eristostatin and Melanoma

Malignant melanoma is difficult to treat due to its resistance to chemotherapeutic regimens. Discovery of new pharmaceuticals with inhibitory potential can be helpful in the development of novel treatments. The snake venom disintegrin eristostatin, from the viper Eristicophis macmahoni, caused immunodeficient mice to be significantly protected from development of lung colonization when melanoma cells and the disintegrin were co-injected in vivo into the lateral tail vein compared to vehicle controls. Cytotoxicity assays suggested that eristostatin makes the melanoma cells a better target for lysis by human natural killer cells. Direct binding assays using atomic force microscopy showed eristostatin does specifically bind the surface of the six melanoma cell lines tested. Eristostatin binding was partially inhibited by the addition of soluble RGDS peptide, suggesting an integrin as one likely, but not the sole, binding partner. Studies done with melanoma cells on a culture dish and natural killer cells attached to a cantilever tip in atomic force microscopy showed four major populations of interactions which exhibited altered frequency and unbinding strength in the presence of eristostatin.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Separation; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Humans; Killer Cells, Natural; Melanoma; Microscopy, Atomic Force; Microscopy, Confocal; Peptides; Platelet Aggregation; Platelet Aggregation Inhibitors; Recombinant Proteins; Viper Venoms

Eristostatin, an RGD-containing disintegrin isolated from the venom of Eristicophis macmahoni, inhibits lung or liver colonization of melanoma cells in a mouse model. In this study, transwell migration and in vitro wound closure assays were used to determine the effect of eristostatin on the migration of melanoma cells. Eristostatin significantly impaired the migration of five human melanoma cell lines. Furthermore, it specifically inhibited cell migration on fibronectin in a concentration-dependent manner, but not that on collagen IV or laminin. In contrast, eristostatin was found to have no effect on cell proliferation or angiogenesis. These results indicate that the interaction between eristostatin and melanoma cells may involve fibronectin-binding integrins that mediate cell migration. Mutations to alanine of seven residues within the RGD loop of eristostatin and four residues outside the RGD loop of eristostatin resulted in significantly less potency in both platelet aggregation and wound closure assays. For six of the mutations, however, decreased activity was found only in the latter assay. We conclude that a different mechanism and/or integrin is involved in these two cell activities.

Topics: Alanine; Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chorioallantoic Membrane; Humans; Melanoma; Mice; Mutagenesis, Site-Directed; Neovascularization, Physiologic; Peptides; Quail; Viper Venoms

To study the molecular mechanism of the disintegrin eristostatin, cellular functional studies were performed using ten recombinant alanine mutants. ADP-induced platelet aggregation revealed critical contributions of seven residues within the 'RGD loop' (R24, R27, G28, N31) and C-terminus (W47, N48, G49) of this disintegrin. Using an in vitro scratch wound healing assay, four human melanoma cell lines yielded similar results when exposed to wildtype eristostatin. All eristostatin-treated cells healed less of the wounded area than control conditions. This phenomenon was reproduced when using fibronectin as the matrix. C8161 cells showed significant delay in wound closure with the N-terminal mutant P4A but not with R24A or G28A. Evidence from our laboratory and others suggests neither alpha IIb, alpha 4 nor alpha 5 integrins are directly involved in eristostatin's interactions. Eristostatin did not affect the number of melanoma cells in culture after 24 h or the development of apoptosis. However, phosphorylation studies performed after these melanoma cells were exposed to eristostatin revealed changes in several tyrosine phosphorylated molecules.

Topics: Alanine; Animals; Binding Sites; Blood Platelets; Cell Line, Tumor; Disintegrins; Humans; Integrins; Melanoma; Mutagenesis; Peptides; Phosphorylation; Viper Venoms; Wound Healing

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