A ROLE FOR THE HYDROXYBENZOIC ACID METABOLITES OF FLAVONOIDS AND ASPIRIN IN THE PREVENTION OF COLORECTAL CANCER BY RANJINI SANKARANARAYANAN A dissertation submitted in partial fulfillment of the requirements for the Doctor of Philosophy Major in Pharmaceutical Sciences South Dakota State University 2021 ii DISSERTATION ACCEPTANCE PAGE Ranjini Sankaranarayanan This dissertation is approved as a creditable and independent investigation by a candidate for the Doctor of Philosophy degree and is acceptable for meeting the dissertation requirements for this degree. Acceptance of this does not imply that the conclusions reached by the candidate are necessarily the conclusions of the major department. Gunaje Jayarama, A00137123 Advisor Date Omathanu Perumal Department Head Date Nicole Lounsbery, PhD Director, Graduate School Date iii ACKNOWLEDGEMENTS The completion of this work would not have been possible without the help and contribution of various people who have in their own kind way supported its accomplishment. I would like to express my heart-felt gratitude to my mentor Dr. Jayarama Bhat Gunaje for having skillfully guided me through this project and for having generously extended his support throughout. Dr. Jay has taught me invaluable lessons about honesty, integrity, and listening to your data when it comes to scientific research. He has always been a pillar of support and has also been kind in sharing his many experiences to help me both professionally and personally. I thank him for this. These four years that I spent in the lab with him will be time I will fondly remember and cherish forever. I would like to thank Dr. Joy Scaria (Department of Veterinary & Biomedical Sciences, SDSU) and Dr. Hemachand Tummala for their valuable advice and for graciously allowing me to use their laboratory facilities and resources for the completion of this work. I would like to thank my committee members Dr. Xingyou Gu and Dr. Suvobrata Chakravarty for their critical inputs and valuable suggestions during the course of this investigation. I am also extremely thankful to Dr. Theresa Seefeldt, Dr. D. Ramesh Kumar (University of Kentucky), and Dr. Severine Van slambrouck (Department of Chemistry and Biochemistry, SDSU) for all their encouragement, timely advice and help for the progression of my project. Each of them in their own kind way have been instrumental to for the completion of this work. This is especially true of Dr. Ramesh Kumar, who helped me out immensely with the bioinformatics section of the project and patiently guided me iv to better understand this field. I would also like to take this opportunity to thank Dr. Puttur Prasad (Medical College of Georgia at Augusta University) for providing us with the transporter-expressing cancer cell line. A large part of my thesis would have been incomplete without the use of this critical cell line. I am extremely grateful to him for his generosity. I am also grateful to Fraunhofer, Center for Molecular Biotechnology, USA, for providing me with an internship opportunity. My experience there helped me understand the nuances of drug delivery and allowed me to apply the theoretical knowledge I gained in my Ph.D. coursework into practice. It was an enriching experience and I thank them for this. I would love to thank my colleagues, seniors and friends for providing an amazing environment to work in, these last 4 years. I would especially like to thank Dr. Rakesh Dachineni, Maria and Linto Antony and their family, Shruti S. Menon, Prabhjot Kaur Sekhon, Ekavali Sharma, Mawuli MacDonald, Chaitanya Valiveti, Dr. Mibin Joseph, Dr. Somshuvra Bhattacharya, Emily Trias and Tana Lick, for their unwavering support and belief in me. My stay in Brookings was made pleasant because of these wonderful individuals who helped me make a home away from home. I would sincerely like to thank Dr. Omathanu Perumal (the Head), Dr. Xiangming Guan (Graduate Research Coordinator), and all the faculty and staff of the Pharmaceutical Sciences Department for their constant support and encouragement during the course of my stay at SDSU. I would also like to thank them for offering me the opportunity to be the laboratory manager for the last three years. It taught me important lessons in people management and leadership. v I would also like to take this opportunity to thank the department of Pharmaceutical Sciences for their financial support and for providing me with all the facilities and resources required for the successful completion of this project. Last but not the least, I would like to thank my family – my grandmother, parents, sister and brother-in-law without whose support and encouragement this journey would not have been complete. I extend my heartfelt gratitude to them all. _________________________________ Ranjini Sankaranarayanan vi CONTENTS ABBREVIATIONS ......................................................................................................... xiii LIST OF FIGURES ........................................................................................................ xvii LIST OF TABLES ............................................................................................................ xx ABSTRACT ..................................................................................................................... xxi CHAPTER 1: INTRODUCTION ....................................................................................... 1 1.1 Colorectal cancer (CRC) ........................................................................................... 3 1.1.1 Tumor suppressor genes and protooncogenes as drivers of CRC development . 4 1.1.1.1 APC and development of CRC .............................................................. 5 1.1.1.2 Tumor protein P53 (P53) and development of CRC: .................................. 5 1.1.1.3 SMAD4 and development of CRC .............................................................. 6 1.1.1.4 RAS and BRAF and development of CRC ................................................... 7 1.1.1.5 PI3K signaling and development of CRC .................................................... 7 1.2 Cell cycle regulation and cancer development .......................................................... 8 1.2.1 Dysregulation of the cell cycle in cancer: ........................................................ 11 1.2.2 CDKs ................................................................................................................ 12 1.2.3 Cyclins .............................................................................................................. 12 1.2.4 CDK activating enzymes .................................................................................. 13 1.2.5 CKIs .................................................................................................................. 13 1.2.6 CDK Substrates ................................................................................................ 14 vii 1.2.7 Checkpoint proteins .......................................................................................... 14 1.3 CDK inhibitors for cancer treatment ....................................................................... 14 1.3.1 Targeted inhibition of CDK4 and CDK6 ......................................................... 15 1.4 Cancer chemoprevention ......................................................................................... 17 1.4.1 Drugs as agents of cancer prevention ............................................................... 17 1.4.2 Phytochemicals as agents of cancer prevention ............................................... 19 1.5 Flavonoids in cancer prevention ............................................................................. 20 1.5.1 Flavonoid effects on signal transduction .......................................................... 21 1.5.2 Antioxidant effects of flavonoids ..................................................................... 23 1.6 Aspirin as a chemopreventive agent........................................................................ 23 1.6.1 Pharmacological effects of Aspirin .................................................................. 24 1.6.2 Mechanisms proposed for aspirin’s chemo-preventive effects. ....................... 25 1.6.2.1 Inhibition of COX enzymes: the platelet hypothesis ................................. 25 1.6.2.2 Inhibition of Nuclear Factor (NF)-κB signaling ........................................ 29 1.6.2.3 Activation of AMP-kinase and inhibition of mTOR signaling .................. 29 1.6.2.4 Inhibition of Wnt signaling and β-catenin phosphorylation ...................... 30 1.6.2.5 Downregulation of c-myc, cyclin A2 and CDK2 ...................................... 30 1.6.2.6 Induction of polyamine catabolism ............................................................ 31 1.6.2.7 Induction of DNA mismatch repair proteins ............................................. 31 viii 1.6.2.8 Acetylation of P53, glucose-6-phosphate dehydrogenase and other proteins ................................................................................................................................ 32 1.6.2.9 Other mechanisms ...................................................................................... 33 1.7 Metabolism of flavonoids ....................................................................................... 34 1.8 Metabolism of aspirin.............................................................................................. 37 1.9 Biotransformation