echistatin and Melanoma

New blood vessel formation is essential for tumor growth and metastatic spread. Integrins alpha(v)beta3 and alpha(v)beta5 are arginine-glycine-aspartic acid-dependent adhesion receptors that play a critical role in angiogenesis. Hence, selective dual alpha(v)beta3 and alpha(v)beta5 antagonists may represent a novel class of angiogenesis and tumor-growth inhibitors. Here, an arginine-glycine-aspartic acid-based peptidomimetic library was screened to identify alpha(v)beta3 antagonists. Selected compounds were then modified to generate potent and selective dual inhibitors of alpha(v)beta3 and alpha(v)beta5 receptors. One of these compounds, SCH 221153, inhibited the binding of echistatin to alpha(v)beta3 (IC50 = 3.2 nM) and alpha(v)beta5 (IC50 = 1.7 nM) with similar potency. Its IC50 values for related alpha(IIb)beta3 and alpha5beta1 receptors were 1294 nM and 421 nM, respectively, indicating that SCH 221153 is highly selective for alpha(v)beta3 and alpha(v)beta5 receptors. In cell-based assays, SCH 221153 inhibited the binding of echistatin to alpha(v)beta3- and alpha(v)beta5-expressing 293 cells and blocked the adhesion of endothelial cells to immobilized vitronectin and fibroblast growth factor 2 (FGF2). SCH 221153, but not the inactive analogue SCH 216687, was effective in inhibiting FGF2 and vascular endothelial growth factor-induced endothelial cell proliferation in vitro with an IC50 equal to 3-10 microM. Angiogenesis induced by FGF2 in the chick chorioallantoic membrane assay was also inhibited by SCH 221153. Finally, SCH 221153 exerted a significant inhibition on tumor growth induced by intradermal or s.c. injection of human melanoma LOX cells in severe combined immunodeficient mice.

Topics: Allantois; Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Cell Adhesion; Cell Division; Chick Embryo; Chorion; Endothelial Growth Factors; Endothelium, Vascular; Female; Fibroblast Growth Factor 2; Growth Inhibitors; Humans; Integrins; Intercellular Signaling Peptides and Proteins; Lymphokines; Melanoma; Mice; Mice, SCID; Molecular Mimicry; Neovascularization, Pathologic; Neovascularization, Physiologic; Oligopeptides; Peptides; Receptors, Vitronectin; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Vitronectin; Xenograft Model Antitumor Assays

Peptides containing the integrin recognition sequence, RGD, can inhibit experimental metastasis of mouse melanoma cells, but the integrin(s) affected in these experiments is unknown. Besides "classical" RGD-binding integrins such as alpha 5 beta 1 and alpha v beta 3, RGD has been reported to bind alpha 4 beta 1, and mAbs to alpha 4 beta 1 can inhibit melanoma metastasis. We investigated the mode of action of the disintegrin eristostatin, an RGD-containing peptide isolated from snake venom, in a human melanoma experimental metastasis model. Lung colonization following i.v. injection of MV3 cells in nude mice was strongly inhibited by eristostatin. MV3 cells bound FITC-eristostatin and adhered to eristostatin-coated wells. This adhesion was partially inhibited by a GRGDSP peptide and by alpha 4 mAb. Binding of FITC-eristostatin to Jurkat cells and adhesion of Jurkat (but not K562) cells to eristostatin-coated wells further suggested that eristostatin binds alpha 4 beta 1, even though, again, alpha 4 mAb only partially inhibited adhesion. Expression of alpha 4 beta 1 was enhanced in metastatic melanoma cells compared to normal melanocytes and nonmetastatic melanoma cells. Finally, eristostatin inhibited adhesion of both MV3 and CHO alpha 4 cells to the alpha 4 beta 1-ligand VCAM-1, while adhesion to other ligands via other integrins was not affected. These findings demonstrate that inhibition of melanoma cell metastasis by RGD-containing peptides such as eristostatin, may be due to interference with alpha 4 beta 1-VCAM binding, in addition to inhibition of the classical RGD-binding integrins.

Topics: Animals; Binding Sites; Humans; Infant, Newborn; Integrin alpha4beta1; Integrins; Intercellular Signaling Peptides and Proteins; Male; Melanocytes; Melanoma; Mice; Mice, Nude; Neoplasm Metastasis; Oligopeptides; Peptides; Platelet Aggregation Inhibitors; Receptors, Lymphocyte Homing; Skin; Skin Neoplasms; Snake Venoms; Viper Venoms

B16-BL6 mouse melanoma cells cultured on fibronectin-coated dishes were detached by treatment with echistatin, an RGD-containing disintegrin. Echistatin was active at micromolar concentrations and was not cytotoxic. Its effect was dose-dependent and reversible. Sequential morphological changes leading to rounding up of the cells were detected by phase-contrast microscopy and by immunofluorescence analysis. A dramatic reduction in the number and size of focal adhesions and loss of cytoplasmic actin filaments were observed well before cell detachment occurred. Echistatin treatment down-regulated the phosphorylation of pp125FAK in fibronectin-adherent cells in a dose- and time-dependent fashion. The reduction of pp125FAK phosphorylation preceded cell detachment and occurred even in the presence of orthovanadate, an inhibitor of protein tyrosine phosphatases. These results suggest that echistatin detaches cells from the fibronectin substratum by inducing a decrease of pp125FAK phosphorylation and that echistatin acts by inhibiting protein tyrosine kinase activity rather than activating protein tyrosine phosphatases.

Topics: Actin Cytoskeleton; Actins; Animals; Cell Adhesion; Cell Adhesion Molecules; Cell Membrane; Enzyme Inhibitors; Fibronectins; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Intercellular Signaling Peptides and Proteins; Melanoma; Mice; Peptides; Phosphorylation; Phosphotyrosine; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Tumor Cells, Cultured; Vanadates; Viper Venoms

Echistatin, an RGD containing peptide isolated from Echis carinatus snake venom, inhibited the in vitro attachment of B16-BL6 mouse melanoma cells to fibronectin, vitronectin and laminin. Its inhibitory activity on cell adhesion was non-cytotoxic, dose-dependent and fully reversible. Kinetic analysis showed a competitive type of inhibition for all the three substrates examined here. Chemical reduction and alkylation of echistatin almost abolished its effect on cell adhesion to extracellular matrix components. Native echistatin was also able to inhibit B16-BL6 cell attachment to IgG antihuman fibronectin receptor-coated wells, thus suggesting that the molecule binds to adhesive receptors on melanoma cell surface. Our results indicate that echistatin is an useful disintegrin for research on cell adhesion.

Topics: Amino Acid Sequence; Animals; Cell Adhesion; Extracellular Matrix Proteins; Intercellular Signaling Peptides and Proteins; Melanoma; Mice; Molecular Sequence Data; Peptides; Platelet Aggregation Inhibitors; Receptors, Fibronectin; Time Factors; Tumor Cells, Cultured