Lili Wang1, Zhiyong Wang1, Xin Zhao1, Ning Ji1, Yu Zhou1, Jing Li2. 1. State Key Laboratory of Oral Disease, Sichuan University, Chengdu 610041, China. 2. State Key Laboratory of Oral Disease, Sichuan University, Chengdu 610041, China; Email: lijing19840108@126.com.
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.
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 buffersaline, 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.