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A Dissertation Entitled Novel Microtubule-Disrupting Indole A Dissertation entitled Novel Microtubule-Disrupting Indole-Based Chalcones That Induce Cell Death in Glioblastoma by Shengnan Du Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy in Medical Science: Cancer Biology ________________________________________ Dr. William A Maltese, Committee Chair ________________________________________ Dr. Randall Ruch, Committee Member ________________________________________ Dr. Kathryn Eisenmann, Committee Member ________________________________________ Dr Jeffrey G Sarver, Committee Member ________________________________________ Dr. Saori Furuta, Committee Member ________________________________________ Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo December 2017 Copyright 2017, Shengnan Du This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Novel Microtubule-Disrupting Indole-Based Chalcones That Induce Cell Death in Glioblastoma by Shengnan Du Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in in Medical Science: Cancer Biology The University of Toledo December 2017 The extremely poor prognosis of Glioblastoma multiforme highlights the urgent need for more efficient drugs to treat this disease. We have developed a series of small molecules that induce a novel non-apoptotic cell death mechanism, methuosis, in cancer cells. During the structure-activity relationship study of methuosis-inducing compounds, we identified a group of unique chalcone derivatives with more potent anti-tumor activity. The lead compound is 3-(6-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene- 1-one (6-MOMIPP). 6-MOMIPP exhibits potent anti-proliferative and cytotoxic activity in glioblastoma, melanoma, and lung cancer cell lines at concentrations in the nanomolar range. However, cells treated with the new class of compounds did not morphologically resemble cells undergoing methuosis. By 24 h, 6-MOMIPP causes the majority of cells to round up from the culture dish and arrest in mitosis. By 48 h, cell viability is greatly reduced, correlating with activation of caspases 9, 7, and 3. Caspase inhibitors block the cell death induced by 6-MOMIPP, suggesting that cell death is caspase-dependent. Phase contrast images show that the few cells remaining alive after treatment with 6-MOMIPP iii are giant, flat, and multiple micronucleated. Whole cell tubulin polymerization assay shows that 6-MOMIPP significantly reduces the amount of polymerized tubulin in living cells. Immunofluorescence staining of microtubules reveals microtubule disruption in U251 cells after treatment with 6-MOMIPP at a concentration of 250 nM or higher. In vitro scintillation proximity assay and EBI cross-linking studies of β-tubulin in intact cells both reveal that 6-MOMIPP binds to tubulin on the colchicine binding site. Blocking Cdk1 activity with BMS-265246 greatly reduces cell death induced by 6-MOMIPP, but has no effect on the mitotic arrest, suggesting that blockage of metaphase-anaphase transition by prolonged Cdk1 activation is responsible for the 6-MOMIPP-induced death. Treatment of cells for 24 h with 6-MOMIPP induces phosphorylation of Bcl-2 and Bcl-xL, which could be eliminated by inhibiting Cdk1, suggesting that Cdk1 plays a key role in phosphorylation of the anti-apoptotic Bcl proteins. Cell death induced by 6-MOMIPP is also accompanied by activation of JNK and its downstream substrate c-Jun. Blocking JNK activity with SP600125 reduces the percentage of dead cells, indicating that JNK activation may contribute to the cell death. Cell viability studies have shown the toxicity of 6-MOMIPP is substantially reduced in quiescent human fibroblasts, HUVEC (human umbilical vein endothelial cells) and rat neuronal progenitor cells. Pharmacokinetic studies in mice show that 6-MOMIPP attains brain concentrations that are nearly equal to plasma concentrations at time points up to 8 h after intraperitoneal (i.p.) injection, suggesting that 6-MOMIPP readily crosses the blood-brain barrier. 6-MOMIPP administration at 20 mg/kg every 12 h for 12-15 days significantly decreases the growth of glioblastoma in subcutaneous and intracranial xenograft models without notable toxicities, suggesting that 6-MOMIPP has good anti-tumor activity in vivo. These preliminary findings suggest that 6-MOMIPP iv merits further preclinical evaluation as a potential therapeutic agent for primary and metastatic brain tumors. v Acknowledgements I am grateful to the many people that have enabled me to endure the journey of my Ph.D. education. This work wouldn’t have been completed without them. I would like to acknowledge my advisor, Dr. William Maltese, for his guidance throughout my Ph.D. training and the life lessons he has taught me, which will benefit me for years to come. He trained me to grow as a scientist and helped me to improve my skills in scientific presentations and writing. He is one of the most patient and brilliant mentors I have ever met. I’m very grateful to have received my training in his lab. I would also like to thank Dr. Overmeyer for teaching me most skills and techniques during the course of my training. She has always been there for her students, making my research project much more efficient. I also appreciate valuable insights of Dr. Ruch and Dr. Eisenmann into my projects; they have challenged my thinking in many ways. I thank Dr. Sarver for his professional training and help with the pharmacokinetic studies. Also, I thank Dr. Furuta for agreeing to be on my committee at the last minute, and her wonderful ideas for my project. I’d like to thank all my friends for supporting and encouraging me through the past years, especially during tough times. We not only supported each other by deliberating over our studies, but also happily talked about things other than just our research. I’m so blessed to have you all in my life. vi Many thanks my beloved parents who offer unconditional love and support, and have always been there for me. Their understanding and help have allowed me to be successful in graduate school. Thank you for letting me find my own way. I would not be where I am today without your support. You are the best parents! Finally, I thank my husband, Sage, for being by my side, he has supported and encouraged me throughout this process. I am truly grateful to have him in my life. Thank you everyone who has supported and inspired me! vii Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ............................................................................................................ vi Table of Contents ............................................................................................................. viii List of Tables ..................................................................................................................... xi List of Figures ................................................................................................................... xii 1 Literature Review.....................................................................................................1 1.1 Current Challenges in Treatments for Brain Cancer ..........................................1 1.1.1 Glioblastoma multiforme (GBM) ....................................................1 1.1.2 Treatment for Glioblastoma ............................................................2 1.1.3 Brain metastases and treatment ........................................................4 1.2 Microtubules as targets for cancer treatment .....................................................5 1.2.1 Why target Microtubules? ................................................................5 1.2.2 Microtubule and their complex dynamics ........................................5 1.2.3 Microtubule targeting agents ...........................................................8 1.2.4 Chalcones synthesis as novel microtubule targeting agents ..........13 1.3 MTA-induced cell death mechanism ..............................................................14 1.3.1 Spindle assembly checkpoint .........................................................14 1.3.2 Cyclin-dependent kinases ..............................................................16 viii 1.3.3 Mitotic catastrophe .........................................................................19 1.3.4 Apoptosis .......................................................................................23 1.3.5 Necrosis..........................................................................................26 1.3.6 Bcl-2 anti-apoptotic family ............................................................28 1.3.7 Involvement of JNK in cell cycle and cell death ...........................30 2 Evaluation of Novel Indole-Based Chalcones as Microtubule- Targeted Agents for Brain Cancer Therapy ................................................................................................32 2.1 Abstract….. ......................................................................................................33 2.2 Introduction….. ................................................................................................34 2.3 Materials and Methods….. ...............................................................................35
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