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Biochimica et Biophysica Acta 1760 (2006) 1657 – 1666 www.elsevier.com/locate/bbagen

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Resveratrol exerts its antiproliferative effect on HepG2 hepatocellular carcinoma cells, by inducing cell cycle arrest, and NOS activation

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George Notas a,1 , Artemissia-Phoebe Nifli b,1 , Marilena Kampa b , Joseph Vercauteren c , Elias Kouroumalis a , Elias Castanas b,⁎ a Laboratories of Gastroenterology, University of Crete School of Medicine, Heraklion, Greece Laboratory of Experimental Endocrinology, University of Crete School of Medicine, P.O. Box 2208, Heraklion, GR-71003, Greece c Laboratory of Pharmacognosy, School of Pharmacy, University of Montpellier I, France

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Received 2 May 2006; received in revised form 25 August 2006; accepted 15 September 2006 Available online 22 September 2006

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Abstract

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The stilbene resveratrol exerts antiproliferative and proapoptotic actions on a number of different cancer cell lines, through diverse mechanisms, including antioxidant effects, enzyme, growth factor and hormone receptor binding, and nucleic acid direct or indirect interactions. Although resveratrol accumulates in the liver, its effect on hepatocellular carcinoma has not been extensively studied. We have used the human hepatocyte-derived cancer cell line HepG2 to address the possible action of resveratrol on cell growth and to examine some possible mechanisms of action. Our results indicate that the stilbene inhibits potently cell proliferation, reduces the production of reactive oxygen species and induces apoptosis, through cell cycle arrest in G1 and G2/M phases. Furthermore it modulates the NO/NOS system, by increasing iNOS and eNOS expression, NOS activity and NO production. Inhibition of NOS enzymes attenuates its antiproliferative effect. These data could be of value in possible prevention or adjuvant treatment of hepatocellular carcinoma, through an increased consumption of resveratrol-rich foods and beverages. © 2006 Elsevier B.V. All rights reserved.

1. Introduction

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Keywords: Resveratrol; Hepatocellular carcinoma; Cell cycle; Apoptosis; Nitric oxide (NO); Nitric oxide synthase (NOS)

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Resveratrol (trans-3,5,4′-trihydroxystilbene) is a phytoalexin produced by a variety of plants such as grapes, peanuts, and berries in response to stress, injury, ultraviolet irradiation, and fungal infection [1]. Resveratrol can be detected in the leaf epidermis and the skin of grapes (containing 50–100 μg per gram), while its concentration in wine ranges from 0.2 mg/l to 7.7 mg/l [2,3]. The “French paradox”, the low incidence of coronary heart diseases in spite of a diet rich in saturated fats [4,5], has been attributed to a number of contained polyphenols, including resveratrol. Since the discovery that the stilbene acts as an inhibitor of tumorigenesis [6], this compound has experienced an increasing attention from the scientific community. Resveratrol has been shown not only to inhibit cancer ⁎ Corresponding author. Tel.: +30 2810 394580; fax: +30 2810 394581. E-mail address: [email protected] (E. Castanas). 1 Authors have equally contributed. 0304-4165/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2006.09.010

initiation, but also to reverse tumor development in several types of cancers (see [7], for a recent review). However, the mechanism by which resveratrol exerts its health promoting effects is not yet fully elucidated, while the concentrations of the reagent used in most in vitro studies are by far higher than those found after in vivo administration. A major problem, not yet fully resolved, is the bioavailability of free resveratrol, after per os administration, either as a pure substance, or in a food vehicle. Bertelli et al. reported that after oral administration of resveratrol in rats, the agent accumulated mainly in heart muscle and presented a strong affinity for liver and kidneys. They also found that resveratrol concentration in plasma was far lower (at the nanomolar range) than the one administered; however it significantly attenuated platelet aggregation [8]. Similarly, 14C-trans-resveratrol accumulated in mice stomach, liver, kidney, and intestine, after oral administration [9], with a parallel formation of glucurono- and sulfo-conjugates, in liver and kidney. In addition, Kaldas et al. [10] found that resveratrol transport across Caco-2 monolayers

G. Notas et al. / Biochimica et Biophysica Acta 1760 (2006) 1657–1666

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In this respect, we have used the human hepatocellular carcinoma-derived cell line HepG2 and we examined the effect of resveratrol on cell proliferation, apoptosis, cell cycle and the NO/NOS system modulation. In addition, reactive oxygen species (ROS) production and scavenging was assayed as a measure of early effects on mitochondrial function and mitochondrial-driven apoptosis, while the NO/NOS system was studied in order to ponder late effects. Our results indicate that resveratrol treatment inhibits cell growth and induces apoptosis, blocking cells in the G1 and G2 phase of the cell cycle. Moreover it inhibits the production of reactive oxygen species and modulates the NO/NOS system, either at the protein (enzymatic) or transcriptional level. 2. Materials and methods 2.1. Chemicals

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All biochemicals were obtained from Sigma-Aldrich (Sigma Hellas, Athens, Greece). Culture media were from Gibco BRL (Life Technologies, Paisley, UK). Trans-resveratrol was prepared from total wine polyphenolic extract, by semipreparative high-pressure liquid chromatography, using an RP18 column. Detection was made by UV-visible spectroscopy. Resveratrol was recrystalized, and its final purity, assayed by analytical HPLC, was >99%. This was also confirmed by proton nuclear magnetic resonance (NMR), at 500 MHz. It was conserved in a dark bottle, at − 20 °C, under nitrogen. For cell treatment, a 10− 2 M fresh resveratrol stock solution was prepared in absolute ethanol. Subsequent dilutions were done in culture medium. In all cases, control corresponds to vehicle-treated cells.

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(monolayers of well differentiated colon cancer cells, reflecting intestinal absorption) was bi-directional, while extended metabolism, involving mainly sulfation and, to a minor extent, glucuronidation, occurred; this effect could explain resveratrol limited bioavailability, in spite of its increased absorption. Goldberg et al. confirmed that, after oral administration to healthy humans (25 mg/70 kg), resveratrol appears in serum and urine predominantly as glucuronide and sulfate conjugates and reaches a maximum serum concentration (10–40 nM) 30 min after consumption [11]. Free resveratrol accounts for 1.7–1.9% of the total serum metabolites, resulting in concentrations at the low nanomolar or picomolar range. This was equally confirmed in another study, in which aglycone compounds were administered in humans, mice, and rats: resveratrol ingestion yielded detectable levels of the agent and its derivatives in human plasma and urine, while, after intragastric administration (2 mg/kg), it reached 1.2 μM in rat plasma [12]. These studies indicate that (a) nanomolar concentrations of free resveratrol may be detected in the blood after oral ingestion of foods and beverages in humans, and (b) that the liver is one of the main sites in which resveratrol concentrates. Hepatocytes have a dual role: they are active players of resveratrol bio-conjugation and elimination, while they represent a major site of resveratrol bioaccumulation. In this respect, and in view of the major reported effects of resveratrol in cancer, it is of interest to elucidate the role of the agent in hepatocyte biology. However, few studies have evaluated the chemopreventive effects of resveratrol in liver cancer. Carbo et al. demonstrated that resveratrol administration in a transplantable rat ascites hepatoma model, decreased significantly tumor size [13]. In treated animals, tumor cell analysis revealed an aneuploid peak (representing 28% of total population) and a G2/ M phase arrest. Sun et al. demonstrated that resveratrol inhibited the growth of the hepatoma cell line H22 in a dose- and timedependent manner, via induction of apoptosis [14]. Furthermore, resveratrol was found to strongly inhibit cell proliferation (at the micromolar range) in a time- and dose-dependent manner of the rat hepatoma (FaO) and the human hepatocyte derived (HepG2) cancer cell lines, by preventing or delaying cell entry to mitosis [15]. In addition, it has been shown that transresveratrol decreased hepatocyte growth factor-induced cell scattering and invasion [16], through modulation of downstream receptor signaling cascade. Sera from rats treated with resveratrol restrained also the invasion of AH109A cells, although no effect on cell proliferation was observed [17]. A lot of epidemiological studies have attributed to resveratrol-rich food and beverages a beneficial role in a number of pathologies. However, the majority of in vitro studies examine the effects of the agent at very high (micromolar or even millimolar concentrations) which are of value in order to decipher underlying biochemical mechanisms of action, but biologically irrelevant. The aim of the present study was to elucidate the role of resveratrol in hepatocellular cancer in vitro and at concentrations compatible with those found in biological fluids after consumption of resveratrol-rich foods or beverages, in an attempt to transpose in vitro (with all limitations of cell culture systems) the beneficial actions of the agent found in vivo.

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2.2. Cell cultures

The human HepG2 cell line was obtained from DSMZ (Braunschweig, Germany). Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, at 37 °C, 5% CO2, and subcultured weekly.

2.3. Cell viability and growth assay HepG2 cells were seeded in 24-well plates, at an initial density of 2 × 104 cells, with 1.0 ml medium per well. All substances were added to cultures 1 day after seeding (designated as day 0), in order to ensure uniform attachment of cells at the onset of the experiments. Cells were grown for a total of 6 days, with a change of the medium and resveratrol on day 3. Growth and viability of cells were measured by the tetrazolium salt assay. Cells were incubated for 4h at 37 °C with the tetrazolium salt (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), and metabolically active cells reduced the dye to purple formazan. Dark blue crystals were dissolved with propan-1-ol. The absorbance was measured at 570 nm. All experiments were performed a minimum of three times, in triplicates [18].

2.4. Determination of reactive oxygen species (ROS) generation, by flow cytometry Reactive Oxygen Species (ROS) production after a short PMA stimulation was assayed by flow cytometry, as described by Rothe and Valet [19]. Briefly, one million cells, treated or not with polyphenols for 24 h, were removed from dishes, loaded with dihydrorhodamine 123 (Molecular Probes Leiden, The Netherlands, 10 μl of a 100 μM solution in a total volume of 1 ml) and incubated for 7 min at room temperature. Thereafter, 10 μl of a solution of 10 μM Phorbol12 myristate 13-acetate (PMA, Sigma) was added, incubated for another 5 min, and counted in a Beckton Dickinson FACSArray cytometer (BecktonDickinson, Franklin Lakes, NJ). In the presence of intracellular ROS, dihydrorhodamine 123 is converted by oxidation to yellow-green rhodamine 123, trapped intracellularly. Measurements were repeated at determined time

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primers (MWG Biotech, Ebersberg, Germany) and annealing temperatures were used: eNOS (forward 5′AAT CCT GTA TGG CTC CGA GA3′ and reverse 5′GGG ACA CCA CGT CAT ACT CA3′) at 58.3 °C, iNOS (forward 5′ACA GGA GGG GTT AAA GCT GC3′ and reverse 5′TTG TCT CCA AGG GAC CAG G3′) at 59.1 °C and actin (forward 5′GGT GGC TTT TAG GAT GGC AAG3’and reverse 5′ACT GGA ACG GTG AAG GTG ACA3′) were added to the PCR mix in a concentration of 100 and 250 nM, for eNOS and iNOS respectively. PCR products were electrophorized in 3% agarose gel in 0.5× TBE buffer at 100 mV, in a horizontal apparatus (Horizon® 11–14, Gibco BRL, Goettingen, Germany) and bands intensity was measured using the Molecular Analyst Software (BioRad, Hercules, CA).

intervals for 1 h, as indicated in Results. Experiments were repeated four times. Results were quantified with the CELLQuest (Beckton-Dickinson) and ModFit LT (Verify Software, Topsham, MN) software, as appropriate.

2.5. Nitric oxide metabolites assay (NOx)

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Supernatant collected from the proliferation assays was cleared by centrifugation and frozen at − 80 °C until use. NO was measured by assaying its stable metabolites NO−2 and NO−3 , as described [20]. Briefly, 100 μl of the culture medium was incubated with nitrate reductase, in 25 μl of 1 M HEPES buffer (pH 7.4), 25 μl of 0.1 mM FAD and 50 μl of 1 mM NADPH, in order to reduce NO−3 to NO−2 . Excess NADPH was removed by lactate dehydrogenase treatment. Subsequently, 1 ml of Griess reagent was added, followed by measurement at 543 nm. Medium background was subtracted in all cases.

2.9. Nitric Oxide Synthase (NOS) activity assay

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Nitric oxide synthase (NOS) activity was assayed by the conversion of radioactive arginine to citrulline [23]. Briefly, cellular homogenate (10 μg/ml) was incubated in 40 μl of 50 mM Tris–HCl pH 7.4, 6 μM tetrahydrobiopterin, 2 μM FAD and 2 μM FMN, 50 μl 10 mM NADPH, 10 μl [3H] Arginine (Amersham), 50 μl 6 mM CaCl2, for 1 h at 37 °C. Non-reacted arginine was eliminated by resin absorption (AG 50Wx*, BioRad). The eluate was measured in a liquid scintillation counter (Tricarb, Packard, Instrument Co., Meriden, CT), with 60% efficiency for tritium.

2.10. Statistics

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Cells were washed with PBS, containing 1% BSA. Then, 3 ml of cold absolute ethanol were added, incubated at 4 °C for 1 h, washed and provided with 1 ml of a 50 μg/ml of propidium iodide in sodium citrate, and 50 μl of a 10 μg/ml RNaseA solution (R&D Systems, Minneapolis, MS; 200 U/ml). Cells were incubated for 3 h at 4 °C, and assayed by flow cytometry, using a BecktonDickinson FACSArray cytometer [23]. Apoptosis was measured with the APOPercentage Apoptosis Assay (Biocolor Ltd., Belfast, N. Ireland). The assay uses a dye, which is imported by cells undergoing apoptosis, and expressing phosphatidylserine to the outer membrane leaflet. The dye has a purple-red colour and the detection of apoptotic cells can be readily observed with a conventional inverted microscope. For apoptosis quantification, the dye that accumulates within labelled cells is released in the supplier's buffer and measured at 540 nm with a reference filter at 620 nm, in a microplate colorimeter (Dynatech MicroElisa reader Chantilly, VA). Previous results have shown that this method measures accurately apoptotic cells, with comparable results to flow cytometry and the detection of apoptotic bodies [24].

2.8. Multiplex RT-PCR

NOS transcripts after stimulation of HepG2 cells with polyphenols for 2, 6, 12 and 24 h, were measured by semi-quantitative multiplex RT-PCR vs. the constitutive actin gene [23]. Cells were cultured in 6-well plates, 24 h prior to the addition of polyphenols. Samples were taken after 2, 6, 12, 24, 48 and 72 h of treatment. Total RNA was extracted with TRIzol® reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol, with an additional step of 70% ethanol wash. For the RT reaction 1 μg of RNA was used: (i) DNA was eliminated with DNase I amplification grade treatment (Invitrogen) for 20 min at 25 °C, followed by heat inactivation for 10 min at 65 °C, (ii) cDNA synthesis was performed using SuperScript™ II RNA H−reverse trascriptase (Invitrogen), 5 μÌ poly d(T) (Amersham Pharmacia Biotech, Buckinghamshire, UK) and 1 μl ribonuclease inhibitor rRNasin® (Promega, Madison, WI), in a total volume of 20 μl, for 1 h at 42 °C, then stopped after incubation for 5 min at 95 °C. Multiplex PCR reactions were performed using 1 μl of the cDNA product (0.05 μg), DNA primers as described below, 200 μÌ of each dNTP (Invitrogen) and 1 unit of DyNAzyme II polymerase (Finzyme, Ozyme, France), in a total volume of 25 μl, for 35 cycles, with a 30 s extension period. Specific set of

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Statistical comparisons were performed using the paired or unpaired Student's t-test or ANOVA, with the residual variance taken for the calculation of a common estimator of SEM, as appropriate, with SYSTAT V10 (SPSS, Chicago, IL) program. Results are expressed as mean ± standard error. p < 0.05 was considered significant. IC50s were calculated by sigmoidal fitting of raw data, with the use of Origin V 4.0 program (SPSS, Chicago, IL).

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2.7. Determination of cell cycle and apoptosis

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2.6. Measurement of NO production by flow cytometry NO production by HepG2 cells was measured using the diaminofluorescein diacetate (DAF) method [21, 22] and flow cytometry. Briefly, DAF-2 DA (Sigma, 0.1 mM final concentration) was added to 106/ml HepG2 cells, cultured in the presence or absence of resveratrol (final concentration 10− 7 M). Under the action of NO, the dye is transformed to the fluorescent 2′,7′-diaminofluorescein, trapped intracellularly. DAF, in neutral solutions as the cellular environment, does not react with other oxidized forms of NO, such as NO2 and NO3, or other reactive nitrogen species [22], being a direct indicator of produced NO. Fluorescence was measured in a Beckton-Dickinson FACSArray apparatus, using an excitation wavelength of 485 nm (20-nm bandwidth) and an emission wavelength of 530 (25-nm bandwidth) at 48 and 72 h.

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3. Results 3.1. Resveratrol inhibits the proliferation of HepG2 cells, modifies cell cycle and induces apoptosis Treatment of HepG2 cells with variable concentrations of resveratrol resulted in a decrease of cell growth by 35% in a dose dependent manner (Fig. 1A), after two cell division periods (6 days). The effect was also time-dependent (not shown). The apparent IC50 was 3.11 × 10− 11 M. In view of these results and in order to achieve a maximal effect, in subsequent experiments, we have used a concentration of 10 − 7 M resveratrol, 60 times higher than the IC50 of the agent. The effect of resveratrol on cell proliferation could be due to its action on cell cycle and the initiation of programmed cell death. In order to explore this possibility, we treated cells with resveratrol and assayed its effect on cell cycle. The agent induced an increase in the number of cells in both G1 and G2 phases of cell cycle (Fig. 1C), while decreased the number of cells in S phase. As the accumulation of cells in either G1 or G2 phases could lead to apoptosis, we assayed apoptotic cells, at different time points after resveratrol (10− 7 M) application. A time-dependent increase of apoptosis was detected, reaching a 2.5-fold increase over the control after 6 days of treatment (Fig. 1B). 3.2. Resveratrol scavenges ROS production by HepG2 cells One way to access the antioxidant activity of a polyphenolic compound is its ability to scavenge reactive oxygen species, produced after acute mitogen stimulation. This method was

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3.3. Effect of resveratrol on the NO/NOS system

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In addition to ROS, hepatic cells possess a very active NOS system. NO has been associated with cell growth control [25]. We have therefore investigated the effect of resveratrol on the production of NOx species, the activity of NOS, and the transcript of iNOS and eNOS in HepG2 cells.

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3.3.1. (a) Production of NOx species The cumulative production of NOx species after 3 days of resveratrol (10− 7 M) treatment of HepG2 cells is presented in Fig. 2B. The agent induced a substantial, dose- and timedependent increase of NOx. The dose-dependence of NOx production parallels that of growth inhibition after 24 and 48 h of incubation, with an apparent activator concentration (50%) of 6.1 × 10− 11 and 5.8 × 10− 11 M, not significantly different from the IC50 on cell growth reported above (not shown). In order to define the underlying mechanism, we have assayed intracellular NO by flow cytometry, after polyphenol pre-incubation for 48 or 72 h (Fig. 2C and D). Resveratrol stimulated NO production (p < 0.05 at 7.5 and 10 min, as compared to control-non treated-cells). This effect could be explained either by an enhancement of NOS activity or by an up-regulation of NOS molecules.

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3.3.2. (b) Expression of NOS isoenzyme mRNA (eNOS and iNOS) and NOS activity Two major NOS-isoenzymes are present in hepatic cells: endothelial, membrane-bound, constitutively active NOS (eNOS), and cytoplasmic inducible NOS (iNOS) [26]. In order to further analyze the effect of resveratrol on NOS isoenzymes, we performed PCR for iNOS and eNOS transcripts after polyphenol incubation. Resveratrol (10− 7 M) produced major, time-related, effects on both iNOS and eNOS (Fig. 3A). A transient effect on iNOS mRNA was observed with a maximum between 2–4 h and a return to basal levels after 12 h. In addition, resveratrol induced a sustained increase of eNOS transcription. Furthermore, NOS activity assay revealed that the agent (10− 7 M) induced a bimodal increase of the enzymes' activity as compared to the control (taken as 1): an early increase at 8 h and then a constant increase for up to 72 h (p < 0.01 at 72 h) (Fig. 3B).

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Fig. 1. Effect of resveratrol on cell growth (A), cell cycle (B), and apoptosis (C). (A) Cells were cultured for 6 days in the presence of the indicated concentrations of resveratrol, with one medium change at 3 days. Mean ± SE of three different experiments performed in triplicates. (B) Time effect of resveratrol on cell apoptosis. Cells were cultured in the presence of 10−7 M of resveratrol for the designated time periods. Apoptosis was assayed by the ApoPercentage kit. Results are expressed as a percent increase of apoptosis in vehicle-treated cells, which was ranging from 8 to 12% in all cases. Mean ± SE of three experiments (*p < 0.05 as compared to control, vehicle-treated-cells). (C) Effect of resveratrol (10−7 M, 3 days of incubation) on cell cycle (assayed after PI-staining and flow cytometry). Data are expressed as the percentage of cells in each phase of cell cycle. Mean ± SE of three experiments.

used in the present study, with PMA being the ROS inducer. Twenty-four-hour pre-incubation with resveratrol (10− 7 M) inhibited significantly the production of ROS by HepG2 cells after 5 min PMA stimulation (Fig. 2A).

3.4. Inhibition of NOS activity attenuates the effect of resveratrol on cell growth As indicated above, resveratrol was a potent inducer of NOx production. In order to investigate whether NO production interferes with cell growth, we incubated HepG2 cells with the general NOS-inhibitor L-NAME. Under basal conditions, L-NAME did not have any significant effect on cellular proliferation, indicating that the reduction of NOS activity and NOx production does not interfere with proliferation in our system. When cells were incubated with resveratrol and increasing concentrations of L-NAME, a dose-dependent attenuation of the antiproliferative effect of resveratrol was observed, with an IC50 of 0.3 mM (Fig. 4). Our results indicate

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Fig. 2. Effect of resveratrol on ROS and NO production. (A) Effect of resveratrol on ROS production. One million treated or untreated cells (3 days, 10−7 M resveratrol) were removed from dishes, loaded with dihydrorhodamine 123 and incubated for 7 min at room temperature. Thereafter, 10 μl of a solution of 10 μM Phorbol-12myristate-13-acetate (PMA) was added and cells were further incubated for another 5 min. ROS positive cells were counted by flow cytometry at the indicated timeframes. Resveratrol inhibited or scavenged the production of ROS by HepG2 cells after PMA stimulation. (B) Cumulative production of NOx by HepG2 cells, after incubation with 10−7 M resveratrol. Cells were cultured in the presence or the absence of resveratrol. At day 3, medium was replaced and fresh polyphenols were provided. Thereafter, incubation was continued for additional 3 days, medium was collected, centrifuged and assayed for NOx species using the Griess reaction. Results were corrected for cell number and divided by the corresponding control (vehicle-treated cells; control = 1 in any case). Mean ± SE of three experiments performed in triplicates. (C and D) Kinetics of NO production after 48 (C) and 72 h (D) of incubation with 10−7 M of resveratrol, assayed with the DAF-2 method, followed by flow cytometry. Results of a typical experiment repeated three times with comparable results.

that the enhanced NO production is a prerequisite for cell growth inhibition induced by resveratrol. 4. Discussion

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A number of phytochemicals have been extensively studied in recent years as possible chemopreventive or anti-tumor agents. Among them, resveratrol holds a predominant role, as: (1) acts as a phytoestrogen; (2) possesses antioxidant and antimutagenic properties; (3) induces phase II drug-metabolizing enzymes (anti-initiation activity); (4) mediates antiinflammatory effects; (5) inhibits cyclooxygenase and hydroperoxidase functions (anti-promotion activity); and (6) induces cell differentiation (anti-progression activity) (see [7], for reviews, [27]). The underlying molecular mechanisms comprise: (1) interaction with membrane growth factor or intracellular receptors (ER and AhR); (2) modulation of kinase signaling cascades; (3) induction of apoptosis through the extrinsic and/or the intrinsic pathway; (4) regulation of DNA synthesis; (5) inhibition of the sirtuin family of NAD +dependent protein deacetylases. Considering this broad spec-

trum of actions, resveratrol was proposed as a new candidate for cancer chemotherapy and a wide array of synthetic analogs are currently under investigation for the possible treatment of neoplastic diseases [28]. However, it should be noted that the effect of resveratrol and its analogs is variable, depending on the cell type, the duration of its application and the concentration studied. Resveratrol is a constituent of a number of plant-derived foods, with wine being a major source. Studies in animals and humans have shown that free (unconjugated) resveratrol may be detected in the general circulation, at the low nanomolar or the picomolar range, and that liver is one of the organs where predominantly it accumulates [8,9,11,12]. In this respect, it is interesting to evaluate its action in liver physiology and hepatocellular cancer. Indeed, the liver has a dual role in resveratrol bioavailability: First, it is the site of resveratrol conjugation during the first passage of the agent after intestinal absorption, and, second, it is one of the major sites of resveratrol bioaccumulation. However, little work has been done on the action of resveratrol in hepatocytes. In addition, if a beneficial action might be attributed to resveratrol-containing foods and

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cell lines [29,30]. Either the purity of resveratrol preparations used in the present study, or a long-term effect of the stilbene could explain this apparent discrepancy. Indeed, a recent paper indicates that, in MCF7 breast cancer cells, even transcriptional effects of estrogen or xenoestrogens (polyphenols) could be initiated after long incubation times, at low concentrations [31]. In this respect, our data argue for a possible biological and functional role of resveratrol in hepatocellular cancer. Resveratrol has been reported to modulate cell cycle and to induce apoptosis. Different mechanisms of action have been proposed: In breast cancer [32] and leukemia cell lines [33], resveratrol induced the redistribution of Fas in cell membranes and activated Fas-mediated apoptotic signaling cascades [34]. In addition, in leukemia cell lines and rat liver crude membrane preparation, resveratrol was found to modify mitochondrial function, through inhibition of mitochondrial F0F1-ATPases [35], caspase release and activation of Bcl-2-family members [36]. A major initiator of this pathway is mitochondrial membrane leakage and ROS release. We have therefore tested whether resveratrol could modulate ROS generation after PMAstimulation. Intreated cells, free ROS were markedly reduced, indicating that resveratrol, through its antioxidant or mitochondrial membrane action(s), may protect cells from mitochondrialinduced apoptosis. Indeed, unpublished observations indicate that resveratrol-rich wine extracts accumulate to mitochondrial structures within minutes and protect their transmembrane potential. Our data show that resveratrol inhibited cell entry in S-phase, with a concomitant increase of cell number in G1 and especially G2 phases of cell cycle. The agent has been reported to be a modulator of cell cycle progression, as treated cells accumulated either in G1 [37], S [38–40] and/or G2/M [15,41,42], depending on the cell line and the conditions used. Indeed, a differential effect on cell cycle was observed, using variable resveratrol concentrations [15]. In our hands, resveratrol (10− 7 M) induced the same actions, as previously described [15] for concentrations

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Fig. 3. Effect of resveratrol on the NO/NOS system. (A) Resveratrol induces the expression of eNOS (squares) and iNOS (circles). Cells were incubated for the indicated time periods in the absence or the presence of resveratrol (10−7 M). Thereafter mRNA was collected and multiplex PCR for iNOS and eNOS/actin transcripts was performed, as described in Materials and methods. Results are normalized by actin amplification and divided by the corresponding control values (vehicle-treated cells; control=1 in all cases). Figure presents a typical experiment repeated three times (in duplicates) with similar results. (B) NOS activity Assay. Cells were treated with resveratrol (10−7 M) for the indicated time periods, and NOS activity was measured by a radio-enzymatic method (see Materials and methods). Results are presented as the activity in the presence of resveratrol (10−7 M) vs. Control (vehicle-treated cells). Mean ±SE of three independent assays performed in duplicates.

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beverages, as was reported in a number of epidemiological studies, the agent might be active at concentrations usually found in biological fluids (nano- or picomolar range). Indeed we have previously reported that, at these concentrations, both resveratrol and flavonoids might exert an antiproliferative action in breast and prostate cancer cell cultures in vitro [29,30]. The aim of the present study was to evaluate the role of resveratrol in the regulation of the proliferation of HepG2 human hepatocellular carcinoma-derived cells, and provide some insights of the molecular mechanisms involved. We have explored the long-term (2 cell cycles) effects of resveratrol at the pico-/nanomolar range, concentrations compatible with those found in biological fluids after moderate wine or food consumption [11]. Our results show that resveratrol inhibits HepG2 cell growth, in a dose- and time-related manner, with an apparent IC50 of 30 pM. These concentrations are lower than those found in the majority of studies dealing with resveratrol effects in cells, but compatible with those reported previously, by our group, on the proliferation of breast and prostate cancer

Fig. 4. Inhibition of NOS activity reverts the effect of resveratrol on cell growth. Cells were incubated for 3 days in the presence of 10−7 M resveratrol, in the absence or the presence of the indicated concentrations of the general NOS inhibitor L-NAME. L-NAME per se, at the three tested concentrations, does not modify cell number (grey bars). Results are presented as a percentage of control, vehicle-treated, cells (cultured in the absence of any agent, =100%) *p < 0.01.

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molecules inhibited cell growth [51], while a stimulation of tumor growth was observed in lymphoma cells and in colon tumor cells transfected with iNOS [52]. Inducible NOS was positively correlated with tumor growth in gynecological tumors [53,54], indicating that the two major determinants for the effect of NO appear to be its production and tumor type. Excessive NO production results in limited angiogenesis and, in some tumor cells, increased apoptosis, while lower amounts can increase vascularity and protect cells from apoptosis. In normal liver, hepatocytes express low levels of endothelial and inducible NOS [26]. Nevertheless, during liver injury, there is a substantial increase in endothelial NOS, followed by induction of the inducible form of the enzyme; the subsequent massive NO production was incriminated to trigger the onset of several hepatopathies [55]. However, moderate NO levels after hepatectomy or redox stress could prevent cell apoptosis [56]. Treatment of HepG2 cells with NO donors revealed that NO could serve as a ROS sensor and subsequently regulate the expression of HIF-1 [57]. Conversely, NOS inhibitors rescue HepG2 cells from apoptosis (a result reported equally in the present study), when co-cocultured with irradiated endothelial cells [58]. The NO-mediated apoptosis was attributed to increased mitochondria permeability [59] or to reduced DNA binding of HNF-4á [60]. Hence, it has been suggested that NO may possess a dual pro- and anti-tumor activity, depending on the local concentration of the molecule [52]. Resveratrol has been reported to either suppress [61,62] or enhance [47,63–65] NO production. It was reported to induce NOS in cultured pulmonary artery endothelial cells [47], and gastric adenocarcinoma cells [66], leading to inhibition of their proliferation, a result equally reported in the present work. Indeed, inhibition of NOS activity reverted resveratrol action, indicating a direct relationship of increased NO production and inhibition of cell growth. In addition, we report that resveratrol might modify the NOS isoenzymes transcript levels. Previous reports presented evidence that resveratrol could modify iNOS expression [30,67–69]. In our system, iNOS expression was transiently induced by resveratrol, during the first 6 h, while a sustained stimulation of eNOS transcripts was detected. We have recently reported a similar finding in T47D breast cancer cells [70]: the agent induced activation of iNOS transcription and further enhanced eNOS expression, although the response was triggered after 12 h of stilbene treatment. The increased expression of NOS enzymes could further activate multiple molecular mechanisms, controlling cell cycle progression. Resveratrol was reported to induce apoptosis in HepG2 (p53 positive) but not in HeP3B (p53 negative) cell line through p53-associated p21 up-regulation, leading to G1 arrest [71]. As p53 has been implicated in the NO-induced apoptosis in breast cancer cells [72], it is possible that resveratrol could inhibit cell proliferation through up-regulation of eNOS transcripts and the subsequent activation of p53. An alternative mechanism could include NF-kB mediated responses, as resveratrol has been found to modulate IkBβ and iNOS expression [73], an effect associated with resveratrol antioxidant activity [74].

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> 50 μM. As discussed above, we have chosen to use low doses of the agent, compatible with its reported concentrations after ingestion of polyphenol-rich foods, or isolated polyphenol molecules [8,9]. Numerous authors have further investigated the effect of the stilbene on cell cycle-associated proteins. In colon cancer cells, a down-regulation of the cyclin D1/Cdk4 complex has been reported [40], while in transplantable liver cancer H22 cells, resveratrol decreased cyclin B1 and Cdc2 protein, although no modification of cyclin D1 was observed. G2 arrest was reported to be related to the inhibition of Cdk7 and Cdc2 [42]. In addition, an S-phase arrest was found in melanoma cells, being related to an up-regulation of cyclins A, E, and B1 [43]. Thus, it is clear that the effects of resveratrol on cell cycle are highly variable, depending on the cell line studied. An additional complexity level occurs, related to a dose-dependent action of resveratrol on DNA synthesis [44], attributed to the modulation of nuclear p21Cip1/WAF1 and p27Kip1 levels. Our data indicate a decrease (at nanomolar resveratrol concentrations) of DNA synthesis, and therefore a decrease of cells entering Sphase, suggesting that the p21 pathway may not be involved in its action. However, the observed accumulation of cells in G1 could also imply the Rb or the p53 pathways. Indeed, it was shown that resveratrol treatment of melanoma cells resulted in reduced hyperphosphorylated Rb and a relative increase of hypophosphorylated Rb. This response was accompanied by down-regulation of the expression of all five E2F family transcription factors and their heterodimeric partners DP1 and DP2, introducing an arrest of cell-cycle progression at the G1/S phase transition, thereby leading to subsequent apoptotic cell death [45]. In addition, in melanoma [46], endothelial [47] and fibroblastic cell lines [48], resveratrol treatment led to an activation of p53 activity, which correlated with suppression of cell progression through the S and G2 phases of the cell-cycle and apoptosis. The effect of resveratrol on the G2-phase of cell cycle could be attributed to the reported action of resveratrol on the cytoskeleton [28]. We have recently reported that catechin oligomers, acting on membrane androgen receptors could, induce apoptosis, through actin filaments rearrangement [49]. Preliminary results of our group further indicate an equal interaction of resveratrol with membrane androgen receptors, suggesting a similar effect on cytoskeleton components and a probable arrest in G2. Although the above results could explain the effect of resveratrol on the proliferation arrest of HepG2 cells, our data indicate another major mechanism of resveratrol action, namely the modulation of the NO/NOS system. Reactive nitrogen species (RNS) contribute to hepatocyte physiology and growth. RNS are by-products of NO production in living cells. Up-regulated RNS production can cause cell damage or death, through nitration of biological target molecules such as DNA, lipids, and proteins [50]. Nevertheless, the role of NO in cancer cell growth remains highly controversial, depending on its available concentration, target cell type, and interaction with reactive oxygen species (ROS), metal ions, and proteins [25]. For example in colon, pancreatic, breast, bladder and gastric cancer, increased NO production or use of NO-donor

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Acknowledgements

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Work partially supported by an EU (COOP-CT-2003508649 Project PARADOX) grant. GN holds a fellowship from the Manassaki Foundation, and APN holds a scholarship from the Public Benefit Foundation “A.S. Onassis”.

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In conclusion, the action of resveratrol on HepG2 hepatocellular carcinoma cells is exerted at different levels. First, the agent induces apoptosis, through a delayed action on the cell cycle, probably involving a number of cyclins or cdk modulation. Second, it acts as an antioxidant, and finally, a major action seems to be mediated through modulation of the transcription and activity of NOS. The interesting finding in our study is that these actions are mediated at nanomolar or picomolar levels, compatible with the concentrations of free resveratrol in biological fluids after ingestion of polyphenolrich foods and beverages, suggesting a possible protective effect of these foods on hepatocarcinogenesis and a potential role of resveratrol as an adjuvant treatment in hepatocellular cancer chemotherapy.

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