In vivo and In vitro effect of a nutrient mixture on human hepatocarcinoma cell line SK-HEP-1
Long-term survival of patients with hepatocellular carcinoma (HCC), a common cancer worldwide, remains poor, due to metastasis and recurrence. Aim: To investigate the effect of a novel nutrient mixture (NM) containing ascorbic acid, lysine, proline, and green tea extract on human HCC cell line Sk-He...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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nasplib_isofts_kiev_ua-123456789-1386042025-02-23T19:57:32Z In vivo and In vitro effect of a nutrient mixture on human hepatocarcinoma cell line SK-HEP-1 Roomi, M.W. Roomi, N.W. Kalinovsky, T. Niedzwiecki, A. Rath, M. Original contributions Long-term survival of patients with hepatocellular carcinoma (HCC), a common cancer worldwide, remains poor, due to metastasis and recurrence. Aim: To investigate the effect of a novel nutrient mixture (NM) containing ascorbic acid, lysine, proline, and green tea extract on human HCC cell line Sk-Hep-1 In vivo and In vitro. Methods: After one week of isolation, 5–6 week old male athymic nude mice were inoculated with 3 x 106 SK-Hep-1 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 on SK-Hep-1 cells, measuring cell proliferation by MTT assay, invasion through Matrigel, apoptosis by green caspase detection kit, MMP secretion by zymography, and morphology by H&E staining. Results: NM inhibited tumor weight and burden of SK-Hep-1 xenografts by 42% and 33% respectively. In vitro, NM exhibited 33% toxicity over the control at 500 and 1000 μg/ml concentration. Zymography demonstrated MMP-2 and MMP-9 secretion which was inhibited by NM in a dose dependent fashion, with virtual total inhibition at 1000 μg/ml. Invasion through Matrigel was inhibited at 100, 500 and 1000 μg/ml by 53%, 83% and 100% respectively. NM induced slight apoptosis at 100 μg/ml, and profound apoptosis at 500 μg/ml and 1000 μg/ml concentration. Conclusions: These results suggest that NM has therapeutic potential in treatment of HCC. The research study was funded by Dr. Rath Health Foundation (Plantation, Florida, USA) a non-profit organization. 2010 Article In vivo and In vitro effect of a nutrient mixture on human hepatocarcinoma cell line SK-HEP-1 / M.W. Roomi, N.W. Roomi, T. Kalinovsky, A. Niedzwiecki, M. Rath // Experimental Oncology. — 2010. — Т. 32, № 2. — С. 84-91. — Бібліогр.: 25 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/138604 en Experimental Oncology application/pdf Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Roomi, M.W. Roomi, N.W. Kalinovsky, T. Niedzwiecki, A. Rath, M. In vivo and In vitro effect of a nutrient mixture on human hepatocarcinoma cell line SK-HEP-1 Experimental Oncology |
| description |
Long-term survival of patients with hepatocellular carcinoma (HCC), a common cancer worldwide, remains poor, due to metastasis and recurrence. Aim: To investigate the effect of a novel nutrient mixture (NM) containing ascorbic acid, lysine, proline, and green tea extract on human HCC cell line Sk-Hep-1 In vivo and In vitro. Methods: After one week of isolation, 5–6 week old male athymic nude mice were inoculated with 3 x 106 SK-Hep-1 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 on SK-Hep-1 cells, measuring cell proliferation by MTT assay, invasion through Matrigel, apoptosis by green caspase detection kit, MMP secretion by zymography, and morphology by H&E staining. Results: NM inhibited tumor weight and burden of SK-Hep-1 xenografts by 42% and 33% respectively. In vitro, NM exhibited 33% toxicity over the control at 500 and 1000 μg/ml concentration. Zymography demonstrated MMP-2 and MMP-9 secretion which was inhibited by NM in a dose dependent fashion, with virtual total inhibition at 1000 μg/ml. Invasion through Matrigel was inhibited at 100, 500 and 1000 μg/ml by 53%, 83% and 100% respectively. NM induced slight apoptosis at 100 μg/ml, and profound apoptosis at 500 μg/ml and 1000 μg/ml concentration. Conclusions: These results suggest that NM has therapeutic potential in treatment of HCC. |
| format |
Article |
| author |
Roomi, M.W. Roomi, N.W. Kalinovsky, T. Niedzwiecki, A. Rath, M. |
| author_facet |
Roomi, M.W. Roomi, N.W. Kalinovsky, T. Niedzwiecki, A. Rath, M. |
| author_sort |
Roomi, M.W. |
| title |
In vivo and In vitro effect of a nutrient mixture on human hepatocarcinoma cell line SK-HEP-1 |
| title_short |
In vivo and In vitro effect of a nutrient mixture on human hepatocarcinoma cell line SK-HEP-1 |
| title_full |
In vivo and In vitro effect of a nutrient mixture on human hepatocarcinoma cell line SK-HEP-1 |
| title_fullStr |
In vivo and In vitro effect of a nutrient mixture on human hepatocarcinoma cell line SK-HEP-1 |
| title_full_unstemmed |
In vivo and In vitro effect of a nutrient mixture on human hepatocarcinoma cell line SK-HEP-1 |
| title_sort |
in vivo and in vitro effect of a nutrient mixture on human hepatocarcinoma cell line sk-hep-1 |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2010 |
| topic_facet |
Original contributions |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/138604 |
| citation_txt |
In vivo and In vitro effect of a nutrient mixture on human hepatocarcinoma cell line SK-HEP-1 / M.W. Roomi, N.W. Roomi, T. Kalinovsky, A. Niedzwiecki, M. Rath // Experimental Oncology. — 2010. — Т. 32, № 2. — С. 84-91. — Бібліогр.: 25 назв. — англ. |
| series |
Experimental Oncology |
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| first_indexed |
2025-11-24T20:28:34Z |
| last_indexed |
2025-11-24T20:28:34Z |
| _version_ |
1849704961571553280 |
| fulltext |
84 Experimental Oncology 32, 84–91, 2010 (June)
The prognosis of hepatocellular carcinoma (HCC),
which is diagnosed in over 500,000 people worldwide
annually, remains dismal despite advances in its clini-
cal study [1, 2]. HCC is much more common in count-
ries in sub-Saharan Africa and Southeast Asia than
in the United States, ranking among the most common
type of cancer in these regions, and is more common
in men than in women [1]. In the U.S., liver and intrahe-
patic bile duct cancers incidence has been stable and
results in 4% of cancer deaths in men and 2% of cancer
deaths in women [2]. Average age at diagnosis for
liver cancer is 64 and over 90% of people diagnosed
with liver cancer are older than 45 years of age [1].
Hepatitis B and C, abuse of alcohol, and exposure
to aflatoxin are the most important etiological factors
implicated in HCC.
The most prevalent causes of death in patients with
HCC include uncontrolled metastasis and recurrence.
In recent years, efforts have been focused on exploring
many molecular markers related to invasion, metas-
tasis, recurrence and survival in HCC: DNA ploidy, the
proliferating activity of tumor cells, tumor suppressor
and promoter genes, cell cycle controllers, protein-
ases that degrade extracellular matrix, adhesion mo-
lecules, angiogenic factors and metabolic genes [3].
Among these factors, the matrix metalloproteinases
(MMPs) and the plasminogen activation system play
crucial roles in cancer invasion and metastasis. Levels
of MMP expression were found correlated to recur-
rence and reduced survival after HCC resection [4, 5].
MMPs are a family of zinc and calcium dependent
proteolytic enzymes, are able to degrade connective
tissue, among other substrates, such as basement
membrane collagen, and have been associated with
cancer metastasis and tumor angiogenesis. The
gelatinases, especially MMP-9 (gelatinase B) and
MMP-2 (gelatinase A), play a key role in degradation
of collagen type IV, a main component of the extracel-
lular matrix (ECM). These gelatinases are expressed
in hepatocellular carcinoma cells and are associated
with progression and invasion of these tumors [4, 6–9].
For example, Guo et al. [9] noted positive correlation of
MMP-9, MMP-2 and vascular endothelial growth factor
(VEGF) expression with recurrence of HCC.
Rath and Pauling [10] postulated that nutrients
such as lysine and ascorbic acid could act as natural
inhibitors of ECM proteolysis and, as such, have the
potential to modulate tumor growth and expansion.
These nutrients can exercise their anti-tumor effect
through the inhibition of MMPs and by strengthening
connective tissue surrounding cancer cells by influ-
encing collagen synthesis. These two processes are
essential for a tumor encapsulating effect. We have
developed strategies to inhibit cancer development
and its spread using naturally occurring nutrients such
as lysine, proline, ascorbic acid and green tea extract
(NM). This nutrient mixture has exhibited synergistic
anticancer activity in vivo and in vitro in a number
of cancer cell lines through inhibition of cancer cell
growth, MMP secretion, invasion, metastasis, and
angiogenesis [11–13]. The present study examines the
effect of NM on human HCC cell line SK-Hep-1, in vivo,
in athymic nude mice bearing SK-Hep-1 xenografts,
and in vitro, evaluating viability, MMP secretion, inva-
sion and induction of apoptosis.
We designed NM by defining critical physiological
targets in cancer progression and metastasis, such
as ECM integrity and MMP activity. ECM formation
IN VIVO AND IN VITRO EFFECT OF A NUTRIENT MIXTURE
ON HUMAN HEPATOCARCINOMA CELL LINE SK-HEP-1
M.W. Roomi, N.W. Roomi, T. Kalinovsky, A. Niedzwiecki*, M. Rath
Dr. Rath Research Institute, 1260 Memorex Drive, Santa Clara, CA 95050
Long-term survival of patients with hepatocellular carcinoma (HCC), a common cancer worldwide, remains poor, due to metastasis
and recurrence. Aim: To investigate the effect of a novel nutrient mixture (NM) containing ascorbic acid, lysine, proline, and green tea
extract on human HCC cell line Sk-Hep-1 in vivo and in vitro. Methods: After one week of isolation, 5–6 week old male athymic nude
mice were inoculated with 3 x 106 SK-Hep-1 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 on SK-Hep-1 cells, measuring cell proliferation by MTT
assay, invasion through Matrigel, apoptosis by green caspase detection kit, MMP secretion by zymography, and morphology by H&E
staining. Results: NM inhibited tumor weight and burden of SK-Hep-1 xenografts by 42% and 33% respectively. In vitro, NM exhibited
33% toxicity over the control at 500 and 1000 μg/ml concentration. Zymography demonstrated MMP-2 and MMP-9 secretion which
was inhibited by NM in a dose dependent fashion, with virtual total inhibition at 1000 μg/ml. Invasion through Matrigel was inhibited at
100, 500 and 1000 μg/ml by 53%, 83% and 100% respectively. NM induced slight apoptosis at 100 μg/ml, and profound apoptosis at
500 μg/ml and 1000 μg/ml concentration. Conclusions: These results suggest that NM has therapeutic potential in treatment of HCC.
Key Words: hepatocellular carcinoma, SK-Hep-1, nutrient mixture, athymic nude mice, apoptosis, invasion.
Received: April 1, 2010.
*Correspondence: Fax: 408-567-5030
E-mail: author@drrath.com
Abbreviations used: ECM — extracellular matrix; HCC – hepato-
cellular carcinoma; H&E — hematoxylin and eosin; NM – nutrient
mixture; MMP – matrix metalloproteinase; VEGF — vascular endo-
thelial growth factor.
Exp Oncol 2010
32, 2, 84–91
Experimental Oncology 32, 84–91, 2010 (June) 85
and structure is dependent upon adequate supplies
of ascorbic acid and the amino acids lysine and proline,
which insure proper synthesis and hydroxylation of col-
lagen fibers. Manganese and copper are also essen-
tial for collagen formation. Lysine, a natural inhibitor
of plasmin-induced proteolysis, plays an important role
in ECM stability [10, 14]. Green tea extract has been
shown to modulate cancer cell growth, metastasis,
angiogenesis, and other aspects of cancer progres-
sion [15–19]. N-acetyl cysteine has been observed to
inhibit MMP-9 activity [20] and invasive activities of
tumor cells [21]. Selenium has been shown to interfere
with MMP secretion and tumor invasion [22], as well as
migration of endothelial cells through ECM [21]. In ad-
dition to addressing ECM properties, some nutrients
are critical in inducing cancer cell death. A recent study
confirmed that ascorbic acid inhibits cell division and
growth through production of hydrogen peroxide [23].
Since 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 [24].
MATERIALS AND METHODS
Cancer cell line and culture. Human hepatoma
cell line SK-Hep-1 was obtained from ATCC (American
Type Culture Collection, Rockville, MD). SK-Hep-1
cells were maintained in minimum essential medium
(MEM) supplemented with 10% fetal bovine serum,
100 U/ml penicillin and 100 μg/ml streptomycin. The
media and sera used were obtained from ATCC, and
antibiotics (penicillin and streptomycin) were from
Gibco BRL, Long Island, NY.
Composition of the nutrient mixture. The nutrient
mixture (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-arginine 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 indi-
cated 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.
IN VIVO STUDIES
Animals. Male athymic mice (NCr-nu/nu), approxi-
mately five weeks of age on arrival, were purchased
from Simonsen Laboratories, Gilroy, CA and main-
tained in microisolator cages under pathogen-free
conditions on a 12-hour light/12-hour dark schedule
for a week. All procedures were performed according
to humane and customary care and use of experi-
mental animals and followed a protocol approved by
internal institutional animal safety review committee.
Experimental design. After housing for a week,
the mice (n = 12) were inoculated subcutaneously
with 3 x 106 SK-Hep-1 cells in 0.2 ml PBS and 0.1 ml
Matrigel (BD Bioscience, Bedford, MA). After injec-
tion, the mice were randomly 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 sac-
rificed and their tumors were excised and processed
for histology. Dimensions (length and width) of tumors
were measured using a digital caliper, and the tumor
burden was calculated using the following formula:
0.5 x length x width. Mean weight of mice at initiation
of study and termination of study did not differ signifi-
cantly between the groups.
Histology. Tissue samples were fixed in 10%
buffe red formalin. All tissues were embedded in pa-
raffin and cut at 4–5 microns. Sections were deparaf-
finized through xylene and graduated alcohol series to
water and stained with hematoxylin and eosin (H&E) for
evaluation using a standard light microscope.
IN VITRO STUDIES
Cell culture. Human SK-Hep-1 cells were grown
in MEM, supplemented with 10% fetal bovine serum,
penicillin (100 U/ml) and streptomycin (100 mg/ml)
in 24-well tissue culture plates (Costar, Cambridge,
MA). Cells were incubated with 1 ml of media at 37 °C
in a tissue culture incubator equilibrated with 95% air
and 5% CO2. At near confluence, the cells were treated
with the nutrient mixture, 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),
100 ng/ml was added to cells to induce MMP-9 secre-
tion. The plates were then returned to the incubator.
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 #M-2128) 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
formazan 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 solu-
tion in each well was considered to be proportional to
the number of cells. The OD570 of the control (treatment
without supplement) was considered 100%.
Gelatinase zymography. Gelatinase zymography
was performed in 10% Novex Pre-Cast SDS Polyacryla-
mide Gel (Invitrogen Corporation) in the presence of
0.1% gelatin under non-reducing conditions. Culture
media (20 mL) were mixed with sample buffer and
loaded for SDS-PAGE with tris glycine SDS buffer,
86 Experimental Oncology 32, 84–91, 2010 (June)
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 en-
zyme, 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, SK-Hep-1 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.
Morphology and apoptosis. Morphology of cells
cultured for 24 h in test concentrations of NM were
evaluated by H&E staining and observed and photo-
graphed by microscopy. At near confluence, SK-Hep-1
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
Caspases Detection Kit 135104, Invitrogen). The cells
were 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 STUDIES
Tumor growth and incidence. NM strongly inhi-
bited the growth and incidence of SK-Hep-1 xenografts
in nude mice. Mean tumor weight was inhibited by 42%
(p = 0.09) with NM 0.5% dietary supplementation, as
shown in Fig. 1, a and tumor burden was inhibited by
36% (p = 0.005), as shown in Fig. 1, b. Tumor incidence
decreased 33% with NM supplementation; all mice in
the control group developed tumors, while only 4 of
6 mice did on the supplemented diet. Representative
gross tumors from both groups are shown in Fig. 2.
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
Control NM 0.5%
M
ea
n
Tu
m
or
W
ei
gh
t,
g
0
5
10
15
20
25
30
Control NM 0.5%
*
M
ea
n
Tu
m
or
B
ur
de
n
a b
Fig. 1. a, effect of NM on mean tumor weight of SK-Hep-1
xenografts in male nude mice inoculated subcutaneously with
3 x 106 SK-Hep-1 cells. Each group was composed of 6 animals.
b, effect of NM on tumor burden of SK-Hep-1 xenografts in male
nude mice inoculated subcutaneously with 3 x 106 SK-Hep-1
cells. Each group was composed of 6 animals. *Indicates sig-
nificance of p = 0.005 with respect to control.
Control Group
NM-Group
Fig. 2. Gross tumors from male nude mice inoculated subcuta-
neously with 3 x 106 SK-Hep-1 cells. Each group was composed
of 6 animals. All mice in the control group developed tumors while only
4 of the 6 mice in the NM-supplemented group developed tumors
Histology. Histologically, the tumors from both
groups were composed of solid nests of irregularly
round to oval epithelioid cells with large irregularly
round nuclei and cytoplasm with indistinct borders.
Stromal spindle-shaped cells that were intermixed
with inflammatory cells, predominantly macrophages,
surrounded tumor nests, as well as the tumor masses.
Focal areas of necrosis involved about 30% of the
tumor tissue (Fig. 3, a–d).
IN VITRO STUDIES
Cytotoxicity. NM exhibited no toxicity to SK-Hep-1
cells in vitro at lower concentrations, but 33%
(p = 0.002) toxicity over control at 500 and 1000 μg/ml,
as shown in Fig. 4. There was significant negative cor-
relation between NM concentration and cell viability,
with coefficient r = –0.8622 (p < 0.0001).
Experimental Oncology 32, 84–91, 2010 (June) 87
0
20
40
60
80
100
120
140
Control NM
10 μg/ml
NM
50 μg/ml
NM
100 μg/ml
NM
500 μg/ml
NM
1000 μg/ml
Ce
ll
Pr
ol
ife
ra
tio
n,
%
o
f C
on
tro
l
* *
Fig. 4. Effect of NM on viability of SK-Hep-1 cells: MTT 24 h
assay performed in triplicates. *Indicates significance of at least
p = 0.002 with respect to control.
Gelatinase zymography. Zymography demon-
strated MMP-2 and MMP-9 secretion by normal and
PMA-treated SK-Hep-1 cells. NM inhibited secretion
of both MMPS in a dose-dependent fashion with virtual
total inhibition at 1000 μg/ml, as shown in Fig. 5, a–d.
Matrigel invasion. NM significantly inhibited SK-
Hep-1 invasion through Matrigel in a dose-dependent
manner, with 53% (p = 0.0001) inhibition at 100 μg/ml,
83% at 500 μg/ml (p < 0.0001) and 100% (p < 0.0001)
at 1000 μg/ml, as shown in Fig. 6 and Fig. 7, a–f. There
was significant negative correlation between NM con-
centration and number of SK-Hep-1 cells that invaded/
migrated through Matrigel: r = –0.8533, p < 0.0001.
a b
c d
Fig. 3. Histopathology of tumors: a, control (100X); b, control (400X); c, NM-supplemented (100X); d, NM-supplemented (400X)
a c
b d1 2 3 4 5 6 7 1 2 3 4 5 6 7
MMP-9 (94 kD)
MMP-2 (67 kD)
MMP-9 (94 kD)
MMP-2 (67 kD)
R2 = 0.9359
-20
0
20
40
60
80
100
120
140
Control NM
10 μg/ml
NM
50 μg/ml
NM
100 μg/ml
NM
500 μg/ml
NM
1000 μg/ml
Ab
so
lu
te
D
en
si
ty
, %
MMP-2
MMP-9
Linear MMP-9
Linear MMP-2
R2 = 0.7421
R2 = 0.8592
0
20
40
60
80
100
120
140
Control NM
10 μg/ml
NM
50 μg/ml
NM
100 μg/ml
NM
500 μg/ml
NM
1000 μg/ml
Ab
so
lu
te
D
en
si
ty
, %
MMP-2
MMP-9
Linear MMP-9
Linear MMP-2
Fig. 5. Effect of NM on SK-Hep-1 cell MMP-2 and MMP-9 secretion (a) and on PMA (100 ng/ml)-treated SK-Hep-1 cell MMP-2
and MMP-9 secretion (b). 1 — markers, 2 — control, 3–7 — NM 10, 50, 100, 500, 1000 μg/ml. Densitometry analysis of untreated
SK-Hep-1 cells (c) and PMA-treated SK-Hep-1 cells (d)
88 Experimental Oncology 32, 84–91, 2010 (June)
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
In
hi
bi
tio
n
of
In
va
si
on
, %
* *
*
*
*
Fig. 6. Effect of NM on Matrigel invasion of SK-Hep-1 cells per-
formed in triplicate. *Indicates significance of at least p = 0.001
with respect to control.
Morphology and apoptosis. H&E staining
showed no morphological changes at lower con-
centrations but revealed significant changes at the
higher concentrations of NM, as shown in Fig. 8, a–d.
Using the live green caspase kit, dose-dependent
apoptosis of SK-Hep-1 cells was evident with NM
challenge, as shown in Fig. 9, a–d. Approximately 16%
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. 10. At
100 μg/ml NM, 84% of cells were viable, 15% in early
apoptosis and 1% in late apoptosis and at 500 μg/ml
NM < 1% of cells were viable, 5% in early apoptosis,
and 95% in late apoptosis. Virtually all cells exposed
to 1000 μg/ml NM were in late apoptosis: < 1% viable,
2% in early apoptosis and 97% in late apoptosis.
DISCUSSION
The results of the in vivo study of human SK-Hep-1
xenografts in immune impaired (athymic) male nude
mice demonstrated significant suppression of SK-Hep-1
tumor growth (42% reduction in tumor weight and 36%
in tumor burden) with NM dietary supplementation. Re-
sults from the cellular proliferation and apoptosis studies
support the in vivo findings, as NM showed increased
toxicity in SK-Hep-1 cells with NM concentration and in-
a b
c d
e f
Fig. 7. Effect of NM on Matrigel invasion, photomicrographs: a, control; b, NM 10 μg/ml; c, NM 50 μg/ml; d, NM 100 μg/ml; e, NM
500 μg/ml; f, NM 1000 μg/ml
Experimental Oncology 32, 84–91, 2010 (June) 89
duced apoptosis in SK-Hep-1 cells in a dose-dependent
manner, with 33% inhibition of cell growth and apoptotic
induction of virtually all cells exposed to 500 μg/ml NM.
Tumor growth depends upon the generation of new
blood vessels to sustain cellular survival and proliferation.
Growing tumors become hypoxic and acidotic beyond the
size of 2 mm and secrete seve ral growth factors to stimu-
late local blood vessels to sprout branches. Activation of
endothelial cells leads to production of matrix metallopro-
teinases, enabling degradation of the surrounding extra-
cellular matrix and eventual development of new blood
vessels. In a previous study, NM significantly (p < 0.05)
reduced bFGF-induced angiogenesis (utilizing a chorioal-
lantoic membrane (CAM) assay in chick embryos, as well
as decreased human osteosarcoma U2OS cell expression
of VEGF, angiopoietin-2, bFGF, PDGF and TGFb-1 [11].
Invasion of host tissues is dependent on tumor cell
adhesion, cell migration, and proteolytic degradation
of the ECM by MMPs [25]. As mentioned previously,
MMPs, especially MMP-2 and MMP-9 are prognostic
for survival and metastatic potential in liver cancers.
Määtä et al. [6] found that MMP-2 and MMP-9 were
elevated in malignant tissue and that increased latent
and active MMP-2 levels predominantly were found in
tumor stroma while MMP-9 was primarily found in the
neoplastic epithelial cells. Furthermore, elevated MMP-2
and MMP-9 mRNA was associated with a tendency to
poorer survival of patients. In examination of gelatinolytic
activity in tissues of HCC patients, Sakamoto et al. [4]
found that overexpression of MMP-9 mRNA correlated
with growth of small hepatocellular carcinoma. In examin-
ing the expression of apoptotic and angiogenic factors
in a group of patients with HCC, Guo et al. [9] found that
expression of MMP-9, MMP-2 and VEGF levels positively
correlated with prognosis of HCC patients, specifically
with recurrence. Jiang et al. [5] found that high MMP-9,
but not VEGF expression, correlated with recurrence
or metastasis of HCC in patients post hepatectomy. In
our study, MMP-2 and MMP-9 secretion and Matrigel
invasion by SK-Hep-1 cells was inhibited by NM in a
dose-dependent manner with virtual total block of MMP
secretion and invasion at 1000 μg/ml.
a b
c d
e f
Fig. 8. Effect of NM on morphology of SK-Hep-1 cells: a, control; b, NM 10 μg/ml; c, NM 50 μg/ml; d, NM 100 μg/ml; e, NM
500 μg/ml; f, NM 1000 μg/ml. H&E staining
90 Experimental Oncology 32, 84–91, 2010 (June)
0
20
40
60
80
100
120
Control NM 100 μg/ml NM 500 μg/ml NM 1000 μg/ml
%
o
f G
ro
up
Live
Early
Late
Fig. 10. Quantitative analysis of live, early and late apoptotic cells
The relative stability of extracellular matrix plays
a key role in the development of cancer. We have
previously shown that the ECM synthesized by normal
fibroblasts treated with NM demonstrated increased
stability and significantly reduced osteosarcoma cell
growth rate, invasive activity (MMP-2 and MMP-9
secretion and Matrigel invasion), and adhesion to
collagen I and other substrates, suppressing tumor
growth independent of the immune system function
and inhibiting critical steps in cancer metastasis [12].
CONCLUSIONS
The results of the present study show that supple-
mentation with NM was effective in inhibiting growth
of SK-Hep-1 xenografts in nude mice and in inhibiting
growth, MMP secretion, and Matrigel invasion as well
as inducing apoptosis in SK-Hep-1 cell culture. Fur-
thermore, in contrast to the toxic side effects of current
chemotherapy, the nutrient mixture has been shown
to be a safe therapeutic agent. In a previous in vivo
study addressing safety issues, we found that gavag-
ing adult female ODS rats (weighing 250–300 gm)
with the nutrient mixture (at 30, 90 or 150 mg per day
for seven 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.
ACKNOWLEDGMENTS
The research study was funded by Dr. Rath Health
Foundation (Plantation, Florida, USA) a non-profit
organization.
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