Internalization of Extracellular ATP in Cancer Cells and Development of New Generations of Anticancer Glucose Transport Inhibitors A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Yanrong Qian December 2014 © 2014 Yanrong Qian. All Rights Reserved. 2 This dissertation titled Internaliztion of Extracellular ATP in Cancer Cells and Development of New Generations of Anticancer Glucose Transport Inhibitors by YANRONG QIAN has been approved for the Department of Chemistry and Biochemistry and the College of Arts and Sciences by Xiaozhuo Chen Associate Professor of Biomedical Sciences Robert Frank Dean, College of Arts and Sciences 3 ABSTRACT QIAN, YANRONG, Ph.D., December 2014, Molecular and Cellular Biology Internalization of Extracellular ATP in Cancer Cells and Development of New Generations of Anticancer Glucose Transport Inhibitors Director of Dissertation: Xiaozhuo Chen The past decade has witnessed remarkable progress toward understanding the reprogrammed metabolism in cancer, an emerging hallmark as well as an active area of basic, translational, and clinical research. About ninety years ago, Otto Warburg pioneered quantitative investigations of cancer metabolism and discovered that cancer cells exhibit a phenotype of increased glycolysis even under aerobic conditions, known as the Warburg effect. Warburg speculated that the reason for the upregulated glycolysis was for compensating the ATP shortage due to dysfunctions of mitochondria in cancer cells. Despite subsequent progresses, the biological reasons for ATP synthesis by aerobic glycolysis in cancer cells are only partially understood. Intriguingly, intratumoral (extracellular) ATP levels are 103 to 104 times higher than those in normal tissues. We showed that although extracellular ATP is not known to cross the plasma membrane by itself, extracellular ATP in the range of the intratumoral ATP levels induced large intracellular ATP concentration increase in A549 human lung cancer cells and promoted cancer cell survival. More importantly, we reported that a nonhydrolyzable fluorescent ATP was internalized by A549 cells through macropinocytosis as visualized by fluorescence microscopy. The induced ATP increase was reduced by the macropinocytosis inhibitor EIPA but persisted even when mitochondrial oxidative 4 phosphorylation and glycolysis were inhibited, without involving transcription or translation. The increases were also observed in several other cancer cell lines, but not in noncancerous cells. Furthermore, extracellular ATP enhanced cancer cell survival under various stress conditions and promoted drug resistance to tyrosine kinase inhibitors that compete with ATP for their anticancer action. Collectively, these results provide the first piece of evidence that extracellular ATP is internalized by cancer cells via macropinocytosis and potentially other endocytic process, which significantly contribute to their growth and to drug resistance. These findings potentially change our understanding of ATP supply and sharing among cancer cells, enhance our understanding of the Warburg effect, and highlight a novel anticancer target. Current understanding of glucose transport and metabolism in cancer, anticancer therapeutics targeting glycolysis and glucose transporters, and daunting challenges are also summarized. Based on the previous discovery on WZB-117, the first generation of glucose transport inhibitor, we screened a library of its derivatives and identified WZB-173 and DRB-18 with higher stabilities and potencies as lead compounds in new generations of novel glucose transport inhibitors. Both compounds can serve as models for further development of glucose transport inhibitors. 5 ACKNOWLEDGMENTS I would like to express my great appreciation to Dr. Xiaozhuo Chen, my advisor, for his inspirational guidance, valuable teaching, strong support, and extraordinary patience during my PhD study in Ohio University. It would be impossible for me to finish this work without his help. I would also like to express my sincere gratitude to Dr. Shiyong Wu, Dr. Stephen Bergmeier, and Dr. Fabian Benencia for being on my committee and giving me valuable suggestions on my research. Moreover, I would like to cordially thank Dr. Robert Colvin, Dr. Yunsheng Li for their critical suggestions and help in my research. I would like to thank Dr. Yi Liu for her help and teaching me basic techniques in the lab. I would like to express my earnest appreciation to Xuan Wang, Yanyang Cao, Lingyu Zhang, Lingying Tong for their assistance in my research. In addition, I would like to thank Dr. Weihe Zhang and Dennis Roberts for the design and synthesis of novel glucose transporter inhibitors studied in my work. I would like to thank Dr. Jeff Wiseman and Dr. Athena Chen for their critical review and suggestions on the manuscript for ATP internalization study in Chapter 2. Also, I would like to thank Dr. John Kopchick and Dr. Elahu Gosney Sustarsic for their support and help on realtime PCR in the study of glucose transport inhibitor in Chapter 4. Furthermore, I would like to express my appreciation of the assistance I received from MCB program, Edison Biotechnology Institution, Department of Chemistry and Biochemistry. Also, I would like to express my great appreciation to the financial and temporal support in my research granted by 2013-2014 Donald Clippinger Graduate 6 Fellowship, 2013 Student Enhancement Award, 2013 Graduate Student Senate Original Work Grant from Ohio University, and 2013 American Association for Cancer Research- Woman In Cancer Research Scholar Award. Also, I would like to acknowledge Elsevier and Baishideng Publishing Group for permission to include the contents from my publications published in the journals of Free Radical Biology & Medicine, Cancer Letters, and World Journal of Translational Medicine in my dissertation. In addition, I am grateful to Edwin Frebault, Marlene Jenkins, Zongqian Yuan, Dr. Komal Garg, and all of my friends for their encouragement and support during my PhD study in Ohio University. Last but not least, I would like to express my sincere appreciation to my parents, Xiang’an Qian, Liqin Hu, and all my family members for their endless care, love, support, and encouragement during my PhD study in Ohio University. 7 TABLE OF CONTENTS Page Abstract ............................................................................................................................... 3 Acknowledgments ............................................................................................................... 5 List of Tables .................................................................................................................... 10 List of Figures ................................................................................................................... 11 Abbreviations .................................................................................................................... 14 Chapter 1 Introduction ...................................................................................................... 19 1.1 Cancer metabolism and the Warburg effect ............................................................ 19 1.2 Original hypothesis and current findings of the Warburg effect ............................ 20 1.3 Brief history and current interpretations of the Warburg effect ............................. 21 1.4 The Warburg effect in normal cells and cancer cells .............................................. 23 1.5 ROS balancing and the Warburg effect .................................................................. 24 1.6 Gene regulation and the Warburg effect ................................................................. 26 1.7 Oxygen supply, ATP synthesis and the Warburg effect ......................................... 30 1.8 Warburg effect at the tumor microenvironment scale ............................................ 33 1.9 The Warburg effect as a potential target for cancer treatment ................................ 36 1.10 Conclusion ............................................................................................................ 43 Chapter 2: Internalization Of Extracellular ATP In Cancer Cells .................................... 45 2.1 Introduction ............................................................................................................. 45 2.2. Materials and methods ........................................................................................... 47 2.2.1 Compounds and cell lines ................................................................................ 47 2.2.2 ATP rescue study and inATP measurement .................................................... 48 2.2.3 Protein analysis, clonogenic and flow cytometry assays of cancer cells ......... 48 2.2.4 Dose- and time-dependence studies of ATP .................................................... 49 2.2.5 Fluorescence microscopy and ATP localization studies .................................. 49 2.2.6 Oligomycin and glucose deprivation assays – time course study .................... 50 2.2.7 Studies with inhibitors of AMPK, transcription, translation ........................... 50 2.2.8 Macropinocytosis inhibitor study .................................................................... 50 2.2.9 Drug resistance study ......................................................................................