osteoprotegerin and Sarcoma--Ewing

Factor VIII-von Willebrand factor (FVIII.vWF) complex, a molecule involved in coagulation, can be physically associated with osteoprotegerin (OPG). OPG is an anti-osteoclastic protein and a soluble receptor for the proapoptotic protein TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), suggesting a potential role of FVIII.vWF complex in bone and cancer biology. We, thus, assessed the effects of FVIII.vWF complex on osteoclastogenesis and cell survival. We first evidenced that FVIII.vWF complex inhibited RANKL-induced osteoclastogenesis and enhanced the inhibitory effect of OPG. Interestingly, we revealed by surface plasmon resonance that FVIII.vWF complex bound to RANKL, whereas recombinant FVIII and vWF did not. By modeling, we showed that the OPG binding domain to the A1 domain of vWF was closely located and partially overlapped to its binding site to RANKL. Then, we demonstrated that FVIII.vWF complex cancelled the inhibitory activity of OPG on TRAIL-induced apoptosis and characterized interactions between these molecules. The present work evidenced a direct activity of FVIII.vWF complex on osteoclasts and on induced cell apoptosis, pointing out its potential involvement in physiological bone remodeling or in bone damages associated with severe hemophilia and cancer development.

Topics: Animals; Apoptosis; Bone Neoplasms; Cell Differentiation; Cell Proliferation; Cell Survival; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Factor VIII; Humans; Mice; Osteoclasts; Osteoprotegerin; Osteosarcoma; Platelet Aggregation; RANK Ligand; Sarcoma, Ewing; Surface Plasmon Resonance; TNF-Related Apoptosis-Inducing Ligand; von Willebrand Factor

Cellular mechanisms that account for tumour osteolysis associated with Ewing's sarcoma are uncertain. Osteoclasts are marrow-derived multinucleated cells (MNCs) that effect tumour osteolysis. Osteoclasts are known to form from macrophages by both receptor activator for nuclear factor-kappaB (RANK) ligand (RANKL)-dependent and -independent mechanisms. In this study, our aim has been to determine whether tumour-associated macrophages (TAMs) isolated from Ewing's sarcoma are capable of differentiating into osteoclasts and to characterise the cellular and humoral mechanisms whereby this occurs. Tumour-associated macrophages were isolated from two Ewing's sarcomas and cultured on both coverslips and dentine slices for up to 21 days with soluble RANKL and macrophage colony stimulating factor (M-CSF). Osteoclast formation from TAMs (CD14+) was evidenced by the formation of tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor (VNR)-positive MNCs, which were capable of carrying out lacunar resorption. This osteoclast formation was inhibited by the addition of bisphosphonates. Both Ewing's sarcoma-derived fibroblasts and some bone stromal cells expressed RANKL and supported osteoclast formation by a contact-dependent mechanism. We also found that osteoclast differentiation occurred when Ewing's TAMs were cultured with tumour necrosis factor (TNF)-alpha in the presence of M-CSF and that TC71 Ewing's sarcoma cells stimulated osteoclast formation through the release of a soluble factor, the action of which was abolished by an antibody to TNF-alpha. These results indicate that TAMs in Ewing's sarcoma are capable of osteoclast differentiation by both RANKL-dependent and TNF-alpha-dependent mechanisms and that Ewing's sarcoma cells produce osteoclastogenic factor(s). Our findings suggest that anti-resorptive and anti-osteoclastogenic therapies may be useful in inhibiting the osteolysis of Ewing's sarcoma.

Topics: Adolescent; Adult; Aged; Bone and Bones; Bone Neoplasms; Bone Resorption; Cell Differentiation; Female; Humans; Macrophages; Male; Middle Aged; Osteoclasts; Osteoprotegerin; RANK Ligand; Sarcoma, Ewing; Stromal Cells; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Zoledronic acid has been shown to be effective in the treatment of osteoporosis, hypercalcemia, and metastatic bone tumors. The efficacy of zoledronic acid on primary bone tumors has not been investigated.. A primary bone tumor mouse model was established. Intratibia injection of TC71 cells resulted in an osteolytic bone tumor. Four days after injection the mice were treated with zoledronic acid alone, paclitaxel alone, or zoledronic acid plus paclitaxel. Control mice were treated with phosphate-buffered saline. Bone tumor growth was assessed using a Faxitron Specimen Radiography System. The gene expression was detected by reverse-transcription polymerase chain reaction (RT-PCR), ELISA, and immunohistochemistry. Osteoclast formation was determined by tartrate-resistant acid phosphatase (TRAP) staining.. Zoledronic acid induced apoptosis in TC71 human Ewing sarcoma cells and inhibited cell proliferation. Five weeks after injection, 89% of mice in the control group developed osteolytic bone tumors. Paclitaxel had little effect on bone tumor growth, with 78% of mice developing tumors. By contrast, 44% of mice treated with zoledronic acid developed bone tumors. The most effective treatment was zoledronic acid plus paclitaxel. Tumor incidence in the combination therapy group was only 22%. Osteoclasts were quantified using TRAP staining. There was a decrease in TRAP-positive osteoclasts in tumor tissues from zoledronic acid-treated animals compared to control animals. RT-PCR, immunohistochemistry, and ELISA assay demonstrated that zoledronic acid up-regulated osteoprotegerin expression.. These results suggest that zoledronic acid induces apoptosis and inhibits primary bone tumor growth in Ewing sarcoma through a mechanism involving the up-regulation of osteoprotegerin. Zoledronic acid may provide a novel therapeutic approach for the treatment of patients with Ewing sarcoma.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Bone Density Conservation Agents; Bone Neoplasms; Cell Proliferation; Diphosphonates; Drug Therapy, Combination; Enzyme-Linked Immunosorbent Assay; Glycoproteins; Humans; Imidazoles; Immunoenzyme Techniques; Mice; Mice, Nude; Osteoclasts; Osteoprotegerin; Paclitaxel; Radiography; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; Sarcoma, Ewing; Zoledronic Acid