自噬在染料木素抑制结肠癌HCT116细胞增殖中的作用

Role of autophagy in genistein inhibition on proliferation of colon cancer cell line HCT116

目的:观察染料木素(genistein)对结肠癌HCT116细胞的自噬诱导作用并探讨其在抗肿瘤增殖中的作用。方法:不同浓度(0、5、10、20、40、60、80 mg/L)染料木素及80 mg/L染料木素联合2 mmol/L自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)分别作用于HCT116细胞24、48、72 h后,CCK8法检测细胞增殖活力。通过双荧光素酶慢病毒载体构建LC3及突变LC3(LC3 Mu)基因过表达的HCT116细胞,经100 mg/L的染料木素处理24 h后,荧光显微镜观测荧光在细胞内聚集情况。不同浓度(0、10、50、100 mg/L)染料木素处理HCT116细胞24 h后,Western blot检测细胞内LC3蛋白Ⅱ/Ⅰ的比例。结果:CCK8法细胞增殖实验的结果表明,染料木素对HCT116细胞具有显著的增殖抑制作用,染料木素对HCT116细胞的增殖抑制呈时间-剂量依赖性(P<0.01)。染料木素对HCT116细胞的24 h、48 h、72 h半数抑制浓度IC50分别为85.70 mg/L、34.44 mg/L、20.56 mg/L,而3-MA可以降低染料木素对HCT116细胞的抗增殖作用(P<0.01)。荧光显微镜观测结果表明,染料木素可以诱导HCT116细胞中LC3荧光蛋白的聚集(P<0.01),从而参与自噬小体的形成;Western blot结果显示,随着染料木素浓度的增加,LC3蛋白Ⅱ/Ⅰ的比例逐渐升高(P<0.01)。结论:染料木素可能通过上调LC3蛋白Ⅱ/Ⅰ的比例诱导细胞内自噬体的产生,从而抑制结肠癌细胞的增殖。

Objective: To observe the effects of genistein on autophagy in colon cancer cell line HCT116,and discuss its role in proliferation inhibition. Methods: The HCT116 cells were cultured in vitro and treated with genistein at different concentrations( 0,5,10,20,40,60 and 80 mg/L) for different time( 24,48 and 72 hours),meanwhile other cells were treated with 80 mg/L genistein and 2 mmol/L 3-methyladenine( 3-MA,autophagy inhibitor). The cell proliferation activity was detected by CCK8. The LC3 and mutant LC3( LC3 Mu) overexpressing HCT116 cells were established by dual luciferase lentivirus vector. Then the overexpressing HCT116 cells were treated with 100 mg/L genistein for 24 hours,and the aggregation of fluorescence in cells was observed by fluorescence microscope. After treatment with genistein at different concentrations of 0,10,50 and 100 mg/L for 24 hours,the protein expression ratio of LC3 Ⅱ/Ⅰ in cells was detected by Western blot. Results: The results of CCK8 assay showed that genistein could significantly inhibit the proliferation of HCT116 cells,and genistein suppressed the proliferation of HCT116 cells in a time-and dose-dependent manner( P < 0.01). The half inhibitory concentration( IC50) of genistein on HCT116 cells at 24 h,48 h and 72 h were 85.70 mg/L,34.44 mg/L,20. 56 mg/L respectively. 3-MA could decrease the inhibiting effects of genistein on the proliferation of HCT116 cells( P < 0.01). The results of fluorescent microscopy showed that genistein could induce the aggregation of LC3 fluorescent protein in HCT116 cells( P< 0.01),which promoted the production of autophagosome. The results of Western blot showed that the protein ratio of LC3Ⅱ/Ⅰwas gradually increased with the increasing concentration of genistein( P < 0.01). Conclusion: Genistein may inhibit the proliferation of colon cancer cells by upregulating the ratio of LC3 Ⅱ/Ⅰand promoting the production of autophagosome.