Peroxisome proliferator-activated receptor-g and growth inhibition

by its ligands in prostate cancer

Daisuke Nagata MDa, Yoshihiro Hashimoto MD, PhDa,*, Makoto Nakanishi MD, PhDb,Hiromichi Naruyama MDa, Shinsuke Okada MDa, Ryosuke Ando MDa,

Keiichi Tozawa MD, PhDa, Kenjiro Kohri MD, PhDa

a Department of Nephro-urology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Japanb Department of Biochemistry and Cell Biology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi,

Mizuho-cho, Mizuho-ku, Nagoya, Japan

Accepted 30 May 2008

Abstract

Background: Peroxisome proliferator-activated receptor-g (PPAR-g) is expressed in certain human cancers. Ligand-induced PPAR-g

activation can result in growth inhibition and differentiation in these cancer cells; however, the precise mechanism for the anti-proliferative

effect of PPAR-g ligands is not clear. Methods: In this study, we examined the expression of PPAR-g in human prostate cancer and the effect of

two PPAR-g ligands, 15 deoxy-D12,14-prostaglandin J2 (15d-PGJ2) and troglitazone, on prostate cancer cell growth. Results: PPAR-g is

frequently over-expressed in androgen independent prostate cancer cell lines and human prostate cancer tissues (22 of 47; 47%). Both 15d-

PGJ2 and troglitazone inhibited proliferation and DNA synthesis of prostate cancer cell lines in a dose-dependent manner, and slightly

increased the proportion of cells with S-phase DNA content. Prostate specific antigen (PSA) promoter reporter assays showed that troglitazone

and 15d-PGJ2 down-regulated androgen stimulated reporter gene activity in prostate cancer cell lines LNCaP. Interestingly, LNCaP with

troglitazone dramatically suppressed PSA protein expression without suppressing AR expression. Conclusions: Taken together, these results

suggest that PPAR-g ligands may be a useful therapeutic agent for the treatment of prostate cancer.

# 2008 Elsevier Ltd. All rights reserved.

Keywords: PPAR-g; Ligand; Prostate cancer; Prostate cancer cell line LNCaP; Prostate cancer cell line PC-3; Prostate cancer cell line DU145; S-phase cell

arrest; Prostate specific antigen promoter; Prostate cancer cell proliferation

www.elsevier.com/locate/cdp

Cancer Detection and Prevention 32 (2008) 259–266

1. Introduction

Prostate cancer is the most common cancer among men

and the second leading cause of male cancer death [1].

Although surgical resection or radiotherapy is potentially

curative for localized disease, advanced prostate cancer is

associated with a poor prognosis. Conventional chemother-

apy and radiotherapy are of limited effectiveness. Blockade

of androgen stimulation often leads to either a partial or full

Abbreviations: 15d-PGJ2, 15-deoxy-D12,14-prostaglandin J2; AR,

androgen receptor; DHT, 5alpha-dihydrotestosterone; FACS, fluores-

cence-activated cell sorter; PPAR, peroxisome proliferator-activated recep-

tor; PSA, prostate specific antigen.

* Corresponding author. Tel.: +81 52 853 8266; fax: +81 52 852 3179.

E-mail address: hashi@med.nagoya-cu.ac.jp (H. Yoshihiro).

0361-090X/$ – see front matter # 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.cdp.2008.05.008

remission; however, subsequent relapse often occurs and the

disease re-emerges within a few years in a poorly

differentiated, androgen-independent form. The shortage

of curative therapies for advanced disease has provided

impetus for the development of novel therapies.

Peroxisome proliferator-activated receptor (PPAR) is a

member of the steroid hormone receptor superfamily

involved in ligand-inducible regulation [2–5]. There are

three types of PPARs: PPAR-a, PPAR-b, and PPAR-g. All

PPAR isoforms require heterodimerization with the retinoid

X receptor for optimal DNA binding and transcriptional

activity [6].

PPAR-g is an important molecule for adipocyte

differentiation and is over-expressed in adipose tissue

[7,8]. In addition to adipose tissue, PPAR-g expression is

D. Nagata et al. / Cancer Detection and Prevention 32 (2008) 259–266260

detected in a wide variety of cancer cells [9–13]. The

receptor requires ligand activation, and several ligands

have been identified including synthetic antidiabetic

thiazolidinedione (TZD) drugs and natural ligands such

as 15d-PGJ2 [14]. Troglitazone is a TZD that has been

widely used for insulin-resistant diabetes mellitus, and has

been identified as a specific ligand for PPAR-g [15–17]. In

cancer cells, PPAR-g activation by its high affinity ligands

can inhibit cell proliferation [18,19]. Thus PPAR-g is

involved in not only lipid metabolism but also cellular

proliferation in cancer cells. Therefore, it is suggested that

PPAR-g is a possible molecular target for cancer

treatment. Although increasing evidence has established

that PPAR-g activation induces growth arrest in cancer

cells, the molecular mechanism of the growth inhibition by

PPAR-g ligands is not well understood. Some researchers

have recently demonstrated that the cyclin-dependent

kinase inhibitor may be a crucial molecule in the cell

growth inhibition by PPAR-g ligands in human cancer

cells [18–21]. In this study, we show that PPAR-g is

expressed in human prostate cancer, and that ligand

activation of this nuclear hormone receptor inhibits

prostate cancer cell proliferation and activity of andro-

gen-responsive gene, such as prostate specific antigen

(PSA).

2. Materials and methods

2.1. Cells and samples

Prostate cancer cell lines (LNCaP, PC-3, and DU145)

were obtained from the American Type Culture Collection

(Rockville, MD). PC-3 and DU145 were maintained in

Dulbecco’s modified eagle’s media (DMEM) with 10%

FCS. LNCaP was grown in RPMI1640 with 10% FCS. 15-

deoxy-D12,14-prostaglandin J2 (15d-PGJ2) and troglita-

zone (Sigma Chemical Co.; St. Louis, MO) were

dissolved in a solution containing 50% dimethyl sulfoxide

(DMSO) and 50% ethanol and applied to cells at a

concentration of <0.1% of the media volume. Prostate

cancer tissue and adjacent normal prostate tissue (n = 47)

were surgically obtained after informed consent from

patients, and were fixed by a standard method for

immunohistochemistry.

2.2. Cell growth assay (cell viability assay)

For experiments, each prostate cancer cell line was plated

at a density of 2 � 103 cm�2. One day later (at day 0), the

medium was changed to the maintenance medium with 15d-

PGJ2 or troglitazone. Adherent cells were harvested by

trypsinization and collected by centrifugation. Nonadherent

cells were collected from spent media by centrifugation.

Cell pellets were resuspended in 100 ml of fresh media, and

trypan blue solution (Sigma) was added at a ratio of 1:1.

Viable cells were counted by trypan blue exclusion using a

hemocytometer as a percentage of a total of 500 cells. Each

experiment was carried out in quadruplicate and repeated at

least three times.

2.3. Cell cycle analysis

After treatment with ligands at varying concentrations

(described in Table 2), cells were washed with chilled

phosphate-buffered saline (PBS) and kept on ice in

phosphate-buffered saline containing 2 mM ethylenediami-

netetraacetate (EDTA) (Sigma Chemical Co.) for 10 min.

Cells were collected using a cell scraper, suspended in PBS,

and fixed with 0.25% paraformaldehyde (Sigma Chemical

Co.). Fixed cells were washed once with PBS, incubated

with 100 mg of RNase A (Sigma Chemical Co.) in PBS

containing 0.2% Tween 20, and stained with 50 mg/ml of

propidium iodide (Sigma Chemical Co.). Cell cycle analysis

was performed with a fluorescence-activated cell sorter

(FACS) Calibur flow cytometer (Becton Dickinson, Mans-

field, MA). The percentage of cells in the G0/G1, S and G2/

M phases of the cell cycle was determined using the Modfit

LT software program (Verity Software House Inc., Topsham,

ME).

2.4. DNA synthesis inhibitory assay

Cell proliferation was determined by measurement of

tritiated thymidine incorporation during DNA synthesis via

a radioactive assay as described previously [22]. All cells

were treated with 0, 2.5, and 10 mM 15d-PGJ2 or 0, 5, and

20 mM toglitazone for 24 h. Additionally these cells were

incubated with tritiated thymidine (1 mCi/ml) solution for

24 h prior to harvesting.

2.5. Western blot analysis

Total cell lysates were prepared by boiling cells in

sodium dodecylsulfate (SDS) sample buffer after treatment

with 15d-PGJ2 and troglitazone. Samples were equalized to

20 mg/lane and run on a 12% SDS-polyacrylamide gel.

Proteins were transferred to nitrocellulose membranes (Bio-

Rad). Blocked membranes were incubated overnight with

either anti-PPAR-g polyclonal antibody (Santa Cruz; 1:2000

dilution), anti-AR polyclonal antibody (Santa Cruz; 1:500

dilution), anti-PSA polyclonal antibody (Santa Cruz; 1:500

dilution), or anti-b-actin polyclonal antibody (Santa Cruz;

1:500 dilution). Three 10-min washes in Tris-buffered saline

(TBS)-Tween 20 (20 mM Tris–HCl, 137 mM NaCl, and

0.1% Tween 20, pH 7.6) were carried out between antibody

steps, followed by incubation with antirabbit secondary

antibody conjugated to horseradish peroxidase (Amersham;

1:2000 dilution) for 1 h at room temperature. The signals

were detected by enhanced chemiluminescence (Amer-

sham). The stripping procedures were performed using

standard methods.

D. Nagata et al. / Cancer Detection and Prevention 32 (2008) 259–266 261

2.6. Immunohistochemistry

Immunohistochemical staining was performed with the

Vectastatin avidin–biotin peroxidase complex kit (Vector,

Burlingame, CA). Formalin-fixed, paraffin-embedded speci-

mens were sectioned onto microscope slide at a thickness of

3 mm, and then deparaffinized in xylene and rehydrated in

graded ethanol. Endogenous peroxide was blocked with

0.3% hydrogen peroxide in methanol for 30 min. Sections

were reacted with the primary antibody (Santa Cruz; anti-

PPAR-g polyclonal antibody diluted at 1:500) for 24 h at

4 8C, washed with PBS, and incubated with biotinylated

secondary antibody for 30 min at room temperature,

followed by treatment with the avidin–biotin peroxidase

complex (DAKO) for 30 min at room temperature. Sections

were washed with PBS, and peroxidase activity was

visualized with 0.03% 3,30-diaminobenzidine tetrahy-

drochloride containing 0.06% H2O2 in ammonium acet-

ate–citric buffer. Sections were counterstained with

hematoxylin. For each slide, the extent and intensity of

staining with PPAR-g antibody was graded on a scale of 0–

3+ by two blinded observers on two separate occasions using

coded slides, and an average score was calculated.

2.7. Transfections and luciferase assay

LNCaP cells were incubated in RPMI 1640 with 10%

FBS until 50–70% confluency. Cells were transfected with

PSA promoter luciferase reporter gene using the Lipofecta-

mine plus (Gibco) under serum-free conditions. A pCMV-b-

galactosidase vector was included as an internal control for

efficiency of transfection. Following transfections, cells

were incubated in RPMI1640 with 10% charcoal-stripped

fetal bovine serum FBS either with or without 10–9 M

Fig. 1. Expression of PPAR-g in prostate cancer cell lines and human prostate tissu

were extracted from four prostate cancer cell lines. The lysates were electrophorese

antibody was removed from the membrane and incubated with an antibody to b

according to actin probing. (B) Immunohistochemistry for PPAR-g in prostate can

area (left: �400). In contrast, we found significant expression of immunoreactiv

5alpha-dihydrotestosterone (DHT) and either with or

without ligands for PPAR-g for 72 h. Cells were collected

with tissue lysis buffer. Luciferase activity of the cell lysates

was measured by luminometry, and activities were normal-

ized by b-galactosidase activities. All transfection experi-

ments were carried out in triplicate wells and repeated

separately at least three times.

3. Results

3.1. Expression of PPAR-g in prostate cancer cell lines

and human prostate tissues

We initially examined the expression levels of PPAR-g in

prostate cancer cell lines using Western blot analysis

(Fig. 1A). We found variable levels of PPAR-g protein with

moderate expression seen in the androgen independent cell

lines, PC-3 and DU145, and low expression in the androgen-

dependent cell line, LNCaP. Expression levels were

corrected for differences in protein loading according to

probing with antibody to b-actin. In addition, we evaluated

the expression levels of PPAR-g in human prostate tissues by

immunohistochemistry. Tumor area was expressed at a

significant level; in contrast, non-tumor prostate area was

expressed at a very low or negligible level (Fig. 1B and

Table 1).

3.2. PPAR-g ligands inhibit proliferation of prostate

cancer cells

Each prostate cancer cell line was plated at a density of

2 � 103 cm�2 and incubated in the maintenance medium

with 15d-PGJ2 or troglitazone. The effect of each ligand on

es. (A) Western blot analysis of PPAR-g in prostate cancer cell lines Lysates

d, blotted on membrane, and reacted with a specific antibody to PPAR-g. The

-actin. Expression levels were corrected for differences in protein loading

cer. Weak expression of immunoreactive PPAR-g was found in non-tumor

e PPAR-g in tumor area (right: �400).

D. Nagata et al. / Cancer Detection and Prevention 32 (2008) 259–266262

Table 1

Immunohistochemistry of PPAR-g in human prostate cancer patients

Intensity Positive (%) Total samples

+ + + + + +

Tumor 5 13 4 22 (46.8%) 47

Non-tumor 6 0 0 6 (12.8%) 47

Tissues were obtained from the center of grossly recognizable nodules of

prostate cancer. The formalin-fixed, paraffin-embedded tissue sections were

deparaffinized, rehydrated and subjected to immunohistochemistry study

with anti-PPAR-g antibody (1:200 dilution, Santa Cruz) according to

standard protocols. The staining intensity was scored on a 0–3 scale from

negative to strong immunoreactivity.

the growth response of the cells was determined by a cell

viability assay using trypan blue. Low concentrations of

troglitazone (5 mM) and 15d-PGJ2 (2.5 mM) blocked cell

proliferation on all prostate cancer cell lines (Fig. 2).

3.3. PPAR-g ligands inhibit DNA synthesis of prostate

cancer cells

To confirm the effect of PPAR-g ligands on cell

proliferation, the incorporation of tritiated thymidine into

DNA was measured in a radioactive DNA synthesis assay.

As shown in Fig. 3, treatment with 15d-PGJ2 or troglitazone

decreases DNA synthesis significantly in all prostate cancer

cell lines at 24 h post-treatment.

3.4. PPAR-g ligands treatment causes S-phase cell

arrest in prostate cancer cells

Because of the profound inhibition of cellular prolifera-

tion caused by PPAR-g ligands, we wanted to determine

whether the cell cycle was altered. LNCaP, PC-3, and

DU145 were treated with 10 mM 15d-PGJ2 or 20 mM

Table 2

Cell cycle analysis of prostate cancer cell lines treated with PPAR-g ligands

Proportion of cells

G0/G1 S

LNCaP

Control 72.23 � 2.08 3.

15d-PGJ2 (10 mM) 68.37 � 0.71 15.

Troglitazone (20 mM) 74.22 � 1.74 9.

PC-3

Control 54.18 � 0.48 24.

15d-PGJ2 (10 mM) 47.16 � 1.59 39.

Troglitazone (20 mM) 58.13 � 2.43 31.

DU145

Control 53.56 � 1.24 17.

15d-PGJ2 (10 mM) 35.27 � 4.59 44.

Troglitazone (20 mM) 56.14 � 5.41 29.

Cells were treated with 10 mM 15d-PGJ2 or 20 mM troglitazone for 48 h. The effec

determined. Data shown is representative of three experiments. Values are mean

troglitazone for 48 h, at which time cell cycle changes were

studied by FACS analysis. These cell lines showed no

significant increase in the proportion of cells in the G0–G1

phase and the apoptotic phase of cell cycle. All cell lines

showed mild accumulation of cells in the S-phase at 48 h

post-treatment with 15d-PGJ2 and troglitazone compared

with control cells (Table 2).

3.5. PPAR-g ligands down-regulate PSA promoter

activity in LNCaP cell

To explore potential mechanisms by which PPAR-g

ligands inhibited levels of PSA as androgen-responsive

gene, we analyzed the effect of 15d-PGJ2 and troglitazone

on the ability of DHT to transactivate the PSA promoter. The

LNCaP prostate cancer cells were cultured with DHT after

being transfected with the PSA promoter luciferase reporter.

The reporter activity increased about 17-fold as compared

with nontreated control LNCaP cells. This result was

consistent with previous observations. When cells were

treated with both troglitazone and DHT, luciferase activity

dramatically reduced by 65% compared with DHT alone

(Fig. 4).

3.6. Effect of PPAR-g ligands on PSA, AR, and PPAR-g

expression in LNCaP cell

Cytoplasmic PSA protein expression was studied by

Western blot analyses of LNCaP cells cultured with or

without troglitazone and DHT for different durations

(Fig. 5). LNCaP cells constitutively expressed PSA protein,

and when these cells were incubated with DHT, PSA

expression increased 1.7-fold after 48 h of culture.

Troglitazone suppressed PSA expression induced by DHT

at each time point. For example, at day 2, troglitazone

G2/M Apoptotic

21 � 0.23 24.57 � 1.86 1.21 � 0.13

17 � 0.11 16.81 � 0.78 1.77 � 0.35

47 � 0.34 16.32 � 0.62 0.94 � 0.26

52 � 0.18 21.31 � 0.60 2.17 � 0.53

71 � 0.58 13.14 � 0.70 4.65 � 1.71

69 � 3.12 10.18 � 1.08 2.93 � 0.42

26 � 1.17 29.20 � 0.12 3.52 � 0.88

38 � 5.45 20.15 � 2.33 6.46 � 1.26

73 � 1.76 14.34 � 0.97 3.19 � 0.79

t of these ligands on the proportion of cells in each phase of the cell cycle was

� S.D.

D. Nagata et al. / Cancer Detection and Prevention 32 (2008) 259–266 263

Fig. 2. Effects of PPAR-g ligands on prostate cancer cell analyzed by cell number. After being plated at 2 � 103 cm�2 in the maintenance medium (described in

Section 2), cells were incubated in maintenance medium with 15d-PGJ2 or troglitazone on the following day. The number of viable cells was counted by trypan

blue exclusion using a hemocytometer. Data are shown as mean of the triplicate dishes.

D. Nagata et al. / Cancer Detection and Prevention 32 (2008) 259–266264

Fig. 3. Effects of PPAR-g ligands on prostate cancer cell analyzed by DNA

synthesis. LNCaP, PC-3 and DU145 were treated with 15d-PGJ2 or

toglitazone for 24 h. Additionally, cells were incubated with tritiated

thymidine (1 mCi/ml) solution for 24 h prior to harvesting. The amount

of synthesized DNA was measured by the method described in Section 2

(n = 3); bars, S.D.

Fig. 5. Western blot analysis of PSA, AR, PPAR-g and b-actin in LNCaP

cells treated with troglitazone. Cells were incubated in medium with 10%

charcoal-striped FBS for 24 h before the addition of 10�9 M DHT either

with (+) or without (�) 10�5 M troglitazone. After the addition of reagents,

cells were incubated for 24 and 48 h. Control: cell lysates harvested before

the addition of reagents. The relative abundance of band intensity was

measured by densitometric scanning.

suppressed PSA levels by 70% as compared with cells

cultured with DHT alone. Moreover, we examined levels of

AR in LNCaP to determine whether troglitazone suppressed

PSA through inhibition of AR expression. Troglitazone did

not significantly affect levels of AR expression compared

Fig. 4. Effect of PPAR-g ligands on transcriptional activity of PSA

promoter in LNCaP cells. LNCaP cells were transfected with PSA-Luc.

DHT (10�9 M) either with or without PPAR-g ligand (15d-PGJ2: 10 mM,

troglitazone: 20 mM) was added. pCMV-b-galactosidase vector was

cotransfected for normalization. S.D.s from three or more repetitions of

the experiment are shown. *P < 0.05 as determined by Student’s t test

difference compared with DHT alone.

with LNCaP cells cultured with DHT alone. Furthermore,

neither DHT nor troglitazone affected levels of PPAR-g

(Fig. 5).

4. Discussion

PPAR-g belongs to the nuclear steroid receptor super-

family, which includes receptors for steroids, thyroid

hormone, retinoid acid and vitamin D. These receptors

are critical for cellular growth and differentiation [2,3].

Previous studies have reported that PPAR-g ligands activate

PPAR-g expressing cells including adipocytes, fibroblasts,

myoblasts, and help to induce terminal differentiation.

PPAR-g forms a heterodimeric complex that functions as a

central regulator of adipocyte differentiation [7,8]. PPAR-g

is activated by several prostanoids, prostaglandin-like

molecules, and arachidonic acid metabolites [3]. Moreover,

PPAR-g ligands induce growth arrest through apoptosis in

macrophages and endothelial cells [23]. It has also been

reported that PPAR-g ligands can inhibit growth of several

cancer cells in vitro and in vivo [24–26].

Our Western blot data show that the three prostate cancer

cell lines studied here, as well as prostate cancer tissues

expressed PPAR-g. We tested the hypothesis that prostate

cancer cells that express PPAR-g may be inhibited in their

proliferation by PPAR-g ligands. We studied the effects of

low and high dose treatments of the PPAR-g ligands, 15d-

PGJ2 and troglitazone on parameters of cell proliferation in

three prostate cancer cell lines, LNCaP, PC-3 and DU145.

Low concentrations of troglitazone (5 mM) and 15d-PGJ2

(2.5 mM) blocked cell proliferation and suppressed DNA

synthesis on all prostate cancer cell lines (Figs. 2 and 3). In

addition, we have utilised the selective PPAR-g antagonist

GW9662 to elucidate the involvement of PPAR-g in the

growth inhibitory effects observed. The inhibitory effects

D. Nagata et al. / Cancer Detection and Prevention 32 (2008) 259–266 265

exerted by PPAR-g ligands were not reversed by the addition

of the GW9662 (supplementary data). Our findings

demonstrate that the effects induced by 15d-PGJ2 and

troglitazone in the prostate cancer cell lines are PPAR-g

independent. The levels of PPAR-g expression varied

significantly amongst the prostate cancer cell lines examined

and these differing levels did not associate with the observed

growth inhibition. All prostate cancer cell lines showed

accumulations of cells in the S-phase after 48 h of treatment

with PPAR-g ligands and no significant increase in the

proportion of cells in the G0-G1 phase and the apoptotic

phase of cell cycle (Table 2). However, the androgen-

dependent cell line, LNCaP, exhibited a lesser effect than the

androgen independent cell lines DU145 and PC-3. LNCaP

cells grow very slowly in culture and therefore may not have

completed an entire cell cycle before the effects of PPAR-g

occur. The completion of cell cycle events in an orderly

manner, from the G1 stage to DNA replication in S-phase or

from the G2 stage to mitosis, is controlled by many factors,

which act as checkpoints to ensure that no mistakes occur

[27]. Therefore, in addition to carrying out FACS analysis to

study the cell cycle, we also studied changes in the protein

expressions of certain cell cycle regulating factors over the

time course of 15d-PGJ2 treatment. The cell cycle regulators

studied were p53, p21 and p27 all are involved in controlling

various checkpoints in the cell cycle. However, no changes

were observed in the expression levels of any of these

proteins over the time period in which cell cycle arrest

occurred (supplementary data).

Additional experiments explored whether PPAR ligands

could also inhibit androgen-induced production of PSA.

Exposure of LNCaP cells simultaneously to DHT and

troglitazone resulted in inhibition of accumulation of PSA

protein compared with exposure to DHT alone, as shown

by Western blotting (Fig. 5). Reprobing of the Western blot

showed that the down-regulation of PSA expression was

not mediated through down-regulation of androgen

receptor (AR). Because troglitazone is able to inhibit

transactivation of a number of secondary signaling

pathways, it may be either inhibiting coactivators or

stimulating corepressors of the activated AR. A recent

report has shown that activated AR requires coactivators

for efficient expression of androgen-responsive genes

[28,29]. Another less likely hypothesis comes from studies

that have shown that induction of immediate early genes

such as c-Jun and c-Myc can be mediated by PPAR-g

ligands and complexes of c-Jun and c-Fos can disrupt

transactivation of the ARs [30,31]. Additional studies are

clearly required to elucidate the actual mechanism by

which PPAR-g ligands mediate their antiandrogen

activities.

The anti-proliferative effects of PPAR-g ligands cannot

totally be explained by inhibition of the androgen-signaling

pathway. Prior studies show that PC-3 cells, which have a

nonfunctional, mutated AR, are inhibited in their prolifera-

tion in vitro and in vivo by PPAR-g ligands [32]. In addition,

studies by others as well as ourselves have demonstrated that

cancer cells from a variety of tissues including colon, breast,

fat, and stomach can be inhibited in their proliferation by

PPAR-g ligands; and these cancers are not under androgen

control. This would be congruent with the hypothesis that

PPAR-g ligands are affecting key cofactors of activated

signaling pathways in these transformed cells.

In this study, we demonstrate that PPAR-g ligands

inhibited proliferation of human prostate cancer cells and

transactivation of PSA, suggesting that troglitazone inhib-

ited PSA induction by a mechanism other than down-

regulation of the AR. Taken together, these ligands may

represent a useful adjuvant therapy for prostate cancer,

particularly for patients who have minimal residual disease

after surgery or radiotherapy with curative intent.

Acknowledgement

This work was supported by a Grant-in-Aid for Scientific

Research (#17591692) from the Ministry of Education,

Culture, Sport, Science and Technology of Japan.

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.cdp.2008.

05.008.

Conflict of interest

None declared.

References

[1] Jemal A, Murray T, Samuels A, Ghafoor A, Ward E, Thun MJ. Cancer

statistics. CA Cancer J Clin 2003; 53:5–26.

[2] Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Ume-

sono K, et al. The nuclear receptor superfamily: the second decade.

Cell 1995; 83:835–9.

[3] Yu K, Bayona W, Kallen CB, Harding HP, Ravera CP, McMahon G,

et al. Differential activation of peroxisome proliferator-activated

receptors by eicosanoids. J Biol Chem 1995; 270:23975–83.

[4] Qi C, Zhu Y, Reddy JK. Peroxisome proliferator-activated receptors,

coactivators, and downstream targets. Cell Biochem Biophys 2000;

32:187–204.

[5] Kliewer SA, Xu HE, Lambert MH, Willson TM. Peroxisome prolif-

erator-activated receptors: from genes to physiology. Recent Prog

Horm Res 2001; 56:239–63.

[6] Kim MJ, Deplewski D, Ciletti N, Michel S, Reichert U, Rosenfield RL.

Limited cooperation between peroxisome proliferator-activated recep-

tors and retinoid X receptor agonists in sebocyte growth and devel-

opment. Mol Genet Metab 2001; 74:362–9.

[7] Soret B, Lee HJ, Finley E, Lee SC, Vernon RG. Regulation of

differentiation of sheep subcutaneous and abdominal preadipocytes

in culture. J Endocrinol 1999; 161:517–24.

D. Nagata et al. / Cancer Detection and Prevention 32 (2008) 259–266266

[8] Fontaine C, Dubois G, Duguay Y, Helledie T, Vu-Dac N, Gervois P,

et al. The orphan nuclear receptor Rev-Erbb is a peroxisome pro-

liferator-activated receptor (PPAR)-g target gene and promotes

PPAR-g-induced adipocyte differentiation. J Biol Chem 2003;

278:37672–80.

[9] Mueller E, Sarraf P, Tontonoz P, Evans RM, Martin KJ, Zhang M, et al.

Terminal differentiation of human breast cancer through PPAR-g. Mol

Cell 1998; 1:465–70.

[10] Allred CD, Kilogore MW. Selective activation of PPARgamma in

breast, colon, and lung cancer cell lines. Mol Cell Endocrinol 2005;

235:21–9.

[11] Nomura S, Nakajima A, Ishimine S, Matsuhashi N, Kadowaki T,

Kaminishi M. Differential expression of peroxisome proliferator-

activated receptor in histologically different human gastric cancer

tissues. J Exp Clin Cancer Res 2006; 25:443–8.

[12] Cesario RM, Stone J, Yen WC, Bissonnette RP, Lamph WW.

Differentiation and growth inhibition mediated via the RXR:PPAR-

gamma heterodimer in colon cancer. Cancer Lett 2006; 240:

225–33.

[13] Kristiansen G, Jacob J, Buckendahl AC, Grutzmann R, Dietel M,

Pilarsky C. Peroxisome proliferator-activated receptor gamma is

highly expressed in pancreatic cancer and is associated with shorter

overall survival times. Clin Cancer Res 2006; 12:6444–51.

[14] Soares AF, Nosjean O, Cozzone D, Lagarde M, Geloen A. Covalent

binding of 15-deoxy-delta12,14-prostaglandin J2 to PPAR gamma.

Biochem Biophys Res Commun 2005; 337:521–5.

[15] Murphy GJ, Holder JC. PPAR-gamma agonists: therapeutic role in

diabetes, inflammation and cancer. Trends Pharmacol Sci 2000;

21:469–74.

[16] Olefsky JM, Saltiel AR. PPAR gamma and the treatment of insulin

resistance. Trends Endocrinol Metab 2000; 11:362–8.

[17] Willson TM, Lambert MH, Kliewer SA. Peroxisome proliferator-

activated receptor gamma and metabolic disease. Annu Rev Biochem

2001; 70:341–67.

[18] Motomura W, Takahashi N, Nagamine M, Sawamukai M, Tanno S,

Kohgo Y, et al. Growth arrest by troglitazone is mediated by p27Kip1

accumulation, which results from dual inhibition of proteasome

activity and Skp2 expression in human hepatocellular carcinoma cells.

Int J Cancer 2004; 108:41–6.

[19] Han S, Sidell N, Fisher PB, Roman J. Up-regulation of p21 gene

expression by peroxisome proliferator-activated receptor gamma in

human lung carcinoma cells. Clin Cancer Res 2004; 10:1911–9.

[20] Radhakrishnan SK, Gartel A. The PPAR-gamma agonist pioglitazone

post-transcriptionally induces p21 in PC3 prostate cancer but not in

other cell lines. Cell Cycle 2005; 4:582–4.

[21] Fujii D, Yoshida K, Tanabe K, Hihara J, Toge T. The ligands of

peroxisome proliferator-activated receptor (PPAR) gamma inhibit

growth of human esophageal carcinoma cells through induction of

apoptosis and cell cycle arrest. Anticancer Res 2004; 24:1409–16.

[22] Nakanishi M, Robetorye RS, Adami GR, Pereira-Smith OM, Smith

JR. Identification of the active region of the DNA synthesis inhibitory

gene p21 Sdi1/CIP1/WAF1. EMBO J 1995; 14:555–63.

[23] Eligini S, Banfi C, Brambilla M, Camera M, Barbieri SS, Poma F, et al.

15-deoxy-delta12,14-Prostaglandin J2 inhibits tissue factor expression

in human macrophages and endothelial cells: evidence for ERK1/2

signaling pathway blockade. Thromb Haemost 2002; 7:524–32.

[24] Kubota T, Koshizuka K, Williamson EA, Asou H, Said JW, Holden S,

et al. Ligand for peroxisome proliferator-activated receptor gamma

has potent antitumor effects against human prostate cancer both in

vitro and in vivo. Cancer Res 1998; 58:3344–52.

[25] Elstner E, Muller C, Koshijuka K, Williamson EA, Park D, Asou H,

et al. Ligands for peroxisome proliferator-activated receptor and

retinoic acid receptor inhibit growth and induce apoptosis of human

breast cancer cells in vitro and in BNX mice. Proc Natl Acad Sci USA

1998; 95:8806–11.

[26] Fulzele SV, Chatterjee A, Shaik MS, Jackson T, Ichite N, Singh M,

et al. 15-Deoxy-Delta12,14-prostaglandin J2 enhances docetaxel anti-

tumor activity against A549 and H460 non-small-cell lung cancer cell

lines and xenograft tumors. Anticancer Drugs 2007; 11:65–78.

[27] Theocharis S, Margeli A, Vielh P, Kouraklis G. Peroxisome prolif-

erator-activated receptor-gamma ligands as cell-cycle modulators.

Cancer Treat Rev 2004; 30:545–54.

[28] He B, Gampe RT, Kole AJ, Hnat AT, Stanley TB, An G, et al.

Structural basis for androgen receptor interdomain and coactivator

interactions suggests a transition in nuclear receptor activation func-

tion dominance. Mol Cell 2004; 16:425–38.

[29] Estebanez-Perpina E, Moore JM, Mar E, Webb P, Fletterick RJ, Guy

RK. The molecular mechanisms of coactivator utilization in ligand-

dependent transactivation by the androgen receptor. J Biol Chem 2005;

280:8060–8.

[30] Edwards J, Krishna NS, Mukherjee R, Bartlett JM. The role of c-Jun

and c-Fos expression in androgen-independent prostate cancer. J

Pathol 2004; 204:153–8.

[31] Chen SY, Cai C, Fisher CJ, Zheng Z, Omwancha J, Hsieh CL, et al. c-

Jun enhancement of androgen receptor transactivation is associated

with prostate cancer cell proliferation. Oncogene 2006; 25:7212–23.

[32] Shappell SB, Gupta RA, Manning S, Whitehead R, Boeglin WE,

Wheeler TM, et al. 15S-Hydroxyeicosatetraenoic acid activates per-

oxisome proliferator-activated receptor gamma and inhibits prolifera-

tion in PC3 prostate carcinoma cells. Cancer Res 2001; 61:497–503.