内质网应激-自噬参与葫芦素E诱导的结肠癌细胞凋亡  

Effects of cucurbitacin E on apoptosis of human colonic cancer cells induced by endoplasmic reticulum stress and autophagy

作　　者：随何欢 赵海霞 朱丽莎 唐春梅 梁婧 苏强 SUI Hehuan;ZHAO Haixia;ZHU Lisha;TANG Chunmei;LIANG Jing;SU Qiang(Department of Pharmacy, Nanchong Central Hospital, The Second Clinical School of North Sichuan Medical College, Nanchong Key Laboratory of Individualized Drug Therapy, Nanchong 637000, Sichuan, China)

机构地区：[1]南充市中心医院·川北医学院第二临床医学院药学部·个体化药物治疗南充市重点实验室,四川南充637000

出　　处：《西部医学》2020年第11期1604-1607,共4页Medical Journal of West China

基　　金：川北医学院科研发展计划项目(CBY18-A-ZD07);南充市市校科技战略合作项目(19SXHZ0319);南充市市校合作重要研发平台科研项目(NSMC20170311)。

摘　　要：目的观察内质网应激和自噬反应对葫芦素E(CuE)诱导人结肠癌细胞系HT-29凋亡的作用。方法采用0.001、0.01、0.1、1、10μmol/L的CuE分别处理培养的HT-29细胞24、48及72 h,对照组仅用DMEM培养液。Annexin V-FITC/PI双染色流式细胞分析法检测细胞凋亡,蛋白免疫印迹法检测内质网应激相关蛋白CHOP、Grp78以及自噬相关蛋白Beclin1、LC3表达水平。结果Annexin V-FITC/PI双染色流式细胞分析结果表明,随着CuE浓度升高,作用时间延长,凋亡细胞占总细胞比例明显升高(P<0.05),呈时间与剂量依赖性。蛋白印迹分析结果显示,用CuE处理HT-29细胞24 h后,与对照组相比,内质网应激相关蛋白CHOP及Grp78蛋白表达明显升高(P<0.05),自噬相关蛋白Beclin1表达逐渐增(P>0.05),胞浆型LC3(LC3-Ⅰ)发生酶解,转变为自噬体膜型LC3-Ⅱ,且LC3-Ⅱ/LC3-Ⅰ比值大小呈现出先上升(P<0.05)后下降(P>0.05)的趋势。结论内质网应激和自噬反应参与介导了CuE引起的人结肠癌细胞HT-29凋亡,其可能是CuE诱导结肠癌细胞凋亡的重要途径。Objective To study the effect of cucurbitacin E(CuE)on apoptosis of human colonic cancer HT-29 cells induced by endoplasmic reticulum stress(ERS)and autophagy in vitro.Methods The cultured HT-29 cells were treated with CuE at 0.001,0.01,0.1,1 and 10μmol/L for 24,48 and 72 hours,respectively.The changes of cell apoptosis were detected by flow cytometry coupled with Annexin V-FITC/PI staining.The Western blot assay was used to detect the protein expression of CHOP,Grp78,Beclin1 and LC3.Results The results of flow cytometry coupled with Annexin V-FITC/PI staining revealed that with the increase of CuE concentration.The proportion of apoptotic cells and total cells was increased significantly.Western blot analysis showed that after treating HT-29 cells with CuE for 24 hours.The expression of endoplasmic reticulum stress-related proteins CHOP and Grp78 was significantly increased(P<0.05).The autophagy-related protein expression of Beclin1 was increased.The cytoplasmic type LC3(LC3-I)had an enzymatic hydrolysis,which was transformed into autophagy film type LC3-II.The ratio of LC3-Ⅱ/LC3-I was decreased(P<0.05)and then decreased(P>0.05).Conclusion ERS and autophagy were involved in the apoptosis of human colon cancer cell line HT-29 induced by CuE,which suggested that were an important pathway of colon cancer cell apoptosis induced by CuE.

关 键 词：内质网应激 自噬 葫芦素E 结肠癌 结肠癌细胞HT-29 凋亡 

分 类 号：R735.35[医药卫生—肿瘤]