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SHORT REPORT
Anethole blocks both early and late cellular responses transduced by tumor
necrosis factor: eect on NF-kB, AP-1, JNK, MAPKK and apoptosis
Gagan BN Chainy
1,2
, Sunil K Manna
1,2
, Madan M Chaturvedi
1
and Bharat B Aggarwal*
,1
1
Cytokine Research Laboratory, Department of Bioimmunotherapy, The University of Texas M.D. Anderson Cancer Center,
Houston, Texas, TX 77030, USA
Anethole, a chief constituent of anise, camphor, and
fennel, has been shown to block both in¯ammation and
carcinogenesis, but just how these eects are mediated is
not known. One possibility is TNF-me diated signaling,
which has also been associated with both in¯ammation
and carcinogenesis. In the present report we show that
anethole is a potent inhibitor of TNF-induced NF-kB
activation (an early response) as monitored by electro-
phoretic mobility shift assay, IkBa phosphorylation and
degradation, and NF-kB reporter gene expression.
Suppression of IkBa phosphorylation and NF-kB
reporter gene expression induced by TRAF2 and NIK,
suggests that anethole acts on IkBa kinase. Anethole
also blocked the NF-kB activation induced by a variety
of other in¯ammatory agents. Besides NF-kB, anethole
also suppressed TNF-induced activation of the transcrip-
tion factor AP-1, c-jun N-terminal kinase and MAPK-
kinase. In addition, anethole abrogated TNF-induced
apoptosis as measured by both caspase activation and
cell viability. The anethole analogues eugenol and
isoeugenol also blocked TNF signaling. Anethol e
suppressed TNF-induced both lipid peroxidation and
ROI generation. Overall, our results demonstrate that
anethole inhibits TNF-induced cellular responses, which
may explain its role in suppression of in¯ammation and
carcinogenesis. Oncogene (2000) 19, 2943 ± 2950.
Keywords: Anethole; TNF; NF-kB; AP-1; JNK; Apop-
tosis
Anethole, 1-methoxy-4-(1-propenyl) benzene, is the
major component in anise oil, fennel oil, and camphor
(Budavari, 1996). This compound and related ones
have striking metabolic eects. For example, anethol e
and its derivative, anethole ditholethione (ADT), have
been shown to increase the intracellular levels of
glutathione (GSH) and glutathione-S-transferase
(GST) (Drukarch et al., 1997; Rompelberg et al.,
1993; Bouthillier et al., 1996). Structurally related
compounds eugenol and isoeugenol, which are found
in clove-oil, also modulate GSH metabolism (Budavari,
1996; Stohs et al., 1986). These compounds act like
anti-oxidants (Rajakuma r and Rao, 1993; Ko et al.,
1995), inhibit lipid-peroxidation (Stohs et al., 1986;
Nagababu and Lakshmaiah. 1994; Mansuy et al.,
1986), and act as hydroxyl radical scavengers (Taira
et al., 1992). Because eugenol and isoeugenol inhibit
arachidonic acid-induced thromboxane B2, they are
extensively used as anti-in¯ammatory compounds
(Naidu, 1995; Sharma et al., 1994). Besides their anti-
in¯ammatory property, anethole and its analogues
exhibit chemopreventive activities as indicated by
suppression of the incidence and multiplicity of both
invasive and noninvasive adenocarcinomas (Reddy et
al., 1993; Reddy, 1996, 1997; Lubert et al., 1997; Al-
Harbi et al., 1995).
The mechanism underlying these eects of anethole
and its derivatives has not been established, but TNF is
a candidate mediator. TNF is a monokine known to
mediate in¯ammation and carcinogenesis in part
through activation of nuclear factor-kappa B (NF-
kB) (for references see Aggarwal and Natarajan, 1996).
Several genes that are involved in in¯ammation and
carcinogenesis are regulated by this transcription factor
(Baeuerle and Baichwal, 1997). TNF is also a potent
activator of transcription facto r AP-1, which is
involved in carcinogenesis (Karin, 1995). AP-1 activa-
tion requires the activation of c-Jun N-terminal kinase
(JNK) and mitogen-activated protein kinase (MAPK)
kinase (MAPKK or MEK) (Karin, 1995). In addition,
TNF is one of the most important growth regulatory
cytokines known to induce apoptosis through activa-
tion of caspases (Aggarwal and Natarajan, 1996).
Since anethole exhibits anti-carcinogenic, and anti-
in¯ammatory properties, we proposed that the eects
of anethole are mediated through modulation of TNF-
induced cellular responses. Anethole, isoeugenol, and
eugenol have methoxybenzene of methoxyphenol ring
and a propenyl substitution. The conjugate double
bonds in anethole and isoeugenol are known to
stabilize phenoloxy and benzyloxy reactivity. Because
of their antioxidant property, they are likely candidates
to interfere with TNF signaling leading to activation of
NF-kB, AP-1, JNK, MEK and apoptosis. The
concentration of the drugs and the time of incubation
used in our experiments had no eect on cell viability
(cell viability was greater than 97%). ML1a cells were
preincubated for 2 h with dierent concentrations of
anethole (from Sigma Chemicals Co.) followed by
treatment with TNF (100 p
M) for 30 min at 378Cand
then examined for NF-kB activation by EMSA. The
results in Figure 1a indicate that 1 m
M anethole
inhibited most of the TNF response. Anethole by itself
did not activate NF-kB. We next tested the kinetics of
inhibition by pre-, co-, and post-incubation of cells
with anethole (1 m
M) for dierent lengths of time and
Oncogene (2000) 19, 2943 ± 2950
ã
2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00
www.nature.com/onc
*Correspondence: BB Aggarwal
2
These two authors contributed equally to this manuscript
Received 28 December 1999; revised 30 March 2000; accepted 4
April 2000
C
A
B
Figure 1 Anethole inhibits TNF-induced NF-kB activation. (a) ML1-a cells (2610
6
/ml) were pre-incubated at 378C for 2 h with
dierent anethole concentrations (0 ± 2 m
M) followed by 30 min incubation with 0.1 nM TNF. Cells were also treated with DMSO
(0.2%), the vehicle control. After these treatments nuclear extracts were prepared and assayed for NF-kB by electrophoretic
mobility shift assay (EMSA) as described (Manna et al., 1998) using double-stranded oligonucleotides having NF-kB consensus
sequences obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). (b) Cells were preincubated at 378C with 1 m
M
Anethole blocks TNF signaling
GBN Chainy et al
2944
Oncogene
anethole for the indicated times and then tested for NF-kB activation at 378C for 30 min either with or without 0.1 nM TNF. (7)
indicates time anethole was added before the addition of TNF, (0) indicates co-incubation with TNF and (+) indicates time
anethole was added after TNF. After these treatments nuclear extracts were prepared and assayed for NF-kB. (c) HeLa cells were
treated with 1 m
M anethole for 2 h and then transiently transfected with indicated plasmids along with NF-kB containing plasmid
linked to SEAP gene. Where indicated, cells were exposed to 1 n
M TNF for 2 h. Cells were assayed for secreted alkaline
phosphatase activity as previously described (Darnay et al., 1998, 1999). Results are expressed as fold activity over the
nontransfected control
Figure 2 Eugenol and isoeugenol inhibit TNF-induced NF-kB activation. ML1-a cells were pretreated with dierent
concentrations of eugenol (a) or isoeugenol (b) for 2 h at 378C and then treated with 0.1 n
M TNF for 30 min. Then cell extracts
were prepared and assayed for NF-kB. (c) ML1-a cells (2610
6
/ml) were pre-incubated for 2 h at 378C with anethole (1 mM)
followed by PMA (25 ng/ml), serum activated-LPS (10 ug/ml), H
2
O
2
(250 uM), okadaic acid (OA) (500 nM), ceramide (10 uM) and
TNF (0.1 n
M) for 30 min and then tested for NF-kB activation. UN is untreated cells
Oncogene
Anethole blocks TNF signaling
GBN Chainy et al
2945
with TNF (100 pM) for 30 min. When the cells were
pretreated for 240 and 120 min with anethol e, NF-kB
activation was almost completely inhibited, and the
inhibition decreased gradually with decreased preincu-
bation time. Co-treatment or post-treatment with
anethole was not eective in inhibiting NF-kB
activation by TNF (Figure 1b). Antibodies to either
subunit of NF -kB shifted the band to a higher m.w.
complex (data not shown), thus suggesting that the
TNF-activated complex consisted of p50 and p65
subunits. Also, a probe in which the three GGG in
NF-kB binding sites were mutated to CTC did not
show any binding activity, indicating the speci®city of
NF-kB. To determine whether anethole also directly
modi®ed NF-kB proteins, we incubated cytoplasmic
extracts (0.8% deoxycholate treated) or nuclear
extracts from TNF-treated cells and then incubated
them in vitro with various concentrations of anethole
(data not shown). We found that anethole did not
modify the DNA-binding ability of NF-kB proteins.
Besides ML1-a cells, we found that anethole inhibited
NF-kB activation in other myeloid (U-937) and in
epithelial (HeLa) cells all cell types, thus suggesting
that this eect of anethole is not cell type speci®c.
NF-kB is known to control the expression of several
genes involved in in¯ammation and carcinogenesis. To
determine if inhibition of NF-kB binding to the DNA
by anethole leads to suppression of gene expression, we
examined the TNF-induced a SEAP reporter gene
expression (Figure 1c). These results demon strate that
anethole represses NF-kB-dependent gene expression
induced by TNF. TNF-induced NF-kB activation is
mediated through sequential interaction of the TNF
receptor with TRADD, TRAF2, NIK, and IKK-b,
resulting in phosphorylation of IkBa (Hsu et al., 1996;
Simeonidis et al., 1999). To de lineate the site of action
of anethole in the TNF-signali ng pathway leading to
NF-kB activation, cells wer e transfected with TRAF2,
NIK an d p65 plasmids, and then NF-kB-dependent
SEAP expression monitored in anethole-untreated and
-treated cells. As shown in Figure 1c, TRAF2, NIK,
and p65 plasmids induced gene expression and ane-
thole suppressed TRAF-2 and NIK-induced but had
little eect on p65-induced NF-kB reporter expression.
RANK (Darnay et al., 1998), another NF-kB-inducing
receptor was minimally aected by anethole, indicating
the speci®city. Speci®city of the assay is indicated by
suppression of the TNF-induced NF-kB reporter
Figure 3 Anethole blocks TNF-induced IkBa phosphorylation and degradation. (a) Cells were incubated at 378C either with media
or 1 m
M anethole for 2 h and then treated with 0.1 nM TNF at 378C for dierent times as indicated, and then cytosolic extracts
were prepared, resolved on 9% SDS ± PAGE, electrotransferred, blotted with IkBa antibodies as described (Chauturvedi et al.,
1997), and detected by chemiluminescence (ECL, Amersham). (b) Cells were incubated at 378C with 1 m
M anethole at 37 8C for
dierent times as indicated, and then cytosolic extracts were prepared, resolved on 9% SDS ± PAGE, electrotransferred and blotted
with either IkBa antibodies (upper panel) or with anti-p65 antibodies (lower panel). (c) Cells were incubated ®rst with 1 m
M
anethole for 2 h and then with ALLN (100 ug/ml) for an additional 1 h. Thereafter cells were treated with TNF (1 nM) for 15 min,
and then analysed by Western blot using antibodies against phosphorylated IkBa (upper panel) obtained from New England
Biolabs, Inc. Same blot was stripped and reprobed with nonphosphorylated IkBa (lower panel). S indicates slow-migrating band and
N is a normal-migrating band
Anethole blocks TNF signaling
GBN Chainy et al
2946
Oncogene
activity by the dominant negative (DN)-IkBa plasmid.
Thus anethole must act at a step downstream from
NIK. Since NIK is known to activate IKK-b, which in
turn phosphorylates I kBa , it appears that anethole
must block the activity of IKK-b.
To determine if eugenol and isoeugenol, which are
structurally related to anethole also inhibit TNF-
induced NF-kB activation, cells were pretreated with
dierent concentrations of eugenol and isoeugenol for
2 h at 378C, then stimulated with TNF (100 p
M) for
30 min, and NF-kB was assayed in nuclear extracts. As
shown in Figure 2, both eugenol (a) and isoeugenol (b)
inhibited TNF-induced NF-kB activation in a dose-
dependent manner, and the maximum inhibition
occurred at 5 m
M concentration.
Besides TNF, NF-kB activation is also induced by
phorbol ester, H
2
O
2
, LPS, okad aic acid, and ceramide.
However, the signal transduction pathways induced by
these agents dier. We therefore examined the eect of
anethole on the activation of the transcription factor
by these various agents. The results shown in Figure 2c
indicate that anethole completely blocked the activa-
tion of NF-kB induced by all these agents except H
2
O
2
,
in which case the inhibition was partial. Since H
2
O
2
directly increases the oxidative load in a cell and since
anethole acts by modulating intermediates in the
pathway of NF-kB activation, a higher concentration
than 1 m
M anethole is probably required to inhibit
completely H
2
O
2
-induced NF-kB activation. These
results suggest that anethole may act at a step co mmon
to all these agents in the signal transduction pathway
of NF-kB activation.
The convergent step in signal-induced activation of
NF-kB is the phosphorylation IkBa leading to its
ubiquitination and degradation. Once the IkBa is
degraded, the active NF-kB is translocated into the
nucleus. Whether anethole inhibits this crucial step was
studied using Western blotting for IkBa (Figure 3).
Upon TNF treatment , the IkBa was degraded rapidly
by 15 min of treatment, leading to the activation of
NF-kB. IkBa was, however, resynthesized, as the
transcription of IkBa gene is under the control of
NF-kB. Pretreatment of cells with anethole inhibited
the degradation of IkBa (Figure 3a). Interestingly,
anethole treatment alone caused the appearance of a
fast migrating form of the IkBa (Figure 3b). However,
anethole and TNF toget her caused the appearance of
an IkBa form that migrated in between the fast and
slow migrating forms. We attribute the dierences in
the mobility of IkBa to the dierential phosphorylation
state of IkBa. We also investigated if anethole inhibits
IkBa degradation by blocking its phosphorylation. The
serine phosphorylation of IkBa induced by TNF was
stabilized by pretreatment of cells for 1 h with ALLN,
a proteosome inhibitor (Whiteside et al., 1995). The
hyperphosphorylated form of IkBa appeared as a slow-
migrating band on SDS ± PAGE (Figure 3c, lower
panel), which disappeared when cells were pretreated
with anethole indicating that anethole blocks IkBa
phosphorylation. This was further examined by the use
of antibodies which detect only serine phosphorylated
form of IkBa. These results shown in Figure 3c (upper
panel) further con®rm that TNF-induces IkBa phos-
phorylation and anethole inhibits it quite eectively.
Besides NF-kB, TNF also potently activates AP-1,
but it requires longer treatment of cells, as TNF
induces the transcription of c-Fos and c-Jun via
activation of JNK (Karin, 1995). In ML1-a cells,
TNF induced AP-1 DNA binding activity in a dose-
dependent manner, the maximum increase (by seven-
fold) being reached with 1 n
M TNF. This increase in
AP-1 binding was inhibited by anethole (Figure 4a).
AP-1 acti vation requires the activation of JNK. To
determine if anethole abolishes TNF-induced c-Jun
kinase activation, the ML1-a cells were pretreated with
dierent concentrations of anethole for 2 h and then
stimulated with 1 n
M TNF for 10 min; activation of
JNK was then measured. About 13-fold activation of
JNK was produced by TNF, which was gradually
decreased with increasing concentration of anethole,
and at 1 m
M anethole the activation of JNK by TNF
was totally abolished (Figure 4b). JNK activation is
dependent on activation of an upstream MAPK kinase.
To determ ine if anethole inhibits TNF-mediated MAP
kinase phosphorylation, ML1-a cells were pretreated
with 1 m
M anethole for 2 h and then stimulated with
TNF (0.01, 0.1 and 10 n
M) for 30 min, and the
phosphorylation form of MAP kinase was examined.
The results in Figure 4c demonstrate that with
increasing concentration of TNF, phosphorylated band
intensity was increased. In anethole-pret reated cells,
however, TNF did not induce the phosphorylation of
MAP kinase. Thus anethole is also a potent inhibitor
of TNF-induced MAPK kinase activation.
Among all the cytokines, TNF is one of the most
potent inducers of apoptosis. Whether anethole inhibits
TNF-mediated ap optosis was investigated. ML1-a cells
were pretreated with dierent concentrations of anet-
hole for 2 h, and then stimulated with 1 n
M TNF for
72 h at 378CinaCO
2
incubator. Then the MTT assay
was done to check the viability of the cells. The results
in Figure 5a show that anethole suppressed TNF-
induced cytotoxicity in a dose-dependent manner. At
0.5 m
M anethol e, the cells were c ompletely protected
from TNF-mediated killing. TNF-mediated cytotoxi-
city is mediated through activation of caspases. When
activated caspases induce the cleavage of poly (ADP)
ribose polymerase (PARP) (Tewari et al., 1995). Hence
the eect of anethole on TNF-induced PARP cleavage
was studied. ML1-a cells were pretreated with dierent
concentrations of anethole for 2 h, and then stimulated
with TNF (1000 p
M) for 2 h in the presence of
cycloheximide (2 mg/ml), and PARP cleavage was
detected by Western blotting (Figure 5b). Anethole
suppressed TNF-induced PARP cleavage in a dose-
dependent manner, and there was a complete inhibition
at 1 m
M concentration.
Because the role of lipid peroxidation has been
implicated in TNF-induced NF-kB activation (Bowie et
al., 1997), we also examined the eect of anethole on
TNF-induced lipid pe roxidation. Results in Figure 5c
show that TNF induced lipid peroxidation in ML1-a
cells and this was completely supp ressed by anethole.
Thus it is quite likely that anethole may block TNF
signaling through suppression of ROI generation and
of lipid peroxidation. Previous reports have shown that
TNF activates NF-kB through generation of ROI
(Bowie et al., 1997; Baeuerle and Baichwal, 1997).
Whether anethole suppresses NF-kB activation
through suppression of ROI generation was examined
by ¯ow cytometry. As shown in Figure 5d, TNF
induced ROI generation in a time-dependent manner
Oncogene
Anethole blocks TNF signaling
GBN Chainy et al
2947
and this was suppressed on pretreatment of cells with
anethole.
How anethole and its structural analogues inhibit
TNF-induced signaling events is not clear at present.
Anethole an d its sulfated analogues (anethole dithio-
lethione and trithione) have been shown to increase the
activity of GST leading to an increase in cellular GSH
levels (Khanna et al., 1998). This increase may be
responsible for inhibiting TNF-mediated cellular
responses. Agents such as N-acetyl cysteine, which
increase intracellular GSH levels, are known to inhibit
TNF-induced activation of NF- kB (Schreck et al.,
1992). Recent studies from our group have indicated
that transfection of cells with g-glutamyl cysteine
synthetase, a rate-limiting enzyme in GSH biosynthesis
pathway, blocks TNF-induced NF-kB activation
(Manna et al., 1999a). Thus anethole could block
TNF-induced NF-kB activation by increasing cellular
Figure 4 Anethole inhibits TNF-dependent activation of AP-1, JNK and MAPK kinase. (a) Cells (2610
6
) were pre-treated with
anethole (1 m
M) for 2 h, then cells were stimulated with dierent concentrations of TNF for 2 h and assayed for AP-1 by EMSA
(Manna et al., 1998) using double-stranded oligonucleotides having AP-1 consensus sequences obtained from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). (b) ML1-a cells were pretreated with dierent concentrations of anethole for 2 h, then
stimulated with 1 n
M TNF at 378C for 10 min and assayed for JNK activation (Kumar and Aggarwal, 1999). (c) Cells were
pretreated with 1 m
M anethole for 2 h, then stimulated with 0.01, 0.1 and 1 nM TNF for 30 min, and assayed for MAPK kinase
activity using the phosphospeci®c p44/42 MAP kinase (Thr 202/Tyr 204) antibody raised in rabbits (Manna et al., 1998) obtained
from New England Biolabs, Inc.
Anethole blocks TNF signaling
GBN Chainy et al
2948
Oncogene
GSH levels. NF-kB activation by TNF is also
regulated by MEKK1 (Lee et al., 1997), and the latter
is known to activate a dual-speci®city kinase MEK.
Because we found that anethole blocked MEK
activation, it is possible that inhibition of NF-kB
activation is due to suppression of MEK. Our results
indicate that anethole blocks NF-kB activation induced
by PMA, LPS, H
2
O
2
, okadaic acid, ceramide, and
TNF. ADT, an analogue of anethole, has been shown
to block NF-kB activation induced by TNF and PMA
in human T cell line (Sen et al., 1996). The partial
suppression of NF-kB reported by Sen et al could have
resulted from their use of a single dose of ADT at
0.1 m
M for 18 h.
Our results indicate that anethole also blocks TNF-
induced JNK, AP-1 and apoptosis activation. This
eect of anethole may be through the same mechanism
as NF-kB. Upon binding of TNF to its receptors, the
adaptor molecules like TRADD and TRAF2 interact
with sp eci®c regions in the cytoplasmic domains of the
receptors. TRADD in turn interacts with FADD to
activate FLICE to commen ce apoptotic pathway.
Besides NF-kB, TRAF2 also mediates JNK activation
(Hsu et al., 1996), which then phosphorylates c-Jun, a
Figure 5 Anethole inhibits TNF-induced cytotoxicity, caspase activation lipid peroxidation and ROI generation. (a) ML1-a cells,
pretreated with dierent concentrations of anethole for 2 h at 378C, were incubated with 1 n
M TNF for 72 h at 378CinaCO
2
incubator. The cell viability was then determined by MTT dye at 590 nm (Manna et al., 1998). The results indicated in ®gure are the
mean O.D. of triplicate assays. Open circles are anethole alone and closed circles are anethole+TNF. (b) Cells were incubated with
dierent concentrations of anethole for 2 h, then treated with 2 mg/ml cycloheximide and TNF (1 n
M) for 2 h at 378C and examined
for caspase-induced PARP cleavage by Western blot using anti-PARP monoclonal antibody (Haridas et al., 1998). (c) Cells (3610
6
in 1 ml) were pretreated with medium (open circles) or 1 mM anethole (closed circles) for 2 h and then incubated with dierent
concentrations of TNF for 1 h and assayed for lipid peroxidation with thiobarbituric acid (TBA)-reactive malondialdehyde (MDA),
an end product of the peroxidation of polyunsaturated fatty acids and related esters (Bowie et al., 1997; Manna et al., 1999b).
Untreated cells showed 0.571+0.126 nmol of MDA equivalents/mg of protein. (d) Cells (5610
5
/ml) were treated with 1 mM
anethole for 2 h, then exposed to TNF (0.1 nM) for indicated times and ROI production was then determined by the ¯ow cytometry
method (Manna et al., 1999b) using the ¯uorescent ROI probe dihydrorhodamine 123 (DHR 123) purchased from Molecular
Probes, Inc. (Eugene, OR, USA). The results shown are representative of two independent experiments
Oncogene
Anethole blocks TNF signaling
GBN Chainy et al
2949
component of AP-1 transcription factor (Karin, 1995).
Our results indicate that the NF-kB report er activity of
TRAF2 is inhibited by anethole. These results may also
explain the suppression of JNK and AP-1 by anethole.
Like anethole, the anti-in¯ammatory drugs sodium
salicylate and aspirin are also known to block the
activation of NF-kB by altering the steady state levels
of IkBa (Kopp and Ghosh, 1994). The eects of
salicylate on NF-kB activation were observed, how-
ever, at suprapharmacological concentration (45m
M).
In contrast anethole in our studies is eective at a
concentration lower than 1 m
M, suggesting that this is
a potent inhibitor. TNF has been shown to mediate
both in¯ammation and carcinogenesis (Aggarwal and
Natarajan, 1996; Robertson et al., 1996; Suganuma et
al., 1996). Various inhibitory eects of anethole on
TNF signaling might explain its anti-in¯ammatory and
anti-carcinogenic eects previously described. Over all,
our results indicate that anethole and its structural
analogues are potent inhibitors of TNF-induced
divergent eects, and they act at an early step in the
cascade of TNF-dependent signal transduction.
Abbreviations
ALLN, N-ace tylleucylleucylnorleucinal; DHR123, dihy-
drorhodamine 123; DOC, deoxycholate; FADD, Fas-
associated death domain; FLICE, FADD-like ICE; I kB,
inhibitory subunit o f NF-kB; IkB-DN, IkB-dominant
negative; IKK, IkBa kinase; E MSA, elec trophoretic mobi-
lity shift assay; JNK, c-jun amino terminal kina se; LPS ,
lipopolysaccharide; MDA, malondialdehyde; MEK, mito-
gen ac tivated protein kinase kinase ; NE, nuclear extracts;
NIK, NF-kB inducin g kinase; NF-kB, nuclear tra nscrip-
tion factor-kB; OA, okadaic acid; PM A, phorbol myristate
acetate; PARP, poly (ADP) ribose polymerase; TRADD,
TNF receptor-associated death domain; TRAF2, TNF
receptor-associated fa ctor 2.
Acknowledgments
This research was conducted by The Clayton Foundation
for Research. We would like to thank D r B Darnay and Dr
NT Van for assistance with IkBa Western blot analysis and
with FACS analysis, respectively.
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Anethole blocks TNF signaling
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