The Effect And Mechanism Of Neurokinin 2 Receptor Antagonist SR48968 On Proliferation Of Myeloid Leukemia Cells

Leukemia is a malignant clonal disease of hematopoietic stem cells.Myeloid leukemia is a type of leukemia,including acute and chronic,accounting for more than 63%of the total incidence of leukemia.The incidence of myeloid leukemia is high and the disease develops rapidly.At present,there are some shortcomings in clinical medicine for myeloid leukemia,such as high toxicity and side effects,low cure rate,and so on.Therefore,it is urgent to develop new therapeutic targets and drugs for myeloid leukemia.SR48968 is a highly selective and specific antagonist of neurokinin receptor 2(NK2R).There are no reports about NK2R involved in the development of myeloid leukemia.In this study,we focused on the effects of NK2R antagonist SR48968 on the proliferation of myeloid leukemia and its potential molecular mechanisms.The results showed that NK2R was highly expressed in peripheral blood leukocytes and myeloid leukemia cell lines of patients with myeloid leukemia.Low concentration of SR48968 could promote the proliferation of K562/HL60 cells,while a high concentration of SR48968 could inhibit the proliferation of K562/HL60 cells and block the cell cycle at G0/G1 phase,and induce cell necrosis in a dose-and time-dependent manner.Treatment of K562/HL60 cells by SR48968 increased LDH release and PI uptake in a dose-dependent manner.Nec-1,a necrosis inhibitor,significantly alleviated the inhibitory effect of SR48968 on K562/HL60 cell proliferation,suggesting that the necrosis induced by SR48968 is programmed.In addition,in the calcium flow experiment,we observed a significant increase in cytoplasmic and mitochondrial calcium flux as the concentration of SR48968 drug increased.Under the action of SR48968,cytoplasmic and mitochondrial ROS increased,while cytosolic Ca2+chelators and mitochondrial Ca2+ inhibitors decreased cytoplasiic and mitochondrial ROS induced by SR48968,suggesting that the increase of cytoplasmic and mitochondrial Ca2+resulted in the increase of ROS.Endoplasmic reticulum calcium channel blockers had no effect on ROS production,and the elimination of extracellular Ca2+by calcium-free mediul also reduced ROS production.These results suggest that SR48968 induces extracellular Ca2+ influx to increase Ca2+in cytoplasm and mitochondria,and further promotes the increase of ROS in cytoplasm and mitochondria.Cell viability in proliferation showed that the inhibitory effect of SR48968 on K562 cell could be alleviated by cytoplasmic Ca2+chelating agent,1itochondrial Ca2+inhibitor and ROS scavenger,suggesting that the increase of ROS caused by calcium elevation which mediated K562 cell necrosis.Immunoblotting results showed that SR48968 increased the phosphorylation of necrosis-related proteins RIP1,RIP3,and MLKL.Quantitative proteomics and transcriptome results showed that SR48968 blocked NK2R which induced differential expression of apoptosis and cyclin-related proteins in K562 cells.In conclusion,this study found that high-dose NK2R antagonist SR48968 could inhibit the proliferation of myeloid leukemia cells in a dose-dependent manner,and elucidated its novel molecular mechanism,which provided a theoretical basis for the development of NK2R as a new target for the treatment of myeloid leukemia,and brought new hope and breakthrough for the clinical treatment of leukemia.