A Retinoid-Related Molecule that Does Not Bind to...

[CANCER RESEARCH 64, 3302–3312, May 1, 2004]

A Retinoid-Related Molecule that Does Not Bind to Classical Retinoid ReceptorsPotently Induces Apoptosis in Human Prostate Cancer Cells through RapidCaspase Activation

Richard G. Keedwell,1 Yi Zhao,4 Lisette A. Hammond,2 Suofu Qin,4 Kwok-Yin Tsang,4 Armin Reitmair,4

Yanira Molina,4 Yumiko Okawa,4 Larissa I. Atangan,4 Dixie-Lee Shurland,4 Kaisheng Wen,1 D. Michael A. Wallace,3

Roger Bird,1 Roshantha A. S. Chandraratna,4 and Geoffrey Brown1

Divisions of 1Immunity and Infection and of 2Cancer Studies, University of Birmingham Medical School, Edgbaston, Birmingham, United Kingdom; 3Department of Urology,Queen Elizabeth Hospital, Birmingham; and 4Retinoid Research, Departments of Chemistry and Biology, Allergan Inc., Irvine, California

ABSTRACT

Synthetic retinoid-related molecules, such as N-(4-hydroxyphenyl)reti-namide (fenretinide) and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naph-thalene carboxylic acid (CD437) induce apoptosis in a variety of malignantcells. The mechanism(s) of action of these compounds does not appear toinvolve retinoic acid receptors (RARs) and retinoid X receptors (RXRs),although some investigators disagree with this view. To clarify whethersome retinoid-related molecules can induce apoptosis without involvingRARs and/or RXRs, we used 4-[3-(1-heptyl-4,4-dimethyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)-3-oxo-E-propenyl] benzoic acid (AGN193198)that neither binds effectively to RARs and RXRs nor transactivates inRAR- and RXR-mediated reporter assays. AGN193198 potently inducedapoptosis in prostate, breast, and gastrointestinal carcinoma cells and inleukemia cells. AGN193198 also abolished growth (by 50% at 130–332nM) and induced apoptosis in primary cultures established from prostaticcarcinoma (13 patients) and gastrointestinal carcinoma (1 patient). Ap-optosis was induced rapidly, as indicated by mitochondrial depolarizationand DNA fragmentation. Molecular events provoked by AGN193198 in-cluded activation of caspase-3, -8, -9, and -10 (by 4–6 h) and the produc-tion of BID/p15 (by 6 h). These findings show that caspase-mediatedinduction of apoptosis by AGN193198 is RAR/RXR-independent andsuggest that this compound may be useful in the treatment of prostatecancer.

INTRODUCTION

Recent reviews have highlighted the potential of retinoids as pre-ventive and therapeutic agents for various cancers (1–5). Moreover,some synthetic retinoids are effective in preclinical studies againstcommon carcinomas, such as prostate (reviewed in Ref. 3), lung (6),and breast (7), for which mortality rates have not been substantiallydecreased by current treatments. Prostate cancer accounts for 13% ofcancer deaths in males in the United States, and late-stage disease isincurable after metastasis (8–10).

Retinoids bind to and activate the following two classes of recep-tors: retinoic acid receptors (RAR�, �, and �) and retinoid X receptors(RXR�, �, and �). All-trans-retinoic acid (ATRA), a natural RARagonist, and synthetic agonists and antagonists of subtypes of RARsinhibit growth and induce apoptosis in prostate cancer cells. However,the prostate cancer cell lines LNCaP, DU145, and PC3 are relativelyinsensitive to ATRA and other RAR agonists, with around 1–10 �M

needed to inhibit colony formation (11–14). Accordingly, clinical

studies of 13-cis-retinoic acid (isotretinoin), with or without IFN-�, inprostate cancer have demonstrated only moderate efficacy (15–17).By contrast to the weak activity of agonists, the RAR pan-antagonist4-2(6-(2,2-dimethyl-(1H)-4-(4-ethylphenyl)-1-benzothiopyran))ethy-nyl] benzoic acid (AGN194310) and the RAR �, �-selective antago-nist 3-fluoro-4-[7-(4,4-dimethyl-3,4-dihydro-1-(p-tolyl)-naphthyl)-2-azo] benzoic acid (AGN194431) are potent growth inhibitors ofprostate carcinoma lines and patients’ cells (3, 14) and inhibit colonyformation at �100 nM. Similarly, the weak RAR antagonist MX781inhibits the growth of breast carcinoma lines more potently thanATRA (7).

Some synthetic retinoid-related molecules (RRMs; 4) appear toinduce apoptosis in malignant cells, including prostate carcinomacells, through mechanisms that do not involve RARs and RXRs.Compounds that display potential as therapeutic agents include N-(4-hydroxyphenyl)retinamide (fenretinide) and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437). Possible tar-gets have been identified for these agents, but their mechanisms ofaction, particularly the involvement of RARs, remain unresolved(reviewed in Refs. 2 and 18). Fenretinide binds poorly to RARs invitro (19), induces apoptosis in, for example, prostate (20, 21) andovarian carcinoma cells (22–25), and is chemopreventive against oralleukoplakias and ovarian and breast carcinomas (26–28). CD437 wassynthesized as an RAR� agonist but also binds to RAR� and to aslight extent to RAR� (29, 30), and RAR-independent mechanisms ofaction have been proposed (31–33). Like fenretinide, CD437 inducesapoptosis in a variety of cancers including lung, cervical, breast, andovarian carcinomas and melanoma (25, 30, 33–39).

Is binding of RRMs to nuclear retinoid receptors, even if weak, andtheir transactivation essential for RRMs to induce apoptosis in cancercells? During the course of screening compound libraries for antican-cer activity, we identified a novel chalcone structure-based RRM(Ref. 40; 4-[3-(1-heptyl-4,4-dimethyl-2-oxo-1,2,3,4-tetrahydroquino-lin-6-yl)-3-oxo-propenyl] benzoic acid (AGN193198). This com-pound neither binds effectively to RARs and RXRs nor transactivatesin RAR- and RXR-mediated reporter assays but yet was effective ininhibiting the growth of prostate cancer and other malignant cell lines.AGN193198 is an extremely potent inducer of apoptosis in primarycultures established from 13 patients with prostatic carcinoma. Theinduction of apoptosis by RRMs, such as fenretinide and CD437,involves mitochondrial depolarization and activation of caspase-3 andcaspase-9 (25, 36). Lopez-Hernandez et al. (41, 42) have proposedthat caspases are the major effectors of RRM action and, together withother workers, have shown caspase-8 activation leading to mitochon-drial depolarization. AGN193198 provokes mitochondrial depolariza-tion, activation of caspase-3, -8, -9, and -10 and BID/p15 production,leading ultimately to DNA fragmentation. These processes seem toinvolve neither RARs nor RXRs, suggesting that RRMs such asAGN193198 that do not bind to these receptors may be useful in thetreatment of a variety of malignancies.

Received 9/3/03; revised 2/26/04; accepted 3/1/04.Grant support: This work was supported by a grant from Allergan Inc., Irvine, CA.The costs of publication of this article were defrayed in part by the payment of page

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

Note: R.G. Keedwell and Y. Zhao contributed equally to this work.Requests for reprints: Geoffrey Brown, Division of Immunity and Infection, Medical

School, University of Birmingham, Birmingham B15 2TT, United Kingdom. Phone:44-121-414-4082; Fax: 44-121-414-3599. Email: [email protected].

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MATERIALS AND METHODS

Retinoids. The following synthetic retinoids and RRMs were synthesizedat Allergan Inc. (Irvine, CA): the RRM 4-[3-(1-heptyl-4,4-dimethyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)-3-oxo-propenyl] benzoic acid (AGN193198),the RAR pan-antagonist 4-2(6-(2,2-dimethyl-(1H)-4-(4-ethylphenyl)-1-benzo-thiopyran))ethynyl] benzoic acid (AGN194310), the RAR �,�-selective antagonist3-fluoro-4-[7-(4,4-dimethyl-3,4-dihydro-1-(p-tolyl)-naphthyl)-2-azo] benzoic acid(AGN194431), the RXR pan-antagonist 7-(2-heptyloxy-3,5-diisopropyl-phenyl)-3-methyl-octa-2Z,4E,6E trienoic acid (AGN195393), and the RXR pan-agonist3,7-dimethyl-6S,7S-methano,7-[1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphth-7-yl]-2E,4E heptadienoic acid (AGN194204). Stock solutions of these compoundsand ATRA were prepared at 10 mM in 50% ethanol/50% dimethylsulphoxide andstored at �20°C. Retinoids were analyzed as described previously (43) for theirability to competitively inhibit binding of 5 nM [3H]ATRA (for RARs) or 10 nM

[3H]9-cis-retinoic acid (for RXRs) to extracts (5–50 �g) of baculovirus/sf9-

expressed RARs (�, �, and �) and RXRs (�, �, and �), respectively. Receptortransactivation assays were performed using CV-1 cells (at 2500 cells/well in a96-well plate) that were LipofectAMINE transfected with 100 ng of luciferasereporters and receptor plasmid DNA (for amounts see Fig. 1; Ref. 44). After 5 h,cells were fed with DMEM containing 10% charcoal-treated fetal bovine serum(FBS, Life Technologies, Inc.). Twenty h after transfection, cells were treated withcompounds for 16 h before measuring luciferase activity and �-galactosidaseactivity using the Promega system. Triplicate wells were used, and relativeluciferase activity was normalized to �-galactosidase activity. For both agonismand antagonism RXR homodimer transactivation assays, wild-type RXRs wereused together with the reporter plasmid pRXRE-tk-Luc that contains five tandemrepeats of a 35-bp sequence (DR-1) from the promoter of the mouse CRBP-II gene(45) inserted immediately upstream of tk-luciferase. For RAR transactivationassays, the ERE-tk-Luc that contains the binding site for estrogen receptors wascotransfected with chimeric estrogen receptor-RARs, in which the DNA-binding

Fig. 1. Structures of retinoids and their transactivation properties. A, structures of all-trans-retinoic acid (ATRA), 9-cis-retinoic acid, and AGN193198. B, transactivation of retinoicacid receptors (RARs) and retinoid X receptors (RXRs) by AGN193198. CV-1 cells were transfected with a 100-ng RAR reporter and each 4-ng chimeric estrogen receptor-RAR ora 100-ng RXR reporter and each 40-ng RXR and then treated for 16 h with the different doses of ATRA or AGN193198 for activities of RARs (panel 1), and 9-cis-retinoic acid orAGN193198 for activities of RXRs (panel 2). Luciferase activity of transfected and treated samples was measured and normalized to the activity of �-galactosidase. C, antagonismof RARs and RXRs by AGN193198. Panel 1, different doses of AGN194310 (a RAR pan-antagonist as the positive control) and AGN193198 were used to antagonize thetransactivation of each 50-ng RAR-progesterone/10-ng RXR� heterodimer by 1 nM ATRA. Panel 2, different doses of AGN195393 (a RXR pan-antagonist as the positive control) andAGN193198 were used to antagonize the transactivation of each 40-ng RXR homodimer by 1 nM AGN194204 (a RXR pan-agonist).

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RETINOID-RELATED MOLECULES AND PROSTATE CANCER



domain of RAR was substituted with that of the estrogen receptor. Antagonism ofATRA transactivation of RARs by AGN193198 or AGN194310 was assayedusing RARs-progesterone/RXR� heterodimers, in which the P-box sequence ofRAR had been changed into the sequence (GSCKV) of glucocorticoid receptor, socalled RARs-progesterone (44). RARs-progesterone/RXR� heterodimers recog-nize the half-site binding motif of glucocorticoid receptor and the half-site bindingmotif of RXR.

Cell Lines and Cell Culture. Cultures of the carcinoma cells of patientswere established and maintained serum-free and, for comparison, cell lineswere grown serum-free or in low serum. Serum-free sub-lines of the prostatecarcinoma cell lines LNCaP, PC-3, and DU-145 (serum stocks from AmericanType Culture Collection, Rockville, MD) and of the promyeloid leukemic cellline HL60 have been described previously (14, 46). These lines are stablebecause they have been maintained in RPMI 1640 (Life Technologies, Inc.,Paisley, United Kingdom) containing antibiotics (100 units/ml penicillin and100 �g/ml streptomycin), insulin (100 �g/ml), transferrin (5.5 �g/ml), sele-nium dioxide (5 �g/ml), linoleic acid (5 ng/ml), and BSA (0.5 mg/ml) (ITS�1;Sigma, Poole, United Kingdom) for �5 years. The three prostate carcinomalines are identical in appearance to serum-grown cells, and their doubling timesand sensitivities to ATRA (inhibition of colony formation and growth in flaskcultures) are similar to those of their serum-grown counterparts (14). Thebreast carcinoma lines MDA-MB-231 and MCF-7 (provided by Dr. K. Col-ston, St. Georges Hospital, London) were adapted for growth in serum-freemedium (MDA-MB-231) and in RPMI 1640 supplemented with antibioticsand 0.5% FBS (MCF-7) as described previously (14). HeLa cells were grownin DMEM, 10% FBS, and antibiotics, and Jurkat cells were grown in RPMI1640, 10% FBS, and antibiotics. Cells were grown at 37°C in a humidifiedatmosphere of 5% CO2 in air, and adherent cells were passaged by trypsinizingwith trypsin-EDTA (Life Technologies, Inc.).

Growth of Primary Carcinoma Cells. Core biopsies were obtained frompatients undergoing investigation for suspected prostatic carcinoma. Consentfor research use was obtained from the patients and the Local Research EthicsCommittee. Histological examination confirmed that the biopsies were tumormaterial. Cultures were established as described by Peehl et al. (47), using thecommercially available serum-free medium prostate epithelial cell growthmedium supplemented with SingleQuots (BioWhittaker, Wokingham, UnitedKingdom). A primary culture was established from a gastro-intestinal carci-noma biopsy in ITS�1 medium. Cells grown from biopsies were subculturedonto plates coated with collagen-1 (Greiner, Stonehouse, United Kingdom),and these plates and prostate epithelial cell growth medium were used forassays.

Analysis of the Effect of AGN193198 on Cell Growth. Trypsinizedsingle-cell suspensions of cells were plated into a 96-well microtiter plate at400 cells/ well in 100 �l of ITS�1 medium (cell lines) or prostate epithelial cellgrowth medium (primary cells). Retinoids were added immediately and at day2 by replacing the medium. Cellular ATP levels were measured at day 5 usingthe Vialight HS High Sensitivity Cell Proliferation/Cytotoxicity kit and aBerthold LB953 luminometer (Lumi Tech, Nottingham, United Kingdom).Wells were set up in triplicate, and at least three experiments were performed.Vehicles, titrated alongside agents, did not substantially affect the growth ofany of the cells tested. Anti-Fas antibody ZB4, used in blocking experiments,was obtained from Upstate (New York).

In colony formation assays, trypsinized single-cell suspensions of cells wereplated at 1000 cells/ 65 mm2 dish in 4 ml of serum-free medium. Retinoids(10�9-10�6 M) were added immediately, and the medium plus retinoids werereplaced at day 2. Plates were incubated for 14 days, fixed using 1% formal-dehyde in 0.9% NaCl, stained with 1% methylene blue in 0.9% NaCl, anddried. Around 200 colonies were counted on each duplicate plate, and at leastthree experiments of each type were performed. Colony formation on agent-treated plates is presented as a percentage (mean � SE) of the number ofcolonies on untreated plates. Vehicle alone did not substantially effect colonyformation (for titrations see Ref. 14).

Measurements of Apoptotic Events. All of the experiments were per-formed in triplicate. LNCaP cells and primary prostate carcinoma cells wereseeded at 5 � 105 cells/ 75 cm2 flask, and AGN193198 was added immedi-ately. Apoptotic cells were identified, within pooled adherent (trypsinized) andsuspension cells, by the terminal deoxynucleotidyltransferase-mediated nickend-labeling assay (48). A FITC-conjugated antibody to BrdUrd (Becton-Dickinson & Co., Mountain View, CA) was used, and fluorescence was

measured by fluorescence-activated cell sorter analysis at 510–550 nM. Acri-dine orange (5 �g/ml) was used to stain nuclear material of apoptotic HL60cells. At least 200 cells were scored using a Zeiss microscope equipped witha mercury burner for incident illumination.

Changes to the mitochondrial membrane potential after treatment of bulkcultures of cells with AGN193198 were measured by incubating harvestedcells with 5 �g/ml of the fluorescent probe JC-1 (5,5�,6,6�-tetrachloro-1,1�,3,3�-tetrabenzimidazolylcarbocyanine iodide; Eastman Kodak Co., Roch-ester, NY) for 20 min at 37°C in an atmosphere of 5% CO2. After washingtwice in PBS for 10 min, cells were analyzed on a Becton-Dickinson fluores-cence-activated cell sorter (49).

Caspase Studies. Stock solutions of the pan-caspase inhibitor Z-VAD-FMK and of the caspase-8 inhibitor Z-IETD-FMK (R & D Systems, Abingdon,United Kingdom) were prepared in 20 mM dimethylsulphoxide. Inhibitors wereadded to bulk cultures of cells 1 h before adding AGN193198, and cells weremonitored for changes to the mitochondrial membrane potential and by usingthe terminal deoxynucleotidyltransferase-mediated nick end-labeling assay.

In fluorometric assays for caspase activity, 50 �l of cell lysate and 50 �l ofassay buffer [20 mM HEPES (pH 7.5), 10% glycerol, 2 mM DTT] containing50 �M peptide substrates for caspase-3 (DEVD-AFC), caspase-8 (IETD-AFC),or caspase-9 (LEHD-AFC; all from Biovision Inc., Mountain View, CA) wereadded to each well of microtiter plates. Backgrounds were measured in wellsthat contained assay buffer, substrate, and lysis buffer. Fluorescence wasmeasured on a CytoFluor 4000 plate reader (Applied Biosystems) set at 400nm excitation and 508 nm emission. Caspase activities were calculated as foldincreases relative to control wells.

Antibodies to caspase-3 (Stressgen), caspase-8 (Cell Signaling Technol-ogy), caspase-9 (Cell Signaling Technology), caspase-10 (MBL, Nagoya,Japan), BID (Cell Signaling Technology), and Bcl-2 (Upstate Biotechnology,clone 100) were used to stain blots. Cells (1 � 107/ml) were lysed in ice-coldbuffer [5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 100 �M Na3VO4, 2 mM

phenylmethylsulfonyl fluoride, 10 �g/ml leupeptin, and 50 mM Tris (pH 7.4)]for 0.5 h with gentle rotation at 4°C. Lysates were clarified (at 16,000 � g for15 min at 4°C), resolved by SDS-PAGE, and transferred onto polyvinylidenedifluoride membranes for immunostaining. Immunoreactive proteins werevisualized by enhanced chemiluminescence.

Analysis of the c-Jun NH2-Terminal Kinase (JNK) Signaling Pathway.Eighty percent confluent LNCaP and Jurkat cells were treated with 2 �M

AGN193198 (0.5 to 8 h) or 10 �g/ml anisomycin (30 min). Cell lysates wereresolved by SDS-PAGE, for immunoblotting with antiphospho-JNK1/2 anti-body (Cell Signaling Technology), and also analyzed using the GST-c-Junpulling-down and kinase activity assay using the stress-activated protein ki-nase/JNK assay kit (Cell Signaling Technology).

Analyses of the Nuclear Factor (NF)-�B Signaling Pathway. Interfer-ence of AGN193198 with tumor necrosis factor (TNF)-�-driven degradationof inhibitor of nuclear factor-�B� (I�B�) was determined as follows. Eightypercent confluent cultures of cells were grown overnight in medium containing10% charcoal-treated FBS. The cells were then treated for 1 h with eitherAGN193198 or the proteosomal inhibitor MG132 (Calbiochem), followed by10 ng/ml TNF-� (R & D Systems) for 10 min. Lysates were resolved bySDS-PAGE, and blots were probed with an antibody to I�B� (Santa CruzBiotechnology). Levels of NF-�B in nuclear extracts were examined using aelectromobility shift assay and a double-stranded oligonucleotide probe con-taining a consensus NF-�B sequence (GGGGACTTTCCC), which was labeledwith �-32P-CTP (Klenow fill-in reaction). Nuclei were isolated cells using aNP40 lysis buffer [0.4% NP40, 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM

EDTA, 0.1 mM EGTA, 1 mM DTT, and protease inhibitors] and then lysed [20mM HEPES (pH 7.9), 0.4 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM

DTT, protease inhibitors]. Five �g of nuclear extract were incubated withprobe for 15 min at room temperature in the presence of 1 �g poly(dI�dC) in20 �l of binding buffer [20 mM HEPES (pH 7.9), 50 mM KCl, 1 mM DTT, 2.5mM MgCl2, and 10% glycerol]. Protein-DNA complexes were separated on a5% nondenaturing gel that was dried for autoradiography.

RESULTS

Binding and Transactivation Properties of AGN193198. In thestudies described below, 1 �M AGN193198 completely inhibited thegrowth of various malignant cells and induced apoptosis. This amount

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of AGN193198 did not bind in any substantial degree to baculovirus-expressed RARs and RXRs; Ki values were as follows: 6.6 �M RAR�;�10 �M RAR�; 3.5 �M RAR�; �10 �M RXR�; 5.6 �M RXR�; and�10 �M RXR�. Compared with the transactivation activities ofATRA on RARs or 9-cis-RA on RXRs in cells expressing RARs orRXRs and reporter constructs, very low efficacies were obtained usingAGN193198 (Fig. 1B). AGN193198 also showed weak antagonismsof 1 nM ATRA-driven transactivation of RARs and of 1 nM

AGN194204-driven transactivation of RXRs (Fig. 1C). One nM of thepan-RAR antagonist AGN194310 blocked ATRA-driven transactiva-tion of RARs, whereas only partial effects were observed for 1 �M

AGN193198.AGN193198 Potently Inhibits the Growth of Patients’ Prostate

Carcinoma Cells. Primary cultures of carcinoma cells were estab-lished from core biopsies of 13 patients with prostatic carcinoma (seeFig. 2 for Gleason scores). After biopsy, the patients received antian-drogen therapy (cyproterone acetate and Zoladex), and have re-sponded as shown by substantial reductions in their serum levels ofprostate-specific antigen (Fig. 2). Hence, the cultures were establishedfrom androgen-dependent tumors. Cultures had epithelial morphol-ogy, confirmed by immunocytochemical staining for cytokeratins andprostate-specific antigen, and contained few fibroblasts.

Primary prostate carcinoma cells were tested, at passage 2, for theirsensitivity to AGN193198 and to ATRA. As measured using themicrotiter plate assay, AGN193198 potently inhibited the growth of13 of 13 patients’ cells, with concentrations that inhibited growth by50% (IC50 values) in the range 130–332 nM (see table in Fig. 2).Complete growth inhibition of each of the patients’ cells was observedat 1 �M. Cultures from patients whose carcinomas had high Gleasongradings appeared to be slightly more sensitive to AGN193198 (Fig.2B), although ANOVA revealed that this relationship was not statis-

tically significant. Whether grading is important to sensitivity willrequire testing of more patients’ cells. ATRA at 2 �M only partiallyinhibited the growth of cells from 11 of the patients (values between5 and 35% and as illustrated in Fig. 2A; see also Ref. 14), and IC50

values of �1 �M were obtained for PrC2 and PrC6.AGN193198 Is Active Against Several Types of Malignant

Cells. AGN193198 potently inhibited the growth of prostate carci-noma cell lines in the microtiter plate assay, with IC50 values of238 � 17 nM for LNCaP cells, 163 � 26 nM for DU-145 cells, and223 � 18 nM for PC-3 cells (Fig. 3A). For comparison, Fig. 3C (rightpanel) shows the relative insensitivity of LNCaP cells to ATRA.Activity of AGN193198 does not relate to an inhibitory effect on cellattachment. Allowing LNCaP cells to adhere to microtiter wells (by6 h) before adding compound did not displace the dose-responsecurve, and complete growth inhibition was observed at 1 �M (data notshown).

The pan-specific RAR antagonist AGN194310 potently inhibits thegrowth of serum-free-grown prostate carcinoma lines, but a compo-nent(s) of serum blocks this effect (14). We, therefore, compared theactivity of AGN193198 against serum-free-grown versus serum-grown LNCaP cells cultured in microtiter wells. AGN193198 sub-stantially inhibited the growth of serum-grown LNCaP cells at 2 �M,as compared with 500 nM for serum-free-grown cells (Fig. 3C, leftpanel). The IC50 value obtained for serum-grown cells was 1.2 � 0.3�M. This 4-fold difference relates to either a slight difference in thesensitivity of serum-free versus serum-grown cells to AGN193198 orbinding of this agent to a component(s) of serum.

In the clonal proliferation assay, 100 nM AGN193198 preventedcolony formation by LNCaP and PC-3; IC50 values were 51 � 4 nM

(n � 9) and 50 � 8 nM (n � 4), respectively (Fig. 3B). For compar-ison, 1 �M ATRA inhibited colony formation by LNCaP and PC3cells by �70%, and IC50 values were 344 � 32 and 419 � 129 nM,respectively. That less AGN193198 was needed for inhibition ofcolony formation (�50 nM) than for inhibition of growth (�230 nM)is commensurate with the widely held view that the colony assay is amore sensitive indicator of agent effects.

To investigate the range of malignant cell types sensitive toAGN193198, we measured activity against microtiter well cultures ofthe breast carcinoma lines MDA-MB-231 and MCF-7, the promyeloidcell line HL60, and a primary culture of gastrointestinal carcinomacells. All these cells were as sensitive as the prostate carcinoma linesand patients’ cells to growth inhibition by AGN193198, with IC50

values in the range 110–236 nM (see table in Fig. 3). HL60 cellstreated with AGN193198 underwent apoptosis without differentiating.At day 1, 20% � 2% of cells had fragmented nuclei, 1% of cellsphagocytosed complement-coated yeast and reduced nitroblue tetra-zolium, and cell death was complete by day 2 (50).

AGN193198 Induces Apoptosis in Prostate Carcinoma Cells viaActivation of Caspases. The time course of the effect of 1 �M

AGN193198 on the growth and survival of LNCaP cells wasdetermined by growing cells in microtiter wells and measuringATP levels at intervals. A 65% � 1% (n � 4) reduction in thegrowth/survival of LNCaP cells was observed after 2 days oftreatment with AGN193198 (Fig. 4A, left panel), and by day 3,survival was 16% � 0.2% (n � 4) of control cultures. Terminaldeoxynucleotidyltransferase end-labeling of DNA strand-breaksand fluorescence-activated cell sorter analysis revealed that cellswere undergoing apoptosis. The rate of appearance of DNA frag-mentation was commensurate with the loss of cell number/viability(Fig. 4A, compare left and middle panels). By day 2, 69% � 15%(n � 4) of the cells in flask cultures of LNCaP cells treated with1 �M AGN193198 had undergone apoptosis, as compared with2% � 0.5% in control cultures, and apoptotic cells had risen to

Fig. 2. AGN193198 potently inhibits the growth of patients’ prostate carcinoma cells.Activities of AGN193198 and all-trans-retinoic acid (ATRA) against carcinomacells from core biopsies of 13 patients with prostatic carcinoma were measured by seed-ing cells into wells of a microtiter plate, treating with agents immediately and at day 2, andmeasuring cellular ATP levels at day 5. A, titration curves obtained for AGN193198 (F)and ATRA (E) against PrC10. Similar experiments were used to obtain the IC50 valuesshown in the table. B, IC50 values for AGN193198 plotted against Gleason grading.

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81% � 2% (n � 4) by day 3 (Fig. 4A, middle panel; illustrated inright panel). We treated a bulk culture of early passage cells of theprimary prostate carcinoma PrC10 with 2 �M AGN193198. By24 h, cells had detached from the flask and 48% were apoptotic, ascompared with 8% in the control culture (data not shown).

Fig. 4B shows that mitochondrial depolarization, as measured bythe fluorescent probe JC-1, preceded DNA fragmentation inAGN193198-treated LNCaP cells. Doses of 0.75 and 1 �M resulted in71% � 11% and 92% � 2% (n � 3), respectively, of cells depolar-izing their mitochondria (Fig. 4B, left panel). Time course studies of1 �M AGN193198-treated cultures showed that this occurred quickly(Fig. 4B, middle panel, experimental data illustrated in right panel).There was an increase at 6 h, from 10% � 4% in untreated culturesto 18% � 5% (n � 5, P � 0.05) in AGN193198-treated cultures, andabout 90% of cells had undergone mitochondrial depolarization by18 h.

AGN193198-stimulated DNA fragmentation and mitochondrial de-polarization are both influenced by caspases. As shown in Fig. 4C,pretreatment of LNCaP cells for 1 h with the pan-caspase inhibitorZ-VAD-FMK before the addition of 1 �M AGN193198 abolished thesubsequent appearance of DNA strand-breaks. The proportion ofapoptotic cells at 72 h in cultures treated with AGN193198 and theinhibitor was 15% � 6% as compared with 78% � 7% in culturestreated with AGN193198 alone (n � 5, P � 0.0004). Pretreatmentwith the inhibitor also reduced the proportion of cells showing mito-chondrial depolarization in response to AGN193198, from 85% � 5%to 30% � 4% (n � 4, P � 0.0001).

We used the selective caspase-8 inhibitor Z-IETD-FMK to inves-

tigate whether caspase-8 activation lies upstream or downstream ofmitochondrial depolarization in LNCaP cells treated withAGN193198. At 72 h, the percentage of apoptotic cells was reducedfrom 95 � 0.5 to 61 � 12 (n � 3, P � 0.05) by the inhibitor, but therewas no change in the proportion of cells at 42 h with depolarizedmitochondria (Fig. 4C). We re-examined the effect of the caspase-8on AGN193198-induced depolarization at 12 h, and there was also noeffect (80% � 4% versus 77% � 9% in the presence of inhibitor,P � 0.4).

AGN193198 Provokes Activation of Caspases-3, -8, -9, and -10in LNCaP and Jurkat Cells. We used Jurkat cells for time coursestudies of activities of individual caspases as suspension cells aremore amenable to rapid harvesting. Jurkat T cells, like other malig-nant cells we tested, were growth-inhibited by 1 �M AGN193198(Fig. 5A, left panel), their mitochondria became depolarized(29% � 9% by 12 h) and, as in LNCaP cells, this was unaffected bythe caspase-8 inhibitor (also 26% � 7%, P � 0.9). Activities ofcaspase-3 and -9, measured using specific substrates, were increased10- and 3-fold, respectively, in extracts from Jurkat cells treated with1 �M AGN193198 (see Fig. 5A, middle and right panels).

As shown in Fig. 5C, treatment of LNCaP and Jurkat cells with 2�M AGN193198 led to the appearance of truncated (active) forms ofcaspase-3, -8, and -9 within around 4–6 h, and maximals at between6–8 h. Parallel time course analyses of enzyme activity within Jurkatcells showed that activities of caspase-3, -8, and -9 increased 25-, 4-,and 8-fold, respectively, within 6 h (Fig. 5B). Cleavages of pro-caspase-10 were detected at 4 to 6 h in LNCaP and Jurkat cells treated

Fig. 3. AGN193198 is active against a variety of cell types. A, the activityof AGN193198 against the prostate carcinoma lines LNCaP (E), DU-145(�), and PC-3 (‚) as determined by seeding cells in microtiter wells, treatingwith agent immediately and at day 2, and measuring cellular ATP levels atday 5. B, the ability of AGN193198 to inhibit colony formation in plates byLNCaP (E) and PC-3 (‚). Plated cells were treated with agent immediatelyand by replacing medium plus agent at day 2. Colonies were enumerated atday 14. IC50 values shown in the table were obtained using the microtiterwell assay. C, left, the activity of AGN193198 against serum-free grownversus serum grown LNCaP cells as measured by seeding cells in microtiterwells. C, right, titrations of all-trans-retinoic acid (ATRA) and vehiclecontrol against serum-free grown LNCaP cells seeded in microtiter wells.Data are means � SE.

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with 2 �M AGN193198 by immunoblotting of extracts (data notshown).

Fig. 6 shows that treatment of Jurkat cells with the caspase-8inhibitor Z-IETD-FMK (1 or 5 �M) for 1 h before adding 2 �M

AGN193198 (for 3 or 6 h) prevented activation of capsases-8, -3 and-9. Similarly, Z-IETD-FMK (5 �M) prevented the appearance ofcleaved forms of caspase-9 and caspase-8 (which can cleave itself) inresponse to AGN193198. A cleaved form of caspase-3 was observedin the inhibitor/AGN193198-treated cells, but this has a higher mo-lecular weight than the forms generated in the presence ofAGN193198 alone.

As shown in Fig. 6B, treatment of Jurkat cells for 6 h with 2 �M

AGN193198 led to the production of BID/p15, and this was com-pletely blocked by pretreating cells with the caspase-8 inhibitor.

We examined whether Fas signal transduction, JNK activationleading to c-Jun phosphorylation, and changes to the level of Bcl-2

play roles in AGN193198-provoked apoptosis. In the ATP end pointplate assay, 250 ng/ml anti-Fas antibody ZB4 blocks agonistic anti-Fas-driven apoptosis of Jurkat cells. ZB4 antibody, at concentrations62.5–1,000 ng/ml, did not affect the growth of LNCaP cells in theplate assay nor interfere with the growth inhibitory effects of 250 nM,500, nM, and 1 �M AGN193198 (data not shown). Fig. 6B (middlepanel) shows that the level of phosphorylated JNK was increasedsubstantially in LNCaP cells treated with the known JNK activatoranisomycin (51), and had increased to a much lesser extent at 8 h incells treated with 2 �M AGN193198. Comparable results were ob-tained for the levels of phosphorylated c-Jun. This weak activation ofJNK by AGN193198 is later than the onset of mitochondrial depo-larization (see time course in Fig. 4), and is, therefore, unlikely tomediate AGN193198-driven mitochondrial depolarization. Resultsfrom similar experiments using Jurkat cells confirmed this finding(data not shown). The level of Bcl-2 was not affected by treatment of

Fig. 4. AGN193198-induced apoptosis of LNCaP cells involves depolarization of the mitochondrial membrane potential. A, left, the time course for the effect of 1 �M AGN193198on the survival of LNCaP cells in microtiter wells as determined by measuring levels of cellular ATP. A, middle, shows the time course for DNA fragmentation in 1 �M

AGN193198-treated cells (F) and vehicle-treated cells (E). Right panels illustrate these data. B, left, the dose-response curve obtained for AGN193198 for the induction ofmitochondrial membrane depolarization (measured using the JC-1 probe). B, middle, the time course for this event when cells were treated with 1 �M AGN193198 (F) and vehiclealone (E). Right panels illustrate these data. C, the effects of pre-treatments for 1 h with the pan-caspase inhibitor Z-VAD-FMK (at 50 �M) and of the caspase-8 inhibitor Z-IETD-FMK(at 5 �M) on the capacity of 1 �M AGN193198 to induce depolarization of the mitochondrial membrane potential (left) and apoptosis (strand breaks, right panel) in LNCaP cells.

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LNCaP and Jurkat cells for up to 48 h with 1 �M AGN193198 (Fig.6B, bottom panel).

AGN193198 Is Not an Inhibitor of the NF-�B Signaling Path-way. The RRM MX781 induces caspase-dependent apoptosis bybinding to and inhibiting activity of the inhibitor of nuclear factor Bkinase (IKK)� and IKK� kinases that phosphorylate inhibitor ofnuclear factor B, and this, in turn, leads to reduced activity of thetranscription factor NF-�B (52). Nonphosphorylated inhibitor of nu-clear factor B sequesters NF-�B in the cytoplasm, whereas phospho-rylated inhibitor of nuclear factor B is polyubiquitinated and de-

graded, by the proteosome-dependent pathway, allowing NF-�B totranslocate to the nucleus.

We examined whether AGN193198 interfered with IKK-provokeddegradation of I�B� when IKK activity was stimulated in HeLa,LNCaP, and Jurkat cells by TNF-� (see Fig. 7). As shown in Fig. 7A,treatment of HeLa cells with 10 or 100 ng/ml TNF-� led to completedegradation of I�B� within 10 min, and its concentration returned tothat of control cells by 2 h. Fig. 7B (left panel) shows that treatmentof HeLa cells for 1 h with up to 2 �M AGN193198 before the additionof 10 ng/ml TNF-� for 10 min did not influence TNF-�-stimulated

Fig. 5. AGN193198 activates caspase-3, -8, and -9. A, Jurkat cells substantially growth inhibited by treatment with 1 �M AGN193198 for 5 days. Cell lysates fromAGN193198-treated (4 h) Jurkat cells showed increased activities of caspase-3 and caspase-9, as measured using specific substrates (middle and right panels). ��, P 0.01 versuscontrols (without treatment). B, time courses obtained for increased enzyme activities of caspase-8, -3 and -9 post-treatment of Jurkat cells with 2 �M AGN193198. C, cleavage ofcaspase-3, -8 and -9 in 2 �M AGN193198-treated LNCaP and Jurkat cells, as revealed by immunostaining blots of cell extracts.

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degradation of I�B�. Micromolar amounts of the proteosomal inhib-itor MG132 did block this process (Fig. 7B, right panel). Similarexperiments using LNCaP cells and Jurkat T cells confirmed thatdoses of AGN193198 up to 2 �M had no effect on the activity of IKK(Fig. 7B).

NF-�B is partially active in untreated LNCaP cells (53). We ex-amined levels of NF-�B in nuclear extracts of LNCaP cells that weretreated with TNF-�, with or without a 1 h prior exposure toAGN193198. Moderate and similar levels of binding of NF-�B to theNF-�B-specific oligonucleotide probe were observed (electromobility

Fig. 6. Caspase-8 inhibitor blocks AGN193198-drivenactivation of caspase-3 and caspase-9. A, pretreatment ofJurkat cells with 5 �M of the caspase-8 inhibitor Z-IETD-FMK (C8-I) blocks 2 �M AGN193198-induced activation(left) and cleavage (right) of caspase-3, -8, and 9. Specificsubstrates were used to measure enzyme activities, andcleaved caspases were revealed by immunostaining blots ofcell extracts. Measurements were at 6 h post-treatment withAGN193198. B, top, 2 �M AGN193198-induced productionof BID/p15 at 6 h in Jurkat cells, and this is prevented bypretreatment of cells for 1 h with the caspase-8 inhibitor.B, middle, the effect of 2 �M AGN193198 on the c-JunNH2-terminal kinase (JNK) pathway in LNCaP cells as meas-ured (immunoblotting) by levels of phosphorylated JNK andphosphorylated c-Jun. For a positive control of JNK activa-tion, LNCaP cells were treated with 10 �g/ml anisomycin for30 min. B, bottom, 2 �M AGN193198 did not affect the levelof Bcl-2 protein in LNCaP cells, as revealed by immuno-blotting.

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shift assay) for extracts from untreated LNCaP cells and from cellspretreated for 1 h with 1 �M AGN193198. Stimulation of the level ofNF-�B in nuclear extracts by 7.5 ng/ml TNF-� was also not affectedby pretreatment of cells for 1 h with AGN193198 (data not shown).

DISCUSSION

If RRMs are to be used as therapeutic agents for multiple cancers,an important question is how RRMs work and, particularly, whetherRARs play any role in their induction of apoptosis. AGN193198potently induces apoptosis in many types of malignant cells, and itseems very unlikely that the mechanism(s) of induction involvesRARs or RXRs. AGN193198 elicited maximum effects at 1 �M in theseveral assays used to measure various activities, and there weresubstantial effects at lower doses. One �M AGN193198 shows littlebinding to RARs or RXRs and does not effectively transactivate inRAR- and RXR-mediated reporter assays. AGN193198 is also not aneffective antagonist of RARs and RXRs. By contrast, effective RARagonists and antagonists generally bind with a Ki of 2–5 nM andagonise and antagonize at nM amounts (14).

AGN193198 (at 1 �M) killed all 13 of the primary cultures estab-lished from prostatic carcinoma. Moreover, AGN193198, like otherRRMs such as fenretinide and CD437, induces apoptosis in diversecancer-derived cell lines. Of particular interest is that CD437 inducesapoptosis in lung cancer cell lines and malignant human epidermalkeratinocytes whereas sparing normal cells (6, 54, 55), although itdoes arrest the growth of normal lung epithelial cells (6). Thesedifferential effects led Sun et al. to advocate the application of CD437for cancer treatment (6).

Induction of apoptosis by AGN193198 involves mitochondrial depo-larization leading to DNA fragmentation. Apoptosis was induced quickly

in AGN193198-treated cells: e.g., mitochondria started to depolarizewithin �6 h, and most cells showed mitochondrial depolarization by12 h. In parallel, caspase-3, -8, -9, and -10 were activated. Activatedcaspase-8 might be needed for activation of capsase-3 and -9, and indeed,blocking caspase-8 activity in AGN193198-treated Jurkat cells preventedactivation of caspase-3 and -9. BID/p15 was produced around the sameperiod as caspases were activated, and the caspase-8 inhibitor alsoblocked the induced appearance of BID/p15. These results led us toconsider a sequential pathway involving caspase-8 activation, BID/p15production, mitochondrial depolarization, cytochrome c release, activa-tion of caspase-3 and -9, and finally DNA fragmentation, as described forsurface death receptors (56, 57). However, the antagonistic Fas antibodydid not block the effect of AGN193198. Also, mitochondrial depolariza-tion in AGN193198-treated LNCaP and Jurkat cells was unaffected bythe caspase-8 inhibitor, and DNA fragmentation occurred even when thecaspase-8 inhibitor was used to block caspase-8-mediated cleavage ofcaspase-3 and -9. By contrast, the pan-caspase inhibitor did preventAGN193198-induced mitochondrial depolarization and DNA strand-breaks, confirming the involvement of at least some caspase-relatedprotease in AGN193198-stimulated mitochondrial depolarization andDNA fragmentation. Caspase-10 is activated by AGN193198, althoughnot driven via Fas signaling. Whether AGN193198 generally activatescaspases or there is a particular activation sequence, the importance ofindividual caspases remains to be determined.

It is widely accepted that mitochondrial depolarization and activa-tion of caspase-3 and -9 are involved in apoptosis induction by CD437or fenretinide (58–62). Moreover, inhibiting mitochondrial depolar-ization with bongkriekic acid blocks activation of caspase-3 and -9and apoptosis (25). The timing of involvement of other caspases inRRM-induced apoptosis may be cell type and/or agent specific. Fen-

Fig. 7. AGN193198 is not an effective inhibitor of tumor necrosis factor (TNF)-�-driven degradation of I�B�. A, the time dependence of TNF-� (at two different doses, 10 ng/mland 100 ng/ml) mediated degradation of inhibitor of nuclear factor B� (I�B�) in HeLa cells. B, treatment of HeLa, LNCaP, and Jurkat cells for 1 h with up to 2 �M AGN193198 beforeaddition of TNF-� for 10 min did not affect TNF-�-driven I�B� degradation (three left panels). In each cell line, the proteosomal inhibitor MG132 blocked TNF-�-driven I�B�degradation (three right panels). I�B� degradation was revealed by immunostaining of blots of cell extracts.

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retinide-stimulated apoptosis in Fas-defective hepatoma cells involvesactivation of caspase-8 that precedes release of cytochrome c frommitochondria and slight caspase-9 activation (42). By contrast, mito-chondrial depolarization is upstream of the activation of caspase-3, -8,and -9 during fenretinide-induced apoptosis in squamous cells (63).However, other workers have suggested that caspase-8 activity is notnecessary for either CD437 or fenretinide induction of apoptosis (25).Other caspases are also likely to be important to RRM-inducedapoptosis as, for example, caspase-6 plays a role in CD437-inducedapoptosis in lung cancer cells (6).

What is the immediate molecular target for the action ofAGN193198? Mitochondrial depolarization and activation of caspasesare well underway within 4–6 h, but we have not yet identified the earlierevents. Bayon et al. (52) have offered evidence that MX781 inducesapoptosis by inhibiting IKK and have argued that RRM structures serveas a lead for the development of IKK inhibitors. However, concentrationsof AGN193198 that trigger apoptosis do not inhibit IKK, so interferencewith IKK activity is very unlikely to play any role in AGN193198-induced apoptosis. Another recently identified putative target for RRMsis the orphan nuclear receptor TR3. CD437, but not fenretinide, inducestranslocation of TR3 to the cytosol and to mitochondria (25). WhetherCD437, fenretinide, MX781, and AGN193198 initially use differentpathways to induce mitochondrial depolarization or share a common, butunidentified target, remains a question for the future.

In summary, we have described a novel RRM that is potent ininducing apoptosis in a variety of malignant cell types, apparentlywithout any involvement of retinoid nuclear receptors. BecauseRRMs, such as AGN193198, show promise as therapeutic agents incancer, identification of their molecular targets is an important aim forfuture studies.

ACKNOWLEDGMENTS

We thank Phil Hughes for help in the preparation of this manuscript andBob Michell for valuable comments.

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2004;64:3302-3312. Cancer Res Richard G. Keedwell, Yi Zhao, Lisette A. Hammond, et al. Prostate Cancer Cells through Rapid Caspase ActivationRetinoid Receptors Potently Induces Apoptosis in Human A Retinoid-Related Molecule that Does Not Bind to Classical

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