In multiple myeloma (MM) blood-borne malignant plasma cells home to bone marrow (BM), where they
accumulate in close contact with stromal cells. Nevertheless, the mechanisms responsible for MM cell
chemotaxis are still poorly defined. In the present study we explored the mechanisms involved in the
chemotaxis of RPMI 8226 cell line, RPMI 8226 cell line was found to express CCR3, CCR5, CCR9, CXCR3
and CXCR4, but these cells were migrated only towards CXCL12 (the ligand for CXCR4). To clarify the
signaling pathways involved in the regulation of MM cell chemotaxis, we therefore analyzed the effect of
various inhibitors targeting intracellular effectors proteins on the CXCL12-mediated RPMI 8226
chemotaxis using flow cytometry and western blot analysis. Using flow cytometry, we observed that the
chemotaxis of RPMI 8226 cell to CXCL12 was completely abrogated by adding AMD (CXCR4
antagonist), PTX (G-protein coupled receptor inhibitor) and U73122 (phospholipase C beta; PLC

inhibitor), moreover, CXCL12-mediated RPMI 8226 chemotaxis was partially inhibited by 1 μM
wortmannin (WM, Class II PI3K inhibitor)), SH5 (AKT inhibitor), Y27632 (Rho-A inhibitor), SN50 (IkB
inhibitor), PD98059 (ERK1/2 MAPK inhibitor) and Na3VO4 (phosphatase inhibitor). These results were
further confirmed by using western blot analysis where we observed that triggering of CXCR4 by
CXCL12 resulted in the activation of PLC
3, PI3K/AKT, RhoA, IB and ERK1/2. In conclusion, our
results revealed that PLC
3, PI3K/AKT, RhoA, IKB and ERK1/2 are crucial effectors for CXCL12-
mediating MM cell chemotaxis.