Anandamide Induces Apoptosis in Human Cells via Vanilloid Receptors
2000; Elsevier BV; Volume: 275; Issue: 41 Linguagem: Inglês
10.1074/jbc.m005722200
ISSN1083-351X
AutoresMauro Maccarrone, Tatiana Lorenzon, Monica Bari, Gerry Melino, Alessandro Finazzi‐Agrò,
Tópico(s)Pharmacological Receptor Mechanisms and Effects
ResumoThe endocannabinoid anandamide (AEA) is shown to induce apoptotic bodies formation and DNA fragmentation, hallmarks of programmed cell death, in human neuroblastoma CHP100 and lymphoma U937 cells. RNA and protein synthesis inhibitors like actinomycin D and cycloheximide reduced to one-fifth the number of apoptotic bodies induced by AEA, whereas the AEA transporter inhibitor AM404 or the AEA hydrolase inhibitor ATFMK significantly increased the number of dying cells. Furthermore, specific antagonists of cannabinoid or vanilloid receptors potentiated or inhibited cell death induced by AEA, respectively. Other endocannabinoids such as 2-arachidonoylglycerol, linoleoylethanolamide, oleoylethanolamide, and palmitoylethanolamide did not promote cell death under the same experimental conditions. The formation of apoptotic bodies induced by AEA was paralleled by increases in intracellular calcium (3-fold over the controls), mitochondrial uncoupling (6-fold), and cytochrome c release (3-fold). The intracellular calcium chelator EGTA-AM reduced the number of apoptotic bodies to 40% of the controls, and electrotransferred anti-cytochrome c monoclonal antibodies fully prevented apoptosis induced by AEA. Moreover, 5-lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid and MK886, cyclooxygenase inhibitor indomethacin, caspase-3 and caspase-9 inhibitors Z-DEVD-FMK and Z-LEHD-FMK, but not nitric oxide synthase inhibitorNω-nitro-l-arginine methyl ester, significantly reduced the cell death-inducing effect of AEA. The data presented indicate a protective role of cannabinoid receptors against apoptosis induced by AEA via vanilloid receptors. The endocannabinoid anandamide (AEA) is shown to induce apoptotic bodies formation and DNA fragmentation, hallmarks of programmed cell death, in human neuroblastoma CHP100 and lymphoma U937 cells. RNA and protein synthesis inhibitors like actinomycin D and cycloheximide reduced to one-fifth the number of apoptotic bodies induced by AEA, whereas the AEA transporter inhibitor AM404 or the AEA hydrolase inhibitor ATFMK significantly increased the number of dying cells. Furthermore, specific antagonists of cannabinoid or vanilloid receptors potentiated or inhibited cell death induced by AEA, respectively. Other endocannabinoids such as 2-arachidonoylglycerol, linoleoylethanolamide, oleoylethanolamide, and palmitoylethanolamide did not promote cell death under the same experimental conditions. The formation of apoptotic bodies induced by AEA was paralleled by increases in intracellular calcium (3-fold over the controls), mitochondrial uncoupling (6-fold), and cytochrome c release (3-fold). The intracellular calcium chelator EGTA-AM reduced the number of apoptotic bodies to 40% of the controls, and electrotransferred anti-cytochrome c monoclonal antibodies fully prevented apoptosis induced by AEA. Moreover, 5-lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid and MK886, cyclooxygenase inhibitor indomethacin, caspase-3 and caspase-9 inhibitors Z-DEVD-FMK and Z-LEHD-FMK, but not nitric oxide synthase inhibitorNω-nitro-l-arginine methyl ester, significantly reduced the cell death-inducing effect of AEA. The data presented indicate a protective role of cannabinoid receptors against apoptosis induced by AEA via vanilloid receptors. anandamide (arachidonoylethanolamide) 2-arachidonoylglycerol N-(4-hydroxyphenyl)arachidonoylamide arachidonoyl-trifluoromethyl ketone capsazepine cannabidiol type 1/2 cannabinoid receptor acetoxymethyl ester enzyme-linked immunosorbent assay 5,8,11,14-eicosatetraynoic acid fatty acid amide hydrolase linoleoylethanolamide Nω-nitro-l-arginine methyl ester oleoylethanolamide phosphate-buffered saline palmitoylethanolamide vanilloid receptor Z-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-fluoromethyl ketone Z-Leu-Glu(OCH3)-His-Asp (OCH3)-fluoromethyl ketone goat anti-mouse alkaline phosphatase programmed cell death Anandamide (arachidonoylethanolamide, AEA)1 belongs to an emerging class of endogenous lipids including amides and esters of long chain polyunsaturated fatty acids and collectively indicated as "endocannabinoids" (1Pop E. Curr. Opin. Chem. Biol. 1999; 3: 418-425Crossref PubMed Scopus (40) Google Scholar). In fact, AEA has been isolated and characterized as an endogenous ligand for cannabinoid receptors in the central nervous system (CB1 subtype) and peripheral immune cells (CB2 subtype). AEA is released from depolarized neurons, endothelial cells and macrophages (2Di Marzo V. Bisogno T. De Petrocellis L. Melck D. Orlando P. Wagner J.A. Kunos G. Eur. J. Biochem. 1999; 264: 258-267Crossref PubMed Scopus (281) Google Scholar), and mimics the pharmacological effects of Δ9-tetrahydrocannabinol, the active principle of hashish and marijuana (3Pertwee R.G. Pharmacol. Ther. 1997; 74: 129-180Crossref PubMed Scopus (1275) Google Scholar). Recently, attention has been focused on the possible role of AEA and other endocannabinoids in regulating cell growth and differentiation, which might account for some pathophysiological effects of these lipids. An anti-proliferative action of AEA has been reported in human breast carcinoma cells, due to a CB1-like receptor-mediated inhibition of the action of endogenous prolactin at its receptor (4De Petrocellis L. Melck D. Palmisano A. Bisogno T. Laezza C. Bifulco M. Di Marzo V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8375-8380Crossref PubMed Scopus (344) Google Scholar). An activation of cell proliferation by AEA has been reported instead in hematopoietic cell lines (5Derocq J.-M. Bouaboula M. Marchand J. Rinaldi-Carmona M. Ségui M. Casellas P. FEBS Lett. 1998; 425: 419-425Crossref PubMed Scopus (73) Google Scholar). Moreover, preliminary evidence that the immunosuppressive effects of AEA might be associated with inhibition of lymphocyte proliferation and induction of programmed cell death (PCD or apoptosis) has been reported (6Schwarz H. Blanco F.J. Lotz M. J. Neuroimmunol. 1994; 55: 107-115Abstract Full Text PDF PubMed Scopus (159) Google Scholar), and growing evidence is being collected that suggests that AEA might have pro-apoptotic activity, both in vitro (7Sarker K.P. Obara S. Nakata M. Kitajima I. Maruyama I. FEBS Lett. 2000; 472: 39-44Crossref PubMed Scopus (149) Google Scholar) and in vivo (8Galve-Roperh I. Sànchez C. Cortes M.L. del Pulgar T.G. Izquierdo M. Guzman M. Nat. Med. 2000; 6: 313-316Crossref PubMed Scopus (556) Google Scholar). This would extend to endocannabinoids previous observations on Δ9-tetrahydrocannabinol, shown to induce PCD in glioma tumors (8Galve-Roperh I. Sànchez C. Cortes M.L. del Pulgar T.G. Izquierdo M. Guzman M. Nat. Med. 2000; 6: 313-316Crossref PubMed Scopus (556) Google Scholar), glioma cells (9Sànchez C. Galve-Roperh I. Canova C. Brachet P. Guzmàn M. FEBS Lett. 1998; 436: 6-10Crossref PubMed Scopus (241) Google Scholar), primary neurons (10Chan G.C.-K. Hinds T.R. Impey S. Storm D.R. J. Neurosci. 1998; 18: 5322-5332Crossref PubMed Google Scholar), hippocampal slices (10Chan G.C.-K. Hinds T.R. Impey S. Storm D.R. J. Neurosci. 1998; 18: 5322-5332Crossref PubMed Google Scholar), and prostate cells (11Ruiz L. Miguel A. Diaz-Laviada I. FEBS Lett. 1999; 458: 400-404Crossref PubMed Scopus (139) Google Scholar). However, the mechanism of AEA-induced PCD is unknown. The various effects of AEA in the central nervous system and in immune system (reviewed in Refs.1Pop E. Curr. Opin. Chem. Biol. 1999; 3: 418-425Crossref PubMed Scopus (40) Google Scholar, 2Di Marzo V. Bisogno T. De Petrocellis L. Melck D. Orlando P. Wagner J.A. Kunos G. Eur. J. Biochem. 1999; 264: 258-267Crossref PubMed Scopus (281) Google Scholar, 3Pertwee R.G. Pharmacol. Ther. 1997; 74: 129-180Crossref PubMed Scopus (1275) Google Scholar), as well as its ability to reduce the emerging pain signals at sites of tissue injury (12Walker J.M. Huang S.M. Strangman N.M. Tsou K. Sañudo-Peña M.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12198-12203Crossref PubMed Scopus (426) Google Scholar), are terminated by a rapid and selective carrier-mediated uptake of AEA into cells (13Maccarrone M. Bari M. Lorenzon T. Bisogno T. Di Marzo V. Finazzi-Agrò A. J. Biol. Chem. 2000; 275: 13484-13492Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), followed by its degradation to ethanolamine and arachidonic acid by the enzyme fatty acid amide hydrolase (FAAH) (14Maccarrone M. Valensise H. Bari M. Lazzarin N. Romanini C. Finazzi-Agrò A. Lancet. 2000; 355: 1326-1329Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Recently, we showed that human neuroblastoma CHP100 cells and human lymphoma U937 cells do have these tools to eliminate AEA (15Maccarrone M. van der Stelt M. Rossi A. Veldink G.A. Vliegenthart J.F.G. Finazzi-Agrò A. J. Biol. Chem. 1998; 273: 32332-32339Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Therefore, these cell lines were chosen to investigate how AEA and related endocannabinoids induce apoptosis and how the removal and degradation of AEA are related to this process. The existence of a neuroimmune axis appears to be confirmed by the finding that endocannabinoids elicit common responses in these two cell types. 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 (15Maccarrone M. van der Stelt M. Rossi A. Veldink G.A. Vliegenthart J.F.G. Finazzi-Agrò A. J. Biol. Chem. 1998; 273: 32332-32339Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). 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). Human neuroblastoma CHP100 cells were cultured as reported (15Maccarrone M. van der Stelt M. Rossi A. Veldink G.A. Vliegenthart J.F.G. Finazzi-Agrò A. J. Biol. Chem. 1998; 273: 32332-32339Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), 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 (15Maccarrone M. van der Stelt M. Rossi A. Veldink G.A. Vliegenthart J.F.G. Finazzi-Agrò A. J. Biol. Chem. 1998; 273: 32332-32339Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). 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 (9Sànchez C. Galve-Roperh I. Canova C. Brachet P. Guzmàn M. FEBS Lett. 1998; 436: 6-10Crossref PubMed Scopus (241) Google Scholar). Cells were maintained at 37 °C in a humidified atmosphere with 5% CO2 and 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 (16Maccarrone M. Veldink G.A. Vliegenthart J.F.G. Eur. J. Biochem. 1992; 205: 995-1001Crossref PubMed Scopus (22) Google Scholar). 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 (16Maccarrone M. Veldink G.A. Vliegenthart J.F.G. Eur. J. Biochem. 1992; 205: 995-1001Crossref PubMed Scopus (22) Google Scholar). 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 (17Maccarrone M. Catani M.V. Finazzi-Agrò A. Melino G. Cell Death Differ. 1997; 4: 396-402Crossref PubMed Scopus (45) Google Scholar). 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 (18Maccarrone M. Nieuwenhuizen W.F. Dullens H.F.J. Catani M.V. Melino G. Veldink G.A. Vliegenthart J.F.G. Finazzi-Agrò A. Eur. J. Biochem. 1996; 241: 297-302Crossref PubMed Scopus (37) Google Scholar). The uptake of [3H]AEA by intact C6 or DAUDI cells was studied as described (15Maccarrone M. van der Stelt M. Rossi A. Veldink G.A. Vliegenthart J.F.G. Finazzi-Agrò A. J. Biol. Chem. 1998; 273: 32332-32339Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). 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 (18Maccarrone M. Nieuwenhuizen W.F. Dullens H.F.J. Catani M.V. Melino G. Veldink G.A. Vliegenthart J.F.G. Finazzi-Agrò A. Eur. J. Biochem. 1996; 241: 297-302Crossref PubMed Scopus (37) Google Scholar), 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 (15Maccarrone M. van der Stelt M. Rossi A. Veldink G.A. Vliegenthart J.F.G. Finazzi-Agrò A. J. Biol. Chem. 1998; 273: 32332-32339Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). 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 (15Maccarrone M. van der Stelt M. Rossi A. Veldink G.A. Vliegenthart J.F.G. Finazzi-Agrò A. J. Biol. Chem. 1998; 273: 32332-32339Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). 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. 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 (19Maccarrone M. Fiorucci L. Erba F. Bari M. Finazzi-Agrò A. Ascoli F. FEBS Lett. 2000; 468: 176-180Crossref PubMed Scopus (74) Google Scholar). 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 (19Maccarrone M. Fiorucci L. Erba F. Bari M. Finazzi-Agrò A. Ascoli F. FEBS Lett. 2000; 468: 176-180Crossref PubMed Scopus (74) Google Scholar). 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 (19Maccarrone M. Fiorucci L. Erba F. Bari M. Finazzi-Agrò A. Ascoli F. FEBS Lett. 2000; 468: 176-180Crossref PubMed Scopus (74) Google Scholar). Data of displacement of 400 pm [3H]CP 55,940 by various concentrations of AEA (in the range 10−12 to 10−6m) were elaborated by the GraphPad program (GraphPad Software for Science), calculating the inhibition constant (K i) as reported (20Deutsch D.G. Goligorsky M.S. Schmid P.C. Krebsbach R.J. Schmid H.H.O. Das S.K. Dey S.K. Arreaza G. Thorup C. Stefano G. Moore L.C. J. Clin. Invest. 1997; 100: 1538-1546Crossref PubMed Scopus (332) Google Scholar). Unspecific binding was determined in the presence of 10 μm AEA (19Maccarrone M. Fiorucci L. Erba F. Bari M. Finazzi-Agrò A. Ascoli F. FEBS Lett. 2000; 468: 176-180Crossref PubMed Scopus (74) Google Scholar, 20Deutsch D.G. Goligorsky M.S. Schmid P.C. Krebsbach R.J. Schmid H.H.O. Das S.K. Dey S.K. Arreaza G. Thorup C. Stefano G. Moore L.C. J. Clin. Invest. 1997; 100: 1538-1546Crossref PubMed Scopus (332) Google Scholar). 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. 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 (21Smiley S.T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3671-3675Crossref PubMed Scopus (1273) Google Scholar). 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 (22Vandenberghe P.A. Ceuppens J.L. J. Immunol. Methods. 1990; 127: 197-205Crossref PubMed Scopus (180) Google Scholar). 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. 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 (23Ushmorov A. Ratter F. Lehmann V. Dröge W. Schirrmacher V. Umansky V. Blood. 1999; 93: 2342-2352Crossref PubMed Google Scholar). 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 (23Ushmorov A. Ratter F. Lehmann V. Dröge W. Schirrmacher V. Umansky V. Blood. 1999; 93: 2342-2352Crossref PubMed Google Scholar) was quantified by enzyme-linked immunosorbent assay (ELISA). Cell extracts (25 μg/well) were prepared as reported (23Ushmorov A. Ratter F. Lehmann V. Dröge W. Schirrmacher V. Umansky V. Blood. 1999; 93: 2342-2352Crossref PubMed Google Scholar) 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 (15Maccarrone M. van der Stelt M. Rossi A. Veldink G.A. Vliegenthart J.F.G. Finazzi-Agrò A. J. Biol. Chem. 1998; 273: 32332-32339Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). 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). 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'st test, elaborating experimental data by means of the InStat program (GraphPad Software for Science). 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 (24Di Marzo V. Sepe N. De Petrocellis L. Berger A. Crozier G. Fride E. Mechoulam R. Nature. 1998; 396: 636Crossref PubMed Google Scholar). 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).Table IEffect of AEA analogues on apoptotic body formation, mitochondrial uncoupling and intracellular calcium concentration in CHP100 cellsCompound (1 μm)Apoptotic bodiesMitochondrial uncouplingIntracellular calcium-fold over control-fold over control-fold over controlNone1.01.01.02-AG1.4 ± 0.2 1-ap > 0.05 compared with control.1.4 ± 0.2 1-ap > 0.05 compared with control.1.4 ± 0.2 1-ap > 0.05 compared with control.LEA1.4 ± 0.2 1-ap > 0.05 compared with control.1.4 ± 0.2 1-ap > 0.05 compared with control.1.3 ± 0.2 1-ap > 0.05 compared with control.OEA1.3 ± 0.2 1-ap > 0.05 compared with control.1.3 ± 0.2 1-ap > 0.05 compared with control.1.2 ± 0.2 1-ap > 0.05 compared with control.PEA1.4 ± 0.2 1-ap > 0.05 compared with control.1.2 ± 0.2 1-ap > 0.05 compared with control.1.3 ± 0.2 1-ap > 0.05 compared with control.Values refer to measurements performed 48 h (apoptotic bodies), 6 h (mitochondrial uncoupling), or 6 min (intracellular calcium) after the addition of each compound. In all analyses, U937 cells showed results superimposable to those obtained with CHP100 cells, omitted for the sake of clarity.1-a p > 0.05 compared with control. Open table in a new tab Values refer to measurements performed 48 h (apoptotic bodies), 6 h (mitochondrial uncoupling), or 6 min (intracellular calcium) after the addition of each compound. In all analyses, U937 cells showed results superimposable to those obtained with CHP100 cells, omitted for the sake of clarity. 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 (3Pertwee R.G. Pharmacol. Ther. 1997; 74: 129-180Crossref PubMed Scopus (1275) Google Scholar). 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 (3Pertwee R.G. Pharmacol. Ther. 1997; 74: 129-180Crossref PubMed Scopus (1275) Google Scholar), 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 (20Deutsch D.G. Goligorsky M.S. Schmid P.C. Krebsbach R.J. Schmid H.H.O. Das S.K. Dey S.K. Arreaza G. Thorup C. Stefano G. Moore L.C. J. Clin. Invest. 1997; 100: 1538-1546Crossref PubMed Scopus (332) Google Scholar). Either the inhibitor of AEA transporter AM404 (25Piomelli D. Beltramo M. Glasnapp S. Lin S.Y. Goutopoulos A. Xie X.Q. Makriyannis A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5802-5807Crossref PubMed Scopus (238) Google Scholar) or the FAAH inhibitor ATFMK (26Koutek B. Prestwich G.D. Howlett A.C. Chin S.A. Salehani D. Akhavan N. Deutsch D.G. J. Biol. Chem. 1994; 269: 22937-22940Abstract Full Text PDF PubMed Google Scholar), 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).Table IIEffect of various compounds
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