Research of the influence and mechanism about metformin on the proliferation of differentiated endometrial carcinoma cells

Abstract

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.