Blocking αvβ3 integrin by a recombinant RGD disintegrin impairs VEGF signaling in endothelial cells

Abstract

Vascular endothelial growth factor (VEGF) and αvβ3 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 αvβ3 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 αvβ3 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.

DisBa-01 inhibits angiogenesis, inflammation and fibrogenesis of sponge-induced-fibrovascular tissue in mice

“…So far, this is the first study to demonstrate such effects concurrently. However, a similarly effect has been reported in DisBa-01 such as inhibition of bFGFinduced angiogenesis using matrigel assay (Ramos et al, 2008) and reduction in vitro of pro-angiogenic cytokines (Montenegro et al, 2012). Those data are in agreement with various reports that demonstrated the inhibitory action of other disintegrins on the processes investigated in this study in different in vitro and in vivo systems.…”

“…N-terminal sequencing confirmed the expected sequence of the 12 kDa fusion protein which was then used in all biological assays. Molecular modeling suggested that the fusion His-tag peptide was not involved in integrin binding and therefore its proteolytic removal could not be needed (Ramos et al, 2008;Montenegro et al, 2012).…”

“…Since the native DisBa-01 disintegrin could not be purified from the venom due to very low yields in protein preparations, recombinant DisBa-01 was produced from a cDNA venom gland library of Bothrops alternatus (Montenegro et al, 2012). The coding region corresponds to a medium disintegrin (78 amino acid residues) with an RGD adhesive motif (GenBank accession AY259516).…”

Integrins

“…Induce angiogenesis in brain tumor [56], gliomas [57], breast cancer [58], and glioblastoma [59]. Induce metastasis in glioblastoma, carcinomas of the breast, prostate, pancreas and lung, osteosarcoma, and melanoma [55,[60][61][62]; promote lung metastasis in renal cell carcinoma and soft tissue sarcoma [63]; promote liver metastasis of murine RAW117 large cell lymphoma [64]; promote invasion of cervical tumor [65]; reduce metastasis in ovarian cancer [66] αvβ5…”

Pharmacological and Clinical Importance of Integrin Antagonists in Treatment of Cancer

“…These in vitro reports demonstrate the anti-adhesive properties of DisBa-01. The ex vivo and in vivo studies demonstrate the antimetastatic and protective effect of disintegrin [61]. All these results describe the essentiality to understand the molecular mechanism underlying that target integrin-mediated processes using novel antimetastatic therapies.…”