European Journal of Pharmacology 746 (2015) 221–232

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Molecular and cellular pharmacology

AM404 inhibits NFAT and NF-κB signaling pathways and impairs migration and invasiveness of neuroblastoma cells Francisco J. Caballero a, Rafael Soler-Torronteras a, Maribel Lara-Chica a, Victor García a, Bernd L. Fiebich b, Eduardo Muñoz a,n, Marco A. Calzado a,n a b

Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC)/ Hospital Universitario Reina Sofía/ Universidad de Córdoba, Córdoba, Spain Department of Psychiatry, University of Freiburg Medical School, Freiburg, Germany

art ic l e i nf o

a b s t r a c t

Article history: Received 21 May 2014 Received in revised form 14 November 2014 Accepted 17 November 2014 Available online 25 November 2014

N-arachidonoylphenolamine (AM404), a paracetamol lipid metabolite, is a modulator of the endocannabinoid system endowed with pleiotropic activities. AM404 is a dual agonist of the Transient Receptor Potential Vanilloid type 1 (TRPV1) and the Cannabinoid Receptor type 1 (CB1) and inhibits anandamide (AEA) transport and degradation. In addition, it has been shown that AM404 also exerts biological activities through TRPV1- and CB1 -independent pathways. In the present study we have investigated the effect of AM404 in the NFAT and NF-κB signaling pathways in SK-N-SH neuroblastoma cells. AM404 inhibited NFAT transcriptional activity through a CB1- and TRPV1-independent mechanism. Moreover, AM404 inhibited both the expression of COX-2 at transcriptional and post-transcriptional levels and the synthesis of PGE2. AM404 also inhibited NF-κB activation induced by PMA/Ionomycin in SK-N-SH cells by targeting IKKβ phosphorylation and activation. We found that Cot/Tlp-2 induced NFAT and COX-2 transcriptional activities were inhibited by AM404. NFAT inhibition paralleled with the ability of AM404 to inhibit MMP-1, -3 and -7 expression, cell migration and invasion in a cell-type specific dependent manner. Taken together, these data reveal that paracetamol, the precursor of AM404, can be explored not only as an antipyretic and painkiller drug but also as a co-adjuvant therapy in inflammatory and cancer diseases. & 2014 Elsevier B.V. All rights reserved.

Keywords: AM404 NFAT COX-2 Tpl2/Cot kinase Neuroblastoma Acetaminophen

1. Introduction Paracetamol (acetaminophen) is a widely used over-thecounter pain reliever and antipyretic drug. The analgesic effect of paracetamol seems to be due to the activation of descending serotonergic pathways (Bonnefont et al., 2003a; Bonnefont et al., 2003b) but its action mechanism is not fully understood. The discovery that paracetamol is metabolized to N-arachidonoylphenolamine (AM404), a potent Transient Receptor Potential Vanilloid type 1 (TRPV1) activator, may shed light on the path to understand the anti-nociceptive effect of paracetamol (Hogestatt et al., 2005). More recently, it has been shown that 4-aminophenol, a de-acetylated metabolism of paracetamol, is also transformed into AM404 through a fatty acid amino hydrolase (FAAH)-dependent mechanism (Barriere et al., 2013). AM404 modulates the endocannabinoid system (ECs) by acting at

Abbreviations: AEA, N-arachidonoyl-ethanolamide; CB1, Cannabinoid receptor-1; NADA, N-arachidonoyl-dopamine; NF-κB, Nuclear Factor kappa B; TRPV1, Transient receptor potential vanilloid n Corresponding authors. Tel.: þ 34 957218267; fax: þ 34 957218266. E-mail addresses: [email protected] (E. Muñoz), [email protected] (M.A. Calzado). http://dx.doi.org/10.1016/j.ejphar.2014.11.023 0014-2999/& 2014 Elsevier B.V. All rights reserved.

different levels. First, AM404 is a dual agonist of TRPV1 and Cannabinoid Receptor type 1 (CB1) (Fegley et al., 2004), and the antinociceptive activity of paracetamol in vivo is blocked by either CB1 or TRPV1 antagonists (La Rana et al., 2006; Mallet et al., 2008; Ottani et al., 2006; Mallet et al., 2010). Second, AM404 inhibits the degradation of anandamide (AEA) by FAAH and its cellular uptake, thus increasing the endogenous levels of AEA (Giuffrida et al., 2001; Glaser et al., 2003). In addition, AM404 also exerted biological activities through CB1 and TRPV1-independent mechanisms (Caballero et al., 2007; Frischbutter et al., 2012; Hogestatt et al., 2005). The NFAT family of transcription factors comprises four classical members called NFAT1, NFAT2, NFAT3 and NFAT4 that are regulated by the calcium/calcineurin signaling pathway and by a calcium independent member referred to as NFAT5 (Hogan et al., 2003; Mancini and Toker, 2009). Classical NFAT are activated through dephosphorylation by calcineurin, a Ca2 þ þ /calmodulindependent phosphastase, and subsequent nuclear translocation (Hogan et al., 2003). In addition NFAT is also activated by calcineurin-independent mechanisms (Kuroda et al., 2008). For instance, it has been shown that Cot/Tpl-2 (cancer Osaka thyroid/ tumor progression locus 2), a MAP3K serine/threonine kinase,

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activates NFAT in the absence of Ca2 þ mobilization and calcineurin activity (Kuroda et al., 2012). Moreover, Cot/Tpl-2 is also involved in NF-κB activation and tumorigenesis (Lin et al., 1999; Miyoshi et al., 1991; Sourvinos et al., 1999). A link between NFAT activity and cancer has been demonstrated in previous studies. Thus, NFAT1 promotes migration and invasion of breast and colon carcinoma cells (Jauliac et al., 2002; Yiu and Toker, 2006) and an elevated expression of NFAT1 is observed in patients with invasive breast cancer (Mancini and Toker, 2009). NFAT interacts with other transcription factors such as AP-1 (Jain et al., 1993) to activate transcription. Adjacent NFAT and AP-1 binding sites are present in the promoter region of inducible genes including COX-2 (Iniguez et al., 2000), and it has been suggested that the mechanism by which NFAT functions as a pro-invasion transcription factor is partially dependent on COX-2 induction and PGE2 release (Yiu and Toker, 2006). Our study showed that AM0404 inhibits NFAT and NF-κB activation on neuroblastoma cells. Moreover, AM404 impaired COX-2 expression, PGE2 release, migration and invasion through Matrigel in a cell specific manner.

COX-2-Luc plasmid includes the COX-2 human promoter (  327 a þ104) fused to the luciferase gene (Duque et al., 2005). The HACot expression plasmid contains the Tpl2/Cot cDNA human and was a gift from Dr. Susana Alemany (IIB-CSIC, Madrid, Spain). SKN-SH and MDA-MB-231 cells were transiently transfected for 24 h with the indicated plasmids using Roti-Fect (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) according to the manufacturer's recommendations, and then stimulated as indicated during 6 h. For the luciferase assay the cells were washed twice in PBS and lysed in 25 mM Tris-phosphate pH 7.8, 8 mM MgCl2, 1 mM DTT, 1% Triton X-100 and 7% glycerol during 15 m at RT in a horizontal shaker. After centrifugation, the supernatant was used to measure luciferase activity using an Autolumat LB 9510 (EG&G Berthold, USA) following the instructions of the luciferase assay kit (Promega, Madison, WI, USA). The background obtained with the lysis buffer was subtracted in each experimental value and the specific transactivation expressed as a percentage of activation relative to control.

2. Material and methods

SK-N-SH cells were treated with the indicated doses of AM404 during 6 h, washed with PBS and incubated with 2.5 μM of calcein-AM (Invitrogen Ltd., San Diego, USA) for 25 min at 37 1C. Fluorescence was determined in a Genios-Pro (Tecan Trading AG, Switzerland) using an excitation/emission filter set of 485/538 nm. Data are expressed as the percentage of living cells (100% untreated cells).

2.1. Cell lines and reagents The human neuroblastoma SK-N-SH cell line, the immortalized mouse hippocampal HT22 cell line, the human oligodendroglial MO3.13 cell line and the breast cancer MDA-MB-231 cell line were maintained at 37 1C with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat inactivated FCS, 2 mM L-glutamine, and penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA). The mouse neuroblastoma N2a cell line was maintained at the same condition in a 1:1 mixture of DMEM and Ham's F-12 Nutrient Mixture. The antibodies anti-phospho-IκBα (5A5) and anti-phospho-IKKβ were obtained from Cell Signaling (Danvers, MA, USA), anti-HA were obtained from Roche (Applied Science, Mannheim, Germany), anti-tubulin from Sigma-Aldrich, anti-COX-2 (M19) and anti-Tpl2/Cot (M20) from Santa Cruz Biotechnology (San Diego, CA, USA). Acetaminophen, p-Aminophenol, NADA (N-arachidonoyl-dopamine), Mitomycin C, Ionomycin and PMA (phorbol 12-myristate 13-acetate) were obtained from Sigma-Aldrich. AM404, URB597 and Anandamide were obtained from Enzo Life Sciences (Farmingdale, NY, USA). Arvanil (Narachidonoylvanillamine) was obtained from Cayman-Chemical (Ann Arbor, MI, USA). The CB1 antagonist SR141716A was obtained from Tocris Biosciences (Bristol, UK). The TRPV1 receptor antagonist 50 -Iodoresiniferatoxin (50 IRTX) was a gift from Prof. Giovanni Appendino (Universitá degli Studi del Piemonte Orientale, Novara, Italy). The solvent vehicle used for the drugs was DMSO and the final concentration in the cell cultures was always lower than 0.1%. 2.2. Plasmids, transient transfections and luciferase assays The NFAT-Luc plasmid contains three copies of the NFAT binding site of the IL-2 promoter fused to the luciferase gene (Durand et al., 1988). The KBF-Luc plasmid contains three copies of the major histocompatibility complex enhancer kB site upstream of the conalbumin promoter followed by the luciferase gene (Yano et al., 1987). The Gal4-Luc reporter plasmid includes five Gal4 DNA binding sites fused to the luciferase gene (Schmitz et al., 1995). The Gal4-NFAT contains the first 1–415 aa of human NFAT1, fused to the DNA binding domain of yeast Gal4 transcription factor and was previously described (Luo et al., 1996). The DSCR1-Luc plasmid contains the internal promoter of human DSCR1 gene (  1664 a þ81) followed by the luciferase gene (Minami et al., 2004). The

2.3. Calcein-AM cell viability assay

2.4. Western blot SK-N-SH cells were stimulated as indicated, washed with PBS and total cell extracts extracted in 50 ml of lysis buffer (20 mM Hepes pH 8.0, 10 mM KCl, 0.15 mM EGTA, 0.15 mM EDTA, 0.5 mM Na3VO4, 5 mM NaF, 1 mM DTT, leupeptin 1 mg/ml, pepstatin 0.5 mg/ml, aprotinin 0.5 mg/ml, and 1 mM PMSF) containing 0.5% NP-40. Cells were incubated for 15 min in ice, and cellular proteins were obtained by centrifugation at 10,000g for 10 min. Protein concentrations were determined by the Bradford assay (Bio-Rad, Richmond, CA, USA), and 20 mg of proteins was boiled in Laemmli buffer and electrophoresed in 10% SDS polyacrylamide gels. Separated proteins were transferred to nitrocellulose membranes (0.5 A at 100 V; 4 1C) for 1 h. Blots were blocked in TBS solution containing 0.1% Tween 20 and 5% non-fat dry milk overnight at 4 1C, and immunodetection of specific proteins was carried out with primary antibodies using an ECL system (GE Healthcare, New Jersey, USA). 2.5. mRNA extraction and qPCR Total RNA was extracted using the kit RNAspin Min (GE Healthcare) according to the manufacturer's instructions. Reverse transcription was performed with the iScript cDNA Synthesis Kit (Bio-Rad, Madrid, Spain). Real-time PCR was employed with GoTaq qPCR Master Mix (Promega) in an iCYCLER detection system (BioRad). The primers used in this study are shown in Table 1. The amplification profile consisted of an initial denaturation for 5 min at 95 1C and then 40 cycles of 30 s at 95 1C, annealing for 30 s at 60 1C, and elongation for 30 s at 72 1C. A cycle of 10 s at 83 1C and a final extension for 1 min was carried out at 72 1C. Amplification efficiencies were validated and normalized against HPRT1 (hypoxanthine phosphoribosyltransferase 1). All experiments were performed in triplicate, and fold change in gene expression was calculated using the delta–delta CT method.

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Table 1 List of primer sequences used in RT-PCR. Genes

Forward

Reverse

MMP1 MMP2 MMP3 MMP7 COX-2 Cot/Tpl2 HPRT1

50 -AGCTAGCTCAGGATGACATTGATG-30 50 -CGCTCAGATCCGTGGTGA-30 50 -CCAGGTGTGGAGTTCCTGAT-30 50 -AAACATGTGGGGCAAAGAGA-30 50 -TCTGCAGAGTTGGAAGCACTCTA-30 50 -GTCTGGACTCTGCCCTCTTG-30 50 -ATGGGAGGCCATCACATTGT-30

50 -GCCGATGGGCTGGACAG-30 50 -CGCCAAATAAACCGGTCCTT-30 50 -AAGGTGGGTTTTCCTCCACT-30 50 -GCCAATCATGATGTCAGCAG-30 50 -GCCGAGGCTTTTCTACCAGA-30 50 -TCCTGTGCACGAAGAATCAG-30 50 -ATGTAATCCAGCAGGTCAGCAA-30

2.6. Wound migration assay

3. Results

The SK-N-SH and MDA-MB-231 cells were seeded into six-well plates and grown to 100% confluence. Confluent cells were wounded by scratching the surface with a sterile pipet tips and cellular debris removed by washing with PBS. The cells were then incubated in the presence of AM404 at the indicated concentrations for 24 h and treated with 2.5 μg/ml mitomycin C in order to remove the influence of cell proliferation. This initial wounding and the movement of the cells in the scratched area were photographically monitored using the Nikon Eclipse microscope for 24 h (Nikon, Tokyo, Japan). The wounding area was analyzed and quantified using ImageJ v1.45 software (http://rsbweb.nih. gov/ij/). The migration of cells towards the wounds was expressed as percentage of wound closure considering the area of wound measured immediately after scratching, and then measured 24 h after scratching. All experiments were performed in triplicate.

3.1. AM404 inhibits NFAT, COX-2 and DSCR1 transcriptional activity induced by PMA/Io in SK-N-SH neuroblastoma cells

2.7. Determination of PGE2 by enzyme immunoassay Supernatants were harvested, centrifuged at 10,000g for 10 min and levels of PGE2 in the media were measured by enzyme immunoassay (EIA) (Cayman-Chemical) according to the manufacturer's instructions. The standards used were in the interval of 78–1000 pg/ml (detection limit of 15 pg/ml).

2.8. Cell invasion assay Cell invasion was determined by a modified microchemotaxis assay (Jauliac et al., 2002). 2.5  105 cells in 250 ml of serum-free DMEM were seeded into the cell culture 24-well inserts (Corning Incorporated, Corning, NY, USA). Inserts of 8 mm pore polycarbonate-filter were coated with Matrigel (0.2 mg/ml) (BD Biosciences Bedford, MA, USA). The lower chambers were filled with DMEM supplemented with 10% FSC and incubated at 37 1C with 5% CO2 for 6 h. The migrated cells were washed twice times with PBS, stained with crystal violet and counted using a Nikon Eclipse microscope. Data are expressed as the percentage of invasion (100% untreated cells) and show the mean 7S.D. of three different experiments.

2.9. Statistical analysis At least three independent experiments were used for data analysis. Differences were analyzed by Student's t test. Po 0.05 was considered significant. Images were analyzed and quantified using ImageJ v1.45 software (http://rsbweb.nih.gov/ij/). Statistical analyses were performed using GraphPad Prism version 6.00 (GraphPad, San Diego, CA, USA). Data are expressed as mean 7 S.D.

Others and we have previously shown that AM404 inhibits NFAT activation in T cells (Caballero et al., 2007; Frischbutter et al., 2012). To study the possible role of AM404 on NFAT activity in neuroblastoma cells we used the SK-N-SH cell line that express CB1 and CB2 receptors (Ramer et al., 2003). The cells were transiently transfected with NFAT-Luc plasmid, and stimulated with PMA plus Ionomycin (PMA/Io) in the absence and presence of AM404 at the indicated concentrations. As shown in Fig. 1A, PMA/ Io-induced NFAT transcriptional activity was markedly inhibited by AM404 in a dose-dependent manner. The inhibitory activity of AM404 was independent of FAAH activity since pre-incubation of the cells with URB597 (1 μM) did not affect AM404-mediated NFAT inhibition. AM404 also inhibited PMA/Io-induced NFAT activation in other brain cell lines such as N2a, HT22 and MO3.13 although less effectively than in SK-N-SH cells (Fig. 1B). URB597 capacity to inhibit FAAH activity was checked in parallel (Supplementary Fig. 1). Next, we decided to investigate the effect of AM404 on NFAT-regulated genes such as COX-2 (Iniguez et al., 2000) or the Down syndrome critical region gene 1 (DSCR1) (Kyttala et al., 2009), which play a key role in tumorigenesis. SKN-SH cells were transiently transfected with the COX-2-Luc or DSCR1-Luc plasmid and stimulated with PMA/Io in the presence or absence of AM404 at different concentrations. As observed in Fig. 1C and D, AM404 could also inhibit the transcriptional activity of both genes in a dose-dependent manner. To rule out the possibility that the observed results were due to a toxic effect of AM404, we performed a cell viability assay. As observed in Fig. 1E, AM404 did not affect significantly the viability of SK-N-SH cells at the concentrations tested. 3.2. Arvanil and N-arachidonoyl-dopamine inhibit NFAT transcriptional activity It has been shown that both paracetamol and 4-aminophenol, a de-acetylated metabolism of paracetamol, undergo fatty acid conjugation in the brain leading to the formation of both AM404 and Arvanil (Barriere et al., 2013). Arvanil is an extremely powerful TRPV1 activator with potency in the subnanomolar range (Ross et al., 2001). Thus, we were interested in investigating if Arvanil was also able to inhibit NFAT activation in SK-N-SH cells. We found that Arvanil inhibited PMA/Io-induced NFAT transcriptional activation with a similar potency to that of AM404 (Fig. 2A). Moreover, N-arachidonoyl-dopamine (NADA), an endocannabinoid structurally related to Arvanil, and AM404 (Fig. 2A), which inhibits NF-κB and NFAT activation in T cells (Sancho et al., 2004), also inhibited NFAT activation in this cell line. In contrast, anandamide (AEA) showed a substantially lower inhibitory ability on the NFAT transcriptional activity. Cell viability assay was performed in parallel to rule out cytotoxic effects. Since arachidonic acid was

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NFAT-Luc AM404 AM404 + URB597

% Activation

100 80

NFAT-Luc

120

60 40

N2a HT22 MO3.13

100

% Activation

120

80 60 40

20

20

0

0

PMA/Io

-

+

+

+

+

PMA/Io

-

+

+

+

+

AM404 ( M)

-

-

5

15

25

AM404 ( M)

-

-

5

15

25

COX-2-Luc

120

100

% Activation

% Activation

100 80 60 40

PMA/Io AM404 ( M)

80 60 40 20

20 0

DSCR1-Luc

120

-

+

+

+

+

PMA/Io

-

-

5

15

25

AM404 ( M)

0

-

+

+

+

+

-

-

5

15

25

120

% Viability

100 80 60 40 20 0 AM404 ( M)

-

5

15

25

Fig. 1. Effects of AM404 on NFAT-dependent transcription. SK-N-SH (A) N2a, HT22 and MO3.13 (B) cells were transiently transfected with the reporter plasmid NFAT-Luc. After 24 h cells were incubated during 30 min with the indicated concentrations of AM404 and stimulated for 6 h with PMA/Io (25 ng/ml and 0.5 μM). Results are presented as the percentage of luciferase activity relative to control (100% for PMA/Io treatment). Data are mean 7 S.D. of n¼ 3 experiments. nnP o 0.001 and nnnPo 0.0001 for AM404 and AM404 þURB597 versus PMA/Io control. (C) SK-N-SH were transiently transfected with the reported plasmids COX-2-Luc or DSCR1-Luc (D), and after 24 h were incubated and stimulated as described above. Data are mean 7S.D. of n¼ 3 experiments. nPo 0.05 and nnnPo 0.0001 for AM404 versus PMA/Io control. (E) SK-N-SH cells were incubated with the indicated doses of AM404 during 6 h and cell viability analyzed by the Calcein-AM assay. Results are expressed as the % of viability (100% for treatment with vehicle). Data are mean7 S.D. of n¼ 3 experiments.

not able to inhibit NFAT (data not shown), our results indicate that the phenolic moiety in the structures analyzed is essential for the NFAT inhibitory activity. Next, we were interested in clarifying whether precursors of AM404 and Arvanil formation (Acetaminophen and p-Aminophenol) affected NFAT transcriptional activity mediated by PMA/Io, and if the effect of AM404 is independent of cannabinoid and vanilloid receptors. SK-N-SH cells were transiently transfected with the NFAT-Luc plasmid, treated with either SR141716A (CB1 receptor antagonist) or 50 -Iodoresiniferatoxin (50 I-RTX; TRPV1 antagonist) for 30 min before adding AM404, and then stimulated with PMA/Io. The specific activity of both compounds was verified in parallel (Supplementary Figs. 2 and 3). As shown in Fig. 2B, neither Acetaminophen nor p-Aminophenol affected NFAT transcriptional activity induced by PMA/Io. In addition, AM404 also inhibited NFAT transcriptional activity in the presence of SR141716A and 50 I-RTX, showing that AM404 inhibited NFAT through a CB1 and TRPV1-independent mechanism. 3.3. AM404 inhibits COX-2 expression and PGE2 release in SK-N-SH cells It has been shown that AM404 inhibits the synthesis of PGE2 in macrophages stimulated with LPS (Hogestatt et al., 2005). These data,

together with our previous observation that AM404 inhibits COX-2 transcriptional activity induced by PMA/Io in human neuroblastoma SK-N-SH cells (Fig. 1C), led us to study in detail the possible action mechanisms of this compound. First, we analyzed the effect of AM404 on COX-2 expression at the mRNA and protein levels. We show in Fig. 3A that AM404 clearly inhibited PMA/Io-induced COX-2 protein expression in a dose-dependent manner, obtaining a nearly complete inhibition at 15 μM. AM404 also showed a significant inhibition of COX-2 mRNA expression induced by PMA/Io. To determine whether COX-2 activity was also affected, we studied the effect of AM404 on PGE2 synthesis and liberation. SK-N-SH cells were incubated for 24 h with PMA/Io in the presence or absence of AM404, and then PGE2 levels were measured in the supernatant. We found that AM404 produced a clear inhibition of the liberation of PMA/Io-induced PGE2 in a dose-dependent manner (Fig. 3B). The concentrations of AM404 required to inhibit PGE2 release were significantly lower compared to those required to inhibit COX-2 expression, and therefore it is likely that AM404 also inhibited COX-2 enzymatic activity as previously described (Hogestatt et al., 2005). To further confirm that the inhibitory effect of AM404 on COX-2 protein expression is independent of cannabinoid and vanilloid receptors, SK-N-SH cells were pre-incubated with either SR141716A or 5´I-RTX. Then, cells were incubated with 15 μM AM404 and stimulated with PMA/Io to induce COX-2 expression.

F.J. Caballero et al. / European Journal of Pharmacology 746 (2015) 221–232

100

100

80

80

60

60

40

40

20

20

0

0

PMA/Io

-

+

+

+

+

Compounds ( M)

-

-

1

5

15

AM404

Arvanil

NADA

AEA

Acetaminophen p-aminophenol AM404 AM404 + SR141716A AM404 + 5' IRTX

NFAT-Luc

120

% Viability

NFAT-Luc

120

% Activation

NADA ARVANIL AEA 120

225

% Activation

100 80 60 40 20 0 PMA/Io

-

+

+

+

+

Compounds ( M)

-

-

1

5

15

Fig. 2. Effects of endocannabinoid/endovanilloid compounds on NFAT-dependent transcription. (A) SK-N-SH cells were transiently transfected with the reporter plasmid NFAT-Luc. After 24 h cells were incubated during 30 min with NADA, Arvanil and AEA at the indicated concentrations and then stimulated for 6 h with PMA/Io (25 ng/ml and 0.5 μM). Cells were harvested and one aliquot was analyzed for NFAT-dependent transcription while another aliquot was used to analyze cell viability. Luciferase activity is represented as % of activation, considering PMA/Io treatment as 100% of activation. Viability results are expressed as the % of viability (100% for treatment with vehicle). Data are mean 7 S.D. of n¼ 3 experiments. nPo 0.05, nnPo 0.001 and nnnPo 0.0001 for NADA, Arvanil and AEA versus PMA/Io control. (B) SK-N-SH cells were transiently transfected with the reporter plasmid NFAT-Luc, after 24 h cells incubated with acetaminophen, p-aminophenol and AM404 in the absence and the presence of SR141716A (1 μM) or 50 IRTX (0.1 μM) at the indicated concentrations, and then stimulated for 6 h with PMA/Io (25 ng/ml and 0.5 μM). Luciferase activity is represented as % of activation, considering PMA/Io treatment as 100% of activation. Results represent the mean7 S.D. of n¼ 3 experiments. nnPo 0.001 and nnnP o0.0001 for AM404 and AM404 þSR141716A versus PMA/Io control.

As shown in Fig. 3C, neither SR141716A nor 50 IRTX affected the inhibitory activity of AM404 on COX-2 expression. This result confirms that this activity of AM404 is also mediated by a mechanism that does not involve CB1 or TRPV1 receptors. 3.4. AM404 does not inhibit NFAT dephosphorylation Among the NFAT family of transcription factors, NFAT1 and NFAT2 are preferentially expressed in peripheral T cells (Lyakh et al., 1997) and NFAT3 and NFAT4 are the most commonly studied isoforms in neuronal cells (Nguyen and Di Giovanni, 2008; Ulrich et al., 2012; Vashishta et al., 2009). In order to study the mechanism of action of AM404 on NFAT signaling pathway we were interested in examining the phosphorylation status of NFAT. We found that SK-N-SH cells did not express NFAT1 and we were not able to detect the endogenous dephosphorylated NFAT4 in these cells with commercially available antibodies (data not shown). Therefore, we overexpressed HA-NFAT4 and the cells were stimulated with PMA/Io in the presence or absence of AM404. As shown in Fig. 4A, cyclosporine A but not AM404 prevented PMA/Io– induced NFAT4 dephosphorylation. Next, to explore the inhibitory mechanisms of AM404 on NFAT activation, a Gal4-derived reporter

system was employed. SK-N-SH cells were co-transfected with the chimeric vector pGal4-NFAT1 (1–415), encoding the Gal4 DBD fused to amino acids 1 through 415 of human NFAT1 along with the reporter plasmid Gal4-Luc. The fusion protein pGal4-NFAT1 (1–415) contains both the calcineurin-binding regulatory and transactivation domains. In Fig. 4B we show that AM404 prevented the transactivation function of NFAT1 induced by PMA/Io in a concentration-dependent manner. It has been previously described that Tpl2/Cot can activate NFAT transcriptional activity through a calcineurin-independent mechanism (de Gregorio et al., 2001; Gomez-Casero et al., 2007). Moreover, Tpl2/Cot increases NFAT1 protein stability through phosphorylation of residues distinct from those required for cytoplasmic–nuclear shuttling, thereby enhancing NFAT1 activation in a calcineurin-independent manner (Kuroda et al., 2012). Thus, we studied the effects of AM404 on Tpl2/Cot-mediated NFAT and COX-2 transactivation in SK-N-SH cells. The cells were transiently co-transfected with the expression plasmid HA-Cot together with the reporter plasmids NFAT-Luc or COX-2-Luc, treated with AM404 for 6 h and luciferase activity measured in the cell lysates. We found that AM404 inhibited Tpl2/Cot-induced NFAT (Fig. 4C) and COX-2 (Fig. 4D) transcriptional activities.

F.J. Caballero et al. / European Journal of Pharmacology 746 (2015) 221–232

PMA/Io

-

+

+

+

+

AM404 (µM)

-

-

5

15

25

120

COX-2

COX-2

Tubulin

Tubulin

QRT-PCR (Relative fold)

1.0

1.0

0.2

0.2

2

0

% PGE Release

226

25

100 80 60 40 20

20 15

PMA/Io

10

AM404 ( M)

0

-

+

+

+

+

+

-

-

1

5

15

25

5 0

PMA/Io µM) SR141716A (1 µM) AM404 (15 µM)

-

+ -

+ +

+ + +

+ + +

+ + -

+ + -

COX-2

COX-2

Tubulin

Tubulin

0

1.0 0.2 0.2 0.2 0.9 0.9

Fig. 3. AM404 inhibits COX-2 expression and PGE2 release induced by PMA/Io in SK-N-SH cells. (A) SK-N-SH cells were treated for 30 min with AM404 at the indicated concentrations and stimulated with PMA/Io for 6 h. Cells were lysed and one aliquot was analyzed for the levels of endogenous protein expression by immunoblot (upper panel) while another aliquot was analyzed by qPCR to measure COX-2 mRNA levels (lower panel). Data are mean 7 S.D. of n ¼3 experiments. nP o 0.05 and nnnP o 0.0001. The values below the gels indicate COX-2 protein signal intensities (quantified using ImageJ) after normalization to tubulin signal intensities. (B) SK-N-SH cells were treated as described before and released PGE2 levels determined in the supernatant. Inhibition was statistically significant. Data are mean 7 S.D. of n ¼ 3 experiments. n P o 0.05 and nnnP o 0.0001 for AM404 versus PMA/Io control. (C) SK-N-SH cells were treated for 30 min in the presence or absence of SR141716A (1 μM) or 50 IRTX (0.1 μM) and then incubated with AM404 (15 μM) for 30 min and stimulated with PMA/Io for 6 h. COX-2 protein expression was determined by Western Blot. All the results are representative of three different experiments. The values below the gels indicate COX-2 protein signal intensities (quantified using ImageJ) after normalization to tubulin signal intensities.

3.5. Effect of AM404 on NF-κB signaling pathways The control of COX-2 expression at the transcriptional level is mediated by the coordinated action of different inducible transcription factors including NFAT, AP-1 and NF-κB. The well-known contribution of NF-κB to inflammatory and tumorigenic processes led us to investigate the effects of AM404 on NF-κB activation in SK-N-SH cells. Cells were transiently transfected with the plasmid KBF-Luc and, after 24 h, stimulated with either PMA/Io or TNFα in the presence or absence of AM404. As observed in Fig. 5A, AM404 inhibited NF-κB transcription activity induced by PMA/Io whereas it showed no effect on TNFα-mediated stimulation. Accordingly, AM404 inhibited PMA/Io-induced but not TNFα-induced IκBα phosphorylation (Fig. 5B). Previous studies have shown the role of Tpl2/Cot kinase in the NF-κB signaling pathway (Lin et al., 1999), and therefore were studied the effect of AM404 on Tpl2/Cot-induced IκBα phosphorylation and its upstream kinase IKKβ. SK-N-SH cells were transiently transfected with the expression plasmid HA-Cot, and 24 h later treated with increasing concentrations of AM404 for 6 h. In Fig. 5C it is shown that AM404 inhibited both IκBα and IKKβ phosphorylation induced by Tpl2/Cot kinase. Next, to determine the mechanism of action of AM404 on Tpl2/Cot kinase, we investigated the impact of AM404 on the endogenous Tpl2/Cot levels. As shown in Fig. 5D, AM404 treatment resulted in a dose-dependent decrease in Tpl2/Cot protein levels without affecting the gene expression. Taken together, these results suggest that AM404 targets Tpl2/Cot at the protein level to inhibit NFAT and NF-κB signaling pathways.

3.6. AM404 inhibits migration and invasiveness in human neuroblastoma cell line SK-N-SH NFAT proteins have been found to be involved in cell cycle regulation, cell differentiation, cell survival, angiogenesis, and tumor cell invasion and metastasis (Mancini and Toker, 2009). In addition, it has been widely described that an increase in PGE2 promotes tumor proliferation and increases the expression of MMPs (Itatsu et al., 2009; Kurihara et al., 2009). Therefore, we decided to study the effect of AM404 on SK-N-SH cell migration, invasiveness and on the expression of MMPs. SK-N-SH is a neuroblastoma cell line established from a bone marrow metastasis that displays epithelial morphology and metastatic capacities. The cells were treated with increasing concentrations of AM404, stimulated with PMA/Io, and the expression of the MMPs analyzed by quantitative real-time PCR. As can be seen in Fig. 6A, PMA/Io treatment increased the mRNA expression of MMP-3, -1 and -7, which were inhibited differently by AM404, while the expression of MMP-2 was not affected. To study the effect of AM404 on cell migration, wound healing assays were performed. AM404 treatment showed a clear inhibitory effect on SK-N-SH cell migration (Fig. 6B) and similar results were obtained in the presence of PMA/Io (data not shown). Next, we investigated the effects of AM404 on the invasive activity using a Matrigel invasion assay. The cell invasion was significantly inhibited (p o0.05) by AM404 even at the lower concentrations tested (Fig. 6C). The inhibition Matrigel invasion suggested that the motility of cells was suppressed by the AM404 treatment.

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Fig. 4. AM404 regulates NFAT transcriptional activity and COX-2 expression mediated by Tpl2/Cot kinase. (A) SK-N-SH cells were transfected with the HA-NFAT plasmid and 24 h later the cells were incubated with either AM404 or cyclosporine A (CsA, 100 ng/ml) 30 min prior stimulation with PMA/Io. The phosphorylation status of NFAT was analyzed by immunoblotting using an anti-HA mAb. The values below the gels indicate NFAT4 protein signal intensities (quantified using ImageJ) after normalization to tubulin signal intensities. (B) SK-N-SH cells were co-transfected with Gal4-Luc reporter plasmid together with the Gal4-NFAT1 (1–415) expression vector. After 24 h, cells were pre-treated or not with the indicated concentrations of AM404 and then stimulated for 6 h with PMA/Io. SK-N-SH cells were co-transfected with HA-Cot expression vector and NFAT-Luc (C) or COX-2-Luc (D) reporter plasmids, and 24 h later incubated for 6 h with AM404 increasing concentrations. Results represent the mean 7 S.D. of n¼ 3 experiments. nnP o 0.001 and nnnPo 0.0001 for AM404 from Gal4-NFAT 1–415 versus PMA/Io control and nPo 0.05, nnPo 0.001 and nnnPo 0.0001 for AM404 versus HACot control.

3.7. Cell type specificity of AM404 activities To determine whether the effects shown by AM404 are cell type specific, we performed experiments in the breast cancer MDA-MB-231 cell line that express TPRV1, CB1 and CB2 receptors (Ligresti et al., 2006). It has been shown that NFAT upregulates COX-2 and drives invasiveness in this cell line (Qamri et al., 2009; Yiu and Toker, 2006). First, we studied the effect of AM404 on NFAT transcriptional activity and on the regulation of COX-2 promoter expression in this cell line. MDA-MB-231 cells were transiently transfected with the plasmids NFAT-Luc or COX-2-Luc, and 24 h later incubated with AM404 and stimulated with PMA/Io. As shown in Fig. 7A, AM404 had no effect, at the concentrations tested, on the induction of NFAT and COX-2 transcriptional activities mediated by PMA/Io. Next, the cells were treated with increasing concentrations of AM404 and stimulated with PMA/Io for 6 h and the mRNA expression of COX-2, MMP-3, MMP-2, MMP1 and MMP-7 determined by qPCR. In Fig. 7B it is shown that the constitutive expression of MMP-2 and MMP-7 was not affected by the stimulation with PMA/Io in the absence or in the presence of AM404. The expression of COX-2, MMP-1 and MMP-3 was upregulated by PMA/Io and this induction was not affected by AM404. Moreover, AM404 did not interfere with migration (Fig. 7C) or invasiveness (7D) of MDA-MB-231 cells. These data clearly show that the effect of AM404 on the control of NFAT activity and cellular migration and invasiveness is cell type specific.

4. Discussion Neuroblastoma is an extracranial solid tumor that remains an important pediatric problem because it accounts for approximately

15% of cancer deaths in children. Current treatment for aggressive forms of neuroblastoma includes chemotherapy, surgery, and radiation, but even with this treatment there is a recurrence of 50%, and therefore there is an urgent need for more effective therapies (Maris et al., 2007). Here we show for the first time that AM404, a paracetamol metabolite, specifically inhibited Cot/Tpl2-induced NFAT transcriptional activity and COX-2 expression in neuroblastoma cells but not in the breast cancer MDA-MB-231 cell line. Moreover, AM404 inhibited migration and Matrigel invasion, the expression of MMP-1 and -7 and the synthesis of PGE2. These data show the possible relevance of paracetamol and 4-aminophenol, through the metabolites AM404 and Arvanil, as a co-adjuvants in the therapy against some type of cancers such as neuroblastoma. Although evidence suggests that paracetamol may mediate some effects through metabolites such as AM404, the concentrations of this metabolite in different tissues are not well known. It has been shown that picomolar concentrations of AM404 are detected in murine brain after a single injection of 4-aminophenol (30 mg/kg) (Barriere et al., 2013), and these concentrations are about a thousand times smaller than those described for activating TRPV1 (Zygmunt et al., 2000). However, paracetamol-mediated antinociceptive effect is not reproduced in trpv1  /  mice (Mallet et al., 2010), and therefore it is possible that AM404 concentrations are higher in some tissue compartments. In addition, the levels of AM404 in the CNS and in the periphery after continued ingestion of paracetamol have not been studied. Although the rate of AM404 generation starting from acetaminophen is not known, it has been shown in humans that acetaminophen concentrations in the cerebrospinal fluid after intravenous administration are around 40 μM, and even higher concentrations were found in plasma (Kumpulainen et al., 2007; Moreau et al., 1993). Thus, it is possible that higher concentrations of AM404 are found in humans after

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Fig. 5. AM404 inhibits NF-κB transcriptional activity mediated by PMA/Io and not by TNFα. (A) SK-N-SH cells were transiently transfected with KBF-Luc plasmid, incubated for 30 min with the indicated concentrations of AM404 and then stimulated for 6 h with PMA/Io or TNFα. Luciferase activity is represented as % activation (100% activation mediated by PMA/Io or TNFα). The graph represents the mean 7 S.D. of three independent experiments. nPo 0.05 and nnP o 0.001 for AM404 versus PMA/Io control. (B) SKN-SH cells were treated for 6 h with different doses of AM404 and then stimulated with PMA/Io or TNFα for 15 or 5 min respectively. The steady state levels of phospho-IκBα and tubulin were measured by Western Blot. The values below the gels indicate phospho-IκBα protein signal intensities (quantified using ImageJ) after normalization to tubulin signal intensities. (C) SK-N-SH cells were transfected with the HA-Cot plasmid and then treated for 6 h with the indicated doses of AM404. The phosphorylation status of IκBα and IKKβ was analyzed by Western Blot analysis. The results are representative of three different experiments. The values below the gels indicate phosphoIκBα and phospho-IKKβ protein signal intensities (quantified using ImageJ) after normalization to tubulin signal intensities. (D) SK-N-SH cells were stimulated for 6 h with the indicated doses of AM404, harvested and one aliquot was analyzed for the levels of the Tpl2/Cot protein by immunoblot (upper panel) while another aliquot was analyzed by qPCR to measure Tpl2/Cot mRNA levels (lower panel). Data are mean7 S.D. of n¼ 3 experiments. The values below the gels indicate Tpl2/Cot protein signal intensities (quantified using ImageJ) after normalization to tubulin signal intensities.

paracetamol intake. In addition, we have found that the anti-NFAT and anti-NF-κB activities of AM404 are enhanced under serum-free culture conditions (data not shown). Nevertheless, the physiological significance of the results presented herein needs further in vivo studies. NFAT proteins reside in the cytoplasm in resting cells in a phosphorylated form. Upon stimulation, NFAT proteins are dephosphorylated through the calcium/calcineurin signaling pathway and translocated into the nucleus where it undergoes post-translational modifications such as phosphorylation of the transactivation domain (TAD) (Macian, 2005). Recent findings have shown that some serine/ threonine kinases such as Cot/Tpl2, PKCζ, Pim-1 and c-Jun kinase are able to modulate NFAT-mediated transcriptional activation through the potentiation of the transactivating function of the N-terminal TAD of NFAT proteins in T cells (de Gregorio et al., 2001; San-Antonio et al., 2002; Ortega-Perez et al., 2005; Rainio et al., 2002). Moreover, it has been shown that Cot/Tpl2, PKCζ and NFAT interact with each other resulting in PKCζ-mediated phosphorylation of Ser53 and Ser56 in the TAD of NFATc2 (Gomez-Casero et al., 2007). In our assays we have observed that AM404 does not alter the cytoplasmic phosphorylation state of NFAT but, on the contrary, it affects the transcriptional activity induced by the fusion protein Gal4-NFAT (1–415). These data suggest

that the inhibitory effect of AM404 on NFAT in SK-N-SH cells is produced by inhibition of a pathway that targets the transactivation domain of this transcription factor. The importance of Tpl2/Cot on the regulation of NFAT activity and on the expression of several proinflammatory mediators such as TNFα (Rousseau et al., 2008; Wu et al., 2009), and the fact that PMA/Io can induce its expression (Sanchez-Gongora et al., 2000), points out that Tpl2/Cot is a likely target of the action of AM404. In this sense, our results demonstrate that AMA404 inhibits the expression of TPL2/Cot at the protein level without affecting the transcription, suggesting a possible alteration of a post-transcriptional regulatory mechanism of this kinase. As a consequence, AM404 inhibits NFAT and COX-2 transcriptional activities induced by the over-expression of Tpl2/Cot. Nonetheless, we cannot exclude the existence of other regulatory mechanisms involved in the effect of AM404 on Tpl2/cot, because although this kinase can activate other transcription factors, such as AP-1 or NF-κB (Das et al., 2005; Lin et al., 1999; Wittwer and Schmitz, 2008), we have not observed any effect of AM404 on AP-1 activation mediated by PMA/Io in SK-N-SH cells (data not shown). These results suggest that Tpl2/Cot may be interacting with other regulatory proteins to activate NFAT as it has been demonstrated for PKCζ (Gomez-Casero et al., 2007). The differential expression of Tpl2/Cot cofactors in different cells could explain

QRT-PCR (Relative fold)

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Fig. 6. AM404 inhibits the expression of some MMPs, cell migration and invasiveness in SK-N-SH cells. (A) SK-N-SH cells were treated for 30 min with the indicated doses of AM404 and then stimulated with PMA/Io for 6 h. MMPs expression was determined by qPCR. The results are representative of three different experiments. ***P o 0.0001 for AM404 versus PMA/Io control. (B) Representative images of AM404-treated and untreated (control) SK-N-SH cells in which cell motility has been analyzed after 24 h of healing (scale bar¼ 1000 μm). The values in the pictures indicate percentage of wound closure (quantified using ImageJ). (C) SK-N-SH cell invasion was determined by Matrigel assay (see Material and Methods). Graph shows the quantification of AM404 effect on SK-N-SH cell invasiveness expressed as the percentage of invasion (100% untreated cells). Data are the mean 7S.D. of three different experiments. ***Po 0.0001 for AM404 versus PMA/Io control.

the lack of effects of AM404 in MDA-MB-231 cells compared to SK-NSH cells. A more thorough study of the differences of both expression and activity of Tpl2/Cot cofactors in different cell lines and tumors would be very interesting in order to support the potential therapeutic application of AM404 and other paracetamol metabolites in cancer. As for the possible role exerted on the NF-κB pathway, we show that AM404 can inhibit IκBα phosphorylation and NF-κB activation in PMA/Io-stimulated cells but not TNFα-stimulated cells. Moreover, AM404 also inhibits IKKβ and IκBα phosphorylation induced by over expression of Tpl2/Cot. Previous reports have

shown that Tpl2/Cot regulates NF-κB pathway at two different levels. First, IKKβ as well as its regulatory subunit IKKγ are required for Tpl2/Cot activation (Waterfield et al., 2004), and second, this kinase directly phosphorylates p65 at ser536 and ser468 (Wittwer and Schmitz, 2008). However, Tpl2/Cot is dispensable for TNFα-triggered IKK activation (Das et al., 2005). Thus, it is likely that AM404 inhibits the activity of Tpl2/Cot induced by PMA/Io preventing NF-κB activation. We also found that AM404 was able to inhibit PMA/Io-induced NF-κB activation in MDA-MB231 cells (data not shown) indicating that NFAT, but not NF-κB,

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Fig. 7. Differential effect of AM404 depending on cell type. AM404 has no effect on the NFAT transcriptional activity, cell migration and invasiveness in MDA-MB-231 cells. (A) MDA-MB-231 cells were transiently transfected with NFAT-Luc or COX-2-Luc plasmids. After 24 h the cells were treated with the indicated doses of AM404 for 30 min and stimulated for 6 h with PMA/Io. Luciferase activity is represented as % of activation, considering PMA/Io treatment as 100% of activation. The results are represented as mean 7S.D. of three different experiments. (B) MDA-MB-231 cells were incubated for 30 min with increasing concentrations of AM404 and then stimulated with PMA/Io for 6 h. The expression of MMPs and COX-2 was determined by qPCR. The results are representative of three different experiments. (C) Representative images of AM404-treated and untreated (control) MDA-MB-231 cells in which cell motility has been analyzed after 24 h of healing (scale bar¼ 1000 μm). (D) MDA-MB-231 cells invasiveness was determined by Matrigel assay and expressed as the percentage of invasion (100% untreated cells). Data are the mean 7S.D. of three different experiments.

may represent the major driving force in the expression of MMP-1, -3 and -7 and in the migration and invasiveness of SK-N-SH cells. Accordingly, the NFAT-COX-2-prostaglandins axis has been shown to be upregulated in a wide variety of tumors (Bernard et al., 2008; Subbaramaiah and Dannenberg, 2003). Interestingly, other Tpl2/ Cot inhibitors have been shown to inhibit COX-2 expression and the release of PGE2, MMP-1 and MMP-3 in human synoviocytes (Hall et al., 2007). Overall AM404, as well as other structurally related compounds such as endocannabinoids and Arvanil, have pleiotropic activities that account for multiple and potential medical applications. The results presented herein show for the first time that Tpl2/Cot is a potential target for AM404, thus indicating that paracetamol, the precursor of AM404, can be explored not only as an antipyretic and painkiller drug but also as co-adjuvant therapy in inflammatory and cancer diseases.

Conflict of interests The authors declare that they have no conflict of interest.

Acknowledgments This work was supported by MICINN (SAF2010-17122) and Consejería de Salud (Junta de Andalucía) (PI-0650-2010 and PI0246-2013) grants to M.A.C., by MICINN (SAF2010-19292) and Junta de Andalucía (P09-CTS-4973) grants to E.M. Finally, we acknowledge Carmen Cabrero-Doncel for her assistance with the article.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ejphar.2014.11.023. References Barriere, D.A., Mallet, C., Blomgren, A., Simonsen, C., Daulhac, L., Libert, F., Chapuy, E., Etienne, M., Hogestatt, E.D., Zygmunt, P.M., Eschalier, A., 2013. Fatty acid amide hydrolase-dependent generation of antinociceptive drug metabolites acting on TRPV1 in the brain. PLoS One 8, e70690.

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Bernard, M.P., Bancos, S., Sime, P.J., Phipps, R.P., 2008. Targeting cyclooxygenase-2 in hematological malignancies: rationale and promise. Curr. Pharm. Des. 14, 2051–2060. Bonnefont, J., Alloui, A., Chapuy, E., Clottes, E., Eschalier, A., 2003a. Orally administered paracetamol does not act locally in the rat formalin test: evidence for a supraspinal, serotonin-dependent antinociceptive mechanism. Anesthesiology 99, 976–981. Bonnefont, J., Courade, J.P., Alloui, A., Eschalier, A., 2003b. Antinociceptive mechanism of action of paracetamol. Drugs 63 (2), 1–4. Caballero, F.J., Navarrete, C.M., Hess, S., Fiebich, B.L., Appendino, G., Macho, A., Munoz, E., Sancho, R., 2007. The acetaminophen-derived bioactive Nacylphenolamine AM404 inhibits NFAT by targeting nuclear regulatory events. Biochem. Pharmacol. 73, 1013–1023. Das, S., Cho, J., Lambertz, I., Kelliher, M.A., Eliopoulos, A.G., Du, K., Tsichlis, P.N., 2005. Tpl2/cot signals activate ERK, JNK, and NF-kappaB in a cell-type and stimulus-specific manner. J. Biol. Chem. 280, 23748–23757. de Gregorio, R., Iniguez, M.A., Fresno, M., Alemany, S., 2001. Cot kinase induces cyclooxygenase-2 expression in T cells through activation of the nuclear factor of activated T cells. J. Biol. Chem. 276, 27003–27009. Duque, J., Fresno, M., Iniguez, M.A., 2005. Expression and function of the nuclear factor of activated T cells in colon carcinoma cells: involvement in the regulation of cyclooxygenase-2. J. Biol. Chem. 280, 8686–8693. Durand, D.B., Shaw, J.P., Bush, M.R., Replogle, R.E., Belagaje, R., Crabtree, G.R., 1988. Characterization of antigen receptor response elements within the interleukin2 enhancer. Mol. Cell. Biol. 8, 1715–1724. Fegley, D., Kathuria, S., Mercier, R., Li, C., Goutopoulos, A., Makriyannis, A., Piomelli, D., 2004. 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