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Carcinogenesis Advance Access originally published online on July 17, 2007
Carcinogenesis 2007 28(9):1978-1984; doi:10.1093/carcin/bgm161
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Green tea selectively targets initial stages of intestinal carcinogenesis in the AOM-ApcMin mouse model

Ala Y. Issa, Suresh R. Volate, Stephanie J. Muga1, Daniela Nitcheva2, Theresa Smith3 and Michael J. Wargovich1,*

Department of Pathology, Microbiology and Immunology, School of Medicine, University of South Carolina, Columbia, SC 29203, USA
1 Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, SC 29245, USA
2 Department of Epidemiology and Biostatistics, School of Public Health
3 Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of Columbia, SC 29208, USA

* To whom correspondence should be addressed at Cancer Chemoprevention Program, Hollings Cancer Center, 86 Jonathan Lucas Street, PO Box 250955, Charleston, SC 29245, USA. Tel: +843 792 7604; Fax: +843 792 3200; Email: wargovic{at}musc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
One of the liabilities of the ApcMin mouse as a model for colon cancer is its lack of a robust tumor response in the large bowel. In our protocol, we treated the ApcMin mouse with azoxymethane, a colon-selective carcinogen. This protocol induced a 4-fold increase in the number of colon tumors. We utilized this protocol to investigate the possible mechanisms of inhibition of colorectal carcinogenesis by green tea. Mice received water or a 0.6% (w/v) solution of green tea as the only source of beverage. Green tea treatment commenced at the eighth week of age and lasted for either 4 or 8 weeks. Green tea significantly inhibited the formation of new adenomas, but was ineffective against larger tumors. Mechanistically, we investigated the effects of green tea on the expression of biomarkers involved in colon carcinogenesis. Western blotting analysis showed that green tea decreased the total levels of the early carcinogenesis biomarker ß-catenin and its downstream target cyclin D1. In contrast, the expression of COX-2 was not altered. Immunohistochemical analysis showed that green tea inhibited the formation of adenomas overexpressing ß-catenin and cyclin D1, but did not reduce the number of COX-2-expressing adenomas. Our results suggest that green tea specifically targets initial stages of colon carcinogenesis; the time of administration of green tea is pivotal for effective chemoprevention. Beverage levels of green tea do not inhibit the progress of any large adenomas or adenocarcinomas existing prior to the tea administration.

Abbreviations: AOM, azoxymethane; EGCG, (–)-epigallocatechin gallate


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Green tea is one of the most common beverages worldwide. The primary active ingredients in green tea are a group of flavan-3-ol polyphenols known as catechins. The major tea catechins are (–)-epigallocatechin gallate (EGCG), (–)-epigallocatechin, (–)-epicatechin gallate and (–)-epicatechin with EGCG comprising >60% of the total catechins (1). In brewed green tea, the water-extractable material accounts for about one-third of the tea leaves in dry weight and contains ~30 to 40% catechins, 3% flavonols (quercetin, kamepferol and rutin), 3–6% caffeine and a mixture of other constituents (2). Tea catechins, particularly EGCG, have been shown to posses a variety of pharmacological effects. These include antioxidant, anticarcinogenic, anti-inflammatory, antimicrobial, antiangiogenic and hypocholesterolemic effects (3–8). In addition, a wealth of epidemiological data suggested an inverse correlation between tea consumption and the risk of cancer (9). Inhibition of colon carcinogenesis by green tea catechins has been extensively investigated (9–15). Colon carcinogenesis is a promising target for dietary intervention since polyphenols such as tea catechins can reach concentrations that far exceed their concentrations in other organs of the body. For colon cancer, the cancer chemopreventive activity of green tea has been attributed mostly to its EGCG component. Several mechanisms of action for EGCG have been reported in a variety of in vivo and in vitro studies (5,16–21). These include antioxidant properties, modulation of cell signaling pathways, modulation of gene expression and modulation of carcinogen metabolism. The overwhelming majority of these studies, however, used EGCG concentrations that were several fold higher that what could be achieved physiologically, even in organs that retain higher levels of catechins such as the colon and small intestine (22). In addition, the contribution of the other tea catechins to the biological activities of green tea was often not investigated. Further controversy rose due to a contradictory epidemiological evidence, the lack of an animal model that genetically and phenotypically truly represent the human colon cancer and variations in the methodology of the published studies (22). Consequently, the exact mechanisms through which physiological levels of green tea might inhibit colon carcinogenesis are still poorly understood.

We undertook the current study in order to investigate the possible chemopreventive effects of a low physiological concentration of green tea in vivo. The objective was to study the effects of green tea on pre-existing tumors and new tumors that would develop during the tea treatment. To achieve this aim, we needed to modify the ApcMin mouse model by introducing a colon-specific carcinogen to maximize tumor yield. Although the majority of tumors still developed in the small intestine, we were able to gain a 4-fold increase in colon tumors thus making this a more attractive model for colon cancer chemoprevention studies. Our results show that green tea significantly reduced the development of new tumors but did not inhibit the pre-existing ones. The data suggest that green tea selectively targets initial stages of colon carcinogenesis.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Animals and treatments
A total of 150 mice were used in the study; 120 male and female C57BL/6J-ApcMin (ApcMin) mice and 30 wild-type C57BL/6J (B6). The mice were housed in an animal research facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) at the University of South Carolina. Treatment protocol was approved by the Institutional Animal Care and Use Committee at the University of South Carolina. The mice were kept on a light/dark (12/12 h) cycle, 20–24°C and 50% humidity. Mice were weaned at 3 weeks of age and fed AIN76A diet ad libitum thereafter. The mice were split into three groups based on the type of analysis conducted on each group (Figure 1A). The groups were further divided into five equal subgroups based on the green tea or azoxymethane (AOM) treatments (Figure 1A). AOM (purchased from Ash Stevens, Detroit, MI) was diluted to a final concentration of 8 mg/kg body wt with 0.9% saline solution on the day of injection and administered to the mice intraperitoneally once a week for 3 weeks (Figure 1B). Green tea was well characterized (Table I) and a generous gift from Dr C.S.Yang (Rutgers University, NJ). The green tea stock was received as a green tea powder and was analyzed by high-performance liquid chromatography (Table I) to determine its composition. The same stock was used throughout the experiment to avoid any variations in the green tea content. A fresh solution of 0.6% (w/v) was prepared every other day by dissolving the green tea powder in the proper volume of ultrapure hot water. The powder completely dissolved in the hot water and no filtration was necessary. The tea solution was then set to cool to room temperature in the dark, poured into light-protective bottles and served to the mice as the only source of beverage. The tea treatment commenced 1 week after the last AOM or saline injection and lasted for 4–8 weeks. All the mice were killed at 16 weeks of age by cervical dislocation except for the group dedicated to the immunohistochemical analyses. The mice in the latter group were killed after 12 weeks (4 weeks of green tea treatment) in order to study the effects of green tea on earlier neoplastic lesions. The mice were closely monitored and weighed weekly. Any mouse that lost >10% of its original body weight was excluded from the experiment.


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  		Table I. High-performance liquid chromatography analysis of green tea polyphenols

 


Figure 1
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  		Fig. 1. Experimental design of the study. (A) Male and female mice were categorized into three groups based on the type of analysis intended for each group as indicated by the schematic. Mice [C57BL/6J-ApcMin (ApcMin) and C57BL/6J (B6)] were randomized into the appropriate subgroups groups as shown in the table. IHC, immunohistochemical analysis. (B) Treatment protocol. Mice in Groups I–V were treated with AOM or 0.9% saline followed by 0.6% (w/v) green tea or water as indicated in the schematic.

 
Tumor count and size comparison
Colons and small intestines were removed, flushed with ice-cold phosphate-buffered saline, slit open along the longitudinal median and fixed flat in 10% buffered formalin for 24 h. The fixed tissues were stained with 0.2% methylene blue (Sigma-Aldrich, St. Louis, MO) dissolved in phosphate-buffered saline. Tumors were scored at x30 magnification using a Nikon dissecting microscope with a fiber optic light source to illuminate the tissues and a calibration scale to determine the tumor size. Tumors with diameters ≤1 mm were classified as small tumors, whereas tumors that exceeded 1 mm in diameter were classified as large tumors. All the tumors were scored by the same investigator who was blind to the treatment groups. No tumors were found in any of the B6 mice. A minimum of a 100 different colon and small intestinal tumors were randomly sampled and further analyzed to determine the histological type.

Immunohistochemical analysis
The colons and small intestines were fixed as described above, but Swiss-rolled before fixation. Fixed tissues were paraffin embedded, cut into 5 µm sections, mounted on slides and processed for immunohistochemistry as described previously (23). Sections were incubated for 45 min with one of the following primary antibodies: Mouse monoclonal anti-ß-catenin antibody (BD Transduction Laboratories, Lexington, KY) diluted 1:300; Rabbit monoclonal anti-cyclin D1 (SP4) antibody (Lab Vision/Neomarkers, Fremont, CA) diluted 1:100 and Mouse polyclonal anti-COX-2 antibody (Caymen Chemical, Ann Arbor, MI) diluted 1:400. Blocking of the sections and detection of the anti-ß-catenin and anti-cyclin D1 antibodies were by ACUITY polymer Detection Kit, which consisted of a special polymer for pre- and post-primary antibody incubation (Signet Laboratories, Dedham, MA) according to the manufacturer's instructions. Detection of the anti-COX-2 antibody was by CSA IIBiotin-Free Catalyzed Signal Amplification System (DakoCytomation, Carpinteria, CA) according to the manufacturer's instructions. Non-specific binding was blocked by incubating the sections with normal goat serum (BioGenex, San Ramon, CA) for 20 min at room temperature. Quantitation of the staining was carried out by dividing the lesions with abnormal staining into three categories (Table II). Small lesions consisted of 1–5 crypts. Medium lesions consisted of 6–10 crypts. Large lesions consisted of >10 crypts. Epithelial cells in these lesions showed overexpression of ß-catenin and cyclin D1. In contrast, COX-2 was only expressed in stromal cells of medium and large lesions and it was not expressed in the epithelial cells. Therefore, quantitation of the COX-2 staining was carried out by counting the number of lesions expressing COX-2 without further classification. All the lesions were scored at x100 magnification by the same investigator who was blind to the treatment groups. The entire small intestines and colons were scored for lesions in each mouse and the average number of each class of lesions per animal was determined.


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  		Table II. Classification of immunohistochemical lesions

 
Western blotting
Mucosal layers of colons and small intestines from 50 mice (10 per subgroup as described above) were scraped and flash frozen in liquid nitrogen immediately after killing and then stored at –80°C. The scrapings were later thawed on ice, total proteins were isolated and their concentrations were determined as described previously (24). Western blotting analysis was carried out as described previously (24), with 50 µg of total proteins loaded into each lane. The primary antibodies for ß-catenin, cyclin D1 and COX-2 were the same as in the previous section, but each was used at a dilution of 1:500. The blots were visualized by incubating the polyvinylidene difluoride membranes with the ECL plus kit (Amersham Biosciences, Piscataway, NJ), according to the manufacturer's instructions; blots were scanned with a StormTM 860 gel and blot imaging system (Amersham Biosciences). Densitometry analysis and quantitation of the antibody-associated protein bands were performed using Quantity OneTM software (Bio-Rad, Hercules, CA). At least three mice per subgroup were analyzed. The data are normalized to the ß-actin loading control. Error bars represent standard error of the mean.

Statistical analysis
All the data were analyzed using Sigmastat V3.0 (SPSS, Chicago, IL) and SAS V8.2e (SAS Institute, Cary, NC) softwares. Descriptive statistics were used to identify the distribution of the data. A two-way analysis of variance test was used to compare the treatment groups in the presence or absence of AOM or green tea. For data that failed the normality test, generalized linear models were used to compare the groups. In this case, the data were modeled after a Poisson distribution when appropriate. Alternatively, a negative binomial distribution model was followed when the data exhibited an overdispersion. The analyses included testing for the main effects of AOM or green tea in the treatment groups and for interactions between the treatments. The data were considered very significant if P < 0.005, significant if P < 0.05, marginally significant if 0.05 ≤ P < 0.1 and insignificant if P ≥ 0.1.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
AOM treatment significantly and selectively augments colon tumorigenesis in the ApcMin mouse
The first objective of the current study was to increase the number of colon tumors in the ApcMin mouse model using a colon-selective carcinogen. Different doses and treatment protocols of AOM injection were tested. An AOM dose of 8 mg/kg body wt, injected intraperitoneally once a week for 3 weeks, was sufficient to significantly induce colon tumors and yet was well tolerated by the mice. All the tumors in the small intestine and colon were scored for each mouse after fixing the tissues and staining with methylene blue as described in the Materials and methods (Figure 2A). A 4-fold increase in the number of colon tumors (0.5–2 colon tumors per mouse, P < 0.005) was observed with the AOM treatment compared with saline-injected mice (Figure 2B). The induction of tumors by AOM was selective to the colon as reflected by only a slight induction (an 18% increase, P > 0.1) of the total number of ApcMin tumors (tumors in the colons and the small intestines) per mouse. AOM significantly increased the numbers of both small and large tumors in the colon.


Figure 2
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  		Fig. 2. (A) Representative ApcMin colon (left) and small intestine (right) tumors. Tumors were fixed with 10% buffered formalin for 24 h and stained with 0.2% methylene blue and scored at a magnification of x30. Tumors with a diameter of ≤1 mm were classified as small tumors whereas those with diameters >1 mm were classified as large tumors. (B) AOM induced the formation of colon tumors. AOM treatment (20 mice scored) induced a statistically significant 4-fold increase in colon tumors (from 0.5 to 2 colon tumors per mouse, **P < 0.005) compared with saline-injected mice (20 mice scored). The induction of tumors by AOM was selective to the colon. Error bars represent standard error of the mean.

 
Green tea inhibits the formation of ApcMin tumors and selectively targets small tumors
Green tea treatment (20 mice scored, 10 per treatment group) induced a 50% reduction in colon tumors (1.8–0.9 tumors per mouse, P < 0.05) compared with water-treated mice (20 mice scored). Green tea treatment caused ~20% reduction in the total number of ApcMin tumors evolving in both the colon and small intestine (41–33 tumors per mouse, P < 0.1) (Figure 3A). The inhibition of colon tumor development by green tea was mainly due to a reduction in the number of small tumors with only slight effect on large tumors (0.7–0.1 tumors per mouse, P < 0.05 and 1.1–0.8, P = 0.2, respectively) (Figure 3B). Likewise, green tea induced a statistically significant reduction in the number of total ApcMin small tumors in the colons and small intestines with little effects on the large ones (17.3–12.5 tumors per mouse, P < 0.05, and 23.4–20.8, P = 0.4, respectively) (Figure 3C).


Figure 3
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  		Fig. 3. (A) Green tea caused a statistically significant reduction in colon tumors. Green tea solution of 0.6% was effective at inhibiting AOM-induced colon tumors in the ApcMin mice. Tea also inhibited the total number of tumors by >20%. Error bars represent standard error of the mean. *P < 0.05. (B) The reduction of colon tumors by green tea was mainly due to reduction in the number of small tumors. The inhibitory effect of green tea was primarily due to reduction in the number of small tumors (≤1 mm). The data indicate that tea was ineffective against large tumors. (C) The reduction in the number of total ApcMin tumors by green tea was mainly due to reduction in the small tumors. In agreement with the colon tumor data, green tea was most effective at inhibiting the formation of small tumors in the ApcMin mice. Large tumors were resistant to the tea treatment.

 
Green tea selectively targets earlier stages of ApcMin intestinal carcinogenesis
Immunohistochemical staining of ß-catenin, cyclin D1 and COX-2 showed a selective inhibition of small lesions by green tea (Figure 4). All the mice in this group were killed after 12 weeks of age in order to study the effects of green tea on the development of early lesions in ApcMin intestinal carcinogenesis. These mice received green tea treatment for only 4 weeks. The aim of the immunohistochemical analysis was to confirm whether or not the inhibitory effects of green tea on ApcMin tumors could be explained by any modulation of biomarkers involved in intestinal carcinogenesis. Lesions were scored as described in Table II. Medium lesions showed a pattern that was indistinguishable from large lesions; therefore, the analysis for large lesions is also representative of the medium lesions. Green tea (20 mice scored) caused a statistically significant reduction in the number of small lesions overexpressing ß-catenin, but no inhibition of large lesions (8.1–5.5 small lesions per mouse, P < 0.05, and 1.1–1.3 large lesions, P > 1.0, respectively) compared with water-treated mice (20 mice scored) (Figure 5A). In correlation with ß-catenin overexpression, only the number of small lesions overexpressing cyclin D1 was significantly reduced by the green tea treatment compared with water (8.3–5.1 small lesions per mouse, P < 0.05, and 2.3–2.6 large lesions, P > 1.0, respectively) (Figure 5B). Green tea at a 0.6% (w/v) concentration was ineffective in reducing the number of lesions expressing COX-2 (4.4–3.9 lesions per mouse with water or green tea treatment, respectively) (Figure 5C). The expression of COX-2 was directly proportional to the size of the adenoma; i.e. the larger the adenoma the more the COX-2 expression. Small lesions (1–5 crypts) were virtually void of COX-2 expression. Western blotting analysis confirmed the immunohistochemical data (Figure 6). Green tea caused statistically significant reduction in the total protein levels of ß-catenin and cyclin D1 in ApcMin mice injected with AOM (P < 0.05). The protective effects of green tea were more evident in the AOM-treated ApcMin mice compared with saline-injected mice, as reflected by a greater difference in the ß-catenin and cyclin D1 levels between mice injected with AOM and received green tea compared with mice that received water. Green tea treatment had only a modest effect on altering COX-2 levels in saline-treated mice given only water. However, green tea supplementation in AOM-treated mice exhibited a statistically significant increase in COX-2 protein levels as compared with saline-treated mice that received green tea.


Figure 4
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  		Fig. 4. (A) Immunohistochemical staining of ß-catenin-overexpressing lesions. (1) ß-catenin is overexpressed in a small lesion with two crypts (refer to Table II for classification of lesions). (2) A medium-sized lesion with <10 crypts overexpressing ß-catenin. (3) ß-catenin overexpression in a large lesion with >10 crypts. (4) A large colon adenoma with extensive nuclear localization of ß-catenin. (B) Immunohistochemical staining of cyclin D1- and COX-2-overexpressing lesions. The expression of cyclin D1 correlated with the overexpression of ß-catenin (1) and (2). Black arrow points to cyclin D1 overexpression in a large lesion adjacent to normal looking epithelia. Cyclin D1 protein was detected in small, medium and large lesions; green tea treatment reduced expression of cyclin D1 protein in small lesions only. COX-2 was predominantly expressed in large adenomas (3) and (4); the black arrows depict COX-2 staining in the stromal rather than epithelial cells. Small lesions were almost void of COX-2 expression.

 


Figure 5
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  		Fig. 5. (A) Quantitative analysis of immunohistochemical staining of ß-catenin in ApcMin mice. All the mice were killed after 12 weeks of age. Lesions were scored based on definitions in Table II. All the lesions were scored at a x100 magnification. Error bars represent standard error of the mean. (B) Quantitative analysis of immunohistochemical staining of cyclin D1 in ApcMin mice. (C) Quantitative analysis of the immunohistochemical staining of COX-2 in ApcMin mice. Green tea had very modest effect on COX-2 expression (4.4–3.9 adenomas per mouse) compared with water-treated mice. The expression of COX-2 was directly proportional to the size of the adenoma; the larger the adenoma the more the COX-2 expression.

 


Figure 6
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  		Fig. 6. (A) Representative western blots of selected biomarkers of intestinal carcinogenesis in the ApcMin mice. Representative western blots of ß-catenin and its downstream target cyclin D1 and COX-2. Each lane contains 50 µg total protein. (B) Western blotting analysis of ß-catenin, cyclin D1 and COX-2 levels in ApcMin mice treated with green tea. Densitometric analyses of these protein western blots show a significant reduction in the total protein levels of ß-catenin and cyclin D1 in AOM- plus green tea-treated mice compared with AOM plus water. The analyses were performed using Bio-Rad imager and Quantity OneTM software. The protein levels were normalized to ß-actin. The figure shows relative levels of ß-catenin or cyclin D1 compared with saline plus green tea group, which was assigned a value of 100%. Error bars represent standard error of the mean. Wild-type B6 mice had no tumors and therefore no detectable levels of COX-2. The saline plus green tea group was assigned a value of 100% and the COX-2 protein levels are relative to that group. AOM-treated mice had a significant increase in COX-2 levels compared with the saline groups.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
A wealth of epidemiological and experimental studies have suggested an inverse correlation between green tea consumption and cancer (9,25–27). Paradoxically, a vast number of epidemiological studies did not find any benefit or negative association between green tea consumption and the risk for cancer (28,29). The overwhelming majority of experimental reports deviate from the epidemiological evidence by studying selected components in the green tea at concentrations far exceeding what could be achieved physiologically in humans (22,30). Further, most of the mechanistic studies were conducted in cancer cell lines in which the cells were in direct contact with the tea catechins (30). In light of these results, the official position of the Food and Drug Administration between green tea and cancer is that there is simply not enough compelling evidence to support health claims made by those in the tea industry (31). The current study investigates the possible cancer-protective mechanisms of green tea in the ApcMin mouse model. The ApcMin mouse model has been a useful model for studying various aspects of colon cancer, but it does have some limitations (32–35). The most important of these is the fact that the majority of tumors develop predominantly in the small intestine with often very few or no tumors in the colon. We and others (36) have optimized a protocol to increase the number of colon tumors by injecting the ApcMin mice with the colon-selective carcinogen AOM, thus applying a carcinogen known to influence mutations in the Apc allele. Our protocol provides a fast and convenient approach to induce the colon tumors. We used this improved model to study if and how physiological concentrations of green tea might inhibit colon carcinogenesis. We chose a concentration of 0.6% of green tea, since it provides similar concentrations of tea catechins to their concentrations in the typical green tea beverage. For example, a 250-ml cup of the 0.6% solution has ~180 mg of EGCG, calculated according to green tea composition in Table I, compared with ~150 mg in the typical tea beverage. We chose to start the green tea treatment after the mice completed 8 weeks of age. ApcMin mice develop tumors as early as the second or third week of age and by the end of the eighth week have definable tumors. The objective was to study the effects of green tea on pre-existing tumors and new tumors that would develop during the period of tea treatment. We (in Materials and methods) chose a diameter of 1 mm as a borderline between small and large tumors. All the ApcMin mice in the ‘tumor analysis and size comparison’ group were killed after 16 weeks of age. The majority of large tumors in the colons or small intestines of these mice had diameters exceeding 4 mm. Interestingly, large colon tumors were often larger than the small intestinal large tumors and had average diameters >5 mm. We have conducted a series of experiments correlating tumor size with time (data not shown). Based on these experiments, we have established that it is unlikely for an ApcMin tumor to grow to >4 mm in diameter within 8 weeks in the absence of any exogenous stimulatory factors. On the other hand, it is probably that the ApcMin tumors will grow to >1 mm in diameter in a period of 8 weeks, in the absence of any interfering factors. Therefore, large tumors probably existed in the ApcMin mice before the green tea treatment started and continued to grow during the treatment. Small tumors, on the other hand, could be those that developed after starting the tea treatment, existing tumors whose growth was halted after the start of green tea treatment, tumors whose size was reduced by the green tea treatment or a mixture of these tumors. We have randomly collected >100 tumors of varying sizes from ApcMin mice in the treatment groups and further classified them histologically. All the analyzed tumors were adenomas. Tumors of similar sizes were very similar morphologically and showed the same patterns of staining when stained for ß-catenin, cyclin D1 and COX-2. We have no reason to believe that ApcMin tumors of equal sizes do not behave similarly or that green tea is differentially targeting some tumors but not the others. On the other hand, if green tea halted the growth of tumors, we should see little or no increase in the number of large tumors in ApcMin mice killed after 4 or 8 weeks of green tea treatment. Indeed, we found a significant increase in the number of large tumors in mice killed after 12 weeks and after 16 weeks of age (1.3 large adenoma per mouse and 12.5, respectively). Therefore, it is probably that small tumors with diameters ≤1 mm were tumors that developed during the green tea treatment and any reduction in their numbers was due to prevention of the development of new adenomas by green tea. The number of small tumors was significantly reduced in colons and small intestines of green tea-treated ApcMin mice with only slight reduction in the number of larger tumors. The data suggest that green tea, at a 0.6% concentration, is selectively targeting the development of new adenomas in the AOM-ApcMin mouse model without affecting the pre-existing ones. Previous reports indicated that treating the ApcMin mice with green tea reduced the total number of tumors (14). These reports, however, did not discriminate between new and pre-existing tumors. Our results indicate that the time of administration of green tea is pivotal to its ability to inhibit colon carcinogenesis; physiological levels of green tea are not probably to inhibit the progress of any large adenomas or adenocarcinomas existing prior to the tea administration.

In order to explain the ability of green tea to inhibit the formation of new adenomas, we examined the possible modulation of several biomarkers known to be involved in colon carcinogenesis. These included ß-catenin, cyclin D1 and COX-2. In accordance with the tumor data, green tea inhibited the formation of small neoplastic lesions overexpressing ß-catenin and its downstream target cyclin D1. This inhibition was observed after only 4 weeks of green tea treatment. In contrast, only modest effects were observed in the population of large lesions that are probably to have pre-existed when the tea treatment started. Surprisingly, we did not observe any significant inhibitory effects of green tea on COX-2 expression, although published literature had previously suggested an inhibition of COX-2 by EGCG in vitro (32,33). Despite its appeal as a target for chemoprevention by green tea, inhibition of COX-2 expression seems to be achieved at concentrations far exceeding the physiological concentrations of catechins present in the green tea beverage. Therefore, inhibition of COX-2 cannot explain the vast number of epidemiological data that show negative association between drinking green tea and the risk for cancers such as colorectal cancer. Further, we observed that COX-2 expression was limited to medium and large neoplastic lesions in the ApcMin colons and small intestines indicating a later involvement of COX-2 in ApcMin tumorigenesis. The expression of COX-2 was directly proportional to the size of the lesions and the degree of dysplasia regardless of the green tea treatment. The expression was only observed in the stromal cells surrounding the epithelial cells in the lesions.

In summary, western blotting and the immunohistochemical analysis showed that a 0.6% green tea treatment reduced the total levels of ß-catenin and its downstream target cyclin D1, but did not inhibit COX-2 expression. Green tea inhibited the formation of new adenomas but not pre-existing ones. Our data suggest that physiological concentrations of green tea target very early events in ApcMin intestinal carcinogenesis with little or no effects on events that take place later in the process.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
National Institutes of Health (CA 96994).


   Acknowledgments
 
We would like to thank Valerie Kennedy and Sharon Cooper for expert assistance with tissue preparation and immunohistochemical analyses.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received May 16, 2006; revised June 29, 2007; accepted June 29, 2007.


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Mouse models for the study of colon carcinogenesis
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