蟾毒灵调控PI3K/Akt 通路对结肠癌HT-29 细胞凋亡及周期的影响
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R285

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河南省中医管理局青苗人才项目(豫中医科教[2018]16号)


Effects of Bufalin Regulating PI3K/Akt Pathway on Apoptosis and Cell Cycle of Colon Cancer HT-29 Cells
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    摘要:

    目的:研究蟾毒灵诱导人结肠癌HT-29 细胞凋亡及周期阻滞的能力并对其分子机制进行探讨。方法:将人结肠癌HT-29 细胞分为对照组(野生型细胞),蟾毒灵(Bufalin) 处理组(0.25,0.5,1 μmol/L) 和顺铂(DDP) 组(1 μg/mL)。通过流式细胞术(FCM) 检测蟾毒灵诱导人结肠癌HT-29 细胞凋亡及周期阻滞的作用;使用蛋白质免疫印迹法检测经蟾毒灵处理HT-29细胞24 h 后磷脂酰肌醇-3 激酶(PI3K)、磷酸化磷脂酰肌醇-3 激酶(p-PI3K)、蛋白激酶B(Akt) 和磷酸化蛋白激酶B(p-Akt)蛋白表达的变化。通过实时荧光定量PCR 法(RT-qPCR) 检测蟾毒灵对HT-29 细胞PI3K、Akt mRNA 表达的影响。结果:FCM结果显示,与对照组比较,蟾毒灵(0.25,0.5,1 μmol/L) 组能诱导结肠癌HT-29 细胞的凋亡(P<0.05),并呈剂量依赖性。与对照组比较,蟾毒灵(0.25,0.5,1 μmol/L) 组能够诱导结肠癌HT-29 细胞的周期阻滞于G2/M 期,并且蟾毒灵浓度越大,周期阻滞效果越显著(P<0.05)。蛋白质印迹实验结果表明,与对照组比较,蟾毒灵能够下调PI3K、p-PI3K、Akt 和p-Akt 的表达水平,并呈一定的剂量依赖(P<0.05)。RT-qPCR 结果显示,与对照组比较,蟾毒灵能够显著下调PI3K 和Akt mRNA 的表达。结论:蟾毒灵能够诱导结肠癌HT-29 细胞凋亡及周期阻滞,其机制可能与抑制PI3K/Akt 信号通路有关。

    Abstract:

    Abstract:Objective:To study the ability of bufalin to induce apoptosis and cycle arrest of human colon cancer HT- 29 cell, and to explore its molecular mechanism. Methods: Human colon cancer HT- 29 cells was divided into control group (wild- type cells) and bufalin treatment group(0.25, 0.5, 1 μmol/L) and cisplatin(DDP) group(1 μg/mL). The apoptosis and cycle arrest of human colon cancer HT- 29 cell induced by bufalin were detected by flow cytometry(FCM). Western blotting was used to detect the changes of protein expression of phosphatidylinositol- 3 kinase(PI3K), phosphorylated phosphatidylinositol- 3 kinase(p- PI3K), protein kinase B(Akt) and phosphorylated protein kinase B(p- Akt) in HT- 29 cells treated with bufalin for 24 hours. The effect of bufalin on the mRNA expression of PI3K and Akt in HT-29 cells was detected by real-time fluorescence quantitative PCR(RT-qPCR). Results:The results of FCM showed that compared with the control group,bufalin(0.25,0.5,1 μmol/L) group could induce apoptosis of colon cancer HT-29 cell in a dose-dependent manner (P<0.05). Compared with the control group,bufalin(0.25,0.5,1 μmol/L) group could induce the cycle arrest of colon cancer cell line HT-29 in G2/M phase,and the greater the concentration of bufalin,the more significant the cycle arrest effect(P< 0.05). Western blot results showed that compared with the control group,bufalin could down regulate the expression levels of PI3K,p-PI3K,Akt and p-Akt in a dose-dependent manner(P<0.05). RT-qPCR results showed that compared with the control group, bufalin could significantly down regulate the expression of PI3K and Akt mRNA. Conclusion: Bufalin can induce apoptosis and cycle arrest of colon cancer HT-29 cell,and its mechanism may be related to the inhibition of PI3K/Akt signal pathway.

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董倩倩,张俊萍.蟾毒灵调控PI3K/Akt 通路对结肠癌HT-29 细胞凋亡及周期的影响[J].新中医,2021,53(23):149-154

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  • 在线发布日期: 2021-12-10
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