An Experimental Study On Combined APE1 SiRNA And P53 Gene Therapy For Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC) is one of the most frequent and devastating malignancies in China,and early diagnosis is very difficult,therefore with poor prognosis and high mortality.Even with the combined use of the three classic treatment modalities: surgery,radiation,chemotherapy,little has changed over the last five years to alter the deathly outcome.The rapid development of molecular biology makes the gene therapy a kind of new treatment mode in clinical application.Because the carcinogenesis and progression of HCC is a complicate process involved many genes and several transforming steps,the combination gene therapy against HCC has emerged as a promising approach.The human apurinic/apyrimidinic endonuclease(APE1) is a ubiquitous multifunctional protein which has both DNA repair activity and redox regulatory activity.APE1 plays an important role in maintaining genomic integrity and regulating gene expression,and its abnormal expression is associated with anti-apoptosis and tumor progression.Wild-type p53 plays a key role in cell cycle control and apoptosis,and the mutation of p53 gene is correlated with the carcinogenesis and progression of HCC.So we hypothesis that the APE1 and mutant p53(mtp53) may contribute to the resistance to apoptosis in HCC.In this study,we first detected the expression of APE1 and investigated its correlation with the expression of mtp53 and clinic-pathological parameters in HCC.Then,we examined the effect of chimeric adenoviral vector Ad5/F35 carrying human APE1 siRNA combined with recombinant adenovirus carrying wild-type p53 on cell proliferation inhibition and apoptosis induction in human hepatocellular carcinoma cells in vitro and in vivo.The present study will present a new strategy for combined gene therapy against cancer.Objective1.To investigate the expression of APE1 and explore its correlation with the expression of mtp53 and clinic-pathological parameters in HCC. 2.To investigate the inhibitory effects of Ad5/F35-APE1 siRNA combined with Ad-p53 on human HCC cells in vitro.3.To investigate the inhibitory effects of Ad5/F35-APE1 siRNA combined with Ad-p53 on human HCC cells in a tumor-bearing nude mice model.Materials and Methods1.Expression feature of APE1 and mtp53 and its significance in HCC:Expression of APE1 and mtp53 was detected by S-P immunohistochemical method in 10 cases of normal liver tissue,40 cases of liver cirrhosis and 103 cases of HCC.2.Study of inhibitory effects of Ad5/F35-APE1 siRNA combined with Ad-p53 on human HCC cells in vitro:The infection efficiency of Ad5/35-EGFP and Ad-EGFP to human HCC SMMC-7721 cell line was measured by flow cytometry and observed by fluorescence microscopy.The expression of human APE1 and p53 protein was detected after Ad5/F35-APE1 siRNA and Ad-p53 were transfered into SMMC-7721 cells by western blot analysis and immunohistochemical technique.The cellular proliferation capacity was observed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay. Cell growth curve was detected by trypanblau,and cell apoptosis was determined by TUNEL.3.Study of inhibitory effects of Ad5/F35-APE1 siRNA combined with Ad-p53 on human HCC cells in a tumor-bearing nude mice model:Nude mice hepatocarcinoma xenograft model was established by using human HCC SMMC-7721 cell line.When the tumor volumes reached 40～50 mm~3 on the sixth day after inoculation of SMMC-7721 cells, sixteen mice were randomly divided into four groups:control group,Ad5/F35-APE1 siRNA group,Ad-p53 group and Ad5/F35-APE1 siRNA combined with Ad-p53 group.The tumor volumes were measured and the tumor growth curves were plotted.The APE1 and p53 expression were observed by immunohistochemistry.Apoptosis index was detected by TUNEL technique.Results1.Expression feature of APE1 and mtp53 and its significance in HCC:In normal liver tissue,APE1 was detected in nucleus of cells,and the shifts of APE1 from nucleus to cytoplasm was observed in liver cirrhosis and HCC tissues.There was no significant difference in the positive nuclear expression rate of APE1,while there was a significant difference(p＜0.01) in the positive cytoplasmic expression rate of APE1 among normal liver tissue,liver cirrhosis and HCC tissues.There was a significant difference(p＜0.01) in the cytoplasmic and nuclear staining intensity of APE1 among normal liver tissue,liver cirrhosis and HCC tissues,and the cytoplasmic and nuclear staining intensity of APE1 was correlated with histological grade of HCC(p＜0.01).The location of APE1 combination with the p53 status was significantly correlated with tumor grade,and the cytoplasmic APE1 expression/p53~+ status implied a higher tumor grade(p＜0.01).2.Study of inhibitory effects of Ad5/F35-APE1 siRNA combined with Ad-p53 on human HCC cells in vitro:The results showed that the infection efficiency of Ad5/35-APE1 siRNA at 20 multiplicity of infection(MOI) and Ad-p53 at 50 MOI to SMMC-7721 cells was 99%and 85%,respectively.Infection of SMMC-7721 cells with Ad5/F35-APE1 siRNA resulted in a dose-dependent suppression of APE1 protein,and the APE1 protein expression lowest on 48～72h.Infection of SMMC-7721 cells with Ad-p53 resulted in a dose-dependent enhancement of p53 protein,and the p53 protein expression peaked on 48～72h.Apoptosis and inhibition of cell growth were induced by infection with Ad5/F35-APE1 siRNA or Ad-p53.Apoptosis index(AI) and inhibition effect of cell growth in Ad5/F35-APE1 siRNA combined Ad-p53 group was significantly higher,compared with Ad5/F35-APE1 siRNA or Ad-p53 alone(p＜0.01).3.Study of inhibitory effects of Ad5/F35-APE1 siRNA combined with Ad-p53 on human HCC cells in a tumor-bearing nude mice model:Ad5/F35-APE1 siRNA suppressed significantly the expression of APE1 protein,and Ad-p53 enhanced significantly the expression of p53 protein in vivo.The tumor growth inhibition ratio and AI in Ad5/F35-APE1 siRNA combined with Ad-p53 group were significantly higher,compared with the control group,Ad5/F35-APE1 APE1 group and Ad-p53 group(p＜0.01).Conclusion1.The shifts of APE1 from nucleus to cytoplasm and overexpression might play a pivotal role in the carcinogenesis and progression of HCC.Cytoplasmic expression of APE1 might be a useful marker for predicting carcinogenesis of hepatocyte.2.APE1 combination with the p53 status may be risk factors for HCC.It suggests that APE1 and p53 are potential molecular therapeutic targets of HCC.3.Ad5/F35-APE1 siRNA can specifically knock down the APE1 protein expression in HCC cells,and the inhibition rate of expression of APE1 protein reached in 95%.Ad-p53 can specifically raise the p53 protein expression in HCC cells,and the enhancement rate of expression of p53 protein reached in 95%.4.Ad5/F35-APE1 siRNA combined with Ad-p53 transfer can significantly inhibit the growth of HCC,promote tumor cells apoptosis.Therefore,the gene therapy with Ad5/F35-APE1 siRNA combined Ad-p53 may be a promising approach to treat HCC in the future.