Abstract: 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. 

Key words: Bufotalin, Acute promyelocytic leukemia, HL-60 cells, Ferroptosis