osteoprotegerin and Osteosarcoma

Receptor activator of NF-kappab ligand (RANKL), a key regulator of osteoclastogenesis, is proteolytically processed and converted to a soluble form. RANKL sheddase and the biologic and pathologic role of RANKL shedding have been undetermined, but the identity of sheddase and its effect on osteoclastogenesis are gradually clarified. The regulatory mechanism and its relevance to some pathologic conditions are to be elucidated.

Topics: Animals; Cell Line; Humans; Matrix Metalloproteinases, Membrane-Associated; NF-kappa B; Osteoblasts; Osteoclasts; Osteogenesis; Osteoprotegerin; Osteosarcoma; RANK Ligand

p53 plays a pivotal role in controlling the differentiation of mesenchymal stem cells (MSCs) by regulating genes involved in cell cycle and early steps of differentiation process. In the context of osteogenic differentiation of MSCs and bone homeostasis, the osteoprotegerin/receptor activator of NF-κB ligand/receptor activator of NF-κB (OPG/RANKL/RANK) axis is a critical signaling pathway. The absence or loss of function of p53 has been implicated in aberrant osteogenic differentiation of MSCs that results in higher bone formation versus erosion, leading to an unbalanced bone remodeling. Here, we show by microCT that mice with p53 deletion systemically or specifically in mesenchymal cells possess significantly higher bone density than their respective littermate controls. There is a negative correlation between p53 and OPG both in vivo by analysis of serum from p53

Topics: Animals; Bone Remodeling; Cell Differentiation; Cell Line, Tumor; Humans; Male; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Mice, Nude; NF-kappa B; Osteogenesis; Osteoprotegerin; Osteosarcoma; Prostatic Neoplasms; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction; Transcription Factor RelA; Tumor Suppressor Protein p53

Osteosarcoma (OS) is the most common primary malignant tumor of the bone. Due to its high heterogeneity and to survival signals from bone microenvironment, OS can resist to standard treatments, therefore novel therapies are needed. c-MET oncogene, a tyrosine-kinase receptor, plays a crucial role in OS initiation and progression. The present study aimed to evaluate the effect of c-MET inhibitor cabozantinib (CBZ) on OS both directly and through its action on bone microenvironment. We tested different doses of CBZ in in vitro models of OS alone or in co-culture with bone cells in order to reproduce OS-tumor microenvironment interactions. CBZ is able to decrease proliferation and migration of OS cells, inhibiting ERK and AKT signaling pathways. Furthermore, CBZ leads to the inhibition of the proliferation of OS cells expressing receptor activator of nuclear factor κB (RANK), due to its effect on bone microenvironment, where it causes an overproduction of osteoprotegerin and a decrease of production of RANK ligand by osteoblasts. Overall, our data demonstrate that CBZ might represent a new potential treatment against OS, affecting both OS cells and their microenvironment. In this scenario, RANK expression in OS cells could represent a predictive factor of better response to CBZ treatment.

Topics: Anilides; Bone and Bones; Bone Neoplasms; Cell Line, Tumor; Humans; MAP Kinase Signaling System; Mesenchymal Stem Cells; Osteoblasts; Osteoprotegerin; Osteosarcoma; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; Pyridines; RANK Ligand; Tumor Microenvironment

Biological treatments have been studied extensively and previous studies have proved that osteoprotegerin (OPG) can inhibit the development and progress of human osteosarcoma. However, the utility of biologic agents for cancer therapy has a short half-life, which can hardly deliver to and function in tumor sites efficiently. Mesenchymal stem cells (MSCs) have the potential to migrate to tumor sites. In this study, MSCs transfected with adenoviruses carrying the OPG gene (MSCs-OPG) were used via the tail vein to treat athymic nude mice (nu/nu) bearing osteosarcoma. In vivo and ex vivo images were used to validate the MSCs homing to tumors. The therapeutic effect for osteosarcoma was evaluated by observations on growth of tumors and bone destruction. The results showed that infected MSCs-OPG labeled with red fluorescent protein (RFP) can migrate to tumor sites and express OPG protein. The treatment by MSCs-OPG reduced the tumor growth and inhibited bone destruction in vivo. All these indicated that MSCs can deliver OPG to tumor sites, which could be a new direction of biological treatment for human osteosarcoma.

Topics: Animals; Bone Neoplasms; Gene Targeting; Gene Transfer Techniques; Humans; Male; Mesenchymal Stem Cell Transplantation; Mice; Mice, Nude; Microscopy, Fluorescence; Neoplasms, Experimental; Osteoprotegerin; Osteosarcoma; Tumor Cells, Cultured

Recent studies suggest that nanosecond pulsed electric field (nsPEF) is a novel minimal invasive and non-thermal ablation method that can induce apoptosis in different solid tumors. But the efficacy of nsPEF on bone-related tumors or bone metastasis is kept unknown. The current study investigates antitumor effect of nsPEF on osteosarcoma MG-63 cells in vitro.. MG-63 cells were treated with nsPEF with different electric field strengths (0, 10, 20, 30, 40, and 50 kV/cm) and different pulse numbers (0, 6, 12, 18, 24, and 30 pulses). The inhibitory effect of nsPEF on the growth of MG-63 cells was measured by Cell Counting Kit-8 (CCK-8) assay at different time points (0, 3, 12, 24, and 48 h post nsPEF treatment). The apoptosis was analyzed by Hoechst stain, in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL), and flow cytometric analysis. The expression of osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), and tumor necrosis factor α (TNF-α) was examined by reverse-transcription polymerase chain reaction (RT-PCR) and western blot.. The CCK-8 assay showed that nsPEF induced a distinct electric field strength- and pulse number-dependent reduction of cell proliferation. For treatment parameter optimizing, the condition 40 kV/cm and 30 pulses at 24 h post nsPEF achieved the most significant apoptotic induction rate. Hoechst, TUNEL, and flow cytometric analysis showed that the cell apoptosis was induced and cells were arrested in the G0/G1 phase. PCR and western blot analysis demonstrated that nsPEF up-regulated OPG expression had no effect on RANKL, increased OPG/RANKL ratio.. NsPEF inhibits osteosarcoma growth, induces apoptosis, and affects bone metabolism by up-regulating OPG, indicating nsPEF-induced apoptosis in osteosarcoma MG-63 cells. NsPEF has potential to treat osteosarcoma or bone metastasis. When nsPEF is applied on metastatic bone tumors, it might be beneficial by inducing osteoblastic differentiation without cancer proliferation. In the future, nsPEF might be one of the treatments of metastatic bone tumor.

Topics: Ablation Techniques; Apoptosis; Cell Line, Tumor; Cell Proliferation; Electromagnetic Fields; Flow Cytometry; Humans; Osteoprotegerin; Osteosarcoma; RANK Ligand; Tumor Necrosis Factor-alpha

Osteosarcoma (OS) is an aggressive and often fatal cancer that afflicts over 1000 humans and 10,000 dogs per year in the United States. Recent evidence suggests deregulation in the signaling triad, receptor activator of nuclear factor kappa B (RANK), its activating ligand (RANKL), and the RANKL inhibitor, osteoprotegerin (OPG) plays a key role in the pathogenesis of OS. This study investigated the expression of RANK and RANKL in osteosarcoma tumors and cell lines and describes an activating effect of OPG on OS cells in vitro.. Canine OS tumors and cell lines co-express mRNA for both RANK and RANKL. Expression of these proteins in OS cell lines was confirmed by Western blot and immunofluorescence microscopy. Expression of the soluble form of RANKL was not detected in media from OS cells. OPG-Fc incubation increased the phosphorylation status of ERK, AKT and the p65 subunit of nuclear factor kappa B (NFκB) and induced NFκB translocation from the cytoplasm to the nucleus in canine OS cells. OPG increased proliferation in both canine and human derived OS cell lines.. RANKL is produced by OS tumors and cell lines that also express RANK. This data provides preliminary evidence for a potential autocrine and or paracrine activation pathway in canine OS. An activating effect of exogenous OPG on signal transduction proteins, NFκB and proliferation in OS is described. These data provide new information concerning aberrant signaling in OS and could be important to those considering OPG as a therapeutic agent for osteosarcoma.

Topics: Animals; Base Sequence; Bone Neoplasms; Dog Diseases; Dogs; Female; Humans; Male; Neoplasm Invasiveness; Osteoprotegerin; Osteosarcoma; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Sequence Analysis, DNA; Sequence Homology, Nucleic Acid; Signal Transduction

Bone morphogenetic protein 9 (BMP9) is a member of the transforming growth factor-β (TGF-β) family, which has been shown to regulate the progression of several tumors. Recent studies indicated that BMP9 affects osteosarcoma (OS) processes, but its specific roles and molecular mechanisms have yet to be fully elucidated. The human OS cell lines 143B and MG63 were used for the present study. We found that BMP9 overexpression suppressed the growth of OS cells, whereas inhibition of BMP9 reversed this effect. Our results also showed that BMP9 overexpression induced G0/G1 phase arrest and apoptosis in OS cells. We further investigated the possible molecular mechanisms mediating the biological role of BMP9. We observed that BMP9 overexpression reduced β-catenin mRNA and protein levels, and also downregulated its downstream proteins c-Myc and osteoprotegerin (OPG) and inhibited the phosphorylation levels of GSK-3β (Ser 9) in OS cells, whereas inhibition of BMP9 reversed these effects. Moreover, the suppressive effects of BMP9 overexpression on OS cells was reversed by exogenous β-catenin expression, but augmented by β-catenin silencing. In conclusion, our results revealed that BMP9 can regulate tumor growth of OS cells through the Wnt/β-catenin pathway. Therefore, BMP9 may be a new therapeutic target in OS.

Topics: Actins; Antibodies; Apoptosis; beta Catenin; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Culture Media, Conditioned; Down-Regulation; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Growth Differentiation Factor 2; Growth Differentiation Factors; HEK293 Cells; Humans; Osteoprotegerin; Osteosarcoma; Phosphorylation; Proto-Oncogene Proteins c-myc; RNA Interference; RNA, Messenger; RNA, Small Interfering; Wnt Signaling Pathway

Osteosarcoma is the most common primary malignant tumor of bone and accounts for around 50% of all primary skeletal malignancies. In addition to novel chemotherapies, there is a need for adjuvant therapies designed to inhibit osteosarcoma proliferation and tumor-induced osteolysis to attenuate tumor expansion and metastasis. As such, studies on the efficacy of bisphosphonates on human osteosarcoma are planned after feasibility studies determined that the bisphosphonate zoledronic acid (ZOL) can be safely combined with conventional chemotherapy. However, the molecular mechanisms responsible for, and means of inhibiting, osteosarcoma-induced osteolysis are largely unknown. We establish that osteosarcoma growth directly correlates with tumor-induced osteolysis and activation of osteoclasts in vivo. In vitro, tumor cells were determined to expresses surface, but not soluble, receptor activator of NF-κB ligand (RANKL) and stimulated osteoclastogenesis in a manner directly proportional to their malignant potential. In addition, an aggressive osteosarcoma cell line was shown to secrete monocyte chemoattractant protein-1 (MCP-1), resulting in robust monocyte migration. Because MCP-1 is a key cytokine for monocyte recruitment and surface-bound RANKL strongly supports local osteoclastogenesis, we suggest that high levels of these signaling molecules are associated with the aggressive potential of osteosarcoma. Consistent with these findings, abundant expression of RANKL/MCP-1 was observed in tumor in vivo, and MCP-1 plasma levels strongly correlated with tumor progression and osteolysis. ZOL administration directly attenuates osteosarcoma production of RANKL/MCP-1, reducing tumor-induced bone destruction. In vivo, these findings also correlated with significant reduction in osteosarcoma growth. ZOL attenuates tumor-induced osteolysis, not only through direct inhibition of osteoclasts, but also through direct actions on tumor expression of osteoclast activators. These data provide insight regarding the effect of ZOL on osteosarcoma essential for designing the planned upcoming prospective randomized trials to determine the efficacy of bisphosphonates on osteosarcoma in humans.

Topics: Animals; Bone Resorption; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Chemokine CCL2; Diphosphonates; Humans; Imidazoles; Mice; Monocytes; Neoplasm Invasiveness; Osteoclasts; Osteogenesis; Osteolysis; Osteoprotegerin; Osteosarcoma; RANK Ligand; Zoledronic Acid

Serum levels of sRANKL, RANK, OPG, IL-8, IL-6, IL-16, MMP-2, and calcitonin were measured by ELISA in patients with malignant, borderline, and benign bone tumors and in healthy individuals (control). Serum levels of RANK, OPG, IL-8, IL-6, and the OPG/sRANKL ratio were significantly higher, while the level of MMP-2 was significantly lower in patients with bone tumors than in controls. Serum concentration of IL-16 in osteosarcoma patients was significantly lower than in chondrosarcoma patients. No significant differences between bone sarcomas of different differentiation were detected for any of the studied markers. Calcitonin level depended on the tumor location and type.

Topics: Adolescent; Adult; Aged; Bone Neoplasms; Calcitonin; Case-Control Studies; Chondrosarcoma; Chordoma; Female; Gene Expression; Humans; Interleukin-16; Interleukin-6; Interleukin-8; Male; Matrix Metalloproteinase 2; Middle Aged; Neoplasms; Osteoprotegerin; Osteosarcoma; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B

The P2X7 receptor (P2X7R) is attracting increasing attention for its involvement in cancer. Several recent studies have shown a crucial role of P2X7R in tumour cell growth, angiogenesis and invasiveness. In this study, we investigated the role of the two known human P2X7R functional splice variants, the full length P2X7RA and the truncated P2X7RB, in osteosarcoma cell growth. Immunohistochemical analysis of a tissue array of human osteosarcomas showed that forty-four, of a total fifty-four tumours (81.4%), stained positive for both P2X7RA and B, thirty-one (57.4%) were positive using an anti-P2X7RA antibody, whereas fifteen of the total number (27.7%) expressed only P2X7RB. P2X7RB positive tumours showed increased cell density, at the expense of extracellular matrix. The human osteosarcoma cell line Te85, which lacks endogenous P2X7R expression, was stably transfected with either P2X7RA, P2X7RB, or both. Receptor expression was a powerful stimulus for cell growth, the most efficient growth-promoting isoform being P2X7RB alone. Growth stimulation was matched by increased Ca(2+) mobilization and enhanced NFATc1 activity. Te85 P2X7RA+B cells presented pore formation as well as spontaneous extracellular ATP release. The ATP release was sustained in all clones by P2X7R agonist (BzATP) and reduced following P2X7R antagonist (A740003) application. BzATP also increased cell growth and activated NFATc1 levels. On the other hand cyclosporin A (CSA) affected both NFATc1 activation and cell growth, definitively linking P2X7R stimulation to NFATc1 and cell proliferation. All transfected clones also showed reduced RANK-L expression, and an overall decreased RANK-L/OPG ratio. Mineralization was increased in Te85 P2X7RA+B cells while it was significantly diminished in Te85 P2X7RB clones, in agreement with immunohistochemical results. In summary, our data show that the majority of human osteosarcomas express P2X7RA and B and suggest that expression of either isoform is differently coupled to cell growth or activity.

Topics: Adenosine Triphosphate; Adolescent; Adult; Bone Neoplasms; Calcification, Physiologic; Cell Line, Tumor; Child; Child, Preschool; Extracellular Space; Female; Gene Expression; Humans; Immunohistochemistry; Male; Middle Aged; NFATC Transcription Factors; Osteoprotegerin; Osteosarcoma; Protein Isoforms; RANK Ligand; Receptors, Purinergic P2X7; Transfection; Young Adult

A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the human receptor activator of nuclear factor κB (RANKL)-osteoprotegerin (OPG) complex was developed by utilizing a monoclonal antibody that recognizes human soluble RANKL as an immobilized capture component and biotinylated human OPG polyclonal antibody. We could quantify the RANKL-OPG complex level (detection limit: 100 pg/mL). Employing this assay system, we demonstrated that the RANKL-OPG complex was constitutively present in the conditioned medium of human osteoblastic sarcoma U2OS, although the complex was not detectable in other human osteoblastic sarcoma cell line, MG-63, HOS, and SaOS-2.

Topics: Cell Line, Tumor; Culture Media, Conditioned; Enzyme-Linked Immunosorbent Assay; Humans; Osteoprotegerin; Osteosarcoma; RANK Ligand

Osteosarcoma is a bone tumor that frequently develops during adolescence. 2-Methoxyestradiol (2-ME), a naturally occurring metabolite of 17beta-estradiol, induces cell cycle arrest and cell death in human osteosarcoma cells. To investigate whether the osteoprotegrin (OPG) protein plays a role in 2-ME actions, we studied the effect of 2-ME treatment on OPG gene expression in human osteosarcoma cells. 2-ME treatment induced OPG gene promoter activity and mRNA levels. Also, Western blot analysis showed that 2-ME treatment increased OPG protein levels in MG63, KHOS, 143B and LM7 osteosarcoma cells by 3-, 1.9-, 2.8-, and 2.5-fold, respectively, but did not affect OPG expression in normal bone cells. In addition, increases in OPG protein levels were observed in osteosarcoma cell culture media after 3 days of 2-ME treatment. The effect of 2-ME on osteosarcoma cells was ligand-specific as parent estrogen, 17beta-estradiol and a tumorigenic estrogen metabolite, 16alpha-hydroxyestradiol, which do not affect osteosarcoma cell cycle and cell death, had no effect on OPG protein expression. Furthermore, co-treating osteosarcoma cells with OPG protein did not further enhance 2-ME-mediated anti-tumor effects. OPG-released in 2-ME-treated cultures led to an increase in osteoblastic activity and a decrease in osteoclast number, respectively. These findings suggest that OPG is not directly involved in 2-ME-mediated anti-proliferative effects in osteosarcoma cells, but rather participates in anti-resorptive functions of 2-ME in bone tumor environment.

Topics: 2-Methoxyestradiol; Alkaline Phosphatase; Animals; Cell Death; Cell Proliferation; Drug Screening Assays, Antitumor; Estradiol; Gene Expression Regulation, Neoplastic; Humans; Osteoblasts; Osteoprotegerin; Osteosarcoma; Peptides; Rats; RNA, Messenger; Transcription, Genetic

The aim of this study was to investigate the expression of bone resorption (RANK/RANKL), bone resorption inhibitor (osteoprotererin [OPG]), and bone formation marker (osteocalcin [OC]) in neoplastic and bone-related lesions (BRL).. Using immunohistochemistry, their expression was evaluated in ossifying fibroma (OF), fibrous dysplasia (FD), simple bone cysts (SBC), central giant cell lesions (CGCL), and osteosarcoma (OS).. Quantitative analyses of the expression of bone markers between all lesions, considering fibroblast-like cells and bone matrix, showed that RANK-RANKL presented higher expression in OF and CGCL, whereas OPG and OC presented higher expression in FD and SBC. There was higher expression of all proteins investigated when OS was the BRL. Moreover, the RANKL expression was greater than OPG in this neoplasm.. Our data indicate that the bone resorption markers are more highly expressed in OF, CGCL, and OS than in FD and SBC, indicating a significant association between these proteins and the clinical behavior of these lesions.

Topics: Adolescent; Adult; Biomarkers; Biomarkers, Tumor; Bone Matrix; Bone Remodeling; Bone Resorption; Child; Female; Fibroblasts; Fibroma, Ossifying; Fibrous Dysplasia of Bone; Giant Cells; Granuloma, Giant Cell; Humans; Jaw Cysts; Jaw Diseases; Jaw Neoplasms; Male; Middle Aged; Osteoblasts; Osteocalcin; Osteoclasts; Osteogenesis; Osteoprotegerin; Osteosarcoma; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Stromal Cells; Young Adult

Osteosarcoma is the most frequent primary bone malignant tumor that develops mainly in children and adolescents. Despite recent improvements in chemotherapy and surgery, survival rate is approximately 50% after 5 years. Osteoprotegerin (OPG) is a potent inhibitor of osteoclast differentiation and activation, but its use as therapeutic agent in cancer-associated osteolysis remains controversial due to its ability to bind and inhibit the apoptotic effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on tumor cells. The therapeutic effects of full-length OPG (1-401) and OPG 1-194 lacking its heparin-binding domain delivered by nonviral gene therapy were compared in a murine model of osteolytic osteosarcoma. Tumor incidence, progression, and associated bone lesions were significantly diminished in the OPG 1-194 group, but not in the OPG 1-401 group, compared with controls. As receptor activator of nuclear factor-kappaB ligand (RANKL), TRAIL, and glycosaminoglycans (GAG) were shown to be overexpressed in osteosarcoma environment compared with control tissue, OPG 1-401 bioactivity may be modulated by one of these protagonists. Surface plasmon resonance analyses performed with OPG, TRAIL, and GAGs revealed that TRAIL binds both forms of OPG with the same affinity. In addition, as OPG 1-194 and OPG 1-401 similarly inhibit TRAIL-induced apoptosis, it suggests that TRAIL is not involved in the modulation of OPG bioactivity. However, as GAGs inhibit OPG 1-401 but not OPG 1-194 binding to TRAIL or to RANKL, they may represent potent regulators of OPG availability and antitumor activity in bone tumor microenvironment.

Topics: Animals; Bone Neoplasms; Bone Resorption; Cell Line, Tumor; DNA; Genetic Therapy; Glycosaminoglycans; Humans; Male; Mice; Mice, Inbred C3H; Osteoprotegerin; Osteosarcoma; Peptide Fragments; RANK Ligand; RNA, Messenger; Surface Plasmon Resonance; TNF-Related Apoptosis-Inducing Ligand; Transgenes

Osteosarcoma is the most prevalent bone tumor in children and adolescents. At present, the mechanisms of initiation, maintenance, and metastasis are poorly understood. The purpose of this study was to identify relevant molecular targets in the pathogenesis of osteosarcoma.. Tumor chemonaive osteoblastic populations and paired control normal osteoblasts were isolated and characterized phenotypically from seven osteosarcoma patients. Global transcriptomic profiling was analyzed by robust microarray analysis. Candidate genes were confirmed by real-time PCR and organized in molecular pathways. EBF2 and osteoprotegerin (OPG) levels were determined by real-time PCR and OPG protein levels were assessed by ELISA. Immunohistochemical analysis was done in a panel of 46 osteosarcoma samples. Silencing of EBF2 was achieved by lentiviral transduction of short hairpin RNA. Apoptosis was determined by caspase-3/7 activity.. A robust clustered transcriptomic signature was obtained in osteosarcoma. Transcription factor EBF2, a known functional bone regulator, was among the most significantly overexpressed genes. Immunohistochemical analysis showed that osteosarcoma is expressed in approximately 70% of tumors analyzed. Because EBF2 was shown previously to act as a transcriptional activator of OPG, elevated levels of EBF2 were associated with high OPG protein levels in osteosarcoma samples compared with normal osteoblastic cells. Knockdown of EBF2 led to stunted abrogation of OPG levels and increased sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis.. These findings suggest that EBF2 represents a novel marker of osteosarcoma. EBF2 up-regulation may be one of the mechanisms involved in the high levels of OPG in osteosarcoma, contributing to decrease TRAIL-induced apoptosis and leading to TRAIL resistance.

Topics: Adolescent; Adult; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Bone Neoplasms; Cells, Cultured; Child; Down-Regulation; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Male; Matched-Pair Analysis; Osteoprotegerin; Osteosarcoma; Survival Analysis; TNF-Related Apoptosis-Inducing Ligand; Young Adult

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

Osteoprotegerin (OPG) expression participates in the pathophysiology of osteoblastic metastasis in prostate cancer.. We investigated whether the expression of OPG of PC-3 prostate cancer cells grown in 3-D collagen gels is stimulated by co-culture with MG-63 osteoblast-like cells. The expression of Runx2 (Cbfa1) and OPG were assessed by reverse transcription-polymerase chain reaction and Western blot analysis.. OPG and Runx2 were expressed in both PC-3 and MG-63 cells. OPG expression was remarkably enhanced in PC-3 cells grown in co-culture with MG-63 cells in a time-dependent manner. Runx2 expression of PC-3 cells was not altered by their co-culture with MG-63 cells. OPG expression of PC-3 cells was altered neither by insulin-like growth factor I (IGF-1), transforming growth factor beta1 (TGFbeta1), interleukin 6 (IL-6) nor by dexamethasone and zoledronic acid exogenously added to PC-3 cells.. The enhancement of the OPG expression in PC-3 cells by MG-63 cells is not mediated by IGF-1, IL-6 and TGFbeta1.

Topics: Bone Neoplasms; Cell Communication; Coculture Techniques; Core Binding Factor Alpha 1 Subunit; Humans; Insulin-Like Growth Factor I; Interleukin-6; Male; Osteoblasts; Osteoprotegerin; Osteosarcoma; Prostatic Neoplasms; Transforming Growth Factor beta1

The aim of this study was to investigate the influence of different implant surface topographies and chemistries on the expression of differentiation/proliferation markers on MG63 cells and primary human alveolar osteoblasts.. Hydrophobic acid-etched (A) and hydrophobic coarse-grit-blasted, acid-etched (SLA) surfaces and hydrophilic acid-etched (modA) and hydrophilic coarse-grit-blasted (modSLA) surfaces were produced. Thereby, modA and modSLA surfaces were rinsed under nitrogen protection and stored in a sealed glass tube containing isotonic NaCl solution at pH 4-6. Tissue culture plates without specimens served as controls. The behavior of MG63 cells and primary human alveolar osteoblasts (AOB) grown on all surfaces was compared through determination of alkaline phosphatase (ALP) activity, cell proliferation ((3)H-thymidin incorporation, MTT colorimetric assay) and expression of osteocalcin (OC), osteoprotegerin (OPG), transforming growth factor-beta1 (TGF-beta(1)) and vascular endothelial growth factor (VEGF), detected with commercial available test kits.. Proliferation of MG63 and primary cells was highest on controls, followed by A surfaces, modA and SLA surfaces being almost on the same level and lowest on modSLA surfaces. modSLA surfaces exhibited highest ALP and OC production, followed by SLA, modA and A surfaces. Proliferation and OC production were comparable for MG63 cells and AOB. OPG, TGF-beta(1) and VEGF produced on primary cells showed a slightly different rank order on different surfaces compared to MG63 cells. modSLA still showed the highest production of OPG, TGF-beta(1) and VEGF, but was followed by modA, SLA and A. Statistical significance was checked by ANOVA (p<0.0035).. MG63 cells and primary human alveolar osteoblasts showed similar proliferation and differentiation characteristics on different titanium surfaces. Only modA surfaces showed enhanced expression of OPG, TGF-beta(1) and VEGF on MG63 cells compared to primary human alveolar osteoblasts. Overall, the lowest proliferation rates and the highest expressions of differentiation markers and growth factor productions were observed on modSLA.

Topics: Acid Etching, Dental; Alkaline Phosphatase; Alveolar Process; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytokines; Humans; Osteocalcin; Osteocytes; Osteoprotegerin; Osteosarcoma; Surface Properties; Titanium; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

K7M2 mouse osteosarcoma cells form lytic tumors and are deficient in osterix (Osx), a zinc finger-containing transcription factor required for osteoblast differentiation and bone formation. Our previous studies showed that replacement of Osx suppresses lytic bone destruction. Cytokines, including interleukin (IL)-1alpha, IL-6, IL-11, and prostaglandin E2, have been shown to stimulate osteoclast activity. We showed that IL-1alpha production by K7M2 cells was significantly suppressed following Osx transfection through a transcription-mediated mechanism. Osx had no effect on IL-6, IL-11, or prostaglandin E2. Site-directed mutagenesis and chromatin immunoprecipitation indicated that Osx down-regulated IL-1alpha through an Sp1-binding site on the IL-1alpha promoter. Inhibiting Osx by small interfering RNA in two cell lines (Dunn and DLM8) that expressed high levels of Osx led to enhanced IL-1alpha promoter activity and protein production and altered the phenotype from blastic to lytic. These data suggest that Osx down-regulates IL-1alpha expression in mouse osteosarcoma cells via transcriptional repression of IL-1alpha and this may in turn affect the lytic activity of the tumor cells.

Topics: Animals; Binding Sites; Bone Neoplasms; Cell Line, Tumor; Down-Regulation; Gene Expression Regulation, Neoplastic; Interleukin-1alpha; Male; Mice; Mice, Inbred BALB C; Mutation; Osteoprotegerin; Osteosarcoma; Phenotype; Promoter Regions, Genetic; RNA, Small Interfering; Sp1 Transcription Factor; Sp7 Transcription Factor; Transcription Factors; Transcription, Genetic

RANK, RANK ligand (RANKL) and osteoprotegerin (OPG) are the key regulators of bone metabolism, both in normal and pathological conditions. Previous data have demonstrated that human osteosarcoma biopsies express RANKL as well as OPG, and functional RANK is expressed in a murine osteosarcoma cell line. As RANK expression in human osteosarcoma remains controversial, the aim of the present study was to analyse its expression in vitro in human osteosarcoma cell lines, ex vivo using pathological tissues, and then to determine its functionality in terms of signal transduction pathways modulated by RANKL. RT-PCR analysis and immunohistochemistry experiments revealed that RANK is expressed at both transcriptional and protein levels in MNNG/HOS, Saos-2 and MG-63 human osteosarcoma cell lines, in contrast to the U-2 OS osteosarcoma cell line and human osteoblasts, which were negative. RANK was also expressed in 57% of osteosarcoma biopsies. Furthermore, western blot experiments clearly demonstrated the functionality of RANK. Thus, RANKL significantly induced the phosphorylation of ERK1/2, p38 and IkappaB in RANK-positive osteosarcoma cells. This study is the first report of functional RANK expression in human osteosarcoma cells: this strengthens the involvement of the RANK-RANKL-OPG axis in primary bone tumour biology and identifies novel therapeutic approaches targeting RANK-positive osteosarcoma.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Bone Neoplasms; Cell Line, Tumor; Child; Cross-Sectional Studies; Female; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Proteins; Osteoblasts; Osteoprotegerin; Osteosarcoma; Phosphorylation; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Signal Transduction

The beneficial effects of melatonin on bone homeostasis have been shown in various diseases. As this indoleamine causes dose-dependent modulation of bone-forming osteoblast and bone-resorbing osteoclast activities by receptor-independent and -dependent pathways, we investigated the expression of G-protein-coupled melatonin receptors (MTs) in malignant and non-malignant human bone lesions. By TaqMan polymerase chain reaction (PCR), we analyzed 30 specimens from osteosarcoma and 11 from benign bone tumors for MT1-mRNA expression. Furthermore, we determined mRNA expression levels of the osteoclast activity-stimulating receptor activator of nuclear factor-kappa B ligand (RANKL) and its counterpart osteoprotegerin (OPG). Although mean MT1-mRNA levels were similar (P = 0.596) in malignant (4.39 +/- 4.98-fold) and benign samples (4.64 +/- 6.81-fold), the highest MT1-mRNA levels (up to 27-fold) were observed in individual osteosarcomas, particularly, in two specimens of patients with local recurrence of the tumor. Moreover, mean RANKL- and OPG-mRNA levels were similar in malignant and benign specimens (RANKL: 7.38 +/- 9.61-fold versus 3.57 +/- 3.11-fold, P = 0.207; OPG: 23.45 +/- 32.76 versus 8.07 +/- 7.23-fold, P = 0.133). Again, highest RANKL- and OPG-mRNA levels (up to 41- and 160-fold, respectively) were observed in individual osteosarcomas. Expression of MT1-mRNA was confirmed in two human osteosarcoma cell lines (HOS, MG63). High expression levels of MT1-mRNA together with low OPG-mRNA were found in both osteosarcoma cell lines, while in normal human osteoblasts and bone marrow stromal cells, high OPG-mRNA levels were associated with low MT1-mRNA levels. These data on the abundant expression of MT1-mRNA in human bone tumors and osteosarcoma cells lines suggest an important role for MT1 in bone pathology.

Topics: Adolescent; Adult; Bone Neoplasms; Cells, Cultured; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Osteoblasts; Osteoprotegerin; Osteosarcoma; RANK Ligand; Receptor, Melatonin, MT1; RNA, Messenger

Osteosarcoma is the most frequent primary bone tumor that develops mainly in the young, the median age of diagnosis being 18 years. Despite improvement in osteosarcoma treatment, survival rate is only 30% at 5 years for patients with pulmonary metastases at diagnosis. This warrants exploration of new therapeutic options, and among them, osteoprotegerin (OPG), a naturally occurring protein that inhibits bone resorption, is very promising in blocking the vicious cycle between bone resorption and tumor proliferation that takes place during tumor development in bone site. As OPG binds and inhibits the activity of tumor necrosis factor-related apoptosis-inducing ligand, the truncated form of murine OPG 1-194 was used. The cDNA encoding OPG was administered by gene transfer using replication-defective adenoviral vector or was associated with an amphiphilic polymer in two models of rodent osteosarcoma. In both models, OPG gene transfer was effective in preventing the formation of osteolytic lesions associated with osteosarcoma development, in reducing the tumor incidence and the local tumor growth, leading to a 4-fold augmentation of mice survival 28 days postimplantation. On the contrary, OPG did not prevent the development of pulmonary metastasis alone, suggesting that bone environment is necessary for OPG therapeutic efficacy. Because OPG has no direct activity on osteosarcoma cells in vitro (cell binding, cell proliferation, apoptosis, or cell cycle distribution), we show that OPG exerts indirect inhibitory effect on tumor progression through the inhibition of RANKL whose production is enhanced in bone tumor environment, leading to osteolysis inhibition as reflected by osteoclast number decrease.

Topics: Adenoviridae; Animals; Apoptosis; Bone Density; Bone Neoplasms; Bone Resorption; Caspases; Cell Cycle; Cell Proliferation; Enzyme-Linked Immunosorbent Assay; Genetic Therapy; Genetic Vectors; Male; Mice; Mice, Inbred C3H; Myoblasts; Osteolysis; Osteoprotegerin; Osteosarcoma; RANK Ligand; Rats; Transduction, Genetic; Xenograft Model Antitumor Assays

Paget's disease is a focal disorder of bone remodelling, in which there is an increase in osteoclast formation. A rare complication of Paget's disease is the development of a sarcoma, most commonly an osteosarcoma. Osteoclast formation occurs in the presence of macrophage-colony stimulating factor and receptor activator for nuclear factor-kappaB ligand (RANKL), and it has been shown that bone stromal cells in Paget's disease can influence osteoclast formation by modulating the expression of RANKL and its decoy receptor, osteoprotegerin (OPG). In this study we show that pagetic bone stromal cells express RANKL and that these cells promote osteoclast formation by a RANKL-dependent mechanism. Osteoclast formation in co-cultures of monocytes and either pagetic bone stromal cells or Paget's sarcoma stromal cells was not only induced by a contact-dependent mechanism but also occurred via the release of a soluble factor. In contrast to bone stromal cells isolated from normal controls, stromal cells isolated from morphologically normal bone in one patient with Paget's disease also stimulated osteoclast formation in this way; this osteoclastogenesis was inhibited by OPG. Our results indicate that Paget's bone stromal cells support osteoclast formation by a RANKL-dependent process which involves not only cell-cell contact but also secretion of soluble RANKL.

Topics: Aged; Aged, 80 and over; Biological Factors; Bone Neoplasms; Carrier Proteins; Cell Communication; Cells, Cultured; Coculture Techniques; Gene Expression; Glycoproteins; Humans; Male; Membrane Glycoproteins; Osteitis Deformans; Osteoclasts; Osteoprotegerin; Osteosarcoma; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Stromal Cells

The high prevalence of osteoblastic bone metastases in prostate cancer involves the production of osteoblast-stimulating factors by prostate cancer cells. Prostate-specific antigen (PSA) is a serine protease uniquely produced by prostate cancer cells and is an important serologic marker for prostate cancer. In this study, we examined the role of PSA in the induction of osteoblast differentiation.. Human cDNA containing a coding region for PSA was transfected into human osteosarcoma SaOS-2 cells. SaOS-2 cells were also treated with exogenously added PSA. We evaluated changes in global gene expression using cDNA arrays and Northern blot analysis resulting from expression of PSA in human osteosarcoma SaOS-2 cells.. SaOS-2 cells expressing PSA had markedly up-regulated expression of genes associated with osteoblast differentiation including runx-2 and osteocalcin compared with the controls. Consistent with these results, the stable clones expressing PSA showed increased mineralization and increased activity of alkaline phosphatase in vitro compared with controls, suggesting that these cells undergo osteoblast differentiation. We also found that osteoprotegerin expression was down-regulated and that the receptor activator of NF-kappaB ligand expression was up-regulated in cells expressing PSA compared with controls.. Modulation of the expression of osteogenic genes and alteration of the balance between osteoprotegerin-receptor activator of NF-kappaB ligand by PSA suggests that PSA produced by metastatic prostate cancer cells may participate in bone remodeling in favor of the development of osteoblastic metastases in the heterogeneous mixture of osteolytic and osteoblastic lesions. These findings provide a molecular basis for understanding the high prevalence of osteoblastic bone metastases in prostate cancer.

Topics: Alkaline Phosphatase; Biomarkers, Tumor; Blotting, Northern; Bone Neoplasms; Bone Remodeling; Calcification, Physiologic; Carrier Proteins; Cell Differentiation; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Membrane Glycoproteins; Oligonucleotide Array Sequence Analysis; Osteoblasts; Osteoprotegerin; Osteosarcoma; Prostate-Specific Antigen; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tumor Cells, Cultured

Platelet-rich plasma (PRP) is widely used to promote tissue healing. However, there is no concrete evidence for the biological effects of PRP. This study evaluated the biological effects of PRP on the proliferation and differentiation of 2 human osteoblast-like cell lines.. Human osteosarcoma cell lines HOS and SaOS-2 were used in this study. PRP was prepared from freshly drawn human venous blood containing a large number of platelets. The MTT assay was used to examine the effects of PRP on osteoblast viability. To evaluate the growth and differentiation, alkaline phosphatase activity was assessed and the expression of procollagen type I, osteopontin, and osteoprotegerin mRNA was measured using semiquantitative reverse transcriptase-polymerase chain reaction. Further, core binding factor alpha 1 (cbfa1/Runx2/AML3/PebpalphaA), a critical regulator of osteoblast differentiation, was also determined.. The administration of PRP enhanced the viability of HOS and SaOS-2 cells in a dose-dependent manner. Alkaline phosphatase activity was suppressed during the cell growth phase, but was strongly enhanced when the cells reached confluence. Semiquantitative reverse-transcription polymerase chain reaction analysis showed that PRP enhanced the levels of procollagen type I, osteopontin, osteoprotegerin, and core binding factor alpha 1 (cbfa1) mRNA.. These results suggest that PRP has a favorable effect on human osteoblast-like cells, and acts both to enhance bone regeneration and as an activator in wound healing.

Topics: Alkaline Phosphatase; Blood Platelets; Bone Regeneration; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Collagen Type I; Coloring Agents; Core Binding Factor alpha Subunits; DNA-Binding Proteins; Glycoproteins; Humans; Osteoblasts; Osteopontin; Osteoprotegerin; Osteosarcoma; Phosphoproteins; Plasma; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Sialoglycoproteins; Tetrazolium Salts; Thiazoles; Transcription Factors; Wound Healing

All-trans-retinoic acid (ATRA) induces bone resorption, but the molecular mechanisms are unknown. We have studied the effect of ATRA on osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) expression in human MG-63 osteosarcoma cells and primary osteoblast-like cultures. ATRA dose-dependently down-regulated protein levels of OPG in MG-63 cells, with a maximum (-56%) observed at a dose of 10(-6)M. This effect was confirmed with quantitative real-time PCR, where OPG mRNA was decreased after 4h (-68%) in primary cultures and after 8h (-87%) in MG-63 cells. The reduction in OPG expression was inhibited by a retinoic acid receptor (RAR)-antagonist and was mimicked by a RARbeta,gamma-agonist, indicating that the ATRA effect is mediated by these receptors. In primary cultures we found a threefold induction of RANKL mRNA expression. Thus, the RANKL/OPG ratio was markedly increased, suggesting a potential mechanism of ATRA-induced bone resorption.

Topics: Carrier Proteins; Cell Line, Tumor; Cells, Cultured; Dose-Response Relationship, Drug; Gene Expression Regulation; Glycoproteins; Humans; Membrane Glycoproteins; NF-kappa B; Osteoblasts; Osteoprotegerin; Osteosarcoma; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Receptors, Tumor Necrosis Factor; Tretinoin

To determine the effect of nylestriol and levonorgestrel on mRNA expression of OPG/OPGL in MG-63 cell lines, and to explore whether the paracrine effect is involved in the regulation course. Methods Semi-quantitive RT-PCR was conducted to analyze mRNA expression of OPG/OPGL in MG-63 cell lines treated with nylestriol and levonorgestrel. Results Both medicines could stimulate MG-63 cell lines to express OPG, and the best action levels of the two medicines were 10(-6) mol/L. Both medicines could decrease mRNA expression levels of OPGL dramatically. In NYL 10(-10) mol/L treatment group, medium renewal has negative effect on mRNA expression of OPG. Conclusion Both the two medicines can promote mRNA expression of OPG in MG-63 cell lines, while decrease mRNA expression of OPGL. The paracrine effect of MG-63 cell lines may be involved in the regulation of mRNA expression of OPG by NYL.

Topics: Carrier Proteins; Female; Glycoproteins; Humans; Levonorgestrel; Membrane Glycoproteins; Osteoprotegerin; Osteosarcoma; Quinestrol; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; RNA, Messenger; Tumor Cells, Cultured

Previous analysis of tumor-specific constitutional LOH had identified a putative tumor-suppressor gene (LOH18CR) active in osteosarcoma tumorigenesis, which mapped to a subregion of chromosome 18q linked to both familial Paget's disease and FEO. Using 9 new polymorphic loci within the previous minimal region of LOH, we have reduced the minimal region of LOH in osteosarcoma tumors to localize the LOH18CR locus to the distal end of chromosome 18q21.33. This new region is approximately 500 kb and contains at least 7 known genes; however, it excludes 2 previous candidate genes: TNFRSF11A (RANK) and BCL2.

Topics: Bone Neoplasms; Chromosome Mapping; Chromosomes, Human, Pair 18; DNA; DNA Primers; Genes, Tumor Suppressor; Genetic Markers; Glycoproteins; Homozygote; Humans; Loss of Heterozygosity; Microsatellite Repeats; Osteoprotegerin; Osteosarcoma; Polymerase Chain Reaction; Proto-Oncogene Proteins c-bcl-2; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Risk Factors

Vitamin K2 is a critical nutrient required for blood clotting that also plays an important role in bone formation. Vitamin K2 supplementation up-regulates the expression of bone markers, increases bone density in vivo, and is used clinically in the management of osteoporosis. The mechanism of vitamin K2 action in bone formation was thought to involve its normal role as an essential cofactor for gamma-carboxylation of bone matrix proteins. However, there is evidence that suggests vitamin K2 also has a transcriptional regulatory function. Vitamin K2 bound to and activated the orphan nuclear receptor SXR and induced expression of the SXR target gene, CYP3A4, identifying it as a bona fide SXR ligand. Vitamin K2 treatment of osteosarcoma cells increased mRNA levels for the osteoblast markers bone alkaline phosphatase, osteoprotegerin, osteopontin, and matrix Gla protein. The known SXR activators rifampicin and hyperforin induced this panel of bone markers to an extent similar to vitamin K2. Vitamin K2 was able to induce bone markers in primary osteocytes isolated from wild-type murine calvaria but not in cells isolated from mice deficient in the SXR ortholog PXR. We infer that vitamin K2 is a transcriptional regulator of bone-specific genes that acts through SXR to favor the expression of osteoblastic markers. Thus, SXR has a novel role as a mediator of bone homeostasis in addition to its role as a xenobiotic sensor. An important implication of this work is that a subset of SXR activators may function as effective therapeutic agents for the management of osteoporosis.

Topics: Alkaline Phosphatase; Animals; Biomarkers; Bone and Bones; Bone Density; Bridged Bicyclo Compounds; COS Cells; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Gene Expression; Glycoproteins; HeLa Cells; Homeostasis; Humans; Mice; Mice, Knockout; Osteoblasts; Osteocalcin; Osteopontin; Osteoprotegerin; Osteosarcoma; Phloroglucinol; Pregnane X Receptor; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Receptors, Tumor Necrosis Factor; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Rifampin; RNA, Messenger; Sialoglycoproteins; Terpenes; Transfection; Tumor Cells, Cultured; Vitamin K 2

To observe the regulative effects of 17 beta-estradiol (17 beta-E(2)) and ICI 182780 on the expression of osteoprotegerin (OPG), the ligand of osteoprotegerin (OPGL) and the related cytokines [macrophage colony-stimulating factor (M-CSF), TRAIL and transforming growth factor beta(TGF beta)] in MG63 cells.. The expression of OPG, OPGL, M-CSF, TRAIL and TGF beta mRNA was examined by reverse transcriptase (RT)-PCR. The expression of OPG and OPGL protein was measured with Western blot.. (1) 17 beta-E(2) up-regulated the expression of OPG and TGF beta but down-regulated OPGL, M-CSF and TRAIL expression in MG63 cells. (2) ICI 182780 showed varied transactivating capability when regulating the expression of OPG, OPGL, TRAIL, M-CSF and TGF beta genes in MG63 cells.. (1) One of the key pathogenetic factors of postmenopausal osteoporosis is that estrogen deficiency leads to the decreasing expression of OPG and TGF beta but the increasing expression of some bone-resorbing cytokines such as OPGL, M-CSF and TRAIL in osteoblasts, then stimulation of osteoclast differentiation and activity which potentiates bone resorption and bone loss; (2) ICI 182780 shows partial transactivating activity in osteoblasts, so it should belong to a kind of selective estrogen receptor modulators.

Topics: Apoptosis Regulatory Proteins; Blotting, Western; Carrier Proteins; Cell Line, Tumor; Cytokines; Estradiol; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Macrophage Colony-Stimulating Factor; Membrane Glycoproteins; Osteoprotegerin; Osteosarcoma; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; TNF-Related Apoptosis-Inducing Ligand; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The aims of this study were (i). to assess the morphological features of osteo-blast-like, osteosarcoma cells (cell line SaOS-2) cultured on implant surfaces of varying alloys and (ii). to evaluate the biological activity of these cells, following their adhesion onto these surfaces.. SaOS-2 cells (6 x 104) were grown on titanium discs (diameter 30 mm), each with a surface of differing composition and roughness (commercially pure titanium, titanium-aluminium-vanadium alloy, oxide-blasted titanium and Astra-Tech special treatment titanium; the alloys are directly comparable with those used to construct implants). The cells were grown for time periods of 1, 3, 5 and 7 days, the media were collected and the cells were fixed with 2.5% glutaraldehyde. The media were then assayed (using enzyme-linked immunosorbant assay) for the levels of interleukin (IL)-1, interleukin-6, interleukin-18 and osteoprotegerin (OPG) produced by the cells. The discs, with the cells fixed on them, were viewed under scanning electron microscopy (SEM, x 2.0 k) to evaluate cell morphology.. Following attachment, the cells changed their morphology and released local factors known to activate osteoclasts. Commercially pure titanium stimulated the cells the most and titanium-aluminium-vanadium alloy the least. All implant materials stimulated production of IL-1, IL-6, IL-18 above that produced by cells grown on Petri dishes (polystyrene). The titanium-aluminium-vanadium alloy allowed cell attachment but levels of IL-1 in this medium were significantly lower (31.5 +/- 5.2 pg/ml on same day) than cultures with pure titanium (201.8 +/- 11.5 pg/ml at day 5). The same pattern was observed with the IL-6, IL-18, and OPG with polystyrene appearing to stimulate most production of OPG. Titanium-aluminium-vanadium produced the least biological response.

Topics: Alloys; Biocompatible Materials; Cell Adhesion; Cell Count; Cell Line; Cell Size; Cells, Cultured; Coated Materials, Biocompatible; Culture Media, Conditioned; Dental Alloys; Dental Implants; Enzyme-Linked Immunosorbent Assay; Glycoproteins; Humans; Interleukin-1; Interleukin-18; Interleukin-6; Interleukins; Microscopy, Electron, Scanning; Osteoblasts; Osteoprotegerin; Osteosarcoma; Oxides; Polystyrenes; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Surface Properties; Time Factors; Titanium

Apo2L/TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines that induces death of cancer cells but not normal cells. Its potent apoptotic activity is mediated through its cell surface death domain-containing receptors, DR4 and DR5. Apo2L/TRAIL interacts also with 3 "decoy" receptors that do not induce apoptosis, DcR1, DcR2, which lack functional death domains, and osteoprotegerin (OPG). The aim of our study was to investigate the cytotoxic activity of Apo2L/TRAIL on established osteogenic sarcoma cell lines (BTK-143, HOS, MG-63, SJSA-1, G-292 and SAOS2) and in primary cultures of normal human bone (NHB) cells. When used alone, Apo2L/TRAIL at 100 ng/ml for 24 hr induced greater than 80% cell death in only 1 (BTK-143) of the 6 osteogenic sarcoma cell lines. In contrast, Apo2L/TRAIL-resistant cells were susceptible to Apo2L/TRAIL-mediated apoptosis in the presence of the anticancer drugs, Doxorubicin (DOX), Cisplatin (CDDP) and Etoposide (ETP) but not Methotrexate (MTX) or Cyclophosphamide (CPM). Importantly, neither Apo2L/TRAIL alone nor in combination with any of these drugs affected primary normal human bone cells under equivalent conditions. Apo2L/TRAIL-induced apoptosis, and its augmentation by chemotherapy in the resistant cell lines was mediated through caspase-8 and caspase-3 activation. Furthermore, Apo2L/TRAIL-induced apoptosis and its augmentation by chemotherapy was effectively inhibited by caspase-8 zIETD-fmk and caspase-3 zDEVD-fmk protease inhibitors and by the pan-caspase inhibitor zVAD-fmk. The pattern of basal Apo2L/TRAIL receptor mRNA expression, or expression of the intracellular caspase inhibitor FLICE-inhibitory protein, FLIP, could not be readily correlated with resistance or sensitivity to Apo2L/TRAIL-induced apoptosis. However, the augmentation of Apo2L/TRAIL effects by chemotherapy was associated with drug-induced up-regulation of death receptors DR4 and DR5 mRNA and protein. No obvious correlation was seen between the expression of OPG mRNA or protein and susceptibility of cells to Apo2L/TRAIL-induced apoptosis. Stable over-expression of a dominant negative form of the Fas-associated death domain protein (FADD) in the Apo2L/TRAIL-sensitive BTK-143 cells completely inhibited Apo2L/TRAIL-induced cell death. Our results indicate that chemotherapy and Apo2L/TRAIL act synergistically to kill cancer cells but not normal bone-derived osteoblast-like cells, which has implications for future therapy of osteosarcoma.

Topics: Apoptosis Regulatory Proteins; Blotting, Western; Bone and Bones; Bone Neoplasms; Caspases; Cell Death; Cell Nucleus; Cell Survival; Dose-Response Relationship, Drug; Flow Cytometry; Glycoproteins; Humans; Membrane Glycoproteins; Osteoprotegerin; Osteosarcoma; Protein Binding; Protein Structure, Tertiary; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; Sarcoma; TNF-Related Apoptosis-Inducing Ligand; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Topics: Bone Neoplasms; Chromosomes, Human, Pair 18; Genetic Linkage; Genetic Predisposition to Disease; Glycoproteins; HLA Antigens; Humans; Mutation; Osteitis Deformans; Osteoprotegerin; Osteosarcoma; Pedigree; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Respirovirus; Respirovirus Infections

Local growth of osteosarcoma involves destruction of host bone by proteolytic mechanisms and/or host osteoclast activation. Osteoclast formation and activity are regulated by osteoblast-derived factors such as the osteoclast differentiating factor, receptor activator of NF-kappaB ligand (RANKL) and the inhibitor osteoprotegerin (OPG). We have investigated the in vitro effects of bisphosphonates on a clonal rat osteosarcoma cell line. The aminobisphosphonate pamidronate was added to UMR 106-01 cell cultures (10(-8)M to 10(-4)M up to 5 days). The non-aminobisphosphonate clodronate was administered for the same time periods (10(-6)M to 10(-2)M). Cell proliferation, apoptosis and mRNA expression was assessed. Both agents inhibited cell proliferation in a time- and dose-dependent manner. ELISA analysis demonstrated an increase in DNA fragmentation although there was no significant dose-related difference between the doses studied. Bisphosphonate-treated cultures had a greater subpopulation of cells exhibiting morphological changes of apoptosis. Expression of mRNA for osteopontin and RANKL was down-regulated by both agents, while the expression of mRNA for alkaline phosphatase, pro-alpha1(I) collagen and OPG was not altered. Out in vitro work suggests the bisphosphonates not only have direct effects on osteosarcoma cell growth and apoptosis, but also, by altering the relative expression of osteoclast-regulating factors, they may inhibit the activity of osteoclasts and their recruitment.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bone Neoplasms; Carrier Proteins; Cell Division; Clodronic Acid; Diphosphonates; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Glycoproteins; Growth Inhibitors; Membrane Glycoproteins; Osteoprotegerin; Osteosarcoma; Pamidronate; RANK Ligand; Rats; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tumor Cells, Cultured

Paget's disease of bone (PDB) is a common disorder characterized by focal areas of increased and disorganized osteoclastic bone resorption, leading to bone pain, deformity, pathological fracture, and an increased risk of osteosarcoma. Genetic factors play an important role in the pathogenesis of Paget's disease. In some families, the disease has been found to be linked to a susceptibility locus on chromosome 18q21-22, which also contains the gene responsible for familial expansile osteolysis (FEO)--a rare bone dysplasia with many similarities to Paget's disease. Insertion mutations of the TNFRSF11A gene encoding Receptor Activator of NF kappa B (RANK) have recently been found to be responsible for FEO and rare cases of early onset familial Paget's disease. Loss of heterozygosity (LOH) affecting the PDB/FEO critical region has also been described in osteosarcomas suggesting that TNFRSF11A might also be involved in the development of osteosarcoma. In order to investigate the possible role of TNFRSF11A in the pathogenesis of Paget's disease and osteosarcoma, we conducted mutation screening of the TNFRSF11A gene in patients with familial and sporadic Paget's disease as well as DNA extracted from Pagetic bone lesions, an osteosarcoma arising in Pagetic bone and six osteosarcoma cell lines. No specific abnormalities of the TNFRSF11A gene were identified in a Pagetic osteosarcoma, the osteosarcoma cell lines, DNA extracted from Pagetic bone lesions, or DNA extracted from peripheral blood in patients with familial or sporadic Paget's disease including several individuals with early onset Paget's disease. These data indicate that TNFRSF11A mutations contribute neither to the vast majority of cases of sporadic or familial PDB, nor to the development of osteosarcoma.

Topics: Adult; Bone Neoplasms; DNA; DNA Mutational Analysis; DNA Primers; Genetic Predisposition to Disease; Genetic Testing; Glycoproteins; Humans; Osteitis Deformans; Osteoprotegerin; Osteosarcoma; Point Mutation; Polymerase Chain Reaction; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Bone cancer pain is common among cancer patients and can have a devastating effect on their quality of life. A chief problem in designing new therapies for bone cancer pain is that it is unclear what mechanisms drive this distinct pain condition. Here we show that osteoprotegerin, a secreted 'decoy' receptor that inhibits osteoclast activity, also blocks behaviors indicative of pain in mice with bone cancer. A substantial part of the actions of osteoprotegerin seems to result from inhibition of tumor-induced bone destruction that in turn inhibits the neurochemical changes in the spinal cord that are thought to be involved in the generation and maintenance of cancer pain. These results demonstrate that excessive tumor-induced bone destruction is involved in the generation of bone cancer pain and that osteoprotegerin may provide an effective treatment for this common human condition.

Topics: Animals; Astrocytes; Behavior, Animal; Bone Demineralization, Pathologic; Glycoproteins; Hindlimb; Male; Mice; Mice, Inbred C3H; Osteoclasts; Osteoprotegerin; Osteosarcoma; Pain; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Sarcoma, Experimental; Spinal Cord

A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for human osteoclast differentiation factor (ODF/RANKL/ OPGL/TRANCE) utilizing a polyclonal antibody that recognizes both human soluble ODF and mouse ODF in combination with a osteoclasogenesis inhibitory factor (OCIF/OPG) was developed. We can quantify the ODF level in not only human ODF (detection limit: 0.05 ng/ml), but also mouse ODF by virtue of cross-reactivity. Employing this assay system, we demonstrated that ODF is constitutively present as a membrane-bound form in both the human osteosarcoma cell lines, MG-63, HOS and SaOS-2, and the mouse osteoblastic cell line MC3T3-E1.

Topics: Animals; Carrier Proteins; Cell Differentiation; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Glycoproteins; Humans; Membrane Glycoproteins; Mice; Osteoblasts; Osteoclasts; Osteoprotegerin; Osteosarcoma; Protein Binding; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tumor Cells, Cultured

Topics: Animals; Bone Neoplasms; Bone Resorption; Carrier Proteins; Glycoproteins; Membrane Glycoproteins; Mice; Osteoclasts; Osteolysis; Osteoprotegerin; Osteosarcoma; Pain; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Osteoprotegerin (OPG) is a recently cloned soluble member of the tumor necrosis factor receptor family. OPG has been shown to inhibit osteoclast recruitment by binding to OPG-ligand, an osteoclast differentiating factor on osteoblastic stromal cells, thereby blocking osteoclastogenesis. In this report we have examined the effect of tumor necrosis factor-alpha (TNF-alpha) and tumor necrosis factor-beta (TNF-beta) on OPG mRNA levels in the human osteosarcoma cell line MG-63. We demonstrate that both TNF-alpha and TNF-beta dose- and time-dependently upregulate the mRNA levels of OPG. The effect is significant at and above 5 pM of TNF-alpha and 1 pM of TNF-beta. The stimulatory effect on OPG mRNA levels in MG-63 cells was detected after 2 hrs of incubation with TNF-alpha or TNF-beta. These data demonstrate that the expression of OPG in osteoblasts, with subsequent effects on osteoclastogenesis, is regulated by TNFs.

Topics: Bone Neoplasms; Glycoproteins; Humans; Kinetics; Lymphotoxin-alpha; Osteoprotegerin; Osteosarcoma; Polymerase Chain Reaction; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; RNA, Messenger; Time Factors; Transcription, Genetic; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Up-Regulation

Osteoprotegerin (OPG) is a soluble receptor for the Osteoprotegerin-Ligand (OPGL) which is expressed on osteoblasts and mediates the signal for osteoclast differentiation. In the present study we demonstrate that OPG mRNA levels in MG-63 cells are increased in a dose-dependent manner after 8 h of treatment with IL-1 alpha (338 +/- 53% over control at 25 U/ml). Interleukin-6 (IL-6), under similar culture conditions, does not affect OPG mRNA levels. Time-course studies show that IL-1 alpha (25 U/ml) causes a transient increase of OPG mRNA levels in MG-63 cells, peaking after 4 h of treatment. An increase of the OPG transcript occurs in hOB cells at 0.5 h which is still present after 24 h of IL-1 alpha treatment. In MG-63 cells neither basal-nor IL-1 alpha-induced OPG mRNA levels are altered by the translational inhibitor cycloheximide. These results suggest that expression of OPG in osteoblasts may be regulated by IL-1 alpha.

Topics: Bone Neoplasms; Cell Differentiation; Cycloheximide; Dose-Response Relationship, Drug; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Interleukin-1; Kinetics; Osteoblasts; Osteoclasts; Osteoprotegerin; Osteosarcoma; Polymerase Chain Reaction; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; RNA, Messenger; Signal Transduction; Transcription, Genetic; Tumor Cells, Cultured