Contents lists available at ScienceDirect European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar Full length article Combination of metformin and VSL#3 additively suppresses western-style diet induced colon cancer in mice Eun-Ju Chung a,1 , Eun-ju Do b,1 , Sang-Yeob Kim b,c , Eun A Cho b , Dong-Hee Kim b , Sehyung Pak b , Sung Wook Hwang d , Hyo Jeong Lee a , Jeong-Sik Byeon d , Byong Duk Ye d , Dong Hoon Yang d , Sang Hyoung Park d , Suk-Kyun Yang d , Jin-Ho Kim d , Seung-Jae Myung b,d, ⁎ a Health Screening & Promotion Center, Seoul, Republic of Korea b Asan Institute for Life Sciences, Asan Medical Center, Seoul, Republic of Korea c Department of Medicine, University of Ulsan, College of Medicine, Seoul, Republic of Korea d Department of Gastroenterology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea ARTICLE INFO Keywords: Colon cancer Chemoprevention Metformin VSL#3 Western-style diet ABSTRACT Western-style diet (WD) and dysbiosis are known to be associated with colonic inflammation, which contributes to carcinogenesis. Metformin (Met) exerts anti-inflammatory effects to induce AMP-activated protein kinase (AMPK), resulting in suppressed protein synthesis and reduced cell proliferation. Probiotic VSL#3 (V) modifies microbial composition. We investigated the chemopreventive mechanisms of Met and V in WD-induced colitis- associated colon carcinogenesis. Male BALB/c mice were randomly divided into five groups: a control diet (CD) group, WD group, WD+ Met (250 mg/kg/day) group, WD+V (1.3 million bacteria/day) group, and WD+Met+V group. All mice were exposed to azoxymethane (10 mg/kg) followed by 2% dextran sodium sulfate (DSS) for 7 days. Using HCT-116 human colon cancer cell line, expression of AMPK, extracellular signal-regulated kinase (ERK), cyclin D1, and Bcl-2 was investigated and cell cycle arrest was assessed. WD enhanced the severity of colitis and tumor growth compared with CD. The combination of Met and V significantly ameliorated colitis and tumor growth by inhibiting macrophage infiltration and maintaining epithelial integrity. In vitro assays showed that the combination therapy promoted late apoptosis by inhibiting cyclin D1 and Bcl-2 and activating pro- apoptotic ERK. A combination therapy with Met and V attenuates tumor growth in a mouse model of WD- induced colitic cancer, suggesting that this strategy could be useful for the chemoprevention of colon cancer. 1. Introduction Colorectal cancer (CRC) is the third leading cause of cancer-related mortality in Western populations (Siegel and Miller, 2015). Recently, the incidence of CRC in Asia has significantly increased, and one of the factors involved is westernization of the diet (Sung and Lau, 2005). Dietary fats influence intestinal inflammation and dysbiosis, which may be associated with risk of inflammatory bowel disease (IBD) (Ananthakrishnan and Khalili, 2014). Chronic inflammation predis- poses the intestine to cancer development (Han and Theiss, 2014). A Western-style diet (WD) contributes to colon inflammation and carcinogenesis via macrophage activation (Kim and Myung, 2010). It is reasonable to predict that switching dietary patterns from a WD to a healthier Mediterranean-style food would reduce the incidence of CRC (O'Keefe and Li, 2015). However, a safe drug that could inhibit the cancer promoting effects of a WD would also be beneficial for the at- risk populations. Therefore, our present study was designed to identify a suitable chemopreventive strategy to suppress the colitis-associated carcinogenesis (CAC) induced by a WD. Among the many known chemopreventive drugs, metformin (Met) and VSL#3 (V) were selected. Met is a widely prescribed antihypergly- cemic agent of the biguanide family. Its potential benefits extend beyond its original usage, including reducing cancer risk and delaying aging (Cabreiro et al., 2013; Dowling and Goodwin, 2011; Kasznicki and Sliwinska, 2014). In mammals, its mode of action is partially mediated by AMP-activated protein kinase (AMPK) activation, which results in downregulation of the mTOR and IGF-1/AKT pathways (Pierotti and Berrino, 2013). Two studies have demonstrated that metformin has an antiproliferative effect associated with cell cycle arrest and apoptosis by down regulation of anti-apoptotic proteins as well as AMPK (Zhuang and Miskimins, 2008). V (VSL#3 Pharmaceuticals, Gaithersburg, MD) is a mixture of eight http://dx.doi.org/10.1016/j.ejphar.2016.11.012 Received 30 June 2016; Received in revised form 31 October 2016; Accepted 7 November 2016 ⁎ Corresponding author at: Department of Gastroenterology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea. 1 Eun-ju Chung and Eun-ju Do contributed equally to this article. E-mail address: sjmyung@amc.seoul.kr (S.-J. Myung). European Journal of Pharmacology 794 (2017) 1–7 Available online 12 November 2016 0014-2999/ © 2016 Elsevier B.V. All rights reserved. MARK