Bortezomib Inhibits Human Osteoclastogenesis.

Enhanced osteoclastogenesis in cancer-induced bone disease may be caused by intercellular interactions between tumor cells and cells of the bone marrow microenvironment. In multiple myeloma, overexpression of RANKL in the bone marrow microenvironment may lead to the activation of TRAF-signaling and in consequence to increased NF-κB and AP-1 transcriptional activities in osteoclastic lineage cells. This results in enhanced osteoclast differentiation, activation and increased bone resorption. In this study, we have examined the effects of two NF-κB inhibitors towards their inhibitory potency on human osteoclastogenesis: proteasome inhibitor bortezomib and selective IKK inhibitor PS-1145. CD14+ osteoclast precursors from peripheral blood were stimulated with RANKL and M-CSF up to four weeks. Using MTT- and TUNEL-assays, cytotoxicity levels of each drug were determined on the differentiation stage day +1 (early osteoclast precursors), day +8 (preosteoclasts) and day +21 (osteoclasts). To evaluate the effects of both drugs considering osteoclast differentiation and -function, 2 sub-apoptotic doses of bortezomib, one sub-apoptotic and one low-apoptotic dose of PS-1145 were used. As revealed by the microscopic quantification of mature osteoclasts (TRAP-positive and multi-nucleated cells), the osteoclast differentiation was diminished by both drugs, whereas the effects were dose- and time-dependent. The microscopic quantification of resorption lacunae on dentine pits revealed that the resorptional activity was reduced by 65% for 0.1 nM bortezomib (p=0.007), by 79% for 1 nM bortezomib (p<0.0005), by 60% for 1 μM PS-1145 (p=0.023) and by 91% for 10 μM PS-1145 (p<0.0005). As shown by immunoblotting and by ELISA-based methods, the subcellular mechanisms of action involved in inhibition of early osteoclast differentiation were found to be related to the inhibition of p38 mitogen-activated protein kinase (MAPK) pathways, whereas the advanced differentiation and activation occurred in course of inhibition of AP-1 and NF-κB activation. The AP-1 blockade contributed to significant reduction of osteoclastic vascular endothelial growth factor (VEGF) production. In conclusion, our data demonstrate that proteasomal inhibition should be considered as a novel therapeutical principle of cancer-induced lytic bone disease.