| OBJECTIVES:To investigate whether our bladder cancer specificity recombinant adenovirus (Ad-UPII-E1A) transfer enhances Mitomycin (MMC) and Hydroxycamptothecin (HCPT) cytotoxicity and inhibit growth synergistically in vitro, and whether chemotherapeutics, which can be incorporated into DNA, may hamper replication of virus and subsequent tumor lysis, in this study, we combined Ad-UPII-E1A with mitomycin (MMC) and hydroxycamptothecin (HCPT) for the first time, tested the inhibit growth effect and evaluated virus multiplication, apoptosis induction, molecular mechanism related in the bladder carcinoma cell line, to provide a theoretical basis of Adenovirus combined with chemotherapy for clinical application in the future.METHODS:Bladder cancer cell lines were treated with Ad-UPII-E1A and MMC or HCPT. Cell viability was quantified by a short-term microculture tetrazolium (MTT) assay. Bladder cancer cells were infected with Ad-UPⅡ-E1A, MMC and HCTP independently or combined under the same concentration, after24,48,72and96h respectively, the cell viability was tested by MTT assay. The synergistic effect was analysed by Jin’s Formula. Gene expression of E1A and transcriptional activation was determined by Western blot analysis. After72h treated with the combined group or adenovirus alone in5637cells, the total soluble proteins was extracted from cells and the El A protein expression was detected by western blot analysis. Cell cycle distribution analysis:5637cells were treated with Ad-UPII-El A only or combiend with chemotherapy drugs respectively for48,72,96h and were harvested. After being washed, the cells were kept4℃in70%ethanol. Then, the cells were collected and resuspended in PBS propidium iodide and RNaseA, incubated at37℃for30min. Cell cycle distribution were evaluated by flow cytometry(Beckman Coulter Epics XL, USA). Apoptosis assays:In accordance with the instruction of Apoptosis Assay Kit#2, and the number of apoptotic cells was evaluated by flow cytometry (Ex=488nm; Em=530nm)(BD, San Diego, CA, USA). For ultrastructural analyses,5637Cells were intervened and incubated for72h. The cultures were washed with PBS, suspended in2.5%glutaraldehyde, post-fixed for1h in1%osmium tetroxide, dehydrated in ethanol and embedded in epoxyresin. Ultrathin sections were cut, stained with lead citrate and uranyl acetate and observed with JEM-1230TEM (Japan).RESULTS:Combination therapy, which used Ad-UPII-E1A with MMC or HCPT resulted in decreased growth and promoted apoptosis in vitro compared with either agent alone, most agents showed higher cell killing effect in combination treatment groups, indicating a synergetic effect of the combination treatment on the viability of bladder cancer cells in vitro. Cell survival was negatively correlated with drug dose, but the decline of cell survival rate has no significant negative correlation with it. Analysis results of synergies by Jin’s formula suggest that chemotherapy combined with adenovirus significant synergistic inhibition proliferation of5637cells, EJ cells followed by and that of BIU-87is not obvious.E1A expression didn’t influenced by chemotherapy. Electron microscope to observe cell submicroscopic structure also proved this point, the virus is widely present in the cells dispersed or ordered after the combined effects, the cells appear early and late apoptotic phenotype, indicating drugs have no effect on viral replication and the induction of apoptotic.CONCLUSIONS:These findings illustrate that combined treatment with Ad-UPII-E1A and MMC or HCPT could be an attractive strategy for inhibiting progression of bladder cancer through effective induction of apoptosis. |
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