Arsenic Induced Pseudohypoxia in Malignant Transformation: the Role of HIF-1A Mediated Metabolism Disturbance Item Type text; Electronic Dissertation Authors Zhao, Fei Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 01/10/2021 14:43:22 Link to Item http://hdl.handle.net/10150/321003 1 ARSENIC INDUCED PSEUDOHYPOXIA IN MALIGNANT TRANSFORMATION: THE ROLE OF HIF-1A MEDIATED METABOLISM DISTURBANCE by FEI ZHAO _____________________ A Dissertation Submitted to the Faculty of the DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 2014 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Fei Zhao entitled: ARSENIC INDUCED PSEUDOHYPOXIA IN MALIGNANT TRANSFORMATION: THE ROLE OF HIF-1A MEDIATED METABOLISM DISTURBANCE and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy _______________________________________________________________________ Date: 04/23/2014 Walter T. Klimecki, DVM, Ph.D. _______________________________________________________________________ Date: 04/23/2014 R. Clark Lantz, Ph.D. _______________________________________________________________________ Date: 04/23/2014 Richard R. Vaillancourt, Ph.D. _______________________________________________________________________ Date: 04/23/2014 Georg T. Wondrak, Ph.D. Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. ________________________________________________ Date: 04/23/2014 Dissertation Director: Walter T. Klimecki, DVM, Ph.D. 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of the requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that an accurate acknowledgement of the source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the author. SIGNED: Fei Zhao 4 ACKNOWLEDGEMENTS I would like to thank my mentor, Dr. Walter Klimecki for his wisdom, patience, support, expertise, advice, and encouragement that he has given me during my time in his laboratory. Thank you for always pushing me toward excellence and for training me into the researcher I am today. I will be forever grateful for having such a wonderful mentor that took the time to mentor me. I would like to also thank my committee members, Dr. Clark Lantz, Dr. Richard Vaillancourt, and Dr. Georg Wondrak, for their support, advice, and expertise. I appreciate all the time you took to evaluate my work and give helpful feedback and criticism that made me take the time to think critically in order to better understand and interpret my work. I would like to thank members of the Klimecki laboratory. Especially, I would like to acknowledge Dr. Paulina Gomez-Rubio, Dr. Alicia Bolt, Sandeep Anne, Samantha Pacheco, Scott Malm, and Alyssa Hinchman for supporting my research in a number of ways. Thank you for being someone I could rely on for resources and scientific discussions. I also want to thank other past and present members of the lab for all of their support, friendship and for working with me on various aspects of my project throughout the years. For critical edits of this dissertation, I would like to thank Dr. Klimekci, Alyssa, Scott, and Jessica Sapiro. I would like to acknowledge Dr. Xiao-jun Wang for intensively training me in all type of molecular techniques during my undergraduate study, which made me very productive during my doctoral studies. I would like to thank the students and faculties in the Department of Pharmacology and Toxicology. Thank you for your support, advice, expertise and friendship. I have made many wonderful friends here, and I hope that our paths cross again in the future. Finally I would like to thank my family for all of their support and encouragement. Thank you to my Mom for always believing in me and being there for me during the challenging times and praying for me every day. I also want to thank my better half Dr. Tongde Wu for always supporting me and inspiring me with brilliant intellectual input. Thank you to all my friends who have been there for me, were my biggest supporters, and were good listeners. I will be forever grateful for all the people who have made a difference and have been there with me through this process. 5 DEDICATION I would like to dedicate my dissertation work to my parents, Dr. Zhifan Zhao and Xiuchan Hu, to our family dog Lucky, who companioned my mom in Beijing for the years while I was studying in the States. Last, but not least, this is a dedication to my better half, Dr. Tongde Wu who believes in my potential and is my best partner in life and career. I know that this dedication may never repay for the million things you have done for me, but I thank you with all my heart. 6 TABLE OF CONTENTS LIST OF FIGURES...……………………………………………………………………10 LIST OF TABLES……………………………………………………………………….17 LIST OF ABBREVIATIONS…………………………………………………………....18 ABSTRACT……………………………………………………………………………...22 CHAPTER 1 INTRODUCTION, RESEARCH QUESTION AND SPECIFIC AIMS....24 1.1 ARSENIC.………………………………………………………………………...24 1.1.1 Historical significance...…………..………………………..…....24 1.1.2 Sources of human exposure...…………………………………....26 1.1.3 Biotransformation..…………………………………………..…..28 1.1.4 Arsenic induced Diseases ...……………………………………..29 1.1.5 Mechanism of action ...…………………………………………..31 1.1.6 Models to study arsenic-induced malignant transformation …….35 1.2 ENERGY METABOLISM………………………………………………………...37 1.2.1 Glycolysis, TCA cycle, and Oxidative Phosphorylation...……....37 1.2.2 The Warburg Effect and its implications in cancer...…………....42 7 STABLE OF CONTENTS – Continued 1.2.3 Chemicals affecting energy metabolism..………………………..45 1.3 THE HYPOXIA INDUCIBLE FACTOR 1-ALPHA………………………………...48 1.3.1 Tight regulation of HIF-1A....…………………………………....49 1.3.2 Functional significance.....…………………………………….....52 1.3.3 Chemicals affecting the HIF-1A stability.…………………….....54 1.4 CENTRAL RESERCH QUESTION AND SPECIFIC AIMS ...……………………...56 1.5 FIGURES.…...……………………...……………………...……………………...60 1.6 TABLES...…...……………………...……………………...……………………...67 CHAPTER 2: ARSENIC EXPOSURE INDUCES THE WARBURG EFFECT IN CULTURED HUMAN CELLS..…..……………………………………………..……...68 2.1 ABSTRACT..…………………………………………………………………….68 2.2 INTRODUCTION....………………………………………………………….…69 2.3 MATERIALS AND METHODS..…………………………………………….....71 2.4 RESULTS....……………………………………………………………………..76 2.5 DISCUSSION....…………………………………………………………………80 2.6 FIGURES….……………………………………………………………………..85 8 TABLE OF CONTENTS – Continued 2.7 TABLES…..……………………………………………………………………..95 CHAPTER 3: ARSENIC-INDUCED PSEUDOHYPOXIA IS ASSOCIATED WITH MALIGNANT TRANSFORMATION IN BEAS-2B.…………………………………100 3.1 ABSTRACT…………………………………………………………………….100 3.2 INTRODUCTION..………………………………………………………….…100 3.3 MATERIALS AND METHODS.……………………………………………....103 3.4 RESULTS..……………………………………………………………………..111 3.5 DISCUSSION......………………………………………………………………114 3.6 FIGURES….……………………………………………………………………121 CHAPTER 4: CULTURECONDITIONS DRAMATICALLY ALTER BEAS-2B PHENOTYPE.....……………………..…………………………………………..…….137 4.1 ABSTRACT.……………………………………………………………………137 4.2 INTRODUCTION..………………………………………………………….…138 4.3 MATERIALS AND METHODS..…………………………………………….. 139 4.4 RESULTS....……………………………………………………………………142 4.5 DISCUSSION.….………………………………………………………………145 9 TABLE OF CONTENTS – Continued 4.6 FIGURES….……………………………………………………………………148 4.7 TABLES…..……………………………………………………………………155 CHAPTER 5: CONCLUSIONS AND FUTURE DIRECTIONS…..…………………160 5.1 CONCLUSIONS..……………………………………………………………...160 5.2 FUTURE DIRECTIONS .……………………………………………………...167 APPENDIX A: AUTOPHAGY REQUIREMENT IN AN ARSENIC-TRANSFORMED BLADDER CANCER MODEL…………….....…………...…………...…………...…172 A.1 ABSTRACT……………………………………………………………………172 A.2 INTRODUCTION.....………………………………………………………….173 A.3 MATERIALS AND METHODS.……………………………………………...175 A.4 RESULTS………..…………………………………………………………….179 A.5 DISCUSSION.…………………………………………………………………183 A.6 FIGURES………………………………………………………………………186 A.7 TABLES.....……………………………………………………………………196 REFERENCES……………………………………………..…………………………..197 10 LIST OF FIGURES CHAPTER 1 INTRODUCTION Figure 1.5.1 U.S. geological distribution of arsenic in groundwater .………………...60 Figure 1.5.2 Arsenic biotransformation in humans……………………………………61 Figure 1.5.3 The glycolysis pathway …...……………………………………………62 Figure 1.5.4 The citric acid cycle ……………………..………………………………63 Figure 1.5.5 Oxidative phosphorylation ……..………………………………………64 Figure 1.5.6 HIF-1A protein domains ………………………………………………65 Figure 1.5.7 HIF-1A regulation ……………………………………………………66 CHAPTER 2 ARSENIC EXPOSURE INDUCES THE WARBURG EFFECT IN CULTURED HUMAN CELLS Figure 2.6.1 Sodium arsenite exposure increases BEAS-2B intracellular