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B, Activator Protein-1, and Apoptosis: Potential Role of Reactive Oxygen Intermediates and Lipid Peroxidation1
Cytokine Research Laboratory, Department of Bioimmunotherapy, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B, a nuclear transcription factor that
regulates the expression of various genes involved in inflammation,
cytoprotection, and carcinogenesis. In this study, we investigated the
effect of resveratrol on NF-B activation induced by various
inflammatory agents. Resveratrol blocked TNF-induced activation of
NF-B in a dose- and time-dependent manner. Resveratrol also
suppressed TNF-induced phosphorylation and nuclear translocation of the
p65 subunit of NF-B, and NF-B-dependent reporter gene
transcription. Suppression of TNF-induced NF-B activation by
resveratrol was not restricted to myeloid cells (U-937); it was also
observed in lymphoid (Jurkat) and epithelial (HeLa and H4) cells.
Resveratrol also blocked NF-B activation induced by PMA, LPS,
H2O2, okadaic acid, and ceramide. The
suppression of NF-B coincided with suppression of AP-1. Resveratrol
also inhibited the TNF-induced activation of mitogen-activated protein
kinase kinase and c-Jun N-terminal kinase and abrogated TNF-induced
cytotoxicity and caspase activation. Both reactive oxygen intermediate
generation and lipid peroxidation induced by TNF were suppressed by
resveratrol. Resveratrol’s anticarcinogenic, anti-inflammatory,
and growth-modulatory effects may thus be partially ascribed to the
inhibition of activation of NF-B and AP-1 and the associated
kinases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In 1997, resveratrol was reported to be one of the most potent chemopreventive agents able to block all three phases of tumor development that includes initiation, promotion, and progression, induced by the aryl hydrocarbon DMBA (14). How resveratrol exerts its anticarcinogenic effects is only partially understood. This polyphenol has been shown to inhibit the growth of a wide variety of tumor cells, including leukemic, prostate, breast, and endothelial cells (10, 15, 16, 17). The ability of resveratrol to induce the expression of CD95L (also called FasL), p53, and p21 may contribute to its growth-inhibitory effects (10, 15). The suppression of cyclooxygenase-2 (COX-2), cytochrome p450, and c-fos expression by resveratrol may account for its ability to inhibit tumor promotion (18, 19, 20). Recently, the drug was reported to be a phytoestrogen that behaves as superagonist of estrogen receptor and thereby an inducer of tumor cell proliferation (21). Its structural similarity with estrogen may also account for its cardioprotective effects.
Because the carcinogenic, inflammatory, and growth-modulatory effects
of many chemicals are mediated by NF-B, we hypothesized that the
suppression of NF-B activation pathway accounts for resveratrol’s
activities. Numerous lines of evidence suggest this possibility. For
example, various agents that promote tumorigenesis are known to
activate NF-B (for references, see 22), including
phorbol ester, okadaic acid, and TNF. Experiments in TNF-deficient mice
showed that TNF is required for tumor promotion (23). In
addition, several genes that are involved in tumorigenesis, metastasis,
and inflammation are regulated by NF-B (22). A critical
role for NF-B in cellular transformation has also been reported
(24).
Most agents that activate NF-B also activate another transcription
factor, AP-1 (25). That AP-1 activation mediates
tumorigenesis and invasiveness has also been described (26
and references therein). The activation of NF-B and AP-1 is
regulated by several protein kinases that belong to the
mitogen-activated protein kinase (MAPK) family (27). The
activation of NF-B and AP-1 and its associated kinases is in most
cases dependent on the production of reactive oxygen species
(28, 29, 30, 31).
In this study, we tested the hypothesis that the anti-inflammatory
and anticarcinogenic effects of resveratrol are mediated through its
modulation of NF-B and AP-1 activation, members of the MAPK, and
caspase-mediated apoptosis. We demonstrated that resveratrol was a
potent inhibitor of NF-B and AP-1 activation. It also inhibited
TNF-induced c-Jun N-terminal protein kinase (JNK) and MAPK kinase (MEK)
activation and caspase-induced apoptosis. Both reactive oxygen
intermediate (ROI) generation and lipid peroxidation induced by TNF
were also suppressed by resveratrol.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Resveratrol, penicillin, streptomycin, RPMI 1640 medium, and FCS
were obtained from Life Technologies (Grand Island, NY). Glycine, PMA,
LPS, ceramide, NaCl, and BSA were obtained from Sigma (St. Louis, MO).
A 5 mM solution of resveratrol (m.w. 228.2) was prepared in
H2O and used directly at this concentration.
Bacteria-derived human rTNF, purified to homogeneity with a sp. act. of
5 x 107 U/mg, was kindly provided by
Genentech (South San Francisco, CA). Abs against IB
and p65 were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Poly(ADP-ribose) polymerase (PARP) Ab was purchased from PharMingen
(San Diego, CA). Phospho-IB (Ser32) Ab was
purchased from New England Biolabs (Beverly, MA). The rat MDR1bCAT
plasmid -243RMICAT containing the chloramphenicol acetyltransferase
(CAT) gene with either wild-type or mutated NF-B binding site was
kindly supplied by Dr. M. Tien Kuo (University of Texas M. D.
Anderson Cancer Center, Houston, TX). The characterization of these
plasmids has been described previously in detail (32).
Cell lines
The cell lines used in this study were as follows: U-937 (human histiocytic lymphoma), HeLa (human epithelial cells), H4 (glioma cells), and T-Jurkat (T cells); they were obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). All cells were free from mycoplasma, as detected by Gen-Probe mycoplasma rapid detection kit (Fisher Scientific, Pittsburgh, PA).
Isolation of PBL
Freshly drawn human blood was incubated with 2.5% gelatin in saline in 1:1 ratio for 30 min at 37°C. The supernatant was layered on Histopaque 1077 (from Sigma) and centrifuged at 1500 rpm for 30 min at room temperature. The cells were then collected from the top layer of Histopaque, diluted with Dulbecco’s PBS and centrifuged at 2000 rpm for 10 min. To get rid of mixed reticulocytes, pellet was suspended in 0.2% NaCl for 1 min, immediately diluted with equal volume of 1.6% NaCl, and centrifuged at 1000 rpm. The pellet was then suspended in RPMI 1640 medium supplemented with 10% FBS and cultured for 2 h at 37°C in a CO2 incubator in a petri dish to remove macrophages by adherence. Then the lymphocytes were harvested from the medium by centrifugation at 1000 rpm.
NF-B activation assays
To determine NF-B activation, EMSA were conducted essentially
as described (33, 34). Briefly, nuclear extracts prepared
from TNF-treated cells (2 x 106/ml) were
incubated with 32P end-labeled 45-mer
double-stranded NF-B oligonucleotide (4 µg protein with 16 fmol
DNA) from the HIV-LTR,
5'-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3'
(bold indicates NF-B binding sites) for 15 min at 37°C, and the
DNA-protein complex formed was separated from free oligonucleotide on
6.6% native polyacrylamide gels. A double-stranded mutated
oligonucleotide,
5'-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3',
was used to examine the specificity of binding of NF-B to the DNA.
The specificity of binding was also examined by competition with the
unlabeled oligonucleotide. The dried gels were visualized, and
radioactive bands were quantitated by a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA) using ImageQuant software.
AP-1 activation assay
The activation of AP-1 was determined as described (28). Briefly, 6 µg of nuclear extract prepared as indicated above was incubated with 16 fmol of the 32P end-labeled AP-1 consensus oligonucleotide 5'-CGCTTGATGACTCAGCCGGAA-3' (bold indicates AP-1 binding site) for 15 min at 37°C and analyzed by using 6% native polyacrylamide gel. The specificity of binding was examined by competition with unlabeled oligonucleotide. Visualization and quantitation of radioactive bands were done as indicated above.
Western blot for IB and p65
To determine the levels of IB, postnuclear (cytoplasmic)
extracts were prepared (33) from TNF-treated cells and
resolved on 10% SDS-polyacrylamide gels. To determine the levels of
NF-B protein p65, nuclear and postnuclear extracts were prepared
from TNF-treated cells and were resolved on 10% SDS-PAGE. After
electrophoresis, the proteins were electrotransferred to nitrocellulose
filters, probed with rabbit polyclonal Abs against IB or p65, and
detected by chemiluminescence (ECL; Amersham, Arlington Heights, IL).
The bands obtained were quantitated using Personal Densitometer Scan
v1.30 using Imagequant software version 3.3 (Molecular Dynamics).
Oct-1 and Sp1 binding
The effect of resveratrol on the binding of Oct-1 and Sp1 was determined by incubating 6 µg of nuclear extracts with 16 fmol of the 32P end labeled with either Oct-1 consensus oligonucleotide 5'-TGTCGAATGCAAATCACTAGAA-3' (bold indicates Oct-1 binding site) or Sp1 consensus oligonucleotide 5'-ATTCGATCGGGGCGGGGCGAGC-3' for 15 min at 37°C and analyzed by using 6% native polyacrylamide gel. Visualization and quantitation of radioactive bands were done as indicated above.
Immunoprecipitation of p65 from orthophosphate-labeled cells
To determine the phosphorylation of p65 subunit of NF-B,
U-937 cells were labeled with
[32P]orthophosphate (Amersham) in
phosphate-free medium for 1 h at 37°C, and then resveratrol (5
µM) was added and incubation continued for another 2 h at
37°C. Then cells were washed and suspended with same medium. Cells
were then treated with 0.1 nM TNF for 30 min at 37°C. The cells were
washed with Dulbecco’s PBS and then lysed on ice for 15 min with
buffer containing 20 mM Tris-Cl, pH 7.9, 2 mM EDTA, 250 mM NaCl, 0.1%
Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM PMSF, 0.5
µg/ml benzamidine, 1 mM DTT, and 1 mM sodium orthovanadate. An
800-µg protein was immunoprecipitated with 0.5 µg anti-p65
polyclonal Ab (Santa Cruz Biotechnology) overnight at 4°C. Immune
complexes were collected by incubation with protein A/G Sepharose beads
for 1 h at 4°C. The beads were extensively washed with lysis
buffer (4 x 400 µl) and wash buffer (2 x 400 µl: 20 mM
HEPES, pH 7.4, 1 mM DTT, 25 mM NaCl). Washed beads were then boiled
with 15 µl of 2x SDS sample buffer for 5 min, and subjected to
SDS-PAGE (9%). The gel was dried, exposed to phospho-screen, and
analyzed by a PhosphorImager (Molecular Dynamics). To determine equal
loading, 50-µg protein was resolved on 10% SDS-PAGE,
electrotransferred to nitrocellulose filters, and probed with the
anti-p65 Ab, and the bands were detected by chemiluminescence (ECL;
Amersham).
Cytotoxicity assay
The TNF-induced cytotoxicity was measured by the MTT assay (35). Briefly, cells (10,000 cells/well) were incubated in the presence or absence of the indicated test sample in a final volume of 0.1 ml for 72 h at 37°C. Thereafter, 0.025 ml of MTT solution (5 mg/ml in PBS) was added to each well. After a 2-h incubation at 37°C, 0.1 ml of the extraction buffer (20% SDS, 50% dimethylformamide) was added. After an overnight incubation at 37°C, the OD at 590 nm were measured using a 96-well multiscanner autoreader (Dynatech MR 5000, Chantilly, VA), with the extraction buffer as a blank.
Immunoblot analysis of PARP degradation
TNF-induced apoptosis was examined by proteolytic cleavage of PARP (35). Briefly, cells (2 x 106/ml) were treated with different concentrations of resveratrol at 37°C for 1 h and then stimulated with 1 mM TNF with cycloheximide (2 µg/ml) for 2 h at 37°C. The cells were then washed and extracted by incubation for 30 min on ice in 0.05 ml buffer containing 20 mM HEPES, pH 7.4, 2 mM EDTA, 250 mM NaCl, 0.1% Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM PMSF, 0.5 µg/ml benzamidine, 1 mM DTT, and 1 mM sodium vanadate. The lysate was centrifuged and the supernatant was collected. Cell extract protein (50 µg) was resolved on 7.5% SDS-PAGE, electrotransferred onto a nitrocellulose membrane, blotted with mouse anti-PARP Ab, and then detected by chemiluminescence (ECL; Amersham). Apoptosis was represented by the cleavage of 116-kDa PARP into a 85-kDa product (36).
MEK assay
Activation of MEK was assayed as described (37). U-937 cells, treated with different concentrations of resveratrol for 1 h and then stimulated with 1 nM TNF for 30 min at 37°C, were washed with Dulbecco’s PBS and then lysed on ice for 15 min with buffer containing 20 mM HEPES, pH 7.4, 2 mM EDTA, 250 mM NaCl, 0.1% Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM PMSF, 0.5 µg/ml benzamidine, 1 mM DTT, and 1 mM sodium orthovanadate. A 50-µg aliquot of protein was resolved on 10% SDS-PAGE, electrotransferred to nitrocellulose filters, and probed with the phospho-specific anti-p44/42 MAPK (Thr202/Tyr204) Ab (New England Biolabs) raised in rabbits (1/3000 dilution). Then the membrane was incubated with peroxidase-conjugated anti-rabbit IgG (1/3000 dilution), and the bands were detected by chemiluminescence (ECL; Amersham).
c-Jun kinase assay
The c-Jun kinase assay was performed by a modified method, as
described earlier (31). Briefly, after treatment of cells
(3 x 106/ml) with TNF for 10 min, cell
extracts were prepared by lysing cells in buffer containing 20 mM
HEPES, pH 7.4, 2 mM EDTA, 250 mM NaCl, 1% Nonidet P-40, 2 µg/ml
leupeptin, 2 µg/ml aprotinin, 1 mM PMSF, 0.5 µg/ml benzamidine, and
1 mM DTT. Cell extracts (150 µg/sample) were immunoprecipitated with
0.3 µg anti-JNK Ab for 60 min at 4°C. Immune complexes were
collected by incubation with protein A/G Sepharose beads for 45 min at
4°C. The beads were extensively washed with lysis buffer (4 x
400 µl) and kinase buffer (2 x 400 µl: 20 mM HEPES, pH 7.4, 1
mM DTT, 25 mM NaCl). Kinase assays were performed for 15 min at 30°C
with GST-Jun 1–79(1–79) as a substrate in 20 mM HEPES, pH 7.4, 10 mM
MgCl2, 1 mM DTT, and 10 µCi
[
-32P]ATP. Reactions were stopped by the
addition of 15 µl of 2x SDS sample buffer, boiled for 5 min, and
subjected to SDS-PAGE (9%). GST-Jun 1–79(1–79) was visualized by staining
with Coomassie blue, and the dried gel was analyzed by a PhosphorImager
(Molecular Dynamics).
Transient transfection and CAT assay
To determine TNF-induced NF-B-mediated reporter gene
transcription, U-937 cells were transiently transfected by the calcium
phosphate method with the plasmids 243RMICAT (contains wild-type
NF-B binding site) and -243 RMICAT-km (mutated binding site),
according to the instructions supplied by the manufacturer (Life
Technologies). After 12 h of transfection, the cells were
stimulated with different concentrations of TNF for 2 h, washed,
and examined for CAT activity, as described (38).
Determination of lipid peroxidation
TNF-induced lipid peroxidation was determined by detection of thiobarbituric acid-reactive malondialdehyde (MDA), an end product of the peroxidation of polyunsaturated fatty acids and related esters, as described (39). Results were normalized with the amount of MDA equivalents/mg of protein and expressed as a percentage of thiobarbituric acid-reactive substances above control values. Untreated cells showed 0.571 ± 0.126 nmol of MDA equivalents/mg of protein.
Measurement of ROI
The production of ROI upon treatment of cells with TNF was determined by flow cytometry, as described (39).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. It is a highly
water-soluble compound. For most studies, U-937 cells were used because
these cells express both types of TNF receptor, and TNF-induced
responses in this cell type are well characterized in our laboratory.
The concentration of resveratrol and its time of exposure had no effect
on TNF receptors or on cell viability (data not shown).
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B activation
U-937 cells were pretreated for 4 h with different
concentrations of resveratrol and then stimulated with 100 pM TNF for
30 min. Nuclear extracts were prepared and assayed for NF-B by EMSA.
As shown in Fig. 2A, TNF
induced 10-fold activation of NF-B, and resveratrol inhibited this
activation in a dose-dependent manner; full inhibition occurred at 5
µM resveratrol. Resveratrol even at 25 µM by itself did not
activate NF-B. We next examined the effect of changes in the length
of incubation with resveratrol on NF-B activation by TNF. Cells were
incubated with 5 µM resveratrol for different times and then
stimulated with 0.1 nM TNF for 30 min and assayed for NF-B. The
results in Fig. 2B show that resveratrol inhibited
TNF-induced NF-B activation with increased time of incubation. At
4 h, complete inhibition was observed.
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B within 5 min, and
this induction is higher in its intensity than that obtained with cells
using a 100-fold lower concentration of TNF for longer times
(40). To determine the effect of resveratrol on NF-B
activation at even higher concentrations, both untreated and
resveratrol-pretreated cells were incubated with various concentrations
of TNF (0–10,000 pM) for 30 min and then assayed for NF-B by EMSA.
Although the activation of NF-B by 10,000 pM TNF was strong (Fig. 2
C), resveratrol completely inhibited it as efficiently as
it did at 0.1 nM concentration. These results show that resveratrol is
a very potent inhibitor of NF-B activation. We also examined the
effect of resveratrol on the kinetics of TNF-induced NF-B
activation. Both untreated and resveratrol-pretreated cells were
incubated with TNF (100 pM) for different times and then assayed for
NF-B. In untreated cells, TNF activated NF-B in a time-dependent
manner with almost maximum activation at 15 min (Fig. 2D).
In resveratrol-pretreated cells, however, a little activation of
NF-B was detected after TNF exposure of up to 60 min (Fig. 2D).
Activated NF-B inhibited by resveratrol consists of p50 and p65
subunits
Various combinations of Rel/NF-B proteins can constitute
an active NF-B heterodimer that binds to specific sequences in DNA.
To show that the retarded band visualized by EMSA in TNF-treated cells
was indeed NF-B, we incubated the nuclear extracts from
TNF-activated cells with Ab to either p50 (NF-BI) or p65 (Rel A)
subunits and then conducted EMSA. Abs to either subunit of NF-B
shifted the band to a higher m.w. (Fig. 3A), thus suggesting that the
TNF-activated complex consisted of p50 and p65 subunits. Neither
preimmune serum nor such irrelevant Abs as anti-c-Rel or anticyclin
DI had any effect on the mobility of NF-B. Excess cold NF-B
(100-fold) almost completely eradicated the band, indicating the
specificity of NF-B. Further specificity is indicated by the
observations that the oligonucleotide probe with labeled mutated
NF-B binding site failed to bind the NF-B protein.
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B proteins
It has been shown that
N-tosyl-L-Phe-chloromethylketone
(TPCK), a serine protease inhibitor, and herbimycin A, a protein
tyrosine kinase inhibitor, and caffeic acid phenylethyl ester
down-regulate NF-B activation by chemical modification of the
NF-B subunits, thus preventing its binding to DNA
(41, 42, 43). To determine whether resveratrol also directly
modifies the ability of NF-B proteins to bind to the DNA, we
incubated the cytoplasmic extracts with deoxycholate (DOC) (0.8%) for
15 min at room temperature. The DOC treatment has been shown to
dissociate the IB subunit, thus releasing NF-B for binding to
the DNA. DOC-treated cytoplasmic extracts were then exposed to various
concentrations of resveratrol and assayed for DNA binding by EMSA. As
shown in Fig. 3B, resveratrol had no effect on the binding
of NF-B to the DNA.
Whether resveratrol modifies the nuclear fraction of NF-B in
TNF-treated cells was also examined. The nuclear extracts from
TNF-pretreated cells were treated with various concentrations of
resveratrol and then examined for DNA-binding activity by EMSA. Our
results in Fig. 3C show that resveratrol did not modify the
DNA-binding ability of NF-B proteins prepared from TNF-treated cells
either. Therefore, resveratrol inhibits NF-B activation through a
mechanism different from that of TPCK, herbimycin A, and caffeic acid
phenylethyl ester (41, 42, 43).
Whether resveratrol suppresses the DNA binding of other transcription
factors, such as Oct-1 and Sp1, was also examined. As shown in Fig. 3, D and E, resveratrol has no effect on Oct-1 or
Sp1, respectively, indicating that the effects of resveratrol are
specific to NF-B.
Inhibition of NF-B activation by resveratrol is not cell type
specific
That distinct signal transduction pathways could mediate NF-B
induction in epithelial and lymphoid cells has been demonstrated
(44). All the effects of resveratrol described above were
conducted with U-937, a myeloid cell line. In another set of
experiments, we found that resveratrol blocks TNF-induced NF-B
activation in T cells (Jurkat) and epithelial (HeLa) and glioma (H4)
cells (Fig. 4). NF-B binding in all
three cell lines was abrogated by a 25-fold molar excess of unlabeled
oligonucleotide. An almost complete inhibition in all the cell types
suggests that this effect of resveratrol is not restricted to myeloid
cells.
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B
Besides TNF, NF-B is also activated by various other tumor
promoters and inflammatory agents, including phorbol ester,
H2O2, LPS, okadaic acid,
and ceramide (22), but by different signal transduction
pathways (44, 45, 46). We found that these five agents
activated NF-B and that resveratrol completely blocked the
activation of NF-B induced by all five inducers (Fig. 5A). These results suggest
that resveratrol may act at a step in which all these agents converge
in the signal transduction pathway leading to NF-B activation.
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B in normal cells was examined.
As shown in Fig. 5B, PMA, LPS, PHA, and TNF, all agents
activated NF-B in normal human PBL, and the pretreatment with
resveratrol (5 µM) abolished the activation.
Resveratrol does not inhibit TNF-dependent phosphorylation and
degradation of IB
The translocation of NF-B to the nucleus is preceded by the
phosphorylation, ubiquitination, and proteolytic degradation of
IB (22). To determine whether the inhibitory action
of resveratrol was due to an effect on IB degradation, the
cytoplasmic level of IB proteins was examined by Western blot
analysis. IB degradation started 5 min after TNF treatment of
U937 cells and was complete within 10 min. The band reappeared by 30
min owing to NF-B-dependent IB resynthesis. The presence of
resveratrol had no significant effect on the TNF-induced IB
degradation (Fig. 6A).
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B
phosphorylation, cells were treated with the proteosome inhibitor
N-acetylleucyl-leucylnorleucinal (42) for
1 h and then assayed by Western blot with Abs against either the
serine-phosphorylated (Fig. 6B, upper panel) or
nonphosphorylated form of IB (Fig. 6B, lower
panel). Resveratrol had neither any effect on the TNF-induced
phosphorylation of IBa (upper panel), nor on the
migration of the hyperphosphorylated form of IB, which appeared
as a slow-migrating band on SDS-PAGE (Fig. 6B, lower
panel) in TNF-treated cells.
Resveratrol inhibits TNF-dependent phosphorylation and nuclear
translocation of p65 subunit of NF-B
Whether resveratrol affects the TNF-induced nuclear translocation
of the p65 subunit of NF-B was also examined by Western blot
analysis. As shown in Fig. 6C, upon TNF treatment, p65
disappeared from the cytoplasm, and resveratrol prevented the
disappearance. In the nuclear fraction, however, p65 appeared after TNF
treatment and resveratrol inhibited the appearance. Resveratrol alone
had no effect in these experiments. These results indicate that
resveratrol blocks the nuclear translocation of NF-B.
Recently, it was reported that mesalamine inhibits IL-1-induced NF-B
activation by blocking the phoshorylation of p65 subunit
(47). Whether resveratrol affects the TNF-induced
phosphorylation of the p65 subunit of NF-B was also examined by
metabolic labeling of cells with
[32P]orthophosphate, followed by
immunoprecipitation of p65 from labeled cells treated with either TNF
or resveratrol or combination. As shown in Fig. 6D, TNF
induced the phosphorylation of the p65 subunit and resveratrol
inhibited it. Resveratrol alone had no effect in these experiments.
These results indicate that resveratrol also blocks the phosphorylation
of p65 subunit of NF-B.
Resveratrol represses TNF-induced NF-B-dependent reporter gene
expression
Although we have shown by EMSA that resveratrol blocks the NF-B
activation and blocks the phosphorylation and nuclear translocation of
p65, DNA binding alone does not always correlate with NF-B-dependent
gene transcription, suggesting the role of additional regulatory steps
(48). To determine the effect of resveratrol on
TNF-induced NF-B-dependent reporter gene expression, we transiently
transfected resveratrol-pretreated or untreated cells with the CAT
reporter construct and then stimulated with TNF. An almost 6-fold
increase in CAT activity over the vector control was noted upon
stimulation with TNF (Fig. 7). The CAT
gene reporter construct with mutated NF-B could not be activated by
TNF, suggesting specificity of action. TNF-induced CAT activity was
almost completely abolished when the cells were pretreated with
resveratrol. These results demonstrate that resveratrol also represses
NF-B-dependent reporter gene expression induced by TNF.
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TNF is one of the most potent activators of various kinases of the
MAPK family (49). There are also reports that some of the
kinases of this family are required for TNF-induced NF-B activation
(50). And TNF is known to be a potent activator of JNK
(51). Whether resveratrol affects any of these kinases was
also examined. The U-937 cells were pretreated with different
concentrations of resveratrol for 4 h and then stimulated with TNF
(1 nM) for 10 min. About a 17-fold activation of c-jun
kinase was detected with 1 nM TNF. This activation gradually decreased
with increasing concentrations of resveratrol, and at 5 µM
resveratrol the activation of JNK by TNF was completely inhibited (Fig. 8A).
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B). Resveratrol inhibits TNF-induced AP-1 activation
The activation of JNK causes the activation of AP-1. TNF is also a
potent activator of AP-1 (52). TNF induced AP-1 expression
by 7-fold in myeloid cells at 1 nM concentration. The activation of
AP-1 was completely inhibited by resveratrol in a dose-dependent
manner, with maximum suppression occurring at 5 µM (Fig. 9A). Supershift analysis with
specific Abs against c-fos and c-jun indicated
that TNF-induced AP-1 consists of c-fos and c-jun
(data not shown). Lack of supershift by unrelated Abs and disappearance
of the AP-1 band by competition with cold oligo show the specificity.
In untreated cells, TNF activated AP-1 in a dose-dependent manner, but
in resveratrol-treated cells, no AP-1 activation was observed (Fig. 9B).
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Among all the cytokines, TNF is one of the most potent inducers of
apoptosis (for references, see 53). Whether resveratrol
modulates TNF-induced apoptosis was also investigated. U-937 cells were
treated with variable concentrations of TNF for 72 h either in the
absence or presence of resveratrol and then examined for cytotoxicity
by the MTT method. Results in Fig. 10A show that the cytotoxic
effects of TNF in U-937 cells were dose dependent, with almost 70%
killing occurring at 5 nM concentration of the cytokine. This
cytotoxicity was completely inhibited by treatment of cells with 5 µM
resveratrol.
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B, TNF
induced complete cleavage of PARP, and this cleavage was inhibited in a
dose-dependent manner by treatment of cells with resveratrol, with
maximum effect at 3 µM concentration. Thus, resveratrol also blocks
TNF-induced apoptosis. Resveratrol blocks TNF-induced ROI generation and lipid peroxidation
Previous reports have shown that TNF activates NF-B, AP-1, JNK,
and apoptosis through generation of ROI (28, 29, 30, 31, 53, 54).
Whether resveratrol mediates its effects through suppression of ROI
production was examined by flow cytometry. As shown in Fig. 11A, TNF induced ROI
generation in a time-dependent manner, but this was suppressed by
pretreatment of cells with resveratrol. Because lipid peroxidation has
also been implicated in TNF-induced NF-B activation and cytotoxicity
(53, 55), we also examined the effect of resveratrol on
TNF-induced lipid peroxidation. Results in Fig. 11B show
that TNF induced lipid peroxidation in U-937 cells, and this was
completely suppressed by resveratrol. Thus, it is quite likely that
resveratrol blocks TNF signaling through suppression of ROI generation
and of lipid peroxidation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation, we tested the
hypothesis that resveratrol directly blocks NF-B activation. We
found that resveratrol is indeed a potent inhibitor of TNF-induced
activation of NF-B, and this inhibition is not cell type specific.
The suppression is observed in both normal and tumor cells. Besides
TNF, resveratrol also blocked NF-B activation induced by a wide
variety of other inflammatory agents. NF-B-dependent reporter gene
transcription was also suppressed by resveratrol. Besides NF-B,
resveratrol blocked activation of AP-1 and the associated kinases MEK
and JNK. TNF-induced cytotoxicity and caspase activation were also
down-regulated by resveratrol. Resveratrol’s ability to block both ROI
generation and lipid peroxidation induced by TNF may account for its
effects on transcription factors and the associated kinases.
Recent evidence indicates that different inflammatory agents may
activate NF-B through mechanisms that consist of some overlapping
and some nonoverlapping steps (44, 45, 46). How resveratrol
blocks NF-B activation by TNF is not clear. Its suppression of
NF-B activation by a wide variety of agents suggests that
resveratrol must act at a step common to all agents. Most inhibitors of
NF-B activation, such as curcumin and silymarin, mediate their
effects through suppression of phosphorylation and degradation of
IBa (39, 56, 57). Resveratrol, however, blocked neither
the phosphorylation nor the degradation of IBa. These results are
similar to that described for caffeic acid phenethyl ester or
mesalamine, which also block NF-B activation without any effect on
IB phosphorylation or degradation (43, 47). Caffeic
acid phenethyl ester, however, modifies the NF-B protein so that it
can no longer bind to DNA. Resveratrol had no effect on the binding of
NF-B proteins to the DNA, but it did block the TNF-induced
translocation of NF-B’s p65 subunit and reporter gene
transcription. These results are similar to those described recently
for mesalamine, which inhibits cytokine-induced and NF-B-dependent
gene expression without degrading IB (47). Egan et
al. (47) reported that mesalamine did not suppress nuclear
translocation of p65. In contrast, resveratrol did block p65
translocation, which may explain how it suppresses reporter gene
expression.
We found that resveratrol blocks TNF-induced phosphorylation of p65,
which is in agreement with the results of Egan et al.
(47), who showed suppression of IL-1-induced
phosphorylation of p65 by mesalamine. Several kinases have been
implicated that could phosphorylate p65, including protein kinase A and
IKK (47, 58 and references therein). Because IKK that
phosphorylates IB can also phosphorylate p65 (58)
and IB phosphorylation is unaffected, our results indicate that
IKK is not inhibited by resveratrol.
Resveratrol also blocked TNF-induced AP-1 activation. The mechanism of
activation of NF-B and AP-1 is very similar. Most agents that
activate NF-B also activate AP-1. Similarly, agents that suppress
NF-B also suppress AP-1 (28, 29). The activation of
AP-1 requires the activation of JNK and the upstream kinase MEK. Both
of these kinases were inhibited by resveratrol, which may explain the
mechanism of suppression of AP-1.
TNF-induced cyotoxicity and caspase activation were also blocked by
resveratrol. Because NF-B activation has been shown to play an
antiapoptotic role (59), the suppression of apoptosis by
resveratrol may seem paradoxical. However, NF-B activation does not
block apoptosis induced by all the agents (28). The
overexpression of the antioxidant enzymes manganous superoxide
dismutase or -glutamyl cysteinyl synthetase has been shown to
suppress TNF-induced apoptosis and NF-B (28, 29),
suggesting that the mechanisms of activation of apoptosis and NF-B
are very similar. Our discovery that resveratrol blocks TNF-induced ROI
generation and lipid peroxidation explains the mechanism by which
resveratrol exerts its effects. The antioxidant properties of
resveratrol have been previously reported (8, 9).
Resveratrol also blocked TNF-induced NF-B-mediated gene
transcription. Previously, it has been shown that PMA-induced COX-2 is
blocked by resveratrol (16). This gene is known to be
regulated by NF-B activation (60, 61). NO synthase gene
is also regulated by NF-B (62). Thus, it is possible
that resveratrol suppresses COX-2 and NO synthase expression by
inhibiting of NF-B activation. Besides COX-2, various other genes,
including those for matrix metalloproteinase-9 (MMP-9) and cell surface
adhesion molecules (e.g., ICAM-1, endothelial leukocyte adhesion
molecule 1 (ELAM-1), and VCAM-1), are also regulated by NF-B
(63, 64, 65). Urokinase-type plaminogen activator, whose gene
is regulated by NF-B (66), is also involved in tumor
growth and metastasis (67). All these proteins have been
implicated in carcinogenesis (68). It is possible that the
anticarcinogeneic properties assigned to resveratrol (14, 15) are due to the suppression of NF-B-mediated expression of
the genes for these enzymes and adhesion molecules. For instance, high
COX-2 expression has been associated with cancer progression and
inhibition of apoptosis, and antioxidants reduce COX-2 expression,
prostaglandin production, and proliferation in colorectal cancer cells
(69). Due to its ability to suppress COX-2 through
NF-B, aspirin is beneficial for preventing colon cancer
(70). This suggests that resveratrol may also prove to be
beneficial for colon cancer. By using TNF-deficient mice, it was shown
that TNF is required for tumor promotion (23), thus
suggesting its role in carcinogenesis, the role of JNK in TNF-induced
cellular transformation, has been documented (71). Thus,
resveratrol’s ability to suppress TNF-induced NF-B, JNK, AP-1, and
other cellular responses may provide the molecular basis for the
anticarcinogenic properties of resveratrol. Recently, resveratrol was
also found to inhibit the expression and function of androgen receptors
in prostate cancer cells (72). In addition,
adenovirus-enforced overexpression of mitochondrial superoxide
dismutase gene therapy has been used to treat ischemia/reperfusion
injury of the liver through the down-regulation of NF-B and AP-1
activation (73). Our results indicate that suppressive
effects of resveratrol on NF-B and AP-1 activation and on other
TNF-mediated cellular responses may also explain its protective effects
on liver and against cardiovascular diseases.
Our results indicate that 5 µM resveratrol is sufficient to suppress most of the TNF-mediated cellular responses by greater than 90%. Previous studies have shown that to block progression of carcinogenesis and to induce terminal differentiation by 50%, 19 µM resveratrol is required (14). Similarly, 98% inhibition of DMBA plus phorbol ester-induced skin tumors in mice occurred by a topical application of 25 µM resveratrol (14). Thus, concentrations used in our studies are comparable with that used in animal studies. Considering that each gram of fresh grape skin contains 50–100 µg (200–400 µM) resveratrol and the red wine has 1.5–3 mg/L (5, 14), this suggests that resveratrol concentration used in our studies is achievable in vivo by consumption of grapes or wine.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bharat B. Aggarwal, Cytokine Research Section, Department of Bioimmunotherapy, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 143, Houston, TX 77030.
3 Abbreviations used in this paper: resveratrol, trans-3,4',5-trihydroxystilbene; DMBA, dimethylbenz(a)anthracene; CAT, chloramphenicol acetyltransferase; COX, cyclooxygenase; DOC, deoxycholate; ECL, enhanced chemiluminescence; IB, inhibitory subunit of NF-B; IKK, IB kinase; JNK, c-Jun N-terminal protein kinase; MAPK, mitogen-activated protein kinase; MDA, malondialdehyde; MEK, MAPK kinase; PARP, poly(ADP-ribose) polymerase; ROI, reactive oxygen intermediate; TPCK, N-tosyl-L-Phe-chloromethylketone.
Received for publication November 29, 1999. Accepted for publication March 29, 2000.
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H. Ferry-Dumazet, O. Garnier, M. Mamani-Matsuda, J. Vercauteren, F. Belloc, C. Billiard, M. Dupouy, D. Thiolat, J. P. Kolb, G. Marit, et al. Resveratrol inhibits the growth and induces the apoptosis of both normal and leukemic hematopoietic cells Carcinogenesis, August 1, 2002; 23(8): 1327 - 1333. [Abstract] [Full Text] [PDF]
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J. Gusman, H. Malonne, and G. Atassi A reappraisal of the potential chemopreventive and chemotherapeutic properties of resveratrol Carcinogenesis, August 1, 2001; 22(8): 1111 - 1117. [Abstract] [Full Text] [PDF]
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 T. O. Cheng, L. M. Blanco-Colio, C. Bustos, M. Ortego, M. A. Hernandez-Presa, P. Cancelas, J. Gomez-Gerique, J. Egido, M. Valderrama, L. A. Alvarez-Sala, et al. Conundrum of the "French Paradox" Response Circulation, June 26, 2001; 103 (25): e132 - e133. [Full Text] [PDF]
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R. van den Berg, T. van Vliet, W. M. R. Broekmans, N. H. P. Cnubben, W. H. J. Vaes, L. Roza, G. R.M.M. Haenen, A. Bast, and H. van den Berg A Vegetable/Fruit Concentrate with High Antioxidant Capacity Has No Effect on Biomarkers of Antioxidant Status in Male Smokers J. Nutr., June 1, 2001; 131(6): 1714 - 1722. [Abstract] [Full Text]
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J. Fan, R. S. Frey, A. Rahman, and A. B. Malik Role of Neutrophil NADPH Oxidase in the Mechanism of Tumor Necrosis Factor-alpha -induced NF-kappa B Activation and Intercellular Adhesion Molecule-1 Expression in Endothelial Cells J. Biol. Chem., January 25, 2002; 277(5): 3404 - 3411. [Abstract] [Full Text] [PDF]
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