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Clinical Cancer Research Vol. 11, 5651-5656, August 1, 2005
© 2005 American Association for Cancer Research

Cancer Prevention

The Chemopreventive Agent Resveratrol Stimulates Cyclic AMP–Dependent Chloride Secretion In vitro

Irina Blumenstein, Benjamin Keserü, Freya Wolter and Jürgen Stein

Authors' Affiliation: Division of Gastroenterology and Clinical Nutrition, 1st Department of Medicine, ZAFES, J.W. Goethe-Universität, Theodor-Stern-Kai 7, Frankfort on the Main, Germany

Requests for reprints: Irina Blumenstein, Division of Gastroenterology and Clinical Nutrition, 1st Department of Medicine, ZAFES, J.W. Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfort on the Main, Germany. Phone: 49-69-6301-5917; Fax: 49-69-6301-83112; E-mail: J.Stein{at}em.uni-frankfurt.de.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Resveratrol and its analogs are promising cancer chemoprevention agents, currently under investigation in clinical trials. However, patients administered other plant polyphenols experienced severe diarrhea, likely due to an increase in intracellular cyclic AMP (cAMP). Resveratrol itself raises intracellular cAMP levels in breast cancer cells in vitro. Its future use as a cancer chemopreventive agent could therefore be compromised by its severe side effects. The aim of the study was (a) to define the influence of resveratrol on intestinal Cl– secretion and (b) to elucidate possible intracellular transduction pathways involved. Resveratrol caused a dose- and time-dependent increase in {Delta}Isc in T84 cells. The specificity of resveratrol was confirmed by using piceatannol 100 µmol/L, the hydroxylated resveratrol analog, which did not alter {Delta}Isc. A significant elevation of [cAMP]i by resveratrol was assessed in T84 cells. In mouse jejunum, resveratrol induced a time- and dose-dependent increase in {Delta}Isc as well. In bilateral Cl–-free medium, as well as after inhibition of protein kinase A, resveratrol-induced {Delta}Isc was reduced significantly. Preincubation of T84 cells with butyrate 2 mmol/L (24 and 48 hours) significantly inhibited resveratrol as well as forskolin-induced Cl– secretion. In summary, the main mechanism of action of resveratrol in intestinal epithelia is cAMP-induced chloride secretion which can be suppressed by butyrate. It can therefore be suggested that in cancer chemoprevention, both agents should be combined to reduce an undesired side effect such as diarrhea and to benefit from the known agonistic effect of both agents on differentiation of colon cancer cells.

Resveratrol (3,4',5-trihydroxystilbene; molecular weight 228.2) is a naturally occurring plant polyphenol able to counteract environmental oxidative stress (1–3). Resveratrol, present in significant levels in grapes, berries, and peanuts, has received wide attention because it has been shown to have antioxidant, antimutagenic, antiinflammatory, and chemopreventive effects. Resveratrol has also been reported to exert substantial cytotoxic effects in different tumor cell lines (4–8). It has been proposed that resveratrol is at least in part responsible for the beneficial effects of moderate red wine consumption on the development of cardiovascular diseases, an effect most probably mediated by its potential to prevent atherosclerosis (9) by modulating the synthesis of hepatic apolipoproteins and lipids (10), inhibiting platelet aggregation (11, 12) and the synthesis of proatherogenic eicosanoids in human platelets and neutrophils (12, 13). Resveratrol is currently under investigation in clinical trials to test the safety and efficiency of resveratrol in the treatment of early stages of cancer (14).

A variety of cellular effectors have been recognized as targets for resveratrol actions: resveratrol activated GC and AC in cardiovascular tissues and blood cells (15, 16). El-Mowafy et al. showed that resveratrol acts as an agonist for the cyclic AMP (cAMP)/protein kinase A (PKA) system, a known proapoptotic and cell cycle suppressor in breast cancer cells (17).

The short-chain fatty acid butyrate is the preferred energy substrate for colonic epithelial cells. It also plays an important role in modulating colonic electrolyte transport. The ability of short-chain fatty acids to stimulate sodium absorption from the mammalian colon has been shown in vivo and in vitro (18, 19). In vivo studies have shown that butyrate inhibits net fluid secretion induced by cholera toxin in the rat colon (20). In rat distal colon mucosa in vitro, as well as in T84 cells, butyrate had an inhibitory effect on cAMP-induced Cl– secretion (15, 21).

Thus, the aim of the present study was to define the influence of resveratrol on intestinal anion secretion and to elucidate intracellular signal transduction pathways involved. Furthermore, we were interested in the influence of butyrate on resveratrol-induced Cl– secretion. In order to gain insight into the epithelial effects of intestinal application of resveratrol, we did electrophysiologic studies in monolayers of T84 colon cancer cells as well as isolated, muscle-stripped mouse jejuni. A better understanding of the mechanisms involved in resveratrol-induced intestinal anion secretion should allow improvement of the therapeutic use of this promising chemopreventive drug.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and solutions. T84 cells were a generous gift from Prof. K.E. Barrett, University of California, San Diego. Cell culture media were obtained from Gibco (Eggenstein, Germany) and newborn calf serum from PAN Biotech (Aidenbach, Germany). All other cell culture supplements were from Sigma (Steinheim, Germany). Permeable supports for Snapwell diffusion chambers were obtained from Costar (Cambridge, MA): Resveratrol, forskolin, tetrodotoxin, indomethacin, bumetanide, and ouabain were purchased from Sigma (Deisenhofen, Germany). The PKA inhibitor H8 was purchased from Biomol (Hamburg, Germany). All chemicals were of the purest quality available.

Cell culture. T84 cells (passages 20-44) were grown in DMEM/F12 mix (1:1), supplemented with 5% newborn calf serum, 100 units/mL penicillin, and 100 µg/mL streptomycin. The medium was changed every other day. Cells were subcultured every 14 days (Dulbecco's PBS containing 0.25% trypsin, 0.1% EDTA). For electrophysiologic measurement, the cells were seeded at a density of 105 cm–2 on permeable supports (Snapwell, 1 cm2 surface area) and grown until confluent.

Measurement of chloride secretion in T84 cells. The permeable supports bearing confluent monolayers were mounted on Snapwell diffusion chambers 12 to 14 days after plating. Only T84 monolayer with initial resistances ≥1,000 {Omega} cm2 were used for experiments. Each experiment was initiated after a 30-minute equilibration period with a stable baseline resistance present. Bicarbonate buffered Ringer solution (115 mmol/L NaCl, 1.2 mmol/L MgCl2, 1.2 mmol/L CaCl2, 2.4 mmol/L K2HPO4, 0.4 mmol/L KH2PO4, 25 mmol/L NaHCO3) supplemented with 10 mmol/L glucose was used in apical and basolateral chambers and was circulated by bubbling with 5% CO2 = 95% O2. Open circuit transepithelial potential differences (Vt) were measured by calomel electrodes immersed in saturated potassium chloride, connected with agarose bridges (4% agarose in 0.9% NaCl solution) to the apical and basolateral reservoir. Studies were conducted under short-circuited conditions except during brief intervals (500 milliseconds) at each time point, when the open circuit potential difference across the monolayer was assessed.

Animals. Male balb6 mice (Charles River) weighing 25 g were maintained under standardized temperature (21-22°C) and light conditions (12:12 hour light/dark cycle) in cages. Mice had access to tap water and pelleted food ad libitum. All animals were allowed to adjust to their new environment for at least 2 weeks. The study conformed to the guiding principles in the care and use of animals and was done with the approval of the ethical committee of Darmstadt, Germany (F134/03).

Tissue isolation. After cervical dislocation with preceding anesthesia (isoflurane), the abdomen was opened and the small bowel was excised. The jejunum was rinsed with ice-cold oxygenated Krebs-Ringer saline. Thereafter, the proximal jejunum was cut open at the insertion of the mesentery and rinsed once more. It was then placed serosal side up into a preparation chamber filled with ice-cold oxygenated standard Krebs-Ringer solution and fixed with insect needles. The serosal layer and the two muscle layers were quickly and carefully removed with a dissecting forceps.

Using chamber experiments. The mucosa was mounted between two lucite half chambers of 0.625 cm2 exposed area and placed into an Ussing apparatus. The bathing solutions were circulated by a gas lift system with 95% O2-5% CO2 at 37°C. The luminal and basolateral solution contained Na+ (140 mmol/L), K+ (4.5 mmol/L), Ca2+ (2 mmol/L), Mg2+ (1.3 mmol/L), Cl– (115 mmol/L), SO42– (1.3 mmol/L), HCO3– (22 mmol/L), HPO42– (1.5 mmol/L), pyruvate (10 mmol/L), and glucose (basolateral, 10 mmol/L) or mannitol (luminal, 10 mmol/L). For ion replacement studies, Cl– was substituted by gluconate. The chloride-free buffer solution contained Na+ (140 mmol/L), K+ (4.5 mmol/L), Ca2+ (2 mmol/L), Mg2+ (1.3 mmol/L), SO42– (1.3 mmol/L), HCO3– (22 mmol/L), HPO42– (1.5 mmol/L), pyruvate (10 mmol/L), gluconate (115 mmol/L), and glucose (basolateral solution, 10 mmol/L), or mannitol (luminal solution, 10 mmol/L). In chloride-free experiments, glucose (25 mmol/L) was applied luminally (to stimulate Na+-glucose cotransport) at the end of each experiment to verify viability of each tissue specimen. Tetrodotoxin (10–6 mol/L) and indomethacin (0.1 mmol/L) were administered to the basolateral solution to abolish the influence of the submucosal plexus and to prevent endogenous prostaglandin production, respectively. Drugs were administered at the indicated times. Ouabain was applied as a standard test for active secretion and viability at the end of each experiment.

Electrophysiology. The open circuit transepithelial electrical potential difference as well as the tissue resistance was recorded (Ussing Chamber System, KMSCI, Aachen, Germany) via AgCl glass electrodes. Under open-circuit conditions, short circuit current (Isc) was calculated from potential difference and tissue resistance. Before the tissue was placed into the chamber, the series resistances of the solutions and the chamber were assessed, and a fluid resistance compensation was done before each experiment. For all electrophysiologic experiments, independent controls were processed in parallel.

Measurement of intracellular cAMP. cAMP levels were measured in the lysates of stimulated or control cell T84 monolayers using a commercially available direct cAMP enzyme immunoassay kit (Sigma). T84 cells grown on Millipore filters (Ø 0.4 µm) were treated by resveratrol (100 and 400 µmol/L) added to the apical compartment. The assay was done according to the instructions manual for nonacetylated probes.

Statistical analysis. All data are expressed as means ± SE for a series of experiments. Statistical significance was calculated using the t test for unpaired values or ANOVA with Bonferroni post hoc test, as appropriate.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Effects and specifity of resveratrol. Resveratrol, applied apically, increased the Isc response of T84 monolayers from 0.16 ± 0.71 (25 µmol/L) up to 76 ± 6.08 µ A/cm2 (400 µmol/L). The Isc was not altered by application of resveratrol in concentrations <25 µmol/L, whereas higher concentrations of resveratrol led to a highly significant increase in {Delta}Isc. This stimulation of {Delta}Isc was time- and dose-dependent (Fig. 1). Short circuit current as well as total epithelial resistance remained unaffected, when resveratrol was applied from the basolateral side (data not shown). The cytotoxicity of resveratrol was excluded by showing that carbachol- or forskolin-stimulated Isc was fully preserved at the end of each experiment (data not shown).



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  		Fig. 1. Resveratrol, applied apically, stimulated a dose-dependent increase in {Delta}Isc (n = 4; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

 
Effects of piceatannol on intestinal Cl– secretion in T84 cells. To exclude any unspecific effect of resveratrol on Isc, we compared effects of resveratrol and its hydroxylated analog piceatannol at concentrations of 100 and 400 µmol/L. Unlike resveratrol, piceatannol did not affect transepithelial resistance nor Isc, whereas subsequent stimulation with resveratrol (100 µmol/L) led to a significantly reduced increase in {Delta}Isc (Fig. 2).



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  		Fig. 2. Effect of piceatannol, a hydroxylated analog of resveratrol, on {Delta}Isc in T84 cells. Piceatannol was applied apically and caused nonsignificant change in {Delta}Isc (n = 4, not significant). The subsequent stimulation of {Delta}Isc by resveratrol (100 µmol/L) was inhibited by ~50% (P < 0.01 versus control, n = 4).

 
Effect of resveratrol on forskolin-induced Cl– secretion in T84 cells. We investigated the influence of forskolin (10 µmol/L) on resveratrol-induced intestinal chloride secretion. Forskolin is a potent stimulator of the cAMP-dependent intestinal Cl– secretion. Incubation of T84 cells with forskolin (10 µmol/L) led to a marked stimulation of {Delta}Isc (60.33 ± 3.66 µA/cm2), subsequent addition of resveratrol (100 µmol/L) to the apical side was not able to further stimulate {Delta}Isc (data not shown). On the contrary, preincubation of cells with resveratrol (100 µmol/L) for 30 minutes did not alter forskolin-induced (10 µmol/L) changes in {Delta}Isc ({Delta}Isc 49.32 ± 3.7 µA/cm2 versus {Delta}Isc 50 ± 4.32 µA/cm2; n = 3, not significant).

Resveratrol-induced intestinal chloride secretion in mouse jejuni. In order to predict the potential of resveratrol to induce secretory diarrhea, we were interested in the effect of resveratrol in intact epithelia. Thus, muscle-stripped mouse jejuni were analyzed in Ussing chambers. After stable tissue parameters were reached, resveratrol was added subsequently in 20-minute intervals in rising concentrations (100, 400, and 1,000 µmol/L) to the luminal bath. A time- and dose-dependent increase in {Delta}Isc could be seen (Fig. 3). Interestingly, forskolin, given consecutively to the serosal bath, increased {Delta}Isc considerably as well, suggesting that resveratrol (1 mmol/L) did not entirely exhaust the intracellular capacity for cAMP generation. Ouabain was applied to the serosal bath at the end of each experiment as a test for active transport processes as well as tissue viability.



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  		Fig. 3. Resveratrol, applied apically, stimulated a time- and dose-dependent increase in {Delta}Isc (n = 3) in mouse jejuni (P < 0.5 for resveratrol 1,000 µmol/L).

 
Influence of resveratrol on [cAMP]i in T84 cells. To elucidate the intracellular signal transduction pathways mediating the effect of resveratrol, we measured intracellular cAMP levels in T84 cells. Figure 4 shows the intracellular cAMP levels after stimulation with resveratrol (100 µmol/L). Basal intracellular cAMP concentration was 3.29 ± 0.61 pmol/mg protein. Incubation with resveratrol (100 µmol/L) led to a significant increase in intracellular cAMP levels after 10 minutes. The peak level was 6.14 ± 0.55 pmol/mg protein (n = 3, P < 0.05). This maximum of intracellular cAMP concentration coincided with the maximum increase in {Delta}Isc after stimulation with resveratrol in cell culture experiments.



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  		Fig. 4. Changes in [cAMP]i after incubation with resveratrol were time-dependent (n = 3; *, P < 0.05). Values are given as picomoles of cAMP per milligram of protein.

 
Ion replacement studies and blocking of downstream targets of the cAMP second messenger system. In order to identify the ion transport processes responsible for the Isc induced by resveratrol in the proximal mouse jejunum, ion replacement studies were done, replacing chloride by gluconate in the luminal and serosal compartment. After stable tissue parameters were reached, resveratrol (400 µmol/L) was applied apically. The increase in {Delta}Isc induced by resveratrol was reduced significantly by 86% ({Delta}Isc 7.97 ± 2.63 µA/cm2 versus {Delta}Isc 57.24 ± 4.02 µA/cm2 in control experiments in chloride-containing buffer, P < 0.001; Fig. 5). The residual {Delta}Isc was insensitive to basolateral application of bumetanide (0.5 mmol/L), applied at the end of each experiment (data not shown), whereas the response to luminal application of glucose (25 mmol/L) was fully preserved (data not shown). To further characterize the intracellular pathway involved in resveratrol stimulation of intestinal chloride secretion, an inhibitor of PKA (H8, 50 µmol/L) was applied on both sides 30 minutes before stimulation with resveratrol. The effect of inhibition of PKA was a significant reduction of {Delta}Isc stimulated by resveratrol of 60% ({Delta}Isc 23.67 ± 2.63 versus {Delta}Isc 57.24 ± 4.02 µA/cm2, P < 0.001; Fig. 5).



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  		Fig. 5. Effect of bilateral Cl–-free bathing solution and the protein kinase inhibitor H8 (50 µmol/L) on short circuit current (Isc) in muscle-stripped mouse jejuni. Columns, means; bars, ± SE; n = 5; ***, P < 0.001 versus control.

 
Effect of butyrate on resveratrol-induced intestinal chloride secretion in T84 cells. T84 monolayers were incubated with 2 mmol/L sodium butyrate for 24 and 48 hours, respectively. Afterwards {Delta}Isc was stimulated with resveratrol (100 µmol/L) apically or with forskolin (10 µmol/L) basolaterally. Forskolin as well as resveratrol-induced stimulation of {Delta}Isc was inhibited at a significant level. The forskolin-induced increase in {Delta}Isc was diminished by 40% after 24 hours of incubation with 2 mmol/L butyrate (30.63 ± 5.13 versus 49.32 ± 3.7 µA/cm2, P < 0.05). After 48 hours, the increase in {Delta}Isc was reduced by 55% (22.5 ± 3.05 µA/cm2 versus control; P < 0.001). The resveratrol-induced change in {Delta}Isc was cut down to ~50% after 24 hours of incubation with 2 mmol/L butyrate (10.56 ± 1.59 versus 21.2 ± 1.0 µA/cm2, P < 0.01). This reduction was not further enhanced after 48 hours of incubation (see Fig. 6).



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  		Fig. 6. Butyrate significantly inhibited resveratrol- and forskolin-induced changes in {Delta}Isc (n = 4-7; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we provided several lines of evidence showing: (a) that resveratrol led to a significant time- and dose-dependent stimulation of {Delta}Isc in T84 intestinal colon cancer cell monolayers as well as in isolated and muscle-stripped mouse jejuni; (b) that this stimulatory process was caused by an increase in intracellular concentration of the second messenger cAMP. This elevation of [cAMP]i drives chloride secretion, as ion replacement studies as well as inhibition of PKA could reveal; and (c) that the resveratrol-induced changes in {Delta}Isc could be prevented by incubation of cells with sodium butyrate (2 mmol/L) for at least 24 hours. This study therefore elucidates the mechanisms by which resveratrol may enhance intestinal secretory processes, thereby narrowing the potential use of resveratrol as a chemopreventive agent in intestinal malignancy.

T84 cells have been widely used as a model system to define regulatory mechanisms for intestinal Cl– secretion (22). By using these cells grown as monolayers, we were able to show that their stimulation with resveratrol increases {Delta}Isc in a dose-dependent manner (Fig. 1). Concordantly, in mouse jejuni, resveratrol elicited a significant time- and dose-dependent increase in {Delta}Isc as well (Fig. 3). Stimulation of Cl– currents by resveratrol in Calu-3 human epithelial airway cells has been reported by Illek et al. (23). In this study, resveratrol stimulated a maximum current of 88.8 µA/cm2, with a half-maximal stimulatory concentration of 39.0 ± 11 µmol/L. Interestingly, high doses of resveratrol had a blocking effect on {Delta}Isc in Calu-3 epithelia with the half-maximal blocking concentration (Kb) being 435 ± 45 µmol/L.

Concerning the bioavailability of resveratrol after oral application, the highest plasma concentration in humans was detected after 30 minutes (7.1 µg/L free resveratrol and 338 µg/L conjugated resveratrol after oral application of 25 mg resveratrol) and returned to the base level after 2 hours. The rate of conjugated resveratrol was 30 to 50 times higher than the concentration of free resveratrol. During 24 hours, 24.6% of the stilbene were excreted with the urine (24). To our knowledge, no data exists about the bioavailability of stilbene glucosides. Because glucosides of flavonoids are absorbable, it is likely that stilbene glucosides are bioavailable as well (25). Thus far, no data are available regarding the absorption and metabolism of piceatannol. The extent of absorption of resveratrol from the normal diet is largely unknown, and may also depend on the degradability of resveratrol-glucosides (e.g. piceid) and resveratrol-polymers (e.g. viniferins) in the gut.

Other known flavonoids, e.g., flavopiridol, genistein and apigenin, are either direct chloride secretagogues at high doses or potentiate responses to other stimuli at low doses in Calu-3 cells (26), T84 colonic epithelial cells (27–30) and in rat distal colon (31, 32).

Stimulation of T84 cells with the hydroxylated resveratrol analogue piceatannol (trans-3,4',3',5-tetrahydroxystilbene) in concentrations up to 100 µmol/L did not affect {Delta}Isc (Fig. 2). It could therefore be suggested that the lacking hydroxyl group at position 3' seems to be crucial for the stimulatory potency of resveratrol. Other flavonoids (e.g., quercetin, luteolin, and genistein), however, having an identical hydroxylation pattern as piceatannol at position 3', are potent stimulators of intestinal Cl– secretion in various systems (29, 31). Quercetin, luteolin, as well as genistein, possess a central pyrone ring, which is a key contributor to the affinity of flavonoids to the stimulatory binding site of CFTR in Calu-3 human epithelial airway cells (23). In this study, kinetics of flavonoids such as apigenin (4',5,7-trihydroxyflavone) and resveratrol were significantly changed in tissues prestimulated with forskolin. We therefore assessed the effect of resveratrol on {Delta}Isc after stimulation with forskolin. In our study, resveratrol failed to elicit a secretory response after prestimulation with 10 µmol/L forskolin, leading to the conclusion that cAMP-dependent activation of CFTR by forskolin leads to either a conformational change of the stimulatory binding site of resveratrol, or alternatively, that resveratrol uses the same intracellular signal transduction pathway indicating Michaelis-Menten saturation kinetics during stimulation with the two substances. The latter hypothesis was further strengthened by the observation that reversal of the stimulatory sequence showed no change in forskolin-induced {Delta}Isc (data not shown). In prestimulated Calu-3 cells, apigenin was able to additionally activate Cl– currents markedly (23). However, resveratrol-induced stimulation of {Delta}Isc was prevented after prestimulation with forskolin, indicating that the pyrone ring is the most significant part of apigenin for stimulation of {Delta}Isc after forskolin treatment. Another interesting fact concerning interaction of flavonoids and common secretagogues appeared as flavopiridol's use in clinical trials as a potent chemotherapeutic agent against refractory cancers was compromised by severe diarrhea (33, 34). In order to elicit the underlying pathophysiologic mechanism, Kahn et al. tested the ability of flavopiridol to modify the chloride secretory response in T84 cells. High concentrations of flavopiridol had a direct stimulatory effect on chloride secretion, probably due to an increase in [cAMP]i. Lower, clinically relevant concentrations of flavopiridol had no effect on chloride secretion by themselves but potientiated the responses to thapsigargin and taurodeoxycholate and reversed the inhibitory effect of carbachol and epidermal growth factor on calcium-dependent chloride secretion (30).

In this study, we aimed to further characterize the particular mechanism of resveratrol-induced changes in {Delta}Isc. From our first series of experiments, it seemed likely that forskolin and resveratrol might use the same intracellular transduction pathway to induce intestinal Cl– secretion. Indeed, resveratrol treatment of T84 cells resulted in a significant increase in [cAMP]i (Fig. 4). In mouse jejunal epithelial membrane, ion replacement studies in bilateral chloride-free medium revealed that the main anion secreted in response to resveratrol seems to be chloride (Fig. 5). In the absence of chloride, the residual {Delta}Isc elicited by resveratrol is most likely due to bicarbonate secretion, as seen in normal mouse jejunum (35, 36). In our study, PKA inhibitor H8 was able to inhibit resveratrol-induced chloride secretion (Fig. 5), indicating that the cAMP/PKA pathway is mediating this secretory process. Accordingly, resveratrol treatment led to an increase of intracellular cAMP in breast cancer cells (17) and polymorphonuclear leukocytes (37). In contrast to our findings, resveratrol had no effect on basal or forskolin-stimulated cAMP levels in coronary artery smooth muscle cells. Instead, short-term treatment with resveratrol substantially inhibited mitogen-activated protein kinase activity, thereby suppressing the influence of endothelin-1, a primary antecedent of coronary heart disease (38). Moreover, resveratrol could interact directly with CFTR as this mode of interaction could be shown for other flavonoids, e.g., genistein and quercetin. Indeed, quercetin-induced Cl– secretion was at least in part mediated by activation of basolateral K+ channels in the rat distal colonic mucosa (39).

The effect of butyrate on Cl– secretion is of particular clinical interest, because it may reduce colonic secretion in secretory diarrhea. In our experiments, butyrate was a potent inhibitor of resveratrol as well as forskolin-dependent stimulation of {Delta}Isc (Fig. 6). As shown earlier, short-chain fatty acids inhibit cAMP-mediated chloride secretion in rat and rabbit colon (15, 40). In Madin-Darby canine kidney cells and Calu-3 cells, butyrate (5 mmol/L) reduced basal as well as cAMP-stimulated Isc (41, 42). In contrast, lower concentrations of phenylbutyrate (<2 mmol/L) had no effect on cAMP-stimulated Cl– secretion across Calu-3 cells (42). In several studies, the effect of butyrate on Cl– secretion in T84 cells was investigated. Butyrate inhibited Cl– secretory responses to prostaglandin E2, forskolin, and cholera toxin by reducing expression and activity of adenyl cyclase and decreasing expression of the basolateral Na-K-2Cl cotransporter (21, 43, 44).

In summary, our findings show that resveratrol is able to stimulate a cAMP-dependent Cl– secretion in T84 colon cancer cells and mouse jejunal epithelial membrane, providing evidence that resveratrol could induce secretory diarrhea. Pretreatment with sodium butyrate prevented the resveratrol-induced Cl– secretion. The underlying mechanism is most probably the above mentioned reduced expression of adenyl cyclase and the decreased expression of the basolateral Na-K-2Cl cotransporter, which leads to an impairment of basolateral Cl– entry into the cell. Thus, this study provides experimental evidence that, in cancer chemoprevention, some additional attention should be paid to a possible combination of both agents in order to reduce diarrhea, as well as to benefit from the known agonistic effect of both agents on differentiation in colon cancer cells.


   Acknowledgments
 
This work is dedicated to the 65th anniversary of Prof. Dr. W.F. Caspary. The authors thank Carolin Daniel for help with the in vivo experiments, and Vladan Milovic for critical reading of the manuscript.


   Footnotes
 
Grant support: Else Kröner-Fresenius-Foundation (Bad Homburg, Germany).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: This study was presented in part at the Digestive Disease Week 2004 in New Orleans.

Received 12/28/04; revised 4/21/05; accepted 5/ 9/05.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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