| Objective: To investigate the effects of Cinobufagin(CBF)on human breast cancer MCF-7 cells and to explore its potential molecular mechanisms as an antitumor agent.Methods: MCF-7 cells were seeded into 96-well plates,the exponentially growing MCF-7 cells were selected to culture for 24 h and then treated with different concentrations of CBF at 0.1,0.2,0.4,0.8 and1.6 ?M.After incubated for 24,48 and 72 h at 37 °C,the proliferation activity of MCF-7 cells were assessed using Cell Counting Kit-8(CCK-8)assay.Different concentrations of taxol at 0.02,0.04,0.08,0.16 and 0.32 ?M were chosed as positive control and the blank group was setted as negative control.Hoechst 33258 staining was performed to observe the morphological changes of apoptotic MCF-7 cells after the cells were cultured with CBF(0,0.2,0.4,0.8 ?M)for 48 h.Flow cytometry was introduced to analyze the apoptotic ratio and cell cycle distribution of CBF-treated(0,0.2,0.4,0.8 ?M)MCF-7 cells.After the MCF-7 cells were cultured with CBF(0,0.2,0.4,0.8 ?M)for 48 h,we used the quantitative real-time PCR(RT-q PCR)to determine the expression of Bax and Bcl-2 genes,and the expression of Bax and Bcl-2 proteins were detected by Western-blot assay.Results: Our CCK-8 assay showed that CBF inhibited the proliferation of MCF-7 cells in a dose-and time-dependent manner(P < 0.05).The IC50(half maximal inhibitory concentration)values at 24,48 and 72 h were 0.94,0.44 and 0.22 ?M,respectively.In taxol-treated MCF-7 cells,the IC50 values at 24,48 and 72 h were 0.20,0.08 and 0.02 ?M,respectively.Also,we observed from our Hoechst 33258 staining that the small bright blue dots,which are representative of condensed or fragmented nuclei,were clearly present in CBF-treated(0.2,0.4 and 0.8 ?M)groups while the nuclei of MCF-7 cells in the control group stained an even blue,signifying MCF-7 cells underwent apoptosis after treated with CBF.In addition,the analysis of Annexin V-APC/7-AAD staining by flow cytometry revealed that the percentage of apoptosis,especially early and late stage apoptosis in CBF-treated MCF-7 cells,significantly increased in a dose-dependent manner(P < 0.05).The proportions of apoptotic MCF-7 cells treated with 0.2,0.4 and 0.8 ?M CBF were 22.94,50.68 and 68.29%,respectively,while the control group was 5.87%.Meanwhile,the analysis of PI staining by flow cytometry demonstrated that cell cycle was arrested in the G1 phase.Also,the percentage of MCF-7 cells in the G1 phase were 49.11,57.54 and 63.75% following treatment with 0.2,0.4 and 0.8 ?M CBF,respectively,while the control group was 39.45%.Besides,further molecular mechanism investigation by RT-q PCR and Western-blot demonstrated that CBF significantly affected the expression of Bax and Bcl-2.Our RT-q PCR analysis showed that Bax gene expression was up-regulated and Bcl-2 gene expression was down-regulated in MCF-7 cells after treatment with 0.2,0.4 and 0.8 ?M CBF for 48 h,which were in accordance with the increased Bax protein expression and decreased Bcl-2 protein expression under identical conditions as demonstrated by Western-blot analysis.Furthermore,the ratio of Bax to Bcl-2 was increased in a dose-dependent manner when compared with the control group(P < 0.05).Conclusion: Our results verify,for the first time,that CBF inhibits growth and induces apoptosis by altering the expression of Bax and Bcl-2 proteins.These findings suggest that CBF has potential for further development and use as an anti-cancer agent. |
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