| Object:Previously we found that TP can inhibit the growth of cisplatin-resistant human epithelial ovarian cancer cell and induce apoptosis.However,TP blongs to the lab supplies which cannot be used in clinic.Tripterygium glycosides(GTW)is widely used in clinic to treat immune diseases,and TP is the main active component of GTW.GTW can inhibit growth of nasopharynx cancer cell and various hematological malignant tumor cells but there is no report that GTW can inhibit growth of cisplatin-resistant human epithelial ovarian cancer cell.So this study is about observing the cisplatin sensitization effect of tripterygium glycosides(GTW)on cisplatin-resistant human epithelial ovarian cancer cell(SKOV3/DDP)and studying its mechanism in virtro to provide experimental basis for GTW becoming adjuvant therapy or chemotherapy-sensitization drug in treating cisplatin-resistant ovarian cancer.Methods:1.SKOV3/DDP cells were treated with different concentrations of DDP(0μg/ml、0.625μg/ml、1.25μg/ml、2.5μg/ml、5μg/ml、10μg/ml、20μg/ml、40μg/ml、80μg/ml)respectively for 24 hours.Cell viability was examined by CCK8.2.SKOV3/DDP cells were treated with different concentrations of GTW(0μg/ml、50μg/ml、200μg/ml、800μg/ml、1600μg/ml、3200μg/ml)respectively for 24 hours.Cell viability was examined by CCK8.3.The SKOV3/DDP cell in log phase was randomly divided into eight groups:(1)blank control group,(2)10μg/ml DDP group,(3)50μg/ml GTW group,(4)800μg/ml GTW group,(5)3200μg/ml GTW group,(6)10μg/ml DDP+50μg/ml GTW group,(7)10μg/ml DDP+800μg/ml GTW group,(8)10μg/ml DDP+3200μg/ml GTW group.After 24 hours drug treating cell viability of each group was examined by CCK8.4.Cell apoptosis of the above eight groups were examined by flow cytometric method.5.Cell wound scratch assay was used to analyze the average migration distance of cells of the above eight groups after drug treating.6.Transwell assay was used to analyze the change of migration ability of cells of the above eight groups after drug treating.7.Transwell invasion assay was used to analyze the change of invasion ability of cells of the above eight groups after drug treating.8.Western blotting was used to detect the expression of GST-pi,MDR1,STAT3,and p-STAT3 of cells of the above eight groups after drug treating.Results:1.SKOV3/DDP cells were cultivated with different dose(0.625,1.25,2.5,5,10,20,40,80μg/ml)of DDP for 24 h,the cell viability was 96.119±3.467%,80.646±3.054%,66.808±3.705%,51.642±1.316%,43.712±1.533%,34.403±2.917%,31.156±1.193%,27.240±2.220% respectively.0.625μg/ml DDP have no effect on the cell viability(P>0.05).The minimum inhibition concentration is 1.25μg/ml(P<0.01)and as the concentration of DDP increasing cell survival rate reduced significantly(P<0.01).2.SKOV3/DDP cells were treated with different dose(50,200,800,1600,3200μg/ml)of GTW for 24 h,the cell viability was 98.064±1.474%,93.942±2.756%,64.748±3.557%,27.883±2.841%,12.899±2.351% respectively.50μg/ml GTW have no effect on the cell viability(P>0.05).The minimum inhibition concentration is 200μg/ml(P<0.05)and with the increasing concentration of GTW cell survival rate reduced(P<0.05).3.SKOV3/DDP cells were treated with DDP and GTW.There was no statistic difference between the cell viability of 10μg/ml DDP+50μg/ml GTW group and that of 10μg/ml DDP group(P>0.05).The cell viability of 800μg/ml or 3200μg/ml GTW combined with 10μg/ml DDP group is significantly lower than that of corresponding single drug group and as the dose of GTW increasing the viability of combined drug group significantly reduced(P<0.01).4.The SKOV3/DDP cell in log phase was randomly divided into eight groups: blank control group,10μg/ml DDP group,50μg/ml GTW group,800μg/ml GTW group,3200μg/ml GTW group,10μg/ml DDP+50μg/ml GTW group,10μg/ml DDP+800μg/ml GTW group,10μg/ml DDP+3200μg/ml GTW group,the apoptosis rates of each group were 3.623±1.608%,12.090±1.418%,3.860±1.434%,13.107±2.231%,17.920±1.136%,13.563±3.133%,19.053±0.963%,34.580±2.722% repectively.There was no statistic difference between the apoptosis rate of 50μg/ml GTW group and that of blank control group(P>0.05)while differences between the apoptosis rates of other drug groups and that of blank control group were all statistically significant(P<0.01).There was no statistic difference between the apoptosis rate of 10μg/ml DDP+50μg/ml GTW group and that of 10μg/ml DDP group.The apoptosis rate of 800μg/ml or 3200μg/ml GTW combined with DDP group is significantly higher than that of corresponding single drug group(P<0.01)and with the increasing dose of GTW the apoptosis rate increased(P<0.01).5.The results of cell wound scratch assay showed average migration distance of cells in each group was(1)87.241±0.135μm,(2)29.266±1.252μm,(3)78.365±2.267μm,(4)24.324±1.947μm,(5)14.608±1.846μm,(6)25.606±1.191μm,(7)13.304±2.943μm,(8)2.901±1.005μm respectively.The migration distance of SKOV3/DDP cell of each drug group was significantly shorter than that of blank control group(P<0.01).The cell migration distance of GTW group became shorter with the increasing dose of GTW(P<0.05).The cell migration distance of DDP and GTW group was shorter than that of corresponding single drug group(P<0.05).6.The average number of migration cells of each group was(1)82.200±1.732,(2)31.800±0.872,(3)73.600±1.039,(4)25.867±0.757,(5)21.067±0.611,(6)23.067±1.222,(7)17.267±0.808,(8)7.867±0.231 respectively.The migration cells of each drug group were much less than that of blank control group(P<0.01).The number of migration cells of GTW group decreased as the increasing dose of GTW(P<0.05).The migration cells of GTW combined with DDP group were much less than that of corresponding single drug group(P<0.01).7.The average number of invasion cells of each group was(1)533.867±2.203,(2)32.733±0.462,(3)393.333±2.715,(4)161.467±0.902,(5)28.467±1.007,(6)29.467±1.102,(7)13.933±0.231,(8)8.600±0.346 respectively.The invasion cells of each drug group were much less than that of blank control group(P<0.01).The number of invasion cells of GTW group decreased as the increasing dose of GTW(P<0.05).The invasion cells of GTW combined with DDP group were less than that of corresponding single drug group(P<0.05).8.The SKOV3/DDP cell in log phase was randomly divided into the above eight groups.There was no difference in the expression of Gst-pi,MDR1,STAT3 in each group.The expression of p-STAT3 of 800μg/ml or 3200μg/ml GTW combined with DDP group was down-regulated than that of corresponding single drug group(P<0.05)and amount of p-STAT3 decreased progressively with increasing dose of GTW(P<0.05).Conclusions:1.GTW have the effect of inhibiting growth of cisplatin-resistant human epithelial ovarian cancer cell SKOV3/DDP and inducing its apoptosis.2.GTW can suppress cisplatin-resistant human epithelial ovarian cancer cell SKOV3/DDP migration and invasion.3.800μg/ml or 3200μg/ml GTW have synergistic effect with DDP to inhibit growth and induce apoptosis of SKOV3/DDP cell and to enhance sensitivity of SKOV3/DDP cell to cisplatin in a concentration dependent manner.4.GTW have synergistic effect with DDP to suppress migration and invasion of SKOV3/DDP cell in a dose dependent manner.5.GTW may inhibit the expression of p-STAT3 of STAT3 signaling pathway in SKOV3/DDP cell,and play a role in cisplatin sensitization effect to SKOV3/DDP cell. |
