Study On The Mechanism Of Triptonide Inducing Apoptosis In Leukemia Cells

ObjectiveTo explore the mechanism of triptonide on the acute myelogenous leukemia cells,we investigated the triptonide on U937 cell proliferation inhibition,cycle distribution,apoptosis,RNA-seq,mRNA and related protein expression;the effect of triptonide of SHI-1 cell proliferation inhibition,cycle distribution,apoptosis and related protein expression and the effect of triptonide of NB4 cell proliferation inhibition and apoptosis.Methods1.The inhibitory effects of triptonide on proliferation of leukemia cells MTT assay was accustomed notice the repressing effects of triptonide with totally different concentrations on the proliferation of leukemia cells U937,SHI-1 and NB4,and the half inhibitory concentration was calculated.2.Effect of triptolide on cell cycle distribution and apoptosis of leukemia cellsThe cycle distribution and cell death of U937,NB4 and SHI-1 cells were detected by flow cytometry,when treated with totally different concentration triptonide severally.3.Effect of triptonide on transcriptome expression in U937 cellsUsing U937 cells as the research object,transcriptome sequencing was performed by high-throughput sequencing method.The effect of triptonide on U937 cell transcriptome expression was investigated by gene quantitative and differential gene analysis,GO enrichment analysis,KEGG pathway enrichment and cluster analysis.4.Effect of triptonide on related mRNA expression in leukemia cellsThe expression of mTOR,TP53,MAPK13,CREB3L3 and SOCS1 in U937 cells were detected by qRT-PCR.5.The effect of triptonide on the expression of related proteins in leukemia cellsWestern blot was accustomed discover the expression of apoptosis-related proteins Caspase-3,Caspase-8 and NF-κB in SHI-1 cells treated with triptonide.6.Effect of triptonide on H9C2 proliferation in normal cardiomyocytesMTT assay was wont to sight the restrictive result of various concentrations of triptonide on the proliferation of H9C2 cells.Results1.Inhibitory effect of triptonide on proliferation of leukemia cellsCompared with the control group,the triptonide considerably smothered the proliferation of U937,SHI-1 and NB4 cells with concentration-dependent(P<0.05).2.Effect of triptonide on cell cycle distribution and apoptosis of leukemia cellsThe cycle distribution of U937 and SHI-1 cells was affected by triptonide(P<0.05).The proportion of apoptotic cells in U937,SHI-1 and NB4 cells treated by triptonide were significantly higher than that in the control group(P<0.05).3.Effect of triptonide on transcriptome expression in U937 cellsAccording to the volcano map,compared with the control group,a total of 652 differential genes were detected in the triptonide group,among which the number of up-regulated genes and down-regulated were 427 and 225,respectively.The expression of various genes within the 2 teams of samples were showed by cluster analysis.The most obvious correlation pathway for GO enrichment analysis of differential genes was negative regulation of JAK-STAT cascade,and the KEGG pathway enrichment analysis showed twenty pathways,among them,MAPK signaling pathway,FOXO signaling pathway,p53 signaling pathway,JAK-STAT signaling pathway,PI3K-Akt signaling pathway,and mTOR signaling pathway are related to tumor apoptosis mechanism.The expression of key genes mTOR,TP53,MAPK13,CREB3L3 and SOCS1,which belong to the above pathway,were significantly different between control group and triptonide group.4.Effect of triptonide on related mRNA expression in U937 cellsCompared with control group,mTOR expression in U937 cells treated with triptonide was decreased(P<0.001),TP53(P<0.01),MAPK13(P<0.05),CREB3L3(P<0.01)and SOCS1(P<0.05)were increased.5.The effect of triptonide on the expression of related proteins in SHI-1 cellsCompared with control group,the expressions of NF-κB(P<0.01),Caspase-3(P<0.001)and Caspase-8(P<0.001)in SHI-1 cells treated with triptonide were decreased.6.Effect of triptonide on H9C2 proliferation in normal cardiomyocytesTriptonide had no significant inhibitory effect on the proliferation of rat normal cardiomyocytes H9C2(P<0.05).Conclusions1.Triptonide can significantly inhibit the proliferation and induce apoptosis of leukemia cells in vitro at the concentration of nM level.2.Interference with the cell cycle progression of leukemia may be one of the mechanisms of triptonide inhibiting the proliferation of leukemia cells.3.The mechanism of triptonide inhibiting U937 cell proliferations may be related to one or many of the following pathways(MAPK signaling pathway,FOXO signaling pathway,p53 signaling pathway,JAK-STAT signaling pathway,PI3K-Akt signaling pathway,mTOR signaling pathway).4.The apoptosis of SHI-1 cells induced by triptonide may be related to NF-κB pathway.5.There was no obvious side effect of triptonide on rat normal cardiomyocytes H9C2.