Abstract
BackgroundMatrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of α2β1 integrin, one of the major collagen I receptors, in MMP activity and expression has not been studied. Alternagin-C (ALT-C), a glutamate-cysteine-aspartate-disintegrin from Bothrops alternatus venom, has high affinity for an α2β1 integrin. Herein, we used ALT-C as a α2β1 integrin ligand to study the effect of ALT-C on MMP-9 and MMP-2 expression as well as on tumor cells, fibroblats and endothelial cell migration.MethodsALT-C was purified by two steps of gel filtration followed by anion exchange chromatography. The α2β1 integrin binding properties of ALT-C, its dissociation constant (Kd) relative to this integrin and to collagen I (Col I) were determined by surface plasmon resonance. The effects of ALT-C (10, 40, 100 and 1000 nM) in migration assays were studied using three human cell lines: human fibroblasts, breast tumor cell line MDA-MB-231, and microvascular endothelial cells HMEC-1, considering cells found in the tumor microenvironment. ALT-C effects on MMP-9 and MMP-2 expression and activity were analyzed by quantitative PCR and gelatin zymography, respectively. Focal adhesion kinase activation was determined by western blotting.ResultsOur data demonstrate that ALT-C, after binding to α2β1 integrin, acts by two distinct mechanisms against tumor progression, depending on the cell type: in tumor cells, ALT-C decreases MMP-9 and MMP-2 contents and activity, but increases focal adhesion kinase phosphorylation and transmigration; and in endothelial cells, ALT-C inhibits MMP-2, which is necessary for tumor angiogenesis. ALT-C also upregulates c-Myc mRNA level, which is related to tumor suppression.ConclusionThese results demonstrate that α2β1 integrin controls MMP expression and reveal this integrin as a target for the development of antiangiogenic and antimetastatic therapies.
Highlights
Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites
Our group has previously demonstrated the binding of ALT-C to α2β1 integrin by inhibition of cell adhesion assays in collagen I (Col I) coating; the kinetics parameters of this association were never determined
To address the question if ALT-C could bind to Col I and to α2β1 integrin, we determined the ALT-C dissociation constant (Kd) values for α2β1 integrin and Col I by surface plasmon resonance (SPR). α2β1 integrin and Col I were immobilized to a carboxymethylated dextran (CM5) sensor chipTM and ALT-C was passed over the layers in phosphate-buffered saline (PBS) buffer
Summary
Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. The role of α2β1 integrin, one of the major collagen I receptors, in MMP activity and expression has not been studied. The ECM components play key roles in intracellular signaling by interacting with adhesion receptors such as integrins [7]. Α2β1 integrin is one of the major receptors for type I collagen (Col I) and it has been demonstrated to be relevant during the process of bone metastasis of prostate and breast cancer [12, 13]. In vitro experiments performed on primary epithelial tumor cells have shown that the deletion of α2β1 integrin increased tumor cell intravasation and anchorage-independent growth [17]
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