Effect of RGD-Disintegrins, Flavoridin, Kistrin and Jarastatin, on B16F10 Melanoma Cells, in vivo and in vitro.

1Oliva IB, 1Barcellos GG, 1Coelho AL, 2Zingali R, 3Marcinkiewicz C, 1De Freitas MS & 1Barja-Fidalgo C.

1 Dept. Farmacologia, IBRAG-UERJ, 2 Dept. Bioquímica Médica, ICB-UFRJ, RJ, Brazil; 3 Dept. Biology, CST-Temple University, PA, USA. 

Integrins are surface adhesion receptors known to play a critical role in different stages of tumor metastasis. Disintegrins are proteins isolated from snake venoms which selectively bind to integrins and can lead to alterations on intracellular signaling pathways. We have investigated whether different RGD-disintegrins could interfere with the capability of B16F10, a murine highly metastatic melanoma cell line, in inducing lung tumor colonization in mice. The experimental metastasis assay was performed in C57BL/6 mice and the number of lung surface metastatic nodules assessed 21 days after i.v. injection of B16F10 cells treated, in vitro, with 1 µM jarastatin (JT), kistrin (KR) or flavoridin (FL). Treatment of melanoma cells with the RGD-disintegrins decreased lung tumor colonization (KR=p<0.001; FL/JT=p<0.01), compared to control mice Although the three peptides present an RGD-motif, they show different affinity for specific integrins. FL binds to alpha5-beta1 and alphav-beta3 integrins. JT, an RGD-disintegrin isolated from Bothrops jararaca venom, binds to the same integrins as FL and also on alpham-beta2 on leukocytes, while KR binds only to alphav-beta3. To understand how these disintegrins interfere with integrin-dependent intracellular signaling in melanoma cells, the dynamic of actin cytoskeleton, the phosphrylation of Focal Adhesion Kinase (FAK) and its association with Src were analysed 5 min after disintegrin association with B16F10. Cells treated with FL and JT showed profound alterations in actin network dynamic. An increase in tyrosine phosphorylation was induced by FL, but no difference was observed in FAK-Src association. On the other hand, although only a slight increase on FAK phosphorylation was observed in JT-treated cells, its association with Src was significantly augmented. In contrast, KR, at the time assessed, did not induced any change in actin polymerization, in FAK phosphorylation or in FAK-Src association when compared to control. The data suggest that alphav-beta3 integrin-binding disintegrins are able to interfere with the ability of B16F10 in inducing lung tumor colonization. This effect is probably related with the alterations in integrin-mediated signaling induced by RGD-disintegrins in melanoma cells.

FAPERJ, CNPq, SR2-UERJ, IFS (Sweden).

Correspondence to: barja-fidalgo@uerj.br