Materials

Chemicals were of the purest analytical grade. Anandamide (arachidonoylethanolamide, AEA), actinomycin D, cycloheximide, 5,8,11,14-eicosatetraynoic acid (ETYA), indomethacin, cytochrome c, cyclosporin A, andNω-nitro-L-arginine methyl ester (L-NAME) were purchased from Sigma. 2-Arachidonoylglycerol (2-AG), arachidonoyltrifluoromethyl ketone (ATFMK) andN-(4-hydroxyphenyl)arachidonoylamide (AM404) were from Research Biochemicals International. EGTA-AM, capsaicin ([N-(4-hydroxy-3-methoxy-phenyl)methyl]-8-methyl-6-nonenamide), capsazepine (N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide, Caps), caspase-3 inhibitor II (Z-Asp(OCH₃)-Glu(OCH₃)-Val-Asp(OCH₃)-fluoromethyl ketone, Z-DEVD-FMK), and caspase-9 inhibitor I (Z-Leu-Glu(OCH₃)-His-Asp(OCH₃)-fluoromethyl ketone, Z-LEHD-FMK) were from Calbiochem. [³H]AEA (223 Ci/mmol) and [³H]CP55,940 (126 Ci/mmol) were purchased from NEN Life Science Products.N-Piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazolecarboxamide (SR141716) andN-[1(S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) were a kind gift from Sanofi Recherche (Montpellier, France). Palmitoylethanolamide (PEA), oleoylethanolamide (OEA), and linoleoylethanolamide (LEA) were synthesized and characterized (purity >96% by gas-liquid chromatography) as reported (15). Cannabidiol (CBD) was a kind gift from Dr. M. Van der Stelt (Utrecht University, The Netherlands). 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide (JC-1) and 1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl)-phenoxy]-2-[2-amino-5-methyl-phenoxy]ethane-N,N,N′,N′-tetraacetoxymethyl ester (Fluo-3 AM) were from Molecular Probes. Anti-cytochromec monoclonal antibodies (clone 7H8.2C12 and clone 6H2.B4) were purchased from PharMingen, and goat anti-mouse alkaline phosphatase conjugates (GAM-AP) were from Bio-Rad. Non-immune mouse serum was from Nordic Immunology (Tilburg, The Netherlands).

Cell Culture and Treatment

Human neuroblastoma CHP100 cells were cultured as reported (15), in a 1:1 mixture of Eagle’s minimal essential medium plus Earle’s salts and Ham’s F-12 media (Flow Laboratories Ltd., Ayrshire, Scotland, UK), supplemented with 15% heat-inactivated fetal bovine serum, sodium bicarbonate (1.2 g/l), 15 mM Hepes buffer, 2 mM L-glutamine, and 1% non-essential amino acids. Human lymphoma U937 and leukemia DAUDI cells were cultured in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 25 mM Hepes, 2.5 mM sodium pyruvate, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated fetal calf serum (15). Rat C6 glioma cells, a kind gift from Dr. Dale G. Deutsch (Department of Biochemistry and Cell Biology, State University of New York, Stony Brook), were cultured in Ham’s F-12 medium supplemented with 10% fetal calf serum as described (9). Cells were maintained at 37 °C in a humidified atmosphere with 5% CO₂and were fed every 3–4 days. Before each treatment, cells were washed twice with sterile, Ca²⁺ and Mg²⁺-free phosphate-buffered saline (PBS), and then they were resuspended in sterile PBS at a concentration of 10⁶cells/ml. Cell suspensions were incubated for 30 min at 37 °C in the presence of various concentrations (up to 10 μM) of endocannabinoids dissolved in methanol, and they were then resuspended in their culture media for the indicated periods. Control cells were incubated with the same volumes of vehicle alone (up to 10 μl/ml PBS). The interference of various compounds with the effects of AEA was assessed by incubating together CHP100 or U937 cells with each substance and AEA.

Electrotransfer of anti-cytochrome c monoclonal antibodies (clone 6H2.B4, which recognizes the native form of cytochromec; 200 μg/test) into U937 cells (10⁶cells/test) was performed with a Gene Pulser II Plus apparatus (Bio-Rad). Exponentially decaying pulses were generated and delivered to cells suspended in PBS (0.7 ml/test) in sterile disposable electroporation cuvettes (Bio-Rad) of 0.4-cm path length (16). U937 cells were electroporated at a capacitance of 125 microfarads and a field strength of 800 V/cm, with a time constant (τ) of 1.5 ± 0.2 ms. Control cells were electroporated under the same experimental conditions, in the presence of non-immune mouse serum (200 μg/test). After electroporation, cells were kept for 5 min at 4 °C, and they were then washed twice in PBS and treated with AEA as described for the non-electroporated cells. Under these experimental conditions, approximately 1.0 pg/cell (2.5 μg/mg protein) of monoclonal antibodies was incorporated (16).

Evaluation of Cell Death

After incubation for the indicated periods in culture medium, floating and enzymatically detached cells were collected together by centrifugation at 200 × gfor 5 min. Viability was estimated by trypan blue dye exclusion in a Neubauer hemocytometer. Apoptosis was estimated in all experiments by cytofluorimetric analysis in a FACScalibur flow cytometer (Becton Dickinson), which quantified apoptotic body formation in dead cells by staining with propidium iodide (50 μg/ml, pretreated also with RNase to reduce noise), as reported (17). Cells were fixed using a methanol:acetone (4:1 v/v) solution, 1:1 in PBS, at −20 °C, and were stored at 4 °C. Cells were excited at 488 nm using a 15-milliwatt argon laser, and the fluorescence was monitored at 570 nm. Events were triggered by the FSC signal and gated for FL2-A/FL2-W to skip aggregates. Ten thousand events were evaluated using the Cell Quest Program. Controls of different cell lines contained less than 4.0 ± 1.0 apoptotic bodies every 100 cells analyzed. PCD was evaluated also by the cell-death detection ELISA kit (Roche Molecular Biochemicals), based on the evaluation of DNA fragmentation by an immunoassay for histone-associated DNA fragments in the cell cytoplasm (18).

Determination of Anandamide Uptake and Fatty Acid Amide Hydrolase (FAAH)

The uptake of [³H]AEA by intact C6 or DAUDI cells was studied as described (15). Cells were washed in PBS and resuspended in the respective serum-free culture media, at a density of 2 × 10⁶ cells/ml. Cell suspensions (1 ml/test) were incubated for different time intervals, at 37 °C, with 200 nM [³H]AEA; then they were washed three times in 2 ml of culture medium containing 1% bovine serum albumin and were finally resuspended in 200 μl of PBS. Membrane lipids were then extracted (18), resuspended in 0.5 ml of methanol, and mixed with 3.5 ml of Sigma-Fluor liquid scintillation mixture for non-aqueous samples (Sigma), and radioactivity was measured in a LKB1214 Rackbeta scintillation counter (Amersham Pharmacia Biotech). To discriminate noncarrier-mediated from carrier-mediated transport of AEA into cell membranes, control experiments were made at 4 °C (15). Incubations (15 min) were also carried out with different concentrations of [³H]AEA, in the range 0–1000 nM, in order to determine apparent K _m andV _max of the uptake by Lineweaver-Burk analysis (in this case, the uptake at 4 °C was subtracted from that at 37 °C). AEA uptake was expressed as picomoles of AEA taken up per min per mg of protein.

Fatty acid amide hydrolase (EC 3.5.1.4; FAAH) activity was assayed in C6 or DAUDI cell extracts by measuring the release of [³H]arachidonic acid from [³H]AEA, using reversed phase high performance liquid chromatography as reported (15). FAAH activity was expressed as picomoles of arachidonate released per min per mg of protein. Kinetic studies were performed using different concentrations of [³H]AEA (in the range 0–25 μM), and the kinetic constants (K _m,V _max) were calculated by fitting the experimental points in a Lineweaver-Burk plot with a linear regression program (Kaleidagraph 3.0.4). Straight lines with r values >0.95 were obtained.

Cannabinoid Receptor Binding Assay

CHP100, U973, C6, or DAUDI cells (2 × 10⁸) were pelleted and resuspended in 8 ml of buffer A (2 mM Tris-EDTA, 320 mMsucrose, 5 mM MgCl₂, pH 7.4) and then were homogenized in a Potter homogenizer and centrifuged at 1000 ×g for 10 min (19). The supernatant was recovered and combined with the supernatants obtained from two subsequent centrifugations at 1000 × g of the pellet. Combined supernatant fractions were centrifuged at 40000 × gfor 30 min, and the resulting pellet was resuspended in assay buffer B (50 mM Tris-HCl, 2 mM Tris-EDTA, 3 mM MgCl₂, pH 7.4), to a protein concentration of 1 mg/ml (19). The membrane preparation was divided in aliquots, quickly frozen in liquid nitrogen, and stored at −80 °C for no longer than 1 week. These membrane fractions, as well as those prepared from the brain of Wistar rats (male, weighting 250–280 g), were used in rapid filtration assays with the synthetic cannabinoid [³H]CP55,940, as described previously (19). Data of displacement of 400 pM [³H]CP 55,940 by various concentrations of AEA (in the range 10^− 12 to 10^− 6 M) were elaborated by the GraphPad program (GraphPad Software for Science), calculating the inhibition constant (K _i) as reported (20). Unspecific binding was determined in the presence of 10 μM AEA (19, 20). Binding of [³H]AEA to cells was assessed using the same membrane preparations and the same filtration assays as those described above for the cannabinoid receptors binding.

Measurement of Mitochondrial Uncoupling and Intracellular Calcium

Mitochondrial uncoupling and intracellular calcium concentration were evaluated by flow cytometric analysis in a FACScalibur Flow Cytometer (Becton Dickinson).

Mitochondrial uncoupling was measured using the fluorescent probe JC-1, as described (21). JC-1 (dissolved in dimethyl sulfoxide) was used at 20 μM final concentration. Control cells were treated with vehicle alone (1% of the final volume). After the treatment, CHP100 or U937 cells were washed in PBS and incubated 20 min at 37 °C. Cells were then analyzed in a FL1/FL2 dot plot (530 nm/570 nm), gating on morphologically normal cells.

Cytoplasmic free calcium was measured using the fluorescent Ca²⁺ indicator Fluo-3 AM, as reported (22). CHP100 or U937 cells were collected by centrifugation and washed twice in Ca²⁺– and Mg²⁺-free PBS. Then, Fluo-3 AM (10 μM dissolved in dimethyl sulfoxide) was added, and cells were incubated 40 min at 37 °C in the dark and frequently shaken manually. Control cells were treated with vehicle alone (1% of the final volume). Cells were then collected by centrifugation and resuspended in culture medium without fetal bovine serum. Fluo-3 AM fluorescence was recorded on a linear scale at 530 nm (bandwidth 30 nm), at a flow rate of approximately 1000 cells/s. Mean fluorescence values for 3000 events were registered every 10 s. Changes in mean fluorescence were plotted versus time.

Immunochemical Analysis

SDS-polyacrylamide gel electrophoresis (12%) under reducing conditions and electroblotting onto 0.45-μm nitrocellulose filters (Bio-Rad) were performed on cell extracts (25 μg/lane), prepared as reported (23). Prestained molecular mass markers (Bio-Rad) were carbonic anhydrase (37 kDa), soybean trypsin inhibitor (27 kDa), and lysozyme (19 kDa). Immunodetection of cytochrome c on nitrocellulose filters was performed with specific anti-cytochrome c monoclonal antibodies (clone 7H8.2C12, which recognizes the denatured form of cytochrome c), diluted 1:250. Goat anti-mouse immunoglobulins conjugated with alkaline phosphatase (GAM-AP) were used as secondary antibody at 1:2000 dilution. The amount of cytochromec released into the cytosol of CHP100 or U937 cells 8 h after treatment (23) was quantified by enzyme-linked immunosorbent assay (ELISA). Cell extracts (25 μg/well) were prepared as reported (23) and were reacted with anti-cytochrome c monoclonal antibodies (clone 7H8.2C12), diluted 1:250. GAM-AP were used as secondary antibody at 1:2000 dilution. Color development of the alkaline phosphatase reaction was recorded at 405 nm, usingp-nitrophenyl phosphate as substrate (15). The absorbance values of the unknown samples were within the linearity range of the ELISA test, assessed by calibration curves with known amounts of cytochrome c (in the range 0–500 ng/well).

Statistical Analysis

Data reported in this paper are the mean (±S.D.) of at least three independent determinations, each in duplicate. Statistical analysis was performed by the Student’sttest, elaborating experimental data by means of the InStat program (GraphPad Software for Science).

AEA-induced PCD Is Not Mediated by CB1 or CB2 Receptors and Is Potentiated by Inhibitors of AEA Degradation

Treatment of human neuroblastoma CHP100 cells and human lymphoma U937 cells with AEA led to apoptotic body formation in a dose- (Fig.1 A) and time- (Fig.1 B) dependent manner. Detection by ELISA of DNA fragments in the cell cytosols under the same experimental conditions confirmed the cytofluorimetric data (Fig. 1 C). Apoptosis at 48 h was significant in both cell lines already at 0.25 μM AEA (approximately 2.5-fold over the control) and reached a level of 6.0-fold over the control at 1 μM AEA (Fig.1 A). These concentrations are in the physiological range (24). Time course experiments showed that 1 μM AEA induced a significant increase in apoptotic bodies 24 h after treatment (2.5-fold over the control) and a maximum of 6-fold the control after 48 h (Fig. 1 B). Unlike AEA, 2-AG and other endocannabinoids failed to induce significant cell death in CHP100 or U937 cells (Table I).

View larger version:

• In this page
• In a new window

• Download as PowerPoint Slide

Figure 1

Induction of apoptosis by AEAin different cell lines. AEA induced apoptotic body formation and DNA fragmentation in human neuroblastoma CHP100 cells (white bars) and human lymphoma U937 cells (gray bars).A, the number of apoptotic bodies was measured at 48 h, and B, it was measured in cells treated with 1 μM AEA. C, DNA fragmentation was measured in the same samples as in B. Rat glioma C6 cells (light gray bars) and human leukemia DAUDI cells (black bars) did not show apoptotic body formation or DNA fragmentation under the same experimental conditions. *, p > 0.05 compared with control; §, p < 0.01 compared with control.

In order to investigate the possible role of cannabinoid receptors on PCD induced in CHP100 or U937 cells by AEA, two specific CBR antagonists were used as follows: SR141716 and SR144528, which bind CB1R and CB2R, respectively (3). Neither SR141716 nor SR144528, even if used at an excess concentration of 5 μM, could significantly prevent the AEA-induced toxicity, suggesting that the effect was not mediated by “classical” CB1 or CB2 receptors (TableII). In line with these data, the synthetic cannabinoid [³H]CP55.940, a high affinity ligand for both CB1 and CB2 receptors (3), did not bind to human neuroblastoma or lymphoma cells, suggesting that they had no functional cannabinoid receptors on their surface (Fig.2). Instead rat brain, used as a positive control, did bind [³H]CP55.940, which was displaced by AEA (Fig. 2, inset) with an inhibition constant (K _i = 30 ± 4 nM) close to that previously reported (20). Either the inhibitor of AEA transporter AM404 (25) or the FAAH inhibitor ATFMK (26), each used at 10 μM, significantly increased (up to approximately 180 or 165% of the control, respectively) the AEA toxicity (Table II). On the other hand, actinomycin D and cycloheximide (both at 10 μg/ml) reduced AEA-induced PCD in CHP100 cells down to approximately 20 or 30% of the control, respectively (Table II). Superimposable results were observed with the human lymphoma U937 cell line (Table II).

View larger version:

• In this page
• In a new window

• Download as PowerPoint Slide

Figure 2

Analysis of functional cannabinoid receptors in different cell lines. Binding of the synthetic cannabinoid [³H]CP55.940 (400 pM) to CHP100 (white bars), U937 (gray bars), C6 (light gray bars), and DAUDI (black bars) cells and displacement by 0.1 μMSR141716, 0.1 μM SR144528, or 1 μM AEA. Unspecific binding was measured in the presence of 10 μM AEA. Values were expressed as percentage of the maximum (100% = 1000 ± 100 cpm). Inset, binding of [³H]CP55.940 (400 pM) by rat brain membranes and displacement by various concentrations of AEA. Values were expressed as percentage of the maximum (100% = 1000 ± 100 cpm).CTR, control. *, p < 0.01 compared with control C6 cells; **, p > 0.05 compared with control C6 cells; ***, p > 0.05 compared with control DAUDI cells; §, p < 0.01 compared with control DAUDI cells.

AEA-induced PCD Is Mediated by Vanilloid Receptors

CHP100 and U937 cells were able to bind [³H]AEA, according to a saturable process with an apparent affinity constant of 75 ± 10 nM (Fig. 3 A). Cold AEA, but not 2-AG, SR141716 or SR144528 (each used at 1 μM), displaced 200 nM [³H]AEA from the binding site (Fig. 3B). Also, 1 μM cannabidiol (CBD), a selective antagonist of a newly discovered type of cannabinoid receptor (27), failed to affect the binding of 200 nM [³H]AEA to CHP100 or U937 cells, whereas 1 μM capsazepine (Caps), a selective antagonist of vanilloid receptors (28), fully displaced it (Fig.3 B). Interestingly, 10 μM CBD did not protect CHP100 or U937 cells against PCD induced by 1 μM AEA, whereas 10 μM Caps led to a 30% reduction of apoptotic bodies in both cell lines (Table II). Moreover, when we treated CHP100 or U937 cells with 1 μM capsaicin, the physiological agonist of vanilloid receptors (28), we found a 5-fold increase in apoptotic bodies after 48 h, which resembled the effect of 1 μM AEA on these cells (Fig. 1 A).

View larger version:

• In this page
• In a new window

• Download as PowerPoint Slide

Figure 3

Binding of AEA to CHP100 and U937 cells. A, represents the binding of [³H]AEA to CHP100 (circles) or U937 (triangles) cells.B, the effect of (cold) AEA, 2-AG, SR141716, SR144528, CBD, or Caps (each used at 1 μM) on the binding of 200 nM [³H]AEA to CHP100 (white bars) or U937 (gray bars) is shown, and values were expressed as percentage of the untreated controls (100% = 170 ± 20 (CHP100) or 160 ± 20 (U937) cpm, respectively). CTR,control. *, p < 0.01 compared with control cells.

Cannabinoid Receptors Prevent AEA-induced PCD

Rat glioma C6 cells and human leukemia DAUDI cells were found to have a specific AEA transporter, with apparent K _m andV _max values for AEA of 0.15 ± 0.02 μM and 40 ± 4 pmol·min^− 1·mg protein^− 1 (C6 cells) and 0.10 ± 0.01 μM and 150 ± 15 pmol·min^− 1·mg protein^− 1 (DAUDI cells). FAAH activity, which has been already reported in C6 cells (29), was found to depend on AEA concentration according to a Michaelis-Menten kinetics in these cells and in DAUDI cells (not shown), with apparent K _m of 5.0 ± 0.5 μM and V _maxof 135 ± 15 pmol·min^− 1·mg protein^− 1 (C6), and 5.0 ± 0.5 μM and 450 ± 50 pmol·min⁻¹·mg protein^− 1 (DAUDI). These kinetic parameters of AEA transporter and FAAH in C6 and DAUDI cells closely resembled those measured in CHP100 or U937 cells, respectively (15). Moreover, C6 cells have been reported to express CB1R on their surface (9), whereas in DAUDI cells the mRNA for the CB2 receptor subtype has been found (30). Consistently, these cells were able to bind [³H]CP55.940, which was displaced by 0.1 μMSR141716 in C6 cells or 0.1 μM SR144528 in DAUDI cells and by 1 μM AEA in both cell types (Fig. 2). Quite interestingly, neither cell line showed apoptotic body formation or DNA fragmentation when treated with 1 μM AEA under the experimental conditions previously tested with CHP100 or U937 cells (Fig. 1). However, at 10 μM AEA concentration a 3.5–4.0-fold increase in PCD after 48 h (TableIII) was observed, an effect that was enhanced in C6 cells by 1 μM SR141716, but not by SR144528, whereas in DAUDI cells the opposite was found (Table III). Inhibition of the AEA transporter by 10 μMAM404 almost doubled apoptotic body formation induced by AEA in both cell lines, and this effect was additive to that of SR141716 in C6 cells and of SR144528 in DAUDI cells (Table III). Apoptosis induced by 10 μM AEA in C6 or DAUDI cells was reduced to approximately 40% by 10 μM Caps, whereas CBD at the same concentration was ineffective (Table III). Remarkably, C6 and DAUDI cells were able to bind [³H]AEA (400 ± 40 or 350 ± 40 cpm respectively, upon incubation with 200 nM[³H]AEA), which was fully displaced by 1 μMSR141716 + 1 μM Caps (C6 cells) or 1 μMSR144528 + 1 μM Caps (DAUDI cells). Caps alone displaced approximately 30% [³H]AEA from each cell type, under the same experimental conditions.

AEA Induces Mitochondrial Uncoupling, Intracellular Calcium Rise, and Cytochrome c Release

AEA led to a dose-dependent mitochondrial uncoupling, which was most evident 6 h after treatment of CHP100 or U937 cells (TableIV). At this time interval, the increase in mitochondrial uncoupling was already significant with 0.25 μM AEA, a concentration that also induced significant apoptotic body formation (Fig. 1). Treatment with AEA also caused a dose-dependent and rapid (within 6 min) increase of intracellular calcium concentration (Table IV). Again, AM404 or ATFMK (both at 10 μM) significantly potentiated, whereas 10 μM Caps inhibited, the effect of AEA on both mitochondrial uncoupling and calcium rise (Table IV). Instead, 2-AG and the other endocannabinoids did not affect mitochondrial integrity or intracellular calcium concentration (Table I). Interestingly, 50 μM EGTA-AM, a permeant calcium chelator (31), reduced the number of apoptotic bodies induced by AEA to approximately 40 or 35% of the control, in CHP100 or U937 cells, respectively (Table II). Also 10 μM ETYA or 10 μM MK886, specific 5-lipoxygenase inhibitors (32, 33), reduced to approximately 40–50% the pro-apoptotic activity of AEA in both cell lines (Table II). Similar results were observed by treating the cells with 10 μM indomethacin, a cyclooxygenase inhibitor (34). Instead, treatment with 10 μM cyclosporin A, a mitochondrial permeability transition pore inhibitor (35), or 50 μM L-NAME, a nitric oxide synthase inhibitor (20), were ineffective (Table II).

Western blot showed that anti-cytochrome c monoclonal antibodies specifically recognized a single immunoreactive band in CHP100 and U937 cell saps, corresponding to a molecular mass of approximately 15 kDa (Fig.4 A). These antibodies were used to quantify cytochrome c release into cell cytosol by ELISA. Treatment of CHP100 or U937 cells with 1 μM AEA led to a 3–3.5-fold increase in cytochrome c release after 8 h, an effect that was not prevented by 5 μMSR141716, 5 μM SR144528, or 10 μM CBD (Fig.4B). 10 μM AM404 or 10 μM ATFMK enhanced cytochrome c release up to approximately 5-fold over the controls, whereas 10 μM Caps fully inhibited cytochrome c release induced by 1 μM AEA in both cell lines (Fig. 4 B). 2-AG and the other endocannabinoids did not stimulate cytochrome c release in CHP100 or U937 cells, when used at 10 μM (Fig.4 Band data not shown). Electrotransfer of anti-cytochromec monoclonal antibodies into U937 cells reduced the number of apoptotic bodies induced by 1 μM AEA after 24 h, from 2.8-fold (Fig. 1 B) to 1.3-fold over the controls, whereas non-immune mouse serum under the same experimental conditions was ineffective. Since the release of cytochrome c can trigger an apoptotic caspase cascade (23, 36, 37), we tested the effect of inhibitors of caspase-3 and caspase-9 on AEA-induced PCD. Table IIshows that the caspase-3 inhibitor Z-DEVD-FMK or the caspase-9 inhibitor Z-LEHD-FMK, each used at 50 μM, reduced PCD induced by 1 μM AEA in CHP100 or U937 cells to 20–30% of the controls.

View larger version:

• In this page
• In a new window

• Download as PowerPoint Slide

Figure 4

Effect of AEA on cytochrome crelease from CHP100 and U937 cells. A shows Western blot analysis of cytochrome c in cell homogenates (25 μg/lane). The arrow indicates the expected molecular size for cytochrome c. Molecular mass markers are shown on the right-hand side. B shows the effect of 1 μM AEA, in the absence or in the presence of 5 μM SR141716, 5 μMSR144528, 10 μM AM404, 10 μM ATFMK, 10 μMCBD, or 10 μM Caps, on cytochrome c release from CHP100 (white bars) or U937 (gray bars) cells, as determined by ELISA at 405 nm (see “Experimental Procedures”). Treatment of either cell line with any of the compounds listed, in the absence of AEA, was ineffective under the same experimental conditions. CTR, control. *, p< 0.01 compared with control cells; **, p > 0.05 compared with control cells; #, p> 0.05 compared with AEA-treated cells; §, p < 0.05 compared with AEA-treated cells; @, p < 0.01 compared with AEA-treated cells.