The Effect Of Bladder Cancer Specific Oncolytic Adenovirus In Combination With TRAIL In Vitro

OBJECTIVES:To investigate whether tumor necrosis factor related apoptosis inducing ligand (TRAIL) can affect the cytotoxicity and cell apoptosis on bladder cancer specificity recombinant adenovirus (Ad-UPII-E1A) in vitro,we use the method of cell and molecular biology test the inhibit growth effect and apoptosis induction of bladder cancer cell after use TRAIL combined Ad-UPII-E1A,and discuss the molecular mechanism related in the bladder carcinoma cell. Expect to provide a theoretical basis of Adenovirus combined with TRAIL for clinical application in the future.METHODS:Bladder cancer cell line of EJ were treated with Ad-UPII-E1A and TRAIL. We determine the concentration of the TRAIL for the experiment through preliminary test. Bladder cancer cells were treated with Ad-UPII-E1A, TRAIL independently or combined under the same concentration, after24,48,72and96h respectively, the cell viability was tested by microculture tetrazolium (MTT) assay. The synergistic effect was analysed by Jin’s Formula. After48h treated with the combined group or adenovirus alone in EJ cells, the total RNA was extracted from cells and Bax was detected by Real-Time RCR. Caspase-3apoptosis assay was applied to show whether the apoptosis of the bladder cancer cells treated with TRAIL increased:In accordance with the instruction of Apoptosis Assay Kit#2, EJ cells were treated with Ad-UPⅡ-E1A only or combined with TRAIL respectively for48h and were harvested. The number of apoptotic cells was evaluated by flow cytometry (Ex=488nm; Em=530nm) Extract total RNA from each treatment group, design and synthesized primers,reverse transcriptase RNA to the target genes according to the instruction.use PCR-Cycler reacting and amplification curve is obtained,then speculated the expression of apoptosis related genes. Using caspase3activity detection kits detection the activity of caspase3.RESULTS:Combination therapy, which used Ad-UPⅡ-E1A with TRAIL, resulted in decreased growth and promoted apoptosis in vitro compared with either agent alone. Cell survival inhibition rate are related with drug dosages, and with the increase of time, the inhibition rate increased gradually. Analysis results of synergies by Jin’s formula suggest that TRAIL combined with adenovirus can synergisticiy enhance inhibition proliferation of EJ cells. Apoptosis analysis by the flow cytometry instrument explain that the combination group have more apoptosis cells compared with TRAIL group and virus group alone. Real-time PCR detected apoptosis related factor gene expression and Capase3activity detection showed, the expression of apoptosis related gene caspase3and Bax in combination group were higher than TRAIL and virus group alone. CONCLUSIONS:TRAIL combined Ad-UPII-E1A adenovirus therapy has synergistic inhibition of bladder cancer cell proliferation and can strengthen the role of apoptosis induced by adenovirus.