The Mechanism Of Ferroptosis And Autophagy Induced By FTY720 Via PP2A/AMPK Pathway In Multiple Myeloma Cells

Objective:Multiple myeloma?MM?is a plasma cell disease that clonal plasma cells secrete monoclonal immunoglobulin and its fragments,causes a series of related symptoms and signs in vivo.At present,MM has developed into the second largrest malignant tumot in hematology after non-Hodgkin's lymphoma,accounts for approximately 10%of hematological malignancies.Fingolimod?FTY720?is a novel immunosuppressant.Because of its immunosuppressant effect,it has been approved as the first-line drug for treatment of multiple sclerosis in 2010.In addition to its immunosuppressant effect,FTY720 also has a wide range of antitumor effects.FTY720 can inhibit proliferation in many tumor cells and multiple myeloma is also found to be sensitive to it.Our previous study shows FTY720 can induce apoptosis and autophagic cell death in MM cells;however,the mechanism of FTY720 induced autophagy needs further study and the apoptosis and autophagy cannot fully explain the high death rate of MM cells.We speculate there are other ways of cell death.Ferroptosis is a newly discovered form of cell death and many reports show it can regulate proliferation,migration,metastasis and resistance in tumor cells.Because it is often accompanied by inhibition of tumor cell proliferation,ferroptosis is expected to become a new direction for tumor treatment.It has been reported the relationship with autophagy.It has been reported that autophagy is closely related to ferroptosis.The study is aimed to explore the mechanism of FTY720-induced autophagy,verify the relationship and mechanism between FTY720 and ferroptosis,finally discuss the relationship between ferroptosis and autophagy,so as to provide further theoretical basis and reference significance for the clinical application of FTY720.Methods:Experiments were performed on 25 human primary cell samples and two MM cell lines including 12 normal human samples and 13 newly diagnosed multiple myeloma patients.1?In order to explore the mechanism of FTY720-induced autophagy,the expression of eEF2K in mRNA level was detected by qRT-PCR in primary MM patients compared with bone marrow transplant donors;the expression of proteins related to PP2A/AMPK/eEF2K signaling pathway was detected by Wesrern Blot;MM cells were treated with PP2A inhibitor/AMPK activator and/or FTY720,the cell viability was detected by CCK8 assay;MM cells were treated with PP2A inhibitor/AMPK activator and/or FTY720 and Western Blot was used to examine the expression of p62 and LC3B.2?In order to explore the relationship between FTY720 and ferroptosis,MM cells were treated with FTY720,flow cytometry and fluorescence microscopy were used to verify change of reactibe oxygen species?ROS?,fluorescence microscopy was also used to obserbe Fe²⁺accumulation and transmission electron microscopy was used to observe mitochondrial changes;MM cells were treated with two ferroptosis inhibitors ferrostatin-1?Fer-1?/deferoxamine mesylate?DFOM?and FTY720,the cell viability was detected by CCK8 assay;the expression of GPX4 and SLC7A11 in mRNA level was detected by qRT-PCR in primary MM patients compared with bone marrow transplant donors;MM cells were treated with FTY720,the expression of GPX4 and SLC7A11 in mRNA level was detected by qRT-PCR in two MM cell lines;MM cells were treated with FTY720 in different doses and at different times,the expression of GPX4 and SLC7A11 in protein level was detected by Western Blot;3?In order to explore the relationship between ferroptosis and autophagy induced by FTY720,MM cells were treated with PP2A inhibitor/AMPK activator and/or FTY720,the expression of GPX4 and SLC7A11 in protein level was detected by Western Blot;MM cells were treated with autophagy inbibitors and ferroptosis inbitors and/or FTY720,the expression of p62,LC3B and ferroptosis related GPX4,SLC7A11 was detected by Western Blot.Results:1?The expression of eEF2K in mRNA level is significantly higher in primary MM patients compared with bone marrow transplant donors;after treatment of FTY720 of MM cells,the expression of p-PP2Ac?Tyr307?,p-AMPK??Thr172?and p-eEF2 is lower than that of the control group,while the total proteins have not changed;after treatment of PP2A inbibitor/AMPK activator and/or FTY720 of MM cells,results show that the cell viability of FTY720 group is lower than that of control group,the cell viability of FTY720and PP2A inhibitor/AMPK activator group is higher than that of FTY720 group,the cell viability of the PP2A inhibitor group is not significantly changed than that of the control group,the cell viability of the AMPK activator group is slightly higher than that of the control group;after treatment of PP2A inbibitor/AMPK activator and/or FTY720,the expression of p62 decreased,LC3?/?increased in FTY720 group compared with that of the control group,while in the group of PP2A inhibitor/AMPK activator and FTY720,the expression of p62 increased,LC3?/?decreased compared with FTY720 group.2?After treatment of FTY720,results show the accumulation of Fe²⁺and reactive oxygen species?ROS?,shrunken mitochondria,mitochondria membrane rupture,mitochondrial cristae destruction in FTY720 group;after treatment of Fer-1 and/or FTY720,the cell viability of FTY720 is lower than that of the control group,the cell viability of FTY720 and Fer-1is higher than FTY720 group,the Fer-1 group is not significantly changed and similar results are obtained after treatment of DFOM to MM cells;the expression of GPX4 and SLC7A11 in mRNA level is significantly higher in primary MM patients compared with bone marrow transplant donors;after treatment of FTY720 of MM cells,the expression of GPX4 and SLC7A11 decreased;after treatment of FTY720 in different doses and at different times,the expression of GPX4 and SLC7A11 in protein level decreased with the increase of concentration and time.3?After treatment of PP2A inhibitor/AMPK activator and/or FTY720,results show the expression of GPX4 and SLC7A11 decreases in FTY720group,while that increases in PP2A inhibitor/AMPK activator and FTY720 group compared with FTY720 group;after treatment of Fer-1 and/or FTY720,the expression of p62 decreased,LC3?/?increased in FTY720 group,the expression of p62 increased and LC3?/?decreased in Fer-1 and FTY720 group compared with FTY720 group;after treatment of bafilomycin A1?Baf-A1?and/or FTY720,the expression of GPX4 and SLC7A11 decreased in FTY720 group while that increased in FTY720 and Baf-1 group.Conclusion:1?FTY720 can induce autophagy via PP2A/AMPK signaling pathway.2?FTY720 can induce ferroptosis in MM cells.3?FTY720 can induce ferroptosis through PP2A/AMPK signaling,ferroptosis and autophagy can reinforce each other.