Effect of genistein on the bioavailability and intestinal cancer chemopreventive activity of (-)-epigallocatechin-3-gallate

Abstract

The green tea (Camellia sinensis) catechin, (-)-epigallocatechin-3-gallate (EGCG), has shown cancer-preventive activity in animal models. Previously, we have reported the bioavailability of EGCG in rats and mice. Here, we report that cotreatment of HT-29 human colon cancer cells with genistein (from soy) increased cytosolic EGCG by 2- to 5-fold compared with treatment with EGCG only. Inclusion of genistein, at non-cytotoxic concentrations, increased the growth inhibitory effects of EGCG against HT-29 cells (up to 2-fold at 20 μM genistein). Intragastric coadministration of EGCG (75 mg/kg) and genistein (200 mg/kg) to CF-1 mice resulted in an increase in plasma half-life (t_1/2 148.7 ± 16.4 versus 111.5 ± 23.4 min) and exposure (AUC_0→∞ 183.9 ± 20.2 versus 125.8 ± 26.4 μg/ml × min) of EGCG. Cotreatment with genistein also increased the maximal concentration (C_max), 6 h exposure (AUC_{0→360 min}), and t_1/2 of EGCG in the small intestine by 2.0-, 4.7- and 1.4-fold, respectively, compared with mice treated with EGCG only. Contrary to our expectations, the combination of 0.01% EGCG in the drinking fluid and 0.2% genistein in the diet enhanced intestinal tumorigenesis in male adenomatous polyposis coli (APC)^min/+ mice. This combination increased the multiplicity of tumors in the medial and distal small intestine: the largest increase was in tumors >2 mm in diameter (4.3-fold compared with controls). This study demonstrates that although genistein can enhance EGCG bioavailability and in vitro growth inhibitory activity, this combination enhances tumorigenesis in the APC^min/+ mouse. These results further show the need for careful cancer prevention studies in animal models and for caution when interpreting data from in vitro studies.

Introduction

Tea (Camellia sinensis) is second only to water in terms of worldwide popularity as a beverage. (-)-Epigallocatechin-3-gallate (EGCG, Figure 1) is the major catechin component of green tea and is believed to be a major active constituent. Studies with animal models have shown that green tea and EGCG have preventive activity against cancer of the oral cavity, esophagus, stomach, intestine, colon, liver, lung, prostate, skin and other sites (1). Studies with human cancer cell lines have shown that EGCG possesses a number of activities related to cancer prevention such as inhibition of mitogen-activated protein kinase cascades, DNA methyltransferase, epidermal growth factor receptor signaling and others. It is not known, however, whether these potential mechanisms of action occur in animals or in humans because of the limited oral bioavailability of EGCG (1,2).

Fig. 1.

Structures of EGCG and genistein.

Previously, we have reported that the absolute bioavailability of EGCG in CF-1 mice and Sprague–Dawley rats is 26.5 and 1.6%, respectively (3,4). EGCG undergoes methylation, glucuronidation and sulfation in vivo and is largely present as the glucuronide in the plasma of treated mice (1,5,6). Studies in our laboratory and others have suggested that EGCG might also be a substrate for multidrug resistance-related protein (MRP) 1 and 2 (7–9). Cotreatment of Madin–Darby canine kidney cells overexpressing human MRP1 and MRP2 with EGCG and selective inhibitors of MRP1 and MRP2 resulted in a 10-fold increase in the cytosolic concentration of EGCG in both cases (8). Zhang et al. (9) have reported that cotreatment of Caco-2 cells with green tea catechins, including EGCG, and MK571, an inhibitor of MRP, resulted in 5.7-fold decrease in basolateral-to-apical flux of EGCG compared with cells treated only with EGCG. Modulation of the factors affecting EGCG bioavailability might increase plasma and tissue levels of this compound and increase its cancer-preventive activity.

Genistein (Figure 1), an isoflavone from soybeans (Glycine max, Fabaceae), is common in the diet and has been studied for a number of potential health effects including cancer prevention and anti-inflammatory activity (10–12). Genistein has also been shown to modulate MRP-mediated efflux (13,14). We have previously reported that cotreatment of MRP-overexpressing cells with [³H]-EGCG and genistein or related isoflavones results in increased cell-associated radioactivity compared with treatment with EGCG alone (8). This measurement includes both membrane-bound and intracellular EGCG and its metabolites: the effect of genistein on the intracellular levels of EGCG, strictly defined, has not been previously reported.

The interaction between EGCG and genistein is also of interest due to the likelihood of co-occurrence in the diet. Whereas laboratory studies have shown that EGCG and green tea prevent cancer, epidemiological studies have been less conclusive (2,15–17). One possible confounding variable is differences in diet among individuals and between populations. Other dietary factors could influence the bioavailability and biological activity of EGCG. Careful studies are needed to understand these potential interactions.

The effects of genistein on EGCG cancer chemopreventive effects and bioavailability have not been previously reported. In the present study, we hypothesized that genistein could increase the cytosolic concentration and growth inhibitory activity of EGCG in vitro and increase the plasma and tissue levels, as well as the cancer-preventive activity, of EGCG in vivo. Herein, we report the results of our studies.

Materials and methods

Chemicals

EGCG (100% pure) was provided by Mitsui Norin Co., Ltd (Fujieda City, Japan). β-D-glucuronidase (G-7896, EC 3.2.1.31, from Escherichia coli with 9 × 10⁶ U/g solid) and sulfatase (S-9754, EC 3.1.6.1, from Abalone entrails with 2.3 × 10 ⁵ U/g solid) purchased from Sigma Chemical Co. (St Louis, MO). Genistein (>95% pure) was purchased from AB Chem Technologies (Franklin Park, NJ). All other reagents were of the highest grade commercially available. Dosing solutions of EGCG and genistein were prepared in 0.9% NaCl. For analytical purposes, a standard stock solution of EGCG, epigallocatechin, epicatechin and epicatechin-3-gallate (10 μg/ml each) was prepared in 11.4 mM ascorbic acid-0.13 mM ethylenediaminetetraacetic acid (pH 3.8) and stored at −80°C. Stock solutions (20 or 100 mM) of genistein and EGCG for cell culture were prepared in dimethyl sulfoxide (DMSO) and stored at −80°C. The final concentration of DMSO in all cell culture experiments was 0.1%. Control cells were grown in the same concentration of DMSO.

Mice and diets

All experiments involving mice were approved by the Institutional Animal Care and Use Committee at Rutgers University (Piscataway, NJ). Male CF-1 mice (25 g) were purchased from Charles River Laboratories (Wilmington, MA) and maintained on AIN76A diet for 1 week prior to the start of experiments. Male adenomatous polyposis coli (APC)^min/+ mice were derived from a colony housed at the Susan L. Cullman Laboratory for Cancer Research at Rutgers University. Mice were genotyped at 3 weeks of age as described previously (18). All mice were housed 10 per cage and maintained in air-conditioned quarters with a room temperature of 20 ± 2°C, a relative humidity of 50 ± 10% and an alternating 12 h light–dark cycle. All mice had ad libitum access to food and water. All experimental and control diets were obtained from Research Diets (New Brunswick, NJ). AIN76A, a soy-free, defined diet, was used as the basal diet (19).

Cell culture and treatment

HT-29 human colon cancer cells (American Type Tissue Culture, Rockville, MD) were maintained at subconfluency in McCoy’s 5A medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin at 37°C in 95% humidity and 5% CO₂.

Cytosolic levels of EGCG in the presence or absence of genistein were determined as previously reported (8). In brief, cells were allowed to grow to 70–90% confluency in six-well plates. The medium was replaced with fresh, serum-complete medium containing 50 μM EGCG with or without genistein (0–20 μM). Medium also contained 5 U/ml superoxide dismutase to stabilize EGCG. The cells were incubated for 1.5–24 h at 37°C, after which the medium was removed, the cells were washed two times with cold phosphate-buffered saline, 2% ascorbic acid was added to each well and the cells were scraped and sonicated. The resulting solution was centrifuged at 16 000g for 10 min. The supernatant was combined with an equal volume of ice-cold methanol and centrifuged for 10 min at 16 000g to precipitate the protein. The resulting supernatant was analyzed by high-performance liquid chromatography. Cytosolic EGCG was normalized to cytosolic protein concentration.

To determine the growth inhibitory activity of EGCG in combination with genistein, HT-29 cells were plated in 96-well plates (12 × 10³ cells/cm²) and allowed to attach for 24 h. The medium was replaced with fresh, serum-free medium containing 0–50 μM of EGCG in the presence or absence of 0, 10 or 20 μM genistein. Incubations were carried out in the presence of 5 U/ml superoxide dismutase to stabilize EGCG. Cells were incubated for 24 h at 37°C. The medium was removed, the cells were washed once with medium containing no EGCG or genistein and the relative number of viable cells was determined using the 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (20). Control cells were treated with the equivalent amount of DMSO.

Treatment of mice and sample collection for bioavailability

EGCG (75 mg/kg, intragastric (i.g.) or EGCG plus genistein (75 and 200 mg/kg, i.g.) was administered to male CF-1 mice (six per group). Mice were then killed at 20, 50, 90, 180 and 360 min posttreatment, blood was collected via cardiac puncture and plasma was isolated by centrifugation at 500g for 15 min. Plasma was combined with 0.1 volume of ascorbate preservative (20% ascorbic acid and 0.1% ethylenediaminetetraacetic acid) and stored at −80°C for later analysis. The small intestine and liver were collected, washed in 0.9% NaCl and frozen at −80°C for later analysis.

Intestinal tumorigenesis studies in APC^min/+ mice

Male APC^min/+ mice (5 weeks old, n = 12) were treated with 0.01% EGCG in the drinking fluid, 0.2% genistein in the diet or the combination of the two agents. Body weight, food and fluid consumption were monitored weekly. After 9 weeks, mice were euthanized and the entire intestinal tract was harvested, flushed with 0.9% NaCl and opened longitudinally. The tissue was flattened on filter paper to expose the tumors and briefly frozen on dry ice to aid visual scoring of tumors. All tumors were scored and subdivided by location (proximal, medial and distal small intestine and colon) and size (<1, 1–2 or >2 mm in diameter). After scoring, a 150 mm segment of the small intestine (beginning at the jejunum) was fixed in 10% formalin for 24 h and then Swiss rolled and paraffin-embedded for pathological analysis. Embedded tissue blocks were cut serially for at least 30 slides and labeled numerically. Slides 1, 10, 20 and 30 were stained with hematoxylin and eosin for histopathological evaluation.

Quantification of EGCG

Plasma levels of EGCG were analyzed as previously reported (4). Tissue samples were homogenized in 2% ascorbic acid and processed as described previously (3). Samples were analyzed by high-performance liquid chromatography with electrochemical detection.

EGCG levels were analyzed using an high-performance liquid chromatography system consisting of two ESA model 580 dual-piston pumps (Chelmsford, MA), a Waters Model 717plus refrigerated autosampler (Milford, MA) and an ESA 5500 coulochem electrode array system. The potentials of the coulochem electrode array system were set at −100, 100, 300 and 500 mV. Separation was achieved using previously described methods (21).

Statistical analysis

Values are means ± SEM or standard deviation. The pharmacokinetic parameters, maximal concentration (C_max), exposure (AUC_0→∞) or AUC_0→360, and half-life (t_1/2) were determined by the Trapezoidal Rule using Microsoft Excel (Redmond, WA). Differences between means were determined using Student’s t-test, one-way analysis of variance with Tukey’s posttest or two-way analysis of variance with Bonferroni’s posttest, as appropriate. Differences were considered significant at P < 0.05.

Results

Effect of genistein on EGCG uptake and growth inhibitory activity

We examined the ability of genistein to increase the intracellular concentrations of EGCG in HT-29 cells. Cotreatment of cells with EGCG (50 μM) and genistein (0–20 μM) resulted in an increase in the intracellular levels of EGCG after 1.5, 3 and 24 h (Figure 2A).

Fig. 2.

Effect of genistein on cytosolic levels (A) and growth inhibitory activity (B) of EGCG. For cell uptake studies, cells were incubated with EGCG in the presence or absence of genistein for 1.5, 3 or 24 h. Cells were fractionated as in the Materials and Methods and levels of cytosolic EGCG were determined by high-performance liquid chromatography (n = 3, error bars represent the standard deviation). G0 = no genistein, G10 = 10 μM genistein and G20 = 20 μM genistein. Growth inhibition by EGCG was determined by 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay after treatment for 24 h (n = 6, error bars represent SEM). Values with different superscripted letters are significantly different (P < 0.05) by one-way analysis of variance with Tukey’s posttest (cell uptake) or repeated measures analysis of variance with Bonferroni’s posttest (3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay).

To determine if this genistein-mediated increase in cytosolic EGCG resulted in enhanced growth inhibitory activity, we used the 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay to assess cell viability after 24 h treatment. Genistein dose dependently increased the growth inhibitory activity of EGCG. The inhibitory concentration 50% of EGCG was 43, 32 and 25 μM in the presence of 0, 5 and 20 μM genistein, respectively (Figure 2B). At 50 μM EGCG, the effect of genistein is lost. Genistein alone, at the concentrations used in the present study, did not significantly affect the number of viable cells (Figure 2B).

Effect of genistein on the bioavailability of EGCG in mice

Treatment of male CF-1 mice with a single dose of EGCG (75 mg/kg, i.g.) in combination with genistein (200 mg/kg, i.g.) resulted in an increase in the AUC_0→∞ and t_1/2, but not the C_max of total EGCG in the plasma compared with treatment with EGCG only (Figure 3). The t_1/2 for EGCG was 111.5 ± 23.4 and 148.7 ± 16.4 min (P < 0.05) in the absence or presence of genistein, respectively. The AUC_0→∞ for EGCG in the plasma was 125.8 ± 26.4 and 183.9 ± 20.2 μg/ml × min (P < 0.05) in the absence or presence of genistein, respectively. The bioavailability of EGCG in the small intestine was also increased by cotreatment with genistein (Figure 3). The AUC_{0→360 min} was 1431.7 ± 585.6 μg/g × min and 6727.5 ± 1823.2 μg/g × min (P < 0.05) in the absence of presence of genistein, respectively. The C_max and t_1/2 of EGCG increased from 16.8 ± 6.1 to 34.4 ± 9.4 μg/g (P = 0.05) and from 80.8 ± 33.0 to 115.0 ± 31.2 min, respectively, in the presence of genistein. The change in EGCG t_1/2 in the small intestine was not statiscally significant. There was no significant effect of genistein on the bioavailability of EGCG in the liver (Figure 3).

Fig. 3.

Plasma, small intestine and liver levels of EGCG following treatment with EGCG or EGCG plus genistein. Male CF-1 mice were given a single dose of EGCG (75 mg/kg, i.g.) alone or in combination with genistein (200 mg/kg, i.g.). Each point represents n = 6. Error bars represent the SEM.

Effect of genistein on the chemopreventive activity of EGCG in APC^min/+ mice

We conducted a chemoprevention study of the combination of EGCG and genistein in the APC^min/+ mouse model of intestinal tumorigenesis to determine if the effects of genistein on EGCG bioavailability and in vitro biological activity translated to improved cancer chemoprevention in vivo. There were minor differences in food intake among the treatment groups: mice treated with genistein diet (either alone or in combination with EGCG) consumed ∼20% less diet than did mice fed AIN76A diet (control- and EGCG-treated mice, Figure 4A). Consequently, the mice fed genistein diet had significantly lower final body weight compared with mice fed AIN76A (17% decrease, Figure 4B). There was no significant difference in fluid consumption between mice treated with distilled water and those treated with 0.01% EGCG (Figure 4A).

Fig. 4.

Food and fluid consumption (A), final body weight (B) and small intestinal tumor histology (C) in male ACP^min/+ mice (n = 12, 5 weeks old) treated with 0.01% EGCG as the sole source of drinking fluid, 0.2% genistein in the diet or both for 9 weeks. Fluid and food consumption were determined weekly for 9 weeks (error bars represent SD, n = 9). Final body weight was determined at time of killing (error bars represent SD, n = 12). Tumor histology is hematoxylin and eosin staining of representative adenomas.

Unexpectedly, treatment of male APC^min/+ mice with 0.01% EGCG in combination with 0.2% dietary genistein for 9 weeks resulted in significantly higher tumor multiplicity compared with control mice or treatment with either agent alone (Table I). The largest increase was in tumors >2 mm in diameter where combination treatment increased tumor multiplicity by 4.3-fold compared with control animals. In contrast, treatment with 0.01% EGCG appeared to decrease the number of tumors >1 mm in both the medial and distal small intestine, although the effect was not statistically significant. Treatment with genistein had no clear effect on tumors at any location in the small intestine. Histopathological analysis showed, as expected, that the tumors present in the mice were adenomas and confirmed the gross trends in tumor multiplicity (Figure 4C). In order to confirm that genistein enhanced the bioavailability of EGCG in the APC^min/+ mouse, we measured the plasma levels of EGCG in both EGCG and EGCG plus genistein-treated mice (Figure 5). We found, as expected, that plasma levels of EGCG were 23% higher (P < 0.01) in the mice cotreated with genistein compared with mice treated with EGCG only.

Table I.

Effect of EGCG and genistein treatment on tumorigenesis in male APC^min/+ mice^a

	Tumor location			Tumor size
	Proximal	Medial	Distal	<1 mm	1–2 mm	>2 mm	Total
Control	2.4 ± 0.61	4.2 ± 0.71	5.1 ± 0.91	4.8 ± 0.81	5.6 ± 0.91	0.8 ± 0.21	12.3 ± 1.61
0.01% EGCG	3.1 ± 0.51,2	3.8 ± 0.61	2.9 ± 0.51	5.2 ± 0.91	3.8 ± 1.51	0.3 ± 0.141	9.9 ± 1.01
0.2% Genistein	2.0 ± 0.61	3.7 ± 0.91	6.5 ± 1.21,2	5.9 ± 1.11	4.8 ± 1.11	0.9 ± 0.21	13.8 ± 1.61
EGCG + genistein	5.5 ± 1.12	8.0 ± 1.81	10.3 ± 1.82	5.4 ± 0.91	14.1 ± 2.42	4.3 ± 1.22	26.0 ± 3.42

^aValues represent the mean ± SEM. Values with different superscripts are different by analysis of variance with Tukey’s posttest (P < 0.05).

Fig. 5.

Plasma levels of total EGCG in male APC^min/+ mice treated with EGCG alone or in combination with genistein. Mice were treated for 9 weeks with 0.01% EGCG as the sole source of drinking fluid and 0.2% genistein in the diet. Each bar represents the mean of n = 5–6. Error bars represent the SEM. **P < 0.01 compared with EGCG treatment only.

Discussion

In the present study, we determined the effect of the soy isoflavone, genistein, on the in vitro and in vivo bioavailability and cancer-preventive activity of the tea catechin, EGCG. Previous cell line studies in our laboratory and others have suggested that EGCG is a substrate for MRP and that cotreatment of MRP1- and 2-overexpressing cells with EGCG and selective inhibitors of those efflux pumps resulted in increased cell-associated and cytosolic levels of EGCG (7,8). Genistein has been reported previously to modulate the activity of MRPs (13,14). Our present results confirmed this phenomenon and additionally extended our previous results showing that cotreatment of MRP-overexpressing cells with [³H]-EGCG and genistein results in increased cell-associated radioactivity (8). Whereas these previous data included both membrane-bound and cytosolic EGCG and its metabolites, the present results represent a precise measurement of cytosolic EGCG. Many of the proposed targets of EGCG are cytosolic (e.g. mitogen-activated protein kinases) or nuclear (e.g. DNA methyltransferase) and precise determinations of the cytosolic levels of EGCG are important for interpreting biological effects observed in vitro (10,22).

The effect of genistein on the growth inhibitory activity of EGCG was also investigated in HT-29 cells. To our knowledge, this is the first report to demonstrate that the dose-dependent increases in cytosolic EGCG induced by genistein are associated with similar dose-dependent increases in EGCG-mediated growth inhibition of human cancer cells. The effect of genistein on EGCG-mediated growth inhibition is lost when the concentration of EGCG reaches 50 μM. This could indicate that maximal growth inhibition by EGCG is reached at 50 μM and that increases in cytosolic EGCG beyond this do not affect cell growth. The genistein-mediated increase in growth inhibitory activity observed in the present experiments was not equivalent in magnitude to the increase in cytosolic concentration of EGCG. This discrepancy appears to be due to the fact that although the effect of genistein on cytosolic EGCG is very strong, the magnitude of this effect decreases as a function of time. Since the 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay is a composite of the effect of EGCG and genistein over the entire course of the 24 h experiment, the results appear to reflect this decreasing effect of genistein on cell uptake. The decreasing effect of genistein on cytosolic EGCG may be the result of genistein and/or EGCG undergoing biotransformation that is independent of the MRPs. Additionally, we cannot rule out that genistein is affecting some target other than, or in addition to, MRP.

The present study also demonstrates that our present and previous data concerning the effects of genistein on EGCG in vitro are recapitulated following oral dosing of EGCG and genistein to mice (8). Although MRP-mediated efflux is not the only factor limiting EGCG bioavailability in vivo, it appears that inhibition of MRP-mediated efflux can improve the plasma and small intestinal pharmacokinetics of EGCG. It is interesting that genistein increased the levels of EGCG in the small intestine and plasma, but not in the liver. This may suggest that other factors play a role in determining the levels of EGCG in the liver: these factors may include additional efflux pumps or more efficient metabolism of EGCG that is not overcome by the addition of genistein. Further studies are needed to clarify these tissue differences.

Based on our cell line and mouse bioavailability studies, we expected to see an enhancement of the antitumorigenic activity of EGCG in the APC^min/+ mouse. We were, therefore, surprised to observe a dramatic increase in tumor multiplicity in mice treated with the combination of EGCG and genistein compared with controls. The effect on tumor multiplicity observed in mice treated with EGCG or genistein as single agents was as expected. EGCG had a non-significant inhibitory effect, whereas genistein, which has previously been shown ineffective in this model, had no effect (18,23–26). The reasons for the increase in tumor multiplicity induced by the combination of 0.01% EGCG and 0.2% genistein remain unclear. Although genistein was originally identified as a chemopreventive agent, more recent studies have shown that genistein can enhance tumorigenesis in a number of models including gastrointestinal cancers. Rao et al. (27) have previously reported that genistein increased the multiplicity of total and non-invasive colon adenocarcinomas in azoxymethane-treated rats by 50.3 and 48.5%, respectively. These authors suggested that the effect may be due to genistein-mediated increases in prostaglandin E₂ levels, but that possibility was not tested experimentally. Such an effect is not expected in the present model since EGCG has been shown previously to inhibit the production of prostaglandin E₂ both in in vitro and in vivo models of colon cancer.

It has also been recently reported that dietary treatment with genistein enhances β-catenin accumulation in colonic crypts of 1,7-dimethylhydrazine-treated rats (28). Again, the underlying mechanisms remain unclear. Genistein has also been shown to enhance N-nitroso-N-methylurea-induced mammary tumorigenesis in both normal and ovarectomized rats (29). These effects seemed to result from the estrogenic activity of genistein.

The mechanism responsible for the enhanced tumorigenesis due to the treatment with the combination of EGCG and genistein remains to be further studied. Similarly, careful dose-response studies are needed to determine if different dose combinations have cancer-preventive effects, whereas others promote tumorigenesis.

From a risk assessment perspective, the present data should be interpreted carefully. Based on allometric scaling, the daily dose of EGCG and genistein used in the present study are 50 and 667 mg/d, respectively (30). These values assume a daily calorie requirement of 12 and 2000 kcal for mice and humans, respectively. Whereas this dose of EGCG is easily achievable through dietary consumption of green tea, the dose of genistein exceeds daily dietary intake by 10- to 50-fold even in Asian countries such as Japan which have a high per capita intake of soy products (31,32). A dose of 667 mg/d genistein is, however, achievable through the use of dietary supplements, particularly if the recommended dose of such supplements is exceeded (33,34). Although there are no reports of increased risk of intestinal cancer associated with intake of genistein and EGCG, the emerging animal data on genistein as a single agent warrants further investigation of the potential procarcinogenic effects of genistein alone or in combination with EGCG.

In summary, the present study for the first time demonstrates that genistein can modulate the cytosolic levels and growth inhibitory activities of EGCG against human colon cancer cells. Moreover, we report for the first time that acute treatment with genistein in combination with EGCG results in higher plasma and intestinal levels of EGCG in mice compared with treatment with EGCG only. Whereas these data indicate that cotreatment with both EGCG and genistein should have enhanced chemopreventive activity against intestinal tumorigenesis, we found exactly the opposite effects. Although the mechanisms for this tumor enhancement remain unclear, these studies reiterate the need for careful in vivo studies to assess the impact of any new chemopreventive regimen, even those based on well-studied dietary components.

Funding

American Institute for Cancer Research (05A047); National Institutes of Health (CA125780 to J.D.L.; CA 88961 and CA120915 to C.S.Y.)

Glossary

Abbreviations

APC

adenomatous polyposis coli

DMSO

dimethyl sulfoxide

EGCG

(-)-epigallocatechin-3-gallate

i.g.

intragastric

MRP

multidrug resistance-related protein

t_1/2

half-life