On the inhibition of 5-lipoxygenase product formation by tryptanthrin: mechanistic studies and efficacy in vivo

Abstract

BACKGROUND AND PURPOSE

Leukotrienes (LTs) are pro-inflammatory mediators produced by 5-lipoxygenase (5-LO). Currently available 5-LO inhibitors either lack efficacy or are toxic and novel approaches are required to establish a successful anti-LT therapy. Here we provide a detailed evaluation of the effectiveness of the plant-derived alkaloid tryptanthrin as an inhibitor of LT biosynthesis.

EXPERIMENTAL APPROACH

We analysed LT formation and performed mechanistic studies in human neutrophils stimulated with pathophysiologically relevant stimuli (LPS and formyl peptide), as well as in cell-free assays (neutrophil homogenates or recombinant human 5-LO) and in human whole blood. The in vivo effectiveness of tryptanthrin was evaluated in the rat model of carrageenan-induced pleurisy.

KEY RESULTS

Tryptanthrin potently reduced LT-formation in human neutrophils (IC₅₀ = 0.6 µM). However, tryptanthrin is not a redox-active compound and did not directly interfere with 5-LO activity in cell-free assays. Similarly, tryptanthrin did not inhibit the release of arachidonic acid, the activation of MAPKs, or the increase in [Ca²⁺]_i, but it modified the subcellular localization of 5-LO. Moreover, tryptanthrin potently suppressed LT formation in human whole blood (IC₅₀ = 10 µM) and reduced LTB₄ levels in the rat pleurisy model after a single oral dose of 10 mg·kg⁻¹.

CONCLUSIONS AND IMPLICATIONS

Our data reveal that tryptanthrin is a potent natural inhibitor of cellular LT biosynthesis with proven efficacy in whole blood and is effective in vivo after oral administration. Its unique pharmacological profile supports further analysis to exploit its pharmacological potential.

Introduction

5-Lipoxygenase (5-LO) is a non-haeme iron-containing dioxygenase, which plays a central role in the biosynthesis of leukotrienes (LTs), a class of potent lipid mediators of inflammation and immunity (Peters-Golden and Henderson, 2007). 5-LO initiates LT biosynthesis by incorporation of molecular oxygen into arachidonic acid (AA) released by cytosolic phospholipase (cPL)A₂, followed by formation of the epoxide LT (LT)A₄. The unstable LTA₄ is further converted to other LTs (i.e. LTB₄ or cysteinyl-LTs) by LTA₄ hydrolase or LTC₄ synthase, depending on the cell type (Radmark et al., 2007). 5-LO products have been established as mediators of asthma and allergic rhinitis, but may also have roles in sepsis, atherosclerosis, cardiovascular disease and cancer (Peters-Golden and Henderson, 2007). Accordingly, pharmacological suppression of 5-LO product synthesis is a major concept for intervention with LT-associated diseases (Werz and Steinhilber, 2006).

Intensive research in the field of LT synthesis inhibitors has led to the identification of different classes of compounds, acting directly on 5-LO enzymatic activity or on other enzymes or proteins involved in LT cellular biosynthesis [e.g. inhibitors of cPLA₂ or of 5-LO-activating protein (FLAP)]. Direct 5-LO inhibitors have been classified on the basis of their mechanism of action. Thus, iron ligand inhibitors chelate the active site iron, redox-active inhibitors uncouple the catalytic cycle of 5-LO, and non-redox-type 5-LO inhibitors compete with AA (Pergola and Werz, 2010). Most of these compounds, however, have failed in preclinical studies and/or in clinical trials due to their lack of efficacy or to toxicity. Until today, the iron ligand zileuton (Figure 1) was the only 5-LO inhibitor that could enter the market (Drazen et al., 1999), but its clinical use is limited by liver toxicity.

Figure 1.

Chemical structure of tryptanthrin and zileuton.

Novel strategies for anti-LT intervention that may circumvent the problems associated with the typical classes of 5-LO inhibitors are therefore needed for steps forward in anti-LT therapy. With this aim, the plant kingdom represents a valuable source of structurally diverse compounds that might offer novel pharmacological concepts for interference with 5-LO (Werz, 2007; Feisst et al., 2009). Although numerous plant-derived substances have been reported to interfere with LT synthesis, detailed investigations of 5-LO inhibition by natural compounds are rare (Werz, 2007). Most of these compounds are reducing agents like polyphenols (e.g. nordihydroguaiaretic acid), quinones or coumarins and seemingly act as redox-type 5-LO inhibitors with limited therapeutic value. Moreover, many analyses have often been conducted in only one type of assay (e.g. cell-free test model) and/or neglected pharmacologically relevant factors (i.e. plasma albumin-binding), which did not allow conclusions to be drawn regarding the pharmacological profile and therapeutic potential of the respective compounds.

Tryptanthrin [indolo-(2,1-b)-quinazoline-6,12-dione] (Figure 1) is a weakly basic alkaloid, with unique structural features in comparison with other LT synthesis inhibitors, that occurs in various plant species such as Isatis (Danz et al., 2001), Strobilanthus (Honda and Tabata, 1979), Calanthe (Murakami et al., 2001) and Couroupita (Bergman et al., 1985). Tryptanthrin has previously been shown to potently inhibit LTB₄ release in isolated granulocytes (IC₅₀ = 0.15 µM) stimulated with ionophore (Danz et al., 2002b). Based on these initial findings, tryptanthrin has generally been designated a 5-LO inhibitor (Danz et al., 2002a; Lee et al., 2007; Jao et al., 2008), although its pharmacological profile is essentially unclear. Also, data on the efficacy of tryptanthrin on LT formation in vivo and corresponding anti-inflammatory effectiveness are lacking. Here we demonstrate the effectiveness of tryptanthrin in an animal model of disease related to LTs (i.e. carrageenan-induced pleurisy in the rat), and we provide a detailed evaluation of the effects of tryptanthrin on 5-LO product formation in diverse assay systems, taking into account essential parameters that may affect its effectiveness in vivo.

Methods

Materials

Tryptanthrin was synthesized and characterized as reported previously (Danz et al., 2001). The cPLA_2α inhibitor N-{(2S,4R)-4-(biphenyl-2-ylmethyl-isobutyl-amino)-1-[2-(2,4-difluorobenzoyl)-benzoyl]-pyrrolidin-2-ylmethyl}-3-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)-phenyl]acrylamide was from Calbiochem (Bad Soden, Germany). Zileuton was from Sequoia Research Products (Oxford, UK). Hyperforin and MK886 were generous gifts by Schwabe AG (Karlsruhe, Germany) and by Dr AW Ford-Hutchinson, Merck Frosst (Quebec, Canada), respectively. The λ-carrageenan type IV (isolated from Gigartina aciculaire and Gigartina pistillata) was from Sigma-Aldrich (Milan, Italy) as well as PGE₂ antibody. [³H]-PGE₂ was from PerkinElmer Life Sciences (Milan, Italy). Antibodies recognizing phosphorylated extracellular signal-regulated protein kinases (pERK)1/2 (Thr202/Tyr204), phosphorylated p38 MAPK (Thr180/Tyr182), IκBα, NF-κB p65 protein, and phosphorylated proteins containing the phosphorylation motif of PKA (phospho-Ser/Thr residue with arginine at the −3 and −2 positions, RRXS/T) (Bryn et al., 2006) were from Cell Signaling Technology (Danvers, MA, USA). HPLC solvents were from Merck (Darmstadt, Germany). All other chemicals were purchased from Sigma-Aldrich (Deisenhofen, Germany), unless stated otherwise.

Cells

Human neutrophils were freshly isolated from human venous blood of healthy adult donors (Blood Center, University Hospital, Tuebingen, Germany) as described previously (Werz et al., 2002b). In brief, venous blood was taken from donors and leucocyte concentrates were prepared by centrifugation at 4000× g, 20 min, 20°C. Neutrophils were immediately isolated by dextran sedimentation, centrifugation on Nycoprep cushions, and hypotonic lysis of erythrocytes. Neutrophils (purity > 96–97%) were finally resuspended in PBS pH 7.4 plus 1 mg·mL⁻¹ glucose (PG buffer), or in PG buffer plus 1 mM CaCl₂ (PGC buffer) as indicated. For incubations with tryptanthrin, dimethyl sulphoxide (DMSO) was used as vehicle, never exceeding 0.1% (v v⁻¹). To exclude toxic effects of tryptanthrin during pre-incubation periods, neutrophil viability was analysed by light microscopy and trypan blue exclusion. Incubation with 30 µM tryptanthrin at 37°C for up to 30 min caused no significant change in neutrophil viability.

Expression and purification of human recombinant 5-LO from Escherichia coli

Human recombinant 5-LO was expressed in E. coli Bl21 (DE3) cells, transformed with pT3–5LO, and purification of 5-LO was performed as described previously (Fischer et al., 2003). In brief, E. coli were harvested and lysed in 50 mM triethanolamine/HCl, pH 8.0, 5 mM EDTA, soybean trypsin inhibitor (STI; 60 µg·mL⁻¹), 1 mM PMSF, 1 mM dithiothreitol (DTT) and lysozyme (1 mg·mL⁻¹), homogenized by sonication (6 × 6 s) and centrifuged at 10 000× g for 15 min followed by centrifugation at 100 000× g for 70 min at 4°C. The supernatant was then applied to an ATP-agarose column (Sigma A2767; Sigma-Aldrich), and the column was eluted as described previously (Brungs et al., 1995). Partially purified 5-LO was immediately used for in vitro activity assays.

Determination of 5-LO product formation

For assays of intact cells, freshly isolated neutrophils (3 × 10⁷ mL⁻¹) were resuspended in 1 mL PGC buffer and primed at 37°C with LPS (1 µg·mL⁻¹) plus adenosine deaminase (Ada; 0.3 U·mL⁻¹) for 30 min. Formation of 5-LO products was started by addition of 1 µM formyl-methionyl-leucyl-phenylalanine (fMLP), and after 5 min the reaction was stopped with 1 mL of methanol and 30 µL of 1 N HCl, 200 ng PGB₁ and 500 µL of PBS were added. Test compounds or vehicle (0.1% DMSO) were added 15 min before fMLP. The samples were centrifuged (800× g, 10 min) and the supernatants were applied to C-18 solid-phase extraction columns (100 mg; UCT, Bristol, PA, USA) preconditioned with 1 mL methanol and 1 mL water. After being washed with 1 mL water and 1 mL 25% (v v⁻¹) methanol, formed 5-LO metabolites were eluted with 300 µL methanol. The eluates were diluted with 120 µL water, and 100 µL diluted extracts were analysed by reversed phase HPLC on a C-18 Radial-Pak® column (Waters, Eschborn, Germany) with 75% (v v⁻¹) methanol, 25% (v v⁻¹) water and 0.1% (v v⁻¹) trifluoroacetic acid as eluent (flow rate of 1.2 mL·min⁻¹) and detection at 280 and 235 nm. 5-LO products include LTB₄, its all-trans isomers, and 5(S)-hydro(pero)xy-6-trans-8,11,14-cis-eicosatetraenoic acid [5-H(P)ETE]. The chromatographic method used does not allow the measurement of cysteinyl-LTs (LTC₄, D₄ and E₄).

For determination of 5-LO product formation in neutrophil homogenates, 1 mM EDTA was added to cells resuspended in PBS. Samples were cooled on ice (5 min), sonicated (3 × 10 s) at 4°C, and 1 mM ATP and 1 mM DTT were added as indicated. For determination of human recombinant 5-LO activity, partially purified 5-LO was added to 1 mL of a 5-LO reaction mix (PBS, pH 7.4, 1 mM EDTA, 1 mM ATP). Samples of either partially purified 5-LO or of cell homogenates (corresponding to 10⁷ neutrophils) were incubated for 10 min at 4°C with vehicle or tryptanthrin, pre-warmed for 30 s at 37°C and 2 mM CaCl₂ and the indicated concentrations of AA were added. The reaction was stopped after 10 min at 37°C by addition of 1 mL ice-cold methanol, and 30 µL of 1 N HCl, 200 ng PGB₁ and 500 µL of PBS were added. Formed metabolites were extracted and analysed by HPLC as described for intact neutrophils.

For assays in whole blood, freshly withdrawn blood from healthy adult donors was obtained by venipuncture and collected in monovettes containing 16 U heparin mL⁻¹. Aliquots of 2 mL (A23187) or 3 mL (LPS and fMLP) were pre-incubated with the test compounds or with vehicle (0.1% DMSO) for 15 min at 37°C, as indicated, and formation of 5-LO products was either started by addition of 1 µM fMLP following 30 min priming with 1 µg·mL⁻¹ LPS, or by addition of 30 µM A23187. The reaction was stopped on ice after 15 (LPS and fMLP) or 10 (stimulation with A23187) min and the samples were centrifuged (600× g, 10 min, 4°C). Aliquots of the resulting plasma (500 µL) were then mixed with 2 mL of methanol and 200 ng PGB₁ were added as internal standard. The samples were placed at −20°C for 2 h and centrifuged again (600× g, 15 min, 4°C). The supernatants were collected and diluted with 2.5 mL PBS and 75 µL 1 N HCl. Formed 5-LO metabolites were extracted and analysed by HPLC as described for intact neutrophils.

Determination of release of ³H-labelled AA from neutrophils

Release of ³H-labelled AA from neutrophils was analysed as previously described (Fischer et al., 2005). Briefly, freshly isolated neutrophils were resuspended at 2 × 10⁶ in 1 mL RPMI 1640 medium containing 5 nM [³H]-AA (corresponding to 1 µCi·mL⁻¹, specific activity 200 Ci·mmol⁻¹) and incubated for 120 min at 37°C in 5% CO₂ atmosphere. Cells were then collected by centrifugation, washed once with PBS and twice with PBS containing 2 mg·mL⁻¹ fatty acid-free bovine albumin, to remove unincorporated [³H]-AA. Labelled neutrophils (10⁷) were resuspended in 1 mL PGC, primed with 1 µg·mL⁻¹ LPS for 30 min and stimulated with 1 µM fMLP for 10 min at 37°C. Test compounds were added 15 min before fMLP. The samples were then placed on ice for 2 min and cells were centrifuged at 400 ×g for 5 min at 4°C. Aliquots (100 µL) of the supernatants were measured by liquid scintillation in a beta-counter (Micro Beta Trilux, Perkin Elmer, Foster City, CA, USA) to detect the amounts of ³H-labelled AA released into the medium.

Analysis of subcellular redistribution of 5-LO, IκBα and p65 by cell fractionation and immunoblotting

For analysis of 5-LO subcellular redistribution, neutrophils (3 × 10⁷ mL⁻¹ PGC buffer) were primed at 37°C with 1 µg·mL⁻¹ LPS plus 0.3 U·mL⁻¹ Ada for 30 min and stimulated by addition of 1 µM fMLP. After 5 min the reaction was stopped on ice and samples were centrifuged (200× g for 5 min at 4°C). Tryptanthrin or vehicle (DMSO, 0.1%) was added 15 min before fMLP. IκBα and p65 subcellular redistribution was analysed as described (Vancurova et al., 2001) with slight modifications. In brief, neutrophils (1 × 10⁷ mL⁻¹ phenol red-free RPMI medium supplemented with 10% low endotoxin fetal calf serum) were pre-incubated at 37°C for 10 min with vehicle (DMSO, 0.1%) or tryptanthrin and then 1 µg·mL⁻¹ LPS or 1 µM fMLP were added for 30 min at 37°C. The reaction was stopped on ice, 1 mM PMSF was added and samples were centrifuged (600× g for 5 min at 4°C) and washed twice with ice-cold PBS containing 1 mM PMSF. Subcellular fractionation was performed by mild detergent (0.1% NP40) lysis, yielding a nuclear and a non-nuclear fraction (Werz et al., 2001).

Briefly, neutrophils were suspended in ice-cold NP-40-lysis buffer (10 mM Tris/HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 1 mM EDTA, 0.1% NP-40, 1 mM PMSF, 60 µg·mL⁻¹ STI, and 10 µg·mL⁻¹ leupeptin), vortexed (3 × 5 s), kept on ice for 10 min and centrifuged (800× g for 10 min at 4°C). The resulting supernatants (non-nuclear fractions) were transferred to a new tube and the pellets (nuclear fractions) were resuspended in ice-cold relaxation buffer (50 mM Tris/HCl, pH 7.4, 250 mM sucrose, 25 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 1 mM PMSF, 60 µg·mL⁻¹ STI, 10 µg·mL⁻¹ leupeptin). Both nuclear and non-nuclear fractions were centrifuged again (800× g, 10 min, 4°C), for further purification. Lysis of cells and integrity of nuclei was confirmed by light microscopy with trypan blue exclusion. Nuclei in relaxation buffer were disrupted by sonication (3 × 5 s).

Nuclear and non-nuclear fractions were immediately mixed with the same volume of SDS-PAGE sample loading buffer (SDS-b; 20 mM Tris/HCl, pH 8, 2 mM EDTA, 5% SDS (w v⁻¹), 10% β-mercaptoethanol) and heated for 6 min at 95°C. Aliquots (20 µL) of these samples were analysed by SDS-PAGE using a Mini Protean system (Bio-Rad, Hercules, CA, USA) on 10% gels. Correct loading of the gel and transfer of proteins to the nitrocellulose membrane was confirmed by Ponceau staining. After electroblotting to nitrocellulose membrane (GE Healthcare, Munich, Germany), membranes were blocked with 5% bovine serum albumin in 50 mM Tris/HCl, pH 7.4 and 100 mM NaCl (TBS) for 1 h at room temperature (RT). Membranes were washed and then incubated with primary antibody overnight at 4°C. 5-LO antiserum (1551, AK7, affinity purified on a 5-LO column, kindly provided by Dr Olof Rådmark, Karolinska Institute, Stockholm, Sweden) was used as 1:100 dilution. The membranes were washed, incubated with 1:1000 dilution of alkaline phosphatase-conjugated IgGs for 2 h at RT, and proteins were visualized with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate in detection buffer (100 mM Tris/HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl₂).

Analysis of subcellular localization of 5-LO by indirect immunofluorescence microscopy

Neutrophils (1.5 × 10⁶ in 500 µL PGC buffer) were primed at 37°C with LPS (1 µg·mL⁻¹) plus Ada (0.3 U·mL⁻¹). After 15 min, test compounds or vehicle (0.1% DMSO) were added and samples were incubated for additional 15 min at 37°C. Cells were then centrifuged at 10× g for 30 s onto poly-L-lysine (MW 150 000–300 000; Sigma-Aldrich)-coated glass coverslips, and activated by addition of 1 µM fMLP for 3 min at 37°C. Cells were fixed in methanol (−20°C, 30 min) and permeabilized with 0.1% Tween 20 in PBS (RT, 10 min), followed by three washing steps with PBS. Samples were blocked with 10% non-immune goat serum (Invitrogen, Darmstadt, Germany) for 10 min at RT, washed with PBS and incubated with anti-5-LO serum (1551, affinity purified) for 30 min at RT. The coverslips were washed ten times with PBS, incubated for 10 min at RT in the dark with Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) diluted 1 to 1500 in PBS, and washed 10 times with PBS. The coverslips were then mounted on glass slides with Mowiol (Calbiochem) containing 2.5% n-propyl gallate (Sigma-Aldrich). The fluorescence was visualized with a Zeiss Axio Observer.Z1 microscope and a Plan-Apochromat 100x/1.40 Oil DIC M27 objective (Carl Zeiss AG, Jena, Germany). An AxioCam MR3 camera was used for image acquisition.

Diphenylpicrylhydrazyl (DPPH) assay

The antioxidant activity of tryptanthrin was assessed by the method of Blois (Blois, 1958), with slight modifications. Briefly, 100 µL of 25, 50, 100 or 200 µM tryptanthrin in ethanol (corresponding to 2.5, 5, 10 or 20 nmol) were added to 100 µL of a solution of the stable free radical DPPH in ethanol (50 µM, corresponding to 5 nmol), buffered with acetate to pH 5.5, in a 96 well plate. The absorbance was recorded at 520 nm after 30 min incubation under gentle shaking in the dark. Ascorbic acid and L-cysteine were used as reference compounds. All analyses were performed in triplicates.

Evaluation of the phosphorylation state of MAPK and of proteins containing the PKA-phosphorylation motif

Neutrophils (10⁷ in 100 µL PGC buffer) were preincubated with the indicated concentrations of tryptanthrin or vehicle for 15 min. Then, 1 µM fMLP was added for 1.5 min at 37°C. Alternatively, cells were directly incubated with tryptanthrin or vehicle for 1.5 min at 37°C. The reaction was stopped by addition of 100 µL of SDS-b, and heated for 6 min at 95°C. Proteins in cell lysates were separated by SDS-PAGE on a 10% gel, electroblotted on Hybond ECL nitrocellulose membranes (GE Healthcare) and analysed for phosphorylated ERK1/2, phosphorylated p38-MAPK and phosphorylated proteins containing the PKA phosphorylation motif (RRXS/T) using an Ettan-DIGE imaging system (GE Healthcare) as described (Pergola et al., 2008).

Reactive oxygen species (ROS) generation assay

Neutrophils (10⁷ mL⁻¹ PG buffer) were preincubated with tryptanthrin or vehicle (0.1% DMSO) for 15 min. Then, the peroxide-sensitive fluorescence dye 2′,7′-dichlorofluorescein-diacetate (1 µg·mL⁻¹) and CaCl₂ (1 mM) were added 2 min before addition of 1 µM fMLP. The fluorescence emission at 530 nm was measured after excitation at 485 nm in a temperature-controlled (37°C) cuvette with continuous stirring in a spectrofluorometer (Aminco-Bowman series 2, Thermo Spectronic, Rochester, NY, USA).

Evaluation of intracellular Ca²⁺ mobilization

The determination of intracellular Ca²⁺ levels was performed as described previously (Werz et al., 2002b). In brief, freshly isolated neutrophils (10⁷) were resuspended in 1 mL PG buffer and incubated with 2 µM Fura-2/AM for 30 min at 37°C. After washing, cells were finally resuspended in 1 mL PG buffer, preincubated with tryptanthrin or vehicle (0.1% DMSO) for 15 min, supplemented with 1 mM CaCl₂, and transferred into a temperature-controlled (37°C) fluorimeter cuvette in a spectrofluorometer (AB-2) with continuous stirring. Mobilization of intracellular Ca²⁺ was initiated by addition of fMLP (0.1 µM). The fluorescence emission at 510 nm was measured after excitation at 340 and 380 nm, respectively. Intracellular Ca²⁺ levels were calculated according to the method of Grynkiewicz et al. (1985). F_max (maximal fluorescence) was obtained by lysing the cells with 0.5% Triton-X 100 and F_min by chelating Ca²⁺ with 10 mM EDTA.

Carrageenan-induced pleurisy in rats

Male Wistar Han rats (220–230 g, Harlan, Udine, Italy) were housed in a controlled environment and provided with standard rodent chow and water. All animal care and experimental procedures complied with Italian regulations on protection of animals used for experimental and other scientific purpose (Ministerial Decree 116192) as well as with the European Economic Community regulations (Official Journal of E.C. L 358/1 12/18/1986). Tryptanthrin (10 mg·kg⁻¹) or vehicle (0.5 mL of 0.5% carboxymethylcellulose and 10% Tween 20) was given p.o. 1 h before carrageenan. Rats were anaesthetized with enflurane 4% mixed with O₂, 0.5 L·min⁻¹, N₂O 0.5 L·min⁻¹ and submitted to a skin incision at the level of the left sixth intercostal space. The underlying muscle was dissected, and saline (0.2 mL) or 1% λ-carrageenan type IV (w v⁻¹) (0.2 mL) was injected into the pleural cavity. The skin incision was closed with a suture, and the animals were allowed to recover. At 4 h after the injection of carrageenan, the animals were killed by inhalation of CO₂. The chest was carefully opened, and the pleural cavity was rinsed with 2 mL saline solution containing heparin (5 U·mL⁻¹). The exudates and washing solution were removed by aspiration, and the total volume was measured. Any exudate that was contaminated with blood was discarded. Leucocytes in the exudates were resuspended in PBS and counted with an optical light microscope in a Burker's chamber after vital trypan blue staining. The amount of LTB₄ in the supernatant of centrifuged exudate (800× g for 10 min) was below the detection limit of the HPLC and was assayed by enzyme immunoassay (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's protocol. The amount of PGE₂ in the supernatant of centrifuged exudate (800× g for 10 min) was assayed by radioimmunoassay. The results are expressed as ng per rat.

Statistics

Results are expressed as mean ± SEM of n observations, where n represents the number of experiments performed on different days in duplicates or the number of animals, as indicated. The IC₅₀ values were determined by interpolation on semi-logarithmic graphs and validated with GraphPad Instat program. Data fit was obtained using the sigmoidal dose-response equation (variable slope) (GraphPad Prism software, GraphPad, San Diego, CA, USA). Statistical evaluation of the data was performed by one-way anova for independent or correlated samples followed by Tukey's HSD post hoc tests. Where appropriate, Student's t-test was applied. A P value < 0.05 (*) was considered significant.

Results

Tryptanthrin inhibits 5-LO product formation in intact neutrophils

Previous studies showed that tryptanthrin suppresses LTB₄ synthesis in neutrophils stimulated with the unphysiological stimulus A23187 ionophore (Danz et al., 2002b). Neutrophils are the major source of LTs in blood (Surette et al., 1993) and stimulation of LT synthesis with the natural agonists LPS and fMLP uses defined cellular pathways to activate 5-LO (i.e. Ca⁺² and MAPK) (Surette et al., 1993; Werz et al., 2002a) and is considered to reflect pathophysiologically relevant conditions at inflammation sites. Tryptanthrin effectively suppressed 5-LO product formation in LPS and fMLP-stimulated neutrophils with an IC₅₀ = 0.6 ± 0.2 µM, and was essentially equipotent to the 5-LO reference inhibitor zileuton (IC₅₀ = 0.7 ± 0.1 µM) (Figure 2A). The formation of the 5-LO metabolites LTB₄ and 5-H(P)ETE was inhibited with a similar potency by tryptanthrin (not shown).The suppression of 5-LO product formation under the assay conditions described earlier could be due to inhibition of substrate supply, for example, by interference with AA release. Stimulation of human neutrophils with LPS and fMLP resulted in a 1.54-fold increase in AA release, which is in agreement with previous findings (Pergola et al., 2008). Tryptanthrin (up to 30 µM) did not significantly inhibit LPS and fMLP-induced increase in AA release, whereas a specific cPLA₂ inhibitor prevented it (Figure 2B).

Figure 2.

Tryptanthrin inhibits 5-LO product formation in intact neutrophils. Effect of (A) tryptanthrin or zileuton on 5-LO product synthesis in neutrophils stimulated with fMLP (1 µM; 5 min, 37°C) after 30 min priming with LPS (1 µg·mL⁻¹) plus Ada (0.3 U·mL⁻¹). Data are expressed as percentage of control, means + SEM; n = 3, duplicates. **P < 0.01, ***P < 0.001 vs. control; anova+ Tukey's test. 5-LO products in 100% controls were (ng 10⁻⁷ cells): 10.9 ± 3.6. (B) Effect of tryptanthrin and of the cPLA_2α inhibitor (15 µM) on the release of [³H]-AA in intact neutrophils, following activation with fMLP (1 µM; 5 min, 37°C) after 30 min priming with LPS (1 µg·mL⁻¹). Data are expressed as percentage of control, means + SEM; n = 3, duplicates. **P < 0.01 vs. control; anova+ Tukey's test.

Tryptanthrin is not a direct 5-LO inhibitor

A major unresolved question is whether inhibition of cellular LT formation by tryptanthrin is due to direct interference with 5-LO enzymatic activity. Most of the plant-derived 5-LO inhibitors possess reducing properties and act by reducing the active site iron of 5-LO (Werz, 2007). Therefore, we evaluated the redox potential of tryptanthrin and we analysed its radical scavenging properties in the well-recognized DPPH assay. Tryptanthrin did not reduce the DPPH radical, whereas L-cysteine and ascorbic acid acted as expected (Figure 3A).

Figure 3.

Tryptanthrin is not a direct 5-LO inhibitor. (A) Reduction of the DPPH radical by tryptanthrin, L-cysteine and ascorbic acid. Data are means + SEM; triplicates. (B) Effect of tryptanthrin on 5-LO activity in neutrophil homogenates (20 µM AA, 1 mM free Ca⁺², 1 mM ATP; ±1 mM DTT, as indicated). Data are expressed as percentage of control, means + SEM; n = 3, duplicates. 5-LO products in 100% controls were (ng mL⁻¹): without DTT, 522.2 ± 104.0, plus DTT: 990.9 ± 63.5. (C) Effect of tryptanthrin at the indicated concentrations and of 3 µM zileuton on the activity of partially purified 5-LO (20 µM AA, 1 mM free Ca⁺², 1 mM ATP). Data are expressed as percentage of control, means + SEM; n = 3, duplicates. ***P < 0.001 vs. control; anova+ Tukey's test. 5-LO products in 100% controls were (ng mL⁻¹): 1312.0 ± 237.5.

We next analysed whether tryptanthrin inhibits the activity of crude 5-LO in cell-free systems. In neutrophil homogenates, 5-LO activity was not inhibited by tryptanthrin up to 30 µM and only 21% (non-significant) inhibition was observed at 100 µM (Figure 3B). An impaired efficacy of 5-LO inhibition in cell-free systems has also previously been observed for certain direct 5-LO inhibitors, which could be restored by varying the assay conditions. For instance, non-redox-type 5-LO inhibitors are less active in cell-free systems due to high peroxide levels, and inclusion of thiols restores their efficacy (Fischer et al., 2004). Also, the potency of hyperforin is strongly attenuated by cellular membranes present in cell homogenates, but is preserved when partially purified 5-LO is analysed (Feisst et al., 2009). Addition of exogenous DTT to neutrophil homogenates in order to decrease the lipid hydroperoxide levels did not significantly restore 5-LO inhibition by tryptanthrin (Figure 3B). In addition, tryptanthrin did not significantly inhibit partially purified human recombinant 5-LO under standard assay conditions (20 µM AA as substrate, 1 mM ATP and 1 mM Ca²⁺ as supplements), whereas the direct 5-LO inhibitor zileuton (used as control) reduced 5-LO product formation by 79% at 3 µM (Figure 3C). Also, no significant direct inhibitory effects of tryptanthrin on 5-LO activity were observed after removal of Ca²⁺ as stimulating cofactor or by lowering the substrate concentration (from 20 to 2 µM AA, not shown).

Analysis of the effect of tryptanthrin on 5-LO subcellular localization

Based on the finding that despite potently suppressing cellular 5-LO product formation, tryptanthrin failed to directly inhibit 5-LO, we investigated possible points of attack of tryptanthrin, which may cause suppression of 5-LO product synthesis in intact cells. A major event governing cellular 5-LO product formation is the localization of 5-LO at the nuclear membrane and access to its substrate (Werz et al., 2001; Werz, 2002). Stimulation of neutrophils by LPS and fMLP caused redistribution of 5-LO from the non-nuclear to the nuclear compartment, as assessed by subcellular fractionation by mild-detergent lysis and 5-LO immunodetection (Figure 4A). Tryptanthrin did not reduce LPS and fMLP-induced 5-LO translocation to the nucleus. Also the FLAP inhibitor MK886 only partially increased the amount of 5-LO in the non-nuclear fraction under these conditions. We therefore further analysed 5-LO subcellular localization by immunofluorescence microscopy (Figure 4B). In resting cells, (a) 5-LO was homogeneously distributed in the cytosol. Interestingly, after incubation with tryptanthrin we observed that (b) 5-LO accumulated within the perinuclear region. After activation with LPS and fMLP, (c) 5-LO was localized at the nuclear membrane, as indicated by the stain tracing out the three-lobulated nuclei of neutrophils, which was only partially altered by tryptanthrin (d) and both nuclear membrane and perinuclear staining were evident. The FLAP inhibitor MK886 (e) prevented the nuclear membrane localization of 5-LO induced by LPS and fMLP but did not restore the homogeneous cytosolic staining of 5-LO observed in resting cells. The compound interfering with the C2-like domain of 5-LO hyperforin (f) also reduced the amount of 5-LO at the nuclear membrane.

Figure 4.

Effect of tryptanthrin on 5-LO subcellular localization in human neutrophils. Effect of tryptanthrin and MK886 (100 nM) on 5-LO subcellular localization following activation of neutrophils with fMLP (1 µM; 5 min, 37°C) after 30 min priming with LPS (1 µg·mL⁻¹) plus Ada (0.3 U·mL⁻¹), analysed by (A) immunodetection of 5-LO in the nuclear (Nuc) and non-nuclear (Non-n) fractions of mild-detergent (0.1% NP-40)-lysed cells and (B) by immunofluorescence microscopy. (a) Vehicle; (b) tryptanthrin (30 µM); (c) vehicle (0.1% DMSO) + LPS/Ada/fMLP; (d) tryptanthrin (30 µM) + LPS/Ada/fMLP; (e) MK886 (100 nM) + LPS/Ada/fMLP; (f) hyperforin (10 µM) + LPS/Ada/fMLP. The results shown are representative of 3 independent experiments. In (B) pictures with staining for 5-LO (green, Alexa Fluor 488) are shown; scale bar, 5 µm.

Analysis of the effect of tryptanthrin on mechanisms governing cellular 5-LO product formation and on NF-κB activation

Cellular activation of 5-LO activity is also regulated by crucial signalling molecules (e.g. MAPKs, Ca²⁺, PKA) and by cellular peroxides (Werz and Steinhilber, 2005). Incubation of human neutrophils with tryptanthrin (30 µM) partially increased the phosphorylation state of p38 MAPK, but did not alter the phosphorylation of ERK1/2 and of PKA substrate proteins containing the phosphorylation motif RRXS/T (Figure 5A,B). However, tryptanthrin (1 to 30 µM) did not influence MAPK phosphorylation induced by fMLP, and also 30 µM tryptanthrin failed to suppress fMLP-stimulated increase in [Ca²⁺]_i (Figure 5C,D). Similarly, fMLP-induced elevation of the cellular peroxide tone was not reduced by tryptanthrin (30 µM, Figure 5E). In previous studies addressing the effects of tryptanthrin on cytokine production by lymphocytes, the inhibition of NF-κB has been hypothesized as a possible mechanism of action (Takei et al., 2003). However, tryptanthrin (30 µM) did not prevent LPS-induced degradation of IκBα in the cytosolic and nuclear fractions of neutrophils [an index of activation of NF-κB in neutrophils (Vancurova et al., 2001)] and/or the LPS-induced increase of p65 in the nucleus (Figure 5F,G).

Figure 5.

Effects of tryptanthrin on MAPK, [Ca⁺²]_i, ROS and NF-κB activation in neutrophils. Effect of vehicle (0.1% DMSO) or tryptanthrin (30 µM, 1.5 min, 37°C) on the phosphorylation state of (A) p38 MAPK (p-p38 MAPK) and ERK1/2 (pERK1/2) and (B) of proteins containing the PKA-phosphorylation motif (RRXS/T, a marker for PKA activation). In (B), phorbol myristate acetate (PMA; 0.1 µM) was used as control. Effect of tryptanthrin on fMLP-induced increase in (C) phosphorylated ERK1/2 (pERK1/2) and p38 MAPK (p-p38 MAPK), (D) intracellular Ca⁺² concentration, and (E) ROS formation in human neutrophils. Effect of 30 µM tryptanthrin on (F) the expression of IκBα in the non-nuclear (Non-n) and nuclear (Nuc) fractions of human neutrophils and (G) on nuclear accumulation of p65 after treatment with 1 µg·mL⁻¹ LPS or 1 µM fMLP for 30 min at 37°C. The results shown are representative of at least three independent experiments.

Tryptanthrin inhibits 5-LO product formation in human whole blood

Despite their strong efficacy in isolated cells, several inhibitors of LT biosynthesis failed to reduce 5-LO product formation in whole blood assays, seemingly due to high binding to plasma albumin or to interference/competition with other blood cells or with blood components (e.g. fatty acids) (Werz, 2002). Accordingly, these compounds also failed in subsequent in vivo studies in terms of both inhibition of LT synthesis and anti-inflammatory effectiveness. The ability of a certain compound to suppress 5-LO product synthesis in whole blood is thus considered to reflect its efficacy in vivo (Siemoneit et al., 2009). Human whole blood was pre-incubated with tryptanthrin for 10 min before stimulation with either LPS plus fMLP or with ionophore A23187. As shown in Figure 6A, tryptanthrin concentration-dependently reduced 5-LO product synthesis in whole blood with an IC₅₀ of about 10 µM for both A23187 and LPS plus fMLP stimulations. Similar to neutrophils, no differences were observed in the potency of tryptanthrin for inhibition of LTB₄ and of 5-H(P)ETE in whole blood (not shown). For the 5-LO inhibitor zileuton, the IC₅₀ values were 0.9 ± 0.1 µM and 2.5 ± 0.5 µM, for LPS/fMLP- and A23187-stimulated blood, respectively (Figure 6B).

Figure 6.

Tryptanthrin inhibits 5-LO product formation in human whole blood. Effect of (A) tryptanthrin and of (B) zileuton on 5-LO product synthesis in whole blood stimulated with fMLP (1 µM; 15 min, 37°C) after 30 min priming with LPS (1 µg·mL⁻¹) or with 30 µM A23187 ionophore (10 min, 37°C), as indicated. Data are expressed as percentage of control, means + SEM; n = 3, duplicates. **P < 0.01, ***P < 0.001 vs. control; anova+ Tukey's test. 5-LO products in 100% controls were (ng·mL⁻¹ plasma): LPS/fMLP, 30.6 ± 10; A23187, 252.5 ± 71.9.

Tryptanthrin inhibits LTB₄ formation in vivo

We evaluated the biological relevance of 5-LO inhibition by tryptanthrin in vivo in a rat model of carrageenan-induced pleurisy. The oral administration of 10 mg·kg⁻¹ tryptanthrin to rats 1 h before carrageenan significantly reduced LTB₄ pleural levels by 46% (Figure 7A). A significant parallel inhibition of the inflammatory reaction was observed, and tryptanthrin reduced PGE₂ levels (by 42%, Figure 7B), exudate volume (by 80%, Figure 7C) and the number of infiltrating cells (by 41%, Figure 7D).

Figure 7.

Tryptanthrin reduces LTB₄ levels in pleural exudates from carrageenan-treated rats. Rats (n = 10 for each experimental group) were treated p.o. with 10 mg·kg⁻¹ tryptanthrin (trypt) or vehicle (0.5 mL of 0.5% carboxymethylcellulose and 10% Tween 20, ctr) 1 h before intrapleural injection of carrageenan. (A) LTB₄ and (B) PGE₂ pleural exudate levels, (C) exudate volume, and (D) inflammatory cell accumulation in pleural cavity were assessed 4 h after carrageenan injection. Data are expressed as mean + SEM, n = 10. **P < 0.01, ***P < 0.001 vs. control; Student's t test.

Discussion and conclusions

Here we investigated the interference by tryptanthrin of 5-LO product formation in both cell-based and cell-free systems, taking into account critical components that are relevant for the in vivo pharmacology of LT synthesis inhibitors. We found marked inhibition of LT formation by tryptanthrin in intact neutrophils, which is in agreement with previous findings (Danz et al., 2002b). However, tryptanthrin was not found to be a direct 5-LO inhibitor, and also it did not inhibit classical signalling pathways and/or factors known to regulate 5-LO product formation in intact cells. These features suggest a novel molecular mechanism for its interference of 5-LO product formation different from other characterized blockers of LT synthesis such as inhibitors of cPLA₂, 5-LO or FLAP. Importantly, tryptanthrin was active in the human whole blood assay, which is considered a suitable system for prediction of the in vivo pharmacodynamics of anti-LTs. Finally, oral administration of tryptanthrin was shown to be effective as an anti-inflammatory agent in the rat model of pleurisy. Our data suggest that tryptanthrin is a promising drug candidate and may represent a new approach for pharmacological intervention with LTs.

LTs are potent mediators of inflammatory and allergic reactions (Peters-Golden and Henderson, 2007), and the development of anti-LT drugs is a major challenge. However, classical concepts for inhibition of 5-LO product synthesis (e.g. interference with the catalytic redox cycle of 5-LO, chelation of the active site iron, or competition with the AA substrate) have essentially failed in drug development. Redox active compounds, and to a lesser extent also iron-ligands, are poorly selective for 5-LO, often present unfavourable pharmacokinetics, and exert severe side effects (McMillan and Walker, 1992). The potency of non-redox-type inhibitors, on the other hand, is strongly impaired by elevated peroxide levels (Werz et al., 1998) and/or by phosphorylation of 5-LO by MAPKs (Fischer et al., 2003), which accompany inflammatory reactions. Also, several FLAP inhibitors show poor activity in whole blood, possibly due to high plasma protein binding or competition with fatty acids (Werz, 2002). To establish new approaches, natural compounds of plant origin offer both structural diversity and privileged scaffolds. In fact, we recently identified the acylphloroglucinol hyperforin from Hypericum perforatum as a novel type of 5-LO inhibitor with efficacy in vivo, acting by interference with the C2-like domain of 5-LO (Feisst et al., 2009).

Nevertheless, most of the plant-derived 5-LO inhibitors that proved effective in cell-free or cell-based systems (i.e. IC₅₀ < 50 µM) are redox-active compounds (e.g. flavonoids, polyphenols, coumarins, quinones) and thus not immediately suitable for drug development (Werz, 2007). Other promising natural products (e.g. boswellic acids from Boswellia species), instead, markedly lost efficacy in whole blood and were not effective in vivo in human subjects (Siemoneit et al., 2009). In comparison with those compounds, tryptanthrin possesses a unique, weakly basic indoloquinazoline scaffold, and our data reveal this substance as one of the most potent natural inhibitors of LT biosynthesis with proven effectiveness in whole blood and in vivo after oral administration. Besides tryptanthrin, the structurally related isaindigotone but also other alkaloids including colchicine, sanguinarine and chelerythrine, goshuyuamide-II, and certain alkyl-/alkylene-quinolone alkaloids have been reported to repress 5-LO product formation in intact cells (Werz, 2007). However, of these inhibitors, only isaindigotone has been found to have a direct effect on 5-LO (Molina et al., 2001), whereas for all the other alkaloids inhibition of 5-LO in cell-free assays was not addressed and their molecular mechanism of action is unknown.

So far, a definite assignment of tryptanthrin to a typical class of LT synthesis inhibitors is yet not possible, but our results imply that tryptanthrin is not a direct inhibitor of 5-LO. In fact, tryptanthrin does not possess redox features and does not inhibit 5-LO in cell-free assays, independently of the peroxide tone [as for non-redox type 5-LO inhibitors (Werz et al., 1998)] and of the interference with endogenous (phospho-)lipids [as for hyperforin (Feisst et al., 2009)]. In addition to 5-LO, cPLA₂ and FLAP are also seemingly not target candidates of tryptanthrin. Thus, tryptanthrin does not inhibit AA release from cPLA₂ and shows different features in comparison with the FLAP inhibitor MK-886 (Kargman et al., 1991; Mancini et al., 1993; Fischer et al., 2007). In fact, tryptanthrin only partially altered the localization of 5-LO at the nuclear membrane in stimulated cells, whereas MK886 (and also hyperforin) prevents this localization of 5-LO at the nuclear membrane. Intriguingly, however, tryptanthrin caused 5-LO redistribution, that is, the accumulation of 5-LO within the perinuclear region, as determined by immunofluorescence microscopy. After tryptanthrin treatment, 5-LO was mainly localized in the membranous area surrounding the nucleus (which is compatible with an endoplasmic reticulum staining) and only partially at the nuclear membrane, as instead observed after LPS/fMLP stimulation. We speculate that such subcellular redistribution of 5-LO may prevent activation of the enzyme and/or reduce the accessibility of 5-LO to stimuli and eventually to its substrate AA provided by FLAP. Interestingly, it has been previously observed that the 5-LO inhibitor HZ52 (Greiner et al., 2011) and dual 5-LO/FLAP inhibitors of the thiopyrano[2,3,4-c,d]indole class (Hutchinson et al., 1995) also cause redistribution of 5-LO from the cytosol towards the perinuclear/membrane region in the absence of stimulation. It is noteworthy that tryptanthrin potently reduced 5-LO product formation in cells stimulated with pathophysiologically-relevant stimuli (i.e. LPS and fMLP), but classical pathways activated by fMLP were not inhibited, indicating a rather specific effect on 5-LO physiology and not a general alteration of neutrophil responses.

In addition to inhibition of 5-LO product synthesis, tryptanthrin has been shown to possess other biological effects that might be beneficial in therapeutic use. On the one hand, the activity of tryptanthrin against a wide spectrum of pathogenic organisms has long been recognized (Schindler and Zahner, 1971), and its therapeutic potential as an antibacterial drug still attracts attention (Bandekar et al., 2010). Sepsis belongs to those diseases that have possible associations with LTs (Peters-Golden and Henderson, 2007), and the effects of tryptanthrin on 5-LO product formation might indeed be beneficial, in particular in the second stage of sepsis, when LTs might have vasculopathic effects (Benjamim et al., 2005). We showed that tryptanthrin is effective on LT synthesis when the bacterial products LPS and fMLP were used as stimuli, which prompts the evaluation of its pharmacological potential in in vivo experimental models of sepsis. Nevertheless, tryptanthrin might also have inhibitory effects on other relevant inflammatory pathways. In fact, we observed a reduction in the levels of both LTB₄ and PGE₂ after tryptanthrin administration in our model of rat pleurisy, which was accompanied by a reduction in exudate volume and infiltrating cells. In a previous study, we observed a similar inhibition of LTB₄ and inflammatory cells by the 5-LO inhibitors zileuton and hyperforin. On the other hand, the COX inhibitor indomethacin failed to suppress LTB₄ levels despite significant inhibition of infiltrating cells (Feisst et al., 2009), which suggests that reduced LTB₄ formation in this pleurisy model is not secondary to a reduction in inflammatory cells. In accordance with the reduction in PGE₂ in the rat model of pleurisy, tryptanthrin was shown to inhibit the activity of isolated COX-2 (IC₅₀ = 0.83 µM) (Danz et al., 2002b) and the LPS-stimulated synthesis of PGs in a variety of cells (IC₅₀ = 0.01–0.25 µM, depending on the cell type) (Danz et al., 2002b). Also, the formation of NO (IC₅₀∼ 7 µM) and the expression of inducible nitric oxide synthase was impaired by tryptanthrin in murine macrophages (Ishihara et al., 2000) and tryptanthrin was shown to have reduce oedema in vivo, in the second phase of carrageenan-induced paw oedema in mice (dosage: 50 mg·kg⁻¹ p.o.) (Recio et al., 2006), and to have beneficial effects in a murine model of inflammatory bowel disease (dosage: 100 mg·kg⁻¹ p.o. for 3 days) (Micallef et al., 2002). Whether or not tryptanthrin attenuates the activity of inflammation-related enzymes other than 5-LO by altering their subcellular location is not yet known. Interestingly, dual or multiple-target inhibitors interfering with both the LT and the PG synthetic pathways represent a major pharmacological strategy to establish a more effective anti-inflammatory therapy, with reduced number and severity of side effects (e.g. higher gastrointestinal tolerance). Tryptanthrin exhibits comparable potencies for inhibition of both LT and PG biosynthesis, which suggest a possible concomitant interference with both pathways of AA metabolism.

Recent studies have shown that tryptanthrin has antitumoural effects in leukaemia cells (Chan et al., 2009) and in a breast cancer cell line MCF-7 (Yu et al., 2007; 2009), indicating its potential as an anticancer agent. Although the role of 5-LO in tumour cell viability still requires further elucidation (Fischer et al., 2010), inhibition of LT formation in neutrophils by tryptanthrin may reduce the inflammatory component that accompanies and sustains the development of neoplasm. Finally, tryptanthrin has two major advantages for now drugs, stability and ease of synthesis (Bandekar et al., 2010). In fact, the potential of natural products in drug development has often been impaired by difficulties in isolation and/or synthesis in sufficient quantities. The synthetic accessibility of tryptanthrin also encourages research into future structural modifications for optimization of its pharmacological properties.

Glossary

5-H(P)ETE

5(S)-hydro(pero)xy-6-trans-8,11,14-cis-eicosatetraenoic acid

5-LO

5-lipoxygenase

AA

arachidonic acid

Ada

adenosine deaminase

cPLA₂

cytosolic phospholipase A₂

DMSO

dimethyl sulphoxide

DPPH

diphenylpicrylhydrazyl radical

DTT

dithiothreitol

FLAP

5-lipoxygenase-activating protein

fMLP

N-formyl-methionyl-leucyl-phenylalanine

LT

leukotriene

PGC

phosphate-buffered saline plus 1 mg·mL⁻¹ glucose and 1 mM CaCl₂

ROS

reactive oxygen species

SDS-b

SDS-PAGE sample loading buffer

STI

soybean trypsin inhibitor

Conflicts of interest

None.