Inhibitory Effect of a Mixture Containing Ascorbic Acid, Lysine, Proline, and Green Tea Extract on Critical Parameters in Angiogenesis

M.W. Roomi, N. Roomi, V. Ivanov, T. Kalinovsky, A. Niedzwiecki, M. Rath
Oncology Reports 2005, 14(4): 807-815


Degradation of extracellular matrix (ECM) is a hallmark of tumor invasion, metastasis and angiogenesis. Based on Rath multitargeted approach to cancer by 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 antimetastatic effects using in vitro and in vivo experimental models.



Angiogenesis was measured using 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 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 a mouse chow diet supplemented with 0.5% NM for four weeks. In vitro studies on cell proliferation (MTT assay), MMP secretion (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 µg/ml in triplicate at each dose.


NM at 250 µg/ml caused 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 did tumors in the control group. In addition, NM exhibited dose-dependent inhibition of osteosarcoma U2OS cell proliferation (up to 60% at 1000 µg/ml), MMP-2 and -9 expression (with virtual total inhibition at 500 µg/ml NM), and invasion through Matrigel (with total inhibition at 100 µg/ml NM). Moreover, NM decreased U2OS cell expression of VEGF, angiopoietin-2, bFGF, PDGF and TGF beta-1.


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.

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