Introduction: Angiogenesis, a process of abnormal new blood vessel development from pre-existing capillaries, is a main prerequisite for tumor growth. It is a complex multi-step process that plays an important role in tumor growth, invasion, and metastasis. We designed a novel formulation of lysine, proline, ascorbic acid and green tea extract (NM), which has shown significant anti-cancer activity against a number of cancer cell lines. The aim of the present study was to determine whether NM also exhibits anti-angiogenic and antimetastatic effects using in vitro and in vivo experimental models. Since angiogenesis depends on the interaction between tumor and endothelial cells, we sought to determine the effect NM on both cell types. Methods: Human osteosarcoma cell lines U2OS and MNNG-HOS, and human umbilical vein endothelial cells (HUVEC), grown to near confluence in 24-well tissue culture plates, were tested with NM at 0, 10, 50, 100, 500 and 1000 μg/ml in triplicate at each dose for proliferation, scratch/migration, MMP expression, and invasion. Cell proliferation was evaluated by MTT assay, invasion potential by Matrigel, MMP expression by gelatinase zymography, and cell migration by a 2 mm wide scratch in plates. For tube formation, HUVEC were cultured in previously polymerized Matrigel. Angiogenesis was measured using chorioallantoic membrane (CAM) assay in chick embryos. To determine the in vivo effect of NM on tumor xenograft growth, male nude mice were inoculated with 3x106 MNNG-HOS cells. Control mice were fed a mouse chow diet, while the test group was fed mouse chow diet supplemented with 0.5% NM for four weeks. Results: NM at 250 μg/ml caused significant (P<0.05) reduction in bFGF-induced angiogenesis in CAM. In vivo, NM inhibited tumor growth of osteosarcoma MNNG-HOS cells xenografts in nude mice by 53%; furthermore, tumors in NM-treated mice were less vascular and expressed lower levels of VEGF, MMP-9 and ki 67 immunohistochemically than did tumors in the control group. In addition, NM inhibited the proliferation, migration, MMP expression and invasion through Matrigel of osteosarcoma U2OS and of HUVEC cells in a dose dependent manner. NM inhibited U2OS proliferation by 60% at 1000 μg/ml and MMP-2 and -9 expression in a dose dependent fashion with virtual total inhibition at 500 μg/ml. Invasion through Matrigel was significantly reduced at 50 μg /ml (74%) and totally inhibited at 100 μg /ml NM. Cell migration by scratch test was also reduced by NM in a dose dependent fashion with total inhibition at 500 μg /ml concentration. Moreover, in vitro NM decreased U2OS cell production of VEGF, angiopoietin, bFGF, PDGF and TGF beta-1. NM also inhibited the tube formation of HUVEC. Conclusions: These results with our earlier findings suggest that NM is a relatively non-toxic formulation that inhibits growth, invasion, metastasis, and angiogenesis of tumor cells.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]