Changes In Radiosensitivity Of Bladder Cancer Cells Induced By Rottlerin And Its Mechanisms

Objective: To examine the mechanisms by which Rottlerin inhibits the growth of bladder cancer, its effect on radiotherapy sensitization of bladder cancer and clarify its mechanisms to provide theoretical basis for the clinical application of radiation treatment of bladder cancer.Methods: The EJ cell viability was evaluated using a MTT assay. The effective siRNA of Atg12 was detected by real-time quantitative PCR(q RT-PCR) assay.The formation of autophagosome after Rottlerin and different doses of radiation processing was determined via Transmission Electron microscopy(TEM).The effect of Rottlerin on cell cycle and cell apoptosis rate was detected by flow cytometry. The expression of autophagy related proteins LC3 Ⅱ and the apoptosis related proteins Caspase 3, PARP, after drug treatment were analyzed via Western blotting. The survival fraction was calculated by cloning formed experiment after Rottler in. Atg12 si RNA and radiation combined prosessing to estimate either promoting or inhibiting autophagy can increase the radiation sensitivity. Autophagy related proteins LC3 Ⅱ expression after joint processing of Rottlerin and Atg12 si RNA was analyzed by Western Blotting. The effects on cell cycle and cell apoptosis rate after combined treatment of Rottlerin and radiation were detected by flow cytometry. The expression of autophagy related proteins LC3 Ⅱ and the apoptosis related proteins Caspase 3, PARP, were analyzed via Western blotting.Results:(1) We treated malignant bladder cancer EJ cells with 0-16μΜ rottlerin for 24 h, 48 h, 72 h, and assessed the viability of EJ cells. rottlerin had a growth inhibitory effect on EJ cells in a dose-dependent and time-dependent manner, indicating that rottlerin has antitumor effects on the EJ cells, but 0、0.5 and 1μM rottlerin had little effect on the EJ cells. But with the concentration increased to more than 2μM, cell survival rate significantly lower than the control group.(2) Dealing with 2μM Rottler in after 48 hours, it found that Rottlerin can reduce the number of EJ cell clone formation. Compared with the control group, it can decrease the ability of cloning forming of EJ cells.(3) Dealing with 2μM Rottler in after 48 hours, autophagy related proteins LC3 Ⅱ increased expression analyzed by Western Blotting and autophagosome formation determined via TEM detection after drug treatment increased.(4) After differenter doses of radiation, it found that Rottlerin can improve the radiation sensitivity of EJ cells by survival fraction. The 2Gy of radiation sensitivity can be mild, but starting from 4Gy, the radiation sensitivity increased obviously. Si RNA and X-ray combined displayed not obvious effect, but has a mild inhibit cell killing effect of radiation.(5) Rottlerin treatment with radiation to EJ cells by 2 Gy, 4Gy X-ray after 24 hours, EJ cells were mainly blocked in G1 phases, compared with simple by 2Gy, 4Gy X-ray to EJ cells. And compared to irradiation cell, the cell cycle of pure radiation blocked occurred in G2.(6) Cell apoptos is by 2Gy, 4Gy exposure rate is 5.55%, 16.88% respectively. To radiation therapy, EJ cell has a certain resistance, but Rottlerin combined with x ray cells after cell apoptosis rate increased signif icantly, 2Gy, 4Gy of apoptosis rate reached 65.5%, 72.43% respectively.(7) Control group and 2μM Rottler in processing EJ cells after 24 hours, cells were 2Gy radiation exposure, and then trained for 24 hours, after the completion, Caspase 3, PARP and activation of Caspase 3, PARP had no obvious change before and after drug treatment. It had no obvious change before and after radiation in the same.Conclusions: These findings provide support for the use of rottlerin inhibit bladder cancer cell growth and improve radiosensitivity of bladder cancer.