Synergistic Effect And Mechanism Of BV6 And Cisplatin On Promoting Apoptosis Of SKOV3 Cells

Objective:To explore the effect and its mechanism of smac mimetic BV6 and cisplatin on the apoptosis of ovarian cancer cell line SKOV3 in vitro. Methods:1. The inhibitory effect of DDP 、BV6 and combination of two drugs on the growth of SKOV3 in different concentration was detected at different times by MTT assay.We calculated the IC50 of DDP and BV6 at different times and chose moderate dose of two drugs to continue combined experiment.2. SKOV3 cells were treated with DDP, different dose of BV6 and combination of two drugs for 48 hours. The growth of cells was observed under inverted microscope and the pictures from which were obtained.3. The mRNA and protein level of NF-κBp65 and caspase-3 in SKOV3 cells was investigated after the treatment of different drugs by RT-PCR and Western Blot.4. The results were analyzed with SPSS19.0. Results:1. MTT assay illustrated that DDP and BV6 could significantly inhibit the growth of SKOV3 cells with dose- time dependent manner respectively and the IC50 of two drugs was inversely proportional to the intervention time. MTT assay also showed that the inhibitory effect of BV6/DDP on SKOV3 cells was significantly higher than that of the two drugs alone and the difference was statistically significant(P<0.01). The combined effect of BV6 and DDP on the inhibition of the growth of SKOV3 cells was also in dose-time dependent manner(P<0.01).2. Inverted microscope showed that the density and size of SKOV3 cells decreased, proliferation activity decreased gradually, cell morphology changed from spindle to irregular, cytoplasm condensed, adherent cells decreased gradually and eventually lead to apoptosis after treatment of two drugs.3. The result of PCR and Western Blot revealed that the mRNA and total protein level of NF-κBp65 was decreased in the experimental group compared with control group and that of caspase-3 was increased,which was similar to BV6 / DDP group compared with the two drugs alone group(P<0.05). The mRNA and total protein level of NF-κBp65 decreased and that of caspase-3 increased when dose of BV6 went up(P<0.05). The mRNA and total protein level of NF-κBp65 was lower in middle and high dose-BV6 group than that in DDP group and that of caspase-3 was higher(P < 0.01),while that of two genes had no significant difference in low dose-BV6 and DDP group(P > 0.05).4. The result of Western Blot also revealed that the nuclear protein level of NF-κBp65 was increased in DDP group compared with control group(P<0.05) while that was decreased in other experiment groups(P<0.01). The nuclear protein level of NF-κBp65 decreased when dose of BV6 went up(P<0.05). The nuclear protein level of NF-κBp65 was lower in BV6/DDP group than that in DDP group(P<0.01) while they had no significant difference compared with BV6 group(P > 0.05).The protein activity of cleaved caspase-3 could be detected in high dose-BV6 group, medium dose-BV6/DDP group and high dose–BV6/DDP group and the protein level of cleaved caspase-3 was greater in high dose–BV6/DDP group than that in the others(P<0.05). Conclusions:1. BV6 and DDP could significantly promote apoptosis of SKOV3 cells with dose- time dependent manner respectively.2. The combination of BV6 and DDP showed a synergistic effect on the apoptosis of SKOV3 cells, which is in dose- time dependent manner.3. The role of BV6 in promoting apoptosis of SKOV3 cells may be related to its inhibition of the nuclear translocation of NF-κBp65 and the promotion of apoptosis cascade triggered by caspase-3.4. DDP may induce drug resistance of SKOV3 cells through the nuclear translocation of NF-κBp65 which could be reversed by the combination of BV6 and DDP.