Carcinogenesis vol.21 no.5 pp.921–927, 2000
Plant phenolics decrease intestinal tumors in an animal model of
familial adenomatous polyposis
Najjia N.Mahmoud1, Adelaide M.Carothers3,
Dezider Grunberger2, Robyn T.Bilinski1,
Matthew R.Churchill1, Charles Martucci2,
Harold L.Newmark4 and Monica M.Bertagnolli1,3,5
1The
New York Hospital–Cornell Medical Center, 525 East 68th Street,
New York, NY 10021, 2College of Physicians and Surgeons, Columbia–
Presbyterian Cancer Center and School of Public Health, Columbia
University, New York, NY 10032, 3The Strang Cancer Prevention Center,
428 East 72nd Street, New York, NY 10021 and 4Rutgers University
Laboratory for Cancer Research, 164 Frelinghuysen Road, Piscataway,
NY 08854, USA
5To
whom correspondence should be addressed
Email: [email protected]
Epidemiological studies consistently indicate that consumption of fruits and vegetables lowers cancer risk in humans
and suggest that certain dietary constituents may be effective in preventing colon cancer. Plant-derived phenolic
compounds manifest many beneficial effects and can potentially inhibit several stages of carcinogenesis in vivo. In this
study, we investigated the efficacy of several plant-derived
phenolics, including caffeic acid phenethyl ester (CAPE),
curcumin, quercetin and rutin, for the prevention of tumors
in C57BL/6J-Min/⍣ (Min/⍣) mice. These animals bear a
germline mutation in the Apc gene and spontaneously
develop numerous intestinal adenomas by 15 weeks of age.
At a dietary level of 0.15%, CAPE decreased tumor
formation in Min/⍣ mice by 63%. Curcumin induced a
similar tumor inhibition. Quercetin and rutin, however,
both failed to alter tumor formation at dietary levels of
2%. Examination of intestinal tissue from the treated
animals showed that tumor prevention by CAPE and
curcumin was associated with increased enterocyte
apoptosis and proliferation. CAPE and curcumin also
decreased expression of the oncoprotein β-catenin in the
enterocytes of the Min/⫹ mouse, an observation previously
associated with an antitumor effect. These data place the
plant phenolics CAPE and curcumin among a growing list
of anti-inflammatory agents that suppress Apc-associated
intestinal carcinogenesis.
Introduction
Numerous epidemiological studies of colorectal cancer suggest
that dietary agents play an important role in the development
of intestinal neoplasia (1). Frequent consumption of fruits,
vegetables and plant fiber is associated with a decrease in
colorectal cancer incidence (2,3), and a high intake of fat or
red meat may increase intestinal neoplasia (4,5). The search
for an effective chemopreventive regimen based upon natural
Abbreviations: Apc/APC, adenomatous polyposis coli; CAPE, caffeic acid
phenethyl ester; CAS, Cell Analysis System; COX, cyclooxygenase; DAB,
diaminobenzidine; Min/⫹, C57BL/6J-Min/⫹; NSAIDs, non-steroidal antiinflammatory drugs; PBS, phosphate-buffered saline; PCNA, proliferating cell
nuclear antigen; ROS, reactive oxygen species.
© Oxford University Press
food substances has identified several plant-derived compounds
with antitumor activity for many different cancers (6). Plant
phenolics are a class of antitumor agents whose beneficial
effects have been characterized in several cell culture and
animal cancer models. These agents inhibit carcinogenesis at
the initiation, promotion and progression stages. For example,
plant phenolics prevent tumor initiation because they are
antimutagenic, they reduce levels of carcinogen–DNA adducts
in chemically treated cells and they suppress the metabolic
activation of carcinogens by inhibiting Phase I monoxygenases
(7). Examples of inhibitory effects on promotion by plant
phenolics include studies showing that they scavenge free
radicals, induce the transcription of Phase II detoxifying
enzymes and reduce the expression of ornithine decarboxylase
(8–10). Finally, plant phenolics both inhibit cell proliferation
and induce cell death or differentiation in tumor cells, suggesting that they may antagonize all phases of carcinogenesis
(7,11–13).
Caffeic acid phenethyl ester (CAPE) and curcumin (Figure
1) characterize plant phenolics that are structurally related to
3,4-dihydroxycinnamic acid (6). CAPE may be obtained from
propolis, a substance produced by the bark of conifer trees
and carried by honeybees to their hives (14). Curcumin is the
yellow pigment of tumeric and mustard that is widely used
for flavoring and coloring in foods. Both CAPE and curcumin
are effective in vitro inhibitors of the growth of transformed
cells (15–20) and in vivo inhibitors of tumor initiation and
promotion in different carcinogen-induced rodent models
(14,21–26).
Flavonoids constitute another promising class of dietary
antioxidants that are ubiquitously found in fruits, vegetables
and tea. Like the cinnamates, these phytochemicals exhibit
anti-inflammatory activity and inhibit tumor cell growth in
culture and animal bioassays. Quercetin is the most common
biologically active flavonoid. The glycosidic derivatives of the
flavonoids are preferentially absorbed in humans (6). Rutin is
the glycoside form of quercetin and is hydrolyzed to quercetin
by colonic microflora, possibly enhancing its bioavailability
in the colon (27). Quercetin and rutin inhibit neoplasia in
carcinogen-induced skin and colon cancer models (25,26,28–
30).
To further characterize the chemopreventive efficacy of
naturally occurring antioxidant compounds, we treated C57BL/
6J-Min/⫹ (Min/⫹) mice with diets containing curcumin,
CAPE, quercetin and rutin. The Min/⫹ mouse develops
multiple intestinal adenomas as a result of a germline mutation
in one adenomatous polyposis coli (Apc) allele (31,32).
Although these animals develop intestinal adenomas, rather
than carcinomas, the role of Apc early in the adenoma–
carcinoma sequence of sporadic colorectal cancer suggests that
Apc-deficient animals such as the Min/⫹ mouse are important
spontaneous tumor models. Consequently, modulation of the
Min/⫹ phenotype has been used as a screen for potential
chemopreventive agents (33–35).
921
N.N.Mahmoud et al.
Fig. 1. Chemical structure of plant phenolics. CAPE and curcumin belong
to a class of plant phenolics known as the hydroxycinnamic acids. Quercetin
and its glycoside rutin are plant phenolics of the flavonoid family.
We found that tumors in Min/⫹ mice were inhibited by
dietary administration of CAPE at a dose of 0.15%. A similar
degree of tumor prevention resulted from addition of 0.10%
curcumin to the diet. In these instances, tumor prevention
was associated with an increase in enterocyte apoptosis and
decreased expression of β-catenin in the intestinal mucosa.
Measurement of proliferation in the intestine of treated animals
also indicated that these effective agents increased the
enterocyte turnover rate. No tumor inhibition was observed in
animals treated with either quercetin or rutin. These data
suggest that the plant phenolics CAPE and curcumin should
be further investigated as chemopreventive agents for colorectal cancer.
Materials and methods
Dietary treatment of Min/⫹ mice
Female Min/⫹ mice were obtained at 5 weeks of age (Jackson Laboratories,
Bar Harbor, ME) and started on experimental feeds on arrival. CAPE was
synthesized by esterification of caffeic acid with phenethyl alcohol as described
elsewhere (15). Curcumin, quercetin and rutin were purchased from Sigma
Chemical Co. (St Louis, MO). The agents were pelleted into an AIN-76A
diet by Research Diets (New Brunswick, NJ) at the following concentrations:
CAPE 0.03% and 0.15%; curcumin 0.1%; quercetin 2%; rutin 2%. The mice
consumed ~2.5 g of feed/day. Control Min/⫹ mice and their wild-type
littermates (⫹/⫹) were fed AIN-76A diet without phenolic supplementation.
Animals and their food were weighed twice weekly. Animals were checked
daily for signs of weight loss or lethargy that may indicate intestinal obstruction
or anemia. 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 examined under 3⫻ magnification to determine the
tumor number. The tumors were counted by an individual blind 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
histological examination. All samples used for the tissue analyses were taken
from the middle portion of the small intestine.
Tissue histology
Specimens of small intestine of ~5 mm in length were formalin fixed,
embedded in paraffin and sectioned at 3 µm. Sections were stained with
hematoxylin and eosin for evaluation of mucosal histology. 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 7.0.
922
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 described previously (33). Mouse lymphoid tissue with a known 2–3% rate
of apoptosis was used as a positive control. For each specimen, eight crypt–
villus units were chosen randomly from serial sections of small intestinal
mucosa by an individual blind to the animal’s treatment group and genetic
status. The percent staining of enterocytes in these crypt–villus units was
measured using the Cell Analysis System (CAS) 200 and CAS 200 Quantitative
Nuclear Analysis Software. To confirm that uniform sampling was achieved,
the nuclear densities were measured and confirmed to be equal throughout
the three study groups.
Measurement of enterocyte proliferation
Small bowel sections were deparaffinized and rehydrated and endogenous
peroxidase activity was blocked by incubating the slides with 0.45% H2O2 in
methanol. Antigen retrieval was achieved by microwaving at 700 W for 10
min in citrate buffer, pH 6.0. Anti-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 (Vector
Laboratories, Burlingame, CA) for 30 min at room temperature. Incubation
for 5 min in diaminobenzidine (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 blind to the animal’s genetic and treatment
status. The percent staining of enterocytes in these crypt–villus units was
measured using the CAS 200.
Determination of tissue 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
by microwaving as described above. The slides were then incubated with a
monoclonal antibody to β-catenin (Transduction Laboratories, Lexington, KY)
at 25°C for 1 h. Horse anti-mouse IgG secondary antibody was added for 30
min at 25°C followed by Vector Elite ABC detection as above. The slides
were stained with DAB for 5 min and counterstained with methyl green. The
amount of β-catenin staining in the middle third of each crypt–villus was
quantified using CAS 2000 with CAS 200 Quantitative Analysis Software.
Results
CAPE and curcumin inhibit tumor formation in Min/⫹ mice
During the course of these experiments there was no difference
in body weight or food consumption among the various study
groups and the animals remained active, suggesting that the
treatments were not toxic. Dietary administration of both
CAPE and curcumin reduced tumors in the intestine of Min/⫹
mice. At a dietary level of 0.15%, or ~7.5 mg/day, CAPE for
10 weeks reduced intestinal tumor formation in Min/⫹ mice
by 63% (Table I). A similar level of tumor inhibition was
observed upon treatment of the animals with 0.1% curcumin
in the diet. The majority of the tumors in both the control
Min/⫹ and the treatment groups were located in the small
intestine, consistent with the standard Min/⫹ phenotype. As
expected, no tumors were found in the wild-type littermates
lacking the germline Apc mutation (⫹/⫹). No decrease in
tumor number or change in tumor distribution was observed
at a CAPE dose of 0.03%.
The flavonoids quercetin and rutin were admininstered to
the Min/⫹ mice at dietary levels of 2%. The decision to use
a 10-fold higher dose than that chosen for curcumin and CAPE
was based upon estimates of the relative bioavailability of
quercetin and rutin in rodents (9,36). At this dose, however,
these agents altered neither the number nor distribution of
tumors relative to that of control animals (Table I).
CAPE and curcumin induce enterocyte apoptosis in Min/⫹ mice
In previous studies of Min/⫹ mice we found that the enterocytes
lining the tumor-prone small intestine exhibited decreased
Plant phenolics prevent Apc-associated tumors
Table I. Tumor number in Min/⫹ mice treated with CAPE and curcumin
Min/⫹ ⫹ control diet
CAPE 0.03%
CAPE 0.15%
Curcumin 0.1%
Quercetin 2%
Rutin 2%
32.5
32.5
33.2
33.2
33.2
⫾
⫾
⫾
⫾
⫾
6.5
6.5
4.5
4.5
4.5
Min/⫹ ⫹ chemopreventive agent
Tumor inhibition (%)
P value
33.0 ⫾ 6.4
12.0 ⫾ 2.6
11.8 ⫾ 3.2
30.3 ⫾ 4.4
26.3 ⫾ 5.1
NA
63
64
NA
NA
0.95
0.005
0.005
0.74
0.44
Beginning at 5–6 weeks of age, Min/⫹ mice were treated with the chemopreventive agents pelleted in AIN-76A diet at the doses listed. Control Min/⫹ mice
were fed AIN-76A diet without additive. At 110 days of age, total intestinal tumor counts were obtained. Values expressed represent the mean number of
tumors per mouse ⫾ SEM. P values were determined by two-sided t-test. For the control groups (⫹/⫹ and Min/⫹), number of animals ⫽ 20; for the
treatment groups, number of animals ⫽ 10 each.
Fig. 2. CAPE increases apoptosis in the small intestine. Specimens of small
intestine from animals at 110 days of age were formalin fixed, embedded in
paraffin and sectioned at 5 µm. Where indicated, animals were treated with
(A) curcumin (0.10%) or (B) CAPE (0.15%) as described in Materials and
methods. Sections of small intestine were analyzed by TUNEL. Values
expressed are percent of cells in the entire crypt–villus that show positive
staining. (A) *P ⬍ 0.0001 compared with ⫹/⫹, **P ⬍ 0.0001 compared
with Min/⫹; (B) *P ⬍ 0.0001 compared with ⫹/⫹, **P ⬍ 0.0001
compared with Min/⫹. For the control groups (⫹/⫹ and Min/⫹), number
of animals ⫽ 20; for the treatment groups, number of animals ⫽ 10 each.
levels of apoptosis when compared with similar tissue from
wild-type littermates (34,37). This difference suggests that
germline Apc mutation alters apoptosis in the villi of the small
intestine, at least as measured by TUNEL (38). Moreover,
these studies showed that administration of agents capable of
inhibiting tumor formation in Min/⫹ mice, such as sulindac
or aspirin, increased enterocyte apoptosis in vivo. Because
CAPE and curcumin induce intestinal tumor cell apoptosis
in vitro (20,39), we predicted a similar response in the Min/
⫹ intestinal mucosa treated with these agents.
To evaluate apoptosis in the Min/⫹ small intestine, samples
of histologically normal small intestine were obtained from
treated and control mice and processed for TUNEL as described
in Materials and methods. In all cases, the cells undergoing
apoptosis were located in the upper half of crypt–villus units.
For the animals treated with curcumin, a different technique
was used for processing the specimens prior to immunohistochemistry, accounting for the higher level of TUNEL staining.
The CAPE-treated animals were assayed at a later time when
our assay was better refined and do not show this increased
background. These studies, however, both yield the same
conclusions. As shown in Figure 2, the small intestine from
the Min/⫹ animals showed a significantly reduced level of
apoptosis in comparison with their wild-type littermates. When
compared with untreated Min/⫹ mice, however, small intestine
from animals whose diets included a tumor-inhibiting dose of
either CAPE or curcumin showed an ~10-fold increase in
apoptosis. The diet containing 0.1% curcumin restored the
level of enterocyte apoptosis to that of wild-type animals,
whereas the diet containing 0.15% CAPE stimulated enterocyte
apoptosis to a level twice that of the wild-type mice (Figure
2). There was no difference in apoptosis between control Min/⫹
mice and Min/⫹ mice treated with CAPE at 0.03% (data not
shown). Thus, the effect of tumor-preventing doses of CAPE
and curcumin on apoptosis in Min/⫹ small intestine was
similar to that observed previously in response to other
effective chemopreventive agents such as sulindac and aspirin
(33,34,37). These data also support previous in vitro studies
suggesting that CAPE and curcumin exert their tumor-preventing activity through induction of apoptosis in abnormal
cells (8,12,20). Because quercetin and rutin were ineffective
in reducing tumor number, further histochemical analyses of
these treated groups were not performed.
CAPE and curcumin normalize enterocyte proliferation in
Min/⫹ mice
Previous studies show that normal migration of enterocytes
from the crypts to the villus tips is inhibited in the Min/⫹
mouse and, consequently, the mucosal enterocytes of these
animals have a reduced turnover rate (38). Consistent with
this observation, we observed a decrease in proliferation of
this cell population in previous studies of the Min/⫹ mouse
(37,38). Effective chemopreventive drugs, such as sulindac
(33) and aspirin (34), normalize intestinal cell proliferation
and restore the enterocyte migration rate to the level of wildtype animals.
In order to evaluate cell proliferation in Min/⫹ mice treated
with CAPE and curcumin, the relative number of cells showing
a nuclear localization of PCNA was measured by standard
immunohistochemistry. PCNA is a cytoplasmic protein that
translocates to the nucleus during S phase of the cell cycle
and is therefore a proliferation marker. Histologically normal
small intestinal specimens from CAPE- and curcumin-treated
animals were stained with an anti-PCNA antibody. The tissue
specimens were compared with similarly stained sections of the
untreated control Min/⫹ mice and their wild-type littermates.
Figure 3 shows that positive PCNA staining was decreased in
Min/⫹ negative control animals when compared with their
wild-type littermates, as expected from previous observations
(34,37,38). Treatment with curcumin completely normalized
enterocyte proliferation (Figure 3A), whereas treatment with
a tumor-suppressing dose of CAPE increased enterocyte proliferation to 88% of the control level (Figure 3B). The finding
that both CAPE and curcumin increased apoptosis, as well as
cell proliferation, is consistent with data obtained following
effective tumor prevention in Min/⫹ mice with non-steroidal
anti-inflammatory drugs (NSAIDs) (34,37,38). These data also
suggest that curcumin and CAPE, like the NSAIDs, increase
the turnover rate of enterocytes in Min/⫹ mice.
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N.N.Mahmoud et al.
Fig. 3. CAPE increases enterocyte proliferation. Specimens of small
intestine from animals at 110 days of age were formalin fixed, embedded in
paraffin and sectioned at 5 µm. Where indicated, animals were treated with
(A) curcumin (0.10%) or (B) CAPE (0.15%) as described in Materials and
methods. Sections of small intestine were stained with antibody to PCNA.
Values expressed are percent of total cells positive ⫾ SEM, with 100%
equal to the entire crypt–villus population. For the control groups (⫹/⫹ and
Min/⫹), number of animals ⫽ 20; for the treatment groups, number of
animals ⫽ 10 each. (A) *P ⬍ 0.0001 compared with ⫹/⫹, **P ⬍ 0.0001
compared with Min/⫹; (B) *P ⬍ 0.0001 compared with ⫹/⫹, **P ⬍
0.0001 compared with Min/⫹.
CAPE and curcumin lower enterocyte β-catenin levels in
Min/⫹ mice
The intestinal mucosa of Min/⫹ mice shows an increased
level of β-catenin expression when compared with tissue from
wild-type animals (37,38). Previous investigations found that
effective tumor inhibition correlated with a decrease in tissue
β-catenin expression by immunohistochemistry. To characterize the in vivo effects of the plant phenolics with regard to
this tumor-associated oncoprotein, we measured β-catenin
expression in the intestinal mucosa of Min/⫹ mice treated
with curcumin and CAPE.
Tissue sections from the mid small intestine 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/⫹ animals exhibited a 2-fold
increase in staining with anti-β-catenin antibody when compared with ⫹/⫹ controls (Figure 4). β-Catenin levels were
unchanged in animals treated with 0.03% CAPE (data not
shown). As shown in Figure 5, a tumor-preventing dose of
curcumin lowered tissue β-catenin levels 20-fold (Figure 5A),
whereas treatment of the animals with a diet containing 0.15%
CAPE reduced intracellular β-catenin expression to a level
equal that of wild-type mice (Figure 5B).
Discussion
These studies confirm a role for orally administered CAPE or
curcumin in prevention of spontaneous tumors resulting from
a germline Apc mutation. The data also suggest that CAPE or
curcumin treatment can reverse the aberrant accumulation of
β-catenin and the alterations in enterocyte growth that are
characteristic of deficient APC function. Although they exhibit
chemopreventive activity in vitro (40,41) and in azoxymethaneinduced rat intestinal tumor models (29,30), quercetin and
rutin did not reduce the incidence of intestinal tumors at the
doses used in this study. The difference in chemical structure
between quercetin and rutin, on the one hand, and curcumin
and CAPE, on the other, may account for the difference in
antitumor effect. The lack of efficacy of these compounds in
our model may also be due to a bioavailability problem.
Quercetin and rutin are poorly absorbed from the gastrointestinal tract, although ingestion of a 0.5–1.0% diet of either
924
compound in rats results in detectable serum levels (36). We
chose a 2% diet for this study because this amount is nontoxic and was used in previous studies of colon tumor inhibition
in mice (29,30). Moreover, this dose has been shown to be
more than sufficient to alter intestinal oxidative status following
oral feeding in rats (9). Species-specific bioavailability differences may account for the lack of effect and it is possible that
higher dietary doses of quercetin or rutin may produce an
antitumor response in Min/⫹ mice. Doses exceeding 2%,
however, would be impractical for further testing in humans.
For the plant phenolics, antitumor effects have been variously
characterized in cell culture systems. By altering the cellular
redox status, CAPE and curcumin modulate several critical
growth-regulating signaling pathways (42,43). For example,
in tumor cell lines, treatment with CAPE or curcumin inhibited
receptor tyrosine kinases (44–46) and blocked the DNA binding
of transcription regulators, such as NF-κB (47,48) and AP-1
(49). Recently, we showed that CAPE abrogates the ability of
p53 to effect sequence-specific DNA binding, a result that
correlated with an alteration in the conformation of p53 protein
and with the induction of p53-dependent apoptosis of colon
carcinoma cells (39).
A possible mechanism of CAPE- or curcumin-induced tumor
suppression involves their antioxidant properties (14,50–53).
A substantial body of data suggests that reactive oxygen
species (ROS) are associated with tumor promotion. ROS and
oxidant defense enzyme activities have been detected in human
normal mucosal biopsies, tumor tissue and colon carcinoma
cells (54,55), as well as in rat azoxymethane-induced colonic
tumors (56). These studies indicate that, compared with nontumor tissues, both colonic tumors in vivo and cultured human
tumor cells maintain higher levels of ROS. These reactive
species are thought to act as second messengers for signal
transduction pathways that regulate cell proliferation (57).
Thus, by reducing intracellular peroxides, antioxidants are
expected to inhibit carcinogenesis.
Compounds that prevent tumors in Apc-deficient animals,
such as sulindac, aspirin, NS-398, SC58635, curcumin and
CAPE, all reduce cyclooxygenase (COX)-2 expression, prostaglandin production and proliferation of cultured colorectal
carcinoma cells (58–60). In HCA-7 cells, a colorectal cancer
cell line that constitutively overexpresses COX-1 and COX-2,
treatment with antioxidants induced G1 growth arrest or
apoptosis (58). Endogenous levels of H2O2 in these cells were
decreased ~4-fold after a 24 h exposure to the antioxidants
(31). Moreover, CAPE- and curcumin-mediated suppression
of proliferation and prostaglandin production was associated
with a reduction in COX2 gene transcription. It is presently
unclear whether the effect on cell growth is directly due to
the inhibition of COX-2 activity, or whether it is a secondary
consequence of decreased prostaglandin synthesis (58–60).
One of the beneficial effects of chemopreventive agents upon
the preneoplastic intestinal mucosa appears to be regulation of
enterocyte growth. Most, if not all, of the effective chemopreventive agents induce apoptosis of colon cancer cells in vitro
(12,61,62) and of the rectal mucosa in human subjects (63).
In the Min/⫹ mouse, tumor-inhibiting compounds produce
characteristic changes in intestinal cell apoptosis, proliferation
and migration (37,38). The present study shows that CAPE
and curcumin increased apoptosis in the enterocytes of Min/⫹
mice as measured by TUNEL, a result similar to that found
previously for sulindac and aspirin (37,38). Tumor-preventing
doses of these plant phenolics also normalized the decreased
Plant phenolics prevent Apc-associated tumors
Fig. 4. β-Catenin expression in Min/⫹ small intestine. Specimens of small intestine from animals at 110 days of age were formalin fixed, embedded in
paraffin and sectioned at 5 µm. Where indicated, animals were treated with curcumin (0.10%) or CAPE (0.15%) as described in Materials and methods.
Sections were stained with antibody to β-catenin. The level of β-catenin staining in the middle third of each crypt–villus unit was measured using the CAS
2000. β-Catenin levels were elevated in Min/⫹ mice when compared with the wild-type controls (⫹/⫹) and decreased by both curcumin and CAPE. All
fields are 400⫻ magnification.
Fig. 5. CAPE lowers enterocyte β-catenin as measured by
immunohistochemistry. Specimens of small intestine from animals at 110
days of age were formalin fixed, embedded in paraffin and sectioned at 5
µm. Where indicated, animals were treated with (A) curcumin (0.10%) or
(B) CAPE (0.15%) 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 blind to the animal’s
genetic or treatment status using the CAS 200 and CAS 200 Quantitative
Analysis Software. Values represented are means ⫾ SEM where n ⫽ 20 for
all treatment groups. (A) *P ⬍ 0.0001 compared with ⫹/⫹, **P ⬍ 0.0001
compared with Min/⫹; (B) *P ⬍ 0.0001 compared with ⫹/⫹, **P ⬍
0.0001 compared with Min/⫹.
proliferation observed in the intestinal mucosa of Min/⫹ mice.
These effects occurred before the development of tumors and
are therefore promising early markers of chemoprevention
efficacy.
Wild-type APC protein associates with GSK-3β kinase and
axin to negatively regulate cytoplasmic levels of free β-catenin
(64). The complex of these three factors binds to β-catenin,
an intracellular protein associated with the actin cytoskeleton,
and facilitate its degradation (65,66). The precise role of β-
catenin in colorectal carcinogenesis is still not entirely clear.
Among other functions, β-catenin is a component of the
adherens junction of intestinal epithelial cells and may therefore
play an important role in intercellular communication and cell
migration (67,68). Recent studies indicate that growth-factorinduced tyrosine phosphorylation of β-catenin together with
its membrane-associated binding partners destabilizes cell–cell
adhesion (69,70). Furthermore, excess β-catenin promotes
accumulation of transcriptionally active p53 that may favor
cell survival rather than apoptosis of enterocytes in Min/⫹
mice (71). Since chemopreventive agents, including CAPE
and curcumin, inhibit receptor tyrosine kinase activity (44–
46), they may also stabilize cell–cell adhesion while inhibiting
cell migration and/or adhesion. Thus, the reduced staining for
β-catenin in the muscosa of Min/⫹ mice treated with these
compounds (Figure 4) may reflect the redistribution of βcatenin from membrane to cytoplasmic pools, if not an actual
lowering of its total concentration.
In conclusion, a significant body of experimental and epidemiological data indicate that NSAIDs such as sulindac and
aspirin reduce the incidence of human colorectal cancer (72).
The studies presented here suggest that CAPE and curcumin
produce similar chemopreventive effects in vivo. To further
develop and implement colon cancer chemoprevention strategies, additional studies are needed to determine whether these
compounds are safe and effective for long-term administration
to humans. Also, studies to characterize the operative molecular
mechanisms of tumor prevention by these compounds should
suggest whether combinations of these agents can achieve
increased antitumor efficacy.
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N.N.Mahmoud et al.
Acknowledgements
Special thanks are due to Dr Robert Goldman for providing the CAPE used
in these studies. This work was supported by American Cancer Society grant
ACS CDA-95010-95, National Cancer Institute grant NCI-1R29CA74162-01
and the Alice Bohmfalk Charitable Trust (M.M.B.), National Institute of
Health NIH Surgical Oncology Research Training Grant 525435 (N.N.M.),
National Cancer Institute grant 5R01CA67944 (R.K.), the Cancer Research
Foundation of America (M.M.B. and N.N.M.) and the American Society of
Colon and Rectal Surgeons (N.N.M.).
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Received June 29, 1999; revised October 13, 1999; accepted November 8, 1999
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