[Regulation of Bub1 mRNA expression in endometrial carcinoma Ishikawa cells by estrogen and paclitaxel].

OBJECTIVE

To explore the regulation of Bub1 mRNA expression in endometrial carcinoma cells by estrogen and paclitaxel.

METHODS

The high differentiated endometrial adenocarcinoma cells (Ishikawa cell line) were cultured in DMED/F12 supplemented with 10% fetal bovine serum (FBS) or phenol red-free DMED/F12 supplemented with 5% dextran-charcoal FBS (dcFBS). Firstly, the cells were stimulated by 10 nmol/L estradiol (17β-E(2)) or no hormonal stimulation as control group, and the cell proliferation was quantified at 24, 48 and 72 hours using cell counting kit-8 (CCK-8) method. Then the cells were stimulated with different concentrations of 17β-E(2) (0.1, 10, 1000 nmol/L) at different periods (5, 15, 30 minutes and 2, 4, 8, 12, 16, 24, 30 hours), the expression of Bub1 mRNA was detected by real-time quantitative PCR. Ishikawa cells were cultured with non-serum DMEM/F12 to be synchronized, and then were treated with different concentrations of paclitaxel (10, 100 nmol/L) for 8 and 24 hours. While, non-synchronized Ishikawa cells were exposed to 100 nmol/L paclitaxel for different periods (4, 8, 16, 24, 48 hours), and real-time quantitative PCR was also used to detect the expression levels of Bub1 mRNA. Data were presented as folds change relative to control group, in which values < 1 were down-regulated, and those > 1 were up regulated.

RESULTS

The proliferation rate of cells in the presence of 17β-E(2) was significantly higher than that of the control group after treated 24 hours (A value: 0.70 ± 0.08 vs. 0.86 ± 0.10, P = 0.049). Time-dependent experiments revealed that addition of 17β-E(2) could increase cell numbers during 72 hours period, while the expression level of Bub1 mRNA was decreased in Ishikawa cell. Dose-dependent experiments revealed maximal estradiol stimulation effects at 10 nmol/L (P = 0.020). After being treated with serum-free culture, Ishikawa cells were exposed to 10 nmol/L paclitaxel for 8 and 24 hours, and the expression of Bub1 mRNA decreased (0.403 ± 0.008 vs. 0.775 ± 0.144, P = 0.251). Compared to the control cells, the mRNA expression levels of Bub1 in cells treated by paclitaxel for 8 hours was significantly decreased (P = 0.009), while there was not significantly decreased at 24 hours (P = 0.396). When exposed to 100 nmol/L paclitaxel for 8 and 24 hours, the expression of Bub1 mRNA was also decreased (0.697 ± 0.017 vs. 0.850 ± 0.004, P = 0.061). Compared to the control cells, Bub1 mRNA expression was also significantly decreased (P = 0.038 and P = 0.019, respectively). While with serum free-treatment culture, when Ishikawa cells exposed to 100 nmol/L paclitxel for 4, 8, 16, 24 or 48 hours, the expression of Bub1 mRNA significantly increased (1.127 ± 0.105 vs. 1.614 ± 0.154 vs. 2.092 ± 0.179 vs. 1.381 ± 0.061 vs. 1.519 ± 0.182, P = 0.002), of which was significantly increased at 16 hours treatment.

CONCLUSION

Bub1 expression could be regulated by estradiol and paclitaxel, in which deregulated Bub1 expression may contribute to chemotherapeutic efficacy of paclitaxel.