Effects Of Superoxide Anion On Gene Therapy Of Tissue-specific Oncolytic Adenovirus For Bladder Cancer

Objective To further enhance the effectiveness of bladder cancer tissue-specific oncolytic adenovirus gene therapy, we study tumor microenvironment as a target for cancer treatment, studies have shown that changes in the intraceliular environment could affect the replication of viral vector and the expression of apoptosis related protein, While in the process of adenovirus infected cells, the cells would response to generate superoxide anion, due to the superoxide anion was used as a second messenger molecule involved in cell signaling pathways regulating gene expression, superoxide anion also could change the intraceliular oxidative stress state which regulates cell growth, proliferation and apoptosis. Therefore, this study is to clarify the role and possible mechanism of O2in gene therapy of oncolytic adenovirus for bladder cancer. Bladder cancer cells transformed with recombinant oncolytic adenovirus Ad/UPII/E1A was as an observation system.Methods EJ and5637bladder cancer cells were cultured, xan oxidase-xan (XO-X) reaction system was applied as the exogenous O2donor, NAC and DDC respectively is O2scavenger and superoxide dismUtase (SOD) inhibitor. MTT method was performed to detect anti-tumor cell proliferation after intervention with recombinant viruses and/or O2, we used micrography observation to observe the cytopathic effect (CPE) of bladder cancer cells; Transmission electron microscopy was used to observe adenovirus particle which infected into cells and the changes of cellular structure and shapes; Western blot analysis was applied to determine the expression of CAR and the key kinase of MAPK signaling pathway, such as P38, ERK, JNK, at the level of phosphorylation. Cell cycle and apoptosis were detected by flow cytometry. Student t-test and ANONA were used for data analysis by Sigmaplot11.0and SPSS17.0softwares.Results Recombinant adenovirus Ad/UPII/E1A has high purity and titer, and exert the oncolytic effect through E1A gene. MTT test in vitro cell poison experiment found that low concentration of O2or reductive stress could promote bladder tumor cell proliferation, however, high concentration of O2or oxidative stress was effective to enhance the bladder cancer cell killing ability of adenovirus, with a dose, time dependence. Micrography observed that bladder tumor cells EJ and5637appeared an obvious cytopathic effect (CPE) after infection of oncolytic adenovirus.Transmission electron microscope observation found that O2could promote adenovirus to enter the bladder tumor cells which cause cancer cells both autophagy and apoptosis. Flow cytometry results showed that bladder tumor cells appeared G1phase retardation after being infected with viruses, however, combination of O2and viruses could make cell cycle block in S phase, results appeared obvious apoptosis peak. Meanwhile O2induce bladder cancer cells apoptosis mainly early apoptosis, O2and viruses increased the percentage of late apoptosis. Western blotting found that in EJ cells, the external O2could raise the expression of CAR which effectively promoted the anti-tumor effect of adenovirus, and this effect has the cell difference, In combination group, along with the increase of O2concentration, the phosphorylation level of ERK and P38increased gradually, while the change of JNK’s phosphorylation level have no obvious difference, suggesting that the mechanism of O2on oncolytic adenovirus gene therapy bladder cancer may be related with the MAPK signaling pathways.Conclusion These findings illustrated that, through regulating the inside and outside microenvironment of tumor cell, external O2or oxidative stress could improve the effectiveness of oncolytic adenovirus gene therapy for bladder cancer. Superoxide anion could enhance adenovirus killing ability on bladder cancer cells, the mechanism maybe related with cell cycle S phase retardation and apoptosis, moreover MAPK signaling pathways involved in the process. The application of a certain dose of O2will improve the effectiveness of oncolytic adenovirus treatment on bladder cancer, which provides the theory for the clinical application of free radical biology treatment.