橙皮苷诱导慢性髓性白血病细胞系K562细胞发生铁死亡的作用及分子机制

Effect and molecular mechanism of hesperadin-induced ferroptosis in chronic myeloid leukemia K562 cells

目的:探讨橙皮苷诱导慢性髓性白血病细胞系K562细胞发生铁死亡的作用及分子机制。方法:通过CCK-8、EDU-594、Transwell法检测橙皮苷对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。采用C11-BODIPY和FerroOrange检测细胞中脂质过氧化和Fe^(2+)水平。通过Western blot法检测细胞中铁死亡相关蛋白溶质载体家族7成员11(SLC7A11)、谷胱甘肽过氧化物酶4(GPX4)表达水平。采用SLC7A11过表达质粒转染细胞后,同样检测脂质过氧化及Fe^(2+)水平。结果:橙皮苷可呈剂量依赖性降低K562细胞活力,IC_(50)值为0.544μmol/L,选取0.4、0.8μmol/L的橙皮苷行后续实验。EDU-594、Transwell法和流式细胞术检测显示,0.4、0.8μmol/L橙皮苷作用24 h后K562细胞增殖和迁移率明显降低,细胞凋亡率明显提高,与对照组比较差异均有统计学意义(P值均<0.05)。同时Western blot法检测显示,抗凋亡蛋白Bcl-2表达下调,促凋亡蛋白Bax及Caspase-3表达升高。与对照组比较,橙皮苷可以提高细胞内脂质过氧化和Fe^(2+)水平(P值均<0.05)。铁死亡抑制剂(Fer-1)与橙皮苷联合给药可以逆转橙皮苷对K562细胞的作用。0.8μmol/L橙皮苷作用组铁死亡相关基因SLC7A11、GPX4 mRNA及蛋白水平明显降低(P值均<0.05)。SLC7A11过表达可以抑制橙皮苷作用,减轻铁死亡。结论:橙皮苷可通过调控SLC7A11/GPX4轴,促进K562细胞铁死亡。

Objective To investigate the effect and molecular mechanism of hesperadin in inducing ferroptosis in chronic myeloid leukemia cell line K562 cells.Methods 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.Results Hesperadin decreased cell viability in a dose-dependent manner with IC_(50) of 0.544μmol/L.Hesperadin concentrations of 0.4 and 0.8μmol/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 0.8μmol/L hesperadin treatment for 24 h,and the apoptosis rate was significantly increased compared with the control group(P<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(P<0.05). The combination of ferroptosis inhibitor(Fer- 1)and hesperadin could reverse theeffect of hesperadin on K562 cells. The mRNA and protein levels of ferroptosis-related genes SLC7A11and GPX4 were significantly decreased in the 0.8 μmol/L hesperadin-treated group(P<0.05). SLC7A11overexpression can inhibit hesperadin effect and alleviate ferroptosis. Conclusion Hesperadin canpromote ferroptosis in K562 cells by regulating the SLC7A11/GPX4 axis.