二甲双胍对雌激素诱导的子宫内膜癌细胞增殖及ER表达的影响

Effects of metformin on the estrogen-induced proliferation and the expression of ER in human endometrial cancer cells

目的 探讨二甲双胍对雌激素诱导的子宫内膜癌细胞增殖及ER表达的影响.方法 不同浓度（分别为0.5、1、5、10、15、20、25、30 mmol/L）二甲双胍作用于子宫内膜癌细胞系Ishikawa和HEC-1A细胞不同时间（分别为24、48、72 h）后,采用四甲基偶氮唑蓝（MTT）比色法检测Ishikawa和HEC-1A细胞的增殖情况.17β雌二醇（1×10-6mol/L）单独及联合二甲双胍（5 mmol/L）分别作用Ishikawa和HEC-1A细胞24 h后,通过5-溴脱氧尿嘧啶核苷（BrdU）标记法检测Ishikawa和HEC-1A细胞的增殖情况;不同浓度（分别为1、5、15 mmol/L）二甲双胍作用24 h后,实时荧光定量PCR技术检测Ishikawa和HEC-1A细胞中原癌基因c-fos和c-myc mRNA的表达水平,蛋白印迹法检测Ishikawa和HEC-1A细胞中ERα和ERβ蛋白的表达水平.结果 MTT比色法检测显示,与对照（仅加入培养基）细胞比较,上述不同浓度的二甲双胍作用不同时间后,Ishikawa和HEC-1A细胞增殖的抑制率均明显增加（P＜0.05）,且随着二甲双胍浓度的增加及作用时间的延长,其抑制作用呈明显的剂量及时间依赖性（P＜0.05）.BrdU标记法检测显示,17β雌二醇联合二甲双胍作用后Ishikawa、HEC-1A细胞的增殖率分别为（62±7）％、（72±6）％,分别与17β雌二醇单独作用后的Ishikawa、HEC-1A细胞的增殖率[分别为（124±16）％、（109±5）％]比较,差异均有统计学意义（P＜0.01）.实时荧光定量PCR技术检测显示,不同浓度（分别为1、5、15 mmol/L）二甲双胍作用后,Ishikawa、HEC-1A细胞中c-fos和c-myc mRNA表达水平均逐渐降低,除HEC-1A细胞中1 mmol/L的二甲双胍作用后c-myc mRNA表达水平与相应对照（即二甲双胍浓度为0）细胞比较差异无统计学意义（P=0.074）外,其余浓度分别与相应对照比较差异均有统计学意义（P＜0.05）;蛋白印迹法检测显示,随着二甲双胍浓度的增加,Ishikawa、HEC-1A细胞中ERα蛋白的表达水平逐渐下降,而ERβ蛋白的表达水平逐渐增加,其浓度为5 mmol/L和15 mmol/L时分别与相应对照细胞比较,差异均有统计学意义（P＜0.05）.结论 二甲双胍能抑制雌激素对子宫内膜癌细胞的促增殖作用,其作用机制可能与二甲双胍调节ER的表达进而影响原癌基因表达有关.

Objective To assess the effects of metformin on estrogen-induced proliferation of human endometrial cancer cell lines and investigate whether metformin could regulate the expression of ER and estrogen-dependent proliferative genes.Methods Human endometrial cancer cell lines Ishikawa and HEC-1A underwent treatment with metformin at various concentrations （0.5,1,5,10,15,20,25 and 30 mmol/L） for different durations （24,48 and 72 hours）,followed by assessment of cell proliferation by methyl thiazolyl tetrazolium （MTT） assay.Ishikawa and HEC-1A cells were exposed to 17β-estradiol （1× 10-6mol/L） alone or in combination with metformin （5 mmol/L） for 24 hours.Cell proliferation was assessed by 5-bromo-2-deoxyuridine （BrdU） assay.Twenty-four hours after metformin treatment at the concentrations of 1,5 and 15 mmol/L,the expression levels of estrogen-dependent proliferative genes c-fos and c-myc were determined by real-time quantitative fluorescent PCR （real-time FQ-PCR）.Western blot analysis was performed to assess the effects of metformin on the expressions of estrogen receptors.Results As revealed by MTT assay,at different time points of metformin treatment at different concentrations,the proliferation rates of both cell lines were inhibited in a dose-dependent and time-dependent manner between metformin groups and the control group （P＜0.05）.BrdU assay showed that the proliferation rate of Ishikawa and HEC-1A cells exposed to 17β-estradiol （1 × 10-6 mol/L） in combination with metformin （5 mmol/L） was （62±7）％ and （72±6）％,respectively,while that in 17[β-estradiol groups was （124± 16）％ and （109±5）％,respectively,with significantly statistical differences （P＜0.01）.By real-time FQ-PCR tested,the expression levels of c-fos and c-myc in both cell lines gradually declined subsequent to metformin treatment at different concentrations （1,5 and 15 mmol/L）.As compared with the control group,the c-myc and c-fos expressions in both cell lines in metformin groups had significant differences （P＜0.05） except for the c-myc expression of the concentration of 1 mmol/L in HEC-1A cell line （P=0.074）.Western blot analyses showed that with the increasing concentrations of metformin,the ERα expression was markedly down-regulated,while ERβ expression was up-regulated in the metformin group at the concentrations of 5 mmol/L and 15 mmol/L,compared to those at the control group,there were significant differences between them,respectively （all P＜0.01）.Conclusion Metformin could inhibit estrogen-mediated proliferation of human endometrial cancer cells,which might be correlated with its regulation of the expressions of estrogen receptors and estrogen-dependent proliferative genes.