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Zheng Zhang,
Xiu-Li Wang, Department of Biochemistry and Molecular Biology,
Qingdao University Medical College, Qingdao 266021, Shandong
Province, China
Yang Song, Nutrition and Food Sanitation Institution, Qingdao
University Medical College, Qingdao 266021, Shandong Province, China
Supported by the Health Bureau Foundation of Province
Shandong, No. 1999CA2CBA2
Correspondence to: Yang Song, Nutrition and Food Sanitation
Institution, Qingdao University Medical College, Qingdao 266021,
Shandong Province, China. qdsongyang@126.com
Telephone: +86-532-2991029
Received: 2004-11-23
Accepted: 2005-01-26
Abstract
Aim: To
investigate the anti-neoplastic effect of inositol hexaphosphate
(InsP6
or phytic acid) on dimethylhydrazine (DMH)-induced colon tumor in
rats and its effect on blood natural killer (NK) cell activity.
Methods:
Healthy Wistar rats, 4 wk old, were divided into control group (fed
with common food) and InsP6
group (fed with common food+2% sodium inositol hexaphosphate in the
drinking water), 15 rats in each group. Both groups were injected
with 1,2-dimethylhydrazine subcutaneously (20 mg/kg body weight)
once a week for 20 wk. Rats were killed after 21 wk. The whole large
intestine was isolated to determine the general condition of tumors
and to test blood NK cell activity by lactate-dehydrogenase-release
assay.
Results:
Administration of InsP6
significantly increased blood NK cell activity in DMH-induced
colorectal tumor in rats. InsP6
group had a smaller tumor size on average and a smaller number of
tumors than the control group. Its mortality was also higher than
that in control. However, the variables of body weight and tumor
incidence were not significantly different between the two groups.
Conclusion:
InsP6
can increase blood NK cell activity in DMH-induced colon tumor in
rats and inhibit tumor growth and metastasis in rats.
ã 2005
The WJG Press and Elsevier Inc. All rights reserved.
Key words: Inositol hexaphosphate; Phytic acid; Natural
killer cell activity; Colon cancer
Zhang Z, Song Y, Wang XL. Inositol hexaphosphate-induced enhancement
of natural killer cell activity correlates with suppression of colon
carcinogenesis in rats. World J Gastroenterol
2005; 11(32): 5044-5046
http://www.wjgnet.com/1007-9327/11/5044.asp
INTRODUCTION
Inositol hexaphosphate (InsP6)
is a naturally occurring compound that has various chemical
properties and biological activities[1].
It is rich in matured plant seeds, particularly in cereals and
legumes, and exists in nature as a salt with monovalent and divalent
cations (Ca2+,
Mg2+,
and K+).
It has the ability to chelate minerals such as iron, copper, zinc,
cobalt, and manganese, most efficiently at neutral pH[2-4].
InsP6
has anti-neoplastic activity on a variety
of experimental models of carcinogenesis, decreases serum
cholesterol level, inhibits renal stone formation, and may find use
in controlling myocardial damage following ischemia. Among these
biological activities, anti-neoplastic activity is one of the most
intriguing properties of InsP6[5,6].
The above facts need clinical trials in human colorectal
cancer. It has been reported that intestinal lipodystrophy can be
prevented by InsP6
treatment[7].
Recent studies demonstrate that InsP6
inhibits experimental colon carcinogenesis in rats[8-10].
There is a correlation between neoplastic diseases and depressed
natural killer (NK) activity[11].
There is evidence that NK cells are involved in the destruction and
growth inhibition of tumor cells in vivo. This study aimed to
study the effect of InsP6
on blood NK cell activity in
dimethylhydrazine (DMH)-induced colon tumor in rats.
MATERIALS AND METHODS
Animals and chemicals
Thirty-four-weeks old male Wistar rats (70-110 g) were purchased
from Animal Center of Henan Medical University. After
acclimatization for 1 wk, the experimental animals were randomly
divided into control group and InsP6
group (15 rats/group). Animals in the control group were fed with
the basal diet and had regular access to drinking water. Rats in
InsP6
group were fed with the basal diet and had access to 2% sodium
inositol hexaphosphate (purchased from Guangdong Qingyun Chemical
Factory) solution. Basal diet was made by American Institute of
Nutrition method.
Animals in both groups were given subcutaneous
injections of DMH (from Sigma) dissolved in normal saline solution
(20 mg/kg body wt) once a week for 20 wk. Body weight was measured
and food consumption was recorded once a week. All surviving animals
were killed under 4.3% trichloraldehyde hydrate anesthesia after 21
wk.
Tissue processing
All animals (including rats that died before the end of experiment)
were autopsied. The colons were removed, flushed with saline, opened
along the longitudinal median axis. Macroscopically, the number of
tumors in each colon was counted. Tumor width and length were
measured with clippers. Simultaneously, peripheral blood was
obtained from the abdominal aorta for testing NK cell activity.
Test of NK cell activity
Peripheral blood mononuclear cells (PBMCs) were separated by
Ficoll-Hypaque density centrifugation from the collected blood.
Lactate-dehydrogenase (LDH)-release assay was used to measure the NK
cell activity. PBMCs were washed and suspended in complete RPMI-1640
medium, counted and diluted to 1.0×106/mL.
The amount of LDH released from the lysed target cells was
determined for NK cell activity measurement. The NK-sensitive cell
line K562
(human erythroleukemia cell line, Shandong Medical Science
Institute, Shandong, China) was used as the target cell. K562
cells were washed with complete RPMI-1640 medium, counted and
finally diluted to 1.0×105/mL
with the medium. An equal volume of K562
cells and PBMCs was added to the wells of 96-round-bottomed
microwell plates (the cell ratio of effector-to-target was 10:1).
Each test was repeated in three wells. To ensure contact between
cells, the plate was centrifuged at a low speed for 2 min. After 2-h
incubation at 37 ℃
in a humidified atmosphere with 50 mL/L CO2,
the plate was centrifuged at 1 000 r/min for 5 min. The supernatant
from each well (100 mL)
was transferred into the corresponding wells of a 96-flat-bottomed
microwell plate. Then 100 mL
of lactic acid hydrogenase substrate mixture was added to each well.
After 3 min, reactions were stopped by adding 50 mL
of cold medium. Finally, a microtiter plate reader (Bio-Rad,
MODE-550) was used for evaluation of changes in the absorbance at a
wavelength of 490 nm. The release of LDH from K562
cells was expressed as absorbance. The percentage of NK cell
activity was calculated by the formula: NK cell activity = (E-S)/(M-S)100%,
where E represents the experimental release of LDH activity
from target cells incubated in the presence of PBMCs, M
represents the maximum release of the LDH activity determined by
lysing the target cells with 1% of NP40, and S is the
spontaneous release of the LDH activity from target cells incubated
in the absence of PBMCs.
Statistical analysis
Results were expressed as mean±SD.
Statistical analyses were performed with SPSS 9.0. The significance
of diff-erences in the average values between the two groups was
analyzed using t-test. P<0.05 was considered
statistically significant.
RESULTS
During the initial period of the experiment, body weight of animals
increased steadily in the first 20 wk. Then both groups began to
loose their body weight. In addition, the animals consumed a less
amount of food. The change in two groups had no difference (Figure
1).
After DMH was injected for 10 wk, death occurred
in rats of control group. By the end of the experiment, six rats
died in the control group. Only one rat died in InsP6
group. The difference in mortality was significant (P<0.01,
Table 1).
Figure
1 (PDF) Body weight change of rats during feeding.
Table 1
Effect of
InsP6
on mortality of rats
| Groups |
Survival |
Death |
Mortality
(%) |
| Control |
9 |
6 |
40.0 |
| InsP6 |
14 |
1 |
7.1b |
bP<0.01
vs control group.
Twelve rats had colon tumor in control group
treated with DMH but not InsP6.
Tumors were found in 11 rats of InsP6
group. The difference in tumor incidence was not significant (P>0.05).
But the number of tumors and their size were significantly less in
InsP6
group than in control group (P<0.05, Table 2).
Table 2 Effect of
InsP6
on total large intestinal carcinomas (mean±SD)
| Groups |
n |
Incidence
of tumor (%) |
Average
tumor number |
Average
sign of tumor (mm3) |
| Control |
15 |
80 |
4.1±1.2 |
1
080.3±463.4 |
| InsP6 |
15 |
73 |
0.9±0.2b |
123.6±29.6a |
aP<0.05,
bP<0.01
vs control group.
Blood NK cell activity was reduced in the two
groups after DMH treatment. But the blood NK cell activity was
significantly higher in InsP6
group than in control group (P<0.01,
Table 3).
Table 3 Effect of
InsP6
on blood NK cell activity (NK-A) (mean±SD)
| Group |
n |
NK-A
(%) |
| Control |
9 |
15.7±1.2 |
| InsP6 |
14 |
42.2±1.1b |
bP<0.01
vs control group.
DISCUSSION
Diet composition is an important etiologic factor in colon
carcinogenesis and has a significant impact on colon cancer
occurrence. InsP6
is a dietary phytochemical present in cereals, soy, legumes, and
fiber-rich foods[12,13].
Epidemiological studies have shown that InsP6
can inhibit the metastasis of tumor[14-16].
But the anti-tumor mechanism of InsP6
awaits further investigation.
Our study demonstrated that InsP6
could significantly increase blood NK cell activity in DMH-induced
colorectal cancer in rats (P<0.01). The number and size of
tumors were smaller in InsP6
group than in control group (P<0.05), indicating that InsP6
can also inhibit tumor growth and metastasis in DMH-induced
colorectal cancer in rats.
InsP6
is degraded into lower polyphosphorylated
forms of inositol (including InsP1-InsP6)
by the enzyme meso-inositol hexaphosphate phosphohydrolase, and
dephosphorylated by acid, acid phosphatase and intestinal alkaline
phosphatase. When InsP6
was administered to rats as a soluble form
in drinking water, it is rapidly absorbed through the upper
gastrointestinal tract and quickly distributed in various organs,
most notably in liver, kidneys, and skeletal muscle[17,18].
Among the lower polyphosphorylated forms of inositol, InsP3
appears to act as a second messenger and
promotes intracellular free calcium (Ca2+)
release, which can induce proliferation of NK cells[19]
as well as the release of NK cell cytotoxicity factor (NKCF). NKCF
can bind to target cells (tumor cells) which are subsequently lysed[20].
Close contact between the plasma membrane of the two types of cells,
affects the cytotoxic reaction. InsP3
can also affect the membrane phosphatidyl inositol proteins, which
may be important in attachment and subsequent fusion with the target
cells, suggesting that InsP6
mediates its chemopreventive and probably chemotherapeutic effect
via InsP3[21,22].
Our data indicate that DMH depresses the NK cell
activity, while InsP6
significantly increases the NK cell
activity and inhibits tumor growth, suggesting that changes in NK
cell activity are related to progressive cancer growth[23].
Since InsP6
enhances the NK cell activity in vivo, it may have potential
application in therapy of cancer and other diseases associated with
depressed NK cytotoxicity.
REFERENCES
1 Singh
RP, Sharma G, Mallikarjuna GU, Dhanalakshmi S, Agarwal
C, Agarwal R. In vivo suppression
of hormone-refractory prostate cancer
growth by inositol hexaphosphate induction of insulin-like growth
factor
binding protein-3 and inhibition of
vascular endothelial growth factor. Clinical Cancer Res 2004;
10 (1 Pt 1): 244-250
2 Singh
A, Singh SP. Postnatal effect of smokeless tobacco on phytic
acid or the butylated
hydroxyanisole-modulated hepatic
detoxication system and antioxidant defense mechanism in suckling
neonates
and lactating mice. Cancer Lett
1998; 122: 151-156
3 Nickel
KP, Belury MA. Inositol hexaphosphate reduces 12-O-tetradecanoylphorbol-13-acetate-induced
ornithine decarboxylase independent
of protein kinase C isoform expression in keratinocytes. Cancer
Lett 1999;
140: 105-111
4 Jenab
M, Thompson LU. Phytic acid in wheat bran affects colon
morphology, cell differentiation and
apoptosis. Carcinogenesis
2000; 21: 1547-1552
5 Singh
RP, Agarwal C, Agarwal R. Inositol hexaphosphate inhibits
growth, and induces G1 arrest and apoptotic death
of prostate carcinoma DU145 cells:
modulation of CDKI-CDK-cyclin and pRb-related protein-E2F
complexes. Carcinogenesis 2003;
24: 555-563
6 Midorikawa
K, Murata M, Oikawa S, Hiraku Y, Kawanishi S. Protective effect
of phytic acid on oxidative DNA damage
with reference to cancer
chemoprevention. Biochem Biophy Res Comm 2001; 288:
552-557
7 Reddy
BS. Role of dietary fiber in colon cancer: An overview.Am J
Med 1999; 106: 16S-19S
8 Singh
A, Singh SP, Bamezai R. Modulatory influence of arecoline on the
phytic acid-altered hepatic
biotransformation system
enzymes,sulfhydryl content and lipid peroxidation in a murine
system. Cancer Lett 1997;
117: 1-6
9 Alabaster
O, Tang Z, Shivapurkar N. Dietary fiber and the chemopreventive
modelation of colon carcinogenesis.
Mutation Res 1996; 350:
185-197
10 Takaba
K, Hirose M, Yoshida Y, Kimura J, Ito N, Shirai T. Effects of n-tritriacontane-16,
18-dione,
curcumin, chlorophyllin,
dihydroguaiaretic acid, tannic acid and phytic acid on the
initiation stage in a rat
multi-organ carcinogenesis
model. Cancer Lett 1997; 113: 39-46
11 Shivapurkar
N, Tang ZC, Frost A, Alabaster O. A rapid dual organ rat
carcinogenesis bioassay for evaluating
the chemoprevention of breast
and colon cancer. Cancer Lett 1996; 100: 169-179
12 Reddy
BS, Hirose Y, Cohen LA, Simi B, Cooma I, Rao CV. Preventive
potential of wheat bran fractions
against experimental colon
carcinogenesis: implications for human colon cancer prevention.
Cancer Res 2000;
60: 4792-4797
13 Ma Q,
Hoper M, Halliday I, Rowlands BJ. Diet and experimental colorectal
cancer. Nutrition Res 1996; 16: 413-426
14 Weber
G, Singhal RL, Prajda N, Yeh YA, Look KY, Sledge GW Jr.
Regulation of signal transductioin. Advan
Enzyme Regul 1995; 35:
1-21
15 Baten
A, Ullah A, Tomazic VJ, Shamsuddin AM. Inositol-phosphate
induced enhancement of natural killer cell
activity correlates with rumor
suppression. Carcinogenesis 1989; 10: 1595-1598
16 Shamsuddin
AM, Ullah A, Chakravarthy AK. Inositol and inositol
hexaphosphate suppress cell proliferation and
tumor formation in CD-1 mice.
Carcinogenesis 1989; 10: 1461-1463
17 Sakamoto
K, Venkatraman G, Shamsuddin AM. Growth inhibition and
differentiation of HT-29 cells in vitro by
inositol hexaphosphate(phytic
acid). Carcinogenesis 1993; 9: 1815-1819
18 Owen
RW, Spiegelhalder B, Bartsch H. Generation reactive oxygen
species by the faecal matrix. Gut 2000; 46: 225-232
19 Challa
N, Rao DR, Reddy AS. Interactive suppression of aberrant crypt
foci induced by azoxymethane in rat
colon by phytic acid and green
tea. Carcinogenesis 1997; 18: 2023-2026
20 Thompson
LU, Zhang L. Phytic acid and minerals:effect on early markers of
risk for mammary and colon
carcinogenesis.
Carcinogenesis 1991; 12: 2041-2045
21 Zi X,
Singh RP, Agarwal R. Impairment of erbB1 receptor and
fluid-phase endocytosis and associated mitogenic
signaling by inositol
hexaphosphate in human prostate carcinoma DU145 cells. Carcinogenesis
2000; 21: 2225-2235
22 Vucenik
I, Shamsuddin AM. Cancer Inhibition by Inositol Hexaphosphate
(IP6)
and Inositol: From Laboratory to Clinic.
J
Nutr 2004; 133: 3778S-3784S
23 Bode AM,
Dong Z. Targeting signal transduction pathways by chemopreventive
agents. Mutation Res
2004; 555: 33-51
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