| Objective: To study on the effect of Xanthoceraside in cell lines of human bladder cancer T24 and 5637,and primarily investigate the antitumor mechanisms of Xanthoceraside in bladder cancer.Methods: 1.Cell Counting Kit-8(CCK-8)was used to assay the proliferation inhibition of Xanthoceraside at different concentrations on the cell lines of human bladder cancer T24 and 5637 after 24 h.2.CCK-8 assay was used to assay the proliferation inhibition of 10?M Xanthoceraside at different times on the cell lines of human bladder cancer T24 and 5637.3.Flow cytometry(FCM)was used to determine the apoptosis rate of bladder cancer T24 and 5637 cells treated with different concentrations of Xanthoceraside after 24 h.4.The optical microscope was used to observe the morphological changes of bladder cancer T24 and 5637 cells treated with different concentrations of Xanthoceraside after 24 h.5.DAPI(4',6-diamidino-2-phenylindole)staining was used to detect the morphological changes of the nucleus of bladder cancer T24 and 5637 cells treated with different concentrations of Xanthoceraside after 24 h.6.q RT-PCR(quantitative real time PCR)was used to detect the expression levels of Caspase-3,Bcl-2 and P53 gene in bladder cancer T24 and 5637 cells treated with different concentrations of Xanthoceraside after 24 h.7.Western blot was performed to detect Caspase-3,Caspase-8,Caspase-9,Bcl-2,Bcl-xl,Bax,P53,PI3 K,p-PI3 k,Akt,p-Akt protein expression in bladder cancer T24 and 5637 cells treated with different concentrations of Xanthoceraside after 24 h.Results:1.The results of CCK-8 assay showed that Xanthoceraside could significantly inhibit the proliferation of bladder cancer T24 and 5637 cells,and the cell survival rate decreased gradually with the increase of drug concentration,which showing a concentration-dependent relationship.The IC50 was 8.39 ?M and 10.77 ?M for T24 and 5637 at 24 h,respectively.2.The survival rate of bladder cancer T24 and 5637 cells decreased gradually,with the extension of drug action time,after treated with 10?M Xanthoceraside,which showing a time-dependent relationship.3.The apoptosis rate of T24 cells was(3.72 ±0.53)%,(18.97 ±1.24)%,(25.30 ±2.02)%,(38.97 ±0.76)%,respectively,the difference was statistically significant(P <0.05);The apoptosis rate of 5637 cells(2.82 ±0.44)%,(4.51 ±0.60)%,(13.60 ±1.11)%,and(23.13±1.99)%,respectively,the difference was statistically significant(P <0.05),after treatment with Xanthoceraside in concentration of 0,6,8 and 10?M for 24 h.4.Bladder cancer T24 and 5637 cell lines were treated with Xanthoceraside in concentration of 0,6,8 and 10 ?M for 24 h,optical microscope showed that T24 and 5637 cells of bladder cancer were dewalled,rounded and retracted,and the space between adjacent cells increased with the increase of the concentration of Xanthoceraside 5.Bladder cancer T24 and 5637 cell lines were treated with Xanthoceraside in concentration of 0,6,8 and 10 ?M for 24 h,and stained with DAPI.Microscopic examination revealed morphological changes of the nucleus with deepened staining and occurrence of apoptotic bodies.6.Bladder cancer T24 and 5637 cell lines were treated with Xanthoceraside in concentration of 0,6,8 and 10 ?M for 24 h,q RT-PCR was used to detect increased Caspase-3,increased P53 and decreased Bcl-2 gene expression,the difference was statistically significant(P <0.05).7.Bladder cancer T24 and 5637 cell lines were treated with Xanthoceraside in concentration of 0,6,8 and 10 ?M for 24 h,Western blot results showed that there was no significant difference in Caspase-3,Caspase-8,Caspase-9 protein compared with the control group.Bcl-2,Bcl-x L,p-PI3 K and p-Akt protein expressions were significantly decreased,while P53 protein expression was significantly increased in bladder cancer T24 cells after treatment with Xanthoceraside in concentration of 8 and 10?M for 24 h,compared with the control group,the difference was statistically significant(P <0.05).Bax protein expressions were significantly increased in bladder cancer T24 cells After 24 h of treatment with 6,8 and 10 ?M Xanthoceraside,compared with the control group,the difference was statistically significant(P <0.05).The protein expression levels of Bcl-2 and Bcl-x L protein expressions were significantly decreased in bladder cancer 5647 cells After 24 h of treatment with 8 and 10 ?M Xanthoceraside,compared with the control group,the difference was statistically significant(P <0.05).p-PI3 K and p-Akt protein expressions were significantly decreased,while the protein expression levels of P53 increased,after treatment with Xanthoceraside in concentration of 10?M for 24 h,compared with the control group,the difference was statistically significant(P <0.05).Conclusion: 1.Xanthoceraside can inhibit the proliferation of bladder cancer cells;2.Xanthoceraside induces cell apoptosis through downregulation of the PI3K/Akt/Bcl-2/Bax signaling pathway in cell lines of human bladder cancer T24 and 5637.3.Xanthoceraside may be a potential active compound for the treatment of bladder cancer. |
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