| Objective:In this study,In vitro experimental model was used to study the effects of cucurbitacin B on proliferation,migration and invasion of bladder cancer cells,as well as on apoptosis and cell cycle distribution.Methods:T24 cells were cultured in vitro,and the direct effect of cucurbitacin Bon tumor cell proliferation was observed using the CCK8 assay.Cell scratch method is used to observe the effect of cell migration.The effect of cucurbitacin B on the invasive ability of bladder cancer T24 cells was detected by Transwell assay.The main function of flow cytometry is to detect the apoptosis rate and cell cycle distribution of bladder cancer cells.The main purpose of Western blot analysis is to accurately detect the expression of active proteins related to apoptosis and cell cycle.Results:CCK8 assay and cell scratch method were used to observe the effect of cucurbitacin B on the proliferation and migration of T24 cells at different drug concentrations(0,400,800,1000 n M).The results showed that cucurbitacin B inhibited the proliferation and migration ability of bladder cancer cells at different time periods(24h,48 h,72h).With the increase of cucurbitacin B concentration in cells,the cell proliferationrate and average migration distance were significantly decrease.This effect is time-dependent.Hoechst 33258 stained cells were observed using an inverted fluorescence microscope.Compared with the control group,the experimental group can be obviously observed more apoptotic bodies and ruptured cell nuclear.Annexin-V-FITC/PI dual staining can directly detect the apoptotic rate of cells,and found that the apoptosis rate of bladder cancer cells increased with the increase of concentration in the experiment(P<0.05).The Western Blot was used to further study the expression of Caspase-3 and Caspase-9 in cells treated with cucurbitacin B.The results showed that both Caspase-3 and Caspase-9 were highly expressed.The results of real-time fluorescence quantitative(RT-PCR)showed that as the concentration of cucurbitacin B increased,the m RNA expression of Bcl-2 decreased significantly,while the expression of Bax increased significantly(P<0.05).In addition,our further research confirmed that the protein expression of Bcl-2 and Bax was consistent with the results of RT-PCR in the experiment.Flow cytometry analysis showed that compared with the control group,the cells in the experimental group decreased in the G1 phase and increased in the G2 phase(P<0.05).At the same time,Western-blot analysis showed that Cdc 25 C and Cyclin B1 were down-regulated.Conclusion:Cucurbitacin B can significantly inhibit cell proliferation,migration and invasion ability,and promote T24 cell cycle arrest and apoptosis. |
