Introduction

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In normal physiological conditions, the amount of free intracellular arachidonic acid (AA) available is very small. However, the activation of phospholipases, mainly phospholipase A₂ (PLA₂) induces the release of AA from membrane phospholipids. Then, free intracellular AA can be metabolized via cyclooxygenase (COX), lipoxygenase, or cytochrome P-450 monooxygenase pathways. Thus, this fatty acid is the precursor for the further metabolism of a large number of biologically active products, named eicosanoids (Needleman et al., 1986).

There are two isoforms of COX that catalyze the formation of prostaglandins (PGs) from AA. COX-1 is a housekeeping gene that is expressed constitutively (Smith et al., 1996), whereas COX-2 is an immediate, early response gene that is highly inducible by mitogenic and inflammatory stimuli (Kujubu et al., 1991; Hla and Neilson, 1992).

Although many studies of eicosanoids have focused on their role as intercellular messengers in physiopathological processes such as inflammation, more recent information provides strong evidence that AA and/or its metabolites play an important role in the regulation of cell proliferation. Thus, considerable evidence indicates that the COX-2 pathway is important for cell proliferation. For example, COX-2 is up-regulated in transformed cells (Kutchera et al., 1996; Subbaramaiah et al., 1996; Sheng et al., 1997). Furthermore, a null mutation for COX-2 markedly reduced the number and size of intestinal tumors in a murine model of familial adenomatous polyposis (Oshima et al., 1996).

Recently, we observed that the multiple cell contact with neighboring cells in a confluent 3T6 fibroblast monolayer inhibits PLA₂ activity and, consequently, PGE₂ release and 3T6 fibroblast growth (Lloret et al., 1996), a situation that can be reversed by a mechanical wound. Thus, wound injury of a confluent monolayer initiates a repair process that restores the integrity of the cell monolayer. In these previous experiments, we demonstrated that the induction of COX-2 and prostanoid synthesis, and specifically PGE₂, plays an important role in induced cell proliferation and wound repair, stimulated by fetal calf serum (FCS) or platelet-derived growth factor (PDGF) in 3T6 fibroblasts cultures (Martinez et al., 1997; Moreno, 1997).

Resveratrol is a phytoalexin found in grapes and other foods that has anticancer and anti-inflammatory effects. Thus, it inhibits the development of preneoplastic lesions in carcinogen-treated mouse mammary glands, and it blocks tumorigenesis in a two-stage model of skin cancer (Jang et al., 1997). In addition, its anti-inflammatory properties were demonstrated by its suppression of carrageenan-induced paw edema, an effect attributed to suppression of PG synthesis (Jang et al., 1997). However, very little is known about the molecular basis for its biological activities. Resveratrol is endowed with antioxidant properties and inhibits the hydrogen peroxidase activity of COX-1 (Jang et al., 1997; Johnson and Madipati, 1998).

This article reports that resveratrol suppressed the production of reactive oxygen species (ROS) stimulated by FCS and PDGF in 3T6 fibroblast cultures, and that this effect was correlated with an impairment of [³H]AA release and the subsequent PGE₂ synthesis, and with a decrease in 3T6 fibroblast growth. Thus, these data provide evidence that ROS and prostanoids could be involved in the antiproliferative action of resveratrol.

	Materials and Methods

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Reagents. [5,6,8,9,11,12,14,15-³H]AA (180-240 Ci/mmol), phosphatidylcholine L--1-palmitoyl 2-arachidonyl[arachidonyl-1-¹⁴C] (60-80 Ci/mmol), and [methyl-³H]thymidine (20 Ci/mmol) were purchased from DuPont-New England Nuclear (Boston, MA). RPMI 1640 medium, FCS, penicillin G, streptomycin, and trypsin/EDTA were obtained from Life Technologies (Gaitherburg, MD). Aprotinin, leupeptin, diethyldithiocarbamic acid, phenylmethylsulfonyl fluoride, AA, PGE₂, PDGF, quercetin, curcumin, and resveratrol were acquired from Sigma Chemical Co. (St. Louis, MO). Polyclonal antiserum specific against COX-2 as well as ovine COX-2 were from Cayman Chemicals (Ann Arbor, MI). Resveratrol was resuspended in dimethyl sulfoxide at 100 mM and stored at 80°C. The final concentration of dimethyl sulfoxide never exceeded 0.1%. All other reagents were of analytical grade.

Fibroblast Culture. Murine 3T6 fibroblasts (CCL96; American Type Culture Collection, Manassas, VA) were grown and maintained as previously described (Lloret et al., 1996). Briefly, cells were grown in RPMI 1640 containing 10% FCS, penicillin (100 U/ml), and streptomycin (100 µg/ml). Cells were harvested with trypsin/EDTA and passed to tissue-culture plates with a surface area of 5 cm²/well (tissue-culture cluster 12; Costar, Cambridge, MA). Cell cultures were maintained in a temperature- and humidity-controlled incubator at 37°C with 95% air, 5% CO₂. Cell viability was tested in all experimental conditions with the trypan blue exclusion test.

Determination of ROS. Intracellular ROS levels were measured with a fluorescent dye, 2',7'-dichlorofluorescein diacetate (DCFL-DA). For assays, medium was replaced with HBS solution (130 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, and 10 mM HEPES, pH 7.4) containing 5 µM DCFL-DA. After 30 min of incubation at room temperature, medium was again replaced with fresh HBS solution. The fluorescent intensity in the absence or presence of stimuli was determined as described in Suzuki et al. (1998).

Cell Growth. 3T6 fibroblasts were plated at 10³ cells/well in 12-well plates (Costar) and cultured for 3 days in RPMI 1640 supplemented with FCS (10%) or PDGF (10 ng/ml) in the presence of resveratrol. Finally, the cells were washed, trypsinized, and counted.

Analysis of DNA Synthesis. DNA synthesis was measured by a [³H]thymidine incorporation assay. This involved culturing 3T6 fibroblasts in 96-well plates in RPMI 1640 with FCS (10%) or PDGF (10 ng/ml) at a density of 400 cells/well. Six hours later, cells were incubated with resveratrol and [³H]thymidine (1 µCi/well) for 24 h. [³H]Thymidine-containing media were aspirated, cells were overlaid with 1% Triton X-100, and then cells were scraped off the dishes. Finally, the radioactivity in the cell fraction was measured by scintillation counting with a Packard Tri-Carb 1500 counter.

Protein Determination. Protein concentration was measured by the Bradford (1976) method by means of the Bio-Rad detergent-compatible protein assay, with BSA as standard.

Determination of PLA₂ Activity. PLA₂ activity was measured in crude membrane and cytosolic fractions from cells. For this purpose, at the end of the experiments, the medium was replaced by cold buffer (20 mM Tris/HCl, pH 8.0; 5% saccharose). The dishes were either kept on ice for 15 min or frozen to 20°C before use. Then, fibroblasts were scraped off with a rubber policeman and briefly sonicated in the cold. To prepare crude membrane and cytosolic fractions, the homogenate was centrifuged at 100,000g for 1 h. The pellets were resuspended in buffer (20 mM Tris/HCl, pH 8.0; 5% saccharose) and PLA₂ activity was determined as described in Lloret et al. (1995) with the substrate proposed by Flesch and Ferber (1986), phosphatidylcholine L--1-palmitoyl 2-arachidonyl[arachidonyl-1-¹⁴C].

Incorporation and Release of [³H]AA. After a period of fibroblast replication (3-4 days) and a period of FCS starvation (16 h), the medium was removed and replaced with 0.5 ml RPMI 1640 containing 0.1% fatty acid-free BSA and 0.1 µCi [³H]AA for 24 h. Cells were then washed three times in medium containing 0.5% BSA to remove unincorporated [³H]AA. After a study period, the medium was removed for analysis of radioactivity released. The amount of [³H]AA released into the medium was expressed as a percentage of cell-incorporated [³H]AA, which was determined in solubilized cells. Background release from untreated cells (9 ± 2% of ³H incorporated) was subtracted from all data.

Measurement of PGE₂ Production by Fibroblasts. An aliquot of culture supernatant medium (0.25 ml) was acidified with 1 ml of 1% formic acid. PGE₂ was extracted in ethyl acetate (5 ml), and after the aqueous phase had been discarded, the organic phase was evaporated in a stream of nitrogen. The presence of PGE₂ was determined with a PGE₂-monoclonal enzyme immunoassay kit (Cayman Chemicals), following the manufacturer's protocol.

Western Blot Analysis of COX-2. Fibroblast cultures were washed twice in ice-cold PBS and scraped off in PBS containing 2 mM EDTA and pelleted. Cells were sonicated in PBS containing 2 mM EDTA, 20 µg/ml phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 20 µg/ml leupeptin, and 200 µg/ml dimethyldithiocarbamic acid. Equal amounts of protein (20 µg) were separated by a 10% SDS-polyacrylamide electrophoresis gel and immunoblot analysis for COX-2 was performed as described in Martinez et al. (1997) with a specific rabbit polyclonal antiserum anti-COX-2 (Cayman Chemicals), which did not cross-react with COX-1. Finally, antibody binding was visualized by the enhancement chemiluminescence technique (Amersham, Arlington Heights, IL).

Statistics and Data Analysis. Results are expressed as mean ± S.E. Differences between control cultures and treated cultures were tested by using either Student's t test or one-way ANOVA followed by the least significant difference test as appropriate.

	Results

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PLA₂ Activity Changes Induced by FCS or PDGF Are Affected by Resveratrol. PLA₂ activity can be induced to associate with natural membranes by growth factors in 3T6 fibroblasts cultures (Sanchez and Moreno, 2000). In quiescent cells only 12.8% of PLA₂ activity was associated with the membrane fraction. However, our data show that FCS and PDGF caused an increase in the percentage of PLA₂ activity associated with the membrane fraction (33.6 and 33.3%, respectively), whereas this percentage decreased in the cytosolic fraction in the same proportion, indicating a translocation process (Figs. 1 and 2). Resveratrol induced a dose-dependent inhibition on the enhancement of PLA₂ activity of membrane fraction stimulated by growth factors (Figs. 1 and 2).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Distribution of PLA2 activity in 3T6 fibroblast cultures with FCS in presence of resveratrol. Preconfluent cells (10,000 cells/cm2) were maintained with FCS starvation overnight. Then, cells were incubated with FCS (10%) in the absence or presence of resveratrol for 2 h and cells were harvested and frozen for the subsequent measurement of PLA2 activity in the cytosolic () and membrane () fraction. The data represent the mean ± S.E. from three experiments performed in triplicate. *P < .05 compared with nontreated group.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Distribution of PLA2 activity in 3T6 fibroblast cultures with PDGF in the presence of resveratrol. Preconfluent cells (10,000 cells/cm2) were maintained with FCS starvation overnight. Then, cells were incubated with PDGF (10 ng/ml) in the absence or presence of resveratrol for 2 h and cells were harvested and frozen for the subsequent measurement of PLA2 activity in the cytosolic () and membrane () fraction. The data represent the mean ± S.E. from three experiments performed in triplicate. *P < .05 compared with nontreated group.

[³H]AA Release and Subsequent PGE₂ Synthesis Stimulated by FCS or PDGF Are Inhibited by Resveratrol. PLA₂ activity translocation to the membrane fraction appears to be essential for AA release. Thus, this event can be correlated with [³H]AA mobilization and the subsequent metabolism through the COX pathway to synthesize prostanoids. Thus, FCS or PDGF induced [³H]AA release, whereas resveratrol at 30 µM, doses that inhibit PLA₂ activity in membrane fraction, significantly reduced this release. Moreover, FCS and PDGF enhanced PGE₂ synthesis, which was inhibited by resveratrol (30 µM; Table 1). Curcumin and quercetin, natural antioxidants (Cohly et al., 1998; Shutenko et al., 1999; Wang and Joseph, 1999) as is resveratrol, also had a significant effect on [³H]AA mobilization and PGE₂ production (Table 1).

Resveratrol Inhibits the Induction of COX-2 Stimulated by PDGF or H₂O₂. PGs are the predominant AA metabolites synthesized by fibroblasts (Mayer et al., 1984). Two isoenzymes of COX catalyze this conversion of AA to PGs and growth factors such as FCS induce markedly the expression of COX-2 in 3T6 fibroblast cultures (Martinez et al., 1997). In this study, PDGF induced COX-2 and the treatment with resveratrol decreased PDGF-mediated induction of COX-2. Furthermore, H₂O₂ significantly induced the cellular COX-2 levels and resveratrol also inhibited the effect of H₂O₂ on COX-2 induction (Fig. 3).

View larger version (61K): [in this window] [in a new window]   Fig. 3.   Western blot analysis of COX-2 isoform in cultured murine 3T6 fibroblasts. A, lane 1, ovine COX-2 (50 ng); lane 2, cell cultured with FCS starvation overnight; lane 3, cell cultured 2 h in RPMI 1640 with PDGF (10 ng/ml); lane 4, cell cultured with PDGF in presence of resveratrol (30 µM). B, lane 1, ovine COX-2 (50 ng); lane 2, cell cultured with FCS starvation overnight; lane 3, cell cultured 2 h in RPMI 1640 with H2O2 (1 mM); lane 4, cell cultured with H2O2 (1 mM) in presence of resveratrol (30 µM). Western blot shown is representative of four experiments with similar results.

Resveratrol Reduces ROS Production Stimulated by FCS or PDGF. ROS such as O₂ and H₂O₂ are involved in many biological processes as secondary messengers and/or as direct mediators, and they may affect cell growth (Arbault et al., 1997; Burch et al., 1997). To test this hypothesis, I monitored intracellular ROS in real time with the dye DCFL-DA, which reacts with free radical-derived oxidants to become 2',7'-dichlorofluorescein, a highly fluorescent compound (Ohba et al., 1994). The overall intracellular levels of the oxidants in the responsive cells began to increase a few minutes after FCS or PDGF was added, reaching a maximum at 30 to 40 min (Fig. 4). Interestingly, this increase was significantly abolished by treatment with resveratrol (Fig. 4).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Changes in intracellular ROS levels of 3T6 fibroblasts after resveratrol treatment. Intracellular ROS levels were measured with DCFH-DA. Cells were untreated (open symbols) or treated (filled symbols) with resveratrol (30 µM) 30 min before assays. For assays, medium was replaced with HBS solution containing 5 µM DCFH-DA. After 40 min of incubation at room temperature, medium was replaced with fresh HBS solution. In the absence () or presence of FCS 10% (, ) or PDGF (10 ng/ml, , ), the fluorescent intensity was monitored. Relative fluorescence intensity was calculated with untreated control cells as standard. Data are pooled from six different experiments.

Resveratrol Impaired 3T6 Fibroblast Proliferation. To analyze the effect of resveratrol on FCS or PDGF-stimulated growth cell, 3T6 fibroblast were incubated in medium containing the growth factors with or without the polyphenol. As shown Fig. 5, resveratrol significantly reduced 3T6 fibroblast growth. Similar data were obtained when I studied the effect of the polyphenol on DNA synthesis induced by FCS or PDGF. Thus, resveratrol reduced significantly the [³H]thymidine incorporation induced by the growth factors (Table 2). Same effects were obtained when curcumin or quercetin were used.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Effect of resveratrol on 3T6 fibroblast growth induced by FCS (10%, ) or PDGF (10 ng/ml, ). The data represent the mean ± S.E. from three experiments performed in triplicate. *P < .05 compared with nontreated group.

	Discussion

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ROS are considered to be toxic to cells. However, the molecular detection and response of cells to ROS are likely to be among the earliest evolved second-messenger systems. Moreover, there is evidence that ROS are not only pathological but also, in many instances, are used as second messengers by the cell in response to growth factors (Sundaresan et al., 1995), considering that ROS are growth promoters. Thus, Shibanuma et al. (1988) reported that the treatment of 3T3 fibroblast with H₂O₂ plus insulin induced progression of the cell cycle from the quiescent state. Moreover, Irani et al. (1997) have reported that activation of H-Ras in fibroblasts increased ROS production, which was associated with increased cellular proliferation.

In addition, a previous report indicated that ROS could be involved in the regulation of PLA₂ activity and the subsequent AA release in 3T6 fibroblasts (Martinez and Moreno, 1996). Furthermore, changes in intracellular distribution of PLA₂ stimulated by growth factors may be controlled by 3T6 fibroblast growth through a mechanism dependent on PG release (Lloret et al., 1996; Moreno, 1997; Sanchez and Moreno, 1999). Thus, the impairment of 3T6 fibroblast growth induced by PLA₂ inhibitors or COX inhibitors was reverted by the addition of PGE₂ to the culture medium (Martinez et al., 1997; Sanchez and Moreno, 1999). On the basis of these findings, I hypothesized that active oxygen species and AA cascade metabolites could be altered in the signal transduction pathways that regulate 3T6 fibroblast growth. Furthermore, I hypothesized that these mechanisms could be interrupted by an antioxidant such as resveratrol.

In this study, I observed that PDGF or FCS increases ROS production and that resveratrol, a natural antioxidant (Frankel et al., 1993), markedly inhibits ROS produced by FCS- and PDGF-stimulated 3T6 fibroblasts. Moreover, treatment with the polyphenol reduced the FCS- or PDGF-induced translocation of PLA₂ activity to the membrane fraction and the subsequent [³H]AA release and PGE₂ synthesis. An 85-kDa cytosolic PLA₂ is expressed in many cell types such as fibroblasts (Bunt et al., 1997) and appears to be involved in the selective AA release. Recently, several studies documented that both calcium-dependent translocation to membranes and phosphorylation are required for AA release by cystolic PLA₂ (Schievella et al., 1995; McNicol and Shibou, 1998). Martinez and Moreno (1996) showed that ROS are potent stimulators of signal-responsive PLA₂ activity, and this study has now provided evidence that resveratrol and other polyphenols such as quercetin and curcumin could interfere in this pathway.

After AA mobilization, COX catalyzes the metabolism of AA in fibroblasts. There are two isozymes, COX-1 and COX-2, involved in PG synthesis (DeWitt, 1991). COX-1 is expressed constitutively in most cells and its activity is regulated by substrate availability, whereas COX-2 is an inducible enzyme expressed in activated 3T6 fibroblasts and involved in the control of 3T6 fibroblast growth (Martinez et al., 1997). Numerous genes and their enzyme products, including COX-2, are regulated by cellular redox status. Thus, I observed the induction of COX-2 by H₂O₂ at extracellular concentrations ranging from 100 µM to 1 mM H₂O₂ (data not shown). These concentrations are similar to the amounts of H₂O₂ needed to mimic the levels of intracellular H₂O₂ generated by PDGF treatments (Sundaresan et al., 1995). Furthermore, I observed that the induction of COX-2 by H₂O₂ or PDGF was significantly inhibited by resveratrol, in agreement with previous results that showed that resveratrol suppressed phorbol-12-myristate-13-acetate-mediated increases in COX-2 mRNA and protein and that the polyphenol also directly inhibits the activity of COX-2 (Subbaramaiah et al., 1998). This suggests that it may be the antioxidant properties of resveratrol that modulates AA release and the subsequent AA metabolism via the COX pathway.

Interestingly, my data also show that resveratrol, curcumin, or quercetin significantly inhibit 3T6 fibroblast proliferation and DNA synthesis induced by FCS or PDGF. A decrease in cell proliferation can be attributed to either growth arrest or cell loss due to apoptosis. Growth of 3T6 fibroblast in the presence of resveratrol followed by propidium iodide staining showed that the antioxidant did not induce apoptosis in these experimental conditions (data not shown). This finding suggests that resveratrol could induce an arrest of the cell cycle as reported by Della Ragione et al. (1998) with HL-60 cells. Furthermore, the effect of resveratrol on 3T6 fibroblast growth was markedly reverted when I added AA or PGE₂ to culture medium.

In summary, these data show that resveratrol inhibits 3T6 fibroblast proliferation in vitro, suggesting a direct relationship between the activity of the antioxidant to alter intracellular redox status, decrease PG synthesis, and affect cell growth. Based on these findings, I suggest that these mechanisms are involved in the effect of the polyphenol on inflammation and cancer. The location of hydroxyl functional groups at 2', 3', or 4' site(s), especially at the 4' site, as in resveratrol, curcumin, or quercetin, seems essential for anti-12-O-tetradecanoylphorbol 13-acetate induced transformatin (Lee and Lin, 1997) and could be involved in these effects on arachidonate cascade and 3T6 fibroblast proliferation. Although, I must consider that resveratrol also is able to inhibit ribonucleotide reductase (Fontecave et al., 1998) and DNA polymerase (Sun et al., 1998), and it might act as a phytoestrogen (Gehm et al., 1997).