RWJ-241947 (MCC-555), A Unique Peroxisome Proliferator- … · RWJ-241947 (MCC-555), A Unique...

RWJ-241947 (MCC-555), A Unique Peroxisome Proliferator-Activated Receptor-� Ligand with Antitumor Activity againstHuman Prostate Cancer in Vitro and in Beige/Nude/X-Linked Immunodeficient Mice and Enhancementof Apoptosis in Myeloma Cells Induced byArsenic Trioxide

Takashi Kumagai,1 Takayuki Ikezoe,1

Dorina Gui,2 James O’Kelly,1 Xiang-Jun Tong,1

Fredric J. Cohen,3 Jonathan W. Said,2 andH. Phillip Koeffler1

Division of Hematology/Oncology, Departments of 1Medicine and2Pathology, Center of Health of Science, University of California atLos Angeles School of Medicine, Los Angeles, California, and3Global Clinical Research, Johnson and Johnson PharmaceuticalResearch and Development, La Jolla, California

ABSTRACTPurpose: RWJ-241947 (MCC-555) is a novel peroxi-

some proliferator-activated receptor-� ligand of the thiazo-lidinedione class that was recently developed as an antidia-betic drug with unique properties. Some thiazolidinedioneshave anticancer activity against solid and hematological ma-lignancies; the anticancer potency of RWJ-241947 has notbeen examined. We, therefore, investigated these effects invitro and in vivo either alone or in combination with othercompounds.

Experimental Design: Tumor growth was examinedby 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide assay, soft agar colony assay in vitro, and xenografts innude mice. Its effects on cell cycle, differentiation, and apo-ptosis were examined.

Results: In vitro studies using various solid and hema-tological tumor cell lines showed that RWJ-241947 hadantiproliferative activity against prostate cancer cells, withthe strongest effect against the androgen-independent PC-3prostate cancer cells. It increased expression of cyclin-de-

pendent kinase inhibitor p21WAF1, deceased cyclin E, andinduced apoptosis in PC-3 cells. It increased E-cadherin andlowered protein expression of prostate-specific antigen with-out down-regulating the androgen receptor in androgen-dependent LNCaP prostate cancer cells. Reporter gene as-says showed that this peroxisome proliferator-activatedreceptor-� ligand inhibited androgen activation of the an-drogen receptor response elements of the prostate-specificantigen gene. Remarkably, in vivo treatment of malebeige/nude/X-linked immunodeficient (BNX) mice withRWJ-241947 profoundly suppressed growth of PC-3 pros-tate cancer xenografts with prominent apoptosis, as well asfibrosis, including inflammatory and giant cell reaction inthe remaining tumor tissue. Notably, the experimented micehad a significantly decreased cholesterol. In addition, westudied the combination of arsenic trioxide (As2O3), whichis used in the treatment of multiple myeloma, and RWJ-241947; these two reagents together prominently inhibitedproliferation and caused apoptosis of multiple myelomacells.

Conclusions: RWJ-241947 has surprisingly potent an-tiproliferative effects against prostate cancer cells in vivo,and it enhances the antitumor activity of As2O3 againstmyeloma cells. Small, well-defined clinical studies usingRWJ-241947 are in order for these cancers.

INTRODUCTIONThe peroxisome proliferator-activated receptor � (PPAR �)

belongs to the nuclear hormone receptor superfamily and has animportant role in adipocyte differentiation. Thiazolidinediones(TZDs) are a class of synthetic PPAR� ligands that induceadipocyte differentiation (1, 2), regulate genes involved in lipo-genesis, and enhance glucose utilization (3, 4). Normal preadi-pocytes can be induced to differentiate in the presence of ligandsfor PPAR�. With this knowledge in hand, investigators haveused TZDs to induce differentiation of human liposarcoma cellsin vitro (5) and treat patients with these tumors (6). Troglita-zone, one of the TZDs, retarded growth and induced differen-tiation of these tumor cells. This has prompted investigations ofTZDs in breast and prostate cancers. Both of these tumors haveprominent expression of PPAR� (7, 8). Studies have shown thatTZDs (10�6 to 10�5

M) can moderately inhibit proliferation andinduce differentiation-like alterations in breast cancer cell lines,both in vitro and in xenografts growing in nude mice (8).Additional studies showed that the combination of a retinoid

Received 3/25/03; revised 11/24/03; accepted 11/25/03.Grant support: NIH grants as well as grants from Parker Hughes Fundand Johnson and Johnson. H. P. K. holds the Mark Goodson endowedChair in Oncology Research and is a member of the Jonsson CancerCenter and Molecular Biology Institute, University of California at LosAngeles.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.Requests for reprints: Takashi Kumagai, Cedars-Sinai Medical Center,Davis Building 5068, 8700 Beverly Boulevard, Los Angeles, CA 90048.Phone: (310) 423-7736; Fax: (310) 423-0225; E-mail: [email protected].

1508 Vol. 10, 1508–1520, February 15, 2004 Clinical Cancer Research

Cancer Research. on October 21, 2020. © 2004 American Association forclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

with a PPAR� ligand synergistically decreased proliferation andinduced apoptosis of several breast cancer cell lines both in vitroand in xenografts growing in nude mice (8). The PPAR� ligandGW7845 was also able to inhibit the development of carcino-gen-induced breast cancer in rats (9). Similarly, troglitazoneprevented the carcinogen-induced transformation of breast tis-sue (10).

Likewise, investigations have found that proliferation ofseveral human prostate cancer cell lines could be inhibited invitro and in vivo and undergo profound morphological changeswhen cultured in the presence of a ligand of PPAR� (7, 11).Clinical study showed that troglitazone was able to retard theprogression of prostate cancer in patients (7, 11).

Troglitazone was voluntarily withdrawn from the market inMarch 2000, because it caused severe idiosyncratic liver injury.Rosiglitazone and pioglitazone have been available since 1999.They also inhibit the growth of cancer cell lines, includingprostate, thyroid, lung, pancreatic, renal cell, hepatocellular, andgastric cancers in vitro (12–19) and in animal models of cancer(15).

RWJ-241947 is a TZD established as an antidiabetic drugin animal models of Type 2 diabetes. Like other TZDs, RWJ-241947 binds to PPAR� and effects its transcriptional activities,but its binding affinity for PPAR� is �0.1 of rosiglitazone. Itstranscriptional properties are unique because it can function as afull or partial agonist or antagonist, depending on the cell typeor DNA-binding site. Its hypoglycemic potency may depend onthe context selectivity of RWJ-241947 (20). RWJ-241947 iscurrently being tested as a treatment for Type 2 diabetes inhumans. We tested for the first time the anticancer activity ofthis new TZD.

Arsenic-containing remedies have long been part of tradi-tional Chinese medicine for a variety of ailments (21). Chronicmyelogenous leukemia was treated in the 1940s and 1950s withFowler’s solution that contained 1% potassium arsenite (22).Administration of arsenic trioxide (As2O3) produced a highcomplete remission rate as well as long-term survival in asignificantly high proportion of individuals with either relapsedor refractory acute promyelocytic leukemia (APL) without se-vere marrow suppression (23–25). Very recently, As2O3 hasbeen shown to have an antitumor effect on human multiplemyeloma cells by induction of apoptosis (26–28). We haveshown previously that the combination of all-trans-retinoic acidand an organic arsenic compound had a profound synergisticgrowth inhibitory effect against prostate and breast cancer celllines (29). This inhibition occurred both in vitro and in vivo andwas associated with profound apoptosis and marked decrease inlevels of BclII. To our knowledge, no one has investigated theanticancer activity of a combination of a PPAR� ligand and anarsenic-containing compound.

Although this unique TZD has been characterized and isexpected to be an antidiabetic drug to improve impaired glucosetolerance (20, 30), its anticancer properties have not been ex-amined. Because PPAR� is widely expressed in many types ofcancer, including prostate, breast, colon, leukemia, and my-eloma, and the receptor is rarely altered in human malignancies(31), we tested RWJ-241947 against a wide range of humancancer cells and found that it inhibited the growth of prostatecancer cells both in vitro and in vivo and synergistically inter-

acted with As2O3 to inhibit the growth of multiple myelomacells.

MATERIALS AND METHODSCells and Samples. All cell lines were obtained from

American Type Culture Collection (Rockville, MD) and main-tained according to their recommendations. DU145 and MCF-7were maintained in DMEM with 10% FCS. LNCaP, PC-3,HTB-182, HL-60, NB-4, U937, RPMI8226, U266, ARH-77,and NCI-H929 were grown in RPMI 1640 with 10% FCS. Toexamine the effect of DHT on LNCaP, the cells were incubatedin RPMI 1640 containing 10% charcoal dextran-treated fetalbovine serum for 24 h before the addition of 10�9

M dihydrotes-tosterone (DHT) either with or without 10�5

M RWJ-241947.RWJ-241947 (MCC-555; Johnson and Johnson PharmaceuticalResearch and Development), rosiglitazone (SmithKlineBeecham Pharmaceuticals, West Sussex, United Kingdom), pio-glitazone (Takeda Chemical Industries, Tokyo, Japan), and15dPGJ2 (Calbiochem, La Jolla, CA) were dissolved in a solu-tion containing 50% DMSO and 50% ethanol.

MTT Assays. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide (MTT; Sigma) was placed in solutionwith PBS (5 mg/ml) and used to measure cellular proliferation.Cells (103) were incubated in culture medium for 96 h in96-well plates, and 10 �l of MTT solution were added. After 4-hincubation, 50 �l of solubilization solution (20% SDS) wereadded and incubated at 37°C for 16 h. In this assay, MTT iscleaved to an orange formazan dye by metabolically active cells.The dye was directly quantified using an enzyme-linked immu-noabsorbent assay reader at 540 nm.

Soft Agar Colony Assay. Trypsinized and washed sin-gle-cell suspensions of cells were enumerated and plated into24-well, flat-bottomed plates using a two-layer soft agar systemwith a total of 1 � 103 cells/well in a volume of 400 �l/well, asdescribed previously (11).

Cell Cycle Analysis. PC-3 cells (5 � 104) were exposedto 10�5

M RWJ-241947 in six-well, flat-bottomed plates for 3days. Total cells, both in suspension and adherent, were col-lected, washed, and suspended in cold PBS. Cells were fixedin chilled 75% methanol and stained with propidium iodine.Cell cycle status was analyzed on a Becton Dickinson FlowCytometer.

Western Blot Analysis. Cells were washed twice inPBS, suspended in lysis buffer [50 mM Tris (pH 8.0), 150 mM

NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40, phen-ylmethylsulfonyl fluoride at 100 �g/ml, aprotinin at 2 �g/ml,pepstatin at 1 �g/ml, and leupetin at 10 �g/ml], and placed onice for 30 min. After centrifugation at 15,000 � g for 15 min at4°C, the suspension was collected. Protein concentrations werequantitated by using the Bio-Rad protein Assay Dye ReagentConcentrate (Bio-Rad Laboratories, Hercules, CA) according tothe manufacturer’s recommendation. Whole cell lysates (40 �g)were resolved by SDS-PAGE in a 4–15% gel, transferred to apolyvinylidene difuride membrane (Immobilon; AmershamCorp., Arlington Heights, IL), and probed sequentially withantibodies against the following proteins: (a) prostate-specificantigen (PSA); (b) androgen receptor (AR); (c) E-cadherin; (d)cyclin E; (e) p21WAF1; ( f ) p27Kip1; (g) Bcl-2; (h) Bcl-XL; (i)

1509Clinical Cancer Research



BAX; ( j) PARP; and (k) GAPDH (Santa Cruz Biotechnology;Inc., Santa Cruz, CA). The blots were developed using theSupersignal West Pico Chemiluminescent Substrate Kit (Pierce,Rockford, IL).

Murine Studies. Beige/nude/X-linked immunodeficient(BNX) nude mice at 8 weeks of age were purchased fromHarlan Sprague Dawley, Inc. (Indianapolis, IN) and maintainedin pathogen-free conditions with irradiated chow. A total of 5 �106 PC-3 cells in 0.1 ml of Matrigel (Collaborative BiologicalProducts, Bedford, MA) was injected s.c. into bilateral flanks ofeach mouse, resulting in the formation of two tumors per mouse.The mice were blindly randomized to the experimental andcontrol groups. Treatment was started on the day after theinjection of PC-3 cells and continued for 6 weeks. The control(eight mice) received dilutant only, and the experimental (sevenmice) received RWJ-241947 (30 mg/kg, p.o. by gavage, 5 daysa week). RWJ-241947 was suspended in 1.5% carboxymethyl-cellulose with 0.2% Tween 20 (Sigma). Tumor sizes weremeasured every week and calculated by the formula: A(length) � B (width) � C (height) � 0.5236. After 6 weeks,blood was collected for serum chemistry and circulating bloodcounts. One mouse (control) died during gavage. All mice wereeuthanized at the end of the study, and tumors, prostates, livers,lungs, and bone marrows were fixed in 10% neutral-bufferedformalin and embedded in paraffin for histological analysis. Thedata were analyzed by Student’s t test.

Measurement of Apoptosis. Terminal deoxynucleotidyltransferase-mediated nick end labeling assay was performed forimmunohistochemical detection and quantification of pro-grammed cell death at the single cell level, based on labeling ofDNA strand breaks using the In Situ Cell Death Detection, POD(Roche, Indianapolis, IN). Early apoptosis was also detected bymeasuring Annexin V protein in the cell membrane using An-nexin V-FITC Kit (Clontech Laboratories, Inc., Palo Alto, CA)followed by flow cytometric analysis.

Measurement of Cell Surface CD36 Antigen on Mono-cytic Cell Lines. Monocytic leukemia cell lines (U937 andTHP-1) were treated with RWJ-241947 or troglitazone (10�6 or10�5

M) for 4 days and examined for CD36 expression by flowcytometry using CD36 antibody (Immunotech, Inc.), as de-scribed previously (32).

Plasmids. One PSA promoter that we examined has the2.4-kb enhancer sequences (�5322 to �2925) fused to the first564-bp promoter sequences, which is attached to the luciferasegene. It contains all known androgen receptor elements (AREs)in the region of the PSA human gene (33, 34). The secondconstruct has the first 496-bp nucleotides of the PSA enhancer(wild type) with six AREs cloned upstream of the luciferasegene in the pGL3 vector (Promega, Chicago, IL; PSA enhancerE4-LUC) and the PSA enhancer (S-All)-E4LUC, which encom-passes the same sequences, but the four major AREs in it havebeen mutated (generous gift of Michael Carey, University ofCalifornia at Los Angeles; Ref. 35).

Transfections and Luciferase Assay. LNCaP cells wereincubated in RPMI 1640 with 10% fetal bovine serum until50–70% confluency. Cells were transfected with the indicatedplasmids using the Superfect (Qiagen, Santa Clarita, CA) underserum-free conditions. A PSV-�-galactosidase vector was in-cluded as an internal control for efficiency of transfections.

After the transfections, cells were incubated with 10% charcoal-stripped fetal bovine serum RPMI 1640 either with or without10�9

M DHT and either with or without RWJ-241947 for 48 h.Cells were collected with tissue lysis buffer (Promega). Lucif-erase activity of the cell lysates was measured by luminometry,and activities were normalized by �-galactosidase activities. Alltransfection experiments were carried out in triplicate wells andrepeated separately at least three times.

RESULTSEffect of RWJ-241947 on the Proliferation and Viabil-

ity of Various Tumor Cell Lines in Vitro. We examined theantitumor effect of RWJ-241947 on various cancer cell lines,including prostate, breast, lung, and hematopoetic tissues invitro. The first screening was done using the rapid MTT assaywith a relative short exposure of 4 days to the RWJ-241947 (Fig.1A). Prostate (LNCaP, PC-3, and DU145) and breast (MCF-7)cancer cells showed some sensitivity to RWJ-241947. Lungcancer cell line (HTB-182), myeloid leukemic cell lines (U937,HL-60, NB-4, and THP-1), and myeloma cell lines (RPMI1882,ARH-77, NCI-H929, and U266) were insensitive toRWJ-241947 in vitro by this assay (Fig. 1A). We then tested theantiproliferative activity of RWJ-241947 against the prostateand breast cancer cell lines using the extremely sensitive softagar colony assay. We compared RWJ-241947 with the knownactive TZD, troglitazone. Both showed similar potency againstall four cancer cell lines (Fig. 1B). The PC-3 human prostatecancer cells were most sensitive, with 1.5 � 10�5

M as theestimated dose of RWJ-241947 that caused a 50% inhibition(ED50) of clonal growth. This is a concentration that is achiev-able in patients.

We compared the antiproliferative activities ofRWJ-241947 versus other PPAR ligands (rosigilitazone, cigli-tazone, and PGJ2) against PC-3, LNCaP, and MCF-7 cancercells. These cells were incubated with 10�5

M these compoundsfor 14 days, and colony assays showed that each had comparableanticlonal proliferative activities (Fig. 1C).

Effect of RWJ-241947 on Prostate Cancer Cells inVitro. The LNCaP prostate cancer cells have a functional AR.Treatment of these cells with DHT (10�9

M, 24 h) enhancedtheir expression of PSA. This increase level was suppressed by�50% when the cells were simultaneously treated with DHTand RWJ-241947 (10�5

M; Fig. 2A). The promoter of the PSAgene contains a number of androgen response elements, and itsexpression is dependent on AR (36). Evaluation of expression ofthe AR by Western blot analysis showed that RWJ-241947suppressed PSA expression without suppressing AR levels inthe LNCaP cells (Fig. 2A).

To examine further the mechanisms by whichRWJ-241947 inhibited levels of PSA, we analyzed the effect ofRWJ-241947 on the ability of DHT to transactivate the PSApromoter/enhancer. The LNCaP prostate cancer cells were cul-tured with DHT (10�9

M) after they were transfected with aluciferase reporter construct that contained a 496-bp fragment ofthe PSA enhancer, which contains six AREs. Dihydrotestoster-one (10�7

M) increased the reporter activity �5-fold as com-pared with nontreated control LNCaP cells. When cells weretreated with both RWJ-241947 (10�5

M) and DHT (10�9M),

1510 Anticancer Effect of RWJ-241947 against Prostate Cancer



Fig. 1 Effect of RWJ-241947 on growth of various tumor cell lines in vitro. In A, a variety of tumor cell lines, including prostate (LNCaP, PC-3,and DU145), breast (MCF-7), lung (HTB-182), acute myeloid leukemia (U937, HL-60, NB-4, and THP-1), and multiple myeloma cells (ARH-77,RPMI8226, U266, and NIH-H929), was treated with either RWJ-241947 at various concentrations (10�8 � 10�5 M) or the diluant (control) for 96 h,and growth (% of control) was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results represent the mean � SDof three independent experiments with triplicate dishes. B, dose-response clonogenic assays of prostate (LNCaP, PC-3, and DU145) and breast(MCF-7) cancer cell lines treated with either RWJ-241947 or troglitazone. Results represent the mean � SD of three independent experiments withtriplicate dishes. C, clonogenic assay of prostate (PC-3 and LNCaP) and breast (MCF-7) cancer cell lines treated with 10�5 M either RWJ-241947,Pioglitazone, Rosiglitazone, or PGJ2. Results represent the mean � SD of three independent experiments with triplicate dishes.




luciferase activity was reduced by 40% compared with DHTalone (Fig. 2B). This indicated that RWJ-241947 inhibited theability of androgens to transactivate the PSA promoter/en-hancer. Mutation of the four most active AREs and repeating theexperiments showed that DHT did not activate andRWJ-241947 did not inhibit luciferase activity (Fig. 2B). Theseexperiments were repeated using a PSA promoter/enhancer thatincluded several additional AREs that were in an upstreamenhancer region (�5322 to �2925 bp), which was attached tothe first 564-bp fragments of the PSA promoter upstream of thestart of transcription. The results were similar (data not shown).

The cell surface-binding protein, E-cadherin, has beenidentified as a differentiation marker of prostate cancer cells(37). RWJ-241947 increased by 220% the expression of E-cadherin in LNCaP cells (Fig. 2C). PPAR� ligands were re-ported to change the expression of cell cycle-related genes,including several cyclin genes (38). We found that RWJ-241947

slightly decreased the level (62%) of cyclin E in LNCaP cells(Fig. 2C).

Additional studies used the RWJ-241947-sensitive PC-3cells. The cell cycle pattern of PC-3 cells after a 96-h exposureto 10�5

M RWJ-241947 was almost identical to control PC-3cells (control: G0-G1 50%, S 32%, G2-M 17%; RWJ-241947-exposed PC-3 cells: G0-G1 51%, S 30% G2-M 18%, data notshown). The expression of the cyclin-dependent kinase inhibitorp21WAF1 was not detectable in PC-3 cells, but low expressionlevels of the protein were observed on the 2nd and 3rd day ofexposure to RWJ-241947 (10�5

M). The expression of p27KIP1

was unchanged after culture with RWJ-241947 (Fig. 3A), andexpression of cyclin E decreased by 46% in PC-3 cells (Fig. 3B).

We performed Annexin V measurements to detect earlyapoptosis of PC-3 cells exposed to RWJ-241947 (10�5

M, 96 h;Fig. 4). A portion (9%) of PC-3 cells (Annexin V/propidiumiodine�) underwent early apoptosis, and 13% were no longer

Fig. 2 The effect of RWJ-241947on expression of prostate-specificantigen (PSA) and androgen recep-tor (AR) in androgen-dependentLNCaP cells. In A, LNCaP cellswere cultured in 10% charcoal-stripped fetal bovine serum for24 h before the addition of 10�9 M

dihydrotestosterone (DHT) eitherwith or without 10�5 M RWJ-241947. After 24 h of treat-ment, cell lysates were harvested,Western blotted, and probed forprotein expression of PSA andAR. The amount of protein wasnormalized to levels of glyceral-dehyde-3-phosphate dehydrogen-ase (GAPDH). B, effect of RWJ-241947 on transactivation ofeither the wild-type or mutantPSA enhancer. The reporter lucif-erase (LUC) constructs [PSA en-hancer (496 bp) and this PSA en-hancer with the major androgenreceptor elements (ARE) mutated]are shown at the top. Wild-typeand mutant ARE are representedby boxes and crosses, respectively.LNCaP cells were transfected withthe reporter construct, and DHT(10�9 M) was added either withor without 10�5 M RWJ-241947.PSV-�-galactosidase vector wascotransfected and measured fornormalization. Means � SD ofthree experiments are shown. InC, LNCaP cells were cultured in10% fetal bovine serum RPMI ei-ther with or without 10�5 M RWJ-241947. After 72 h of treatment,cell lysates were made, Westernblotted, and probed with antibod-ies for cyclin E and E-cadherin.The amount of protein was nor-malized by GAPDH.




viable (Annexin V/propidium iodine) after exposure to10�5

M RWJ-241947 for 4 days. In addition, at 10�6M RWJ-

241947 for 4 days, 5% underwent early apoptosis, and 4% wereno longer viable. Control cultures had 2% of the cells showingearly apoptosis, and 3% were dead. We also examined expres-sion of genes associated with apoptosis (Bcl-2, BclXL, and Bax)by Western blot analysis (Fig. 3B). Their levels of expressiondid not change after exposure of PC-3 cells to RWJ-241947(10�5

M, 72 h).Morphological change after exposure to RWJ-241947

(10�5M, 96 h) was observed by electron microscopy, but the

changes were minor (data not shown). Protein expression of

E-cadherin, a marker of epithelial differentiation, was notclearly altered by RWJ-241947 in PC-3 cells (data not shown).

Antitumor Effect of RWJ-241947 against PC-3 ProstateCancer Cells in Vivo. We evaluated the effect ofRWJ-241947 in vivo on PC-3 human prostate tumor cells grow-ing in nude mice. The PPAR� ligand was given by gavage.Tumor volumes were measured weekly. All mice were eutha-nized after 6 weeks, and tumors were dissected and weighed.RWJ-241947 significantly suppressed both the growth of pros-tate tumors (P 0.0035; Fig. 5A) and their mean weights (P 0.0027) at autopsy (Fig. 5B), as compared with diluant controls.

Histological analysis of PC-3 tumors from untreated micerevealed poorly differentiated infiltrating adenocarcinoma (Fig.6A). Tumors from mice receiving RWJ-241947 had markedfibrosis, including an inflammatory and giant cell reaction (Fig.6B). Some tumor cells treated with RWJ-241947 showed py-knotic nuclei and cytoplasmic shrinkage with eosinophilia, in-dicating that they were undergoing apoptosis (Fig. 6C). Noincrease in macrophages were noted in the tumors of the exper-imental compared with the control mice.

The weights, hematopoetic values, and blood chemistrieswere not different between the two groups except, interestingly,a significant decrease in the serum cholesterol occurred in theexperimental group (Table 1). No elevation of transaminases(glutamic oxalacetic transminase and glutamic pyruvic transmi-nase) was seen in the experimental group (Table 1). A variety oforgans (lung, liver, prostate, and bone marrow) was histologi-cally examined in the experimental and control mice. All of thetissues, including the prostates, looked normal (data not shown).We also performed immunohistochemistry of the experimentaland control tumors, including Ki-67, a marker of cellular pro-liferation in vivo. A total of 32 and 20% of cells was Ki-67positive in the control and experimental tumors, respectively.The expression of p21WAF1 and Bcl-2 was unchanged in bothcontrol and experimental tumors (data not shown).

Effect of RWJ-241947 on Expression of Cell SurfaceCD36 Antigen on Human Monocytic Cell Lines. Becausethe serum cholesterol level was markedly decreased in the nudemice receiving RWJ-241947 (Table 1), we examined the effectof RWJ-241947 on the expression of CD36 on the humanmonocytic cell lines, U937 and THP-1. CD36 is also known asthe scavenger receptor, which is responsible for uptake of theoxidized low-density lipoprotein in macrophages (39–41). Lev-els of CD36 expression increased on both cell lines in a dose-dependent manner, with levels increasing by 21-fold on THP-1and 8-fold on U-937 compared with control cells, after theywere cultured with RWJ-241947 (10�5

M) for 4 days. Comparedwith RWJ-241947, troglitazone was a more potent inducer ofCD36 expression (Fig. 7).

RWJ-241947 Enhanced Apoptosis Induced by ArsenicTrioxide in Myeloma Cells in Vitro. Arsenic trioxide is oneof the treatments for multiple myeloma (42). In the course oflooking for agents that enhanced antiproliferative activitiesof RWJ-241947, we examined the combined effect of RWJ-241947 and As2O3 on myeloma cell lines in vitro. Arsenictrioxide suppressed the cell growth of myeloma cells in adose-dependent manner (data not shown). We chose a dose ofAs2O3 (10�6

M for ARH-77 and 2 � 10�7M for RPMI8226,

U266, and NCI-H-929, for 4 days) or RWJ-241947 (10�5M, 4

Fig. 3 Western blot analysis of p21Waf1, p27Kip1, cyclin E, Bcl-2,Bcl-XL, and Bax in PC-3 cells cultured with RWJ-241947. In A, thePC-3 cells were treated with RWJ-241947 (10�5 M), and cell lysateswere harvested after 1, 2, and 3 days for Western blot, which was probedsequentially for expression of p21WAF1, p27KIP1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Control cells were treated withvehicle alone. The amount of protein was normalized by GAPDH. In B,the PC-3 cells were treated with RWJ-241947 (10�5 M), and cell lysateswere harvested after 3 days for Western blot, which was probed sequen-tially for levels of cyclin E, Bcl-2, Bcl-XL, Bax, and GAPDH. Controlcells were treated with vehicle alone. The amount of protein wasnormalized by GAPDH.




days) that had little or no effect alone on growth of the myelomacells. However, the combination of RWJ-241947 and As2O3

significantly suppressed the proliferation of the myeloma cellscompared with either RWJ-241947 or As2O3 alone, e.g., thegrowth of ARH-77 myeloma cell line was not suppressed byRWJ-241947 and only inhibited 15% by As2O3. Their combi-nation suppressed growth by �45% (Fig. 8A).

Because As2O3 induces apoptotic cell death of myelomacells in vitro, we examined the effect of RWJ-241947 plusAs2O3 on the induction of apoptosis. Flow cytometric analysisshowed that As2O3 (10�6

M, 4 days) alone caused apoptosis of18% myeloma cells, and RWJ-241947 induced 3% apoptosis.But the apoptotic (subG1) cell population markedly increased(49% cells) when RWJ-241947 was added to As2O3 (Fig. 8B).The quantitative analysis of apoptosis by the combination ofthese two agents in ARH-77, RPMI8226, U266, and NCI-H929cell lines was also analyzed by terminal deoxynucleotidyl trans-ferase-mediated nick end labeling assay (Fig. 8C). The combi-nation of both agents markedly increased apoptosis comparedwith either alone. Enhancement of apoptosis was further con-firmed by the detection of PARP, which is a target of caspases.The cleaved form of PARP was detected only in the cells treatedwith both RWJ-241947 and arsenic trioxide for 2 days (Fig.8D). The expression of several apoptosis-related genes, includ-ing Bcl-2 and BclXL, did not change after culture with RWJ-241947 (10�5

M) and arsenic trioxide (10�6M) for 4 days in

ARH cells (data not shown).We also examined the effect of the combination of RWJ-

241947 and arsenic trioxide on prostate and breast cancer cells.LNCaP, PC-3, DU145, and MCF-7 cells were treated withRWJ-241947 (10�5

M), arsenic trioxide (10�6M), or both for 4

days, and their cell growth was measured by MTT assay. Thiscombination had a slightly enhanced antiproliferative effect onPC-3 cells and, to a lesser extent, on MCF-7 cells comparedwith either agent alone. (Fig. 8E).

The PPAR� heterodimerizes with retinoid X receptor-�.

Both receptors of this heterodimer can simultaneously combinewith their respective ligands; and in certain circumstances, thiscan result in enhanced biological activity (8, 43–46). All-transretinoic acid is the treatment of choice for APL (47). We alsoexamined whether combining RWJ-241947 with either all-transretinoic acid or 9-cic-retinoic acid in vitro could have either anadditive or synergistic effects. The inhibition of growth ofLNCaP, PC-3, MCF-7, HTB-182, NB-4 (APL cells), THP-1,NCI-H929, U266, and RPMI8226 cell lines was not enhancedby the combination of the TZD and a retinoid (data not shown).

DISCUSSIONOur studies found that RWJ-241947 was able to inhibit the

clonal proliferation of three prostate cancer cell lines in vitroand profoundly inhibited the in vivo proliferation of the humanprostate cancer cell line, PC3, in nude mice. Prostate cancercells express prominent levels of PPAR�, and this receptor doesnot appear to be mutated in prostate cancer (7, 11, 31). Inaddition, we have shown previously that the androgen-indepen-dent prostate cancer cell line PC3 could be inhibited in itsgrowth in nude mice by treatment with troglitazone. Our in vitrostudies showed that RWJ-241947 increased the protein expres-sion of the cyclin-dependent kinase inhibitor p21Waf1, decreasedlevels of cyclin E, and induced mild apoptosis of the PC-3 cells.Previous studies found that the expression of both p21Waf1 andp27Kip1 increased in cancer cells exposed to PPAR� ligands;this may have been mediated in part by inhibition of the ubiq-uitin–proteosome protein degradation pathway (48, 49). Theincrease in p21WAF1 and decrease of cyclin E could be associ-ated with a decreased expression of phospholylated-retinoblas-toma, but that was not the case in our experiments (data notshown). The blunting of expression of a variety of inflammatorycytokines and transcription factors, such as tumor necrosis fac-tor, interleukin-1, interleukin-4, and nuclear factor-��, has beenreported in the transformed cells exposed to PPAR� ligand. This

Fig. 4 Induction of apoptosis of PC-3 prostate cancer cells cultured with RWJ-241947. PC-3 cells were cultured either with or without RWJ-241947(10�6 or 10�5 M) for 4 days followed by measurement of Annexin V protein in the cell membrane by flow cytometry using FITC-conjugated AnnexinV antibody. The cells were also stained with propidium iodine (PI). Control panel showed diluant-treated PC-3 cells. The bottom right quadrant ofeach panel displays V-FITC-positive and PI-negative cells, indicating that the cells were in an early stage of apoptosis (control, 2%; RWJ-24194710�6 M, 3%; 10�5 M, 9%). In addition, cells in the top right quadrant stained with both PI and Annexin V, indicating that the cells were no longerviable (control, 3%; RWJ-241947 10�6 M, 4%; 10�5 M, 13%).




may be associated with slowing the growth of transformed cells.On occasions, the PPAR� ligand can induce apoptosis of cellswith activation of caspase 3.

The PSA protein is something regarded as a differentiationmarker. It is also clearly an androgen-responsive gene. Wefound that RWJ-241947 can partially inhibit androgen-stimu-lated expression of PSA. We noted previously that TZDs candown-regulate PSA production in androgen-responsive LNCaPprostate cancer cells and also demonstrated that TZDs caninhibit androgen activation of the androgen response element inthe regulatory region of the PSA gene (36). Here, we also show

Fig. 5 Effect of RWJ-241947 on the growth of PC-3 human prostatetumor cells in BNX immunodeficient mice. PC-3 cells were bilaterallyinjected s.c into nude mice, forming two tumors per mouse. The micewere divided randomly into a control and experimental group. RWJ-241947 (30 mg/kg) was administered by gavage for 5 days a week in theexperimental groups. A, time course of tumor volumes. Tumor volumeswere measured every week. The mean volume � SD of 14 tumors ineach group is shown. Tumor volumes were significantly different be-tween the experimental and control group (P 0.0035). B, tumorweights at autopsy. After 6 weeks of therapy, tumors were removedfrom each group. Their weights were significantly different in the twogroups (P 0.0027).

Fig. 6 Histology of PC-3 human prostate tumors from mice treatedwith RWJ-241947 in vivo. After 6 weeks of growth in nude mice eitherwith or without treatment with RWJ-241947, PC-3 tumors were re-moved, fixed in formalin, and stained with H&E. In A, control tumorsfrom mice that received diluant control displayed poorly differentiatedadenocarcinoma (�200). In B, tumors from mice treated with RWJ-241947 (30 mg/kg/day) for 5 days a week showed marked fibrosis,including an inflammatory and giant cell reaction (�200). Arrows, giantcells. C, furthermore, tumors from mice treated with RWJ-241947 hada prominent population of cells with pyknotic nuclei (arrows) andcytoplasmic eosinophilia, indicating apoptosis (�400).




by reporter gene assays that RWJ-241947 can inhibit androgenactivation of the ARE of the PSA gene. This inhibition ofandrogen responsiveness may in part explain the antiprolifera-tive effect of RWJ-241947 in LNCaP cells, but this cannot bethe entire explanation because growth of AR-independent PC-3cells was also inhibited by this PPAR� ligand. Patients withprostate cancer have received troglitazone after they had pros-tectomies with curative intent and subsequently developed arising PSA, indicating a return of their prostate cancer. Thelargest study showed that 1 of 41 patients had a 50% decrease inserum PSA, and 7 of 41 individuals had a �51% decrease intheir PSA. Of those 7 individuals, 4 had androgen-independentand 3 had androgen-dependent prostate cancer. Many of theseresponses appeared to last for �18 months.

We showed that RWJ-241947 may increase the differenti-ation of LNCaP cells because it did increase the expression of

E-cadherin in LNCaP cells. This protein has been associatedwith cell differentiation of prostate cancer cells (37).

We found that the ability of RWJ-241947 to inhibit pros-tate tumor growth in experimental mice was particularly dra-matic. It induced marked fibrosis, including an inflammatoryand giant cell reaction of the tumor. This may represent a hostreaction to the tumor or a secondary reaction to tumor necrosis.Apoptotic cell death was also detected in the experimentaltumor. We noted previously that troglitazone was able potentlyto inhibit the growth of PC-3 tumors in nude mice. Usingimmunohistochemistry, we looked for difference of expressionof growth-related genes in the tumor cells of the experimentalmice. Slightly fewer dividing cells (Ki-67) were present in thetumors of the experimental mice, but levels of expression ofp21WAF1 and Bcl-2 in the tumor cells of both cohorts of micewere similar. Probably the remaining tumor cells in the exper-imental mice are not as sensitive to the antiproliferative effectsof PPAR ligand, as were the original responsive tumor cells.

The reason for the profound antitumor activity of RWJ-241947 in vivo is not clear. In this study, we showed thatRWJ-241947 affects macrophage differentiation, as shown bythe induction of CD36, as well as by stimulating an inflamma-tory and giant cell reaction in the experimental tumors. OtherPPAR� ligands have been shown to promote macrophage dif-ferentiation and enhance transcriptional induction of the scav-enger receptor CD36 by ligand activation of the PPAR:retinoidX receptor heterodimers (39).

The RWJ-241947 may mediate some of its antiproliferativeeffects by inhibition of angiogenesis. Rosiglitazone has beenshown to suppress the primary tumor growth and metastasis byboth direct and indirect antiangiogenetic effects (50). ActivatedPPAR� down-regulates the production of vascular endothelialgrowth factor that is involved in the regulation of angiogenesis(51, 52). This may explain some of the anticancer effects of

Fig. 7 Induction of expressionof CD36 on the cell surfaceof human monocytic cells cul-tured with RWJ-241947. Cells(U937 and THP-1) were cul-tured either with or withoutRWJ-241947 (10�6 and 10�5

M) or troglitazone (TG 10�6

and 10�5 M) for 4 days andexamined for CD36 expressionby flow cytometry using FITC-conjugated CD36 antibody.Percentage of CD36-positivecells is shown. Diluant-treatedcells are the control. Resultsrepresent the mean � SD ofthree independent experiments.

Table 1 Blood analysis and weights of BNX triple immunodeficientmice with PC-3 tumors after treatment of RWJ-241947a

Control mice Experimental mice

Weight (gms) 25.5 � 2.4 25.7 � 1.3WBC (103/mm2) 5.5 � 0.1 5.5 � 3.0Hemoglobin (gms/dl) 17.3 � 1.9 14.4 � 2.3Platelet (103/mm2) 1582 � 731 2134 � 611Alkaline phosphatase (units/liter) 91.5 � 24.6 106 � 15.5GOT (units/liter)b 61.8 � 14.3 67.8 � 8.7GPT (units/liter) 26.5 � 3.4 21.0 � 5.8Glucose (mg/dl) 231 � 33.4 203 � 52.9Cholesterol (mg/dl)c 78.5 � 13.1 40.0 � 17.3Total protein 4.9 � 0.3 4.4 � 0.1

a Blood analysis and weight measurement were performed justpreceeding the termination of the experiment (6 weeks).

b GOT, glutamic oxalacetic transminase; GPT, glutamic pyruvictransminase.

c P �0.05.




Fig. 8 RWJ-241947: enhancement of As2O3-induced apoptosis in myeloma cells. A, myeloma (ARH-77, RPMI8226, NIH-H929, and U266), prostate(LNCaP, PC-3, and DU-145), and breast MCF-7 cancer cell lines were treated with RWJ-241947 (10�5 M), As2O3 (10�6 M for ARH-77, LNCaP,PC-3, DU-145, and MCF-7; 2 � 10�7 M As2O3 for RPMI8226, NIH-H929, and U266), or their combination for 4 days. Growth (% of control) wasmeasured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results represent the mean � SD of three independent experimentswith triplicate dishes. B, cell cycle analysis of ARH-77 cells by flow cytometry. ARH-77 (multiple myeloma cells) after being cultured with eitherRWJ-241947 (10�5 M), As2O3 (10�6 M), or both for 4 days was harvested and stained with propidium iodine. Each histogram displays propidiumiodine (X axis), as a reflection of the cellular DNA content and cell number (Y axis). Arrowheads, positions of the apoptotic subG1 cell population.A total of 0% (control), 3% (RWJ-241947), 18% (As2O3), and 49% (As2O3 RWJ-241947) of the cells showed an apoptotic DNA pattern. In C,quantitative analysis of apoptosis in each myeloma cell line treated with RWJ-241947 (10�5 M), As2O3 (10�6 M for ARH-77 and 2 � 10�7 M forRPMI8226, U266, and NCI-H929), or both for 4 days was also analyzed by terminal deoxynucleotidyl transferase-mediated nick end labeling assayafter culture for 4 days. Results represent the mean � SD of three independent experiments. In D, ARH-77 cells (myeloma cell line) were treatedwith either RWJ-241947 (10�5 M), As2O3 (10�6 M), or both; cellular lysates were Western blotted and probed with antibody for poly(ADP-ribose)polymerase (PARP). Intact form of PARP is 116 kb, and cleaved form is 85 kb.




RWJ-241947 in our model system. Clearly, additional studiesare required to understand why TZDs appear to have more invivo cancer activity than might have been predicted from the invitro data.

Previous studies have shown that the in vitro bindingaffinity for PPAR� by RWJ-241947 is much less than that ofother TZDs, although it seems to have a similar anticanceractivity. This might be explained by several pieces of data whichsuggest that some of the activities of PPAR� ligands can occurindependently of PPAR�, including experiments on cells fromhomozygous PPAR� (PPAR��/�) null mice. Exposure of nor-mal activated macrophages to TZDs can decrease their releaseof inflammatory cytokines, such as tumor necrosis factor, inter-leukin-1, interleukin-6, COX-2, and inducible NO-synthatase(53–55). Surprisingly, PPAR��/� macrophages when acti-vated and exposed to TZDs also exhibited decreased productionof these anti-inflammatory molecules (41). In a second series ofexperiments using embryonic stem cells from PPAR��/�mice, TZDs inhibited their proliferation and DNA synthesis andcaused them to arrest in the G1 phase of the cell cycle. Inaddition, the TZDs inhibited the growth of PPAR��/� embry-onic stem cell “tumors” growing in syngenic mice (56).

We found that RWJ-241947 markedly reduced the totalserum cholesterol in mice. Troglitazone also has cholesterol-lowering ability in vivo. But other TZDs, including rosiglita-zone, pioglitazone, and 15d-PGJ2, showed only weak activity orno inhibition (57). The mechanism for these differences isunclear at this time, but RWJ-241947 and troglitazone appear tobe similar in their effect on serum lipids. The activated PPAR�plays a critical role in the regulation of cholesterol homeostasisby modulating the lipid metabolism by the macrophages.PPAR� is required for basal expression of CD36, the scavengerreceptor responsible for uptake of the oxidized low-densitylipoprotein in macrophages (39–41). We found thatRWJ-241947 was able to induce CD36 expression on monocyticcell lines in vitro, suggesting that it also can modulate the lipidmetabolism via macrophages. The strong cholesterol-loweringactivity of RWJ-241947 should be further investigated for po-tential therapeutic applications for atherosclerosis.

In general, arsenic compounds can be considered a poisonand potential environmental carcinogen for the development ofboth lung and skin cancers (58, 59). However, the inorganicAs2O3 is able to induce apoptosis of APL cells as well as avariety of other cancer cell types, including multiple myeloma.Clinical studies have shown that As2O3 is an efficacious drugfor patients with multiple myeloma and APL (23–28). Previ-ously, we showed that a ligand for the nuclear hormone receptorretinoic acid receptor plus an organic arsenical had a synergisticinhibitory activity against prostate and breast cancer cells (29).These findings prompted us in this study to look at the potentialsynergy of a PPAR� ligand with an arsenical. We showed that,together, they had an enhanced antiproliferative activity againstPC-3 prostate cells and a variety of myeloma cell lines. Arsenictrioxide has a fair amount of toxicity (60–62); perhaps bycombining it with RWJ-241947, its dose can be lowered, reduc-ing its side effects while enhancing its clinical efficacy.

In conclusion, our data demonstrate a surprisingly potentantiproliferative effect of RWJ-241947 against prostate cancercells in vivo. It also enhanced the antiproloferative, proapoptotic

activity of As2O3 against myeloma cells. This oral drug appearsto have few side effects and may lower serum cholesterol levels.Together, the data suggest the possibility that RWJ-241947 mayhave a therapeutic role in cancer, perhaps in a chemopreventionrole or clinical setting when the tumor burden is low.

ACKNOWLEDGMENTSWe thank Kim Burgin for her excellent secretarial and administra-

tive support.

REFERENCES1. Tontonoz, P., Hu, E., and Spiegelman, B. M. Stimulation of adipo-genesis in fibroblasts by PPAR gamma 2, a lipid-activated transcriptionfactor. Cell, 79: 1147–1156, 1994.2. Rosen, E. D., Sarraf, P., Troy, A. E., Bradwin, G., Moore, K.,Milstone, D. S., Spiegelman, B. M., and Mortensen, R. M. PPARgamma is required for the differentiation of adipose tissue in vivo andin vitro. Mol. Cell, 4: 611–617, 1999.3. Ciaraldi, T. P., Gilmore, A., Olefsky, J. M., Goldberg, M., andHeidenreich, K. A. In vitro studies on the action of CS-045, a newantidiabetic agent. Metabolism, 39: 1056–1162, 1990.4. Nolan, J. J., Ludvik, B., Beerdsen, P., Joyce, M., and Olefsky, J.Improvement in glucose tolerance and insulin resistance in obese sub-jects treated with troglitazone. N. Engl. J. Med., 331: 1188–1193, 1994.5. Tontonoz, P., Singer, S., Forman, B. M., Sarraf, P., Fletcher, J. A.,Fletcher, C. D., Brun, R. P., Mueller, E., Altiok, S., Oppenheim, H.,Evans, R. M., and Spiegelman, B. M. Terminal differentiation of humanliposarcoma cells induced by ligands for peroxisome proliferator-acti-vated receptor gamma and the retinoid X receptor. Proc. Natl. Acad. Sci.USA, 94: 237–241, 1997.6. Demetri, G. D., Fletcher, C. D., Mueller, E., Sarraf, P., Naujoks, R.,Campbell, N., Spiegelman, B. M., and Singer, S. Induction of solidtumor differentiation by the peroxisome proliferator-activated receptor-gamma ligand troglitazone in patients with liposarcoma. Proc. Natl.Acad. Sci. USA, 96: 3951–3956, 1999.7. Mueller, E., Smith, M., Sarraf, P., Kroll, T., Aiyer, A., Kaufman,D. S., Oh, W., Demetri, G., Figg, W. D., Zhou, X. P., Eng, C.,Spiegelman, B. M., and Kantoff, P. W. Effects of ligand activation ofperoxisome proliferator-activated receptor gamma in human prostatecancer. Proc. Natl. Acad. Sci. USA, 97: 10990–10995, 2000.8. Elstner, E., Muller, C., Koshizuka, K., Williamson, E. A., Park, D.,Asou, H., Shintaku, P., Said, J. W., Heber, D., and Koeffler, H. P.Ligands for peroxisome proliferator-activated receptorgamma and reti-noic acid receptor inhibit growth and induce apoptosis of human breastcancer cells in vitro and in BNX mice. Proc. Natl. Acad. Sci. USA, 95:8806–8811, 1998.9. Suh, N., Wang, Y., Williams, C. R., Risingsong, R., Gilmer, T.,Willson, T. M., and Sporn, M. B. A new ligand for the peroxisomeproliferator-activated receptor-� (PPAR-�), GW7845, inhibits rat mam-mary carcinogenesis. Cancer Res., 59: 5671–5673, 1999.10. Mehta, R. G., Williamson, E., Patel, M. K., and Koeffler, H. P. Aligand of peroxisome proliferator-activated receptor gamma, retinoids,and prevention of preneoplastic mammary lesions. J. Natl. Cancer Inst.(Bethesda), 92: 418–423, 2000.11. Kubota, T., Koshizuka, K., Williamson, E. A., Asou, H., Said,J. W., Holden, S., Miyoshi, I., and Koeffler, H. P. Ligand for peroxi-some proliferator-activated receptor � (troglitazone) has potent antitu-mor effect against human prostate cancer both in vitro and in vivo.Cancer Res., 58: 3344–3352, 1998.12. Shappell, S. B., Gupta, R. A., Manning, S., Whitehead, R., Boeglin,W. E., Schneider, C., Case, T., Price, J., Jack, G. S., Wheeler, T. M.,Matusik, R. J., Brash, A. R., and Dubois, R. N. 15S-Hydroxyeicosatet-raenoic acid activates peroxisome proliferator-activated receptor gammaand inhibits proliferation in PC3 prostate carcinoma cells. Cancer Res.,61: 497–503, 2001.




13. Ohta, K., Endo, T., Haraguchi, K., Hershman, J. M., and Onaya, T.Ligands for peroxisome proliferator-activated receptor gamma inhibitgrowth and induce apoptosis of human papillary thyroid carcinomacells. J. Clin. Endocrinol. Metab., 86: 2170–2177, 2001.

14. Satoh, T., Toyoda, M., Hoshino, H., Monden, T., Yamada, M.,Shimizu, H., Miyamoto, K., and Mori, M. Activation of peroxisomeproliferator-activated receptor-gamma stimulates the growth arrest andDNA-damage inducible 153 gene in non-small cell lung carcinomacells. Oncogene, 21: 2171–2180, 2002.

15. Itami, A., Watanabe, G., Shimada, Y., Hashimoto, Y., Kawamura,J., Kato, M., Hosotani, R., and Imamura, M. Ligands for peroxisomeproliferator-activated receptor gamma inhibit growth of pancreatic can-cers both in vitro and in vivo. Int. J. Cancer, 94: 370–376, 2001.

16. Goke, R., Goke, A., Goke, B., El-Deiry, W. S., and Chen, Y.Pioglitazone inhibits growth of carcinoid cells and promotes TRAIL-induced apoptosis by induction of p21waf1/cip1. Digestion, 64: 75–80,2001.

17. Inoue, K., Kawahito, Y., Tsubouchi, Y., Kohno, M., Yoshimura, R.,Yoshikawa, T., and Sano, H. Expression of peroxisome proliferator-activated receptor gamma in renal cell carcinoma and growth inhibitionby its agonists. Biochem. Biophys. Res. Commun., 287: 727–732, 2001.18. Rumi, M. A., Sato, H., Ishihara, S., Kawashima, K., Hamamoto, S.,Kazumori, H., Okuyama, T., Fukuda, R., Nagasue, N., and Kinoshita, Y.Peroxisome proliferator-activated receptor gamma ligand-inducedgrowth inhibition of human hepatocellular carcinoma. Br. J. Cancer, 84:1640–1647, 2001.19. Takahashi, N., Okumura, T., Motomura, W., Fujimoto, Y., Kawa-bata, I., and Kohgo, Y. Activation of PPARgamma inhibits cell growthand induces apoptosis in human gastric cancer cells. FEBS Lett., 455:135–139, 1999.20. Reginato, M. J., Bailey, S. T., Krakow, S. L., Minami, C., Ishii, S.,Tanaka, H., and Lazar, M. A. A potent antidiabetic thiazolidinedionewith unique peroxisome proliferator-activated receptor gamma-activat-ing properties. J. Biol. Chem., 273: 32679–32684, 1998.21. Mervis, J. Ancient remedy performs new tricks. Science (Wash.DC), 273: 578, 1996.22. Donofrio, P. D., Wilbourn, A. J., Albers, J. W., Rogers, L., Salanga,V., and Greenberg, H. S. Acute arsenic intoxication presenting asGuillain-Barre-like syndrome. Muscle Nerve, 10: 114–120, 1987.23. Sun, H. D., Ma, L., and Hu, X. C. Arsenic trioxide treated 32 casesof acute promyelocytic leukemia. Journal of Integration of Chinese andWestern Medicine, 12: 170–171, 1992.24. Soignet, S. L., Maslak, P., Wang, Z. G., Jhanwar, S., Calleja, E.,Dardashti, L. J., Corso, D., DeBlasio, A., Gabrilove, J., Scheinberg,D. A., Pandolfi, P. P., and Warrell, R. P., Jr. Complete remission aftertreatment of acute promyelocytic leukemia with arsenic trioxide.N. Engl. J. Med., 339: 1341–1348, 1998.25. Shen, Z. X., Chen, G. Q., Ni, J. H., Li, X. S., Xiong, S. M., Qiu,Q. Y., Zhu, J., Tang, W., Sun, G. L., Yang, K. Q., Chen, Y., Zhou, L.,Fang, Z. W., Wang, Y. T., Ma, J., Zhang, P., Zhang, T. D., Chen, S. J.,Chen, Z., and Wang, Z. Y. Use of arsenic trioxide (As2O3) in thetreatment of acute promyelocytic leukemia (APL): II. Clinical efficacyand pharmacokinetics in relapsed patients. Blood, 89: 3354–3360, 1997.26. Chen, G. Q., Zhu, J., Shi, X. G., Ni, J. H., Zhong, H. J., Si, G. Y.,Jin, X. L., Tang, W., Li, X. S., Xong, S. M., Shen, Z. X., Sun, G. L., Ma,J., Zhang, P., Zhang, T. D., Gazin, C., Naoe, T., Chen, S. J., Wang,Z. Y., and Chen, Z. In vitro studies on cellular and molecular mecha-nisms of arsenic trioxide (As2O3) in the treatment of acute promyelo-cytic leukemia: As2O3 induces NB4 cell apoptosis with downregulationof Bcl-2 expression and modulation of PML-RAR alpha/PML proteins.Blood, 88: 1052–1061, 1996.27. Rousselot, P., Labaume, S., Marolleau, J. P., Larghero, J., Noguera,M. H., Brouet, J. C., and Fermand, J. P. Arsenic trioxide and melarso-prol induce apoptosis in plasma cell lines and inplasma cells frommyeloma patients. Cancer Res., 59: 1041–1048, 1999.28. Park, W. H., Seol, J. G., Kim, E. S., Hyun, J. M., Jung, C. W., Lee,C. C., Kim, B. K., and Lee, Y. Y. Arsenic trioxide-mediated growthinhibition in MC/CAR myeloma cells via cell cycle arrest in association

with induction of cyclin-dependent kinase inhibitor, p21, and apoptosis.Cancer Res., 60: 3065–3671, 2000.

29. Koshiuka, K., Elstner, E., Williamson, E., Said, J. W., Tada, Y., andKoeffler, H. P. Novel therapeutic approach: organic arsenical melarso-prol) alone or with all-trans-retinoic acid markedly inhibit growth ofhuman breast and prostate cancer cells in vitro and in vivo. Br. J.Cancer, 82: 452–458, 2000.30. Liu, L. S., Tanaka, H., Ishii, S., and Eckel, J. The new antidiabeticdrug MCC-555 acutely sensitizes insulin signaling in isolated cardiom-yocytes. Endocrinology, 139: 4531–4539, 1998.31. Ikezoe, T., Miller, C. W., Kawano, S., Heaney, A., Williamson,E. A., Hisatake, J., Green, E., Hofmann, W., Taguchi, H., and Koeffler,H. P. Mutational analysis of the peroxisome proliferator-activated re-ceptor � gene in human malignancies. Cancer Res., 61: 5307–5310,2001.32. Elstner, E., Linker-Israeli, M., Said, J., Umiel, T., de Vos, S.,Shintaku, I. P., Heber, D., Binderup, L., Uskokovic, M., and Koeffler,H. P. 20-epi-vitamin D3 analogues: a novel class of potent inhibitors ofproliferation and inducers of differentiation of human breast cancer celllines. Cancer Res., 55: 2822–2830, 1995.33. Pang, S., Dannull, J., Kaboo, R., Xie, Y., Tso, CL., Michel, K.,deKernion, J. B., and Belldegrun, A. S. Identification of a positiveregulatory element responsible for tissue-specific expression of prostate-specific antigen. Cancer Res., 57: 495–499, 1997.34. Abreu-Martin, M. T., Chari, A., Palladino, A. A., Craft, N. A., andSawyers, C. L. Mitogen-activated protein kinase kinase kinase 1 acti-vates androgen receptor-dependent transcription and apoptosis in pros-tate cancer. Mol. Cell. Biol., 19: 5143–5154, 1999.35. Huang, W., Shostak, Y., Tarr, P., Sawyers, C., and Carey, M.Cooperative assembly of androgen receptor into a nucleoprotein com-plex that regulates the prostate-specific antigen enhancer. J. Biol.Chem., 274: 25756–25768, 1999.36. Hisatake, J. I., Ikezoe, T., Carey, M., Holden, S., Tomoyasu, S., andKoeffler, H. P. Down-regulation of prostate-specific antigen expressionby ligands for peroxisome proliferator-activated receptor � in humanprostate cancer. Cancer Res., 60: 5494–5498, 2000.37. Otto, T., Rembrink, K., Goepel, M., Meyer-Schwickerath, M., andRubben, H. E-cadherin: a marker for differentiation and invasiveness inprostatic carcinoma. Urol. Res., 21: 359–362, 1993.38. Koeffler, H. P. Peroxisome proliferator-activated receptor � andcancers. Clin. Cancer Res., 9: 1–9, 2003.39. Tontonoz, P., Nagy, L., Alvarez, J. G., Thomazy, V. A., and Evans,R. M. PPARgamma promotes monocyte/macrophage differentiation anduptake of oxidized LDL. Cell, 93: 241–252, 1998.40. Moore, K. J., Rosen, E. D., Fitzgerald, M. L., Randow, F., Anders-son, L. P., Altshuler, D. Milstone, D. S., Mortensen, R. M., Spiegelman,B. M., and Freeman, M. W. The role of PPAR-gamma in macrophagedifferentiation and cholesterol uptake. Nat. Med., 7: 41–47, 2001.41. Chawla, A., Barak, Y., Nagy, L., Liao, D., Tontonoz, P., and Evans,R. M. PPAR-gamma dependent and independent effects on macroph-age-gene expression in lipid metabolism and inflammation. Nat. Med.,7: 48–52, 2001.42. Ryoo, J. J., Cole, C. E., and Anderson, K. C. Novel therapies formultiple myeloma. Blood Rev., 16: 167–174, 2002.43. Elstner, E., Linker-Israeli, M., Umiel, T., Le, J., Grillier, I., Said, J.,Shintaku, I. P., Krajewski, S., Reed, J. C., Binderup, L., and Koeffler,H. P. Combination of a potent 20-epi-vitamin D3 analogue (KH 1060)with 9-cis-retinoic acid irreversibly inhibits clonal growth, decreasesbcl-2 expression, and induces apoptosis in HL-60 leukemic cells. CancerRes., 56: 3570–3576, 1996.44. Elstner, E., Linker-Israeli, M., Le, J., Umiel, T., Michl, P., Said,J. W., Binderup, L., Reed, J. C., and Koeffler, H. P. Synergistic decreaseof clonal proliferation, induction of differentiation, and apoptosis ofacute promyelocytic leukemia cells after combined treatment with novel20-epi vitamin D3 analogs and 9-cis retinoic acid. J. Clin. Investig., 99:349–360, 1997.45. Stio, M., Celli, A., and Treves, C. Synergistic anti-proliferativeeffects of vitamin D derivatives and 9-cis retinoic acid in SH-SY5Y




human neuroblastoma cells. J. Steroid Biochem. Mol. Biol., 77: 213–222, 2001.

46. Danielsson, C., Torma, H., Vahlquist, A., and Carlberg, C. Positiveand negative interaction of 1,25-dihydroxyvitamin D3 and the retinoidCD437 in the induction of human melanoma cell apoptosis. Int. J.Cancer, 81: 467–470, 1999.

47. Parmar, S., and Tallman, M. S. Acute promyelocytic leukaemia: areview. Exp. Opin. Pharmacother., 4: 1379–1392, 2003.

48. Joyce, D., Albanese, C., Steer, J., Fu, M., Bouzahzah, B., andPestell, R. G. NF-kappaB and cell-cycle regulation: the cyclin connec-tion. Rev. Cytokine Growth Factor Rev., 12: 73–90, 2001.

49. Karin, M., and Ben-Neriah, Y. Phosphorylation meets ubiquitina-tion: the control of NF-[kappa]B activity. Rev. Annu. Rev. Immunol.,18: 621–663, 2000.

50. Panigrahy, D., Singer, S., Shen, L. Q., Butterfield, C. E., Freedman,D. A., Chen, E. J., Moses, M. A., Kilroy, S., Duensing, S., Fletcher, C.,Fletcher, J. A., Hlatky, L., Hahnfeldt, P., Folkman, J., and Kaipainen, A.PPARgamma ligands inhibit primary tumor growth and metastasis byinhibiting angiogenesis. J. Clin. Investig., 110: 923–932, 2002.

51. Jozkowicz, A., Dulak, J., Piatkowska, E., Placha, W., and Dembin-ska-Kiec, A. Ligands of peroxisome proliferator-activated receptor-gamma increase the generation of vascular endothelial growth factor invascular smooth muscle cells and in macrophages. Acta Biochim. Pol.,47: 1147–1157, 2000.

52. Fauconnet, S., Lascombe, I., Chabannes, E., Adessi, G. L., Desvergne,B., Wahli, W., and Bittard, H. Differential regulation of vascular endothelialgrowth factor expression by peroxisome proliferator-activated receptors inbladder cancer cells. J. Biol. Chem., 277 : 23534–23543, 2002.

53. Ricote, M., Li, A. C., Willson, T. M., Kelly, C. J., and Glass, C. K.The peroxisome proliferator-activated receptor-gamma is a negativeregulator of macrophage activation. Nature (Lond.), 391: 79–82, 1998.

54. Jiang, C., Ting, A. T., and Seed, B. PPAR-gamma agonists inhibitproduction of monocyte inflammatory cytokines. Nature (Lond.), 391:82–86, 1998.55. Maggi, L. B., Jr., Sadeghi, H., Weigand, C., Scarim, A. L.,Heitmeier, M. R., and Corbett, J. A. Anti-inflammatory actions of15-deoxy-delta 12,14-prostaglandin J2 and troglitazone: evidence forheat shock-dependent and -independent inhibition of cytokine-in-duced inducible nitric oxide synthase expression. Diabetes, 49: 346 –355, 2000.56. Palakurthi, S. S., Aktas, H., Grubissich, L. M., Mortensen, R. M.,and Halperin, J. A. Anticancer effects of thiazolidinediones areindependent of peroxisome proliferator-activated receptor � and me-diated by inhibition of translation initiation. Cancer Res., 61: 6213–6218, 2001.57. Wang, M., Wise, S. C., Leff, T., and Su, T. Z. Troglitazone, anantidiabetic agent, inhibits cholesterol biosynthesis through a mecha-nism independent of peroxisome proliferator-activated receptor-gamma.Diabetes, 48: 254–260, 1999.58. Jaafar, R., Omar, I., Jidon, A. J., Wan-Khamizar, B. W., Siti-Aishah, B. M., and Sharifah-Noor-Akmal, S. H. Skin cancer caused bychronic arsenical poisoning–a report of three cases. Med. J. Malaysia,48: 86–92, 1993.59. Dong, J. T., and Luo, X. M. Effects of arsenic on DNA damage andrepair in human fetal lung fibroblasts. Mutat. Res., 315: 11–15, 1994.60. Barbey, J. T. Cardiac toxicity of arsenic trioxide. Review: discus-sion. Blood, 98: 1633–1634, 2001.61. Singer, J. W. Cardiac toxicity of arsenic trioxide. Discussion.Blood, 98: 1633–1634, 2001.62. Westervelt, P., Brown, R. A., Adkins, D. R., Khoury, H., Curtin, P.,Hurd, D., Luger, S. M., Ma, M. K., Ley, T. J., and DiPersio, J. F. Suddendeath among patients with acute promyelocytic leukemia treated witharsenic trioxide. Blood, 98: 266–271, 2001.




2004;10:1508-1520. Clin Cancer Res Takashi Kumagai, Takayuki Ikezoe, Dorina Gui, et al. Arsenic TrioxideEnhancement of Apoptosis in Myeloma Cells Induced byBeige/Nude/ X-Linked Immunodeficient Mice and

and inin VitroActivity against Human Prostate Cancer Ligand with AntitumorγProliferator-Activated Receptor-

RWJ-241947 (MCC-555), A Unique Peroxisome

Updated version

http://clincancerres.aacrjournals.org/content/10/4/1508

Access the most recent version of this article at:

Cited articles

http://clincancerres.aacrjournals.org/content/10/4/1508.full#ref-list-1

This article cites 62 articles, 25 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/10/4/1508.full#related-urls

This article has been cited by 5 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

SubscriptionsReprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://clincancerres.aacrjournals.org/content/10/4/1508To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/content/10/4/1508.full#ref-list-1

http://clincancerres.aacrjournals.org/content/10/4/1508.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



RWJ-241947 (MCC-555), A Unique Peroxisome Proliferator- … · RWJ-241947 (MCC-555), A Unique...

Documents

Transcript of RWJ-241947 (MCC-555), A Unique Peroxisome Proliferator- … · RWJ-241947 (MCC-555), A Unique...