Antiangiogenic properties of a nutrient mixture in a model of hemangioma
The pathogenesis of hemangiomas is still largely unknown and the current therapy, such as systemic corticosteroid, vincristine, and interferon-alpha, is toxic and remains unsatisfactory. A nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract has shown significant ant...
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irk-123456789-1382072018-06-19T03:02:54Z Antiangiogenic properties of a nutrient mixture in a model of hemangioma Roomi, M.W. Kalinovsky, T. Niedzwiecki, A. Rath, M. Original contributions The pathogenesis of hemangiomas is still largely unknown and the current therapy, such as systemic corticosteroid, vincristine, and interferon-alpha, is toxic and remains unsatisfactory. A nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract has shown significant anti-angiogenic and anti-tumor effect against a number of cancer cell lines. Aim: Using a mouse hemangioendothelioma model, we investigated the efficacy of NM. We also tested the effect of NM in vitro, evaluating cell viability, MMP secretion, invasion, morphology and apoptosis. Methods: Athymic nude mice, 5–6 weeks old, were inoculated with 3 x106 EOMA cells subcutaneously and randomly divided into two groups; group A was fed a regular diet and group B — a regular diet supplemented with 0.5% NM. Four weeks later, the mice were sacrificed and their tumors were excised, weighed and processed for histology. We also tested the effect of NM in vitro. Results: NM inhibited the growth of tumors by 50%. In vitro, NM exhibited dose response cytotoxicity with 10%, 30% and 55% at 10, 100 and 1000 μg/ml. Invasion through Matrigel was inhibited at 50, 100 and 500 μg/ml by 25%, 30% and 100% respectively. NM induced dose-dependent apoptosis of EOMA cells. Conclusions: These results suggest that NM may have therapeutic potential in treating infantile hemangioendotheliomas and, perhaps, other cutaneous vascular tumors. 2009 Article Antiangiogenic properties of a nutrient mixture in a model of hemangioma / M.W. Roomi, T. Kalinovsky, A. Niedzwiecki, M. Rath // Experimental Oncology. — 2009. — Т. 31, № 4. — С. 214-219. — Бібліогр.: 33 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/138207 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions Roomi, M.W. Kalinovsky, T. Niedzwiecki, A. Rath, M. Antiangiogenic properties of a nutrient mixture in a model of hemangioma Experimental Oncology |
| description |
The pathogenesis of hemangiomas is still largely unknown and the current therapy, such as systemic corticosteroid, vincristine, and interferon-alpha, is toxic and remains unsatisfactory. A nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract has shown significant anti-angiogenic and anti-tumor effect against a number of cancer cell lines. Aim: Using a mouse hemangioendothelioma model, we investigated the efficacy of NM. We also tested the effect of NM in vitro, evaluating cell viability, MMP secretion, invasion, morphology and apoptosis. Methods: Athymic nude mice, 5–6 weeks old, were inoculated with 3 x106 EOMA cells subcutaneously and randomly divided into two groups; group A was fed a regular diet and group B — a regular diet supplemented with 0.5% NM. Four weeks later, the mice were sacrificed and their tumors were excised, weighed and processed for histology. We also tested the effect of NM in vitro. Results: NM inhibited the growth of tumors by 50%. In vitro, NM exhibited dose response cytotoxicity with 10%, 30% and 55% at 10, 100 and 1000 μg/ml. Invasion through Matrigel was inhibited at 50, 100 and 500 μg/ml by 25%, 30% and 100% respectively. NM induced dose-dependent apoptosis of EOMA cells. Conclusions: These results suggest that NM may have therapeutic potential in treating infantile hemangioendotheliomas and, perhaps, other cutaneous vascular tumors. |
| format |
Article |
| author |
Roomi, M.W. Kalinovsky, T. Niedzwiecki, A. Rath, M. |
| author_facet |
Roomi, M.W. Kalinovsky, T. Niedzwiecki, A. Rath, M. |
| author_sort |
Roomi, M.W. |
| title |
Antiangiogenic properties of a nutrient mixture in a model of hemangioma |
| title_short |
Antiangiogenic properties of a nutrient mixture in a model of hemangioma |
| title_full |
Antiangiogenic properties of a nutrient mixture in a model of hemangioma |
| title_fullStr |
Antiangiogenic properties of a nutrient mixture in a model of hemangioma |
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Antiangiogenic properties of a nutrient mixture in a model of hemangioma |
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antiangiogenic properties of a nutrient mixture in a model of hemangioma |
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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2009 |
| topic_facet |
Original contributions |
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http://dspace.nbuv.gov.ua/handle/123456789/138207 |
| citation_txt |
Antiangiogenic properties of a nutrient mixture in a model of hemangioma / M.W. Roomi, T. Kalinovsky, A. Niedzwiecki, M. Rath // Experimental Oncology. — 2009. — Т. 31, № 4. — С. 214-219. — Бібліогр.: 33 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
AT roomimw antiangiogenicpropertiesofanutrientmixtureinamodelofhemangioma AT kalinovskyt antiangiogenicpropertiesofanutrientmixtureinamodelofhemangioma AT niedzwieckia antiangiogenicpropertiesofanutrientmixtureinamodelofhemangioma AT rathm antiangiogenicpropertiesofanutrientmixtureinamodelofhemangioma |
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2025-07-10T05:20:34Z |
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2025-07-10T05:20:34Z |
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1837236052769636352 |
| fulltext |
214 Experimental Oncology 31, 214–219, 2009 (December)
Hemangiomas, the most common vascular tumors
in Caucasian infants, occur in approximately 1% of
normal neonates [1] and 12% of normal year-old child-
ren [2]; the incidence increases to 20% in premature
infants weighing less than 1000 g [3]. In a recent study,
Dr Drolet et al. [4] reported an increased rate of he-
mangiomas (an increase of 40% in the last 20 years) in
the United States and found the increase to be linked
to the rise in the frequency of low birth weight infants.
These lesions are characterized by rapid proliferation
of capillaries during the first year of life (proliferative
stage), followed by a slowed growth and regression of
the tumor over the next 5–6 years (involuting stage),
with complete regression of the lesion in 90% of af-
fected individuals by the age of 10–12 years [1, 5].
Though the majority have minor vascular birthmarks
that resolve without treatment, 10% of hemangiomas
cause severe skin distortion or problems with vision
or brea thing [6]. Such hemangiomas are generally
treated with agents, such as systemic corticosteroid,
vincristine, and interferon-alpha, which are associ-
ated with toxic effects. In addition, when these tumors
obstruct the airway or deform the cornea, surgical
removal becomes necessary.
Although the pathomechanism of hemangioma
is unknown, the rapid proliferation of endothelial
cells suggests the importance of angiogenesis. He-
mangiomas originate from a single endothelial cell
precursor and tumors are composed of microves-
sels lined by mitotically active endothelial cells and
pericytes [7]. Proliferating hemangiomas are highly
angiogenic with urinary basic fibroblast growth factor
levels 25–50 times the level seen in normal controls
[6]. In studying the cellular markers during the phases
of hemangioma, Takahashi et al. [6] found that the
proliferating phase was defined by high expression of
type IV collagenases and vascular endothelial growth
factor (VEGF) and the involuting phase — by elevated
expression of tissue inhibitor of metalloproteinases
(TIMP)1. High expression of bFGF and urokinases were
seen in both the proliferating and involuting phases.
These results may be used to evaluate therapeutic
agents [6].
Consumption of a plant-based diet has been as-
sociated with prevention of the development and
progression of cancer [8, 9]. Antiangiogenic proper-
ties of edible plant products, such as flavonoids, have
been reported [10, 11]. In previous studies we found
that a NM containing lysine, proline, arginine, ascorbic
acid, and green tea extract demonstrated significant
antiangiogenic activity utilizing the chorioallantoic
membrane (CAM) assay in chick embryos and bFGF-
induced vessel growth in C57BL/6J female mice in the
mouse Matrigel plug assay [12]. Furthermore, in vitro
we observed that NM decreased osteosarcoma U2OS
cell expression of VEGF, angiopoietin-2, bFGF, PDGF
and TGF beta-1 [12]. In a study on human umbilical
vein endothelial cells (HUVEC), we found NM inhibited
HUVEC migration, MMP expression, Matrigel invasion
and capillary tube formation [13].
In this study we investigated the effect of an
NM-supplemented diet on tumor growth of murine
hemangioendothelioma (EOMA) cells administered
subcutaneously in male athymic mice. We chose
a mouse EOMA model as this model has been utilized
to test the efficacy of antiangiogenic medications in
inhibiting vascular tumor proliferation [14]. Further,
we tested the effect of NM on EOMA cells in vitro,
inclu ding cell proliferation, MMP secretion, Matrigel
invasion and apoptosis.
ANTIANGIOGENIC PROPERTIES OF A NUTRIENT MIXTURE
IN A MODEL OF HEMANGIOMA
M.W. Roomi, T. Kalinovsky, A. Niedzwiecki*, M. Rath
Dr. Rath Research Institute, Oncology Division, Santa Clara, CA 95050
The pathogenesis of hemangiomas is still largely unknown and the current therapy, such as systemic corticosteroid, vincristine, and
interferon-alpha, is toxic and remains unsatisfactory. A nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea
extract has shown significant anti-angiogenic and anti-tumor effect against a number of cancer cell lines. Aim: Using a mouse heman-
gioendothelioma model, we investigated the efficacy of NM. We also tested the effect of NM in vitro, evaluating cell viability, MMP
secretion, invasion, morphology and apoptosis. Methods: Athymic nude mice, 5–6 weeks old, were inoculated with 3 x106 EOMA cells
subcutaneously and randomly divided into two groups; group A was fed a regular diet and group B — a regular diet supplemented with
0.5% NM. Four weeks later, the mice were sacrificed and their tumors were excised, weighed and processed for histology. We also
tested the effect of NM in vitro. Results: NM inhibited the growth of tumors by 50%. In vitro, NM exhibited dose response cytotoxicity
with 10%, 30% and 55% at 10, 100 and 1000 μg/ml. Invasion through Matrigel was inhibited at 50, 100 and 500 μg/ml by 25%, 30%
and 100% respectively. NM induced dose-dependent apoptosis of EOMA cells. Conclusions: These results suggest that NM may have
therapeutic potential in treating infantile hemangioendotheliomas and, perhaps, other cutaneous vascular tumors.
Key Words: hemangioma, hemangioendothelioma, nutrients, tumor growth, athymic nude mice, Matrigel invasion, cytotoxicity.
Received: October 12, 2009.
*Correspondense: Fax: 408 567 50 30
E-mail: author@drrath.com
Abbreviations used: CAM — chorioallantoic membrane; EOMA —
hemangioendothelioma; HUVEC — human umbilical vein endothe-
lial cells; NM — nutrient mixture.
Exp Oncol 2009
31, 4, 214–219
Experimental Oncology 31, 214–219, 2009 (December) 215
MATERIALS AND METHODS
In vivo studies. Male athymic mice (NCr-nu/nu),
approximately five weeks of age on arrival, were pur-
chased from Simonsen Laboratories, Gilroy, CA and
maintained in microisolator cages under pathogen-
free conditions on a 12-h light/12-h dark schedule for
a week. All procedures were performed according to
humane and customary care and use of experimental
animals and followed a protocol approved by internal
institutional animal safety review committee.
After housing for a week, the mice (n = 12) were
inoculated subcutaneously with 3x106 EOMA cells
(ATCC) in 0.2 ml PBS and 0.1 ml Matrigel (BD Biosci-
ence, Bedford, MA). After injection, the mice were ran-
domly divided into two groups; group A mice were fed
regular Purina mouse chow and group B — the regular
diet supplemented with 0.5% NM (w/w). During the
study, the mice consumed, on the average, 4 g of their
respective diets per day. Thus, the supplemented mice
received approximately 20 mg of NM per day. After four
weeks, the mice were sacrificed and their tumors were
excised and processed for histology. Mean weight of
mice at initiation of study and termination of study did
not differ significantly between the groups.
Tissue samples were fixed in 10% buffered forma-
lin. All tissues were embedded in paraffin and cut at
4–5 microns. Sections were deparaffinized through
xylene and graduated alcohol series to water and
stained with hematoxylin and eosin (H & E) for evalu-
ation using a standard light microscope.
Cell line and culture. EOMA cell line was obtained
from ATCC (American Type Culture Collection, Rockville,
MD). EOMA cells were maintained in DME (Dulbecco’s
modified Eagle’s) medium supplemented with 10% fetal
bovine serum, 100 U/ml penicillin and 100 μg/ml strep-
tomycin in 24-well tissue culture plates (Costar, Cam-
bridge, MA). The media and sera used were obtained
from ATCC, and antibiotics (penicillin and streptomycin)
were from Gibco BRL, Long Island, NY.
At near confluence, the cells were treated with the
NM, dissolved in media and tested at 0, 10, 50, 100,
500, and 1000 μg/ml in triplicate at each dose. Phorbol
12-myristate 13-acetate (PMA), 200 ng/ml was added
to cells to induce MMP-9 secretion. The plates were
then returned to the incubator.
Composition of the NM. The NM was composed
of the following in the ratio indicated: Vitamin C (as
ascorbic acid and as Mg, Ca, and palmitate ascorbate)
700 mg; L-lysine 1000 mg; L-proline 750 mg; L-argin-
ine 500 mg; N-acetyl cysteine 200 mg; standardized
green tea extract (derived from green tea leaves,
was obtained from US Pharma Lab; the certificate of
analysis indicated the following characteristics: total
polyphenol 80%, catechins 60%, epigallocatechin
gallate (EGCG) 35%, and caffeine 1.0%); 1000 mg;
selenium 30 μg; copper 2 mg; manganese 1 mg.
MTT assay. Cell viability was evaluated by MTT
assay, a colorimetric assay based on the ability of
viable cells to reduce a soluble yellow tetrazolium
salt [3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetra-
zolium bromide] (MTT) to a blue formazan crystal by
mitochondrial succinate dehydrogenase activity of
viable cells. This test is a good index of mitochondrial
activity and thus of cell viability. After 24 h incubation,
the cells were washed with phosphate buffered saline
(PBS) and 500 μl of MTT (Sigma, USA) 0.5 mg/ml in
media was added to each well. After MTT addition
(0.5 mg/ml) the plates were covered and returned to
the 37 °C incubator for 2 h, the optimal time for for-
mazan product formation. Following incubation, the
supernatant was carefully removed from the wells,
the formazan product was dissolved in 1 ml DMSO,
and absorbance was measured at 570 nm in Bio Spec
1601, Shimadzu spectrometer. The OD570 of the DMSO
solution in each well was considered to be proportional
to the number of cells. The OD570 of the control (treat-
ment without supplement) was considered 100%.
Gelatinase zymography. Gelatinase zymography
was performed in 10% Novex Pre-Cast SDS Polyacryl-
amide Gel (Invitrogen Corporation) in the presence of
0.1% gelatin under non-reducing conditions. Culture
media (20 μL) were mixed with sample buffer and
loaded for SDS-PAGE with Tris glycine SDS buffer,
as suggested by the manufacturer (Novex). Samples
were not boiled before electrophoresis. Following
electrophoresis the gels were washed twice in 2.5%
Triton X-100 for 30 min at room temperature to remove
SDS. The gels were then incubated at 37 °C overnight in
substrate buffer containing 50 mM Tris-HCl and 10 mM
CaCl2 at pH 8.0 and stained with 0.5% Coomassie Blue
R250 in 50% methanol and 10% glacial acetic acid
for 30 min and destained. Upon renaturation of the
enzyme, the gelatinases digested the gelatin in the
gel, producing clear bands against an intensely stained
background. Protein standards were run concurrently
and approximate molecular weights were determined
by plotting the relative mobilities of known proteins.
Matrigel invasion. Invasion studies were con-
ducted using Matrigel (Becton Dickinson) inserts in
24-well plates. Suspended in medium, EOMA cells
were supplemented with nutrients, as specified in the
design of the experiment and seeded on the insert in
the well. Thus both the medium on the insert and in the
well contained the same supplements. The plates with
the inserts were then incubated in a culture incubator
equilibrated with 95% air and 5% CO2 for 24 h. After
incubation, the media from the wells were withdrawn.
The cells on the upper surface of the inserts were
gently scrubbed away with cotton swabs. The cells that
had penetrated the Matrigel membrane and migrated
onto the lower surface of the Matrigel were stained with
hematoxylin and eosin and visually counted under the
microscope.
Apoptosis. At near confluence, EOMA cells were
challenged with NM dissolved in media at 0, 100, 500,
and 1000 μg/ml and incubated for 24 h. The cell culture
was washed with PBS and treated with the caspase
reagent as specified in the manufacturer’s protocol
(Molecular Probes Image-IT™ Live Green Poly Cas-
pases Detection Kit 135104, Invitrogen). The cells were
216 Experimental Oncology 31, 214–219, 2009 (December)
photographed under a fluorescence microscope and
counted. Green-colored cells represent viable cells,
while yellow orange represents early apoptosis and
red, late apoptosis.
Statistical analysis. The results were expressed as
means + SD, as indicated in the results, for the groups.
Data was analyzed by independent sample «t» test.
Pearson’s correlation coefficients were determined for
toxicity and invasion correlations to NM concentration
using MedCalc Software (Markakerke, Belgium).
RESULTS
In vivo
Tumor growth. NM strongly inhibited the growth
of EOMA xenografts in nude mice. Mean tumor weight
was significantly inhibited (by 50%, p = 0.0001) with NM
0.5% dietary supplementation, as shown in Fig. 1.
0
0.05
0.1
0.15
0.2
0.25
0.3
Control NM 0.5%
Treatment
Tu
m
or
W
ei
gh
t (
in
g
)
*
Fig. 1. Effect of NM on growth of EOMA xenografts in male nude
mice. NM inhibited the growth of tumors by 50% (p = 0.0001).
*Indicates significance of p = 0.0001 with respect to control.
Histology. Histologically, the tumors from both
groups were round, highly vascular invasive tumors
consistent with cavernous hemangioma with thrombo-
sis. Specimens consisted of two sections from similar
morphologically round highly vascular invasive tumors
composed of large, blood-filled cavitary spaces, lined
by highly anaplastic endothelial cells, which in many
locations were multilayered. Mitotic figures were infre-
quent. Fibrin thrombi and associated mild neutrophilic
infiltration were common in the cavernous spaces.
Tissue from control group showed more extensive
thrombosis with associated necrosis and inflamma-
tory cell infiltration than did the supplemented group
specimen (Fig. 2, a–d).
In vitro
Cytotoxicity. NM exhibited dose response toxicity
on EOMA cells in vitro, with maximum toxicity of 57%
(p < 0.0001) over the control at 1000 μg/ml, as shown
in Fig. 3. There was significant negative correlation
between NM concentration and cell viability, with coef-
ficient r = –0.8323 (p < 0.0001).
Gelatinase zymography. Zymography showed no
MMP-2 or MMP-9 secretion by normal or PMA-treated
EOMA cells (not shown).
Matrigel invasion. NM significantly inhibited EOMA
invasion through Matrigel in a dose-dependent manner,
with 28% (p = 0.0006) inhibition at 100 μg/ml and 100%
(p < 0.0001) at 500 μg/ml, as shown in Fig. 4 and 5.
There was significant negative correlation between NM
a
b
c
d
Fig. 2. Histopathology of tumors. Histologically, tumor specimens
from both groups were consistent with cavernous hemagioma with
thrombosis. Specimens consisted of two sections from similar
morphologically round highly vascular invasive tumors composed
of large, blood-filled cavitary spaces, lined by highly anaplastic
endothelial cells, which in many locations were multilayered. Mi-
totic figures were infrequent. Fibrin thrombi and associated mild
neutrophilic infiltration were common in the cavernous spaces.
Tissue from control group showed more extensive thrombosis with
associated necrosis and inflammatory cell infiltration than did the
supplemented group specimen. a, control 40x; b, control 200x;
c, NM-supplemented 40x; d, NM-supplemented 200x
Experimental Oncology 31, 214–219, 2009 (December) 217
concentration and number of EOMA cells that invaded/
migrated through Matrigel: r = –0.8984, p < 0.0001.
0
20
40
60
80
100
120
Control NM 10
μg/ml
NM 50
μg/ml
NM 100
μg/ml
NM 500
μg/ml
NM
1000
μg/ml Treatment
Ce
ll
Pr
ol
ife
ra
tio
n,
%
o
f C
on
tro
l
* *
* *
Fig. 3. Effect of NM on viability of EOMA cells. NM exhibited dose
response toxicity on EOMA cells in vitro, with maximum toxicity
of 57% (p < 0.001) over the control at 1000 μg/ml. There was
significant negative correlation between NM concentration and
cell viability, with coefficient r = –0.8323 (p < 0.0001). *Indicates
significance of at least p = 0.002 with respect to control.
0
20
40
60
80
100
120
Control NM 50 μg/ml NM 100 μg/ml NM 500 μg/ml
Treatment
In
hi
bi
tio
n
of
In
va
si
on
, %
o
f C
on
tro
l
*
*
*
Fig. 4. Effect of NM on Matrigel invasion of EOMA cells. NM
significantly inhibited EOMA invasion through Matrigel in a
dose-dependent manner, with 28% (p = 0.0006) inhibition at
100 μg/ml and 100% (p < 0.00001) at 500 μg/ml. There was
significant negative correlation between NM concentration and
number of EOMA cells that invaded/migrated through Matrigel:
r = –0.8984, p < 0.0001. *Indicates significance of at least p =
0.05 with respect to control.
Apoptosis. Using the live green caspase kit, dose-
dependent apoptosis of EOMA cells was evident with
NM challenge, as shown in Fig. 6, a–d. Approximately
50% of cells exposed to 100 μg/ml NM were apoptotic;
the number of apoptotic cells increased significantly
with increased NM concentration. Quantitative analysis
of live, early and late apoptotic cells is shown in Fig. 7.
At 100 μg/ml NM, 49% of cells were viable, 16% in early
apoptosis and 35% in late apoptosis and at 500 μg/ml
NM 2% of cells were viable, 9% in early apoptosis, and
89% in late apoptosis. Virtually all cells exposed to
1000 μg/ml NM were in late apoptosis: 1% viable, 7%
in early apoptosis and 92% in late apoptosis.
DISCUSSION
Using the EOMA mouse model, dietary supplemen-
tation with 0.5% NM resulted in a dramatic reduction
(57%) in tumor growth in immune impaired (athymic)
male nude mice after subcutaneous administration of
3 x106 EOMA cells. Results from the cellular prolifera-
tion and apoptosis studies support the in vivo findings,
as NM showed dose-dependent toxicity in EOMA cells
and induced apoptosis in a dose-dependent manner,
with 55% inhibition of cell growth and apoptotic induc-
tion of virtually all cells exposed to 500 μg/ml NM.
Thus, mechanisms of NM-induced inhibition of mouse
EOMA growth involved both inhibition of proliferation
and increased tumor cell apoptosis, as seen in the
natural involution of the more common vascular tumor
hemangiomas of infancy [15].
a
b
c
d
Fig. 5. Effect of NM on Matrigel invasion: photomicrographs.
a, control; b, NM 50 μg/ml; c, NM 100 μg/ml; d, NM 500 μg/ml
218 Experimental Oncology 31, 214–219, 2009 (December)
Invasion of EOMA through Matrigel was also found
to be significantly inhibited by NM in vitro. As mentioned
previously, the proliferating phase of hemangioma is
associated with high expression of type IV collagenases
and vascular endothelial growth factor (VEGF) [6].
Degradation of the ECM by migrating endothelial cells
and their subsequent invasion of the underlying stroma
of neighboring tissues where they organize into new
capillary structures are critical in angiogenesis. Regula-
tion of angiogenesis is achieved through a balance of
pro- and anti-angiogenic stimuli. The two major pro-
angiogenic factors are MMPs that degrade the ECM and
vascular endothelial growth factor, a stimulatory factor
for cell migration. In our previous study on HUVEC, NM
demonstrated a dose-dependent inhibition of capillary
tube formation on Matrigel, with completed disruption
of tubules at 500 μg/ml [13]. Capillary tube formation
in the basement membrane like matrix of Matrigel is
a complex process requiring cell-matrix interactions,
intercellular communications and cell motility.
0
20
40
60
80
100
120
Control NM 100 μg/ml NM 500 μg/ml NM 1000 μg/ml
Treatment
%
o
f G
ro
up
Live
Early
Late
Fig. 7. Quantitative analysis of live, early and late apoptotic cells. At
100 μg/ml NM, 49% of cells were viable, 16% in early apoptosis and
35% in late apoptosis and at 500 μg/ml NM 2% of cells were viable,
9% in early apoptosis, and 89% in late apoptosis. At 1000 μg/ml NM:
1% viable, 7% in early apoptosis and 92% in late apoptosis
The NM was formulated by selecting nutrients that
act on critical physiological targets in cancer progres-
sion and metastasis. Adequate supplies of ascorbic acid
and the amino acids lysine and proline, are essential for
optimal ECM formation and structure as these nutrients
insure proper synthesis and hydroxylation of collagen
fibers. Manganese and copper are also essential for
collagen formation. Lysine also contributes to ECM
stability as a natural inhibitor of plasmin-induced pro-
teolysis [16, 17]. Green tea extract has been shown to
control cancer cell growth, metastasis, angiogenesis,
and other aspects of cancer progression [18–24].
N-acetyl cysteine and selenium have been observed
to inhibit MMP-9 and invasive activities of tumor cells,
as well as migration of endothelial cells through ECM
[25–29]. Ascorbic acid has been shown to inhibit tumor
growth via antiangiogenic activity [30] and to inhibit cell
division and growth in vitro through production of hydro-
gen peroxide [31]. Arginine is a precursor of nitric oxide
(NO); any deficiency of arginine can limit the production
of NO, which has been shown to predominantly act as an
inducer of apoptosis, as in breast cancer cells [32].
CONCLUSIONS
Current therapy for hemangiomas, such as cor-
ticosteroids, interferon-α and vincristine, all require
systemic administration and are associated with toxic
side effects. Thus, they are only used in severe cases
a
b
c
d
Fig. 6. Effect of NM on apoptosis of EOMA cells: live-green cas-
pases. Using the live green caspase kit, dose-dependent apopto-
sis of EOMA cells was evident with NM challenge. Approximately
50% of cells exposed to 100 μg/ml NM were apoptotic; virtually all
cells exposed to 500 μg/ml NM were in late apoptosis. a, control;
b, NM 100 μg/ml; c, NM 500 μg/ml; d, NM 1000 μg/ml
Experimental Oncology 31, 214–219, 2009 (December) 219
of hemangiomas and most cases are left untreated.
The results of the present study show that supple-
mentation with NM was effective in inhibiting growth of
EOMA xenografts in nude mice and in inhibiting growth
and Matrigel invasion, as well as inducing apoptosis in
EOMA cell culture. Furthermore, in contrast to the toxic
side effects of current treatments, the NM has been
shown to be a safe therapeutic agent. In a previous
in vivo study addressing safety issues, we found that
gavaging adult female ODS rats (weighing 250–300 g)
with the NM (at 30, 90 or 150 mg per day for 7 days),
had neither adverse effects on vital organs (heart, liver
and kidney), nor on the associated functional serum
enzymes, indicating that this mixture is safe to use
even at these high doses, which far exceed the normal
equivalent dosage of the nutrient [33]. While clinical
studies are necessary to better determine the efficacy
of nutrient therapy in hemangiomas, the results of this
study suggest the NM may have therapeutic potential
in treating infantile hemangioendotheliomas and other
cutaneous vascular tumors with minimal toxic effect.
ACKNOWLEDGMENTS
The research study was funded by Dr. Rath Health
Foundation (Plantation, Florida, USA), a non-profit
organization.
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