CAJ | 학술논문

背景：芬维A胺可提高细胞内活性氧（ ROS）水平，其抗肿瘤活性已在众多体内外实验和临床化学预防试验中得到验证。前期研究发现芬维A胺能体外诱导人肝细胞癌（ HCC）细胞凋亡，但确切机制不明。目的：探讨ROS在芬维A胺诱导人HCC细胞凋亡中的作用及其可能机制。方法：分别以抗氧化剂维生素E、芬维A胺单独或联合处理人HCC细胞株Huh-7，共聚焦显微镜和流式细胞术检测活细胞ROS，CellTiter-Glo发光法细胞活力检测试剂盒和Caspase-Glo3/7检测试剂盒分别检测细胞活力和细胞凋亡，免疫荧光染色和蛋白质印迹法分别检测核受体Nur77的表达、定位和应激诱导转录因子GADD153表达。结果：维生素E预处理能充分阻断芬维A胺诱导的ROS产生。在经维生素E预处理的Huh-7细胞中，芬维A胺的促凋亡效应显著降低（ P<0.05）。维生素E预处理能显著降低芬维A胺诱导的GADD153表达，对芬维A胺诱导的Nur77表达和出核则无明显影响。结论：以维生素E清除ROS可抑制芬维A胺促Huh-7细胞凋亡的效应，提示ROS参与了芬维A胺诱导的人HCC细胞凋亡，其机制可能与诱导GADD153蛋白表达有关。
배경：분유A알가제고세포내활성양（ ROS）수평，기항종류활성이재음다체내외실험화림상화학예방시험중득도험증。전기연구발현분유A알능체외유도인간세포암（ HCC）세포조망，단학절궤제불명。목적：탐토ROS재분유A알유도인HCC세포조망중적작용급기가능궤제。방법：분별이항양화제유생소E、분유A알단독혹연합처리인HCC세포주Huh-7，공취초현미경화류식세포술검측활세포ROS，CellTiter-Glo발광법세포활력검측시제합화Caspase-Glo3/7검측시제합분별검측세포활력화세포조망，면역형광염색화단백질인적법분별검측핵수체Nur77적표체、정위화응격유도전록인자GADD153표체。결과：유생소E예처리능충분조단분유A알유도적ROS산생。재경유생소E예처리적Huh-7세포중，분유A알적촉조망효응현저강저（ P<0.05）。유생소E예처리능현저강저분유A알유도적GADD153표체，대분유A알유도적Nur77표체화출핵칙무명현영향。결론：이유생소E청제ROS가억제분유A알촉Huh-7세포조망적효응，제시ROS삼여료분유A알유도적인HCC세포조망，기궤제가능여유도GADD153단백표체유관。
Background:Fenretinide,which is capable of generating reactive oxygen species( ROS ),has emerged as a promising antineoplastic agent based on numerous in vitro and in vivo studies and clinical chemoprevention trials. Preliminary studies showed that fenretinide could induce apoptosis in human hepatocellular carcinoma( HCC)cells in vitro, however,the precise mechanism was not clarified. Aims:To elucidate the effect of ROS on apoptosis of human HCC cells induced by fenretinide and the underlying mechanism. Methods:Human HCC cell line Huh-7 was treated with antioxidant vitamin E,fenretinide or their combination,respectively. ROS in live cells was evaluated by confocal microscopy and flow cytometry;cell viability and apoptosis were assessed by CellTiter-Glo Luminescent Cell Viability Assay Kit and Caspase-Glo3/7 Assay Kit;expression and intracellular localization of nuclear receptor Nur77,as well as expression of stress-induced transcription factor GADD153 were measured by immunofluorescence staining and Western blotting,respectively. Results:Vitamin E pretreatment fully blocked the fenretinide-induced ROS production. In Huh-7 cells pretreated with vitamin E,cell apoptosis induced by fenretinide was significantly reduced(P<0. 05). Furthermore,effect of vitamin E pretreatment was noteworthy on reducing fenretinide-induced GADD153 expression, while no significant impact on fenretinide-induced Nur77 expression and translocation was observed. Conclusions:Elimination of ROS by vitamin E can abrogate the pro-apoptotic effect of fenretinide on Huh-7 cells,which indicates the participation of ROS in fenretinide-induced apoptosis of human HCC cells. Its mechanism might be associated with induction of GADD153 protein expression.