Degradation of extracellular matrix (ECM) is a hallmark of
tumor invasion,
metastasis and angiogenesis. Based on the Rath multitargeted approach to
cancer using natural substances to control ECM stability and enhancing its strength, we developed a novel formulation (NM) of
lysine,
proline,
ascorbic acid and
green tea extract that has shown significant anti-
cancer activity against a number of
cancer cell lines. The aim of the present study was to determine whether NM exhibits anti-angiogenic and anti-metastatic effects using in vitro and in vivo experimental models. Angiogenesis was measured using a chorioallantoic membrane (CAM) assay in chick embryos and bFGF-induced vessel growth in C57BL/6J female mice. To determine the in vivo effect of NM on the
tumor xenograft growth, male nude mice were inoculated with 3 x 10(6)
MNNG-HOS cells. Control mice were fed a mouse chow diet, while the test group was fed a mouse chow diet supplemented with 0.5% NM for 4 weeks. In vitro studies on cell proliferation (MTT assay),
MMP expression (zymography) and
Matrigel invasion were conducted on human
osteosarcoma U2OS, maintained in McCoy medium, supplemented with 10% FBS,
penicillin and
streptomycin in 24-well tissue culture plates and tested with NM at 0, 10, 50, 100, 500, and 1000 microg/ml in triplicate at each dose. NM at 250 microg/ml caused a significant (p<0.05) reduction in bFGF-induced angiogenesis in CAM. NM inhibited
tumor growth of
osteosarcoma MNNG-HOS cell xenografts in nude mice by 53%; furthermore,
tumors in NM-treated mice were less vascular and expressed lower levels of
VEGF and MMP-9 immunohistochemically than
tumors in the control group. In addition, NM exhibited a dose-dependent inhibition of
osteosarcoma U2OS cell proliferation (up to 60% at 1000 microg/ml), MMP-2 and -9 expression (with virtual total inhibition at 500 microg/ml NM), and invasion through
Matrigel (with total inhibition at 100 microg/ml NM). Moreover, NM decreased U2OS cell expression of
VEGF,
angiopoietin-2, bFGF, PDGF and TGFbeta-1. These results together with our earlier findings suggest that NM is a relatively non-toxic formulation, which inhibits growth, invasion,
metastasis, and angiogenesis of
tumor cells.