Data from Attenuation of Tumor Growth by Formation of Antiproliferative Glycosaminoglycans Correlates with Low Acetylation of Histone H3

<div>Abstract<p>Glycosaminoglycan (GAG) chains anchored to core proteins form proteoglycans, widely distributed cell-surface macromolecules with multiple functions, such as regulation of growth factor and cytokine signaling, cell-cell interactions, and uptake of biomolecules. The biosynthesis of GAG can be manipulated by xylosides attached to various hydrophobic groups, and we have earlier reported that a naphthoxyloside, 2-(6-hydroxynaphthyl) β-d-xylopyranoside (XylNapOH), which serves as a primer for GAG synthesis, reduces tumor load up to 97% <i>in vivo</i>, despite lower efficiency <i>in vitro</i>. Here we show, using radiolabeled xylosides and coculture experiments, that XylNapOH-treated bladder and breast carcinoma cells secrete antiproliferative GAG chains that are taken up by both normal and cancer cells and transported to the cell nuclei where they induce an antiproliferative effect, accompanied by apoptosis. We also show that XylNapOH treatment lowers the level of histone H3 acetylation selectively in bladder and breast carcinoma cells without affecting expression of histone H3. However, XylNapOH-primed GAG chains from normal cells are not internalized and do not cause growth retardation. Using <i>in vitro</i> and <i>in vivo</i> C6 glioma cell and tumor models, we show that XylNapOH is much more effective <i>in vivo</i> than <i>in vitro</i>. We propose that, <i>in vivo</i>, the antiproliferative XylNapOH-primed GAG chains produced by tumor cells inhibit tumor growth in an autocrine fashion by formation of antiproliferative GAG chains on the xyloside prodrug, whereas no antiproliferative GAG chains are produced by surrounding normal cells. This is a novel mechanism for targeting tumor cells, making these xylosides promising drug candidates for antitumor therapy. Cancer Res; 70(9); 3771–9. ©2010 AACR.</p></div>

Glycosaminoglycan (GAG) chains anchored to core proteins form proteoglycans, widely distributed cell-surface macromolecules with multiple functions, such as regulation of growth factor and cytokine signaling, cell-cell interactions, and uptake of biomolecules. The biosynthesis of GAG can be manipulated by xylosides attached to various hydrophobic groups, and we have earlier reported that a naphthoxyloside, 2-(6-hydroxynaphthyl) beta-D-xylopyranoside (XylNapOH), which serves as a primer for GAG synthesis, reduces tumor load up to 97% in vivo, despite lower efficiency in vitro. Here we show, using radiolabeled xylosides and coculture experiments, that XylNapOH-treated bladder and breast carcinoma cells secrete antiproliferative GAG chains that are taken up by both normal and cancer cells and transported to the cell nuclei where they induce an antiproliferative effect, accompanied by apoptosis. We also show that XylNapOH treatment lowers the level of histone H3 acetylation selectively in bladder and breast carcinoma cells without affecting expression of histone H3. However, XylNapOH-primed GAG chains from normal cells are not internalized and do not cause growth retardation. Using in vitro and in vivo C6 glioma cell and tumor models, we show that XylNapOH is much more effective in vivo than in vitro. We propose that, in vivo, the antiproliferative XylNapOH-primed GAG chains produced by tumor cells inhibit tumor growth in an autocrine fashion by formation of antiproliferative GAG chains on the xyloside prodrug, whereas no antiproliferative GAG chains are produced by surrounding normal cells. This is a novel mechanism for targeting tumor cells, making these xylosides promising drug candidates for antitumor therapy.

Osteosarcoma is the most common bone tumor associated with childhood and adolescence. The main characteristics of the osteosarcoma are the malignant transformation of the osteoblasts and the excessive accumulation of the partially mineralized extracellular matrix (ECM). The abundant content of the ECM modulates the microenvironment of the bone tissue and affects the normal function of the osteoblasts as well as the differentiation of the malignant cells. Proteoglycans (PGs) are one of the major classes of macromolecules located both at the cell membrane and the ECM. They consist of a protein core to which one or more sulfated glycosaminoglycan (GAG) chains are covalently bound. GAG chains are linear polymers composed of repeating dissacharide units consisting of a hexosamine and an uronic acid. The specific physicochemical characteristics with which PGs are endowed enable them to affect the organization of the ECM, as well as to participate in the regulation of several cellular events, including cell proliferation, adhesion and migration. These roles of PGs are perpetrated through specific interactions with target macromolecules or growth factors and these interactions may involve both their GAG chains and/or protein cores. PGs are major non-collagen component of the bone ECM. They are synthesized and secreted by osteoblasts and participate in the regulation of bone calcification. Furthermore, it has been suggested that cell membrane (mainly heparan sulfate containing) as well as secreted PGs participate in cell-cell and cell-ECM interactions and play important roles in the in the formation and the renewal of the bone tissue. Both the physicochemical characteristics and the alternations in the composition of the osteosarcoma ECM as well as the association of these changes with its differentiation and prognosis have not been adequately studied. The role of PGs in the mechanisms of osteosarcoma development is suggested to be important. The fine structural analysis of the proteoglycans’ GAG chains may contribute to the better understanding of these glycocomplexes, which participate in the regulation of numerous cellular events as well as in the cell malignant transformation. Therefore, the aim of this study was to contribute to the understanding of the role specific PGs / GAGs have in the pathology of the osteosarcoma, which is one of the most important human primary bone tumors. MG-63 and Saos 2 human osteosarcoma cell lines, endowed with high and low metastatic capability, and differing in their differentiation level, being medium and well differentiated, respectively were utilized. The synthesis and the distribution of GAGs among the cell membrane and the culture medium by these cell lines were studied. The obtained results showed that both cell lines synthesized extracellular hyaluronan (HA) and both extracellular and cell-associated galactosaminoglycans (GalAGs) and heparin sulfate (HS). Even though both cell lines synthesized considerable amounts of PGs, the Saos 2 cells produced HA, GalAGs and HS at considerably lower rates than the MG-63 cells. The role of genistein on the synthesis of these macromolecules has also been studied. Genistein is a well known specific inhibitor of the protein tyrosine kinase (PTK) and affects the proliferation of both cancer and normal cells. The inhibitory effect of genistein on the synthesis of both extracellularly secreted and cell-associated GAGs / PGs in Saos 2 cells was found to be dose-dependent and mediated most probably through a PTK mechanism. The synthesis of GAGs / PGs by the MG-63 cells in the presence of genistein was dependent on their type and localization suggesting that a more complex mechanism regulates their PG synthesis. It has been shown that growth factors such as the transforming growth factor (TGF-β2), basic fibroblast growth factor (bFGF) and the platelet derived growth factor...

Effect of glypican-1 covalently attached chains on turkey myogenic satellite cell proliferation, differentiation, and fibroblast growth factor 2 responsiveness

Beta-D-Xylosides are known to initiate or prime free glycosaminoglycan (GAG) chain synthesis in cell and tissue culture. As such, the effect of the venous antithrombotic beta-D-xyloside, naroparcil, was investigated on the plasma GAG profile in the rabbit after oral administration. Using dose-response experiments, we showed that antithrombin activity via antithrombin III and heparin cofactor II was increased in parallel with GAG plasma levels compared to control. A more detailed qualitative examination of plasma GAGs by cellulose acetate electrophoresis and ion-exchange chromatography, following oral administration of naroparcil at 400 mg/kg, revealed the presence of higher density charged molecules compared to control. The extracted GAGs were found to activate inhibition of thrombin by heparin cofactor II and contained approximately 25% of a dermatan sulfate-like compound (undetectable in control), which could be responsible for the antithrombotic effect. Using radiolabeled naroparcil, we found radiolabeled GAG fractions and the fact that naroparcil was a substrate for galactosyltransferase I, the second enzyme responsible for GAG chain polymerization, suggested that the compound could initiate in vivo the biosynthesis of antithrombotic free GAG chains. This is, to our knowledge, the first description of the in vivo effect of a beta-D-xyloside on GAG biosynthesis; furthermore, this is correlated with an antithrombotic action.

Proteoglycans are important components of the extracellular matrix of all tissues. Proteoglycans are comprised of a core protein and one or more covalently attached glycosaminoglycan (GAG) chains. The major chondroitin sulfate (CS) and dermatan sulfate (DS) proteoglycans are aggrecan, versican, biglycan and decorin. Cells synthesize GAGs of natural or basal lengths and the GAG chains are subject to considerable growth factor, hormonal and metabolic regulation to yield longer GAG chains with altered structure and function. The mechanism by which the CS/DS GAG chains are polymerized is unknown. Recent work has identified several monosaccharide transferases which when co-expressed yield GAG polymers and the length of the polymers depends upon the pair of enzymes coexpressed. The further extension of these chains is regulated by signaling pathways. Inhibition of these latter pathways may be a therapeutic target to prevent the elongation which is associated with increased binding of atherogenic lipids and the disease process of atherosclerosis.

Biosynthesis and functions of glycosaminoglycan(GAG) chains arecomplex and remain elusive. To better understand the factors thatregulate the biosynthesis and functions, fluorophore-tagged xylosidescarrying two different linkages between fluorophore and xylose residuewere synthesized and evaluated for their ability to prime GAG chainssuch as heparan sulfate (HS), chondroitin sulfate (CS), and dermatansulfate (DS) in various cell lines. These in vitro studies resultedin the identification of fluorophore-tagged xylosides that prime highmolecular weight GAG chains. Primed GAG chains carrying a fluorophoregroup has several advantages for studying the factors that regulatethe biosynthesis, analyzing intact fine structures at low detectionlimits, and setting the stage for studying structure–functionrelations of GAG chains of cellular origin.

Collagen fibrils in the dermis are bundled by glycosaminoglycan (GAG) chains of decorin, which contribute to its strength. The three-dimensional structure of collagen fibrils and GAG chains has been discussed on the basis of observations and experiments. This study uses scanning transmission electron microscope (STEM) tomography with high Z-axis resolution to analyze the three-dimensional structure of GAG chains in the dermis from a healthy individual and a patient with musculocontractural Ehlers-Danlos syndrome caused by pathogenic variants in CHST14 (mcEDS-CHST14). This observation revealed that the dermis from a healthy individual featured multiple GAG chains that wrapped around collagen fibrils and formed incomplete ring structures. However, in the dermis from a patient with mcEDS-CHST14, GAG chains were linear and did not form rings. Based on the relationship between collagen fibrils and GAG chains, we suggest the three-dimensional structure of normal GAG chains in a new model named the 'segmented ring-mesh model'. The interactions between collagen fibrils and GAG chains in this model also apply to the dermis of mcEDS-CHST14 patients, in which the GAG chain composition changes to become CS-rich and more linear. This change leads to an increased inter-fibrillar space, which inhibits the dense packing of collagen fibrils. These findings suggest that this phenomenon contributes to the skin fragility observed in mcEDS-CHST14 patients. Our study suggests the 'segmented ring-mesh model' of GAG chains is essential for the dense packing of collagen fibrils in normal dermis. STEM tomography is highly effective in analyzing the three-dimensional structure of collagen fibrils and GAG chains.

Syndecan-4 is a cell membrane heparan sulfate proteoglycan that is composed of a core protein and covalently attached glycosaminoglycans (GAG) and N-linked glycosylated (N-glycosylated) chains. Syndecan-4 has been shown to function independent of its GAG chains. Syndecan-4 may derive its biological function from the N-glycosylated chains due to the biological role of N-glycosylated chains in protein folding and cell membrane localization. The objective of the current study was to investigate the role of syndecan-4 N-glycosylated chains and the interaction between GAG and N-glycosylated chains in turkey myogenic satellite cell proliferation, differentiation, and fibroblast growth factor 2 (FGF2) responsiveness. The wild type turkey syndecan-4 and the syndecan-4 without GAG chains were cloned into the expression vector pCMS-EGFP and used as templates to generate syndecan-4 N-glycosylated one-chain and no-chain mutants with or without GAG chains. The wild type syndecan-4, all of the syndecan-4 N-glycosylated chain mutants were transfected into turkey myogenic satellite cells. Cell proliferation, differentiation, and responsiveness to FGF2 were measured. The overexpression of syndecan-4 N-glycosylated mutants with or without GAG chains did not change cell proliferation, differentiation, and responsiveness to FGF2 compared to the wild type syndecan-4 except that the overexpression of syndecan-4 N-glycosylated mutants without GAG chains increased cell proliferation at 48 and 72 h post-transfection. These data suggest that syndecan-4 functions in an FGF2-independent manner, and the N-glycosylated and GAG chains are required for syndecan-4 to regulate turkey myogenic satellite cell proliferation, but not differentiation.

Proteoglycans consist of a core protein substituted with one or more glycosaminoglycan (GAG) chains and execute versatile functions during many physiological and pathological processes. The biosynthesis of GAG chains is a complex process that depends on the concerted action of a variety of enzymes. Central to the biosynthesis of heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS) GAG chains is the formation of a tetrasaccharide linker region followed by biosynthesis of HS or CS/DS-specific repeating disaccharide units, which then undergo modifications and epimerization. The importance of these biosynthetic enzymes is illustrated by several severe pleiotropic disorders that arise upon their deficiency. The Ehlers-Danlos syndromes (EDS) constitute a special group among these disorders. Although most EDS types are caused by defects in fibrillar types I, III, or V collagen, or their modifying enzymes, a few rare EDS types have recently been linked to defects in GAG biosynthesis. Spondylodysplastic EDS (spEDS) is caused by defective formation of the tetrasaccharide linker region, either due to β4GalT7 or β3GalT6 deficiency, whereas musculocontractural EDS (mcEDS) results from deficiency of D4ST1 or DS-epi1, impairing DS formation. This narrative review highlights the consequences of GAG deficiency in these specific EDS types, summarizes the associated phenotypic features and the molecular spectrum of reported pathogenic variants, and defines the current knowledge on the underlying pathophysiological mechanisms based on studies in patient-derived material, in vitro analyses, and animal models.

Glypican-1 Is Frequently Overexpressed in Human Gliomas and Enhances FGF-2 Signaling in Glioma Cells

Xylosides are a group of compounds that can induce glycosaminoglycan (GAG) chain synthesis independently of a proteoglycan core protein. We have previously shown that the xyloside 2-(6-hydroxynaphthyl)β-D-xylopyranoside has a tumor-selective growth inhibitory effect both in vitro and in vivo, and that the effect in vitro was correlated to a reduction in histone H3 acetylation. In addition, GAG chains have previously been reported to inhibit histone acetyltransferases (HAT). To investigate if xylosides, or the corresponding xyloside-primed GAG chains, can be used as HAT inhibitors, we have synthesized a series of naphthoxylosides carrying structural motifs similar to the aromatic moieties of the known HAT inhibitors garcinol and curcumin, and studied their biological activities. Here, we show that the disubstituted naphthoxylosides induced GAG chain synthesis, and that the ones with at least one free phenolic group exhibited moderate HAT inhibition in vitro, without affecting histone H3 acetylation in cell culture. The xyloside-primed GAG chains, on the other hand, had no effect on HAT activity, possibly explaining why the effect of the xylosides on histone H3 acetylation was absent in cell culture as the xylosides were recruited for GAG chain synthesis. Further investigations are required to find xylosides that are effective HAT inhibitors or xylosides producing GAG chains with HAT inhibitory effects.

Proteoglycans (PGs) are complex glycoconjugates that are composed of a core protein and glycosaminoglycan (GAG) chains. The GAG chains are covalently bound to the serine residue of the core protein via a common core tetrasaccharide (glucuronic acid-galactose-galactose-xylose) as the linkage region. The endo-type glycosidases were investigated with the aim of performing enzymatic synthesis of PG. It is known that many glycosidases catalyze a transglycosylation reaction as a re-verse reaction in addition to their main hydrolysis reaction. Therefore, the transglycosylation mechanism of testicular hyaluronidase, which is an endo-β-N-acetylhexosaminidase, was investi-gated. It was found that disaccharides are successively released from the nonreducing terminal of a donor hyaluronic acid (HA) and rapidly transferred to the nonreducing terminal of an acceptor HA. It was also found that testicular hyaluronidase also acted on chondroitin (Ch), chondroitin 4-sulfate (Ch4S), chondroitin 6-sulfate (Ch6S), and other GAGs as well as HA. Therefore, by repeating the transglycosylation using suitable combinations of Ch, Ch4S, Ch6S, and other GAGs as acceptors and donors, it was possible to custom synthesize GAGs. It is likely that application of this system would facilitate artificial reconstruction of GAG moieties of PG. Subsequently, we found that an endo-β-xylosidase activity was present in rabbit liver. This enzyme specifically hydrolyzed the xylose-serine linkage between the core protein and GAG chains of PG, thereby allowing intact GAGs to be obtained. At present, we are studying the enzymatic transfer of the reconstructed GAG chains to the core protein using the transglycosylation activity of this enzyme.

The secretory pathway is blocked between the trans-Golgi and the plasma membrane during meiotic maturation in Xenopus oocytes

Proteoglycans consist of a core protein and an associated glycosaminoglycan (GAG) chain of heparan sulfate, chondroitin sulfate, dermatan sulfate or keratan sulfate, which are attached to a serine residue. The core proteins of cell surface proteoglycans may be transmembrane, e.g., syndecan, or GPI-anchored, e.g., glypican. Many different cell surface and matrix proteoglycan core proteins are expressed in the mammary gland and in mammary cells in culture. The level of expression of these core proteins, the structure of their GAG chains, and their degradation are regulated by many of the effectors that control the development and function of the mammary gland. Regulatory proteins of the mammary gland that bind GAG include many growth factors and morphogens (fibroblast growth factors, hepatocyte growth factor/scatter factor, members of the midkine family, wnts), matrix proteins (collagen, fibronectin, and laminin), enzymes (lipoprotein lipase) and microbial surface proteins. Structural diversity within GAG chains ensures that each protein-GAG interaction is as specific as necessary and a number of sequences of saccharides that recognize individual proteins have been elucidated. The GAG-protein interactions serve to regulate the signal output of growth factor receptor tyrosine kinase and hence cell fate as well as the storage and diffusion of extracellular protein effectors. In addition, GAGs clearly coordinate stromal and epithelial development, and they are active participants in mediating cell-cell and cell-matrix interactions. Since a single proteoglycan, even if it carries a single GAG chain, can bind multiple proteins, proteoglycans are also likely to act as multireceptors which promote the integration of cellular signals.