The Regulative Role Of Nitric Oxide In Gene Therapy Of Tissue-specific Oncolytic Adenovirus For Bladder Cancer

Objective:Some studies show that free radical nitric oxide (NO) plays a wide range of regulative role in gene expression, but the role of NO on exogenous gene expression after transfection of tumor tissue-specific oncolytic virus and its mechanism is unclear. This study was designed to clarify the role and possible mechanism of NO in gene therapy of oncolytic adenovirus for bladder cancer. Bladder cancer cells transfected with recombinant oncolytic adenovirus Ad-UPII-E1A was as observation system.Methods:BIU87 and 5637 bladder cancer cell lines were cultured; sodium nitroprusside (SNP), PTIO and L-NMMA were applied respectively as exogenous NO donor, endogenous NO scavenger and nitric oxide synthase (NOS) inhibitor. NO level in cell culture medium before and after SNP and/or virus intervention was detected by Nitrate/Nitrite Assay Kit. By TCID50 method and determined plaque forming unit (PFU), we detected the titer and purity of recombinant virus Ad-UPII-E1A. MTT assay was applied to detect the proliferation of bladder cancer cells before and after intervention by recombinant viruses and/or SNP; transmission electron microscopy (TEM) was used to observe adenovirus particles within cells and the changes of subcellular structure; real-time quantitative PCR and Western blot were used to detect the expression of E1A, iNOS and the Ras/MAPK pathway genes, such as Ras, P38, ERK, JNK at the level of transcription and translation; DNA contents of bladder tumor cells cycle and apoptosis were also detected by flow cytometry. Student t-test and ANONA were used for data analysis by Sigmaplot 11.0 and SPSS 13.0 softwares.Results:Low levels of NO were detected in BIU87 or 5637 cell culture medium itself, but NO concentration increased with time when cells exposed to exogenous NO donor SNP. Recombinant virus Ad-UPII-E1A has high purity and titer, and plays the oncolytic effect through E1A gene. NO can promote the virus transfer into BIU87,5637 cells, SNP at 50uM or 100uM combined with Ad-UPII-E1A at 30 MOI can enhance tumor cells proliferation, while SNP at 200uM combined with adenovirus can promote tumor cells death, and the role of NO on Ad-UPII-E1A is time-dependent. Observation by TEM showed that the recombinant virus Ad-UPII-E1A can enter and replicate within bladder tumor cells, NO can increase the transfection efficiency of virus and also cause tumor cell autophagy and apoptosis. After tumor cells exposed 72 hours of NO combined with adenovirus, cell cycle was arrested at G2/M phase. SNP at 50uM or 100uM can inhibit E1A gene expression in transcription and translation levels, whereas SNP at 200uM can increase the expression of E1A. After administration of exogenous NO, the expression of iNOS increased, while giving PTIO and or L-NMMA can reduce the expression of iNOS. By detecting the Ras/MAPK signal pathway genes expression in transcription and translation levels, we found that the expression of Ras, JNK and ERK increased when cells were exposed 72 hours in the environment of NO combined with adenovirus compared with adenovirus alone, while the expression of P38 reduced.Conclusion:Free radical nitric oxide can promote oncolytic adenovirus Ad-UPII-E1A transfected into bladder cancer cells, but NO plays a bidirectional regulation (positive and negative) role in the gene therapy of oncolytic adenovirus for bladder cancer according to different NO concentrations. In this study, we found that low doses NO can reduce the E1A gene expression of recombinant virus so as to promote tumor cell proliferation, while high doses NO can increase E1A expression thus to play a oncolytic effect which may be associated with cell cycle G2/M arrest and cell apoptosis. Exogenous NO and endogenous iNOS-derived NO can both play a regulative role in viral gene therapy for bladder cancer, and the mechanism may be related to Ras/MAPK signal pathway.