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Mediated Multidrug Resistance (MDR) in Cancer A Thesis Entitled: Differential Effects of Dopamine D3 Receptor Antagonists in Modulating ABCG2 - Mediated Multidrug Resistance (MDR) in Cancer by Noor A. Hussein Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science in Pharmaceutical Sciences Degree in Pharmacology and Toxicology _________________________________________ Dr. Amit K. Tiwari, Committee Chair _________________________________________ Dr. Frank Hall , Committee Member _________________________________________ Dr. Zahoor Shah, Committee Member _________________________________________ Dr. Caren Steinmiller, Committee Member _________________________________________ Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo May 2017 Copyright 2017, Noor A. Hussein This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Differential Effects of Dopamine D3 Receptor Antagonists in Modulating ABCG2 - Mediated Multidrug Resistance (MDR) by Noor A. Hussein Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science in Pharmaceutical Sciences Degree in Pharmacology and Toxicology The University of Toledo May 2017 The G2 subfamily of the ATP-binding cassette transporters (ABCG2), also known as the breast cancer resistance protein (BRCP), is an efflux transporter that plays an important role in protecting the cells against endogenous and exogenous toxic substances. The ABCG2 transporters are also highly expressed in the blood brain barrier (BBB), providing protection against specific toxic compounds. Unfortunately, their overexpression in cancer cells results in the development of multi-drug resistance (MDR), and thus, chemotherapy failure. Dopamine3 receptor (D3R) antagonists were shown to have excellent anti-addiction properties in preclinical animal models but produced limited clinical success with the lead molecule. Thus, other more potent D3R antagonists, notably NGB2904, SB-277011A, and U99194, and PG01037 were dropped from further studies. Whether ABCG2 transporters limited D3R antagonists’ efficacy or whether these D3R antagonists could modulate ABCG2-mediated MDR has not been evaluated before. The present study was designed a) in a quest to understand whether ABCG2 transporters might be a limiting factor in D3R antagonists’ accumulation in the brain; b) if these D3R antagonists could be repurposed as anticancer chemo adjuvants to iii reverse MDR mediated by ABCG2 transporters. Interestingly, the structure of various D3R antagonists is similar to that of substrates for the ABCG2 transporter. We found that the D3R antagonists (PG01037, NGB2904, SB-277011A, and U99194), alone up to 50µM, do not produce significant toxicity towards normal or cancer cells. In contrast, the D3R antagonists (PG010037, NGB2904, SB-277011A, and U99194) significantly sensitized ABCG2 overexpressing HEK293/ABCG2; H460/MX-20, S1-M1-80 and A549-MX-10 cells to well-known anticancer agent’s mitoxantrone (MX) and doxorubicin (DOX) that are substrates of ABCG2 transporters. As shown by accumulation and efflux assay D3R antagonists combination enhanced accumulation of in MX in ABCG2 overexpressing H460-MX20 cells. Additionally, D3R antagonists (PG01037 and NGB2904) at a concentration of only 5µM significantly downregulated the expression of ABCG2 when incubated for 24 and 48 hours with ABCG2 overexpressing cells, suggesting that D3R antagonists reverse ABCG2 mediated drug resistance by not only inhibiting its function but also downregulating its expression. Furthermore, D3R antagonists were found to produce synergistic anticancer activity when combined with MX and DOX. In conclusion, this is the first study to show interaction of D3R antagonists with ABCG2 transporters suggesting that D3R antagonists might be ABCG2 substrates, and combining D3R antagonists with certain anticancer agents that are substrates of ABCG2 transporters (i.e. MX and DOX) could produce beneficial results in ABCG2 overexpressing MDR cancer cells. iv I would like to dedicate my thesis to my dearest friend Qaswaa who passed away last year. She was a source of inspiration throughout all the way back since my undergraduate years, where we did thesis project together. I am sure that she would be very proud of my success and me reaching a higher degree. Acknowledgements I would like to express my sincere gratitude to my advisor and mentor Dr. Tiwari for his tremendous support during my master’s journey. Truly, without his guidance, I could not have successfully completed my scientific research and discovered this new finding. Also, I thank, from the bottom of my heart, my beloved husband Abdullah, who has not only supported me in the difficult times, but also, always motivated me to give the best I can do to complete my mission and achieve my dreams. There are not enough words to describe how grateful I am to have my lovely family: mother, father, brothers and sister in my life. Their endless love and encouragement motivated me from early childhood to explore and excel in science. While I was on this journey to accomplish my master’s thesis, my little baby boy, Yamin, arrived, motivating me even more to accept the challenge of being a successful mother and researcher. Thus, I dedicate my success to my lovely family and to the souls of my lovely grandmother (Sabeha), who passed away few months before my thesis defense. Also, I thank Dr. Ashby (St. John’s University, NY) for his tremendous efforts guiding me and revising my thesis. Furthermore, I thank Dr. Liu for introducing me to the basics of scientific research. Also, I would like to thank my committee members Dr. Hall, Dr. Shah, and Dr. Steinmiller. Lastly, I thank the Higher Committee for Education Development in Iraq (HCED) for their financial support and for giving me the opportunity to complete my higher education path abroad. v Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ..............................................................................................................v Table of Contents ............................................................................................................... vi List of Tables……………………………………………………………………………. ix List of Figures ......................................................................................................................x List of Abbreviations ........................................................................................................ xii List of Symbols ..................................................................................................................xv 1. Introduction 1.1 Multi Drug Resistance (MDR) in Cancer ......................................................1 1.2 Adenosine Tri-Phosphate Binding Cassette (ABC) Transporters .................3 1.3 The Role of ABC Transporters in Cancer Chemotherapy Resistance ........10 1.3.1 The Role of ABCB1 Transporter in Producing MDR …………….11 1.3.2 The Role of ABCC1 Transporter in Producing MDR …………...12 1.3.3 The Role of ABCG2 Transporter in Producing MDR……………..13 1.4 Strategies to Surmount ABC- Mediated MDR……………………………14 1.4.1 Generations of MDR Modulators…………………………………14 1.4.2 Natural MDR Modulators…………………………………………18 vi 1.4.3 Miscellaneous MDR Modulators………………………………. ...18 1.4.4 Compounds Known to Modulate ABCG2 Transporter-Mediated MDR………………………………………………………………………19 1.5 Dopamine D3 Receptor Antagonists………….……….…………………….21 1.6 Objective and Aims………………………………………………………….25 2. Materials and Methods ...................................................................................................26 2.1 Materials……………………………………………………………………. 26 2.2 Cell lines and Cell Culture…………………………………………………. .27 2.3 Determination of Cell Cytotoxicity by MTT Assay………………………. ..28 2.4 Cell Morphological Analysis………………………………………………..29 2.5 Protein Estimation: Cell lysate Preparation and Bicinchoninic acid (BCA) Analysis…………………………………………………………………………....29 2.6 Western Blot Analysis……………………………………………………... 30 2.7 Immunocytochemistry…………………………………………………….. .31 2.8 Rhodamine 123 Accumulation and Efflux Assay…………………………..32 2.9 The Effect of D3 Receptor Antagonists on the Efficacy of Mitoxantrone and Doxorubicin……………………………………………………………………….33 2.10 Molecular Docking Studies…………………………………………………..33 2.10.1 Ligand Structure Preparation…………………………………… 33 2.10.2 Protein Structure Preparation……………………………………..34 vii 2.10.3 Docking Protocol………………………………………………… ...35 2.11 Statistical Analysis…………………………………………………………. .35 3. Results…………………………………………………………………… ....................36 3.1 The Effect of D3R Antagonists on the Efficacy of Mitoxantrone and Doxorubicin in Cell Lines Overexpressing ABCG2 Transporters………………………………...36 3.2 D3 Receptor Antagonists Synergistically Increase the Efficacy of Mitoxantrone and Doxorubicin ……………………………………………...................................38 3.3 PG01037 and NGB2904 Significantly Decrease the Protein Expression levels of the ABCG2 Transporter ………. .................................................................................40 3.4 PG01037 and NGB2904 Significantly Inhibit the Efflux Function of the ABCG2 Transporter ………………………………………………………………...41 3.5 Molecular Docking of SB277011A, NGB2904, PG01037, and U99194A as Determined Using a Homology Model of the ABCG2 Transporter…………………42 4. Tables
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