二甲双胍对口腔鳞状细胞癌抑制作用的研究

The inhibitory effect of metformin on oral squamous cell carcinoma

目的 研究二甲双胍对口腔鳞状细胞癌（oral squamous cell carcinoma,OSCC）细胞增殖和凋亡的影响.方法 采用3-（4,5-二甲基噻唑-2）-2,5-二苯基四氮唑溴盐法[3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide,MTT]、流式细胞术检测,使用不同浓度二甲双胍（2.5、5、10、20、40 mmol/L）处理OSCC细胞系（HSC-3、HSC-4）24、48、72 h后,检测药物对细胞增殖、克隆形成的影响;检测体内二甲双胍不同浓度（2、20、50 mmol/L）对细胞周期和凋亡的影响.采用小鼠皮下移植瘤模型,对比二甲双胍处理对小鼠肿瘤生长影响.共42只BALB/c小鼠分为对照组（水或磷酸盐缓冲液）和实验组（同时口服或预先口服、腹腔注射共6个亚组）,每组各6只.从接种肿瘤开始,3d测量1次肿瘤大小,至实验结点共35 d,取出肿瘤组织制作切片并通过DNA末端转移酶（terminal deoxynucleotidyl transferase,TdT）介导的dUTP缺口末端标记（TdT-mediated dUTP nick-end labeling,TUNEL）实验和免疫组织化学检测增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）的表达,评价二甲双胍对OSCC凋亡和增殖的影响.结果 分别采用5、10、20和40 mmol/L二甲双胍处理HSC-3和HSC-4细胞48和72h后MTT检测结果显示细胞增殖均减少,72h后克隆形成被明显抑制;TUNEL结果显示经50 mmol/L二甲双胍药物处理后细胞凋亡率增加到30％（对照组低于5％）;小鼠体内实验结果显示,预先或同时口服二甲双胍的小鼠肿瘤体积较对照组分别下降了82.5％和63.9％,腹腔注射组比对照组肿瘤体积减小62.8％.小鼠肿瘤组织TUNEL检测结果显示,使用过二甲双胍小鼠的肿瘤组织细胞凋亡率增加,口服药物组分别为50％（同时口服）和70％（预先口服）,腹腔注射组为25％,而两组对照均低于10％;免疫组织化学染色检测PCNA的表达下降,以对照组为100％计算,实验组PCNA阳性细胞均低于60％.以上结果实验组与对照组相比差异均有统计学意义（P＜0.05）.结论 二甲双胍可通过抑制OSCC细胞的增殖和克隆形成、促进细胞凋亡,在体外内抑制HSC-3和HSC-4细胞系的生长和增殖,具有作为肿瘤临床治疗辅助用药的潜力.

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.