ascorbic-acid and Osteosarcoma

Vitamin C and iron are both important nutrients for humans and involved in several physiological processes. The biological activities of vitamin C and iron are based on their abilities to accept or donate electrons. Although vitamin C is well known as an excellent electron donor in physiological conditions, it also has pro-oxidant properties, especially with catalytic metal iron. Cancer cells have a higher iron requirement than normal cells, which allows pharmacological ascorbate to kill cancer cells selectively. In this study, we demonstrated that the levels of H

Topics: Ascorbic Acid; Bone Neoplasms; Cell Line, Tumor; Humans; Iron; Osteosarcoma; Oxidative Stress

Osteosarcoma (OS) is the most common malignant bone tumor and a leading cause of cancer-related deaths in children and adolescents. Current standard treatments for OS are a combination of preoperative chemotherapy, surgical resection, and adjuvant chemotherapy. Cisplatin is used as the standard chemotherapeutic for OS treatment, but it induces various adverse effects, limiting its clinical application. Improving treatment efficacy without increasing the cisplatin dosage is desirable. In the present study, we assessed the combined effect of ascorbate on cisplatin treatment using cultured human OS cells. Co-treatment with ascorbate induced greater suppression of OS cell but not nonmalignant cell proliferation. The chemosensitizing effect of ascorbate on cisplatin treatment was tightly linked to ROS production. Altered cellular redox state due to increased ROS production modified glycolysis and mitochondrial function in OS cells. In addition, OS cell sphere formation was markedly decreased, suggesting that ascorbate increased the treatment efficacy of cisplatin against stem-like cells in the cancer cell population. We also found that enhanced MYC signaling, ribosomal biogenesis, glycolysis, and mitochondrial respiration are key signatures in OS cells with cisplatin resistance. Furthermore, cisplatin resistance was reversed by ascorbate. Taken together, our findings provide a rationale for combining cisplatin with ascorbate in therapeutic strategies against OS.

Topics: Antineoplastic Agents; Ascorbic Acid; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Cisplatin; Drug Resistance, Neoplasm; Humans; Osteosarcoma; Oxidation-Reduction

The objective of this study is to understand the effect of sustained release of vitamin C from β-tricalcium phosphate (β-TCP) scaffold on proliferation, viability and differentiation of human fetal osteoblast cells (hFOB). The influence of pH, drug concentration, and presence of polymer on the sustained release of vitamin C from polycaprolactone (PCL) coated β-TCP scaffolds are studied. Prolonged and sustained release of vitamin C, over 60 days is observed in PCL coated β-TCP scaffolds compared to uncoated scaffolds. Presence of PCL helps to minimize the burst release of vitamin C from β-TCP scaffolds in the initial 24 h of release. To evaluate the osteogenic potential of vitamin C incorporated β-TCP scaffolds, osteoblast cells are cultured and cell morphology, proliferation, viability, and differentiation are assessed. Morphological characterization shows layer like osteoblast cell attachment in the presence of vitamin C compared to the control. MTT cell viability assay shows 2 folds increase in osteoblast cell density in the presence of vitamin C after 3,7 and 11 days of culture. Furthermore, increased ALP activity at 11 days of culture indicates the possible role of vitamin C on osteoblast differentiation. Additionally, a preliminary study shows vitamin C loaded scaffolds suppress osteosarcoma (MG-63) cell proliferation to 4 folds after 3 days compared to control. These results show a sustained release of vitamin C from PCL coated β-TCP scaffolds improve proliferation, viability, and differentiation of osteoblasts cell as well as mitigate osteosarcoma cell proliferation, suggesting its potential application as synthetic bone graft substitutes in tissue engineering application.

Topics: Ascorbic Acid; Bone Neoplasms; Calcium Phosphates; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Delayed-Action Preparations; Humans; Osteoblasts; Osteosarcoma

To evaluate the potential involvement of redox-specific signalling pathways in cold atmospheric plasma (CAP)-induced apoptosis on human osteosarcoma cells.. Osteosarcoma cell lines were treated with CAP with or without antioxidative agents and seeded in cell culture plates. Cell proliferation was determined by counting viable cells. Carrier gas-treated cells served as control. Peroxiredoxin (PRX) 1-3 expression and secretion were assessed.. CAP treatment exhibited strongly attenuated proliferation rates. This effect was significantly attenuated by the addition of N-acetylcysteine (NAC). CAP-treated cells exhibited an increase of PRX 1 and 2 10 sec after treatment. The ratio of oxidized to reduced PRX1 and PRX2 was significantly altered with increasing cellular concentration of the oxidized dimer.. Antioxidant supplementation with NAC increases proliferation of CAP-treated osteosarcoma cells, implicating an involvement of redox signalling. Activation of PRX1 and -2 indicate CAP affects redox homeostasis.

Topics: Apoptosis; Ascorbic Acid; Atmosphere; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Homeostasis; Humans; Osteosarcoma; Oxidation-Reduction; Peroxiredoxins; Plasma Gases; Protein Multimerization; Signal Transduction

Osteosarcoma originates from mesenchymal stem cells with impaired bone differentiation. In the present study we investigated the effect of ascorbic acid (AsA) on osteogenic differentiation and apoptosis of the MG-63 osteosarcoma cell line.. We evaluated the expression of runt-related transcription factor-2 (RUNX2) and secreted phosphoprotein 1 (SPP1) genes by real-time Polymerase Chain Reaction (PCR) and of endogenous bone morphogenetic protein-2 (BMP2) and osteocalcin proteins by immunohistochemistry. We analyzed osteoblast maturation by phosphatase alkaline synthesis and calcium deposition, and apoptosis by (TUNEL) test and Annexin staining.. Our results showed that RUNX2 and SPP1 gene expression was increased in cells treated with low concentrations of AsA with respect to untreated cells. At higher concentrations, AsA induced apoptosis of osteosarcoma cells, possibly with the involvement of p21.. Our findings support the ability of AsA to induce both differentiation, by affecting the target involved in early and late phases of osteogenic maturation, and apoptosis in poorly-differentiated osteosarcoma cells.

Topics: Antineoplastic Agents; Antioxidants; Apoptosis; Ascorbic Acid; Bone Morphogenetic Protein 2; Bone Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Lineage; Cell Survival; Core Binding Factor Alpha 1 Subunit; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Osteopontin; Osteosarcoma

Osteosarcoma (OS) is a highly aggressive bone cancer affecting children and young adults. Growing evidence connects the invasive potential of OS cells with their ability to form invadopodia (structures specialized in extracellular matrix proteolysis).. In this study, we tested the hypothesis that commonly used in vitro stimulators of mineralization limit the invadopodia formation in OS cells. Here we examined the invasive potential of human osteoblast-like cells (Saos-2) and osteolytic-like (143B) OS cells treated with the stimulators of mineralization (ascorbic acid and B-glycerophosphate) and observed a significant difference in response of the tested cells to the treatment. In contrast to 143B cells, osteoblast-like cells developed a mineralization phenotype that was accompanied by a decreased proliferation rate, prolongation of the cell cycle progression and apoptosis. On the other hand, stimulators of mineralization limited osteolytic-like OS cell invasiveness into collagen matrix. We are the first to evidence the ability of 143B cells to degrade extracellular matrix to be driven by invadopodia. Herein, we show that this ability of osteolytic-like cells in vitro is limited by stimulators of mineralization.. Our study demonstrates that mineralization competency determines the invasive potential of cancer cells. A better understanding of the molecular mechanisms by which stimulators of mineralization regulate and execute invadopodia formation would reveal novel clinical targets for treating osteosarcoma.

Topics: Ascorbic Acid; Bone Neoplasms; Calcification, Physiologic; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Surface Extensions; Cell Survival; Collagen Type I; Glycerophosphates; Humans; Neoplasm Invasiveness; Osteosarcoma; Phenotype; Proteolysis

To further understand the synergistic mechanism of As2O3 and asscorbic acid (AA) in human osteosarcoma MG-63 cells by systems biology analysis.. Human osteosarcoma MG-63 cells were treated by As2O3 (1 µmol/L), AA (62.5 µmol/L) and combined drugs (1 µmol/L As2O3 plus 62.5 µmol/L AA). Dynamic morphological characteristics were recorded by Cell-IQ system, and growth rate was calculated. Illumina beadchip assay was used to analyze the differential expression genes in different groups. Synergic effects on differential expression genes (DEGs) were analyzed by mixture linear model and singular value decomposition model. KEGG pathway annotations and GO enrichment analysis were performed to figure out the pathways involved in the synergic effects.. We captured 1987 differential expression genes in combined therapy MG-63 cells. FAT1 gene was significantly upregulated in all three groups, which is a promising drug target as an important tumor suppressor analogue; meanwhile, HIST1H2BD gene was markedly downregulated in the As2O3 monotherapy group and the combined therapy group, which was found to be upregulated in prostatic cancer. These two genes might play critical roles in synergetic effects of AA and As2O3, although the exact mechanism needs further investigation. KEGG pathway analysis showed many DEGs were related with tight junction, and GO analysis also indicated that DEGs in the combined therapy cells gathered in occluding junction, apical junction complex, cell junction, and tight junction.. AA potentiates the efficacy of As2O3 in MG-63 cells. Systems biology analysis showed the synergic effect on the DEGs.

Topics: Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Ascorbic Acid; Bone Neoplasms; Cell Line, Tumor; Drug Synergism; Drug Therapy, Combination; Humans; Osteosarcoma; Oxides; Systems Biology

Enhanced supplementation with certain naturally occurring nutrients and vitamins has been associated with a reduction in occurrence and progression of human cancer. The exact mechanisms of this action are still under investigation. Extracellular matrix (ECM) plays a key role in the development of cancer. Therefore, we studied whether nutrients could exert anticancer effects through alteration of ECM biological properties. Confluent cultures of normal human dermal fibroblasts were allowed to produce and deposit ECM during a 7 d incubation period in the presence of tested compounds. Subsequently, fibroblasts were removed and the growth rates of freshly placed cultures of human osteosarcoma cells (lines U2-OS, MMNG, or SK.ES1) on pre-formed ECM were assayed in plain cell growth medium. In addition, ECM was deposited by fibroblasts on the upper surface of a porous plastic membrane and the subsequent migration of osteosarcoma cells to the other side was assayed in non-supplemented medium. The results demonstrated that the ECM produced by normal fibroblasts treated with a mixture of ascorbic acid, lysine, proline, arginine, cysteine, and green tea polyphenols significantly reduced the growth rate and invasive activity of osteosarcoma cells in contrast to the non-supplemented control. The changes in ECM properties were accompanied by significant changes in ECM protein and glycosaminoglycan composition as assayed immunochemically. We conclude that anticancer effects of nutrients involve beneficial changes in ECM biological properties. The role of ECM components in ECM-dependent regulation of tumor cell activities is discussed.

Topics: Amino Acids; Antineoplastic Combined Chemotherapy Protocols; Ascorbic Acid; Bone Neoplasms; Camellia sinensis; Cell Line, Tumor; Cell Proliferation; Collagen; Extracellular Matrix; Fibroblasts; Humans; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Osteosarcoma; Plant Extracts

Structural changes in the extracellular matrix (ECM) are necessary for cell migration during tissue remodeling. MMPs, VEGF, Ki-67 (proliferative protein), and constituents of ECM play a critical role in angiogenesis and underlie neoplastic invasion and metastasis. This prompted us to investigate the effect of a diet containing lysine, proline, arginine, ascorbic acid, and green tea extract (NM) on the growth of tumors induced by implanting human osteosarcoma MNNG in athymic nude mice and the expression of MMPs, VEGF, Ki-67 and fibronectin in these tumors, as well as the production of mucin (by PAS staining). We also investigated the effect of the supplemented diet on serum ascorbic acid, total protein content, alkaline phosphatase activity, and liver enzymes. Athymic male nude mice (n = 12) were inoculated with 3 x 10(6) osteosarcoma cells MNNG-HOS and randomly divided into group A (fed a regular diet) and group B (fed a regular diet supplemented with 0.5% NM). Four weeks later, the mice were sacrificed. Results showed that NM inhibited the growth and reduced the size of tumors in nude mice. Histological evaluation revealed increased mitotic index, MMP-9, and VEGF secretion in the control group tissues. Results demonstrate that the nutrient mixture of lysine, proline, arginine, ascorbic acid, and green tea extract tested strongly suppressed the growth of tumors without adverse effects in nude mice, suggesting potential as an anticancer agent.

Topics: Animals; Antioxidants; Ascorbic Acid; Cell Line, Tumor; Humans; Immunohistochemistry; Lysine; Male; Mice; Mice, Nude; Neoplasm Transplantation; Osteosarcoma; Plant Extracts; Proline; Tea

Current treatment of osteosarcoma is associated with poor prognosis, especially due to the increased risk of developing other cancers with chemotherapy. Therefore, new, safe and effective treatment strategies are needed. We investigated the effect of a unique mixture of nutrients containing lysine, proline, arginine, ascorbic acid, and epigallocatechin gallate (EGCG) on human osteosarcoma cell lines U-2OS, MNNG-HOS, and Ewing's sarcoma SK-ES-1 by measuring: cell proliferation, expression of matrix metalloproteinase-2 (MMP-2), MMP-9, and invasive and angiogenesis potential. Cell proliferation was evaluated by MTT assay, matrix metalloproteinases (MMP) expression by gelatinase zymography, VEGF expression by ELISA, and invasion through Matrigel. Cells were also treated with phorbol 12-myristate 13-acetate (PMA) to study enhanced MMP and VEGF expression. The invasion of osteosarcoma U-2OS and MNNG-HOS cells through Matrigel was significantly reduced in a dose-dependent fashion, with 100% inhibition of invasion of U-2OS cells at 100 microg/ml, and MNNG cells at 50 microg/ml concentration of the synergistically acting nutrient mixture. Ewing's sarcoma SK-ES-1 cells were not invasive. Nutrient synergy (NS) exhibited a dose response antiproliferative effect on osteosarcoma U-2OS cells, reaching 67% at 1000 microg/ml of NS; no significant suppression of cell proliferation was seen with MNNG or Ewing's sarcoma cells. Zymography showed dose-dependent inhibition of MMP secretion by all three cell lines in the presence of NS. VEGF secretion by U-2OS cells was completely blocked at 500 microg/ml of NS. Our results suggest NS is an excellent candidate for therapeutic use in the treatment of osteosarcoma, by inhibiting cancer cell invasion, and secretion of MMPs and VEGF, all critical parameters for cancer control and prevention.

Topics: Arginine; Ascorbic Acid; Bone Neoplasms; Catechin; Cell Proliferation; Dose-Response Relationship, Drug; Humans; Lysine; Matrix Metalloproteinases; Neovascularization, Pathologic; Nutritional Physiological Phenomena; Osteosarcoma; Proline; Sarcoma, Ewing; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A

Degradation of extracellular matrix (ECM) is a hallmark of tumor invasion, metastasis and angiogenesis. Based on the Rath multitargeted approach to cancer using natural substances to control ECM stability and enhancing its strength, we developed a novel formulation (NM) of lysine, proline, ascorbic acid and green tea extract that has shown significant anti-cancer activity against a number of cancer cell lines. The aim of the present study was to determine whether NM exhibits anti-angiogenic and anti-metastatic effects using in vitro and in vivo experimental models. Angiogenesis was measured using a chorioallantoic membrane (CAM) assay in chick embryos and bFGF-induced vessel growth in C57BL/6J female mice. To determine the in vivo effect of NM on the tumor xenograft growth, male nude mice were inoculated with 3 x 10(6) MNNG-HOS cells. Control mice were fed a mouse chow diet, while the test group was fed a mouse chow diet supplemented with 0.5% NM for 4 weeks. In vitro studies on cell proliferation (MTT assay), MMP expression (zymography) and Matrigel invasion were conducted on human osteosarcoma U2OS, maintained in McCoy medium, supplemented with 10% FBS, penicillin and streptomycin in 24-well tissue culture plates and tested with NM at 0, 10, 50, 100, 500, and 1000 microg/ml in triplicate at each dose. NM at 250 microg/ml caused a significant (p<0.05) reduction in bFGF-induced angiogenesis in CAM. NM inhibited tumor growth of osteosarcoma MNNG-HOS cell xenografts in nude mice by 53%; furthermore, tumors in NM-treated mice were less vascular and expressed lower levels of VEGF and MMP-9 immunohistochemically than tumors in the control group. In addition, NM exhibited a dose-dependent inhibition of osteosarcoma U2OS cell proliferation (up to 60% at 1000 microg/ml), MMP-2 and -9 expression (with virtual total inhibition at 500 microg/ml NM), and invasion through Matrigel (with total inhibition at 100 microg/ml NM). Moreover, NM decreased U2OS cell expression of VEGF, angiopoietin-2, bFGF, PDGF and TGFbeta-1. These results together with our earlier findings suggest that NM is a relatively non-toxic formulation, which inhibits growth, invasion, metastasis, and angiogenesis of tumor cells.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Ascorbic Acid; Cell Line, Tumor; Cell Proliferation; Chick Embryo; Chorioallantoic Membrane; Collagen; Dose-Response Relationship, Drug; Drug Combinations; Extracellular Matrix; Fibroblast Growth Factor 2; Formaldehyde; Humans; Immunohistochemistry; In Vitro Techniques; Laminin; Lysine; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; Osteosarcoma; Plant Extracts; Platelet-Derived Growth Factor; Proline; Proteoglycans; Tea; Tetrazolium Salts; Thiazoles; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

Bone morphogenetic protein-7 (BMP-7) affects differentiation of preosteoblasts enabling the resultant cells to respond optimally to acutely acting regulators. As the phosphoinositide cascade and, particularly, the calcium-mobilizing inositol 1,4,5-trisphosphate (InsP3) receptor are integral to stimulus-secretion coupling in osteoblasts, the hypothesis that BMP-7 affects InsP3 receptor expression was examined in the G-292 human osteosarcoma cell line and in primary cultures of human osteoblasts. G-292 osteosarcoma cells were found to be a valid experimental model for primary human osteoblasts, expressing osteoblastic mRNAs encoding osteocalcin, bone sialoprotein, alkaline phosphatase, alpha1-collagen, epidermal growth-factor receptor, and BMP type II receptor. When cultured long term in the presence of ascorbic acid and beta-glycerophosphate, G-292 cells underwent further osteoblastic differentiation, forming nodules and exhibiting restricted mineralization. G-292 cells responded to BMP-7 with an increase in InsP3 receptor density. Ligand-binding studies established that BMP-7 (50 ng/ml) treatment of G-292 cells increased InsP3 receptor density 2.4-fold with no apparent change in affinity. Immunoblot analysis with antibodies specific for type I, type II, and type III InsP3 receptors revealed that BMP-7 (50 ng/ml) treatment resulted in a specific increase (206+/-8%) in the type I receptor. Reverse transcription-polymerase chain reaction and Northern blot analyses of G-292 and primary human osteoblasts confirmed an increase in type I InsP3 receptor mRNA upon BMP-7 treatment. These results demonstrate that G-292 cells respond to BMP-7 with an increase InsP3 receptor density, consistent with the enhanced capacity of these cells to respond to Ca2+-mobilizing secretory hormones during osteoblast differentiation.

Topics: Alkaline Phosphatase; Ascorbic Acid; Blotting, Northern; Bone Morphogenetic Protein 7; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Proteins; Calcification, Physiologic; Calcium Channels; Cell Differentiation; Cells, Cultured; Collagen; ErbB Receptors; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Glycerophosphates; Humans; Immunoblotting; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Integrin-Binding Sialoprotein; Osteoblasts; Osteocalcin; Osteosarcoma; Phosphatidylinositols; Polymerase Chain Reaction; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Receptors, Growth Factor; RNA, Messenger; Sialoglycoproteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta1 (TGF-beta1) induces increased extracellular matrix deposition. Bone morphogenetic protein-1 (BMP-1) also plays key roles in regulating vertebrate matrix deposition; it is the procollagen C-proteinase (PCP) that processes procollagen types I-III, and it may also mediate biosynthetic processing of lysyl oxidase and laminin 5. Here we show that BMP-1 is itself up-regulated by TGF-beta1 and that secreted BMP-1, induced by TGF-beta1, is either processed to an active form or remains as unprocessed proenzyme, in a cell type-dependent manner. In MG-63 osteosacrcoma cells, TGF-beta1 elevated levels of BMP-1 mRNA approximately 7-fold and elevated levels of mRNA for mammalian tolloid (mTld), an alternatively spliced product of the BMP1 gene, to a lesser extent. Induction of RNA was dose- and time-dependent and cycloheximide-inhibitable. Secreted BMP-1 and mTld, induced by TGF-beta1 in MG-63 and other fibrogenic cell cultures, were predominantly in forms in which proregions had been removed to yield activated enzyme. TGF-beta1 treatment also induced procollagen N-proteinase activity in fibrogenic cultures, while expression of the procollagen C-proteinase enhancer (PCPE), a glycoprotein that stimulates PCP activity, was unaffected. In contrast to fibrogenic cells, keratinocytes lacked detectable PCPE under any culture conditions and were induced by TGF-beta1 to secrete BMP-1 and mTld predominantly as unprocessed proenzymes.

Topics: Amino Acid Sequence; Ascorbic Acid; Bone Morphogenetic Protein 1; Bone Morphogenetic Proteins; Enhancer Elements, Genetic; Humans; Keratinocytes; Metalloendopeptidases; Metalloproteases; Molecular Sequence Data; Osteosarcoma; Procollagen N-Endopeptidase; Proteins; RNA, Messenger; Tolloid-Like Metalloproteinases; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

The biosynthesis of type VI collagen was studied in human glioblastoma cell line, U-87 MG. The effects of ascorbic acid on type VI collagen synthesis and secretion were investigated. After ascorbic acid treatment, type VI collagen in cell layers increased from 4.48% in control to 6.63% in the ascorbic acid treated cultures, an increase of 48%. The effect of ascorbic acid on type VI collagen synthesized by glioblastoma cells was lower than that reported for osteosarcoma cells (Engvall et al., 1986). The reason for these differences is still under investigation. The function of type VI collagen in glioblastoma cells is still unknown. We utilized the collagen gel system to elucidate the possible roles of type VI collagen in glioblastoma cells in vitro. Glioblastoma cells in collagen gels showed a stellate shape with long, branched processes in all directions. The strong positive reactivity of type VI collagen detected on cell bodies and cell processes by anti-type VI collagen antibody indicated that this specific collagen was associated with cell surfaces and processes, without releasing or diffusing into the gels. Type VI collagen was directly involved in the cell process extension. When living cells were treated with anti-type VI collagen antibody, a variation of cell morphology was observed. Instead of a stellate shape with processes, cells formed clusters without or with very short processes. These data suggest that type VI collagen, synthesized and secreted by glioblastoma cells, may play a role in tumor cell adhesion and spreading, and enhance cell process extension, penetration, and invasion into collagen gels.

Topics: Antibodies; Artificial Organs; Ascorbic Acid; Bone Neoplasms; Brain Neoplasms; Cell Adhesion; Cell Membrane; Cell Shape; Cell Surface Extensions; Collagen Type VI; Extracellular Matrix; Gels; Glioblastoma; Humans; Models, Biological; Neoplasm Invasiveness; Osteosarcoma; Up-Regulation

Mineralization occurred both in fetal rat calvarial cells and UMR 106 osteoblastic cells when they were cultured in medium containing L-ascorbate and beta-glycerophosphate as evidenced by von Kóssa staining as well as deposition of calcium ions and inorganic phosphate in the cells. When compared with corresponding non-mineralized cell cultures, both the mineralized cultures of calvarial cells and UMR 106 cells did not exhibit any change in intracellular bone-specific alkaline phosphatase activities which were measured by wheatgerm lectin precipitation method. Our results support the hypothesis that mineralization may not exert any direct negative feedback on matrix protein synthesis in osteoblasts during bone formation.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Calcification, Physiologic; Calcium; Culture Media; Fetus; Glycerophosphates; Osteoblasts; Osteosarcoma; Phosphates; Rats; Skull; Tumor Cells, Cultured

We compared the procollagen synthetic properties of MG-63 osteosarcoma cells with those of cultured human skin fibroblasts. In both cells, the expressions of type I and III procollagens are largely dependent on the constant presence of ascorbate and coordinately decreased by the neutral polymer dextran T-40. The amino-terminal propeptides of pro-alpha 1 and pro-alpha 2 chains of type I procollagen are phosphorylated and those of the pro-alpha 1 and pN-alpha 1 chains of type III procollagen both phosphorylated and sulfated, there being no difference in net charge in the propeptides between these cell types. The major differences between MG-63 and normal fibroblasts are the exceptionally high relative synthesis of type III procollagen by MG-63 cells, up to about 40% of the total of types I and III (6% in cultured skin fibroblasts), and the inability of ascorbate-supplemented MG-63 cells to deposit collagens into an insoluble pericellular matrix. A longer dextran treatment shifts up to one-fourth of the proline-labeled extracellular macromolecules into the matrix fraction within 4 days (in control 4%). Despite processing of the procollagens to the respective collagens in the matrix, neither control matrices nor those induced by dextran induced increased production of alkaline phosphatase. In cultures up to 4 days postconfluence the proportion of type III collagen produced tended to increase over that in early confluent cultures. With respect to collagen production, the MG-63 cell line is not a representative of the osteoblast lineage but rather resembles a proliferative wound fibroblast.

Topics: Ascorbic Acid; Cell Line; Extracellular Matrix; Fibroblasts; Humans; Osteosarcoma; Procollagen; Skin; Tumor Cells, Cultured

Osteoblasts possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na(+)-dependent ascorbate transporter located in the plasma membrane. The transporter is specific for ascorbate and stereoselective for L-ascorbate over D-isoascorbate. The present study examined the effects of ascorbate supplementation and deprivation on the activity of this transport system. L-ascorbate transport activity was determined by measuring uptake of the vitamin by ROS 17/2.8 osteosarcoma cells during 1 minute incubations with 5 microM L-[14C]ascorbate. The initial rate of L-[14C]ascorbate uptake by ROS 17/2.8 cells grown for 18 h in L-ascorbate-replete medium was 89 +/- 8 nmol/g protein per minute. Following removal of L-ascorbate from the growth medium, the initial rate of uptake increased within 6 h to 126 +/- 13 nmol/g protein per minute. Conversely, the initial rate of uptake by cells grown in ascorbate-free medium decreased following the addition of L-ascorbate, but not D-isoascorbate, to the medium. The effect of ascorbate pretreatment was specific for ascorbate transport in that preincubation of cultures with L-ascorbate did not affect uptake of 2-deoxy-D-glucose. Kinetic analysis revealed that modulation of ascorbate transport arose from changes in the apparent maximum rate of transport (Vmax) without changes in the affinity of the transport system for L-ascorbate. These experiments are the first to show that ascorbate transport by osteoblastic cells responds to vitamin C deprivation and supplementation. Adaptation of transport activity to substrate availability may play an important role in the physiological regulation of intracellular ascorbate levels.

Topics: Ascorbic Acid; Biological Transport, Active; Humans; Osteoblasts; Osteosarcoma; Tumor Cells, Cultured

Ascorbate (reduced vitamin C) is required for bone formation. We have shown previously that both the osteoblast-like cell line ROS 17/2.8 and primary cultures of rat calvarial cells possess a saturable, Na(+)-dependent uptake system for L-ascorbate (J Membr Biol 111:83-91, 1989). The purpose of the present study was to investigate the specificity of this transport system for organic anions and its sensitivity to transport inhibitors. Initial rates of ascorbate uptake were measured by incubating ROS 17/2.8 cells with [L-14C]ascorbate at 37 degrees C. Uptake of [L-14C]ascorbate (5 microM) was inhibited 98 +/- 1% by coincubation with unlabeled L-ascorbate (3 mM) and 48 +/- 4% by salicylate (3 mM), but it was not affected by 3 mM formate, lactate, pyruvate, gluconate, oxalate, malonate, or succinate. Uptake of the radiolabeled vitamin also was not affected by acute (1 minute) exposure of the cells to the Na+ transport inhibitors amiloride and ouabain or the glucose transport inhibitor cytochalasin B. In contrast, anion transport inhibitors rapidly (less than 1 minute) and reversibly blocked [L-14C]ascorbate uptake. In order of potency, these drugs were 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) approximately equal to sulfinpyrazone greater than furosemide approximately equal to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). These findings indicate that the ascorbate transporter is relatively specific for the ascorbate anion, since other organic anions (with the exception of salicylate) did not compete with ascorbate for uptake. Rapid and reversible inhibition by the impermeant antagonists DIDS and SITS suggests that they interact directly with the ascorbate transporter, consistent with location of the transport system in the plasma membrane.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Ascorbic Acid; Biological Transport; Cells, Cultured; Furosemide; Kinetics; Osteoblasts; Osteosarcoma; Salicylates; Salicylic Acid; Sulfinpyrazone; Tumor Cells, Cultured

Among several bioactive substances known as coupling factors, transforming growth factor-beta (TGF-beta), interleukin-1 (IL-1), and prostaglandin (PG) E1 and E2 increased not only the activity of alkaline phosphatase but also the rate of incorporation of 45Ca2+ into ROS 17/2.8 during a 3-day culture: the former two factors are known to be formed at the site where bone is resorbed, while PG's are known as one of the factors involved in bone resorption. Parathyroid hormone, another hormone that affects bone metabolism, elevated the incorporation of 45Ca2+ by and decreased the alkaline phosphatase activity of the cells. The facts indicate the possibility that the osteoblastic cells are involved in the transport of calcium ions when bones are being resorbed. On the other hand, when these osteosarcoma cells were cultured in DMEM containing ascorbate and beta-glycerophosphate, followed by staining with silver nitrate by the procedure of von Kossa, there appeared many groups of cells that were positively stained as dark brown spots. Cells were then cultured under the same conditions in the presence of radioactive calcium, and the radioactivity accumulated was measured. The result showed that the presence of both ascorbate and beta-glycerophosphate in the culture medium dramatically increased the accumulation of 45Ca2+. It appears from these facts that ROS 17/2.8 cells are capable of incorporating and/or accumulating calcium ion if they are cultured under appropriate conditions. These cells will probably be able to produce a calcified matrix in vitro.

Topics: Alkaline Phosphatase; Ascorbic Acid; Bone Resorption; Calcium; Culture Media; Glycerophosphates; Humans; Osteosarcoma; Tumor Cells, Cultured

Induction by interferon-gamma of indoleamine 2,3-dioxygenase (a tryptophan degradation enzyme) was examined with 11 human cell lines. The enzyme induction was demonstrated in 7 of the 11 cell lines. The induced enzyme in each of the 7 cell lines was identical to the enzyme purified from human placenta, as evidenced by immunoblot analysis with a monoclonal antibody specific to the placental one. The extent of the induction varied largely with the cell line; a relatively high induction was observed with HEL (lung fibroblasts), NY (osteosarcoma), and A-431 (epidermoid carcinoma). The enzyme induction was dependent on the concentration of interferon-gamma and occurred 12-18 h after addition of interferon-gamma to the cultures. Interferon-alpha or -beta was completely ineffective in this induction. Interferon-gamma inhibited the growth of the 7 cell lines observed with the enzyme induction, and this growth inhibition was accompanied with a complete deletion of tryptophan (less than 1 microM) in the culture medium by the induction of the enzyme. For two of these cell lines, the inhibition was partially reversed by an addition of exogenous tryptophan to the medium not to be depleted. These findings indicated that the growth inhibition by interferon-gamma was in part explained by the tryptophan depletion in the medium caused by the enzyme induction.

Topics: Ascorbic Acid; Carcinoma, Squamous Cell; Enzyme Induction; Humans; Immunosorbent Techniques; Interferon-gamma; Lung; Methylene Blue; Osteosarcoma; Oxygenases; Recombinant Proteins; Tryptophan; Tryptophan Oxygenase; Tumor Cells, Cultured

L-ascorbic acid at physiological concentrations (10 micrograms/ml) increased alkaline phosphatase activity in the osteoblastlike rat osteosarcoma cell line, UMR-106. The increase was dose-dependent and detectable at 6 hours after the addition of 100 micrograms/ml ascorbic acid to the medium. Treatment of the cells with 100 micrograms/ml ascorbic acid potentiated the response of cAMP to both PTH and PGE1, while cell growth was inhibited. Furthermore, the number of colonies formed by the cells grown in the soft agar was significantly reduced by increasing concentrations of ascorbic acid. These results indicate that ascorbic acid might play some role in the differentiation of osteoblasts.

Topics: Alkaline Phosphatase; Alprostadil; Animals; Ascorbic Acid; Cell Division; Cell Line; Cyclic AMP; Kinetics; Osteosarcoma; Parathyroid Hormone; Rats

Monoclonal antibodies reactive with the tissue form of type VI collagen were used to isolate the type VI collagen polypeptides from cultured fibroblasts and muscle cells. Two [35S]methionine-labeled polypeptides of 260 and 140 kD were found intracellularly, in the medium, and in the extracellular matrix of metabolically labeled cells. These polypeptides were disulfide cross-linked into very large complexes. The 260- and 140-kD polypeptides were intimately associated and could not be separated from each other by reduction without denaturation. In the absence of ascorbic acid, both polypeptides accumulated inside the cell, and their amounts in the medium and in the matrix were decreased. These results suggest that both the 260- and the 140-kD polypeptides are integral parts of the type VI collagen molecule. Examination of type VI collagen isolated from the intracellular pool by electron microscopy after rotary shadowing revealed structures corresponding to different stages of assembly of type VI collagen. Based on these images, a sequence for the intracellular assembly of type VI collagen could be discerned. Type VI collagen monomers are approximately 125 nm long and are composed of two globules separated by a thin strand. The monomers assemble into dimers and tetramers by lateral association. Only tetramers were present in culture media, whereas both tetramers and multimers were found in extracellular matrix extracts. The multimers appeared to have assembled from tetramers by end-to-end association into filaments that had prominent knobs and a periodicity of approximately 110 nm. These results show that, unlike other collagens, type VI collagen is assembled into tetramers before it is secreted from the cells, and they also suggest an extracellular aggregation mechanism that appears to be unique to this collagen.

Topics: Animals; Antibodies, Monoclonal; Ascorbic Acid; Cells, Cultured; Collagen; Extracellular Matrix; Fibroblasts; Humans; Leiomyosarcoma; Muscles; Osteosarcoma; Protein Processing, Post-Translational; Rats; Rhabdomyosarcoma