RNA-dependent protein kinase is required for interferon-γ-induced autophagy in MG63 osteosarcoma cells.

Osteosarcoma is a bone tumor that mainly affects children and adolescents. Interferons (IFNs) have been shown to exert antitumor effects in osteosarcoma cells, although the molecular mechanisms have not been fully realized. We investigated IFN-γ actions on osteosarcoma cells. Our results show that IFN-γ induces the accumulation of autophagosomes in osteosarcoma cells. IFN-γ treatment leads to the conversion of autophagy marker light chain 3 (LC3)-I to LC3-II in osteosarcoma cells, and this conversion is accompanied by puncta formation. Also, IFN-γ-mediated induction of autophagosome formation and autophagic flux require RNA-dependent protein kinase (PKR) activity. In addition, our findings show that IFN-γ-mediated osteosarcoma cell death is not dependent on PKR. Our study suggests that IFN-γ has differential effects that lead to induction of cell death and autophagy in osteosarcoma cells. Further evaluation of the IFN-γ-mediated molecular mechanism could lead to improved understanding of and targeted treatment strategies for osteosarcoma.

The inhibitory effects of vancomycin on rat bone marrow-derived mesenchymal stem cell differentiation.

OBJECTIVE: The use of intrawound vancomycin powder in spine surgery has been shown to decrease the rate of surgical site infections; however, the optimal dose is unknown. High-dose vancomycin inhibits osteoblast proliferation in vitro and may decrease the rate of solid arthrodesis. Bone marrow-derived mesenchymal stem cells (BMSCs) are multipotent cells that are a source of osteogenesis in spine fusions. The purpose of this study was to determine the effects of vancomycin on rat BMSC viability and differentiation in vitro. METHODS: BMSCs were isolated from the femurs of immature female rats, cultured, and then split into two equal groups; half were treated to stimulate osteoblastic differentiation and half were not. Osteogenesis was stimulated by the addition of 50 µg/mL l-ascorbic acid, 10 mM ß-glycerol phosphate, and 0.1 µM dexamethasone. Vancomycin was added to cell culture medium at concentrations of 0, 0.04, 0.4, or 4 mg/mL. Early differentiation was determined by alkaline phosphatase activity (4 days posttreatment) and late differentiation by alizarin red staining for mineralization (9 days posttreatment). Cell viability was determined at both the early and late time points by measurement of formazan colorimetric product. RESULTS: Viability within the first 4 days decreased with high-dose vancomycin treatment, with cells receiving 4 mg/mL vancomycin having 40%-60% viability compared to the control. A gradual decrease in alizarin red staining and nodule formation was observed with increasing vancomycin doses. In the presence of the osteogenic factors, vancomycin did not have deleterious effects on alkaline phosphatase activity, whereas a trend toward reduced activity was seen in the absence of osteogenic factors when compared to osteogenically treated cells. CONCLUSIONS: Vancomycin reduced BMSC viability and impaired late osteogenic differentiation with high-dose treatment. Therefore, the inhibitory effects of high-dose vancomycin on spinal fusion may result from both reduced BMSC viability and some impairment of osteogenic differentiation.

Detection of macromolecular inversion-induced structural changes in osteosarcoma cells by FTIR microspectroscopy.

Fourier transform infrared (FTIR) microspectroscopy provides a biochemical fingerprint of the cells. In this study, chemical changes in 143B osteosarcoma cells were investigated using FTIR analysis of cancer cells after their treatment with polymeric invertible micellar assemblies (IMAs) and curcumin-loaded IMAs and compared with untreated osteosarcoma cells. A comprehensive principal component analysis (PCA) was applied to analyze the FTIR results and confirm noticeable changes in cell surface chemical structures in the fingerprint regions of 1480-900 cm-1. The performed clustering shows visible differences for all investigated groups of cancer cells. It is demonstrated that a combination of FTIR microspectroscopy with PCA can be an efficient approach in determining interactions of osteosarcoma cells and drug-loaded polymer micellar assemblies. Graphical abstract.

STIM1 expression is associated with osteosarcoma cell survival.

OBJECTIVE: To examine the role of store-operated calcium entry (SOCE) and stromal interaction molecule 1 (STIM1) in survival and migration of osteosarcoma cells and investigate what blockade of store-operated Ca2+ contributes to the regulation of osteosarcoma cells. METHODS: First, we examined the expression levels of STIM1 in osteosarcoma cell lines by Western analysis and in tissue specimens by immunohistochemistry. Second, we investigated the effect of SOCE and STIM1 on osteosarcoma cell viability using MTS assays and on cell proliferation using colony formation. Third, we investigated the role of SOCE and STIM1 in cell migration using wound healing assays and Boyden chamber assays. Finally, we studied the effect of SOCE on the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) activity by luciferase assays. RESULTS: STIM1 was overexpressed in osteosarcoma cell lines and tissue specimens and was associated with poor survival of osteosarcoma patients. Also, inhibition of SOCE and STIM1 decreased the cell viability and migration of osteosarcoma cells. Furthermore, our results showed that blockade of store-operated Ca2+ channels involved down-regulation of NFATc1 in osteosarcoma cells. CONCLUSIONS: STIM1 is essential for osteosarcoma cell functions, and STIM1 and Ca2+ entry pathway could be further explored as molecular targets in the treatment of osteosarcoma.

Inhibition of STAT3 blocks protein synthesis and tumor metastasis in osteosarcoma cells.

BACKGROUND: Osteosarcoma is the most common bone cancer. Despite advances, molecular mechanisms associated with osteosarcoma have not been fully understood. Hence, an effective treatment for osteosarcoma has yet to be developed. Even though signal transducer and activator of transcription3 (STAT3) has been implicated, its role in pathogenesis of osteosarcoma is not fully determined. In this study, we investigated the antitumor effect of napabucasin (NP) (BBI608), an inhibitor of STAT3 on osteosarcoma in vitro and in vivo and studied the underlying molecular mechanism. METHODS: Cell viability, colony formation, apoptosis, tumor growth and metastasis assays were performed to examine the effect of NP on osteosarcoma in vitro and in vivo. Real-time RT-PCR, western analysis, immunofluorescence and reporter assays were used to monitor the expression and activity of proteins and underlying molecular pathways. Protein synthesis, co-immunoprecipitation and CAP binding assays were carried out to understand NP-mediated mechanism of actions in osteosarcoma cells. RESULTS: Our results show that NP treatment decreases cell viability and induces apoptosis in several osteosarcoma cell lines. NP treatment suppresses both expression and phosphorylation of STAT3 in addition to blocking STAT3-mediated transcription and downstream target proteins in osteosarcoma cells. Furthermore, NP inhibits protein synthesis through regulation of the eukaryotic initiation factor 4E (eIF4E) and eIF4E-binding protein 1 (4E-BP1). NP also inhibits the progression of osteosarcoma tumors and metastasis in vivo in an orthotopic tibial model of osteosarcoma. CONCLUSIONS: Taken together, our investigation reveals that NP acts through a novel mechanism and inhibits osteosarcoma growth and metastasis, and could be investigated clinically for treating osteosarcoma patients alone or in combination with other drugs.

Decreased local and systemic levels of sFRP3 protein in osteosarcoma patients.

Osteosarcoma is a malignant bone tumor that occurs mainly in children and adolescents. Because Wnt signaling has been implicated in the pathogenesis of osteosarcoma, we have investigated the circulating and local levels of the Wnt antagonist protein, Secreted Frizzled Related Protein (sFRP) 3, in osteosarcoma patients. Enzyme linked immunosorbent assay (ELISA) analysis of 67 osteosarcoma and age-matched non-diseased control sera showed that sFPR3 protein levels were significantly lower in osteosarcoma than in normal. Analysis of tumor and adjacent normal tissues (9 pairs) from osteosarcoma patients showed a decrease in sFRP3 expression in 5 out of 9 tumor samples compared to normal tissues. Furthermore, immunohistochemical analysis of tissue microarray revealed a significant decrease in sFRP3 levels in tumor compared to normal bone. RNA sequencing analysis in osteosarcoma cells shows suppression of sFRP3 and concomitant expression of multiple Wnt family members mediating canonical or non-canonical Wnt signaling. Taken together, our findings show that the systemic and local levels of sFRP3 protein are downregulated in osteosarcoma and sFRP3 levels could be explored further in the diagnosis and the care of osteosarcoma patients.

FH535 Suppresses Osteosarcoma Growth In Vitro and Inhibits Wnt Signaling through Tankyrases.

Osteosarcoma (OS) is an aggressive primary bone tumor which exhibits aberrantly activated Wnt signaling. The canonical Wnt signaling cascade has been shown to drive cancer progression and metastasis through the activation of ß-catenin. Hence, small molecule inhibitors of Wnt targets are being explored as primary or adjuvant chemotherapy. In this study, we have investigated the ability of FH535, an antagonist of Wnt signaling, to inhibit the growth of OS cells. We found that FH535 was cytotoxic in all OS cell lines which were tested (143b, U2OS, SaOS-2, HOS, K7M2) but well tolerated by normal human osteoblast cells. Additionally, we have developed an in vitro model of doxorubicin-resistant OS and found that these cells were highly responsive to FH535 treatment. Our analysis provided evidence that FH535 strongly inhibited markers of canonical Wnt signaling. In addition, our findings demonstrate a reduction in PAR-modification of Axin2 indicating inhibition of the tankyrase 1/2 enzymes. Moreover, we observed inhibition of auto-modification of PARP1 in the presence of FH535, indicating inhibition of PARP1 enzymatic activity. These data provide evidence that FH535 acts through the tankyrase 1/2 enzymes to suppress Wnt signaling and could be explored as a potent chemotherapeutic agent for the control of OS.

Three-Dimension-Printed Porous Poly(Propylene Fumarate) Scaffolds with Delayed rhBMP-2 Release for Anterior Cruciate Ligament Graft Fixation.

Anterior cruciate ligament (ACL) ruptures reconstructed with tendon grafts are commonly fixed with bioabsorbable implants, which are frequently complicated by incomplete bone filling upon degradation. Bone regeneration after ACL reconstruction could be enhanced by utilizing tissue engineering techniques and three-dimensional (3D) printing to create a porous bioabsorbable scaffold with delayed delivery of recombinant-human bone morphogenetic protein 2 (rhBMP-2). The first aim of this study was to design a 3D poly(propylene fumarate) (PPF) porous scaffold that maintained suitable pullout strength for future testing in a rabbit ACL reconstruction model. Our second aim was to determine the release kinetics of rhBMP-2 from PPF scaffolds that utilized both calcium-phosphate coatings and growth factor delivery on microspheres, both of which have been shown to decrease the initial burst release of rhBMP-2 and increase bone regeneration. To determine the degree of scaffold porosity that maintained suitable pullout strength, tapered scaffolds were fabricated with increasing porosity (0%, 20%, 35%, and 44%) and pullout testing was performed in a cadaveric rabbit ACL reconstruction model. Scaffolds were coated with carbonate hydroxyapatite (synthetic bone mineral [SBM]), and radiolabeled rhBMP-2 was delivered in four different experimental groups as follows: Poly(lactic-co-glycolic acid) microspheres only, microspheres and collagen (50:50), collagen only, and saline solution only. rhBMP-2 release was measured at day 1, 2, 4, 8, 16, and 32. The microsphere delivery groups had a smaller burst release and released a smaller percentage of rhBMP-2 over the 32 days than the collagen and saline only groups. In conclusion, a porous bioabsorbable scaffold with suitable strength for a rabbit ACL reconstruction was developed. Combining a synthetic bone mineral coating with microspheres had an additive effect, decreasing the initial burst release and cumulative release of rhBMP-2. Future studies need to evaluate this scaffold's fixation strength and bone filling capabilities in vivo compared to traditional bioabsorbable implants.

2-Methoxyestradiol-Mediated Induction of Frzb Contributes to Cell Death and Autophagy in MG63 Osteosarcoma Cells.

Osteosarcoma is a bone tumor that mainly affects children and adolescents. Although its pathogenesis is still not fully understood, activation of Wnt signaling has been implicated in the development and metastasis of osteosarcoma. In this report, we have investigated the effect of the anti-tumor compound, 2-methoxyestradiol (2-ME) on Wnt antagonist frizzled-related protein b (Frzb), also known as secreted frizzled-related protein (sFRP)3 in human osteosarcoma (MG63) cells. Our results show that 2-ME treatment induces Frzb gene promoter activity, and increases Frzb mRNA and protein levels in osteosarcoma cells. In addition, 2-ME treatment regulates downstream Wnt signaling, increasing the cytoplasmic levels of ß-catenin, and blocking ß-catenin-mediated Wnt activation in osteosarcoma cells. 2-ME-mediated induction of Frzb protein expression is specific to osteosarcoma cells, as it does not affect Frzb expression in normal primary human osteoblasts. Furthermore, 2-ME-induced apoptosis and autophagy are blocked in osteosarcoma cells transfected with Frzb siRNAs. Taken together, these studies demonstrate that Frzb protein plays an important role in 2-ME-mediated anti-tumor mechanisms in osteosarcoma cells. J. Cell. Biochem. 118: 1497-1504, 2017. © 2016 Wiley Periodicals, Inc.

The estrogen metabolite 2-methoxyestradiol regulates eukaryotic initiation factor 4E (eIF4E) and inhibits protein synthesis in MG63 osteosarcoma cells.

Osteosarcoma is a primary bone tumor that affects children and young adults. The estrogen metabolite 2-methoxyestradiol (2-ME) induces cell death in osteosarcoma cells. To determine whether 2-ME actions involve the control of protein synthesis, we studied the effect of 2-ME on eukaryotic initiation factor 4E (eIF4E) and eIF4E-binding protein 1 (4E-BP1) in MG63 osteosarcoma cells. Our results show that 2-ME treatment increases the association of eIF4E with 4E-BP1 in osteosarcoma cells. Also, 2-ME decreases the binding of eIF4E protein to 7-methyl-guanosine cap structure, indicating that 2-ME treatment results in the inhibition of translational initiation. These findings are further supported by the inhibition of protein synthesis in 2-ME-treated osteosarcoma cells. Taken together, our studies show that 2-ME-mediated antitumor effects in osteosarcoma cells involve the regulation of protein synthesis, and translational machinery could serve as a target in the treatment of osteosarcoma.

Inhibition of Autophagy Increases 2-Methoxyestradiol-Induced Cytotoxicity in SW1353 Chondrosarcoma Cells.

Chondrosarcoma is a cartilage tumor and is the second most common malignant bone cancer. Unlike many tumors, chondrosarcomas are resistant to conventional chemotherapy and radiotherapy. Autophagy is a homeostatic mechanism through which cellular proteins and organelles are subjected to lysosomal degradation and recycling. Autophagy could play a dual role in cancer by facilitating either cell death or cell survival. To determine whether autophagy plays a role in cell death in chondrosarcoma, we have studied the effect of the anti-tumor compound 2-methoxyestradiol (2-ME) in chondrosarcoma cells in culture. Transmission electron microscopy imaging indicates that 2-ME treatment leads to the accumulation of autophagosomes in human chondrosarcoma (SW1353 and Hs819T) cells. Also, 2-ME induces the conversion of microtubule-associated protein LC3-I to LC3-II, a protein marker that is correlated with the formation of autophagosomes. Our results show that siRNAs directed against ATG3 blocks 2-ME-induced autophagosome formation in chondrosarcoma cells. In addition, treatment with Bafilomycin A1 (Baf) and 3-methyladenine (3-MA), the inhibitors of autophagy, further increased the cell death in 2-ME-treated chondrosarcoma cells. Taken together, our studies demonstrate that autophagy causes resistance to cytotoxicity in chondrosarcoma cells, and the efficacy and anti-tumor effects of drugs in chondrosarcoma could be enhanced by modulating autophagy.

Invertible micellar polymer nanoassemblies target bone tumor cells but not normal osteoblast cells.

AIM: To demonstrate the capability of the invertible micellar polymer nanoassemblies (IMAs) to deliver and release curcumin using the recently discovered mechanism of macromolecular inversion to treat bone tumor cells. MATERIALS & METHODS: The effect of IMA-mediated delivery of curcumin on osteosarcoma cell survival was investigated using MTS assays. To assess the effect of IMAs-delivered curcumin on osteosarcoma cell growth, fluorescence-activated cell sorting was performed. The uptake of micellar nanoassemblies was followed using confocal microscopy. RESULTS & DISCUSSION: IMAs-delivered curcumin is effective in blocking osteosarcoma cell growth. It decreases cell viability in human osteosarcoma (MG63, KHOS, and LM7) cells while having no effect on normal human osteoblast cells. It indicates that curcumin-loaded IMAs provide a unique delivery system targeted to osteosarcoma cells.

MicroRNA-34c inversely couples the biological functions of the runt-related transcription factor RUNX2 and the tumor suppressor p53 in osteosarcoma.

Osteosarcoma (OS) is a primary bone tumor that is most prevalent during adolescence. RUNX2, which stimulates differentiation and suppresses proliferation of osteoblasts, is deregulated in OS. Here, we define pathological roles of RUNX2 in the etiology of OS and mechanisms by which RUNX2 expression is stimulated. RUNX2 is often highly expressed in human OS biopsies and cell lines. Small interference RNA-mediated depletion of RUNX2 inhibits growth of U2OS OS cells. RUNX2 levels are inversely linked to loss of p53 (which predisposes to OS) in distinct OS cell lines and osteoblasts. RUNX2 protein levels decrease upon stabilization of p53 with the MDM2 inhibitor Nutlin-3. Elevated RUNX2 protein expression is post-transcriptionally regulated and directly linked to diminished expression of several validated RUNX2 targeting microRNAs in human OS cells compared with mesenchymal progenitor cells. The p53-dependent miR-34c is the most significantly down-regulated RUNX2 targeting microRNAs in OS. Exogenous supplementation of miR-34c markedly decreases RUNX2 protein levels, whereas 3'-UTR reporter assays establish RUNX2 as a direct target of miR-34c in OS cells. Importantly, Nutlin-3-mediated stabilization of p53 increases expression of miR-34c and decreases RUNX2. Thus, a novel p53-miR-34c-RUNX2 network controls cell growth of osseous cells and is compromised in OS.

RNA-dependent protein kinase is essential for 2-methoxyestradiol-induced autophagy in osteosarcoma cells.

Osteosarcoma is the most common primary malignant bone tumor in children and young adults. Surgical resection and adjunctive chemotherapy are the only widely available options of treatment for this disease. Anti-tumor compound 2-Methoxyestradiol (2-ME) triggers cell death through the induction of apoptosis in osteosarcoma cells, but not in normal osteoblasts. In this report, we have investigated whether autophagy plays a role in 2-ME actions on osteosarcoma cells. Transmission electron microscopy imaging shows that 2-ME treatment leads to the accumulation of autophagosomes in human osteosarcoma cells. 2-ME induces the conversion of the microtubule-associated protein LC3-I to LC3-II, a biochemical marker of autophagy that is correlated with the formation of autophagosomes. Conversion to LC3-II is accompanied by protein degradation in 2-ME-treated cells. 2-ME does not induce autophagosome formation in normal primary human osteoblasts. In addition, 2-ME-dependent autophagosome formation in osteosarcoma cells requires ATG7 expression. Furthermore, 2-ME does not induce accumulation of autophagosomes in osteosarcoma cells that express dominant negative mutant RNA-dependent protein kinase (PKR) and are resistant to anti-proliferative and anti-tumor effects of 2-ME. Taken together, our study shows that 2-ME treatment induces PKR-dependent autophagy in osteosarcoma cells, and that autophagy could play an important role in 2-ME-mediated anti-tumor actions and in the control of osteosarcoma.

Hydrogel-PLGA delivery system prolongs 2-methoxyestradiol-mediated anti-tumor effects in osteosarcoma cells.

Osteosarcoma is a bone tumor that affects children and young adults. 2-Methoxyestradiol (2-ME), a naturally occurring estrogen metabolite, kills osteosarcoma cells, but does not affect normal osteoblasts. In order to effectively target osteosarcoma and improve the therapeutic index of the drug 2-ME, we have encapsulated 2-ME in a composite of oligo-(polyethylene glycol) fumarate (OPF) hydrogel and poly (lactic-co-glycolic acid) (PLGA) microspheres and investigated the effect of polymer composition on 2-ME release kinetics and osteosarcoma cell survival. The in vitro study shows that 2-ME can be released in a controlled manner over 21-days. The initial burst releases observed on day 1 were 50% and 32% for OPF and OPF/PLGA composites, respectively. The extended release kinetics show that 100% of the encapsulated 2-ME is released by day 12 from OPF, whereas the OPF/PLGA composites showed a release of 85% on day 21. 2-ME released from the polymers was biologically active and blocked osteosarcoma cell proliferation in vitro. Also, comparison of 2-ME delivery in osteosarcoma cells in culture, shows that direct treatment has no effect after 3 days, whereas polymer-mediated delivery produces anti-tumor effects that could be sustained for 21 days. These findings show that the OPF and PLGA polymeric system may prove to be useful in controlled and sustained delivery of 2-ME and could be further explored in the treatment of osteosarcoma.

Induction of angiogenesis and osteogenesis in surgically revascularized frozen bone allografts by sustained delivery of FGF-2 and VEGF.

Large conventional bone allografts are susceptible to fracture and nonunion due to incomplete revascularization and insufficient bone remodeling. We aim to improve bone blood flow and bone remodeling using surgical angiogenesis combined with delivery of fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF). Frozen femoral allografts were heterotopically transplanted in a rat model. The saphenous arteriovenous bundle was implanted within the graft medullary canal. Simultaneously, biodegradable microspheres containing phosphate buffered saline (control), FGF-2, VEGF, or FGF-2 + VEGF were placed within the graft. Rats were sacrificed at 4 and 18 weeks. Angiogenesis was determined by quantifying bone capillary density and measuring cortical bone blood flow. Bone remodeling was assessed by histology, histomorphometry, and alkaline phosphatase activity. VEGF significantly increased angiogenesis and bone remodeling at 4 and 18 weeks. FGF-2 did not elicit a strong angiogenic or osteogenic response. No synergistic effect of FGF-2 + VEGF was observed. VEGF delivered in microspheres had superior long-term effect on angiogenesis and osteogenesis in surgically revascularized frozen bone structural allografts as compared to FGF-2 or FGF-2 + VEGF. Continuous and localized delivery of VEGF by microencapsulation has promising clinical potential by inducing a durable angiogenic and osteogenic response in frozen allografts.

Regulation of interferon pathway in 2-methoxyestradiol-treated osteosarcoma cells.

BACKGROUND: Osteosarcoma is a bone tumor that often affects children and young adults. Although a combination of surgery and chemotherapy has improved the survival rate in the past decades, local recurrence and metastases still develop in 40% of patients. A definite therapy is yet to be determined for osteosarcoma. Anti- tumor compound and a metabolite of estrogen, 2-methoxyestradiol (2-ME) induces cell death in osteosarcoma cells. In this report, we have investigated whether interferon (IFN) pathway is involved in 2-ME-induced anti-tumor effects in osteosarcoma cells. METHODS: 2-ME effects on IFN mRNA levels were determined by Real time PCR analysis. Transient transfections followed by reporter assays were used for investigating 2-ME effects on IFN-pathway. Western blot analyses were used to measure protein and phosphorylation levels of IFN-regulated eukaryotic initiation factor-2 alpha (eIF-2α). RESULTS: 2-ME regulates IFN and IFN-mediated effects in osteosarcoma cells. 2 -ME induces IFN gene activity and expression in osteosarcoma cells. 2-ME treatment induced IFN-stimulated response element (ISRE) sequence-dependent transcription and gamma-activated sequence (GAS)-dependent transcription in several osteosarcoma cells. Whereas, 2-ME did not affect IFN gene and IFN pathways in normal primary human osteoblasts (HOB). 2-ME treatment increased the phosphorylation of eIF-2α in osteosarcoma cells. Furthermore, analysis of osteosarcoma tissues shows that the levels of phosphorylated form of eIF-2α are decreased in tumor compared to normal controls. CONCLUSIONS: 2-ME treatment triggers the induction and activity of IFN and IFN pathway genes in 2-ME-sensitive osteosarcoma tumor cells but not in 2-ME-resistant normal osteoblasts. In addition, IFN-signaling is inhibited in osteosarcoma patients. Thus, IFN pathways play a role in osteosarcoma and in 2-ME-mediated anti-proliferative effects, and therefore targeted induction of IFN signaling could lead to effective treatment strategies in the control of osteosarcoma.

2-methoxyestradiol-mediated anti-tumor effect increases osteoprotegerin expression in osteosarcoma cells.

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.

Osteitis fibrosa is mediated by Platelet-Derived Growth Factor-A via a phosphoinositide 3-kinase-dependent signaling pathway in a rat model for chronic hyperparathyroidism.

Abnormal secretion of PTH by the parathyroid glands contributes to a variety of common skeletal disorders. Prior studies implicate platelet-derived growth factor-A (PDGF-A) as an important mediator of selective PTH actions on bone. The present studies used targeted gene profiling and small-molecule antagonists directed against candidate gene products to elucidate the roles of specific PTH-regulated genes and signaling pathways. A group of 29 genes in rats continuously infused with PTH and cotreated with the PDGF receptor antagonist trapidil were differentially expressed compared with PTH treatment alone. Several of the identified genes were functionally clustered as regulators of fibroblast differentiation and extracellular matrix modeling, including the matrix cross-linking enzyme lysyl oxidase (LOX). Treatment with beta-aminopropionitrile, an irreversible inhibitor of LOX activity, dramatically reduced diffuse mineralization but had no effect on PTH-induced fibrosis. In contrast, the receptor tyrosine kinase inhibitor Gleevec and the phosphoinositide 3-kinase inhibitor wortmannin each reduced bone marrow fibrosis. In summary, the present studies support the hypotheses that PTH-induced bone marrow fibrosis is mediated by PDGF-A via a phosphoinositide 3-kinase-dependent signaling pathway and that increased LOX gene expression plays a key role in abnormal mineralization, a hallmark of chronic hyperparathyroidism.

2-methoxyestradiol-induced cell death in osteosarcoma cells is preceded by cell cycle arrest.

2-Methoxyestradiol (2-ME), a naturally occurring mammalian metabolite of 17beta-Estradiol (E2), induces cell death in osteosarcoma cells. To further understand the molecular mechanisms of action, we have investigated cell cycle progression in 2-ME-treated human osteosarcoma (MG63, SaOS-2 and LM7 [corrected]) cells. At 5 microM, 2-ME induced growth arrest by inducing a block in cell cycle; 2-ME-treatment resulted in 2-fold increases in G1 phase cells and a decrease in S phase cells in MG63 and SaOS-2 osteosarcoma cell lines, compared to the appropriate vehicle controls. 2-ME-treatment induced a threefold increase in the G2 phase in LM7 [corrected] osteosarcoma cells. The results demonstrated steroid specificity, as the tumorigenic metabolite, 16alpha-hydroxyestradiol (16-OHE), did not have any effect on cell cycle progression in osteosarcoma cells. The cell cycle arrest coincided with an increase in expression of the cell cycle markers p21, p27 and p53 proteins in 2-ME-treated osteosarcoma cells. Also, MG63 cells, transiently transfected with cDNA for a 'loss of function mutant' RNA-dependent protein kinase (PKR) protein, were resistant to 2-ME-induced cell cycle arrest. These results suggest that 2-ME works in concert with factors regulating cell cycle progression, and cell cycle arrest precedes cell death in 2-ME-treated osteosarcoma cells.