三氧化二砷诱导肝癌细胞凋亡的机制研究

The Apoptotic Mechanism of Hepatocellular Carcinoma Cell Line (HepG2) Induced by Arsenic Trioxide

中文关键词:  三氧化二砷 凋亡 γ-谷氨酰半胱氨酸合成酶 核因子E2相关因子2（Nrf2）

英文关键词:Arsenic trioxide Apoptosis γ-GCS Nrf2

中文摘要:

      目的 通过研究三氧化二砷对人肝癌细胞株HepG2活性氧（ROS）、谷胱甘肽（GSH）水平、γ-谷氨酰半胱氨酸合成酶(γ-GCS)和核因子E2相关因子2(Nrf2)表达的影响，探讨三氧化二砷诱导肝癌细胞凋亡的机制。方法 分别采用0、 2.5、5和10 μmol/L三氧化二砷处理HepG2达24 h后，MTT实验测定细胞存活率（另增25、50 μmol/L浓度组），流式细胞术检测细胞凋亡水平，DCFH-DA荧光探针测定细胞内ROS水平，5,5-二硫代二硝基苯甲酸（DTNB）比色法检测细胞中GSH含量，蛋白印记检测γ-GCS催化亚基GCLC和调节亚基GCLM以及核因子E2相关因子2（Nrf2）蛋白的表达水平。结果 随着三氧化二砷染毒浓度的增加，HepG2细胞凋亡率、ROS水平和Nrf2蛋白表达均增加(P<0.05)，而细胞存活率、GSH含量、GCLC和GCLM蛋白表达量均降低(P<0.05)。结论 三氧化二砷通过诱导细胞产生ROS，抑制γ-GCS的表达以及减少GSH合成，从而诱导人肝癌细胞凋亡。

英文摘要:

      Objective To explore the apoptotic mechanism of human hepatic carcinoma cell line HepG2 induced by arsenic trioxide (As2O3). Methods The human hepatoma cell line HepG2 was treated with 0, 2.5, 5 and 10 μmol/L arsenic trioxide for 24 h. Cytotoxicity was tested by MTT assay (additional 25 and 50 μmol/L arsenic trioxide treatment groups), cellular apoptosis were detected by flow cytometry, reactive oxygen species (ROS) level were quantified by DCFH-DA fluorescent probe staining and glutathione content were measured by DTNB method with commercial kits. Western blot assay was used to detect the protein expression of γ-glutamylcysteine synthetase (γ-GCS, GCLC and GCLM subunits) and nuclear factor erythroid 2-related factor 2 (Nrf2). Results With the increase of arsenic trioxide concentration, cellular survival, glutathione content and γ-GCS (GCLC and GCLM subunits) protein expression level decreased (P<0.05); while cellular apoptotic rate, reactive oxygen species level and Nrf2 protein expression increased (P<0.05). Conclusion Arsenic trioxide induces the apoptosis of human hepatoma cell line HepG2 through ROS induction, γ-GCS expression inhibition and cellular glutathione content depletion.