Bortezomib is widely used for the treatment of multiple myeloma, based on findings that it induces apoptosis and strongly inhibits proliferation of myeloma cells. Bone marrow stromal cells (BMSCs) endow myeloma cells with survival and growth advantages. However, the influence of bortezomib on BMSCs is not well elucidated. In the present study, we examined the role of bortezomib in the survival and growth of BMSCs and its implications in myeloma cell biology.
First, the effects of bortezomib on the proliferation and survival of the BMSCs were investigated. In a 3-day incubation of MS-5 murine BMSCs, the addition of 5 nM bortezomib significantly decreased cell proliferation as compared with controls (relative proliferation index, 2.1 ± 0.5 vs.1.6 ± 0.3 at 48 h; 3.5 ± 0.6 vs. 1.5 ± 0.2 at 72 h; both P < 0.05) and ≥ 50 nM bortezomib abolished cell proliferation. Consistent with these findings, bortezomib diminished the percentages of cells in S phase in a dose-dependent manner. Bortezomib (up to 500 nM) did not affect the survival of MS-5 cells for initial 24 h of incubation, whereas it markedly increased the percentages of annexin V-positive apoptotic cells after 72 h (54.7 ± 7.2% for 50 nM; 73.0 ± 10.4% for 500 nM; both P < 0.05 as compared with controls). Similar results were obtained with BMSCs from both healthy individuals (n = 4) and myeloma patients (n = 5). Next, we tested our hypothesis that downregulation of the chemokine CXCL12 may be involved in these phenomena. Knock-down of CXCL12 mRNA, using a short interfering RNA strategy, in BMSCs from both healthy individuals and myeloma patients resulted in a significant decrease in the survival and spontaneous proliferation of cells, indicating that CLCX12 is an autocrine survival and growth factor. Treating BMSCs with bortezomib at concentrations of 5, 50, and 500 nM for 24 h reduced CXCL12 mRNA expression to 50%, 20%, and < 10% of the control, respectively. In parallel, bortezomib decreased CXCL12 production in a dose-dependent manner, as evidenced in Western blotting and ELISA analysis. Additionally, CXCL12 serum levels in myeloma patients (n = 5) placed on bortezomib treatment were significantly decreased after 3 days of administration (2,256 ± 664 pg/mL vs. 1,283 ± 482 pg/mL; P < 0.05). The inhibition of BMSC survival and proliferation induced by 5 nM bortezomib was partially reversed by the addition of CXCL12. Finally, we tested whether bortezomib-induced alterations in BMSCs affect myeloma cell biology. Supernatants from 5 nM bortezomib-treated MS-5 cells induced chemotaxis of RPMI8226 myeloma cells to a much lower extent as compared with supernatants from non-treated cells (migration index, 7.3 ± 1.5 vs. 3.4 ± 1.1; P < 0.05). Additionally, short-term bortezomib treatment of MS-5 monolayers resulted in decreased localization of RPMI8226 cells under the monolayers as compared with non-treated ones.
These results indicate that bortezomib inhibits the survival and growth of BMSCs via downregulation of CXCL12. The anti-myeloma effects of bortezomib may be exerted at least in part via modulation of BMSCs.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.