Ascorbate depletion increases growth and metastasis of melanoma cells in vitamin C deficient mice
Aim: Our main objective was to determine the effect of ascorbate supplementation in mice unable to synthesize ascorbic acid (gulo KO) when challenged with murine B16FO cancer cells. Methods: Gulo KO female mice 36–40 weeks of age were deprived of or maintained on ascorbate in food and water for 4 we...
Gespeichert in:
| Datum: | 2011 |
|---|---|
| Hauptverfasser: | , , , , , |
| Format: | Artikel |
| Sprache: | English |
| Veröffentlicht: |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2011
|
| Schriftenreihe: | Experimental Oncology |
| Schlagworte: | |
| Online Zugang: | http://dspace.nbuv.gov.ua/handle/123456789/138671 |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Zitieren: | Ascorbate depletion increases growth and metastasis of melanoma cells in vitamin C deficient mice / J. Cha, M.W. Roomi, V. Ivanov, T. Kalinovsky, A. Niedzwiecki, M. Rath // Experimental Oncology. — 2011. — Т. 33, № 4. — С. 226-230. — Бібліогр.: 20 назв. — англ. |
Institution
Digital Library of Periodicals of National Academy of Sciences of Ukraine| id |
irk-123456789-138671 |
|---|---|
| record_format |
dspace |
| spelling |
irk-123456789-1386712018-06-20T03:10:38Z Ascorbate depletion increases growth and metastasis of melanoma cells in vitamin C deficient mice Cha, J. Roomi, M.W. Ivanov, V. Kalinovsky, T. Niedzwiecki, A. Rath, M. Original contributions Aim: Our main objective was to determine the effect of ascorbate supplementation in mice unable to synthesize ascorbic acid (gulo KO) when challenged with murine B16FO cancer cells. Methods: Gulo KO female mice 36–40 weeks of age were deprived of or maintained on ascorbate in food and water for 4 weeks prior to subcutaneous injection of 2.5×106 B16FO murine melanoma cells in the right flank of mice. A control group of wild type mice were also injected with the melanoma cells and maintained on a regular murine diet. Mice were continued on their respective diets for another 2 weeks after injection. The mice were then sacrificed, blood was drawn and their tumors were measured, excised and processed for histology. Results: Mean weight of animals decreased significantly (30%, p < 0.0001) in the ascorbate-restricted group but increased slightly, but insignificantly, in the ascorbate-supplemented group. The mean tumor weight in ascorbate supplemented mice was significantly reduced (by 64%, p = 0.004) compared to tumor weight in ascorbate-deprived gulo mice. The mean tumor weight of wild type mice did not differ significantly from the ascorbate-supplemented mice. Gulo KO mice supplemented with ascorbate developed smaller tumors with more collagen encapsulation and fibrous capsule interdigitation, while gulo KO mice deprived of ascorbate hosted large tumors with poorly defined borders, showing more necrosis and mitosis. Ascorbate supplementation of gulo KO mice resulted in profoundly decreased serum inflammatory cytokine IL-6 (90% decrease, p = 0.04) and IL-1β (62% decrease) compared to the levels in gulo KO mice deprived of ascorbate. Conclusion: Ascorbate supplementation modulated tumor growth and inflammatory cytokine secretion as well as enhanced encapsulation of tumors in scorbutic mice. 2011 Article Ascorbate depletion increases growth and metastasis of melanoma cells in vitamin C deficient mice / J. Cha, M.W. Roomi, V. Ivanov, T. Kalinovsky, A. Niedzwiecki, M. Rath // Experimental Oncology. — 2011. — Т. 33, № 4. — С. 226-230. — Бібліогр.: 20 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/138671 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
English |
| topic |
Original contributions Original contributions |
| spellingShingle |
Original contributions Original contributions Cha, J. Roomi, M.W. Ivanov, V. Kalinovsky, T. Niedzwiecki, A. Rath, M. Ascorbate depletion increases growth and metastasis of melanoma cells in vitamin C deficient mice Experimental Oncology |
| description |
Aim: Our main objective was to determine the effect of ascorbate supplementation in mice unable to synthesize ascorbic acid (gulo KO) when challenged with murine B16FO cancer cells. Methods: Gulo KO female mice 36–40 weeks of age were deprived of or maintained on ascorbate in food and water for 4 weeks prior to subcutaneous injection of 2.5×106 B16FO murine melanoma cells in the right flank of mice. A control group of wild type mice were also injected with the melanoma cells and maintained on a regular murine diet. Mice were continued on their respective diets for another 2 weeks after injection. The mice were then sacrificed, blood was drawn and their tumors were measured, excised and processed for histology. Results: Mean weight of animals decreased significantly (30%, p < 0.0001) in the ascorbate-restricted group but increased slightly, but insignificantly, in the ascorbate-supplemented group. The mean tumor weight in ascorbate supplemented mice was significantly reduced (by 64%, p = 0.004) compared to tumor weight in ascorbate-deprived gulo mice. The mean tumor weight of wild type mice did not differ significantly from the ascorbate-supplemented mice. Gulo KO mice supplemented with ascorbate developed smaller tumors with more collagen encapsulation and fibrous capsule interdigitation, while gulo KO mice deprived of ascorbate hosted large tumors with poorly defined borders, showing more necrosis and mitosis. Ascorbate supplementation of gulo KO mice resulted in profoundly decreased serum inflammatory cytokine IL-6 (90% decrease, p = 0.04) and IL-1β (62% decrease) compared to the levels in gulo KO mice deprived of ascorbate. Conclusion: Ascorbate supplementation modulated tumor growth and inflammatory cytokine secretion as well as enhanced encapsulation of tumors in scorbutic mice. |
| format |
Article |
| author |
Cha, J. Roomi, M.W. Ivanov, V. Kalinovsky, T. Niedzwiecki, A. Rath, M. |
| author_facet |
Cha, J. Roomi, M.W. Ivanov, V. Kalinovsky, T. Niedzwiecki, A. Rath, M. |
| author_sort |
Cha, J. |
| title |
Ascorbate depletion increases growth and metastasis of melanoma cells in vitamin C deficient mice |
| title_short |
Ascorbate depletion increases growth and metastasis of melanoma cells in vitamin C deficient mice |
| title_full |
Ascorbate depletion increases growth and metastasis of melanoma cells in vitamin C deficient mice |
| title_fullStr |
Ascorbate depletion increases growth and metastasis of melanoma cells in vitamin C deficient mice |
| title_full_unstemmed |
Ascorbate depletion increases growth and metastasis of melanoma cells in vitamin C deficient mice |
| title_sort |
ascorbate depletion increases growth and metastasis of melanoma cells in vitamin c deficient mice |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2011 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/138671 |
| citation_txt |
Ascorbate depletion increases growth and metastasis of melanoma cells in vitamin C deficient mice / J. Cha, M.W. Roomi, V. Ivanov, T. Kalinovsky, A. Niedzwiecki, M. Rath // Experimental Oncology. — 2011. — Т. 33, № 4. — С. 226-230. — Бібліогр.: 20 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
AT chaj ascorbatedepletionincreasesgrowthandmetastasisofmelanomacellsinvitamincdeficientmice AT roomimw ascorbatedepletionincreasesgrowthandmetastasisofmelanomacellsinvitamincdeficientmice AT ivanovv ascorbatedepletionincreasesgrowthandmetastasisofmelanomacellsinvitamincdeficientmice AT kalinovskyt ascorbatedepletionincreasesgrowthandmetastasisofmelanomacellsinvitamincdeficientmice AT niedzwieckia ascorbatedepletionincreasesgrowthandmetastasisofmelanomacellsinvitamincdeficientmice AT rathm ascorbatedepletionincreasesgrowthandmetastasisofmelanomacellsinvitamincdeficientmice |
| first_indexed |
2025-07-10T06:19:56Z |
| last_indexed |
2025-07-10T06:19:56Z |
| _version_ |
1837239788896256000 |
| fulltext |
226 Experimental Oncology 33, 226–230, 2011 (December)
ASCORBATE DEPLETION INCREASES GROWTH AND METASTASIS
OF MELANOMA CELLS IN VITAMIN C DEFICIENT MICE
J. Cha, M.W. Roomi, V. Ivanov, T. Kalinovsky, A. Niedzwiecki*, M. Rath
Dr. Rath Research Institute, Santa Clara, CA 95050
Aim: Our main objective was to determine the effect of ascorbate supplementation in mice unable to synthesize ascorbic acid
(gulo KO) when challenged with murine B16FO cancer cells. Methods: Gulo KO female mice 36–40 weeks of age were deprived
of or maintained on ascorbate in food and water for 4 weeks prior to subcutaneous injection of 2.5×106 B16FO murine mela-
noma cells in the right flank of mice. A control group of wild type mice were also injected with the melanoma cells and maintained
on a regular murine diet. Mice were continued on their respective diets for another 2 weeks after injection. The mice were then
sacrificed, blood was drawn and their tumors were measured, excised and processed for histology. Results: Mean weight of animals
decreased significantly (30%, p < 0.0001) in the ascorbate-restricted group but increased slightly, but insignificantly, in the ascor-
bate-supplemented group. The mean tumor weight in ascorbate supplemented mice was significantly reduced (by 64%, p = 0.004)
compared to tumor weight in ascorbate-deprived gulo mice. The mean tumor weight of wild type mice did not differ significantly
from the ascorbate-supplemented mice. Gulo KO mice supplemented with ascorbate developed smaller tumors with more collagen
encapsulation and fibrous capsule interdigitation, while gulo KO mice deprived of ascorbate hosted large tumors with poorly de-
fined borders, showing more necrosis and mitosis. Ascorbate supplementation of gulo KO mice resulted in profoundly decreased
serum inflammatory cytokine IL-6 (90% decrease, p = 0.04) and IL-1β (62% decrease) compared to the levels in gulo KO mice
deprived of ascorbate. Conclusion: Ascorbate supplementation modulated tumor growth and inflammatory cytokine secretion as well
as enhanced encapsulation of tumors in scorbutic mice.
Key Words: ascorbate, gulo (-/-) mice, melanoma tumor growth, tumor capsule, cytokines.
Melanoma causes the majority of skin cancer-re-
lated deaths, due to metastasis to other areas of the
body, such as lymph nodes, lungs, liver, brain or bone.
Though often curable in its early stages, metastatic
malignant melanoma is an extremely aggressive can-
cer with no current viable treatment. Thus, any suc-
cessful treatment for melanoma has to target metas-
tasis. Invasion of host tissues by cancer cells requires
alteration of cancer cell adhesion, cell migration and
proteolytic degradation of the extracellular matrix
(ECM) [1]. Stromal resistance is dependent upon the
ability of the host to encapsulate tumor cells in a dense
barrier of fibrous tissue. Rath and Pauling postulated
that essential nutrients needed for optimum synthesis
and structure of collagen, such as vitamin C and lysine
are critical components in natural control of spread
and invasion of cancer cells in the body [2]. Ascorbic
acid, needed for synthesis and hydroxylation of col-
lagen and optimal stability of the extracellular matrix
(ECM), is internally produced by most animals but not
by humans [2]. Humans must obtain ascorbate by diet
and cancer patients have been shown to have very low
reserves of vitamin C [3, 4].
Dr. McCormick was the first to note that the gen-
eralized stromal changes of scurvy were identical with
the local stromal changes observed in cancer in the im-
mediate vicinity of invading neoplastic cells [5]. Scurvy,
which results from severe dietary lack of ascorbate,
is characterized by generalized tissue disintegration,
including the intercellular ground substance and the
collagen bundles, and lysis of the interepithelial and
interendothelial cements. This condition leads to ul-
ceration, secondary bacterial infection, vascular dis-
organization (edema and interstitial hemorrhage) and
generalized cellular proliferation, with specialized cells
through the tissue reverting to a primitive form. Pa-
tients with advanced cancer exhibit symptoms virtually
identical with those shown by humans with advanced
scurvy: anemia, cachexia, extreme lassitude, hemor-
rhages, ulceration, susceptibility to infections and
abnormally low tissue, plasma and leukocyte ascor-
bate levels, and terminal adrenal failure. Suggestions
have been proposed to supplement cancer patients
with ascorbate to reverse their scorbutic symptoms
and treat the cancer, as well as improve the patients’
quality of life [6, 7].
Our main objective was to determine the effect
of ascorbate supplementation in mice unable to syn-
thesize ascorbic acid, gulonolactone oxidase (gulo)
knockout (KO) mice, on the development of tumors
when challenged with murine B16FO cancer cells.
METHODS AND MATERIALS
Animals. Female Balb/C wild type mice and mice
heterozygous for the sfx mutation, a deletion in the L-
gulono-gamma lactone oxidase gene (gulo) were ob-
tained from The Jackson Laboratory (Bar Harbor, ME).
The gulo mice were bred to develop a homozygous
gulo (-/-) colony and were maintained on Vitamin C for-
tified food and water. Genotyping of litters was per-
formed through Transnetyx (Cordova, TN) and female
gulo (-/-) mice were selected from the homozygous
colony for the study. Wild type and gulo (-/-) mice se-
lected for the study were approximately 36–40 weeks
Received: October 3, 2011.
Correspondence: Fax: 408-567-5030
E-mail: author@drrath.com
Abbreviation used: Gulo KO mice - gulonolactone oxidase knockout
mice.
Exp Oncol 2011
33, 4, 226–230
Experimental Oncology 33, 226–230, 2011 (December)33, 226–230, 2011 (December) (December) 227
of age. They were acclimated for a week before
treatments, housed in standard separator cages with
bedding on a 24-hour light/dark schedule. All animals
were cared for in accordance with institutional guide-
lines for the care and use of experimental animals.
Diet. Gulo (-/-) mice were divided into two groups:
group 1 (n = 11) and 2 (n = 11) and wild type mice
(n = 6) were allocated to group 3. Prior to injection with
melanoma cells, the groups of mice were maintained
for 4 weeks on the following diets. Group 1 (vitamin
C deprived gulo (-/-) mice) and Group 3 (wild type
mice) were maintained on a regular diet (Laboratory
Rodent Diet 5001 from Purina Mills, LLC/Test Diet)
and distilled water. Group 2 (vitamin C-supplemented
gulo (-/-) mice) were provided the regular diet supple-
mented with 500 ppm L-ascorbyl-2-polyphosphate
and distilled water with 150 mg/L ascorbic acid,
0.01 mM EDTA. The ascorbate-supplemented nutrient
mix diet was milled and pressed by Purina Mills, LLC.
Experimental design. After mice had been
on their respective diets for 4 weeks, 2.5 × 106 murine
melanoma B16FO cells in 0.2 ml PBS and 0.1 ml Matri-
gel (BD Bioscience, Bedford, MA) were injected
subcutaneously into the shaven right flank of each
mouse. Mice were continued on their respective di-
ets for another 2 weeks. Body weight measurements
(in triplicates) of all mice were taken every 3 to 4 days
beginning 1 week before harvest. The mice were then
sacrificed, blood was drawn and their tumors were
measured, excised, weighed, photographed and pro-
cessed for histology. All procedures were conducted
under protocols approved by the Internal Animal Care
and Use Committee (IACUC).
Serum analysis. Serum was processed from
whole blood and stored at -80°C until analyzed. Cy-
tokine analyses, including interleukin (IL)-6, IL-1β,
granulocyte macrophage colony-stimulating factor
(GM-CSF), tumor necrosis factor (TNFα), IL-2 and
vascular endothelial growth factor (VEGF), were run
in duplicates by Procarta® Cytokine Assay Service us-
ing Procarta® Cytokine kit mouse 8-plex at Affymetrix
(Santa Clara, CA). Serum ascorbate analysis was per-
formed using the Ferric Reducing Ascorbate (FRASC)
Assay Kit form Biovision (Mountain View, CA).
Histology. Tumors were placed in a formalin cas-
sette and sent to IDEXX for analysis. Formalin fixed
samples of tumors, skin, kidneys and livers were
trimmed, processed, blocked, sectioned and stained
with H&E and EVG stains, and evaluated microscopi-
cally by IDEXX Pathology.
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 using MedCalc Software (Markakerke, Belgium).
RESULTS
Effect of dietary ascorbate on tumor growth
in mice. The mean tumor weight in ascorbate supple-
mented mice (0.114 ± 0.096 g) was significantly re-
duced (by 64%, p = 0.004 ) compared to tumor weight
in ascorbate-deprived gulo KO mice (0.317 ± 0.155 g),
as shown in Fig. 1. The mean tumor weight of wild type
mice (0.109 ± 0.064 g) was not statistically significant
from the ascorbate-supplemented mice. Among
the gulo (-/-) mice deprived of ascorbate two mice
developed metastasis and one a premature death,
while neither the wild type nor ascorbate supple-
mented gulo (-/-) mice developed metastases. Gross
tumor photographs of representative tumors from the
gulo KO groups is shown in Fig. 2. (Note: Among the
supplemented group, 3 data points were removed due
to 2 gravidities and 1 anesthesia death before tumor
inoculation).
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
0,4
Wild Type GULO KO
no ascorbate
GULO KO
with ascorbate
M
ea
n
Tu
m
or
W
ei
gh
t (
g)
Fig. 1. Effect of ascorbate on B16FO melanoma growth in gulo
KO mice (SEM bars)
Fig. 2. Effect of ascorbate on B16FO melanoma growth in gulo
KO mice: gross tumor photographs of groups
Effect of vitamin C supplementation on tumor
histology. Gulo KO mice supplemented with ascor-
bate hosted smaller tumors with more collagen en-
capsulation and fibrous capsule interdigitation (Fig. 3,
a). The smallest tumor had an unusually thick fibrous
capsule of collagen and a few elastic fibers. In contrast,
gulo KO mice deprived of ascorbate hosted large
tumors with poorly defined borders, showing more
necrosis and mitosis (Fig. 3, b). These conditions co-
incided with severe metastasis from the primary tumor
to the colon and/or spleen in a couple of the ascorbate
deprived gulo KO mice (a more dynamic tumor charac-
terized by cell death and turnover and poorly defined
borders correlates with metastasis). Wild type mice
challenged with melanoma cells formed well-defined
tumors, encapsulated with a fibrous capsule of colla-
gen. An inflammatory infiltrate was observed in some
wild type tumors.
Effect of dietary ascorbate on weight of mice.
The mean weight of mice in each group did not sig-
nificantly differ at onset or at 7 days post injection.
However, the mean weight of ascorbate restricted gulo
(-/-) mice significantly decreased with time, showing
a 30% (p < 0.0001) decrease in weight at 14 days
post injection compared to the mean weight at 7 days
post injection. Furthermore, the weight of ascorbate-
228 Experimental Oncology 33, 226–230, 2011 (December)
supplemented gulo (-/-) mice continued to increase
slightly over time, but did not reach statistical signifi-
cance. However, the final mean weight of gulo (-/-)
ascorbate-deprived mice was 36% lower than the
mean final weight of gulo (-/-) mice supplemented
with ascorbate, as shown in Fig. 4.
a
b
Fig. 3. Effect of ascorbate on B16FO melanoma tumors in gulo
KO mice: representative histology of tumors in the gulo KO mice
groups. a — ascorbate-supplemented gulo KO mice showing
strong tumor capsules (pink); b — scorbutic gulo KO mice show-
ing weak or absent tumor capsules
0
5
10
15
20
25
30
35
40
45
7 days 9 days 11 days 14 days
Days after injection
M
ea
n
we
ig
ht
o
f m
ic
e
(g
)
GULO KO no ascorbate
GULO KO with ascorbate
Fig. 4. Effect of ascorbate on animal weights (in g ± SD) in mela-
noma bearing mice: comparison of the mean weights of wild
type and gulo (-/-) ascorbate depleted and supplemented
mouse groups
Effect of ascorbate supplementation on cyto-
kines. Serum inflammatory cytokines IL-6 and IL-1β
were substantially higher in gulo KO mice deprived
of ascorbate than in gulo KO mice supplemented with
ascorbate, as shown in Fig. 6. For the gulo KO scorbu-
tic mouse group, the mean IL-6 level per mouse was
6.97 ± 7.85 pg/mL compared to 0.715 ± 1.08 in the
ascorbate supplemented gulo KO group (90% de-
crease, p = 0.04). In regard to IL-1β, ascorbate supple-
mentation resulted in a 62% decrease from the level
in the restricted gulo KO group, but the difference did
not reach statistical significance. Mean IL-1β levels per
mouse were 2.82 ± 5.68 and 7.25 ± 6.74 pg/mL in the
ascorbate supplemented and deprived gulo KO mice,
respectively. The mean IL-1β level in wild type mice was
significantly lower (11%, p = 0.04) than in the ascor-
bate deprived gulo KO mice but did not significantly
differ from the mean level in ascorbate supplemented
gulo KO mice. Mean GM-CSF, TNFα, IL-2 and VEGF
levels did not significantly differ between ascorbate
supplemented and deprived gulo KO mice. See Table
and Fig. 5 for cytokine levels in groups.
Table. Mean serum cytokine levels (pg/mL ± SD) in melanoma bearing mice
Cytokine Wild Type (Control) GULO KO — Ascorbate GULO KO + Ascorbate
IL-1β 0.772 ± 1.89 7.25 ± 6,74 2.82 ± 5.68
IL-6 0.592 ± 0.292 6.97 ± 7.84 0.715 ± 1.08
GM-CSF 2.31 ± 2.67 6.76 ± 4.71 8.95 ± 13.6
TNF-α 2.31 ± 2.50 2.07 ± 0.962 2.38 ± 2.79
IL-2 0.607 ± 0.181 0.641 ± 0.144 0.65 ±0.265
VEGF 17.8 ±8.85 13.8 ± 7.01 17.0 ± 8.59
Notes: IL-1β — interleukin-1β, IL-6 — interleukin-6, GM-CSF — granulocyte
macrophage colony-stimulating factor, TNF-α — tumor necrosis factor-α,
IL-2 — interleukin-2, VEGF — vascular endothelial growth factor.
WT Control IL-1B
GU LO КО - Ascorbate IL-1B
GU LO КО + Ascorbate IL-1B
WT Control IL-6
GULO КО-Ascorbate IL-6
GULO КО + Ascorbate IL-6
WT Control GM-CSF
GULO КО-Ascorbate GM-CSF
GU LO КО + Ascorbate GM-CSF
WTControlTNF-a
GU LO КО - Ascorbate TNF-a
GU LO КО + Ascorbate TNF-a
WT Control IL-2
GULO КО-Ascorbate IL-2
GULO КО + Ascorbate IL-2
WT Control VEGF
GU LO КО - Ascorbate VEG F
GU LO КО + Ascorbate VEG F
0 5 10 15 20 25
pg/mL
Fig. 5. Comparative mean serum cytokine levels in melanoma
bearing groups of mice
0
2
4
6
8
10
12
Wild Type GULO KO
no ascorbate
GULO KO
with ascorbate
Cy
to
ki
ne
in
p
g/
m
L
IL-6
IL-1β
Fig. 6. Effect of ascorbate supplementation on mean IL-6 and
IL-1β levels in gulo KO mice (using SEM bars)
Experimental Oncology 33, 226–230, 2011 (December)33, 226–230, 2011 (December) (December) 229
Effect of ascorbate supplementation on serum
ascorbate levels in gulo KO mice. As expected,
ascorbate-supplemented gulo KO mice were found
to have profoundly higher ascorbate levels than did the
unsupplemented gulo KO mice (Fig. 7). Mean serum
ascorbate level in ascorbate restricted mice (7.72 ±
15.5 µM) was 9% (p < 0.001) of the mean ascorbate
level in supplemented mice (85.8 ± 22.6 µM). Wild type
mice also showed significantly higher ascorbate levels
than the restricted mice (68.6 ± 17.1 µM). The mean
ascorbate level in wild type mice was found to be 80%
of that in ascorbate supplemented mice, but the dif-
ference did not reach statistical significance.
0
10
20
30
40
50
60
70
80
90
100
Wild Type GULO KO
no ascorbate
GULO KO
with ascorbate
As
co
rb
at
e
(µ
M
)
Fig. 7. Comparative mean serum ascorbate levels in groups
of melanoma bearing mice (using SEM bars)
DISCUSSION
Numerous studies report that cancer patients
exhibit abnormally low plasma ascorbate levels sec-
ondary to the disease and/or treatment [3, 4, 8, 9].
Studies have demonstrated a correlation between de-
ficient plasma ascorbate levels in cancer patients and
decreased survival, as well as increased expression
of inflammatory markers [8]. A correlation between
tumor aggressiveness and low ascorbate content has
also been reported [9]. Ascorbate has been implicated
in host resistance to neoplasia, at both the stromal
and systemic levels. Invasiveness, characteristic
of melanoma and other metastatic cancers, depends
upon the structural integrity of the ground substance
matrix as a barrier to invasiveness. Degradation of ad-
jacent matrix in the vicinity of invading neoplastic cells
is dependent upon continual release of lysosomal
glycosidases (hyaluronidases) [6], proteases and
collagenases from the invading cells [1, 10].
In our study, ascorbate supplementation of gulo
KO mice resulted in significant reduction (by 64%,
p = 0.004) in the mean tumor weight compared to that
in ascorbate-deprived gulo KO mice, In addition, gulo
KO mice supplemented with ascorbate hosted smaller
tumors with enhanced collagen encapsulation and fi-
brous capsule interdigitation. In contrast, gulo KO mice
deprived of ascorbate hosted large tumors with poorly
defined borders, showing more necrosis and mitosis.
Tumors in scorbutic mice with systemic metastasis
showed multi-focal collagen capsule breaks or absent
collagen capsules. Tumors in ascorbate supplemented
gulo KO mice had much thicker collagen capsules
and were of similar size to those in wild type vitamin
C generating mice.
Interestingly, Moriarty et al. [11] reported that
a shift of ascorbate occurs from reserves to the tu-
mor stroma, resulting in ascorbate levels in human
tumors to be higher than that in the surrounding tis-
sues, resulting in significant depletion of circulating
reserves. The ascorbate is reported to concentrate
at the periphery of the tumor, against the actively
growing invasive margin [12]. Although most animals
can synthesize ascorbic acid, humans cannot and are
totally dependent upon dietary intake. Ascorbic acid
is known to be required for the structural integrity of the
intercellular matrix and closely related to glucoronic
acid, an essential building block of the matrix structure.
Increasing amounts of ascorbic acid are needed for
protective encapsulation of tumors. Stromal resistance
is dependent upon the host’s ability to encapsulate the
neoplastic cells by forming a practically impenetrable
barrier of dense fibrous tissue [6]. Highly anaplastic
invasive tumors are associated with a scanty, poorly
defined collagenous barrier. Collagenous barriers
are found to be more defined in tumors of moderate
rapidity of growth and very abundant in slow-growing
“contained” atrophic scirrhous tumors [6]. Our studies
have shown that a combination of ascorbate with other
micronutrients have profound anti-cancer effects
mediated through increased stability of connective
tissue, including suppression of metastasis, tumor
growth, angiogenesis in more than 40 types of human
tumor cells [13].
Cancer patients exhibit chronic low-grade inflam-
mation that causes a variety of pathologies, such
as cachexia and poor postsurgical outcome [14, 15].
Levels of serum cytokines, such as IL-6, IL-1β, IL-1α,
IL-8, IL-12p40 IL-13, GM-CSF, monocyte chemoat-
tractant protein- (MCP-1), macrophage inflammatory
protein (MP)-1α, MP-1β, IFNα, tumor necrosis factor
(TNF)-α, epidermal growth factor, VEGF and TNF re-
ceptor II are reported to be significantly higher in pa-
tients with resected high-risk melanoma than in healthy
controls [16]. These factors promote tumor cell growth
and stimulate endothelial cell proliferation, migration
and angiogenesis, which lead to melanoma growth
and metastasis [17]. Clinical studies have shown sig-
nificantly increased levels of serum IL-6 in malignant
metastatic melanoma patients and have been found
to be correlated to the tumor burden in these patients
[18–20]. Since IL-6 promotes melanoma progression
and resistance to chemotherapy, new approaches
to treatment have focused on targeting IL-6. In our
study, ascorbate supplementation of scorbutic mice,
showed significantly reduced levels of IL-6 than the
ascorbate deprived scorbutic mice. Of interest, weight
loss occurred in the gulo (-/-) mice deprived of ascor-
bate but not in those supplemented with ascorbate.
The weight loss observed in cancer patients, second-
ary to the inflammation-associated cachexia, mirrors
the weight loss observed in the scorbutic mice with
high IL-6 and IL-1β levels.
230 Experimental Oncology 33, 226–230, 2011 (December)
The comparative ascorbate serum levels in the
respective groups of mice support the claim that the
beneficial effects observed in the ascorbate-supple-
mented mice were due to significantly increased serum
ascorbate levels. Among the melanoma bearing mice
in this study, ascorbate-supplemented gulo KO mice
were found to have profoundly higher (an increase
of 1111%) ascorbate levels than did the restricted gulo
KO mice and slightly higher (125%) levels than the
ascorbate producing wild type mice.
In conclusion, ascorbate supplementation resulted
in decreased tumor growth and enhanced encapsula-
tion of tumors elicited by melanoma challenge in scor-
butic mice. Tumor encapsulation is critical in curbing
invasion and metastasis of malignant cells. In addition,
ascorbate supplementation modulated inflammatory
cytokine secretion.
ACKNOWLEDGEMENTS
Special thanks to Earl Rainey for animal colony
maintenance.
The research study was funded by Dr. Rath Health
Foundation (Santa Clara, CA, USA), a non-profit or-
ganization.
REFERENCES
1. Fidler IJ. Molecular biology of cancer: Invasion and
metastasis. In: Cancer: Principles and practice of oncology
(ed 5). De Vita VT, Hellman S, Rosenberg SA, eds. Philadel-
phia, PA, Lippincott-Raven, 1997, 135–152.
2. Rath M, Pauling L. Plasmin-induced proteolysis and
the role of apoprotein(a), lysine and synthetic analogs. Ortho-
molecular Medicine 1992; 7: 17–23.
3. Nunez C, Ortiz de Apodaca Y, Ruiz AL. Ascorbic acid
in the plasma and blood cells of women with breast cancer.
The effect of consumption of food with an elevated content
of this vitamin. Nutr Hosp 1995; 10: 368–72.
4. Anthony HM, Schorah CJ. Severe hypovitaminosis
C in lung-cancer patients: the utilization of vitamin C in surgi-
cal repair and lymphocyte-related host resistance. Br J Cancer
1982; 46: 354–67.
5. McCormick WJ. Cancer: a collagen disease, secondary
to nutritional deficiency? Arch Pediatr 1959; 78: 166–171.
6. Cameron E, Pauling L, Leibovitz B. Ascorbic acid and
cancer: a review. Cancer Res 1979; 39: 663–81.
7. Ichim TE, Minev B, Braciak T, et al. Intravenous
ascorbic acid to prevent and treat cancer-associated sepsis?
J Translational Med 2011; 9: 25–38.
8. Mayland CR, Bennett MI, Allan K. Vitamin C defi-
ciency in cancer patients. Palliat Med 2005; 19: 17–20.
9. Kuiper C, Molenaar IG, Dachs GU, et al. Low ascor-
bate levels are associated with increased hypoxia-inducible
factor-1 activity and an aggressive tumor phenotype in endo-
metrial cancer. Cancer Res 2010; 70: 5749–58.
10. Stetler-Stevenson WG, Hewitt R, Corcoran M. Matrix
metalloproteinases and tumor invasion from correlation and
causality to the clinic. Semin Cancer Biol 1996; 7: 174–54.
11. Moriarty MJ, Mulgrew S, Malone JR, et al. Results
and analysis of tumour levels of ascorbic acid. Ir J Med Sci
1977; 146: 74–78.
12. Chinoy NJ. Histochemical studies on ascorbic acid
in human cancerous tissue and its significance. J Anim Mor-
phol Physiol 1972; 19: 238–40.
13. Niedzwiecki A, Roomi MW, Kalinovsky T, et al. Mi-
cronutrient synergy — a new tool in effective control of metas-
tasis and other key mechanisms of cancer. Cancer Metastasis
Rev 2010; 29: 529–43.
14. Argiles JM, Busquets S, Toledo M, et al. The role
of cytokines in cancer cachexia. Curr Opin Support Palliat
Care 2009; 3: 263–8.
15. Deans C, Wigmore SJ. Systemic inflammation, ca-
chexia and prognosis in patients with cancer. Curr Opin Clin
Nutr Metab Care 2005; 8: 265–9.
16. Yurkovetsy ZR, Kirkwood JM, Edington HD, et al.
Multiplex analysis of serum cytokines in melanoma patients
treated with interferon-α2b. Clin Cancer Res 2007; 13: 2422–8.
17. Lazar-Molnar E, Hegyesi H, Toth S, et al. Autocrine
and paracrine regulation by cytokines and growth factors
in melanoma. Cytokine 2000; 12: 547–54.
18. Mouawad R, Benhammouda A, Rixe O, et al. Endo-
genous interleukin 6 levels in patients with metastatic malig-
nant melanoma: correlation with tumor burden. Clin Cancer
Res 1996; 2: 1405–9.
19. Mouawad R, Rixe O, Meric JB, et al. Serum interleu-
kin 6 concentration as predictive factor of time to progression
in metastatic malignant melanoma patients treated by bioche-
motherapy: a retrospective study. Cytokines Cell Mol Ther
2002; 7: 151–6.
20. Tawara K, Oxford JT, Jorcyk CL. Clinical significance
of interleukin (IL)-6 in cancer metastasis to bone: potential
of anti-IL-6 therapies. Cancer Management Research 2011;
3: 177–89.
Copyright © Experimental Oncology, 2011
|