Zhang Jingbo, Zhang Bei, Yin Zeyuan, Chen Feng, Liu Tong, Xu Hui, Liu Yuqing, Zhou Xueyan
Major in Obstetrics and Gynecology, Graduate School, Xuzhou Medical College, Xuzhou 221004, China.
Department of Obstetrics and Gynecology, Clinical School, Xuzhou Medical College, 221009 Xuzhou. Email:
Zhonghua Fu Chan Ke Za Zhi. 2014 Dec;49(12):932-7.
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.
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(-6) mol/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.
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 17β-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 ERa 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).
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.
评估二甲双胍对雌激素诱导的人子宫内膜癌细胞系增殖的影响，并研究二甲双胍是否能调节雌激素受体（ER）及雌激素依赖性增殖基因的表达。
人子宫内膜癌细胞系Ishikawa和HEC-1A分别用不同浓度（0.5、1、5、10、15、20、25和30 mmol/L）的二甲双胍处理不同时间（24、48和72小时），然后通过甲基噻唑基四氮唑（MTT）法评估细胞增殖。Ishikawa和HEC-1A细胞单独暴露于17β-雌二醇（1×10⁻⁶ mol/L）或与二甲双胍（5 mmol/L）联合暴露24小时。通过5-溴-2-脱氧尿苷（BrdU）法评估细胞增殖。在1、5和15 mmol/L浓度的二甲双胍处理24小时后，通过实时定量荧光PCR（实时FQ-PCR）测定雌激素依赖性增殖基因c-fos和c-myc的表达水平。进行蛋白质免疫印迹分析以评估二甲双胍对雌激素受体表达的影响。
MTT法显示，在不同浓度二甲双胍处理的不同时间点，二甲双胍组与对照组相比，两种细胞系的增殖率均呈剂量依赖性和时间依赖性抑制（P < 0.05）。BrdU法显示，暴露于17β-雌二醇（1×10⁻⁶ mol/L）联合二甲双胍（5 mmol/L）的Ishikawa和HEC-1A细胞的增殖率分别为（62±7）%和（72±6）%，而在17β-雌二醇组中分别为（124±16）%和（109±5）%，差异有统计学意义（P < 0.01）。通过实时FQ-PCR检测，在不同浓度（1、5和15 mmol/L）的二甲双胍处理后，两种细胞系中c-fos和c-myc的表达水平逐渐下降。与对照组相比，除HEC-1A细胞系1 mmol/L浓度的c-myc表达外（P = 0.074），二甲双胍组两种细胞系中的c-myc和c-fos表达均有显著差异（P < 0.05）。蛋白质免疫印迹分析显示，随着二甲双胍浓度的增加，ERα表达明显下调，而在5 mmol/L和15 mmol/L浓度的二甲双胍组中ERβ表达上调，与对照组相比，它们之间分别有显著差异（均P < 0.01）。
二甲双胍可抑制雌激素介导的人子宫内膜癌细胞增殖，这可能与其对雌激素受体及雌激素依赖性增殖基因表达的调节有关。
