SURAMIN AS A CHEMO- AND RADIO-SENSITIZER: PRECLINICAL TRANSLATIONAL STUDIES DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yan Xin, M.S. ***** The Ohio State University 2006 Dissertation Committee: Dr. M. Guillaume Wientjes, Adviser Approved by Dr. Jessie L.-S. Au ____________________________________ Adviser Dr. Duxin Sun Graduate Program in Pharmacy ABSTRACT Previous studies in our laboratory showed that low-dose suramin, an inhibitor of fibroblast growth factor action, enhances sensitivity of various human tumors in preclinical and clinical studies to chemotherapy. Chemosensitization required apoptosis, and increased the extent and duration of the induction of apoptosis. The primary focus of this dissertation research was to explore, in preclinical studies, therapeutic approaches for therapy enhancement based on this mechanism. A phase III clinical trial in superficial bladder cancer, which emanated from our laboratory, showed that optimizing mitomycin C delivery nearly doubled the recurrence- free survival of treated patients to 40%. Tumor sensitization with suramin might yield further therapeutic improvements, and was investigated in in vitro and in vivo studies. Studies in Chapter 2 demonstrated enhanced antitumor activity of mitomycin C, administered at subtherapeutic and therapeutic regimens. Various preclinical and clinical studies determined that suramin sensitized tumor tissue at low but not at high concentrations, presumably due to additional pharmacologic effects at elevated concentrations. Studies to overcome this limitation, especially in tumors containing high fibroblast growth factor concentrations, used pentosan polysulfate, another nonspecific FGF inhibitor. This agent was also a chemosensitizer, but combined use with suramin did not increase the overall efficacy (Chapter 3). ii Radiotherapy depends on induction of apoptosis for its anticancer effect, as is the case for many forms of chemotherapy, and led to our evaluation of suramin as radiosensitizer. Results in Chapter 4 and Chapter 5 showed that low-dose suramin sensitized the radiation response of both radiosensitive (prostate PC-3) and relatively radioresistant (pharynx FaDu, pancreatic Hs 766T) xenograft tumors, thereby further extending the clinical application of suramin to modulate radiotherapy. More importantly, the radiosensitizaiton effect of suramin had tumor type specific dose dependence, with a narrow sensitiziting window in FaDu and a wider range in Hs 766T (pancreatic) xenograft tumors (Chapter 5), suggesting the necessity of drug target-specified dosing regimen for suramin. To better understand the biphasic effect of suramin in combination with radiation, pharamacodynamic studies at a microscopic level were conducted (Chapter 6). The results indicated that low-dose but not high-dose suramin enhanced radiation-induced apoptosis, inhibiting radiation-induced upregulation of phospho-ERK and survivin, two survival signals involved in radioresistance. iii DEDICATION Dedicated to my dear parents, my brothers, sisters-in-law, and nephews iv ACKNOWLEDGMENT I wish to express my sincere appreciation to my advisor, Dr. M. Guillaume Wientjes, who has made this dissertation come true. His scientific enthusiasm, integrity and dedication have been inspiring me throughout my graduate study. His patience and encouragement helped me through the frustrated moments. He is a great mentor and friend. My deep appreciation also goes to Dr. Jessie L-S. Au, for all her invaluable contribution to my overall scientific training and growth, as well as her kind friendship in daily life. Being a successful scientist, a wife, and a mother, she is definitely an amazing role model for me. Special thanks go to Dr. Yong Wei for his invaluable scientific suggestions, discussions, and input into my thesis work. His enthusiasm and dedication to science have greatly inspired me. I appreciate his encouragement and friendship. A special recognition is extended to our collaborators, Dr. John C. Grecula and Dr. Nilendu Gupta, in the College of Medicine and Public Health. It is their dedication that made our cooperation productive. Everyone in the lab has played a special role in my training. I feel really grateful to have such an enjoyable environment to finish the most important training in my career. I want to thank each of them for the sweet memorable days. In particular, my sincere v gratitude goes to Dr. Yuebo Gan and Dr. SaeHeum Song, who walked me through the starting stage in this laboratory. I would also like to recognize the following individuals for their direct experimental contribution to my thesis: Dr. Ling (Lucy) Chen for the collaboration in phospho-ERK and survivin staining and discussion in pathology, Dr. Greg Lyness for the coopperation of patient tissue culture, Jianning Yang for the help with PK modeling, Tong Shen for assisting me with the suramin concentration analysis, Dr. Ling Chen and Adrianne Lovelace for the help of tissue sectioning. In addition, I want to express my gratitude to the following labmates for their friendship and help: Dr. Xiao (Shelley) Hu, Dr. Jie (Jack) Wang, Dr. Ze Lu, Bei Yu, Dan Lu, Jing Li, Jia Ji, Dr. Leijun Hu, Dr. Colin Walsh, Dr. Danny Chen, Dr. Liang Zhao, Dr. Mingjie Liu, and Dr. Zancong Shen. My appreciation also goes to Kathy Brooks, Sharon Palko, and Emily Noble for their patient help during my study. My sincere thanks also extend to my dear roommate, Xiaohui (Tracey) Wei, for her friendship and encouragements during the last few years. Thanks to all my other friends for their support during the whole training. Last but not the least, my deepest appreciation goes to my parents, my brothers and sisters-in-law. Without their endless love and support, I would not be able to go through all the way here. vi VITA September 12, 1975 ................................................ Born – Hubei, P.R. China July, 1998 ...........................................….…............ B.S., Pharmacy School of Pharmacy Beijing Medical University July, 2001 ....................................................….…... M.S., Biochemical Engineerin Tsinghua University September, 2001 – present .....………...........…...... Graduate Research Associate College of Pharmacy The Ohio State University PUBLICATIONS Papers Xin Y., Lyness G., Chen D., Song S., Wientjes M.G., and Au J. L.-S., Low dose suramin as a chemosensitizer of bladder cancer to mitomycin C. J Urol. 2005 Jul;174(1):322-7. Xin Y., Li Q., and Cao Z.. Investigation of glutamine metabolism in hybridoma cell C50. Chinese Journal of Biotechnology, Vol. 17(4), 2001. Presentations Xin. Y., Grecula J.C., Gupta N., Wei Y., Chen L., Au J. L.-S., and Wientjes M.G., Pharmacodynamics of the chemosensitizer suramin in radiotherapy. Proceedings of the American Association for Cancer Research Annual Meeting 97, 2006. Xin Y., Chen D., Song S., Lyness G., Wientjes M.G., and Au J. L.-S., Low-dose suramin enhances antitumor activity of mitomycin C in bladder tumors. Proceedings of the American Association for Cancer Research Annual Meeting 95, 2004. vii FIELDS OF STUDY Major field: Pharmacy viii TABLE OF CONTENTS Page ABSTRACT.................................................................................................................... ii ACKNOWLEDGMENT................................................................................................. v VITA............................................................................................................................. vii LIST OF TABLES........................................................................................................ xv LIST OF FIGURES .................................................................................................... xvii Chapter 1 ........................................................................................................................... 1 BACKGROUND INFORMATION ............................................................................... 1 1.1 Introduction......................................................................................................... 1 1.2 Chemoresistance and modulation strategies ....................................................... 1 1.2.1 Chemoresistance ......................................................................................... 1 1.2.2 Growth factor signaling and resistance to cancer chemotherapy................ 4 1.2.3 Other targeted therapies to modulate chemoresistance............................... 8 1.3 Radioresistance and modulation strategies ....................................................... 10 1.3.1 Overview................................................................................................... 10 1.3.2 Combination of conventional cytotoxic agents with radiation ................. 11 ix 1.3.3 Modulating radiotherapy with molecular targeted agents ........................ 13 1.4 Modulation of hypoxia in chemo- and radiotherapy......................................... 17 1.5 Protection of normal tissue in cancer therapy................................................... 18 1.6 Suramin............................................................................................................. 19 1.7 Overview of dissertation................................................................................... 22 Chapter 2 ........................................................................................................................