亚硝酸钠通过线粒体自噬增强人肝癌SMMC-7721细胞迁移和侵袭能力  

Sodium nitrite enhanced the potentials of migration and invasion of human hepatocellular carcinoma SMMC-7721 cells through induction of mitophagy

作　　者：归冠 孟珊珊[2] 李鲁娟[3] 刘彬[1] 梁红霞[1] 皇甫超申[2] 

机构地区：[1]河南大学护理学院,河南开封475004 [2]河南大学环境医学研究所,河南开封475004 [3]河南大学第一附属医院,河南开封475004

出　　处：《药学学报》2016年第1期59-67,共9页Acta Pharmaceutica Sinica

基　　金：国家环保公益项目专项资助项目(200809115);省部共建河南大学科研项目(SBGJ090702)

摘　　要：亚硝酸盐对细胞具有独特的氧化还原作用,可以促进肝癌细胞侵袭和转移,但是具体机制尚不清楚。癌细胞通过线粒体自噬,清除多余的线粒体,维持细胞的恶性特征。本文旨在探讨亚硝酸盐对肝癌细胞线粒体自噬的作用以及活性氧(ROS)和缺氧诱导因子-1α(HIF-1α)在其中的作用。用系列浓度的亚硝酸钠在常氧下孵育人肝癌SMMC-7721细胞24 h,用MTT法测定吸光度值反映细胞活力,发现16 mg·L^(-1)亚硝酸钠对细胞活力的促进作用最强。Transwell小室实验显示,亚硝酸钠孵育细胞24 h,可以明显增强细胞迁移和侵袭能力;用微丝示踪剂和Mito-Tracker Red共染色,荧光显微镜下发现,亚硝酸钠促进应力纤维和伪足增加,线粒体呈现核周分布;以上现象均可以被活性氧清除剂N-乙酰半胱氨酸(N-acetylcysteine,NAC)抑制。用ROS示踪剂DCFH-DA孵育,在荧光显微镜下发现,亚硝酸钠明显增强细胞内ROS水平。LC3免疫荧光和Western blot结果显示,亚硝酸钠明显增强细胞的自噬流;透射电镜观察发现,亚硝酸钠诱导细胞自噬溶酶体形成,NAC阻止自噬体降解。RT-PCR结果显示,亚硝酸钠明显降低线粒体标志分子COXⅠ和COXⅣ基因的表达;Mito-Tracker Green染色,激光共聚焦显微镜下发现,亚硝酸钠明显减少线粒体质量;Western blot结果显示,HIF-1α、线粒体自噬标志分子Beclin-1和Bnip3蛋白表达水平升高,这些现象同样被NAC所逆转。结果表明,亚硝酸钠(16 mg·L^(-1))增强SMMC-7721细胞ROS水平,通过HIF-1α诱导线粒体自噬,促进细胞迁移和侵袭。Nitrites play multiple characteristic functions in invasion and metastasis of hepatic cancer cells, but the exact mechanism is not yet known. Cancer cells can maintain the malignant characteristics via clearance of excess mitochondria by mitophagy. The purpose of this article was to determine the roles of nitrite, reactive oxygen species（ROS） and hypoxia inducing factor 1 alpha（HIF-1α） in mitophagy of hepatic cancer cells. After exposure of human hepatocellular carcinoma SMMC-7721 cells to a serial concentrations of sodium nitrite for 24 h under normal oxygen, the maximal cell vitality was increased by 16 mg·L^（-1） sodium nitrite. In addition, the potentials of migration and invasion for SMMC-7721 cells were increased significantly at the same time. Furthermore, sodium nitrite exposure displayed an increase of stress fibers, lamellipodum and perinuclear mitochondrial distribution by cell staining with Actin-Tracker Green and Mito-Tracker Red, which was reversed by N-acetylcysteine（NAC, a reactive oxygen scavenger）. DCFH-DA staining with fluorescent microscopy showed that the intracellular level of ROS concentration was increased by the sodium nitrite treatment. LC3 immunostaining and Western blot results showed that sodium nitrite enhanced cell autophagy flux. Under the transmission electron microscopy（TEM）, more autolysosomes formed after sodium nitrite treatment and NAC could prevent autophagosome degradation. RT-PCR results indicated that the expression levels of COXⅠ and COXⅣ m RNA were decreased significantly after sodium nitrite treatment. Meanwhile, laser scanning confocal microscopy showed that sodium nitrite significantly reduced mitochondrial mass detected by Mito-Tracker Green staining. The expression levels of HIF-1α, Beclin-1 and Bnip3（mitophagy marker molecular） increased remarkably after sodium nitrite treatment, which were reversed by NAC. Our results demonstrated that sodium nitrite（16 mg·L^（-1）） increased the potentials of invasion and migration of hepatic

关 键 词：亚硝酸盐 人肝癌细胞 侵袭 活性氧 线粒体自噬 

分 类 号：R965[医药卫生—药理学]