| Objective:Bruton Tyrosine Kinase(BTK)is an important component of B cell receptor signaling(BCR)and mediates the migration,chemotaxis and adhesion of B cells and promotes proliferation,survival and drug resistance of B cell malignancy.Ibrutinib is a small molecule inhibitor of BTK and has been approved by the FDA for the first-line treatment of chronic lymphocytic leukemia and the second-line treatment of mantle cell lymphoma.This study aims to explore the mechanisms of ibrutinib inducing apoptosis and overcoming drug-resistance of DLBCL Cells via DLBCL cell lines and primary DLBCL cells.Methods:1.By pre-treatment with different doses of ibrutinib,the cell proliferation inhibition rate of DLBCL cells was measured by MTS assay,and cell apoptosis was detected and analyzed by annexin V/iodinated pyridine staining and flow cytometry.DAPI staining was used to observe morphological changes of the cell nuclei.Western blot was used to detect the expression of BCL-2 in the lysates of DLBCL cells.2.DLBCL cells were co-cultured with MSCs,the amounts of DLBCL cells that migrated and adhered to MSCs were counted under microscope.Secretion of CXCL-12 by MSCs was detected by ELISA,expression of CXCR4 on DLBCL cells was detected by flow cytometry.DLBCL cells were transfected with the lentivirus carrying CXCR4,then co-cultured with MSC,and mitoxantrone and/or ibrutinib were used to treat the co-cultured DLBCL cells,Annexin V / iodinated pyridine staining was used to detect the apoptosis of DLBCL cells.3.DLBCL cells were co-cultured with MSCs on MethoCult methylcellulose medium and pre-treated with ibrutinib.The clonogenesis of DLBCL cells in vitro was observed by inverted microscope.DLBCL cells and MSCs were co-injected into NOD/SCID mouse models subcutaneously and treated with ibrutinib 2.5 mg/kg/day intraperitoneally.Measurement of tumor growth was adopted regularly.Results:1.After treatment with Ibrutinib,cell proliferation inhibition and apoptosis of DLBCL cells were observed in a dose-dependent manner.The cell nuclei were condensation and shrinkage.The pro-apoptotic proteins,BAD and BAX were upregulated and the anti-apoptotic proteins,BCL-2,BCL-CL and MCL1 were inhibited,leading to high activation of caspase-3 and the intracellular proteolytic cascades,PARP was cleaved and invalid.The expression and phosphorylation of BTK,AKT and ERK were down-regulated,the BCR signaling pathway was blocked.2.MSCs promoted the migration and adhesion of DLBCL cells to MSCs,ibrutinib inhibited the migration and adhesion of DLBCL cells to MSCs in a dose-dependent manner.MSCs up-regulated the expression of CXCR4 in DLBCL cells,DLBCL cells promoted the secretion of CXCL12 by MSCs,ibrutinib inhibited the secretion of CXCL12 promoted by DLBCL cells and the expression of CXCR4 upregulated by MSCs.DLBCL cells had CXCR4 overexpression after transfection,after co-cultured with MSCs,the proportion of mitoxantrone-induced apoptosis of DLBCL cells was decreased.As treatment with ibrutinib,the apoptotic rate of co-culture system was increased,indicating that ibrutinib overcame the MSC-mediated drug resistance.3.MSC promoted the clonogenesis in vitro and the proliferation in vivo of DLBCL cells.Ibrutinib inhibited the clonogenesis of DLBCL cells in vitro in co-culture system(P < 0.01)and inhibited proliferation of DLBCL cells in vivo(P < 0.05).Conclusion:Ibrutinib has a direct anti-tumor effect by activating caspase-3 and upregulating the expression of pro-apoptotic proteins such as BAD and BAX,and inhibiting the expression of anti-apoptotic proteins such as BCL-2,BCL-CL,MCL1,leading to proteins hydrolysis cascades,cleaving PARP and inducing apoptosis of DLBCL cells.The CXCL12/CXCR4 axis plays a key role in MSC-mediated drug resistance in DLBCL microenvironment.Ibrutinib inhibits the secretion and expression of CXCL12 and CXCR4,thereby inhibits the axis overcomes drug resistance. |
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