藤黄酸联合肿瘤坏死因子相关凋亡诱导配体诱导人结肠癌HT-29细胞凋亡的效果和机制  

Effects of gambognic acid combined with tumor necrosis factor-related apoptosis-inducing ligand on apoptosis of human colon cancer HT-29 cells and their mechanisms

作　　者：叶记林[1] 吴爱莲[1] 王冬艳[1] 彭建明[1] 于有江[1] 刘延庆[2] YE Jilin;WU Ailian;WANG Dongyan;PENG Jianming;YU Youjiang;LIU Yanqing(Medical Science Department, Yangzhou Polytechnic College, Yangzhou 225009, Jiangsu Province, China;Institute of Medical and Pharmaceutical Sciences, Yangzhou University, Yangzhou 225001, Jiangsu Province, China)

机构地区：[1]扬州市职业大学医学院,江苏扬州225009 [2]扬州大学医药研究所,江苏扬州225001

出　　处：《中国癌症杂志》2018年第4期256-262,共7页China Oncology

基　　金：江苏省卫生职业技术教育研究科研课题基金(J201311)

摘　　要：背景与目的:肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)能选择性地杀伤肿瘤细胞,但多种肿瘤对其耐药。该研究旨在探讨藤黄酸(gambognic acid,GA)与TRAIL联合对人结肠癌HT-29细胞裸鼠移植瘤生长的影响及GA联合TRAIL抗结肠癌的作用机制。方法:建立人结肠癌HT-29细胞裸鼠皮下移植瘤模型,测定TRAIL和(或)GA对裸鼠移植瘤的影响,采用H-E染色观察肿瘤组织形态结构改变,TUNEL法检测细胞凋亡状况;HT-29细胞经过siRNA干扰Nrf2表达后,通过AnnexinⅤ-FITC/PI双染检测细胞凋亡情况,流式细胞术检测细胞内活性氧(reactive oxygen species,ROS)含量,RT-PCR法检测Nrf2、Bcl-2、Bax和DR5 mRNA的表达水平。结果:联合应用GA显著促进了TRAIL对裸鼠皮下移植瘤的生长抑制(抑瘤率达67.0%)和诱导凋亡作用,下调了组织中Nrf2、Bcl-2的表达,增强了Bax和DR5的表达;与阴性对照siRNA相比,Nrf2干扰能明显上调TRAIL诱导HT-29细胞的凋亡率,提高ROS含量,下调Nrf2和Bcl-2表达,上调Bax和DR5表达。结论:GA可能是通过下调Nrf2表达,使ROS水平升高而激活线粒体凋亡途径与死亡受体途径,从而逆转人结肠癌HT-29细胞体内外对TRAIL的耐药性。Background and purpose： Tumor necrosis factor-related apoptosis-inducing ligand（TRAIL） can selectively kill tumor cells, but many tumors are resistant to it. The aim of our study was to investigate the effects of gambognic acid（GA） combining TRAIL on the growth of subcutaneous tumor xenografts in nude mice established from human colon cancer cell line HT-29, and to explore the mechanisms related to the effects of GA combined with TRAIL on apoptosis of human colon cancer HT-29 cells. Methods： A nude mouse model of colon cancer was established by subcutaneous inoculation of human colon cancer cell line HT-29. The effects of TRAIL or/and GA on transplanted tumor in nude mice were measured. The histopathological changes of tumor tissues were observed by H-E staining, and the tumor cell apoptosis was detected by TUNEL assay. Transfection of HT-29 cells with the si RNA caused a significant reduction in Nrf2 protein expression. Then Annexin Ⅴ-FITC apoptosis kit was used to detect the cell apoptosis, and the generation of reactive oxygen species（ROS） was assayed using flow cytometry. The mRNA expressions of Nrf2, Bcl-2, Bax and DR5 were determined by RT-PCR. Results： TRAIL combined with GA significantly promoted the inhibitory effect of TRAIL on the growth of transplanted tumor in nude mice（the inhibition rate reached 67.0%）, increased the apoptosis of HT-29 cells, promoted the decrease in the expression of Nrf2 and Bcl-2, and potentiated the expression of Bax and DR5. Compared with control siRNA, Nrf2 interference markedly increased the apoptosis of HT-29 cells induced by TRAIL, enhanced the ROS level, down-regulated the expression of Nrf2 and Bcl-2, and up-regulated the expression of Bax and DR5. Conclusion： GA reverses the TRAIL resistance in HT-29 cells in vivo and in vitro by upregulating Nrf2 and promoting ROS-activated mitochondrial apoptosis pathway and death receptor pathway.

关 键 词：藤黄酸 肿瘤坏死因子相关凋亡诱导配体 HT-29细胞 Nrf2 Bcl-2 Bax DR5 

分 类 号：R730.23[医药卫生—肿瘤]