| Objective: To study the relationship of the expressions of ERCCl BRCA1 hMLH1 and HSP27 gene proteins and cispatin resistance in ovarian cancer tissues and celll lines. The enhanced cisplatin cytotoxity in vitro and in vivo by mifepristone in ovarian cancer cells were evaluated and its machanisms were analyzed. The alterations of sensitivity to cisplatin by RNA interfering ERCC1 gene were explored in ovarian cancer cell lines.Methods: The expressions of those four proteins were examined in 58 ovarian cancer tissues by immunohistochemistry technique and their relationships to cislpatin resistance were analyzed. The RT-PCR method was used to assess the mRNA levels of those four genes in ovarian cancer cell lines ES-2 SKOV3 hMLHl and HSP27, and their changes were also observed after cisplatin of different concentration treatment. The enhanced sensitivity of drug-resistance ovarian cancer cell line COC1/DDP to cisplatin induced by mifepristone and its machanisms were investigated by means of MTT assay, flow cytometry, RT-PCR, western blot analysis. Meanwhile the subcutanous xenograft model in nude mouse of COC1/DDP cell line were established and the reversal effect on drug resistance of mifepristone was observed in vivo. The siRNA targeting ERCC1 gene mRNA were synthesized by in vitro transcription and transfected into ES-2 SKOV3 COC1/DDP cell lines respectively, which means by RNAi technique to close ERCC1 gene, then the efficacy of interference was evaluated by means of RT-PCR,western blot, and immunocytochemistry, the alterations of cisplatinsensitivity in those three cell lines were observed by MTT assay. Results:1. The positive rate of ERCC1 BRCA1 hMLHl and HSP27 proteins in 58 ovarian cancer tissues were 37.9%, 32.8%, 75.9%, 58.6% respectively, and the expressions of ERCC1 , BRCA1 in drug-resistant patients were significantly higher than that of drug-sensitive(p<0.05). There was no difference in the expressions of hMLHl and HSP27 between drug-resistant and sensitive patients (p>0.05) .2. The expressions of ERCC1 gene in those four ovarian cancer cell lines were associated with cisplatin IC50 values, while the other three genes were not. Low concentration (2.5ug/ml) of cisplatin could up-regulate ERCC1 gene in those four cell lines, while high concentration down-regulate it. The levels of BRCAl mRNA was higher in COC1/DDP cell line than sensitive COC1 cell line and their levels both were up-regulated induced by cisplatin. Cisplatin could up-regulate the hMLH1 genes in the cell lines whose basal hMLHl level was low, and the hMLHl gene were down-regulated with the increase of cisplatin concentration. Cisplatin could down-regulate the HSP27 gene in ES-2 and SK0V3 cell lines.3. 2.5uM,5.0uM,10.0uM mifepristone could increase cisplatin sensitivy in COC1/DDP cell line by 1.16, 1.95, 5.78 times respectively, cisplatin combined with mifepristone treatment could increase apoptosis rate, percentage of G0/G1 phase and up-regulate the ERCCK BRCAK hMLHl, HSP27 genes in COC1/DDP cell line.4. The rate of successfully inoculation of subcutanous xenograft was 96%, the percentage of inhibition of tumor volume in mifepristone , cisplatin. mifepristone combined cisplatin groups were 6.50% , 22.53% , 70.08% respectively, the combined treament groups had significant difference with thecontrol groups (p<0.01) The weights of nude mouse in each groups had no difference, and the livers and kidneys had no pathological alterations 5. 24,48,72 hours after transfection of the siRNA targeting ERCC1 mRNA, the levels of ERCC1 gene mRNA and protein both were down-regulated, and the sensitivity to cisplatin of ES-2, SKOV3, COC1/DDP cell lines increased by 53.88, 5.07, 3.75 times respectively. Conclusions:1. The expressions of ERCC1, BRCA1 proteins were associated with cisplatin sensitivity in ovarian epithelial carcinoma tissues, while the expressions of hMLH1, HSP27 were not.2. The levels of ERCC1 mRNA were associated with cisplatin sensitivity in ovarian cancer ce...
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