蛇葡萄素通过抑制自噬促进结肠癌HCT116细胞凋亡

Ampelopsin promotes apoptosis of colon cancer HCT116 cells by inhibiting autophagy

目的:研究蛇葡萄素(ampelopsin,AMP)对人结肠癌细胞增殖、自噬及凋亡的影响及可能的机制,并观察自噬在结肠癌细胞凋亡过程中的作用。方法:采用慢病毒感染法构建绿色荧光蛋白(green fluorescent protein,GFP)和红色荧光蛋白(red fluorescent protein,RFP)双标记的微管相关蛋白轻链3(microtubule-associated protein light chain 3,LC3)过表达的重组结肠癌HCT116-RFP-GFP-LC3细胞;荧光显微镜下观察标记有GFP和RFP LC3的表达情况,同时采用蛋白质印迹法检测HCT116-RFP-GFP-LC3细胞中LC3的表达水平。CCK-8法检测不同质量浓度的AMP(0、50、100、175、250、350、400和450μg/mL)对重组HCT116-RFP-GFP-LC3与亲本HCT116细胞增殖的影响,同时观察对2株细胞形态的影响。用AMP(0、25、50和75μg/mL)处理亲本HCT116细胞后,采用FCM法检测细胞的凋亡率,蛋白质印迹法检测凋亡通路相关蛋白Bax、核因子-κB(nuclear factor-kappa B,NF-κB)及Bcl-2的表达水平。通过激光共聚焦显微镜观察用AMP(0、25、50和75μg/mL)单药或联合自噬晚期抑制剂巴佛洛霉素A1(bafilomycin A1,Baf A1)(20 nmol/L)处理后,HCT116-RFP-GFP-LC3细胞中自噬点状聚集数量的变化情况;AMP(25μg/mL)单药或联合Baf A1(20 nmol/L)处理HCT116细胞后,再用FCM法检测细胞凋亡率,蛋白质印迹法检测HCT116细胞中相关自噬通路蛋白LC3Ⅱ、p62及凋亡相关蛋白的表达。结果:RFP-GFP-LC3在HCT116-RFP-GFP-LC3细胞中稳定表达,成功构建重组HCT116-RFP-GFP-LC3细胞株。CCK-8法检测结果显示,AMP可以显著抑制重组株和亲本株细胞的存活率并呈浓度依赖性,且AMP对HCT116重组株和亲本株增殖抑制率及细胞形态的影响均无明显差异。AMP能明显促进HCT116细胞的凋亡,呈浓度依赖性(P值均<0.05);促凋亡蛋白Bax及核转录因子NF-κB的表达水平上调,抑凋亡蛋白Bcl-2的表达水平下调(P值均<0.01)。经不同质量浓度的AMP处理后,HCT116-RFP-GFP-LC3细胞中自噬点数量明显增加(P<0.01),同时自噬蛋白LC3Ⅱ表达水平有略微上调,说明AMP诱导结肠癌细胞发生自噬。相较于AMP单药组,AMP联合Baf A1组HCT116细胞的凋亡率明显提高(P<0.05),促凋亡蛋白Bax表达水平明显上调(P<0.05),抑凋亡蛋白Bcl-2的表达水平明显下调(P<0.01),提示自噬过程的阻断能够促进细胞的凋亡。结论:AMP可抑制结肠癌HCT116细胞的增殖,诱导其发生自噬及凋亡;自噬过程的抑制可以促进AMP诱导的结肠癌细胞的凋亡。

Objective:To investigate the inhibitory effects of ampelopsin(AMP)on proliferation,autophagy and apoptosis to human colon cancer cells and its possible mechanism.To observe the function of autophagy in the apoptosis of colon cancer cells as well.Methods:The lentivirus infection method was adopted here to construct double fluorescence[green fluorescent protein(GFP)and red fluorescent protein(RFP)]labeled microtubule-associated protein light chain 3(LC3)overexpressed recombinant HCT116-RFP-GFP-LC3 cells.The GFP and RFP labeled LC3 expression in HCT116-RFP-GFP-LC3 cells were observed by fluorescence microscope,and Western blotting was used to detect the expression of LC3 in HCT116-RFP-GFP-LC3 cells at the same time.The proliferation inhibition of recombination HCT116-RFP-GFP-LC3 cells and parental HCT116 cells treated with different concentrations of AMP(0,50,100,175,250,350,400 and 450μg/mL)was detected by CCK-8 assay and the cell morphology was observed.The apoptotic rate of HCT116 cells treated with AMP(0,2550,and 75μg/mL)was detected by FCM method,and the apoptotic-related proteins Bax,nuclear factor-kappa B(NF-κB)and Bcl-2 expression levels were detected by Western blotting.Autophagy induction was detected by counting fluorescence dots triggered by transformation of LC3Ⅰto LC3Ⅱvisualized by laser confocal microscopy after HCT116-RFP-GFP-LC3 cells treated with AMP(0,2550,and 75μg/mL)alone or combined with bafilomycin A1(Baf A1)(an autophagy inhibitor)(20 nmol/L).After the HCT116 cells were treated with AMP(25μg/mL)alone or combined with Baf A1(20 nmol/L),the apoptotic rate was detected by FCM method,and the expression level of autophagy-related proteins LC3Ⅱand p62 as well as apoptotic-related proteins were detected by Western blotting.Results:RFP-GFP-LC3 could stably express in HCT116-RFP-GFP-LC3 cells,indicating that HCT116-RFP-GFP-LC3 recombinant cells were successfully constructed.The results of CCK-8 assay suggested that AMP could significantly inhibit the proliferation of recombinant and parental cells in a concentration-dependent manner.Meanwhile,there was no significant difference in cell morphology or cell proliferation inhibition rate between these two cells(P>0.05).The apoptosis rate of HCT116 cells increased remarkably along with the enhancement of AMP concentration which means there is a concentration-dependent manner(all P<0.05).The expression levels of pro-apoptotic protein Bax and NF-κB were increased,and the apoptosis inhibitory protein Bcl-2 was down-regulated(all P<0.01).The confocal microscopy results showed that with the increase of AMP concentration,the numbers of autophagy points were significantly increased(P<0.01),and the expression level of autophagy protein LC3Ⅱwas up-regulated,indicating that AMP could trigger autophagy on colon cancer cells.Comparing with AMP alone group,AMP combined with Baf A1 could increase the apoptosis rate of HCT116 significantly(P<0.05),and the expression level of apoptotic protein Bax promoted remarkably(P<0.05).The expression level of the apoptosis inhibitory protein Bcl-2 demoted as well.All these results suggest that autophagy blocking could promote AMP induced apoptosis.Conclusion:AMP can inhibit the proliferation of colon cancer cells HCT116,inducing autophagy and apoptosis;inhibition of autophagy can promote AMP-induced apoptosis on colon cancer cells.