The Effect Of Raf Kinase Inhibitor Protein On The Sensitivity Of Ovarian Cancer Cells To Cisplatin Treatment

Objective:Ovarian cancer SKOV-3 cells was transfected with Raf kinase inhibitor protein (RKIP) gene. we observed cells proliferation and apoptosis after treatment with cisplatin, to detect the RKIP on the sensitivity of SKOV-3 ovarian cancer to chemotherapy, and to discuss the underline molecular mechanism.Method:1. The recombinant plasmids expressing sense RKIP cDNA or empty vector was transfected into human ovarian cancer cell line SKOV-3 by lipofectamine, the positive cell clone was selected by G418.2. The expression level of RKIP protein in ovarian cancer cells were detected by Western Blot analysis.3. The expression level of RKIP gene in ovarian cancer cells were detected by Semi-Quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysis.4. Single-value-added detection solution (MTS) assay was performed to investigate the effect of RKIP on cell inhibition after treatment with cisplain.5. Flow cytometric (FCM) analysis was performed to observe the effect of RKIP on cell cycle and apoptosis after treatment with cisplain.Results:1. SKOV-3 clones stably expressing full-length recombinant pcDNA3.1(+)-ssRKIP and their corresponding empty vector pcDNA3.1(+)-transfected cells were obtained.2. Treatment with cisplatin for 24hrs,48hrs or 72hrs inhibited each group cell proliferation, but the RKIP transfected showed significant differences with other groups (P<0.05), the inhibition rate was significantly increased.3. After treatment of cisplatin at 2.5μg/ml for 24hrs, transfected cells showed a significantly higher percentage of apoptosis (0.86±0.73)% than control cells [(4.27±0.67)% for non-transfected cells and (4.02±0.43)% for cells transfected with an empty vector)]. Without cisplatin treatment, the percentage of apoptosis for cells transfected with RKIP is (3.11±0.78)%, which is still significantly higher than non-transfected cells (1.51±0.13)% and cells transfected with an empty vector (1.97±0.46)%(p<0.01).4. After treatment of cisplatin at 2.5μg/ml for 24hrs, there are fewer cells in G0/G1 phase and more cells in S phase in RKIP-transfected cells compared with control cells(P<0.05), suggesting that cisplatin caused more efficient S phase arrest in transfected cells.Conclusion:RKIP overexpression affected a series of influence on ovarian cancer cells, increase tumor cell inhibition ratio after cisplatin treatment, RKIP overexpression sensitize ovarian cancer cells to chemotherapy with cisplatin, and much more SKOV-3 cells were arrested in S phase, effectively delayed synthesis and repair of DNA.