title: In vitro and in vivo anti-tumor effect of metformin as a novel therapeutic agent in human oral squamous cell carcinoma.
abstract: BACKGROUND
  Metformin, which is widely used as an antidiabetic agent, has recently been reported to reduce cancer risk and improve prognosis in certain malignancies. However, the specific mechanisms underlying the effect of metformin on the development and progression of several cancers including oral squamous cell carcinoma (OSCC) remain unclear. In the present study, we investigated the effects of metformin on OSCC cells in vitro and in vivo.
  
  
  METHODS
  OSCC cells treated with or without metformin were counted using a hemocytometer. The clonogenic ability of OSCC cells after metformin treatment was determined by colony formation assay. Cell cycle progression and apoptosis were assessed by flow cytometry, and the activation of related signaling pathways was examined by immunoblotting. The in vivo anti-tumor effect of metformin was examined using a xenograft mouse model. Immunohistochemistry and TUNEL staining were used to determine the expression of cyclin D1 and the presence of apoptotic cells in tumors from mice treated with or without metformin.
  
  
  RESULTS
  Metformin inhibited proliferation in the OSCC cell lines CAL27, WSU-HN6 and SCC25 in a time- and dose-dependent manner, and significantly reduced the colony formation of OSCC cells in vitro. Metformin induced an apparent cell cycle arrest at the G0/G1 phase, which was accompanied by an obvious activation of the AMP kinase pathway and a strongly decreased activation of mammalian target of rapamycin and S6 kinase. Metformin treatment led to a remarkable decrease of cyclin D1, cyclin-dependent kinase (CDK) 4 and CDK6 protein levels and phosphorylation of retinoblastoma protein, but did not affect p21 or p27 protein expression in OSCC cells. In addition, metformin induced apoptosis in OSCC cells, significantly down-regulating the anti-apoptotic proteins Bcl-2 and Bcl-xL and up-regulating the pro-apoptotic protein Bax. Metformin also markedly reduced the expression of cyclin D1 and increased the numbers of apoptotic cells in vivo, thus inhibiting the growth of OSCC xenografts.
  
  
  CONCLUSIONS
  Our data suggested that metformin could be a potential candidate for the development of new treatment strategies for human OSCC.
authors.affiliation: Department of Clinical Laboratories, Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai, 200011, China.
authors.forename: Qingqiong
authors.initials: Q
authors.lastname: Luo
authors.forename: Dan
authors.initials: D
authors.lastname: Hu
authors.forename: Shuiqing
authors.initials: S
authors.lastname: Hu
authors.forename: Ming
authors.initials: M
authors.lastname: Yan
authors.forename: Zujun
authors.initials: Z
authors.lastname: Sun
authors.forename: Fuxiang
authors.initials: F
authors.lastname: Chen
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D008687:Metformin / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009062:Mouth Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D014410:Tumor Stem Cell Assay; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D008687:Metformin