Proteomics Study On ONE-AE-248-induced Non-apoptotic Programmed Cell Death Of Neutrophils

Objective: To explore the different mechanisms betweenONO-AE-248 (a selective agonist of neutrophils)-induced non-apoptosisprogrammed cell death of polymorphonuclear neutrophils (PMN) andnormal apoptosis of PMN on the basis of proteomics, mass spectrometryand bioinformatics. Methods: To make the models of non-apoptoticprogrammed cell death and normal apoptosis, PMN isolated from healthyvolunteers by Ficoll were incubated in the presence or absence ofONO-AE-248 for the indicated periods of 2 h,6 h,12 h and 24 hrespectively at 37℃ in a humidified 5% CO2, 95% air incubator. Themorphological changes of PMN were evaluated by light microscopy atdifferent indicated time. PMN proteins were extracted at the indicatedtime and determined with Bradford method. The proteins wereisoelectrically focused on IPG Drystrip pH 4-7 linear gels, subsequentlyseparated by second-dimensional SDS-PAGE (10% gel concentration)and then stained by silver staining. Two-dimensional electrophoresismaps were acquired by scanning and then analyzed by PDQuest2-DEsoftware. The pI and MW of proteins were identified by the lineargradient of IPG and the MW of standard marker. At the same time, 20differentially expressed proteins in 12h-experimental group and12h-control group were found after being compared by PDQuest2-DE.These 20 hand-cut protein spots were analyzed by MALDI-TOF-MS.Finally, differentially expressed proteins between the experimental groupof non-apoptotic programmed cell death and the control group of normalapoptosis were searched and matched by Mascot and theProteinProspector software, and then identified in Swiss-Protdatabase .The time courses of some important differentially expressedproteins were plotted according to a semi-quantitative analysis. Results:â‘  The proteomes of ONO-AE-248-induced non-apoptotic programmedcell death of PMN at 2 h,6 h,12 h and 24 h were changed remarkably.â‘¡ The proteomes between ONO-AE-248-induced non-apoptoticprogrammed cell death and normal apoptosis of PMN were obviouslydifferent. â‘¢ By MALDI-TOF-MS and bioinformatics analysis, 12differentially expressed proteins were identified. They were CK-10,YALI0D26950g, SfriDRAFT₃533, B2N18.040, stsE, oxidoreductase,CCR4, RPEDRAFT₂200, Bone marrow macrophagecDNA, MA₂085, ASC, DVU₂490. â‘£ The proteins only expressed inthe PMN of ONO-AE-248-induced non-apoptotic programmed cell deathwere CK-10, YALI0D26950g and SfriDRAFT₃533. The proteins onlyexpressed in the PMN of normal apoptosis were B2N18.040, stsE,oxidoreductase, CCR4 and RPEDRAFT₂200. The proteins expressed inboth groups but differentially were bone marrow macrophage cDNA,MA₂085, ASC and DVU₂490. ⑤ The time courses of ASC andDVU₂490 according to quantitative analysis shown that these twoproteins were expressed highly in the PMN of normal apoptosis andincreased to a peak at 12h. Comparatively, DVU₂490 was expressedlowly in the group of PMN treated by ONO-AE-248, and it began todecline at 12 h and was not detected any more at 24 h. The time course ofASC in the group of PMN treated by ONO-AE-248 was similar toDVU₂490. With modest expression before 6 h, ASC began to decline at6 h and almost not expressed any more at 24 h. Conclusion: â‘  Theexpression of proteins of ONO-AE-248-induced non-apoptoticprogrammed cell death of PMN is changed remarkably as the time goesby. â‘¡The mechanism between ONO-AE-248-induced non-apoptoticprogrammed cell death and normal apoptosis is different. â‘¢ It issuggested that the mechanism of ONO-AE-248-induced non-apoptoticprogrammed cell death of PMN is involved with some transcriptionfactors, catalytic enzymes and cytoskeleton proteins. â‘£ Caspase-9induced by ASC may not be involved in ONO-AE-248-inducednon-apoptotic programmed cell death of PMN, but it needs to be provedby more experimental proofs. ⑤DVU₂490 and ASC have differentexpression formats in ONO-AE-248-induced non-apoptotic programmedcell death and normal apoptosis.