Benzyl isothiocyanate but not benzyl nitrile from Brassicales plants dually blocks the COX and LOX pathway in primary human immune cells

Journal of Functional Foods 23 (2016) 135–143

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Benzyl isothiocyanate but not benzyl nitrile from Brassicales plants dually blocks the COX and LOX pathway in primary human immune cells Corinna Herz a,1, Melinda-Rita Márton a, Hoai Thi Thu Tran a,1, Carsten Gründemann b, Jana Schell a, Evelyn Lamy a,1,* a

Molecular Preventive Medicine, Institute of Environmental Health Sciences, University of Freiburg – Medical Center, Breisacher Strasse 115b, 79106 Freiburg, Germany b Center for Complementary Medicine, Institute of Environmental Health Sciences, University of Freiburg – Medical Center, Breisacher Strasse 115b, 79106 Freiburg, Germany

A R T I C L E

I N F O

A B S T R A C T

Article history:

The main products formed from glucosinolates from Brassicales plants are isothiocyanates

Received 5 August 2015

(ITCs) and nitriles. ITCs are known for their diverse bioactive potential, knowledge about

Received in revised form 16

the bioactivity of food-borne nitriles is poor, though. Anti-inflammation by benzyl ITC (BITC)

February 2016

and its nitrile has not so far been studied in human immune cells. Here, the potential of

Accepted 17 February 2016

BITC to inhibit the lipopolysaccharide (LPS) induced inflammatory response in human pe-

Available online

ripheral mononuclear blood cells (PBMC) was analysed in comparison to benzyl nitrile (BCN).

Keywords:

sponse in terms of dual COX and LOX pathway inhibition and reduced the activation and

Benzyl isothiocyanate

function of activated T lymphocytes. In contrast, BCN did not influence prostaglandin PGE2

BITC

release from PBMC. The inactivity of the glucosinolate product BCN must be considered when

Brassicales

Brassicales plants are intended for use against inflammatory diseases.

Our data show that BITC strongly interferes with the cellular pro-inflammatory re-

Anti-inflammatory effect in primary

© 2016 Elsevier Ltd. All rights reserved.

human PBMC Benzyl nitrile Dual LOX and COX pathway inhibitor

Authorship contribution statement HTTT, MRM and JS performed the research. CG and EL designed the research study. CH, MRM, HTTT, CG, JS, and EL analysed the data. CH, CG and EL wrote the paper. Authorship contribution is given without weighting. * Corresponding author. Molecular Preventive Medicine, Institute for Prevention and Cancer Epidemiology, University of Freiburg – Medical Center, Elsässerstr. 2, 79110 Freiburg, Germany. Tel.: +49(0)761 270 82150; fax: +49(0)761 270 82130. E-mail address: [email protected] (E. Lamy). 1 Current address: University of Freiburg – Medical Center, Institute for Prevention and Cancer Epidemiology, Molecular Preventive Medicine, Elsässerstr. 2, 79110 Freiburg, Germany. Abbreviations: AA, arachidonic acid; BCN, benzyl nitrile; BITC, benzyl isothiocyanates; CFSE, carboxyfluorescein disacetate succinimidyl ester; COX, cyclooxygenase; DMSO, dimethylsulfoxid; ELISA, enzyme linked immunosorbent assay; fMLP, N-formyl-methionyl-leucylphenylalanine; GLS, glucosinolates; ITC, isothiocyanates; LOX, lipoxygenase; LPS, lipopolysaccharide; LTB4, leukotriene B4; mAB, monoclonal antibody; MAPK, mitogen-activated protein kinase; PBMC, primary human peripheral mononuclear cells; PBS, phosphate buffered saline; PE, phycoerythrin; PeCN, 3-phenylpropanenitrile; PGE, prostaglandins; PMA, phorbol 12-myristate 13-acetate), SFN (sulforaphane), Temed (N,N,N’,N’-tetramethyl-1-,2-diaminomethane) http://dx.doi.org/10.1016/j.jff.2016.02.034 1756-4646/© 2016 Elsevier Ltd. All rights reserved.

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1.

Journal of Functional Foods 23 (2016) 135–143

Introduction

Glucosinolates (GLS) are secondary metabolites present in vegetables of the order Brassicales such as broccoli sprouts or broccoli which are available on the international market as dietary supplements. There is a strong interest in consumers and patients in the use of these products due to their potential health value. It is widely accepted though that GLSs are biologically inactive precursors and that their degradation products are responsible for their positive bioactive effects (Gerhäuser, 2013). Following tissue disruption, these β-dthioglucoside-N-hydroxysulfates encounter myrosinase, a β-dthioglucosidase that cleaves β-d-glucose. The resulting aglucon spontaneously decomposes to form an isothiocyanate (ITC) or nitrile. At typical plant pH values (pH 5-6), ITCs would be the main degradation products (Gil & MacLeod, 1980). However, enzymatic degradation might preferentially lead to the formation of nitriles if plants contain additional proteins such as the epithiospecifier or nitrile specifier proteins (Matusheski, Juvik, & Jeffery, 2004; Williams, Critchley, Pun, Chaliha, & O’Hare, 2009; Wittstock & Burow, 2010). Nitriles are also the main thermally induced degradation products of GLS in Brassica vegetables (Choi et al., 2009; Williams et al., 2009). So far ITCs are the most intensively studied GLS degradation products and are well recognized for their pleiotropic pharmacological activity (Lamy et al., 2013; Lamy & Mersch-Sundermann, 2009; Rose, Won, Ong, & Whiteman, 2005; Tang & Zhang, 2004; Zhang, 2012). ITCs are also well known for their anti-microbial potential (Conrad, Kolberg, Engels, & Frank, 2006; Halbeisen, 1954). Besides the direct bacteriostatic or bactericidal effect, ITC were found to interact with pro-inflammatory pathways in cells of the immune system, which are triggered by bacteria upon release of lipopolysaccharide (LPS) from their membrane. But these properties of ITCs were studied so far mostly in animal cell cultures or permanent cancer cell lines (Keum, Jeong, & Kong, 2004; Nakamura, 2009). In contrast to ITCs, knowledge about the bioactivity of food-borne nitriles is poor and inconsistent (Keck, Staack, & Jeffery, 2002; Matusheski & Jeffery, 2001). It was shown that the potential of sulforaphane nitrile to induce activity of quinone reductase was about 1000-fold less than sulforaphane (Matusheski & Jeffery, 2001). 3-Hydroxy4-pentenenitrile in contrast yielded an induction of hepatic quinone reductase similar to sulforaphane in Fischer 344 rats (Keck et al., 2002). No data have so far been published so far on the interaction of Brassica plant derived nitriles with pro-inflammatory pathways. Arachidonic acid (AA) is the main precursor of the inflammatory response triggered by bacteria upon release of LPS from their membrane which can be further metabolized by two major pathways: the cyclooxygenase (COX) and lipoxygenase (LOX) pathway. Three isoforms of COX have been identified so far. COX-2 is one of the important enzymes in the inflammatory cell response as it is the crucial step for the formation of prostaglandins (PGE), whereas PGE 2 presents one important determinant for symptoms like fever and pain. In recent years, dual inhibition of both, the COX and LOX pathway have been considered as a promising new approach for inflammation inhibition with no or low toxicity (Burnette, 1981; Laemmli, 1970).

COX pathway inhibition was found by ITCs in LPS stimulated mouse macrophage Raw 264.7 cells, human colon or bladder cancer cells. ITCs then blocked COX-2, PGE2, iNOS, IL1β, IL-6 and TNF-alpha (Brown et al., 2009; Heiss, Herhaus, Klimo, Bartsch, & Gerhauser, 2001; Reddy et al., 2015). Only very recently it was shown that sulforaphane (SFN) from broccoli inhibits the LPS-stimulated inflammatory response in human monocytes through the COX pathway (Reddy et al., 2015). In silico analysis also indicated that ITCs could possibly bind to both, COX-2 and 5-LOX enzyme (Misra et al., 2014). Additionally, ITC were found to inhibit mitogen-activated protein kinase (MAPK) cascades which lead to downstream activation of proinflammatory pathways (Kaminska, 2005; Reddy et al., 2015). So far no information is available on the dual COX/LOX pathway inhibitory potential of benzyl ITC (BITC) or its nitrile counterpart on primary cells of the human immune system. This was addressed in the present study on activated primary human peripheral blood mononuclear cells (PBMC). Our data show that BITC but not benzyl nitrile or phenylethyl nitrile strongly interferes with the cellular pro-inflammatory response in terms of dual COX and LOX pathway inhibition.

2.

Methods

2.1.

Materials

LymphoPrepTM gradient was purchased from Progen (Heidelberg, Germany). RPMI 1640 medium, foetal calf serum, trypsin 10x (25 mg/ml), trypsin-EDTA 10x (5 mg/ml, respectively 2.2 mg/ ml) and phosphate buffered saline (PBS, without Ca and Mg) were from PAA Laboratories GmBH (Coelbe, Germany). L-glutamine, penicillin and streptomycin were from Invitrogen (Karlsruhe, Germany). Camptothecin was from Tocris (Eching, Germany), triton-X 100, milk powder and N,N,N’,N’-tetramethyl1-,2-diaminomethane (Temed) from Carl Roth (Karlsruhe, Germany), dimethyl sulfoxide (DMSO), lipopolysaccharide (LPS), phorbol, 12-myristate 13-acetate, 5(6) carboxyfluorescein disacetate succinimidyl ester (CFSE), ammoniumpersulfate, phorbol 12-myristate 13-acetate (PMA), bovine serum albumin, ethanol absolute, hydrochloric acid (37%), leupeptin hemisulfate, p-coumaric acid, pepstatin A, ponceau S, trypan blue, Tween 20, N-formyl-methionyl-leucyl-phenylalanine (fMLP), and ionomycin and 3-phenylpropanenitrile (2-phenylethyl-CN, PeCN, ≥ 99%) from Sigma-Aldrich (Taufkirchen, Germany). Cytofix/Cytoperm solution was from BD Bioscience (Heidelberg, Germany). Benzyl-ITC and methanol were purchased from Fluka (Buchs, Switzerland). Phenylacetonitrile (benzyl-CN, ≥ 98%) was from Lancaster Synthesis Ltd. (Morecambe, UK). Butylbenzene and n-hexane were purchased from Merck KGaA (Darmstadt, Germany); dodecyl sulfate-sodium-salt from Serva (Heidelberg, Germany). Anti-human CD3 (clone OKT3), CD28 (clone 28.2), CD4, CD8, CD25, CD62L and CD69, IFN-γ and IL-2 monoclonal antibody (mAb), and propidium iodide were purchased from eBioscience (Frankfurt, Germany). Antibodies against p-38, p-ERK, p-JNK, p-c-Jun and the horseradish peroxidase (HRP)-labelled secondary antibodies, anti-mouse and anti-rabbit were from Cell Signaling Technology (Boston, USA). Anti-human COX-2 was from R&D System (Wiesbaden, Germany); anti-human COX-1 and anti-human LOX from Santa

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Cruz Biotechnology (Heidelberg, Germany), and mAb against β-actin from Sigma Aldrich (Taufkirchen, Germany). Brefeldin A and Cytofix/Cytoperm solution was from Becton Dickinson (Franklin Lakes, NJ).

2.2.

Preparation of human PBMC and cell culture

The study was approved by the Ethical Committee of the University of Freiburg. Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coats obtained from the Blood Transfusion Centre (Freiburg University Medical Center, Germany) or fresh blood from donors by centrifugation on a LymphoPrepTM gradient (density: 1.077 g/cm3, 20 min, 500 x g). Isolated PBMC were then washed twice with pre-warmed PBS and cell viability, and concentration was determined using the trypan blue exclusion test. Then the cells were supplemented with fresh RPMI 1640 medium supplemented with 10% heatinactivated foetal calf serum, 2 mM L-glutamine and 100 U/ mL penicillin/streptomycin and cultured at 37 °C in a humidified incubator with a 5% CO2/95% air atmosphere.

2.3.

Activation and treatment of PBMC

Peripheral blood mononuclear cells (105 or 2x 106 cells/ml) were either treated with vehicle or different concentrations of the test compound and stimulated with either anti-human CD3/ CD28 mAbs or LPS (1 µg/mL) and/or fMLP (1 µM) for different time points in the presence of culture medium. Afterwards, cells were washed with PBS and prepared for the bioassays as described below.

2.4. Determination of cell viability, apoptosis and necrosis of PBMC Cell viability rate was determined by using the trypan blue exclusion test, based on the principle that intact cell membranes are impermeable to the dye. Furthermore, the WST-1 assay (Roche Diagnostics Deutschland GmbH, Mannheim, Germany) was used to assess cell viability by quantification of mitochondrial dehydrogenase activity which cleaves the tetrazolium salt WST-1 to form formazan. After compound exposure, the assay was performed following the manufacturer’s instructions. Apoptosis and necrosis were determined using the Annexin V-FITC Apoptosis Detection kit (eBioscience, Frankfurt, Germany) according to the manufacturer’s instructions. After annexin V staining, propidium iodide was added and the cells were incubated for additional 10 min in the dark, followed by a flow cytometry analysis to determine the amount of apoptosis and necrosis.

2.5.

Protein analysis by immunoblotting

Proteins were analysed by immunoblotting after a modified protocol of Burnette (1981). Protein concentration was determined as described by Bradford (1976). For immunoblotting, 15 µg of protein were mixed with ready-made SDS-containing sample buffer, supplemented with 0.5% β-mercaptoethanol. Electrophoresis was performed according to the method of Laemmli (1970). The gel was then transferred to a nitrocellulose membrane by wet blotting (0.7 mA/cm2, 90 min.), blocked with 5%

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low fat milk in TBS/Tween 0.1 % and incubated with primary antibody for 1 h at RT or overnight at 4 °C, and subsequently horseradish peroxidase labelled secondary antibody for 1 h at RT each. After antibody incubation, the proteins of interest were detected by chemiluminescence technique. A digital image of the western blot was captured by the gel documentation system Molecular Imager® ChemiDocTM XRS system (Bio-Rad, Munich, Germany). Size approximations were taken by comparing the stained bands to that of a protein standard loaded during electrophoresis. The process was repeated for the structural protein β-actin as loading control.

2.6.

Quantification of TNF-α

Release of TNF-α was quantified in the supernatant of treated PBMC using a TNF-α Enzyme Linked Immunosorbent Assay (ELISA) ELISA kit from eBioscience (Frankfurt, Germany) according to the protocol of the manufacturer.

2.7.

Quantification of PGE2

Release of PGE2 was quantified using the PGE2 ELISA kits from R&D System (Wiesbaden, Germany) and Cayman (Hamburg, Germany) according to the protocol of the manufacturer.

2.8.

Quantification of Leukotriene B4

Release of LTB4 was quantified using the Leukotriene B4 EIA ELISA kit from cayman (Hamburg, Germany) according to the protocol of the manufacturer.

2.9.

Cell division tracking using CFSE

For monitoring cell proliferation, the dye CFSE was used. This assay is based on the progressive halving of CFSE fluorescence within daughter cells following each cell division. 5 × 106 cells/mL were incubated with 5 µM CFSE for 10 min at 37 °C. The staining reaction was stopped by washing twice with culture medium. Afterwards, PBMC were additionally stained with antibodies to anti-human CD4 mAbs and CD8 mAbs analysed using flow cytometry.

2.10. Determination of lymphocyte activity by surface antigen expression and intracellular cytokine staining Peripheral blood mononuclear cells (105) were stained for surface expression of CD4+ and CD8+ cells using antibodies to antihuman CD4 and anti-human CD8 mAbs, additionally stained with either phycoerythrin (PE)-labelled anti-human CD25, CD62L and CD69 mAbs and subsequent analysed by flow cytometry. For intracellular staining, cells were re-stimulated with 40 nM PMA and ionomycin (1 µg/mL) for 2 h, following 4 h incubation time in the presence of 10 µg/mL brefeldin A. Intracellular cytokine staining was performed with PE-conjugated mAb specific for anti-human IFN-γ mAbs or anti-human IL-2 mAbs using Cytofix/Cytoperm solution and flow cytometry.

2.11.

Data analysis

Results were calculated by using Graphpad Prism 5.0 software (La Jolla, CA). Data are presented as mean and standard

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error of the mean. Statistical significance was determined by Student’s paired t-test. P values <0.05 (*) were considered significant and <0.01 (**) were considered highly significant, compared to the respective controls.

3.

Results

3.1.

BITC, but not BCN inhibits PGE2 release

To analyse whether BITC has toxic effects on human immune cells in the presence of LPS, WST-1 assay was first used. BITC did not significantly affect the cells at concentrations < 25 µM after 18h or 36h incubation (Fig. 1a). Using specific cell staining for apoptosis and necrosis, it was confirmed that below a concentration of 25 µM, no cytotoxicity in terms of apoptosis and necrosis induction was evident (Fig. 1b). Apoptosis was detected at concentration >25 µM; however no necrosis was detected even at 100 µM. Based on these results, cells were pre-incubated with noncytotoxic concentrations of BITC (0.1–10 µM) for 3 h or 6 h and subsequently LPS-stimulated. As shown in Fig. 2a (left panel), BITC suppressed COX-2 expression in a concentrationdependent manner. The expression of COX-1 was not influenced by BITC (Fig. 2a, right panel). ELISA measurements confirmed the inhibition of PGE2 release from the cells by BITC pretreatment, at a concentration exceeding 1 µM (Fig. 2b). Increase in pre-treatment time with BITC intensified the inhibition (Fig. 2b). In contrast, BCN did not inhibit PGE2 release, even at high concentrations (Fig. 2c). Also PeCN, another nitrile, did not inhibit PGE2 release (Fig. 2d). A diminished release of TNF-alpha upon BITC treatment was also detected (Fig. 2e). This was significant at 3 µM.

3.2.

BITC inhibits LTB4 release from human PBMC

In order to determine the effect of BITC on 5-LOX pathway, we then analysed the impact of BITC on LTB4 release from PBMC, stimulated with LPS and fMLP. Whereas BITC pre-treatment of cells at 3 µM had no effect, 10 µM significantly attenuated the release of LTB4. The LTB4 levels were then comparable with non-stimulated control cells (Fig. 3).

3.3. BITC differentially modulates LPS-stimulated MAPK signalling pathways Next, we analysed the MAPK signalling pathway that is one of the important early upstream cascades involved in inflammatory responses triggered by LPS. One hour BITC preincubation of PBMC led to a concentration dependent activation of p38 beyond the effect triggered by LPS alone (Fig. 4a). At 10 µM BITC, suppression in LPS-induced p38 activation was detected after 3 h or 6 h (Fig. 4a). ERK kinase activation showed a gradual concentration-dependent inhibition, detected after 1 h and 3 h of BITC treatment (Fig. 4b). After 6 h, this effect was again only evident at 10 µM BITC; at lower concentrations, a co-stimulatory effect on ERK was evident. We then investigated the response of c-Jun activation in PBMC upon BITC. At early time points (0.5 h) of BITC pre-incubation, a reduced phosphorylation of c-Jun could be detected at 3 µM as compared to LPS control.

3.4. Impact of BITC on CD4+ and CD8+ T cell activation We finally determined the effect of BITC on CD4 + and CD8+ T cell activation and function. First, we assessed cell

Fig. 1 – Effect of BITC on cell viability. (a) Peripheral blood mononuclear cells were stimulated for 18 and 36 h with 1 µg/mL LPS followed by 2 h BITC treatment, and the cell viability was determined by WST-1 assay. Bars are mean ± SEM (n = 4). (b) PBMC were stimulated with LPS (1 µg/mL) for 36 h followed by 2 h BITC treatment. Apoptosis and necrosis were determined by Annexin V/propidium iodide assay. CPT (300 µM) and Triton X-100 (0.005%) were used as positive controls; bars are mean ± SEM (n = 3).

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Fig. 2 – Effect of BITC and BCN on inflammatory factors in LPS stimulated human PBMC. (a) Peripheral blood mononuclear cells were pre-treated with BITC for 3 or 6 h and subsequently stimulated with 1 µg/mL LPS for another 3 h. Cells were lysed and total lysate subjected to immunoblotting. The picture depicts representative immunoblots of COX-2 (left panel) and COX-1 (right panel). β-actin was used as loading control. (b) Peripheral blood mononuclear cells were pre-treated with BITC for 3 or 6 h and subsequently stimulated with 1 µg/mL LPS for another 24 h. Supernatants were then used for photometric quantification of PGE2 release. PBMC were pre-treated with BCN (c) or PeCN (d) for 6h and subsequently stimulated with 1 µg/mL LPS for another 24h. Supernatants were then used for photometric quantification of PGE2 release. PGE2 release was calculated relative to the LPS control; bars are mean ± SEM (n = 3). (e) Peripheral blood mononuclear cells were treated with BITC for 1 or 3 h and subsequently stimulated with 1 µg/mL LPS for another 3 h. Supernatants were then used for photometric quantification of TNF-alpha release. TNF-alpha release was calculated relative to the LPS control; bars are mean ± SEM (n = 4).

proliferation using CFSE-based flow cytometry on day three post-stimulation with non-toxic concentrations of BITC. As demonstrated in Fig. 5, BITC pre-treatment of anti-CD3/CD28 stimulated CD4+ cells did not impact proliferation at any concentration tested, as compared to vehicle control. In contrast, proliferation of CD8+ T cells was significantly reduced by 10 µM BITC. We next quantified the impact of BITC on expression of the markers CD25, CD69, CD62L as well as endogenous release of interleukin 2 (IL-2) and interferon-gamma (IFN-γ). BITC pretreatment of stimulated cells significantly reduced CD25 expression, but only at a concentration of 10 µM in both populations (Fig. 5b). At 3 µM, BITC increased CD25 expression in CD8+ T cells. At this concentration, the expression of CD69 was also significantly increased in CD8 + cells. CD62L was increased by BITC at 10 µM, but this was only significant in CD4+ T cells. No change was observed in endogenous IL-2 release (Fig. 5c) upon pre-treatment with BITC. Finally, it was of interest to determine whether BITC influences the effector function of cells, which would directly relate to changes in IFN-γ

levels. Therefore, its production was analysed in activated CD4+ and CD8+ T cells following treatment with BITC. Again, at 10 µM, the IFN-γ concentration was significantly blocked by BITC in both cell populations, but this was significant only in CD8+ cells (Fig. 5c).

4.

Discussion

In Brassicales one group of secondary plant compounds which are made accountable for bioactivity are GLS or more precisely their breakdown products in the form of ITCs. The stability of GLS can be low, and they are easily hydrolysed either by the plant-endogenous enzyme myrosinase after disruption of the plant tissue (Lamy & Mersch-Sundermann, 2009) or thermallyinduced degradation (e.g. during plant processing) (Oerlemans, Barrett, Suades, Verkerk, & Dekker, 2006). ITCs are the most intensively studied breakdown products, and by now they are well known for their pleiotropic mechanisms in human cells.

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Fig. 3 – Effect of BITC on LTB4 release in LPS stimulated human PBMC. Peripheral blood mononuclear cells were first exposed to BITC for 3 h followed by 15 min treatment of 1 µg/ml LPS, and subsequently stimulated with 1 µM fMLP for 15 min. Supernatants were then used for photometric quantification of LTB4 release. LTB4 release was calculated relative to the LPS control; bars are mean ± SEM (n = 3).

However, enzymatic degradation might also lead to the formation of significant nitrile amounts, especially if plants contain additional proteins, such as the epithiospecifier or nitrile specifier proteins, which can modify enzymatic degradation in favour for nitriles (Williams et al., 2009; Wittstock & Burow, 2010). Moreover, several studies showed that thermally-induced degradation might even result in a predominant formation of nitriles, which are comparatively more stable than ITCs (Hanschen et al., 2012a; Hanschen, Rohn, Mewis, Schreiner, & Kroh, 2012b). The role and relevance of food-borne nitriles for the efficacy of pharmaceutical preparations or dietary supplements from Brassicales plants has so far been barely addressed. In order to judge this, better knowledge on their bioactivity is essential. In this study, LPS stimulated human PBMC were treated with non-toxic concentration of BITC, BCN or PeCN, and strong in-

hibition of PGE2 synthesis was seen but only with BITC. This was then preceded by inhibition of COX-2 protein expression. Our results show good transferability of the observations made in animal or human cancer cells upon ITC treatment. So far, COX-2 inhibition by ITCs was reported in studies using mouse macrophage RAW cells, peritoneal macrophages and human T24 bladder cancer cells at concentration ranges between 0.25–20 µM (Cheung & Kong, 2010; Dey, Ribnicky, Kurmukov, & Raskin, 2006; Lee, Seon, Cho, Kim, & Park, 2009; Lin et al., 2008; Prawan et al., 2009; Rose et al., 2005; Tsai, Liu, & Chen, 2010). As an example, in LPS stimulated RAW cells, COX-2 expression was suppressed by 65% after 30 minutes of treatment with 5 µM BITC (Murakami, Song, & Ohigashi, 2007). However, in many studies a strong anti-inflammatory effect was observed only at concentrations exceeding 10 µM (Heiss et al., 2001; Lin et al., 2008; Shan et al., 2009). So, it was reported that SFN at 5 to 20 µM decreased COX-2 mRNA expression by 28–87% after 10 h treatment and by 18–85% after 24 h treatment (Shan et al., 2009). Besides COX pathway inhibition, we found BITC (10 µM) as inhibitor of LTB4 release in human primary PBMC. So far, LTB4 inhibition mediated by ITC was only shown in RBL-2H3 rat basophilic leukaemia cells at a quite high concentration of 50 µM (Hsu, Hsu, Lee, & Ko, 2006). The activation of the MAPK cascade is a major pathway to signal an inflammatory response, triggered by LPS. In our study, a concentration-dependent suppression was observed with short-term exposure of PBMC to BITC at ≥ 3 µM in the case of LPS-activated ERK1/2. The same effect was found in RAW cells pre-treated with BITC at a concentration between 1–5 µM (Lee et al., 2009). Downstream signalling of ERK1/2 has been reported to lead to elevated COX-2 expression in the way of stabilizing COX-2 mRNA (Tsatsanis, Androulidaki, Venihaki, & Margioris, 2006). The temporary inhibitory effect on ERK1/2 could therefore possibly provide an explanation for the reduced levels of COX-2 protein levels upon pre-treatment with the compound. Although ERK1/2 was blocked by BITC, the LPS-induced activation of p38 as well as JNK was even further elevated by BITC. Furthermore, BITC treatment blocked c-Jun activation. c-Jun forms the early response transcription factor AP-1 which starts the transcription of proinflammatory cytokines, including TNF-alpha when localized in the nucleus (Tak & Firestein, 2001). Finally, the results with activated human T lymphocytes demonstrate that BITC has immunosuppressive influence

Fig. 4 – Effect of BITC on MAPK pathway and AP-1 activation. Peripheral blood mononuclear cells were pre-treated with BITC for the indicated time points and subsequently stimulated with 1 µg/mL LPS for 15 min. Cells were lysed and total lysate subjected to immunoblotting. The picture depicts representative immunoblots of phosphorylated p-38 (Thr180/Tyr182) (a), ERK1/2 (Thr202/Tyr204) (b) and c-JUN (Ser73) (c). β-actin was used as loading control.

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Fig. 5 – Effect of BITC on T cell activation. Peripheral blood mononuclear cells were stimulated for 3 h with 100 ng/ml antiCD3/CD28 mAbs and treated with BITC for 2 h (a), (b). (a) Effect of BITC on proliferation capacity of T cells as determined by CFSE. (b) Effect of BITC on activation markers (CD25, CD69, CD62L) was analysed by antibody staining and flow cytometry. (c) The endogenous release of IL-2 or IFN-γ was analysed after cell stimulation for 18h with 100 ng/ml anti-CD3/CD28 mAbs and treatment with BITC for 2 h by antibody staining and flow cytometry. Quantification was done using flow cytometry analysis and Modfit software. The graphs are mean ± SEM (n = 3).

on these cells but only at higher concentrations. So the activation, function and proliferation of CD8+ T cells was reduced at 10 µM BITC. This immunosuppressive effect on human CD8+ cells was not reported when the ITC erucin was used in another study recently published (Grundemann, Garcia-Kaufer, Lamy, Hanschen, & Huber, 2015).This is in line with the assumption that structure dependent differences in ITC reactivity and consequently bioactivity exist, see e. g. (Lamy, Scholtes, Herz, & Mersch-Sundermann, 2011). The available literature demonstrates that after consumption of glucotropaeolin-containing plants, quite high BITC plasma levels can be reached. After consumption of 10 g Indian cress (tropaeolum majus) containing 1 mM glucotropaeolin, a mean peak plasma level of 16 µM BITC, quantified as BITCglutathione metabolite, was present in human volunteers (Platz et al., 2013). Quantification of the corresponding nitrile has not so far been done. Therefore, it seems likely that high GLScontaining preparations could result in BITC plasma levels which are capable of inducing an anti-inflammatory response in humans, but evidence for this should be obtained. In the future, plant breeding efforts or food technology tools

could try to optimize plant preparations for preferential ITC but not nitrile formation.

5.

Conclusion

This study provides for the first time evidence for a dual inhibition of the arachidonic acid metabolism by BITC in an in vitro model using primary human PBMC. Based on the present results it can be hypothesized that inhibition of LPS-activated ERK1/2 and downstream signalling could partly account for this observation. Some of the effects were observed only at higher BITC concentrations, but could possibly be achieved by consuming GLS/ITC-rich dietary supplements or phytopharmaceuticals. The preference of nitrile formation instead of ITC under certain conditions and the absence of antiinflammatory activity of nitriles should be considered carefully, because it could be most relevant for the efficacy of dietary supplements or phytopharmaceutical Brassicales plant preparations containing the pro-drug GLS.

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Acknowledgements E. L. is funded by an academic grant from the European Social Fund and the Ministry of Science, Research and Arts BadenWürttemberg, Germany. C.G. received financial support from Software AG foundation and DAMUS-DONATA e V.

Conflict of interest The study was partly supported by a grant from Repha GmbH, Langenhagen, Germany. Repha GmbH was not involved in the interpretation of the results or writing of the manuscript. REFERENCES

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