receptor - Alpha (FRA). Selenite appeared to cleave the protein β-actin, in addition to generating intracellular superoxide.

Similar to normal cells, cancer cells rely on an adequate supply of energy and nutrients to support cellular events. In the case of cancer cells, the most prominent energy utilization appears to support proliferation, migration and invasion of both surrounding and distal tissues. One of the most frequently studied and distinctive attributes observed in carcinoma cells is that the preferred method of energy generation is Anaerobic (Glycolysis and Fermentation- 4 ATP per Glucose molecule). Based on studies of hypoxia and the tumor microenvironment, carcinoma cells are known to switch from

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Texas Tech University, Soni Khandelwal, May 2018 relying heavily on Aerobic Respiration (tricarboxylic acid or TCA cycle) and Mitochondrial Electron Transport Chain (ETC - 38 ATP per glucose molecule) to Glycolysis. This is also referred to as the Warburg effect. The end product of the anaerobic path is lactic acid, which results in local acidosis around the cancer cells, further promoting the hypoxic milieu [221]. However, tumors are composed of different types of cells (i.e., fibroblast, adipocytes, and macrophages) which can display different metabolic requirements. Geographic location of cells within the tumor will contribute to these changes. For example, cancer cells situated near blood vessels have access to oxygen and nutrients. Therefore, energy can be generated from both glycolysis and oxidative phosphorylation. In contrast, cells located furthest away, within the tumor mass, resort to aerobic glycolysis to survive the prevalent hypoxic environment.

Evidence for this is based on studies showing that hypoxia promotes the stabilization of HIF (hypoxia-inducible factor) 1 and 2. Normally, the von Hippel-Lindau (VHL) protein degrades HIF but many cancer cells do not express this protein. HIF-1α also extends its stimulatory effects to the TCA cycle by activating pyruvate kinase and increased expression of glucose transporters as compared to normal cells. In normal cells, pyruvate kinase catalyzes an irreversible reaction in glycolysis i.e., conversion of phosphophenol pyruvate to pyruvate. Pyruvate is a critical step in production of ATP and lactate and glycolysis to TCA cycle. There are two isoforms of pyruvate kinase (PyK)- one is a highly active tetramer and other is a less active dimer. Cancer cells switch between these isoforms; when energy is needed for proliferation they use both, involving oncogenes and tumor suppressor genes. Additionally, for their more rapid proliferation requirement, hexokinase II isoform binds to mitochondria and utilizes newly generated ATP from ATP synthase system as a substrate [222].

The oncogene, c-myc, is overexpressed in cancer cells and upregulates genes for metabolic enzymes. Another oncogene, ras also promotes glycolysis. Inhibition of genes expressing cytochrome c oxidase occurs due to loss of tumor suppressor genes such as p53 in cancer cells. In normal cells, cytochrome c oxidase is involved in the ETC. Thus, oncogene activation in cancer cells drives changes in metabolism in contrast to normal cells [222].

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It is this difference in metabolic requirements and pathways that may give us a crucial clue why the selenium conjugates are more effective in the cancer cells and demonstrate significantly lower cell death in the normal cells. Taking the FRA experiment as an example, Selenofolate was most effective in the MDA-MB-231 cells, then the MDA-MB-468 cells with little (no significant cell death) in the normal HME50- 5E cells. In comparing Western results for the TNBC lines, it is likely that the effectiveness of the Selenofolate is based on relative FRA expression levels (e.g. more in MDA-MB-231). However, this same argument cannot be made for the HME50-5E because the levels of FRA were comparable or greater than the TNBC levels. Therefore, another mechanism has to be involved. Based, on the ROS generation, differences in metabolism between normal and cancer cells is the most likely reason.

Glutamine is the most abundant amino acid in the body, being used in several pathways, including those of the nucleotide synthesis, oxidation in the Krebs cycle to produce ATP, and as a lipogenic and gluconeogenic precursor. GA (glutaminases) converts glutamine to glutamate. Glutamate is used to synthetize nucleotides, hexosamines and . There are 2 types of GA, K and L. Type K is encoded by the GLS gene and expresses KGA and GAC isoforms. L type (encoded by the GLS2 gene) expresses LGA and GAB isoforms. The GAC isoform is highly expressed in many types of cancer cells whereas normal cells use glutamine for synthesis of amino acids from the amide and amino groups, and nitrogen for de novo nucleotide formation. Activation of c-myc and inactivation of p53 causes the cancer cells to utilize glutamine through GAC. In cancer cells, Glutamine is then metabolized by the TCA cycle and converted into lactate, producing NADPH for lipid biosynthesis and oxaloacetate for replenishment of Krebs cycle intermediates [222]. DMEM high glucose media used for culturing TNBC cells contained 4 mM of glutamine in contrast to Human Mammary Epithelial Media used for culturing HME50-5E cells contained 6 mM of glutamine.

This goals of the study were to investigate efficacy of selenium to kill TNBC cells by delivery to the cells of Se via different targets; vitamin (Folate), a ligand (VEGF) and a transmembrane protein receptor (HER2) as shown in Figure VI-1. In our current study, selenium acted as the cytotoxic agent with folate and two monoclonal antibodies serving

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Texas Tech University, Soni Khandelwal, May 2018 the dual role of acting a carrier for selenium and as targeting agents to deliver selenium to TNBC cell lines. Organic diselenides, as shown by Chaudière et al [92], selenocyanates, and isoselenocyanates as shown by Crampsie et al [93], may continuously undergo the redox cycle and generate superoxides. In the present study, organic selenocyanate (RSeCN) was attached to Folate, Avastin® and Herceptin® to investigate whether these current anticancer drugs can be re-purposed and utilized to target TNBC cell lines. Selenocyanate reacts with thiols and generates selenoate anions (RSe-) via a seleneylsulfide intermediate (RSe-SG) as illustrated in the reactions below

RSeCN + GSH → RSeSG + HCN

RSeSG + GSH → RSe- + GSSG

.- RSe- + O2 → O2

The selenoate anions subsequently undergo redox cycling and produce superoxide. The presence of superoxide is the most likely reason for the catastrophic effects of Se observed in the cells regardless of introduction.

Another important observation from this work was that in each approach to selenium conjugation and delivery to the cells, the selenium was being internalized (again, evidenced by ROS formation). How the selenium is being internalized is the focus of ongoing research projects. Development of a drug-carrier, targeting approach, by using a dietary nutrient result in reduced systemic drug toxicity towards normal cells makes this study different. Furthermore, the study demonstrates that the method of delivery to the TNBC cells (thought to lack specific markers for targeting strategies) can be piggy- backed onto current clinical therapies effectively, while demonstrating less cytotoxicity in the normal cells. These studies help profound implications for future drug design and efficacy with less side effects, impacting both treatment outcomes and quality of life in patients with TNBC.

The studies presented demonstrate some of the considerations and potential therapeutic possibilities underlying the design of targeted cancer therapy with Selenofolate, and seleno-immunoconjugates, and that likely small redox active selenium

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Texas Tech University, Soni Khandelwal, May 2018 compounds and the more than 70 ADCs in varies phases of clinical trials may be clinically effective in cancer treatment. The overriding consideration following the experimental results for this therapeutic technology is the simplicity with which redox selenium targeting conjugates can be formulated and how effectively they inhibit cell proliferation by generating superoxide and another ROS.

Figure VI-1: Summarized diagrammatic representation of the study.

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182. Folkman, J., Fundamental concepts of the angiogenic process. Current molecular medicine, 2003. 3(7): p. 643-651. 183. Miller, K., et al., Paclitaxel plus bevacizumab versus paclitaxel alone for metastatic breast cancer. New England Journal of Medicine, 2007. 357(26): p. 2666-2676. 184. Miles, D., et al., Randomized, double-blind, placebo-controlled, phase III study of bevacizumab with docetaxel or docetaxel with placebo as first-line therapy for patients with locally recurrent or metastatic breast cancer (mBC): AVADO. Journal of Clinical Oncology, 2008. 26(15_suppl): p. LBA1011-LBA1011. 185. O'Shaughnessy, J., et al., Comparison of Subgroup Analyses of PFS from Three Phase III Studies of Bevacizumab in Combination with Chemotherapy in Patients with HER2-Negative Metastatic Breast Cancer (MBC). 2009, AACR. 186. von Minckwitz, G., et al., Neoadjuvant chemotherapy and bevacizumab for HER2-negative breast cancer. New England Journal of Medicine, 2012. 366(4): p. 299-309. 187. Bear, H.D., et al., Bevacizumab added to neoadjuvant chemotherapy for breast cancer. New England Journal of Medicine, 2012. 366(4): p. 310-320. 188. Winer, E., et al., Clinical cancer advances 2008: major research advances in cancer treatment, prevention, and screening—a report from the American Society of Clinical Oncology. Journal of clinical oncology, 2008. 27(5): p. 812-826. 189. Tanne, J.H., FDA cancels approval for bevacizumab in advanced breast cancer. BMJ: British Medical Journal (Online), 2011. 343. 190. Rose, S., FDA pulls approval for avastin in breast cancer. Cancer discovery, 2011. 1(7): p. OF1-2. 191. Sikov WM, e.a., Impact of the addition of carboplatin and/or bevacizumab to neoadjuvant weekly paclitaxel followed by dose-dense AC on pathologic complete response rates in triple-negative breast cancer. 2013 San Antonio Breast Cancer Symposium., in 2013 San Antonio Breast Cancer Symposium. 2013: San Antonio. 192. Goswami, D., CYTOTOXIC EFFECTS OF SELENIUM CONJUGATED TRANSFERRINS ON LEUKEMIA CELL LINES, in Nutritional Sciences. May 2014, Texas Tech University: Lubbock, TX, USA. 193. Bapat, P., Cytotoxic effects of selenium conjugated trastuzumab on HER2+ breast cancer cell lines., in Nutritional Sciences. 2015-05, Texas Tech University: Lubbock. 194. Darius, J.R. and D.R. Richardson, William Hunter and radioiodination: revolutions in the labelling of proteins with radionuclides of iodine. Biochem. J., 2011: p. 1-4. 195. Kim, K.J., et al., The vascular endothelial growth factor proteins: identification of biologically relevant regions by neutralizing monoclonal antibodies. Growth Factors, 1992. 7(1): p. 53-64. 196. Prewett, M., et al., Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse and human tumors. Cancer research, 1999. 59(20): p. 5209-5218. 197. Gerber, H.-P. and N. Ferrara, Pharmacology and pharmacodynamics of bevacizumab as monotherapy or in combination with cytotoxic therapy in preclinical studies. Cancer research, 2005. 65(3): p. 671-680.

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198. Fasler, S., F. Skvaril, and H.U. Lutz, Electrophoretic properties of human IgG and its subclasses on sodium dodecyl-sulfate-polyacrylamide gel electrophoresis and immunoblots. Analytical biochemistry, 1988. 174(2): p. 593-600. 199. Arakawa, T., J.S. Philo, and Y. Kita, Kinetic and thermodynamic analysis of thermal unfolding of recombinant erythropoietin. Bioscience, biotechnology, and biochemistry, 2001. 65(6): p. 1321-1327. 200. Kaja, S., et al., Effects of dilution and prolonged storage with preservative in a polyethylene container on Bevacizumab (Avastin™) for topical delivery as a nasal spray in anti-hereditary hemorrhagic telangiectasia and related therapies. Human antibodies, 2011. 20(3, 4): p. 95-101. 201. Trail, P.A., G.M. Dubowchik, and T.B. Lowinger, Antibody drug conjugates for treatment of breast cancer: novel targets and diverse approaches in ADC design. Pharmacology & therapeutics, 2017. 202. Carpenter, G., L. King, and S. Cohen, Epidermal growth factor stimulates phosphorylation in membrane preparations in vitro. Nature, 1978. 276(5686): p. 409-410. 203. Padhy, L.C., et al., Identification of a phosphoprotein specifically induced by the transforming DNA of rat neuroblastomas. Cell, 1982. 28(4): p. 865-871. 204. Schechter, A.L., et al., The neu oncogene: an erb-B-related gene encoding a 185,000-Mr tumour antigen. Nature, 1984. 312(5994): p. 513-516. 205. Brandt-Rauf, P.W., M.R. Pincus, and W.P. Carney, The c-erbB-2 protein in oncogenesis: molecular structure to molecular epidemiology. Critical Reviews™ in Oncogenesis, 1994. 5(2-3). 206. Iqbal, N. and N. Iqbal, Human epidermal growth factor receptor 2 (HER2) in cancers: overexpression and therapeutic implications. Molecular biology international, 2014. 2014. 207. Hicks, D.G. and S. Kulkarni, HER2+ breast cancer: review of biologic relevance and optimal use of diagnostic tools. American Journal of Clinical Pathology, 2008. 129(2): p. 263-273. 208. Rosen, L.S., H.L. Ashurst, and L. Chap, Targeting signal transduction pathways in metastatic breast cancer: a comprehensive review. The oncologist, 2010. 15(3): p. 216-235. 209. Ross, J.S., et al., The Her-2/neu gene and protein in breast cancer 2003: biomarker and target of therapy. The oncologist, 2003. 8(4): p. 307-325. 210. Gusterson, B.A., et al., Prognostic importance of c-erbB-2 expression in breast cancer. International (Ludwig) Breast Cancer Study Group. Journal of Clinical Oncology, 1992. 10(7): p. 1049-1056. 211. Oostra, D.R. and E.R. Macrae, Role of trastuzumab emtansine in the treatment of HER2-positive breast cancer. Breast Cancer: Targets and Therapy, 2014. 6: p. 103. 212. Verma, S., et al., Trastuzumab emtansine for HER2-positive advanced breast cancer. New England Journal of Medicine, 2012. 367(19): p. 1783-1791. 213. Foulkes, W.D., I.E. Smith, and J.S. Reis-Filho, Triple-negative breast cancer. New England journal of medicine, 2010. 363(20): p. 1938-1948.

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214. Alley, S.C., N.M. Okeley, and P.D. Senter, Antibody–drug conjugates: targeted drug delivery for cancer. Current opinion in chemical biology, 2010. 14(4): p. 529-537. 215. Xu, C., C.Y.-T. Li, and A.-N.T. Kong, Induction of phase I, II and III drug metabolism/transport by xenobiotics. Archives of pharmacal research, 2005. 28(3): p. 249. 216. Oraki Kohshour, M., et al., Ablation of Breast Cancer Cells Using Trastuzumab‐ Functionalized Multi‐Walled Carbon Nanotubes and Trastuzumab‐Diphtheria Toxin Conjugate. Chemical biology & drug design, 2014. 83(3): p. 259-265. 217. Leamon, C.P. and J.A. Reddy, Folate-targeted chemotherapy. Advanced drug delivery reviews, 2004. 56(8): p. 1127-1141. 218. Marchetti, C., et al., Targeted drug delivery via folate receptors in recurrent ovarian cancer: a review. OncoTargets and therapy, 2014. 7: p. 1223. 219. Gordon, M.S., et al., Phase I safety and pharmacokinetic study of recombinant human anti-vascular endothelial growth factor in patients with advanced cancer. Journal of Clinical Oncology, 2001. 19(3): p. 843-850. 220. Herceptin https://www.accessdata.fda.gov/drugsatfda_docs/label/1998/trasgen092598lb.pdf . 221. Hsu, P.P. and D.M. Sabatini, Cancer cell metabolism: Warburg and beyond. Cell, 2008. 134(5): p. 703-707. 222. Amoêdo, N.D., et al., How does the metabolism of tumour cells differ from that of normal cells. Bioscience reports, 2013. 33(6): p. e00080.

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APPENDICIES

APPENDIX A

QUALITY CONTROL AND MICRO-PHOTOGRAPHS OF DIFFERENT

TREATMENTS

Figure A-1: Folate did not auto-fluoresce under UV light spectrum. Folate was run through the Attune™ NxT Flow Cytometer (ThermoFisher Scientific, Carlsbad, CA) as a quality control to detect if there was any kind of fluorescence emitted by Folate, because this would if present effect fluorescent assays.

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Figure A-2: Selenoavastin exhibited elemental fluorescence under UV light spectrum. Selenoavastin was run through the Attune™ NxT Flow Cytometer (ThermoFisher Scientific, Carlsbad, CA) as a quality control to detect if there was any kind of fluorescence emitted by Selenoavastin, because this would if present effect fluorescent assays.

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Figure A-3: Morphological Changes of Control, Selenite (10 µM), Folate (75 µM) and Selenofolate (75 µM) Treatments in MDA-MB-231 Cells. Treatment of MDA-MB-231 Cells with Selenite and Selenofolate morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-231 cells. Cells were visualized under 20X magnification.

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Figure A-4: Morphological Changes of Control, Selenite (20 µM), Folate (100 µM) and Selenofolate (100 µM) Treatments in MDA-MB-231 Cells. Treatment of MDA-MB-231 Cells with Selenite and Selenofolate morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-231 cells. Cells were visualized under 20X magnification.

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Figure A-5: Morphological Changes of Control, Selenite (10 µM), Folate (75 µM) and Selenofolate (75 µM) Treatments in MDA-MB-468 Cells. Treatment of MDA-MB-468 Cells with Selenite and Selenofolate morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-468 cells. Cells were visualized under 20X magnification.

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Figure A-6: Morphological Changes of Control, Selenite (20 µM), Folate (100 µM) and Selenofolate (100 µM) Treatments in MDA-MB-468 Cells. Treatment of MDA-MB-468 Cells with Selenite and Selenofolate morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-468 cells. Cells were visualized under 20X magnification.

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Figure A-7: Morphological Changes of Control, Selenite (10 µM), Folate (75 µM) and Selenofolate (75 µM) Treatments in HME50-5E Cells. Treatment of HME50-5E cells with Selenite, Folate and Selenofolate did not induce severe morphological change comparable to those seen in TNBC cells. Representative fields of view of HME50-5E cells. Cells were visualized under 20X magnification.

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Figure A-8: Morphological Changes of Control, Selenite (20 µM), Folate (100 µM) and Selenofolate (100 µM) Treatments in HME50-5E Cells. Treatment of HME50-5E cells with Selenite, Folate and Selenofolate did not induce severe morphological change comparable to those seen in TNBC cells. Representative fields of view of HME50-5E cells. Cells were visualized under 20X magnification.

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Figure A-9: Morphological Changes of Control, Selenite (5 µg as Se), Avastin® and Selenoavastin Treatments in MDA-MB-231 Cells. Treatment of MDA-MB-231 Cells with Selenite and Selenoavastin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-231 cells. Cells were visualized under 20X magnification.

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Figure A-10: Morphological Changes of Control, Selenite (5 µg as Se), Avastin® and Selenoavastin Treatments in MDA-MB-468 Cells. Treatment of MDA-MB-468 Cells with Selenite and Selenoavastin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-468 cells. Cells were visualized under 20X magnification.

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Figure A-11: Morphological Changes of Control, Selenite (5 µg as Se), Avastin® and Selenoavastin Treatments in HME50-5E Cells. Representative fields of view of HME50-5E cells. Cells were visualized under 20X magnification.

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Figure A-12: Morphological Changes of Control, Selenite (10 µg as Se), Avastin® and Selenoavastin Treatments in MDA-MB-231 Cells. Treatment of MDA-MB-231 Cells with Selenite and Selenoavastin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-231 cells. Cells were visualized under 20X magnification.

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Figure A-13: Morphological Changes of Control, Selenite (10 µg as Se), Avastin® and Selenoavastin Treatments in MDA-MB-468 Cells. Treatment of MDA-MB-468 Cells with Selenite and Selenoavastin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-468 cells. Cells were visualized under 20X magnification.

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Figure A-14: Morphological Changes of Control, Selenite (5 µg as Se), Avastin® and Selenoavastin Treatments in HME50-5E Cells. Representative fields of view of HME50-5E cells. Cells were visualized under 20X magnification.

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Figure A-15: Morphological Changes of Control, Selenite, Herceptin® and Selenoherceptin Treatments in MDA-MB-231 Cells. Treatment of MDA-MB-231 Cells with Selenite and Selenoherceptin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-231 cells. Cells were visualized under 20X magnification.

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Figure A-16: Morphological Changes of Control, Selenite, Herceptin® and Selenoherceptin Treatments in MDA-MB-468 Cells. Treatment of MDA-MB-468 Cells with Selenite and Selenoherceptin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-468 cells. Cells were visualized under 20X magnification.

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Figure A-17: Morphological Changes of Control, Selenite, Herceptin® and Selenoherceptin Treatments in HME50-5E Cells. Representative fields of view of HME50-5E cells. Cells were visualized under 20X magnification.

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Figure A-18: Morphological Changes of Control, Selenite, Herceptin® and Selenoherceptin Treatments in MDA-MB-231 Cells. Treatment of MDA-MB-231 Cells with Selenite and Selenoherceptin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-231 cells. Cells were visualized under 20X magnification.

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Figure A-19: Morphological Changes of Control, Selenite, Herceptin® and Selenoherceptin Treatments in MDA-MB-468 Cells. Treatment of MDA-MB-468 Cells with Selenite and Selenoherceptin morphological changes indicative of membrane disruption with decreased cell viability. Representative fields of view of MDA-MB-468 cells. Cells were visualized under 20X magnification.

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Figure A-20: Morphological Changes of Control, Selenite, Herceptin® and Selenoherceptin Treatments in HME50-5E Cells. Representative fields of view of HME50-5E cells. Cells were visualized under 20X magnification.

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Figure A-21: Standard Curve prepared from a serial dilution of Bovine Gamma Globulin used for protein determination of Avastin®, Selenoavastin, Herceptin® and Selenoherceptin.

Figure A-22: Time dependent superoxide generation as measured by lucigenin chemiluminescence (CL) for blank, sodium selenite.

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Figure A-23: HME50-5E MTT Plate Photo on Day 3 Post Treatment showing the insoluble formazan salt.

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APPENDIX B

CELL DIAMETER FROM DIFFERENT TREATMENTS.

Table B-1: MDA-MB-231 Cell Diameter (microns). Data is expressed as mean (n=3).

Day Control Selenite Avastin Selenoavastin Herceptin Selenoherceptin Folate Selenofolate 2 µg as as 30.7 as 2 µg Se as 26.2 µg as 2 µg as 26.2 100 µM 100 µM Se µg and 30.7 µg protein µg protein protein protein 0 19.37 18.00 18.30 18.90 19.57 19.42 19.43 18.76 1 18.80 18.38 17.11 16.81 16.31 19.82 18.53 18.62 2 17.32 16.36 17.97 19.31 19.03 19.02 18.25 18.67 3 16.59 17.65 18.01 18.38 17.97 18.78 17.74 18.01 4 17.04 16.59 13.35 19.41 17.67 19.37 16.94 18.91 5 17.02 16.51 17.74 18.64 17.26 18.59 16.61 18.05 6 16.58 16.33 17.08 18.69 17.43 18.94 16.40 18.11 7 16.26 16.30 11.04 18.41 16.74 19.32 16.99 18.60

Table B-2: MDA-MB-231 Cell Circularity (microns). Data is expressed as mean (n=3).

Day Control Selenite Avastin Selenoavastin Herceptin Selenoherceptin Folate Selenofolate 2 µg as as 30.7 as 2 µg Se as 26.2 µg as 2 µg as 26.2 100 µM 100 µM Se µg and 30.7 µg protein µg protein protein protein 0 0.84 0.84 0.74 0.83 0.86 0.84 0.81 0.82 1 0.84 0.78 0.31 0.33 0.38 0.85 0.85 0.78 2 0.80 0.77 0.81 0.74 0.80 0.80 0.85 0.82 3 0.73 0.82 0.83 0.79 0.83 0.73 0.84 0.77 4 0.85 0.81 0.78 0.78 0.82 0.79 0.80 0.78 5 0.83 0.79 0.76 0.82 0.78 0.84 0.78 0.80 6 0.81 0.82 0.68 0.78 0.82 0.82 0.71 0.78 7 0.80 0.79 0.74 0.83 0.80 0.86 0.75 0.82

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Texas Tech University, Soni Khandelwal, May 2018

Table B-3: MDA-MB-468 Cell Diameter (microns). Data is expressed as mean (n=3).

Day Control Selenite Avastin Selenoavastin Herceptin Selenoherceptin Folate Selenofolate 2 µg as as 30.7 as 2 µg Se as 26.2 µg as 2 µg as 26.2 100 µM 100 µM Se µg and 30.7 µg protein µg protein protein protein 0 19.36 19.86 19.84 19.97 19.92 20.26 17.77 17.99 1 18.35 19.37 18.78 20.63 19.19 19.59 16.91 17.48 2 17.80 18.24 18.20 19.58 18.74 18.64 16.39 16.58 3 16.99 17.36 18.02 18.67 18.48 18.96 16.35 16.47 4 16.70 16.95 17.16 18.08 17.43 17.86 15.95 16.60 5 17.37 16.97 16.91 17.43 17.40 17.54 15.62 16.22 6 16.76 16.79 16.49 17.05 16.62 17.44 15.32 16.11 7 16.81 16.91 16.08 16.38 16.45 17.03 15.07 15.91

Table B-4: MDA-MB-468 Cell Circularity (microns). Data is expressed as mean (n=3).

Day Control Selenite Avastin Selenoavastin Herceptin Selenoherceptin Folate Selenofolate 2 µg as as 30.7 as 2 µg Se as 26.2 µg as 2 µg as 26.2 100 µM 100 µM Se µg and 30.7 µg protein µg protein protein protein 0 0.86 0.88 0.86 0.81 0.89 0.86 0.91 0.91 1 0.88 0.88 0.90 0.88 0.87 0.89 0.90 0.90 2 0.87 0.90 0.89 0.88 0.90 0.88 0.91 0.86 3 0.85 0.89 0.87 0.87 0.88 0.82 0.88 0.85 4 0.89 0.80 0.81 0.81 0.85 0.81 0.88 0.83 5 0.86 0.74 0.81 0.82 0.73 0.82 0.85 0.84 6 0.81 0.72 0.81 0.81 0.80 0.80 0.80 0.85 7 0.77 0.71 0.78 0.83 0.79 0.81 0.80 0.84

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