T. Tsegenidis

Glycosaminoglycans: key players in cancer cell biology and treatment

Glycosaminoglycans are natural heteropolysaccharides that are present in every mammalian tissue. They are composed of repeating disaccharide units that consist of either sulfated or non‐sulfated monosaccharides. Their molecular size and the sulfation type vary depending on the tissue, and their state either as part of proteoglycan or as free chains. In this regard, glycosami‐noglycans play important roles in physiological and pathological conditions. During recent years, cell biology studies have revealed that glycosaminoglycans are among the key macromolecules that affect cell properties and functions, acting directly on cell receptors or via interactions with growth factors. The accumulated knowledge regarding the altered structure of glycosaminoglycans in several diseases indicates their importance as biomarkers for disease diagnosis and progression, as well as pharmacological targets. This review summarizes how the fine structural characteristics of glycosaminoglycans, and enzymes involved in their biosynthesis and degradation, are involved in cell signaling, cell function and cancer progression. Prospects for glycosaminoglycan‐based therapeutic targeting in cancer are also discussed.

Ion-pair high-performance liquid chromatography for determining disaccharide composition in heparin and heparan sulphate.

In this report we describe a convenient and sensitive HPLC method for separating and determining the non- and variously sulphated delta-disaccharides derived from heparan sulphate, heparin and Fragmin, using heparin- and heparan sulphate lyases. This method is superior to others since it can separate and determine twelve different non-, mono-, di- and trisulphated delta-disaccharides containing either N-sulphated, N-acetylated or unsubstituted glucosamine in a single HPLC run. The various types of delta-disaccharides are separated by an ion-pair reversed-phase chromatographic procedure on a Supelcosil LC-18 column, using a binary acetonitrile gradient system with tetrabutylammonium as the ion-pairing reagent. The eluted peaks were recorded by dual wavelength at 232 and 226 nm and a linear detector response was obtained over the entire interval tested, i.e., to 50 micrograms of delta-disaccharides. As little as 0.8-5 ng of delta-disaccharides can be reliably detected and accurately determined. Following separate digestion with the heparin- and heparan sulphate lyases (heparin lyases I, II and III), the characteristic heparin delta-disaccharides in the heparan sulphate chain, as well as the heparan sulphate delta-disaccharides in the heparin polymer, can be identified. Using combined digestions with these three lyases, the glycosaminoglycan chains are degraded almost completely (> 90%) to delta-disaccharides, which are then determined by direct injections into the HPLC system and thus an almost complete spectrum of disaccharide composition can be obtained. By this method, it is possible to analyse and confirm that the heparan sulphate chain is defined as a glycosaminoglycan dominated by GlcNAc(+/- 6S)-GlcA disaccharides and by some copolymeric disaccharides, such as GlcNS-IdoA2S and GlcNS6S-IdoA2S, otherwise most common in heparin. Fragmin, which is a controlled cepolymerized heparin fragment of M(r) 5000, is made up mainly of trisulphated disaccharides of the GlcNS6S-IdoA2S type (88.8%). Using separate digestions with the specific heparin lyases, one can also distinguish between heparin and heparan sulphate.

Determination of iduronic acid and glucuronic acid in glycosaminoglycans after stoichiometric reduction and depolymerization using high-performance liquid chromatography and ultraviolet detection.

The reduction of uronic acids in glycosaminoglycans (GAGs) prior to depolymerization reactions is one way in which the uronic acid content of polysaccharides can be studied without major losses. The obtained monosaccharides can be recovered from the subsequent depolymerization with a yield better than 95%. Following reduction, depolymerization, and lyophilization, D-glucuronic acid is converted to D-Glc and L-iduronic acid to 1,6-anhydro-idose. Per-O-benzoyl derivatives of these monosaccharides can be separated and detected in nanogram amounts using reversed phase HPLC. A linear detector response was obtained for injections up to 22 nmol (4 micrograms) of Glc and 1,6-anhydro-idose and the detection limit was 5 and 7 pmol, respectively. Reduction, depolymerization, and derivatization with subsequent chromatography of various GAGs can be readily performed in the 1- to 30-micrograms range.

Human abdominal aortic aneurysm is closely associated with compositional and specific structural modifications at the glycosaminoglycan level

Human abdominal aortic aneurysm (AAA) is a commonly occuring disease of blood vessels and is related to alterations in extracellular matrix molecules. In this study we report on the type and fine structural characterization of glycosaminoglycans (GAGs) present in AAA as compared with those present in normal abdominal aorta. Hyaluronan (HA), the galactosaminoglycans-chondroitin sulfate (CS) and dermatan sulfate (DS) with average molecular size (Mr) of 35-kDa-as well as heparan sulfate (HS) with Mr of 40-kDa were identified in both tissues. No significant intrabatch differences in total GAG content were identified in normal and aneurysmal aortas. Comparing, however, tissue composition and structure of GAGs between AAAs and normal aortas, significant differences (P < or = 0.001) were found. The overall GAG content in AAAs was approx. 60% lower than the normal ones. A 90% decrease in HS content, and 65 and 73% in CS and HA, respectively, were also recorded. In contrast, only a slight decrease in the amount of DS was noted (8%). Structural alterations in disaccharide composition of GAGs correspond mainly to significant decreases (P < or = 0.001) of HS-derived N-sulfated disaccharides, CS-derived 6-sulfated disaccharide and DS-derived disulfated disaccharides. These results demonstrate that the development of AAA is related to dramatic quantitative and structural modifications at the GAG level and this may well be attributed to the destruction of arterial wall architecture and further significant functional inadequacies of the tissue.

Effects of glycosaminoglycans on proliferation of epithelial and fibroblast human malignant mesothelioma cells: a structure–function relationship

Abstract. Proteoglycans interact with other effective macromolecules regulating a variety of cellular events via their glycosaminoglycan (GAG) chains. The effects of all known glycosaminoglycans (GAGs) produced by normal cells and tissues on the proliferation of two human malignant mesothelioma cell lines, one with fibroblast‐like morphology and the other with epithelial differentiation – both able to produce hyaluronan (HA), galactosaminoglycans (GalAGs) and heparan sulphate (HS) containing proteoglycans – have been studied. Cell proliferation was assessed by measuring [3H]thymidine incorporation and cell number. GalAGs, i.e. chondroitin sulphates (CSs) and dermatan sulphate (DS), strongly stimulate the proliferation of fibroblast‐like cells in a dose‐dependent manner (170–250% at 100 μg/ml), independently of their sulphation pattern. In epithelial cells, however, only DS stimulates cell proliferation. The effects of CSs on proliferation of epithelial cells are not depended on their sulphation pattern. Thus, CSs either with ‐[GlcA‐GalNAc‐(‐6‐O‐SO3−)]‐ or ‐[GlcA‐GalNAc‐(‐4‐O‐SO3−]‐ as the commonest unit, had no significant effect. l‐Iduronic acid (IdoA)‐rich heparin and fast‐moving HS (fm‐HS), a HS fraction with a heparin‐like structure, had significant antiproliferative effects on mesothelioma cells of both types (30–70% at 1.0 μg/ml and 85–90% at 100 μg/ml, respectively). GlcA‐rich HS, however, had no significant effects. HA inhibits only the proliferation of fibroblast‐like cells by 25% at 50 and 100 μg/ml. Keratan sulphate suppresses cell proliferation (10–30%) in both cell lines. In the view of these findings, a structure–function relationship of GAGs on cell proliferation of the two human malignant mesothelioma cell lines is discussed. Other factors, such as chain conformation and geometry, as well as interactions of growth factors with GAGs, possibly involved in the regulation of cell proliferation, are also discussed.

Analysis of neutral sugars as dinitrophenyl-hydrazones by high-performance liquid chromatography

Abstract A new method for the separation and determination of neutral sugars as 2,4-dinitrophenyl derivatives is presented. After a simple derivatization procedure at 65°C for 90 min and purification of the reaction mixture from excess of reagents, the sugar derivatives were separated on a LiChrosorb Si 100 column using methanolwater in chloroform as the eluent and UV detection at 352 nm. The yield of the products is affected by several parameters. However, when the proposed conditions are closely adhered to an excellent precision is obtained. Calibration graphs are linear in the range of 0.05–3.35 nmol of each sugar injected. The method was used for the determination of neutral sugars normally found in glycoconjugates, namely fucose, xylose, mannose, galactose and glucose. Hexosamines, uronic acids, alditoles and amino acids do not interfere with the analysis. Thus, the derivatization procedure can be applied directly to unfractionated acid hydrolysate residues of glycoconjugates.

Analysis of the acid polysaccharides from squid cranial cartilage and examination of a novel polysaccharide

The polysaccharides of cranical cartilage were isolated by ethanol precipitation after papain digestion and beta-elimination procedures and were fractionated chromatographically on CPC-cellulose. In addition to the previously described, heavily oversulphated chondroitin sulphate, the tissue contained small amounts of hyaluronic acid, which, however, co-eluted with the chondroitin sulphate from the CPC-cellulose. Approx. 20% of the isolated polysaccharides consisted of an acidic polysaccharide which to our knowledge is not previously described. This polysaccharide consists mainly of glucuronic acid, galactose and mannose in a molar ratio of 1:2:1. Gel chromatography of the preparation indicated a polydisperse molecule with an apparent average molecular weight of 39 200 on weight basis (Mw) and 31 400 on number basis (Mn).

Imatinib as a key inhibitor of the platelet‐derived growth factor receptor mediated expression of cell surface heparan sulfate proteoglycans and functional properties of breast cancer cells

Cell surface heparan sulfate proteoglycans (HSPGs), syndecans and glypicans, play crucial roles in the functional properties of cancer cells, such as proliferation, adhesion, migration and invasion. Platelet‐derived growth factor (PDGF)/PDGF receptor (PDGF‐R) mediated signaling, on the other hand, is highly associated with cancer progression. Specifically, PDGF‐Rα and PDGF‐Rβ expressions documented in breast cancer tissue specimens as well as breast cancer cell lines are correlated with tumor aggressiveness and metastasis. Imatinib (Glivec®) is a tyrosine kinase inhibitor specific for PDGF‐Rs, c‐ΚΙΤ and BCR‐ABL. In this study we evaluated the effects of imatinib on the properties of breast cancer cells as well as on the expression of HSPGs in the presence and absence of PDGF‐BB. These studies have been conducted in a panel of three breast cancer cell lines of low and high metastatic potential. Our results indicate that imatinib exerts a significant inhibitory effect on breast cancer cell proliferation, invasion and migration as well as on the cell surface expression of HSPGs even after exposure of PDGF. These effects depend on the aggressiveness of breast cancer cells and the type of HSPG. It is suggested that imatinib may be of potential therapeutic usefulness in breast cancer regimes.

Extraction and fractionation of proteoglycans from squid skin

The extractability of squid skin proteoglycans with solutions of varying concentrations of guanidine-HCl, urea and SDS was studied; 4 M guanidine-HCl, being the best extractant, removed 95% of the tissue proteoglycans (glycosaminoglycan uronic acid). The proteoglycans in the 4 M guanidine-HCl extract were fractionated by repeated ion exchange and gel chromatography on Sepharose CL-4B to give three main populations, all being present in about equal proportions. Two populations (Kd 0.34 and 0.56) contained only chondroitin (proteochondroitin) and the other (Kd 0.50) only oversulphated chondroitin sulphate (oversulphated proteochondroitin sulphate). Two minor populations, one containing chondroitin and chondroitin sulphate and the other chondroitin sulphate and oversulphated chondroitin sulphate, were also identified.

Heparan sulfate: biological significance, tools for biochemical analysis and structural characterization

Heparan sulfate (HS) and heparin (HP) are functionally important glycosaminoglycans, which interact with a plethora of proteins and participate in several cellular events. They form specific proteoglycans, which are ubiquitously distributed at both extracellular and cellular levels. HS and HP chains vary in the sulfation pattern and the degree of C-5 epimerization of d-glucuronic acid to l-iduronic acid. These modifications are not uniformly distributed within the chain, providing functional oligomeric domains interacting specifically with various effective proteins. The utilization of specific lyases and chemical depolymerization are the commonest procedures used for structural analysis. Di- and oligosaccharide composition of HS can be accurately and sensitively determined by HPLC, CE and MS. Ultraviolet detection is satisfactory enough for unsaturated saccharides and pre-column derivatization with fluorophores and detection with laser-induced fluorescence results in even higher sensitivity. Solid-phase assays can also be used for monitoring interactions with other molecules. In this article the biological significance of HS and HP in health and disease as well as the portfolio of analytical methods that may help to a deeper understanding of their roles in various pathological processes is presented. Such methodologies are of crucial importance for disease diagnosis and the design of novel synthetic sugar-based drugs.