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The Effects of ɤ-Glutamyl Hydrolase Modulation on Cancer Treatment and Dna Methylation in Human Cancer Cells

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The Effects of ɤ-Glutamyl Hydrolase Modulation on Cancer Treatment and Dna Methylation in Human Cancer Cells THE EFFECTS OF ɤ-GLUTAMYL HYDROLASE MODULATION ON CANCER TREATMENT AND DNA METHYLATION IN HUMAN CANCER CELLS by Sung-Eun Kim A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Nutritional Sciences University of Toronto © Copyright by Sung-Eun Kim 2013 THE EFFECTS OF ɤ-GLUTAMYL HYDROLASE MODULATION ON CANCER TREATMENT AND DNA METHYLATION IN HUMAN CANCER CELLS Sung-Eun Kim Doctor of Philosophy Graduate Department of Nutritional Sciences University of Toronto 2013 Abstract Folate and antifolates are retained in cells by polyglutamylation mediated by folylpolyglutamate synthase (FPGS), and are exported from cells after hydrolysis to monoglutamates by γ-glutamyl hydrolase (GGH). Polyglutamylated (anti)folates are retained in cells longer and are better substrates than their monoglutamate counterparts for intracellular folate-dependent enzymes. GGH modulation may therefore affect chemosensitivity of cancer cells to antifolates and 5- fluorouracil by altering polyglutamylation of antifolates and a specific target intracellular folate cofactor for 5-fluorouracil (5,10-methylenetetrahydrofolate), respectively. We generated an in vitro model of GGH modulation in HCT116 and MDA-MB-435 cells with predictable functional consequences and investigated chemosensitivity to 5-fluorouracil and antifolates. Overall, GGH overexpression decreased chemosensitivity to 5-fluorouracil+ leucovorin and methotrexate, while GGH inhibition increased chemosensitivity to 5-fluorouracil +leucovorin. However, these effects appeared to depend not only on the GGH modulation- induced changes in polyglutamylation of 5,10-methylenetetrahydrofolate and antifolates but also on intracellular folate levels as well as adaptive and compensatory changes in other enzymes ii involved in intracellular folate and antifolate accumulation and metabolism in response to GGH modulation. Polyglutamylation is also important in DNA methylation as polyglutamylated folates are better substrates for methylenetetrahydrofolate reductase and methionine synthase involved in the generation of S-adenosylmethionine, which is a substrate for DNA methylation. We hypothesized that GGH and FPGS modulation may affect DNA methylation at global and gene- specific levels with consequent functional ramifications. GGH and FPGS modulation demonstrated the inverse relationship between GGH activity and global DNA methylation as well as was associated with differential gene expression and altered CpG promoter DNA methylation involved in important biological pathways. Some of the observed altered gene expression appear to be regulated by DNA methylation. Whether or not GGH modulation may be an important determinant of chemosensitivity to 5-fluorouracil- and antifolate-based chemotherapy, and the potential roles of GGH and FPGS modulation in DNA methylation need further exploration. iii Acknowledgments First and foremost, I would like to express my sincere gratitude to my supervisor, Dr. Young-In Kim for the opportunity to complete a Ph.D. degree under his supervision. I truly appreciate his encouragement, guidance and invaluable assistance throughout the project. His knowledge and enthusiasm towards my research have been a true inspiration. I would also like to thank my committee members, Dr. Robert Bruce, Dr. Deborah O’Connor and Dr. Andrew Bognar for their expert knowledge and guidance throughout the course of my research. I would like to extend my gratitude to the collaborators - Dr. Toshinori Hinoue for the bioinformatic analysis of our microarray data, Dr. Daniel Weisenberger and Dr. Peter Laird for advice on the methylation analysis, and Ruth Croxford for the detailed explanations and guidance on the statistics behind the data. I am also grateful to all the past and present lab members for their support and for all the memories we have shared. I am thankful to my friends from the Department of Nutritional Sciences for their support and encouragement throughout my doctorate degree. My deepest thanks are reserved to people dearest and nearest to me. I would like to thank Dr. Mi-Kyung Sung for her continued moral support towards pursuing a graduate degree. I am grateful to all my friends in Toronto and Korea for their attention, encouragement, and support. Special thanks to my family for their love and inspiration throughout my studies. Lastly, to my parents, words cannot describe the gratitude you deserve. I will always appreciate your unwavering love, understanding, and support that have made me who I am today. I would also like to express my gratitude to my grandparents who were always by my side and wished my success. Again, my sincere and lasting gratitude to my father and mother, and it is to them that I dedicate this thesis. iv Table of Contents ACKNOWLEDGMENTS .......................................................................................................... IV TABLE OF CONTENTS .............................................................................................................V LIST OF ABBREVIATIONS .................................................................................................. VII LIST OF TABLES ........................................................................................................................X LIST OF FIGURES ................................................................................................................... XV LIST OF APPENDICES ......................................................................................................... XIX CHAPTER 1: INTRODUCTION .................................................................................................... 1 CHAPTER 2: LITERATURE REVIEW .......................................................................................... 5 2.1 Folate .............................................................................................................................. 6 2.1.1 Chemistry of Folate ................................................................................................. 6 2.1.2 Folate Metabolism and Biochemical Function ....................................................... 7 2.1.3 Intracellular Homeostasis of Folate ..................................................................... 16 2.1.4 Folate and Health ................................................................................................. 27 2.2 Folate and Cancer Risk ................................................................................................ 30 2.3 Folate and Cancer Treatment ....................................................................................... 31 2.3.1 Folate and Chemotherapeutic Agents ................................................................... 31 2.3.2 5-Fluorouracil ....................................................................................................... 34 2.3.3 Methotrexate ......................................................................................................... 37 2.3.4 Pemetrexed ............................................................................................................ 39 2.3.5 Trimetrexate .......................................................................................................... 42 2.3.6 Drug Resistance .................................................................................................... 44 2.4 Epigenetics and Cancer Treatment ............................................................................... 46 2.4.1 Epigenetics ............................................................................................................ 46 2.4.2 DNA Methylation and Cancer .............................................................................. 47 2.4.3 Inhibition of DNA Methylation as Chemotherapeutic Strategy ............................ 52 2.4.4 Epigenetic Silencing and Response to Chemotherapeutic Agents ........................ 53 2.4.5 Analysis of DNA methylation ................................................................................ 54 2.4.6 Folate and Epigenetics ......................................................................................... 56 CHAPTER 3: RATIONALE, HYPOTHESES, AND OBJECTIVES ..................................... 58 CHAPTER 4: STUDY 1 – THE DEVELOPMENT AND VALIDATION OF AN IN VITRO MODEL OF GGH MODULATION AND INVESTIGATION OF THE EFFECT OF GGH v MODULATION ON CHEMOSENSITIVITY OF HUMAN COLON AND BREAST CANCER CELLS TO CHEMOTHERAPEUTICS (PROOF-OF-PRINCIPLE) ...................... 63 4.1 Abstract ......................................................................................................................... 64 4.2 Introduction ................................................................................................................... 65 4.3 Materials and Methods ................................................................................................. 67 4.4 Results... ........................................................................................................................ 73 4.5 Discussion ..................................................................................................................... 87 CHAPTER 5: STUDY 2 – THE EFFECTS OF GGH MODULATION AND FOLATE ON CHEMOSENSITIVITY OF HUMAN COLON AND BREAST CANCER CELLS TO CHEMOTHERAPEUTICS IN IN VITRO AND IN VIVO MODELS ....................................... 93 5.1 Abstract ........................................................................................................................
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