NEW MECHANISM-BASED ANTICANCER DRUGS THAT ACT AS ORPHAN NUCLEAR RECEPTOR AGONISTS A Dissertation by SUDHAKAR REDDY CHINTHARLAPALLI 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 May 2006 Major Subject: Biochemistry NEW MECHANISM-BASED ANTICANCER DRUGS THAT ACT AS ORPHAN NUCLEAR RECEPTOR AGONISTS A Dissertation by SUDHAKAR REDDY CHINTHARLAPALLI 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 Safe Committee Members, David Peterson James Sacchettini Robert Burghardt Head of Department, Gregory Reinhart May 2006 Major Subject: Biochemistry iii ABSTRACT New Mechanism-Based Anticancer Drugs That Act as Orphan Nuclear Receptor Agonists. (May 2006) Sudhakar Reddy Chintharlapalli, B.V.M., College of Veterinary Science, Acharya N.G.Ranga Agricultural University, India; M.S., Texas A&M University-Kingsville Chair of Advisory Committee: Dr. Stephen Safe 1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes containing p- trifluoromethyl (DIM-C-pPhCF3), p-t-butyl (DIM-C-pPhtBu), and phenyl (DIM-C- pPhC6H5) substituents have been identified as a new class of peroxisome proliferator- activated receptor γ (PPARγ) agonists that exhibit antitumorigenic activity. In this study, the PPARγ-active compounds decreased HT-29, HCT-15, RKO, HCT116 and SW480 colon cancer cell survival and KU7 and 253JB-V33 bladder cancer cell survival. In HT- 29, HCT-15, SW480 and KU7 cells, the PPARγ agonists induced caveolin-1 expression and this induction was significantly downregulated after cotreatment with the PPARγ antagonist GW9662. Since overexpression of caveolin-1 is known to suppress cancer cell and tumor growth, the growth inhibitory effects of the DIM compounds in these cell lines are associated with PPARγ-dependent induction of caveolins. These PPARγ-active compounds did not induce caveolin-1 in HCT-116 cells. However, these compounds induced NSAID-activated gene-1 (NAG-1) and apoptosis in this cell line. This represents a novel receptor-independent pathway for C-DIM-induced growth inhibition iv and apoptosis in colon cancer cells. In SW480 colon cancer cells 2.5-7.5 μM C-DIMs induced caveolin-1 whereas high concentrations (10 μM) induced pro-apoptotic NAG-1 expression. In athymic nude mice bearing SW480 cell xenografts DIM-C-pPhC6H5 inhibited tumor growth and immunohistochemical staining of the tumors show induction of apoptosis and NAG-1 expression. Thus, the PPARγ-active compounds induce both receptor-dependent and-independent responses in SW480 cells which are separable over a narrow range of concentrations and this dual mechanism of action enhances their antiproliferative and anticancer activities. Similar results were obtained for another structural class of PPARγ agonists namely 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) and the corresponding methyl (CDDO-Me) and imidazole (CDDO-Im) esters. Structure-activity studies show that 1,1-bis(3'-indolyl)-1-(p- substitutedphenyl)methanes containing p-trifluoromethyl (DIM-C-pPhCF3), hydrogen (DIM-C-pPh) and p-methoxy (DIM-C-pPhOCH3) substituents activate Nur77 and induce apoptosis in pancreatic, prostate, and breast cancer cell lines. Nur77 agonists activate the nuclear receptor, and downstream responses include decreased cell survival, induction of cell death pathways including tumor necrosis factor related apoptosis-inducing ligand (TRAIL) and PARP cleavage. Nur77 agonists also inhibit tumor growth in vivo in athymic nude mice bearing Panc-28 cell xenografts. v DEDICATION This dissertation is dedicated to my parents C. ASWATHAPPA & C. LEELAVATHI Their constant love and care are the cornerstones for where I am and what I am. My gratitude and my love to them are beyond words. vi ACKNOWLEDGEMENTS No one walks alone on the journey of life. Just where do you start to thank those that joined you, walked beside you, and helped you along the way. It is my chair Dr. Stephen Safe that I owe the most overwhelming debt of gratitude for his guidance, assistance and suggestions that have been paramount for the success of the work. His mentorship made me perceive new horizons of wisdom. I would like to thank all my committee members, Dr. David Peterson, Dr. Robert Burghardt, Dr. James Sacchettini, and Dr. Sumana Datta, for their scholarly guidance and valuable suggestions. I would like to thank my father, Sri Chintharlapalli Aswathappa Reddy, for giving me courage and strength needed to achieve my goals. I also realize that the blessing of my mother Chintharlapalli Leelavathi made it possible for me to do my best. It is true that time, knowledge, skill and support are needed for a successful project. I am proud and lucky to have a person who contributed all these factors, my adorable wife, Dr. Sabitha. She is awesome and in these two years of wedded life she made an impact in my life. I love her very much. I also want to thank my only brother, Chintharlapalli Rama Krishna Reddy, for his love and encouragement through all my endeavors. I must also, thank my sister-in- law, Chintharlapalli Madhavi Reddy, and my chirpy toddler, Chintharlapalli Aryanth Reddy. My heartfelt gratitude goes to my cousins, Konda Lakshminarayana and his wife Sreevani, two little angels, Alekya and Eesha Konda. I would also like to mention the vii love and care from my mother-in-law Papineni Lakshmi, my father-in-law, Papineni Penchal Reddy, my sister-in-law Dr. Papineni Babita, co-brother, Dr. Konda Venkata SubbaReddy, brother-in-law, Papineni Kishore Reddy and co-sister, Dr. Papineni Sapna. Words are not adequate to express my thanks to Dr. Maribel from whom I learned the quintessence of science. I am very grateful to her. I feel honored to be molded into a successful researcher in the lab of Dr. Maribel. Thanks are also due to my friends, Leela Kotha and Sharon Ngwenya, for having fun in the lab and for always welcoming me with their cheery and jovial personalities. I should not forget the help of Indira Jutooru and Gayathri Chadalapaka in formatting my dissertation. I also want to thank my dearest friends, Archana and Srikanth. I would also like to thank all the present and past members of the Safe lab for their continuing support and cooperation. A special thanks to the staff, Lorna Safe, Kathy Mooney, Pat Swigert 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........................................................................................................... xi LIST OF TABLES ........................................................................................................... xv CHAPTER I INTRODUCTION.......................................................................................... 1 Nuclear Receptors.....................................................................................1 Orphan Nuclear Receptors ......................................................................30 II 1,1-BIS(3’-INDOLYL)-1-(p-SUBSTITUTEDPHENYL)METHANES INDUCE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR γ-MEDIATED GROWTH INHIBITION, TRANSACTIVATION AND DIFFERENTIATION MARKERS IN COLON CANCER CELLS........................................................................... 63 Introduction .............................................................................................63 Materials and Methods ............................................................................65 Results .....................................................................................................68 Discussion ...............................................................................................85 ix CHAPTER Page III INHIBITION OF BLADDER TUMOR GROWTH BY 1,1-BIS(3’- INDOLYL)-1-(p-SUBSTITUTEDPHENYL)METHANES: A NEW CLASS OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR γ AGONISTS ........................................................................... 91 Introduction .............................................................................................91 Materials and Methods ............................................................................93 Results .....................................................................................................99 Discussion .............................................................................................114 IV 1,1-BIS(3’-INDOLYL)-1-(p-SUBSTITUTEDPHENYL)METHANES ARE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR γ AGONISTS BUT DECREASE HCT-116 COLON CANCER CELL SURVIVAL THROUGH RECEPTOR-INDEPENDENT ACTIVATION OF EARLY GROWTH RESPONSE-1 AND NAG-1....... 118 Introduction ...........................................................................................118 Materials and Methods ..........................................................................120 Results ...................................................................................................125