Serglycin is a proteoglycan expressed by some malignant cells. It promotes metastasis and protects some tumor cells from complement system attack. In the present study, we show for the first time the in situ expression of serglycin by breast cancer cells by immunohistochemistry in patients’ material. Moreover, we demonstrate high expression and constitutive secretion of serglycin in the aggressive MDA-MB-231 breast cancer cell line. Serglycin exhibited a strong cytoplasmic staining in these cells, observable at the cell periphery in a thread of filaments near the cell membrane, but also in filopodia-like structures. Serglycin was purified from conditioned medium of MDA-MB-231 cells, and represented the major proteoglycan secreted by these cells, having a molecular size of ∼250 kDa and carrying chondroitin sulfate side chains, mainly composed of 4-sulfated (∼87%), 6-sulfated (∼10%) and non-sulfated (∼3%) disaccharides. Purified serglycin inhibited early steps of both the classical and the lectin pathways of complement by binding to C1q and mannose-binding lectin. Stable expression of serglycin in less aggressive MCF-7 breast cancer cells induced their proliferation, anchorage-independent growth, migration and invasion. Interestingly, over-expression of serglycin lacking the glycosaminoglycan attachment sites failed to promote these cellular functions, suggesting that glycanation of serglycin is a pre-requisite for its oncogenic properties. Our findings suggest that serglycin promotes a more aggressive cancer cell phenotype and may protect breast cancer cells from complement attack supporting their survival and expansion.

No abstract

Proteoglycans participate and regulate several physiological processes via their glycosaminoglycan constituents. For a deeper understanding of how they interact with extracellular ligands as well as with cell bound effector molecules, the fine chemical structures of their glycosaminoglycan chains must be elucidated. Lately developed capillary electrophoretic techniques is a powerful analytical tool for the analysis of glycosaminoglycans, combining a high resolving power with sensitive detection. In this review we describe how depolymerized and intact glycosaminoglycans/proteoglycans can be characterized by capillary electrophoresis, relating these analyses to their possible biological significance. Conditions for running these separations and the detection systems for particular applications are also summarized.

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 microg/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-SO(3)-)]- or -[GlcA-GalNAc-(-4-O-SO(3)-]- 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 microg/ml and 85-90% at 100 microg/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 microg/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.

The synthesis and distribution of glycosaminoglycans (GAGs) were studied in two human malignant mesothelioma cell lines: one with fibroblast-like morphology and the other with epithelial differentiation. Analyses using highly sensitive high-pressure liquid chromatography techniques and agarose gel electrophoresis showed that these cells produce not only hyaluronan (HA) but also galactosaminoglycans (GalAGs, chondroitin sulfate and (or) dermatan sulfate) and heparan sulfate (HS). In both cell lines most of the HA (87-90%) and GalAGs (57-66%) are secreted into the extracellular matrix. Although HS is mainly bound to the cell surface in fibroblast-differentiated cells (75%), in epithelial type cells only 40% occurs in the cell-associated fraction. The amounts of secreted GAGs are 6- to 8-fold higher in epithelial than in fibroblast-like mesothelioma cultures. In cells with the fibroblast phenotype, the beta-homodimer of platelet-derived growth factor (PDGF) in a concentration of 1.5 ng/mL stimulates HA and GalAG synthesis 5-fold and that of HS 10-fold, whereas higher concentrations suppress this stimulatory effect. The stimulatory effect, observed at low concentrations of this growth factor, was completely blocked by the addition of antibodies against this factor. In epithelially differentiated cells, the production of all GAGs was suppressed after addition of this factor, even at low concentrations. We therefore suggest that mesothelioma cells can produce GAGs, the synthesis of which is dependent on the presence and concentration of PDGF beta-homodimer. The differences between the two cell lines regarding the effect of this growth factor on GAG synthesis indicates that the regulation of this synthesis is complex, other factors also being important.