Regulation of claudin-4 gene expression in endometrial adenocarcinoma Ishikawa cell line by progesterone

Abstract

To explore the regulation of claudin-4 expression in endometrial adenocarcinoma cell lines by progesterone. Ishikawa cells were treated with various concentrations of megestrol acetate (MA: 2, 5, 10, 15, 20 mg/L). After cultured for 24, 48 and 72 hours, cells growth were measured by methyl thiazolyl tetrazolium (MTT). The group of Ishikawa cells incubated with MA at the 50% inhibitory concentration (IC(50)) was selected for cell apoptosis assay by using transmission electron microscopy and flow cytometry method. Real-time PCR and western blot were used for detecting the mRNA and protein expression levels of claudin-4. The localization of claudin-4 was examined by immunofluorescent staining. The inhibitory effects of megestrol acetate on the growth of Ishikawa cells were dose-dependent and time-dependent. IC(50) of MA on Ishikawa cells was 15 mg/L after incubated for 72 hours. After MA treatment, Ishikawa cells showed shrinkage, nuclear chromatin condensation, fractures of nuclear membrane and endoplasmic reticulum expansion, even round apoptotic bodies were found. The apoptosis rate of cells before MA treatment was (0.076 ± 0.024)%, and the rate was (3.934 ± 0.816)% by MA treated for 72 hours, in which there were signicant difference (P < 0.05). The relative quantification of claudin-4 mRNA and protein of the cells before MA treatment were 0.64 ± 0.20 and 0.94 ± 0.18, while they were 0.47 ± 0.15 and 0.62 ± 0.15 after MA treated. The expression of claudin-4 was significantly decreased after MA treatment (P < 0.05). The localization of claudin-4 transferred from cytomembrane to cytoplasm and nucleus after MA treatment. MA could inhibite the growth of Ishikawa cells, in which the mechanism may be decrease the expression of claudin-4 and the apoptosis of cells. The distribution change of claudin-4 may be related to the anti-cancer effect of progesterone.