M.W. Roomi, V. Ivanov, T. Kalinovsky, A. Niedzwiecki, M. Rath Dr. Rath Research Institute, 1260 Memorex Drive, Santa Clara, CA 95050
10th World Congress on Advances in Oncology and 8th International Symposium on Molecular Medicine, Crete, Greece, October 13-15, 2005.
International Journal of Molecular Medicine, abstract #121, page S10.
Introduction: MMPs, especially –2 and –9 have been identified as key players in tumor invasion, metastasis and angiogenesis. We developed a novel formulation (NM) of ascorbic acid, lysine, proline and green tea extract that has shown significant anti-cancer activity against a number of cancer cell lines.
Objective: 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. Since angiogenesis depends on the interaction between tumor cells and endothelial cells, the present study focused on both of these cell types.
Methods: Human osteosarcoma cell lines U2OS and MNNG-HOS, and human umbilical endothelial cells (HUVECs) 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, HUVECs 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.
Results: 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, 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 NM. 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 NM. 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 HUVECs.
Conclusion: 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.
Degradation of extracellular matrix (ECM) is a hallmark of tumor invasion, metastasis and angiogenesis. Based on a 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. Using various in vitro and in vivo experimental models, we found that NM significantly inhibited angiogenesis and metastasis of various cancer cell lines. These results suggest that the nutrient mixture (NM) has strong therapeutic potential treating various cancers by blocking angiogenesis and tumor invasion and metastasis.
Chicken CAM Angiogenesis Assay
The nutrient mixture caused a significant (P<0.50) reduction (from 22 to 10 blood vessel branch points within the confined region of the filter disc) in bFGF-induced angiogenesis as compared to no treatment (bFGF only), as shown in Figure 1. The number of blood vessel branch points is relative to the number of newly sprouting angiogenic vessels
Figure 1 – Effect of NM on bFGF-induced angiogenesis in chick CAM assay In Vivo bFGF Induced Vessel Growth
To investigate the anti-angiogenic potential of NM, an extract of basement membrane proteins (Matrigel) impregnated with bFGF, an inducer of neovascularization was injected subcutaneously into C57BL/6J female mice. The test group of mice received NM in the injection mixture and the control mice received just the vehicle. After 7 days, red blood cells were abundant within the lumen of numerous vessels in the control mice (Figure 2A,B). In contrast, NM strongly suppressed the bFGF-stimulated angiogenesis in supplemented mice (Figures 2C,D).