Article pubs.acs.org/crt
Lysosomes as a Possible Target of Enniatin B-Induced Toxicity in Caco-2 Cells L. Ivanova,*,† W. M. Egge-Jacobsen,‡ A. Solhaug,† E. Thoen,† and C. K. Fæste† †
Norwegian Veterinary Institute, Oslo, Norway Department of Molecular Biosciences, University of Oslo, Oslo, Norway
‡
ABSTRACT: Enniatins are cyclic hexadepsipeptidic mycotoxins with ionophoric, antibiotic, and insecticidal activity. Enniatin B (EnnB), the most important analogue, is produced by many Fusarium species and is a common contaminant in grain-based foods. The compound’s cytotoxic potential has been shown in different experiments; however, the mode of action has not been detailed so far. In the present study, several mutually confirmative experiments have been performed indicating that EnnB-initiated cytotoxicity could be connected with lysosomal membrane permeabilization (LMP). Lysosomal functionality, as assessed by the Neutral Red assay, was already affected after 3 h of toxin exposure. After 24 h, cell proliferation was decreased, and there was indication for a cell cycle arrest in the G2/M phase leading to the initiation of apoptosis or necrosis. Intracellular ROS-production was observed. However, antioxidants did not alter the observed EnnB-induced loss of lysosomal functionality leading to the conclusion that ROS was not an initial factor but one produced later in the event cascade. The collected data suggested that lysosomal destabilization is an upstream event in EnnB-initiated cytotoxicity followed by a certain extent of translocation of cathepsins into the cytosol, which was observed using immunological and proteomic methods. It appeared that cell death induced by EnnB was delayed and occurred not as a massive lysosomal breakdown but was probably progressing and leading to partial and selective LMP, starting a nonapoptotic cell death pathway with morphological features that had been previously considered as necrotic. The molecular mechanism of EnnB-triggered lysosomal destabilization, and the cellular processes leading to mitochondrial permeabilization and cell death are still unknown. They may, however, be connected to the compound’s ionophoric properties.
1. INTRODUCTION Enniatins are secondary fungal metabolites that are mainly produced by Fusarium strains growing on field grains.1 Enniatin B (EnnB), one of the most important analogues of this mycotoxin group, has a molecular structure composed of an alternating sequence of three N-methyl-L-valine (N-Me-Val) and three D-α-hydroxyisovaleric acid (Hiv) molecules forming an 18-membered cyclic depsipeptide: (N-Me-Val-Hiv)3 (Figure 1).2
transport mono- and divalent cations either in sandwiched complexes or by creating channels in biological membranes. EnnB and other enniatins possess a wide range of biological activities primarily connected to their ionophoric properties. They have shown antibacterial, antifungal, antihelmintic, insecticidal, and phytotoxic potencies.1,3 In conventional cytotoxicity assays, the effect concentrations (EC50) of individual enniatins or enniatin mixtures are commonly in the lower micromolar range.4−7 It is presently assumed that the cytotoxic activity of the enniatins, leading to apoptotic cell death, is related to the induction of mitochondrial membrane depolarization and cell cycle disruption.7 Additionally, it has been shown that DNA damage and oxidative stress may play only minor roles in enniatins-induced cytotoxicity.4,8 However, mitochondrial dysfunction might not be the only or initial step to trigger enniatin-caused programmed cell death.7,9 Mitochondrial dysfunction and the destabilization of the lysosomal system appear to be connected suggesting lysosomal− mitochondrial cross-talk during cell death.10 Recently, the lysosomal system has been increasingly linked to cell death processes.11−13 Lysosomes are acidic, hydrolase-rich
Figure 1. Chemical structure of enniatin B.
The cyclopeptidic enniatins form ionophores with hydrophobic groups on the outside and polar groups in the core, resembling a disk in the three-dimensional conformation.1,3 They can © 2012 American Chemical Society
Received: March 16, 2012 Published: June 25, 2012 1662
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Lysosomes may therefore often be involved in necrotic, apoptotic, apoptotic-like, and autophagic cell responses.21 The lysosomal cell death pathway thus offers a promising alternative to damage malignant cells, which often have enlarged lysosomal systems.12 In this context, we decided to evaluate the relevance of LMP in EnnB-induced cytotoxicity in Caco-2 cells focusing on the potential cathepsin leakage. In the same cell line, we had previously observed changes in the lysosomal morphology in the presence of EnnB.22 Since enniatins are under consideration as potential anticancer drug candidates,7 more information on their cellular targets would be advantageous.
organelles that have a major role in intracellular protein recycling.12 Lysosomes contain numerous proteases that catalyze the cleavage of peptide bounds in proteins.13 A major class of lysosomal proteases are the cathepsins, which are acknowledged as potential mediators of cell death.11,14−16 Cathepsins share common catalytic folds with other proteases. On the basis of the amino acid sequences of their active sites, they can be subdivided into papain-like cysteine proteases such as the cathepsins B, C, F, H, K, L, N, O, S, T, U, W, and X, aspartate proteases such as the cathepsins D and E, and serine proteases such as the cathepsins A and G.13 Cathepsins are synthesized as inactive preproenzymes in the endoplasmic reticulum, transported into the lysosomes, and post-translationally modified into catalytically active enzymes.14,15 Consequently, lysosomal destabilization, subsequently referred to as lysosomal membrane permeabilization (LMP), leads to the release of active cathepsins into the cytosol, eventually triggering apoptosis-related reactions. Although cathepsins work optimally at low pH within the lysosomes, they retain some activity at the neutral pH of the cytosol.16 Moreover, lysosomal impairment implies also the efflux of hydrogen ions and acidification of contiguous cytosolic zones. It has been widely discussed as to which cellular mechanisms could lead to LMP. The best studied candidates for causing LMP are tumor necrosis factor family proteins (TNF), p53, caspase-8, reactive oxygen species (ROS), intracellular sphingosine, and lysosome-associated apoptosis-inducing protein (LAPF).11,12,17,18 The existence of an amplification loop has been suggested, by which released cathepsins may contribute to increased lysosomal leakage via the enhancement of cytosolic ROS formation and activation of LMP-inducing phospholipase A2.11,12 Cell death can occur through different cellular processes.13 Apoptotic phenotypes follow programmed processes and proceed in a self-determined manner after initiation, whereas necrotic cell death appears to be a more catastrophic event.19 However, it has been recently demonstrated that the response to a particular death stimulus leads often to a continuum of apoptosis and necrosis in the same cell showing coexisting features of both.19 Apoptotic processes can get started by two major molecular pathways, and several of the potential LMPmediators have been associated with the extrinsic death pathway, which can be initiated by external factors such as TNF.10,18 However, LMP interacts also with the intrinsic apoptosis pathway involving mitochondrial outer membrane permeabilization (MOMP).12 A specific sign for LMP is the release of soluble lysosomal hydrolases (including cathepsins) from the lysosomal lumen into the cytosol. The leakage of cathepsins can bring about a variety of cell death-associated morphologies ranging from classical apoptosis to necrosis.10−17 In the case of massive lysosomal breakdown, the cytosolic acidification causes degradation of cellular compartments leading to uncontrolled cell death by necrosis.12,19 If the LMP is partial, cell death through apoptosis can be mediated by cathepsins either through the cleavage of Bid and the subsequent release of apoptogenic factors from the mitochondria or in a mitochondrion-independent way by upregulating levels of cytosolic ROS and sphingosine as well as by degradation of the lysosome-associated membrane proteins 1 (LAMP-1) and 2 (LAMP-2).10,11,18,20 In addition, cathepsins may also be involved in effecting LMP by disturbing the lysosomal trafficking after activation by different agents.18 The interference of microtubule and actin dynamics causes a dramatic lysosomal swelling, which damages the organelle membrane.
2. MATERIALS AND METHODS 2.1. Materials. EnnB was isolated and purified from rice cultures of the fungus Fusarium avenaceum.5 Neutral Red (NR) cytotoxicity assay was obtained from Xenometrix (Allschwil, Switzerland). Alamar blue was purchased from Biosource (Nivelles, Belgium). ProteoExtract Subcellular proteome extraction kit was delivered by Merck Chemicals LTD (Nottingham, UK). CytoTox-ONE Homogeneous Membrane Integrity kit was purchased from Promega (Madison, USA). Propidium iodide (PI, 1 mg/mL), Hoechst 33342 (10 mg/mL), RNase (Purelink RNase A), LysoTracker Red DND-99 (1 mM, DMSO), dihydroethidium (DHE, 5 mM, DMSO), and gels and buffers for Western blotting were obtained from Invitrogen (Paisley, UK). Bio-Rad DC protein assay was purchased from Bio-Rad Laboratories Inc. (Hercules, CA, USA). Dulbecco’s Modified Eagle's Medium (DMEM), trypsin−versene (EDTA) mixture, penicillin−streptomycin mixture, fetal bovine serum EU standard (FBS), and MEM non-essential amino acid solution (10 mM) were purchased from Lonza (Verviers, Belgium). BD BioCoat Cellware poly-L-lysine 12 mm coverslips were obtained from BD Biosciences (Bedford, MA, USA). The lysosomotropic reagent leucineleucyl-O-methyl HBr (LeuLeuOMe) was purchased from Bachem AG (Bubendorf, Switzerland). Antibodies for cathepsin B (CB, FL-339, rabbit polyclonal antibody, 200 μg/mL), cathepsin D (CD, C-20, goat polyclonal antibody, 200 μg/mL), and cathepsin L (CL, C-18, goat polyclonal antibody, 200 μg/mL), and donkey antirabbit IgG-HRP and donkey antigoat IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cysteine protease inhibitor E-64d, a general inhibitor of aspartic proteases pepstatin A, protease inhibitor cocktail, and dimethyl sulfoxide (DMSO) were delivered by Sigma-Aldrich (St Louis, MO, USA). All other chemicals were of analytical grade. 2.2. Cell Culture. The human colon adenocarcinoma cell line Caco-2 was obtained from the European Collection of Cell Cultures (ECACC, Porton Down, Salisbury, UK) and was used between passages 48 and 64. The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2 in growth medium (Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, EU standard), 2 mM L-glutamine, and 1% nonessential amino acids, all from Lonza (Verviers, Belgium). Cell passaging was done by trypsination. All experiments were based on cells cultured to 70−80% confluence. 2.3. Morphological Changes and Proliferation. The cells were seeded at a density of 6.4 × 104 cells/cm2 the day before the experiments. After 24 h exposure to different concentrations of EnnB (1 μM, 5 μM, 10 μM, and 25 μM), changes in cell morphology were observed using a light microscopy (Leica DMIL, Solms, Germany). Proliferation was determined by cell counting. Adherent cells were harvested with trypsin-EDTA (Lonza) and combined with floating cells, collected by centrifugation. The number of cells was determined by direct counting using a light microscopy (Leica DMIL, Solms, Germany) and compared to an untreated control, which was used as the 100% reference value. The same procedure was performed in the presence of 20 μM E-64d and 40 μM Pep A, general inhibitors of cysteine and aspartate proteases, respectively. 1663
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33342 (10 μg/mL, DMEM) for 30 min at room temperature. All liquid was removed, and the stained cells were resuspended in 20 μL of FBS, spread on slides, and quickly air-dried. Small cells with a strong bright Hoechst 33342-blue color and characteristic patterns of nuclear condensation and fragmentation were counted as apoptotic, while cells with uniform blue appearance were defined as normal healthy cells. Furthermore, cells with an increased cellular volume that were stained red by PI were counted as necrotic. Microscopic observations and photography were performed with a fluorescence microscope (Leica DM 5000 B, Nikon digital camera DXM1200). For each sample, at least 300 cells were examined to determine the percentage of normal, apoptotic, and necrotic cells on the basis of cell nuclei morphology and PI-indicated cell-membrane damage. Separate experiments were performed in the presence of 20 μM E-64d or 40 μM Pep A to investigate the involvement of cathepsins in the observed cell death. Caco-2 cells were preincubated for 1 h at 37 °C prior to EnnB exposure (10 μM, 25 μM). The same cathepsin inhibitor concentrations were present during the experiments. The DMSO concentrations in the incubation medium were 0.14% (v/v) and 0.55% (v/v), respectively. Additionally, EnnB-induced effects on Caco-2 cells were evaluated in the presence of 500 μM ascorbic acid, which was freshly prepared in DMEM (2.2 mg/mL) and administrated to Caco-2 cells 30 min prior to EnnB treatment. 2.7. Lysosomal Stability Assessment by Confocal Microscopy Using LysoTracker Red DND-99 Staining. Loss of lysosomal membrane integrity was measured by the uptake of the red-fluorescent dye LysoTracker Red (LTR, Invitrogen). Briefly, cells were grown on glass coverslips overnight at normal cell culture conditions in 12-well plates (6.4 × 104 cells/cm2). On the next day, cells were exposed to EnnB for 3 h (10 μM, 25 μM, and 50 μM) and 24 h (1 μM, 5 μM, 10 μM, and 25 μM) in FBS-containing medium at 37 °C. The medium was then replaced with fresh prewarmed medium containing 75 nM LTR, and the cells were further incubated for 30 min at 37 °C. After washing two times with ice-cold PBS, the live cultures were examined by confocal microscopy (Zeiss LSM 710, Jena, Germany) using a 40× objective with oil immersion. Fluorescence from LTR was excited at 561 nm by He−Ne laser. Emitted fluorescence was detected from 590 to 650 nm. The lysosomotropic agent LeuLeuOMe (Bachem) was dissolved in methanol at a final concentration of 2.5 mM and used as positive fluorescence control. The appropriate concentration of LeuLeuOMe was determined by using the NR assay (Xenometrix). 2.8. Measurement of Mitochondrial Membrane Potential Changes (ΔΨm) by Flow Cytometry. Mitochondrial membrane permeabilization (MOMP) was determined using the lipophilic cationic probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1 Guava MitoPotential kit, Millipore, USA). Briefly, cells (5.5 × 104 cells/cm2) were cultured in 12-well plates for 18−24 h prior to exposure with EnnB (5 μM and 10 μM) for 3 and 24 h. Subsequently, both adherent and floating cells were collected by trypsination combined with centrifugation and loaded with JC-1 and 7-AAD staining solution for 30 min at 37 °C. After the incubation was complete, samples were centrifuged at 400g for 5 min. Following centrifugation, the cells were resuspended in 200 μL of fresh complete growth medium and analyzed immediately for orange−red and green fluorescence using an Accuri C6 flow cytometer on FL1 (530 nm) and FL2 (585 nm) channels, respectively. A total of 25 000 events was acquired per sample and analyzed by Accuri CFlow software. The mean fluorescence intensities at 585 nm (MFI585) and 525 nm (MFI525) were measured, and the ratio MFI585/MFI525 was calculated. Data were presented as percentage compared to the control, which was assumed as 100%. The same assay was performed in the presence of cathepsin inhibitors to elucidate the role of cathepsins in the mitochondrial stress development. Cells were pretreated with 20 μM E-64d and 40 μM Pep A for 1 h followed by exposure to EnnB (5 μM, 10 μM) for 24 h. 2.9. Measurement of ROS Production by Flow Cytometry. Intracellular ROS production was determined by using the oxidationsensitive fluorescent probe dihydroethidium (DHE), which is rapidly oxidized to ethidium (a red fluorescent compound) by intracellular
Differences in cell morphology after 24 and 48 h of treatment with 5 μM EnnB were observed using a light microscope (Leica DMIL) and compared to control cells. Photographs were taken with Moticam 1000. 2.4. Combination of Alamar Blue, LDH Leakage, and the Neutral Red Assay. EnnB-induced cytotoxicity in Caco-2 cell cultures was determined using a combined bioassay for the simultaneous measurement of metabolic activity (Alamar Blue assay, AB), membrane integrity (LDH leakage assay), and lysosomal activity (Neutral Red assay, NR) as described by Ivanova and Uhlig23 with some modifications. Briefly, cells were seeded into 96-well culture plates at 2 × 104 cells/well. On the next day, the cells were treated with EnnB concentrations ranging from 56 pM to 437.2 μM. After 3 or 24 h of incubation, 100 μL of medium from each well was used for the determination of LDH activity. Prior to the measurement of lysosomal and metabolic activities, the remaining culture medium was discarded and 200 μL of fresh medium containing 0.1% NR and 8 μL of AB-solution were added to the wells of the test plate. The cells were incubated for an additional three hours at 37 °C. The product of the metabolic reduction of AB (resorufin) was quantified by fluorometry using a Victor2 multilabel counter (Perkin-Elmer Inc., Boston, MA, USA) at 530 nm excitation wavelength and 580 nm emission wavelength. The NR- and AB-containing medium was discarded and the cells subsequently treated with 100 μL of fixing reagent and 200 μL of solubilization solution that are included in the commercial kit (NR, Xenometrix). Finally, the absorbance of the dissolved NR was measured at 540 nm using the Victor2 multilabel counter with 690 nm as the reference wavelength. Cytotoxicity was expressed as EC50 values from full dose−response curves. When EnnB cytotoxicity was studied in the presence of the cathepsins inhibitors E-64d and Pep A, the inhibitors were applied to the cells 1 h prior to EnnB exposure. E-64d and Pep A (both 5 mg/mL stock in DMSO) were added at a final concentration of 20 μM and 40 μM, respectively.24 The same concentration of inhibitor was present during the experiment. The DMSO concentration in the incubation medium was 0.14% and 0.55%, respectively (v/v). In a separate set of experiments, cells were exposed to EnnB (5 μM, 10 μM) in the presence of the potent antioxidant ascorbic acid (500 μM; vitamin C). Ascorbic acid was freshly prepared in DMEM (2.2 mg/mL) and administrated to Caco-2 cells 30 min prior to EnnB treatment. The same concentration of vitamin C was present during the experiment. As a control, the sensitivity of Caco-2 cells to ascorbic acid was investigated under the same assay conditions. The effects of prolonged exposure to 5 μM, 10 μM and 25 μM EnnB on cell fate were investigated in the NR assay by comparing 24 and 48 h incubations. Furthermore, in an additional experiment to study potential cell recovery after removal of the toxin, the cell culture medium was changed back to standard conditions after 24 h of EnnB-exposure, and the cells were incubated for further 24 h. The results were compared to the data obtained for 48 h continuous exposure. 2.5. Cell Cycle Analysis by Flow Cytometry after Propidium Iodide (PI) Staining. Briefly, Caco-2 cells (6.4 × 104 cells/cm2) were cultured in 12-well plates. After 24 h of exposure to EnnB (1 μM, 5 μM, 10 μM, and 25 μM), the cells were harvested by trypsination, centrifuged, washed with ice-cold PBS, and fixed with ice-cold 70% EtOH overnight at −20 °C. Afterward, the cells were stained with PI (10 μg/mL)/RNase A (20 μg/mL) in PBS for 30 min at 37 °C. Flow cytometric measurements were performed using an Accuri C6 flow cytometer (Accuri Cytometers Inc., Ann Arbor, MI, USA). Single cells were gated, and PI fluorescence was measured using a 585 ± 20 nm band-pass filter (FL2). For each experiment, 10 000 events were acquired and analyzed by CFlow software (CFlow Plus version 1.0.227.4). 2.6. Cell Death Analysis. Early apoptosis, late apoptosis, and necrosis in Caco-2 cells after exposure to EnnB were distinguished by using a method based on PI/Hoechst 33342 double-staining.25 Briefly, Caco-2 cells (6.4 × 104 cells/cm2) were cultured in 12-well plates (18−24 h) and treated with increasing concentrations of EnnB (1 μM, 5 μM, 10 μM and 25 μM). After 24 h of exposure, both adherent and floating cells were harvested by trypsination and centrifugation. The cells were subsequently double-labeled with PI (10 μg/mL, DMEM) and Hoechst 1664
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superoxide. Briefly, cells (6.4 × 104 cells/cm2) were cultured in 12-well plates for 24 h prior to the exposure with EnnB (1 μM, 5 μM, 10 μM, and 25 μM) for 3 and 24 h. Subsequently, both adherent and floating cells were collected by trypsination combined with centrifugation and incubated with DHE (5 μM, DMEM) for 20 min at 37 °C. The cells were washed twice with ice-cold PBS, and fluorescence intensity was assessed using an Accuri C6 flow cytometer on FL3 channel (>670 nm). A total of 10 000 events were acquired per sample and analyzed by Accuri C Flow software. The same experiment was additionally carried out in the presence of an antioxidant investigating potential changes of EnnB-induced ROSproduction. Ascorbic acid (500 μM) was added to the cells 30 min before incubating with EnnB for 24 h. The cells were collected and analyzed as before by flow cytometry. The appropriate vitamin C assay concentration was determined using the combined bioassay for the simultaneous measurement of metabolic activity (AB), membrane integrity (LDH leakage assay), and lysosomal activity (NR assay) as described above (section 2.4.). 2.10. Preparation of Cytosolic Extract. Caco-2 cells were seeded into 60 mm culture dishes at 6.4 × 104cells/cm2 and grown overnight to 70−80% confluence. After exposure to EnnB (1 μM, 5 μM, and 10 μM) for 24 h, cells were harvested by trypsination and washed twice with icecold PBS buffer. The cells were then incubated on ice for 30 min in hypotonic buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, and 0.5 mM DTT) containing 1% (v/v) protease inhibitor (Sigma-Aldrich). Thereafter, cells were opened by homogenization on ice with a Dounce tissue grinder in 25 passages (7 mL, Sigma). Homogenization time and passage numbers had been previously optimized based on the release of cytosolic lactate dehydrogenase. The homogenates were centrifuged at 500g, and cellular debris was discarded. The supernatant was centrifuged again at 20 000g for 40 min at 4 °C for separation of the cytosolic fraction (MicroCL 21R, Thermo Scientific, Waltham, MA, USA). The extraction achieved was comparable to results obtained by using ProteoExtract subcellular proteome extraction kit (Merck). The total protein concentration was determined by the Lowry method using the Bio-Rad protein assay kit. 2.11. Gel Electrophoresis, Western Blot, and Image Analysis. Electrophoretic separations of proteins were carried out using the NuPage Novex Gel system according to the manufacturer’s instructions (Invitrogen). Samples containing equal amounts of total protein were separated on SDS−polyacrylamide gel. Proteins were either stained with SimplyBlueTM Safe Stain (Invitrogen) and used for in-gel digestion and mass spectrometry experiments, or transferred by Western blot onto nitrocellulose membrane (Bio-Rad) in an XCell II Blot Module (Invitrogen). For immunostaining, membranes were blocked in Tris buffer saline Tween20 (TBS-T; 0.1% Tween20) containing 3% BSA (blocking buffer) at room temperature (RT) for 1 h. After incubating overnight at 4 °C with primary antibodies against CB (Santa Cruz, rabbit, 1:500), CD (Santa Cruz, goat, 1:500), or CL (Santa Cruz, goat, 1:500), the membranes were washed three times (20 min each) in TBS-T and incubated with adequate secondary antibodies for 1 h. Secondary antibodies had been diluted in blocking buffer (1:5000) and were either horseradish peroxidase-conjugated donkey antigoat IgG or donkey antirabbit IgG (Santa Cruz). Finally, the membranes were washed three times with TBS-T and once with PBS buffer. Bands were developed using TMB solution. All washing steps were performed at RT under gentle shaking. Immunoblots were scanned and processed using GelPro 4.5 Analyzer Image Analysis (MediaCybernetics, Bethesda, MD). Signal intensities were determined by applying Standard Optical Density Fitting correlating (second order polynomial) the number of pixels measured and the optical density (OD). The measured maximum OD values were used for data analysis. 2.12. Sample Preparation for MS Experiments. Protein bands of interest were excised from the SDS−PAGE gels, and the gel slices were placed in microfuge tubes, sliced into pieces, and destained by shaking with acetonitrile (ACN)/50 mM NH4HCO3 (50/50) at room temperature (RT) for 10 min. The liquid was discarded, and the procedure was repeated twice. After drying in a speedvac centrifuge
(Savant speedvac concentrator, Fisher Scientific, Atlanta, GA, USA) for about 10 min, proteins in the gel were reduced with 1.5 mg/mL dithiothreitol (Sigma Chemicals, St. Louis, MD) in 25 mM NH4HCO3 at 56 °C for 1 h in a heating block (Dri-Block, Techne, Duxford, UK). The incubation mixture was cooled to RT, and the supernatant was discarded following centrifugation at 13 000g for 2 min (Eppendorf GmBH, Hamburg, Germany). Proteins were alkylated by incubation with 10 mg/mL iodoacetamide (Sigma Chemicals) in 25 mM NH4HCO3 at RT for 45 min in the dark under shaking (Shuttler MTS 4I, IKA-Werke GmbH, Staufen, Germany). The supernatant was discarded, and the gel pieces were washed once with 100 mM NH4HCO3 and twice with ACN/50 mM NH4HCO3 (50/50), each time for 10 min at RT under shaking. Gel pieces were dried by Speed-Vac centrifugation. Proteins were digested in situ in the gel pieces by adding a few microliters of 0.1 μg/mL trypsin solution (Trypsin Gold mass spectrometry grade, Promega, Madison, WI, USA), which had been prepared by diluting 1 mg/mL trypsin in 50 mM acetic acid with 25 mM NH4HCO3. The gel pieces were covered with ACN/10 mM NH4HCO3 (10/90) and incubated at 37 °C overnight. The tryptic protein fragments in the supernatant were collected by centrifugation. Additionally, two extractions were performed by shaking the gel pieces with ACN/1% formic acid (10/90) for 10 min at RT and then ACN/1% formic acid (50/50), with collection of the supernatant each time. Finally, the volume of the pooled supernatants was reduced to about 7 μL in a Speed-Vac centrifuge. The tryptic peptides were mixed with 0.3 μL of concentrated formic acid and 0.1% trifluoroacetic acid (TFA) to a final volume of 15 μL and analyzed by mass spectrometry. 2.13. Identification of Cytosolic Proteins by Nano LC-ESIOrbitrap-MS/MS. Reverse phase (C18) nano online liquid chromatographic MS/MS analysis of tryptic peptides was performed using a system consisting of two Agilent 1200 HPLC binary pumps (nano and capillary) with autosampler, column heater, and integrated switching valve (Agilent, Waldbronn, Germany). This LC system was coupled via a nanoelectrospray ion source to a LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Peptide solutions (4 μL) were injected onto the 5 × 0.3-mm extraction column filled with Zorbax 300 SB-C18 of 5-μm particle size (Agilent). Samples were washed with a mobile phase consisting of 97% 0.1% formic acid and 3% acetonitrile. The flow rate was 4 μL/min provided by the capillary pump. After 7 min, the integrated switching valve was activated, and peptides were eluted in the back-flush mode from the extraction column onto a 150 × 0.075-mm C18, 3-μm resin column (GlycproSIL C18−80 Å, Glycpromass, Stove, Germany). Chromatographic separation was achieved using an acetonitrile/water (0.1% formic acid) binary gradient from 5 to 55% acetonitrile in 70 min and a flow rate of 0.2 μL min−1 provided by the nanoflow pump. Mass spectra were acquired in the positive ion mode applying a datadependent automatic switch between survey scan and tandem mass spectra (MS/MS) acquisition. Peptide samples were analyzed with a high energy collisional dissociation (HCD) fragmentation method, acquiring one Orbitrap survey scan in the mass range of m/z 300−2000 followed by MS/MS of the three most intense ions in the Orbitrap. The target value in the LTQ-Orbitrap was 1,000,000 for survey scan at a resolution of 30,000 at m/z 400 using lock masses for recalibration to improve the mass accuracy of precursor ions. Collision-induced fragmentation was performed with a target value of 5,000 ions. The ion selection threshold was 500 counts. Selected sequenced ions were dynamically excluded for 180 s. Mass spectrometric data were first analyzed by generating msf-files from raw MS and MS/MS spectra using the Proteome Discoverer 1.0 software (Thermo Fisher Scientific). Database searches were either performed by using the NCBI-database applying the taxonomy filter for human proteins or by comparison to an in-house maintained targeted protein sequence database. Both, the SEQUEST search engine (La Jolla CA, USA) involving the criteria enzyme name; trypsin; missed cleavage sites, 2; precursor mass tolerance, 10 ppm; fragment mass tolerance, 0.6 Da; fixed modifications, carbamidomethyl; variable modification, oxidation; and the MASCOT search engine (Matrix Science Inc., Boston, MA) with the criteria enzyme name, trypsin; fixed modifications, 1665
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Figure 2. Effect of enniatin B on the general morphology and viability of Caco-2 cells after exposure to different concentrations of EnnB at 24 and 48 h. (A) Light microscopy (Leica DMIL) with a 10× objective. The representative images from one out of two experiments with exposure to 5 μM EnnB are presented. (B) Recovery experiment. Cell viability was measured in the Neutral Red assay after 24 h exposure to EnnB and additional 24 h incubation in EnnB-free medium. The results are represented as a percentage of absorbance relative to control cells (100%). The data are presented as mean ± SEM for three independent experiments with triplicate measurements. *, significant at P < 0.05.
Table 1. Cell Viability after Exposure to Enniatin B
carbamidomethyl; variable modifications, oxidation; mass values, monoisotopic; protein mass, unrestricted; peptide mass tolerance, ± 7 ppm; fragment mass tolerance, ± 0.6 Da; and max missed cleavages, 1, were used. Proteins were considered as significant hits if the XCorr was higher than 1.5 (SEQUEST) or if the Score was higher than 30 (MASCOT).
3. RESULTS 3.1. EnnB Affects Morphology and Proliferation of Caco-2 Cells. The number of cells with changed morphology increased with increasing concentrations of EnnB (data not shown). Only very few cells rounded off or floated after exposure to 1 μM EnnB, whereas a considerable reduction of cell proliferation, but without noteworthy cell loss, was observed after exposure to 5 μM EnnB (Figure 2A). Moreover, the treatment of Caco-2 cells with higher concentrations of EnnB (>5 μM) resulted in an increasing detachment of cells from the culture dishes and a considerable loss of cell−cell contacts. These observations were most evident at 25 μM EnnB. Direct counting of cells after EnnB treatment (1 μM, 5 μM, 10 μM, and 25 μM) demonstrated that cell proliferation decreased in a concentrationdependent manner (Table 1). In the presence of aspartate cathepsins inhibiting 40 μM Pep A, the ability of EnnB to decrease cell proliferation was reduced. Compared to treated cells (5 μM, 10 μM, or 25 μM EnnB) without the inhibitor, cell proliferation was increased in the presence of Pep A by 11 ± 2% (mean ± SD, n = 3), 23 ± 9% (mean ± SD, n = 3) and 18 ± 7% (mean ± SD, n = 3),
sample
%
±SD
control 1 μM EnnB 5 μM EnnB 10 μM EnnB 25 μM EnnB
100 85 67 69 50
0 7 6 11 5
respectively, after incubation with 5 μM, 10 μM, or 25 μM EnnB for 24 h. However, the cell proliferation was still reduced compared to that in untreated cells (control). Thus, Pep A partly reduced the ability of EnnB to decrease cell proliferation. Similar effects were not observed when cysteine cathepsins inhibiting E-64d (20 μM) were used in the assays. 3.2. Characterization of EnnB-Induced Cytotoxicity in Caco-2 Cells. The combined bioassay for simultaneous measurement of metabolic activity (AB), cell membrane integrity (LDH), and lysosomal activity (NR) was performed using an EnnB standard curve starting at pmolar concentrations and two exposure times. Significant cytotoxicity was only observed in connection with lysosomal functionality resulting in effect concentrations (EC50) of 10 ± 3.8 μM (mean ± SD, n = 3) and 2.1 ± 0.4 μM (mean ± SD, n = 4) after exposure for 3 and 24 h, respectively (graphical abstract: 24 h exposure). On the contrary, EnnB apparently had no effect in the AB and LDH bioassays (data not shown). 1666
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Chemical Research in Toxicology
Article
The inclusion of the cathepsin inhibitors E-64d (20 μM) and Pep A (40 μM) in the NR-assay led to a slight but not significant reduction of the EnnB-induced effects (data not shown). When the NR-assay was performed in presence of the antioxidant ascorbic acid, reduction of the EnnB-induced cytotoxic effect was not observed (data not shown). Caco-2 cells showed a significant increase (P < 0.05) in cell viability after changing the medium back to standard conditions after 24 h treatment to EnnB concentrations of 1−10 μM (Figure 2B). A similar result could not be demonstrated for 25 μM EnnB, indicating that the lysosomal damage that had already occurred was irreversible. After prolonged (48 h) exposure of the Caco-2 cells to EnnB, a small increase in toxicity as compared to the result after 24 h was observed in the NR assay for the two lowest concentrations (Figure 2B). 3.3. Shift in Cell Cycle Distribution. The potential association of the observed morphological changes and viability decrease with the induction of cell cycle arrest and/or apoptosis was investigated by determining the distribution of cells in the different phases using PI-staining and flow cytometry. It could be demonstrated that the exposure of Caco-2 cells with EnnB for 24 h led to a slight shift in the cell cycle distribution with a noticeable increase of the cells in the G2/M phase (Figure 3). Exposure to
Figure 4. Enniatin B-induced apoptosis and necrosis in Caco-2 cells. (A) Representative pictures of apoptotic cell (a), normal cell (b), and necrotic cell (c) (magnification 10 × 100) after PI/Hoechst 33342 double-staining. (B) Fluorescence microscopy histogram showing incidences of the different cell populations after 24 h exposure to increasing concentrations of EnnB. The data are presented as the mean ± SEM for three independent experiments.
Necrotic cells (Nec) with lost membrane integrity were entered by PI and colored red (Figure 4A, panel c). “Post apoptotic necrosis” (Pan) cells were recognized by a combination of distinctive nuclear condensation and PI-red stain due to membrane fragmentation (not shown). Apparently normal cells were blue-colored and had uncondensed nuclei (Figure 4b). The population of necrotic cells was increasing in a dosedependent manner reaching a ratio of about 13% after exposure with 25 μM EnnB for 24 h (Figure 4B). In contrast, the amount of apoptotic cells accounted for maximal 2% of the cells at 1 μM EnnB and decreased at higher concentrations. Only a low proportion (
