Zhu Xueqin, Luo Lei, Xiong Yanyan, Jiang Nan, Wang Yurun, Lv Yuan, Xie Ying
Key Laboratory of Molecular Epidemiology of Hunan Province, School of Medicine, Hunan Normal University, Changsha, China.
Changsha Center for Disease Control and Prevention, Beijing, China.
FEBS Open Bio. 2022 Feb;12(2):516-522. doi: 10.1002/2211-5463.13359. Epub 2022 Jan 14.
Cisplatin (DDP)-based chemotherapy is a preferred treatment for a broad spectrum of cancers, but the precise mechanisms of its hepatotoxicity are not yet clear. Recently, the role of voltage-dependent anion channel protein 1 (VDAC1) in mitochondrial activity and cell apoptosis has attracted much attention. Our aim was to investigate the effects of mitochondrial outer membrane protein VDAC1 oligomerization in DDP-induced hepatocyte apoptosis. L-02 hepatocytes were divided into 4 groups: (a) control group, (b) 4,4'diisothiocyanate-2,2'-disulfonic acid (DIDS; 40 μm) group, (c) DDP (5 μm) group, and (d) DDP and DIDS combination group. Cell apoptosis was tested by Annexin V/FITC assay, protein expression of caspase-3, γH2AX and NDUFB6 were observed by western blot assay, reactive oxygen species (ROS), and mitochondrial superoxide anion radical (O ) were detected by DCFH-DA and MitoSOX probe, and DNA damage was assessed by comet assay. Moreover, the activity of mitochondrial respiratory chain complex I was determined by the colorimetry method. Compared with the control group, apoptosis rate and activated cleaved-caspase-3 protein, ROS and O generation, DNA damage marker comet tail length, and γH2AX protein level increased in the DDP treatment group (P < 0.05). Activity of mitochondrial COXI decreased after DDP treatment (P < 0.05). DIDS, as a VDAC1 oligomerization inhibitor, antagonized DDP-induced apoptosis by diminishing oxidative stress and DNA damage and protecting mitochondrial complex protein. These results show that VDAC1 oligomerization may play an important role in DDP-induced hepatocyte apoptosis by increasing ROS and mtDNA leakage from VDAC1 pores, exacerbating oxidative stress and mtDNA damage.
基于顺铂（DDP）的化疗是多种癌症的首选治疗方法，但其肝毒性的确切机制尚不清楚。最近，电压依赖性阴离子通道蛋白1（VDAC1）在线粒体活性和细胞凋亡中的作用备受关注。我们的目的是研究线粒体外膜蛋白VDAC1寡聚化在DDP诱导的肝细胞凋亡中的作用。将L-02肝细胞分为4组：（a）对照组，（b）4,4'-二异硫氰酸-2,2'-二磺酸（DIDS；40μm）组，（c）DDP（5μm）组，和（d）DDP与DIDS联合组。通过Annexin V/FITC法检测细胞凋亡，通过蛋白质印迹法观察caspase-3、γH2AX和NDUFB6的蛋白表达，通过DCFH-DA和MitoSOX探针检测活性氧（ROS）和线粒体超氧阴离子自由基（O），并通过彗星试验评估DNA损伤。此外，采用比色法测定线粒体呼吸链复合体I的活性。与对照组相比，DDP处理组的凋亡率、活化的裂解型caspase-3蛋白、ROS和O生成、DNA损伤标志物彗星尾长以及γH2AX蛋白水平均升高（P<0.05）。DDP处理后线粒体COXI活性降低（P<0.05）。DIDS作为一种VDAC1寡聚化抑制剂，通过减轻氧化应激和DNA损伤以及保护线粒体复合蛋白来拮抗DDP诱导的凋亡。这些结果表明，VDAC1寡聚化可能通过增加ROS和mtDNA从VDAC1孔的泄漏，加剧氧化应激和mtDNA损伤，在DDP诱导的肝细胞凋亡中起重要作用。
