The proteasome inhibitor bortezomib stimulates osteoblastic differentiation of human osteoblast precursors via upregulation of vitamin D receptor signalling.

Interactions of myeloma cells with the bone marrow microenvironment lead to enhanced osteoclast recruitment and impaired osteoblast activity. Recent evidence revealed that the proteasome inhibitor bortezomib stimulates osteoblast differentiation, but the mechanisms are not fully elucidated. We hypothesised that bortezomib could influence osteoblastic differentiation via alteration of vitamin D signalling by blocking the proteasomal degradation of the vitamin D receptor (VDR). This is of clinical importance, as a high rate of vitamin D deficiency was reported in patients with myeloma. We performed cocultures of primary human mesenchymal stem cells (hMSCs) and human osteoblasts (hOBs) with myeloma cells, which resulted in an inhibition of the vitamin D-dependent differentiation of osteoblast precursors. Treatment with bortezomib led to a moderate increase in osteoblastic differentiation markers in hMSCs and hOBs. Importantly, this effect could be strikingly increased when vitamin D was added. Bortezomib led to enhanced nuclear VDR protein levels in hMSCs. Primary hMSCs transfected with a VDR luciferase reporter construct showed a strong increase in VDR signalling with bortezomib. In summary, stimulation of VDR signalling is a mechanism for the bortezomib-induced stimulation of osteoblastic differentiation. The data suggest that supplementation of vitamin D in patients with myeloma treated with bortezomib is crucial for optimal bone formation.

Synergistic action of the novel HSP90 inhibitor NVP-AUY922 with histone deacetylase inhibitors, melphalan, or doxorubicin in multiple myeloma.

Heat shock protein 90 (HSP90) is a promising target for tumor therapy. The novel HSP90 inhibitor NVP-AUY922 has preclinical activity in multiple myeloma, however, little is known about effective combination partners to design clinical studies. Multiple myeloma cell lines, OPM-2, RPMI-8226, U-266, LP-1, MM1.S, and primary myeloma cells were exposed to NVP-AUY922 and one of the combination partners histone deacetylase inhibitor NVP-LBH589, suberoylanilide hydroxamic acid (SAHA), melphalan, or doxorubicin, either simultaneously or in sequential patterns. Effects on cell proliferation and apoptosis were determined. Synergistic effects were evaluated using the method of Chou and Talalay. Combined sequential incubation with NVP-AUY922 and SAHA showed that best synergistic effects were achieved with 24 h preincubation with SAHA followed by another 48 h of combination treatment. Combination of NVP-AUY922 with SAHA, NVP-LBH589, melphalan, or doxorubicin resulted in synergistic inhibition of viability, with strong synergy (combination index < 0.3) in the case of melphalan. Importantly, resistance of the RPMI-8226 cell line and relative resistance of some primary myeloma cells against NVP-AUY922 could be overcome by combination treatment. These data show impressive synergistic action of the novel HSP90 inhibitor NVP-AUY922 with melphalan, doxorubicin, NVP-LBH589, and SAHA in multiple myeloma and build the frame work for clinical trials.

Curcumin diminishes human osteoclastogenesis by inhibition of the signalosome-associated I kappaB kinase.

PURPOSE: Curcumin is a natural polyphenolic derogate extracted from spice turmeric, exhibiting anti-inflammatory and chemopreventive activities. It was described to interact with the signalosome-associated kinases and the proteasome-ubiquitin system, which both are involved in the osteoclastogenesis. Thus, we hypothesized that curcumin could diminish osteoclast differentiation and function. METHODS: For the experiments considering osteoclast differentiation and resorptional activities, preosteoclasts were cultured for 4 weeks and treated with curcumin at subapoptotic dosages. Derived mature osteoclasts were identified as large, multinucleated cells with expression of tartrate-resistant acid phosphatase activity. Formation of resorption lacunae, a hallmark of osteoclast activity, was quantified using dentine pits and light microscopy. The signaling pathways were examined by ELISA-based methods and by immunoblotting. RESULTS: Both 1 and 10 microM curcumin abrogated osteoclast differentiation (by 56 and 81%) and function (by 56 and 99%) (P < 0.05) dose-dependently. The effects were accompanied by the inhibition of I kappaB phosphorylation and NF-kappaB activation. In contrast, subtoxic doses did not have any significant effects on proteasome inhibition. CONCLUSION: This manuscript is the first report that describes the effects of curcumin toward human osteoclastogenesis, and builds the framework for clinical trials of curcumin in the treatment of cancer-induced lytic bone disease.

Histone deacetylase inhibitors reduce VEGF production and induce growth suppression and apoptosis in human mantle cell lymphoma.

OBJECTIVES: Mantle cell lymphoma (MCL) is an incurable disease with an aggressive course and novel treatment strategies are urgently needed. The purpose of this study was to evaluate the effects of histone deacetylase (HDAC) inhibitors, a new group of antiproliferative agents, on human MCL cells. METHODS: Three MCL cell lines (JeKo-1, Hbl-2 and Granta-519) were exposed to different concentrations of the HDAC inhibitors sodium butyrate (NaB) and suberoylanilide hydroxamic acid (SAHA) for 8-72 h. Their effects on cell viability, apoptosis induction and cell cycle proliferation were studied. Moreover, the influence of SAHA on the expression of cyclin D1, the cell cycle regulators p21 and p27 and the production of vascular endothelial growth factor (VEGF) were analyzed. RESULTS: The HDAC inhibitors induced accumulation of acetylated histones in MCL cells. MTT assays and Annexin-V staining showed that they potently inhibited viability in a dose-dependent manner and induced apoptosis in all cell lines tested. Cell cycle analysis indicated that their exposure to SAHA or NaB decreased the proportion of cells in S phase and increased the proportion of cells in the G0/G1 and/or G2/M phases. Incubation with the two HDAC inhibitors resulted in downregulation of cyclin D1. SAHA lead to an upregulation of p21 in all cell lines and an upregulation of p27 in JeKo-1 and Granta-519 cells, while expression of p27 in Hbl-2 was not altered. In addition, SAHA inhibited the production of the angiogenic cytokine VEGF. Treatment with NaB increased the expression of p21 in JeKo-1 and Hbl-2 cells, while in Granta 519 cells no effect was noted. The expression of p27 remained constant in all three cell lines after exposure to NaB. CONCLUSION: Based on these findings, we provide evidence that HDAC inhibitors have antiproliferative effects in MCL and may represent a promising therapeutic approach.