黄芪皂苷Ⅳ对食管癌细胞凋亡、干细胞样特性和PI3K/AKT通路的影响

Effects of AstragalosideⅣon apoptosis,stem cell-like characteristics and PI3K/AKT pathway of esophageal carcinoma cells

目的探讨黄芪皂苷Ⅳ(astragalosideⅣ,AS-Ⅳ)对食管癌TE-1细胞凋亡、干细胞样特性和PI3K/AKT通路的影响。方法将TE-1细胞分为四组:空白对照组、黄芪皂苷Ⅳ20μg/ml组、黄芪皂苷Ⅳ40μg/ml组和黄芪皂苷Ⅳ80μg/ml组。给予各组细胞不同的黄芪皂苷Ⅳ药物浓度处理48 h后采用CCK-8法检测细胞存活率,成球实验检测干细胞成球情况,流式细胞术检测细胞凋亡,Western印迹法检测凋亡相关蛋白、干细胞标志蛋白和PI3K/AKT通路蛋白,以及免疫荧光检测AKT膜定位情况。结果与空白对照组比较,黄芪皂苷Ⅳ40、80μg/ml组显著增加细胞凋亡率,显著上调caspase-3和caspase-9蛋白表达(P<0.05);黄芪皂苷Ⅳ40、80μg/ml组显著减小干细胞成球球体直径及球体数量(P<0.05);黄芪皂苷Ⅳ40、80μg/ml组显著下调干细胞标志蛋白SOX2、OCT4和CD44的表达(P<0.05);黄芪皂苷Ⅳ40、80μg/ml组显著下调通路蛋白PI3K和AKT磷酸化水平,显著抑制AKT膜定位(P<0.05)。结论黄芪皂苷Ⅳ可诱导TE-1细胞凋亡,抑制干细胞样特性,其作用机制可能与抑制PI3K/AKT通路活化有关。

Objective To investigate the effects of astragalosideⅣ(AS-Ⅳ)on apoptosis,stem cell-like characteristics and PI3K/AKT pathway of esophageal cancer TE-1 cells.Method TE-1 cells were divided into four groups:blank control group,astragalosideⅣ20μg/ml group,astragalosideⅣ40μg/ml group and astragalosideⅣ80μg/ml group.After 48 h of treatment with different concentrations of astragalosideⅣ,the cell survival rate was detected by CCK-8 method,the pellet formation of stem cells was detected by pellet formation assay,cell apoptosis was detected by flow cytometry,apoptosis-related proteins,stem cell marker proteins and PI3K/AKT pathway proteins were detected by Western Blot.And the localization of AKT membrane was detected by immunofluorescence.Result Compared with blank control group,astragalosideⅣ40 and 80μg/ml groups significantly increased the apoptosis rate,and significantly up-regulated the protein expression of caspase-3 and caspase-9(P<0.05).AstragalosideⅣ40 and 80μg/ml groups significantly decreased the diameter and number of stem cell spherules(P<0.05);AstragalosideⅣ40 and 80μg/ml groups significantly down-regulated the expression of stem cell marker proteins SOX2,OCT4 and CD44(P<0.05).AstragalosideⅣ40 and 80μg/ml groups significantly down-regulated the phosphorylation levels of pathway protein PI3K and AKT,and significantly inhibited AKT membrane localization(P<0.05).Conclusion AstragalosideⅣcan induce apoptosis of TE-1 cells and inhibit stem cell-like properties,which may be related to inhibition of PI3K/AKT pathway activation.