Effect Of APE1on Radiation-induced Angiogenesis In Lung Cancer

Lung cancer is the common malignancy and the leading cause of cancer deathworldwide. The survival of lung cancer has shown the least improvement for the past30years. More than85%of all lung cancers are NSCLC. Surgery, radiotherapy, andchemotherapy are the three common useful treatment modalities for the patients withNSCLC. With the development of radiation oncology, radiotherapy has become animportant approach of local and regional therapy for many types of cancer and it is also ahighly effective treatment method for NSCLC at the present. Nonetheless, the outcome ofradiotherapy alone is undesirable and many tumors are poorly controlled due to radiationresistance. What’s more vital is that radiotherapy would stimulate cancer invasion andmetastasis. Many studies have reported radiotherapy can stimulate multiple signaltransduction pathways simultaneously and alter the expression of proangiogenic moleculesincluding VEGF in surviving cancer cells and host cells, and the angiogenesis andproangiogenic molecules are essential for radiation resistance, tumor progression andmetastasis. But the exact mechanism is still not fully understood. The radiotherapycombined with gene therapy against tumor is an extremely important new modality.The human APE1is a multifunctional protein, which has DNA repair and redoxfunctions with two definitive DNA domains. APE1is an upstream effector of VEGF as wellas other molecules that relate to angiogenesis. Our previous studies have also indicated thatAPE1is a crucial angiogenic regulator and can regulate VEGF expression in osteosarcoma.APE1is high expression in NSCLC, which is associated with response to chemotherapy orradiotherapy. Based on these results and conclusion, it is reasonable to consider that APE1has the effect on radiation-induced angiogenesis in lung cancer through VEGF. In thepresent study, we first investigated the expression feature of APE1and VEGF, and thecorrelation with angiogenesis and the prognostic significance in patients with NSCLC, then,we observed that the expression of the APE1and VEGF in human lung adenocarcinoma A549cells after various doses X-ray irradiation, finally, we significantly suppressed theexpression of APE1in A549cells by Ad5/F35-APE1siRNA and investigated the role in theexpression of VEGF, the endothelial cells immigration and capillary-like structureformation induced by X-ray irradiation in vitro.Objective1. To investigate the relationship between the expression of APE1and angiogenesis inNSCLC, and determine that APE1is an effective and important gene therapeutic targetagainst angiogenesis in NSCLC.2. To investigate the effect of APE1on radiation-induced angiogenesis in lung cancer.3. To investigate the significance of radiotherapy combined with gene therapy thatunder-expression of APE1decreases radiation-induced angiogenesis in NSCLC.Materials and Methods1. The expression feature of APE1and VEGF and the relationship with angiogenesisand prognosis in NSCLC: The expression of APE1and VEGF was detected by SPimmunohistochemical technique in136cases of NSCLC. MVD was evaluated byimmunohistochemistry with anti-human CD34antibody. The relationship among APE1,VEGF and MVD was analyzed, and the correlation with DFS was analyzed.2. The dose-effect relationship of X-ray irradiation and the expression of APE1andVEGF in A549cells: The A549cells were irradiated using different doses8MV X-ray atroom temperature. Western blot analysis was used to examine the protein levels of APE1inlysates of irradiated and non-irradiated A549cells. The VEGF mRNA levels were detectedby RT-PCR. The VEGF protein levels in the A549cells culture supernatant were measuredby ELISA and the concentration of VEGF was normalized by the number of A549cells.3. Study of Ad5/F35-APE1siRNA decreasing angiogenesis induced by X-rayirradiation in A549cells: A549cells were infected with recombinant adenovirus vectorAd5/F35-APE1siRNA or Ad5/F35. Western blot analysis detected the APE1protein levelsto confirm the gene transduction efficiencies mediated by adenovirus. The A549cells wereirradiated using different doses X-ray. ELISA was applied to detect the VEGF protein levelsin the A549cells culture supernatant. The VEGF mRNA levels were detected by RT-PCR.The transwell chamber was used to determine the effect of Ad5/F35-APE1siRNA on HUVECs immigration induced by X-ray irradiation in vitro. The HUVECs tube formationassay was used to determine the effects of Ad5/F35-APE1siRNA on capillary-likestructures formation induced by X-ray irradiation in vitro as an indirect measure ofangiogenesis.Results1. The expression feature of APE1and VEGF and the relationship with angiogenesisand prognosis in NSCLC: The high expression rates of APE1and VEGF were77.94%and66.18%in NSCLC, respectively. The expression of APE1was not associated with gender,age, pathological type, pathological grade, tumor size and lymphatic metastasis (P>0.05).The expression of VEGF was associated with tumor size and lymphatic metastasis (P<0.05),and not associated with gender, age, pathological type, pathological grade (P>0.05). APE1,VEGF and MVD showed significant correlations (r=0.369, P=0.000; r=0.256, P=0.003;r=0.387, P=0.000). Tumor size, lymphatic metastasis, MVD, APE1and VEGF weresignificantly related with DFS by Kaplan-Meier survival curve (P<0.01). Multivariateanalysis by COX regression model showed that tumor size, lymphatic metastasis and VEGFexpression level were the important independent prognosis factors of DFS.2. The dose-effect relationship of X-ray irradiation and the expression of APE1andVEGF in A549cells: X-ray irradiation caused the same expression level change of APE1and VEGF in A549cells. X-ray irradiation enhanced the protein expression levels of APE1in A549cells in a dose-dependent manner, and the normalized concentration of VEGF inthe A549cells culture supernatant by the number of A549cells as well as the expressionlevels of VEGF mRNA showed dose-dependent increase with increasing doses of X-ray.The non-normalized concentration of VEGF in the A549cells culture supernatant began todecrease at the dose more than4Gy.3. Study of Ad5/F35-APE1siRNA decreasing angiogenesis induced by X-rayirradiation in A549cells: The APE1protein expression in A549cells was significantlysuppressed at48h after infection with Ad5/F35-APE1siRNA, and the inhibition rate ofAPE1protein expression was approximately85%compared with the non-infected A549cells. Ad5/F35-APE1siRNA significantly inhibited the expression of APE1induced byX-ray irradiation, too. RT-PCR and ELISA demonstrated that VEGF expression in A549 cells infected with Ad5/F35-APE1siRNA was significantly downregulated. The inhibitionrates of VEGF mRNA and normalized concentration of VEGF protein in culturesupernatant were approximately88%and60%compared with the control group,respectively. The inhibition rates in the cells treated with Ad5/F35-APE1siRNA combinedwith4Gy X-ray irradiation were approximately88%and64%compared with the cellstreated with4Gy X-ray irradiation alone, respectively. The numbers of immigratedHUVECs and capillary-like structure in the cells treated with Ad5/F35-APE1siRNA weremuch less compared with the non-treated cells. Ad5/F35-APE1siRNA also decreasedHUVECs immigration and HUVECs capillary-like structures formation induced by X-rayirradiation in vitro. Two-way ANOVA test proved the interaction betweenAd5/F35-APE1siRNA and X-ray irradiation on VEGF expression, HUVECs immigrationand HUVECs capillary-like structures formation in vitro.Conclusion1. The high expression of APE1may be correlated with angiogenesis in NSCLC.APE1is not an independent prognosis factor of DFS in NSCLC. APE1may increaseangiogenesis and be related to tumor local recurrence and metastasis through regulating theexpression of VEGF.2. Ad5/F35-APE1siRNA can significantly suppress the expression of VEGF in humanlung adenocarcinoma A549cells and inhibit the endothelial cells immigration andcapillary-like structure formation in vitro. It suggests that APE1might be a potential genetherapeutic target against angiogenesis in NSCLC.3. The high expression of APE1and VEGF is concurrently induced by X-rayirradiation in a dose-dependent manner in A549cells. X-ray irradiated A549cells culturesupernatant induces the endothelial cells immigration and capillary-like structuresformation. Ad5/F35-APE1siRNA can significantly suppress the expression of VEGF inX-ray irradiated A549cells and inhibit the endothelial cells immigration and capillary-likestructure formation induced by X-ray irradiation in vitro. Therefore, APE1may play a veryimportant role in the radiation-induced angiogenesis in lung cancer.4. Administration of Ad5/F35-APE1siRNA or inhibitor of APE1during radiotherapycould decrease angiogenesis induced by X-ray irradiation, which might be a potent adjuvant therapeutic approach to effectively eliminate metastasis and improve the efficacy ofradiotherapy for NSCLC.