[cancer research 59, 6257–6266, december 15, 1999] the … · (35), cinnarizine, flunarizine...

[CANCER RESEARCH 59, 6257–6266, December 15, 1999]

The Novel Retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphtalene CarboxylicAcid Can Trigger Apoptosis through a Mitochondrial Pathway Independentof the Nucleus1

Philippe Marchetti, Naoufal Zamzami, Bertrand Joseph, Susanna Schraen-Maschke, Claude Méreau-Richard,Paola Costantini, Didier Métivier, Santos A. Susin, Guido Kroemer,2 and Pierre Formstecher2,3

Institut National de la Santé et de la Recherche Médicale U459, Faculte´ de Médecine, F- 59045 Lille Cedex [P. M., B. J., S. S-M., C. M-R., P. F.], and Centre National de laRecherche Scientifique-Unité Propre de Recherche 420, Villejuif [N. Z., P. C., D. M., S. A. S., G. K.], France

ABSTRACT

The novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphtalenecarboxylic acid (AHPN/CD437), a retinoic acid receptor (RAR)g activa-tor, has been found to inhibit the growth and to induce apoptosis of a widevariety of malignant cell types including solid tumors and various leuke-mias. Interestingly, CD437 is able to induce apoptosis in some all-trans-retinoic acid (ATRA)-resistant models. In a number of experimentalsystems, the early apoptotic stage that precedes nuclear chromatinolysisconsists in mitochondrial alterations, including a disruption of the innermitochondrial transmembrane potential (Dcm) mediated by the mito-chondrial permeability transition (MPT). Similarly CD437 causes RPMI8226, a human myeloma cell line, to undergo a rapidDcm disruption thatprecedes other apoptotic alterations such as the generation of reactiveoxygen species and DNA fragmentation. The same sequence of events isobserved during the CD437-induced apoptosis in L363, a RARg-negativehuman myeloma cell line, as well as RPMI 8226 cytoplasts (anucleatecells). Indeed, RPMI 8226 cells and cytoplasts manifest a similar degree inDcm loss, phosphatidylserine exposure, and caspase activation in responseto CD437, which indicates that nuclear effects cannot account for theapoptogenic potential of CD437. The mitochondrial release of cytochromec, the activation of caspases as well as nuclear signs of CD437-inducedapoptosis are fully prevented by the MPT inhibitory compound cyclo-sporin A. Purified mitochondria can be directly induced to undergo MPTwith CD437 but not with ATRA. In a cell-free in vitro system consisting ofexposing mitochondrial supernatants to isolated nuclei, only supernatantsfrom CD437-treated mitochondria provoke chromatin condensation,whereas supernatants from mitochondria treated with ATRA, or with thecombination of CD437 and cyclosporin A, remain inactive. In conclusion,these results suggest that the rapid execution of CD437-induced apoptosisis a nucleus-independent (and probably RARg-independent) phenomenoninvolving mitochondria and MPT.

INTRODUCTION

Retinoids have been described as displaying pleiotropic activitiesincluding inhibition of growth, induction of differentiation, and ap-optosis in a wide variety of tumor cell lines (1). On the basis of theseeffects, retinoic acid was used with some efficiency in the chemopre-vention and treatment of various malignancies (2). The molecularmechanism of action of retinoic acid involves the binding and acti-vation of specific nuclear receptors, RARs and retinoic X receptors,

which modulate gene expression (3). During the last few years,synthetic retinoids, specific for different receptor types (RAR orretinoic X receptor) and subtypes (a, b, g) of RARs have beendiscovered, and some already seem very promising for the treatmentof certain cancers (4).

Among them, one new compound, AHPN/CD437,4 is particularlyinteresting. First described as a RARg selective retinoid (5), thismolecule has been found to inhibit growth and promote apoptosis inan extremely wide variety of tumor cell lines (6–19). Moreoverseveral other synthetic retinoids structurally related to CD437 are alsoable to induce apoptosis in many human cancer cell lines (20, 21).Compared with retinoic acid and other RARg selective syntheticretinoids, CD437 is characterized by unique properties. It is active notonly in retinoic acid-sensitive cell lines but also in retinoic acid-resistant cells (6–9, 11–14) and in RARg-negative cells (17). More-over, its antiproliferative and apoptogenic activities in cancer cells arenot inhibited by either RARb, or RARg or by pan-RAR selectiveantagonists (11, 14, 19). Structure-activity relationship studies com-paring CD437 with various RARg selective synthetic retinoids did notshow any correlation between antiproliferative activator protein ac-tivity and RARg-dependent transactivating activities of the testeddrugs (9, 13, 22). A significantly higher concentration of CD437 wasrequired to inhibit cell proliferation and to induce apoptosis than totrigger RARg-dependent transcriptional activity (5, 6, 8, 11–13, 18).Taken together, these data suggest that the antiproliferative effects ofCD437 cannot be related to only its RARg selectivity but probablyinvolve other, still unknown, mechanisms.

CD437 arrests cells in Go-G1 (8, 18, 23, 24) by increasing thecyclin-dependent kinase inhibitor p21cip1/waf1(8, 12, 16, 17, 24). Thisincrease has been related to a posttranscriptional stabilization ofp21cip1/waf1 mRNA (8, 25). CD437 induces both p53-dependent and-independent apoptosis in human cancer cells (26). Conflicting datahave been reported about the effects of CD437 on the c-jun/activatorprotein pathway (6, 8, 24). Nur77 has also been proposed to play arole in the CD437-induced apoptosis (24), whereas the effects ofCD437 on the expression of Bcl-2, Bcl-x, and Bax seemed variableaccording to the cell model studied (6, 8, 11, 27). However, overex-pression of Bcl-2 retards or inhibits CD437-triggered apoptosis ofcancer cells (18, 23). Recent observations suggest that CD437-in-duced apoptosis can be dissociated from the effects of CD437 ongrowth arrest and could involve both caspase-dependent and -inde-pendent pathways (18, 19).

From all of these data, it seems that a clear identification of themolecular targets of CD437 that are specifically responsible for itsunique proapoptotic activities is still lacking. Surprisingly, whereas

Received 6/11/99; accepted 10/19/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from Institut National de la Santé et de la Recherche Médicale(INSERM), Université de Lille II, Association Regionale pour l’Enseignement et laRecherche Scientifique (ARERS), Association de Recherche Contre le Cancer (ARC),Ligue Nationale Contre le Cancer [(LNC) to P. F.], and ARC, LNC, Centre National dela Recherche Scientifique, Association Nationale de Recherche sur la SIDA, Fondationpour le Recherche Medicale (to G. K.). INSERM U459 belongs to IFR 22 (CHU de Lille,COL, INSERM and Université de Lille II).

2 G. K. and P. F. share senior coauthorship of this article.3 To whom requests for reprints should be addressed, at Institut National de la Santé

et de la Recherche Médicale U 459, Faculte´ de médecine, 1, place Verdun, F- 59045 LilleCedex, France. Phone: 33-3-20-62-69-52; Fax: 33-3-20-62-68-84; E-mail: [email protected].

4 The abbreviations used are: AHPN/CD437, 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid; ATRA, all-trans-retinoic acid; AIF, apoptosis inducingfactor; Dcm, inner mitochondrial transmembrane potential; MPT, mitochondrial perme-ability transition; PTPC, permeability transition pores complex; CMX-Ros, chloromethylX-rosamine; ROS, reactive oxygen species; CsA, cyclosporin A; RAR, retinoic acidreceptor; ANT, adenine nucleotide translocator; DAPI, 49-6-diamidino-2-phenylindoledihydrochloride; BA, bongkrekik acid; ER, endoplasmic reticulum; HE, dihydroethidium.

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growing attention is paid to the effects of CD437 on caspase activa-tion (18, 19, 23, 28), very few data have been reported about theinvolvement of mitochondria in CD437-induced apoptosis.

The contribution of mitochondria in the execution phase of apo-ptosis is now well-established (29). Two interspace membrane pro-teins, cytochromec and a 57-kDa protein called AIF (30), are releasedin the cytosol during apoptosis and stimulate pathways of apoptosis.The early stage of apoptosis is characterized by a rupture in theDcmpreceding signs of DNA fragmentation (for review, see Ref. 31). Thisreduction inDcm is thought to result from the opening of megachan-nels or pores, referred to as the MPT pore. PTPCs are located at thecontact sites between the inner and outer mitochondrial membranes.The PTPCs include (a) the ANT in the inner membrane, whichcooperates with the proapoptotic molecule Bax to increase mitochon-drial membrane permeability and to trigger cell death (32); (b) cyclo-philin D in the matrix; (c) porin and the peripheral benzodiazepinereceptor in the outer membrane; and (d) possibly other proteins (33).In isolated mitochondria or in intact cellsin vitro, MPT is blockedspecifically by (a) CsA (a ligand of matrix cyclophilin D); (b) BA (aligand of ANT); (c) CMX-Ros, which acts on the matrix thiols, (d)phenylglyoxal and 2,3, butanedione, which are thought to modifyarginines of inner membrane proteins (34); and (e) trifluoperazine(35), cinnarizine, flunarizine (36), and trimetazidine (37) by acting onyet unknown receptors. CsA (38, 39), BA (40), CMX-Ros (41),trimetazidine (37), and trifluoperazine (35) can also prevent celldeath, at least in some models of apoptosis.

We recently demonstrated that the induction of apoptosis by reti-noic acid in a human myeloma cell line, RPMI 8226, required theactivation of both classes of retinoid receptors, RARs and retinoic Xreceptors (42). During this previous study, we observed that CD437

had a unique behavior and was able by itself to trigger a fast andmassive apoptosis with peculiar morphological and molecular fea-tures.

On the basis of the above findings, we decided to evaluate thepossibility that CD437 might induce apoptosis via an effect on mito-chondria, rather than via an effect on nuclear RARg receptors.

MATERIALS AND METHODS

Chemicals and Modulation of Apoptosis.ATRA was purchased fromSigma (St. Louis, MO) and retinoid-derivatives CD437, CD336, CD666,CD2665, and CD3126 were obtained from Galderma Research and Develop-ment (Sophia Antipolis, France). Properties of retinoids used in this study aresummarizeded in Table 1. Retinoids were dissolved in DMSO at an initialstock concentration of 10 mM and stored at220°C in the dark. Subsequentdilutions were performed in PBS or in RPMI 1640. Cells were cultured withthese reagents alone or in combination with the following inhibitors of MPT:(a) 1 mM CsA (Sandoz, Hannover, Germany; Ref. 39); (b) 1 mM CMX-Ros(Molecular Probes, Eugene, OR; Ref. 41); (c) 50mM BA (kindly provided byDr. Duine, Delft University of Technology, Delft, the Netherlands; Ref. 40);(d) 30 mM trifluoperazine (Sigma; Ref. 35); (e) 30mM each, cinnarizine andflunarizine (Sigma; Ref. 36); (f) 1 mM Phenylglyoxal (Sigma; Ref. 34); (g) 4mM 2,3-butanedione (Sigma Chemical Co; Ref. 34); and (h) 2mg/ml trimeta-zidine (Servier Laboratories, Neuilly, France; Ref. 37). FiftymM Z-Asp-Glu-Val-Asp-chloromethyl-ketone (DEVD.cmk; Bachem, Basel, Switzerland) wasused as inhibitor of DEVDase activity.

Cell Lines and Culture Conditions. RPMI 8226, a human myeloma cellline (CCL-155 American Type Culture Collection), and 2B4.11 T-cell hybri-doma cell lines (kindly provided by Jonathan Ashwell, NIH, Bethesda, MD)were routinely cultured in RPMI 1640 supplemented withL-glutamine, anti-biotics, and 10% heat-inactivated FCS. Human carcinoma HeLa cells, as wellas mouse embryonal carcinoma P19 cells, were cultured in DMEM1 10%

Table 1 Structure, binding constants, and transactivating properties of the retinoids used

Compound Structure

kDaa (nM)

ActivityRARa RARb RARg

ATRA 16 7 3 Pan-RAR Agonist

CD336 (Am580) 6 130 827 RARa Agonist

CD437 (AHPN) 6500 2480 77 RARg Agonist

CD666 2240 2300 68 RARg Agonist

CD2665 .2250 306 110 RARb, g Antagonist

CD3126 b b b Inactive

a kDa values are from Szondyet al. (63).b No binding (U. Reichert, CIRD Galderma, personal communication).

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AHPN/CD437, APOPTOSIS, AND MPT



FCS. L363, another human myeloma cell line (Deutsche Sammlung vonMikroorganismen und Zellkulturen DSM ACC 49), was characterized for theabsence of RARg and controlled by reverse transcription-PCR (data notshown) and cultured in the same condition as RPMI 8226. Thymocytes andhepatocytes were obtained from female BALB/c mice, 6–8 weeks of age. Forapoptosis modulation, cells were resuspended at 500,000/ml in RPMI 1640 orDMEM without FCS but supplemented with 1% of insulin-transferrin-sodiumselenite (Sigma). Rat-1 fibroblasts, constitutively expressing a Myc-estrogenreceptor fusion protein and stably overexpressing the different versions ofBcl-2, were a gift from David W. Andrews (Department of Medicine, Mc-Master University, Hamilton, Ontario, Canada). Rat-1/myc cell culture andapoptosis induction by serum starvation have been detailed elsewhere (43).

Anucleate Cells. RPMI 8226 cells were enucleated following proceduresdescribed previously (44). Briefly, cells (23 107/ml) were cultured in RPMI16401 5% FCS in the presence of cytochalasin B (10mg/ml; Sigma) for 45min at 37°C and then were subjected to centrifugation on a discontinuousFicoll (Pharmacia) density gradient (2 ml of 25%, 2 ml of 17%, 0.5 ml of 16%,0.5 ml of 15%, and 2 ml of 12.5% Ficoll in RPMI 1640 containing 10mg/mlcytochalasin B preequilibrated 24 h at 37°C1 CO2 5%). Two ml of the cellsuspension were gently applied to the gradient and were centrifuged in aprewarmed rotor SW41 Beckman at 25,000 rpm for 1 h at33°C. Cytoplasts(anucleate cells) were collected from the interface between 15 and 17% Ficolllayers. Then, cytoplasts were washed with RPMI 16401 10%FCS andresuspended in RPMI 16401 1% ITS (insulin, transferrin, and selenium,Sigma). Control staining with trypan blue revealed.99% viability in thisfraction, and microscopy analysis using May-Grünwald-Giemsa dye showedthat more than 90% of the cells had lost their nuclei (data not shown).

Cytofluorometric Analysis. To evaluateDcm and superoxide generation,a procedure described elsewhere was followed (45). Briefly, cells (53 105/ml)were incubated for 15 min at 37°C with 40 nM 3–39-dihexyloxacarbocyanineiodide [DiOC6(3)] in PBS (Molecular Probes) and with 2mM HE in PBS(Molecular Probes). DEVD cleavage was assayed by using Phi-Phi LuxDEVD-Rhodamine substrate according to the manufacturer’s recommanda-tions (Oncoimmunin Inc., College Park, MD) at the final concentration of 10mM. After incubation, cells were immediately analyzed on a XL cytofluorom-eter (Coulter). For determination of the external exposition of phosphatidyl-serine residues, staining with Annexin-V-FITC (1:20 dilution; PharMingen,San Diego, CA) was performed in binding buffer containing 10 mM HEPES-NaOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2 and was analyzed bycytometry within 1 h.

Immunofluorescence.Cells were fixed with 4% paraformaldehyde and0.19% picric acid in PBS for 60 min and were washed in PBS for 10 min. Thecells were further permeabilized with 0.1% SDS in PBS1 1% FCS for 5 minbefore incubation with 2mg/ml 6H2-B4 mouse IgG1 anticytochromec anti-body (PharMingen) for 1 h. After three washes in PBS1 1% FCS, cells wereincubated in 1:125 FITC-conjugated antimouse [F(ab9)2, Sigma) for 30 min.The cells were washed three times in PBS1 1% FCS followed by mountingin 80% glycerol in PBS. All of the samples were viewed and photographedwith a 3100 lens using fluorescence microscopy.

Analysis of Nuclear Apoptosis.The frequency of hypoploid cells (sub-G1cells) was assessed by studying the cell cycle after fixation overnight at 4°Cwith 70% ice-cold ethanol-PBS followed by propidium iodine (50mg/ml)staining and analysis in a Coulter XL cytofluorometer (45). DNA fragmenta-tion (1 3 106 cells/lane) was determined by horizontal agarose gel electro-phoresis following published methods (46).

In Vitro Tests of MPT and Generation of Mitochondrial Supernatants.Mitochondria were purified from the livers of female (ages, 6–8 weeks)BALB/c mice on a Percoll gradient (47) and were resuspended on ice in buffercontaining 200 mM sucrose, 10 mM Tris/4-morpholinepropanesulfonic acid, 1mM NaPO4, 10 mM EGTA, 2 mM rotenone, 5 mM succinate [(pH 7.4) reagentsfrom Sigma Chemical Co] at the concentration of 0.5mg protein/ml of bufferbefore manipulation. For determination of swelling, mitochondria were dilutedat 1:10 in the same buffer and adsorption was recorded at 540 nm in aBeckman DU 7400 spectrophotometer, as described previously (48). Forcell-free system analysis, mitochondrial supernatants and the resulting pelletscontaining mitochondria were separated after centrifugation at 150,0003 g for1 h at 4°C, were aliquoted, and were frozen at280°C.

Western Blot Analysis. From 1 3 107 intact cells for each sample,mitochondrial supernatants (called cytosolic fraction) and the resulting pellets

containing mitochondria (designated as mitochondrial fraction) were aliquotedat280°C. Twenty-fivemg of protein from both fractions for each sample wereloaded on a 12% polyacrylamide gel. After electrophoresis, the gels wereblotted onto nitrocellulose membrane (Amersham Life Science) which wasthen probed with a mouse monoclonal antibody 7H8.2C12 (1:500; PharMin-gen) specifically recognizing the denaturated form of cytochromec. Primaryantibody binding was detected with a goat antimouse IgG conjugated withhorseradish peroxidase (1:1000; Sigma Chemical Co) and visualized by en-hanced chemiluminescence (ECL) following the manufacturer’s instructions(Amersham).

Fluorimetric Detection of Caspase-3 and Caspase-3-like Activity.DEVD-dependent activity was assayed fluorometrically using Ac-DEVD-amino-4-methyl-coumarine (Bachem, Basel, Switzerland) as a substrate.Five 3 106 RPMI 8226-treated cells were washed with PBS and disrupted byfreeze-thawing in 1 ml of lysate buffer containing 10 mM digitonin, 1 mMEDTA, and 10 mM EGTA in 50 mM Tris-HCl (pH 7.5) as described previously(49). After 3 min of centrifugation at 10,000 xg, the supernatant was assayedfor protein concentration. Aliquots (50-ml) were further incubated with 50mlof substrate (final concentration, 20mM) in 50 mM Tris-HCl (pH 7.5) buffercontaining 1 mM EDTA and 10 mM EGTA at 37°C for 1 h. The reaction wasstopped by adding an equal volume of 0.2M glycine-HCl [(pH 2.8) Ref. 50].Fluorescence of the released aminomethylcoumarine was read on a microplatefluorometer Fluorocount (Packard Instrument Company, Meriden, CT) at anexcitation/emission wavelength of 380/460 nm.

Cell-free System of Apoptosis.Nuclei from HeLa cells were purified on asucrose gradient, as explained previously (51) and were diluted in HNB buffer(10 mM PIPES; 10 mM KCl; 2 mM MgCl2, 1 mM DTT, 1 nM cytochalasin B,and 0.1 mM PMSF (pH 7.4). Nuclei were added to the supernatants frommitochondria at the final concentration of 13 103/ml and were incubated for 90min at 37°C, then stained with 10mM of DAPI , and examined by fluorescencemicroscopy at 365 nm.

RESULTS

CD437 Has the Unique Property of Inducing Rapid Cytoplas-mic and Nuclear Signs of Apoptosis.CD437, ATRA, CD666 (aRARg agonist structurally unrelated to CD437), and CD3126 (themethyl ester of CD437 inactive on RARs; Table 1), were comparedfor their ability to induce rapid apoptosis in various cell lines. After12 h of incubation, CD437, at 3mM, induced significant hypoploidy inRPMI 8226 (a human myeloma cell line) as well as in 2B4.11 mouseT-cells hybridoma, in mouse embryonal carcinoma P19 cells, or inhuman carcinoma HeLa cells (Fig. 1). In contrast to CD437, the otherretinoids tested did not induce, or had little effect on, apoptosis after

Fig. 1. CD437 induces rapid nuclear signs of apoptosis. Induction of apoptosis byCD437 and other retinoids in different tumor cell lines. Cells (500,000/ml) were treatedwith 3 mM CD437, CD666, CD3126, or ATRA for 12 h. Apoptosis was assessed bycytofluorometric analysis of propidium iodide-stained, permeabilized tumor cells: humanmyeloma (RPMI 8226), mouse T-cell leukemia (2B4.11), mouse embryonal carcinoma(P19), and human carcinoma (HeLa). Data (6SD) are representative of three independentexperiments.

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a short incubation time (Fig. 1). We reported previously that theinduction of apoptosis by ATRA in RPMI 8226 follows slow kinetics,becoming detectable only after 72 h and becoming maximum after 6days (42). The lack of activity of CD666 and CD3126 stronglysuggests that CD437 acts via a RARg-independent pathway butrequires a free carboxylic group. The effects of CD437 seemeddose-dependent with an ED50 at 3mM for the increase in sub-G1 cellsobserved after 8 h of treatment (data not shown). In most cell types,the first detectable sign of apoptosis is a loss ofDcm followed by thesubsequent generation of ROS resulting from the uncoupling of therespiratory chain (45). We performed a kinetic analysis of the appear-ance of the main signs of apoptosis in the CD437-treated cells. Asshown in Fig. 2A, CD437 induces a rapid (within 1 h)Dcm disruptionmeasured by the reduction of the uptake of the potential-sensitive dyeDiOC6(3) in RPMI 8226 cells (increase in the % of DiOC6(3)

low HE2

cells). No effect was observed with the other retinoids tested in thisstudy (data not shown). The loss ofDcm in CD437-treated cells wasfollowed by an enhanced ROS formation detected by the oxidation ofHE to the fluorescent product ethidium (HE1 cells) concomitant withthe appearance of the first signs of nuclear apoptosis (hypoploidy).Agarose gel electrophoresis confirms the apoptotic nature of CD437-triggered cell death (not shown). Caspase-3 and caspase-3-like activ-ities were assessed by using a specific fluorogenic substrate (DEVDcoupled to amino methyl coumarine). The increase in DEVDaseactivity was detected 1 h after CD437 exposure,i.e. slighty delayedwhen compared with theDcm disruption (Fig. 2B). Thus, in theCD437-induced apoptosis, theDcm reduction precedes the down-

stream events of the apoptotic cascade including caspase-3 activation,ROS generation, and nuclear apoptosis.

CD437 Cytotoxicity Is Observed in the Absence of RARs, Tran-scription, and Even Nucleus.To explore the role of RAR in theCD437-induced apoptosis, we preincubated RPMI 8226 cells, whichexpress RARa, b, andg (42), with various synthetic retinoids. Cellspresaturated with these specific ligands were then treated with CD437.None of the compounds used for the preincubation step (CD666, aRARg agonist; CD2665, a RARg antagonist; CD336, a RARa ago-nist, or CD3126, a transcriptionnally inactive methyl ester of CD437),was able to protect RPMI 8226 cells against the apoptotic effect ofCD437 (Fig. 3A). This experiment suggests that the proapoptoticeffect of CD437 is not influenced by the pharmacological modulationof the functional status of the RARs. An additional series of experi-ments confirmed that CD437-induced apoptosis can occur in the

Fig. 2. CD437 induces dissipation ofDcm before nuclear apoptosis and caspasesactivation.A, kinetics of the CD437-induced mitochondrial and nuclear signs of apoptosis.RPMI 8226 cells were cultured in the presence of CD437 (1mM, 3 mM), and, at theindicated times, the percentage of DiOC6(3)

lowHE2, DiOC6(3)lowHE1, and subdiploid

cells was determined (SD was,10%). Data are representative of three independentexperiments.B, time course of caspase-3 activity. At the indicated interval after CD437treatment, RPMI 8226 cells were lysed and assayed for DEVDase activity. Data arepresented as an average of three determinations of arbitrary fluorescence units/mg ofprotein (6SD).

Fig. 3. CD437 induces apoptosis through a retinoic acid nuclear receptor-independentpathway.A, RPMI 8226 cells were either treated with the various retinoids tested alone(left column part of the panel) or preincubated for 2 h with CD666 (3mM), CD336 (3mM),CD3126 (3mM) or CD2665(3mM) before the exposure to CD437 (1mM). After 12 h, cellswere analyzed by flow cytometry for their DNA content. Data represent the percentage ofhypoploid cells of three independent experiments (mean6 SD). B, effect of CD437 onL363 cells. L363 cells (a RARg-negative cell line) as well as RPMI 8226 control cellswere cultured in the presence of CD437 (1mM) for 8 h and submitted to doublecolor-staining with DiOC6(3)/HE for the simultaneous assessment ofDcm and superoxideanion generation.Numbers in dark circlesrefer to the percentage of hypoploid cellsdetermined by propidium iodide-staining cells from parallel cultures. Results are repre-sentative of two independent experiments.

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absence of functional RARg. Indeed, we compared the onset ofapoptosis in RPMI 8226 and L363 cells, a RARg negative humanmyeloma cell line (42). As shown in Fig. 3B, L363 cells—exposed 8 hto 1 mM CD437—exhibitedDcm reduction and generation of ROSassociated with chromatolysis. Control experiments confirmed thatRPMI 8226 cells undergo mitochondrial dysfunction and nuclearapoptosis to the same extent (Fig. 3B).

Inhibition of mRNA or protein synthesis by actinomycin D orcycloheximide, respectively, did not significantly affect the CD437-induced apoptosis, whereas substantial inhibition was observed for theglucocorticoid-induced apoptosis, used as control, in the 2B4.11mouse T-cells model, both at the mitochondrial and at the nuclearlevels (Fig. 4). To further document that the cell nucleus was dispen-sable for the triggering of apoptosis by CD437, we generated anucle-ate RPMI 8226 cells. Control cells and cytoplasts behaved similarly

Fig. 4. mRNA and protein synthesis are dispensable for CD437-induced apoptosis.2B4.11 T cells were cultivated with 1mM CD437 or 1mM dexamethasone in the presenceof actinomycin D (30 nM) or cycloheximide (1mM). After 12 h of culture, mitochondrialparameters were assessed by DiOC6(3)/HE staining (A), and DNA hypoploidy wasmeasured (B). Results are representative of three independent experiments.

Fig. 5. Induction of apoptosis in CD437-treated cytoplasts. RPMI 8226 cells wereenucleated as described in “Materials and Methods.” Anucleate cells (cytoplasts) andcontrol cells were incubated for 2 h in the presence or absence of 3mM CD437,followed by (A) determination of mitochondrial signs of apoptosis using fluoro-chromes (DiOC6(3)/HE); (B) detection of DEVDase activity with DEVD-Rhodaminefluorochrome; and (C) determination of the cell surface exposure of phosphatidyl-serine residues with FITC-Annexin-V. Results are representative of three differentexperiments.

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regarding the apoptotic manifestations observed (Fig. 5). CD437 notonly disrupted theDcm and triggered the generation of ROS in bothintact and anucleated cells (Fig. 5A), but it also induced a DEVDaseactivity detectable by cytofluorometry irrespective of the presence ofnucleus (Fig. 5B). Moreover, CD437 provoked the aberrant exposureof phosphatidylserine residues to the outer plasma membrane leaflet(Fig. 5C), an early apoptotic event allowing for the recognition ofapoptotic cells and their phagocytosis by adjacent cells (52).

Inhibition of the MPT Pore Prevents CD437-induced Apopto-sis. Because CD437 induces a rapid disruption of theDcm, weaddressed the question of the exact role of MPT in the CD437-provoked apoptosis by pharmacological modulation of this mitochon-drial event using well-known inhibitors (for a review, see Ref. 53). Asshown in Fig. 6A, inhibitors of MPT were able to efficiently suppressboth the mitochondrial depolarization and the nuclear apoptosis in-duced by CD437. Thus, CsA, the prototype inhibitor of MPT that actson mitochondrial matrix cyclophilin D, inhibits the mitochondrialeffects of CD437. In contrast, the immunosuppressor FK506, whichacts via calcineurin but not via cyclophilin D, is totally devoid of any

effect on MPT or on the nuclear apoptosis induced by CD437 (Fig.6A). MPT inhibitors other than CsA were also efficient in preventingCD437 effects. Thus, 2,3-butanedione and phenylglyoxal, two re-agents used to modify accessible arginine residues in native proteinsand reported to inhibit the MPT of isolated mitochondria (34), im-peded theDcm reduction and subsequent nuclear hypoploidy inducedby CD437 (Fig. 6A). These effects were also observed with variousknown agents, recently shown to be effective MPT inhibitors, includ-ing trimetazidine (37) trifluoperazine (35), and cinnarizine (36).BA—an inhibitor of ANT, which is located in the inner mitochondrialmembrane of the PTPC—and the thiol reactive reagent, CMX-Ros,were also protective against the dysfunction induced by CD437.In contrast, flunarizine, reported to be effective in inhibiting MPTat low concentrations (,50 mM; Ref. 36), was totally inefficient inour model. Contrasting with the effects of these MPT inhibitors,DEVD.cmk—an inhibitor of caspase-3 activities—impeded nuclearapoptosis without affecting theDcm suppression (Fig. 6A), whichconfirms that MPT precedes the appearance of the DEVDase activity,itself upstream of the nuclear events.

Fig. 6. MPT inhibitors prevent CD437-triggered apoptosis.A, pharmacological inhibition of CD437-induced hypoploidy. RPMI 8226 cells were cultured in the presence of 1mMCD437 and various inhibitors of MPT: CsA (1mM); phenylglyoxal (1 mM); 2,3 butanedione (4 mM); BA (50 mM); CMX-Ros (1mM); trifluoperazine (30mM); cinnarizine (30mM);trimetazidine (2mg/ml); or flunarizine (30mM). FK-506 (1mM)—an immunosuppressive drug devoid of any MPT activity—and DEVD.cmk (50mM)—a caspase-3 inhibitor—were alsoused. After 6 h,Dcm and sub-G1 cells were determined by flow cytometry using DiOC6(3) and propidium iodide, respectively. Results obtained in samples treated with CD437 andthe various compounds tested were compared with those obtained after treatment with CD437 alone (45%Dcm

low cells; 296 3% hypoploid cells) and expressed as the percentageof suppression of this control response [% of suppression5 100 (Y2 X) 4 Y, whereX andY correspond to the observed % ofDcm or sub-G1 cells in the samples treated with CD437alone (Y) or in association (x).] Results were corrected for the very low amount of spontaneous apoptosis occurring in untreated cells.B and C, release of cytochromec frommitochondria of intact cells. RPMI 8226 cells were treated for 4 h with CD437 (1mM) and/or CsA (1mM). CD437 induces the release of cytochromec (B, Western blot;C,immunofluorescence) into the cytosol of intact cell. CsA impedes cytochromec (B, C) leakage from mitochondria of intact CD437 treated-cells.D, CsA blocks CD437-dependentDEVDase activity. After 4 h ofincubation with CD437 (1mM, 3 mM) and/or CsA (1mM), RPMI 8226 cells were harvested and lysed for fluorimetric detection of caspase activity(DEVDase activity). Data are presented as the average of arbitrary fluorescence units per milligram of protein (6SD).

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Next, we tested the effects of CsA, the reference inhibitor of MPT,on different manifestations of CD437-induced apoptosis, includingthe release of mitochondrial proapoptotic factors and the activation ofcaspases. CD437 treatment resulted in cytochromec release frommitochondria. Immunoblot analysis of subcellular fractions revealedan increase of cytochromec in the cytosol concomitant to its decreasein the mitochondrial fraction (Fig. 6B). Immunofluorescence detectionof cytochromec in intact cells (Fig. 6C) confirms that control cellsshowed a bright spotted cytoplasmic staining consistent with a mito-chondrial location (Fig. 6C, upper left), whereas the immunostainingof CD437-treated cells was diffuse, which indicated cytochromecrelease from mitochondria (Fig. 6C, upper right). Interestingly, CsAprevented the mitochondria-cytosolic redistribution of cytochromecinduced by CD437 (Fig. 6,B and C). Moreover, CsA blocked thecaspase-3-like activation induced by CD437 (Fig. 6D). Taken to-gether, these data indicate that the MPT is mandatory for all of theCD437-induced apoptotic events, including the activation of down-stream caspases.

CD437 Provokes the Release of Apoptogenic Factors from Mi-tochondria in a Cell-free System.To further correlate the directeffect of CD437 on MPT and nuclear apoptosis, we used a cell-freesystem of apoptosis (54), in which isolated mitochondria were treatedwith CD437, CD666, CD3126, or ATRA alone or in combination withCsA and then centrifuged. The mitochondrial supernatants were re-covered and added to isolated nuclei to determine their effect onchromatin condensation. Proapoptotic activity was detected only inthe supernatant of mitochondria treated with the MPT-inducer CD437but not in the supernatants of mitochondria treated with CD666,CD3126, or ATRA (Fig. 7A). Moreover, the addition of CsA reducedthe release of apoptosis-inducing activity by CD437-treated mito-chondria. None of the retinoids were able to induce apoptosis inisolated nuclei on its own in the absence of mitochondria (Fig. 7A).Typical morphology of the nuclei submitted to these various condi-tions is presented in Fig. 7B. These results, which suggested a directaction of CD437 on mitochondrial fractions to induce the release ofproapoptotic factors, prompted us to test its ability to induce MPT inisolated mitochondria. MPT gives rise to the colloidosmotic swellingof isolated mitochondria resuspended in a protein-free buffer. Thisswelling causes a reduction in theA540 nm(33). As shown in Fig. 8, 1mM CD437 (but neither CD666 nor ATRA) induced the permeabilitytransition-dependent colloidosmotic swelling of mitochondria, andthis effect was inhibited by CsA. Hence, CD437 can exert a directeffect on mitochondria that involves MPT.

CD437-induced Apoptosis Is Inhibited by Mitochondrial-targeted but not by ER-targeted Bcl-2. Rat-1/myc fibroblasts are awell established model for examining apoptosis triggered by serumdeprivation and concomitant treatment withb-estradiol to induce Mycactivity (55). We used Rat-1/myc fibroblasts that stably expressedBcl-2 mutants with restricted subcellular location (43). In the mutatedBcl-2, the COOH-terminal tail has been replaced by heterologoussignal peptides specifically targetting Bcl-2 either to the ER (Bcl-cb5

Fig. 7. CD437 induces the release of proapo-ptotic activities in a cell-free system of apoptosis.A, proapoptotic activity in a cell-free system ofapoptosis. Mitochondria isolated from BALB/cmice liver were exposed to CD437 (1mM, 3 mM),ATRA (3 mM), CD666 (3mM), CD3126 (3mM), orthe combination of CD437 and CsA (1mM) for 20min and then ultracentrifuged. The supernatantswere tested for apoptosis-inducing activity on iso-lated HeLa nuclei. In control samples, mitochon-dria were omitted, and isolated nuclei were directlyexposed to the various retinoids and to CsA. Aftera 90-min incubation period at 37°C, HeLa nucleiwere stained with the DNA dye DAPI and wereexamined by fluorescence microscopy. Two hun-dred nuclei were counted for each point by twoindependent experimentators. Results represent themeans6 SD of three independent experiments.B,typical morphology of the counted nuclei.

Fig. 8. CD437-induced permeability transition of isolated mitochondria. Permeabilitytransition of isolated mitochondria. Mitochondria from mice hepatocytes were monitoredfor changes in the absorbance (A540 nm) indicative of mitochondrial swelling.Arrows,CD437 (1 mM or 3 mM) 6 CsA (1 mM), ATRA(3 mM), or CD666 (3 mM) were added tomitochondria. One hundred % maximum value was determined as the result of theatractyloside (Atr)-induced drop inA540 nmmeasured after 5 min of monitoring.

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mutation) or to the outer mitochondrial membrane (Bcl-ActA muta-tion). Rat-1/myc cells with the empty vector were used as control. Asshown in Fig. 9, treatment of control cells by either CD437 alone orestradiol associated with serum deprivation resulted in comparablemassive apoptosis, observed at the mitochondrial (Fig. 9A), plasmamembrane (Fig. 9B), and nuclear (Fig. 9C) steps. CD437-inducedapoptosis was prevented in Bcl-ActA cells but not in Bcl-cb5 cells,which suggested that mitochondrial location was required for Bcl-2 tocounteract CD437 action. As previously reported, the serum depriva-tion-induced apoptosis was prevented in both Bcl-ActA- and Bcl-cb5-expressing cells,i.e., by both mitochondrial- and ER-localized formsof Bcl-2 (43). The fact that the mitochondrial location of Bcl-2correlates with the prevention of the apoptosis triggered by CD437strongly suggests that the mitochondrion is one of the targets of theapoptogenic action of CD437.

DISCUSSION

In the present report, we have demonstrated the pivotal role ofmitochondria in CD437-induced apoptosis and have established that

CD437 can act directly on these organelles. We first confirmed theability of CD437 to rapidly induce apoptosis in different cell types.Our results document the fact that CD437 is active in cells displayingno, or very low, sensitivity to retinoic acid and to synthetic retinoids,including CD666 (a RARg selective compound) and CD3126 (themethyl ester of CD437). These data are in agreement with structure-activity relationship studies performed by others, which comparedCD437 and related compounds to ATRA and synthetic retinoids (7, 9,13, 20). The importance of structural features like the adamantylgroup and the presence of free carboxylic group have already beenemphasized. The lack of activity of CD3126 in the four cell lines used(Fig. 1) suggests that there was no significant hydrolysis of the esterbond, in accordance with the recent report demonstrating that such anhydrolysis, yielding free CD437, occurs only in macrophage cell lines(56). The cell death induced by CD437 is particularly rapid whencompared with the slow kinetics observed with ATRA (42). The fastkinetics of CD437-induced apoptosis has also been observed in cer-vical carcinoma, HL-60, and human lymphoma cell lines (11, 17, 18,28). Kinetic analysis of the apoptotic events triggered by CD437indicate that MPT preceded the activation of downstream caspasesand nuclear apoptosis (Fig. 2). CD 437 has been shown to inducecytochromec leakage into cytoplasm in different cell types (19, 23).One of the possible consequences of the MPT is the release ofintermembrane proteins—AIF and/or cytochromec—implicated inthe activation of downstream caspases. However, in some models ofapoptosis, it has been suggested that mitochondrial release of cyto-chromec occurs independently of MPT (57). In contrast, we demon-strated in this study that the inhibition of MPT prevents the mitochon-drial leakage of cytochromec and subsequent caspase-3 activation.Moreover, CD437 is also able to release AIF from mitochondria inRat-1 cells, and this effect is fully prevented by CsA (data not shown).In our model, the identification of MPT as the first step governing theexecution of CD437-mediated apoptosis was unambiguously estab-lished by the demonstration that MPT inhibitors prevent all of themanifestations of CD437-induced apoptosis including caspase activa-tion. In contrast, the inhibition of downstream caspases preventednuclear apoptosis without affecting MPT (Fig. 6). The implication ofcaspases 3 and 7 in the CD437-mediated apoptosis has already beenmentioned (18, 19, 28). Interestingly, pro-caspase-3 has been reportedto have both a cytosolic and a mitochondrial distribution, the latterbeing coupled to the Bcl-2-sensitive apoptotic pathway (58).

Bcl-2 and other members of the family are predominantly localizedin the outer mitochondrial membrane but also are found in the nuclearmembrane and the ER (59). Recently, it has been found that Bcl-2 andBax interact with the adenine nucleotide translocator of the innermembrane, one of the proteins contained in the PTPC (32). Experi-ments involving purified PTPC indicate that at least part of thefunction of Bax, Bcl-2, and Bcl-xL is to facilitate or inhibit MPT. Inthis study, we have demonstrated that overexpression of Bcl-2 pre-vents the apoptosis that is triggered by CD437 only when Bcl-2 istargeted to mitochondria (Fig. 9), which underlines the implication ofthese organelles in the pathway activated by CD437. Bcl-2 has beenreported to prevent the induction of apoptosis by CD437 in Molt-4cells, as indicated by the inhibition of caspase-3 like activity and DNAfragmentation (18). However, CD437 was still able to inhibit cellproliferation, and cells finally died. In another model, overexpressionof Bcl-2 or Bcl-xL failed to inhibit apoptosis mediated by CD437 (23).In fact the appearance of apoptotic manifestations was delayed in cellsoverexpressing Bcl-2, and higher concentrations of CD437 were re-quired to achieve maximal apoptosis. The apparent variability in theability of Bcl-2 to inhibit CD437-triggered apoptosis could be ex-plained by differences in Bcl-2 expression levels and the cellularcontext. Nevertheless, our results highlight the importance of the

Fig. 9. CD437-induced apoptosis is affected differentially by mitochondrial-versusER-targeted Bcl-2. Effect of mitochondrialversusER localization of Bcl-2 on apoptosisinduction by CD 437 or serum deprivation and myc activation. Rat-1 fibroblasts stablytransfected with retroviral vectors expressing Bcl-2 mutants or control vector werecultured during 6 h in thepresence of 3mM of CD 437 or for 18 h in the presence orabsence of 0.1% estradiol and 5% FCS (serum starvation), followed by the determinationof several apoptosis-associated parameters:A, loss ofDcm determined by the potential-sensitive dye DIOC6(3);B, phosphatidylserine exposure on the plasma membrane de-tected by Annexin-V-FITC conjugate;C, propidium iodide-detectable loss of nuclearDNA (hypoploidy). Results are representative of three independent experiments(mean6 SD).

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subcellular location of Bcl-2 in mitochondria for any anti-CD437effect. Interestingly, it was recently shown that PK 111–95, a specificligand of the peripheral benzodiazepine receptor (60), one of thePTPC components, was able to prevent the protective action of Bcl-2against apoptosis (61). Similarly, CD437, by acting on PTPC, couldinduce a perturbation of the mitochondrial Bcl-2/Bax complexes andalter the efficiency of Bcl-2 to prevent apoptosis.

Previous reports have documented that CD437 is able to induceapoptosis in retinoic acid-resistant and RARg-negative cells, and thatthis effect is not inhibited by antiretinoids acting at the RARs level(see “Introduction”). Here, we not only confirmed this observation inour model, but we also extended it to the mitochondrial events ofapoptosis and demonstrated that the CD437-mediated apoptosis oc-curred in the presence of inhibitors of transcription and protein syn-thesis (Figs. 3 and 4). This observation prompted us to investigate fora proapoptotic effect of CD437 on cytoplasts (Fig. 5) and to explorethe possibility of CD437 to act on mitochondria. For that purpose, wehave developed subcellular fractionation and a cell-free system ofapoptosisin vitro. First, we have shown that CD437 is able to induceMPT of purified mitochondria at the same concentration used forintact cells. Secondly, only supernatants from supernatants from mi-tochondria that have undergone MPT display apoptogenic activity ina cell-free system. Here again, CD437 was the only active compound,whereas ATRA, CD666, and CD3126 remained inactive. These re-sults confirm the relevance of the cell-free system and identify mito-chondria as a direct target of CD437. Interestingly, other chemother-apeutic agents have also been reported to act on mitochondria and toinduce MPT in cancer cells (62).

An important point to be stressed is that the effects of CD437 onapoptosis and on isolated mitochondria in this study were observed atrather high concentrations of CD437,i.e. in the micromolar range.This is in accordance with most of the data reported to date about theproapoptotic activity of CD437 in cancer cells (6, 8, 11–13) with theexception of HL-60 cells (17) and normal mouse thymocytes, whichare sensitive to low-CD437 concentrations. In addition, normal mousethymocytes undergo apoptosis in a RARg-dependent way (63).

Furthermore, CD437 and related compounds have been demon-strated to be able to inhibit the growth of xenografts of retinoicacid-resistant human tumors in nude micein vivo (6, 10, 20). Hereagain, high doses—in the 25-mmoles/kg-range—were necessary.

Taken together, our results suggest that a significant part of theproapoptotic effects of CD437 reported in the literature could beexplained by a direct action of CD437 on the mitochondria of targetcells. However, according to the cell context, RARg and other factorslike p21, p53, Bcl-2, and related proteins could also be involved in avariable manner. However, this finding suggests that CD437 may bea particularly useful cancer-cell death-inducer when “classical” reti-noids requiring the nuclear action of specific receptors fail to act astherapeutic agents.

ACKNOWLEDGMENTS

We especially thank Anne-Marie Thomas and Edith Dhuiege for technicalassistance, Dr. Duine (Delft University of Technology, Delft, the Netherlands)for the gift of bongkrekik acid, and Dr. W. Andrews (Department of Medicine,McMaster University, Hamilton, Ontario, Canada) for the gift of Rat-1 fibro-blasts.

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1999;59:6257-6266. Cancer Res Philippe Marchetti, Naoufal Zamzami, Bertrand Joseph, et al. Independent of the NucleusAcid Can Trigger Apoptosis through a Mitochondrial Pathway6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphtalene Carboxylic The Novel Retinoid

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