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
1 The New York HospitalCornell Medical Center, 525 East 68th Street, New York, NY 10021,
2 College of Physicians and Surgeons, ColumbiaPresbyterian Cancer Center and School of Public Health, Columbia University, New York, NY 10032,
3 The Strang Cancer Prevention Center, 428 East 72nd Street, New York, NY 10021 and
4 Rutgers University Laboratory for Cancer Research, 164 Frelinghuysen Road, Piscataway, NY 08854, USA
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Abstract
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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 ß-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.
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 anti-inflammatory drugs; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; ROS, reactive oxygen species.
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Introduction
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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 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 carcinogenDNA 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 (810). 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 (7,1113).
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 (1520) and in vivo inhibitors of tumor initiation and promotion in different carcinogen-induced rodent models (14,2126).

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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.
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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 (25,26,2830).
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 adenomacarcinoma 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 (3335).
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 ß-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.
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Materials and methods
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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 3x 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-DeTM (Fisher Scientific, Pittsburgh, PA) and an alcohol series, followed by washing in phosphate-buffered saline (PBS), pH 7.0.
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 ApopTagTM Kit (Oncor, Gaithersburg, MD) as described previously (33). Mouse lymphoid tissue with a known 23% 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 blind to the animal's treatment group and genetic status. The percent staining of enterocytes in these cryptvillus 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 cryptvillus 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 cryptvillus units was measured using the CAS 200.
Determination of tissue ß-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 ß-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 ß-catenin staining in the middle third of each cryptvillus was quantified using CAS 2000 with CAS 200 Quantitative Analysis Software.
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Results
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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 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 cryptvillus 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.

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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 cryptvillus 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.
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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 wild-type 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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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 cryptvillus 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/+.
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CAPE and curcumin lower enterocyte ß-catenin levels in Min/+ mice
The intestinal mucosa of Min/+ mice shows an increased level of ß-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 ß-catenin expression by immunohistochemistry. To characterize the in vivo effects of the plant phenolics with regard to this tumor-associated oncoprotein, we measured ß-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 ß-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-ß-catenin antibody when compared with +/+ controls (Figure 4
). ß-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 ß-catenin levels 20-fold (Figure 5A
), whereas treatment of the animals with a diet containing 0.15% CAPE reduced intracellular ß-catenin expression to a level equal that of wild-type mice (Figure 5B
).

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Fig. 4. ß-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 ß-catenin. The level of ß-catenin staining in the middle third of each cryptvillus unit was measured using the CAS 2000. ß-Catenin levels were elevated in Min/+ mice when compared with the wild-type controls (+/+) and decreased by both curcumin and CAPE. All fields are 400x magnification.
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Fig. 5. CAPE lowers enterocyte ß-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 ß-catenin. The percent staining of enterocytes in these cryptvillus 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/+.
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Discussion
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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 ß-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 azoxymethane-induced 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 0.51.0% diet of either compound in rats results in detectable serum levels (36). We chose a 2% diet for this study because this amount is non-toxic 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 (4446) and blocked the DNA binding of transcription regulators, such as NF-
B (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 (14,5053). 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 non-tumor 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 (5860). 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 (5860).
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 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-3ß kinase and axin to negatively regulate cytoplasmic levels of free ß-catenin (64). The complex of these three factors binds to ß-catenin, an intracellular protein associated with the actin cytoskeleton, and facilitate its degradation (65,66). The precise role of ß-catenin in colorectal carcinogenesis is still not entirely clear. Among other functions, ß-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-factor-induced tyrosine phosphorylation of ß-catenin together with its membrane-associated binding partners destabilizes cellcell adhesion (69,70). Furthermore, excess ß-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 (4446), they may also stabilize cellcell adhesion while inhibiting cell migration and/or adhesion. Thus, the reduced staining for ß-catenin in the muscosa of Min/+ mice treated with these compounds (Figure 4
) may reflect the redistribution of ß-catenin 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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Acknowledgments
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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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Notes
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5 To whom correspondence should be addressed Email: mbertagnolli{at}partners.org 
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Received June 29, 1999;
revised October 13, 1999;
accepted November 8, 1999.