The Inhibitory Effect Of Zanubrutinib On The Proliferation Of Acute Myeloid Leukemia Cells And Its Related Mechanisms

Objective:To analyze the inhibitory effect of Zanubrutinib on the proliferation of acute myeloid leukemia cells and its related mechanism and hope to provide a new method for the clinical treatment of acute myeloid leukemia.Methods:1.Bioinformatics techniques were used to explore the expression of BTK in AML patients and normal controls,and to analyze the relationship between BTK expression and overall survival(OS)in AML patients.2.Proliferation inhibition of Zanubrutinib on HL-60 and U937 AML cell lines was detected by CCK-8 assay and Trypan blue rejection assay.3.Fluorescence probe and flow cytometry were used to detect the apoptotic induction of Zanubrutinib in AML cells.4.The clonogenesis ability of AML cells treated with Zanubrutinib was detected by methylcellulose method.5.mRNA and protein levels of BTK molecule and downstream PI3K/Akt signaling pathway after Zanubrutinib treatment were detected by quantitative PCR and Western blot.6.The subcutaneous tumor bearing model of human AML mice was constructed to observe the killing effect of Zanubrutinib on AML cells in vivo.Results:1.Data analysis showed that BTK high expression was found in AML patients,and BTK high expression was associated with overall survival(OS).2.Zanubrutinib could inhibit the proliferation of HL-60 and U937 AML cell lines.Compared with blank control and solvent control group,Zanubrutinib could inhibit theproliferation of HL-60 and U937 in a time and concentration dependent manner(P <0.05).3.Fluorescence probe method and flow cytometry were used to analyze the effect of Zanubrutinib on apoptosis of HL-60 and U937 cells.The results showed that the apoptosis rate of the experimental group increased gradually with time(P < 0.05).4.Effect of Zanubrutinib on cell clonogenesis: The effect of Zanubrutinib on cell clonogenesis of HL-60 and U937 was analyzed by using methylcellulose culture method.The results showed that Zanubrutinib treatment reduced cell clonogenesis ability.5.Quantitative PCR and Western blot were used to detect the expression levels of BTK and important signal molecules in downstream signaling pathways in HL-60 and U937 cells after Zanubrutinib treatment.The results showed that the downstream protein levels of m TOR,Akt and m RNA of BTK signaling pathway were not changed and the protein expression was down-regulated after Zanubrutinib treatment.6.The tumor bearing mice of AML were established and injected with normal saline,solvent group(DMSO)and Zanubrutinib,respectively.The tumor size,survival time and leukemia cells in the tumor and spleen of the mice were observed.The results showed that Zanubrutinib could effectively eliminate AML cells in the tumor bearing mice.Conclusion:Zanubrutinib can effectively inhibit the proliferation of AML cells,and its mechanism may be related to inducing apoptosis and regulating the expression of BTK molecule and downstream signaling pathway.