Decitabine Induces Ferroptosis In Myelodysplastic Syndrome Cells And Its Mechanism

Part 1 Decitabine Induces Ferroptosis in Myelodysplastic syndrome Cells and Its MechanismObjectives Myelodysplastic syndrome(MDS)is the most common acquired adult bone marrow failure syndrome which is often accompanied by iron overload.Ferroptosis is a form of regulated cell death which has independent morphological,biochemical and genetic characteristics.Ferroptosis may participate in the occurrence and development of MDS,which may be a new target for treatment.Decitabine is one of the classical demethylation drugs for the treatment of MDS.This study was to investigate whether decitabine can induce ferroptosis in MDS cells and its mechanism.Methods 1.Cell lines SKM-1 and MUTZ-1 were co-cultured with decitabine and ferroptosis inhibitor Fer-1,respectively.CCK-8 assay was used to detect the effects of drugs on cell viability at different times and concentrations.At the same time,we observed whether Fer-1,necrostatin-1(necroptosis inhibitor),z-vad-fmk(apoptosis inhibitor)and DFO(iron chelating agent)could reverse the inhibitory effect of decitabine on MDS cells.CCK-8 assay was used to detect the effects of erastin(ferroptosis inducer)combined with decitabine on MUTZ-1 cell viability.2.Flow cytometry was performed to detect the ROS level of cells treated with Fer-1,DFO,erastin alone or in combination with decitabine.3.Detected the level of glutathione treated with Fer-1,DFO alone or in combination with decitabine by biochemical methods.4.Detected the level of glutathione perxidases treated with decitabine、Fer-1、GSH、necrostatin-1、z-vad-fmk by biochemical methods.Results 1.Necrostatin-1 and z-vad-fmk could increase the cell viability of MUTZ-1 significantly,necrostatin-1 could increase the cell viability of SKM-1 significantly(P<0.05).The growth-inhibitory effect of decitabine on SKM-1 and MUTZ-1 could bepartially reversed by Fer-1,DFO and necrostatin-1.The effect of Fer-1 is the most significant(P<0.05).Erastin could increase the cytotoxicity of decitabine at different concentrations in MUTZ-1 cells,although the synergistic effect was not significant(Q<1.15).2.The intracellular ROS levels increased when treated with decitabine.Fer-1 and DFO could inhibit the increasing of ROS level caused by decitabine.Erastin in combination with decitabine could further increase the ROS level.3.When treated with decitabine,the intracellular GSH level of MUTZ-1 could be depressed,so was the GPXs activity.The decrease of GSH level could be reversed by Fer-1.The decrease of GPXs activity could be reversed by Fer-1,necrostatin-1,z-vad-fmk and GSH.Conclusion 1.The pathogenesis of MDS may mainly related to necroptosis and apoptosis.Necroptosis and ferroptosis may be the main mechanisms of inducing the death of MDS cells by decitabine;2.Decitabine can increase ROS level related to ferroptosis;3.Decitabine may induce ferroptosis in MDS cells by decreasing GSH level and GPXs activity.Part 2 Iron Overload Induces Ferroptosis in Bone Marrow Mononuclear Cells of MiceObjectives MDS patients are often accompanied by iron overload.The iron overload model of C57BL/6 mice was constructed to observe whether iron overload can induce ferroptosis,aiming at providing new ideas for the treatment of iron overload patients.Methods 1.The iron overload model of C57BL/6 mice was established by intraperitoneal injection of iron dextran.2.The level of ferritin in peripheral blood of mice was detected by ELISA.Iron deposition in liver and spleen pathological section was observed to verify the establishment of iron overload model.Confocal microscopy was used to show the amount of ferrous ions in bone marrow mononuclear cells of mice.Flow cytometry was used to detect the fluorescence intensity quantitatively;3.The amount of hemoglobin in peripheral blood was detected to observe whether there is a linear relationship between the degree of iron overload and anemia.4.Fer-1(ferroptosis inhibitor),necrostatin-1(necroptosis inhibitor),z-vad-fmk(apoptosis inhibitor),erastin(ferroptosis inducer)and DFO(iron chelator)were treated with the bone marrow mononuclear cells of mice respectively.Cell viability was detected by CCK-8 method,ROS level was detected by flow cytometry.The level of glutathione(GSH)and the activity of glutathione peroxidases(GPXs)were detected by biochemical method.Results 1.The iron overload model of mice was established successfully.2.Iron overload caused iron deposition in liver,spleen and bone marrow,and also decreased hemoglobin(Hb)in peripheral blood;Iron overload increased the number of ferrous ions in bone marrow mononuclear cells of mice significantly compared with the control group.The concentration of hemoglobin in peripheral blood of mice was negatively correlated with intracellular Fe2 + level and ferritin concentration;3.Iron overload led to decreased cell viability,which was negatively correlated with intracellular Fe2 + level.4.Fer-1 and necrostatin-1 partially reversed the decline of cell viability in iron overload groups,erastin promoted the proliferation of bone marrow mononuclear cells in iron overload mice(P<0.05);5.The ROS level in bone marrow mononuclear cells of iron overload mice increased significantly(P<0.05).The level of GSH and the activity of GPXs decreased.Iron chelating agent could increase the level of GSH(P<0.05).Ferroptosis inhibitor,apoptosis inhibitor and iron chelating agent could increase the GPXs activity of BMMNCs in high dose group significantly(P<0.05).Conclusions 1.Iron overload can induce anemia in mice,and the degree of anemia is positively correlated with the degree of iron overload;2.Necroptosis and ferroptosis may play a role in the decrease of cell viability in iron overload mice.3.With the increase of iron overload,the level of intracellular Fe2 + and ROS increased;4.Erastin could promote the proliferation of bone marrow mononuclear cells in iron overloaded mice;5.Iron overload led to the increase of ROS level related to necroptosis and ferroptosis.6.Iron chelating agent could increase the level of GSH in iron overloaded groups.7.The decrease of GPXs activity caused by iron overload may related to ferroptosis and apoptosis.Part 3 Decitabine Induces Ferroptosis in Bone Marrow Mononuclear Cells of patients with MDSObjectives To explore the role of ferroptosis in the pathogenesis of low-risk and high-risk MDS patients respectively and provide treatment strategy for MDS.Methods 1.The bone marrow mononuclear cells(BMMNCs)were obtained from low-risk MDS,high-risk MDS and lymphoma patients respectively and co-cultured with decitabine.Then we observed whether the cytotoxicity of decitabine could be reversed by Fer-1(ferroptosis inhibitor),z-vad-fmk(apoptosis inhibitor),necrostatin-1(necroptosis inhibitor),or DFO(iron chelator).CCK-8 assay was used to detect the effects of ferroptosis inducer erastin combined with decitabine on the activity of three groups.2.Flow cytometry was used to detect the ROS level in BMMNCs of the three groups,biochemical method was used to detect the intracellular glutathione(GSH)level and glutathione peroxidase(GPXs)activity.Results 1.Fer-1,necrostatin-1,z-vad-fmk could significantly reverse the inhibitory effect of decitabine on low-risk BMMNCs(P<0.05),necrostatin-1 and Fer-1 could also reverse the inhibitory effect of decitabine on high-risk BMMNCs,although the difference was not significant(P>0.05);2.Decitabine could significantly increase the ROS level in MDS patients(P<0.05).Fer-1 could significantly inhibit the inhibitory effect of decitabine on low-risk BMMNCs(P < 0.05).Erastin could increase the inhibitory effect of decitabine(P>0.05 in low-risk group,P<0.05 in high-risk group).3.For low-risk MDS patients,the level of GSH in Fer-1+decitabine,necrostatin-1+decitabine and z-vad-fmk +decitabine groups were significantly higher than those in decitabine group(P<0.05).For high-risk MDS patients,Fer-1 and necrostatin-1 could significantly reverse the inhibitory effect of decitabine on GSH level(P<0.05).Erastin combined with decitabine could further reduce the GSH level,the difference was significant in high-risk MDS group.4.For low-risk MDS group,GPXs activity of Fer-1+decitabine and z-vad-fmk+decitabine groups was significantly higher than that of decitabine group(P<0.05).Erastin could further decrease the activity of GPXs(P>0.05).For high-risk MDS group,the activity of GPXs of Fer-1+decitabine and necrostatin-1+decitabine groups were significantly higher than that of decitabine group(P<0.05),Erastin could further decrease the activity of GPXs(P <0.05).Conclusions 1.The mechanism of decitabine in low-risk and high-risk MDS patients may be different.Decitabine mainly induced ferroptosis,apoptosis in low-risk MDS patients and necroptosis,ferroptosis in high-risk MDS patients.2.Decitabine induced ferroptosis by increasing ROS level in BMMNCs cells of MDS patients.3.The inhibition of the level of GSH caused by decitabine was mainly induced by necroptosis,ferroptosis and apoptosis in low-risk group and by necroptosis,ferroptosis in high-risk group.4.The inhibition of the activity of GPXs caused by decitabine was mainly induced by ferroptosis,apoptosis in low-risk group and necroptosis,ferroptosis in high-risk group.