叶黄素通过PI3K/AKT信号通路调控胃癌干细胞增殖与凋亡

Lutein regulates proliferation and apoptosis of gastric cancer stem cells through PI3K/AKT signaling pathway

背景:叶黄素可抑制多种肿瘤细胞的增殖并促进凋亡,但其对胃癌干细胞增殖与凋亡的影响及机制尚未见相关报道。目的:观察叶黄素对胃癌干细胞增殖与凋亡的影响,探讨其可能机制。方法:体外培养人胃癌细胞株SGC-7901获得胃癌干细胞,流式细胞术鉴定细胞表面标志物CD44、CD24。用0,20,40,80,160mmol/L叶黄素分别处理人胃癌干细胞24,48,72h,CCK-8法检测细胞增殖活性,确定最佳作用浓度和作用时间。然后,将人胃癌干细胞分为4组:对照组、80mmol/L叶黄素组、胰岛素样生长因子1组、叶黄素+胰岛素样生长因子1组,分别干预细胞48h,流式细胞术检测细胞凋亡,实时定量PCR检测PI3K和Akt mRNA的表达,Western blot检测PI3K、p-PI3K、Akt、p-Akt蛋白的表达。结果与结论:①体外培养人胃癌SGC7901细胞获得的胃癌干细胞表达CD44和CD24;②80和160mmol/L叶黄素作用48h对人胃癌干细胞增殖活力的抑制作用优于其他作用浓度和时间(P<0.05),叶黄素作用48h的IC50为91.58mmol/L,选取80mmol/L叶黄素作用48h进行后续实验;③胰岛素样生长因子1组细胞增殖活力高于其他3组,叶黄素组细胞增殖活力低于其他3组,差异有显著性意义(P<0.05);④叶黄素组细胞凋亡率高于其他3组,胰岛素样生长因子1组细胞凋亡率低于其他3组,差异有显著性意义(P<0.05);⑤叶黄素组PI3K和Akt mRNA的表达低于其他3组,胰岛素样生长因子1组PI3K和Akt mRNA的表达高于其他3组,差异有显著性意义(P<0.05);⑥叶黄素组p-PI3K和p-Akt蛋白的表达低于对照组和胰岛素样生长因子1组,胰岛素样生长因子1组p-PI3K和p-Akt蛋白表达高于其他3组,差异有显著性意义(P<0.05);⑦结果提示,叶黄素通过抑制PI3K/Akt信号通路抑制人胃癌干细胞增殖和诱导凋亡。

BACKGROUND:Lutein can inhibit proliferation and promote apoptosis in cancer cells of various origins.However,there are no reports on the effect and mechanism of lutein on the proliferation and apoptosis of gastric cancer stem cells.OBJECTIVE:To observe the effect of lutein on proliferation and apoptosis of gastric cancer stem cells and explore its possible mechanism.METHODS:Gastric cancer stem cells were obtained from human gastric cancer cell line SGC-7901 in vitro.Flow cytometry was used to identify cell surface markers CD44 and CD24.Human gastric cancer stem cells were treated with 0,20,40,80,and 160 mmol/L lutein for 24,48,and 72 hours,respectively.Cell proliferation activity was detected by cell counting kit-8 method,to determine the optimal concentration and action time of lutein.Human gastric cancer stem cells were then divided into four groups:control group,80 mmol/L lutein group,insulin-like growth factor 1 group,lutein+insulin-like growth factor 1 group,and given the corresponding treatments for 48 hours.Flow cytometry was used to detect cell apoptosis.The expressions of PI3K and Akt mRNA were detected by quantitative real-time PCR,and the expressions of PI3K,p-PI3K,Akt and p-Akt proteins were detected by western blot assay.RESULTS AND CONCLUSION:(1)Gastric cancer stem cells derived from human gastric cancer SGC7901 cells cultured in vitro expressed CD44 and CD24.(2)Lutein at 80 and 160 mmol/L acting for 48 hours could achieve the best inhibitory effect(P<0.05).As the half maximal inhibitory concentration(IC50)of lutein for 48 hours was 91.58 mmol/L,80 mmol/L lutein acting for 48 hours was selected for subsequent experiments.(3)The proliferation activity of the cells was highest in the insulin-like growth factor 1 group and lowest in the lutein group,and there were significant differences between groups(P<0.05).(4)The apoptotic rate was highest in the lutein group and lowest in the insulin-like growth factor 1 group,and there were significant differences between groups(P<0.05).(5)The expressions of PI3K and Akt mRNA were highest in the lutein group and lowest in the insulin-like growth factor 1 group,and there were significant differences between groups(P<0.05).(6)The expressions of p-PI3K and p-Akt protein in the lutein group were significantly lower than those in the control group and insulin-like growth factor 1 group,while the expressions of p-PI3K and p-Akt protein in the insulin-like growth factor 1 group were significantly higher than those in the other three groups(P<0.05).These results suggest that lutein inhibits the proliferation and induces apoptosis of human gastric cancer stem cells by inhibiting the PI3K/Akt signaling pathway.