Molecular Mechanism Of PFR Peptide Inducing Necrosis Of AML Cells

Leukemia is a kind of malignant hematological tumor with abnormal proliferation of hematopoietic stem cells,which still cannot fully achieve the expected therapeutic effect.Recent studies have found that antimicrobial peptides have anti-tumor activity,which can play an anti-tumor effect from destroying cell membrane,inducing apoptosis,destroying the skeleton system,and enhancing the immune response.The PFR peptide is a 9-amino acid alpha-helical small molecule peptide derived from lactoferrin which has antibacterial activity.We found that PFR peptides caused necrosis of acute myeloid leukemia cell line HL60 in a dose-dependent manner.The results of flow cytometry showed that HL60cells were distributed in the necrotic area of PI-positive staining after PFR peptides treatment.The PI uptake and LDH release experiment further confirmed that PFR peptides treatment of HL60 cells could cause dose-dependent necrosis.The necrosis inhibitor Nec-1 can significantly reduce PFR-mediated necrosis,suggesting that PFR peptides-induced HL60 cell necrosis is programmed named necroptosis.Further studies have found that PFR peptides cause pores larger than 1 nm on the surface of HL60 cell membranes.The results of laser confocal microscopy showed that the fluorescently labeled PFR peptides could enter HL60 cells and localize to the endoplasmic reticulum,causing an endoplasmic reticulum stress response.The PFR peptides did not activate the caspase-12-mediated apoptotic pathway after localization to the endoplasmic reticulum.In addition,the PFR peptides destroyed the cell membrane structure of HL60 cells and caused extracellular Ca²⁺influx,together with the Ca²⁺released after endoplasmic reticulum stress reaction promoted the increase of cytosolic Ca²⁺.Blocking the endoplasmic reticulum Ca²⁺release,scavenging extracellular Ca²⁺or chelation of cytosolic Ca²⁺significantly alleviated the decrease in cell proliferative viability caused by PFR peptides,while blocking mitochondrial Ca²⁺has no significant effect,indicating that the increase in cytosolic Ca²⁺mediates necroptosis of HL60 cell after treatment with PFR peptides.PFR peptides also caused an increase of ROS in mitochondrial to mediate necroptosis in HL60 cells accompanied by the phosphorylation of necroptosis-related proteins RIP1,RIP3,MLKL and activated PARP-1,which could aggravate cell membrane damage and destroy mitochondria.In conclusion,we clarify the specific molecular mechanism of necrosis in acute myeloid leukemia HL60 cells after treatment with PFR peptides,which provides new ideas and possibilities for new drug selection for the treatment of acute myeloid leukemia in clinic.