Abstract 408: Intermittent hypoxia effect on osteoclastogenesis stimulated by neuroblastoma cells
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Abstract Neuroblastoma is the most common extracranial pediatric solid tumor that is derived from the developing sympathetic nervous system and results from the improper differentiation of neural crest cells. Neuroblastomas show a tremendous clinical heterogeneity ranging from benign ganglioneuromas to highly aggressive immature tumors. Hypoxia is a common feature of solid tumors and is associated with their malignant phenotype. Intermittent hypoxia (IH) which is characterized by cyclic periods of hypoxia and reoxygenation, occurs in tumor cells that are dependent on tumor blood vessels having intermittent perfusion fluctuations in blood flow. The occurrence of IH episodes varies significantly in rapidly growing malignant tumors. Bone is one of the target organs of metastasis in advanced neuroblastoma. To invade the bone, tumor cells produce osteoclast-activating factors that increase bone resorption by the osteoclast. The present study focuses on how IH-conditioned neuroblastoma cells modulate the differentiation of osteoclasts. IH-conditioned cells were derived by exposing tumor cells to 10 repeated cycles of hypoxia followed by reoxygenation. In order to assess the role of HIF-1α overexpression in osteolysis, we stably transfected human neuroblastoma cells with an expression vector containing a HIF-1α cDNA. Additionally, cells were stably transfected with a plasmid expressing HIF-1α shRNA or luciferase shRNA and selected in neomycin. The expression of HIF-1α protein was analyzed by western blotting in IH-conditioned and stable transfectants of neuroblastoma cells. Changes in gene expression were determined by real-time PCR for IL-8, MCP-1α, PTHrP, CXCR-4 and RANKL and the expression of osteoclastogenic factors was found increased in IH-conditioned and HIF-1α protein overexpressing cells. Conditioned medium collected from IH-conditioned and HIF-1α cDNA overexpressing neuroblastoma cells has shown enhanced osteoclast formation capabilities in the tartrate-resistant acid phosphatase (TRAP) assay. IH-conditioned and HIF-1α protein overexpressing neuroblastoma cells secrete increased amounts of VEGF protein, which has been found to promote osteoclast activity. Calcium-sensing receptor (CaR) plays a pivotal role in osteoclast differentiation. We found enhanced expression of CaR in RAW 264.7 (osteoclast precursors) cells treated with conditioned medium collected from IH-conditioned and HIF-1α protein overexpressing neuroblastoma cells compared with the control. Thus, IH was found to enhance osteolytic capabilities of neuroblastoma cells in vitro in part through HIF-1α protein stabilization. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 408. doi:1538-7445.AM2012-4081958 Objectives The goal of this study was to validate 64Cu-CB-TE2A-c(RGDyK) (64Cu-RGD) as a biomarker for osteoclast number. A change in osteoclast number was monitored by biodistribution and microPET imaging with CT in mice treated with osteoprotegerin (OPG) and Receptor Activator for Nuclear Factor κ B Ligand (RANKL). OPG is a negative regulator of osteoclasts and should decrease osteoclast number. RANKL is a positive regulator of osteoclast differentiation and leads to an increase in osteoclast number. Methods C57BL/6 mice were treated with OPG at 0.2, 1.0 and 5.0 mg/kg twice a week for two weeks or with RANKL at 0.1 and 0.3 mg/kg twice a day for 10 days. On day 15 (OPG) or day 11 (RANKL) treated and control mice were injected with 64Cu-RGD followed by PET imaging and biodistribution. Standard uptake values (SUVs) were determined. Bones were collected for histology. Results Mice treated with 5 mg/kg of OPG or 0.3 mg/kg of RANKL showed a significant difference compared to control. The normal tissue biodistribution of the tracer did not vary greatly between the control and treated mice. PET images of animals treated with 0.3 mg/kg RANKL demonstrated significantly increased SUVs as compared to the control group. Histology and TRAP5b levels supported the biodistribution, PET, and TRAP5b data. Conclusions Analysis of the PET images showed that 64Cu-RGD bone uptake correlated with the osteoclast number and could be quantified as SUVs in leg bones. Taken together, these data support the use of 64Cu-RGD as the osteoclast turnover imaging biomarker.
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RANK Ligand
Imaging biomarker
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Osteoporosis is a debilitating disease caused by decreased bone density. Compounds with anti-osteoclastic activity, such as bisphosphonates, may help in the prevention and treatment of osteoporosis. Herein, we determined the inhibitory effects of ginger hexane extract (GHE) on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells. The results showed that GHE (1) suppressed osteoclast differentiation and the formation of actin rings; (2) inhibited the expression of Nfatc1, a master transcriptional factor for osteoclast differentiation, in a dose-dependent manner (10-20 μg/mL); and (3) inhibited other osteoclastogenesis-related genes, such as Oscar, Dc-stamp, Trap, and Mmp9. These findings suggest that GHE may be used to prevent and treat osteoporosis by inhibiting osteoclast differentiation.
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Although tumor necrosis factor alpha (TNF‑α) is known to serve a critical role in the pathogenesis of inflammatory osteolysis, the exact mechanisms underlying the effects of TNF‑α on osteoclast recruitment and differentiation remain unclear. To investigate the mechanisms by which TNF‑α influences osteoclast differentiation, mouse bone marrow‑derived macrophages (BMMs) were used as osteoclast precursors, and osteoclastogenesis was induced by macrophage colony‑stimulating factor and receptor activator of nuclear factor (NF)‑κB ligand (RANKL) with or without TNF‑α for 4 days. Then, NF‑κB was inhibited using the inhibitor, BAY 11‑7082. The results indicated that treatment with TNF‑α alone did not induce osteoclastogenesis of BMMs. However, TNF‑α in combination with RANKL dramatically stimulated the differentiation of osteoclasts and positively regulated the expression of mRNA markers of osteoclasts. Finally, treatment of BMMs with BAY 11‑7082 prevented the formation of mature osteoclasts by BMMs treated with TNF‑α only or with RANKL, as well as the upregulation of osteoclast marker genes. Therefore, although TNF‑α does not induce osteoclastogenesis alone, it does work with RANKL to induce osteoclastic differentiation, and the NF‑κB pathway may serve an important role in this process.
RANK Ligand
Osteolysis
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Osteoclast differentiation/activation is involved in orthodontic tooth movement at the compression sites of the alveolar bone. RANKL, a member of the TNF family expressed in osteoblasts, binds to RANK, a member of the TNF receptor family expressed on preosteoclasts, resulting in differentiation of preosteoclasts into mature osteoclasts. Several members of the TNF family, such as TNF and Fas ligand, can induce apoptosis by activation of caspase-3. We have investigated whether caspase-3 be involved in the late stage of RANKL-induced osteoclast differentiation. Increased active caspase-3 was found in mouse monocytic RAW264 cells differentiated into mature osteoclasts by treatment with RANKL for 3 days. Co-treatment with Z-Asp-CH₂-DCB, a caspase-3-specific inhibitor, augmented RANKL-induced osteoclast differentiation in RAW264 cells, also seen in mouse bone marrow macrophages. This suggests that activation of caspase-3 may play an inhibitory role at the late stage of RANKL-induced osteoclast differentiation.
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Cathepsin K
Tartrate-resistant acid phosphatase
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RANK Ligand
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Bone remodeling
Tartrate-resistant acid phosphatase
RANK Ligand
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Objectives: Receptor-activator of nuclear factor-κB ligand (RANKL) is an essential stimulating factor for inducing osteoclast differentiation, feasibly resulting in osteoporosis. The bioactive extract of Agrocybe chaxingu mushroom, CHX, has been shown to have osteoclastic inhibitory activity. Accordingly, we investigated if CHX would inhibit or disrupt RANKL-induced osteoclast differentiation, implying possible prevention of osteoporosis. Materials and Methods: Monocyte/macrophage RAW264.7 cells (RAW cells) were employed as our experimental model and treated with varying concentrations of RANKL alone or with CHX for 5 days. Formation of osteoclasts was then assessed using tartrateresistant acid phosphatase (TRAP) assay, counting stained cells as osteoclasts under a light microscope. The inhibitory mechanism of CHX was also explored by examining the RANKLmediated signaling pathways, oxidative stress (OXS), antioxidant enzymes, and apoptosis. Results: RANKL (100 ng/mL)-induced osteoclast differentiation in RAW cells was significantly (~20%) inhibited with 10 μg/mL of CHX. This was accompanied by the downregulation of two key signaling pathways and activation of antioxidant enzymes that likely led to the reduction in OXS. Moreover, CHX ultimately induced undifferentiated or RANKLunresponsive cells to apoptosis, indicated by the modulation of apoptotic regulators. These findings may then account for a disruption of osteoclast differentiation with CHX. Conclusion: CHX appears to have the inhibitory effect on RANKL-induced osteoclast differentiation in RAW cells. It is thus plausible that CHX may have potential clinical implications in osteoclast-mediated bone diseases/disorders, including osteoporosis.
Multinucleate
RANK Ligand
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