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Department of Nutrition and * Department of Pathology, University of Tennessee, Knoxville, TN
2To whom correspondence should be addressed. E-mail: mmcentee{at}utk.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • cancer • resveratrol • Apc • phytochemical • COX-2
Polyphenolic phytochemicals have been shown to be effective antitumorigenic agents in several in vitro models (1,2), and efficacy in vivo has also been demonstrated when these compounds are provided in the diet (3–5). Resveratrol, a resorcinol-containing polyphenol found most commonly in red wine, inhibits the growth of several immortalized human colorectal cancer cell lines (6,7). When provided orally, resveratrol reduced the tumor load in Lewis Lung carcinoma–bearing mice (8) and reduced the number of aberrant crypt foci in an azoxymethane-induced rat model of colorectal cancer (9). Quercetin, curcumin and caffeic acid phenyl ester, chemical analogs of resveratrol, inhibit intestinal tumorigenesis in vitro (10–12) and aberrant crypt foci formation in vivo (5,13). These polyphenolic phytochemicals share a common resorcinol-containing structure, and this structure is believed to be responsible in part for the reported effects (14,15).
Resveratrol’s antitumorigenic effects have been linked in part to inhibition of cyclooxygenase (COX)2 -catalyzed reactions (15–18). COX catalyzes the committed step in the conversion of arachidonic acid to protumorigenic eicosanoids, such as prostaglandin (PG)E2, which are involved in the maintenance of tumor integrity (19). COX-2 expression is upregulated in 80–85% of human adenocarcinomas and colonic tumors (20,21) and in 80–85% of ApcMin/+ mouse adenomas (22). Upregulation was observed in 
60% of the tumors. Resveratrol was shown to inhibit COX-2 promoter activity (15), downregulate COX-2 expression (15,17) and inhibit COX-2 activity in vitro (16,23).
Numerous studies have demonstrated the ability of COX inhibitors to decrease tumor multiplicity in the ApcMin/+ mouse, a murine model possessing a germline mutation in the adenomatous polyposis coli (Apc) tumor-suppressor gene (24–27). These mice spontaneously develop a mean of 40 tumors throughout the intestinal tract that are sensitive to COX inhibition. Murine Apc is homologous to human APC (28), and patients with familial adenomatous polyposis (FAP) have an autosomal dominant disorder with a germline mutation in one allele of the APC tumor suppressor gene (29). Adenoma formation results when a somatic mutation is acquired in the remaining functional allele. By the time they reach their twenties, FAP patients typically develop hundreds to thousands of intestinal adenomas, mostly in the colorectal mucosa, which are also sensitive to COX inhibition (30). Similarly, at least one allele of APC is mutated in 80% of sporadic colorectal cancers, making the ApcMin/+ mouse a relevant model for studying the early stages of colorectal cancer (31,32).
The purpose of this study was to evaluate the dose-dependent effects of dietary resveratrol on intestinal tumorigenesis in ApcMin/+ mice, and to determine whether tumor COX-2 and/or PGE2 levels are affected by this treatment.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Diets. AIN-93G powdered diet (Dyets, Bethlehem, PA) was used as the base diet. Experimental diets were prepared weekly by thoroughly mixing resveratrol (Sigma Chemical, St. Louis, MO) with the base diet to homogeneity. Resveratrol concentrations in the diets were adjusted weekly, on the basis of food consumption and body weights to achieve doses of 0, 4, 20 and 90 mg/kg body weight. Diets were prepared under yellow light and stored at -80°C under an atmosphere of nitrogen to minimize degradation of resveratrol. Fresh diet was provided daily.
Experimental design
Pharmacokinetics and absorption of resveratrol. To establish the stability of resveratrol in the diet, dietary samples containing resveratrol at 750 µg/g (dose equivalent to the mice fed 90 mg/kg body weight) (n = 2) were stored under an atmosphere of nitrogen at -80°C (controls), or exposed to experimental conditions (light and room temperature for 24 h). Samples were extracted four times with ethyl acetate (Sigma), evaporated under an atmosphere of nitrogen, resuspended in the reversed-phase HPLC (RP-HPLC) mobile phase and analyzed for resveratrol content via RP-HPLC.
To establish that resveratrol reaches the intestinal tract intact and is subsequently absorbed, 10-wk-old male C57BL/6J mice (n = 15) were administered resveratrol in the diet at a dose of 200 mg/kg body weight. Before killing, the mice were divided into five groups (n = 3/group). After 6 d of consuming the diet, blood was collected postprandially via cardiac puncture (under general anesthesia) at 5, 15, 30, 60 and 90 min, and plasma resveratrol concentration was measured by RP-HPLC. In a follow-up experiment, 10-wk-old C57BL/6J wild-type mice (n = 3) were fed resveratrol at 90 mg/kg body weight. After 6 d of feeding, blood was collected 30 min postprandially and samples were analyzed for resveratrol content via RP-HPLC.
Effect of resveratrol on intestinal tumorigenesis. Male C57BL/6J ApcMin/+ mice (n = 25; 43 d of age) were assigned to four groups (n = 6–7/group) and fed diets supplemented with resveratrol at 0, 4, 20 and 90 mg/kg body weight for 7 wk, until 85 d of age. Tumor number, size and location were determined with the use of a dissecting microscope. Whole blood was collected via cardiac puncture and immediately placed on ice protected from light. Plasma was isolated and stored at -80°C for analysis of resveratrol via RP-HPLC.
Effects of resveratrol on COX-2 and PGE2 levels in tumors. To establish the effect of resveratrol on levels of tumor COX-2 protein and PGE2, male C57BL/6J ApcMin/+ mice (n = 16) were assigned to a control or experimental group (n = 8) and fed resveratrol at a dose of 0 or 90 mg/kg body weight for 7 wk. At 87 d of age, the mice were killed, and excised intestinal tumors were analyzed for COX-2 expression and basal PGE2 levels.
RP-HPLC analysis of trans-resveratrol. Resveratrol levels were analyzed by RP-HPLC using a Hewlett Packard 1090 Series-2 Liquid Chromatograph (Hewlett Packard, Palo Alto, CA) equipped with a 25 cm, 4.6 mm KR100–5C18 Kromasil RP-HPLC column (Akzo Nobel-EKA Chemicals, Bohus, Sweden) with an isocratic mobile phase consisting of 70% CH3CN, 30% methanol, 0.1% glacial acetic acid (Sigma), at a flow rate of 0.5 mL/min. Samples were monitored at 306 nm using a photodiode array detector. Maximum absorption of resveratrol is reported at 306 nm (34). Detection and spectrophotometric identification limits were 2 and 3 ng, respectively.
Extraction of resveratrol from plasma. To determine relative levels of free vs. glucuronidated resveratrol in the plasma, whole blood was collected via cardiac puncture, using EDTA (Life Technologies, Grand Island, NY) at a concentration of 7.5% as an anticoagulant, and immediately placed on ice, protected from light. Plasma was isolated by centrifugation at 500 x g, 4°C for 15 min. To determine relative levels of resveratrol circulating in the free form, 200-µL aliquots of plasma were placed in Eppendorf tubes, diluted 1:10 with methanol, adjusted to a pH of 3.5 with formic acid and subjected to solid-phase extraction using a conditioned C18 cartridge (Burdick and Jackson, MI). Resveratrol was eluted from the column twice with 2 mL of 100% HPLC-grade methanol (Sigma), evaporated under an atmosphere of nitrogen, resuspended in the RP-HPLC mobile phase and analyzed via RP-HPLC.
To determine relative levels of resveratrol circulating as the glucuronide derivative, 200-µL aliquots of plasma were placed in Eppendorf tubes for enzymatic hydrolysis via ß-glucuronidase. Powdered H-1 ß-glucuronidase (Sigma) was solubilized at 50 g/L in 0.2 mol/L sodium acetate buffer, pH 5.0, at 37°C. This enzyme solution was added to plasma at 5:1, to a final concentration of 0.04 g/mL, and final specific activity of 5 mg/(min · mg protein). Samples were incubated for 2 h at 37°C in a rocking incubator. The reaction was terminated with 100% methanol, pH 3.0 with formic acid, to a final volume of 10% methanol. Samples were extracted three times with equal volumes of ethyl acetate (Sigma), and pooled organic phases were evaporated under nitrogen, resuspended in 1 mL of a 10% aqueous methanol solution and passed through a 0.45-µm filter (Pall, Ann Arbor, MI). We do not report specific plasma levels due to lack of an appropriate internal standard. We report an estimated plasma value based on comparison to a standard curve of authentic resveratrol. Resveratrol was isolated via solid phase extraction as described previously and analyzed via RP-HPLC.
Immunohistochemistry for COX-2 expression. Tissues were formalin-fixed, embedded in paraffin and sectioned at 4 µmol/L. Tissues sections were then heated, deparaffinized in xylene, rehydrated in graded alcohols to PBS (3 times, 5 min each) and pretreated at or just below boiling with 10 mmol/L citrate buffer, pH 6, for 10 min. Endogenous peroxidase activity was quenched by incubation in 3% H2O2 PBS for 15 min at room temperature and incubated for 30 min at room temperature with protein block (Biogenex USA, San Ramon, CA). Slides were then incubated at 20°C for 1 h with affinity-purified rabbit polyclonal anti-mouse COX-2 IgG (1:200) (Cayman Chemical, Ann Arbor, MI), followed by biotinylated anti-rabbit IgG (30 min at room temperature) and streptavidin/biotin-horseradish peroxidase complex (30 min at room temperature), which was localized by reaction with 3,3'-diaminobenzidine tetra-hydrochloride (0.7 g/L; Biogenex) and 0.05% H2O2 in 80 mmol/L Tris-HCl, pH 7.6, for 10 min. Slides were lightly counterstained with Mayer’s hematoxylin. Staining for COX-2 protein was evaluated from coded slides and scored on the bases of distribution (extent) and relative staining intensity by a pathologist who was unaware of the treatments.
Relative staining distribution was scored as follows: 0 = < 10 stained cells per high power field (0.2 mm2 at X400 magnification); 1 = 10–20 stained cells per high power field; 2 = 20–30 stained cells per high power field; and 3 = > 30 stained cells per high power field. Both positive and negative tumors (i.e., distribution = 0) were included in calculating mean scores for each group. Relative staining intensities were graded as follows: 0 = no staining; 1 represented perinuclear staining; 2 represented stain extending to the cytoplasm; and 3 indicated a stain that obscured the cell nucleus. Intensity and distribution scores were then added to generate a final value corresponding to COX-2 expression. The reported staining scores represent the sum of intensity and distribution ± SEM.
Basal PGE2 Levels. To determine the effect of resveratrol on PGE2 levels in tumors, intestinal tumors from ApcMin/+ mice fed resveratrol in the diet at 0 or 90 mg/kg body weight were excised on dry ice, snap-frozen in liquid nitrogen and stored at -80°C. At the time of analysis, tumors were pooled from each mouse and immediately homogenized at 4°C using a Polytron handheld homogenizer at 10:1 in ice-cold 0.1 mol/L Tris-HCl buffer, under acidic conditions (pH 3.5) in the presence of methanol (10%) to eliminate any possibility of ex vivo PG synthesis. An 50-µL aliquot from each group was used for protein determination via the Bradford assay (35). PGE2 levels were determined by enzyme immunoassay (EIA) using the Correlate-EIA PGE2 Kit (catalog # 900–001; Assay Designs, Ann Arbor, MI) according to the manufacturer’s instructions.
Statistical analyses. Statistical analyses were conducted using the SAS and JMP software (SAS Institute, Cary, NC). Data were examined for normality of distribution and homogeneity among variances. Differences in body weight, diet intake, tumor number and basal PGE2 levels were analyzed by student’s t test or one-way ANOVA. Differences among groups were detected using Fisher’s least significant difference at P < 0.05. Data are expressed as mean ± SEM.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Analysis of the diets revealed that resveratrol remained stable during the time of feeding, and plasma pharmacokinetic data indicated adequate absorption. Resveratrol and other polyphenols are absorbed in the small intestines where they undergo glucuronidation before appearing in the blood (38). In the circulation, 100% of resveratrol exists as the glucuronide conjugate. However, there is much debate concerning whether this form of resveratrol is biologically active. This is important because in vitro studies utilize the free form of resveratrol and derivatization in vivo could result in a less biologically active compound. Furthermore, no studies exist on the adequacy of ß-glucuronidase activity to generate sufficient levels of free resveratrol in vivo to mimic the in vitro results. We observed no measurable levels of free resveratrol in the plasma of resveratrol-treated mice, and resveratrol was detected only after enzymatic hydrolysis, indicating 100% glucuronidation. Human and rat small intestines have sufficient luminal ß-glucuronidase activity to hydrolyze naturally occurring glucuronide derivatives of polyphenols found in the diet, but it does not appear that this activity is sustained within the circulation (39–41). Resorcin-containing polyphenols such as resveratrol, quercetin and catechins display pharmacokinetics similar to the kinetics observed here, with peak plasma levels occurring at 0.5–1 h postingestion; others confirmed that >95% of all of these compounds circulate in the plasma in their conjugated forms (42–44).
We did not observe any reduction in tumor load with dietary levels of resveratrol that ranged from 4 to 90 mg/kg body weight. These results are in contrast to a previous paper reporting a 74% reduction in tumor frequency in that model when resveratrol was provided in the drinking water dissolved in ethanol at a dose of 12–16 mg/kg body weight (45). We were unable to dissolve resveratrol in the drinking water under experimental conditions similar to those described in that paper. It is possible that a bolus dose (10 mg) of resveratrol could have had the reported effects. We considered whether the presence of ethanol in the drinking water might have had any effect on free resveratrol levels in the plasma. However, it does not appear that the use of ethanol as a vehicle has any influence on altering the availability of free resveratrol (44–49) and therefore cannot explain the disparate results observed between the two studies. We did, however, duplicate our results to recapitulate our earlier findings. We repeated the study using the highest dose (90 mg/kg body weight) and again observed no significant decreases in tumor load with dietary resveratrol.
Furthermore, our results confirm that resveratrol was absorbed and reached the target tissue as evidenced by reductions in PGE2 levels in tumors of resveratrol-fed mice compared with controls. The doses administered were comparable to doses administered in a previous study (45) and the length of the study (7 wk) should have been adequate to observe any decreases in tumor load. Our estimated concentration of 131 µmol/L for total plasma resveratrol (90 mg/kg body weight group) far exceeds the plasma levels (1.5 µmol/L) from a volunteer given a 25-mg oral dose (0.4 mg/kg body weight for a 70 kg individual) (40). To fully appreciate these oral doses, the amount of resveratrol in a liter of table wine varies from 0.1 to 7.7 mg (36,50). Most wines, however, contain concentrations that are <0.5 mg/L (36).
Disagreement exists concerning resveratrol’s ability to modify tumorigenesis, particularly when comparing results in vitro to those in vivo. Resveratrol inhibited the in vitro growth of 32Dp210 mouse myeloid leukemia cells and resveratrol-pretreated cells implanted subcutaneously; however, when the same cell line was implanted subcutaneously without pretreatment and mice were given resveratrol orally, no inhibition of growth was observed (51). Similarly, orally administered resveratrol did not inhibit tumorigenesis in a chemically induced lung tumor model (52). Yet, resveratrol has been shown to inhibit tumorigenesis in a carcinogen-induced rat model of colon cancer (9), Lewis Lung carcinoma–bearing mice (53) and in a dimethylbenz[a]anthracene–induced mammary tumor mouse model (23). Similarly, conflicting results have been reported with other structurally related polyphenolic compounds. For example, quercetin and its conjugated derivative rutin, did not reduce the number of tumors in ApcMin/+ mice but effectively reduced tumor load in a murine model of carcinogen-induced colon cancer (54,55). In the same vein, genistein inhibited the growth of MDA-MB-231 cells (a human mammary tumor cell line) in vitro, but was ineffective when the cells were implanted in mice that were given genistein orally (56). These conflicting results indicate that it is difficult to predict the in vivo effects of this compound, particularly at human equivalent doses.
We investigated the effect of resveratrol on PGE2 levels and COX-2 expression in intestinal tumors because we and others have established that COX inhibition reduces the growth of intestinal tumors and regresses established tumors in this animal model [as reviewed by Whelan and McEntee (57)]. Chemopreventive agents containing a common resorcin moiety (e.g., resveratrol, quercetin, genistein) were shown to suppress COX-2 transcription and activity in a human colon cancer cell line (15), and selective inhibition of COX-2 inhibited tumorigenesis in ApcMin/+ mice (26). Resveratrol inhibits COX-2 transcription and directly inhibits COX-2 activity in vitro (16,17,58). We did not observe any difference in COX-2 expression as a result of resveratrol treatment, although we did detect PGE2 levels that were 41% lower in tumors from the resveratrol-fed mice; however, this difference was apparently insufficient to affect tumor number. Inhibition of PGE2, viewed as an isolated event, is not always associated with a reduction in tumors (37,59).
In summary, resveratrol was shown to be stable in the diet, was absorbed intact and reached the target tissue, but had no effect on intestinal tumorigenesis at three doses and in two independent experiments. These results may be explained in part by the fact that resveratrol was ineffective as a modulator of COX-2 expression in tumors, the decreases in PGE2 levels were insufficient to modify tumor integrity and the levels of free resveratrol did not attain a sufficient concentration to duplicate the antitumorigenic effects observed in vitro. Further experimentation is required to investigate the seemingly inconsistent effects of resveratrol using in vivo compared with in vitro models.
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FOOTNOTES |
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3 Abbreviations used: APC, human adenomatous polyposis coli; Apc, murine adenomatous polyposis coli; ApcMin/+, multiple intestinal neoplasia mouse; COX, cyclooxygenase; EIA, enzyme immunoassay; FAP, familial adenomatous polyposis; PGE2, prostaglandin E2; RP-HPLC, reversed-phase HPLC.
Manuscript received 2 July 2003. Initial review completed 23 July 2003. Revision accepted 12 September 2003.
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LITERATURE CITED |
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