| INHIBITORY
EFFECT OF A NOVEL MIXTURE CONTAINING ASCORBIC ACID, LYSINE, PROLINE
AND GREEN TEA EXTRACT ON CRITICAL PARAMETERS IN CANCER PROGRESSION
M.W. Roomi, V. Ivanov, T. Kalinovsky, A. Niedzwiecki, M. Rath
Dr. Rath Research Institute, 1260 Memorex Drive, Santa Clara,
CA 95050
Presented at: 10th World Congress on Advances
in Oncology and 8th International Symposium on Molecular Medicine,
Crete, Greece, October 13-15, 2005.
Published in: International Journal of Molecular
Medicine, abstract #121, page S10.
Abstract
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.
Comment:
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).
|
