摘要
目的:探讨索拉非尼(sorafenib)是否能通过诱导线粒体的功能紊乱激活肝癌细胞氧化应激损伤,最终导致肝癌细胞死亡。方法:不同浓度索拉非尼处理肝癌细胞Huh7、HCC-LM3,应用CCK-8法进行细胞活力测定;利用TMRM(tetramethylrhodamine)探针测定线粒体膜电位(mitochondrial membrane potential, MMP)变化;利用Seahorse细胞能量代谢检测仪监测线粒体耗氧速率;采用MitoSOX(mitosox mitochondrial superoxide indicator)荧光探针测定线粒体活性氧(reactive oxygen species, ROS)水平;应用DCF-DA(dichloro-fluorescein diacetate)探针测定细胞内总ROS;通过谷胱甘肽(glutathione, GSH)预处理,观察其对索拉非尼诱导的氧化损伤及细胞死亡的恢复。结果:随着索拉非尼浓度升高,肝癌细胞Huh7、HCC-LM3的活力逐渐降低,并且抑制线粒体氧呼吸,导致氧化磷酸化的减弱及MMP的下降,最终导致线粒体及细胞质中ROS堆积从而激发细胞氧化应激损伤。而非酶抗氧化剂GSH可有效逆转索拉非尼诱导的氧化损伤和细胞死亡。结论:索拉非尼可通过诱导肝癌细胞线粒体功能紊乱,导致ROS累积,激活细胞氧化损伤,而GSH可恢复索拉非尼诱导的氧化应激损伤。因此,通过诱发肝癌细胞谷胱甘肽的缺乏可作为增强索拉非尼疗效的治疗新方案。
AIM: To investigate the role of sorafenib in promoting ferroptosis in HCC, and whether cell death can be induced by activating mitochondrial oxidative stress and consequent mitochondrial dysfunction. METHODS: Hepatocellular carcinoma cell lines Huh7 and HCC-LM3 were treated with different concentrations of sorafenib, the cell viability was determined by CCK-8 assay;mitochondrial membrane potential(MMP) was measured by Tetramethylrhodamine(TMRM) staining;The mitochondrial oxygen consumption rate was monitored by the Seahorse XF24 Analyzer;mitochondrial superoxide indicator(Mitosox) was used to determine the level of reactive oxygen species(ROS) in mitochondria;the formation of total ROS was determined by dichlorofluorescein diacetate(DCF-DA) staning. Finally, The recovery of oxidative damage and cell death induced by sorafenib was observed after pretreated by glutathione(GSH). RESULTS: With the increasing concentration of sorafenib, the survival of the Huh7 and HCC-LM3 was significantly decreased. Sorafenib also inhibited the oxygen consumption rate and decreased oxidative phosphorylation, which results in the depolarization of MMP, ROS accumulation and eventually ferroptosis of HCC cells. However, the occurrence of oxidative stress induced by sorafenib in HCC cells can be effectively reversed by the pretreatment of GSH. CONCLUSION: The ferroptosis can be induced by sorafenib through inducing mitochondrial dysfunction and ROS accumulation in HCC cells. However, the GSH can restore oxidative damage. Therefore, induction of the GSH deficiency in HCC may be a potential therapeutic option to enhance the efficacy of sorafenib.
作者
胡婉晔
袁晨
胡嘉钰
王海瑞
李焕娟
王莹
HU Wanye;YUAN Chen;HU Jiayu;WANG Hairui;LI Huanjuan;WANG Ying(Bengbu Medical College,Bengbu,Bengbu 233030,Anhui,China;Clinical Research Institute,Zhejiang Provincial People's Hospital,People's Hospital of Hangzhou Medical College,Hangzhou 310014,Zhejiang,China;School of Laboratory Medicine and Life Science,Wenzhou Medical University,Wenzhou 325035,Zhejiang,China)
出处
《中国临床药理学与治疗学》
CAS
CSCD
2021年第12期1352-1359,共8页
Chinese Journal of Clinical Pharmacology and Therapeutics
基金
国家科技重大新药创制专项(2017ZX301033)
国家自然科学基金资助项目(81570198)
浙江省级共建项目(WKJ-ZJ-1709)。
关键词
索拉非尼
线粒体
氧化应激
铁死亡
谷胱甘肽
sorafenib
mitochondria
oxidative stress
ferroptosis
glutathione
