澳洲茄胺诱导肠癌HCT-116细胞凋亡的实验研究

Study on Apoptosis Mechanism of Human Colon Cancer Cell Lines HCT-116 Induced by Solasodine

  • 摘要: 目的 通过研究中药灌肠方中的有效成分澳洲茄胺对肠癌HCT-116细胞凋亡及相关基因表达的影响,探讨澳洲茄胺抑制肠癌细胞增殖的作用机制。方法 通过实时无标记细胞分析仪检测澳洲茄胺对HCT-116细胞增殖的影响;流式细胞仪检测HCT-116细胞凋亡;qPCR法检测凋亡相关基因Bax、Survivin的mRNA表达水平;Western blot实验检测凋亡相关蛋白Bcl-2、Bax、Bcl-xl、Caspase-3、Caspase-8、Caspase-9、PARP以及PI3K/Akt信号通路蛋白PI3K、Akt表达情况。结果 实时无标记细胞分析检测法证实澳洲茄胺对肠癌HCT-116细胞的生长有抑制作用,且随药物浓度、给药时间的增加而增强;流式检测发现随药物浓度的增加,其凋亡增多;qPCR法证实澳洲茄胺能上调Bax、下调Survivin的mRNA表达;Western blot法检测证实澳洲茄胺上调Bax蛋白,促进Caspase-3、Caspase-8、Caspase-9、PARP蛋白活化,下调Bcl-2、Bcl-xl蛋白表达,升高Bax/Bcl-2表达比值,并且抑制PI3K、Akt蛋白活化。结论 澳洲茄胺抑制肠癌HCT-116细胞增殖,通过抑制PI3K/Akt信号通路及调控Caspase家族、Bcl-2家族、Survivin、PARP的表达,诱导细胞凋亡,对凋亡的2条途径均有作用,即线粒体途径和死亡受体途径。

     

    Abstract: OBJECTIVE To explore the effects of Solasodine on the apoptosis of human colon cancer cell line HCT-116 and the expression of associated genes,and to investigate the mechnism of the inhibition of HCT-116 cells proliferation. METHODS After treatment, the inhibitory effect of HCT-116 cell proliferation was determined by Real time non-label cell analyzer. Flow cytometry was employed to detect cell apoptosis. The qPCR method was used to determine the mRNA levels of Bax and Survivin. Protein levels of Bcl-2, Bax, Bcl-xl, Caspase-3, Caspase-8, Caspase-9, PARP, PI3K and Akt were determined by Western blot . RESULTS Real time non-label cell analysis result showed that Solasodine inhibited the growth of HCT-116 in vitro, and the potency enhanced with the increase of drug dose and treat time. Flow cytometry found that the cell apoptosis increased with the increase of drug concentration. The qPCR showed that the mRNA levels of apoptosis-related genes Bax and Survivin changed. Western blot results revealed that Bax protein levels increased, while Bcl-2 and Bcl-xl protein levels decreased, and the ratio of Bax/Bcl-2 was also increased. It showed that Solasodine also promoted the Caspase-3, Caspase-8, Caspase-9 and PARP activities, and inhibited the activation of PI3K and Akt . CONCLUSION Solasodine can inhibit HCT-116 cells proliferation and induce the cell apoptosis via blocking PI3K/Akt signaling pathway and regulating Caspase family, the Bcl-2 family, and the expression of Survivin and PARP. It can regulate these two apoptotic pathways, mitochondrial pathway and death receptor pathway.

     

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