An Experimental Study On Combined Treatment Of Adenoviral Vector Ad5/F35-mediated APE1 SiRNA With Photodynamic Therapy For Non-small Cell Lung Carcinomas

Lung cancer is one of the most common malignancies,and is the leading cause of cancer death world wide.Moreover,the incidence rate and mortality of lung cancer tend to increase.The 3 main types of treatment for lung cancer are surgery,radiation therapy and chemotherapy.Although there is a great progress in treatment of lung cancer,5-year survival rates have not significantly improved,owing to complicated molecular mechanism involved in the carcinogenesis and development of lung cancer.Therefore,it is urgent to explore new approach for treatment of lung cancer.Photodynamic therapy(PDT) involves the application of a photosensitizer activated by visible light to generate reactive oxygen species(ROS),which can result in anti-cancer effects through direct lethal direct lethal effects on tumor cells and vascular impairment which limit blood supply to tumor region. But DNA damage and oxidative damage self-repairing capability of tumor cells to PDT decrease sensitivity of tumor cells to PDT.The human apurinic/apyrimidinic endonuclease(APE1) play important role in DNA repair functions and response to oxidative stress and regulation of transcription factors.In this study,we investigated the effect of adenoviral vector Ad5/F35 carrying human APE1 siRNA on the sensitivity of PDT to human NSCLC in vivo and in vitro.Objective1.To research the expression of APE1 protein in NSCLC ceils,analyze the relationship between APE1 expression and prognosis of NSCLC patients,and determine that APE1 is an effective therapeutic target gene of NSCLC.2.To investigate the perspective of clinical application of combined treatment of adenoviral vector Ad5/F35-mediated APE1 siRNA with photodynamic therapy for lung cancer.Materials and Methods1.The expression of APE1 protein in NSCLC cells and its significance.The expression of APE1 in NSCLC patients was detected by immunohistochemical staining.To demonstrate that APE1 is an effective therapeutic target gene of NSCLC,the relationship between expression of APE1 and prognosis of NSCLC patients was determined.2.Efficacy of combination of Ad5/F35-APE1 siRNA with PDT therapy on A549 cells. The efficiency of infection of Ad5/F35-APE1 siRNA on A549 cells was assessed by flow cytometry and fluorescence.After transfection with Ad5/F35-APE1 siRNA,the protein levels of APE1 in A549 cells were evaluated by western blot The AP incision enzyme activity was evaluated by[γ-³²p]ATP-marked oligonucleotide assays.The effect of combination of PDT and Ad5/F35-APE1 siRNA transfection of A549 cell proliferation was assessed by MTT assay.DNA damage was investigated by comet analysis.The cell apoptosis was assessed by flow cytometry.The expression of APE1 and VEGF were detected by western blot3.The models of naked mice bearing transplantation tumor were established.Then,the sizes of the tumors were measured and the relevant growth curves were created.Afterwards, cell apoptosis was assessed by TUNEL assay.Results1.APE1 was detected in both lung tissues and NSCLC.While in the lung tissue,it was mainly observed in nuclear,whereas in NSCLC tussues it was observed in plasma.As shown by Kaplan-Meier survival analysis,the survival time of APE1-high expression group (median survival time:35±5.196 months) was longer than that of APE1-low group(median survival time:.26±0.761 months).2.The infection efficiency of 10 MOI Ad5/F35-APE1 siRNA on A549 cells is 98%. Ad5/F35-APE1 siRNA transfection may inhibit APE1 protein expression in a dose-dependent manner.In the meantime,AP incision enzyme activity can be significantly inhibited.3.Application of PDT with Ad5/F35-APE1 siRNA transfection can significantly decrease the cell proliferation and increase the apoptosis rate of A549 cells.The results of comet assay showed that Ad5/F35-APE1 siRNA transfection can inhibit A549 cell DNA repairing ability after application of PDT.Ad5/F35-APE1 siRNA transfection can inhibit the increase in APE1 and VEGF protein induced by PDT.4.The results of in vivo study showed that tumor control rate in Ad5/F35-APE1 siRNA+PDT mice(93.97%) was higher than that of Ad5/F35-APE1 siRNA mice(34.85%) or PDT mice(52.19%).The results of TUNEL assay showed that apoptosis rate of Ad5/F35-APE1 siRNA+PDT group(24.68±2.47%) was significantly higher than that of Ad5/F35-APE1 siRNA group(6.24±1.32%),PDT group(12.12±1.76%) or control group (3.37±1.28%).Conclusion1.Aberrant expression of APE1 in NSCLC may have a correlation with prognosis of lung cancer.APE1 seems to be a target for treating lung cancer,which may be of great clinical significance.2.Ad5/F35-APE1 siRNA transfection can infect A549 cells easily,which might specifically knockdown their APE1 expression.Also,it can markedly weaken the activity of incision enzymes.3.Ad5/F35-APE1 siRNA transfection significantly enhances sensitivity of A549 cells to PDT treatment,possibly through inhibition of DNA repairing capability after treatment with PDT,or via suppression of oxidation repairing of A549 cells in response to PDT.4.PDT might upregulate the expression of APE1 in A549 cells.However, Ad5/F35-APE1 siRNA might reverse this elevation.5.Combination of Ad5/F35-APE1 siRNA transfection with PDT can significantly inhibit the growth of transplantation tumors in naked mice.