The Molecular Mechanisms Of Apoptosis Induced By EHMT2 Inhibitor BIX-01294 In Human Bladder Cancer Cells

ObjectiveThe bladder cancer is one of the ten most common systemic cancer, which ranks first in urinary tract cancer incidence of China. In 2012, the world’s new cases of bladder cancer were 430 000, of which there were 165000 death cases. Moreover, according to National Cancer Registry, the incidence of bladder cancer was 6.61 / 100,000 in the regions that have registered in 2012, which was the ninth highest cancer incidence in our country.The choice of clinical treatment for bladder cancer depends on the degree of tumor invasion into the bladder wall. Normally, bladder cancer patients are with poor prognosis and suffer from high recurrence rate after electrocision, total cystectomy and partial resection of bladder. At present, combined chemotherapy based on cisplatin (methotrexate, vinblastine, doxorubicin and cisplatin, M-VAC) and cisplatin (gemcitabine and cisplatin, GC) treatment have become the standard treatment of who can tolerate cisplatin. However, not only M-VAC but also GC can not avoid the side effects of chemotherapy drugs. Notably, targeted therapy is expert in reducing side effects and improving patient tolerance, which has a great prospect in clinical application. Unfortunately, some targeted drugs for bladder cancer treatment, including cetuximab and bevacizumab, did not show obvious therapeutic effect. Until now, development and test for new targeted drugs for bladder cancer treatment become an urgent problem.However, the specific mechanism of apoptosis mediated by BIX-01294 in bladder cancer cells remains unknown. The goal of our research was to investigate which pathway BIX-01294 follows to induce human bladder cancer cell apoptosis and which kinds of proteins play important roles in this process.Methods1. Sulfonyl rhodamine B (SRB) colorimetry to detect the proliferation of human bladder cancer cells.2. Western Blot experiments to test the levels of proteins related with apoptosis induced by BIX-01294 in human bladder cancer cells.3. Flow cytometry to detect cell apoptosis changed by BIX-01294.4. RNA interference or plasmid transient transfection to suppress or overexpress the expression of EHMT2、PMAIPl、MCL1、USP9X、DDIT3、 TNFRSF10A genes.Results1. BIX-01294 inhibited the cell proliferation in a concentration-dependent manner in T24 and 5637 cells. BIX-01294 also can induce CASP-dependent apoptosis in human bladder cancer cell lines, which was in concentration-and time-dependent manner.2. BIX-01294 induces up-regulation of PMAIP1 and down-regulation of MCL1 in human bladder cancer cells. In addition, in the cells transfected with PMAIP1 siRNA or overexpressed MCL1, the apoptosis was reduced compared with that in control siRNA knockdown cells after BIX-01294 exposure.3. BIX-01294 reduced the level of USP9X in the dose-and time-dependent manner and MCL1 was degraded more in USP9X siRNA-transfected cells compared with the CTRL siRNA-transfected cells.4. DDIT3 is up-regulated by BIX-01294 in the dose- and time-dependent manner. In addition, in the cells transfected with DDIT3 siRNA, the apoptosis was reduced compared with that in control siRNA knockdown cells after BIX-01294 exposure.5. BIX-01294 incresed the level of ER stress ralted proteins:HSPA5、 ERNland ATF4, which was in the dose- and time-dependent manner.6. In the cells transfected with CASP8 siRNA, the apoptosis was reduced compared with that in control siRNA knockdown cells after BIX-01294 exposure. BIX-01294 incresed the level of TNFRSF10A in the dose- and time-dependent manner, in the cells transfected with TNFRSF10A siRNA, the apoptosis was reduced compared with that in control siRNA knockdown cells after BIX-01294 exposure.7. The low level of DDIT3 in T24 and 5637 rescued the surge of TNFRSF10A induced by BIX-01294.Conclusions1. BIX-01294 induces CASP-dependent apoptosis in human bladder cancer cell lines.2. BIX-01294 activates ER stress3. BIX-01294 induce endogenous apoptosis in human bladder cancer cells through PMAIP1-USP9X-MCLlpathway by activating ER stress.4. BIX-01294 induce extrinsic apoptosis in human bladder cancer cells through activating TNFRSF10A.In summary, we demonstrated that BIX-01294 induces CASP-dependent apoptosis in human bladder cancer cell and figured out the molecular mechanism of this process. BIX-01294 stimulates endoplasmic reticulum stress and mediates apoptosis via mitochondrial apoptotic pathway and extrinsic apoptosis pathway in bladder cancer cells.