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The Apoptotic and Inhibitory Effects of Phylloquinone in the U937 Cell Line Tesha E

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The Apoptotic and Inhibitory Effects of Phylloquinone in the U937 Cell Line Tesha E East Tennessee State University Digital Commons @ East Tennessee State University Electronic Theses and Dissertations Student Works 5-2016 The Apoptotic and Inhibitory Effects of Phylloquinone in the U937 Cell Line Tesha E. Blair East Tennessee State Universtiy Follow this and additional works at: https://dc.etsu.edu/etd Part of the Cancer Biology Commons, and the Cell Biology Commons Recommended Citation Blair, Tesha E., "The Apoptotic and Inhibitory Effects of Phylloquinone in the U937 Cell Line" (2016). Electronic Theses and Dissertations. Paper 3028. https://dc.etsu.edu/etd/3028 This Thesis - Open Access is brought to you for free and open access by the Student Works at Digital Commons @ East Tennessee State University. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Digital Commons @ East Tennessee State University. For more information, please contact [email protected]. The Apoptotic and Inhibitory Effects of Phylloquinone in the U937 Cell Line _____________________ A thesis presented to the faculty of the Department of Biological Sciences East Tennessee State University In partial fulfillment of the requirements for the degree Master of Science in Biology _____________________ by Tesha Blair May 2016 _____________________ Hugh Miller III, Ph.D., Chair Aruna Kilaru, Ph.D. Leonard Robertson, Ph.D. Keywords: apoptosis, phylloquinone, vitamin K, cell death, U937 cells ABSTRACT The Apoptotic and Inhibitory Effects of Phylloquinone in the U937 Cell Line by Tesha Blair Phylloquinone is a natural analog of vitamin K that has been shown to both inhibit cancer cell growth and induce apoptosis in several cancer cell lines. This study examined these effects in a non-Hodgkin lymphoma cell line, known as U937. Cell growth inhibition and apoptosis were assessed through the quantification of cell density and area, following treatment with several concentrations of phylloquinone. In addition, apoptosis was detected and quantified using immunofluorescent markers of apoptosis (i.e. annexin V, APO-BrdU). Treatment with phylloquinone resulted in reduced overall cell density, increased overall cell area, and an increased frequency of apoptosis in U937 cells. Increasing both phylloquinone concentration and treatment time enhanced these effects. These results are significant because they document the anti-cancer effects of this analog of vitamin K, as well as provide insight into the morphological changes that occur during apoptosis in U937 cells. 2 ACKNOWLEDGEMENTS I would like to thank my mentor and advisor Dr. Hugh Miller for his support and guidance throughout my time in graduate school at East Tennessee State University. I would also like to thank my committee members Dr. Aruna Kilaru and Dr. Leonard Robertson for their advisement and input on my thesis project. I would also like to thank Dr. Thomas Jones and Dr. Lev Yampolsky, as well as fellow graduate student Adam McCullough. Special thanks go out to my mom and dad, as well as the rest of my family and friends for supporting and encouraging me in everything I do. 3 TABLE OF CONTENTS Page ABSTRACT ...……………………………………………………………………………………………………......2 ACKNOWLEDGEMENTS ..…………………………………………………………………………………......3 LIST OF TABLES ...……………………………………………………………………………………………......7 LIST OF FIGURES ..……………………………………………………………………………………………….8 Chapter 1. INTRODUCTION ..…………………………………………………..………………………………….......11 Apoptosis ..……………………………………………………………………………………………...11 Morphology...………………………………………………………………………………..13 Programmed Cell Death and Cell Suicide – Incidence of Apoptosis.....14 Biochemical Mechanisms....……………………………………………………………17 Apoptosis and Cancer ...……..…………………………………………………………………….19 Apoptosis and Cancer Therapy ..………………………………..……………………………..22 Vitamins and Cancer Therapy.……………………………………………………….25 Vitamin K...…………………………………………………………………...……26 Phylloquinone, Apoptosis, and Cancer Therapy ………………………………………..27 Hypothesis, Rationale, and Specific Aims .....……………………………………………...31 2. MATERIALS AND METHODS ...……………………………………………………..………………...33 Experimental Design ...…………………………………………………………..………………...33 U937 Cell Line and Cell Culture...…………………………………………………...35 Experimental Culture Conditions...…………………………………………...……35 Phylloquinone Solution....………………………………………………………………36 4 Cell Culture Preparation.………………………………………………………….……37 Data Collection ..………………………………………………….……………………………….….38 Cell Density and Area Determination...………………………………………......38 Determination of Apoptosis…...……………………………………………………...40 Annexin V Assay....……………………………………………………………...40 APO-BrdU Assay...………………………………………………………………41 Analysis ..….…………………………………………………………………………………………….44 Occurrence of Apoptosis Analysis....……………………………………………….44 Cell Area Analysis…………………………………………………………………….…...44 Cell Density Analysis.…………………………………………………………………….44 3. RESULTS ...……………………………...……………………………………………………………………..46 Occurrence of Apoptosis ..……………………………………………..…………………….......46 Cell Area ...……………………………………………………..……………………………………......48 Cell Density ..…………………………………………………………………………………….……67 4. DISCUSSION ..…………………………………...……………………………………………………………77 The Frequency of Apoptosis is Significantly Higher in U937 Cell Populations Following Treatment with Phylloquinone ...………………….……………………….….77 Apoptotic and Non-Apoptotic U937 Cell Areas are Larger Following Treatment with Increasing Concentrations of Phylloquinone ……………………77 Early-Stage Apoptotic Cells are Smaller than Late-Stage Apoptotic Cells ......78 Phylloquinone Inhibits U937 Cell Growth and These Effects are Enhanced Over Time ………………………………………………………………………………………….…...79 5. CONCLUSION AND FUTURE DIRECTIONS ..………………...………………………………......81 5 REFERENCES ..………………………………………………………………………………………………......83 VITA ...……………………………………………………………………………………………………………….94 6 LIST OF TABLES Table Page 1. Experiment List ..………………………………………………………………………………..................34 2. Phylloquinone Treatment Solution ……………...…………………………………………………37 3. Occurrence of Apoptosis Following Treatment with 0, 100, or 500 μM Phylloquinone for 48 Hours …….…………………………………………………………………….47 7 LIST OF FIGURES Figure Page 1. Structure of Phylloquinone …………………………………………………………………...............27 2. Members of the Intrinsic and Extrinsic Apoptotic Pathways Affected by Phylloquinone in Liver and Pancreatic Cancers ……………………………………..............30 3. U937 cells at 20x magnification ……………………………………………………………………..36 4. Cell Density, Cell Area, and Percent Apoptosis Flowchart ………………………………..39 5. Annexin V and APO-BrdU Assay Flowchart …………………………………………………….43 6. U937 Cells Binding Annexin V Following Treatment with 0, 100, or 500 μM Phylloquinone for 48 Hours …………………………………………………………………………..47 7. Mean Cell Area Following Treatment with 0, 100, or 500 μM Phylloquinone for 48 Hours …………………………………………………………………………………………………………...49 8. Distribution of Cell Area Following Treatment with 0, 100, or 500 μM Phylloquinone for 48 Hours ...………………………………………………………………………...50 9. Mean Area of Annexin V Binding Cells Following Treatment with 0, 100, or 500 μM Phylloquinone for 48 Hours ..…………………………………………………………………...52 10. Distribution of Annexin V Binding Cell Area Following Treatment with 0, 100, or 500 μM Phylloquinone for 48 Hours ………………………………………………………………53 11. Mean Area of Annexin V Non-Binding Cells Following Treatment with 0, 100, or 500 μM Phylloquinone for 48 Hours ………………………………………………………………55 12. Distribution of Annexin V Non-Binding Cell Area Following Treatment with 0, 100, or 500 μM Phylloquinone for 48 Hours …………………………………………………..56 8 13. Mean Area of APO-BrdU Binding Cells Following Treatment with 0, 100, or 500 μM Phylloquinone for 48 Hours ……………………………………………………………………..58 14. Distribution of APO-BrdU Binding Cell Area Following Treatment with 0, 100, or 500 μM Phylloquinone for 48 Hours ………………………………………………………………59 15. Mean Area of APO-BrdU Non-Binding Cells Following Treatment with 0, 100, or 500 μM Phylloquinone for 48 Hours ………………………………………………………………61 16. Distribution of APO-BrdU Non-Binding Cell Area Following Treatment with 0, 100, or 500 μM Phylloquinone for 48 Hours ………………………………….……………….63 17. Distribution of Annexin V and APO-BrdU Binding Cell Area Following Treatment with 0, 100, or 500 μM Phylloquinone for 48 Hours ………………………………………..64 18. Distribution of Annexin V and APO-BrdU Non-Binding Cell Area Following Treatment with 0, 100, or 500 μM Phylloquinone for 48 Hours ………………………65 19. Distribution of Annexin V and APO-BrdU Binding and Non-Binding Cell Area Following Treatment with 0, 100, or 500 μM Phylloquinone for 48 Hours ……….67 20. Mean Cell Density Following Treatment with 0, 100, or 500 μM Phylloquinone for 48 Hours …………………………………………………………………………………………………69 21. Mean Cell Density Following Treatment with 0, 10, 50, 100, or 500 μM Phylloquinone for 7 days ……………………………………………………………………………….70 22. Abnormal Mean Cell Density Following Treatment with 0, 10, 50, 100, or 500 μM Phylloquinone for 7 days ...……………………………………………………………………….71 23. Mean Cell Density Following Treatment with 0, 100, or 500 μM Phylloquinone for 24, 48, 72, or 96 Hours ……………………………………………………………………………..73 9 24. Estimated Marginal Means of Cell Density Following Treatment with 0, 100, or 500 μM Phylloquinone Over Time (24, 48, 72, or 96 Hours) ……………………………74 25. Estimated Marginal Means of Cell Density By Phylloquinone Treatment Concentration (0, 100, or 500 μM) for 24, 48, 72, or 96 Hours ………………………...75 26. Mean Cell Density Prior to Treatment with 0, 100, or 500 μM Phylloquinone for 48 Hours ………………………………………………………………………………………………………76 10 CHAPTER 1 INTRODUCTION The National Institute of Health
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