i(3)so3-galactosylceramide and Melanoma

A laminin-binding peptide (peptide G), predicted from the cDNA sequence for a 33-kDa protein related to the 67-kDa laminin receptor, specifically inhibits binding of laminin to heparin and sulfatide. Since the peptide binds directly to heparin and inhibits interaction of another heparin-binding protein with the same sulfated ligands, this inhibition is due to direct competition for binding to sulfated glycoconjugates rather than an indirect effect of interaction with the binding site on laminin for the 67-kDa receptor. Direct binding of laminin to the peptide is also inhibited by heparin. This interaction may result from contamination of the laminin with heparan sulfate, as binding is enhanced by the addition of substoichiometric amounts of heparin but inhibited by excess heparin and two heparin-binding proteins. Furthermore, laminin binds more avidly to a heparin-binding peptide derived from thrombospondin than to the putative receptor peptide. Adhesion of A2058 melanoma cells on immobilized peptide G is also heparin-dependent, whereas adhesion of the cells on laminin is not. Antibodies to the beta 1-integrin chain or laminin block adhesion of the melanoma cells to laminin but not to peptide G. Thus, the reported inhibition of melanoma cell adhesion to endothelial cells by peptide G may result from inhibition of binding of laminin or other proteins to sulfated glycoconjugate receptors rather than from specific inhibition of laminin binding to the 67-kDa receptor.

Topics: Amino Acid Sequence; Binding, Competitive; Cell Adhesion; Cell Line; Heparin; Humans; Laminin; Melanoma; Molecular Sequence Data; Molecular Weight; Peptides; Platelet Membrane Glycoproteins; Protein Precursors; Receptors, Immunologic; Receptors, Laminin; Sulfoglycosphingolipids; Thrombospondins; Tumor Cells, Cultured

Peptides from the three type I repeats of human endothelial cell thrombospondin, containing the consensus sequence-Trp-Ser-Xaa-Trp-, bind to sulfated glycoconjugates including heparin and sulfatide. The peptides are potent inhibitors for the binding of thrombospondin, laminin, or apolipoprotein E to these ligands. The thrombospondin peptides that inhibit heparin binding, but not adjacent peptides from the thrombospondin sequence containing the previously identified adhesive motif Val-Thr-Cys-Gly, promote melanoma cell adhesion when immobilized on plastic. Melanoma cell adhesion to the immobilized peptides is inhibited by soluble recombinant heparin-binding fragment of thrombospondin. The peptides also inhibit heparin-dependent binding of thrombospondin or laminin to human melanoma cells. The active peptides lack any previously identified heparin-binding consensus sequences and most do not contain any basic amino acids. Studies with homologous peptides showed that the tryptophan residues are required for binding. Adjacent basic residues in the second type I repeat enhance binding to heparin but not to sulfatide. Thus the type I peptides of thrombospondin define a distinct class of heparin-binding peptides.

Topics: Amino Acid Sequence; Apolipoproteins E; Binding Sites; Binding, Competitive; Cell Adhesion; Cell Adhesion Molecules; Cells, Cultured; Heparin; Humans; In Vitro Techniques; Laminin; Melanoma; Molecular Sequence Data; Peptides; Platelet Membrane Glycoproteins; Sulfoglycosphingolipids; Thrombospondins

Suramin is a polysulfonated drug with several biological activities including inhibition of binding of some growth factors to cells, inhibition of tumor cell growth, and of glycosaminoglycan metabolism. We report here that suramin also inhibits binding of the adhesive glycoproteins, thrombospondin and laminin, to immobilized sulfatide with 50% inhibitory doses of 220 and 470 micrograms/ml, respectively. Sulfated glycoconjugates on melanoma cells mediate spreading on thrombospondin by binding to the amino-terminal heparin- and sulfatide-binding domain. This domain is also required for chemotaxis on thrombospondin. We therefore examined the effect of suramin on human melanoma cell spreading and migration. Suramin at 50-400 micrograms/ml specifically inhibited G361 melanoma cell spreading on thrombospondin without affecting cell attachment. Suramin also inhibited spreading of A2058 melanoma cells on thrombospondin and laminin and partially inhibited cell attachment. However, suramin had no effect on G361 or A2058 cell attachment or spreading on fibronectin. Chemotaxis of A2058 and G361 melanoma cells to thrombospondin and laminin were also specifically inhibited by suramin, as was haptotaxis of A2058 melanoma cells to laminin. However, suramin only weakly inhibited haptotaxis of G361 melanoma cells to thrombospondin, which is not mediated by the amino-terminal domain, and did not inhibit haptotaxis to fibronectin. These results suggest a new mechanism for the observed antitumor activity of suramin based on its ability to inhibit interactions of tumor cells with laminin or thrombospondin in the extracellular matrix.

Topics: Binding Sites; Cell Adhesion; Cell Movement; Heparin; Humans; Laminin; Melanoma; Membrane Glycoproteins; Protein Binding; Sulfoglycosphingolipids; Suramin; Thrombospondins; Tumor Cells, Cultured

Sulfatides (galactosylceramide-I3-sulfate) but not neutral glycolipids or gangliosides adsorbed on plastic promote adhesion of the human melanoma cell line G361. Direct adhesion of G361 cells requires densities of sulfatide greater than 1 pmol/mm2. In the presence of laminin, however, specific adhesion of G361 cells to sulfatide or seminolipid (galactosylalkylacyl-glycerol-I3-sulfate) but not to other lipids is strongly stimulated and requires only 25 fmol/mm2 of adsorbed lipid. The effects of laminin and sulfatide on adhesion are synergistic, suggesting that laminin is mediating adhesion by cross-linking receptors on the melanoma cell surface to sulfatide adsorbed on the plastic. Although thrombospondin binds to sulfatides and G361 cells, it does not enhance, but rather inhibits direct and laminin-dependent G361 cell adhesion to sulfatide. In contrast, C32 melanoma cells also adhere specifically to sulfatide, but adhesion of these cells is not enhanced by laminin or inhibited by antibodies to laminin that block laminin-dependent adhesion of G361 cells. Thrombospondin is a potent inhibitor of C32 cell adhesion to sulfatide. Fucoidan, which inhibits laminin binding to sulfatide, inhibits laminin-dependent adhesion of G361 cells by 50% at 0.2 micrograms/ml. Several other tumor cell lines also attach directly on sulfatide-coated surfaces. Laminin stimulates adhesion to sulfatide of three of the six cell lines tested. The ability of laminin to promote adhesion of tumor cells to sulfatide suggests that binding to sulfatide could participate in laminin-mediated cell-cell adhesion. Thus, many tumor cell lines can attach on sulfatide substrates using endogenous sulfatide binding proteins, and in some cells laminin but not thrombospondin can promote tumor cell adhesion to sulfatide.

Topics: Adsorption; Cell Adhesion; Glycoproteins; Humans; Laminin; Lipid Metabolism; Melanoma; Polysaccharides; Receptors, Immunologic; Receptors, Laminin; Sulfoglycosphingolipids; Thrombospondins; Tumor Cells, Cultured

Gangliosides from benign and malignant melanomas and from normal skin of the fish genus Xiphophorus were isolated and analyzed by thin-layer chromatography. Individual ganglioside components were characterized by mapping according to their sialic acid content and by cleavage with neuraminidases. In all three tissues examined, sulfatide and the gangliosides NeuAc-GalCer (GM4), II3NeuAc-LacCer (GM3), II3NeuAc-GgOse3Cer (GM2), and II3(NeuAc)2-LacCer (GD3) were found. Ganglioside GD3 yielded a positive reaction, following immunoadsorption with mouse monoclonal antibody R24 on thin-layer plates. Two alkali-labile disialoganglioside species were specifically recognized by mouse monoclonal antibody D1.1, thus indicating the presence of O-acetyl-neuraminic acid residues. One of them, a major ganglioside component of the malignant melanoma, was identified as O-acetyl-GD3, since it could be converted to the R24-positive GD3 ganglioside after alkaline saponification. The other one appears to be restricted to the malignant tumor and represents a novel melanoma-associated ganglioside derivative. It was characterized as O-acetyl(NeuAc)2-nLc4Cer by exoglycosidase cleavage, by proving its neutral carbohydrate backbone as type II-chain lacto-series oligosaccharide using mouse monoclonal antibody 1B2, and by its cross-reaction with antibody R24 following alkaline treatment. Using antibody R24 and cryopreserved tissue sections of both benign and malignant amelanotic melanomas from albino fishes, it was demonstrated that one of the main melanoma-associated gangliosides, GD3, was exposed predominantly in the malignant tumor. Thus, the chemical nature and even the immunohistochemical localization of the gangliosides in fish melanomas proved to be very similar to those of the known gangliosides in the phylogenetically distant human melanomas.

Topics: Animals; Antigens, Neoplasm; Chromatography, Thin Layer; Cyprinodontiformes; Fish Diseases; Gangliosides; Immunohistochemistry; Melanoma; Skin; Sulfoglycosphingolipids