粉防己碱对人结直肠癌HCT116细胞株生物功能的影响及其机制

Effect of tetrandrine on human colorectal cancer HCT116 cell line and its potential mechanism

目的:探究粉防己碱对人结直肠癌HCT116细胞株生物功能的影响及其可能机制。方法:采用CCK-8检测粉防己碱对HCT116细胞活力的影响及其半数抑制浓度;用不同浓度粉防己碱(0、2.5、5和10μmol/L)处理细胞,细胞集落形成实验检测细胞增殖;流式细胞术、线粒体膜电位检测技术和Hoechst 33342细胞核染色技术检测细胞凋亡;细胞迁移实验和划痕实验检测细胞迁移能力;蛋白印迹技术检测细胞上皮间质转化标志蛋白表达水平及PI3K/AKT/NF-κB信号通路中关键分子活化程度。结果:HCT116细胞活力随粉防己碱处理浓度增加而降低,粉防己碱半数抑制浓度为9.441μmol/L;与0μmol/L组相比,2.5、5和10μmol/L粉防己碱组细胞形成集落数明显减少(P均<0.05);5、10μmol/L粉防己碱组G0/G1期细胞比例增加,S期和G2/M期细胞比例下降,凋亡细胞比例增加(P均<0.05);2.5、5、10μmol/L粉防己碱组细胞线粒体膜电位降低,细胞核荧光增强,迁移能力降低(P均<0.05);与0μmol/L组相比,10μmol/L粉防己碱组细胞多呈圆形,有更多上皮细胞形态;2.5、5和10μmol/L粉防己碱组细胞上皮标志蛋白E-钙黏蛋白表达增加,而间充质标志物N-钙黏蛋白和波形蛋白表达减少,且PI3K/AKT/NF-κB信号通路中关键分子AKT和NF-κB的磷酸化水平降低(P均<0.05)。结论:粉防己碱可能通过抑制PI3K/AKT/NF-κB信号途径逆转HCT116细胞上皮间质转化,进而降低细胞增殖迁移能力,诱导其凋亡。

Objective:To explore the effect of tetrandrine(TET)on human colorectal cancer HCT116 cell line and its potential mechanism.Methods:CCK-8 assay was used to assess cells in viability and its 50%inhibitory concentration following treatment with TET.HCT116 cells were treated with different concentrations of TET(0,2.5,5 and 10μmol/L),and cell colony formation assay was used to detect cell proliferation.Cell apoptosis was determined by using flow cytometry,mitochondrial membrane potential(MMP)assay and Hoechst 33342 fluorescent staining,and cell migration was determined by migration assay and Wound healing assay.The protein expression level of epithelial-mesenchymal transition marker protein and PI3K/AKT/NF-κB signal pathway after TET treatment was determined by Western blotting.Results:The activity of HCT116 cells decreased with the increase of TET concentration,and the 50%inhibition concentration of TET was 9.441μmol/L.Compared with 0μmol/L group,the number of colony forming cells in 2.5,5 and 10μmol/L TET groups decreased(all P<0.05),and the proportion of cells in G0/G1 phase increased,the proportion of cells in S phase and G2/M phase decreased in 5 and 10μmol/L TET groups(all P<0.05).Compared with 0μmol/L group,2.5,5 and 10μmol/L TET group had more apoptosis and less migration ability(all P<0.05),and cells appeared round in 10μmol/L TET group.Compared with 0μmol/L group,the expression of E-cadherin was increased in 2.5,5 and 10μmol/L TET groups,while N-cadherin and Vimentin expression were decreased,and the phosphorylation of AKT and NF-κB(p65),the key molecules in PI3K/AKT/NF-κB signaling pathway was inhibited(all P<0.05).Conclusion:TET may reverse the epithelial-mesenchymal transformation of HCT116 cells by inhibiting PI3K/Akt/NF-κB signal pathway,and then reduce the ability of cell proliferation and migration and induce apoptosis.