Figure 4.

Metformin downregulates ERα and upregulates ERβ expression in Ishikawa and HEC-1-A cells in an AMPK-dependent manner. Reverse transcription-quantitative polymerase chain reaction analysis of ERα and ERβ expression in Ishikawa and HEC-1-A cells following treatment with (A) metformin (0, 1, 5 or 15 mM) or (B) 5 mM metformin pre-treatment with or without 5 µM compound C for 24 h. ERα and ERβ mRNA levels in each sample were calculated from a standard curve and normalized using GAPDH mRNA levels. Results represent the mean ± standard error of the mean of three independent experiments, each performed in triplicate (C) Western blot analysis of ERα and ERβ expression in Ishikawa and HEC-1-A cells following treatment with metformin (5 mM) and/or pre-treatment with compound C (5 µM) for 24 h. β-actin was used as a loading control. Results represent the mean ± standard error of the mean of three independent experiments. *P<0.05 compared with control. ^#P<0.05, 5+ Com group compared with the 5 mM metformin group. Con, control; 5, 5 mM metformin; 5+ Com, 5 mM metformin combined with pre-treatment with 5 µM compound C; Met, metformin; ER, estrogen receptor; AMPK, 5′ AMP-activated protein kinase.