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A Dissertation for the Degree of Doctor of Philosophy Effects of metformin on Sirt1, Nrf2 and AhR expression in cancer cells Department of Pharmacy Graduate School Chungnam National University By Minh Truong Do Advisor Hye Gwang Jeong August 2014 Effects of metformin on Sirt1, Nrf2 and AhR expression in cancer cells Advisor Hye Gwang Jeong Submitted to the Graduate School in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy April, 2014 Department of Pharmacy Graduate School Chungnam National University By Minh Truong Do Contents Contents i List of Figures vi List of Abbreviations x Abstract - I Introduction Metformin and reduced risk of cancer - Role of the AhR, CYP1A1 and CYP1B1 in carcinogenesis and mechanisms of regulation of gene expression Regulation of gene expression and role of Nrf2 and HO-1 in tumorigenesis and chemoresistance Role of Sirt1 in tumorigenesis and chemoresistance - 11 II Materials & Methods 15 Materials - 15 Cell culture and treatment - 16 Measurement of cell viability and cytotoxicity - 17 BrdU incorporation assay 18 i RNA preparation and reverse transcription-polymerase chain reaction (RT-PCR) - 19 Quantitative real-time RT-PCR (qRT-PCR) - 20 Luciferase and β-galactosidase assays - 22 Western blotting - 23 Preparation of nuclear and cytosolic extracts - 24 10 Immunoprecipitation (IP) 24 11 Chromatin immunoprecipitation (ChIP) 25 12 Small interfering RNA transfection - 26 13 Sp1, HO-1, Sirt1, Pgc-1 and PPAR overexpression - 26 14 miR-34a inhibitor and mimic transfection - 27 15 Measurement of intracellular reactive oxygen species (ROS) 27 16 Statistical analysis - 28 III Results - 29 Metformin suppresses CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating aryl hydrocarbon receptor expression - 29 1.1 Metformin inhibits CYP1A1 and CYP1B1 expression in breast cancer cells - 29 1.2 Down-regulation of AhR expression is required for the ii suppression of CYP1A1 and CYP1B1 by metformin - 33 1.3 Down-regulation of Sp1 by metformin inhibits AhR transcriptional activity in breast cancer cells - 38 1.4 Inhibition of CYP1A1 and CYP1B1 expression by metformin is independent of ER 41 1.5 Metformin suppresses endogenous AhR-ligand-induced CYP1A1 and CYP1B1 expression by reducing TDO expression in breast cancer cells 43 1.6 Metformin suppresses TDO expression by down-regulating the Sp1/glucocorticoid receptor (GR) signaling pathway - 47 Metformin inhibits heme oxygenase-1 expression in cancer cells through inactivation of Raf/ERK/Nrf2 signaling and AMPK-independent pathways - 52 2.1 Metformin suppresses HO-1 expression in cancer cells - 52 2.2 Metformin suppresses Nrf2 expression through a Keap1independent mechanism in cancer cells - 54 2.3 Metformin suppresses Nrf2 expression in cancer cells via Raf-ERK inactivation 58 2.4 Down-regulation of HO-1 expression by metformin is independent of AMPK 61 iii 2.5 Reduction of HO-1 contributes to anti-proliferative effects of metformin in cancer cells 65 Metformin induces microRNA-34a to down-regulate Sirt1/Pgc-1/Nrf2 pathway leading to increased susceptibility of cancer cells to oxidative stress and therapeutic agents 71 3.1 Metformin suppresses Sirt1 expression in p53 wild-type cancer cells 71 3.2 p53-dependent induction of miR-34a is required for the reduction of Sirt1 by metformin 73 3.3 Down-regulation of Sirt1 by metformin inhibits Nrf2 expression and increases susceptibility of wild-type p53 cancer cells to oxidative stress - 77 3.4 Metformin inhibits Nrf2 expression mediated by suppression of Pgc-1 - 83 3.5 Metformin suppresses Nrf2 expression by inhibiting PPAR transcriptional activity and attenuating PPAR binding to the Nrf2 promoter - 86 3.6 Up-regulation of DR5 expression by metformin sensitizes wild-type p53 cancer cells to TRAIL-induced apoptosis - 90 iv IV Discussion - 97 V Conclusion 118 VI References - 120 Abstract in Korean - 147 Appendix 150 v List of Figures Metformin suppresses CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating aryl hydrocarbon receptor expression Fig Metformin down-regulates CYP1A1 and CYP1B1 transcription in MCF-7 breast cancer cells 31 Fig Metformin down-regulates AhR expression in MCF-7 breast cancer cells 35 Fig Down-regulation of AhR expression is required for the reduction of CYP1A1 and CYP1B1 by metformin in MCF-7 cells 37 Fig The reduction in Sp1 protein levels mediated by metformin suppresses AhR transcriptional activity in MCF-7 breast cancer cells - 39 Fig Metformin down-regulates CYP1A1 and CYP1B1 expression in ER-negative MDA-MB-231 breast cancer cells - 42 Fig Metformin attenuates endogenous AhR ligand-induced CYP1A1 and CYP1B1 expression by reducing tryptophan-2,3-dioxygenase expression in MCF-7 breast cancer cells - 45 Fig The down-regulation of TDO expression by metformin is mediated via down-regulation of Sp1 and GR proteins - 49 vi Fig Proposed signaling pathways underlying the effects of metformin on down-regulation of CYP1A1 and CYP1B1 expression in breast cancer cells - 51 Metformin inhibits heme oxygenase-1 expression in cancer cells through inactivation of Raf/ERK/Nrf2 signaling and AMPK-independent pathways Fig Metformin down-regulates HO-1 expression in various cancer cells - 53 Fig 10 Effects of metformin on Nrf2 and Keap1 expression in cancer cells - 56 Fig 11 Inactivation of Raf-ERK signaling by metformin is required for down-regulation of Nrf2 expression in cancer cells 59 Fig 12 Metformin suppresses HO-1 expression in cancer cells in an AMPK-independent manner - 63 Fig 13 Effects of metformin on proliferation of cancer cells - 67 Fig 14 Role of HO-1 suppression in anti-proliferative effect of metformin in cancer cells 69 Fig 15 Proposed signaling pathways underlying the effects of metformin on down-regulation of HO-1 expression in cancer cells 70 vii 153 154 155 156 157 158 159 160 161 162 163 164 ACKNOWLEDGMENT Successful completion of this milestone in my graduate studies would have not been possible without the encouragement and support from my professional colleagues as well as social well-wishers I would like to take this opportunity to extend my sincere gratitude towards them for helping me achieve my goal With an immense sense of gratitude and respect, I would like to thank my advisor, Prof Hye Gwang Jeong for guiding me to the field of scientific research Without his continuous support, scientific as well as financial, motivation to improve and persuasion to make use of the right opportunities for progress, I would not have been able to accomplish this work Thank you for your confidence in me, believing in my scientific ideas and helping me bring those ideas into successful experiments You will always inspire me to be the best I can etc and more In particular, I would like to thank Prof Luong Thi Hong Van, Prof Nguyen Thi Bich Thu, and Dr Tran Thi Hien for recommendation to study under Prof Hye Gwang Jeong Additionally, I would like to acknowledge all of the Professors and staffs at College of Pharmacy, Chungnam National University I also would like to thank College of Sciences, Thai Nguyen University, Vietnam where I am working presently 165 I extend my gratitude towards my dissertation committee members, Prof Bong Hee Kim, Prof Sang Kyum Kim, Prof Tae Cheon Jeong, and Prof Kwang Youl Lee for validating and supporting the scientific proposal and the results obtained My success would not have been possible without the unquestioned and complete support from my parents, Mr Do Ba Thang and Mrs Tran Thi Lan, and my brothers Do Minh Thanh and Do Minh Dung Dad and mom, your constant, everlasting support and belief in me encourage me to achieve greater heights, professionally and personally You have taught me innumerous values that I cannot even begin to describe etc I always keep them close to me forever I would not have been able to complete my graduate studies without the constant support and guidance of my present and past lab members: Dr Yong Pil Hwang, Dr Hyung Gyun Kim, Dr Jae ho Choi, Dr Eun Hee Han, Dr Tilak Khanal Special thanks to other my lab-mates including Bong Hwan Park, Tran Thi Thu Phuong, Sun Woo Jin, Seok Hoon Oh, Seong Su Won, Se Jong Kim and Eun Ji Lee I got a special emotion in Korea, the moment with Korean friends and many foreigners during four years my stay in Korea is unforgettable, and I gained an opportunity to learn varieties of Korean culture I would like to 166 thank all of my Vietnamese friends and Korean friends for the friendly environment which I got I thank you all kindly Daejeon, June, 2014 Minh Truong Do 167 ... Fig 11 Inactivation of Raf-ERK signaling by metformin is required for down-regulation of Nrf2 expression in cancer cells 59 Fig 12 Metformin suppresses HO-1 expression in cancer cells in an... suppress Nrf2 expression in cancer cells Inhibition of Raf-ERK signaling by PD98059 decreased Nrf2 mRNA expression in HepG2 cells, confirming that the inhibition of Nrf2 expression is mediated... factor–related apoptosis-inducing ligand XRE Xenobiotic responsive elements xii ABSTRACT* Effects of metformin on Sirt1, Nrf2 and AhR expression in cancer cells Minh Truong Do Department of Pharmacy, Graduate
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