A study on the mechanism of Bufotalin-induced ferroptosis in acute promyelocytic leukemia HL-60 cells
Objective To investigate the effect and mechanism of Bufotalin on ferroptosis in acute promyelocytic leukemia(APL)HL-60 cells.Methods APL HL-60 cells were treated with Bufotalin at different concentrations(0.1,0.5,1.0,2.0,and 4.0 μg·mL-1,respectively),and the morphological changes of cells were observed using a light microscope.The survival rate of cells was detected by the CCK-8 method.The expression levels of ferroptosis-related proteins CD71,xCT,FTH1 and GPX4 in HL-60 cells were detected by Western blot.The changes of contents of intracellular GSH and GSSG were detected by GSH/GSSG detection kit.The changes of contents of intracellular MDA in HL-60 cells were determined by MDA detection kit.HL-60 cells were treated with 0.5 μg·mL-1 Bufotalin 0.1 μmol·L-1 Fer-1 and 0.5 μg·mL-1 Bufotalint 0.1 μmol L-1 Fer-1,respectively,and the morphological changes of cells were observed using a light microscope,the expression levels of ferroptosis-related proteins were detected by Western blot.Results After treatment with different concentrations of Bufotalin,HL-60 cells showed irregular shapes,such as dumbbell shape and spindle shape,and the survival rate of the cells decreased in a time-dose dependent relationship.Compared with the control group,Bufotalin could reduce the expression levels of ferroptosis-related proteins GPX4,xCT and FTH1,as well as the content of total glutathione(all P＜0.01),and increase the expression level of CD71 protein and the content of MDA(all P＜0.05).Compared with the control group,the growth number and morphology of HL-60 cells were not significantly changed after combined treatment of Bufotalin and Fer-1,the protein expression levels of CD71,xCT,FTH1 and GPX4 were not statistically significant(all P＞0.05).Conclusions Bufotalin can inhibit the proliferation of APL HL-60 cells and induce ferroptosis,the underlying molecular mechanism is probably realized through GPX4-mediated antioxidant pathway and iron metabolism pathway.
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