Original Contribution Metformin induces microRNA-34a to downregulate the Sirt1/Pgc-1α/Nrf2 pathway, leading to increased susceptibility of wild-type p53 cancer cells to oxidative stress and therapeutic agents Minh Truong Do, Hyung Gyun Kim, Jae Ho Choi, Hye Gwang Jeong n Q1 Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea article info Article history: Received 25 March 2014 Received in revised form 10 June 2014 Accepted 14 June 2014 Keywords: Metformin MiR-34a Sirt1 Nrf2 Apoptosis Oxidative stress Free radicals abstract Sirtuin 1 (Sirt1) plays an important role in cellular redox balance and resistance to oxidative stress. Sirt1 exhibits oncogenic properties in wild-type p53 cancer cells, whereas it acts as a tumor suppressor in p53-mutated cancer cells. Here, we investigated the effects of metformin on Sirt1 expression in several cancer cell lines. Using human cancer cell lines that exhibit differential expression of p53, we found that metformin reduced Sirt1 protein levels in cancer cells bearing wild-type p53, but did not affect Sirt1 protein levels in cancer cell lines harboring mutant forms of p53. Metformin-induced p53 protein levels in wild-type p53 cancer cells resulted in upregulation of microRNA (miR)-34a. The use of a miR-34a inhibitor confirmed that metformin-induced miR-34a was required for Sirt1 downregulation. Metformin suppressed peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (Pgc-1α) expression and its downstream target Nrf2 in MCF-7 cells. Genetic tools demonstrated that the reduction of Sirt1 and Pgc-1α by metformin caused Nrf2 downregulation via suppression of PPARγ transcriptional activity. Metformin reduced heme oxygenase-1 and superoxide dismutase 2 but upregulated catalase expression in MCF-7 cells. Metformin-treated MCF-7 cells had no increase in basal levels of reactive oxygen species but were more susceptible to oxidative stress. Furthermore, upregulation of death receptor 5 by metformin-mediated Sirt1 downregulation enhanced the sensitivity of wild-type p53 cancer cells to TRAIL-induced apoptosis. Our results demonstrated that metformin induces miR-34a to suppress the Sirt1/Pgc-1α/Nrf2 pathway and increases susceptibility of wild-type p53 cancer cells to oxidative stress and TRAIL-induced apoptosis. & 2014 Elsevier Inc. All rights reserved. Sirtuin 1 (Sirt1) 1 , a mammalian NAD þ -dependent histone deace- tylase, is involved in diverse cellular processes such as metabolism, cellular redox balance, resistance to oxidative stress, aging, oncogen- esis, and cancer development [1,2]. Sirt1 regulates important tran- scription factors such as p53 [3,4], peroxisome proliferator-activated receptor γ coactivator 1α (Pgc-1α) [5], forkhead homeobox type O (FOXO) proteins [6], and nuclear erythroid factor 2-related factor 2 (Nrf2) [7], which regulates the transcription of pro- and antioxidant enzymes, by which the cellular redox state is affected [1]. Sirt1 plays a critical role in cancer initiation, progression, and drug resistance by blocking senescence and apoptosis and pro- moting cancer cell growth and angiogenesis [2,8] through inhibi- tion of the tumor suppressor p53, FOXO1, and Ku70-mediated functions [3,4,9,10]. Sirt1 is overexpressed in human breast, colon, non-small-cell lung, and prostate cancer cells [9,11] and a sirtinol- or nicotinamide-specific inhibitor of Sirt1 increased senescence- like growth arrest in human breast, lung, and prostate cancer cells [9,12]. Downregulation of Sirt1 by antisense oligonucleotides inhibited the growth and viability of human prostate cancer [9], induced apoptosis, and enhanced radiation-induced antiprolifera- tive effects in human lung cancer cells [13]. Moreover, pharmaco- logical inhibition of Sirt1 or Sirt1 knockdown induced apoptosis in leukemia stem cells and suppressed growth in vitro and in vivo [14,15]. Sirt1-knockout mice exhibited p53 hyperacetylation, and Sirt1-deficient cells enhanced radiation-induced thymocyte apoptosis, suggesting that Sirt1 can facilitate tumor growth by suppressing p53 function [16]. However, several studies have shown that Sirt1 has tumor-suppressive effects. Activation of Sirt1 by resveratrol inhibited growth of BRCA1-deficient and 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/freeradbiomed Free Radical Biology and Medicine http://dx.doi.org/10.1016/j.freeradbiomed.2014.06.010 0891-5849/& 2014 Elsevier Inc. All rights reserved. Abbreviations: ChIP, chromatin immunoprecipitation; CHOP, C/EBP homology protein; DR5, death receptor 5; HO-1, heme oxygenase-1; IP, immunoprecipitation; miR-34a, microRNA-34a; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- lium bromide; Nrf2, nuclear erythroid factor 2-related factor 2; PARP, poly(ADP ribose) polymerase; Pgc-1α, peroxisome proliferator-activated receptor γ coactivator-1α; PPARγ, peroxisome proliferator-activated receptor γ; PPRE, PPAR- responsive element; ROS, reactive oxygen species; SOD2, superoxide dismutase 2; Sirt1, sirtuin 1; TNF, tumor necrosis factor; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand n Corresponding author. E-mail address: hgjeong@cnu.ac.kr (H. Gwang Jeong). Please cite this article as: Truong Do, M; et al. Metformin induces microRNA-34a to downregulate the Sirt1/Pgc-1α/Nrf2 pathway, leading to increased.... Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.06.010i Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎