title: Metformin suppresses hepatocellular carcinoma cell growth through induction of cell cycle G1/G0 phase arrest and p21CIP and p27KIP expression and downregulation of cyclin D1 in vitro and in vivo.
abstract: Metformin is used as a first-line therapy for type 2 diabetes, with reports of its usefulness for the prevention and control of several types of cancers. This study investigated the effects of metformin on hepatocellular carcinoma (HCC). The human HCC cell lines HepG2 and PLC/PRF/5 were cultured and treated with metformin or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an activator of adenosine monophosphate (AMP)-activated protein kinase. Changes in cell viability and cell cycle distribution were evaluated by MTT and flow cytometry, respectively. Apoptosis was assessed by fluorescent-dye staining. An HCC model was established in 6- to 8-week-old BALB/c-nu mice by subcutaneous injection of PLC/PRF/5 cells. After 1 week, mice were treated intragastrically with metformin or vehicle. Tumor xenograft tissues were examined using immunohistochemistry for evaluation of the the expression of cyclin D1, p21CIP and p27KIP. HCC cells and tissues from the in vitro and in vivo experiments, respectively, were subjected to protein extraction and western blotting. We found that metformin treatment reduced HCC cell viability in a dose-dependent manner similar to AICAR treatment. In addition, metformin treatment induced HCC cell cycle arrest at G1/G0 phase and apoptosis. Intragastric treatment of the mouse PLC/PRF/5 cell xenograft model with metformin showed that metformin not only blocked tumor progression, but also reduced tumor morbidity. Treatment with metformin upregulated the expression of p21CIP and p27KIP, but downregulated cyclin D1 levels, both in vitro and in vivo. Metformin treatment also upregulated the expression of phosphorylated AMPK protein in xenograft tissues. These findings indicate that metformin warrants further evaluation as a novel therapeutic and preventive strategy against HCC.
authors.affiliation: Department of Gastroenterology, The First Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong 515041, P.R. China.
authors.forename: Xianbin
authors.initials: X
authors.lastname: Cai
authors.forename: Xi
authors.initials: X
authors.lastname: Hu
authors.forename: Bozhi
authors.initials: B
authors.lastname: Cai
authors.forename: Qinjia
authors.initials: Q
authors.lastname: Wang
authors.forename: Youfeng
authors.initials: Y
authors.lastname: Li
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Tan
authors.forename: Hui
authors.initials: H
authors.lastname: Hu
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Chen
authors.forename: Jiexiong
authors.initials: J
authors.lastname: Huang
authors.forename: Jidong
authors.initials: J
authors.lastname: Cheng
authors.forename: Xubin
authors.initials: X
authors.lastname: Jing
mesh_terms: D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D002470:Cell Survival / Q000187:drug effects; D019938:Cyclin D1 / Q000096:biosynthesis / Q000235:genetics*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000096:biosynthesis* / Q000235:genetics; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000096:biosynthesis* / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D008687:Metformin / Q000494:pharmacology; D051379:Mice; D012265:Ribonucleotides / Q000494:pharmacology; D023041:Xenograft Model Antitumor Assays
chemical_list: C436291:CDKN1B protein, human; C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D012265:Ribonucleotides; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; D000620:Aminoimidazole Carboxamide; D008687:Metformin; C031143:AICA ribonucleotide