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Nutrition and Cancer

Red Wine Polyphenols Inhibit the Growth of Colon Carcinoma Cells and Modulate the Activation Pattern of Mitogen-Activated Protein Kinases¹

Institute for Nutritional Physiology, Federal Research Center for Nutrition, Karlsruhe, Germany

²To whom correspondence should be addressed. E-mail: karlis.briviba{at}bfe.uni-karlsruhe.de.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Red wine is a rich source of polyphenols, which exhibit a number of biological effects in different in vitro and in vivo systems. The bioavailability of polyphenols is poor and the plasma concentrations of major red wine polyphenols are usually low after consumption of dietary relevant amounts of red wine. In contrast to most organ systems, the gastrointestinal tract (particularly the epithelial cells of this organ system) is exposed to high concentrations of polyphenols. Here, we show that the total polyphenol pool isolated from a red wine (varity Lemberger, vintage 1998) at micromolar concentrations inhibited the proliferation of transformed colon epithelial cells HT 29 clone 19A induced by epidermal growth factor (EGF). Inhibition of proliferation was also associated with modulation of activation of mitogen-activated protein kinases (MAPK). Stress activated c-Jun N-terminal kinases 1/2 (JNK) and p38 MAPK were significantly activated by red wine polyphenols (6 mmol/L). Maximum phosphorylation of both MAPK was observed after a 1-h treatment with red wine polyphenols. In contrast, activation of extracellular signal regulated kinase (ERK) 1/2 by EGF (1 nmol/L) was significantly inhibited by red wine polyphenols (6 mmol/L). This signaling pattern, activation of JNK 1/2 and p38 MAPK and inhibition of ERK 1/2, is typical for antiproliferative compounds, indicating that red wine polyphenols may inhibit the proliferation of colon carcinoma cells by modulating MAPK intracellular signal transduction pathways.

KEY WORDS: • red wine polyphenols • intracellular signaling • mitogen-activated protein kinases

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Consumption of fruit and vegetables is associated with a reduced risk of cancer especially tumors of the gastrointestinal tract (1 ). It has been suggested that phytochemicals including polyphenols may be responsible for these effects. Numerous phenolic compounds have been reported to exhibit chemopreventive effects in different in vitro and animal model systems by affecting the induction or promotion phase of carcinogenesis (2 ). Red wine contains >200 different polyphenolic compounds and can be an important dietary source of polyphenols (3 ). Some red wine polyphenols such as resveratrol or catechins have been shown to inhibit in vitro and in vivo carcinogenesis (4 ,5 ).

It has been shown that polyphenols isolated from red wine inhibit the growth of different cancer cells in vitro (6 ,7 ). The molecular mechanisms of these effects of red wine polyphenols are poorly understood. Mitogen-activated protein kinases (MAPK)³ , such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK, are involved in signal transduction from the cell surface to the nucleus and regulate cellular processes, including proliferation, differentiation, cell growth arrest and apoptosis, which are also important for the promotion phase of carcinogenesis (Fig. 1 ). The importance of modulation of MAPK for colon carcinogenesis has been also demonstrated in animal experiments. A synthetic polyphenol (flavonoid) PD98059, which is a specific inhibitor of ERK upstream activators MAPK kinase (MKK) 1 and MKK 2, inhibited tumor growth in mice with colon carcinomas of both mouse and human origin by 80% (8 ).

View larger version (22K): [in this window] [in a new window]   FIGURE 1 Signal transduction from the cell surface through mitogen-activated protein kinase (MAPK) cascades. The scheme is largely based on the work by Pearson et al., Whitmarsh and Davis, and Lewis et al. (26 –28 ). ASK1, apoptosis signal-regulated kinase 1; MEKK1, MAP kinase kinase kinase 1; TAK1, TGF ß-activated kinase 1; ERK, extracellular signal-regulated kinase; JNK, Jun N-terminal kinase.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Chemicals.

Unless otherwise stated, all chemicals were purchased from Merck (Darmstadt, Germany). Dulbecco’s modified Eagle’s minimum essential medium (DMEM), glutamine, penicillin, streptomycin and phosphate-buffered saline (PBS) without Mg²⁺ and Ca²⁺ were purchased from Life Technologies (Eggenstein, Germany). Fetal calf serum and 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was from Roche (Mannheim, Germany). Protein assay was from Bio-Rad (München, Germany).

Malvidin-3-glucoside was purchased from Polyphenols AS (Sandnes, Norway). The dry red wine grape variety (Lemberger, vintage 1998) was provided by State Winery Weinsberg (Weinsberg, Baden-Württemberg, Germany).

Isolation of red wine polyphenols.

Polyphenols in red wine were extracted using a solid-phase extraction cartridge as described previously (9 ) with slight modifications. Briefly, the cartridge (Sep-Pak C18, 2 g; Waters, Milford, MS) was washed with 15 mL of methanol and equilibrated with 10 mL water. To reduce ethanol content, 30 mL red wine was incubated at 30°C under N₂ stream for 45 min. The red wine was then applied to the equilibrated cartridge. The water-soluble compounds were removed by 10 mL water. Polyphenols were eluted with 12 mL methanol. The methanol phase was collected and the solvent was removed under N₂. The samples were redissolved in 1 mL PBS containing 12% alcohol. An aliqout was used to estimate the total polyphenol concentration by the Folin-Ciocalteu assay (10 ) and the concentration of some major polyphenols such as resveratrol, catechin, epicathehin and malvidin-3-glucoside were estimated by HPLC (9 ). The red wine polyphenols were then diluted to achieve a polyphenol concentration 10-fold higher than in red wine.

Detection of red wine polyphenols by HPLC.

Red wine or extracted red wine polyphenols were diluted with HPLC mobile phase: solution A, [4/4/92 CH₃OH/CH₃CN/87 mmol/L H₃PO₄ in H₂O (v/v/v)] and centrifuged at 14000 x g for 10 min. One hundred microliters were used for HPLC analysis. The gradient cycle consisted of an initial 15-min isocratic segment (solution A, 100%). Then the linear gradient was changed progressively by increasing solution B (100% CH₃CN) to 11% at 25 min and 14% at 32 min. It was maintained at 86% solution A and 14% solution B from 32 to 40 min and then solution B was increased progressively to 20% at 50 min and finally changed back to 0% solution B.

Polyphenols were determined by reverse-phase HPLC using a Nova-Pak C18 column (4 µm, 4.6 x 250 mm) from Waters. Samples were analyzed using a Shimadzu photodiode detector at 280, 350 and 520 nm and a Shimadzu fluorescence detector (_ex280/_em320 nm) for catechin. A binary gradient and a total flow rate of 1 mL/min were used. Polyphenolic compounds were identified by comparing their retention time and UV-vis spectra with those of standards. Catechin and epicatechin were also identified by fluorescence detection. The concentrations of polyphenols were calculated from the calibration curves made with standard solutions.

Detection of total polyphenols.

The concentration of total polyphenols in red wine and extracts was measured by the Folin-Ciocalteu assay (10 ) using gallic acid as the standard. Results are expressed as millimoles per liter gallic acid equivalents.

Cell culture.

A clone HT29 19A was isolated from the parent cell line HT29 derived from a human colon adenocarcinoma (11 ). HT clone 19A was terminally differentiated with 5 mmol/L sodium butyrate and was a gift from L. Laboisse (Institut Nartional de la Santé et de la Recherche Méedicale, Paris, France) (12 ). Compared with HT 29 stem cells, HT 29 clone 19A cells exhibit morphological cell polarity and are able to form domes representing active transepithelial transport.

The culture medium consisted of DMEM (with 4.5 g/L glucose), supplemented with 2 mmol/L glutamine, 25,000 IU/L penicillin, 25 mg/L streptomycin, and 10% (v/v) fetal calf serum. Cells were maintained at 37°C in a humidified atmosphere of 5% CO₂ in air. The culture medium was replaced three times a week, and cells were used 1 d after change of the culture medium.

Cell proliferation assay.

For the determination of cellular proliferation, cells were seeded into microtiter plates (24 wells) at a concentration of 6 x 10⁴ cells/well and incubated for 48 h with serum-free culture medium in the presence and absence of EGF and compounds tested. Living and metabolically active cells were determined via the reduction of MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide) to form a blue-colored formazan. For this, the assay was performed according to the manufacturer’s protocol. Cells were incubated with MTT for 2 h and the formed formazan was measured using a microplate reader (SpectraFluor Plus; Tecan Deutschland GmbH, Crailsheim, Germany) set to read the difference between the absorption at 560- and 690-nm wavelengths.

5-Bromo-2'deoxyuridine-Test (BrdU-Test).

Cells were seeded into microtiter plates (96 wells) at a concentration of 2 x 10⁴ cells/well and incubated for 48 h with serum-free culture medium in the presence and absence of EGF and compounds tested. BrdU was added at a concentration of 10 µmol/L during the last 3 h of incubation. After removing the labeling medium, cells were fixed and DNA was denaturated. Incorporated BrdU was labeled by a monoclonal anti-BrdU antibody conjugated with peroxidase. The immune complexes were detected by the subsequent substrate reaction and quantified by measuring the absorbance at 450 nm using a microplate reader (SpectraFluor Plus; Tecan Deutschland GmbH).

Western blotting.

HT 29 clone 19A cells were grown to 90% confluency in 30-mm dishes. Cells were then washed twice with PBS and incubated with serum-free medium for an additional 2 d to avoid a high background activation of MAPK by growth factors present in serum. In experiments to determine red wine polyphenol effects on JNK and p38 MAPK, cells were treated with red wine polyphenols for different lengths of time. In experiments to determine effects on ERK phosphorylation, cells were preincubated with red wine polyphenols or an inhibitor of MAP kinase kinase 1, PD-98059 for 1 h and then exposed to EGF for 10 min.

After treatment, cells were washed with PBS and lysed by scraping in 2X SDS-PAGE lysis buffer (125 mmol/L Tris, 150 mmol/L SDS, 350 mmol/L glycerol, 100 mmol/L DTT, and 0.29 mmol/L bromophenol blue, pH 6.8). The lysates were heated at 95°C for 5 min and used for SDS-polyacrylamide gel electrophoresis or frozen until use. Samples (25 µL) were subjected to gel electrophoresis on 12% SDS-polyacrylamide gels and blotted onto PVDF membranes (Hybond-P; Amersham Pharmacia Biotech Europe GmbH, Freiburg, Germany). Immunodetection of phosphorylated JNK, p38, and ERK 1/2 were with -phospho-JNK, -phospho-p38, and -active-MAPK (New England Biolabs, Schwalbach, Germany) antibodies, respectively, using the enhanced chemifluorescence Western blotting kit (Amersham Pharmacia Biotech Europe GmbH). After stripping, the membrane was reprobed with -JNK or -p38 or polyclonal MAPK (New England Biolabs) antibodies, which served as gel loading and protein controls.

Statistics.

Results are reported as means ± SD Results were analyzed by one-way ANOVA and the Dunnett’s test to identify significant differences from the control. Differences with P values < 0.05 were considered significant. Analyses were performed using StatView (SAS Institute, Cary, NC).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The concentrations of the main polyphenols in dry red wine from the grape variety Lemberger (vintage 1998) used in our experiments were in the concentration range typical for red wines (Table 1 ).

EGF (1 nmol/L) stimulated growth of HT 29 clone 19A cells compared with cells that were cultured in serum-free medium. EGF induced a fivefold increase as assessed by the BrdU test, which assesses the DNA synthesis activity (Fig. 2A ). In the MTT test, EGF induced cell proliferation by 60%, indicating a lower sensitivity of this assay (Fig. 2 B).

View larger version (27K): [in this window] [in a new window]   FIGURE 2 Effect of red wine polyphenols on proliferation of the colon carcinoma cell line HT29 clone19A induced by EGF. Cell proliferation was induced by EGF (1 nmol/L) and determined 48 h after incubation of cells with the respective additives (red wine polyphenols) by the BrdU-test (A) and by the MTT test (B). Control experiments were done in the absence of EGF and tested compounds and were set to 100%. Data are means ± SD, n > 6. Columns with index letter a differ significantly from control, P < 0.05; columns with index letter b differ significantly from EGF (1 nmol/L), P < 0.05.

A mixture of four major red wine polyphenols (malvidin-3-glucoside, catechin, epicathehin, resveratrol) prepared at the concentration ratio estimated in Lemberger red wine had no effect on cell growth when tested up to 60 µmol/L, while the total red wine polyphenol pool at this concentration had a strong inhibitory effect in the MTT-test (Fig. 2 B). Thus, the antiproliferative effect of red wine polyphenols can not be explained by these four compounds.

Modulation of MAPK.

Incubation of serum-starved HT29 clone 19A cells with red wine polyphenols activated JNK and p38 MAPK phosphorylation (Fig. 3, A and B ). JNK and p38 MAPK were activated (P < 0.05) by 6 mmol/L red wine polyphenols, a concentration corresponding with undiluted red wine. Maximum phosphorylation of both MAPK was observed after a 1-h treatment. Red wine polyphenols at 0.6 mmol/L weakly activated JNK 1/2 (P = 0.226, 30 min).

View larger version (19K): [in this window] [in a new window]   FIGURE 3 Red wine polyphenols induced phosphorylation of JNK (A) and p38 MAPK (B). Serum-starved HT29 clone 19A cells were exposed to red wine polyphenols for the times indicated. Activity was assessed as the phosphorylation of JNK and p38 MAPK in Western blots using phospho-specific antibodies. Immunodetection of total JNK and p38 MAPK served as control. As a positive control, cells were treated with anisomycin (10 ng/L) for 1 h. Control treatments were with mitogen vehicle (ethanol 1.2%) and control is set equal to 1. Data are means ± SD, n = 3.*Different from ethanol, P < 0.05. A representative Western blot of three independent experiments is shown.

View larger version (21K): [in this window] [in a new window]   FIGURE 4 Red wine polyphenols inhibit phosphorylation of ERK induced by EGF. Cells were preincubated with 6 mmol/L red wine polyphenols (RWP) for 60 min and exposed to EGF (1 nmol/L) for 10 min in the presence of polyphenols. Activity was assessed as the phosphorylation of ERK in Western blots using a phospho-specific antibody. Densitometric data were standardized to total ERK. Control treatments were with mitogen vehicle (ethanol 1.2%) and control is set to 1. Data are means ± SD, n = 3. *Different from EGF (1 nnol/L), P < 0.05; #different from control, P < 0.05. A representative Western blot of three independent experiments is shown.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Polyphenols from vegetable foods and some beverages such as tea are believed to be responsible for the observed reduced risk of cancer. Evidence supporting this hypothesis is based on epidemiological observations, animal studies and cell culture experiments. Red wine can also be an important dietary source of polyphenols (3 ). It has been proposed that red wine polyphenols can inhibit the initiation of carcinogenesis due to their antioxidative or anti-inflammatoric properties (4 ,13 ). Polyphenols can also act as suppressing agents by inhibition of growth of transformed cells or by inducing apoptosis (14 –17 ).

In this study we have shown that red wine polyphenols inhibit the growth of colon carcinoma cells at micromolar concentrations that seem to be dietary relevant for the gastrointestinal tract. The concentration of polyphenols in red wines has been estimated to be within the millimolar range. Absorbance of most red wine polyphenols is low and these compounds can arrive in the intestine at relatively high concentrations (9 ,18 ).

The molecular mechanism of the antiproliferative action of red wine polyphenols is poorly understood. There is evidence that inhibition of proliferation of vascular smooth muscle cell proliferation is associated with the down-regulation of expression of cyclin A (7 ). Furthermore, red wine polyphenols did not modulate expression of the p53 gene, indicating that other mechanisms are responsible for the anticarcinogenic effects in breast cancer cells (19 ). The red wine stilbene resveratrol is a remarkable inhibitor of ribonucleotide reductase and DNA synthesis in mammalian cells (20 ). The antitumor and antimetastatic activities of resveratrol glucoside, piceid, were proposed to be due to the inhibition of DNA synthesis in Lewis lung carcinoma tumor cells and angiogenesis of human umbilical vein endothelial cells (21 ). This observation is also in line with our data demonstrating effective inhibition of DNA synthesis by red wine polyphenols.

Here, we investigated the modulation of one important signal transduction cascade involved in controlling mitogenesis. Three MAPK cascades appear to transduce the majority of extracellular signals from the cell membrane to the nucleus and regulate cellular processes such as proliferation, differentiation and cell death. These cascades are the ERK cascade, the JNK cascade and the p38 MAP kinase cascade. Generally, the ERK signal transduction pathway is activated by growth factors and is important for proliferation (22 ). In contrast, JNK and p38 MAPK are activated by cytokines mediating growth arrest and apoptosis (23 –25 ). In this study, EGF-induced phosphorylation of ERK MAP kinase was inhibited by red wine polyphenols. Furthermore, JNK and p38 MAPK were activated by red wine polyphenols. This signaling pattern is typical for antiproliferative compounds, indicating that red wine polyphenols may inhibit proliferation colon carcinoma cells by modulating MAPK intracellular signal transduction pathways.

	ACKNOWLEDGMENTS

 
We appreciate the technical assistance of Renate Lambertz.

	FOOTNOTES

 
¹ Supported by the Federal Ministry of Consumer Protection, Nutrition and Agriculture, Germany.

³ Abbreviations used: BrdU, 5-bromo-2'deoxyuridine; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinases; MKK, MAPK kinase; MTT, 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide; RWP, red wine polyphenols.

Manuscript received 27 February 2002. Initial review completed 7 April 2002. Revision accepted 5 June 2002.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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