Matrix metalloproteinases are involved in both type I (apoptosis) and type II (autophagy) cell death induced by sodium phenylacetate in MDA-MB-231 breast tumour cells.

The effects of sodium phenylacetate (NaPa), an antitumoral molecule, on cell death and matrix metalloproteinase (MMP) activities and synthesis were investigated in two metastatic breast tumour cell lines, MDA-MB-231 and MDA-MB-435, cultured on three-dimensional type I collagen gels (3-D cultures). In both cell lines, NaPa inhibited cell proliferation and induced apoptotic cell death as measured by TUNEL assay, with an IC(30) of 20 mM and 10 mM for MDA-MB-231 and MDA-MB-435 cells, respectively. In MDA-MB-231 cells, NaPa also induced (i) an autophagic process evidenced by the appearance of autophagic vacuoles and an increased phosphatase acid activity, (ii) the formation of pseudopodia and (iii) an increase in MMP-1 and MMP-9 secretion without affecting MT1-MMP. In NaPa-treated MDA-MB-435 cells, no autophagic vacuoles were formed but F-actin depolymerisation was observed. MMP-1, MMP-9 and MT1-MMP levels were strongly enhanced in these cells but MMPs were not secreted and accumulated intracellularly. When breast cancer cells were treated with NaPa in the presence of an MMP inhibitor (GM6001), apoptotic cell death decreased and the induction of autophagic vacuoles in MDA-MB-231 cells was inhibited. Taken together, these data suggest that MMPs are involved in the autophagic cell death and/or apoptosis of breast tumour cells.

Breast tumor cells transendothelial migration induces endothelial cell anoikis through extracellular matrix degradation.

Mutual interactions between human breast cancer cells and endothelial cells were studied in a model mimicking tumor cell intravasation. MDA-MB-231 tumor cells and human umbilical vein endothelial cells (HUVEC) were cocultured on opposite sides of a Transwell filter allowing tumor cell contacts with the basement membrane of the HUVEC forming endothelium and tumor cell transendothelial migration. Confocal microscopy analysis showed that transmigrating MDA-MB-231 cells lay under the HUVEC, thereby inducing HUVEC detachment and tumor cell-HUVEC contact-dependent apoptosis. GM6001 a matrix metalloproteinase (MMP) inhibitor inhibited almost completely, the MDA-MB-231 cell transendothelial migration and the anoikis process. In this intravasation model, a tumor cell invasive mechanism was demonstrated (i) induction of extensive endothelial anoikis induced by degradation of the extracellular matrix (ECM) components, (ii) activation of pro-matrix metalloproteinase (MMP)-2 into MMP-2 by the MT1-MMP-TIMP (tissue inhibitor metalloproteinase) 2-pro-MMP-2 membrane complex and (iii) attraction and migration of metastatic cell through apoptotic endothelium. These interactions could partly explain the necrosis-angiogenesis relationship in tumor angiogenesis.

Modulation of in vitro and in vivo angiogenesis by alternagin-C, a disintegrin-like protein from Bothrops alternatus snake venom and by a peptide derived from its sequence.

We have previously demonstrated that alternagin-C (ALT-C), a disintegrin-like protein from the venom of the Brazilian snake Bothrops alternatus, induces human vascular endothelial cell (HUVEC) proliferation by up-regulating the expression of vascular endothelial growth factor (VEGF). Here, we show that ALT-C is also able to induce in vivo angiogenesis using the model of matrigel plug in nude mice. Fibroblast growth factor (FGF) alone or supplemented with ALT-C was mixed with melted matrigel and subcutaneously injected in nude mice. After two weeks, the matrigel plugs were removed and analyzed to verify endothelial cell migration and new vessel formation. ALT-C (1 and 10 ng) strongly induced endothelial cell migration as well as the formation of new vessels. However, in higher concentrations, ALT-C strongly inhibited angiogenesis. In low concentrations (1 and 10nM), ALT-C also up-regulates the expression of VEGF receptor 2 (VEGFR2, KDR) mostly after 48 h, but it did not affect VEGFR1 (Ftl-1) in HUVEC cells as demonstrated by real-time PCR analysis. However, in higher concentrations (100 nM) the expression of both receptors is down-regulated. A peptide derived from ALT-C primary structure also affects HUVEC proliferation in vitro and angiogenesis in vivo. In conclusion, the present study shows for the first time the in vivo angiogenesis induced by a disintegrin-like molecule and the modulation of VEGFRs as well.