Evaluation of bioreductively-activated Tirapazamine (TPZ) prodrugs for the management of hypoxic solid tumors by Sindhuja Pattabhi Raman A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Cancer Sciences Department of Oncology University of Alberta © Sindhuja Pattabhi Raman, 2019 Abstract Solid tumors often have large areas with low levels of oxygen (termed hypoxic regions), which are associated with poor prognosis and treatment response. Tirapazamine (TPZ), a hypoxia targeting anticancer drug, started as a promising candidate to deal with this issue. However, it was withdrawn from the clinic due to severe neurotoxic side effects and poor target delivery. Hypoxic cells overexpress glucose transporters (GLUT) - a key feature during hypoxic tumor progression. Our project aims at conjugating TPZ with glucose to exploit the upregulated GLUTs for its delivery, and thereby facilitate the therapeutic management of hypoxic tumors. We hypothesized that glucose-conjugated TPZ (G6-TPZ) would be selectively recruited to these receptors, facilitating its entrapment in poorly oxygenated cells only, with minimal damage to their oxygenated counterparts. However, our results reveal that the addition of the glucose moiety to TPZ was counterproductive since G6-TPZ displayed selective hypoxic cytotoxicity only at very high concentrations of the compound. We speculate that the reduced cytotoxicity of G6-TPZ might be due to the fact that the compound was not taken up by the cells. In order to monitor the cellular uptake of TPZ, we developed a click chemistry-based approach by incorporating an azido (N3) group to our parent compound (N3-TPZ). We observed that the azido-conjugated TPZ was highly hypoxia selective and the compound successfully tracks cellular hypoxia. Using a similar methodology, we went on to isolate and identify the proteins that are modified by N3-TPZ under hypoxia in order to obtain a better understanding of the potential underlying molecular mechanism of TPZ cytotoxicity. In addition, we exploited the property of monitoring TPZ uptake by N3-TPZ to study the uptake and localization of G6-TPZ by synthesizing a new derivative of TPZ, which incorporated both glucose and azido moieties. Overall, we carried out the above specified structural modifications on TPZ as an attempt to overcome the limitations of TPZ therapy. ii Acknowledgements I owe sincere gratitude to my co-supervisors, Dr. Michael Weinfeld and Dr. Piyush Kumar for their constant support and motivation without which this dissertation would not be possible. I wish to thank them for providing me the opportunity to work in their team and providing mentorship, motivation, invaluable guidance and encouragement throughout the project, which set the direction for my research. I would like to sincerely thank Dr. Lynne Postovit for kindly letting me work in her laboratory for a year and supervising me in my earlier days. I am indebted to her for all her constructive suggestions and advice in various meetings throughout the research. I also would like to extend my thanks to her lab members who were kind and friendly towards me. My sincere thanks to Dr. Richard Fahlman for letting us perform mass spectrometry in his laboratory and am deeply grateful for his kind assistance and valuable suggestions with the data analysis. I also would like to extend my gratitude to Jack Moore who helped us with the proteomics. My special thanks to Dr. Hassan Elsaidi for designing and synthesizing all the drugs that have been used in this project. Without his effort, it would have been impossible to carry out this work smoothly. I also owe a lot to Faisal Bin Rashed, who is a very good friend and has been a great constant support throughout the entire project. His friendly talks, continual encouragement, valuable discussions and timely help made this journey considerably easier. Most importantly, I would like to thank all the CRIO group members for their warmth of kindness and interesting discussions in our monthly meetings which have been very beneficial. I am delighted and indebted to Dr. Dawn McDonald and Xiao-Hong Yang who constantly helped iii and guided me with their technical expertise. I heartily thank Mesfin Fanta and Xiaoyan Yang who were always welcoming, amicable and willing to help. Furthermore, I am immensely pleased to thank my best friends in the lab Zahra Shire, Diana Diaz, Bingcheng Jiang and Wisdom Kate who made my research experience cheerful and uplifting by providing a great positive working environment. In addition, I would like to thank Dr. Feridoun Karimi-Busheri and Dr. Aghdass Rasouli-Nia for their genuine support and advice. I also extend my thanks to the imaging facility, especially Geraldine Barron. Lastly, I take great pleasure to acknowledge my parents and my husband for always being there patiently by my side throughout this endeavour and for their immense love, moral support and persistent prayers which made this journey worthwhile. 1. iv 2. Table of Contents 1. Chapter I - Introduction ......................................................................................................... 1 1.1. Tumor Hypoxia ................................................................................................................ 2 1.1.1. Different types of tumor hypoxia ............................................................................... 2 1.1.2. Head and Neck Cancer............................................................................................... 5 1.1.3. Tumor hypoxia as a therapeutic barrier in HNC ........................................................ 6 1.1.3.1. Hypoxia induced radioresistance…………………………………………………6 1.1.3.2. Hypoxia induced chemoresistance………………………………………………..7 1.2. Cellular response to hypoxia at the molecular level ........................................................ 8 1.2.1. Hypoxia Inducible Factor dependent and independent mechanism……………….9 1.2.1.1. HIF-dependent mechanism…………………………………………………….....9 1.2.1.2. HIF-independent mechanism……………………………………………………11 1.3. Hypoxia responsive gene: Glucose transporter (GLUT) ............................................... 13 1.3.1. Regulation of glucose transport by hypoxia ............................................................ 17 1.4. Evaluation of hypoxia .................................................................................................... 18 1.4.1. Invasive and Non-invasive methods ........................................................................ 18 1.4.1.1. Invasive methods to assess tumor hypoxia………………………………………18 1.4.1.2. Non-invasive methods to assess tumor hypoxia………………………………....19 1.5. Radiosensitization of hypoxic cells ............................................................................... 21 1.5.1. Increase in oxygen delivery ..................................................................................... 21 1.5.2. Hypoxic cell radiosensitizers ................................................................................... 22 v 1.6. Hypoxic-cell cytotoxins ................................................................................................. 24 1.6.1. Different classes of bioreductive drugs.................................................................... 24 1.6.2. Drug of Interest: Tirapazamine (TPZ) ................................................................ ….28 1.6.2.1. Molecular mechanism of preferential toxicity………………………………….. 28 1.6.2.2. Early studies of TPZ……………………………………………………………..31 1.6.2.3. TPZ clinical trials and the setbacks……………………………………………..32 1.6.2.4. Recent advancements in TPZ studies…………………………………………..34 1.7. Research objectives ........................................................................................................ 36 2. Chapter II - Materials and Methods ..................................................................................... 40 2.1. Cell Culture .................................................................................................................... 41 2.2. Hypoxic condition .......................................................................................................... 41 2.3. Drug and/or chemical compounds ................................................................................. 41 2.4. Cytotoxicity assays ........................................................................................................ 42 2.4.1. MTT assay ............................................................................................................... 42 2.4.2. Crystal Violet Staining (CVS) assay........................................................................ 42 2.4.3. Colony formation assay ........................................................................................... 43 2.5. Western blot analysis ..................................................................................................... 43 2.6. Real-time reverse transcription polymerase chain reaction (Real-Time RT-PCR) ....... 44 2.7. Click Chemistry ............................................................................................................. 45 2.8. Affinity Purification assay ............................................................................................. 46 2.9. Statistical analysis…………………………………………………………………….....47 vi 3. Chapter III - Results ............................................................................................................. 48 3.1. Determination of optimum