A novel mixture containing ascorbic acid, lysine, proline, and green tea extracts inhibits critical parameters in angiogenesis

M.W. Roomi, V. Ivanov, T. Kalinovsky, A. Niedzwiecki, M. Rath
In Anti-Angiogenic Functional and Medicinal Foods, 2007, Losso JN, Shahidi F, Bagchi D (eds), CRC Press, Boca Raton, London, New York, 561-580.

Angiogenesis, the formation of new capillaries from existing blood vessels, is necessary for tumor growth and metastasis to distal organs. MMPs have been recognized as critical to this process secondary to their ability to digest basement membrane and ECM components. The prevention of ECM degradation through the inhibition of MMP activity and increasing its integrity and strength has been shown to be a promising therapeutic approach to block the invasion that occurs during angiogenesis. We developed 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. Our objective was to determine whether NM exhibits anti-angiogenic and antimetastatic effects using in vitro and in vivo experimental models. Since angiogenesis depend on interaction between tumor and endothelial cells, the present study was aimed to determine the effect of NM on both these cells types.


Human osteosarcoma cell lines U2OS, MNNG-HOS, and human umbilical vein endothelial cells (HUVEC) were maintained in their recommended media supplemented with 10% FBS, penicillin and streptomycin in 24-well tissue culture plates. At near confluence, the U2OS cell cultures 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 invasion, MMP expression by gelatinase zymography, and cell migration by a 2mm 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 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.

NM at 250 µg/ml caused significant (P<0.05) reduction in bFGF-induced angiogenesis in CAM assay as compared to the control, also in bFGF induced vessels formation in the mouse Matrigel plug assay. 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, 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 cells in a dose-dependent manner. NM inhibited U2OS proliferation by 60% over the control at 1000 µg/ml. Zymography showed dose-dependent inhibition of MMP-2 and-9 expression with virtual total inhibition at 500 µg/ml concentration. Invasion through Matrigel was significantly reduced at 50 µg/ml (74%) and totally inhibited at 100 µg/ml NM. NM also reduced cell migration by scratch test in a dose dependent fashion with total inhibition at 500 µg/ml concentration. Moreover, in vitro NM decreased U2OS cell expression of VEGF, angiopoietin-2, bFGF, PDGF and TGF beta-1. NM also inhibited the tube formation of HUVEC.

In conclusion, these results corroborate with our earlier findings suggesting that NM is effective in inhibiting cancer cells growth, invasion, metastasis, and angiogenesis of tumor without toxicity.

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