Wang Jinglu, Shi Huirong, Xie Ya, Peng Zheng
Department of Obstetrics and Gynecology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.
Department of Obstetrics and Gynecology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China; Email:
Zhonghua Fu Chan Ke Za Zhi. 2015 Sep;50(9):685-91.
To investigate the effects of metformin on cell proliferation in differentiation degree of endometrial carcinoma cells and related mechanisms.
The endometrial cancer cell lines Ishikawa and AN3CA were used. Cell proliferation was assessed after exposure to metformin with or without epithelial growth factor receptor (EGFR) inhibitor AG1478 by cell counting kit-8 (CCK-8) method. EGFR mRNA was determined by reverse transcription (RT)-PCR. The expression of phosphorylation EGFR (p-EGFR) and total EGFR (t-EGFR) and phosphorylation extracellular signal-regulated kinase 1/2 (p-ERK1/2) and total ERK1/2 (t-ERK1/2) were examined by western blot.
(1) CCK-8 experiment showed that metformin could inhibit the proliferation of endometrial cancer cells in a time-dependent manner and a dose-dependent manner (P < 0.05), but the inhibition of well differentiated cell line Ishikawa was lower than that in poorly differentiated cells AN3CA (P < 0.05). AG1478 also could inhibit the proliferation of endometrial cancer cells in a time-dependent manner and in a dose-dependent manner (P < 0.05), but the inhibition rate of well differentiated cell line Ishikawa was higher than that in poorly differentiated cells AN3CA (P < 0.05). Metformin + AG1478 also could inhibit the proliferation of endometrial cancer cells in a time-dependent manner and in a dose-dependent manner (P < 0.05), and the inhibition of combined with metformin and AG1478 was stronger than that with a single application of drugs, but the inhibition rate of Ishikawa was higher than that in AN3CA (P < 0.05). (2) RT-PCR method showed that different concentrations of metformin (0.01, 0.1, 1, 5, 10 mmol/L, respectively) for 24 hours, the expression level of EGFR mRNA in Ishikawa cells were respectively 0.74 ± 0.03, 0.61 ± 0.04, 0.46 ± 0.03, 0.31 ± 0.03 and 0.23 ± 0.03, the expression level of EGFR mRNA in AN3CA cells were respectively 0.79 ± 0.20, 0.61 ± 0.03, 0.50 ± 0.05, 0.32 ± 0.03 and 0.26 ± 0.04, the inhibition effect showed a significant concentration-dependent manner (all P < 0.01). (3) Western blot method displayed that the effect of metformin treated respectively 2, 4, 6 or 8 hours, there were not significant difference in the expression levels of t-EGFR protein and t-ERK1/2 between Ishikawa and AN3CA cells (all P > 0.05). But the expression levels of p-EGFR and p-ERK1/2 protein were significantly lower between two groups (P < 0.01), which showed a time-dependent manner (P < 0.01).
Metformin could inhibit the proliferation of endometrial cancer cells, the inhibition is associated with the differentiation degree of cancer cells. Metformin could enhance the EGFR signaling pathway inhibitor AG1478 inhibition of endometrial cancer cells, which may inhibit EGFR expression of phosphorylated proteins to inhibit the phosphorylation of ERK1/2 proteins and then inhibit proliferation of endometrial cancer cells.
探讨二甲双胍对子宫内膜癌细胞增殖及分化程度的影响及其相关机制。
采用子宫内膜癌细胞系Ishikawa和AN3CA。通过细胞计数试剂盒-8（CCK-8）法评估在有或无表皮生长因子受体（EGFR）抑制剂AG1478存在的情况下，二甲双胍对细胞增殖的影响。通过逆转录（RT）-PCR测定EGFR mRNA。采用蛋白质印迹法检测磷酸化EGFR（p-EGFR）、总EGFR（t-EGFR）以及磷酸化细胞外信号调节激酶1/2（p-ERK1/2）和总ERK1/2（t-ERK1/2）的表达。
（1）CCK-8实验表明，二甲双胍能以时间和剂量依赖的方式抑制子宫内膜癌细胞的增殖（P<0.05），但对高分化细胞系Ishikawa的抑制作用低于低分化细胞AN3CA（P<0.05）。AG1478也能以时间和剂量依赖的方式抑制子宫内膜癌细胞的增殖（P<0.05），但对高分化细胞系Ishikawa的抑制率高于低分化细胞AN3CA（P<0.05）。二甲双胍+AG1478同样能以时间和剂量依赖的方式抑制子宫内膜癌细胞的增殖（P<0.05），且联合使用二甲双胍和AG1478的抑制作用强于单一用药，但Ishikawa的抑制率高于AN3CA（P<0.05）。（2）RT-PCR法显示，分别用不同浓度的二甲双胍（0.01、0.1、1、5、10 mmol/L）处理24小时后，Ishikawa细胞中EGFR mRNA的表达水平分别为0.74±0.03、0.61±0.04、0.46±0.03、0.31±0.03和0.23±0.03，AN3CA细胞中EGFR mRNA的表达水平分别为0.79±0.20、0.61±0.03、0.50±0.05、0.32±0.03和0.26±0.04，抑制作用呈显著的浓度依赖性（均P<0.01）。（3）蛋白质印迹法显示，二甲双胍分别处理2、4、6或8小时后，Ishikawa和AN3CA细胞中t-EGFR蛋白和t-ERK1/2的表达水平无显著差异（均P>0.05）。但两组间p-EGFR和p-ERK1/2蛋白的表达水平显著降低（P<0.01），呈时间依赖性（P<0.01）。
二甲双胍可抑制子宫内膜癌细胞的增殖，该抑制作用与癌细胞的分化程度有关。二甲双胍可增强EGFR信号通路抑制剂AG1478对子宫内膜癌细胞的抑制作用，可能是通过抑制EGFR磷酸化蛋白的表达，进而抑制ERK1/2蛋白的磷酸化，最终抑制子宫内膜癌细胞的增殖。
