This article was originally published in a journal published by
Elsevier, and the attached copy is provided by Elsevier for the
author’s benefit and for the benefit of the author’s institution, for
non-commercial research and educational use including without
limitation use in instruction at your institution, sending it to specific
colleagues that you know, and providing a copy to your institution’s
administrator.
All other uses, reproduction and distribution, including without
limitation commercial reprints, selling or licensing copies or access,
or posting on open internet sites, your personal or institution’s
website or repository, are prohibited. For exceptions, permission
may be sought for such use through Elsevier’s permissions site at:
http://www.elsevier.com/locate/permissionusematerial
Biochimica et Biophysica Acta 1760 (2006) 1657 – 1666
www.elsevier.com/locate/bbagen
py
Resveratrol exerts its antiproliferative effect on HepG2 hepatocellular
carcinoma cells, by inducing cell cycle arrest, and NOS activation
b
co
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
al
Received 2 May 2006; received in revised form 25 August 2006; accepted 15 September 2006
Available online 22 September 2006
on
Abstract
pe
rs
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
th
or
's
Keywords: Resveratrol; Hepatocellular carcinoma; Cell cycle; Apoptosis; Nitric oxide (NO); Nitric oxide synthase (NOS)
Au
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
co
py
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
al
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.
Au
th
or
's
pe
rs
(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.
on
1658
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
G. Notas et al. / Biochimica et Biophysica Acta 1760 (2006) 1657–1666
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)
py
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
co
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
th
or
's
on
pe
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
Au
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).
rs
2.7. Determination of cell cycle and apoptosis
al
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.
1659
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
1660
G. Notas et al. / Biochimica et Biophysica Acta 1760 (2006) 1657–1666
3.3. Effect of resveratrol on the NO/NOS system
py
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.
on
al
co
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.
th
or
's
pe
rs
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).
Au
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
1661
rs
on
al
co
py
G. Notas et al. / Biochimica et Biophysica Acta 1760 (2006) 1657–1666
th
or
's
pe
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
Au
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
G. Notas et al. / Biochimica et Biophysica Acta 1760 (2006) 1657–1666
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
rs
on
al
co
py
1662
th
or
's
pe
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.
Au
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.
G. Notas et al. / Biochimica et Biophysica Acta 1760 (2006) 1657–1666
on
al
co
py
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].
Au
th
or
's
pe
rs
> 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
1663
G. Notas et al. / Biochimica et Biophysica Acta 1760 (2006) 1657–1666
[15]
[16]
[17]
[18]
[19]
Acknowledgements
[20]
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”.
[23]
Au
th
or
's
pe
rs
[1] G.J. Soleas, E.P. Diamandis, D.M. Goldberg, Wine as a biological fluid:
history, production, and role in disease prevention, J. Clin. Lab. Anal. 11
(1997) 287–313.
[2] P. Langcake, R. Pryce, The production of resveratrol by Vitis vinifera and
other members of the Vitaseae as a response to infection or injury, Physiol.
Plant Pathol. 9 (1976) 77–86.
[3] L. Creasy, M. Coffee, Phytoalexin production potential of grape berries, J. Am.
Soc. Hortic. Sci. 113 (1988) 230–234.
[4] P. Kopp, Resveratrol, a phytoestrogen found in red wine. A possible
explanation for the conundrum of the ‘French paradox’? Eur. J. Endocrinol.
138 (1998) 619–620.
[5] A.Y. Sun, A. Simonyi, G.Y. Sun, The “French Paradox” and beyond:
neuroprotective effects of polyphenols, Free Radical Biol. Med. 32 (2002)
314–318.
[6] M. Jang, L. Cai, G.O. Udeani, K.V. Slowing, C.F. Thomas, C.W.
Beecher, H.H. Fong, N.R. Farnsworth, A.D. Kinghorn, R.G. Mehta, R.C.
Moon, J.M. Pezzuto, Cancer chemopreventive activity of resveratrol, a
natural product derived from grapes, Science 275 (1997) 218–220.
[7] B.B. Aggarwal, A. Bhardwaj, R.S. Aggarwal, N.P. Seeram, S. Shishodia,
Y. Takada, Role of resveratrol in prevention and therapy of cancer:
preclinical and clinical studies, Anticancer Res. 24 (2004) 2783–2840.
[8] A. Bertelli, A.A. Bertelli, A. Gozzini, L. Giovannini, Plasma and tissue
resveratrol concentrations and pharmacological activity, Drugs Exp. Clin.
Res. 24 (1998) 133–138.
[9] X. Vitrac, A. Desmouliere, B. Brouillaud, S. Krisa, G. Deffieux, N. Barthe,
J. Rosenbaum, J.M. Merillon, Distribution of [14C]-trans-resveratrol, a
cancer chemopreventive polyphenol, in mouse tissues after oral administration, Life Sci. 72 (2003) 2219–2233.
[10] M.I. Kaldas, U.K. Walle, T. Walle, Resveratrol transport and metabolism
by human intestinal Caco-2 cells, J. Pharm. Pharmacol. 55 (2003)
307–312.
[11] D.M. Goldberg, J. Yan, G.J. Soleas, Absorption of three wine-related
polyphenols in three different matrices by healthy subjects, Clin. Biochem.
36 (2003) 79–87.
[12] X. Meng, P. Maliakal, H. Lu, M.J. Lee, C.S. Yang, Urinary and plasma
levels of resveratrol and quercetin in humans, mice, and rats after ingestion
of pure compounds and grape juice, J. Agric. Food Chem. 52 (2004)
935–942.
[13] N. Carbo, P. Costelli, F.M. Baccino, F.J. Lopez-Soriano, J.M. Argiles,
[22]
on
References
[21]
py
[14]
Resveratrol, a natural product present in wine, decreases tumour growth in a
rat tumour model, Biochem. Biophys. Res. Commun. 254 (1999) 739–743.
Z.J. Sun, C.E. Pan, H.S. Liu, G.J. Wang, Anti-hepatoma activity of
resveratrol in vitro, World J. Gastroenterol. 8 (2002) 79–81.
D. Delmas, B. Jannin, M. Cherkaoui Malki, N. Latruffe, Inhibitory effect
of resveratrol on the proliferation of human and rat hepatic derived cell
lines, Oncol. Rep. 7 (2000) 847–852.
V. De Ledinghen, A. Monvoisin, V. Neaud, S. Krisa, B. Payrastre, C.
Bedin, A. Desmouliere, P. Bioulac-Sage, J. Rosenbaum, Transresveratrol, a grapevine-derived polyphenol, blocks hepatocyte growth
factor-induced invasion of hepatocellular carcinoma cells, Int. J. Oncol.
19 (2001) 83–88.
Y. Kozuki, Y. Miura, K. Yagasaki, Resveratrol suppresses hepatoma cell
invasion independently of its anti-proliferative action, Cancer Lett. 167
(2001) 151–156.
T. Mosmann, Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays, J. Immunol. Methods
65 (1983) 55–63.
G. Rothe, G. Valet, Flow cytometric assays of oxidative burst activity in
phagocytes, Methods Enzymol. 233 (1994) 539–548.
G. Notas, C. Xidakis, V. Valatas, A. Kouroumalis, E. Kouroumalis, Levels
of circulating endothelin-1 and nitrates/nitrites in patients with virusrelated hepatocellular carcinoma, J. Viral Hepatitis 8 (2001) 63–69.
K.K. Kopec, R.T. Carroll, Phagocytosis is regulated by nitric oxide in
murine microglia, Nitric Oxide 4 (2000) 103–111.
H. Kojima, N. Nakatsubo, K. Kikuchi, S. Kawahara, Y. Kirino, H.
Nagoshi, Y. Hirata, T. Nagano, Detection and imaging of nitric oxide with
novel fluorescent indicators: diaminofluoresceins, Anal. Chem. 70 (1998)
2446–2453.
M. Kampa, V. Alexaki, G. Notas, A. Nifli, A. Nistikaki, A. Hatzoglou, E.
Bakogeorgou, E. Kouimtzoglou, G. Blekas, D. Boskou, A. Gravanis, E.
Castanas, Antiproliferative and apoptotic effects of selective phenolic
acids on T47D human breast cancer cells: potential mechanisms of action,
Breast Cancer Res. 6 (2004) R63–R74.
V.I. Alexaki,I.Charalampopoulos, M. Kampa, H.Vassalou, P.Theodoropoulos,
E.N. Stathopoulos, A. Hatzoglou, A. Gravanis, E. Castanas, Estrogen exerts
neuroprotective effects via membrane estrogen receptors and rapid Akt/NOS
activation, FASEB J. 18 (2004) 1594–1596.
L.J. Hofseth, S.P. Hussain, G.N. Wogan, C.C. Harris, Nitric oxide in cancer
and chemoprevention, Free Radical Biol. Med. 34 (2003) 955–968.
L. McNaughton, L. Puttagunta, M.A. Martinez-Cuesta, N. Kneteman, I.
Mayers, R. Moqbel, Q. Hamid, M.W. Radomski, Distribution of nitric
oxide synthase in normal and cirrhotic human liver, Proc. Natl. Acad. Sci.
U. S. A. 99 (2002) 17161–17166.
M.H. Aziz, R. Kumar, N. Ahmad, Cancer chemoprevention by resveratrol:
in vitro and in vivo studies and the underlying mechanisms (review), Int. J.
Oncol. 23 (2003) 17–28.
Y. Schneider, P. Chabert, J. Stutzmann, D. Coelho, A. Fougerousse, F.
Gosse, J.F. Launay, R. Brouillard, F. Raul, Resveratrol analog (Z)-3,5,4′trimethoxystilbene is a potent anti-mitotic drug inhibiting tubulin
polymerization, Int. J. Cancer 107 (2003) 189–196.
A. Damianaki, E. Bakogeorgou, M. Kampa, G. Notas, A. Hatzoglou, S.
Panagiotou, C. Gemetzi, E. Kouroumalis, P.M. Martin, E. Castanas, Potent
inhibitory action of red wine polyphenols on human breast cancer cells, J.
Cell. Biochem. 78 (2000) 429–441.
M. Kampa, A. Hatzoglou, G. Notas, A. Damianaki, E. Bakogeorgou, C.
Gemetzi, E. Kouroumalis, P.M. Martin, E. Castanas, Wine antioxidant
polyphenols inhibit the proliferation of human prostate cancer cell lines,
Nutr. Cancer 37 (2000) 223–233.
T. Shioda, J. Chesnes, K.R. Coser, L. Zou, J. Hur, K.L. Dean, C.
Sonnenschein, A.M. Soto, K.J. Isselbacher, Importance of dosage
standardization for interpreting transcriptomal signature profiles: evidence
from studies of xenoestrogens, Proc. Natl. Acad. Sci. U. S. A. 103 (2006)
12033–12038.
M.V. Clement, J.L. Hirpara, S.H. Chawdhury, S. Pervaiz, Chemopreventive agent resveratrol, a natural product derived from grapes, triggers CD95
signaling-dependent apoptosis in human tumor cells, Blood 92 (1998)
996–1002.
co
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.
al
1664
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
G. Notas et al. / Biochimica et Biophysica Acta 1760 (2006) 1657–1666
on
al
co
py
[50] G.C. Yen, H.H. Lai, Inhibition of reactive nitrogen species effects in vitro
and in vivo by isoflavones and soy-based food extracts, J. Agric. Food
Chem. 51 (2003) 7892–7900.
[51] H. Iishi, M. Tatsuta, M. Baba, R. Yamamoto, H. Uehara, A. Nakaizumi,
Inhibition of experimental gastric carcinogenesis, induced by N-methyl-N
′-nitro-N-nitrosoguanidine in rats, by sodium nitroprusside, a nitric oxide
generator, Eur. J. Cancer 34 (1998) 554–557.
[52] D.C. Jenkins, I.G. Charles, L.L. Thomsen, D.W. Moss, L.S. Holmes, S.A.
Baylis, P. Rhodes, K. Westmore, P.C. Emson, S. Moncada, Roles of nitric
oxide in tumor growth, Proc. Natl. Acad. Sci. U. S. A. 92 (1995)
4392–4396.
[53] W.J. Thomas, D.L. Thomas, J.A. Knezetic, T.E. Adrian, The role of
oxygen-derived free radicals and nitric oxide in cytokine-induced
antiproliferation of pancreatic cancer cells, Pancreas 24 (2002) 161–168.
[54] L.L. Thomsen, F.G. Lawton, R.G. Knowles, J.E. Beesley, V. RiverosMoreno, S. Moncada, Nitric oxide synthase activity in human gynecological cancer, Cancer Res. 54 (1994) 1352–1354.
[55] P. Martin-Sanz, S. Hortelano, N.A. Callejas, N. Goren, M. Casado, M.
Zeini, L. Bosca, Nitric oxide in liver inflammation and regeneration,
Metab. Brain Dis. 17 (2002) 325–334.
[56] Y. Vodovotz, P.K. Kim, E.Z. Bagci, G.B. Ermentrout, C.C. Chow, I. Bahar,
T.R. Billiar, Inflammatory modulation of hepatocyte apoptosis by nitric
oxide: in vivo, in vitro, and in silico studies, Curr. Mol. Med. 4 (2004)
753–762.
[57] J. Genius, J. Fandrey, Nitric oxide affects the production of reactive
oxygen species in hepatoma cells: implications for the process of oxygen
sensing, Free Radical Biol. Med. 29 (2000) 515–521.
[58] M. Hirakawa, M. Oike, K. Masuda, Y. Ito, Tumor cell apoptosis by
irradiation-induced nitric oxide production in vascular endothelium,
Cancer Res. 62 (2002) 1450–1457.
[59] X.M. Jiang, D.L. Zheng, J.Y. Lin, Effects of nitric oxide on mitochondrial
permeability transition and cytochrome C of human hepatocellular
carcinoma cell lines, Zhongguo Yixue Kexueyuan Xuebao 26 (2004)
519–523.
[60] S. Lucas Sd, J.M. Lopez-Alcorocho, J. Bartolome, V. Carreno, Nitric oxide
and TGF-beta1 inhibit HNF-4alpha function in HEPG2 cells, Biochem.
Biophys. Res. Commun. 321 (2004) 688–694.
[61] E.Y. Chung, B.H. Kim, M.K. Lee, Y.P. Yun, S.H. Lee, K.R. Min, Y. Kim,
Anti-inflammatory effect of the oligomeric stilbene alpha-Viniferin and its
mode of the action through inhibition of cyclooxygenase-2 and inducible
nitric oxide synthase, Planta Med. 69 (2003) 710–714.
[62] T. Kageura, H. Matsuda, T. Morikawa, I. Toguchida, S. Harima, M.
Oda, M. Yoshikawa, Inhibitors from rhubarb on lipopolysaccharideinduced nitric oxide production in macrophages: structural requirements of stilbenes for the activity, Bioorg. Med. Chem. 9 (2001)
1887–1893.
[63] O. Holian, S. Wahid, M.J. Atten, B.M. Attar, Inhibition of gastric cancer
cell proliferation by resveratrol: role of nitric oxide, Am. J. Physiol.:
Gastrointest. Liver Physiol. 282 (2002) G809–G816.
[64] T. Wallerath, G. Deckert, T. Ternes, H. Anderson, H. Li, K. Witte, U.
Forstermann, Resveratrol, a polyphenolic phytoalexin present in red wine,
enhances expression and activity of endothelial nitric oxide synthase,
Circulation 106 (2002) 1652–1658.
[65] R. Hattori, H. Otani, N. Maulik, D.K. Das, Pharmacological preconditioning with resveratrol: role of nitric oxide, Am. J. Physiol.: Heart Circ.
Physiol. 282 (2002) H1988–H1995.
[66] O. Holian, S. Wahid, M.J. Atten, B.M. Attar, Inhibition of gastric cancer
cell proliferation by resveratrol: role of nitric oxide, Am. J. Physiol.:
Gastrointest. Liver Physiol. 282 (2002) G809–G816.
[67] M.M. Chan, J.A. Mattiacci, H.S. Hwang, A. Shah, D. Fong, Synergy
between ethanol and grape polyphenols, quercetin, and resveratrol, in the
inhibition of the inducible nitric oxide synthase pathway, Biochem.
Pharmacol. 60 (2000) 1539–1548.
[68] D.I. Cho, N.Y. Koo, W.J. Chung, T.S. Kim, S.Y. Ryu, S.Y. Im, K.M. Kim,
Effects of resveratrol-related hydroxystilbenes on the nitric oxide
production in macrophage cells: structural requirements and mechanism
of action, Life Sci. 71 (2002) 2071–2082.
[69] S.H. Tsai, S.Y. Lin-Shiau, J.K. Lin, Suppression of nitric oxide synthase
Au
th
or
's
pe
rs
[33] D. Bernhard, I. Tinhofer, M. Tonko, H. Hubl, M.J. Ausserlechner, R. Greil,
R. Kofler, A. Csordas, Resveratrol causes arrest in the S-phase prior to Fasindependent apoptosis in CEM-C7H2 acute leukemia cells, Cell Death
Differ. 7 (2000) 834–842.
[34] D. Delmas, C. Rebe, S. Lacour, R. Filomenko, A. Athias, P. Gambert, M.
Cherkaoui-Malki, B. Jannin, L. Dubrez-Daloz, N. Latruffe, E. Solary,
Resveratrol-induced apoptosis is associated with Fas redistribution in the
rafts and the formation of a death-inducing signaling complex in colon
cancer cells, J. Biol. Chem. 278 (2003) 41482–41490.
[35] J. Zheng, V.D. Ramirez, Piceatannol, a stilbene phytochemical, inhibits
mitochondrial F0F1-ATPase activity by targeting the F1 complex,
Biochem. Biophys. Res. Commun. 261 (1999) 499–503.
[36] J. Dorrie, H. Gerauer, Y. Wachter, S.J. Zunino, Resveratrol induces
extensive apoptosis by depolarizing mitochondrial membranes and
activating caspase-9 in acute lymphoblastic leukemia cells, Cancer Res.
61 (2001) 4731–4739.
[37] A. Kotha, M. Sekharam, L. Cilenti, K. Siddiquee, A. Khaled, A.S. Zervos,
B. Carter, J. Turkson, R. Jove, Resveratrol inhibits Src and Stat3 signaling
and induces the apoptosis of malignant cells containing activated Stat3
protein, Mol. Cancer Ther. 5 (2006) 621–629.
[38] Z. Estrov, S. Shishodia, S. Faderl, D. Harris, Q. Van, H.M. Kantarjian, M.
Talpaz, B.B. Aggarwal, Resveratrol blocks interleukin-1beta-induced
activation of the nuclear transcription factor NF-kappaB, inhibits
proliferation, causes S-phase arrest, and induces apoptosis of acute
myeloid leukemia cells, Blood 102 (2003) 987–995.
[39] T.C. Hsieh, P. Burfeind, K. Laud, J.M. Backer, F. Traganos, Z.
Darzynkiewicz, J.M. Wu, Cell cycle effects and control of gene expression
by resveratrol in human breast carcinoma cell lines with different
metastatic potentials, Int. J. Oncol. 15 (1999) 245–252.
[40] F. Wolter, B. Akoglu, A. Clausnitzer, J. Stein, Downregulation of the
cyclin D1/Cdk4 complex occurs during resveratrol-induced cell cycle
arrest in colon cancer cell lines, J. Nutr. 131 (2001) 2197–2203.
[41] N. Ahmad, V.M. Adhami, F. Afaq, D.K. Feyes, H. Mukhtar, Resveratrol
causes WAF-1/p21-mediated G(1)-phase arrest of cell cycle and induction
of apoptosis in human epidermoid carcinoma A431 cells, Clin. Cancer
Res. 7 (2001) 1466–1473.
[42] Y.C. Liang, S.H. Tsai, L. Chen, S.Y. Lin-Shiau, J.K. Lin, Resveratrolinduced G2 arrest through the inhibition of CDK7 and p34CDC2 kinases
in colon carcinoma HT29 cells, Biochem. Pharmacol. 65 (2003)
1053–1060.
[43] M. Larrosa, F.A. Tomas-Barberan, J.C. Espin, Grape polyphenol
resveratrol and the related molecule 4-hydroxystilbene induce growth
inhibition, apoptosis, S-phase arrest, and upregulation of cyclins A, E, and
B1 in human SK-Mel-28 melanoma cells, J. Agric. Food Chem. 51 (2003)
4576–4584.
[44] N. Kuwajerwala, E. Cifuentes, S. Gautam, M. Menon, E.R. Barrack, G.P.
Reddy, Resveratrol induces prostate cancer cell entry into s phase and
inhibits DNA synthesis, Cancer Res. 62 (2002) 2488–2492.
[45] V.M. Adhami, F. Afaq, N. Ahmad, Involvement of the retinoblastoma
(pRb)-E2F/DP pathway during antiproliferative effects of resveratrol in
human epidermoid carcinoma (A431) cells, Biochem. Biophys. Res.
Commun. 288 (2001) 579–585.
[46] C. Huang, W.Y. Ma, A. Goranson, Z. Dong, Resveratrol suppresses cell
transformation and induces apoptosis through a p53-dependent pathway,
Carcinogenesis 20 (1999) 237–242.
[47] T.C. Hsieh, G. Juan, Z. Darzynkiewicz, J.M. Wu, Resveratrol increases
nitric oxide synthase, induces accumulation of p53 and p21(WAF1/CIP1),
and suppresses cultured bovine pulmonary artery endothelial cell
proliferation by perturbing progression through S and G2, Cancer Res.
59 (1999) 2596–2601.
[48] J. Lu, C.H. Ho, G. Ghai, K.Y. Chen, Resveratrol analog, 3,4,5,4′tetrahydroxystilbene, differentially induces pro-apoptotic p53/Bax gene
expression and inhibits the growth of transformed cells but not their normal
counterparts, Carcinogenesis 22 (2001) 321–328.
[49] A.P. Nifli, A. Bosson-Kouame, N. Papadopoulou, C. Kogia, M. Kampa, C.
Castagnino, C. Stournaras, J. Vercauteren, E. Castanas, Monomeric and
oligomeric flavanols are agonists of membrane androgen receptors, Exp.
Cell Res. 309 (2005) 329–339.
1665
1666
G. Notas et al. / Biochimica et Biophysica Acta 1760 (2006) 1657–1666
breast cancer cells and is transcriptionally regulated by p53, J. Biol. Chem.
274 (1999) 37679–37684.
[73] Y. Uchida, H. Yamazaki, S. Watanabe, K. Hayakawa, Y. Meng, N.
Hiramatsu, A. Kasai, K. Yamauchi, J. Yao, M. Kitamura, Enhancement of
NF-kappaB activity by resveratrol in cytokine-exposed mesangial cells,
Clin. Exp. Immunol. 142 (2005) 76–83.
[74] E. Ilan, O. Tirosh, Z. Madar, Triacylglycerol-mediated oxidative stress
inhibits nitric oxide production in rat isolated hepatocytes, J. Nutr. 135
(2005) 2090–2095.
Au
th
or
's
pe
rs
on
al
co
py
and the down-regulation of the activation of NFkappaB in macrophages by
resveratrol, Br. J. Pharmacol. 126 (1999) 673–680.
[70] A.-.P. Nifli, M. Kampa, V. Alexaki, G. Notas, E. Castanas, Polyphenol
interaction with the T47D human breast cancer cell line, J. Dairy Res. 72
(S1) (2005) 44–50.
[71] P.L. Kuo, L.C. Chiang, C.C. Lin, Resveratrol-induced apoptosis is mediated by
p53-dependent pathway in Hep G2 cells, Life Sci. 72 (2002) 23–34.
[72] K. Mortensen, J. Skouv, D.M. Hougaard, L.I. Larsson, Endogenous
endothelial cell nitric-oxide synthase modulates apoptosis in cultured