Wei J Y, Li L, Liu H M
Department of Hematology, the Second Hospital of Shanxi Medical University, Taiyuan 030001, China Key Laboratory of Cellular Physiology (Shanxi Medical University), Ministry of Education, Taiyuan 030001, China.
Zhonghua Xue Ye Xue Za Zhi. 2024 Jun 14;45(6):577-585. doi: 10.3760/cma.j.cn121090-20231218-00323.
To investigate the effect and molecular mechanism of hesperadin in inducing ferroptosis in chronic myeloid leukemia cell line K562 cells. The effects of hesperadin on the viability, proliferation, and migration of K562 cells were detected though CCK8, EDU-594, and Transwell assays, and the apoptotic rate of K562 cells was detected by flow cytometry. In addition, C11-BODIPY and FerroOrange were utilized to detect intracellular lipid peroxidation and Fe(2+) levels. Meanwhile, the expression levels of ferroptosis-associated protein solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) in cells were detected through Western blot. Lipid peroxidation and Fe(2+) levels were also detected after transfection of cells with SLC7A11 overexpression plasmid. Hesperadin decreased cell viability in a dose-dependent manner with IC(50) of 0.544 μmol/L. Hesperadin concentrations of 0.4 and 0.8 μmol/L were selected for follow-up experiments. EDU-594, Transwell, and flow cytometry showed significantly decreased proliferation and migration rate of K562 cells after 0.4 and 0.8 μmol/L hesperadin treatment for 24 h, and the apoptosis rate was significantly increased compared with the control group (<0.05). Western blot indicated a downregulated expression of the antiapoptotic protein Bcl-2 and an elevated expression of proapoptotic proteins Bax and Caspase-3. Moreover, hesperadin increased intracellular lipid peroxidation and Fe(2+) levels compared with the control treatment (<0.05). The combination of ferroptosis inhibitor (Fer-1) and hesperadin could reverse the effect of hesperadin on K562 cells. The mRNA and protein levels of ferroptosis-related genes SLC7A11 and GPX4 were significantly decreased in the 0.8 μmol/L hesperadin-treated group (<0.05). SLC7A11 overexpression can inhibit hesperadin effect and alleviate ferroptosis. Hesperadin can promote ferroptosis in K562 cells by regulating the SLC7A11/GPX4 axis.
探讨海丝帕啶诱导慢性髓系白血病细胞系K562细胞铁死亡的作用及分子机制。通过CCK8、EDU-594和Transwell实验检测海丝帕啶对K562细胞活力、增殖和迁移的影响，采用流式细胞术检测K562细胞的凋亡率。此外，利用C11-硼二吡咯烷和FerroOrange检测细胞内脂质过氧化和Fe(2+)水平。同时，通过蛋白质免疫印迹法检测细胞中铁死亡相关蛋白溶质载体家族7成员11（SLC7A11）和谷胱甘肽过氧化物酶4（GPX4）的表达水平。用SLC7A11过表达质粒转染细胞后，也检测了脂质过氧化和Fe(2+)水平。海丝帕啶以剂量依赖方式降低细胞活力，IC(50)为0.544 μmol/L。选择0.4和0.8 μmol/L的海丝帕啶浓度进行后续实验。EDU-594、Transwell和流式细胞术显示，0.4和0.8 μmol/L海丝帕啶处理24小时后，K562细胞的增殖和迁移率显著降低，与对照组相比凋亡率显著升高（<0.05）。蛋白质免疫印迹法表明抗凋亡蛋白Bcl-2表达下调，促凋亡蛋白Bax和Caspase-3表达上调。此外，与对照处理相比，海丝帕啶增加了细胞内脂质过氧化和Fe(2+)水平（<0.05）。铁死亡抑制剂（Fer-1）与海丝帕啶联合使用可逆转海丝帕啶对K562细胞的作用。在0.8 μmol/L海丝帕啶处理组中，铁死亡相关基因SLC7A11和GPX4的mRNA和蛋白水平显著降低（<0.05）。SLC7A11过表达可抑制海丝帕啶的作用并减轻铁死亡。海丝帕啶可通过调节SLC7A11/GPX4轴促进K562细胞铁死亡。
