The Effect And Mechanism Of Berberamine Combined With Ibrutinib On Acute Myeloid Leukemia Cells

Background Acute myeloid leukemia（AML）is a malignant tumor derived from myeloid progenitor cells with high invasiveness and poor prognosis.The etiology of most AML patients is unknown,and risk factors include older age,smoking,chemical exposure,and myelodysplastic syndromes.Bruton tyrosine kinase（BTK）and Ca²⁺/calmodulin-dependent protein kinase IIγ（Ca MKIIγ）are important tumor promoters in the pathogenesis of AML.Ibrutinib can inhibit tyrosine Acid kinase thus blocks the proliferation and migration of AML cells.Berbamine（BBM）is a natural inhibitor of Ca MKII,which can compete with Ca MKIIγto combine with ATP,thereby inhibiting the proliferation of leukemia cells and promoting differentiation.Combination drugs are widely used in the treatment of various diseases including cancer.In this study,the combination of berberamine and ibrutinib was attempted to initially explore the mechanism of their combined action and provide theoretical and experimental basis for the exploration of new treatments for AML.Objective To clarify the effect of BBM and ibrutinib combination on the proliferation,apoptosis and cycle of acute myeloid leukemia cells,and to explore the mechanism of the combination.Methods The AML cell lines KG-1、MOLM13、KG-1a and THP-1 were treated with ibrutinib（0,0.5,1.0,2.0 umol/L）and berberamine（0,20,40,80 umol/L）,respectively,and the cell viability was detected by CCK method.Half maximal inhibitory concentration IC₅₀.According to the molar concentration ratio of IC₅₀（berberamine）and IC₅₀（ibrutinib）,the combined drug was cultured for 48 hours,and the survival rate of cells after single drug and double drug effects was compared,and the combination index CI（combination index）was calculated by Calcusyn software.After 48 hours of treatment with IC₅₀ concentration ratio,flow cytometry was used to compare the apoptosis ratio of single drug group and combination group,and Western Blot was used to detect the expression of apoptotic proteins（PARP,Caspase9 and Cleaved-caspase3）.After treatment with 1/2×IC₅₀ concentration ratio for 24h,the cell cycle of the single drug group and the combination group was compared by flow cytometry.Bioinformatics methods were used to verify the differential expression of BTK in AML patients and its relationship with the prognosis of AML patients.GO and KEGG enrichment analysis of BTK-related signaling molecular pathways.Western Blot verified the expression of BTK-related molecular proteins（P-BTK,P-AKT,CREB,GSK3βand BCL-XL）.Results The cell viability in the combined group was significantly decreased（P<0.05）,and the combined index of ED50/ED75/ED90 was less than 1.Flow cytometry showed that the cell apoptosis rates in the combination group were KG-1:98.9%,MOLM-13:40.8%,and the cell apoptosis rates in the single drug group were respectively Berberamine（KG-1:45.8%,MOLM-13:6.06%）and ibrutinib（KG-1:2.70%,MOLM-13:6.11%）,the apoptosis rate of the combined group was significantly increased（P<0.05）.The ratios of cells in G0/G1 phase in the combination group were KG-1:47.57%,MOLM-13:78.59%,respectively,while the ratios of cells in G0/G1phase in the single drug group were berberamine（KG-1:38.70%,MOLM-13:47.95%）and ibrutinib（KG-1:38.51%,MOLM-13:63.64%）,the ratio of G0/G1 phase cells in the combination group was significantly higher than that in the single drug group（P<0.05）.Bioinformatics analysis found that BTK was abnormally high expressed in AML patients and was significantly associated with shortened OS in AML patients（P<0.05）.GO and KEGG gene enrichment analysis found that the interaction between BTK and ECM receptors,PI3K-AKT signaling pathway and B cell receptor signaling pathway were closely related.Western Blot detection showed that the expressions of apoptotic proteins（PARP,Caspase9 and Cleaved-caspase3）in the combination group were significantly up-regulated（P<0.05）,while the expressions of P-BTK,P-AKT,CREB,GSK3βand BCL-XL were significantly down-regulated（P<0.05）.Conclusion Berberamine and ibrutinib have synergistic effects in inhibiting the proliferation of AML cells and promoting apoptosis,and block the cell cycle of AML.BTK is abnormally highly expressed in AML patients and is closely related to signaling pathways such as the PI3K-AKT signaling pathway.Berberamine combined with ibrutinib can exert a synergistic effect against AML cells through the P-BTK/P-AKT/CREB and GSK3β/BCL-XL signaling pathways.