ABSTRACT
The connective tissue formed by extracellular matrix (ECM) rich in fibronectin and collagen consists a barrier that cancer cells have to overpass to reach blood vessels and then a metastatic site. Cell adhesion to fibronectin is mediated by αvß3 and α5ß1 integrins through an RGD motif present in this ECM protein, thus making these receptors key targets for cell migration studies. Here we investigated the effect of an RGD disintegrin, DisBa-01, on the migration of human fibroblasts (BJ) and oral squamous cancer cells (OSCC, SCC25) on a fibronectin-rich environment. Time-lapse images were acquired on fibronectin-coated glass-bottomed dishes. Migration speed and directionality analysis indicated that OSCC cells, but not fibroblasts, showed significant decrease in both parameters in the presence of DisBa-01 (1µM and 2µM). Integrin expression levels of the α5, αv and ß3 subunits were similar in both cell lines, while ß1 subunit is present in lower levels on the cancer cells. Next, we examined whether the effects of DisBa-01 were related to changes in adhesion properties by using paxillin immunostaining and total internal reflection fluorescence TIRF microscopy. OSCCs in the presence of DisBa-01 showed increased adhesion sizes and number of maturing adhesion. The same parameters were analyzed usingß3-GFP overexpressing cells and showed that ß3 overexpression restored cell migration velocity and the number of maturing adhesion that were altered by DisBa-01. Surface plasmon resonance analysis showed that DisBa-01 has 100x higher affinity for αvß3 integrin than forα5ß1 integrin. In conclusion, our results suggest that the αvß3 integrin is the main receptor involved in cell directionality and its blockage may be an interesting alternative against metastasis.
Subject(s)
Carcinoma, Squamous Cell/metabolism , Cell Adhesion/drug effects , Cell Movement/drug effects , Integrin alphaVbeta3/metabolism , Mouth Neoplasms/metabolism , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Crotalid Venoms/pharmacology , Disintegrins/pharmacology , Extracellular Matrix/metabolism , Fibroblasts/metabolism , Fibronectins/metabolism , Humans , Mouth Neoplasms/pathologyABSTRACT
Vascular endothelial growth factor (VEGF) and αvß3 integrin are key molecules that actively participate in tumor angiogenesis and metastasis. Some integrin-blocking molecules are currently under clinical trials for cancer and metastasis treatment. However, the mechanism of action of such inhibitors is not completely understood. We have previously demonstrated the anti-angiogenic and anti-metastatic properties of DisBa-01, a recombinant His-tag RGD-disintegrin from Bothrops alternatus snake venom in some experimental models. DisBa-01 blocks αvß3 integrin binding to vitronectin and inhibits integrin-mediated downstream signaling cascades and cell migration. Here we add some new information on the mechanism of action of DisBa-01 in the tumor microenvironment. DisBa-01 supports the adhesion of fibroblasts and MDA-MB-231 breast cancer cells but it inhibits the adhesion of these cells to type I collagen under flow in high shear conditions, as a simulation of the blood stream. DisBa-01 does not affect the release of VEGF by fibroblasts or breast cancer cells but it strongly decreases the expression of VEGF mRNA and of its receptors, vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2) in endothelial cells. DisBa-01 at nanomolar concentrations also modulates metalloprotease 2 (MMP-2) and 9 (MMP-9) activity, the latter being decreased in fibroblasts and increased in MDA-MB-231 cells. In conclusion, these results demonstrate that αvß3 integrin inhibitors may induce distinct effects in the cells of the tumor microenvironment, resulting in blockade of angiogenesis by impairing of VEGF signaling and in inhibition of tumor cell motility.
Subject(s)
Cell Adhesion/drug effects , Disintegrins/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Integrin alphaVbeta3 , Snake Venoms/pharmacology , Vascular Endothelial Growth Factor A , Animals , Bothrops , Breast Neoplasms/metabolism , Cell Line, Tumor , Collagen Type I/metabolism , Disintegrins/chemistry , Disintegrins/genetics , Endothelial Cells/metabolism , Female , Fibroblasts/drug effects , Humans , Integrin alphaVbeta3/antagonists & inhibitors , Integrin alphaVbeta3/metabolism , Neovascularization, Physiologic , Peptides/chemistry , Peptides/genetics , Protein Binding/drug effects , Signal Transduction/drug effects , Snake Venoms/chemistry , Tumor Microenvironment , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-1/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Cell migration is a key process for the defense of pluricellular organisms against pathogens, and it involves a set of surface receptors acting in an ordered fashion to contribute directionality to the movement. Among these receptors are the integrins, which connect the cell cytoskeleton to the extracellular matrix components, thus playing a central role in cell migration. Integrin clustering at focal adhesions drives actin polymerization along the cell leading edge, resulting in polarity of cell movement. Therefore, small integrin-binding proteins such as the snake venom disintegrins that inhibit integrin-mediated cell adhesion are expected to inhibit cell migration. Here we review the current knowledge on disintegrin and disintegrin-like protein effects on cell migration and their potential use as pharmacological tools in anti-inflammatory therapy as well as in inhibition of metastatic invasion.
