The Effect Of MTOR Inhibitors On Urothelial Bladder Cancer

Objective To evaluate the expression of p-mTOR, p-p70S6K contained in mTOR signal pathway and related proteins of p53and PTEN in urothelial bladder cancer and to provide theoretical evidence for the application of mTOR inhibitors in the treatment for bladder cancer; to evaluate the effect of CCI-779, an inhibitor of mTOR, on baldder T24and BIU-87cells and to provide experimental evidence for bladder cancer therapy; and to evaluate the effect of CCI-779on bladder cancer treated by DDP and the synergistic mechanism.Methods Immunohistochemistry combined with tissue microarray technology was used to detect the expression of p-mTOR, p-p70S6K, p53and PTEN in201tissue samples of urothelial bladder cancer and40tissue samples of normal bladder mucosa. The function of mTOR signal pathway in carcinogenesis, cancer progression, recurrence and prognosis was evaluated. MTT array was used to test the proliferation of T24and BIU-87cells treated by CCI-779. Wound healing test was used to detect the ability of migration of T24and BIU-87cells. Flow cytometry was used to test the effect of CCI-779on cell cycle and apoptosis of T24and BIU-87cells. Transwell invasion was used to test the invasion ability of T24and BIU-87cells treated by various concentration of CCI-779. Western blot analysis was used to test the phosphorylation status of mTOR and p70S6K treated by CCI-779. Then the effect of CCI-779on T24cells was evaluated in vivo. Furthermore, the effect of CCI-779, DDP and CCI-779combined with DDP was compared by MTT to evaluate the synergistic mechanism. The enhancement effect of CCI-779on bladder tumor treated by DDP was evaluated in vivo. The expression of Ki-67in xenogenic tumors was tested by immunohistochemistry and apoptosis was evaluated by TUNEL. P21was tested by western blot to investigate the mechanism of the synergistical effect.Results No correlation was found between p53and PTEN. P53and PTEN could activate mTOR signal pathway in different manners. mTOR/p70S6K pathway was the common downstream effector of p53and PTEN. The expression of p-mTOR and p-p70S6K was higher in urothelial bladder cancer than in normal bladder tussue, indicating the correlation between mTOR/p70S6K pathway and bladder cancer carcinogenisis. The positive correlation of p-mTOR and p-p70S6K expression with histological grade, tumor stage and number of bladder tumor indicated that activation of mTOR pathway might result in tumor progression. Expression of p-mTOR and p-p70S6K indicated shorter recurrence free survival. In the multiplicity, p-mTOR but not p-p70S6k was an independent prognositic factor for bladder cancer, just as tumor stage and tissue grade. The activation of mTOR pathway was an indication of application of mTOR inhibitors, no matter of the expression of p53and PTEN. In the in vitro study, CCI-779can inhibit the proliferation of T24and BIU-87cells, in a concentration and time dependent manner. Compared with the control group, the migration of T24and BIU-87cells was inhibited by CCI-779. The inhibition of proliferation by CCI-779was attributable to cell cycle arrest at G0/G1but not apoptosis. In addition, CCI-779also inhibited the invasion ability. By western blot analysis, CCI-779could inhibit the phosphorylation of mTOR and p70S6K. Results of in vivo study was just consistent with those of in vitro study, CCI-779could inhibit the xenogenic tumros by inhibiting the proliferation but not apoptosis. The inhibitory effect was enhanced when treated with CCI-779combined with DDP, indicating the synergism of CCI-779on DDP treatment. In the in vitro study, the tumor weight and Ki-67expression in the CCI-779and DDP combination group were lower than those in other groups, and the apoptosis rate was higher than that in other groups.Conclusion mTOR signal pathway is activated extensively in urothelial baldder cancer and correlated with carcinogenesis, tumur progression, number of tumors and tumor recurrence. The activation of mTOR is an indication of application of mTOR inhibitors, no matter the expression of p53or PTEN. Because CCI-779can inhibit proliferation of T24and BIU-87cells, CCI-779may be a promising drug for urothelial bladder cancer therapy. CCI-779and DDP is synergistical in bladder cancer therapy in vitro. CCI-779can enhance the function of DDP on bladder cancer therapy in the in vivo study by inhibiting cell cycle protein p21.