Study On Apoptosis Of Cisplatin-resistant Human Epithelial Ovarian Cancer SKOV-3/DDP Cells Induced By Diallyl Trisulfide

Objective:To investigate the apoptosis-inducing and proliferation inhibition mechanism,which are induced by Diallyl Trisulfide (DT) or DT combined with cisplatin (DDP)on cisplatin-resistant human epithelial ovarian cancer cell lines SKOV-3/DDPconstructed in vitro.The results provide the experimental basis for clinical treatmentof cisplatin-resistant ovarian cancer.Methods:Cultured cisplatin-resistant human epithelial ovarian cancer SKOV-3/DDP cellswere randomly divided into the control group, DT-treated groups(5,10,20,40μg/ml),DDP-treated groups(5,10,20,40μg/ml), DDP(5μg/ml)＋DT(20μg/ml) treatedgroup.The inhibitory rate was evaluated by MTT assay, morphological changes ofapoptosis were observed with Optical microscope、Giemsa Staining and fluorescencemicroscope. Cell apoptosis was determined by Flow Cytometry. Caspase-3activitywas tested by spectrophotometer. RT-PCR and Western blot were used to detectmRNA and protein of PUMA, Bax, Bcl-2and Bcl-xl respectively.Results:1. DT and DDP displayed certain degree proliferation inhibitory effect in a dose-and-time-dependent manner against SKOV-3/DDP cells. There was significant difference(P<0.05or P<0.01) to compare control group OD value of cells after treated withvaried concentration of DT and DDP. Moreover the OD value after treated with DTwas significantly lower than that after treated with DDP in identical concentration andworking time, and there was significant difference(P<0.05or P<0.01). The inhibitionratio on the cell growth of SKOV-3/DDP after treated with DT was higher than thatafter treated with DDP.2. DDP combined with DT group displayed proliferation inhibitory effect againstSKOV-3/DDP cells. The inhibitory rate was significant difference (P<0.01) tocompared with either DT or DDP alone. DDP combined with DT has a synergisticeffect. 3. SKOV-3/DDP cells treated with DT and DDP combined DT showed specific andtypical morphology of apoptosis.4. DT induces to apoptosis of SKOV-3/DDP. DT could increase apoptosis rate. Therate of early apoptosis (25.7±4.41）%of the40μg/ml48h group was significantlyhigher than that of control group（4.5±1.12）%（P<0.01）. Early apoptosis of the DTcombined with DDP group higher than that of these two kinds of drug usingseparately (P<0.05or P<0.01).5. DT could increase Caspase-3activity. Caspase-3activity（2.78±0.16）of the40μg/ml48h group was significantly higher than that of control group（P<0.01）.Caspase-3activity of DT combined with DDP group higher than that of these twokind of drug using separately(P<0.01).6. RT-PCR and Western blot results indicated that DT group and DT combined withDDP group up-regulated the apoptosis related PUMA and Bax mRNA and its proteinexpression（P<0.01), and down-regulated the Bcl-2and Bcl-xl mRNA and its proteinexpression（P<0.01). Moreover, DT combined with DDP group up-regulated theexpression of PUMA and Bax higher than use alone（P<0.01）and down-regulated theexpression of Bcl-2and Bcl-xl lower than use alone（P<0.01).Conclusions:1. DT can inhibit proliferation of the cisplatin-resistant human epithelial ovariancancer cell lines and enhance the cell chemosensitization to DDP.2. DT induces to apoptosis of the SKOV-3/DDP cells. The possible molecularmechanism of apoptosis by regulating expression of PUMA、Bax、Bcl-2and Bcl-xland strengthening the activity of Caspase-3.3. DT combined with low-dose DDP can induce to apoptosis higher than use alonewhich can reduce the dose of cisplatin, consequently reduce the side effects ofcisplatin and improve the outcome.