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Effect of ursodeoxycholic
acid on azoxymethane-induced
aberrant crypt foci formation
in rat colon:in vitro potential role of intracellular Ca2+
Mohammed Abdul Momen1, Yasumasa Monden1,
Hitoshi Houchi2 and
Atsushi Umemoto1
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1Second Department of Surgery, and 2Division
of Pharmacy, University Hospital, The University of Tokushima
School of Medicine, Tokushima, Japan
Abstract: The studies were conducted to examine the precise
nature of the suppressive effect of ursodeoxycholic acid (UDCA)
on colonic aberrant crypt foci (ACF) formation. Fischer 344
rats were treated with a single dose of azoxymethane (AOM)
(20 mg/kg, s.c.) and fed basal diet (MF) supplemented with
UDCA (0.4%) during an initiation or a post-initiation stage.
ACF were enumerated at the 2nd, 5th and 8th weeks after AOM
administration (15-18 rats/group). The number of ACF in the
UDCA treated group was decreased significantly in the initiation
and post-initiation stages at the 2nd (P<0.01, P<0.0001)
and 8th weeks (P<0.001, P<0.0001), respectively, compared
with untreated controls. In the time-course experiments, the
effect of continuous feeding of UDCA (0.4%) on ACF formation
was evaluated. ACF number was decreased significantly (P<0.005)
until the 16th week. UDCA showed a significant dose-dependent
suppression of ACF number from a range of 0.1-0.4% UDCA. To
approach the subcellular mechanisms of the effect of bile
acids, the intracellular free Ca2+ concentration ([Ca2+]i)
of bile acid-treated rat colonic cancer cells (ACL-15) was
examined. DCA and CDCA, which are promotive on ACF formation,
induced a rapid increase in [Ca2+]i, while UDCA and CA, which
are suppressive or non-effective on ACF formation, did not.
These findings suggest that the promotive effect of bile acids
may involve intracellular Ca2+ signaling.
J. Med. Invest. 49:67-73, 2002
Keywords:ursodeoxycholic acid, aberrant crypt foci, colon
cancer, bile acid, intracellular Ca2+
INTRODUCTION
The incidence of colorectal cancer in Japan has increased
over the past four decades, and it was suggested that this
increase is linked to increased consumption of Western foods
characterized by high-fat and less fiber diets. Such dietary
habits are known to be correlated with high fecal concentrations
of bile acids. In agreement with this, epidemiological studies
showed that fecal concentrations of bile acids were high in
patients with colon cancer as well as in people from countries
with a high incidence of colon cancer (1, 2). Thus, all these
findings suggest that a strong relation between fecal bile
acids and human colon carcinogenesis. Bile acids have also
been studied extensively in animal models of colon cancer
(3-5). In our previous studies, the effects of human bile
acids on azoxymethane (AOM)-induced aberrant crypt foci (ACF),
which is a good surrogate marker for colon carcinogenesis,
were investigated in the rat colon (6, 7). In these studies
using dietary intake and intracolonic administration of bile
acids, ursodeoxycholic acid (UDCA) showed a significant suppressive
effect on ACF formation, whereas other bile acids, i.e. cholic
acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA)
and lithocholic acid (LCA), showed either promotive effects
or no effect on the colonic mucosa. The preventive effect
of UDCA on colonic tumor formation was also demonstrated in
rats (5). Although controversies continue with respect to
finding a correlation between the ACF parameters and tumor
outcome, as a precursor lesion of the colon cancer (8, 9),
it has been established that ACF is a good biomarker to assess
the effects of colon carcinogens as well as chemopreventive
agents (10, 11). UDCA may be one of the hopeful chemopreventive
agents for high-risk groups for colon cancer. In the present
study, the effects of UDCA in different stages of colon carcinogenesis
by AOM treatment were examined in Fischer 344 (F344) rat colon
and the time-course and dose-response of the effects of UDCA
on ACF formation were also examined.
Although, the bile acids are greatly involved in the development
of colon tumors and ACF, the exact mechanism of the bile acids
action remains unclear. Increasing evidence suggests that
colonic malignant transformation involves various mutations
activating proto-oncogenes such as K-ras (12), inactivating
tumor suppressor genes such as p53 and APC (13), and inactivating
genes involved in DNA mismatch repair. On the other hand,
epigenetic changes in signal transduction elements, such as
protein kinase C (PKC) may also be involved in multistage
processes of colon carcinogenesis (14, 15). Bile acids, which
have various effects on colonic ACF formation, are also known
activators of PKC that have been implicated in the development
of colonic carcinoma (16). Since PKC is activated by intracellular
calcium ions, it is possible to have an association between
the effect of bile acids and their intracellular calcium induction.
It was also reported that calcium ions (Ca2+) are a versatile
mediator of many cellular biological processes, including
cell proliferation (17). The free Ca2+ acts as a proliferative
signal for many normal cells (18). Cytoplasmic calcium is
also a pluripotent regulator of cell function including the
cell cycle and apoptosis (19, 20). Thus, the agents that affect
the intracellular calcium level, could be considered as important
substances for cell functions, and we hypothesized that bile
acid activity on cellular proliferation leading to ACF formation
may be in part through the alteration of the intracellular
calcium level. Therefore, we aimed to assess the intracellular
Ca2+ induction in rat a colon cancer cell line (ACL-15) by
bile acids, which are either promotive or suppressive/ineffective
in colonic ACF formation in rats.
MATERIALS AND METHODS
Chemicals
CA, CDCA and DCA were purchased from Sigma Chemical Co. (St.
Louis, MO). UDCA was from Mitsubishi-Tokyo Pharmaceutical
Co. Ltd. (Tokyo, Japan). All the bile acids were sodium salt
forms and these were at least 97-99% pure according to the
manufacturer's indications. AOM was purchased from Sigma Chemicals
Co. (St. Louis, MO). Fura-2-AMTM was obtained from Dojindo
Laboratories (Kumamoto, Japan). Micro-cover glass (13 mm in
diameter) was obtained from Matsunami Glass Ind. Ltd. (Osaka,
Japan).
Animals
Six-week-old, male, F344 rats were purchased from SLC Co.
(Hamamatsu, Japan) and housed in polycarbonate cages with
wood chips in a specific pathogen-free laboratory of our animal
facilities, three rats/cage. They were kept under constant
conditions of temperature (22±2°C) and
humidity (55±5%) with a 13 hr light/11 hr dark
cycle. All the rats were provided with laboratory chow basal
diet (MF) (Oriental Yeast Co. Ltd., Tokyo, Japan) unless specified
and the beds were changed twice a week. The arrival weight
and food consumption of the animals were recorded.
Study design
Study-1(Initiation and post-initiation)
Male F344 rats (6 weeks old) were treated with a single dose
of AOM (20 mg/kg, 10 mg/ml in sterile saline) subcutaneously.
Rats were allocated to one of 3 experimental groups, the control
and two UDCA groups (initiation and post-initiation, 15-18
rats/group). In the initiation group, the rats were fed MF
(Oriental Yeast, Co., Ltd., Tokyo, Japan) supplemented with
the UDCA (0.4%) ad libitum from four days before to three
days after the AOM injection (for 1 week), and thereafter
the rats were fed MF alone (Fig. 1A). In the post-initiation
group, the MF was changed to a UDCA-supplemented diet (0.4%)
one week after AOM injection (Fig. 1B). The control group
was fed the MF during the entire experimental period (Fig.
1C). Rats (n=5-7) were sacrificed at the 2nd, 5th and 8th
weeks. The isolated colons were collected and washed with
0.9% NaCl water solution. The lumen of the colon was filled
with 10% phosphate-buffered formalin (pH 7.4) for 3 min. These
were incised in a longitudinal direction and fixed flat between
two pieces of filter paper in the buffered formalin. The colons
were stained with 0.2% methylene blue (Sigma Chemical Co.,
St. Louis, MO) and the number of ACF along the colon was counted
by their position in every 2 cm under light microscope (21)
from the anus to proximal colon.
Study-2 (Time-course)
In this study, the rats (6 weeks old) were treated with AOM
as Study-1. One week later, the rats were divided into two
groups and were fed ad libitum the MF diet supplemented with
UDCA (0.4%) or MF alone, until the end of the experiment (Fig.
1D). Rats (n=4-5) were sacrificed at the 2nd, 4th, 8th, 16th
and 32nd weeks to count the colonic ACF.
Study-3 (Dose-response)
After treatment with AOM as a single dose and MF feeding for
1 week, the rats were divided into five groups (6 rats/group).
From the beginning of the second week rats were fed ad libitum
the MF supplemented with different concentrations of UDCA
(0.05%, 0.1%, 0.2% and 0.4%) or MF alone (Fig. 1E). At the
end of 5th week the rats were sacrificed to evaluate the colonic
ACF.
Statistical analyses in all studies were carried out using
analysis of variance (ANOVA), and P<0.05 was considered
significant.
Cell culture
The rat colon cancer cell line (ACL-15 cells) was obtained
from Riken Cell Bank (Tsukuba, Japan). ACL-15 cells were grown
in a 5% CO2-95% air humidified atmosphere at 37°C
and maintained for 3-5 days as monolayer cultures in Dulbecco's
modified Eagle's minimum essential medium (DMEM ; Nissui,
Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS),
2 mM glutamine, penicillin (100 units/ml), streptomycin (100
µg/ml) and gentamycin (40 µg/ml). The
cells were plated on 13 mm-diameter round cover glasses in
35-mm culture dishes at a density of 1×106 cells/dish.
Measurement of intracellular free [Ca2+]i
The cells were incubated at 37°C for 30 min in 1 ml
of calcium containing phosphate-buffered saline PBS(+)/4 µM
fura-2/acetoxy methyl ester, a fluorescent Ca2+ indicator.
Then, the cells on cover glasses were transferred to a small
incubation bath (approx. 0.5 ml) that was continuously rinsed
with PBS(+) on the platform of a microscope. Together with
the onset of the intracellular free Ca2+ concentration ([Ca2+]i)
measurement, PBS(+) rinsing the cells was immediately replaced
by 1 mM bile acid-containing PBS(+). Fluorescence of the cells
was measured using a fluorescence spectromicroscope (excitation,
340/380 nm ;emission, 510 nm). About ten cells that showed
apparently normal morphological features, that were isolated
from surrounding cells were selected for measurement in each
bile acid case. The [Ca2+]i was calculated using the previously
reported equation (22).
RESULTS
Effect of UDCA on ACF formation
In Study-1, the UDCA (0.4%) supplemented diet decreased the
ACF number both during the initiation and post-initiation
stages (Fig. 2A). After two weeks of UDCA treatment, the ACF
numbers in the initiation and post-initiation groups decreased
significantly to 83.8% and 64.7% of the control group (average:56.8
ACF/colon), respectively (P<0.01, P<0.0001). At the
5th week of enumeration, the numbers in the initiation and
post-initiation groups decreased to 82.3% and 87.0% of the
control (average :74.4 ACF/colon), respectively, but these
suppressions were not significant. At the 8th week, the ACF
numbers in the initiation and post-initiation groups decreased
significantly to 74.6% and 65.0% of the controls (average:92.0
ACF/colon), respectively (P<0.001, P<0.0001). At the
end of 8th week, the total numbers of aberrant crypts (AC)
in the initiation and post-initiation groups showed decreasing
tendencies by 77.8% and 66.2% of the control (average:283.0
AC/colon), respectively, but they were not significantly different
from controls.
In the time-course study (Study-2), the numbers of ACF in
the UDCA supplemented diet group were 46.0%, 70.7%, 85.1%,
66.4% and 82.5% of controls in the 2nd, 4th, 8th, 16th and
32nd weeks, respectively (Fig. 2B). The numbers of ACF decreased
significantly in the UDCA supplemented diet group at the 2nd
(P<0.001), 4th (P<0.005) and 16th weeks (P<0.005),
but at the 32nd week, the ACF number was not significantly
decreased. The maximum suppressive effect of UDCA was observed
at the 2nd week (46.0% of controls), whereas it was minimum
(82% of controls) at the 32nd week, the end of the experiment.
In the dose-response study (Study-3), dietary administration
of UDCA decreased the number of ACF in a dose-dependent manner.
The suppressive effect of UDCA on ACF formation was significant
in UDCA of 0.1% (P<0.05), 0.2% (P<0.01) and 0.4% (P<0.005).
UDCA of 0.05% did not decrease the numbers of ACF significantly
(Fig. 2C).
Effect of bile acids on [Ca2+]i of ACL-15 cells
Measurement of intracellular Ca2+ by fluorescence microscopy
revealed that CDCA and DCA induced a rapid increase in [Ca2+]i
even when extracellular Ca2+ was abolished (Fig. 3C-D). CDCA
and DCA (1 mM) increased [Ca2+]i to a peak of approximately
120 nM and 100 nM, respectively, followed by its gradually
decrease to a steady level. In contrast, UDCA and CA did not
induce an increase in [Ca2+]i (Fig. 3A-B).
DISCUSSION
In our previous studies, we found that UDCA suppressed the
carcinogen-induced colonic ACF formation in rats (6, 7). In
the present study, we also observed an ACF-suppressive effect
of UDCA. To observe the ACF suppressive effect of UDCA in
detail, we investigated the effect of UDCA on ACF using different
protocols. In study-1A of the present study, it was shown
that UDCA could decrease the AOM-induced ACF number during
the initiation as well as during the post-initiation, suggesting
that UDCA affects both stages of colon carcinogenesis. In
the time-course study that examined the long-term effect of
UDCA, we found that UDCA continued to significantly suppress
the ACF number compared with controls until the 16th week
of the experiment (70.8 vs. 106.6), at the32nd week the ACF
number of UDCA and MF fed groups (60.0 vs. 72.7 ACF/colon)
did not differ so much as the early period of the experiment.
In the present dose-response study of UDCA we observed that
the MF diet supplemented with 0.05% and 0.1% of UDCA suppressed
the ACF number gradually, but unexpectedly this suppressive
effect of UDCA did not increase so markedly at doses higher
than 0.1%. The exact cause(s) of these variable effects of
UDCA remains to be clarified. However, it was reported that
orally supplemented bile acids are frequently converted to
other bile acids by the enterohepatic circulation as well
as by enzymatic action of gut flora (7, 23, 24). In our previous
study with dietary administration of UDCA and other bile acids,
the fecal bile acids analysis revealed that similar to other
bile acids, UDCA was also converted to muricholic acid (50.8%),
LCA (10.2%), hydodeoxycholic acid (8.7%), DCA (4.1%), CDCA
(0.4%) and others (7). These converted acids are known as
ACF promoting bile acids and this conversion was suggested
to be changed in terms of quality and quantity in long-term
administration. This may be one of the causes for UDCA not
to continue its ACF suppressive effect beyond a certain dose
and time. Moreover, rat colonic mucosal cells may change their
response to UDCA after long-term administration.
The mechanism(s) involved in tumor promotion by bile acids
remains unclear. Similarly, the mechanisms involved in the
anticarcinogenic effects of UDCA on experimental colonic cancer
also remain to be clarified. Alterations in protein kinase
C (PKC) are involved in colonic malignant transformation in
humans (14) and in animals (15). PKC constitutes a gene family
of serine/threonine protein kinases that play central roles
in transmembrane signaling events, and are involved in diverse
biological processes, including cellular proliferation and
differentiation (25). Bile acids, which are known as activators
of PKC (16), can also stimulate the membrane phospholipase
C that converts the phosphatidylinositol biphosphate to inositol
triphosphate (IP3) and diacylglycerol (DAG). IP3 acts on endoplasmic
reticulum and this released calcium ions into the cytoplasm.
These calcium ions in addition to the DAG, ultimately activate
the PKC for further activities. Therefore, bile acids can
activate the PKC via the induction of intracellular calcium
ions. In the present study, CDCA and DCA which were previously
reported to increase the number of ACF in the rat colon (6,
7), induced a rapid increase in the [Ca2+]i in ACL-15 cells.
Thus, the ACF promotive effect of CDCA and DCA in animal model
studies agrees with the present in vitro finding of their
calcium ion induction. From this result we can say that the
effect of CDCA and DCA may be, in part, related to the increase
in [Ca2+]i. However, CA, which showed no effect (7) or suppressive
effect (26) on ACF formation, and UDCA, which suppressed the
ACF number (6, 7), did not show any effect on [Ca2+]i. Although
the ineffective nature of the CA on colonic ACF formation
is consistent with its effect of unchanged [Ca2+]i, the suppressive
effect of CA and UDCA on ACF formation is not correlated with
their [Ca2+]i induction. Other mechanism(s) in this suppression
process may be involved and remain to be clarified.
In conclusion, regardless of the exact mechanism(s) of bile
acids to produce colonic ACF, the present findings demonstrated
that the ACF promotive activity of bile acids in rat colonic
mucosa may be through the involvement of PKC that is activated
partly by intracellular calcium ions. Therefore, it is suggested
that there is a possible relation between the intracellular
calcium ion induction by bile acids and the effects of bile
acids on ACF formation.
ACKNOWLEDGEMENTS
This study was supported by a Grant-in-Aid for Cancer Research
from the Ministry of Health and Welfare, Japan. A doctoral
fellowship to Md. Abdul Momen from the Ministry of Education,
Science, Sports and Culture of Japan is gratefully acknowledged.
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Received for publication January 5, 2002;accepted January
31, 2002.
Address correspondence and reprint requests to Atsushi Umemoto,
Second Department of Surgery, The University of Tokushima
School of Medicine, Kuramoto-cho, Tokushima 770-8503, Japan
and Fax:+81-88-633-7144.
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