title: Inhibitory effect of a mixture containing ascorbic acid, lysine, proline and green tea extract on critical parameters in angiogenesis.
abstract: 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.
authors.affiliation: Matthias Rath Research Institute, Cancer Division, 1260 Memorex Drive, Santa Clara, CA 95050, USA. t.kalinovsky@drrath.com
authors.forename: M Waheed
authors.initials: MW
authors.lastname: Roomi
authors.forename: Nusrath
authors.initials: N
authors.lastname: Roomi
authors.forename: Vadim
authors.initials: V
authors.lastname: Ivanov
authors.forename: Tatiana
authors.initials: T
authors.lastname: Kalinovsky
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Niedzwiecki
authors.forename: Matthias
authors.initials: M
authors.lastname: Rath
mesh_terms: D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D001205:Ascorbic Acid / Q000008:administration & dosage / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000378:metabolism; D003094:Collagen / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D005109:Extracellular Matrix; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D005557:Formaldehyde / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007797:Laminin / Q000494:pharmacology; D008239:Lysine / Q000008:administration & dosage / Q000494:pharmacology*; D020782:Matrix Metalloproteinases / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic; D012516:Osteosarcoma / Q000378:metabolism; D010936:Plant Extracts; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D011392:Proline / Q000008:administration & dosage / Q000494:pharmacology*; D011509:Proteoglycans / Q000494:pharmacology; D013662:Tea; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000378:metabolism; D053773:Transforming Growth Factor beta1; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D004338:Drug Combinations; D007797:Laminin; D010936:Plant Extracts; D010982:Platelet-Derived Growth Factor; D011509:Proteoglycans; C508960:TGFB1 protein, human; D013662:Tea; D013778:Tetrazolium Salts; C508961:Tgfb1 protein, mouse; D013844:Thiazoles; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2; C059485:matrigel; D005557:Formaldehyde; D003094:Collagen; D011392:Proline; D020782:Matrix Metalloproteinases; C022616:thiazolyl blue; D008239:Lysine; D001205:Ascorbic Acid