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Anethole blocks both early and late cellular responses transduced by tumor necrosis factor: Effect on NF-??B, AP-1, JNK, MAPKK and apoptosis

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Anethole, a chief constituent of anise, camphor, and fennel, has been shown to block both inflammation and carcinogenesis, but just how these effects are mediated is not known. One possibility is TNF-mediated signaling, which has also been associated with both inflammation and carcinogenesis. In the present report we show that anethole is a potent inhibitor of TNF-induced NF-kappaB activation (an early response) as monitored by electrophoretic mobility shift assay, IkappaBalpha phosphorylation and degradation, and NF-kappaB reporter gene expression. Suppression of IkappaBalpha phosphorylation and NF-kappaB reporter gene expression induced by TRAF2 and NIK, suggests that anethole acts on IkappaBalpha kinase. Anethole also blocked the NF-kappaB activation induced by a variety of other inflammatory agents. Besides NF-kappaB, anethole also suppressed TNF-induced activation of the transcription 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. Anethole 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 inflammation and carcinogenesis. Oncogene (2000).
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SHORT REPORT
Anethole blocks both early and late cellular responses transduced by tumor
necrosis factor: eect 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 eects 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 eects. 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 eects 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 eects
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 eect on cell viability
(cell viability was greater than 97%). ML1a cells were
preincubated for 2 h with dierent 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 dierent 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
dierent 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 dierent
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 eective 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 eect 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 eect on p65-induced NF-kB reporter expression.
RANK (Darnay et al., 1998), another NF-kB-inducing
receptor was minimally aected 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 dierent 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
dierent 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
dierent 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 dier. We therefore examined the eect 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 dierences in
the mobility of IkBa to the dierential 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 eectively.
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
dierent 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 dierent 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 eect of anethole on TNF-induced PARP cleavage
was studied. ML1-a cells were pretreated with dierent
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 eect 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 dierent 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 dierent 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
eect 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 dierent 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
dierent 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 dierent
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 eects of
salicylate on NF-kB activation were observed, how-
ever, at suprapharmacological concentration (45m
M).
In contrast anethole in our studies is eective 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 eects of anethole on
TNF signaling might explain its anti-in¯ammatory and
anti-carcinogenic eects previously described. Over all,
our results indicate that anethole and its structural
analogues are potent inhibitors of TNF-induced
divergent eects, 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
GBN Chainy et al
2950
Oncogene
... Chronic hyperglycemia triggers inflammatory responses mediated by cytokines such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1β), which contribute to neuroinflammation and subsequent nerve injury [51]. It has already been demonstrated that anethole has great anti-inflammatory activity, inhibiting various inflammatory mediators and pathways, such as cyclooxygenase enzymes, TNF-alpha, and IL-6 [52,53]. Additionally, the neuroprotective effect of anethole on the electrophysiology and morphology of mouse sciatic nerves with chronic constriction has been demonstrated, and the authors attribute this neuroprotection to anethole's anti-inflammatory activity [7]. ...
... The dose of anethole (300 mg/kg) used here was chosen based on our previous studies about the effects of EOCz on DPN [9,10]. Although this dose could be considered high, its toxicity is considered low [8] since rats treated with 250 mg/kg anethole or EOCz for 70 days did not present behavior disturbances or changes in plasma biochemical parameters [52]. It is important to note that anethole is effective anti-inflammatory in doses (3-30 mg/kg) lower than used in this work, and a maximum anti-inflammatory effect may be reached at 30 mg/kg [8]. ...
Article
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Anethole is a terpenoid with antioxidant, anti-inflammatory, and neuronal blockade effects, and the present work was undertaken to study the neuroprotective activity of anethole against diabetes mellitus (DM)-induced neuropathy. Streptozotocin-induced DM rats were used to investigate the effects of anethole treatment on morphological, electrophysiological, and biochemical alterations of the sciatic nerve (SN). Anethole partially prevented the mechanical hyposensitivity caused by DM and fully prevented the DM-induced decrease in the cross-sectional area of the SN. In relation to electrophysiological properties of SN fibers, DM reduced the frequency of occurrence of the 3rd component of the compound action potential (CAP) by 15%. It also significantly reduced the conduction velocity of the 1st and 2nd CAP components from 104.6 ± 3.47 and 39.8 ± 1.02 to 89.9 ± 3.03 and 35.4 ± 1.56 m/s, respectively, and increased the duration of the 2nd CAP component from 0.66 ± 0.04 to 0.82 ± 0.09 ms. DM also increases oxidative stress in the SN, altering values related to thiol, TBARS, SOD, and CAT activities. Anethole was capable of fully preventing all these DM electrophysiological and biochemical alterations in the nerve. Thus, the magnitude of the DM-induced neural effects seen in this work, and the prevention afforded by anethole treatment, place this compound in a very favorable position as a potential therapeutic agent for treating diabetic peripheral neuropathy.
... The antioxidant potential of Croton grewioides essential oil (CGEO) was previously reported, while anethole, which is a phenylpropanoid, is the main molecule present in CGEO [13]. In vitro investigations have shown that anethole reduces the negative effects of ROS in goat ovarian follicles cultured in vitro [14,15] and also inhibits tumor necrosis factor-mediated inflammatory effects [16]. The cellular antioxidant defense system involves transcription factors such as nuclear factor erythroid 2-related factor 2 (NRF2) that acts to regulate the expression of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and peroxirredoxin (PRDX). ...
... In addition, their effects on stromal cell density, collagen fibers in the extracellular matrix, levels of thiol, the activity of antioxidant enzymes (SOD, CAT, and GPx1) and the levels of mRNA for NFR2, PRDX1, SOD, CAT, and GPx1 in cultured bovine ovarian tissues were also evaluated. 16.152 g of essential oil was obtained from 540 g of leaves (yield ± 2.99%). ...
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Objectives This study aims to evaluate the effects of Croton grewioides essential oil (CGEO) and anethole on follicle survival, growth, and oxidative stress in cultured bovine ovarian tissues. Methods Ovarian tissues were cultured for 6 days in a medium supplemented with different concentrations (1, 10, 100, or 1000 µg mL–1) of CGEO or anethole and then, follicular survival and growth, collagen content, and stromal cell density in ovarian tissues cultured in vitro were evaluated by histology. The mRNA levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase 1 (GPX1), peroxirredoxin 6 (PRDX6), and nuclear factor erythroid 2-related factor 2 (NRF2) were evaluated by real-time PCR. The activity of SOD, CAT, glutathione peroxidase (GPx), and thiol concentrations were investigated. Key findings Ovarian tissues cultured with 1 µg mL–1 CGEO or anethole had a higher percentage of healthy follicles than those cultured in a control medium (P < .05). The 1 µg mL–1 CGEO also increased the number of stromal cells, collagen fibers, and thiol levels. Anethole (1 µg mL–1) increased CAT activity and reduced that of GPx. The activity of SOD was reduced by CGEO. In contrast, 1 µg mL–1 anethole reduced mRNA for CAT, PRDX1, and NRF2 (P < .05). In addition, 1 µg mL–1 CGEO reduced mRNA for CAT, PRDX6, and GPx1 (P < .05). Conclusions The presence of 1 µg mL–1 anethole or CGEO in a culture medium promotes follicle survival and regulates oxidative stress and the expression of mRNA and activity of antioxidant enzymes in cultured bovine ovarian tissues.
... Other studies show that AN in monotherapy inhibits the acute and chronic inflammatory response in rats Ritter et al. 2017), and that when associated with ibuprofen (traditional anti-inflammatory), it has greater antiinflammatory activity at much lower doses when compared to monotherapy (Wisniewski-Rebecca et al. 2015). Such AN activity seems to be due to its inhibitory action on the production/release of some inflammatory mediators (NO, TNF) Ritter et al. 2013;Wisniewski-Rebecca et al. 2015;Ritter et al. 2017;Chainy et al. 2000). ...
... Previous studies have shown that AN inhibits the production/release of several cytokines involved in cell recruitment and migration to lesion area, such as tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β) and interleukin 17 (IL-17) Chainy et al. 2000;Ponte et al. 2012). AN treatment has also been shown to decrease the expression of certain intercellular adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1) (Sung et al. 2013) and it increased interleukin concentration 10 (IL-10) . ...
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In this study, the effect of treatment with anethole (AN) and the combination anethole+itraconazole (AN+IT) compared to IT, on infectious arthritis induced by Paracoccidiodes brasiliensis, was evaluated. The animals were treated for 14 days at doses of anethole (AN - 62.5, 125 and 250 mg/kg), itraconazole (IT - 12.5, 25 and 50 mg/kg) and the combination of anethole+itraconazole (AN+ IT - 62.5 and 12.5 mg/kg). The parameters evaluated were: the development of knee edema, the number of leukocytes recruited into the joint cavity, the body weight of the animals, the walking capacity, the plasmatic concentration of nitric oxide, the concentration of TNF in the knee joint exudate, the production of anti-Pb antibodies and the activity of plasma transaminases (AST and ALT). Histological changes in the right knee joint of the hind paw were evaluated using Hematoxylin-eosin and Grocott staining. The results showed that treatment with monotherapies and combinations reduced knee joint edema, the number of leukocytes recruited into the synovial cavity and improved the animals' gait. The concentration of plasma NO, tissue TNF and anti-Pb were reduced by treatment with IT at all doses tested and with the AN+IT combination. Treatment with AN only reduced the plasma concentration of NO and tissue TNF at a high dose. Altogether, the data showed that treatments with IT monotherapy and the combination of AN + IT showed a similar inhibitory effect on the development of arthritis.
... For instance, our present study lacked detailed investigations of the signaling steps that explain how transcription factors STAT3 and NF-κB are activated in I/R injury but suppressed in trans-Anethole co-treated animals. Previous observations suggest that trans-Anethole is a potential TNF-α inhibitor that can suppress NF-κB, the STAT superfamily, and apoptosis, as TNF-α and other inflammatory cytokines are primary activators of apoptotic cell death [39]. Additionally, it has been shown that anethole-containing plant extracts of Illicium verum possess anti-inflammatory effects by blocking TNF-α release and NF-κB activation [40]. ...
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Testicular ischemia reperfusion (I/R) injury is a significant urological problem where clinical interventions may be inadequate, and the antioxidants might be potential co-treatment modalities. This study examined the gonadoprotective effect of trans-Anethole in testicular I/R injury. Twenty-eight male rats were divided into four groups. Rats in the I/R, I/R + t100, I/R + t200 groups underwent bilateral testicular I/R injury. The I/R + t100 and I/R + t200 groups received 100 or 200 mg/kg trans-Anethole at the 2nd hour of ischemia. Microscopic evaluations demonstrated that testicular I/R injury leads to severe testicular degeneration. Tissue oxidative stress, pro-apoptotic Bcl-2 associated X (Bax) and Caspase 3, pro-inflammatory Tumor necrosis factor-alpha (TNF-α), Interleukin-1 beta (IL-1β) and Interleukin 6 (IL-6) cytokines levels were significantly (p < 0.05) upregulated when compared to the Control group. Additionally, transcription factors Signal transducer and activator of transcription 3 (STAT3) and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) levels increased significantly (p < 0.05) compared to the Control group. Tissue disrupted parameters in the I/R + t200 group were significantly different (p < 0.05) from the I/R group, contrasting with the slight improvement in the I/R + t100 group. The STAT3 and NF-κB expression levels in the I/R + t200 group were significantly suppressed (p < 0.05) compared to the I/R group. In conclusion, our study indicates that trans-Anethole could enhance gonadoprotective activity in testicular I/R injury, potentially involving transcription factors STAT3 and NF-κB. However, before the consumption of trans-Anethole-containing natural or manufactured goods, the potential benefits and side effects should be carefully evaluated.
... Monoterpenes present in F. vulgare are considered to be associated with the prevention of several disorders induced by oxidative stress, such as cardiovascular disease, cancer, and inflammation. In particular, Chainy et al. [108] showed that trans-anethole is responsible for the suppression of both inflammation and carcinogenesis (Table 6). This bioactive compound was reported to act at an early step in the cascade of TNF-dependent signal transduction, so inhibiting cytokine-induced cellular response was associated with both diseases. ...
Article
Full-text available
Research studies on plant secondary metabolites have increased over the last decades as a consequence of the growing consumer demand for natural products in pharmaceutics and therapeutics, as well as in perfumery and cosmetics. In this perspective, many Mediterranean plant species could be an appreciated source of bioactive compounds with pharmacological and health-promoting properties, including antioxidant, antimicrobial, antiviral, anti-inflammatory, and antitumor ones. Calendula officinalis and Foeniculum vulgare are commercially important plants of the Mediterranean flora, with great therapeutic use in the treatment of many disorders since ancient times, and are now listed in several world pharmacopoeias and drug agencies. The present review offers an overview of the main phytochemicals, phenols, terpenes, and alkaloids, biosynthesized in C. officinalis and F. vulgare, both species endemic to the Mediterranean region. Further, all current knowledge and scientific data on taxonomic classification, botanical description, traditional uses, pharmacological studies, and potential toxicity of both species were reported. The principal aim of this review is to point out the prospective use of C. officinalis and F. vulgare as valuable reservoirs of beneficial plant-derived products with interesting biological properties, also providing suggestions and future challenges for the full exploitation of these two Mediterranean species for human life improvement.
... The compound can also effectively inhibit lipid peroxidation, further highlighting its potent antioxidant effects. 48 . These results indicate that the Anethole has effectively reduced brain hippocampus MDA. ...
Article
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The occurrence of major depressive disorder is widespread and can be observed in individuals belonging to all societies. It has been suggested that changes in the NO pathway and heightened oxidative stress may play a role in developing this condition. Anethole is a diterpene aromatic compound found in the Umbelliferae, Apiaceae, and Schisandraceae families. It has potential pharmacological effects like antioxidant, anxiolytic, analgesic, anti-inflammatory, antidiabetic, gastroprotective, anticancer, estrogenic, and antimicrobial activities. This study aimed to investigate the potential antidepressant properties of Anethole in a mouse model experiencing maternal separation stress while also examining its impact on oxidative stress and nitrite levels. The research involved the participation of 40 male NMRI mice, separated into five distinct groups to conduct the study. The control group was administered 1 ml/kg of normal saline, while the MS groups were given normal saline and Anethole at 10, 50, and 100 mg/kg doses. The study comprised various behavioural tests, including the open field test (OFT), forced swimming test (FST), and splash test, to assess the effects of Anethole on the mice. In addition to the behavioural tests, measurements were taken to evaluate the total antioxidant capacity (TAC), malondialdehyde (MDA), and nitrite levels in the hippocampus of the mice. According to the findings, maternal separation stress (MS) led to depressive-like conduct in mice, including a rise in immobility duration during the FST and a reduction in the duration of grooming behaviour in the splash test. Additionally, the results indicated that MS correlated with an increase in the levels of MDA and nitrite and a reduction in the TAC in the hippocampus. However, the administration of Anethole resulted in an increase in grooming activity time during the splash test and a decrease in immobility time during the FST. Anethole also exhibited antioxidant characteristics, as demonstrated by its ability to lower MDA and nitrite levels while increasing the TAC in the hippocampus. The results suggest that Anethole may have an antidepressant-like impact on mice separated from their mothers, likely partly due to its antioxidant properties in the hippocampus.
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Purpose: This study investigated the pharmacological effects of topical trans-anethole, a natural compound found in anise, star anise, and fennel essential oils, and its relationship with the transient receptor potential of ankyrin 1 (TRPA1). Methods: The effects of topical anethole were assessed by eye wiping, nociceptive behaviour, and ear oedema in mice. Histological evaluations were performed on the ears of the animals topically treated with anethole. Results: Anethole caused less eye irritation than capsaicin (a TRPV1 agonist) and allyl isothiocyanate (a TRPA1 agonist). Anethole (250 and 500 nmol/20 µL/paw) promoted neurogenic nociception in the paw (20.89 ± 3.53 s and 47.56 ± 8.46 s, respectively) compared with the vehicle (0.88 ± 0.38 s). HC030031 (56.1 nmol/20 µL/paw), a TRPA1 antagonist, abolished this nociceptive response. Anethole (4, 10, and 20 µmol/20 µL/ear) induced ear oedema (30.25 ± 4.78 μm, 78.00 ± 3.74 μm, and 127.50 ± 27.19 μm, respectively) compared with the vehicle (5.00 ± 0.5 μm). HC030031 (56.1 nmol/20 µL/ear) inhibited the oedema induced by anethole (10 µmol/20 µL/ear). Ears pre-treated with anethole or allyl isothiocyanate on the first day and re-exposed to these compounds on the third day showed a reduction in oedema (68.16 ± 6.04% and 38.81 ± 8.98.9%, respectively). Cross-desensitisation between anethole and allyl isothiocyanate was observed. Histological analysis confirmed the beneficial effects of anethol. Conclusion: As repeated topical applications of anethole induce the desensitisation of TRPA1, we suggest its clinical application as a topical formulation for treating skin diseases or managing pain associated with this receptor. Anethole may also have advantages over capsaicin and allyl isothiocyanate because of its low pungency and pleasant aroma.
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Aromatherapy is a medical practice that uses aromatic compounds or essential oils to influence mood and health. Essential oils used in aromatherapy are created from a wide variety of medicinal plants, flowers, herbs, roots, and trees that are found all over the world and have significant, well-documented benefits on enhancing physical, emotional, and spiritual wellbeing. This book is a comprehensive reference on aromatic compounds present in essential oils and their therapeutic use. Starting from fundamentals of essential oil biosynthesis the book guides the reader through their basic biochemistry, toxicology, profiling, blending and clinical applications. The concluding chapters also present focused information about the therapeutic effects of essential oils on specific physiological systems, plant sources, skin treatment and cancer therapeutics. The combination of basic and applied knowledge will provide readers with all the necessary information for understanding how to develop preclinical formulations and standard clinical therapies with essential oils. This is an essential reference for anyone interested in aromatherapy and the science of essential oils.
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NF-kappa B is a multiprotein complex that can activate a great variety of genes involved in early defence reactions of higher organisms. In nonstimulated cells, NF-kappa B resides in the cytoplasm in an inactive complex with the inhibitor I kappa B. Pathogenic stimuli cause release of I kappa B and allow NF-kappa B to enter the nucleus, bind to DNA control elements and, thereby, induce the synthesis of mRNA. A puzzling feature of NF-kappa B is that its activation is triggered by a great variety of agents. These include the cytokines interleukin-1 and tumor necrosis factor, viruses, double-stranded RNA, endotoxins, phorbol esters, UV light and ionizing radiation. We recently found that also low concentrations of H2O2 activate NF-kappa B and that various antioxidants prevent the induction by H2O2. Subsequent analysis revealed that antioxidants not only suppress the activation of NF-kappa B by H2O2 but by all other inducers tested so far. In this review, we will discuss the evidences that NF-kappa B is an oxidative stress-responsive transcription factor of higher eukaryotic cells.