扁蓄苷对人乳腺癌MDA-MB-231细胞凋亡、周期及PI3K/AKT信号通路的影响

Effect of avicularin on the apoptosis,cell cycle and PI3K/AKT signaling pathway in human breast cancer MDA-MB-231 cells

目的探讨扁蓄苷对乳腺癌MDA-MB-231细胞的凋亡、周期及PI3K/AKT信号通路的影响。方法以不同浓度的萹蓄苷作用于体外生长至对数期的MDA-MB-231细胞48 h后,采用噻唑蓝染色(MTT)法检测扁蓄苷对细胞增殖的影响,Hoechst 33258染色观察细胞染色质固缩情况,流式细胞术检测细胞周期,Western-blot检测PI3K/AKT信号通路核心蛋白PI3K、p-PI3K、AKT、p-AKT表达水平,RT-PCR技术检测通路下游细胞凋亡蛋白Bcl-2、Bcl-xl及周期相关蛋白CDK2基因表达情况。结果不同浓度的扁蓄苷(6.25、12.5、25、50、100μmol·L-1)作用于MDA-MB-231细胞48 h后,细胞增殖活性受到抑制(P<0.01),且具有浓度依赖性;扁蓄苷(12.5、50、100μmol·L-1)作用细胞48 h后,细胞核染色质固缩;当扁蓄苷浓度为50μmol·L-1时,S期细胞所占百分比显著增高(P<0.05),当扁蓄苷浓度为100μmol·L-1时,G2/M期细胞所占百分比显著增高(P<0.05);随着扁蓄苷浓度的增加,通路核心位点PI3K、p-PI3K、AKT、p-AKT蛋白表达水平明显降低(P<0.01),细胞凋亡相关蛋白Bcl-2和Bcl-xl、细胞周期蛋白CDK2基因表达水平下调(P<0.05)。结论扁蓄苷可抑制乳腺癌MDA-MB-231细胞增殖,使细胞周期阻滞于S期、G2/M期,并诱导细胞凋亡。其机制可能是通过抑制PI3K/AKT信号通路下调Bcl-2、Bcl-xl、及CDK2基因表达进而诱导人乳腺癌MDA-MB-231细胞凋亡。

Objective To determine the effect of avicularin on the apoptosis,cell cycle and PI3K/AKT signaling pathway in breast cancer MDA-MB-231 cells.Methods MDA-MB-231 cells were grown in vitro in different concentrations of avicularin for 48 h.The effect of avicularin on the cell proliferation was detected by MTT assay.Hoechst 33258 staining was used to observe the chromatin condensation.Flow cytometry was used to detect the cell cycle,and Western-blot was used to detect PI3K/AKT signaling pathway core protein PI3K,p-PI3K,AKT,and p-AKT expression levels.RT-PCR detected the downstream apoptosis protein Bcl-2,Bclxl and cycle-related protein CDK2 gene expression.Results The proliferation of MDA-MB-231 cells was inhibited by avicularin at 6.25,12.5,25,50,and 100μmol·L-1 after 48 h(P<0.01),in a concentrationdependent manner.After the treatment with cells at 12.5,50,and 100μmol·L-1 after 48 h,chromatin condensation was observed.When the concentration of avicularin was 50μmol·L-1,the percentage of cells in S phase was significantly increased(P<0.05).When the concentration of avicularin was 100μmol·L-1,the percentage of cells in G2/M phase was significantly increased(P<0.05).With the increase of avicularin concentration,the core sites of PI3K,p-PI3K,AKT,and p-AKT protein was significantly decreased(P<0.01),and the expression levels of apoptosis-related proteins Bcl-2,Bcl-xl and cyclin CDK2 were down-regulated(P<0.05).Conclusion Avicularin can inhibit the proliferation of breast cancer MDA-MB-231 cells,arrest the cell cycle in the S phase,G2/M phase,and induce the apoptosis.The mechanism may be to induce the apoptosis of human breast cancer MDA-MB-231 cells by inhibiting the expression of Bcl-2,Bcl-xl,and CDK2 by downregulating PI3K/AKT signaling pathway.