Department of Medicine, Division of Gastroenterology, Cook County Hospital and Hektoen Institute for Medical Research, Chicago, Illinois 60612
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ABSTRACT |
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Resveratrol is a dietary phytochemical that
has been shown to inhibit proliferation of a number of cell lines, and
it behaves as a chemopreventive agent in assays that measure the three
stages of carcinogenesis. We tested for its chemopreventive potential against gastric cancer by determining its interaction with signaling mechanisms that contribute to the proliferation of transformed cells.
Low levels of exogenous reactive oxygen (H2O2)
stimulated [3H]thymidine uptake in human gastric
adenocarcinoma SNU-1 cells, whereas resveratrol suppressed both
synthesis of DNA and generation of endogenous O
nitric oxide synthase; reactive oxygen species; gastric adenocarcinoma cells
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INTRODUCTION |
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A VARIETY OF NONPHAGOCYTIC cells generate low levels of reactive oxygen species in response to cytokines and peptide growth factors (10, 35), and the reactive oxygen generated by this miniburst interacts with guanine nucleotide binding proteins and with the Ras pathway to act as an intracellular messenger (17, 18). In transformed cells, the interaction between reactive oxygen and cellular signaling pathways results in transcription factor activation and modulation of gene expression, culminating in enhanced proliferation (5). On the basis of these observations, reactive oxygen species have been considered as procarcinogenic, whereas inhibition of reactive oxygen generation is now considered a plausible approach to suppress cancer development and progression. Resveratrol is a naturally occurring phytoalexin that was shown to inhibit the induction, promotion, and progression of experimentally induced cancer (19). Moreover, resveratrol inhibits transcription and activity of cyclooxygenase-2 (38, 39), an enzyme found to be upregulated in a number of transformed cells and various forms of cancer. Resveratrol also possesses antioxidant and anti-inflammatory activities, as evidenced by its ability to inhibit superoxide generation in stimulated neutrophils (33) and macrophages (19) and by its ability to protect low-density lipoproteins against oxidative damage (41, 46). Increased cellular levels of antioxidants function by 1) directly scavenging reactive oxygen radicals, 2) preventing the formation of cellular reactive oxygen, and/or 3) increasing cellular detoxification mechanisms. The mechanism whereby resveratrol suppresses generation of reactive oxygen species in transformed cells has not yet been addressed.
Although the incidence of gastric cancer is on the decline, this disease remains a major health problem and a common cause of cancer mortality worldwide, because the disease is usually detected at an advanced stage, and the currently available chemotherapeutic agents are not highly effective. Development of gastric cancer is believed to be a slow process, with primary etiological determinants for gastric cancer being exposure to chemical carcinogens and/or infection with Helicobacter pylori (12, 21, 30). Several key events that follow a chemical insult or infection with H. pylori are 1) an inflammatory response in the host gastric mucosa with release of numerous cytokines and reactive oxygen species, 2) glandular atrophy, and eventually 3) cellular proliferative changes such as metaplasia and dysplasia. Therefore, agents like resveratrol that can suppress neutrophil reactivity and the inflammatory response (33) and simultaneously inhibit proliferation of transformed gastric epithelial cells while remaining relatively nontoxic to the host (1) may constitute a new and effective defense against gastric carcinogenesis.
Prevention, suppression, or reversal of cancer induction through long-term use of naturally occurring compounds available in the diet is designated as chemoprevention. Epidemiological evidence indicates a protective effect of fruits and vegetables against gastric cancer, and this protection has been ascribed to their rich source of antioxidant vitamins and polyphenols. Among the polyphenolic compounds tested and proven somewhat effective against gastric cancer is curcumin (28, 36), shown to inhibit carcinogen-induced formation of gastric tumors by interacting with cellular signal transduction pathways to suppress cellular proliferation and induce apoptosis in the targeted cell. Some of the intermediate molecular events associated with the action of curcumin have been addressed (31).
We (1) have shown that resveratrol inhibits proliferation of gastric adenocarcinoma cells and reverses the stimulatory action of carcinogenic nitrosamines through a protein kinase C-mediated mechanism. Here we report on the antiproliferative action of resveratrol toward gastric adenocarcinoma SNU-1 cells through its efficacy as a modulator of reactive nitrogen and oxygen generation in these cells.
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MATERIALS AND METHODS |
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Cells. The human gastric adenocarcinoma cell line SNU-1 (ATTC: CRL-5971) was routinely cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 0.1 mM nonessential amino acids, 10 U/ml of streptomycin, and 0.25 µg/ml of amphotericin B at 37°C in a humidified incubator containing 5% CO2. Freshly plated cells were always allowed to equilibrate 2-3 h before the addition of resveratrol (a kind gift from Pharmascience, Montreal, PQ, Canada), phorbol 12-myristate 13-acetate (PMA), sodium nitroprusside (SNP), or hydrogen peroxide (H2O2). PMA, SNP, and H2O2 were obtained from Sigma (St. Louis, MO). SNP and H2O2 were added as aqueous solutions, whereas resveratrol was dissolved in 95% ethanol, and the concentration of ethanol to which control and treated cells were exposed was always maintained at 0.1%. Because resveratrol is described as a light-sensitive and somewhat labile compound, its exposure to light was minimized, and fresh solutions were prepared on a weekly basis. PMA was dissolved in DMSO, and the concentration of DMSO to which cells were exposed was always maintained at 0.01%.
DNA synthesis. Synthesis of DNA was used as an index of cellular proliferation and was determined by the incorporation of [3H]thymidine into cellular DNA. Cells at a density of 0.5 × 106 cells/5 ml of medium were plated in triplicate in six-well plates. Cells were allowed to equilibrate for 2 h at 37°C before the addition of 1 µCi of methyl-[3H]thymidine ([3H]thymidine; Amersham/Pharmacia Biotech, Piscataway, NJ) and the appropriate concentrations of resveratrol or SNP and were then incubated at 37°C for an additional 24 h. To determine the effects of H2O2 on DNA synthesis, SNU-1 cells were transferred to 0.1% FBS-supplemented medium for 48 h before the addition of H2O2 and [3H]thymidine and were then incubated in 0.1% FBS-containing media with H2O2 in the absence and presence of resveratrol for an additional 24 h. At the end of treatment, cells were harvested in 5 ml PBS, centrifuged at 250 g for 4 min at 4°C, the PBS removed, and the resulting cell pellet suspended in 5 ml of 10% trichloroacetic acid (TCA) at 4°C for 30 min to precipitate protein-bound DNA. Formed precipitate was centrifuged at 1,500 g for 5 min at 4°C, washed once with 5 ml of ice-cold 10% TCA and solubilized in 0.25 ml of 0.1 N NaOH at 60°C, and added to 10 ml of scintillant. Amounts of incorporated [3H]thymidine were quantified by liquid scintillation counting and are expressed as a percentage of their respective control.
Measurement of NO synthase activity. To determine the action of resveratrol on NO synthase (NOS) activity in SNU-1 cells, cells were incubated at 37°C for 16 h in CO2-air with and without the specified concentrations of agonists in 25-cm2 flasks at a density of 2.5 × 106 cells/10 ml media. At the end of treatment, cells were harvested, washed with 5 ml of PBS, resuspended in ice-cold 0.5 ml of 25 mM Tris · HCl (pH 7.4) containing 10 mM EDTA and 10 mM EGTA, and quick frozen in liquid nitrogen where they were stored for further use. Immediately before assay cells were lysed by two freeze-thaw cycles, NOS activity in cell lysates was determined by measuring the conversion of L-[2,3-3H]-arginine to L-[3H]citrulline by employing the NOS assay kit from Calbiochem (La Jolla, CA). Briefly, to 10 µl of cell lysate were added 40 µl of buffer to yield final concentrations of reagents as follows: 25 mM Tris · HCl, pH 7.4, 3 µM tetrahydrobiopterin, 1 µM FAD, 1 µM flavin mononucleotide, 1 mM NADPH (freshly prepared in 10 mM Tris · HCl, pH 7.4), 0.6 mM CaCl2, and 1 µCi of L-[2,3-3H]-arginine (New England Nuclear Life Science Products, Boston, MA). After incubation at 37°C for 60 min, the reaction was terminated by the addition of 400 µl of 50 mM HEPES, pH 5.5, containing 5 mM EDTA, followed by the addition of 100 µl of Dowex AG-50 WX-8 resin suspension in the above HEPES buffer. Samples were mixed, transferred to spin cups, and centrifuged at full speed for 20 s in an Eppendorf microfuge. Eluate, containing the formed L-[3H]citrulline, was transferred to scintillation vials, and the amounts of generated L-[3H]citrulline were quantified in a liquid scintillation counter. All individual treatment groups were performed in triplicate, and all findings were confirmed in at least three independent experiments. The amount of L-[3H]citrulline generated was calculated per milligram lysate protein [protein content was determined by the method of Lowry (25)] and expressed as relative values, with NOS activity in PMA-treated cells assigned an arbitrary value of 1.0.
Measurement of superoxide release.
The action of resveratrol on superoxide (O
Measurement of apoptosis. The percentage of apoptotic SNU-1 cells was determined using a photometric ELISA assay from Boehringer-Mannheim (Cell Death Detection ELISAPLUS) that measures cytoplasmic histone-associated DNA fragments. SNU-1 cells plated in 96-well plates at a density of 1 × 104 cells/100 µl media were treated with resveratrol (10 and 100 µM) for 24 h or with 50 µM camptothecin for 48 h. Optimal apoptotic response (100% apoptotic cells) was observed after cell treatment with 50 µM camptothecin for 48 h, and this value was used to calculate the percentage of apoptotic SNU-1 cells after resveratrol treatment.
Measurement of cellular NADPH levels. The cellular levels of NADPH in resveratrol and ionomycin-treated cells were determined by utilizing bioreduction of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium (MTS) tetrazolium compound (Owen's reagent from Promega, Madison, WI) to its colored formazan product. The quantity of formazan, directly dependent on the amount of NADPH in live cells, was measured by absorbance at 490 nm. To 104 cells in 100 µl of media were added the specified concentrations of resveratrol and ionomycin, and the cells were incubated in 96-well plates at 37°C in 5% CO2-humidified air for 2 h. At the end of the 2-h incubation period were added 20 µl of the MTS reagent, and the plates were incubated for an additional 3 h to develop the colored formazan product, after which time absorbance at 490 nm was recorded using a 96-well plate reader from Molecular Devices (Sunnyvale, CA). Media alone were used as the blank and were subtracted from all other values. Results, expressed as absorbance (optical density at 490 nm), were derived from at least five individual experiments with each experimental value obtained from quadruplicate measurements.
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RESULTS |
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Resveratrol inhibits DNA synthesis in gastric adenocarcinoma SNU-1
cells.
Gastric adenocarcinoma SNU-1 cells proliferate in culture at a fairly
rapid rate with a doubling time of ~24 h. Resveratrol treatment of
these cells resulted in marked suppression of
[3H]thymidine incorporation into cellular DNA (Fig.
1) that was concentration-dependent with
a calculated IC50 value of 25 µM. Treatment with 100 µM
resveratrol, the highest concentration of resveratrol used in this
study, resulted in 97% inhibition of [3H]thymidine
incorporation into SNU-1 cells.
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Resveratrol stimulates NOS activity in SNU-1 cells.
Under basal conditions, SNU-1 cells did not express measurable NOS
activity. However, treatment of these cells for 16 h with PMA
and/or resveratrol resulted in stimulation of NOS activity. NOS
activity obtained after treatment with 0.1 µM PMA was designated as
baseline activity and was assigned a value of 1.0 within each experiment to account for interexperimental variations. Cell treatment with resveratrol resulted in concentration-dependent stimulation of NOS
activity with over threefold activation obtained after treatment with
100 µM resveratrol (Fig. 2). The
stimulatory action of 0.1 µM PMA was found to be additive in the
presence of 10 µM resveratrol, but there was no further response
to 0.1 µM PMA when cells were treated with 100 µM resveratrol.
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Action of SNP on DNA synthesis in SNU-1 cells.
Because resveratrol stimulated NOS activity in SNU-1 cells, we explored
the role of its product, NO, on DNA synthesis using SNP as a NO donor.
As shown in Fig. 3, treatment of SNU-1
cells with low concentrations of SNP (0.05 and 0.1 mM) had no effect on
[3H]thymidine incorporation, whereas cell treatment with
higher SNP concentrations (0.5-5 mM) resulted in significant
(P < 0.001) and dose-related suppression of
[3H]thymidine uptake into cellular DNA, with total
inhibition of [3H]thymidine uptake at 5.0 mM SNP.
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Resveratrol inhibits H2O2-stimulated
proliferation of SNU-1 cells.
To determine the action of reactive oxygen on cellular DNA synthesis,
serum-deprived SNU-1 cells were cultured in the absence and presence of
H2O2. Incubation of SNU-1 cells with varying
concentrations of H2O2 resulted in a
concentration-dependent stimulation of
[3H]thymidine incorporation that peaked with a
2.5-fold increase at 108 M
H2O2, remained elevated over the
corresponding controls at 10
7 and 10
6 M
H2O2, but declined to control levels at
10
5 M H2O2 (Fig.
4). Cell treatment with 100 µM
resveratrol inhibited incorporation of [3H]thymidine into
SNU-1 cell DNA regardless of the presence of stimulating concentrations
of H2O2. Resveratrol, therefore, effectively inhibited both basal (in the absence of exogenous
H2O2) and
H2O2-stimulated DNA synthesis.
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SNP suppresses superoxide generation by SNU-1 cells.
Quiescent human gastric adenocarcinoma SNU-1 cells do not release
measurable amounts of endogenous reactive oxygen species. However,
similar to several other cell types, they responded to ionomycin
treatment with a small but measurable oxidative burst detectable only
when the oxidation of luminol was enhanced by the addition of
lucigenin. Subsequently, generation of reactive oxygen species by SNU-1
cells was routinely stimulated by treatment of the cells with 1 µM
ionomycin and was measured in the presence of luminol and lucigenin as
described in MATERIALS AND METHODS. To
determine the action of NO on O
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Resveratrol inhibits superoxide generation by SNU-1 cells.
Ionomycin-stimulated oxidative burst in SNU-1 cells was also inhibited
in a concentration-dependent manner by resveratrol (Fig.
6). The oxidative burst in response to 1 µM ionomycin decayed very rapidly, and resveratrol enhanced this
decay with nearly total suppression of reactive oxygen generation
observed within 3 min after the addition of 100 µM resveratrol.
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Resveratrol and ionomycin deplete cellular NADPH.
Generation of O
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High-resveratrol concentration induces apoptosis in SNU-1
cells.
Inhibition of cellular proliferation by exogenous agents often
culminates in an apoptotic response. Therefore, we determined the
apoptotic response of SNU-1 cells to treatment with resveratrol for
the duration of time in which we observed inhibition of
[3H]thymidine incorporation and NOS activation. The
effect of resveratrol on SNU-1 cell apoptosis is presented in
Fig. 7. Normally proliferating SNU-1
cells contained <5% apoptotic cells, and cell treatment with 10 µM resveratrol for 24 h, conditions that result in significant stimulation of NOS activity, did not increase the percentage of apoptotic cells. However, when cells were treated for 24 h
with 100 µM resveratrol, there was a significant increase in
apoptotic cells with nearly half of the cells exhibiting DNA
fragmentation.
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DISCUSSION |
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Chemoprevention, defined as the use of nontoxic substances to
inhibit or reverse the process of carcinogenesis, is now considered an
essential approach to cancer prevention and/or treatment. Because gastric cancer is known to have epigenetic origins, such as infection with H. pylori and/or exposure to carcinogenic nitrosamines
(12, 30), it is thought to be preventable through
appropriate intervention. However, at present, there are limited
experimental data regarding specific agents that prevent or retard
gastric carcinogenesis. Several polyphenolic compounds, among them
curcumin, have demonstrated anticarcinogenic activities in experimental
animal cancer models, and their potential as chemopreventive agents
against gastric cancer has been discussed (28, 31, 36,
45). Recent evidence indicates that dietary intervention through
supplementation with antioxidants like ascorbic acid and -carotene
results in regression of H. pylori-induced gastric
dysplasia, a precursor event in gastric carcinogenesis
(7). These studies indicate that polyphenolic compounds
and antioxidants exert a protective action against gastric cancer.
However, there is a paucity of experimental data regarding the cellular
mechanisms engaged by these compounds in their chemopreventive action
against gastric cancer.
We addressed the chemopreventive potential of resveratrol against
gastric cancer by determining the action of resveratrol on the
proliferation of gastric adenocarcinoma SNU-1 cells and on some of the
molecular events associated with cellular proliferation and survival.
There are sufficient data demonstrating that low levels of reactive
oxygen act as intracellular messengers and promote the growth of
transformed cells (5, 10, 17, 18, 35). In addition to
reactive oxygen species, reactive nitrogen in the form of NO has also
been implicated in regulation of cellular proliferation, but its role
as a proliferative signal is not well defined, because it appears to
depend on the cell type responsible for its release and the NOS
isoforms within that cell, as well as on the concentration of released
NO and on the composition of the intracellular milieu (22, 34,
44). The neuronal and endothelial isoforms are thought to be
responsible for production of low levels of NO (37) and
both isoforms have been identified in gastric mucosa (3,
32). NO is a lipophilic radical that, when produced in small
amounts, can exert beneficial effects by reacting with
O
Because resveratrol behaves as an antioxidant and can affect cellular
NO production, we questioned whether its chemopreventive potential
might result, in part, from its action on the generation of these two
reactive species. We tested the antioxidant action of resveratrol by
directly measuring its effect on ionomycin-stimulated reactive oxygen
generation and by its action against
H2O2-stimulated proliferation. Ionomycin
induces generation of reactive oxygen in several cell types, and there
is data indicating that ionomycin-stimulated generation of reactive
oxygen is dependent on its action as a calcium ionophore
(8). Normally, proliferating SNU-1 cells did not generate
measurable levels of reactive oxygen, but treatment with ionomycin
resulted in generation of low levels of reactive oxygen, and this was
suppressed by resveratrol. SNU-1 cells responded to low concentrations
of H2O2 with increased DNA synthesis, findings in line with reports showing that transformed cells respond to low
levels of reactive oxygen species with increased proliferation. Resveratrol reversed the proliferative effect of
H2O2, and at high concentration (100 µM)
totally suppressed [3H]thymidine uptake, suggesting that,
in addition to its antioxidant action, resveratrol may also use other
pathways to exert effective antiproliferative control. The response of
SNU-1 cells to ionomycin treatment resulted in depletion of cellular
NADPH, suggesting that in these cells, ionomycin activates an NADPH
oxidase-like complex. Although resveratrol inhibited
ionomycin-triggered generation of O-stimulated NOS in macrophages (26),
resveratrol stimulates NOS activity and inhibits proliferation of
pulmonary artery endothelial cells (15), suggesting a
different mode of action in nonphagocytic cells. SNU-1 cells also
responded to resveratrol treatment with increased NOS activity. PMA,
which is known to stimulate all three NOS isoforms in a variety of cell
types (2, 24, 29) also stimulated NOS in SNU-1 cells, and
its action was additive with that elicited by low concentration of
resveratrol. However, PMA had no further effect on NOS activity when
cells were treated with high resveratrol concentration, indicating that 100 µM resveratrol elicits a maximal response with respect to both
NOS activation and suppression of DNA synthesis.
To test the action of NO on cellular proliferation, we measured
[3H]thymidine incorporation over a range of SNP
concentrations and observed that low concentrations of SNP had no
effect on DNA synthesis in SNU-1 cells, whereas higher concentrations
exerted an antiproliferative effect. Although we have no direct
measurements of NO levels after cell treatment with SNP, our data
indicate that production of low levels of NO is not detrimental to
SNU-1 cells, but higher levels suppress cellular proliferation. This
observation corroborates findings showing that exposure of SNU-1 cells
to high resveratrol concentration (100 µM), which results in maximal
NOS activation, also results in significant apoptotic response,
whereas cell treatment for the same duration of time with lower
concentrations of resveratrol (10 µM) does not induce
apoptosis. Apoptosis at high resveratrol concentration
may result from accumulation of peroxynitrite arising from production
of significant levels of NO that react with endogenously generated
O
We have shown that resveratrol treatment inhibits protein kinase C activity, induces cell cycle arrest, and suppresses nitrosamine-stimulated proliferation of gastric adenocarcinoma cells (1). The current data indicate that the antioxidant action of resveratrol resides, in part, in its ability to stimulate NOS and enhance production of NO that would interact with endogenously produced reactive oxygen to inhibit SNU-1 proliferation and eventually induce cell death by apoptosis. These observations lend further credence to the intermediary action of NO in resveratrol-elicited cellular responses (6, 16, 40), support existing evidence that the chemopreventive potential of resveratrol results from its interaction with cell signaling mechanisms that control cellular proliferation and apoptotic death, and argue that consumption of a resveratrol-rich diet may be protective against gastric cancer.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. H. Lum for critical commentary of the manuscript and M. Zopel for technical assistance and help in preparation of the figures.
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FOOTNOTES |
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This work was supported, in part, by American Cancer Society, Illinois Division, Grant 99-43.
Address for reprint requests and other correspondence: O. Holian, Dept. of Medicine, Division of Gastroenterology, Cook County Hospital, 627 S. Wood St., Rm. 765, Chicago, IL 60612 (E-mail: oholian{at}aol.com).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 30, 2002;10.1152/ajpgi.00193.2001
Received 9 May 2001; accepted in final form 24 January 2002.
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