Carcinogenesis vol.20 no.2 pp.299–303, 1999
Administration of an unconjugated bile acid increases duodenal
tumors in a murine model of familial adenomatous polyposis
Najjia N.Mahmoud1,2, Andrew J.Dannenberg1,2,
Robyn T.Bilinski1, Juan R.Mestre2, Amy Chadburn1,
Matthew Churchill1, Charles Martucci2 and
Monica M.Bertagnolli1,2,3
1The
New York Hospital-Cornell University Medical Center,
525 East 68th Street, New York, NY 10021 and 2Strang Cancer Prevention
Center, 428 East 72nd Street, New York, NY 10021, USA
3To
whom correspondence should be addressed
Email: [email protected]
Intestinal carcinogenesis involves the successive accumulation of multiple genetic defects until cellular transformation
to an invasive phenotype occurs. This process is modulated
by many epigenetic factors. Unconjugated bile acids are
tumor promoters whose presence in intestinal tissues is
regulated by dietary factors. We studied the role of the
unconjugated bile acid, chenodeoxycholate, in an animal
model of familial adenomatous polyposis. Mice susceptible
to intestinal tumors as a result of a germline mutation in
Apc (Min/1 mice) were given a 10 week dietary treatment
with 0.5% chenodeoxycholate. Following this, the mice
were examined to determine tumor number, enterocyte
proliferation, apoptosis and β-catenin expression. Intestinal
tissue prostaglandin E2 (PGE2) levels were also assessed.
Administration of chenodeoxycholate in the diet increased
duodenal tumor number in Min/1 mice. Promotion of
duodenal tumor formation was accompanied by increased
β-catenin expression in duodenal cells, as well as increased
PGE2 in duodenal tissue. These data suggest that unconjugated bile acids contribute to periampullary tumor
formation in the setting of an Apc mutation.
Introduction
(14). Apc is mutated in .85% of sporadic human colorectal
cancers (15). Humans with a germline mutation of Apc, a
condition known as Familial Adenomatous Polyposis (FAP),
develop multiple tumors in the colorectum and the duodenum.
Through the characterization of Apc protein as a modulator
of intestinal cell growth, FAP is now recognized as a model
relevant to sporadic as well as familial colorectal cancer.
Epigenetic factors, such as diet and exposure to chemical
carcinogens, modulate the process of intestinal carcinogenesis.
In patients with FAP, duodenal tumors are clustered around
the ampulla of Vater, suggesting that biliary secretions play a
role in tumor formation. The C57BL/6J-Min/1 (Min/1) mouse
is an animal model of FAP. Min/1 mice carry a germline
mutation of Apc that produces a truncated Apc protein (16).
These animals develop multiple tumors, located primarily in
the small intestine, and exhibit altered growth kinetics in the
pre-neoplastic intestinal epithelium (17). The stable genetic
background of the Min/1 mouse provides an excellent model
for studying epigenetic modulation of intestinal carcinogenesis.
To study the role of bile acids in Apc-associated carcinogenesis, we determined the effect of dietary administration of
an unconjugated bile acid, chenodeoxycholate, upon tumor
formation in the Min/1 mouse. We also measured the effect of
chenodeoxycholate upon duodenal cell proliferation, apoptosis
and β-catenin expression. We found that dietary administration
of chenodeoxycholate increased duodenal tumor formation in
Min/1 mice. This effect was associated with elevated
enterocyte β-catenin expression and increased levels of
prostaglandins in duodenal tissue. These results suggest that
unconjugated bile acids contribute to duodenal tumor formation
in the setting of Apc mutation.
Materials and methods
A substantial body of evidence suggests that bile acids play a
role in the development of intestinal tumors. In humans,
epidemiological studies indicate that colon cancer is increased
following cholecystectomy (1,2) and by consumption of a high
fat, low fiber diet (3,4), conditions that are associated with
bile acid excess. Secondary bile acids are also increased in
patients with colorectal adenomas (5). In rodent models,
dietary administration of bile acids increases intestinal neoplasia (6–11). Bile acids induce AP-1 mediated gene transcription in colon cancer cell lines (8), and are potent activators of
protein kinase C isoenzymes (10,12,13).
Colorectal carcinoma is a multistage process characterized
by the successive acquisition of mutations in the genes
governing cellular growth and differentiation. The Apc gene
is a key regulator of epithelial growth and mutation of Apc is
one of the earliest events in the adenoma–carcinoma sequence
Treatment of Min/1 mice with bile acids
Female Min/1 mice were obtained at 5 weeks of age from Jackson Laboratories
(Bar Harbor, ME) and started on experimental feeds on arrival. Pelleted feeds
were prepared by Research Diets (New Brunswick, NJ). Chenodeoxycholic
acid (98% pure) was obtained from Aldrich Chemical (St Louis, MO). Test
diets consisted of AIN-76A supplemented with 0.5% chenodeoxycholic acid
(w/w). Control Min/1 mice and their wild-type littermates (1/1) were fed
AIN-76A diet without bile acid supplementation. Animals and their food were
weighed twice weekly and checked daily for signs of weight loss or lethargy
that may indicate intestinal obstruction or anemia. During the course of the
experiment there was no difference in body weight or food consumption
among the various study groups, and the animals remained active, suggesting
that there was no toxicity in the treatment group. At 110 days of age, all mice
were killed by CO2 inhalation, and their intestinal tracts were removed from
esophagus to distal rectum, opened, flushed with saline, and the entire length
was examined under 33 magnification to obtain tumor counts. The tumors
were counted by an individual blinded to the animal’s genetic status and
treatment. Multiple samples of normal-appearing full-thickness small intestine
were harvested and fixed in 10% formalin for histologic examination. Samples
used for the analyses were taken from the duodenum, the mid small intestine
and the middle portion of the colon.
Abbreviations: Cox-2, cyclooxygenase-2; FAP, familial adenomatous
polyposis; Min/1, C57BL/6J-Min/1; PCNA, proliferating cell nuclear antigen;
PGE2, prostaglandin E2; PBS, phosphate-buffered saline; TUNEL, terminal
deoxynucleotide nick end labeling.
Tissue histology
For each animal, 10–15 sections of small intestinal mucosa were examined.
Specimens of small intestine of ~5 mm in length were formalin fixed,
embedded in paraffin and sectioned at 3 µm. Sections were stained with
© Oxford University Press
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N.N.Mahmoud et al.
hematoxylin and eosin for evaluation of mucosal histology. For studies of
nuclear density, Fuelgen staining was performed. To prepare sections for
immunohistochemistry, sections of small intestine were deparaffinized and
dehydrated by processing the slides with Hemo-De (Fisher Scientific,
Pittsburgh, PA) and an alcohol series, followed by washing in phosphatebuffered saline (PBS; pH 7).
Measurement of prostaglandin E2 (PGE2)
PGE2 measurements were performed as described previously (18). Intact small
intestine, rather than intestinal mucosa only, was used for this study because
a relatively large volume of material was required for analysis. Samples were
homogenized, transferred to microcentrifuge tubes and centrifuged at 4°C for
10 min at 10 000 g. The supernatant was decanted, and a 10 µl aliquot of
supernatant was used to determine protein concentration with a Bicinchoninic
Acid Protein Assay Kit (Sigma, St Louis, MO). Determination of PGE2 levels
by EIA was accomplished using a Prostaglandin E2-Monoclonal Enzyme
Immunoassay Kit (Cayman Chemical, Ann Arbor, MI). Plates were read at
410 nm with a UV max Kinetic Plate Reader (Molecular Devices, San Jose,
CA). Data were computerized with DeltaSoft 3 and statistics performed with
InStat 2.00 software.
Measurement of enterocyte proliferation
Small bowel sections were deparaffinized and rehydrated and endogenous
peroxidase activity was blocked by incubating the slides with 0.45% methanol/
H2O2. Antigen retrieval was achieved by microwaving. Proliferating cell
nuclear antigen (PCNA) antibody (Dako, Carpinteria, CA) was applied and
incubated for 1 h at room temperature. Indirect detection was performed by
incubating with secondary biotinylated horse anti-mouse IgG followed by
Vector Elite ABC for 30 min at room temperature. Incubation for 5 min in
DAB was utilized for color development. The specimens were counterstained
with methyl green. For each specimen, eight crypt-villus units were chosen
randomly from serial sections of small bowel mucosa by an individual blinded
to the animal’s genetic status. The percent staining of enterocytes in these
crypt-villus units was measured using the Cell Analysis System 200 and CAS
200 Quantitative Nuclear Analysis Software (CAS 200).
In situ detection of apoptosis
To determine the percentage and distribution of epithelial cells undergoing
cell death, we employed an in situ direct immunoperoxidase technique for
determining cell death using the ApopTag Kit (Oncor, Gaithersburg, MD) as
previously described (18). Mouse lymphoid tissue with a known 2–3% rate
of apoptosis was used as a positive control. For each specimen, eight cryptvillus units were chosen randomly from serial sections of small intestinal
mucosa by an individual blinded to the animal’s treatment group and genetic
status. The percent staining of enterocytes in these crypt-villus units was
measured using the CAS 200. To confirm that uniform sampling was achieved,
nuclear density was measured and confirmed to be equal throughout the three
study groups.
Determination of β-catenin expression
Slides were deparaffinized in xylene for 10 min followed by alcohol rehydration. After quenching endogenous peroxidases with 0.45% H2O2 in
methanol, the slides were rinsed in PBS and an antigen retrieval step was
carried out in a 700 W microwave oven for 10 min in pH 6.0 citrate buffer.
The slides were then incubated with a monoclonal antibody to β-catenin
(Transduction Laboratories, Lexington, KY) at 25°C for 1 h. Horse antimouse IgG secondary antibody was added for 30 min at 25°C followed by
the Vector Elite ABC kit according to the manufacturer’s instructions. The
slides were stained with diaminobenzidine for 5 min and counterstained with
methyl green. Analysis was carried out using the CAS 200.
Results
Chenodeoxycholic acid increases duodenal tumor formation
in Min/1 mice
To determine the effect of increased intestinal bile acids on
Apc-mediated carcinogenesis, we administered 0.5% chenodeoxycholate to Min/1 mice as a dietary additive. After 10
weeks of bile acid supplemented diet, the animals were killed
and their intestines examined to obtain macroscopic tumor
counts. For the small and large intestines, there were no
significant differences in tumor number between treatment
and control animals (Table I). In the duodenum, however,
chenodeoxycholic acid induced a 9-fold increase in tumor
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Table I. Effect of chenodeoxycholate on tumor number
Study group
Duodenum
Small intestine Colon
Total
Min/1
Min/1 CD
1/1
0.3 6 0.6
3.0 6 1.6*
060
31.8 6 20.0
21.3 6 12.5**
060
32.5 6 20.3
25.2 6 13.3***
060
0.4 6 0.6
0.6 6 0.9
060
Beginning at 5–6 weeks of age, Min/1 mice were fed AIN-76A chow diet
supplemented with 0.5% chenodeoxycholic acid (Min/1 CD). As controls,
Min/1 mice and C57BL/6J-1/1 non-affected littermates (1/1) were fed
AIN-76A diet without additives. Each treatment group contained 20 mice,
except the chenodeoxycholate treatment group, which contained 26 animals.
At 110 days of age (10 weeks of treatment), all mice were killed and their
intestinal tracts were removed and examined under 33 magnification to
obtain tumor counts. Tumors were counted by an individual blinded to the
animal’s genetic status and treatment. Values expressed represent mean
number of tumors per mouse 6 SD where n 5 26 (Min/1CD) and n 5 20
(1/1, Min/1). Values for tumor distribution indicate total number of
tumors per mouse per intestinal segment. Statistical analysis by analysis of
variance (ANOVA) demonstrated no significant difference in tumor
distribution between the three groups in the small intestine and colon. For
tumors located in the duodenum, there was a significant increase in tumor
number in the chenodeoxycholate treatment group.
*P , 0.0001; **P 5 0.10; ***P 5 0.34 compared with Min/1.
number compared with that seen in untreated Min/1 mice
(Table I).
Previous studies show that pre-neoplastic intestinal mucosa
in the Min/1 mouse exhibits reduced levels of apoptosis, as
well as a compensatory decrease in enterocyte proliferation
(17). The net result of these changes is a decrease in enterocyte
migration rate. We therefore measured proliferation and
apoptosis in our study animals using immunohistochemical
techniques. Enterocyte proliferation was assessed by staining
actively dividing cells with an antibody to PCNA. Apoptosis
was measured by terminal transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL), an in
situ measure of the genomic DNA degradation induced by
programmed cell death. When Min/1 mice were compared
with their wild-type littermates, duodenal enterocyte proliferation and apoptosis were significantly reduced, consistent with
that found in other studies of the small intestine of Min/1
mice (15–19) (Figures 1 and 2). There was no change in the
general location of proliferating cells or of cells undergoing
apoptosis, i.e. the proliferating cells were found in the upper
third of the crypts, with apoptotic cells generally confined to
the tips of the villi. Although there was a trend toward a
further decrease in apoptosis and proliferation in the duodenum
of bile acid-treated animals, this was not significantly different
from that already produced by the Min mutation (Figures 1
and 2).
Chenodeoxycholic acid increases intracellular β-catenin
expression in Min/1 duodenal mucosa
The Apc protein regulates intracellular levels of β-catenin, a
component of the actin cytoskeleton that also tracks to the
nucleus where it may facilitate transcription of genes governing
cell growth. We therefore assessed cytoplasmic β-catenin levels
in Min/1 mice treated with dietary bile acids. Tissue sections
from the duodenum of study mice were stained with a
polyclonal antibody to β-catenin. In agreement with previous
studies, we found that the histologically normal mucosa of
Min/1 animals exhibited increased staining with anti-β-catenin
antibody when compared with 1/1 controls (Figure 3).
Treatment of the animals with chenodeoxycholate caused an
Bile acids increase Apc-associated duodenal tumors
Fig. 1. Duodenal cell proliferation is decreased in Min/1 mice. Specimens
of duodenum from animals at 110 days of age were formalin fixed,
embedded in paraffin and sectioned at 5 µm. Where indicated, animals were
treated with dietary chenodeoxycholic acid (0.5% diet by weight) as
described in Materials and methods. Sections of duodenum were stained
with antibody to PCNA, and the percent staining of enterocytes in these
crypt-villus units was measured by an observer blinded to the animal’s
genetic status using the CAS 200. Values expressed are % of total cells
positive 6 SEM, with 100% equal to the entire crypt-villus population. For
1/1, n 5 20; Min/1, n 5 20; Min/1 CD, n 5 26. *P , 0.001 compared
with Min/1; **P 5 0.12 compared with Min/1.
Fig. 2. Apoptosis is decreased in the duodenum of Min/1 mice. Specimens
of duodenum from animals at 110 days of age were formalin fixed,
embedded in paraffin and sectioned at 5 µm. Where indicated, animals were
treated with dietary chenodeoxycholate (0.5% by weight) as described in
Materials and methods. Sections of duodenum were analyzed by TUNEL.
The percent staining of enterocytes in these crypt-villus units was measured
by an observer blinded to the animal’s genetic status using the CAS 200.
Values expressed are % apoptotic cells 6 SEM, with 100% equal to the
entire crypt-villus population. For 1/1, n 5 20; Min/1, n 5 20; Min/1
CD, n 5 26. *P , 0.0001 compared with Min/1; **P 5 0.09 compared
with Min/1.
additional 6-fold increase in cytoplasmic β-catenin expression
(Figure 3).
Chenodeoxycholic acid increases duodenal tissue PGE2 levels
Cyclooxygenases catalyze the synthesis of prostaglandins
from arachidonic acid. Overexpression of cyclooxygenase-2
(Cox-2) is observed in both human and murine colon cancers
(18,20) and eliminating the function of Cox-2 dramatically
decreases tumor number in Apc-deficient mice (21). In previous
studies, we found increased tissue PGE2 in the small intestines
of Min/1 mice. We therefore studied the effect of chenodeoxycholate administration on levels of PGE2 in duodenal
tissue. As expected, PGE2 levels were increased 6-fold in the
duodenum of Min/1 animals when compared with wild-type
controls (Figure 4). Dietary administration of chenodeoxy-
Fig. 3. Chenodeoxycholate increases β-catenin expression in Min/1
duodenum. Specimens of duodenum from animals at 110 days of age were
formalin fixed, embedded in paraffin and sectioned at 5 µm. Where
indicated, animals were treated with chenodeoxycholate (0.5% by weight) as
described in Materials and methods. Sections were stained with antibody to
β-catenin. The percent staining of enterocytes in these crypt-villus units was
measured by an observer blinded to the animal’s genetic or treatment status
using the CAS 200. Values represented are 6 SEM where n 5 20 animals
(1/1 and Min/1); n 5 26 animals (Min/1 CD). *P , 0.0001 compared
with Min/1; **P , 0.0001 compared with Min/1.
Fig. 4. Duodenal tissue PGE2 levels are increased by dietary
chenodeoxycholate. Levels of PGE2 were determined in the duodenal tissue
from chenodeoxycholate-treated Min/1 mice (Min/1 CD) and in control
animals. Values represent means 6 SEM where n 5 20 (Min/1 and 1/1),
n 5 26 (Min/1 CD). *P , 0.0001 compared with Min/1; **P . 0.025
compared with Min/1.
cholate further increased tissue PGE2 to nearly seven times
that of wild-type animals and 54% greater than that of
Min/1 mice (Figure 4). These data suggest that chenodeoxycholate increases arachidonic acid metabolism in the duodenum, providing a possible mechanism of tumor promotion
by this compound.
Discussion
Clustering of tumor surrounding the ampulla of Vater is a
characteristic of patients with FAP, suggesting that bile plays
a role in the pathogenesis of Apc-related duodenal tumors.
Bile contains many different substances, including bile acids,
cholesterol and pigments such as bilirubin. In vitro studies
show that bile from both normal individuals and patients
with FAP can react with intracellular DNA to form covalent
DNA adducts (22). It is unclear whether bile-induced DNA
adduct formation occurs in vivo, or whether the component of
bile causing this effect is a bile acid (23,24). Compared with
normal controls, bile from patients with FAP has a higher
concentration of bile acids and a greater proportion of chenodeoxycholic acid (25). In this study, we found that dietary
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administration of chenodeoxycholic acid increased tumors in
the duodenum of animals with a germline Apc mutation. This
study does not allow us to identify whether this results from
a genotoxic interaction, or whether the bile acid acts primarily
as a tumor promoter. The bile acids that are secreted into the
duodenum are primarily conjugated with glycine or taurine.
The activity of intestinal bacteria in the lower gastrointestinal
tract converts these bile acids to their unconjugated forms.
Most studies suggest that it is the unconjugated form of a bile
acid that promotes intestinal tumor formation. Our model is
therefore different from the usual in vivo state, and may be more
relevant to carcinogenesis in the lower gastrointestinal tract.
The exact mechanism of bile acid-mediated tumor promotion
is unknown. In animal studies, direct application of unconjugated bile acids to the intestinal epithelium results in
epithelial damage, followed by increased enterocyte proliferation (26–29). Bile acids can activate protein kinase C (12,13),
as well as induce AP-1-mediated gene transcription in cultured
colon cancer cells (30). Recent studies show that bile acids
upregulate Cox-2 activity in vitro (31), providing yet another
link between arachidonic acid synthesis and tumor promotion.
The products of Cox-2 activity, e.g. prostaglandins such as
PGE2, can inhibit enterocyte apoptosis, decrease tissue immune
surveillance and increase tumor invasiveness (32–34). We
found that the duodenal tissue of Min/1 mice contained
elevated PGE2, and that this arachidonic acid metabolite was
further increased by chenodeoxycholate to seven times that of
normal animals. Our results are therefore consistent with the
theory that a bile acid-induced activation of prostaglandin
synthesis enhances tumor formation. In light of this, it is
interesting that humans with FAP that are treated with inhibitors
of prostaglandin synthesis, such as sulindac, show decreased
tumor formation in the large bowel but not the duodenum
(35,36). One explanation for this may be that the extraordinarily
high prostaglandin levels induced by bile cannot be overcome
by the non-selective inhibitors of cyclooxygenase presently
used to treat tumors in FAP patients.
In order for normal mucosal architecture to be maintained
in the intestine, the opposing processes of enterocyte proliferation and apoptosis must be balanced. In previous studies of
small intestinal mucosa from Min/1 mice, we found that both
apoptosis and proliferation were decreased as a result of the
Min Apc mutation (17). In this study, we extend this observation
to the duodenal mucosa. Although there was a slight further
decrease in apoptosis and proliferation in chenodeoxycholate
treated animals, this was not statistically significant. Decreased
duodenal cell turnover, therefore, may not be a cause of the
tumor-promoting effect of this bile acid.
Apc protein normally associates with GSK-3β, a serinethreonine glycogen synthase kinase (37). This complex binds
to and facilitates the degradation of β-catenin, an intracellular
protein associated with the actin cytoskeleton (38). The role
of β-catenin in colorectal carcinogenesis is unclear. β-catenin
is a component of the adherens junction of intestinal epithelial
cells, and may therefore be a modulator of cell–cell adhesion
or contact-associated growth regulation. β-catenin also binds
to the DNA binding proteins, Tcf and Lef. In association with
β-catenin, Tcf and Lef alter expression of genes that may
govern cell proliferation and apoptosis (37,38).
In previous studies, we found that pre-malignant cells in
the small intestine of Min/1 mice demonstrate increased
cytoplasmic β-catenin expression (17). The present study
suggests that increased β-catenin expression is also seen in the
302
duodenum of Apc-mutant mice. Furthermore, administration of
chenodeoxycholate as a tumor promoter resulted in an additional increase in β-catenin expression. Although the exact
role of β-catenin in intestinal tumorigenesis is unknown, these
data show that mucosal β-catenin expression correlates with
tumor development.
Dietary administration of chenodeoxycholate significantly
increased duodenal tumors in Min/1 mice, but had no significant effect upon overall tumor number in the small or large
intestines of treated animals. This result may be due to an
increased sensitivity of the duodenum of Min/1 mice to bile
acid-induced tumors. A more likely interpretation is that this
distribution of tumors resulted from a dosing effect, as dietary
administration of the unconjugated bile acid produces a higher
concentration of bile acid in the proximal gastrointestinal tract.
In summary, the data presented here indicate that locally
acting unconjugated bile acids increase tumor formation in the
setting of Apc mutation. Furthermore, this effect is associated
with increased PGE2 production, suggesting that effective
inhibition of prostaglandin synthesis in the duodenum may
inhibit bile acid-associated tumor promotion.
Acknowledgements
Special thanks to the Histopathology Technicians of the New York HospitalCornell Medical Center. This work was supported by American Cancer
Society (grant no. ACS CDA-95010–95), National Cancer Institute (grant no.
NCI-1R29CA74162–01) and Alice Bohmfalk Charitable Trust (M.M.B.), also
the National Institute of Health Surgical Oncology Research Training grant no.
25435 (N.N.M.) and Cancer Research Foundation of America (N.N.M., J.M.).
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Received April 30, 1998; revised October 1, 1998; accepted October 9, 1998
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