Inhibition of Tumor Necrosis Factor-induced Cell Death inMCF7 by a Novel Inhibitor of Neutral Sphingomyelinase*

Received for publication, July 8, 2002, and in revised form, July 31, 2002Published, JBC Papers in Press, August 1, 2002, DOI 10.1074/jbc.M206747200

Chiara Luberto‡, Daniel F. Hassler§, Paola Signorelli‡¶, Yasuo Okamoto‡�, Hirofumi Sawai‡**,Eric Boros§, Debra J. Hazen-Martin‡‡, Lina M. Obeid§§, Yusuf A. Hannun‡¶¶, and Gary K. Smith§

From the Departments of ‡Biochemistry and Molecular Biology, §§Medicine, and ‡‡Pathology and Laboratory Medicine,Medical University of South Carolina, Charleston, South Carolina 29425 and the §Divisions of Discovery Chemistry andBiology, GlaxoSmithKline, Research Triangle Park, North Carolina 27709

A high throughput screen for neutral, magnesium-de-pendent sphingomyelinase (SMase) was performed. Oneinhibitor discovered in the screen, GW4869, functionedas a noncompetitive inhibitor of the enzyme in vitrowith an IC50 of 1 �M. It did not inhibit acid SMase at upto at least 150 �M. The compound was then evaluated forits ability to inhibit tumor necrosis factor (TNF)-in-duced activation of neutral SMase (N-SMase) in MCF7cells. GW4869 (10 �M) partially inhibited TNF-inducedsphingomyelin (SM) hydrolysis, and 20 �M of the com-pound was protected completely from the loss of SM.The addition of 10–20 �M GW4869 completely inhibitedthe initial accumulation of ceramide, whereas this effectwas partially lost at later time points (24 h). These datatherefore support the inhibitory action of GW4869 onN-SMase not only in vitro but also in a cellular model.The addition of GW4869 at both 10 and 20 �M did notmodify cellular glutathione levels in response to TNF,suggesting that the action of GW4869 occurred down-stream of the drop in glutathione, which was shownpreviously to occur upstream of the activation ofN-SMase. Further, whereas TNF treatment also caused a75% increase of de novo synthesized ceramide after 20 h ofincubation, GW4869, at either 10 or 20 �M, had no effect onthis pathway of ceramide generation. In addition, GW4869did not significantly impair TNF-induced NF-�B translo-cation to nuclei. Therefore, GW4869 does not interferewith other key TNF-mediated signaling effects. GW4869was able, in a dose-dependent manner, to significantlyprotect from cell death as measured by nuclear conden-sation, caspase activation, PARP degradation, and trypanblue uptake. These protective effects were accompaniedby significant inhibition of cytochrome c release frommitochondria and caspase 9 activation, therefore local-izing N-SMase activation upstream of mitochondrialdysfunction. In conclusion, our results indicate that N-SMase activation is a necessary step for the full devel-opment of the cytotoxic program induced by TNF.

Programmed cell death is a necessary requirement duringdevelopment, and an altered regulation of this process is alsothought to contribute to the occurrence of serious illnesses suchas cancer and neurodegenerative diseases. Significant progresshas been made in the identification of crucial events that con-tribute to defining this process (mitochondrial dysfunction,chromatin fragmentation, and membrane blebbing) as well asin the identification of molecular mechanisms and effectorsthat regulate and/or execute these events (exposure of phos-phatidylserine on the plasma membrane, depolarization of theouter mitochondrial membrane, formation of permeabilitytransition pores in the mitochondrial membrane, cytochrome crelease, caspase activation, and alteration of calcium homeo-stasis, Bcl2 family members, apoptosis-inducing factor, apo-ptosis activating factor-1, Smac/Diablo, IAPs, and others).

The sphingolipid ceramide has been shown to induce cellularfeatures characteristic of programmed cell death (membraneblebbing, mitochondrial dysfunction, and nuclear fragmenta-tion). Moreover, many agents known to cause apoptosis (suchas TNF,1 CD95 cross-linking, daunorubicin, heat stress,growth factor withdrawal, UV-B and �-radiation, bacterial in-fections, and others) have also been found to increase theintracellular levels of ceramide, with emerging evidence sug-gesting an important role for ceramide in regulating/mediatingthe apoptotic response to these agents (for reviews, see Refs.1–3). A major current challenge is to define the role of thepathway promoted by ceramide and integrate it with othermolecular mechanisms that lead to cell death.

Two main routes have been defined for the generation ofceramide: hydrolysis of sphingomyelin and de novo biosynthe-sis. The first occurs by the action of sphingomyelinases(SMases), which operate at different pH optima (acid or neutralSMase) and have different metal requirements (magnesiumdependence) (2, 4). Activation of acid sphingomyelinase (A-SMase) has been observed after treatment with UV-A radiation(5) and stimulation of the p75 neurotropin receptor (6), CD28(7), TNF receptor, and CD95 (8), although some of these latterconclusions have been recently questioned (9, 10). NeutralSMase (N-SMase) activation has been also observed after stim-ulation of the p75 neurotropin receptor (11), after ligation ofCD95 and TNF receptor (12, 13), and after irradiation (14) butalso upon heat stress and serum starvation (15), treatment

* This work was supported in part by National Institutes of HealthGrants GM 43825 and CA 87584 (to Y. A. H.) and AG 16583 (toL. M. O.). The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

¶ Present address: Dept. of General Pathology, Universita’ degliStudi di Milano, 20133 Milano, Italy.

� Recipient of the Merck Company Foundation and Banyu FellowshipAwards in Lipid Metabolism and Atherosclerosis.

** Present address: Dept. of Medicine, Osaka Dental University, 8-1Kuzuhahanazonocho, Hirakata, Osaka 573, Japan.

¶¶ To whom correspondence may be addressed: Dept. of Biochemistryand Molecular Biology, 173 Ashley Ave., Charleston, SC 29425. Tel.:843-792-4321; Fax: 843-792-4322; E-mail: hannun@musc.edu.

1 The abbreviations used are: TNF, tumor necrosis factor �; SMase,sphingomyelinase; N-SMase, neutral sphingomyelinase; A-SMase, acidsphigomyelinase; PARP, poly(ADP-ribose) polymerase; PS, phosphati-dylserine; SM, sphingomyelin; PAF, platelet-activating factor; PLC,phospholipase C; FBS, fetal bovine serum; MSA, methane sulfonic acid;PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothio-cyanate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 43, Issue of October 25, pp. 41128–41139, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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with vitamin D (16), and CD40 ligation (17). The de novopathway is regulated primarily by the action of serine palmi-toyltransferase, the first enzyme along the biosynthetic path-way. Accumulation of ceramide via de novo biosynthesis wasoriginally shown after treatment with retinoic acid in GH4C1(18) and then with daunorubicin in P388 and U937 cells (19),angiotensin II stimulation of PC12W cells (20), etoposide treat-ment of Molt-4 cells (21), IgM-induced receptor cross-linking inRamos B cells (22), loading of palmitate in beta cells (23), andTNF/cycloheximide treatment of bovine cerebral endothelialcells (24).

The study of the de novo pathway has benefited tremen-dously from the availability of highly specific inhibitors: fumo-nisin B1 for ceramide synthase (25) and myriocin/ISP1 forserine palmitoyltransferase (26). On the other hand, very fewtools have been available to dissect the neutral sphingomyeli-nase pathway. One molecule, scyphostatin, has been previouslydescribed to exert inhibitory activity versus a N-SMase (27, 28),and it has been used to implicate N-SMase in the outgrowthprocess of hippocampal neurons in response to nerve growthfactor (29). We set out to develop specific inhibitors of N-SMaseand to determine the role of N-SMase in apoptosis and how itinteracts with other key regulators of apoptosis. A highthroughput assay was developed for N-SMase, and we success-fully identified a molecule, GW4869 (Fig. 1), that exhibitedsignificant and specific inhibitory activity on N-SMase. Weprovide evidence of the specificity of GW4869 in vitro. Next, theeffects of this inhibitor were determined in MCF7 breast cancercells treated with TNF, which has emerged as one of the bestcharacterized models of cytokine-induced cell death and of ce-ramide function. It has been previously shown that TNF in-duces activation of N-SMase in these cells and that this acti-vation is a consequence of the drop of glutathione that followsthe activation of the death receptor and caspase 8 (13). Also,this activation couples to processing of effector caspases (30).

Using this newly characterized inhibitor, we were able toconfirm N-SMase activation upon TNF treatment of MCF7 andits effects on effector caspases and cell death. Second, we pro-vide evidence for the first time of the participation of N-SMasein the processes that lead to cytochrome c release and mito-chondrial dysfunction, indicating the requirement for N-SMaseactivation as a necessary step for the complete development ofthe cytotoxic program induced by TNF.

EXPERIMENTAL PROCEDURES

Materials—RPMI 1640 medium was from Invitrogen. Fetal bovineserum (FBS) was from Summit Technology. TNF was from Peprotech(Rocky Hill, NJ). [�-32P]ATP, [methyl-3H]choline chloride, andEN3HANCETM spray were from PerkinElmer Life Sciences. [choline-methyl-14C]SM was provided by Alicja Bielawska (Medical University ofSouth Carolina, Charleston, SC). SM and PS were from Avanti PolarLipids, Inc. Silica Gel 60 thin layer chromatography plates were fromWhatman. Scintillation mixture Safety Solve was from Research Prod-ucts International. Poly(dI-dC) and poly(dN6) were from AmershamBiosciences. Rabbit anti-p65 NF-�B antibodies were from Rockland;monoclonal anti-cytochrome c and anti-human caspase 6 antibodieswere from Becton Dickinson Co.; and rabbit anti-PARP, anti-mouseIgG-horseradish peroxidase, and anti-rabbit IgG-horseradish peroxi-dase were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

In Vitro Sphingomyelinase Activity Assays—Partially purified anddelipidated rat brain neutral sphingomyelinase was prepared and as-sayed as described (31) with variable amounts of SM or PS incorporatedinto the assay via solubilization into Triton X-100 micelles. Human acidsphingomyelinase, overexpressed in SF-9 insect cells using a baculovi-ral system, was similarly assayed in a final reaction volume of 50 �lcontaining 100 mM sodium acetate (pH 5), 0.1 mM zinc acetate, 0.13%Triton X-100 and lacking PS or dithiothreitol. The reaction was allowedto proceed for 10, 30, and 150 min at room temperature. At the appro-priate time, the reaction was quenched with 75 �l of 5% bovine serumalbumin followed by 75 �l of 8% trichloroacetic acid. After centrifuga-tion, 100 �l of supernatant were counted for 14C with 150 �l of Op-

tiphase Supermix. All rate measurements obtained were in the linearrange and represented conversion of less than 20% of substrate toproduct.

Partial Purification of the Lyso-PAF Phospholipase C (PLC)—HEK293, expressing the lyso-PAF PLC (32), were lysed in 25 mM Tris(pH 7.4), 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 2 �g/mleach of chymostatin, leupeptin, antipain, and pepstatin A in the ab-sence of detergents, and the postnuclear extract was centrifuged for 1 hat 100,000 � g at 4 °C. The pellet, containing the total membranes, wasresuspended in lysis buffer containing 0.1% Triton X-100 and incubatedon a rotating platform for 1 h at 4 °C. The suspension was then centri-fuged for 1h at 100,000 � g at 4 °C, and the supernatant was applied toa HiTrap Q column equilibrated with 20 mM Tris-HCl, pH 7.4, 0.1%Triton X-100, 1 mM EGTA, 1 mM EDTA. The lyso-PAF PLC was theneluted with a linear salt gradient (1.5 M NaCl). Fractions were collected,N-SMase activity was measured as previously indicated (33), and theprotein level was determined by Western blot analysis using anti-His6

monoclonal antibodies against the histidine-FLAG of the protein. Thefraction containing peak activity (which also showed most of the proteinby western) was used for determining the effect of GW4869 in vitro.

Specificity of GW4869 on Enzyme Inhibition—Partially purified ratbrain N-SMase and lyso-PAF PLC were incubated in the absence orpresence of GW4869 and PS (100 �M), and SM hydrolysis was deter-mined as previously described (30). In the case of Bacillus cereus N-SMase (Sigma), PS was not included in the reaction mixture, since itdoes not affect the bacterial enzymatic activity. B. cereus phosphatidyl-choline-PLC (Sigma) was incubated in the presence or absence ofGW4869 in a reaction mixture containing 100 mM Tris, pH 7.2, 25%glycerol, 20 mM p-nitrophenyl/phosphorylcholine, and production of p-nitrophenol was quantified spectrophotometrically at 410 nm. Proteinphosphatase 2A from bovine kidney (Calbiochem) was incubated in thepresence or absence of GW4869 in buffer containing 50 mM Tris, pH 7.4,1 mM dithiothreitol, 100 �M MnCl2, and 20% glycerol, and phosphataseactivity was measured as described by Jones and Hannun (34). In allassays, 10 �M GW4869 was used, and 30-min preincubation with theenzymes preceded the addition of the substrate.

Cell Culture and GW4869 Treatment—MCF7 human breast cancercells were routinely cultured in RPMI 1640 containing 10% FBS at37 °C in 5% CO2. Unless otherwise indicated, for treatment, cells wereseeded at 1.7 � 106 cells/10-cm culture dish in 10 ml of completemedium; after 24 h, the medium was replaced with 7 ml of RPMI 1640containing 2% FBS and 25 mM Hepes, pH 7.5, and the cells were restedfor 2 h prior to treatment. GW4869 was routinely stored at �80 °C as a1.5 mM stock suspension in Me2SO. Right before use, the suspensionwas solubilized by the addition of 5% methane sulfonic acid (MSA) (2.5�l of 5% MSA in sterile double-distilled H2O were added to 50 �l ofGW4869 stock suspension; therefore, the concentration of the GW4869stock solution at the time of the experiments was 1.43 mM). The sus-pension was mixed and warmed up at 37 °C until clear. Cells werepreincubated with the inhibitor for 30 min prior to treatment with TNF.Control cells were treated with Me2SO containing 5% MSA, similarly tothe samples receiving the GW4869 solution. When different doses ofGW4869 were tested, amounts of vehicle solution were added in order toequal the volume of GW4869 used for the highest dose.

Sphingomyelin Measurement—Cells were seeded at 0.1 � 106 cells/10-cm dish in 8 ml of complete growth medium. After 48 h, the cellswere labeled with [methyl-3H]choline chloride (1 �Ci/ml final concen-tration in 10 ml of growth medium/plate). After �60 h, the cells werechased with 10 ml of complete medium for 90 min. Then the cells werewashed once with 5 ml of PBS, and 7 ml of medium containing 2% FBSand 25 mM Hepes, pH 7.5, were added. After resting the cells forapproximately 1 h, preincubation for 30 min with GW4869 was started,and TNF treatment followed. At the appropriate time points, the me-dium from each plate was collected, and the cells were washed oncewith 2 ml of ice-cold PBS. Cells were scraped on ice in 2 ml of PBS, andeach plate was washed with an additional 2 ml of PBS. Cells andwashes were pooled with the medium and centrifuged for 5 min at2000 � g (4 °C). The cell pellets were stored at �80 °C. On the day of themeasurement, the pellets were resuspended in 600 �l of double-distilledH2O by vortexing and sonication. Aliquots of cell lysates were used forprotein determination, and 250 �l in duplicate were used for SM deter-mination as described by Andrieu et al. (35).

Measurements of Mass Levels of Ceramide—Cells were harvested inmethanol, and lipids were extracted by the method of Bligh and Dyer(36). The chloroform organic phase was divided into aliquots (in dupli-cates) and dried down for ceramide and phosphate measurements.Ceramide levels were evaluated using the Escherichia coli diacylglyc-erol kinase assay. Ceramide was quantitated by using external stand-

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ards and normalized to phosphate content (37).Measurement of Ceramide Generated by de Novo Biosynthesis—Cells

were seeded at 1.7 � 106 cells/plate in complete growth medium. After24 h, the medium was replaced with 6 ml of RPMI 1640 containing 2%FBS and 25 mM Hepes, pH 7.5. Right before the addition of GW4869,[3H]palmitate was added to the cells in 1 ml of the same medium to afinal activity of 1 �Ci/ml. After �21 h of treatment with TNF, themedium was collected, and the plates were washed once with PBS thatwas combined with the medium and centrifuged at 2000 � g for 5 minat 4 °C to collect floating cells. Cells were scraped off the plate withmethanol and combined with the floaters. Lipids were extracted by themethod of Bligh and Dyer (36). One ml of the organic phase was used forceramide determination by separation of lipids through thin layer chro-matography (chloroform, methanol, 4.2 N ammonium hydroxide; 4:1:0.1; v/v/v), and 0.35 ml in duplicates were used for determination ofinorganic phosphate used to normalize the ceramide values. The cer-amide band was identified by comparison with an authentic standard,and radioactivity was quantified in a scintillation counter.

Electrophoretic Mobility Shift Assay of NF-�B—The assay was per-formed as previously described (38). Briefly, after treatments, cells werewashed and harvested by scraping in PBS. The pellet was resuspendedin 400 �l of lysis buffer (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM

EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfo-nyl fluoride) and incubated for 10 min on ice. Just prior to centrifuga-tion, 20 �l of 10% Nonidet P-40 was added, and the suspension wasmixed by pipetting up and down three times. Nuclei were pelleted bymicrocentrifugation at 1300 � g for 10 min. The supernatant wasremoved, and the nuclei were resuspended in 20 �l of extraction buffer(20 mM Hepes (pH 7.9), 0.4 M NaCl, 25% (v/v) glycerol, 1 mM EDTA, 1mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride).The suspension was mixed gently for 30 min and pelleted at 13,000 �g for 15 min. The supernatant was flash-frozen and stored at �80 °C.Protein concentrations were determined using the Bio-Rad assay. Nu-clear extracts (10 �g) were incubated in HDKE buffer (20 mM Hepes, pH7.9, 50 mM KCl, 5% (v/v) glycerol, 1 mM EDTA, 5 mM dithiothreitol)containing 1 �g of poly(dI-dC), 1 �g of poly(dN6), and 10 �g of bovineserum albumin. One �l of radiolabeled oligonucleotide probe (60,000–100,000 cpm) was added to each reaction and incubated at room tem-perature for 20 min. The reaction was terminated by the addition of 6�l of 15% Ficoll solution containing indicator dyes (bromphenol blueand xylene cyanol). Equal amounts (20 �l) of reaction mixture wereloaded on a 5% nondenaturing polyacrylamide gel in 1� TBE and runat 200 V. Gels were placed onto Whatman filter paper, dried, andautoradiographed. The specificity of NF-�B activation upon TNF stim-ulation in the absence and in the presence of GW6948 was assayed aspreviously described (34).

Immunocytochemistry of NF-�B and Cytochrome c and NuclearStaining—For immunocytochemical analysis, cells were plated at adensity of 1.7 � 106 cells/100-mm plate that contained 22-mm glasscoverslips. Following treatment, cells were fixed for 15 min in 4%paraformaldehyde and subsequently permeabilized during a 15-minincubation in 4% paraformaldehyde and 0.2% Triton X-100 in PBS,washed, blocked and treated for 4 h with a rabbit anti-p65 NF-�Bantibody (1:200) or a mouse anti-cytochrome c antibody (1:200). Thecoverslips were then washed and incubated for 1 h with a fluoresceinisothiocyanate-conjugated donkey anti-rabbit IgG (1:100; Jackson Im-munoResearch) or a rhodamine (TRITC)-conjugated goat anti-mouseIgG (1:200; Jackson ImmunoResearch), respectively. Nuclei were visu-alized by a 5-min incubation with Hoechst dye (5 �g/ml bisbenzimide;Roche Molecular Biochemicals). The coverslips were mounted on micro-scope slides and stored protected from light at �20 °C. The cells stainedfor NF-�B were photographed with a Dage-MTI 100 video cameramounted on a Zeiss Axiovert microscope equipped with fluorescenceoptics, using a video digitizer (Snappy Video Snapshot, Play Inc.,Rancho Cordova) operated through Adobe Photoshop. The cells stainedfor cytochrome c were observed with a confocal microscope (Olympus IX70), PerkinElmer Biosciences Ultraview software, spinning disk, usingan Olympus �40, 1.4 numerical aperture, oil immersion lens. Fluores-cence signals were collected after single line excitation at 543 nm (red).

Glutathione Measurements—At the indicated time points, adherentcells were collected by trypsinization and combined with floaters in themedium. After centrifugation, cells were washed twice with ice-coldPBS and resuspended in 0.4 ml of ice-cold double-distilled H2O. Thefollowing steps were performed at 4 °C using a modified procedure fromTietze F (39, 40). Briefly, 0.1 ml of lysate were used for protein deter-mination using the Bio-Rad assay, and the remaining 0.3 ml of lysatewere added to 75 �l of 10% (v/v) 5-sulfosalicylic acid, mixed, andincubated on ice for 10 min to allow protein precipitation. Samples were

then centrifuged, and supernatants were stored at �80 °C. For deter-mination of the total glutathione content, 5 �l from the supernatantswere added to 35 �l of a buffer containing 6.3 mM EDTA in 125 mM

sodium phosphate (GAB). Then 200 �l of 0.315 mM NADPH solutionprepared in GAB (final concentration, 0.21 mM), 0.55 units of glutathi-one reductase, and 50 �l of 12 mM 5,5�-dithiobis-(2-nitrobenzoic acid)solution prepared in GAB (final concentration, 2 mM) were added, andthe reaction was followed for 5 min by reading the OD at 412 nm.Concentration values were obtained using a standard curve with knownglutathione values (0.1–100 nM) from a stock solution prepared in GAB,and they were normalized to protein concentrations.

FIG. 1. Structure of GW4869.

FIG. 2. Inhibition of Rat Brain N-SMase by GW4869: Effect ofvaried [SM]. Delipidated rat brain N-SMase was assayed for activityin the presence or absence of GW4869 as described under “Experimen-tal Procedures.” A, double-reciprocal plot for the effects of GW4869(0–30 �M) on substrate (SM). B, inhibition of N-SMase by GW4869 atdifferent SM concentrations.

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In Vitro Caspase Activity Assay—Caspase activity was analyzed withthe ApoAlert CPP32/caspase 3 assay kit according to the manufactur-er’s protocol (CLONTECH, Palo Alto, CA). At the indicated time points,adherent cells were collected by trypsinization and combined with float-ers in the medium. After centrifugation, cells were washed twice withice-cold PBS and stored as a pellet at �80 °C. On the day of theanalysis, cell pellets were solubilized in lysis buffer and centrifuged toremove debris, and equal amounts of protein were incubated with 50 �M

N-acetyl-Asp-Glu-Val-Asp-AFC (7-amino-4-trifluoromethyl coumarin)for 1 h at 37 °C. The samples were analyzed using an enzyme-linkedimmunosorbent assay plate reader with excitation of 360 � 40 andemission of 530 � 25.

MTT Assay—5 � 103 cells/well were seeded in a 96-well plate in 75�l of RPMI containing 2% FBS and 25 mM Hepes, pH 7.5. After 24 h,first GW4869 was added in 15 �l of medium/well and incubated for 30min and then TNF was added in 10 �l/well (total volume of 100 �l/well).At the indicated time points, 25 �l of MTT stock solution (5 mg/ml inPBS) were added to each well and incubated at 37 °C in 5% CO2 for 3 h.Subsequently, cells were solubilized by the addition of 100 �l of lysisbuffer (20% SDS (w/v), 50% N,N-dimethylformamide (v/v), 0.8% aceticacid (v/v), pH 4.6–4.8) to each well. The production of the formazan dyewas quantitated by measuring the OD at 595 nm with a multiwell platereader. We have noticed that the efficacy of TNF to induce morpholog-ical changes was significantly slower in experiments carried out in the96-well plates (as for the MTT) compared with those performed in 10-cmPetri dishes (all other experiments).

Preparation of Cytosolic Extracts—At the indicated time points,plates were washed once with ice-cold PBS, and floaters in the mediumand from the washes were collected by centrifugation. Adherent cellswere scraped in lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 10 mM

EGTA, 2 mM EDTA, 10 mM sodium orthovanadate, 20 mM sodiumfluoride, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 0.5%

Nonidet P-40, and 10 �g/ml each of chymotrypsin, leupeptin, aprotinin,and pepstatin). After combining with floaters, cells were incubated onice for 10 min. Lysates were then centrifuged at 1000 � g for 10 min toremove unbroken cells and nuclei, and supernatants were centrifugedat 100,000 � g for 1 h to obtain cytosolic fractions that were stored at�80 °C. Protein concentrations from postnuclear lysates and cytosolswere determined using the Bio-Rad assay.

Western Blotting—Equal amounts of protein, usually 80 �g, wereresolved by 7, 12, and 15% SDS-PAGE for analysis of PARP andcaspases 6 and 9, respectively. After transfer to a nitrocellulose mem-brane, the proteins were incubated with anti-PARP rabbit polyclonalIgG (1:2500; Santa Cruz Biotechnology), anti-human caspase 6 mono-clonal IgG (2 �g/ml; Pharmingen), or anti-caspase 9 rabbit polyclonalIgG (1:500; Santa Cruz Biotechnology) and secondary anti-rabbit (1:4000) and anti-mouse (1:5000) antibodies (Santa Cruz Biotechnology).The signal was visualized by enhanced chemiluminescence (AmershamBiosciences).

Electron Microscopy Analysis—Cells were seeded at a concentrationof 0.4 � 106 cells/25-cm2 flask in growth medium. After 48 h, mediumwas changed with RPMI 1640 containing 2% FBS and 25 mM Hepes, pH7.5, and cells were rested for 1.5 h. Cells were pretreated with GW4869and treated with TNF in a total volume of 4 ml. After 14 h, cells werefixed in 2% glutaraldehyde in 0.1 M cacodylate buffer for 30 min andrinsed overnight in cacodylate buffer with 7% sucrose. Cells were thendehydrated in graded ethyl alcohol as follows: 5 min in 50% ethylalcohol, 5 min in 70% ethyl alcohol, 5 min in 95% ethyl alcohol, 10 minin 100% ethyl alcohol, 10 min in 100% ethyl alcohol. Samples wereinfiltrated with a 1:1 solution of 100% ethyl alcohol and Embed 812embedding resin for 30 min and embedded (in the flask) with Embed812 embedding resin. Samples were polymerized for 24 h and capped ina 60 °C oven, and polymerization was allowed to continue for an addi-tional 24 h (caps off). The plastic flask was broken away from thehardened resin, and representative blocks were sawed out using a Searselectric scroll saw. The blocks were re-embedded for orientation andpolymerized for 24 h at 60 °C. Thick sections were cut using the Rie-chert Ultramicrotome. These 0.5-�m sections were stained with tolui-dine blue and examined with the light microscope. Those blockscontaining the appropriate monolayer of cells were chosen for thin

FIG. 3. A, inhibition of Rat brain N-SMase by GW4869: effect ofvaried [PS]. Delipidated rat brain N-SMase was assayed for activity asdescribed under “Experimental Procedures” in the presence or absenceof GW4869 at different PS concentrations. B, inhibition of rat brainN-SMase by GW4869: effects of varied [Mg]. Delipidated rat brainN-SMase was assayed for activity as described under “ExperimentalProcedures” in the presence or absence of GW4869 at different Mg2�

concentrations.

FIG. 4. Lack of inhibition of GW4869 on A-SMase. Clonedhuman A-SMase was incubated in the absence and in the presence ofincreasing concentrations of GW6948 as described under “Experi-mental Procedures.”

TABLE ISpecificity of GW4869 on enzyme inhibition

Enzyme Origin Inhibition

%

N-SMase (�PS) Rat brain 89.4N-SMase (�PS) Rat brain 64.3N-SMase (�PS) B. cereus 92Lyso-PAF PLC (�PS) Human recombinant 17.2Lyso-PAF PLC (�PS) Human recombinant 18.6PC-PLCa B. cereus 0PP2Ab Bovine kidney 15.1

a PC-PLC, phosphatidylcholine-specific phospholipase C.b PP2A, protein phosphatase 2A.

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sectioning. Thin sections were cut using the Riechert UltramicrotomeUltracut E. These 70-nm thin sections were picked up on copper gridsand double-stained with uranyl acetate and lead citrate. Thin sectionswere viewed in the JEOL 1210 transmission electron microscope, andrepresentative digital images were saved.

RESULTS

In Vitro Studies

GW4869 was discovered during a high throughput screenwith a preparation of delipidated rat brain neutral SMase. Thecompound structure is shown in Fig. 1, and inhibition of N-SMase by the compound is shown in Fig. 2. GW4869 acted as anoncompetitive inhibitor with the substrate sphingomyelin(Fig. 2A), and the IC50 for the interaction is 1 �M (Fig. 2B). TheKm for sphingomyelin under these conditions was found to be13 �M (0.4 mol % at 0.2% Triton X-100, 0.02% PS, and 10 mM

MgCl2). The compound showed competitive characteristics to-ward the activator PS (Fig. 3A); however, at high PS concen-

trations, the inhibition was more complex, since at these highPS levels, total inhibition was not observed under standardassay conditions. Since PS, an anionic lipid, could bind Mg2�,we evaluated the effects of varied Mg2� levels under high PSconditions. Fig. 3B shows that at higher Mg2� levels, inhibitionagain became complete. The effect of the compound on theA-SMase was also determined. At up to 150 �M, GW4869 didnot inhibit the cloned human A-SMase (Fig. 4). Additionally,the effect of GW4869 on other hydrolytic enzymatic activitieswas also tested. As reported in Table I, only rat brain andbacterial neutral SMases were efficiently inhibited by GW4869,whereas no inhibitory activity was observed for the bacterialphosphatidylcholine-specific PLC, and minimal inhibition wasobserved for the mammalian lyso-PAF PLC, an enzyme thatalso hydrolyzes SM in vitro. Finally, minor inhibitory effectswere observed for bovine protein phosphatase 2A activity, a

FIG. 5. Effects of GW4869 on cellular activation of N-SMase. A, SM hydrolysis. After metabolic labeling with [methyl-3H]choline, cells werepreincubated for 30 min in the absence or presence of GW4869 (10–20 �M), and treatment with TNF (3 nM) followed. After 14 h, cells were collected,and SM levels were determined as described under “Experimental Procedures.” The results are representative of two separate experiments. B,ceramide levels. MCF7 cells (2 � 106) were preincubated in the absence or presence of GW4869 (10 �M). After 30 min, TNF (3 nM) was added, andcells were further incubated for 6, 12, and 24 h. At these time points, ceramide measurements were determined using the E. coli diacylglycerolkinase assay as described under “Experimental Procedures.” The results are representative of three separate experiments.

FIG. 6. Effects of GW4869 on TNF-induced depletion of GSH.MCF7 cells (2 � 106) were preincubated in the absence or presence ofGW4869 (10–20 �M). After 30 min, TNF (3 nM) was added, and cellswere incubated for an additional 14 h. At this time, cells were collected,and total glutathione levels were measured as described under “Exper-imental Procedures.” The results are representative of three separateexperiments.

FIG. 7. Lack of effects of GW4869 on ceramide generatedthrough the de novo biosynthetic pathway after TNF treatment.[3H]Palmitate (1 �Ci/ml) was added to MCF7 cells (1.7 � 106 cells in 7ml of medium) just before preincubation of the cells in the absence orpresence of GW4869 (10–20 �M). After 30 min, TNF was added at afinal concentration of 3 nM, and cells were collected after 21 h. Lipidswere extracted and analyzed as described under “Experimental Proce-dures.” The results are representative of three separate experiments.

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target for ceramide. Overall, the results show that GW4869 isa selective inhibitor of N-SMase, especially among enzymesknown to act on SM.

Cell Studies

Effects of GW4869 on Cellular Activation of N-SMase—Toverify the effect of GW4869 on N-SMase activity in cells, MCF7breast cancer cells were treated with 3 nM TNF, since in thiscell line it has been previously shown that treatment with TNFwould cause activation of N-SMase as evaluated by changes inSM and ceramide (13). As expected, the addition of TNF causedsphingomyelin hydrolysis (Fig. 5A) and ceramide accumulation(Fig. 5B) after 12–14 h of incubation. Importantly, the additionof 10 �M GW4869 significantly inhibited TNF-induced SM hy-drolysis, whereas 20 �M of the compound protected completelyfrom the loss of SM. In the same time frame, ceramide levelsstarted to increase (12–14 h), and they continued to build upduring the incubation as measured by the diacylglycerol kinaseassay. The addition of GW4869 (10 and 20 �M) completelyinhibited the initial accumulation of ceramide, whereas thiseffect was partially lost at later time points (24 h). These data

therefore support the inhibitory action of GW4869 on N-SMasenot only in vitro but also in a cellular model.

Effects of GW4869 on TNF-induced Depletion of GSH—Tolocalize the site of action of GW4869 in cells, the effects of thecompound on molecular events involved in the action of TNF onN-SMase were evaluated. One of the proposed mechanisms forthe activation of N-SMase upon TNF signaling implicates thedrop in GSH levels after induction of early caspases (1). There-fore, MCF7 cells were treated with TNF in the presence orabsence of GW4869, and GSH levels were measured (Fig. 6). Asexpected, TNF induced a significant decrease of glutathionelevels (60%), which occurred in the same time frame of thepreviously observed SM hydrolysis (Fig. 5A). The addition ofGW4869 at both 10 and 20 �M concentrations (effective ininhibiting SM hydrolysis) did not modify glutathione levelssignificantly. These results suggest that the action of GW4869occurred downstream of the drop in glutathione.

Specificity of the Cellular Action of GW4869 to the N-SMasePathway of Ceramide Generation—It has been recently shownthat, in MCF7 cells treated with TNF, ceramide accumulationcould result from both early activation of N-SMase and later

FIG. 8. Lack of effects of GW4869 on TNF-induced activation of NF-�B. A and B, immunocytochemical analysis of NF-�B translocationupon TNF treatment. Cells were grown onto 22-mm glass coverslips for 2 days. Medium was changed, and preincubation of cells with GW4869(10–20 �M) followed. After 30 min, TNF (3 nM) was added to some samples (B), and all of the cells were incubated for an additional 30 min. Cellswere then fixed and processed for NF-�B immunostaining as described under “Experimental Procedures.” C, electrophoretic mobility shift assayof NF-�B upon TNF treatment. MCF7 cells (1.7 � 106) were preincubated with GW4869 (10–20 �M) for 30 min, and TNF treatment followed foran additional 30 min. Equal amounts of nuclear proteins were incubated with prelabeled NF-�B consensus oligonucleotides, and binding wasstudied using a 5% nondenaturing polyacrylamide gel as described under “Experimental Procedures.” The results are representative of threeseparate experiments. The right panel shows supershift studies of NF-�B using antibodies to p65 and p50 components of NF-�B and cold probe forcompetition.

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activation of the de novo biosynthetic pathway, inhibitable byfumonisin B1 (41). Therefore, we wondered whether the inhib-itory effect of GW4869 on ceramide accumulation (Fig. 5B) alsoextended to this second ceramide-generating pathway. MCF7

cells were treated with TNF and concomitantly pulsed withradioactive palmitate, a precursor for ceramide synthesis. In-corporation of radioactivity into ceramide was evaluated in thepresence or absence of GW4869. As shown in Fig. 7, TNF

FIG. 9. Effects of N-SMase activa-tion in TNF-induced apoptosis. A, ef-fects of GW4869 on TNF-induced celldeath. MCF7 cells (1.7 � 106) were prein-cubated with GW4869 (10–20 �M) for 30min, and TNF treatment followed for anadditional 20 h. At this time, floating cellswere stained with trypan blue solution,and positive (dead) cells were counted. B,effects of GW4869 on TNF-induced chro-matin condensation. MCF7 cells (1.7 �106) were preincubated with GW4869(10–20 �M) for 30 min, and TNF treat-ment followed for an additional 20 h.Cells were fixed with paraformaldehydesolution (4%), stained with Hoechst,washed with PBS, and analyzed by fluo-rescent microscopy. Ten random fieldsfrom each sample were counted. The re-sults are representative of at least threeseparate experiments.

FIG. 10. GW4869 does not protectfrom ceramide- induced mitochon-drial damage. MCF7 cells (1.7 � 106)were preincubated with GW4869 (10–20�M) for 30 min, and treatment with TNF(3 nM) (A) or C6-ceramide (30 �M) (B) fol-lowed. After 48 or 18 h, respectively, MTTsolution was added, and cells were incu-bated for 3 h at 37 °C in 5% CO2. Cellswere subsequently solubilized, and ODwas measured at 595 nm using a platereader.

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treatment caused a 75% increase of newly synthesized ceram-ide after 21 h of incubation. On the other hand, the addition ofGW4869, at either 10 or 20 �M, did not affect ceramide accu-mulation significantly. These results have two important im-plications: first, they further support the specificity of action ofGW4869 to N-SMase-mediated ceramide formation. Second,they show that de novo synthesis of ceramide, which is alsoinduced by TNF, is independent of activation of N-SMase.

Lack of Effect of GW4869 on TNF-induced Activation ofNF-�B—Several molecular events triggered by TNF stimula-tion have been suggested to be independent of TNF-inducedaccumulation of ceramide. The rapid and sustained activationof NF-�B after TNF treatment and its translocation to thenucleus appears to be a major ceramide-independent effect(42). To further verify that the effects observed during TNFtreatment in the presence of GW4869 were due to specificinteraction of the compound with the ceramide-mediated path-way, the effects of GW4869 on TNF-induced NF-�B transloca-tion were studied. MCF7 cells were treated with TNF for 30

min in the absence or presence of GW4869 (10 and 20 �M), andNF-�B activation and translocation to the nucleus were moni-tored by immunocytochemical methods (Fig. 8B). As shown inthe figure, control cells exhibited a diffuse NF-�B localizationboth in the cytosol and in the nucleus, and the presence of theN-SMase inhibitor at both concentrations did not affect thispattern. Thirty-minute treatment with TNF induced a robusttranslocation of the cytosolic fraction to the nucleus, and thepresence of the N-SMase inhibitor did not interfere with thisphenomenon.

TNF-induced translocation of NF-�B was also evaluated byelectrophoretic mobility shift assay (Fig. 8C). Cells weretreated with TNF for 30 min with or without GW4869, andnuclei and nuclear proteins were extracted and processed asdescribed under “Experimental Procedures.” As shown in thegel (left panel), the amount of NF-�B in the nuclei greatlyincreased upon TNF treatment, and the presence of GW4869did not significantly impair it. Supershift assays (right panel)demonstrate the specificity of this translocation in the presenceor absence of GW6948 and the involvement of the p65 subunitof NF-�B. Therefore, these results show that GW4869 does notinterfere with key TNF signaling effects, and they furthersuggest that the activation of NF-�B by TNF is independent ofN-SMase.

Biological Implications of N-SMase Activation in TNF-induced Cell Death—The above results suggest specific cellulareffects of GW4869 on activation of N-SMase but not on otherkey cellular effects such as activation of NF-�B or depletion ofGSH or even de novo generation of ceramide. Since both thedrop of GSH and the elevation in ceramide have been corre-lated with TNF-induced cell death, the effects of GW4869 oncell viability were next examined. Initially, the effects ofGW4869 on TNF-induced cytotoxicity were evaluated bytrypan blue exclusion assay. As shown in Fig. 9A, the presenceof the N-SMase inhibitor at 10 and 20 �M effectively reducedthe number of trypan blue-positive floating cells. Similarly,GW4869 was also able, in a dose-dependent manner, to signif-icantly protect from nuclear condensation after TNF treat-ment, as evaluated by DNA staining with Hoechst (Fig. 9B). Infact, after 20 h of treatment with TNF, 81% of the cells werepositive for chromatin condensation, whereas the presence of10 and 20 �M GW4869 during the incubation reduced thispercentage to 57.2 and 34%, respectively.

To further verify the action of GW4869 on N-SMase, weinvestigated whether exogenous ceramide can bypass the ef-fects of the inhibitor. Therefore, MCF7 cells were treated witheither TNF or C6-ceramide, in the presence or absence ofGW4869, and cell viability was evaluated using the MTT assay.As shown in Fig. 10A, TNF treatment significantly affected cellviability, reducing it by 35% after 48 h. The addition ofGW4869 efficiently protected cell viability at both 10 and 20�M. MCF7 cells were then treated with C6-ceramide. In re-sponse to treatment with C6-ceramide, cell viability was sig-nificantly impaired (34%), but in this case the presence ofGW4869 was unable to prevent this effect (Fig. 10B). There-fore, these results show that the GW4869 does not interferewith molecular events leading to cell death downstream ofceramide formation, further supporting its action at the N-SMase level.

To gain more insight on the specific components of the ap-optotic pathway regulated by GW6948, cell morphology wasevaluated by EM studies conducted in cells treated with TNF inthe absence and in the presence of the N-SMase inhibitor (Fig.11). As shown in the figure, cells treated with TNF (Fig. 11, Eand F) showed massive chromatin condensation compared withcontrol cells (A and B), whereas the presence of the inhibitor

FIG. 11. Protective effects of GW4869 on TNF-induced changesof cell morphology evaluated by electron microscopy. 60–70%confluent cells were preincubated with GW4869 (20 �M) for 30 min, andtreatment with TNF (3 nM) followed. After 14 h, cells were fixed andprocessed as described under “Experimental Procedures.” Pictures oftransverse sections of cells representative of each experimental condi-tion are shown. A and B, vehicle control; C and D, 10 �M GW4869; E andF, 3 nM TNF; G and H, preincubation with GW4869 (10 �M) andtreatment with TNF (3 nM).

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significantly reduced this condensation (G and H). It is inter-esting to note that in this latter condition, the nuclear changeswere variable among the cellular population. In fact, some cellsshowed no signs of chromatin condensation (G), whereas someothers showed partially condensed chromatin only at periph-eral areas of the nuclei (H). From these studies, other morpho-logical alterations in TNF-treated cells could be also clearlyappreciated, such as swollen mitochondria (E and F), vacuol-ization of the cytoplasm (E and F), extensive blebbing of theplasma membrane (F), and disruption of cellular junctions (E).In the presence of the N-SMase inhibitor, these changes weremarkedly reduced, in particular the blebbing of the plasmamembrane (G and H). It was important to note, however, thatthe inhibitor was not able to prevent the formation of the largemolecular weight fragments of condensed chromatin peripheralto the nuclei (H).

In order to verify that these morphological changes were partof an ongoing apoptotic process, we evaluated the effect of TNFtreatment on activation of late/effector caspases, thought to beresponsible for nuclear fragmentation. As shown in Fig. 12 andin agreement with previous reports (13, 30, 43, 44), treatmentof MCF7 cells with TNF caused cleavage of PARP, in MCF7 asubstrate for active caspase 7, with the formation of the char-acteristic 85-kDa fragment. The presence of the N-SMase in-hibitor prevented, in a dose-dependent manner, the TNF-in-duced PARP cleavage. In fact, after 12 h of treatment,preincubation with 10 �M GW4869 was able to partially blockthe appearance of the 85-kDa fragment, whereas 20 �M of theinhibitor completely prevented the cleavage. These resultsclearly suggest a role for ceramide generated through N-SMasein activating downstream caspases.

To further investigate the effects of GW4869 late/effectorcaspases, we evaluated the effect of pretreatment with theN-SMase inhibitor on caspase 6 processing. Therefore, MCF7cells were treated with TNF in the presence or absence of 20 �M

GW4869. After 14 h, cells were collected, and Western blottingwas performed on cytosolic proteins using antibodies recogniz-ing the full-length caspase 6. As shown in Fig. 12, TNF inducedprocessing of caspase 6, whereas the addition of the N-SMaseinhibitor effectively blocked it. These results suggest that in-hibition of N-SMase activation prevents activation of late

caspases. Next, in vitro activity of DEVDase caspases wasassayed after treating cells with TNF in the presence or ab-sence of the N-SMase inhibitor in order to evaluate a moregeneralized activation of effector caspases. As shown in Fig. 12,the presence of increasing concentrations of GW4869 (10 and20 �M) resulted in almost complete inhibition of activation ofDEVDase caspases induced by TNF, confirming and extendingthe results obtained from the Western blots.

One of the receptor-mediated mechanisms for the activationof late caspases involves mitochondrial dysfunction, character-ized by release of cytochrome c and subsequent activation ofcaspase 9 (45–48). Thus, we wondered if N-SMase activity wasinvolved in mediating these events. Therefore, MCF7 cells weretreated with TNF in the presence or absence of GW4869, andits effects on cytochrome c release were evaluated by immuno-cytochemistry and confocal microscopy. As shown in Fig. 13A,untreated cells exhibited a punctate pattern characteristic ofthe mitochondrial network, whereas treatment with TNF in-duced the appearance of a diffuse staining characteristic ofcytosolic localization. Importantly, the presence of theN-SMase inhibitor significantly and in a dose-dependent man-ner protected the release of cytochrome c from mitochondriainto the cytosol; 10 and 20 �M of GW4869 reduced by 46 and62% the number of positive cells, respectively. In support of theeffects on cytochrome c release, the presence of the N-SMaseinhibitor also protected from activation of caspase 9 (Fig. 13B).Treatment with TNF resulted in the appearance of the char-acteristic proapoptotic fragment and the presence of the N-SMase inhibitor efficiently prevented processing of caspase 9.

DISCUSSION

Here we report the identification of GW4869 as a novelinhibitor of the Mg2�-dependent N-SMase and its character-ization and biological effect in a cellular system of cytokine-induced apoptosis. Importantly, evidence is provided of speci-ficity of action of GW4869 on N-SMase activity both in vitro andin vivo. Finally, the use of this novel N-SMase inhibitor pro-vided strong evidence implicating activation of N-SMase in theregulation of a number of biological processes induced by TNF.

During a high throughput screen with a preparation of de-lipidated rat brain N-SMase, we successfully identified

FIG. 12. Protective effects of GW4869on activation of effector caspases in-duced by TNF treatment. MCF7 cells(1.7 � 106) were preincubated withGW4869 (10 or 20 �M) for 30 min, andtreatment with TNF (3 nM) followed. Cy-tosolic proteins (80 �g) were resolved by7% (PARP cleavage) or 12% (caspase 6)SDS-PAGE (upper panels). Western blotanalysis was performed using anti-PARP rabbit polyclonal IgG or anti-human caspase 6 monoclonal IgG. Theeffect of GW4869 on activation of DEVD-cleaving caspases was also evaluated invitro (lower panel). Equal amounts ofproteins from cell lysates were incu-bated with 50 �M N-acetyl-Asp-Glu-Val-Asp-AFC (7-amino-4-trifluoromethylcoumarin) for 1h at 37 °C in a 96-wellplate, and samples were analyzed usingan enzyme-linked immunosorbent assayplate reader with excitation of 360 � 40nm and emission of 530 � 25 nm. Theresults are representative of two inde-pendent experiments in quadruplicate.

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GW4869, a molecule that exhibited significant and specificinhibitory activity. The compound is a noncompetitive inhibitorof the enzyme with an IC50 of 1 �M. GW4869 showed no orminor inhibitory activity versus other hydrolytic enzymes, suchas bacterial phosphatidylcholine-PLC and bovine protein phos-phatase 2A, and it showed significantly higher activity versusthe rat brain enzyme compared with the human lyso-PAF PLC(Table I). Importantly, GW6948 showed no inhibition of thehuman acid SMase.

The inhibitory activity of GW4869 against N-SMase ob-served in vitro (Figs. 2–4 and Table I) was also confirmed at acellular level. GW4869 prevented SM hydrolysis induced byTNF treatment and partially inhibited ceramide accumulation(Fig. 5). These effects have been observed using concentrationsranging from 5 to 20 �M. The requirement for higher concen-trations for cellular action could be due to limitation in perme-ability through the plasma membrane.

Specificity of action of GW4869 to the N-SMase was alsoconfirmed in vivo by multiple lines of evidence. 1) GW4869 onlytargeted ceramide formation from the N-SMase pathway. InMCF7 cells, TNF treatment activates at least two major routesfor ceramide generation, the N-SMase and the de novo biosyn-thetic pathways (38). GW4869 completely inhibited ceramideaccumulation only at an early phase of incubation (12–18 h),when at the same time it also inhibited SM hydrolysis (Fig. 5).At a later time, corresponding to the activation of the de novo

pathway, the inhibitor no longer prevented the rise of ceramidelevels. When the effects of the inhibitor were directly tested onthis later ceramide-generating pathway, no effects were ob-served on de novo incorporation of palmitate into ceramide(Fig. 7). Since the de novo pathway requires many enzymesincluding serine palmitoyl transferase and ceramide synthase,these results demonstrate that GW6948 does not affect theseenzymes. It is also interesting to note that, in addition todemonstrating specificity of action of GW4869 on N-SMase-generated ceramide, these results also indicate that the twopathways of ceramide generation are independent. 2) GW4869did not inhibit the drop in glutathione induced by TNF treat-ment, which has been recently shown to precede the activationof N-SMase in response to TNF (13, 49–51). These results alsoadd to the cellular specificity of action of GW4869 (Fig. 6). 3)GW4869 did not inhibit ceramide-mediated cell death. Thisindicates that indeed the target of GW4869 is localized up-stream of ceramide generation and that the N-SMase inhibitordoes not affect directly any of the targets downstream of cer-amide action (such as caspases, cytochrome c, nucleases, etc.).4) GW4869 did not affect other molecular events triggered byTNF that are known to be independent of ceramide accumula-tion. Thus, the lack of effects on NF-�B demonstrates thatGW4869 does not interfere with initial events in TNF action(including interaction with the TNF receptors, activation ofNik, Ikk, or the proteosome). Additionally, GW4869 did not

FIG. 13. Effects of GW6948 on mitochondrial-related events induced by TNF. A, GW4869 partially protects cytochrome c release frommitochondria induced by TNF treatment as evaluated by immunocytochemistry analysis. Cells were grown onto 22-mm glass coverslips for 2 days.Medium was changed, and preincubation of cells with GW4869 (10–20 �M) followed. After 30 min, TNF was added (3 nM), and the cells wereincubated for an additional 14 h. All cells were fixed and processed for cytochrome c immunostaining as described under “ExperimentalProcedures.” Ten to fifteen random fields from each experimental condition were counted, and the percentage of cells that showed releasedcytochrome c over the total number of counted cells is represented in the bar graph. The results are representative of three separate experiments.B, GW6948 protects from activation of caspase 9 induced by TNF. MCF7 cells (1.7 � 106) were preincubated with GW4869 (20 �M) for 30 min, andtreatment with TNF (3 nM) followed. Western blots were performed using anti-caspase 9 rabbit polyclonal IgG.

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affect TNF-induced prostaglandin production in both A549lung cancer cells and L929 murine fibroblasts, thus excluding apossible effect of the compound on either cPLA2 orcyclooxygenase.2

The use of GW4869 as a specific inhibitor generated specificinsight into the molecular mechanisms that are dependent onactivation of N-SMase. Thus, inhibition of N-SMase not onlyblocked PARP cleavage (target for caspase 7) but also pre-vented the activation of other effector caspases such as caspase6 and caspase 9. Processing of procaspase 9 occurs by auto-cleavage as the result of the formation of the so-called “apo-ptosome,” a complex constituted by cytochrome c released frommitochondria, apoptotic protease-activating factor-1, and pro-caspase 9 (52, 53). Therefore, the possibility that activation ofN-SMase leads to the induction of effector caspases by target-ing specific mitochondrial events was verified and confirmed,since inhibition of N-SMase partially, but significantly, pre-vented the release of cytochrome c from the mitochondria (Fig.13). The observation that the activation of N-SMase promotescell death through mitochondrial dysfunction is in agreementwith results obtained in MCF7 overexpressing the Bcl2 protein.Indeed, the overexpression of Bcl2 has been shown to almostcompletely prevent TNF-induced cell death (27) and PARPproteolysis (13), only moderately blocking ceramide formation(27), and without inhibition of either the drop of glutathione orSM hydrolysis (13). In addition, these results are in agreementwith some studies that previously reported the ability of shortchain ceramide analogs to induce cytochrome c release in bothisolated mitochondria (54) and intact cells (55, 56).

Although these results start to shed light on the possiblebiological targets of ceramide generated through the N-SMasepathway, they also raise a number of important questions thatrequire further evaluation. First, how does the ceramide fromthe N-SMase signal to mitochondria? It has been recently dem-onstrated that targeting of an N-SMase to mitochondria in-duced accumulation of ceramide that was sufficient to inducemitochondrial dysfunction and signal cell death (57). There-fore, one possible scenario implicates the activation by TNF ofan N-SMase isoform, which resides in mitochondria or in closeproximity. Second, what are the direct effectors of the mito-chondrial dysfunction induced by accumulation of ceramide?Recent studies suggest a few candidates. For example, it hasbeen shown that ceramide induces conformational changes ofthe Bax protein and synergizes with Bax, altering mitochon-drial functions (58). On the other hand, ceramide was found tospecifically activate a mitochondrial protein phosphatase 2A,which rapidly and completely dephosphorylated Bcl-2, leadingto cell death (59).

It is also important to note that inhibition of N-SMase didnot completely prevent either the formation of ceramide (Fig.5B) or some of the biological effects induced by TNF in MCF7,such as cytochrome c release (Fig. 13A) and nuclear condensa-tion (Fig. 9B). Interestingly, the predominant peripheral con-densation of the chromatin observed in MCF7 treated withTNF in the presence of the N-SMase inhibitor (Fig. 11H) closelyresembles the pattern of nuclear changes induced by activationof the apoptosis-inducing factor-mediated pathway (60). Theseresults suggest that this specific process, which is known to beindependent of caspases, may also be independent of N-SMase.

In conclusion, by using a newly characterized inhibitor ofN-SMase, we show for the first time that activation of theN-SMase leads to mitochondrial dysfunction and that it isrequired for the full development of the cytotoxic programinduced by TNF.

Acknowledgments—We thank Dr. Charles Chalfant and PatrickRoddy for technical assistance with the protein phosphatase assay.

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Eric Boros, Debra J. Hazen-Martin, Lina M. Obeid, Yusuf A. Hannun and Gary K. SmithChiara Luberto, Daniel F. Hassler, Paola Signorelli, Yasuo Okamoto, Hirofumi Sawai,

Inhibitor of Neutral SphingomyelinaseInhibition of Tumor Necrosis Factor-induced Cell Death in MCF7 by a Novel

doi: 10.1074/jbc.M206747200 originally published online August 1, 20022002, 277:41128-41139.J. Biol. Chem. 

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