Lonidamine Combined With RSL3 Synergically Induces Ferroptosis In AML Cells

Background: Acute myeloid leukemia(AML)is a genetically heterogeneous clonal disease that occurs most commonly in adults.Chemotherapy and hematopoietic stem cell transplantation are the main treatment methods,and some patients can achieve long-term disease-free survival or even cure after treatment.However,there is still serious untoward effect in the management of AML,which is a burning question to be solved.Therefore,it is necessary to further understand AML pathogenesis and develop new treatment regimens through the combination of existing drugs sequentially.According to previous studies of the research group,through ferritin degradation dihydroartemisinin induces ferroptosis of leukemia cells and inhibits their proliferation.However,it is not clear whether there is another mechanism for the occurrence of AML and ferroptosis.Objective: To investigate the mechanism of ferroptosis in AML cells induced by Lonidamine(LND)synergy(1S,3R)-RSL3(RSL3).This study aims to further understand the therapeutic mechanism of ferroptosis induction in AML cells and to provide a theoretical basis for the treatment strategy of ferroptosis against AML.Methods: In this study,AML cell lines HL-60 and Kasumi-1 as research objects.Firstly,CCK-8 kit was used to detect the inhibitory effect of LND on cell viability of HL-60 and Kasumi-1,and western blot analysis was used to detect whether the downregulation of x CT expression in cells was dose-dependent and time-dependent on LND.The synergy scores of LND combined with RSL3 on AML cells were evaluated by Zero-inflated poisson(ZIP)regression model.Secondly,the killing effect of the regimen of LND and RSL3 on HL-60 cells was evaluated using Calcein/PI staining kit and Annexin V-FITC/PI double staining method.Then,Mito SOX probe staining method was used to observe the changes in mitochondrial superoxide levels in HL-60 cells caused by the single or combined treatment of LND and RSL3 by flow cytometry and laser confocal microscopy.Furthermore,mitochondrial targeting antioxidant,Mitoquinol mesylate(Mito Q),was added to the combination of LND and RSL3 to observe the rescue of mitochondrial damage and changes in mitochondrial membrane potential(MMP)in HL-60 cells.To verify whether ferroptosis is involved in cell damage caused by LND and RSL3.After HL-60 and Kasumi-1 cells were treated with LND and RSL3,the change in intracellular glutathione(GSH)level was measured by microplate reader.And the intracellular lipid peroxide(LPO)level was detected by flow cytometry using BODIPY 581/591 C11 probe staining.The intracellular reactive oxygen species(ROS)were detected by laser confocal microscopy with DCFH-DA probe staining.Finally,based on the regimen of LND and RSL3,small molecule inhibitors were added to further determine the cell death caused by ferroptosis by observing the vitality of HL-60 cells and the changes in intracellular ROS and LPO levels.These small molecule inhibitors include Ferrostatin 1(Fer-1),Deferoxamine(DFO),GSH,Z-VAD-FMK,and Necrosulfonamide(Necro).Western blot was used to detect the changes in the expression levels of protein x CT and GPX4 after LND,RSL3,and Fer-1 treatment.Results: Firstly,after LND treatment,HL-60 and Kasumi-1 cells showed dosedependent inhibition of cell viability,and LND could down-regulate the expression of protein x CT in cells.ZIP model scores showed that LND combined with RSL3 had a significant synergistic effect on AML cells,and the combination of the two drugs had a synergistic killing effect on HL-60 cells.Secondly,LND combined with RSL3 significantly increased mitochondrial superoxide level of HL-60 cells,but Mito Q alleviated the toxic effects of LND and RSL3 and inhibited MMP hyperpolarization.It is noteworthy that treatment of AML cells with LND and RSL3 regiments not only reduced the intracellular GSH level but also increased the intracellular ROS and LPO levels.To further clarify the mechanism of the above phenomenon,inhibitors such as Fer-1 were added based on the regimen,and it was found that the cell vitality was restored,the levels of ROS and LPO were decreased,and the expression level of intracellular protein x CT was up-regulated.Conclusion: LND combines with RSL3 to reduce the GSH level of AML cells,increase the LPO,ROS,and mitochondrial superoxide levels of AML cells,downregulate the expressions of x CT and GPX4 of AML cells,and ultimately induce ferroptosis in AML cells.