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紅酒多 酚.ABSTRACT

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.

INTRODUCTION

Consumption of fruit and vegetables is associated with a reduced risk of cancer especially tumors of the gastrointestinal tract . 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. Red wine contains  different polyphenolic compounds and can be an important dietary source of polyphenols. Some red wine polyphenols such as resveratrol or catechins have been shown to inhibit in vitro and in vivo carcinogenesis.

It has been shown that polyphenols isolated from red wine inhibit the growth of different cancer cells in vitro. 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 p 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. The importance of modulation of MAPK for colon carcinogenesis has been also demonstrated in animal experiments. A synthetic polyphenol (flavonoid) 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 .

In this study, total polyphenolic pool from red wine was isolated by solid-phase extraction and the effects of red wine polyphenols on proliferation and MAPK in human colon carcinoma cells were investigated.

MATERIALS AND METHODS

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 with slight modifications. Briefly, the cartridge as washed with of methanol and equilibrated with 10 mL water. To reduce ethanol content, 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  and the concentration of some major polyphenols such as resveratrol, catechin, epicathehin and  malvidin- 3-glucoside were estimated by HPLC . 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 . One hundred microliters were used for HPLC analysis. The gradient cycle consisted of an initial-min isocratic segment . Then the linear gradient was changed progressively by increasing solution It was maintained  solution A and solution B from  min and then solution B was increased progressively finally changed back to  solution B.

Polyphenols were determined by reverse-phase HPLC using a Nova-Pak C18 column from Waters. Samples were analyzed using a Shimadzu photodiode detector  Shimadzu fluorescence detector 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  using gallic acid as the standard. Results are expressed as millimoles per liter gallic acid equivalents.

Cell culture.

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

The culture medium consisted of DMEM , supplemented with glutamine, penicillin, streptomycin, fetal calf serum. Cells were maintained at 37°C in a humidified atmosphere of  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  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 L during the last 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  using a microplate reader (SpectraFluor Plus; Tecan Deutschland GmbH).

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.

FIGURE 2 Effect of red wine polyphenols on proliferation of the colon carcinoma cell line HT29 clone induced by EGF. Cell proliferation was induced by EGF (1 nmol/L) and determined 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.

Red wine polyphenols inhibited the growth  clone cells in both test systems in a concentration-dependent manner. However, again red wine polyphenols showed a more pronounced inhibitory effect in the BrdU than in the MTT test, indicating that DNA synthesis is more sensitive than cellular metabolism to red wine polyphenols.

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 , while the total red wine polyphenol pool at this concentration had a strong inhibitory effect in the MTT-test (Fig. 2B). Thus, the antiproliferative effect of red wine polyphenols can not be explained by these four compounds.

Modulation of MAPK.

Incubation of serum-starved clone  cells with red wine polyphenols activated JNK and MAPK phosphorylation . JNK and MAPK were activated  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 a weakly activated .

FIGURE 3 Red wine polyphenols induced phosphorylation of JNK (A) and MAPK (B). Serum-starved clone  cells were exposed to red wine polyphenols for the times indicated. Activity was assessed as the phosphorylation of JNK  MAPK in Western blots using phospho-specific antibodies. Immunodetection of total JNK and MAPK served as control. As a positive control, cells were treated with anisomycin  for 1 h. Control treatments were with mitogen vehicle  and control is set equal to 1. Data are means ± SD, n = 3.*Different from ethanol, P < . A representative Western blot of three independent experiments is shown.

FIGURE 4 Red wine polyphenols inhibit phosphorylation of ERK induced by EGF. Cells were preincubated with 6 mmol/L red wine polyphenols (RWP) for  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 ) 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

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 . It has been proposed that red wine polyphenols can inhibit the initiation of carcinogenesis due to their antioxidative or anti-inflammatoric properties. Polyphenols can also act as suppressing agents by inhibition of growth of transformed cells or by inducing apoptosis.

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.

    The grape polyphenol mainly is all a type of flavonoid with red wine polyphenol

As the plain quercetin and small quantity resveratrol of the skin(resveratrol)