| Objective: To study the antitumor effect of the recombinant adenoviruses Ad/PSCAE/UPâ…¡/E1A-AR, Ad/PSCAE/UPâ…¡/E1A, Ad/PSCAE/UPâ…¡/Luc in vitro and in vivo, to explore the mechanism involved in, to provide basic therory and basic data for clinical trails, and to provide new treatment strategies for bladder caner.Methods:The recombinant adenoviruses were amplified by HEK293, purified through chlorinated cesium density gradient centrifugation. TCID50was used to determine the titer of adenoviruses. The gene fragment of recombinant adeonvirus was identified by PCR. Bladder cancer cell lines5637, BIU87, EJ, T24, normal bladder epithelial cell line SV-HUC-1and hepatoma cell line SMMC-7721were infected with adenovirus (MOI=20), in addition, androgen receptor agonist R1881and androgen receptor anagonist flutamide were supplemented. Cell morphology was observed, MTT assay was used to determine cell proliferation, cytotoxicity was detected by crystal violet staining, RT-PCR and western blot were used to detect the expression of E1A. Additionally, we studied the possible mechanisms involved in the above processes, cell cycle was detected by flow cytometry, the activity of caspase3was detected, ultrastructural changes of bladder cancer cell lines infected with adenovirus was observed through transmission electron microscopy, apoptosis was observed by TUNEL assay. Subcutaneous bladder tumor model was established, and divided into four groups:Ad/PSCAE/UPII/EIA-AR treatment group, Ad/PSCAE/UPII/E1A treatment group, Ad/PSCAEAE/UPâ…¡/Luc treatment group and saline group. Adenoviruses were intratumorally multi-point injected. At the end of the experiment, nude mice were sacrificed, tumor tissue, and major organs were obtained and dealed with HE staining to observe pathological changes. E1A mRNA and protein were obtained and detected through RT-PCR and western blot respectively. The virus particles in tumor tissue were observed through electron microscope.Results: High titer, high purity recombinant adenoviruses were obtained, which could inhibit the proliferation of bladder cancer cells in a dose and time dependent, and has little effect for T24, while sparing normal baldder epithelial cell line SV-HUC-1and non-bladder cancer cell line SMMC-7721. R1881could strengthen the inhibitory effect, while flutamide had no influence. Flow cytometry showed that cell cycle arrested in G1phase, especially for the Ad/PSCAE/UPâ…¡/E1A-AR treatment group. There is much higher activity in bladder tumor cells treated with Ad/PSCAE/UPII/ElA, Ad/PSCAE/UPII/E1A-AR than that treated with Ad/PSCAE/UPII/Luc. Transmission electron microscopy showned that there are a large number of adenovirus particles, chromatin condensation and nuclear fragmentation in bladder cancer cells infected with recombinant adenovirus, while there is no above phenomenon in the control group. Immunofluorescence satining showed that there is a strong fluorescent signal of AR in the nucleus, and there is a strong fluorescent signal of CAR in the cell surface. TUNEL assay showed that a large number of apoptotic cells could be seen in Ad/PSCAE/UPâ…¡/E1A-AR treatment group, while apoptotic cells were not observed in Ad/PSCAE/UPII/Luc group. The results of real-time PCR showed that there was a higher E1A expression in EJ, BIU87,5637than in T24, the expression of E1A gene was not observed in SV-HUC-1. The results of western blot showed that the expression of E1A is much higher in bladder cancer cell lines EJ, BIU87,5637infected with Ad/PSCAE/UPâ…¡/E1A-AR than that infected with Ad/PSCAE/UPâ…¡/E1A. In addition, R1881could boost the expression of E1A protein, while flutamide has no influence. Comparing with Ad/PSCAE/UPâ…¡/Luc and saline, Ad/PSCAE/UPâ…¡/E1A-AR and Ad/PSCAE/UPII/E1A could significantly inhibit the growth of tumor, the inhibition ratio is77%, and could also extend the life-expectancy of tumor-bearing nude mice. HE staining showed that a large number of necrotic foci could be seen in the tumor tissue of Ad/PSCAE/UPâ…¡/E1A-AR treatment group, wheras necrotic foci could not seen in liver, lung and brain.Conclusion:Recombinant adenoviruses were amplified by HEK293, and purified, identified, high titer and high purity adenoviruses were obtained. Recombinant adenovirus carrying E1A-AR could significantly inhibit the proliferation of bladder cancer cells. R1881could promote the effect, while flutamide has no influence on the effect. Recombinant adenoviruses could induce bladder cancer cells apoptosis, arrest cell cycle at G1phase. Human bladde cancer cell line EJ was used to establish bladder cancer xenograft nude mice model, which provide a reliable basis for the study of bladder cancer. Recombinant adenovirus could inhibit the growth of tumor, and extend the life-expencacy of tumor-bearing nude mice.This is the first study of recombinant adenovirus carrying E1A-AR applied in human bladder cancer, which provide novel treatement strategies for bladder cancer. |
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