Disruption of the Crosstalk CXCL12/CXCR4 Chemotactic Pathway To Enhance Melphalan-Induced Apoptosis in Multiple Myeloma Cell Lines.

INTRODUCTION:

Multiple myeloma cells display functional CXCR4 chemokine receptor that stimulates the migration of these cells toward their natural ligand, CXCL12 (stromal-derived factor, SDF-1a). CXCL12 is secreted by bone marrow stroma. Consistent with their CXCR4 expression, myeloma cells home to the marrow microenvironment, where adhesive interactions promote growth, survival, and confer cell adhesion-mediated drug resistance.

METHODS

U266-B1 cells (ATCC myeloma cell line) were pre-treated with recombinant CXCL12 for 30 minutes prior to the addition of melphalan for up to 72 hours. Both melphalan and CXCL12 were added at 24 hour intervals. Cell lines alone and cell lines with only melphalan or only CXCL12 were used as controls. We have also tested the influence of adding AMD3100, a reversible inhibitor of CXCR4, on myeloma cell survival. U266-B1 cells in media alone were pre-treated with AMD3100 for 24 hours prior to treatment with melphalan for 16 hours. Cell viability following treatment was quantified by flow cytometry assay using Annexin-V-FITC staining. Western blot analysis was used to quantify the apoptotic activity of the cell lines using 4 apoptotic markers: PARP (poly ADP-ribose polymerase), caspase-3, Bcl-2, and Mcl-1.

RESULTS:

Recombinant CXCL12 conferred a protective effect to myeloma cell lines during melphalan treatment. This effect was more pronounced at 72 hours of treatment. Western blot analysis showed diminished expression of the apoptotic markers, cleaved PARP (poly ADP-ribose polymerase) and active caspase-3 in the melphalan-treated cell lines with prior exposure to CXCL12. Additionally, pretreatment with AMD3100 resulted in enhanced apoptosis following melphalan treatment.

CONCLUSION:

Our data showed that CXCL12, a naturally occurring cytokine secreted by bone marrow stromal cells confers a protective effect on myeloma cells against apoptosis. Disruption of this effect by AMD3100 resulted in enhanced melphalan-induced apoptosis of myeloma cells. We are continuing to study this effect with the potential future utility of AMD3100 as a melphalan chemo-sensitizer in the treatment of multiple myeloma.