INTRODUCTION
Xanthohumol (Xan) and resveratrol (Res) (Fig. 1) are naturally occurring substances and constituents of human nutrition. Res is formed as a phytoalexin in plants, including grapes to defend against parasitic and fungal infections. Chemically, Res is a stilbene and belongs to the group of polyphenols and accumulates up to 14 mg L−1 (62 µ m) in red wine (Stervbo et al. Reference Stervbo, Vang and Bonnesen2007). Likewise Xan belongs to the group of polyphenols but is structurally different and constitutes a prenylated chalcone. Xan is present as a hop extract in beers responsible for the bitter taste. In certain types of beer, Xan concentration is high and may exceed 1 mg L−1 (3 µ m) (Walker et al. Reference Walker, Lence and Biendl2008). The oral bioavailability of Xan measured in rats is about 30% (Legette et al. Reference Legette, Ma, Reed, Miranda, Christensen, Rodriguez-Proteau and Stevens2012). In contrast, Res has an even lower bioavailability of <1% due to extensive metabolism in intestine and liver (Walle, Reference Walle2011).
Molecular structures of resveratrol (Res) and xanthohumol (Xan).
For Res and related derivatives, several studies demonstrated that antioxidant, antiinflammatory and anticancer activities strongly depend on their chemical structure (Murias et al. Reference Murias, Jäger, Handler, Erker, Horvath, Szekeres, Nohl and Gille2005; Szekeres et al. Reference Szekeres, Fritzer-Szekeres, Saiko and Jäger2010). Furthermore, it was suggested that Res also has a beneficial effect on glucose and lipid metabolism; however, clinical trials did not always confirm these findings (Cottart et al. Reference Cottart, Nivet-Antoine and Beaudeux2014). For Xan, a broad-spectrum activity against Gram-positive bacteria, viruses, fungi and plasmodia was observed (Gerhäuser, Reference Gerhäuser2005). In humans, the oestrogenic activity of hop extracts is well documented (Liu et al. Reference Liu, Burdette, Xu, Gu, van Breemen, Bhat, Booth, Constantinou, Pezzuto, Fong, Farnsworth and Bolton2001a ) showing that this effect is not due to Xan but to related derivatives (Milligan et al. Reference Milligan, Kalita, Heyerick, Rong, De Cooman and De Keukeleire1999; Zanoli and Zavatti, Reference Zanoli and Zavatti2008). Furthermore, Xan also demonstrated antiproliferative and proapoptotic effects in various human cancer cell lines (Miranda et al. Reference Miranda, Stevens, Helmrich, Henderson, Rodriguez, Yang, Deinzer, Barnes and Buhler1999) as well as chemopreventive properties [reviewed by (Zanoli and Zavatti, Reference Zanoli and Zavatti2008)]. Xan and Res also interfere with mitochondrial functions in mammalian cancer cells thereby triggering apoptosis (Madan et al. Reference Madan, Prasad, Roy, George and Shukla2008; Strathmann et al. Reference Strathmann, Klimo, Sauer, Okun, Prehn and Gerhäuser2010). Inhibition of individual mitochondrial complexes and transient mitochondrial superoxide production by Xan in mammalian cells was suggested (Strathmann et al. Reference Strathmann, Klimo, Sauer, Okun, Prehn and Gerhäuser2010). In addition, inhibition of mitochondrial ATPase by targeting the F 1 subunit was reported for Res (Zheng and Ramirez, Reference Zheng and Ramirez1999).
In the present study, we were specifically interested in the role of mitochondria in the antileishmanial activity of Xan using Res as a reference compound since it was found to be effective against Leishmania amazonensis (Ferreira et al. Reference Ferreira, Soares, Nascimento, Pinto da Silva, Sarzedas, Tinoco and Saraiva2014). For Res a study demonstrated that the antiamastigote effect against Leishmania major inside macrophages is more related to host cell toxicity than to direct amastigote killing (Lucas and Kolodziej, Reference Lucas and Kolodziej2013). For Xan, an antiplasmodial effect was observed (Gerhäuser, Reference Gerhäuser2005).
Although these publications provide some information about modes of action of Xan and Res on Leishmania, to our knowledge no studies exist directly comparing effects of Xan and Res in Leishmania.
MATERIAL AND METHODS
Chemicals
Diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), glucose, KCN, K2HPO4, KH2PO4, Na2HPO4, NaCl, KCl, HCl, NaN3, succinate, sucrose, dinitrophenol (DNP) and tris(hydroxymethyl)aminomethane (TRIS) were obtained from Merck (Germany). Bovine serum albumin (BSA), cytochrome c3+ (cyt c3+), decylubiquinone (dUQ), heat-inactivated fetal bovine serum (HFBS), Giemsa stain, haemin, 2,6-dichlorophenol–indophenol (DCPIP), 3-carboxy-proxyl (CP•), sorbitol, dithiothreitol (DTT), penicillin–streptomycin solution, resazurin, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium] bromide (MTT), pentamidine isethionate (Pen), antimycin A (AA), rotenone (Rot), valinomycin (Val), streptomycin, penicillin, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), Brain–heart infusion (BHI) medium, RPMI 1640 and Schneider's medium were purchased from Sigma-Aldrich. Dubelco's Modified Eagle Medium (DMEM) was from Thermo Fisher Scientific and low-endotoxin FCS from Bio&Sell (Germany). Dimethyl sulfoxide (DMSO), desferal (DFO) and triethanolamine (TEA) were from Roth (Germany), Novartis Pharma (Germany) and Fluka (Germany), respectively; while 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine–HCl (CMH) was from Noxygen (Germany). Yeast extract powder was supplied by Amresco (USA). Idebenone (IQ) was obtained from Takeda (Japan). Oligomycin (Oligo) and 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) was purchased from Calbiochem. Decylubiquinol (dUQH2) was prepared from dUQ as described previously (Gille et al. Reference Gille, Staniek and Nohl2001). Res and Xan were obtained from Sigma-Aldrich and their purity was >98%.
Macrophages
Peritoneal macrophages from mouse (PMM) were isolated immediately before use from adult female BALB/c mice in ice-cold RPMI 1640 medium supplemented with antibiotics (100 µg streptomycin mL−1 and 100 U penicillin mL)−1.
The monocyte macrophage cell line J774A.1 (J774, Life Technologies cat. no. 12800-017) was grown in DMEM (high glucose, 1·5 g L−1 NaHCO3) with 50 000 U L−1 penicillin and 50 mg L−1 streptomycin in 50 mL TubeSpin bioreactors using a roller culture apparatus (5 rpm) in an incubator at 37 °C and 5% CO2 (Lam et al. Reference Lam, Herant, Dembo and Heinrich2009). J774 cells were passaged twice a week in a ratio of 1:10.
Leishmania
Leishmania amazonensis strain WHOM/77BR/LTB0016 provided by the Department of Immunology, Oswaldo Cruz Foundation (Brazil) was used. Leishmania amazonensis parasites were maintained in BALB/c mice's footpads (as amastigotes, LaA). All animal experiments were performed according to the ‘Guideline on the Care and Use of Laboratory Animals’ (National Research Council Washington, USA). The protocol for animal use was reviewed and approved by the Institutional Ethical Committee of the Institute of Tropical Medicine ‘Pedro Kouri’ (ID: CEI-IPK 14-12). Healthy animals were obtained from the National Centre of Laboratory Animals Production (CENPALAB), Havana, Cuba and maintained under standard conditions. LaA were isolated by aspiration with a needle and maintained as promastigote form [Leishmania amazonensis promastigotes (LaP)] in Schneider's medium containing 10% HFBS and antibiotics (100 µg streptomycin mL−1 and 100 U penicillin mL−1) at 28 °C.
Leishmania tarentolae promastigotes (LtP) strain P10 from Jena Bioscience (Germany) were used. Parasites were cultivated at 26 °C in yeast extract medium (YEM) consisting of 20·7 g L−1 yeast extract powder, 1·2 g L−1 K2HPO4, 0·2 g L−1 KH2PO4, 2·9 g L−1 glucose or in BHI medium (37 g L−1) supplemented each with 5 mg L−1 haemin and 50 000 U L−1 penicillin and 50 mg L−1 streptomycin in 50 mL Saarstedt tubes with gas-permeable caps and agitation in a tube shaker (0·05 s−1).
Influence of Res and Xan on cell viability
To analyse the activity of Res and Xan in intracellular LaA, experiments were carried out to determine the viability of parasites after treatment. In a 24-well plate with cover slips, a monolayer of PMM was obtained. Afterwards, LaP in stationary-phase were added at a 4:1 parasite/macrophage ratio. The cultures were incubated for further 4 h and free parasites were removed. Then, 1995 µL of RPMI medium and 5 µL of compounds were added. Five 1:2 dilutions were carried out passing each time 1000 µL to the next well. The plate was incubated for 48 h and the experiment was carried out in triplicate (Torres-Santos et al. Reference Torres-Santos, Moreira, Kaplan, Meirelles and Rossi-Bergmann1999). The parasites were then fixed with methanol, stained with Giemsa and examined using light microscopy at a magnification of 1000×, applying immersion oil. The number of intracellular amastigotes was determined by counting the amastigotes in 100 macrophages per sample. The results were expressed as percentage decrease in the infection rate compared with that of the controls. The infection rates were obtained by multiplying the percentage of infected macrophages by the mean number of amastigotes per infected macrophage (Torres-Santos et al. Reference Torres-Santos, Moreira, Kaplan, Meirelles and Rossi-Bergmann1999; Delorenzi et al. Reference Delorenzi, Attias, Gattass, Andrade, Rezende, da Cunha, Henriques, Bou-Habib and Saraiva2001). In parallel, cytotoxicity of compounds was determined in PMM. Macrophages were harvested, distributed to 96-well plates (1–6 × 105 PMM mL−1) and incubated at 37 °C under an atmosphere of 5% CO2 for 2 h. Non-adherent cells were removed and 2 µL of Res or Xan dissolved in DMSO were added to 98 µL of RPMI medium containing 10% HFBS and antibiotics (100 µg streptomycin mL−1 and 100 U penicillin mL−1). Following, 1:2 serial dilutions were performed and the plate was incubated during 48 h at same conditions. Then, cytotoxicity was determined using the colorimetric assay based on MTT reduction. MTT solutions were prepared at 5 mg mL−1 in saline solution, sterile-filtered immediately before use, and 15 µL were added to each well. After incubation for additional 4 h, supernatant was eliminated and the accumulated formazan crystals were dissolved by addition of 100 µL DMSO per well. The optical density was determined using an EMS Reader MF Version 2.4–0 (Pharmacia, LKB, USA), at a wavelength of 560 nm and 630 nm as reference wavelength. The influence of the vehicle DMSO was negligible in used concentrations.
In addition, the viability of LaP was determined. Fifty microlitres of Schneider's medium with HFBS and antibiotics were distributed in each well of a 96-well plate. In the first one, additional 48 µL of medium were added and 2 µL of Res or Xan. Subsequently, five 1:2 dilutions were carried out. Afterwards, 50 µL of parasites at 2 × 105 promastigotes mL−1 in logarithmic phase were added to each well. Plates were sealed with parafilm and incubated at 26 °C during 72 h. Then, 20 µL of MTT was added to each well and cell viability was measured as described above.
In analogy, LtP in YEM/Phosphate-buffered saline (PBS) (1:1) were distributed in 96-well plates at 4 × 106 cells mL−1. Compounds were added and five 1:3 serial dilutions were performed. Control rows with YEM/PBS (no activity) and with untreated (vehicle, DMSO) LtP (100% activity) were loaded. Then, 50 µL of resazurin stock solution was added in each well (20 µ m final concentration) and the plate was incubated at 26 °C for 48 h. Finally, the absorption of resazurin was measured at 600 and 570 nm using a plate reader (Enspire, PerkinElmer, Waltham, MA, USA). From these data, the viability of cells in the absence and presence of drugs was calculated. In analogy to LtP resazurin assays, the J774 resazurin assays were performed with the exception that 8 × 104 cells mL−1 in DMEM were used. In all cases, Pen was used as reference drug and tested in parallel against LaA, LaP, LtP, J774 and PMM.
Influence of Res and Xan on oxygen consumption
To assay the effect of compounds on oxygen consumption of LtP, a Clark-type oxygen electrode (Hansatech, Germany) and software MCREC were used. LtP at 108 cells mL−1 in YEM (25 °C) were added and treated with increasing concentrations of test compounds between 10 and 200 µ m. Each concentration was assayed in quadruplicates and the results were expressed as percentage of oxygen consumption in comparison with the untreated control LtP. The highest concentration of the vehicle (1% DMSO) caused only 2% inhibition.
OxoPlates are U-shaped 96-well plates (OP96U PreSens, Germany) with integrated fluorescence oxygen sensors. Oxygen concentrations were measured using a Perkin Elmer Enspire fluorescence plate reader using excitation wavelength 540 nm and two emission wavelengths (reference dye 590 nm, I Ref, O2-sensing dye 650 nm, I Ind). The fluorescence intensity ratio I R = I Ind/I Ref was used in the following formula to calculate the oxygen concentration (μ m O2) according to the instructions of the manufacturer:
$$C_{{\rm O}_ {2}} = 2.48 \times 100 \times \displaystyle{{(k_0 /I_{\rm R} - 1)} \over {(k_0 /k_{100} - 1)}}.$$
OxoPlates were calibrated with air-saturated buffer (I R = k 100) and sodium dithionite-treated buffer (1% w/v) (I R = k 0). Measurements with LtP and J774 were performed in air-saturated PBS/YEM (1:1) and DMEM (without FCS), respectively. After calibration of the plates, they were loaded either with 200 µL medium (medium controls for drift corrections), 50 µL medium in wells for untreated control cells or 50 µL medium with test substances (Xan, Res, inhibitors/uncouplers of mitochondrial respiration) to wells for treated cells. Immediately before the measurement 150 µL of well-aerated cell suspensions were added to the respective wells to give final cell counts of 0·75–1 × 108 LtP mL−1 and 4–5 × 106 J774 cells mL−1, respectively. Finally on the top of each well, 50 µL paraffin oil was layered. Two min after mixing the fluorescence measurements at 27 °C were started and eight measurements at 5 min intervals were performed. From the linear part of the O2 decay the slopes were calculated and corrected for the medium drift for further statistic evaluation. All measurements were performed at least in triplicate. For inhibition studies the concentration ranges explored were: in LtP Xan, Res, Pen (0·41–100 µ m) and in J774 cells Xan, Res (0·82–200 µ m) and Pen (0·41–100 µ m).
For bioenergetic characterization following inhibitor mixtures were used: 5 µ m Oligo only, 5 µ m Oligo + 100 µ m DNP, 5 µ m Oligo + 0·5 µ m CCCP, 5 µ m Oligo + 1 µ m CCCP and 4 µ m Rot + 4 µ m AA. All concentrations given above are final concentrations in the OxoPlate wells. Derived bioenergetic parameters were calculated according to Brand and Nicholls (Reference Brand and Nicholls2011).
Influence of Res and Xan on mitochondrial complexes
Isolation of the mitochondrial fraction from LtP
To isolate the mitochondrial fraction from LtP (LtP-Mit), a culture of 2700 mL of LtP was centrifuged for 10 min at 1900 × g and 20 °C and the supernatant was discarded. The cell pellet was resuspended in buffer (10 mm TRIS–HCl, 0·3 m sucrose, 0·2 mm EDTA, 0·2% BSA, pH 7·4) and washed in two extra centrifugation steps for 10 min at 1900 × g and 20 °C. The washed cell pellet was incubated in lysis buffer (5 mm TRIS–HCl, pH 7·4) for 10 min at 20 °C and subsequently homogenized in a Dounce homogenizer. Cell debris was removed by centrifugation for 10 min at 1900 × g and 4 °C. The supernatant was centrifuged for 20 min at 13 200 × g and 4 °C to sediment the crude mitochondrial fraction. LtP-Mit were resuspended in 1 mL buffer (250 mm sucrose, 50 mm KH2PO4, 0·2 mm EDTA, pH 7·2) and stored in liquid nitrogen until use. Protein content was assayed by the Biuret method using BSA as standard (Gornall et al. Reference Gornall, Bardawill and David1949).
Isolation of mitochondrial fractions from other species
Bovine heart submitochondrial particles (BH-SMP) were obtained from bovine heart mitochondrial suspensions (Nohl and Hegner, Reference Nohl and Hegner1978). Mitochondrial suspensions were sonicated ten times for 30 s with 1 min interruption at 4 °C in buffer (0·25 m sucrose, 10 mm TRIS, 1 mm EDTA, pH 9·0) using a Branson Sonifier (USA) set to 40 W energy output. Remaining mitochondria were removed by centrifugation at 6500 × g for 10 min at 4 °C and afterwards BH-SMP were sedimented from the supernatant by centrifugation at 95 000 × g for 30 min. Pellets were resuspended in buffer (0·25 m sucrose, 10 mm TRIS, 1 mm EDTA, pH 7·4) and stored in liquid nitrogen until use.
The preparation of the yeast mitochondria (YM) was performed according to the method of Daum et al. (Reference Daum, Bohni and Schatz1982). Yeast cells (W303 strain) were harvested by centrifugation at 1464 × g for 5 min at room temperature. The pellets were resuspended in 100 mL buffer I (10 mm TRIS, 10 mm DTT, pH 9·4) and transferred to centrifugation tubes. After 15 min incubation at 37 °C, cells were centrifuged again. Subsequently centrifugation tubes were tared and filled with cells resuspended in 60 mL buffer II (1·2 mm sorbitol, 20 mm potassium phosphate, pH 7·4). After a third centrifugation, the weight of the cell pellets was determined. To prepare spheroblasts the pellets were suspended in buffer II and 2 mg zymolyase were added per gram yeast cells. Then the suspension was incubated for 45 min at 28 °C. The following steps were performed at 4 °C. Spheroblasts were collected by centrifugation at 1464 × g for 5 min, resuspended in 60 mL buffer II, sedimented by another centrifugation and homogenized in as little buffer III (0·6 m sorbitol, 20 mm TRIS, pH 7·4) as possible. Then the suspension was homogenized by 20 strokes in a Wheaton 15 mL Dounce tissue grinder (tight-fitting pestle). Cells and cell debris were removed by two centrifugations at 1464 × g for 5 min. YM were collected from the supernatant by centrifugation at 11 952 × g for 10 min. Mitochondrial fractions from rat liver (RLM) of Sprague-Dawley rats were previously isolated by consecutive differential centrifugation steps as described in Staniek et al. (Reference Staniek, Rosenau, Gregor, Nohl and Gille2005) and stored in liquid nitrogen.
Inhibition of mitochondrial electron transfer activities
Succinate:ubiquinone oxidoreductase (complex II) activities were measured using DCPIP as terminal electron acceptor. In a 1 mL cuvette buffer (250 mm sucrose, 20 mm TEA, 1 mm EDTA, pH 7·4), DCPIP (20 µ m), KCN (1 mm), BSA (1·25 mg mL−1), IQ (62·5 µ m) and BH-SMP (0·159 mg mL−1) or RLM (0·318 mg mL−1) or YM (W303 strain, 0·0292 mg mL−1) or LtP-Mit (0·08 mg protein mL−1) were added. The reaction was started by adding succinate (4 mm). The reduction of DCPIP was followed photometrically at 600 nm using 780 nm as a reference wavelength with a Shimadzu Multispec 1501 diode array photometer in the presence of test compounds or the corresponding amount of vehicle (DMSO). DCPIP reduction rates were calculated using an extinction coefficient of 19 100 L mol−1 cm−1 at 600 nm. For each individual concentration triplicates were measured.
To measure the inhibition of ubiquinol:cytochrome c oxidoreductase (complex III) dUQH2 (75 µ m) and cyt c3+ (100 µ m) were used as substrates. The reduction of cyt c3+ in the presence of dUQH2 was monitored at 550 nm using 540 nm as reference wavelength in a buffer consisting of 250 mm sucrose, 50 mm KH2PO4, 0·2 mm EDTA, 2 mm KCN, 4 mm NaN3, pH 7·2 (Müllebner et al. Reference Müllebner, Patel, Stamberg, Staniek, Rosenau, Netscher and Gille2010). Test compounds were added at different concentrations 120 s prior to addition of dUQH2 starting the enzymatic reaction. In the presence of test compounds, the residual dUQH2:cyt c3+ oxidoreductase activity of BH-SMP (0·0032 mg mL−1), RLM (0·0425 mg mL−1), YM (0·0291 mg mL−1) and LtP-Mit (0·04 mg mL−1) was obtained and expressed in percentage of the non-inhibited rates (100%). For all test compounds, the absence of significant reactions with dUQH2 and cyt c3+ was verified in the absence of mitochondrial preparations. All compound concentrations were tested in triplicate and the reduction rates for cyt c3+ were calculated from the time trace of the absorption difference at 550 nm minus 540 nm using the extinction coefficient ε 550–540 nm = 19 000 L mol−1 cm−1.
Influence of Res and Xan on superoxide radical production in LtP
Detection of superoxide radicals was performed using the cyclic hydroxyl amine CMH as reaction partner and measuring the formed stable nitroxyl radicals by electron spin resonance (ESR) spectroscopy. Measurements were performed in PBS buffer (136 mm NaCl, 1·15 mm KH2PO4, 14 mm Na2HPO4, 2·7 mm KCl, pH 7·4) containing 100 µ m DFO and 25 µ m DTPA. Before measurements LtP suspensions were washed twice with PBS to remove YEM. Samples contained 5 × 108 LtP mL−1 and were supplemented with 16 mm glucose. Simultaneously drug stock solutions in DMSO were added. Prior to the ESR measurement 400 µ m CMH was added. For ESR measurements 17 µL of suspension was aspirated in a gas-permeable Teflon tube (ø 0·7 mm). This capillary tube was placed in a resonator (split ring, Bruker MD5) of the ESR instrument (Bruker EMX) and ten sequential measurements were performed. Following instrument settings were used: microwave frequency 9·682 GHz, modulation frequency 100 kHz, modulation amplitude 1 G, time constant 0·082 s, centre field 3446 G, scan rate 71 G min−1, sweep width 100 G, scan time 84 s and attenuation 7·96 × 103. From the ESR spectra, the middle peak intensity was retrieved and concentrations of oxidized CMH were obtained by comparison with a standard curve prepared from CP• solutions with defined concentrations. In an additional experiment, the influence of Res (100 µ m) and Xan (100 µ m) on the CP• (3 µ m) signal was studied in PBS (pH 7·4) containing 100 µ m DFO and 25 µ m DTPA using same conditions as for CMH.
Influence of Res and Xan on mitochondrial membrane potential in LtP
JC-1 was used to investigate the changes in mitochondrial membrane potential of LtP. From a stock solution of JC-1 (6·1 mm) in DMSO a diluted solution (15 µ m JC-1) in PBS (pH 7·4) and 15 mm glucose was prepared. The cell density of LtP was adjusted to approximately 5 × 107 LtP cells mL−1. LtP cells cultured in YEM were centrifuged in a sterile 50 mL tube for 10 min at 2000 × g at 20–25 °C. After the centrifugation, the supernatant was discarded and the cell pellet was resuspended in a corresponding volume of the diluted JC-1 solution. To load cells with JC-1 they had to be incubated for 30 min under dark conditions in the incubator (26 °C). Then, a washing step with 10 min centrifugation at 2000 × g at 20–25 °C was included. The supernatant was removed and the pellet was resuspended in a PBS/glucose solution (without JC-1). Subsequently, 200 µL cell suspension loaded with JC-1 were placed in each well of a black 96-well culture plate. In the third row, 100 µL extra suspension were added and in the respective wells Res (200 µ m), Xan (200 µ m), Val (10 µ m), CCCP (0·5 µ m), oligomycin (Oligo, 10 µ m) and AA (0·1 µ m) were placed. Fluorescence measurements (Perkin Elmer Enspire) were started after 4 h of incubation using an excitation wavelength of 485 nm and an emission wavelength of 590 nm for JC-1 aggregates. The fluorescence intensity of control cells (including DMSO) was set to 100% and fluorescence intensities of other samples were expressed in relation to this value.
Statistical analyses
Percentages of residual activity in relation to control experiments (100%) were calculated at each concentration of compounds. Resulting activity-concentration plots were used to determine the half inhibitory concentration (IC50) values by a non-linear regression according to a four-parameter logistic model (4PL, Hill-Slope model) (Müllebner et al. Reference Müllebner, Patel, Stamberg, Staniek, Rosenau, Netscher and Gille2010) using Origin 6·1 (OriginLab Corporation). In case of PMM, LaP and LaA, the IC50 value was obtained by fitting a sigmoidal E max model to dose–response curves (Bodley and Shapiro, Reference Bodley and Shapiro1995). Selectivity indices were calculated by dividing the IC50 values for PMM by the IC50 values for LaA. Statistically significant differences were identified using Student's t-test.
RESULTS
In this work, two natural products which are also present in certain beverages were compared for their effects on Leishmania parasites and their mitochondrial functions. Both, Res and Xan, are polyphenols but nevertheless structurally different (Fig. 1). While Res can be classified as stilbene derivative, Xan is a chalcone substituted by an isoprenyl residue. For the lipophilicity of Res and Xan, the logPOW values were predicted to 3·1 ± 0·8 and 5·0 ± 1·5, respectively. The water solubility (in cell free systems) of Res was calculated to be 15·8 ± 4·6 µ m and that of Xan to be 3·9 ± 1·0 µ m (ALogPS 3·0, Ochem Predictor) (Sushko et al. Reference Sushko, Novotarskyi, Korner, Pandey, Rupp, Teetz, Brandmaier, Abdelaziz, Prokopenko, Tanchuk, Todeschini, Varnek, Marcou, Ertl, Potemkin, Grishina, Gasteiger, Schwab, Baskin, Palyulin, Radchenko, Welsh, Kholodovych, Chekmarev, Cherkasov, Aires-de-Sousa, Zhang, Bender, Nigsch and Patiny2011).
In a first set of experiments, Res and Xan were tested for their antileishmanial activity. The pathologically relevant model systems consisted of PMM and pathogenic LaA. In the viability assay, the anti- leishmanial activity of Xan against amastigotes was confirmed with an IC50 value in the low micromolar range (Table 1), while Res required about 2-fold higher concentrations for amastigote killing. With respect to PMM viability Res was found to be less cytotoxic than Xan. In addition, the antileishmanial activity was tested in LaP and LtP (model for mechanistic studies). In both LaP and LtP (Table 2) Res showed the highest IC50 values, while Pen as reference compound was effective at 1 µ m in both leishmanial species. Xan was more effective than Res in all systems, however, with different IC50 values. For mechanistic studies in mammalian cells the macrophage cell line J774A.1 was used. The IC50 values of J774 cells in the resazurin viability assay (Table 2) corresponded to the IC50 values of PMM (Table 1). Specifically an excellent agreement for Xan and Pen was observed, while the IC50 value of Res was lower than in PMM.
Influence of pentamidine isethionate (Pen, reference drug), resveratrol (Res) and xanthohumol (Xan) on the viability of L. amazonensis amastigotes (LaA) and peritoneal macrophages from mouse (PMM)
IC50 values for LaA were obtained from a direct count in stained cultures by Giemsa using serial dilutions of the compounds. Cytotoxicity in PMM was determined by an MTT assay. Both experiments had an endpoint at 48 h. Data represent means ± s.d. from three independent cell batches.
Influence of pentamidine isethionate (Pen, reference drug), resveratrol (Res) and xanthohumol (Xan) on the viability of L. amazonensis promastigotes (LaP), L. tarentolae promastigotes (LtP) and J774 macrophages
IC50 values for LaP were obtained from an MTT assay using serial dilutions of the compounds with an endpoint at 72 h using 105 cells mL−1. IC50 values for LtP and J774 were obtained from a resazurin viability assay with an endpoint at 48 h using 4 × 106 cells mL−1 (LtP) or 1×105 cells mL−1 (J774). Data represent means ± s.d. from three independent cell batches.
Since mitochondrial genome homology between different Leishmania species is high (Yatawara et al. Reference Yatawara, Le, Wickramasinghe and Agatsuma2008) we used the LtP model to evaluate the role of mitochondria in the leishmanicidal activity of Res and Xan.
For an overview, the influence of Res and Xan on LtP oxygen consumption using a Clark electrode was studied (Fig. 2). Neither Res nor the reference antileishmanial agent Pen inhibited LtP oxygen consumption strongly up to 200 µ m. In contrast, Xan exhibited an IC50 value of 16 µ m and therefore a strong inhibition of mitochondrial oxygen consumption in Leishmania. To extend these experiments oxygen consumption measurements were performed in the 96-well format using Oxoplates (Table 3). Again, in LtP Res and Pen did not show strong inhibition, while Xan inhibited oxygen consumption with an IC50 of about 7 µ m. This confirms the measurement with the Clark electrode for LtP. Likewise, in J774 macrophages no strong inhibition of oxygen consumption by Res and Xan was observed which fits to the viability results in PMM and J774 cells (Tables 1 and 2).
Oxygen consumption of L. tarentolae promastigotes in yeast extract medium and its inhibition by resveratrol (Res), xanthohumol (Xan) and pentamidine (Pen). Xan exhibited an IC50 value of 16 ± 1 µ m, while Res and Pen showed 16 and 28% inhibition at 200 µ m. Non-inhibited oxygen consumption of cells was 9·3 nmol O min−1 (108 cells)−1, which was set to 100%. Data represent mean ± s.e. from four independent experiments.
Influence of pentamidine isethionate (Pen, reference drug), resveratrol (Res) and xanthohumol (Xan) on the oxygen consumption of L. tarentolae promastigotes (LtP) and J774 macrophages
IC50 values for LtP and J774 were measured in 96-well Oxoplates using 1 × 108 and 8 × 104 cells mL−1, respectively. Data represent means ± s.d. from three independent cell batches.
Next experiments addressed the question of the specific target of Xan in the electron transfer chain (ETC) of Leishmania. Therefore, we studied the influence of both compounds on the succinate:ubiquinone oxidoreductase (complex II) activity in mitochondria from LtP (Fig. 3). In agreement with results from oxygen consumption in LtP, we observed a strong inhibition with Xan, but only small effects with Res. Comparing the complex II inhibition in LtP-Mit with that in mitochondria from other species (Table 4) the inactivity of Res is observed in all mitochondria, while Xan inhibited mitochondria from mammalian cells in a similar range (RLM) or even stronger (BH-SMP). Only in YM Xan was less effective.
Complex II activity of L. tarentolae promastigote mitochondria and its inhibition by resveratrol (Res) and xanthohumol (Xan). Xan exhibited an IC50 value of 45·1 ± 3·3 µ m. Res showed an inhibition of 25% at 150 µ m. Non-inhibited complex II activity of mitochondria (0·08 mg protein mL−1) was 6·95 nmol min−1 mL−1, which was set to 100%. Data represent mean ± s.d. from three independent experiments.
Inhibition of complex II activities in mitochondrial fractions from different species by resveratrol (Res) and xanthohumol (Xan)
Data represent means ± s.d. from three independent experiments. Protein concentrations were: submitochondrial particles from bovine heart (BH-SMP) 0·159 mg mL−1, rat liver mitochondria (RLM), 0·318 mg mL−1, yeast mitochondria, W303 strain (YM) 0·0292 mg mL−1. Non-inhibited activities of mitochondrial fractions were: BH-SMP 4·5 nmol min−1 mL−1, RLM 16·4 nmol min−1 mL−1 and YM 2·98 nmol min−1 mL−1.
In analogy, the influence of both compounds on the ubiquinol:cytochrome c oxidoreductase (complex III) activity was tested. In these experiments, the difference between Xan and Res was even more pronounced (Fig. 4). While Res had almost no effect, Xan showed an IC50 value in the micromolar range. For both, complex II and III activities, the IC50 values of Xan with 45·1 ± 3·3 µ m and 58·2 ± 3·7 µ m, respectively, were rather similar. Comparison of complex III inhibition in LtP mitochondria to that in mitochondria from other species (Table 5), shows that also in these species Res had no strong effect, while Xan inhibited the complex III activity even stronger.
Complex III activity of L. tarentolae promastigote mitochondria and its inhibition by resveratrol (Res) and xanthohumol (Xan). Xan exhibited an IC50 value of 58·2 ± 3·7 µ m. Res showed no inhibition at 300 µ m. Non-inhibited complex III activity of mitochondria (0·04 mg protein mL−1) was 6·31 nmol min−1 mL−1, which was set to 100%. Data represent mean ± s.d. from three independent experiments.
Inhibition of complex III activities in mitochondrial fractions from different species by resveratrol (Res) and xanthohumol (Xan)
Data represent means ± s.d. from three independent experiments. Protein concentrations were: submitochondrial particles from bovine heart (BH-SMP) 0·0032 mg mL−1, rat liver mitochondria (RLM) 0·0425 mg mL−1, yeast mitochondria, W303 strain (YM) 0·0291 mg mL−1. Non-inhibited activities of mitochondrial fractions were: BH-SMP 15·7 nmol min−1 mL−1, RLM 11·2 nmol min−1 mL−1 and YM 25·2 nmol min−1 mL−1.
The influence of Res and Xan on mitochondrial superoxide radical formation was tested by ESR experiments with LtP cells using the conversion of CMH to 3-methoxycarbonyl-proxyl (CM•) for superoxide radical detection (Fig. 5). While the negative (Bu, buffer) and the positive controls (+AA) yielded reasonable values in comparison with cells alone, Res and even stronger Xan diminished CMH oxidation in the presence of cells. Control experiments revealed that 100 µ m Res or Xan did not directly diminish the slope of the ESR signal change of CP• (an analogue of CM•, which is available as pure substance) over the measurement time for CMH-mediated superoxide radical detection.
CMH oxidation by L. tarentolae promastigotes (5 × 108 cells mL−1, 17 µL) in the presence of resveratrol (Res, 100 µ m), xanthohumol (Xan, 100 µ m) and AA (5 µ m) was measured by ESR spectroscopy in oxygen-permeable Teflon tubes. ‘Cells only’ samples contained the corresponding amount of vehicle (max. 2% DMSO) used for xenobiotics. Buffer (Bu) samples containing vehicle were prepared without cells. Relative changes of CMH oxidation in comparison with ‘Cells only’ samples (100%) were: Res (72 ± 5%), Xan (32 ± 2%) and AA (145 ± 27%). Data represent mean ± s.d. from quadruplicates and * indicates statistically significant differences on the level P < 0·05 with respect to ‘Cells only’ samples.
A major mitochondrial function is the production of ATP, which requires the mitochondrial membrane potential to drive ATP synthase. Therefore, the influence of Xan and Res on the mitochondrial membrane potential was measured using the JC-1 method (Fig. 6). As expected the ionophore Val, the mitochondrial uncoupler CCCP and the complex III inhibitor AA strongly decreased the amount of JC-1 aggregates. In contrast, Oligo, an inhibitor of ATP synthesis, increased the concentration of JC-1 aggregates. Incubation with Res and Xan for 4 h resulted in a significant decline of the aggregates and therefore of the mitochondrial membrane potential.
Mitochondrial membrane potential in L. tarentolae promastigotes. 5 × 107 cells mL−1 were suspended in the presence of resveratrol (Res, 200 µ m), xanthohumol (Xan, 200 µ m), valinomycin (Val, 10 µ m), carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 0·5 µ m), oligomycin (Oligo, 10 µ m) and antimycin A (AA, 0·1 µ m). Aggregates of JC-1 (15 µ m) were measured by fluorescence (λ Ex = 485 nm, λ Em = 590 nm). The fluorescence of the control cells was set to 100%. Data represent mean ± s.d. from quadruplicates and * indicates statistically significant differences on the level P < 0·05 with respect to control.
To extend the understanding why Xan inhibits isolated mitochondria both in mammalian cells and Leishmania but nevertheless shows some selectivity for LtP in whole cell models, we compared the bioenergetic properties of mitochondria in LtP and J774 cells (Fig. 7, Table 6). Basal oxygen consumption was assessed in LtP and J774 cells within glucose-containing media in the absence of inhibitors/uncouplers. After addition of a mixture of potent mitochondrial inhibitors (AA and Rot) remaining oxygen consumption can be only of extra-mitochondrial origin. Surprisingly LtP exhibit virtually no extra-mitochondrial oxygen consumption. In contrast, in J774 cells about 30% of basal oxygen consumption corresponds to extra-mitochondrial respiration (possibly arising from NADPH oxidases). In the presence of Oligo, an inhibitor of the ATP synthase, oxygen consumption utilized for ATP synthesis was blocked. In J774 cells, about 60% of mitochondrial respiration is used for ATP production (ATP turnover). In LtP, this ATP turnover amounts only to about 30% of mitochondrial respiration. In the presence of the uncoupler DNP or CCCP, the maximal mitochondrial respiration was determined. In J774 cells, this uncoupler-accelerated respiration is more than 200% of mitochondrial respiration, while in LtP maximal uncoupled mitochondrial respiration is barely above normal mitochondrial respiration. The coupling efficiency (LtP: 0·3 vs J774: 0·6) and respiratory control ratios (LtP: 1·4 vs J774: 5·9) clearly indicate that LtP has a low efficiency in ATP production and cannot increase mitochondrial electron transfer under stress conditions as deduced from the lacking spare capacity (maximum uncoupler-stimulated mitochondrial respiration minus coupled mitochondrial respiration). This is a possible explanation why a comparable complex II and III inhibition by Xan in isolated mitochondria results in more severe consequences in LtP than in J774 cells.
Oxygen consumption of L. tarentolae promastigotes (LtP) and J774 macrophages in the absence and presence of inhibitors for bioenergetic characterization. Measurements were performed in Oxoplates with 1 × 108 cells mL−1 for LtP (A) or with 8 × 104 cells mL−1 for J774 (B). Basal oxygen consumption was assessed in the absence of inhibitors/uncouplers (cells). The proton leak rate of mitochondrial membranes was determined in the presence of the ATP synthase inhibitor oligomycin (Oligo, 5 µ m). To obtain the maximally uncoupled respiration different Oligo/uncoupler mixtures were applied (5 µ m Oligo + 100 µ m DNP, 5 µ m Oligo + 0·5 µ m CCCP, 5 µ m Oligo + 1 µ m CCCP). Non-mitochondrial oxygen consumption was measured in the presence of 4 µ m rotenone (Rot) + 4 µ m antimycin A (AA). All measurements were performed in quadruplicates and data represent mean ± s.d.
Bioenergetic parameters of L. tarentolae promastigotes (LtP) and J774 macrophages derived from experiments shown in Fig. 7 using the highest uncoupler-stimulated oxygen consumption rates (LtP: at 1 µ m CCCP, J774: at 100 µ m DNP) for calculation
DISCUSSION
Xan and Res are significant bioactive ingredients of widely consumed beverages. When ingested in moderate amounts (Liu et al. Reference Liu, Wang, Lam and Xu2008) their polyphenol ingredients are considered to have beneficial effects on the cardiovascular and other organ systems (Magalhaes et al. Reference Magalhaes, Carvalho, Cruz, Guido and Barros2009; Nabavi et al. Reference Nabavi, Li, Daglia and Nabavi2014; Yang et al. Reference Yang, Li and Ren2014; Yao et al. Reference Yao, Zhang, Ge, Peng and Fang2015). Due to these findings beers with high Xan content were identified or even specifically produced (Wunderlich et al. Reference Wunderlich, Zurcher and Back2005; Walker et al. Reference Walker, Lence and Biendl2008). Likewise methods for increasing the Res content of red wines were proposed (Sun et al. Reference Sun, Liang, Kang, Huang, Huang, Ye, Guo and Lin2015). In parallel, the intake of food supplements of Res and Xan is on the rise. These compounds may also have a beneficial effect in the treatment of infectious protozoal diseases. Due to the climate change, endemic regions for leishmaniasis tend to spread globally. The fast growing beverage market in the affected areas with endemic leishmaniasis, such as in Brazil, generates an increased intake of Xan via beer consumption (de Araujo et al. Reference de Araujo, Pinheiro, Almeida, de Menezes, Morais, Reis, Assuncao and Carneiro2013). Although there were a few studies on the in vitro antiprotozoal activity of Res and Xan derivatives, so far there was no study, which directly compared the activity of both compounds in the same model using the same methods. Therefore, we set up this study to compare the qualitative and quantitative effects of Res and Xan on Leishmania and their mitochondria as a major target.
Res was shown to be active against L. amazonensis parasites (Ferreira et al. Reference Ferreira, Soares, Nascimento, Pinto da Silva, Sarzedas, Tinoco and Saraiva2014). However, another study demonstrated that the antiamastigote effect of Res against L. major inside macrophages is more related to host cell toxicity than to direct amastigote killing (Lucas and Kolodziej, Reference Lucas and Kolodziej2013). Particularly, this polyphenol did not only target Leishmania, but also caused inhibition of arginase in macrophages, which provides ornithine for polyamine synthesis in Leishmania (Ferreira et al. Reference Ferreira, Soares, Nascimento, Pinto da Silva, Sarzedas, Tinoco and Saraiva2014).
For Xan, antiplasmodial activity in the low micromolar range was described (Gerhäuser, Reference Gerhäuser2005). Although the exact mechanism is still not clear, it was suggested that Xan could inhibit cysteine protease activity in plasmodia (Liu et al. Reference Liu, Wilairat and Go2001b ). Alternatively, the interference of Xan derivatives in the GSH-catalysed haemin degradation, an important haeme detoxification pathway in plasmodia, was proposed (Frölich et al. Reference Frölich, Schubert, Bienzle and Jenett-Siems2005).
Viability tests for Xan and Res in pathogenic and non-pathogenic Leishmania in their promastigote and amastigote forms give evidence for an antileishmanial activity. Although IC50 values were different in the different Leishmania forms and species the ranking of the compounds was similar in LaA (Table 1), LtP and LaP (Table 2). In all cases, Xan was more effective as Res, while Pen as the reference drug was highly active in all Leishmania. Taking into account that Pen is a drug and Xan is a nutritional compound, this was rather unexpected. Xan shows a slightly higher toxicity against PMM (Table 1) and J774 cells (Table 2) than Res. Based on the high selectivity, both Xan and Res, possessing selectivity indices of 10 and 31, respectively, have potential as antileishmanial ingredients although higher concentrations compared to clinically used drugs may be required.
Major efforts in the present work were taken to identify mitochondrial effects of Res and Xan in Leishmania and mammalian cells. The single mitochondrion in Leishmania is an attractive target since it is essential for parasite survival. Many anti- leishmanial drugs were shown to affect this organelle (Fidalgo and Gille, Reference Fidalgo and Gille2011), which has several unique properties, partly different from mammalian mitochondria (Monzote and Gille, Reference Monzote and Gille2010). Furthermore, inhibition of mitochondria in Leishmania was shown to promote apoptotic cell death in these parasites (Mehta and Shaha, Reference Mehta and Shaha2004). Clarification of the question whether a compound has a direct protein target is a prerequisite of structure-based drug modification as performed for other mitochondrial targets by in silico methods (Müllebner et al. Reference Müllebner, Patel, Stamberg, Staniek, Rosenau, Netscher and Gille2010). Previously, we have shown that ascaridole has an effect on the mitochondrial membrane potential without strongly inhibiting the ETC as primary event (Monzote et al. Reference Monzote, Garcia, Pastor, Gil, Scull, Maes, Cos and Gille2014). For Res and Xan this has not yet been elucidated for Leishmania.
Res and other polyphenols were shown to inhibit the F 1-ATPase activity in rat liver mitochondria (Zheng and Ramirez, Reference Zheng and Ramirez1999). Furthermore, in carcinoma cells Res was shown to trigger apoptosis both via JAK/STAT pathway and the intrinsic pathway affecting mitochondria and resulting in cytochrome c release, an increase of Bax and a decrease of Bcl-2 (Madan et al. Reference Madan, Prasad, Roy, George and Shukla2008). Additionally, in human cancer cell lines, a transient increase of superoxide radical production by mitochondria was triggered in low micromolar concentrations of Xan as deduced from the increased oxidation of dihydroethidium (Strathmann et al. Reference Strathmann, Klimo, Sauer, Okun, Prehn and Gerhäuser2010). These authors also studied the inhibition of ETC complexes by Xan in BH-SMP and observed significant inhibition of complex I–III only at concentrations >100 µ m, while complex IV was not affected (Strathmann et al. Reference Strathmann, Klimo, Sauer, Okun, Prehn and Gerhäuser2010).
Effects of Xan on mammalian mitochondrial complex I in cancer cells (Zhang et al. Reference Zhang, Chu, Wei, Liu and Wei2015) could differ from those in Leishmania mitochondria due to strong structural differences of complex I in these organisms. In mammalian cell types, the increased reactive oxygen species formation was accompanied by a rapid decline of the mitochondrial membrane potential [measured by DiOC6(3) or JC-1] (Yang et al. Reference Yang, Della-Fera, Rayalam and Baile2007; Strathmann et al. Reference Strathmann, Klimo, Sauer, Okun, Prehn and Gerhäuser2010).
In this work for mechanistic studies on mitochondrial involvement in Xan actions LtP were used because L. tarentolae were successfully validated as non-pathogenic drug-screening model in the past (Taylor et al. Reference Taylor, Munoz, Cedeno, Velez, Jones and Robledo2010). A high-sequence homology between mitochondrial genomes of Leishmania exists (Yatawara et al. Reference Yatawara, Le, Wickramasinghe and Agatsuma2008) suggesting similar functions and drug sensitivity. In addition, LtP have the advantage that they can be cultivated in high cell densities and analytical techniques outside class II laboratories can be used.
Assaying individual complex II (Fig. 3) and III (Fig. 4) activities in LtP-Mit gives strong evidence for inhibition of complex II/III by Xan but not by Res. Whether this inhibition is based on covalent modification by the reaction with thiol groups of complex proteins [as shown for mammalian enzymes (Brodziak-Jarosz et al. Reference Brodziak-Jarosz, Fujikawa, Pastor-Flores, Kasikci, Jirasek, Pitzl, Owen, Gerhäuser, Amslinger and Dick2016)] remains to be elucidated. Complex I activity assays in LtP-Mit using NADH as donor and DCPIP as acceptor resulted largely in a Rot-insensitive NADH consumption (possibly not linked to the respiratory chain) and was therefore not included in our study. Surprisingly, we observed also significant inhibition of complex II and III activities in mammalian mitochondria (Tables 4 and 5). These findings are qualitatively similar to findings of Strathmann et al. (Strathmann et al. Reference Strathmann, Klimo, Sauer, Okun, Prehn and Gerhäuser2010), but we observed for complex III in BH-SMP and RLM IC50 values in the low micromolar range (Table 5). A possible explanation for this discrepancy are different assay conditions, since absolute (not relative) IC50 values can strongly depend on the artificial substrate used for measurement of complex II/III activity. This raises the question why Xan in spite of inhibition of complex II/III in leishmanial (Figs 3 and 4) and mammalian mitochondria (Tables 4 and 5) shows higher selectivity for Leishmania in viability assays (Tables 1 and 2).
To assess this question, inhibition of respiration by Xan and Res in whole cells (LtP, J774) was studied. Oxygen consumption in LtP-Mit was not strongly stimulated by typical complex I substrates (data not shown) and is, therefore, unlikely to contribute to the activity in whole LtP (Fig. 2). Furthermore, it is known that the complex I equivalent in protozoa is different from mammalian complex I with respect to structure, inhibitor sensitivity and function (Chen et al. Reference Chen, Bennedsen, Zhai and Kharazmi2001; Vaidya, Reference Vaidya2004). Measurement of oxygen consumption by glucose-consuming LtP gives a clear evidence that Xan [Clark electrode (Fig. 2): IC50 = 16 ± 1 µ m, OxoPlate (Table 3): IC50 = 6·74 ± 0·27 µ m], but neither Res nor Pen inhibited ETC in Leishmania. Unexpectedly for the respiration in J774 macrophages the IC50 values for Xan and Res were above 150 µ m (Table 3). Only Pen inhibited respiration in J774 cells with an IC50 value of about 40 µ m. The discrepancy between inhibition of mammalian complex II/III by Xan, but no strong effect on respiration of J774 cells may have several possible reasons: (i) Maximal rates of individual complex activities determined by artificial substrate concentrations exceeding Michaelis constant often exceed the respiration rates of the whole respiratory chain. (ii) Inhibition of individual complex activities measured by these assays may or may not result in inhibition of oxygen consumption depending on the flux control coefficient of the respective mitochondrial complex (Lenaz and Genova, Reference Lenaz and Genova2007), which in turn is different in different cell types and between mitochondria from different species (Rossignol et al. Reference Rossignol, Malgat, Mazat and Letellier1999). (iii) At certain concentrations of test compounds possible uncoupling may reverse decreased oxygen consumption. Therefore, inhibition of oxygen consumption can be explained by inhibition of individual complex activities, but not necessarily the other way round.
To study the bioenergetic performance of LtP and J774 cells several cell respiratory control experiments were performed (Brand and Nicholls, Reference Brand and Nicholls2011) (Fig. 7, Table 6). The major differences between both cell types are lower coupling efficiency, lower respiratory control ratios and the absence of any spare respiratory capacity in LtP in contrast to J774 macrophages. This gives a possible explanation why inhibition of complex II/III in LtP has more severe consequences than in J774 macrophages.
For many proapoptotic drugs the triggering of mitochondrial superoxide radical production is an essential step. Therefore, we determined the superoxide radical production by the ESR/CMH method (Dikalov et al. Reference Dikalov, Kirilyuk, Voinov and Grigor'ev2011) in LtP cells in the presence of xenobiotics. Although the positive control with AA (a complex III inhibitor) triggered additional superoxide radical production, neither Xan nor Res caused higher CMH oxidation rates. In contrast, for both substances and especially for Xan a decrease was observed (Fig. 5) indicating that Xan and Res can scavenge superoxide radicals. Indeed, Res was shown in a previous study to compete with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide for superoxide radicals (Murias et al. Reference Murias, Jäger, Handler, Erker, Horvath, Szekeres, Nohl and Gille2005). For Xan, however, superoxide radical-scavenging was questioned (Schempp et al. Reference Schempp, Vogel, Huckelhoven and Heilmann2010). An alternative explanation for Xan could be its strong inhibition of the ETC at complex II/III and if no oxygen is consumed also no superoxide radicals are produced at complex III. An interference of Xan and Res in the CMH-mediated superoxide radical detection was excluded.
Res and Xan had an effect on the membrane potential (Fig. 6). Both Res and Xan (200 µ m) decreased the mitochondrial membrane potential detected by the fluorescence of JC-1 aggregates. Our detection system was verified by the positive controls Val, an ionophore, CCCP, a protonophore, and AA, a complex III inhibitor. All of them caused a breakdown of the membrane potential. As expected inhibition of ATP synthesis by Oligo increased the potential. The effect of Res and Xan on the membrane potential could have been mediated in part by protonophoric effects of phenolic OH groups in analogy to the classical uncoupler DNP. However, changes of the mitochondrial membrane potential by Res could be also related to indirect effects by apoptotic processes and do not necessarily imply direct mitochondrial targets for Res.
In summary, both natural products, Xan and Res, exhibited an antileishmanial potential in the lower micromolar range. However, only the effects of Xan are directly mediated by mitochondrial targets and the specific vulnerability of oxidative phosphorylation in Leishmania.
ACKNOWLEDGEMENTS
We are grateful for the support of Professor Veronika Sexl and BSc Matthias Tonner.
FINANCIAL SUPPORT
The award of an Ernst Mach scholarship to Dr Lianet Monzote by the Austrian Exchange Office is gratefully acknowledged. The work was supported by the FWF (grant no. P 27814-B22).
