Abstract
Background: Multiple myeloma (MM) is an incurable hematologic cancer, accompanied by excessive osteoclast formation and inflammatory cytokine secretion. The mechanisms by which bromodomain and extra-terminal domain (BET) protein inhibitor I-BET151 regulates osteoclast differentiation and inflammatory cytokine secretion in MM are largely unknown. Methods: The isolated peripheral blood mononuclear cells from normal or patients with MM were treated with receptor activator of NF-κB ligand (RANKL) and M-CSF to induce osteoclast differentiation. RAW 264.7 cells were treated with RANKL. I-BET151 was applied to investigate the effects of BRD4 inhibition on osteoclast formation and inflammatory cytokine secretion. Osteoclast formation was determined by tartrate-resistant acid phosphatase (TRACP) staining. The expression of osteoclast-specific genes TRACP, matrix metalloproteinase-9 (MMP-9), cathepsin K (Ctsk), and c-Src was tested using quantitative real-time PCR. And the level of inflammatory cytokines TNF-α, IL-1β, and IL-6 was assessed by ELISA. Tumor necrosis factor receptor-associated factor 6 (TRAF6), BRD4, nuclear and cytoplasm p65, IκB-α, nuclear factor of activated T cells cytoplasmic (NFATc1), and osteoprotegerin (OPG) expression were measured by Western blotting. RNAi technology was applied to knock down BET family member BRD4. Results: I-BET151 dose-dependently suppressed osteoclast formation, inhibited the levels of osteoclast-specific genes TRACP, MMP-9, Ctsk, and c-Src and inflammatory cytokines TNF-α, IL-1β, and IL-6 secretion in peripheral blood mononuclear cells and RAW 264.7. I-BET151 inhibited the protein levels of BRD4 and NFATc1, increased OPG expression, and suppressed IκB-α degradation and p65 nuclear translocation. Further, the effects of I-BET151 on osteoclast formation, osteoclast-specific genes expression, inflammatory cytokine secretion, and NF-κB inhibition were promoted by BRD4 knockdown. Conclusion: I-BET151 inhibits osteoclast formation and inflammatory cytokine secretion by targetting BRD4-mediated RANKL-NF-κB signal pathway and BRD4 inhibition might be beneficial for MM treatment.
Highlights
Multiple myeloma (MM) is an incurable cancer developing almost exclusively in the bone marrow and produces focal or diffuse bone destruction [1,2]
The isolated mononuclear cells were treated with induction factors receptor activator of NF-κB ligand (RANKL) and macrophage colony stimulating factor (M-CSF) in the absence or presence of I-BET151 at indicated concentrations
RANKL and M-CSF stimulated the expression of osteoclast-specific genes including tartrate-resistant acid phosphatase (TRACP), matrix metalloproteinase-9 (MMP-9), cathepsin K (Ctsk) and C-Src, which were down-regulated by the co-administration of I-BET151 in a dosage-dependent manner in both groups (Figure 1C)
Summary
Multiple myeloma (MM) is an incurable cancer developing almost exclusively in the bone marrow and produces focal or diffuse bone destruction [1,2]. Cytokines, such as TNF-α, IL-6, and IL-1β, and the interaction between myeloma cells and bone microenvironment modulate osteoclast differentiation and osteoblast inhibition [6]. All these contribute to bone destruction and loss, initiating MM development. I-BET151 was applied to investigate the effects of BRD4 inhibition on osteoclast formation and inflammatory cytokine secretion. Results: I-BET151 dose-dependently suppressed osteoclast formation, inhibited the levels of osteoclast-specific genes TRACP, MMP-9, Ctsk, and c-Src and inflammatory cytokines TNF-α, IL-1β, and IL-6 secretion in peripheral blood mononuclear cells and RAW 264.7. The effects of I-BET151 on osteoclast formation, osteoclast-specific genes expression, inflammatory cytokine secretion, and NF-κB inhibition were promoted by BRD4 knockdown. Conclusion: I-BET151 inhibits osteoclast formation and inflammatory cytokine secretion by targetting BRD4-mediated RANKL-NF-κB signal pathway and BRD4 inhibition might be beneficial for MM treatment
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