Anti-angiogenic effects of a nutrient mixture on human umbilical vein endothelial cells

Matrix metalloproteinases (MMPs) have been recognized as key players in the degradation of the extracellular matrix (ECM) by migration and proliferation of endothelial cells and their subsequent invasion of the underlying stroma. The prevention of ECM degradation through the inhibition of MMP activity has been shown to be a promising therapeutic approach to block the invasion that occurs during angiogenesis. In previous studies, we demonstrated the anti-tumor effect of a nutrient mixture (NM) containing ascorbic acid, lysine, proline, green tea extract, arginine, N-acetyl cysteine, selenium, copper and manganese on various tumor cell lines in vivo and in vitro. The aim of the present study was to determine whether this mixture has anti-angiogenic effects on human umbilical vein endothelial cells (HUVECs). At near confluence, the HUVEC cell cultures were tested with NM at 0, 10, 50, 100, 500, and 1000 microg/ml in triplicate at each dose for proliferation, migration, MMP expression, and invasion. Cell proliferation was evaluated by MTT assay, invasion potential by Matrigel invasion, MMP expression by gelatinase zymography, and cell migration by a 2 mm-wide scratch in plates. For tube formation, HUVECs were cultured in previously polymerized Matrigel. NM inhibited HUVEC migration, MMP expression and invasion through Matrigel in a dose-dependent manner. Zymography showed a dose-dependent inhibition of MMP-2 expression with virtual total inhibition at a 500 microg/ml concentration. Invasion through Matrigel was totally inhibited at 500 microg/ml NM. NM reduced cell migration by scratch test in a dose-dependent fashion with total inhibition at a 500 microg/ml concentration. NM also inhibited the tube formation of HUVECs, but did not significantly inhibit cell proliferation. These results together with our earlier findings suggest that NM is a relatively non-toxic formulation with anti-angiogenic effects, such as inhibiting vascular tube formation and endothelial cell invasion and migration.