The Role Of CXCR4 In Bone Marrow Miroenvironment-Mediated AML Cells Drug Resistance

ObjectivesIn his study,we co-cultrued AML cells with bone marrow mesenchymal stromal cells(MSCs)to build co-culture model,compared the effect of co-cultrue model on AML cells apoptosis and CXCR4 expression.And then silence the AML cells CXCR4 expression with AMD3100,while evaluate the effect of AMD3100 on drug-induced AML cells apoptosis in co-cultrue model.The aim of this study was to evaluate that CXCR4 signaling plays a key role in leukemia/bone marrow microenvironment interactions which known as environment-mediated drug resistance.Methods1.Cells co-culture:AML cells were co-cultured with bone marrow MSCs to build co-culture model.2.The changes of mitoxantrone-induced AML cells apoptosis and spontaneous apoptosis when co-cultured with MSCs or cultured without MSCs were detected by flow cytometry with Annexin V/PI staining and DAPI nucleus staining.3.The changes of CXCR4 expression in AML cells after co-cultured(0h,24h,48h)with MSCs were detected by Real-time PCR,western blot and flow cytometry.4.In the AML/MSC-based microenvironment co-culture model,AML cells surface CXCR4 expression was detected by flow cytometry after a pre-treatment with different concentration CXCR4 antagonist AMD3100.5.The effect of CXCR4 antagonist AMD3100 on mitoxantrone-induced AML cells apoptosis when co-culture with MSCs was detected by flow cytometry.6.All datas were processed with SPSS for windows software version 19.0.The means of different samples were compared by using one-way ANOVA.If the difference was statistically significant,further LSD-t tests were used to analyzed the means.The level of significance was set at 0.05.Results1.AML cells adhered to the surface of MSCs after co-cultured with MSCs.2.Mitoxantrone-induced AML cells apoptosis and pontaneous apoptosis statistically decreased after co-cultrued with MSCs comparing to culture without MSCs were detected by by flow cytometry with Annexin V/PI staining and DAPI daining nuclears(p<0.05).3.The up-regulation of AML cells CXCR4 mRNA and CXCR4 protein which is in time-dependence after co-culture with MSCs was detected by Real-time PCR and western blot,while we observed by flow cytometry that the percentage of CXCR4 positive AML cells accounted for 94.3 ± 0.7%,and AML cells surface CXCR4 expression(mean fluorescence intensity ratio,MFIR)increased which is in time-dependence after co-cultured with MSCs,4.Flow cytometry observed that AMD3100 with different concentrations from 5ug/mL to 0.0005ug/ml induced down-regulation of CXCR4 positive U937 cells in dose-dependence from 97.0%± 1.0%to 9.6 ± 0.8%(p<0.05).5.In this co-cultrue model,CXCR4 antagonist AMD3100 significantly increased the degree of mitoxantrone-induced AML cells apoptosis,that is,AMD3100 restores apoptosis in the presence of an MSC-based microenvironment.ConclusionAML cells spontaneous and drug-induced apoptosis decreased,while CXCR4 expression increased after co-cultured with MSCs.In the AML/MSC-based co-culture model,CXCR4 antagonist AMD3100 decreased AML cells surface CXCR4 expression and restored mitoxantrone-induced AML cells apoptosis.Taken together,these datas demonstrate that CXCR4 signaling plays a key role in leukemia/bone marrow microenvironment interactions which known as environment-mediated drug resistance.Combining AMD3100 with conventional chemotherapy may,therefore,represent a promising therapeutic approach to kill AML cells.