The Expression Of APE1 And Its Correlation With Sensitivity Of Platinum Chemotherapy In Non-small-cell Lung Cancer

Non-small-cell lung cancer(NSCLC) accounts for 80%of all lung cancer cases and is the leading cause of cancer mortality.The treatment of advanced NSCLC is based on the combination of platinum and one of the following agents:paclitaxel,docetaxel, gemcitabine or NVB.There are no significant differences in efficacy among these combinations suggesting that the outcome of platinum therapy on NSCLC have reached a plateau.Therefore,the biological mechanisms of cisplatin action need to be understood in order to overcome the treatment plateau on NSCLC.The molecular target of platinum action is the cellular DNA,which hampers DNA replication and transcription,resulting in cell death.Moreover,the development of resistance is a hurdle in the use of this drug.The molecular mechanisms that underlie this chemoresistance are largely unknown.Possible mechanisms of acquired resistance to platinum include reduced intracellular accumulation of platinum,enhanced drug inactivation by metallothionein and glutathione,increased repair activity of DNA damage,and formation of cisplatin-DNA adducts,et al.DNA-repair systems,as the molecular basis of defending against environmental damage to cellular DNA,play an important role in protecting the genomic stabilization and integrity.However,an elevated DNA repair capacity in tumor cells leads to drug resistance and severely limits the efficacy of platinum.The human apurinic/apyrimidinic endonuclease (APE1),is abundant in human cells and accounts for nearly all of the abasic site cleavage activity observed in cellular extracts.In addition to its DNA repair functions,APE1 is also a multifunctional protein that participates in other crucial cellular processes,including the response to oxidative stress and regulation of transcription factors.The transcription factors are associated with chemoresistance.Therefore,targeting inhibition of APE1 display an emerging cancer therapeutic opportunity.In this study,we first investigate the expression of APE1 and its correlation with sensitivity of platinum chemotherapy in NSCLC patients. Then,we investigated the effect of adenoviral vector Ad5/F35 carrying human APE1 siRNA on the sensitivity of cisplatin in human NSCLC cells.Objective1.To research the expression of APE1 protein in NSCLC cells,analyze the relationship between APE1 expression and NSCLC clinical effect,and determinate that APE1 is an effective therapeutic target gene of NSCLC.2.To investigate the perspective of clinical application of gene therapy targeting APE1 gene enhancing the sensitivity of NSCLC cells to DDP.Materials and Methods1.The expression of APE1 protein in NSCLC cells and its significanceNSCLC patients and their medical records were collected.The expression of APE1 in NSCLC patients and NSCLC cell lines(A549) were detected by immunocytochemical staining and Western blot,then analyzed relationship between expression of APE1 and clinical classification,clinical effect of NSCLC.2.Study of Ad5/F35-APE1 siRNA enhancing sensitivity of human NSCLC cells to DDPCells were treated with DDP at various concentrations 48 hours after Ad5/F35-APE1 siRNA or Ad5/F35-EGFP was transfered into A549 cells,and the cellular proliferation capacity was observed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.Cell apoptosis was determined by TUNEL.The expression of human APE1 protein was detected by western blot analysis.Results1.The expression of APE1 protein in NSCLC cells and its significanceProtein expression of APE1 was noticed in all non-small cell lung carcinomas and normal lung tissue.The APE1 expression of normal lung tissue mainly located in the nucleus,while APE1 expression of non-small cell lung carcinomas tissue mainly located in cytoplasm.In addition,APE1 expression was not related with the age and gender of patients,tumor size,lymphonode metastasis,nor the pathologic classification.It was not statistical significance that the patient one-year survival rate between APE1- high expression group and the low expression group(x~2=53.03,P＞0.05),but in the 2 and 3-years survival rates of APE1 low expression higher than the high expression(P＜0.05).In the 12 examples platinum drug fast group,APE1- high expression was 10 examples(83.33%),and in the 24 examples platinum sensitiveness group,APE1- high expression was 2 examples (8.33%),they were also notable statistical slgnlficance(x~2=9.06,P＜0.01).To detect gene APE1 will be conduced to judge the therapeutic effect and prognostic of NSCLC.2.Study of Ad5/F35-APE1 siRNA enhancing sensitivity of human NSCLC cells to DDPThe protein expression of APE1 in A549 cells was induced by DDP in a dosedependent manner.Infection of A549 cells with Ad5/F35-APE1 siRNA resulted in a dose-dependent suppression of APE1 protein in vitro.The result of MTT showed that Ad5/F35-APE1 siRNA enhanced sensitivity of A549 cells to DDP.The 50%inhibitory concentration(IC50) value for A549 cells pretreatmented with Ad5/F35-APE1 siRNA and Ad5/35-EGFP at 72 h after DDP treatment was 0.26μg/ml and 1.54μg/ml,respectively. Ad5/F35-APE1 siRNA also increased cell apoptosis induction by DDP.Conclusion1.The shifts of APE1 from nucleus to cytoplasm might play a pivotal role in the carcinogenesis,progression and metastasis of NSCLC.High expression of APE1 protein may indicate poor prognosis,and correlated with the DDP drug resistance.It suggests that APE1 is a potential molecular therapeutic target of NSCLC.2.The protein expression of APE1 in NSCLC cells was induced by DDP in a dose-dependent manner,which suggests that elevated DNA repair capacity may partially contribute to the resistance of DDP in NSCLC cells.3.Inhibiting APE1 expression could improve sensitivityof DDP remarkably and induce apoptosis in NSCLC cells.Therefore,gene therapy targeting APE1 gene shows a promising approach in enhancing the sensitivity of NSCLC to chemotherapy.