Study On The Relationship Between APE1 Expression And Platinum Resistance In Ovarian Carcinoma

Ovarian carcinoma is one of the most frequent malignancies in women,and the leading cause of death from gynecological cancer. Early diagnosis is very difficult because of hiding symptom in early stage.Even with the combined use of surgery and chemotherapy,the five year survival rate of the patients is about 30%～50%.The major reason for the high mortality of ovarian carcinoma is the high metastatic ability and the resistance to platinum-based chemotherapy. The drug-resistant patients more tend to high relapse,high metastases and high mortality eventually.Identifying the molecules mechanism of the drug-resistance in tumor cells is a hot field for exploring the effective target for new eversal agent of drug resistance and new avaliable chemotherapeutics.The human apurinic/apyrimidinic endonuclease (APE1) is a ubiquitous multifunctional protein which has both DNA repair activity and redox regulatory activity. APE1 participates in some crucial cellular processes, including the response to oxidative stress, regulation of transcription factors. The transcription factors particpate with cellular proliferation,cell differentiation and apoptosis,such as NF-κB,p53,AP-1 etc.Meanwhile,these transcription factors are associated with chemoresistance and radioresistance. In this study, we detected the expression of APE1 protein in 120 cases ovarian carcinoma patients and two ovarian carcinoma cell lines.A2780 cell line is sensitive to cisplatin,and CP70 cell line is resistance to cisplatin.We investigated the relationship between the expression of APE1 and clinic-pathological parameters especially cisplatin- resistance of ovarian carcinoma. Then we used chimeric adenoviral vector Ad5/F35 carrying human APE1 siRNA to investigate its effect on the cispatin sensitivity in human ovarian carcinoma cells. After suppression APE1 protein,we investigated the cell proliferation inhibition and apoptosis induced by cisplatin in two kinds of human ovarian carcinoma cells lines in vitro, and presume that the APE1 protein may contribute to the cisplatin-resistance in ovarian carcinama.As a result,we offered the experiment evidence that the inhibiton of APE1 by chimeric adenoviral vector Ad5/F35 carrying human APE1 siRNA can influence the chemoresistance of ovarian carcinoma cells to cisplatin. Therefore,APE1 siRNA is a potiential new eversal agent of chemoresistance.OBJECTIVES:1. To investigate the expression of APE1 protein in ovarian carcinoma cases.To identify APE1 gene is potential molecules target of gene therapy against cancer.2. To investigate the relationship between APE1 expression and the clinical cisplatin resistance cases in ovarian carcinoma. To investigate the expression of APE1 protein in ovarian carcinoma A2780 and CP70 cell lines, and analysis the role of APE1 gene in platinum-resistance of ovarian carcinoma.3. To investigate the inhibitory effects of Ad5/F35-APE1 siRNA on human ovarian carcinoma in vitro, and explore the effect of enhancing chemosensitivity of ovarian carcinoma by inducing APE1 gene silence. METHODS:1. Expression of APE1 was examined by SP immunohistochemical technique in 120 cases of ovarian carcinoma. The medical records of the ovarian carcinoma patients were collected. The data was analyzed to explore the correlation between APE1 expression and clinical pathological parameters.2. Expression of APE1 in A2780 and CP70 cells was detected by western blot and SP immunohistochemical technique. Then we detected the change of APE1 protein expression of A2780 and CP70 cells which were pretreated with cisplatin by western blot and immunocytochemical staining.It was analyzed that relationship between APE1 expression and cisplatin resistance in ovarian carcinoma.3. Cells were treated with cisplatin after Ad5/F35-APE1 siRNA transient transfection into A2780 and CP70 cells, and the cellular proliferation capacity was observed with MTT assay. To further verify that the effect of Ad5/F35-APE1 siRNA,cellular apoptosis was determined by TUNEL .RESULTS:1. Statistic analysis showed that APE1 expression was related with maligancy biological behaviour such as pathologic classification ,FIGO stage and cell differentiated classify (P<0.05). The intensity and subcellular of APE1 protein expression is related with platinum-based chemotherapy sensetivity.2. We found that APE1 almost localized in cytoplasmic in A2780 cell, but in cytoplasmic and nucleus in CP70 cell,and the APE1 expression in CP70 cell is higher than A2780 cell. The result of Western blot showed that expression of APE1 gene was high in both of the two cell lines pretreated by DDP, the degree of expression of APE1 protein was a prositive corretation with DDP dose and treating time. 3. The influence of Ad5/F35-APE1 siRNA to platinum sensitivity in human ovarian carcinoma cells:(1)The result of MTT showed that Ad5/F35-APE1 siRNA enhanced platinum sensitivity of A2780 and CP70 cell lines.Pretreatmented with 40MOI Ad5/F35-APE1 siRNA ,the 50% inhibitory concentration (IC50) value of cisplatin in A2780 and CP70 cells was 6.60μM and 39.73μM, respectively, decreased by 61.89% and 41.47% compared by pretreatmented with control group.(2)Ad5/F35-APE1 siRNA increased cell apoptosis induction by DDP. Apoptotic index of A2780 and CP70 cell lines respectively significantly increased by transfecting APE1siRNA. it had significant statistics difference(P<0.01).CONCLUSION1.Expression of APE1 and subcellular location was correlated with ovrian carcinogenesis. To detect gene APE1 will be helpful to judge the therapeutic effect and prognostic of ovarian carcinoma.2.The level of APE1 expression and the subcellular location were notable different between A2780 and CP70 cell line.After DDP treated, APE1 expression was increased remarkably in both A2780 and CP70 cell lines.We presumed APE1 may partially contribute to drugresistance of ovarian carcinoma cells.3. Inhibiting APE1 expression improved cisplatin chemotherapy sensitivity remarkably and increase apoptosis in ovarian carcinoma cells. We hypothesis that the main molecular mechanisms involved may be downregulation of base excision repair capacity , regulate the cell generation cycle by its oxidoreduction function, and induce transcription activity of apoptosis pathway of ovarian carcinoma cells. Targeting inhibition of APE1 may be a promising approach to a new eversal platinum resistance agent in ovarian carcinoma.