伊利司莫-铜诱导急性髓系白血病细胞发生铜死亡

Elesclomol-Cu Induces Cuproptosis in Human Acute Myeloid Leukemia Cells

目的:研究伊利司莫-铜(ES-Cu)对人急性髓系白血病(AML)细胞的增殖抑制和铜死亡的诱导作用。方法:采用CCK-8试剂盒检测ES-Cu诱导的AML细胞存活率以及AML细胞经铜螯合剂TTM预处理后ES-Cu诱导的细胞存活率;Calcein/PI检测试剂盒检测ES-Cu诱导引起的细胞活性与细胞毒性变化;采用DCFH-DA荧光探针检测细胞内活性氧水平;GSH和GSSG检测试剂盒检测细胞内谷胱甘肽(GSH)含量;Western blot检测细胞铜死亡相关蛋白ATP7B、FDX1、DLAT、DPYD的表达。结果:ES-Cu对人急性髓系白血病细胞系Kasumi-1及HL-60的毒性呈现出明显的剂量依赖性(r_(Kasumi-1)=-0.99,r_(HL-60)=-0.98)。随着ES-Cu浓度升高,细胞内活性氧水平随之升高(P<0.01-0.001)。铜螯合剂TTM可以显著逆转ES-Cu对细胞增殖的抑制作用以及对活性氧的促进作用。随着ES-Cu浓度升高,Kasumi-1细胞内GSH含量逐渐降低(r=-0.98),ATP7B、FDX1、DLAT和DPYD蛋白的表达均显著降低(P<0.05),最终促发铜死亡。结论:ES-Cu能够诱导急性髓系白血病细胞发生铜死亡,这为急性髓系白血病提供了新的治疗思路。

Objective:To investigate the effects of elesclomol-Cu(ES-Cu)on the proliferation and cuproptosis of human acute myeloid leukemia(AML)cells.Methods:The effects of ES-Cu on the proliferation of AML cells and the AML cells pre-treated with ammonium tetrathiomolybdate(TTM)were examined by CCK-8 assay.The Calcein/PI kit was used to detected the changes in activity and cytotoxicity of AML cells induced by ES-Cu.Flow cytometry and Cytation3 fully automated cell imaging multifunctional detection system were used to analyze DCFH-DA fluorescence intensity,so as to determine the level of reactive oxygen species(ROS).The GSH and GSSG detection kits were used to measure the intracellular GSH content.Western blot was used to detected the expression of cuproptosis-related proteins ATP7B,FDX1,DLAT and DPYD.Results:ES-Cu inhibited the proliferation of Kasumi-1 and HL-60 cells in a concentration-dependent manner(r_(Kasumi-1)=-0.99,r_(HL-60)=-0.98).As the concentration of ES-Cu increased,the level of intracellular ROS also increased(P<0.01-0.001).TTM could significantly reverse the inhibitory effect of ES-Cu on cell proliferation and its promoting effect on ROS.With the increase of ES-Cu concentration,the content of GSH was decreased(r=-0.98),and Western blot showed that the protein expressions of ATP7B,FDX1,DLAT and DPYD were significantly reduced(P<0.05).Conclusion:ES-Cu can induce cuproptosis in AML cells,which provides a new idea for the treatment of AML.