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.