MECHANISMS OF ACTION OF NON-STEROIDAL ANTI-INFLAMMATORY DRUGS (NSAIDs) IN COLON CANCER A Dissertation by SATYA SREEHARI PATHI Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY August 2012 Major Subject: Toxicology MECHANISMS OF ACTION OF NON-STEROIDAL ANTI-INFLAMMATORY DRUGS (NSAIDs) IN COLON CANCER A Dissertation by SATYA SREEHARI PATHI Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Chair of Committee, Stephen H. Safe Committee Members, Robert C. Burghardt Timothy Phillips Yanan Tian Interdisciplinary Faculty Chair of Toxicology, Weston Porter August 2012 Major Subject: Toxicology iii ABSTRACT Mechanisms of Action of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) in Colon Cancer. (August 2012) Satya Sreehari Pathi, B.V.M., Acharya N. G. Ranga Agricultural University, India; M.S., Texas A&M University, Kingsville Chair of Advisory Committee: Dr. Stephen H. Safe Non-steroidal anti-inflammatory drugs (NSAIDs) and their NO derivatives (NO- NSAIDs), and synthetic analogs are highly effective as anticancer agents that exhibit relatively low toxicity compared to most clinically used drugs. However, the mechanisms of action for NSAIDs and NO-NSAIDs are not well defined and this has restricted their clinical applications and applications for combined therapies. Earlier studies from our laboratory reported that specificity protein (Sp) transcription factors (Sp1, Sp3 and Sp4) are overexpressed in several types of human cancers including colon cancer and many Sp-regulated genes are pro-oncogenic and individual targets for cancer chemotherapy. Based on published results showing that NSAIDs downregulate several putative Sp- regulated genes, we hypothesized that the anticancer properties of NSAIDs may be due, in part, to downregulation of Sp transcription factors. NSAIDs and nitro derivatives of NSAIDs have been investigated in colon cancer cells and in vivo xenograft models. Aspirin and TA induced apoptosis and decreased colon cancer cell growth and tumor growth in vivo and downregulated genes associated with cell growth, survival, and angiogenesis. Previous RNA interference studies in this laboratory have shown that many of these genes are regulated, in part, by Sp iv transcription factors Sp1, Sp3 and Sp4 that are overexpressed in colon and other cancer cell lines. Not surprisingly, these NSAIDs also decreased Sp1, Sp3 and Sp4 proteins and Sp-regulated gene products in colon cancer cells and this was due to caspase-dependent proteolysis of Sp1, Sp3 and Sp4. Aspirin-induced activation of caspases and degradation of Spproteins was due to sequestration of zinc and could be reversed by addition of zinc sulphate, whereas TA mediated induction of caspases was independent of zinc ions and is currently being investigated. GT-094 is a novel NO chimera-containing NSAID, which also inhibited colon cancer cell proliferation and induced apoptosis; these effects were accompanied by decreased mitochondrial membrane potential (MMP) and induction of reactive oxygen species (ROS), and were reversed after cotreatment with the antioxidant glutathione. GT-094 also downregulated Sp and Sp-dependent gene products and was due to decreased expression of microRNA-27a (miR-27a) and induction of ZBTB10, an Sp transcriptional repressor that is regulated by miR-27a in colon cancer cells. Moreover, the effects of GT-094 on Sp1, Sp3, Sp4, miR-27a and ZBTB10 were also inhibited by glutathione suggesting that the anticancer activity of GT-094 in colon cancer cells is due, in part, to ROS-dependent disruption of miR-27a:ZBTB10. The importance of ROS induction in targeting Sp transcription factors was also confirmed using pro-oxidants such as ascorbic acid, hydrogen peroxide and t-butyl hydroperoxide and similar results have been observed in collaborative studies with other ROS inducers in colon cancer cells. Many cancer cell lines and tumors exhibit addiction to non-oncogenes such as Sp1, Sp3 and Sp4 for maintaining the oncogenic phenotype and future research will focus on the mechanisms of ROS-mediated targeting of Sp transcription factors which represents a novel approach for cancer chemotherapy. v DEDICATION I dedicate my dissertation to my parents Pathi Satya Narayana Vara Prasad and Pathi Venkata Subbamma. They are my strength and without their support I would not stand here. My gratitude and my love to them are beyond words. vi ACKNOWLEDGEMENTS It‘s my pleasure to thank the many people who made this dissertation possible. Firstly, I would like to express my gratitude to Dr. Stephen Safe, the chair of my advisory committee, for giving me the precious opportunity to work with him and without him; I never would have made it through this challenge. I am very much thankful for his constant support, guidance, and encouragement, which allowed me to venture into new areas and grow as a researcher. He is very understanding, patient and a great person to work with and his precious suggestions will always be remembered. I would like to thank all my committee members, Dr. Robert Burghardt, Dr. Timothy Phillips and Dr. Yanan Tian, for their scholarly guidance and valuable suggestions during my doctoral program. I would like to thank my father and mother, Pathi Satya Narayana Varaprasad and Pathi Venkata Subbamma, for giving me courage and strength needed to achieve my goals. I am deeply grateful to my adorable wife, Mrs. Harika for her consistent support in our newly wedded life. She is awesome and with in short span of wedded life she made an impact in my life. I love her very much. My heartfelt thank to her for helping me in writing this dissertation. I also want to thank my brothers, Pathi Satya Srinivasulu and Pathi Satya Manohar, for their love and encouragement through all my endeavors. I must also, thank my sister-in-laws, Haritha and Vijaya and and my chirpy toddlers, Chinnu and Minnu. vii I would also like to mention the love and care from my mother-in-law Gundlapalli Padmavathi and my father-in-law Gundlapalli Venkateswra Prasad and brother-in-law, Gundlapalli Jagadeesh. I would like to extend my thanks to all Safe lab members Indira Jutooru and Gayathri Chadalapaka, Xi Li, Sandeep Sreevalsan, Vijaya lekshemi, Shane for their constant support and help during my research, and my special thanks to Sudhakar Chintarllapalli and Sabitha Papineni for helping me to join this lab. I also want to thank my dearest friends Subbu, Ram, Praveena and Radhika for their friendship. I also take this opportunity to thank Dr. Jagadeesh for being helpful and for his moral support. A special thanks to Lorna Safe, Kathy Mooney, and Kim Daniel. Finally I thank the almighty for providing me strength and courage to be successful in all my endeavors. viii TABLE OF CONTENTS Page ABSTRACT ........................................................................................................... iii DEDICATION ........................................................................................................ v ACKNOWLEDGEMENTS ...................................................................................... vi TABLE OF CONTENTS ......................................................................................... viii LIST OF FIGURES ................................................................................................ x LIST OF TABLES .................................................................................................. xiv CHAPTER I INTRODUCTION ............................................................................... 1 Cancer ......................................................................................... 1 Colon cancer ............................................................................... 14 Epigenetic changes in colon cancer ............................................. 29 Inflammation and colorectal cancer ............................................. 34 Diagnosis and staging of colorectal cancer .................................. 49 Treatment of colorectal cancer..................................................... 52 Mechanism based anticancer drugs ............................................ 56 Transcription factors as drug targets ............................................ 68 Specificity protein (Sp) transcription factors as drug targets ......... 74 NSAIDs as anti-cancer agents ..................................................... 85 NO-NSAIDs and cancer ............................................................... 100 Ascorbic acid and cancer ............................................................. 108 II ASPIRIN INHIBITS COLON CANCER CELL AND TUMOR GROWTH AND DOWNREGULATES SPECIFICITY PROTEIN (Sp) TRANSCRIPTION FACTORS ................................... 116 Introduction .................................................................................. 116 Materials and methods ................................................................ 118 Results ........................................................................................ 123 Discussion ................................................................................... 137 ix CHAPTER Page III TOLFENAMIC ACID (TA) INHIBITS COLON CANCER CELL AND TUMOR GROWTH AND DOWNREGULATES SPECIFICITY PROTEIN (Sp) TRANSCRIPTION FACTORS ............ 142 Introduction .................................................................................