Study On Ad-PUMA Sensitizing Drug Resistant Choriocarcinoma Cells To Chemotherapeutic Agents

Objective Choriocarcinoma is a highly malignant tumor,which is relatively common in child-bearing ages of females.Drug resistance in choriocarcinoma contributes to poor clinical outcomes and it remains a significant obstacle to successful chemotherapy.Most anticancer drugs exert their cytotoxic effects by inducing apoptosis.Defects in one or more components of the apoptotic pathway may result in the resistance of cells to drug-induced apoptosis.PUMA,p53 up-regulated modulator of apoptosis,can cause rapid p53-dependent apoptosis and growth inhibition.Exogenous expression of PUMA enhances the sensitivity of some cancer cell lines to DNA-damage agents.In the present study,we sought to determine whether exogenous expression of PUMA by adenovirus could inhibit the growth of drug-resistant chriocarcinoma cells and enhance sensitivity of drug-resistant cells to chemotherapeutic agents.Method An adenovirus expressing PUMA(Ad-PUMA),alone or in combination with chemotherapeutic agents(5-Fu,Vp16,MTX),was used to treat drug resistant choriocarcinoma cells jeg-3/vp16 and parental jeg-3 respectively.The growth inhibitory and pro-apoptotic effects of PUMA in vitro and in vivo were examined. The mechanisms of PUMA-mediated growth suppression and apoptosis were investigated by analysis of Caspase 3 activation and the change of mitochondrial membrane potential.The levels of PUMA,p53 and Caspase 3 were detected by Western Blotting.Result PUMA was expressed lower in jeg-3/vp16 than in jeg-3.In addition to p-gp/MDRl induction,lower expression of PUMA contributed the drug resistance of choriocarcinoma cell.As a response to 5-Fu and Vp16 treatment,jeg-3 responded much more sensitively than jeg-3/vp16 did,though PUMA were both up-regulated, which suggested that the level of PUMA is a crucial factor in apoptosis induced by anticancer drugs.Ad-PUMA significantly inhibited the growth of jeg-3 and jeg-3/vp16 cells,and there was no significant difference in the effects of Ad-PUMA between parental and drug resistant cells.The cell proliferation was inhibited more significantly in both cell sublines when treated with combination of Ad-PUMA and the anticancer drugs(5-Fu, Vp16,MTX) than those treated with anticancer drugs alone.When PUMA 10MOI was combined with 5-Fu,Vp16 or MTX,IC₅₀ of drugs was decreased by 8.66,18.66 and 13.06 folds compared with those used with anticancer drugs alone in jeg-3/vp16, while in jeg-3,IC₅₀ was decreased only by 1.80,1.78 and 2.76 folds.The similar results could be seen in vivo.In the drug-resistant group,the inhibitory rate was increased from 14.57%to 78.93%(P＜0.05) in Vp16 and Vp16 combined with Ad-PUMA subgroups.While in the parental group,the inhibitory rate was increased more slightly,from 66.39%to 71.56%.Exogenous PUMA expression resulted in potent growth suppression of drug resistant choriocarcinoma cells through induction of apoptosis.Ad-PUMA alone induced apoptosis of jeg-3 and jeg-3/vp16.The results were confirmed by sub-G1 measurement and Annexin V staining.Rh 123 staining showed that Caspase 3 activated and△ψm decreased with Ad-PUMA infection in jeg-3,but it wasn't so much evident as seen in jeg-3/vp16.The percentage of apoptosis increased significantly when 5-Fu or Vp16 were combined with Ad-PUMA together observed from sub-G1 measurement and DAPI staining.From TUNEL staining,Ad-PUMA enhanced the apoptosis induced by Vp16 in xenograft tumors.Conclusion PUMA is an important player in the therapeutic responses to chemotherapeutic agents of choriocarcinoma cells.In addition to its role in inhibiting tumor growth,low dose of Ad-PUMA significantly restored the sensitivity of choriocarcinoma cells to chemotherapeutic agents in vitro and in vivo.The effect was more obvious when combing anticancer drugs with Ad-PUMA in drug resistant jeg-3/vp16.Adenoviral delivery of PUMA may be a valuable gene therapy tool for killing tumor cells and sensitizing drug-resistant choriocarcinoma cells to chemotherapeutic agents.