| Background:Acute myeloid leukemia(AML)is malignant clonal disease originating from myeloid hematopoietic stem cells or progenitor cells,accounting for 31%of all adult leukemia cases.Currently,the clinical treatment strategies for AML mainly consist of "3+7"intensive induction chemotherapy with anthracycline antibiotics combined with cytarabine and allogeneic hematopoietic stem cell transplantation,but half of patients still relapse after complete clinical remission.Recently,many effective and safe immunotherapies have been developed for the problems of abnormal proliferation and high recurrence rate of AML.Interleukin-33(IL-33)is a pre-inflammatory cytokine that binds to Interleukin-1 receptor like-1(IL1RLI),then involved in a variety of inflammatory and immune response processes.In solid tumors,such as esophageal squamous cell carcinoma,glioma,renal cell carcinoma,etc.,IL-33 has been reported about promoting tumor development,migration,drug resistance,etc.In hematologic malignancies,our team’s previous study confirmed that IL1RL1 is highly expressed on the surface of AML cells,and the addition of exogenous IL-33 can activate p38 MAPK and NF-κB in both primary mouse leukemia cells and human AML cells.In addition,IL-33 could reduce the sensitivity of AML cells to chemotherapeutic drugs.This study aims at exploring the effect of IL-33/IL1RL1 axis on apoptosis,cycle and chemotherapy drug sensitivity of AML cells,as well as the potential underlying mechanisms,which provides new insights into therapeutic strategies for treating AML.Objective:To investigate the effect of IL-33 on apoptosis,the cell cycle,chemotherapy drug sensitivity and possible mechanism of action of acute myeloid leukemia cells.Methods:1.AML cell lines HL-60 and NB4 were treated with exogenous IL-33 and P38 MAPK specific inhibitor SB203580 with Annexin V stained flow cytometry to detect apoptosis.2.The cell cycle assay kit detects HL-60,NB4 cell cycles cultured with IL-33 and SB20358.3.IL-33 and SB20358 after treatment of HL-60,NB4 cells,CDK1 protein expression and p38 MAPK phosphorylation levels detected by western blot.4.Exogenous IL-33 acts on HL-60,NB4 cells at different times(15,30,60,90 minutes),western blot detects whether NF-κB occurs to phosphorylation.After the addition of NF-κB specific inhibitor Bay 11-7085,western blot detects IL-33 can reverse the effects of Bay 11-7085.5.HL-60 and NB4 were treated with exogenous IL-33 and Bay 11-7085 to detect apoptosis by Annexin V/DAPI double-staining flow cytometry,CFSE probe staining to detect cell proliferation,cell cycle assay kit to detect the cell cycle.6.IL-33 and Bay 11-7085 after treatment of HL-60,NB4 cells,western blot detects active caspase 3,BIRC2,Bcl-2,Bax protein expression,and NF-κB phosphorylation levels.7.IL-33 and Bay 11-7085 treated cells from AML patients to detect apoptosis by Annexin staining flow cytometry,western blot detects active caspase 3,BIRC2 protein expression,and NF-κB phosphorylation levels.8.After treatment of HL-60,NB4 and AML patients with exogenous IL-33 and Doxorubicin,apoptosis was detected by Annexin V staining flow cytometry,and NFκB phosphorylation levels were detected by western blot.Results:1.Compared with the control group,HL-60 and NB4 cells in the IL-33 group had a reduced apoptosis level after the action of IL-33,during the cell cycle increased proportion of stage S.The apoptosis level of SB+IL-33 group,treatment with SB203580 and IL-33,was higher than the IL-33 group,the percentage of cells in the S phase was lower than the IL-33 group.2.The results of the western blot assay showed that the level of p38 MAPK phosphorylation(p-p38 MAPK)and CKD1 protein in the IL-33 group was significantly increased,compared to the control group.While the levels of protein pp38 MAPK and CDK1 decreased in the SB+IL-33 group compared with the IL-33 group.3.After IL-33 treatment of HL-60 and NB4 cells for 60 minutes,the level of NF-κB phosphorylation was highest relative to other times,and IL-33 can reverse the inhibition of NF-κB phosphorylation by Bay 11-7085.4.Compared with the control group,IL-33 can reduce the apoptosis level,increase cell proliferation,and increase the proportion of G2/M phase for HL-60 and NB4 cells.In contrast,the Bay+IL-33 group,the apoptosis level is increased,the number of cell proliferation is reduced,and the percentage of cells in the G2/M phase is higher than in the IL-33 group.5.The results of the western blot assay showed a significant decrease in the level of active caspase 3 protein in cells in the IL-33 group compared to the control group,and the levels of BIRC2,Bcl-2 and Bax proteins were significantly increased.The Bay+IL-33 group,the level of active caspase 3 protein was significantly increased compared with the IL-33 group,and the levels of BIRC2,Bcl-2 and Bax proteins decreased significantly.And the expression of Bcl-2 protein exceeded that of Bax.6.In cells of patients with acute myeloid leukemia in clinical samples,compared with the control group,the level of apoptosis in the IL-33 group was reduced,and the expression of BIRC2 and p-p65 protein levels was increased,active caspase 3 expression was decreased.The combination of Bay 11-7085 and IL-33 reverses the biological effects of IL-33.7.Compared with the control group,the apoptosis level of HL-60,NB4 cell lines and primary AML cells in the Dox group increased after Doxorubicin,while the apoptosis level of cells in the Dox+IL-33 group was lower than that in the Dox group after the combination of Doxorubicin and IL-33.The results of the western blot assay showed that cells in the Dox group had lower levels of p-p65 compared to the control group,while cells in the Dox+IL-33 group had higher levels of p-p65 than cells in the Dox group.Conclusions:1.IL-33 inhibits apoptosis of AML cells and promotes cell survival by activating p38 MAPK and NF-κB pathways.2.IL-33 reduces the sensitivity of AML cells to chemotherapy drugs by activating NFκB pathway. |
