藤黄酸增敏TRAIL诱导人结肠癌HT-29细胞凋亡  

Sensitization of human colon cancer HT-29 cells to TRAIL-induced apoptosis by gambognic acid

作　　者：叶记林[1] 于有江[1] 吴爱莲[1] 王冬艳[1] 刘永春[2] 刘延庆[3] 

机构地区：[1]扬州市职业大学医学院,江苏扬州225009 [2]江苏省苏北人民医院,江苏扬州225001 [3]扬州大学医学院,江苏扬州225001

出　　处：《药学学报》2015年第10期1252-1257,共6页Acta Pharmaceutica Sinica

基　　金：江苏省卫生职业技术教育研究科研课题基金(J201311)

摘　　要：研究藤黄酸(GA)与肿瘤坏死因子相关凋亡诱导配体(TRAIL)联合作用对人结肠癌HT-29细胞的影响。采用TRAIL、GA单独及联合作用的方法诱导HT-29细胞凋亡,MTT法检测细胞增殖的抑制;琼脂糖凝胶电泳法和流式细胞术判断细胞凋亡;通过流式细胞术检测胞浆内的活性氧自由基(ROS)水平;用分光光度法检测Caspase-3、-8、-9相对活性;免疫印迹实验分析c-FLIP、CHOP、DR4和DR5蛋白表达水平。联合应用1μg·m L-1GA 12 h,显著促进40 ng·m L-1 TRAIL对HT-29细胞的生长抑制和诱导凋亡作用;使细胞出现了清晰的、凋亡特有的DNA ladder条带,细胞凋亡率达(45.5±4.9)%;明显升高胞浆内ROS含量;显著提高CHOP、DR4和DR5的表达,分别至对照组的(7.38±0.41)、(5.41±0.60)和(4.85±0.31)倍,使细胞内Caspase-3、-8、-9活性明显升高(P<0.05);下调抗凋亡蛋白c-FLIP表达,仅为对照组的(0.22±0.08)倍。GA可能是通过ROS升高激活内质网应激(ERS)凋亡途径、上调DR4、DR5和下调c-FLIP表达来增敏TRAIL诱导人结肠癌HT-29细胞的凋亡。To investigate the effects of gambognic acid （GA） on TRAIL-induced apoptosis of cancer cells, human colon HT-29 cancer cells were treated with GA to promote apoptosis. Inhibition of the cell proliferation was measured with MTT assay and cell apoptosis was detected with formation of DNA ladders in agarose gel electrophoresis, and activation of caspase activity. The content of cytosolic reactive oxygen species （ROS） was measured with flow cytometry. The activities of Caspase-3, -8, -9 were detected using spectrophotometric assay. The levels of c-FLIP, CHOP, DR4 and DR5 in cells were tested by Western blot. Combination of GA （1 μg-mL-l） and TRAIL （40 ng＇mL 1） significantly reduced proliferation and increased apoptosis of HT-29 cells over those induced by each agent alone. Percentage of apoptotic cells was increased to 45.5%. GA markedly enhanced the intracellular ROS generation. Expression of CHOP, DR4 and DR5 was up-regulated to 7.38, 5.41, and 4.85 times of the control group, respectively. GA promoted activation of Caspase-3, -8, and -9 by TRAIL （P〈0.05）. Furthermore, the expression of anti-apoptotic protein c-FLIP was down-regulated to 0.22±0.08 times of the control group. In conclusion, GA sensitizes HT-29 cells to TRAIL-induced apoptosisby promoting ROS-activated ERS pathways, up-regulating of DR4 and DR5, and inhibiting c-FLIP expression.

关 键 词：肿瘤坏死因子相关凋亡诱导配体 HT-29细胞 细胞凋亡 藤黄酸 

分 类 号：R966[医药卫生—药理学]