| The incidence of bladder cancer is seventh in male malignant tumors and more than tenth in female malignant tumors in the world. While in our country, the incidence of bladder cancer is eighth in male, is the most common malignant tumor of the urinary tract. The incidence is higher in urban than that in rural areas, in men than that women, and it is gradually rising trend. At present, the treatments of bladder cancer are mainly traditional surgery, chemotherapy, and radiotherapy. However, the recurrence rate is higher. Therefore, a new effective treatment of bladder cancer is urgently needed. Our previous research work showed that histone acetylation enzyme inhibitor is a kind of high-efficiency and low toxicity drug for bladder tumor. Therefore, it is very important for exploring the relevant pathways of TSA for bladder cancer.Purpose:Through the expression of autophagy and apoptosis markers before and after trichostatin A (TSA) on bladder cancer cells, we investigated the role of autophagy and apoptosis in the process of TSA treatment. It is unclear whether TSA can increase the coxsackie and adenovirus receptor (CAR) expression level to improve the transfection efficiency oncolytic adenovirus Ad-UPII-EI A on lower CAR expression of bladder cancer cells or not.Methods:The cell vitality was detected by MTT assay; Cell morphology and cell ultrastructure were observed by optical microscopy and electron microscopy; The titer and purity of recombinant adenovirus can be determined through TCID50experiment; Real-time quantitative PCR were used to detect the expression of Caspase3, Bcl-2, ATG5and Beclinl genes; Flow cytometry detected cell apoptosis and cycle; Western blotting test the expression of celluar HDAC、P62、LC3B、p-ERK、ERK、P53、Caspase-3、Bax、Bcl-2、CAR and E1A, respectively.Results:(1) TSA can effectively inhibit the proliferation of bladder cancer T24cells and EJ cells by dose-dependent; Transmission electron microscopy showed that T24cells and EJ cells appeared to be a massive autophagosome after24h TSA treatment. After48h200nmol/1TSA, early apoptosis were found in bladder tumor cells by Flow cytometry; Compared with control group, there was no significant change in expression level of Caspase-3(P>0.05), the expression level of Bcl-2were significantly decreased (P<0.05) and the ATG5were significantly increased (P<0.05) after24h TSA treatment. However, after48h. the expression levels of Caspase3, BCL-2and ATG5and Beclin were significantly decreased compared with control group (P<0.05). Western blotting showed that compared with the control group, pro-apoptotic protein P53、Bax、 Caspase-3were significantly increased; anti-apoptosis protein Bcl-2protein expression were significantly reduced; Autophagy related protein P62were gradually increased with the increase of dose; LC3-Ⅰ/LC3-Ⅱ ratio gradually increased; p-ERK and ERK expression were significantly reduced; Likely, the role is consistent with specific inhibitor PD98059on ERK1/2pathway.(2) High titer, high purity recombinant adenoviruses were obtained, which could inhibit the proliferation of CAR postive bladder cancer cells in a dose-dependent; However, there was no obvious effect on the CAR negative T24bladder cancer cells. The optical microscopy showed that bladder tumor cells become big, round and fell off after recombinant adenoviruses infection. Transmission electron microscopy showed that there were a large number of adenovirus particles in bladder cancer cells infected with recombinant adenovirus, while there is no above phenomenon in the control group. Bladder tumor cells infected with recombinant adenovirus appeared early apoptosis by Flow cytometry; cell cycle were blocked in G2/M phase. Western blotting showed that there was a higher E1A expression in bladder tumor cells with the infection of recombinant adenovirus, and compared with the control group, pro-apoptotic protein P53、Bax、Caspase-3were significantly increased; anti-apoptosis protein Bcl-2protein expression were significantly reduced. Although the TSA can up-regulate the expression level of CAR in T24cells, the TSA didn’t improve the transfection efficiency and killing effect of oncolytic adenovirus Ad-UPII-ElA on T24cells.Conclusion:The TSA can obviously inhibit the growth of bladder cancer cells; The TSA can promote bladder cancer cell apoptosis by inhibiting cell autophagy; The TSA inhibited cell autophagy by blocking RAS/RAF/MEK/ERK signaling pathway. Oncolytic adenovirus Ad-UPⅡ-ElA can targeting kill the CAR high expression bladder tumor cells5637and EJ, have an weak effect on CAR lower expression T24cells; Oncolytic adenovirus Ad-UPⅡ-ElA may induce apoptosis in5637and EJ cells; The mechanism of Ad-UPⅡ-ElA killing bladder tumor cells may be associated with induction of cell apoptosis. However, TSA didn’t improve the transfection efficiency and killing effect of oncolytic adenovirus Ad-UPⅡ-ElA on lower CAR expression of bladder cancer cells. |
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