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Clinical Cancer Research Vol. 10, 1508-1520, February 2004
© 2004 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

RWJ-241947 (MCC-555), A Unique Peroxisome Proliferator-Activated Receptor-{gamma} Ligand with Antitumor Activity against Human Prostate Cancer in Vitro and in Beige/Nude/ X-Linked Immunodeficient Mice and Enhancement of Apoptosis in Myeloma Cells Induced by Arsenic Trioxide

Takashi Kumagai1, Takayuki Ikezoe1, Dorina Gui2, James O’Kelly1, Xiang-Jun Tong1, Fredric J. Cohen3, Jonathan W. Said2 and H. Phillip Koeffler1

Division of Hematology/Oncology, Departments of1 Medicine and 2 Pathology, Center of Health of Science, University of California at Los Angeles School of Medicine, Los Angeles, California, and 3 Global Clinical Research, Johnson and Johnson Pharmaceutical Research and Development, La Jolla, California


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: RWJ-241947 (MCC-555) is a novel peroxisome proliferator-activated receptor-{gamma} ligand of the thiazolidinedione class that was recently developed as an antidiabetic drug with unique properties. Some thiazolidinediones have anticancer activity against solid and hematological malignancies; the anticancer potency of RWJ-241947 has not been examined. We, therefore, investigated these effects in vitro and in vivo either alone or in combination with other compounds.

Experimental Design: Tumor growth was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, soft agar colony assay in vitro, and xenografts in nude mice. Its effects on cell cycle, differentiation, and apoptosis were examined.

Results: In vitro studies using various solid and hematological tumor cell lines showed that RWJ-241947 had antiproliferative activity against prostate cancer cells, with the strongest effect against the androgen-independent PC-3 prostate cancer cells. It increased expression of cyclin-dependent kinase inhibitor p21WAF1, deceased cyclin E, and induced apoptosis in PC-3 cells. It increased E-cadherin and lowered protein expression of prostate-specific antigen without down-regulating the androgen receptor in androgen-dependent LNCaP prostate cancer cells. Reporter gene assays showed that this peroxisome proliferator-activated receptor-{gamma} ligand inhibited androgen activation of the androgen receptor response elements of the prostate-specific antigen gene. Remarkably, in vivo treatment of male beige/nude/X-linked immunodeficient (BNX) mice with RWJ-241947 profoundly suppressed growth of PC-3 prostate cancer xenografts with prominent apoptosis, as well as fibrosis, including inflammatory and giant cell reaction in the remaining tumor tissue. Notably, the experimented mice had a significantly decreased cholesterol. In addition, we studied the combination of arsenic trioxide (As2O3), which is used in the treatment of multiple myeloma, and RWJ-241947; these two reagents together prominently inhibited proliferation and caused apoptosis of multiple myeloma cells.

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


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The peroxisome proliferator-activated receptor {gamma} (PPAR {gamma}) belongs to the nuclear hormone receptor superfamily and has an important role in adipocyte differentiation. Thiazolidinediones (TZDs) are a class of synthetic PPAR{gamma} ligands that induce adipocyte differentiation (1 , 2) , regulate genes involved in lipogenesis, and enhance glucose utilization (3 , 4) . Normal preadipocytes can be induced to differentiate in the presence of ligands for PPAR{gamma}. With this knowledge in hand, investigators have used TZDs to induce differentiation of human liposarcoma cells in vitro (5) and treat patients with these tumors (6) . Troglitazone, one of the TZDs, retarded growth and induced differentiation of these tumor cells. This has prompted investigations of TZDs in breast and prostate cancers. Both of these tumors have prominent expression of PPAR{gamma} (7 , 8) . Studies have shown that TZDs (10-6 to 10-5 M) can moderately inhibit proliferation and induce 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 with a PPAR{gamma} ligand synergistically decreased proliferation and induced apoptosis of several breast cancer cell lines both in vitro and in xenografts growing in nude mice (8) . The PPAR{gamma} ligand GW7845 was also able to inhibit the development of carcinogen-induced breast cancer in rats (9) . Similarly, troglitazone prevented the carcinogen-induced transformation of breast tissue (10) .

Likewise, investigations have found that proliferation of several human prostate cancer cell lines could be inhibited in vitro and in vivo and undergo profound morphological changes when cultured in the presence of a ligand of PPAR{gamma} (7 , 11) . Clinical study showed that troglitazone was able to retard the progression of prostate cancer in patients (7 , 11) .

Troglitazone was voluntarily withdrawn from the market in March 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, including prostate, thyroid, lung, pancreatic, renal cell, hepatocellular, and gastric cancers in vitro (12, 13, 14, 15, 16, 17, 18, 19) and in animal models of cancer (15) .

RWJ-241947 is a TZD established as an antidiabetic drug in animal models of Type 2 diabetes. Like other TZDs, RWJ-241947 binds to PPAR{gamma} and effects its transcriptional activities, but its binding affinity for PPAR{gamma} is <0.1 of rosiglitazone. Its transcriptional properties are unique because it can function as a full or partial agonist or antagonist, depending on the cell type or DNA-binding site. Its hypoglycemic potency may depend on the context selectivity of RWJ-241947 (20) . RWJ-241947 is currently being tested as a treatment for Type 2 diabetes in humans. We tested for the first time the anticancer activity of this new TZD.

Arsenic-containing remedies have long been part of traditional Chinese medicine for a variety of ailments (21) . Chronic myelogenous leukemia was treated in the 1940s and 1950s with Fowler’s solution that contained 1% potassium arsenite (22) . Administration of arsenic trioxide (As2O3) produced a high complete remission rate as well as long-term survival in a significantly high proportion of individuals with either relapsed or refractory acute promyelocytic leukemia (APL) without severe marrow suppression (23, 24, 25) . Very recently, As2O3 has been shown to have an antitumor effect on human multiple myeloma cells by induction of apoptosis (26, 27, 28) . We have shown previously that the combination of all-trans-retinoic acid and an organic arsenic compound had a profound synergistic growth inhibitory effect against prostate and breast cancer cell lines (29) . This inhibition occurred both in vitro and in vivo and was associated with profound apoptosis and marked decrease in levels of BclII. To our knowledge, no one has investigated the anticancer activity of a combination of a PPAR{gamma} ligand and an arsenic-containing compound.

Although this unique TZD has been characterized and is expected to be an antidiabetic drug to improve impaired glucose tolerance (20 , 30) , its anticancer properties have not been examined. Because PPAR{gamma} is widely expressed in many types of cancer, including prostate, breast, colon, leukemia, and myeloma, and the receptor is rarely altered in human malignancies (31) , we tested RWJ-241947 against a wide range of human cancer cells and found that it inhibited the growth of prostate cancer cells both in vitro and in vivo and synergistically interacted with As2O3 to inhibit the growth of multiple myeloma cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Samples.
All cell lines were obtained from American Type Culture Collection (Rockville, MD) and maintained according to their recommendations. DU145 and MCF-7 were 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. To examine the effect of DHT on LNCaP, the cells were incubated in RPMI 1640 containing 10% charcoal dextran-treated fetal bovine serum for 24 h before the addition of 10-9 M dihydrotestosterone (DHT) either with or without 10-5 M RWJ-241947. RWJ-241947 (MCC-555; Johnson and Johnson Pharmaceutical Research and Development), rosiglitazone (SmithKline Beecham Pharmaceuticals, West Sussex, United Kingdom), pioglitazone (Takeda Chemical Industries, Tokyo, Japan), and 15dPGJ2 (Calbiochem, La Jolla, CA) were dissolved in a solution containing 50% DMSO and 50% ethanol.

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

Soft Agar Colony Assay.
Trypsinized and washed single-cell suspensions of cells were enumerated and plated into 24-well, flat-bottomed plates using a two-layer soft agar system with a total of 1 x 103 cells/well in a volume of 400 µl/well, as described previously (11) .

Cell Cycle Analysis.
PC-3 cells (5 x 104) were exposed to 10-5 M RWJ-241947 in six-well, flat-bottomed plates for 3 days. Total cells, both in suspension and adherent, were collected, washed, and suspended in cold PBS. Cells were fixed in chilled 75% methanol and stained with propidium iodine. Cell cycle status was analyzed on a Becton Dickinson Flow Cytometer.

Western Blot Analysis.
Cells were washed twice in PBS, suspended in lysis buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40, phenylmethylsulfonyl fluoride at 100 µg/ml, aprotinin at 2 µg/ml, pepstatin at 1 µg/ml, and leupetin at 10 µg/ml], and placed on ice for 30 min. After centrifugation at 15,000 x g for 15 min at 4°C, the suspension was collected. Protein concentrations were quantitated by using the Bio-Rad protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s recommendation. Whole cell lysates (40 µg) were resolved by SDS-PAGE in a 4–15% gel, transferred to a polyvinylidene difuride membrane (Immobilon; Amersham Corp., Arlington Heights, IL), and probed sequentially with antibodies against the following proteins: (a) prostate-specific antigen (PSA); (b) androgen receptor (AR); (c) E-cadherin; (d) cyclin E; (e) p21WAF1; (f) p27Kip1; (g) Bcl-2; (h) Bcl-XL; (i) BAX; (j) PARP; and (k) GAPDH (Santa Cruz Biotechnology; Inc., Santa Cruz, CA). The blots were developed using the Supersignal 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 from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and maintained in pathogen-free conditions with irradiated chow. A total of 5 x 106 PC-3 cells in 0.1 ml of Matrigel (Collaborative Biological Products, Bedford, MA) was injected s.c. into bilateral flanks of each mouse, resulting in the formation of two tumors per mouse. The mice were blindly randomized to the experimental and control groups. Treatment was started on the day after the injection of PC-3 cells and continued for 6 weeks. The control (eight mice) received dilutant only, and the experimental (seven mice) received RWJ-241947 (30 mg/kg, p.o. by gavage, 5 days a week). RWJ-241947 was suspended in 1.5% carboxymethylcellulose with 0.2% Tween 20 (Sigma). Tumor sizes were measured every week and calculated by the formula: A (length) x B (width) x C (height) x 0.5236. After 6 weeks, blood was collected for serum chemistry and circulating blood counts. One mouse (control) died during gavage. All mice were euthanized at the end of the study, and tumors, prostates, livers, lungs, and bone marrows were fixed in 10% neutral-buffered formalin and embedded in paraffin for histological analysis. The data were analyzed by Student’s t test.

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

Measurement of Cell Surface CD36 Antigen on Monocytic Cell Lines.
Monocytic leukemia cell lines (U937 and THP-1) were treated with RWJ-241947 or troglitazone (10-6 or 10-5 M) for 4 days and examined for CD36 expression by flow cytometry using CD36 antibody (Immunotech, Inc.), as described previously (32) .

Plasmids.
One PSA promoter that we examined has the 2.4-kb enhancer sequences (-5322 to -2925) fused to the first 564-bp promoter sequences, which is attached to the luciferase gene. It contains all known androgen receptor elements (AREs) in the region of the PSA human gene (33 , 34) . The second construct has the first 496-bp nucleotides of the PSA enhancer (wild type) with six AREs cloned upstream of the luciferase gene in the pGL3 vector (Promega, Chicago, IL; PSA enhancer E4-LUC) and the PSA enhancer (S-All)-E4LUC, which encompasses the same sequences, but the four major AREs in it have been mutated (generous gift of Michael Carey, University of California at Los Angeles; Ref. 35 ).

Transfections and Luciferase Assay.
LNCaP cells were incubated in RPMI 1640 with 10% fetal bovine serum until 50–70% confluency. Cells were transfected with the indicated plasmids using the Superfect (Qiagen, Santa Clarita, CA) under serum-free conditions. A PSV-ß-galactosidase vector was included 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 without 10-9 M DHT and either with or without RWJ-241947 for 48 h. Cells were collected with tissue lysis buffer (Promega). Luciferase activity of the cell lysates was measured by luminometry, and activities were normalized by ß-galactosidase activities. All transfection experiments were carried out in triplicate wells and repeated separately at least three times.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of RWJ-241947 on the Proliferation and Viability of Various Tumor Cell Lines in Vitro.
We examined the antitumor effect of RWJ-241947 on various cancer cell lines, including prostate, breast, lung, and hematopoetic tissues in vitro. The first screening was done using the rapid MTT assay with a relative short exposure of 4 days to the RWJ-241947 (Fig. 1A)Citation . Prostate (LNCaP, PC-3, and DU145) and breast (MCF-7) cancer cells showed some sensitivity to RWJ-241947. Lung cancer 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 to RWJ-241947 in vitro by this assay (Fig. 1A)Citation . We then tested the antiproliferative activity of RWJ-241947 against the prostate and breast cancer cell lines using the extremely sensitive soft agar colony assay. We compared RWJ-241947 with the known active TZD, troglitazone. Both showed similar potency against all four cancer cell lines (Fig. 1B)Citation . The PC-3 human prostate cancer cells were most sensitive, with 1.5 x 10-5 M as the estimated dose of RWJ-241947 that caused a 50% inhibition (ED50) of clonal growth. This is a concentration that is achievable in patients.



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  		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 ± SD of 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 with triplicate 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.

 
We compared the antiproliferative activities of RWJ-241947 versus other PPAR ligands (rosigilitazone, ciglitazone, and PGJ2) against PC-3, LNCaP, and MCF-7 cancer cells. These cells were incubated with 10-5 M these compounds for 14 days, and colony assays showed that each had comparable anticlonal proliferative activities (Fig. 1C)Citation .

Effect of RWJ-241947 on Prostate Cancer Cells in Vitro.
The LNCaP prostate cancer cells have a functional AR. Treatment of these cells with DHT (10-9 M, 24 h) enhanced their expression of PSA. This increase level was suppressed by ~50% when the cells were simultaneously treated with DHT and RWJ-241947 (10-5 M; Fig. 2ACitation ). The promoter of the PSA gene contains a number of androgen response elements, and its expression is dependent on AR (36) . Evaluation of expression of the AR by Western blot analysis showed that RWJ-241947 suppressed PSA expression without suppressing AR levels in the LNCaP cells (Fig. 2A)Citation .



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  		Fig. 2. The effect of RWJ-241947 on expression of prostate-specific antigen (PSA) and androgen receptor (AR) in androgen-dependent LNCaP cells. In A, LNCaP cells were cultured in 10% charcoal-stripped fetal bovine serum for 24 h before the addition of 10-9 M dihydrotestosterone (DHT) either with or without 10-5 M RWJ-241947. After 24 h of treatment, cell lysates were harvested, Western blotted, and probed for protein expression of PSA and AR. The amount of protein was normalized to levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B, effect of RWJ-241947 on transactivation of either the wild-type or mutant PSA enhancer. The reporter luciferase (LUC) constructs [PSA enhancer (496 bp) and this PSA enhancer with the major androgen receptor elements (ARE) mutated] are shown at the top. Wild-type and mutant ARE are represented by boxes and crosses, respectively. LNCaP cells were transfected with the reporter construct, and DHT (10-9 M) was added either with or without 10-5 M RWJ-241947. PSV-ß-galactosidase vector was cotransfected and measured for normalization. Means ± SD of three experiments are shown. In C, LNCaP cells were cultured in 10% fetal bovine serum RPMI either with or without 10-5 M RWJ-241947. After 72 h of treatment, cell lysates were made, Western blotted, and probed with antibodies for cyclin E and E-cadherin. The amount of protein was normalized by GAPDH.

 
To examine further the mechanisms by which RWJ-241947 inhibited levels of PSA, we analyzed the effect of RWJ-241947 on the ability of DHT to transactivate the PSA promoter/enhancer. The LNCaP prostate cancer cells were cultured with DHT (10-9 M) after they were transfected with a luciferase reporter construct that contained a 496-bp fragment of the PSA enhancer, which contains six AREs. Dihydrotestosterone (10-7 M) increased the reporter activity ~5-fold as compared with nontreated control LNCaP cells. When cells were treated with both RWJ-241947 (10-5 M) and DHT (10-9 M), luciferase activity was reduced by 40% compared with DHT alone (Fig. 2B)Citation . This indicated that RWJ-241947 inhibited the ability of androgens to transactivate the PSA promoter/enhancer. Mutation of the four most active AREs and repeating the experiments showed that DHT did not activate and RWJ-241947 did not inhibit luciferase activity (Fig. 2B)Citation . These experiments were repeated using a PSA promoter/enhancer that included several additional AREs that were in an upstream enhancer region (-5322 to -2925 bp), which was attached to the first 564-bp fragments of the PSA promoter upstream of the start of transcription. The results were similar (data not shown).

The cell surface-binding protein, E-cadherin, has been identified as a differentiation marker of prostate cancer cells (37) . RWJ-241947 increased by 220% the expression of E-cadherin in LNCaP cells (Fig. 2C)Citation . PPAR{gamma} ligands were reported 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)Citation .

Additional studies used the RWJ-241947-sensitive PC-3 cells. The cell cycle pattern of PC-3 cells after a 96-h exposure to 10-5 M RWJ-241947 was almost identical to control PC-3 cells (control: G0-G1 50%, S 32%, G2-M 17%; RWJ-241947-exposed PC-3 cells: G0-G1 51%, S 30% G2-M 18%, data not shown). The expression of the cyclin-dependent kinase inhibitor p21WAF1 was not detectable in PC-3 cells, but low expression levels of the protein were observed on the 2nd and 3rd day of exposure to RWJ-241947 (10-5 M). The expression of p27KIP1 was unchanged after culture with RWJ-241947 (Fig. 3A)Citation , and expression of cyclin E decreased by 46% in PC-3 cells (Fig. 3B)Citation .



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  		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, the PC-3 cells were treated with RWJ-241947 (10-5 M), and cell lysates were harvested after 1, 2, and 3 days for Western blot, which was probed sequentially for expression of p21WAF1, p27KIP1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Control cells were treated with vehicle 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 lysates were harvested after 3 days for Western blot, which was probed sequentially for levels of cyclin E, Bcl-2, Bcl-XL, Bax, and GAPDH. Control cells were treated with vehicle alone. The amount of protein was normalized by GAPDH.

 
We performed Annexin V measurements to detect early apoptosis of PC-3 cells exposed to RWJ-241947 (10-5 M, 96 h; Fig. 4Citation ). A portion (9%) of PC-3 cells (Annexin V+/propidium iodine-) underwent early apoptosis, and 13% were no longer viable (Annexin V+/propidium iodine+) after exposure to 10-5 M RWJ-241947 for 4 days. In addition, at 10-6 M RWJ-241947 for 4 days, 5% underwent early apoptosis, and 4% were no longer viable. Control cultures had 2% of the cells showing early apoptosis, and 3% were dead. We also examined expression of genes associated with apoptosis (Bcl-2, BclXL, and Bax) by Western blot analysis (Fig. 3B)Citation . Their levels of expression did not change after exposure of PC-3 cells to RWJ-241947 (10-5 M, 72 h).



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  		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 Annexin V antibody. The cells were also stained with propidium iodine (PI). Control panel showed diluant-treated PC-3 cells. The bottom right quadrant of each panel displays V-FITC-positive and PI-negative cells, indicating that the cells were in an early stage of apoptosis (control, 2%; RWJ-241947 10-6 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 longer viable (control, 3%; RWJ-241947 10-6 M, 4%; 10-5 M, 13%).

 
Morphological change after exposure to RWJ-241947 (10-5 M, 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 not clearly altered by RWJ-241947 in PC-3 cells (data not shown).

Antitumor Effect of RWJ-241947 against PC-3 Prostate Cancer Cells in Vivo.
We evaluated the effect of RWJ-241947 in vivo on PC-3 human prostate tumor cells growing in nude mice. The PPAR{gamma} ligand was given by gavage. Tumor volumes were measured weekly. All mice were euthanized after 6 weeks, and tumors were dissected and weighed. RWJ-241947 significantly suppressed both the growth of prostate tumors (P = 0.0035; Fig. 5ACitation ) and their mean weights (P = 0.0027) at autopsy (Fig. 5B)Citation , as compared with diluant controls.



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

 
Histological analysis of PC-3 tumors from untreated mice revealed poorly differentiated infiltrating adenocarcinoma (Fig. 6A)Citation . Tumors from mice receiving RWJ-241947 had marked fibrosis, including an inflammatory and giant cell reaction (Fig. 6B)Citation . Some tumor cells treated with RWJ-241947 showed pyknotic nuclei and cytoplasmic shrinkage with eosinophilia, indicating that they were undergoing apoptosis (Fig. 6C)Citation . No increase in macrophages were noted in the tumors of the experimental compared with the control mice.



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  		Fig. 6. Histology of PC-3 human prostate tumors from mice treated with RWJ-241947 in vivo. After 6 weeks of growth in nude mice either with or without treatment with RWJ-241947, PC-3 tumors were removed, fixed in formalin, and stained with H&E. In A, control tumors from mice that received diluant control displayed poorly differentiated adenocarcinoma (x200). 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 (x200). Arrows, giant cells. C, furthermore, tumors from mice treated with RWJ-241947 had a prominent population of cells with pyknotic nuclei (arrows) and cytoplasmic eosinophilia, indicating apoptosis (x400).

 
The weights, hematopoetic values, and blood chemistries were not different between the two groups except, interestingly, a significant decrease in the serum cholesterol occurred in the experimental group (Table 1)Citation . No elevation of transaminases (glutamic oxalacetic transminase and glutamic pyruvic transminase) was seen in the experimental group (Table 1)Citation . A variety of organs (lung, liver, prostate, and bone marrow) was histologically examined in the experimental and control mice. All of the tissues, including the prostates, looked normal (data not shown). We also performed immunohistochemistry of the experimental and control tumors, including Ki-67, a marker of cellular proliferation in vivo. A total of 32 and 20% of cells was Ki-67 positive in the control and experimental tumors, respectively. The expression of p21WAF1 and Bcl-2 was unchanged in both control and experimental tumors (data not shown).


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

 
Effect of RWJ-241947 on Expression of Cell Surface CD36 Antigen on Human Monocytic Cell Lines.
Because the serum cholesterol level was markedly decreased in the nude mice receiving RWJ-241947 (Table 1)Citation , we examined the effect of RWJ-241947 on the expression of CD36 on the human monocytic cell lines, U937 and THP-1. CD36 is also known as the scavenger receptor, which is responsible for uptake of the oxidized low-density lipoprotein in macrophages (39, 40, 41) . Levels of CD36 expression increased on both cell lines in a dose-dependent manner, with levels increasing by 21-fold on THP-1 and 8-fold on U-937 compared with control cells, after they were cultured with RWJ-241947 (10-5 M) for 4 days. Compared with RWJ-241947, troglitazone was a more potent inducer of CD36 expression (Fig. 7)Citation .



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  		Fig. 7. Induction of expression of CD36 on the cell surface of human monocytic cells cultured with RWJ-241947. Cells (U937 and THP-1) were cultured either with or without RWJ-241947 (10-6 and 10-5 M) or troglitazone (TG 10-6 and 10-5 M) for 4 days and examined for CD36 expression by flow cytometry using FITC-conjugated CD36 antibody. Percentage of CD36-positive cells is shown. Diluant-treated cells are the control. Results represent the mean ± SD of three independent experiments.

 
RWJ-241947 Enhanced Apoptosis Induced by Arsenic Trioxide in Myeloma Cells in Vitro.
Arsenic trioxide is one of the treatments for multiple myeloma (42) . In the course of looking for agents that enhanced antiproliferative activities of RWJ-241947, we examined the combined effect of RWJ-241947 and As2O3 on myeloma cell lines in vitro. Arsenic trioxide suppressed the cell growth of myeloma cells in a dose-dependent manner (data not shown). We chose a dose of As2O3 (10-6 M for ARH-77 and 2 x 10-7 M for RPMI8226, U266, and NCI-H-929, for 4 days) or RWJ-241947 (10-5 M, 4 days) that had little or no effect alone on growth of the myeloma cells. However, the combination of RWJ-241947 and As2O3 significantly suppressed the proliferation of the myeloma cells compared with either RWJ-241947 or As2O3 alone, e.g., the growth of ARH-77 myeloma cell line was not suppressed by RWJ-241947 and only inhibited 15% by As2O3. Their combination suppressed growth by ~45% (Fig. 8A)Citation .



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  		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 x 10-7 M As2O3 for RPMI8226, NIH-H929, and U266), or their combination for 4 days. Growth (% of control) was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results represent the mean ± SD of three independent experiments with triplicate dishes. B, cell cycle analysis of ARH-77 cells by flow cytometry. ARH-77 (multiple myeloma cells) after being cultured with either RWJ-241947 (10-5 M), As2O3 (10-6 M), or both for 4 days was harvested and stained with propidium iodine. Each histogram displays propidium iodine (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 x 10-7 M for RPMI8226, U266, and NCI-H929), or both for 4 days was also analyzed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay after culture for 4 days. Results represent the mean ± SD of three independent experiments. In D, ARH-77 cells (myeloma cell line) were treated with 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.

 
Because As2O3 induces apoptotic cell death of myeloma cells in vitro, we examined the effect of RWJ-241947 plus As2O3 on the induction of apoptosis. Flow cytometric analysis showed that As2O3 (10-6 M, 4 days) alone caused apoptosis of 18% 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)Citation . The quantitative analysis of apoptosis by the combination of these two agents in ARH-77, RPMI8226, U266, and NCI-H929 cell lines was also analyzed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay (Fig. 8C)Citation . The combination of both agents markedly increased apoptosis compared with either alone. Enhancement of apoptosis was further confirmed by the detection of PARP, which is a target of caspases. The cleaved form of PARP was detected only in the cells treated with both RWJ-241947 and arsenic trioxide for 2 days (Fig. 8D)Citation . The expression of several apoptosis-related genes, including Bcl-2 and BclXL, did not change after culture with RWJ-241947 (10-5 M) and arsenic trioxide (10-6 M) 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 with RWJ-241947 (10-5 M), arsenic trioxide (10-6 M), or both for 4 days, and their cell growth was measured by MTT assay. This combination had a slightly enhanced antiproliferative effect on PC-3 cells and, to a lesser extent, on MCF-7 cells compared with either agent alone. (Fig. 8E)Citation .

The PPAR{gamma} heterodimerizes with retinoid X receptor-{alpha}. Both receptors of this heterodimer can simultaneously combine with their respective ligands; and in certain circumstances, this can result in enhanced biological activity (8 , 43, 44, 45, 46) . All-trans retinoic acid is the treatment of choice for APL (47) . We also examined whether combining RWJ-241947 with either all-trans retinoic acid or 9-cic-retinoic acid in vitro could have either an additive or synergistic effects. The inhibition of growth of LNCaP, PC-3, MCF-7, HTB-182, NB-4 (APL cells), THP-1, NCI-H929, U266, and RPMI8226 cell lines was not enhanced by the combination of the TZD and a retinoid (data not shown).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our studies found that RWJ-241947 was able to inhibit the clonal proliferation of three prostate cancer cell lines in vitro and profoundly inhibited the in vivo proliferation of the human prostate cancer cell line, PC3, in nude mice. Prostate cancer cells express prominent levels of PPAR{gamma}, and this receptor does not appear to be mutated in prostate cancer (7 , 11 , 31) . In addition, we have shown previously that the androgen-independent prostate cancer cell line PC3 could be inhibited in its growth in nude mice by treatment with troglitazone. Our in vitro studies showed that RWJ-241947 increased the protein expression of the cyclin-dependent kinase inhibitor p21Waf1, decreased levels of cyclin E, and induced mild apoptosis of the PC-3 cells. Previous studies found that the expression of both p21Waf1 and p27Kip1 increased in cancer cells exposed to PPAR{gamma} ligands; this may have been mediated in part by inhibition of the ubiquitin–proteosome protein degradation pathway (48 , 49) . The increase in p21WAF1 and decrease of cyclin E could be associated with a decreased expression of phospholylated-retinoblastoma, but that was not the case in our experiments (data not shown). The blunting of expression of a variety of inflammatory cytokines and transcription factors, such as tumor necrosis factor, interleukin-1, interleukin-4, and nuclear factor-{kappa}ß, has been reported in the transformed cells exposed to PPAR{gamma} ligand. This may be associated with slowing the growth of transformed cells. On occasions, the PPAR{gamma} ligand can induce apoptosis of cells with activation of caspase 3.

The PSA protein is something regarded as a differentiation marker. It is also clearly an androgen-responsive gene. We found that RWJ-241947 can partially inhibit androgen-stimulated expression of PSA. We noted previously that TZDs can down-regulate PSA production in androgen-responsive LNCaP prostate cancer cells and also demonstrated that TZDs can inhibit androgen activation of the androgen response element in the regulatory region of the PSA gene (36) . Here, we also show by reporter gene assays that RWJ-241947 can inhibit androgen activation of the ARE of the PSA gene. This inhibition of androgen responsiveness may in part explain the antiproliferative effect of RWJ-241947 in LNCaP cells, but this cannot be the entire explanation because growth of AR-independent PC-3 cells was also inhibited by this PPAR{gamma} ligand. Patients with prostate cancer have received troglitazone after they had prostectomies with curative intent and subsequently developed a rising PSA, indicating a return of their prostate cancer. The largest study showed that 1 of 41 patients had a 50% decrease in serum PSA, and 7 of 41 individuals had a <51% decrease in their PSA. Of those 7 individuals, 4 had androgen-independent and 3 had androgen-dependent prostate cancer. Many of these responses appeared to last for ~18 months.

We showed that RWJ-241947 may increase the differentiation of LNCaP cells because it did increase the expression of E-cadherin in LNCaP cells. This protein has been associated with cell differentiation of prostate cancer cells (37) .

We found that the ability of RWJ-241947 to inhibit prostate tumor growth in experimental mice was particularly dramatic. It induced marked fibrosis, including an inflammatory and giant cell reaction of the tumor. This may represent a host reaction to the tumor or a secondary reaction to tumor necrosis. Apoptotic cell death was also detected in the experimental tumor. We noted previously that troglitazone was able potently to inhibit the growth of PC-3 tumors in nude mice. Using immunohistochemistry, we looked for difference of expression of growth-related genes in the tumor cells of the experimental mice. Slightly fewer dividing cells (Ki-67) were present in the tumors of the experimental mice, but levels of expression of p21WAF1 and Bcl-2 in the tumor cells of both cohorts of mice were similar. Probably the remaining tumor cells in the experimental mice are not as sensitive to the antiproliferative effects of 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 that RWJ-241947 affects macrophage differentiation, as shown by the induction of CD36, as well as by stimulating an inflammatory and giant cell reaction in the experimental tumors. Other PPAR{gamma} ligands have been shown to promote macrophage differentiation and enhance transcriptional induction of the scavenger receptor CD36 by ligand activation of the PPAR:retinoid X receptor heterodimers (39) .

The RWJ-241947 may mediate some of its antiproliferative effects by inhibition of angiogenesis. Rosiglitazone has been shown to suppress the primary tumor growth and metastasis by both direct and indirect antiangiogenetic effects (50) . Activated PPAR{gamma} down-regulates the production of vascular endothelial growth factor that is involved in the regulation of angiogenesis (51 , 52) . This may explain some of the anticancer effects of RWJ-241947 in our model system. Clearly, additional studies are required to understand why TZDs appear to have more in vivo cancer activity than might have been predicted from the in vitro data.

Previous studies have shown that the in vitro binding affinity for PPAR{gamma} by RWJ-241947 is much less than that of other TZDs, although it seems to have a similar anticancer activity. This might be explained by several pieces of data which suggest that some of the activities of PPAR{gamma} ligands can occur independently of PPAR{gamma}, including experiments on cells from homozygous PPAR{gamma} (PPAR{gamma}-/-) null mice. Exposure of normal activated macrophages to TZDs can decrease their release of inflammatory cytokines, such as tumor necrosis factor, interleukin-1, interleukin-6, COX-2, and inducible NO-synthatase (53, 54, 55) . Surprisingly, PPAR{gamma}-/- macrophages when activated and exposed to TZDs also exhibited decreased production of these anti-inflammatory molecules (41) . In a second series of experiments using embryonic stem cells from PPAR{gamma}-/- mice, TZDs inhibited their proliferation and DNA synthesis and caused them to arrest in the G1 phase of the cell cycle. In addition, the TZDs inhibited the growth of PPAR{gamma}-/- embryonic stem cell "tumors" growing in syngenic mice (56) .

We found that RWJ-241947 markedly reduced the total serum cholesterol in mice. Troglitazone also has cholesterol-lowering ability in vivo. But other TZDs, including rosiglitazone, pioglitazone, and 15d-PGJ2, showed only weak activity or no inhibition (57) . The mechanism for these differences is unclear at this time, but RWJ-241947 and troglitazone appear to be similar in their effect on serum lipids. The activated PPAR{gamma} plays a critical role in the regulation of cholesterol homeostasis by modulating the lipid metabolism by the macrophages. PPAR{gamma} is required for basal expression of CD36, the scavenger receptor responsible for uptake of the oxidized low-density lipoprotein in macrophages (39, 40, 41) . We found that RWJ-241947 was able to induce CD36 expression on monocytic cell lines in vitro, suggesting that it also can modulate the lipid metabolism via macrophages. The strong cholesterol-lowering activity of RWJ-241947 should be further investigated for potential therapeutic applications for atherosclerosis.

In general, arsenic compounds can be considered a poison and potential environmental carcinogen for the development of both lung and skin cancers (58 , 59) . However, the inorganic As2O3 is able to induce apoptosis of APL cells as well as a variety of other cancer cell types, including multiple myeloma. Clinical studies have shown that As2O3 is an efficacious drug for patients with multiple myeloma and APL (23, 24, 25, 26, 27, 28) . Previously, we showed that a ligand for the nuclear hormone receptor retinoic acid receptor plus an organic arsenical had a synergistic inhibitory activity against prostate and breast cancer cells (29) . These findings prompted us in this study to look at the potential synergy of a PPAR{gamma} ligand with an arsenical. We showed that, together, they had an enhanced antiproliferative activity against PC-3 prostate cells and a variety of myeloma cell lines. Arsenic trioxide has a fair amount of toxicity (60, 61, 62) ; perhaps by combining it with RWJ-241947, its dose can be lowered, reducing its side effects while enhancing its clinical efficacy.

In conclusion, our data demonstrate a surprisingly potent antiproliferative effect of RWJ-241947 against prostate cancer cells in vivo. It also enhanced the antiproloferative, proapoptotic activity of As2O3 against myeloma cells. This oral drug appears to have few side effects and may lower serum cholesterol levels. Together, the data suggest the possibility that RWJ-241947 may have a therapeutic role in cancer, perhaps in a chemoprevention role or clinical setting when the tumor burden is low.


   ACKNOWLEDGMENTS
 
We thank Kim Burgin for her excellent secretarial and administrative support.


   FOOTNOTES
 
Grant support: NIH grants as well as grants from Parker Hughes Fund and Johnson and Johnson. H. P. K. holds the Mark Goodson endowed Chair in Oncology Research and is a member of the Jonsson Cancer Center and Molecular Biology Institute, University of California at Los Angeles.

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 with 18 U.S.C. Section 1734 solely to indicate 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: kumamed1_2001{at}yahoo.co.jp

Received 3/25/03; revised 11/24/03; accepted 11/25/03.


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 INTRODUCTION
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 RESULTS
 DISCUSSION
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