Studies On Inhibitory Effect Of Icariin On Bladder Cancer T24 Cells And Its Mechanism Of Action In Vitor

Objective: Effects of icariin (ICA) on proliferation, cell cycle and apoptosis of bladder cancer cell line T24 were investigated in vitor. Meanwhile, we approached Anti-tumor mechanism of ICA initially. For the final purpose, this study provides some theoretical bases to clinical application of ICA to cancer chemoprevention.Methods: 1 The effect of ICA on human bladder cancer T24 cells was evaluated with varying concentration of ICA (0, 5, 10, 20, 40, 80, 160 and 320μg/ml) treatment for 24, 48 and 72 hours by MTT assay. Morphologic changes were studied with optic microscope and Wright-Giemsa's staining. 2 The cell cycle and apoptosis of T24 cell treated by ICA (0, 5, 10, 20 and 40μg/ml) for 24 hours were detected by flow cytometry (FCM). 3 The role of PCNA, BAX and BCL-2 family proteins in apoptosis on T24 cell, which were treated by ICA (0, 5, 10, 20 and 40μg/ml) for 24 hours was analyzed by Western blotting. 4 Statistics Methods: Statistic analysis was performed with SPSS 16.0. All experiment data were indicated Mean±STD.Results: 1 Using the T24 human bladder cancer cell line, we found that ICA treatment ecerted a significant cytotoxic effect upon T24 cells in dose-dependent (0, 5, 10, 20, 40, 80, 160 and 320μg/ml) and time-dependent (24, 48 and 72hours) manner. ICA inhibited T24 cells growth with IC50:63.22μg/ml. Treated with 40μg/ml ICA for 24h, some T24 cells condensed with membrane buding, the nuclear chromatin Compaction and membrane-closed apopototic bodies formation by optic microscope and Wright-Giemsa's staining. 2 The treatment by ICA (5, 10, 20 and 40μg/ml) for 24 hours, compared with the vehicle-treated controls, resulted in an appreciable arrest T24 cells in G1 phase of cell cycle by FCM. Meanwhile, ICA treatment ecerted a significant apoptosis effect and PI value upon T24 cells in dose-dependent. After incubated with 40μg/ml quercetin for 24 h, apoptosis rate of T24 cells increased from 1. 94%±0.44% of control group to 6.67%±0.62% (P<0.01). 3 ICA can significantly inhibit the PCNA protein, down-regulate BCL-2 protein, and up-regulate Bax protein. After 24 hours treatment of ICA, the protein levels of PCNA was significantly decreased. meanwhile, ICA treatment of T24 cell lines resulted in a decrease in antiapoptosis Bcl-2 protein levels, whereas a concomitant increase in proapoptosis Bax proteins. The levels of these proteins are dose-dependent in response to ICA. The ratio of Bax/Bcl-2 was significantly increased in a dose-dependent manner with ICA treatment.Conclusion: Our data suggested ICA treatment resulted in a significant dose and time-dependent inhibition on proliferation, cell cycle and apoptosis of bladder cancer cell line T24. Our study also suggests that ICA causes cell cycle arrest in G1 phase. in turns, ICA can significantly inhibit the expression of PCNA, down-regulate BCL-2 protein, and up-regulate Bax protein. Then, the apoptosis of T24 cells is initiated.