Effects And Mechanisms Of Proliferation And Apoptosis Of Human Ovarian Cancer HO8910 Cells Lines Induced By Antioxidant Pyrrolidine Dithiocarbamate

ObjectiveTo investigate the regulatory effect and the possible mechanisms of antioxidant pyrrolidine dithiocarbamate(PDTC) on proliferation and apoptosis in ovarian carcinoma cell line HO8910 in vitro. in order to evaluate PDTC as a potential drug for chemotherapy of ovarian cancer.Methods1. The cytotoxicity of PDTC, DDP, or PDTC combined with DDP to HO8910 cell line were detected by MTT chromatometry.2. Determination of cellular apoptosis by acridine orange/ ethidium bromide(AO/EB) double fluorescent staining.3. The apoptotic ratios were detected by flowcytometry using PI/Annexin staining method.4. Immunohistochemical assessment was performed for alterations in expression of IKK,NF-κB,XIAP,Bcl-2 and caspase-3.Results1. After exposure to 12,24,36hrs, PDTC (25μM,50μM,100μM,200μM) could significantly inhibit the proliferation of HO8910 cells in a dose-dependent and time-dependent fashion(P<0.01).The IC50 in this assay for PDTC was 393.2836,340.9378,275.7231μmol/L after exposure to 12,24,36 hrs.2. Administration of DDP combined with PDTC significantly improved cytotoxicity of DDP to HO8910. The inhibition rate was only 9.22%,19.9%,37.39%and 66.72% when cells were treated with 0.75μg/ml,1.5μg/ml,3.0μg/ml and 6.0μg/ml DDP alone for 24hrs. The inhibition rate increased to18.61%,37.23%,65.78%and 88.03% when cells were pretreated with 25μM PDTC 1h before exposed to 0.75μg/ml,1.5μg/ml,3.0μg/ml and 6.0μg/ml DDP. The differences between both methods were significant at different concentrations of DDP, P<0.05.3. After exposure to PDTC, staining with acridine orange and ethidium bromide determined apoptotic cells. Orange or red colors could be seen in the middle or late stages of apoptotic nuclei exhibited with a feature of highly condensed and uniformly stained chromatin. Those apoptotic nucleus were always found to become smaller than the normal cells. Somewhere, apoptotic bodies might be detected among the cells.4. In contrast to the control, PDTC could significantly induce apoptosis of human ovarian cancer HO8910 cells by flowcytometry. After exposure to 24hrs, the apoptotic ratios was 4.38% in 25μM PDTC group,8.17% in 50μM PDTC, 17.16% in 100μM PDTC. There was significant differences between the four groups with different concentration of PDTC, P<0.01.5. The results of Immunohistochemical assessment showed that following 24 hours of treatment with PDTC (10,100μM ),the expression level of caspase-3 increased,the expression level of NF-κB in nucleus and Bcl-2,XIAP in cytoplasm decreased, significantly p<0.01,in HO8910 cell compared with the control group,and the expression level of NF-κB and IKKβin cytoplasm has no change.Conclusions1. Antioxidant PDTC could significantly inhibit the proliferation of HO8910 cells.2. Antioxidant PDTC could significantly induce apoptosis of HO8910 cells. The most possible anti-tumor mechanism of PDTC suppresses tumor growth and induces apoptosis by inhibiting activation of NF-κB, decreased the expression level of XIAP,Bcl-2 and increased the expression level of caspase-3.3. Antioxidant PDTC could significantly enhance the effect of DDP for chemotherapy and increase the therapeutic effect.4. Antioxidant PDTC might provide novel prospect for treatment of ovarian cancer.