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Therapeutic Potential of Withaferin a Against Non-Small Cell Lung Cancer

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Therapeutic Potential of Withaferin a Against Non-Small Cell Lung Cancer University of Louisville ThinkIR: The University of Louisville's Institutional Repository Electronic Theses and Dissertations 12-2019 Therapeutic potential of withaferin A against non-small cell lung cancer. Al Hassan Kyakulaga University of Louisville Follow this and additional works at: https://ir.library.louisville.edu/etd Part of the Pharmacology, Toxicology and Environmental Health Commons Recommended Citation Kyakulaga, Al Hassan, "Therapeutic potential of withaferin A against non-small cell lung cancer." (2019). Electronic Theses and Dissertations. Paper 3311. https://doi.org/10.18297/etd/3311 This Doctoral Dissertation is brought to you for free and open access by ThinkIR: The University of Louisville's Institutional Repository. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of ThinkIR: The University of Louisville's Institutional Repository. This title appears here courtesy of the author, who has retained all other copyrights. For more information, please contact [email protected]. THERAPEUTIC POTENTIAL OF WITHAFERIN A AGAINST NON-SMALL CELL LUNG CANCER By Al Hassan Kyakulaga MSc. Pharmacology, Makerere University, 2013 MS Pharmacology & Toxicology, University of Louisville, 2017 A Dissertation Submitted to the Faculty of the School of Medicine of the University of Louisville In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Pharmacology and Toxicology Department of Pharmacology and Toxicology School of Medicine, University of Louisville Louisville, KY, USA December, 2019 THERAPEUTIC POTENTIAL OF WITHAFERIN A AGAINST NON-SMALL CELL LUNG CANCER By Al Hassan Kyakulaga A Dissertation Approved on August 29, 2019 By the following Dissertation Committee: Ramesh C. Gupta, Ph.D. Jason Chesney, M.D., Ph.D. Jun Yan, M.D., Ph.D. Frederick A. Luzzio., Ph.D Farrukh Aqil, Ph.D. ii DEDICATION This doctoral thesis is dedicated to Elianah, Elynn, and Nancy for their love, support and perseverance. iii ACKNOWLEDGEMENTS I would like to extend my sincere gratitude to my mentor, Dr. Ramesh C. Gupta for his enthusiasm, guidance, support, and interest in my academic and professional training at the University of Louisville. I am truly indebted for the amazing opportunity he has provided with me the past five years to work in his laboratory. My acknowledgments also go to Drs. Farrukh Aqil and Radha Munagala for their tireless efforts towards teaching me skills that have enabled me to accomplish this work. Also, I would like to thank my lab former mates - Jeyaprakash Jeyabalan, Ashley Mudd, Divya Karukonda and Dr. Ashish Agrawal for the continuous support they have rendered to me during the conduct of these experiments. I am very grateful with the financial support that I received the Department of Pharmacology and Toxicology, Integrated Programs in Biomedical Sciences (IPBS) program, the Agnes Brown Duggan Endowment, and the School of Graduate Studies at the University of Louisville for the Graduate Fellowship. My special thanks go to Dr. David Hein (Chair, Pharmacology & Toxicology), Dr. Christopher States, and Dr. Brian Ceresa for their encouragement, advice, and professional support towards my Ph.D. training. iv ABSTRACT THERAPEUTIC POTENTIAL OF WITHAFERIN-A AGAINST NON-SMALL CELL LUNG CANCER: Al Hassan Kyakulaga August 29th, 2019 Lung cancer remains the leading cause of cancer-related deaths among both men and women worldwide, and in the United States. Non-small cell lung cancer (NSCLC) represents about 85-90% of all lung cancer cases and has an overall five-year survival rate of only 17%. Since 60-70% of NSCLC patients are diagnosed when the tumors are at an advanced stage, chemotherapy remains the most viable treatment option for NSCLC. Recently, the development of molecularly targeted drugs and the breakthrough immunotherapies have shifted the front-line therapies for NSCLC from cytotoxic chemotherapy towards an era of personalized medicine. However, due to the high genetic and cellular heterogeneity of NSCLC tumors, these revolutionary therapies are not applicable in >70% of NSCLC patients. Moreover, regardless of treatment approach, dose-limiting toxicity and drug resistance are major therapeutic challenges in treatment of advanced NSCLC. Therefore, there is a significant unmet clinical need to develop highly efficacious therapeutic options for the treatment of advanced NSCLC. In this dissertation, studies were performed with the overall objective of determining the therapeutic potential of withaferin A (WFA), a plant-derived steroidal lactone anticancer compound against NSCLC. v Briefly, the preclinical efficacy, toxicity and pharmacokinetic properties of WFA were investigated using in vitro and in vivo models. Two human NSCLC cell lines, H1299, and A549 were used to investigate the antitumor efficacy of WFA, alone or in combination with paclitaxel (PAC), a standard chemotherapeutic agent used for the treatment of various types of cancer. Firstly, we found that WFA alone significantly inhibited (IC50: 0.3-0.6 µM) the growth, proliferation, colony formation, adhesion, migration, and invasion of both H1299 and A549 cells. As reported elsewhere, the antitumor activities of WFA were both time- and concentration-dependent, but were completely abrogated in the presence of thiol-containing compounds such as N-acetyl cysteine (NAC) and dithiothreitol (DTT). Importantly, the combination of PAC and WFA at 1:40, 1:20, and 1:10 ratios, respectively, resulted in synergistic inhibition of tumor cell growth and proliferation for both H1299 and A549 cell lines. Comparatively, the PAC+WFA combination displayed greater anticancer efficacy and potency than cisplatin (cis-Pt) alongside PAC (cis-Pt+PAC) or cis-Pt+WFA, and it resulted in a >40-fold decrease in the IC50 of PAC against both H1299 and A549 cells. In addition, using PAC or taxol resistant cell lines (TR-A549), we demonstrated that WFA was active against the growth and proliferation of chemotherapy resistant human NSCLC cells both in vitro and in vivo. Mechanistically, WFA induced apoptosis, modulated the expression of MDR-1, as well as inhibited the induction of epithelial-to-mesenchymal transition (EMT) in A549 NSCLC cells via the regulation of Smad2/3 and NFκB signaling. In the toxicity evaluation, we found that oral administration of up to 12 mg/kg of WFA was well tolerated with no signs of systemic and histopathologic toxicity in male SD rats. Also, a highly sensitive, reproducible and robust HPLC-MS/MS method for the detection and quantification of WFA in plasma and tissues was developed. Pharmacokinetically, WFA displayed a high plasma protein binding capacity (>95%) and high in vitro intrinsic clearance (Clint: 88µL/min/mg) in SD rat liver microsomes. Following a single intravenous bolus dose (10 mg/kg), plasma vi concentration-time profile of WFA showed a very rapid clearance from plasma (t1/2 = 37 min). Together, the findings presented in this dissertation demonstrate the therapeutic potential of WFA, and provide a strong rationale for the further studies to develop WFA alone or its combination with standard chemotherapeutic agents such as PAC for the treatment of advanced NSCLC and other cancer types. vii TABLE OF CONTENTS DEDICATION .................................................................................................................. III ACKNOWLEDGEMENTS .............................................................................................. IV ABSTRACT ....................................................................................................................... V LIST OF FIGURES ........................................................................................................... X CHAPTER ONE ................................................................................................................. 1 INTRODUCTION ......................................................................................................... 1 CHAPTER TWO .............................................................................................................. 19 SYNERGISTIC COMBINATIONS OF PACLITAXEL AND WITHAFERIN A AGAINST HUMAN NON-SMALL CELL LUNG CANCER CELLS ........................... 19 INTRODUCTION ....................................................................................................... 19 RESULTS .................................................................................................................... 29 DISCUSSION .............................................................................................................. 48 CHAPTER THREE .......................................................................................................... 53 WITHAFERIN A INHIBITS EPITHELIAL TO MESENCHYMAL TRANSITION IN NON-SMALL CELL LUNG CANCER CELLS ............................................................. 53 INTRODUCTION ....................................................................................................... 53 viii MATERIALS AND METHODS ................................................................................. 56 RESULTS .................................................................................................................... 62 DISCUSSION .............................................................................................................. 74 CHAPTER FOUR ............................................................................................................. 79 REPEATED DOSING TOXICITY
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