| Objective:To observe the effects of miferpritone on drug resistance in ovarian cancer cell lines SKOV3/DDP, and the possible mechanisms were discussed in vitro.Methods:1. The cytotoxicity of mifepristone on SKOV3/DDP was detected by MTT assay, and then drew the dose-response curve to found out the nontoxic dose as the reversal dose (inhibition rate<5%). After using reversal dose's mifepristone combined with DDP, we examined the changes of cisplatin-resistance in SKOV3/DDP at different time points.2. The cell cycle changes at different time points were detected by Flow cytometry (FCM) after applying reversal dose's mifepristone on SKOV3/DDP.3. mRNA and protein levels of Smac in SKOV3 and SKOV3/DDP cell lines were assessed by RT-PCR and Western Blot, respectively, and the changes on SKOV3/DDP were also investigated after the treatment of reveral dose's mifepristone.Results:1. MTT assay results showed that mifepristone inhibited the proliferation of SKOV3 and SKOV3/DDP cells in a dose-dependent manner. The inhibition rate increased with the rising concentration. Also, it can be found that, when the consentration of mifepristone was<5μM, it could not obviously inhibit the proliferation of the two cell lines, and by variance analysis, there were significant differences between 5μM and the lower ones in the inhibition rate. So 5μM was chosen as the reversal dose, which combined with DDP was been used to SKOV3/DDP for 24,48 and 72 hrs, and the reversal fold were 1.36,1.91 and 2.48 respectively.2. FCM assay results showed that, after being treated by 5μM mifepritone for 24,48 and 72 hrs, the propotion of G0/G1 phase increased with time (48.7%,53.9%,55.1%, 59.0%), meanwhile, the propotion of S phase decreased (13.5%,9.6%,7.7%,5.8%). The cell cycle was blocked at G0/G1 phase.3. RT-PCR results showed that the Smac mRNA/β-actin mRNA value of SKOV3 and SKOV3/DDP were significantly different [1.00±0.05 vs 0.45±0.04 (P<0.01)]. After being treated with 5μM mifepristone for 24,48 and 72 hrs, the expression of Smac mRNA in SKOV3/DDP cells increased with time (0.50±0.03,0.65±0.06,0.73±0.04), the differences between the three groups were statistically significant (P<0.05), and the values were higher than before treatment(P<0.05), but lower than that of SKOV3.4. Westen Blot results showed that the Smac protein/β-actin protein value of SKOV3 and SKOV3/DDP were 0.67±0.03 and 0.40±0.03 respectively (P<0.01), the difference was significant. After being treated with 5μM mifepristone for 24,48,72 hrs, the protein expression level of the apoptotic protein Smac in SKOV3/DDP cells increased with time (0.42±0.01,0.46±0.01,0.50±0.02), the difference between the groups was statistically significant (P<0.05), and the values were higher than before treatment except 24 h, but lower than that of SKOV3 (P<0.05).Conclusions:1. Mifepristone could inhibit the proliferation of SKOV3 and SKOV3/DDP cells in a dose-dependent manner.5μM mifepristone has some reversal effect on cisplatin-resistance in SKOV3/DDP cells, and the reversal effect presents in a time-dependent manner.2. The possible mechanism of mifepristone, which reverse the cisplatin-resistance in SKOV3/DDP cells, was that it can block the cell cycle of SKOV3/DDP at the G0/G1 phase, and up-regulate the expression of Smac mRNA and protein, which could contribute the SKOV3/DDP cells to apoptosis, and then reverse the drug resistance. |
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