Alphavbeta3 integrin blocking inhibits apoptosis and induces autophagy in murine breast tumor cells.

Integrins are cell receptors that mediate adhesion to the extracellular matrix (ECM) and regulate cell migration, a crucial process in tumor invasion. The αvß3 integrin recognizes the arginine-glycine-aspartic acid (RGD) motif in ECM proteins and it can be antagonized by RGD-peptides, resulting in decreased cell migration and invasion. RGD-based drugs have shown disappointing results in clinical trials; however, the reasons for their lack of activity are still obscure. Aiming to contribute to a better understanding of the molecular consequences of integrin inhibition, we tested a recombinant RGD-disintegrin (DisBa-01) in two types of murine cell lines, breast tumor 4T1BM2 cells and L929 fibroblasts. Only tumor cells showed decreased motility and adhesion, as well as morphologic alterations upon DisBa-01 treatment (100 and 1000 nM). This result was attributed to the higher levels of αvß3 integrin in 4T1BM2 cells compared to L929 fibroblasts making the former more sensitive to DisBa-01 blocking. DisBa-01 induced cell cycle arrest at the S phase in 4T1BM2 cells, but it did not induce apoptosis, which was consistent with the decrease in caspase-3, 8 and 9 expression at mRNA and protein levels. DisBa-01 increases PI3K, Beclin-1 and LC3B expression in tumor cells, indicators of autophagic induction. In conclusion, αvß3 integrin blocking by DisBa-01 results in inhibition of adhesion and migration and in the activation of an autophagy program, allowing prolonged survival and avoiding immediate apoptotic death. These observations suggest new insights into the effects of RGD-based inhibitors considering their importance in drug development for human health.

Alternagin-C, a disintegrin-like protein from Bothrops alternatus venom, attenuates inflammation and angiogenesis and stimulates collagen deposition of sponge-induced fibrovascular tissue in mice.

Alternagin-C (ALT-C), a disintegrin-like protein obtained from the venom of Bothrops alternatus, is able to modulate cellular behaviors such as adhesion, migration and proliferation, as well as the production of various growth factors via α2ß1 integrin, important processes during inflammation, angiogenesis and fibrogenesis, which although appear as distinct events, act concomitantly in several chronic inflammatory diseases. Our objective was to investigate the effects of ALT-C on components of the sponge-induced inflammatory response in balb/c mice. The polyester-polyurethane sponges were implanted in mice's subcutaneous layer of the dorsal region and daily injected with saline (control group) or ALT-C (10, 100 or 1000 ng). Nine days after implantation the implants were removed and processed. ALT-C inhibited the inflammatory response, observed through mast cell reduction, NAG-activity and also by the inhibition of TNF-α, CXCL-1 and CCL2/JE/MCP-1 cytokines. ALT-C was also able to reduce hemoglobin content, number of vessels and the concentrations of VEGF and FGF cytokines. Finally, at its highest dose (1000 ng), ALT-C increased all evaluated markers associated with fibrogenesis (collagen production and TGF-ß1 levels). All these factors reveal that ALT-C is a strong candidate to be exploited in the development of anti-inflammatory and anti-angiogenic therapies in chronic inflammatory processes.

Alternagin-C binding to α2ß1 integrin controls matrix metalloprotease-9 and matrix metalloprotease-2 in breast tumor cells and endothelial cells.

BACKGROUND: 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. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of α2ß1 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 α2ß1 integrin. Herein, we used ALT-C as a α2ß1 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. METHODS: ALT-C was purified by two steps of gel filtration followed by anion exchange chromatography. The α2ß1 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. RESULTS: Our data demonstrate that ALT-C, after binding to α2ß1 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. CONCLUSION: These results demonstrate that α2ß1 integrin controls MMP expression and reveal this integrin as a target for the development of antiangiogenic and antimetastatic therapies.