Mutation Research 496 (2001) 171–180
Resveratrol, a natural phenolic compound, inhibits cell proliferation and prevents oxidative DNA damage Alessandro Sgambato∗ , Raffaele Ardito, Beatrice Faraglia, Alma Boninsegna, Federica I. Wolf, Achille Cittadini Centro di Ricerche Oncologiche “Giovanni XXIII” — Istituto di Patologia Generale, Catholic University, Largo Francesco Vito 1, 00168 Rome, Italy Received 23 October 2000; received in revised form 31 January 2001; accepted 31 January 2001
Abstract Resveratrol (3,4 ,5-trihydroxystilbene) is a naturally occurring phenolic compound which is present at high levels in wine and has been recently proposed as a potential cancer chemopreventive and chemoterapeutic agent. In this study, we evaluated the antiproliferative activity of resveratrol on a panel of cell lines of various histogenetic origin, including normal rat fibroblasts and mouse mammary epithelial cells compared to human breast, colon and prostate cancer cells. The concentration of resveratrol inhibiting cell growth by 50% (IC50 ) ranged from about 20 to 100 M. At such concentration, we were unable to detect a significant increase in the apoptotic index in most of the cell lines analyzed. We also studied the effects of resveratrol on cell cycle distribution. The most striking effect was a reduction in the percentage of cells in the G2/M phase which was most frequently associated with an increase of cells in the S phase of the cell cycle. We also found that resveratrol is able to prevent the increase in reactive oxygen species (ROS) following exposure to oxidative agents (i.e. tobacco-smoke condensate (TAR) and H2 O2 ). Resveratrol also reduced nuclear DNA fragmentation, as assessed by single cell gel electrophoresis (comet test). Taken together our results suggest that resveratrol can act as an antimutagenic/anticarcinogenic agent by preventing oxidative DNA damage which plays a pivotal role in the carcinogenic activity of many genotoxic agents. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Resveratrol; Cell proliferation; Oxidative DNA damage; Chemoprevention; Comet test
1. Introduction Resveratrol (3,4 ,5-trihydroxystilbene) is a naturally occurring phenolic compound found in the berry skins of most grape cultivars and in a variety of medicinal plants where it functions as a phytoalexin protecting against fungal infections [1]. Recent evidence suggests that this molecule can affect many biological activities. Indeed, it has been proposed that ∗ Corresponding author. Tel.: +39-06-301-6619; fax: +39-06-301-2753. E-mail address:
[email protected] (A. Sgambato).
resveratrol, which is present at high levels in wine, may explain for the reduced risk of coronary heart disease associated with moderate wine consumption [2]. This effect has been attributed to the inhibition of oxidation of low-density lipoprotein cholesterol [3], platelet aggregation and coagulation [4,5]. Resveratrol has also been reported to compete with 17-estradiol for estrogen receptor and activate estrogen-responsive reporter genes in vitro [6] but these results have not been confirmed by in vivo studies [7]. It has been reported that diet supplementation with dealcoholized red wine solids can inhibit tumor formation in mice [8]. Several reports suggest that
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resveratrol might mediate this protective effect of moderate wine consumption. Indeed, it has been shown to inhibit cellular events associated with tumor initiation, promotion and progression [9]. In fact, resveratrol inhibits free-radical formation and has an antimutagenic activity [9,10]. It also has an anti-inflammatory effect mainly due to its ability to inhibit cyclooxigenase and hydroperoxidase functions [9,11] and it has been shown to induce differentiation of human promyelocitic leukemia [9] and osteoblastic cells [12]. Resveratrol also inhibits, in a dose-dependent manner, the development of preneoplastic lesions in a mouse mammary gland culture model of chemical carcinogenesis [9] and reduces tumor formation in a two-stage mouse skin cancer model [9]. Moreover, it inhibits tumor cell growth in a rat model of ascites hepatoma [13] and has been shown to induce cell cycle arrest [14] and apoptosis in human promyelocitic leukemia cells [15]. These data have suggested that resveratrol might be effective as both cancer chemopreventive and chemoterapeutic agent and support the need for further studies on the effects of this molecule in both normal and tumor cells. In the present study, we evaluated the effects of resveratrol on the growth and cell cycle parameters of a series of human cancer cell lines. We found that resveratrol inhibits the in vitro growth of different types of human cancer cells and that it has specific effects on cell cycle parameters. We also found that resveratrol prevents oxidative DNA damage induced by different stimuli. The implications of these findings are discussed.
2. Materials and methods 2.1. Cell culture The HC11 mouse mammary epithelial cells were clonally derived from a spontaneously immortalized mammary epithelial cell culture originally established from a mid-term pregnant BALB/c mouse [16] and were grown and maintained in RPMI-1640 medium (RPMI) (Gibco, Merelbeke, Belgium) supplemented with 10% heat inactivated fetal bovine serum (FBS). The Rat-1 normal rat fibroblasts were grown and maintained in Eagle’s minimum essential medium
(EMEM) (Gibco) supplemented with 10% heat inactivated FBS. All the other cell lines used for this study were obtained from the American Type Culture Collection and cultured according with the instructions of the supplier. 2.2. Chemicals Purified resveratrol was purchased from Sigma (St. Louis, MO) and a stock solution (10 mg/ml) was prepared in 100% ethanol and kept protected from light at −80◦ C. The particulate phase of cigarette tobacco smoke (TAR) was provided by the Ente Tabacchi Italiano (ETI, Rome, Italy) and was obtained by mechanically smoking cigarettes. TAR was dissolved in DMSO and diluted in culture medium to obtain the working concentration before being added to cell cultures. 2.3. Cell proliferation assay Cells were plated in triplicate in 24-well plates at a density of 2×104 cells per well and incubated 24–36 h. They were then rinsed and grown in medium supplemented with the indicated concentration of resveratrol. After 48 h, medium was removed and cultures were incubated with medium containing 1 mg/ml MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma) for 2 h at 37◦ C. Medium was then discarded and 500 l acid–isopropanol (0.04N HCl in isopropanol) was added to each well to stop the cleavage of the tetrazolium ring by dehydrogenase enzymes which convert MTT to an insoluble purple formazan in living cells. Plates were then kept in agitation at room temperature for about 15–20 min and the level of the colored formazan derivative was determined on a multiscan reader at a wavelength of 540 nm (reference wavelength 630 nm). 2.4. Detection of DNA fragmentation Cytosolic DNA fragments were quantified with a cell death detection ELISA assay (Boehringer Mannheim Italia, Monza, Italy). Briefly, cells were plated in triplicate in 24-well plates at a density of 2 × 104 cells per well and were exposed to resveratrol. Floating and attached cells were then harvested
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and assayed with a quantitative sandwich-enzymeimmunoassay using mouse monoclonal antibodies directed against DNA and histones, respectively. This allows the specific determination of mono- and oligo-nucleosomes in the cytoplasmatic fraction of cell lysates. The occurrence of apoptosis in the cultures is expressed as enrichment of nucleosomes in the cytoplasm of treated cells compared with control cells using the following formula: absorbance of the sample/absorbance of the corresponding control = enrichment factor. Values represent mean ± S.D. from six experiments. 2.5. Determination of DNA content by FACS analysis Cells were plated in duplicate in 6 cm dishes at a density of 5×105 cells per dish and incubated 24–36 h. Medium was then changed with fresh medium containing a concentration of resveratrol corresponding to the IC50 for each cell lines. After 48 h, cells were trypsinized, collected and washed twice with PBS. Cell pellets were resuspended in 1 ml PBS and fixed in 5 ml of 70% ethanol and stored at 4◦ C. For the analysis, cells were collected by centrifugation and the pellets were resuspended in 0.2 mg/ml of propidium iodide in HBSS containing 0.6% Nonidet P-40. RNase (1 mg/ml) was added and the suspension was incubated in the dark at room temperature for 30 min. The cell suspension was then filtered and analyzed for DNA content on a Coulter EPICS 753 flow cytometer. The percent of cells in different phases of the cell cycle was determined using the Multicycle software version 2.53.
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2.7. Comet assay The comet assay was performed as described by Sestili et al. [18] with slight modifications. Briefly, the cells were resuspended at 1.0 × 104 cells/100 l in 0.5% low-melting agarose in phosphate-buffered saline (PBS) and immediately pipetted onto agarosecoated slides (1% in PBS containing 5 mM EDTA). Cells were then covered with a layer of agarose (0.5% in PBS) and allowed to solidify briefly. The slides were immersed in ice-cold lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Sarkosyl, 5% dimethyl sulfoxide and 1% Triton X-100 (pH 10.0)) for 60 min at 4◦ C. They were then placed on an electrophoretic tray with an alkaline buffer (0.3 NaOH, 1 mM EDTA) and allowed to equilibrate for 20 min at room temperature before the electrophoresis performed at 300 mA for 20 min in the same buffer. The slides were then washed, stained for 5 min with 10 mg/ml ethydium bromide (EB), covered with a coverslip and analyzed with a fluorescence microscope Eclipse E600 (Nikon Corporation, Tokyo, Japan). Images were acquired with a camera coupled with a computer and were analyzed using the software Image-Pro Plus 4.1 (Media Cybernetics, Silver Spring, MD). The extent of DNA damage is expressed as tail/nucleus ratio, as previously reported [18]. In each experiment, the tail/nucleus ratio were calculated from at least 50 randomly selected cells and are expressed as mean ± S.D. of at least three independent experiment. 3. Results
2.6. Measurement of ROS production
3.1. Effects of resveratrol on cell proliferation and cell cycle parameters on cell lines of various histogenetic origin
Intracellular reactive oxygen species (ROS) production was measured in resveratrol-treated and in control cells using the dichlorodihydrofluorescein diacetate (DCF) (Molecular Probes, Inc., Eugene, OR) as described [17]. Fluorescent units were measured in each well at 15 min interval for 45 min following incubation with DCF (10 M), using a CytofluorTM 2300/2350 Fluorescence Measurement System (Millipore Corp., USA) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
In a first feasibility study, we aimed to determine the concentration of resveratrol inhibiting cell growth by 50% (IC50 ) in a series of cell lines including diploid rodent fibroblasts (Rat-1), normal mouse mammary epithelial cells (HC11), three human breast (MCF-7, T47-D and BT-549), three colon (HCT116, HT-29 and SW-620), two prostate (PC3 and DU145) and one cervix (HeLa) cancer cell lines. Cytotoxicity assays were carried out by use of the MTT test. Exponentially growing cultures of each cell line were
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Table 1 IC50 of resveratrol in cell lines of various histogenetic origin Cell line
Source
IC50 a (M)
Rat-1 HC11 MCF-7 T47-D BT-549 HCT116 HT-29 SW-620 PC3 DU145 HeLa
Rat fibroblasts Mouse mammary epithelium Human breast adenocarcinoma Human breast adenocarcinoma Human breast adenocarcinoma Human colon adenocarcinoma Human colon adenocarcinoma Human colon adenocarcinoma Human prostate adenocarcinoma Human prostate adenocarcinoma Human cervix adenocarcinoma
90.12 22.85 101.48 69.08 72.19 89.13 94.52 109.06 67.11 51.12 82.60
a
± ± ± ± ± ± ± ± ± ± ±
5 5 11 5 9 4 3 10 2 3 6
Concentration of resveratrol (M) inhibiting cell growth by
50%.
exposed to increasing concentration (0–200 M/l) of commercially available resveratrol (trans-resveratrol) for 48 h. A dose-dependent decrease in cell number was observed in all of the tested cell lines with a IC50 which ranged from 22 to 109 M depending on the cell line (Table 1 and data not shown). The MCF-7 (IC50 = 101.5 M) and SW-620 (IC50 = 109.1 M) cells were the most resistant followed by the HCT116 (IC50 = 89.1 mM) and HT-29 (IC50 = 94.5 M) and the T47-D (IC50 = 69.1 M). The PC3 and DU145 prostate cancer cell lines showed the highest sensitivity (IC50 = 67.1 and 51.1 M, respectively). We then performed FACS analyses to determine the effects of resveratrol on cell cycle parameters in various cell lines. Exponentially growing cultures of each cell line were exposed to a concentration of resveratrol corresponding to the IC50 and the distribution of cells in the different phases of the cell cycle was determined after 48 h in both treated and parallel untreated cultures. As shown in Table 2, resveratrol markedly affected cell cycle distribution. The most striking effect observed was a reduction in the percentage of cells in the G2/M phase of the cell cycle which occurred in most of the cell lines tested with the only exception of the MCF-7 and HeLa cells. The reduction of cells in the G2/M phase was most frequently associated with an increase of cells in the S phase of the cell cycle. However, in three of the cell lines (T47-D, HT-29 and DU145) the reduction of cells in the G2/M was compensated by an accumulation of cells in the G0/G1 phase (Table 2).
Table 2 Effects of resveratrol on cell cycle distribution in cell lines of various histogenetic origina Cell line
Untreated
Resveratrol-treated
G0/G1
S
G2/M
G0/G1
S
G2/M
Rat-1 HC11 MCF-7 T47-D HCT116 HT-29 PC3 DU145 HeLa
75.6 55.7 58.2 58.2 57.2 62.8 50.4 56.0 67.7
13.7 34.5 32.3 22.2 28.6 18.3 36.6 30.7 20.6
10.7 9.8 9.5 19.6 14.2 18.9 13.0 13.3 11.7
67.4 43.5 50.7 66.9 46.8 76.4 45.8 69.0 39.4
31.0 52.3 39.9 28.3 50.6 17.0 44.6 29.4 46.3
1.6 4.2 9.4 4.8 2.6 6.6 9.5 1.6 11.4
a
Exponentially growing cultures of the indicated cell lines were analyzed by flow cytometry. Parallel cultures were incubated with a concentration of resveratrol corresponding to the IC50 before analysis. The values represent the percentage of the total cell population in each phase of the cell cycle. All assays were performed in triplicate, and all experiments were repeated at least twice. The data reported are the results of a typical experiment for a repre- sentative cell line. Similar results were obtained in replicate experiments.
To extend this analysis, we analyzed cell cycle distribution in the Rat-1, HC11 and MCF-7 cells at various time points after resveratrol exposure. Exponentially growing cultures were exposed to resveratrol (IC50 ) and the cell cycle distribution was analyzed, at various times thereafter, by flow cytometry. As shown in Fig. 1, both the Rat-1 and HC11 cell lines, which can be considered as models of normal non-transformed cells, showed a progressive reduction in the percentage of cells in the G2/M phase of the cell cycle which was associated with a progressive parallel accumulation of cells in the S phase. In fact, incubation of the HC11 cells, a model of normal non-transformed mammary epithelial cells, at 20 M for 48 h caused a marked decrease in the percentage of cells in the G2/M phase of the cell cycle from about 10 to 4%. This effects was associated with an increase of cells in the S phase (from about 35 to 54%) and a decrease of cells in the G0/G1 (from about 58 to 43%). This effect was already evident after 12 h exposure to resveratrol (Fig. 1). On the other hand, the MCF-7 cells, which are a model of transformed mammary epithelial cells, also showed an accumulation of cells in the S phase of the cell cycle (from about 32 to 40%) with a reduction of cells in G1 (from about 58 to 51%), after exposure to 100 M of resveratrol for 48 h. However, unlike the
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Fig. 1. Flow cytometric analysis of resveratrol-induced cell cycle arrest. Exponentially growing cultures of HC11, Rat-1 and MCF-7 cells were exposed to the corresponding IC50 of resveratrol (see Table 1). Cells were subsequently collected at the indicated times, stained with propidium iodide and analyzed by flow cytometry, as described in Section 2. Cell cycle distribution of parallel untreated cultures remained unchanged throughout the time course (not shown).
HC11 cells, they showed no change in the percentage of cells in the G2/M phase (Fig. 1 and Table 2). 3.2. Effects of resveratrol on cell growth in normal and transformed mammary epithelial cells To further investigate the effects of resveratrol on epithelial cells growth we evaluated how this molecule influences the proliferation rate of the HC11
and MCF-7 cell lines, considered as a model of normal and transformed mammary epithelial cells, respectively. Cells were incubated in media at different concentration of resveratrol and cell growth was analyzed up to 72 h (Fig. 2). HC11 cells were very sensitive to resveratrol. In fact, the doubling time was about 22.3 h in the regular medium and increased to 29.1 and 40.8 h when cells were incubated in medium containing 10 and 20 M
Fig. 2. Effects of resveratrol on the growth of the HC11 (A), and the MCF-7 (B) cells. Exponentially growing cultures were exposed to the indicated concentration of resveratrol (M) and the number of cells per well was determined every day by cell counting. Data are derived from an experiment (mean values) performed in triplicate. Similar results were obtained in replicate experiments.
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A. Sgambato et al. / Mutation Research 496 (2001) 171–180 Table 3 Resveratrol-induced apoptosis in cell lines of various histogenetic origina Cell lines
DNA fragmentation (enrichment factor)
HC11 MCF-7 T47-D HCT116 SW-620 DU145
1.10 1.13 2.21 1.37 1.76 1.17
± ± ± ± ± ±
0.12 0.72 1.07 0.65 0.76 0.22
P-value 0.069 0.667 0.019∗ 0.193 0.034∗ 0.087
a Exponentially growing cultures of the indicated cell lines were incubated for 48 h with a concentration of resveratrol corresponding to the IC50 before analysis. The values represent the enrichment of nucleosomes in the cytoplasm of cells compared to control untreated cells (mean ± S.D., n = 6). ∗ Significant increase as compared to control cells.
resveratrol, respectively. Both cell growth and survival of the HC11 cells were compromised at higher concentration of resveratrol (Fig. 2A). MCF-7 were more resistant to the resveratrolmediated inhibition of cell growth. In fact, the doubling time was about 32.4 h in the regular medium and increased to 38.4, 58.9 and 112.9 h when cells were
Fig. 3. Effects of resveratrol on intracellular ROS production in Rat-1 cells, using DCF as fluorescent probe. Cells were pre-incubated with the indicated concentration of resveratrol for 24 h. In the upper panel DCF fluorescence was measured at 15 min interval for 45 min following incubation with DCF (10 M). In the middle and lower panels the cells were exposed to 100 and 200 M H2 O2 , respectively, before the analysis; the fluorescence value at time zero was subtracted to all values (mean ± S.D. of three different experiments performed in triplicate).
Fig. 4. Effect of resveratrol on DNA strand breakage induced by TAR as assessed by the comet assay. Control cells and resveratrol-treated cells (30 M for 24 h) were exposed to the indicated concentration of TAR for 15 min and then analyzed by the comet assay. Results are expressed as tail/nucleus ratio and represent the mean ± S.D. of three separate experiments (see Section 2 for details).
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incubated in medium containing 10, 30 and 100 M resveratrol, respectively. Cell growth and survival of the MCF-7 cells were only inhibited at concentration of resveratrol higher than 100 M (Fig. 2B). We also determined the concentration of resveratrol inhibiting cell growth by 50% (IC50 ) by use of the thymidine incorporation assay. Again, the HC11 cells showed a high sensitivity to resveratrol, the IC50 being 18.88 ± 2.23 M while for the MCF-7 cells the IC50 was 92.63 ± 5.67 M, respectively. These observation were consistent with the results obtained by the MTT test (Table 1). 3.3. Resveratrol does not cause apoptosis in most of the cell lines analyzed We then aimed to evaluate whether resveratrolinduced inhibition of cell growth is associated with
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the induction of apoptotic cell death. Exponentially growing cultures of representative cell lines were exposed to resveratrol (IC50 ) for 48 h and apoptosis was evaluated by quantifying cytosolic DNA fragments. As shown in Table 3, resveratrol caused only a slight increase in cytosolic DNA fragments in most of the cell lines analyzed, compared with control untreated cells. This increase was statistically significant (P < 0.05) only for the T47-D breast (P = 0.02) and the SW-620 colon (P = 0.03) cancer cells. 3.4. Resveratrol prevents ROS production and DNA damage induced by oxidative stresses To investigate the anti-oxidative effect of resveratrol, we evaluated its ability to prevent the production of ROS in normal rat fibroblasts (Rat-1) exposed to oxidative stresses.
Fig. 5. Representative images of agarose-embedded nuclei from both control (A, B and C) and resveratrol-treated (D, E and F) cells. Exponentially growing cultures of Rat-1 cells were exposed to 1 mg/ml (A, D), 2.5 mg/ml (B, E) and 5 mg/ml (C, F) of TAR for 15 min and then analyzed by the comet assay. Resveratrol-treated cells were pre-incubated in medium containing 30 M resveratrol for 24 h.
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As shown in Fig. 3A, incubation of cells with resveratrol at 30 and 90 M for 24 h, slightly decreased the levels of endogenous ROS, as assessed using the fluorescent probe DCF. When cells were exposed to H2 O2 , we observed a marked increase in the intracellular ROS level which was prevented by resveratrol. In fact, when cells were pre-incubated with 30 and 90 M resveratrol for 24 h before being exposed to 100 M H2 O2 , the levels of ROS observed after 45 min were about 26.4 and 57.3% lower than control untreated cells, respectively (Fig. 3B). When the same resveratrol-pretreated cells were exposed to 200 M H2 O2 the levels of ROS observed after 45 min were 10.3 and 45.6%, respectively, lower than control untreated (Fig. 3C). We also tested the ability of resveratrol to prevent the increase in intracellular ROS caused by cigarette-smoke condensate (TAR). When cells were pre-incubated with 30 and 90 M resveratrol for 24 h before being exposed to 0.5 mg/ml TAR, the increase in ROS levels after 45 min was about 73.9 and 44.3%, respectively, compared with control untreated cells. Exposure to 1 mg/ml TAR caused an increase in ROS levels which was about 82.8 and 49.1%, respectively, than control untreated cells (data not shown). Nuclear DNA is one of the major biological target of oxidative stress [19]. ROS can also induce genotoxic damage, including single- and double-strand breaks, and are known to play an important role in the pathogenesis of cancer and other degenerative diseases [19,20]. We used the single cell gel electrophoresis (SCGE) assay, also known as “comet test”, to test the ability of resveratrol to prevents DNA breaks induced by TAR. As shown in Fig. 4, pretreatment of cells with 30 M resveratrol caused a marked inhibition of DNA fragmentation induced by TAR which was significant (P < 0.05) for concentration of TAR up to 5.0 mg/ml (Figs. 4 and 5).
4. Discussion Epidemiological studies have suggested that dietary factors play an important role in cancer development in humans and the preventive effects of plant-based diets is well documented [21,22]. Resveratrol is a stilbene (3,4 ,5-trihydroxystilbene) with a relatively broad
distribution in plants and is present in various human foods, including red wines, peanuts and mulberries [23]. It is an antioxidant and has been shown to inhibit various stages of tumor development [9]. In this study, we investigated the biological properties of resveratrol and obtained data which shed lights on the mechanisms underlying its proposed cancer chemopreventive and chemotherapeutic activity. We found that resveratrol inhibits proliferation of both normal and cancer cells of various histogenetic origin with a IC50 ranging between 20 and 100 M (Table 1 and Fig. 2). This effect was mainly due to an inhibition of cell proliferation rather than to a direct cytotoxic effect, as suggested by the limited amount of dead cells detectable in the culture by the Trypan Blue exclusion test (data not shown) and was associated with an inhibition of cell cycle progression, mainly at the S/G2 boundary. In fact, we observed by flow cytometry an increase in the percentage of cells in the S phase of the cell cycle with a reduction of cells in the G0/G1 and the G2/M phase in most of the cell lines analyzed (Fig. 1 and Table 2). We are currently investigating the molecular mechanisms of these effects and we obtained preliminary data suggesting that resveratrol can inhibit the activity of DNA polymerase (unpublished data). It is noteworthy that the HC11 normal mouse mammary cells displayed the highest sensitivity to resveratrol which is probably related to the high proliferation rate of these cells which have a doubling time (about 16 h) shorter than all the other cell lines tested in this study. Our results also suggest that induction of apoptosis is not a major determinant of the observed antiproliferative effect of resveratrol in most of the cell lines analyzed (Table 3). We observed, however, a different susceptibility of various cell lines to the resveratrol-induced apoptosis. These differences are likely due to a different genetic background of the cells analyzed. Studies are ongoing to identify the molecular determinants of these effects. Resveratrol has been proposed as a potential chemopreventive agent and its antimutagenic and anticarcinogenic activity has been demonstrated in several models. The mechanisms responsible of these effects, however, remain to be elucidated. To our knowledge this is the first study analyzing the ability of resveratrol to prevent the accumulation of ROS and oxidative DNA damage in cells exposed to
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oxidative agents, including TAR. Indeed, we demonstrated that pretreatment of cells with resveratrol is able to prevent the accumulation of intracellular ROS and DNA breaks induced by TAR (Figs. 4 and 5 and data not shown). The antioxidant properties of resveratrol have already been described [3,24]. For the first time, however, we demonstrated that these properties are also effective on a potent human carcinogen, such as TAR. These results are of interest in view of the proposed chemopreventive activity of resveratrol. Antileukemic activity of stilbenes has been associated with their effect on arachidonate metabolism in leukocytes [25]. However, resveratrol has been also shown to inhibit solid tumor development [13] and a more general mechanism has to be responsible for this activity. We believe that our data provide support to the proposed chemopreventive properties of resveratrol and underlines the need for further studies in order to finally define the possible beneficial outcomes of its use as dietary supplement for the general population.
Acknowledgements This work was supported in part by a grant from “Co-finanziamento MURST” 1998. We thank O. Cantoni and P. Sestili (Università di Urbino, Italy) for initial assistance with the comet test.
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