| Purpose:Bladder cancer is one of the most common malignant tumors of the urinary system. Treatments are wide ranging, and include surgical resection, chemotherapy, radiotherapy, and immunotherapy. However, the therapeutic effect is far from satisfactory. Gene therapeutic approaches to bladder cancer have shown research promise with the advancements of molecular biology and virology. A lot of researches have reported that the combination of virotherapy with chemotherapy gained good therapeutic effect in cancer therapy. And, the combination of virotherapy and chemotherapy is a new and promising approach to cancer therapy. In this study, we constructed a bladder cancer-specific oncolytic adenovirus (Ad/PSCAE/UPII/E1A) which carries E1A gene regulated by human Uroplakin Ⅱ (UPII) promoter and prostate stem cell antigen enhancer (PSCAE). Ad/PSCAE/UPII/E1A can express EIA gene selectively in bladder cancer cells and thereby replicate there efficiently. Ad/PSCAE/UPII/E1A can kill bladder tumor cells preferentially and has no effect in normal cells. Cisplatin (DDP) and gemcitabine (GEM) are well known chemotherapeutic agent that are routinely used for the treatment of bladder cancer. The aim of this study was to examine the effects of Ad/PSCAE/UPII/E1A combined with DDP or GEM on human bladder cancer cells and to identify the underlying mechanisms. Our results will provide a potential and promising strategy for bladder cancer therapy.Methods:We constructed a bladder cancer-specific oncolytic adenovirus, Ad/PSCAE/UPII/ E1A, by inserting human UPII promoter and PSCAE upstream of the E1A gene to regulate E1A gene expression, which has shown potent antitumor effects in bladder cancer cells in our previous studies. Also, we constructed recombinant adenovirus vector, Ad/PSCAE/UPII/Luc, by replacing E1A gene with luciferase (Luc) reporter gene. To assess the infectivity of Ad/PSCAE/UPII/Luc and Ad/PSCAE/UPII/E1A, the activity of luciferase in the bladder cancer cells was analyzed by luciferase assay and the express of E1A in the bladder cancer cells was determined by western blot. The combined effects of Ad/PSCAE/UPII/E1A and DDP or GEM on human bladder cancer cells and human normal urinary cells were evaluated by MTT cell proliferation assay. The interaction between Ad/PSCAE/UPII/E1A and chemotherapeutic agents were assessed by calculating combination index (CI). The cell apoptosis and cell cycle distribution were detected by flow cytometry. The activation of the caspase pathway and the expression of Bcl-2 family proteins were determined by qRT-PCR and western blot assay. The effect of GEM on adenovirus infectivity to T24 cells were assessed by luciferase assay and western blot assay. The influence of GEM on the express of CAR in T24 cells were determined by western blot assay.Results:(1) After amplification and purification, high titer and high purity recombinant adenoviruses Ad/PSCAE/UPII/Luc and Ad/PSCAE/UPII/E1A were obtained. (2) Ad/PSCAE/UPII/Luc could effectively infect CAR highly expressed bladder cancer cell lines EJ、 5637 and BIU87, and dose-dependently increase the activity of luciferase in these cells; However, Ad/PSCAE/UPII/Luc could not effectively infect CAR lowly expressed bladder cancer cell line T24. (3) Western blot results showed that there was a high expression of E1A in CAR highly expressed bladder cancer cells and a poor expression of E1A in CAR lowly expressed bladder cancer cells T24 after the infection of recombinant adenovirus Ad/PSCAE/UPII/E1A. (4) MTT results showed that Ad/PSCAE/UPII/E1A or DDP alone induced dose-dependent cell death in CAR highly expressed bladder cancer cells. The combination of Ad/PSCAE/UPII/E1A and DDP synergistically inhibited bladder cancer cells growth. (5) MTT results showed that Ad/PSCAE/UPII/E1A or GEM alone induced dose-dependent cell death in CAR highly expressed bladder cancer cells. The combination of Ad/PSCAE/UPII/E1A and GEM synergistically inhibited bladder cancer cells growth. (6) Ad/PSCAE/UPII/E1A alone didn’t inhibit the growth of CAR lowly expressed T24 cells. There was no enhanced inhibition of growth in T24 cells when Ad/PSCAE/UPII/ElA combined with DDP. But, the combination of Ad/PSCAE/UPII/El A and GEM synergistically inhibited the growth of T24 cells. (7) Ad/PSCAE/UPII/E1A or DDP alone could arrest 5637 cells cycle in G1 phase. And, compared with Ad/PSCAE/UPII/E1A or DDP alone, their cooperation induced G1 phase arrest more significantly. (8) Ad/PSCAE/UPII/El A could arrest EJ cells cycle in G1 phase and GEM could arrest EJ cells cycle in S phase. The combination of Ad/PSCAE/UPII/E1A and GEM could significantly arrest EJ cells cycle in S phase. (9) Ad/PSCAE/UPII/E1A or DDP alone could induce 5637 cells apoptosis. And, compared with Ad/PSCAE/UPII/E1A or DDP alone, their cooperation induced more 5637 cells apoptosis. (10) Ad/PSCAE/UPII/E1A or GEM alone could induce EJ cells apoptosis. And, compared with Ad/PSCAE/UPII/E1A or GEM alone, their cooperation induced more EJ cells apoptosis. (11) qRT-PCR results showed that Ad/PSCAE/UPII/E1A combined with DDP could increase the mRNA expression of Fas and Bax, and decrease the mRNA expression of Bcl-2; Western blot results showed that Ad/PSCAE/UPII/E1A combined with DDP could increase the protein expression of Fas, Bax, Bak, cleaved Bid, cleaved caspase 8, cleaved caspase 9, cleaved caspase 3 and cleaved PARP, and decrease the protein expression of Bcl-2 and Bcl-XL. Above results indicated that Ad/PSCAE/UPII/ElA plus DDP synergistically induced 5637 cells apoptosis closely related to the activation of extrinsic and intrinsic apoptotic pathways. (12) qRT-PCR results showed that Ad/PSCAE/UPII/E1A combined with GEM could increase the mRNA expression of Bax, and decrease the mRNA expression of Bcl-2; Western blot results showed that Ad/PSCAE/UPII/E1A combined with GEM could increase the protein expression of Bax, Bak, cleaved caspase 9, cleaved caspase 3 and cleaved PARP, and decrease the protein expression of Bcl-2 and Bcl-XL. These results indicated that Ad/PSCAE/UPII/EIA plus GEM synergistically induced EJ cells apoptosis closely related to the activation of intrinsic apoptotic pathway. (13) GEM could increase the infection efficiency of Ad/PSCAE/UPII/Luc and Ad/PSCAE/UPII/E1A to T24 cells. (14) GEM increased the expression of CAR in T24 cells, which contributed to the enhanced infection efficiency of Ad/PSCAE/UPII/Luc and Ad/PSCAE/UPII/E1A to T24 cells. (15) The combination of Ad/PSCAE/UPII/E1A with low doses of DDP or GEM had no obvious cytotoxic effect against human normal urinary cell line SV-HUC-1. The combined treatment didn’t result additive cytotoxic effect against SV-HUC-1 cells.Conclusions:(1) Ad/PSCAE/UPII/Luc and Ad/PSCAE/UPII/E1A could effectively infect CAR highly expressed bladder cancer cell lines EJ、5637 and BIU87. However, these two recombinant adenoviruses could not effectively infect CAR lowly expressed bladder cancer cell line T24. (2) Ad/PSCAE/UPII/E1A could inhibited the growth of CAR highly expressed bladder cancer cells in a dose- and time-dependent manner. But, Ad/PSCAE/UPII/E1A didn’t inhibit the growth of CAR lowly expressed T24 cells. (3) The combination of Ad/PSCAE/UPII/E1A and DDP synergistically inhibited CAR highly expressed bladder cancer cells growth. The combined treatment had no synergistical effect on CAR lowly expressed T24 cells growth. (4) The combination of Ad/PSCAE/UPII/E1A and GEM synergistically inhibited CAR highly and lowly expressed bladder cancer cells growth. (5) Ad/PSCAE/UPII/E1A could arrest bladder cancer cells cycle in G1 phase. The combined treatment with DDP could induce G1 phase arrest more significantly and with GEM could induce S phase arrest. (6) Ad/PSCAE/UPII/E1A could induce bladder cancer cells apoptosis. The combined treatment with DDP or GEM could induce more cells apoptosis. (7) Ad/PSCAE/UPII/EIA plus DDP synergistically induced bladder cancer cells apoptosis closely related to the activation of extrinsic and intrinsic apoptotic pathway. (8) Ad/PSCAE/UPII/E1A plus GEM synergistically induced bladder cancer cells apoptosis closely related to the activation of intrinsic apoptotic pathway. (9) The enhanced infection efficiency of Ad/PSCAE/UPII/E1A to T24 cells elicited by GEM was closely related to the increased expression of CAR in T24 cells. (10) Ad/PSCAE/UPII/E1A could exert selective killing activity against bladder cancer cells. The combination with low doses of DDP or GEM had no additive cytotoxic effect against human normal cells. |
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