Alloimperatorin Induced The Apoptosis Of HL-60 Cells Through MAPK Signaling Pathway

Objective: Alloimperatorin is a kind of coumarin extracted from the herbal plant Angelica dahurica,coumarin exhibits anti-tumor activity in various cancer models,such as lung cancer,stomach cancer and breast cancer.However,to the best of our knowledge,the direct effect and mechanism of Alloimperatorin on HL-60 cells has not been reported.This study investigated the anti-tumor effect of Alloimperatorin on HL-60 cells in vitro and explored its possible mechanism of action.Methods: 1.Using CCK-8 and Trypan blue dying method to determine the effect of Alloimperatorin on the proliferation of HL-60 cells;2.The effect of Alloimperatorin on apoptosis and cell cycle of HL-60 cells was observed by flow cytometry.3.Western blot and RT-PCR were used to detect the expression of Bcl-2,Bax,cleaved-caspase8,-9,-3 in HL-60 cells induced by Alloimperatorin;4.Oncomine analysis of IARS2 gene expression of bone marrow of non-AML patients and non-AML patients;5.Ualcan database analysis,the correlation between the level of IARS2 expression and patient survival in AML patients;6.Western blot and RT-PCR were used to verify the expression of differential gene IARS2 the drug group and the control group;7.Microarray analysis,differential expression of Alloimperatorin group and DMSO group;8.The protein expression of P-JNK,JNK,P-P38,P38,P-ERK and ERK in HL-60 cells treated with Alloimperatorin at different concentrations or different time was analyzed by Western blot.SP600125,SCH772984 and SB203580 were used to inhibit the expression of P-JNK,P-P38 and P-ERK in HL-60 cells,and the expression of cleaved-caspase3 was observed.10.Expression of PML-RARA in HL-60 cells after treatment of HL-60 cells with Alloimperatorin.Result:1.The results of CCK-8 showed that the proliferation of HL-60 cells treated with Alloimperatorin was significantly restricted,and the effect of inhibiting the growth of HL-60 cells gradually increased with the increase of drug concentration at a concentration of 50?g/ml;2.With the increase of the concentration of Alloimperatorin,the late apoptosis rate of HL-60 cells increased dramatically,and HL-60 cells were arrested at S period;3.The results of RT-PCR showed that the expression of BAX in the Alloimperatorin groups was significantly higher than that of the control group,while the expression of Bcl-2 was significantly inhibited.The results of Western blot were consistent with RT-PCR,meanwhile,the expression of cleaved-caspase8,-9,-3 were considerably increased;4.Oncomine database analysis showed that compared with the bone marrow of AML patients,the expression of IARS2 gene in the bone marrow of AML patients was significantly increased that healthy people(P=2.11E-4),and the high expression of IARS2 in AML patients often indicates a poor prognosis;5.Western blot and RT-PCR analysis showed that the expression of IARS2 decreased with the increase of drug concentration and time;6.Microarray analysis suggested that IARS2,IER3,HK3,ETV1,NTHL1,SOX4,and PAOX were differentially expressed in Alloimperatorin group and DMSO group;7.Western blot analysis showed that the ratio of P-JNK/JNK,P-ERK/ERK,P-P38/P38 increased gradually with the increase of drug concentration(P<0.05),when P-ERK,P-P38 was inhibited,the expression of Cleaved-caspase-3 was significantly down-regulated compared with the drug-only group,After inhibition of P-JNK expression,the expression of Cleaved-caspase-3 was not significantly different from that of Alloimperatorin group.8.After treatment of HL-60 cells with Alloimperatorin,the expression of PML-RARA in cells decreased significantly.Conclusion: 1.Alloimperatorin can inhibit the proliferation of HL-60 cells and induce apoptosis;2.Alloimperatorin induces apoptosis of HL-60 cells mainly through the death receptor pathway and the mitochondrial pathway;3.The IARS2 gene is highly reflected in the bone marrow of AML patients,Meanwhile,the higher expression of IARS2,the worse prognosis for the patient.4.Alloimperatorin can improve the survival time of AML patients by inhibiting the expression of IARS2.5.Activation of ERK and P38 MAPK signaling pathway plays a major role in the induction of apoptosis in HL-60 cells by Alloimperatorin;6.Alloimperatorin can induce further differentiation of HL-60 cells.