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

Abstract

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-κB activation (an early response) as monitored by electrophoretic mobility shift assay, IκBα phosphorylation and degradation, and NF-κB reporter gene expression. Suppression of IκBα phosphorylation and NF-κB reporter gene expression induced by TRAF2 and NIK, suggests that anethole acts on IκBα kinase. Anethole also blocked the NF-κB activation induced by a variety of other inflammatory agents. Besides NF-κB, 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.

Main

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 effects. For example, anethole 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 (Rajakumar 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-inflammatory compounds (Naidu, 1995; Sharma et al., 1994). Besides their anti-inflammatory 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 effects of anethole and its derivatives has not been established, but TNF is a candidate mediator. TNF is a monokine known to mediate inflammation and carcinogenesis in part through activation of nuclear factor-kappa B (NF-κB) (for references see Aggarwal and Natarajan, 1996). Several genes that are involved in inflammation and carcinogenesis are regulated by this transcription factor (Baeuerle and Baichwal, 1997). TNF is also a potent activator of transcription factor AP-1, which is involved in carcinogenesis (Karin, 1995). AP-1 activation 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 activation of caspases (Aggarwal and Natarajan, 1996).

Since anethole exhibits anti-carcinogenic, and anti-inflammatory properties, we proposed that the effects 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-κB, AP-1, JNK, MEK and apoptosis. The concentration of the drugs and the time of incubation used in our experiments had no effect on cell viability (cell viability was greater than 97%). ML1a cells were preincubated for 2 h with different concentrations of anethole (from Sigma Chemicals Co.) followed by treatment with TNF (100 pM) for 30 min at 37°C and then examined for NF-κB activation by EMSA. The results in Figure 1a indicate that 1 mM anethole inhibited most of the TNF response. Anethole by itself did not activate NF-κB. We next tested the kinetics of inhibition by pre-, co-, and post-incubation of cells with anethole (1 mM) for different lengths of time and with TNF (100 pM) for 30 min. When the cells were pretreated for 240 and 120 min with anethole, NF-κB activation was almost completely inhibited, and the inhibition decreased gradually with decreased preincubation time. Co-treatment or post-treatment with anethole was not effective in inhibiting NF-κB activation by TNF (Figure 1b). Antibodies to either subunit of NF-κB 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-κB binding sites were mutated to CTC did not show any binding activity, indicating the specificity of NF-κB. To determine whether anethole also directly modified NF-κB 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-κB proteins. Besides ML1-a cells, we found that anethole inhibited NF-κB activation in other myeloid (U-937) and in epithelial (HeLa) cells all cell types, thus suggesting that this effect of anethole is not cell type specific.

Figure 1
figure1

Anethole inhibits TNF-induced NF-κB activation. (a) ML1-a cells (2×106/ml) were pre-incubated at 37°C for 2 h with different anethole concentrations (0–2 mM) 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-κB by electrophoretic mobility shift assay (EMSA) as described (Manna et al., 1998) using double-stranded oligonucleotides having NF-κB consensus sequences obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). (b) Cells were preincubated at 37°C with 1 mM anethole for the indicated times and then tested for NF-κB activation at 37°C for 30 min either with or without 0.1 nM TNF. (−) 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-κB. (c) HeLa cells were treated with 1 mM anethole for 2 h and then transiently transfected with indicated plasmids along with NF-κB containing plasmid linked to SEAP gene. Where indicated, cells were exposed to 1 nM 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

NF-κB is known to control the expression of several genes involved in inflammation and carcinogenesis. To determine if inhibition of NF-κB 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 demonstrate that anethole represses NF-κB-dependent gene expression induced by TNF. TNF-induced NF-κB activation is mediated through sequential interaction of the TNF receptor with TRADD, TRAF2, NIK, and IKK-β, resulting in phosphorylation of IκBα (Hsu et al., 1996; Simeonidis et al., 1999). To delineate the site of action of anethole in the TNF-signaling pathway leading to NF-κB activation, cells were transfected with TRAF2, NIK and p65 plasmids, and then NF-κB-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 effect on p65-induced NF-κB reporter expression. RANK (Darnay et al., 1998), another NF-κB-inducing receptor was minimally affected by anethole, indicating the specificity. Specificity of the assay is indicated by suppression of the TNF-induced NF-κB reporter activity by the dominant negative (DN)-IκBα plasmid. Thus anethole must act at a step downstream from NIK. Since NIK is known to activate IKK-b, which in turn phosphorylates IκBα, it appears that anethole must block the activity of IKK-β.

To determine if eugenol and isoeugenol, which are structurally related to anethole also inhibit TNF-induced NF-κB activation, cells were pretreated with different concentrations of eugenol and isoeugenol for 2 h at 37°C, then stimulated with TNF (100 pM) for 30 min, and NF-κB was assayed in nuclear extracts. As shown in Figure 2, both eugenol (a) and isoeugenol (b) inhibited TNF-induced NF-κB activation in a dose-dependent manner, and the maximum inhibition occurred at 5 mM concentration.

Figure 2
figure2

Eugenol and isoeugenol inhibit TNF-induced NF-κB activation. ML1-a cells were pretreated with different concentrations of eugenol (a) or isoeugenol (b) for 2 h at 37°C and then treated with 0.1 nM TNF for 30 min. Then cell extracts were prepared and assayed for NF-κB. (c) ML1-a cells (2×106/ml) were pre-incubated for 2 h at 37°C with anethole (1 mM) followed by PMA (25 ng/ml), serum activated-LPS (10 ug/ml), H2O2 (250 uM), okadaic acid (OA) (500 nM), ceramide (10 uM) and TNF (0.1 nM) for 30 min and then tested for NF-κB activation. UN is untreated cells

Besides TNF, NF-κB activation is also induced by phorbol ester, H2O2, LPS, okadaic acid, and ceramide. However, the signal transduction pathways induced by these agents differ. We therefore examined the effect 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 activation of NF-κB induced by all these agents except H2O2, in which case the inhibition was partial. Since H2O2 directly increases the oxidative load in a cell and since anethole acts by modulating intermediates in the pathway of NF-κB activation, a higher concentration than 1 mM anethole is probably required to inhibit completely H2O2-induced NF-κB activation. These results suggest that anethole may act at a step common to all these agents in the signal transduction pathway of NF-κB activation.

The convergent step in signal-induced activation of NF-κB is the phosphorylation IκBα leading to its ubiquitination and degradation. Once the IκBα is degraded, the active NF-κB is translocated into the nucleus. Whether anethole inhibits this crucial step was studied using Western blotting for IκBα (Figure 3). Upon TNF treatment, the IκBα was degraded rapidly by 15 min of treatment, leading to the activation of NF-κB. IκBα was, however, resynthesized, as the transcription of IκBα gene is under the control of NF-κB. Pretreatment of cells with anethole inhibited the degradation of IκBα (Figure 3a). Interestingly, anethole treatment alone caused the appearance of a fast migrating form of the IκBα (Figure 3b). However, anethole and TNF together caused the appearance of an IκBα form that migrated in between the fast and slow migrating forms. We attribute the differences in the mobility of IκBα to the differential phosphorylation state of IκBα. We also investigated if anethole inhibits IκBα degradation by blocking its phosphorylation. The serine phosphorylation of IκBα 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 IκBα 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 IκBα phosphorylation. This was further examined by the use of antibodies which detect only serine phosphorylated form of IκBα. These results shown in Figure 3c (upper panel) further confirm that TNF-induces IκBα phosphorylation and anethole inhibits it quite effectively.

Figure 3
figure3

Anethole blocks TNF-induced IκBα phosphorylation and degradation. (a) Cells were incubated at 37°C either with media or 1 mM anethole for 2 h and then treated with 0.1 nM TNF at 37°C for different times as indicated, and then cytosolic extracts were prepared, resolved on 9% SDS–PAGE, electrotransferred, blotted with IκBα antibodies as described (Chauturvedi et al., 1997), and detected by chemiluminescence (ECL, Amersham). (b) Cells were incubated at 37°C with 1 mM anethole at 37°C for different times as indicated, and then cytosolic extracts were prepared, resolved on 9% SDS–PAGE, electrotransferred and blotted with either IκBα antibodies (upper panel) or with anti-p65 antibodies (lower panel). (c) Cells were incubated first with 1 mM 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 IκBα (upper panel) obtained from New England Biolabs, Inc. Same blot was stripped and reprobed with nonphosphorylated IκBα (lower panel). S indicates slow-migrating band and N is a normal-migrating band

Besides NF-κB, 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 sevenfold) being reached with 1 nM TNF. This increase in AP-1 binding was inhibited by anethole (Figure 4a). AP-1 activation requires the activation of JNK. To determine if anethole abolishes TNF-induced c-Jun kinase activation, the ML1-a cells were pretreated with different concentrations of anethole for 2 h and then stimulated with 1 nM 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 mM anethole the activation of JNK by TNF was totally abolished (Figure 4b). JNK activation is dependent on activation of an upstream MAPK kinase. To determine if anethole inhibits TNF-mediated MAP kinase phosphorylation, ML1-a cells were pretreated with 1 mM anethole for 2 h and then stimulated with TNF (0.01, 0.1 and 10 nM) 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-pretreated cells, however, TNF did not induce the phosphorylation of MAP kinase. Thus anethole is also a potent inhibitor of TNF-induced MAPK kinase activation.

Figure 4
figure4

Anethole inhibits TNF-dependent activation of AP-1, JNK and MAPK kinase. (a) Cells (2×106) were pre-treated with anethole (1 mM) for 2 h, then cells were stimulated with different 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 different concentrations of anethole for 2 h, then stimulated with 1 nM TNF at 37°C for 10 min and assayed for JNK activation (Kumar and Aggarwal, 1999). (c) Cells were pretreated with 1 mM 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 phosphospecific p44/42 MAP kinase (Thr 202/Tyr 204) antibody raised in rabbits (Manna et al., 1998) obtained from New England Biolabs, Inc.

Among all the cytokines, TNF is one of the most potent inducers of apoptosis. Whether anethole inhibits TNF-mediated apoptosis was investigated. ML1-a cells were pretreated with different concentrations of anethole for 2 h, and then stimulated with 1 nM TNF for 72 h at 37°C in a CO2 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 mM anethole, the cells were completely protected from TNF-mediated killing. TNF-mediated cytotoxicity is mediated through activation of caspases. When activated caspases induce the cleavage of poly (ADP) ribose polymerase (PARP) (Tewari et al., 1995). Hence the effect of anethole on TNF-induced PARP cleavage was studied. ML1-a cells were pretreated with different concentrations of anethole for 2 h, and then stimulated with TNF (1000 pM) for 2 h in the presence of cycloheximide (2 μg/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 mM concentration.

Figure 5
figure5

Anethole inhibits TNF-induced cytotoxicity, caspase activation lipid peroxidation and ROI generation. (a) ML1-a cells, pretreated with different concentrations of anethole for 2 h at 37°C, were incubated with 1 nM TNF for 72 h at 37°C in a CO2 incubator. The cell viability was then determined by MTT dye at 590 nm (Manna et al., 1998). The results indicated in figure are the mean O.D. of triplicate assays. Open circles are anethole alone and closed circles are anethole+TNF. (b) Cells were incubated with different concentrations of anethole for 2 h, then treated with 2 μg/ml cycloheximide and TNF (1 nM) for 2 h at 37°C and examined for caspase-induced PARP cleavage by Western blot using anti-PARP monoclonal antibody (Haridas et al., 1998). (c) Cells (3×106 in 1 ml) were pretreated with medium (open circles) or 1 mM anethole (closed circles) for 2 h and then incubated with different 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 (5×105/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 flow cytometry method (Manna et al., 1999b) using the fluorescent ROI probe dihydrorhodamine 123 (DHR 123) purchased from Molecular Probes, Inc. (Eugene, OR, USA). The results shown are representative of two independent experiments

Because the role of lipid peroxidation has been implicated in TNF-induced NF-κB activation (Bowie et al., 1997), we also examined the effect of anethole on TNF-induced lipid peroxidation. Results in Figure 5c show that TNF induced lipid peroxidation in ML1-a cells and this was completely suppressed 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-κB through generation of ROI (Bowie et al., 1997; Baeuerle and Baichwal, 1997). Whether anethole suppresses NF-κB activation through suppression of ROI generation was examined by flow cytometry. As shown in Figure 5d, TNF induced ROI generation in a time-dependent manner 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 and its sulfated analogues (anethole dithiolethione 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-κB (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-κB activation (Manna et al., 1999a). Thus anethole could block TNF-induced NF-κB activation by increasing cellular GSH levels. NF-κB activation by TNF is also regulated by MEKK1 (Lee et al., 1997), and the latter is known to activate a dual-specificity kinase MEK. Because we found that anethole blocked MEK activation, it is possible that inhibition of NF-κB activation is due to suppression of MEK. Our results indicate that anethole blocks NF-κB activation induced by PMA, LPS, H2O2, okadaic acid, ceramide, and TNF. ADT, an analogue of anethole, has been shown to block NF-κB activation induced by TNF and PMA in human T cell line (Sen et al., 1996). The partial suppression of NF-κB reported by Sen et al could have resulted from their use of a single dose of ADT at 0.1 mM for 18 h.

Our results indicate that anethole also blocks TNF-induced JNK, AP-1 and apoptosis activation. This effect of anethole may be through the same mechanism as NF-κB. Upon binding of TNF to its receptors, the adaptor molecules like TRADD and TRAF2 interact with specific regions in the cytoplasmic domains of the receptors. TRADD in turn interacts with FADD to activate FLICE to commence apoptotic pathway. Besides NF-κB, TRAF2 also mediates JNK activation (Hsu et al., 1996), which then phosphorylates c-Jun, a component of AP-1 transcription factor (Karin, 1995). Our results indicate that the NF-κB reporter 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-inflammatory drugs sodium salicylate and aspirin are also known to block the activation of NF-κB by altering the steady state levels of IκBα (Kopp and Ghosh, 1994). The effects of salicylate on NF-κB activation were observed, however, at suprapharmacological concentration (>5 mM). In contrast anethole in our studies is effective at a concentration lower than 1 mM, suggesting that this is a potent inhibitor. TNF has been shown to mediate both inflammation and carcinogenesis (Aggarwal and Natarajan, 1996; Robertson et al., 1996; Suganuma et al., 1996). Various inhibitory effects of anethole on TNF signaling might explain its anti-inflammatory and anti-carcinogenic effects previously described. Overall, our results indicate that anethole and its structural analogues are potent inhibitors of TNF-induced divergent effects, and they act at an early step in the cascade of TNF-dependent signal transduction.

Abbreviations

ALLN:

N-acetylleucylleucylnorleucinal

DHR123:

dihydrorhodamine 123

DOC:

deoxycholate

FADD:

Fas-associated death domain

FLICE:

FADD-like ICE

IκB:

inhibitory subunit of NF-κB

IκB-DN:

IκB-dominant negative

IKK:

IκBα kinase

EMSA:

electrophoretic mobility shift assay

JNK:

c-jun amino terminal kinase

LPS:

lipopolysaccharide

MDA:

malondialdehyde

MEK:

mitogen activated protein kinase kinase

NE:

nuclear extracts

NIK:

NF-κB inducing kinase

NF-κB:

nuclear transcription factor-κB

OA:

okadaic acid

PMA:

phorbol myristate acetate

PARP:

poly (ADP) ribose polymerase

TRADD:

TNF receptor-associated death domain

TRAF2:

TNF receptor-associated factor 2

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Acknowledgements

This research was conducted by The Clayton Foundation for Research. We would like to thank Dr B Darnay and Dr NT Van for assistance with IκBα Western blot analysis and with FACS analysis, respectively.

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Correspondence to Bharat B Aggarwal.

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Chainy, G., Manna, S., Chaturvedi, M. et al. Anethole blocks both early and late cellular responses transduced by tumor necrosis factor: effect on NF-κB, AP-1, JNK, MAPKK and apoptosis. Oncogene 19, 2943–2950 (2000). https://doi.org/10.1038/sj.onc.1203614

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Keywords

  • Anethole
  • TNF
  • NF-κB
  • AP-1
  • JNK
  • Apoptosis

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