title: [The inhibitory effect of metformin on oral squamous cell carcinoma].
abstract: OBJECTIVE
  To investigate the effect of metformin on the proliferation and cell apoptosis of oral squamous cell carcinoma (OSCC) (HSC-3, HSC-4) in vitro and in vivo.
  
  
  METHODS
  HSC-3, HSC-4 cells were treated with metformin at different concentration (2-50 mmol/L) for 24, 48 or 72 hours. In vitro cell proliferation ability was determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and colony formation assay. Cell cycle progression was assessed by flow cytometry. Cell apoptosis was tested by both TdT-mediated dUTP nick-end labeling (TUNEL) assay and flow cytometry. The activation of related cell markers was examined by immunohistochemistry. Xenograft mouse model was used to demonstrate the in vivo anti-tumor effect of metformin. A total of 30 BALB/c mice were randomly divided into control groups (water + phosphate buffer saline, PBS) and treatment groups (pre-oral, oral or intraperitoneal injection). Each group had 6 mice. The tumor size was measured once every three days until endpoint (35 days). After sacrificing the mice, tumor tissue was removed, sectioning and then analyzed by TUNEL or immunohistochemistry (IHC) assays.
  
  
  RESULTS
  Metformin inhibited proliferation and colony formation of HSC-3, HSC-4 in a time- and dose-dependent manner. The cell proliferation was significantly reduced when treated with 5, 10, 20 and 40 mmol/L metformin for 48 and 72 hours (P < 0.05).The colony formation of OSCC cells treated with metformin for 72 hours in vitro had the same result. Treated with 2, 20 and 50 mmol/L metformin for 24 hours increased the ratio of G0/G1 1.2-1.8 fold compared with the control group on HSC-4 cell. The percentage of apoptosis cell rose from 10% (control) to around 30% (treatment) in vitro. Metformin also decreased the size of xenografts by 82.5% (pre-oral), 63.9% (oral), and 62.8% (oral or intraperitoneal injection). The percentage of apoptosis cell rose from lower than 10% (control) to 70% (pre-oral), 50% (oral), and 25% (oral or intraperitoneal injection). The percentage of PCNA positive cell was lower than 60% (control group was normalized to 100%).
  
  
  CONCLUSIONS
  Metformin could inhibit the growth of OSCC cell line (HSC-3, HSC-4) by reducing cell proliferation and increasing cell apoptosis in vitro and in vivo. Therefore metformin could be a potential new treatment candidate for human OSCC.
authors.affiliation: State Key Laboratory of Oral Disease, Sichuan University, Chengdu 610041, China.
authors.forename: Lili
authors.initials: L
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Oral Disease, Sichuan University, Chengdu 610041, China.
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Oral Disease, Sichuan University, Chengdu 610041, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Oral Disease, Sichuan University, Chengdu 610041, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Ji
authors.affiliation: State Key Laboratory of Oral Disease, Sichuan University, Chengdu 610041, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Oral Disease, Sichuan University, Chengdu 610041, China; Email: lijing19840108@126.com.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy* / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D005434:Flow Cytometry; D005562:Formazans; D006801:Humans; D066298:In Vitro Techniques; D008687:Metformin / Q000008:administration & dosage / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009062:Mouth Neoplasms / Q000188:drug therapy* / Q000473:pathology; D011897:Random Allocation; D013778:Tetrazolium Salts; D013997:Time Factors; D014410:Tumor Stem Cell Assay
chemical_list: D005562:Formazans; D013778:Tetrazolium Salts; C079782:MTT formazan; D008687:Metformin