Please cite this article in press as: Ripoli, M., et al., Hypermethylated levels of E-cadherin promoter in Huh-7 cells expressing the HCV core protein. Virus Res. (2011), doi:10.1016/j.virusres.2011.05.014 ARTICLE IN PRESS G Model VIRUS-95455; No. of Pages 8 Virus Research xxx (2011) xxx–xxx Contents lists available at ScienceDirect Virus Research jo u r n al hom epa ge: www.elsevier.com/locate/virusres Hypermethylated levels of E-cadherin promoter in Huh-7 cells expressing the HCV core protein Maria Ripoli a,1 , Raffaela Barbano b,1 , Teresa Balsamo b , Claudia Piccoli c , Virgilio Brunetti d , Michelina Coco b , Gianluigi Mazzoccoli e , Manlio Vinciguerra f , Valerio Pazienza a,∗ a Gastroenterology Unit, I.R.C.C.S. “Casa Sollievo della Sofferenza” Hospital, viale dei Cappuccini n.1, 71013 San Giovanni Rotondo (FG), Italy b Oncology Research Laboratory, I.R.C.C.S. “Casa Sollievo della Sofferenza”, San Giovanni Rotondo (FG), Italy c Department of Biomedical Sciences, University of Foggia, Italy d Italian Institute of Technology, Center for Bio-Molecular Nanotechnology (LE), Italy e Department of Internal Medicine and Chronobiology Unit, I.R.C.C.S. “Casa Sollievo della Sofferenza” Hospital, San Giovanni Rotondo (FG), Italy f Mouse Biology Unit, European Molecular Biology Laboratory (EMBL), Monterotondo (RM), Italy a r t i c l e i n f o Article history: Received 5 January 2011 Received in revised form 16 May 2011 Accepted 16 May 2011 Available online xxx Keywords: HCV core CDH1 DNA methylation HCC a b s t r a c t Background and aim: The mechanisms of hepatocarcinogenesis induced by hepatitis C virus remain unclear. Our aim was to investigate the effect of the HCV core protein on the promoter methylation status of selected genes potentially involved in the hepatocellular carcinoma (HCC). Materials and methods: We evaluated the promoter methylation levels of the E-cadherin (CDH1), the glutathione S-transferase p1 (GSTP1), adenomatosis polyposis coli (APC), tissue inhibitor of metallo- proteinase 3 (TIMP3), catenin (cadherin-associated protein) beta 1 (CNNTB1) genes by a quantitative methylation-specific polymerase chain reaction (QMSP) in the in vitro model of Huh-7 cells expressing the HCV core protein of genotype 1b. Results: We found that CDH1 promoter was hypermethylated in genotype 1b HCV core protein-positive cells as compared to control cells expressing the GFP protein alone (HCV core 1b vs GFP p = 0.00; HCV core 1b vs Huh-7 p = 0.03). This resulted in reduced levels of CDH1 protein as evaluated by immunoblot and by immunofluorescence. On the other hand no significant changes were observed for the other genes investigated. Furthermore, we present evidence that genotype 1b HCV core protein expression induces SIRT1 upregulation and that treatment with SIRT1 inhibitor sirtinol decreases the methylation levels of CDH1 promoter (1b + sirtinol vs 1b p = 0.05; 1b + sirtinol vs GFP + sirtinol p = NS) resulting in 1.7-fold increased CDH1 mRNA expression (1b + sirtinol vs 1b p = 0.05). Conclusions: Our findings suggest that HCV core protein could play a role in HCC at least in part by altering the methylation status of CDH1 promoter. These findings could also suggest a novel therapeutic approach for HCC. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Hepatitis C Virus (HCV) is one of the leading causes of chronic liver disease worldwide and it is estimated that approximately 3% of the world population is infected. Chronic hepatitis ultimately results in the development of hepatocellular carcinoma (Saito et al., 1990; Simonetti et al., 1992). However, the mechanism of hepa- tocarcinogenesis in chronic HCV infection is still unclear. HCV is an enveloped, positive strand RNA virus, belonging to Flaviviri- dae family genus Hepacivirus (Tellinghuisen and Rice, 2002). Its genome encodes a polyprotein of more than 3000 aminoacids that ∗ Corresponding author. Tel.: +39 0882 416281; fax: +39 0882 416271. E-mail address: pazienza valerio@yahoo.it (V. Pazienza). 1 These authors have contributed equally to this work. is cleaved post-translationally by host and viral proteases yield- ing 3 structural (Core, E1 and E2) and 7 non structural (p7, NS2 to NS5B) proteins. Increasing experimental evidence suggests that HCV contributes to HCC by directly modulating pathways that pro- mote the malignant transformation of hepatocytes (Thorgeirsson and Grisham, 2002). The ability of the HCV core protein to inter- fere with metabolic pathways and to modulate gene transcription, cell proliferation and cell death may be involved in the pathogen- esis of HCC (Moriya et al., 1998; Yoshida et al., 2002). Genotype 1b particularly has been demonstrated to increase of 2 fold the risk of developing HCC in vivo (Raimondi et al., 2009). HCC is due to a multistep process with accumulation of genetic and epigenetic alterations in regulatory genes, leading to activation of oncogenes and inactivation or loss of tumor suppressor genes (TSGs) (Levrero, 2006). Several studies have suggested that DNA hypermethyla- tion of multiple tumor suppressor genes may contribute to the 0168-1702/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2011.05.014