# Peroxisome Proliferator-Activated Receptor-γ Is a Potent Target for Prevention and Treatment in

Authors: Masahide Matsuyama; Rikio Yoshimura
Journal: PPAR Research
 (2008)

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## Abstract

Peroxisome proliferator-activated receptor-γ (PPAR)-γ is a ligand-activated transcriptional factor belonging to steroid receptor superfamily. PPAR-γ plays a role in both adipocyte differentiation and tumorigenesis. Up to date, PPAR-γ is expressed in various cancer tissues, and PPAR-γ ligand induces growth arrest of these cancer cells. In this study, we examined the expression of PPAR-γ in prostate cancer (PC) and testicular cancer (TC) by RT-PCR and immunohistochemistry, and we also examined the effect of PPAR-γ ligand in these cells by MTT assay, hoechest staining, and flow cytometry. PPAR-γ expression was significantly more extensive and intense in malignant tissues than in normal tissues. PPAR-γ ligand induced the reduction of malignant cell viability through apoptosis. These results demonstrated that the generated PPAR-γ in PC and TC cells might play an important role in the tumorigenesis. PPAR-γ may become a new target in the treatment of PC and TC.


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## Body

## 1. Introduction

Prostate cancer (PC) comprises
32% of all cancers in American men and is on the increase worldwide.  Because of
increased screening, PC is frequently diagnosed at a clinically localized
stage, making it amenable to the therapy. Nevertheless, it remains the second
most common cause of cancer death in men. These patients generally respond to
androgen deprivation therapy, but the vast majority eventually experience disease
progression and become refractory to sustained hormonal manipulation.
Typically, such patients progress with a rise in their serum prostate-specific
antigen level. Unfortunately, standard therapeutic options at this stage of
disease are limited, and while there has been some success with chemotherapy
for hormone-refractory prostate cancer patients, the response is generally
short lived [1].Testicular cancer (TC) is very rare with over 90% of
all TC being germ cell tumors (seminoma and nonseminoma), and the remaining
percentage nongerminal tumors. The survival rate of TC has improved in recent
years, reflecting the development and refinement of effective combination
chemotherapy. However, it is still necessary to improve the treatment of TC.Angiogenetic
factors play an important role in prostate and testis as in other organs [2], and although various potential angiogenetic factors
have been identified in PC and TC, it is still unclear by which process PC and
TC cells become angiogenic. Thus, the challenge is to discover new treatment
strategies that target androgen-independent PC and TC. The identification of molecular
targets involved in the tumorigenesis and progression of PC and TC provide opportunities
for the development of new agents with greater therapeutic potential and better
specificity.  Patients with advanced or recurrent disease are suitable
candidates for studies that test the efficacy of these new agents.Peroxisome proliferator-activated receptors (PPARs) are
lipid-activated transcription factors that function as important regulars of
lipid and glucose metabolism,  adipocyte differentiation, and energy homeostasis. PPAR
subtypes (α, β, and γ) have been found. Both PPAR-α and -γ mediate the action
of the hypolipidemic fibrates and antidiabetic thiazolidinediones. PPARs
therefore play a role in metabolic conditions such as dyslipidemia and type 2 diabetes, leading to
atherosclerosis development [3].
PPARs also have regulatory role in inflammation.PPAR-γ provides a strong link between lipid metabolism
and regulation of gene transcription [4].
PPAR-γ acts in adipose tissue and promotes lipogenesis under anabolic
conditions. Recently, the receptor has also been implicated in inflammation and
tumorigenesis. Significant evidence from many experimental systems suggests that PPAR-γ is important
in carcinogenesis.PPAR-γ is up regulated in malignant tissue, and PPAR-γ
ligands induce terminal differentiation in human breast and colon cancer cells [5, 6], and inhibit the growth of human lung
and gastric cancer cells [7, 8]. In addition,
PPAR-γ ligands induce growth arrest through apoptosis in macropharge,
fibrobrasts, and endothelial cells [3, 9, 10]. Our research
elucidates the expression of PPARs in urological cancers and administration of
PPAR-γ ligands as an anticancer therapy [11–15]. Several reports support the expression of PPAR-γ and the efficacy of PPAR-γ ligands in
PC [16–18]. However,  no further data on TC and PPAR-γ
have been documented in other
reports.Our research focuses on the relationship between PPAR-γ
and male reproductive system (prostate and testis) and on the anticancer effect
of PPAR-γ ligands.


## 2. Methods

### 2.1. Tumor Specimens

Prostate specimens were obtained from 156 patients
with PC; 15 with prostatic intraepithelial neoplasia (PIN); 20 with benign
prostatic hyperplasia (BPH), who underwent biopsy due to serum prostate-specific antigen increase; and 12 patients with normal prostate (NP)
tissues who underwent total cystectomy due to bladder cancer.Testis specimens were obtained from 72 TC patients,
and from 20 NT patients who underwent orchiectomy for PC. Tumor tissues,
nontumor tissues, vascular endothelium, and interstitial tissues from the
subjects were preserved in 10% formalin and embedded in paraffin, serially
sectioned onto microscope slides at a thickness of 4μm.


### 2.2. Antibodies

PPAR-α, -β, and -γ are affinity-purified goat
polyclonal antibodies. We purchased these antibodies from Santa Cruz
Biotechnology Inc (Santa Cruz, Calif, USA). They demonstrated about the source
of these antibodies, PPAR-α and -γ are affinity-purified goat polyclonal
antibodies raised against a peptide mapping at the amino terminus of PPAR-α and
-γ of human origin (α differs from corresponding mouse sequence by amino acids;
γ is identical to the corresponding mouse sequence). PPAR-β is an
affinity-purified goat polyclonal antibody raised against a peptide mapping at
the carboxy terminus of PPAR-β of human origin (differs from corresponding
mouse sequence by two amino acids). About specificity of these antibodies,
PPAR-α and -β react with those of mouse, rat and human origin by Western
blotting and immunohistochemistry. PPAR-γ also reacts with PPAR-γ1 and PPAR-γ2
of mouse, rat, and human origin by Western blotting and immunohistochemistry.
These specific antibodies do not cross-match either each other, nor do they
cross-react with each other.


### 2.3. RT-PCR

Total RNA was isolated from PC tissues, BPH and NP
tissues (fresh tissues) by guanidium thiocyanate-phenol-chloroform method. We
performed an RT-PCR
procedure to determine the PPAR-α, -β, and -γ mRNA expression as described
previously [19]. In short,
total RNA was used as a template for DNA synthesis using a superscript preamplification
system (GIBCO-BRL) according to the manufacturer instructions. PCR was
performed with each cDNA; PPAR-α, -β, and -γ; or G3PDH
primer and Taq DNA polymerase (NIPPON GENE, Toyama, Japan). The synthetic
oligonucleotides were obtained from Nippon Flour Mills (Kanazawa, Japan). We
used G3PDH mRNA as a control.The primers used were as follows:(a)
PPAR-α: sense; 5′-CCAGTATTTAGGACGCTGTCC-3′  and
antisense 5′-AAGTTCTTCAAGTAGGCCAGC-3′;(b)
PPAR-β: sense; 5′-AACTGCAGATGGGCTGTAAC-3′  and antisense 5′-GTCTCGATGTCGTGGATCAC-3′;(c)
PPAR-γ: sense; 5′-TCTCTCCGTAATGGAAGACC-3′  and antisense
5′-GCATTATGAGACATCCCCAC-3′;(d)
human G3PDH: sense;5′-CCACCCATGGCAAATT CCATGGCA-3′ and
antisense; 5′-TCTAGAGGGCAG  GTCAGGTCCACC-3′.The primer sets yield PCR products of 492, 484, 474,
and 598 base pair for PPAR-α, -β, and -γ or G3PDH, respectively. Reactions were
incubated in an automatic heat-block for 30 cycles of denaturation 40 seconds,
94°C ; annealing for
50 seconds, 50°C; extension for
50 seconds, 72°C [19]. PCR products were run on 2% agarose gel
in TAE buffer (40 mM Tris acetate, 1 mM EDTA) and visualized by ethidium
bromide staining.


### 2.4. Immunohistochemistry

Tissues sections (4μm thick) were incubated with
anti-PPAR-α, -β, and -γ antibody (2 μg/mL) or purified normal goat IgG (2 μg/mL)
in a humid chamber for 24 hours, and further incubated with biotinylated rabbit
antigoat IgG (Vector Laboratories, Inc. Burlingame, Calif, USA) for 30 minutes.
After washing with PBS, the sections were incubated with the vectastatin
avidin-biotin peroxidase complex kit (Vector, Burlingame, Calif, USA) [20] for 45 minutes. Color was developed by immersing the
sections in a solutions of 0.05% wt/vol 3,3′-diaminobenzidine
tetrahydrochloride (Sigma-Aldrich, St. Louis, Mo, USA). The sections were
counterstained with hematoxylin (Sigma-Aldrich, St. Louis, Mo, USA).


### 2.5. Statistical Analysis

The extent and intensity of staining with PPAR-α, -β,
and -γ antibodies were graded on a scale of 0 to 4 (+) by two blind observers
on two separate occasions using coded slides, and an average score was
calculated [21]. Staining was
classified into 5 grades from 0 to 4 (+) according to the intensity of staining
and the number of positive cells. The observers assessed all tissues on the
slides to assign the score. A 4 (+) grade implies that all staining was
maximally intense throughout the specimen, whereas 0 implies that staining was
absent throughout the specimen. The microanatomical sites of staining were also
recorded. To quantify the expressions of PPAR-α, -β, and -γ, the same two
pathologists made assessments throughout the study, staining control specimens
simultaneously. This method, therefore, increases the credibility of data. In
addition, all specimens were reassessed, which also contributed to the exclusion
of any subjective variability.


### 2.6. Cell Cultures

The human PC cell lines (LNCaP, PC3, DU-145) and TC
cell line (NEC-8) were obtained from Health Science Research Resources Bank
(Osaka, Japan). Cells were grown in culture flask (Nunc, Roskilde, Denmark) in
RPMI 1640 supplemented with 10% FBS, 100 U/mL of penicillin and 100μg/mL of
streptomycin in a humidified 5% CO2 atmosphere at 37°C. The media were
changed every 3 days and the cells were separated via trypsinization, using
trypsin/EDTA when they reached subconfluence.


### 2.7. Cell Proliferative Studies

Troglitazone (thiazolidinedione compounds) was
obtained from Sankyo Pharmaceuticals (Tokyo, Japan). 15-deoxy-Δ12,14-prostaglandin
J2 (15-d-PGJ2) was purchased from Cayman Chemical Company
(St. Louis, Mo, USA). GW9662 was purchased from BIOMOL Research Laboratories
Inc. (Pa, USA). Approximately 1.0×104 cells (all PC and TC cell lines) placed onto 8×8 mm diameter multichamber slides
(Nunc, Copenhagen, Denmark) were treated with troglitazone and 15-d-PGJ2 (5–40 μM) dissolved in ethanol. The final concentration of ethanol was <0.05%.
Cell viability was measured after 48 hours by a microplate reader using a
modified 3-[4,5-dimethylthiazol-2-thiazolyl]-2,5-diphenyltetrazolium bromide
(MTT) assay (WST-1 assay; Dojindo, Kumamoto, Japan) and presented as the
percentage of control-culture conditions (N=6).


### 2.8. Flow Cytometry (Annexin V and Propidium Iodide Staining)

The effects of PPAR-γ ligands on PC (PC3) and TC
(NEC-8) cell lines were determined by dual staining with Annexin V-FITC and
propidium iodide using Annexin V-FITC Apoptosis Detection Kit I (Biosciences Pharmingen, Calif, USA). Annexin
V-FITC and propidium iodide (PI) were added to the cellular suspension as in
the manufacturer instruction, and sample fluorescence of 1.0×104 cells was analyzed
flow cytometry. Flow cytometry was with FACScan (Becton Dickinson, Heidelberg, Germany). Cell which were Annexin V-FITC positive and PI
negative were identified as early apoptosis. Cell which were Annexin V-FITC
positive and PI positive were identified as late apoptosis or necrosis.


### 2.9. Flow Cytometry (Identification of  DNA Fragmentation)

The assay was performed by TdT-mediated dUTP Nick End
Labelling (TUNEL) method using APO-DIRECT kit (Becton Dickinson). Following the
experiments, PC (PC3) and TC (NEC-8) cell lines in suspension (1×106/mL)
were fixed with 1% PBS, washed in PBS, and suspended in 70% (v/v) ice-cold
ethanol. The cells were stored in ethanol at −20°C until use. The positive and negative controls and the
sample were stained with FITC-dUTP by incubation in terminal deoxynucleotidyl
transferase buffer as in the manufacturer instruction, and sample fluorescence
of 1×104 cells was analyzed by flow cytometry (Becton Dickinson). Results are given as
% of TUNEL-positive cells.


### 2.10. Detection of Apoptosis by Hoechst Staining

DNA chromatin morphology was assessed using Hoechst
staining. PC (PC3) and TC (NEC-8) cell (5×105  cells) were incubated
with 20 μM PPAR-γ ligands for 24 hours. Cells were washed by RPMI-1640 and
labeled with 8 mg/mL of hoechest 33342 (Sigma-Aldrich Japan K.K. Tokyo, Japan) for 10 minutes; PI (Sigma-Aldrich Japan K.K. Tokyo, Japan) was added (10 mg/mL final concentration), and the cells
were examined by fluorescence microscopy.


### 2.11. Statistical Analysis

All results are presented as the mean± SD. Analysis
of data was performed using the analysis of variance (ANOVA) [22].


## 2.1. Tumor Specimens

Prostate specimens were obtained from 156 patients
with PC; 15 with prostatic intraepithelial neoplasia (PIN); 20 with benign
prostatic hyperplasia (BPH), who underwent biopsy due to serum prostate-specific antigen increase; and 12 patients with normal prostate (NP)
tissues who underwent total cystectomy due to bladder cancer.Testis specimens were obtained from 72 TC patients,
and from 20 NT patients who underwent orchiectomy for PC. Tumor tissues,
nontumor tissues, vascular endothelium, and interstitial tissues from the
subjects were preserved in 10% formalin and embedded in paraffin, serially
sectioned onto microscope slides at a thickness of 4μm.


## 2.2. Antibodies

PPAR-α, -β, and -γ are affinity-purified goat
polyclonal antibodies. We purchased these antibodies from Santa Cruz
Biotechnology Inc (Santa Cruz, Calif, USA). They demonstrated about the source
of these antibodies, PPAR-α and -γ are affinity-purified goat polyclonal
antibodies raised against a peptide mapping at the amino terminus of PPAR-α and
-γ of human origin (α differs from corresponding mouse sequence by amino acids;
γ is identical to the corresponding mouse sequence). PPAR-β is an
affinity-purified goat polyclonal antibody raised against a peptide mapping at
the carboxy terminus of PPAR-β of human origin (differs from corresponding
mouse sequence by two amino acids). About specificity of these antibodies,
PPAR-α and -β react with those of mouse, rat and human origin by Western
blotting and immunohistochemistry. PPAR-γ also reacts with PPAR-γ1 and PPAR-γ2
of mouse, rat, and human origin by Western blotting and immunohistochemistry.
These specific antibodies do not cross-match either each other, nor do they
cross-react with each other.


## 2.3. RT-PCR

Total RNA was isolated from PC tissues, BPH and NP
tissues (fresh tissues) by guanidium thiocyanate-phenol-chloroform method. We
performed an RT-PCR
procedure to determine the PPAR-α, -β, and -γ mRNA expression as described
previously [19]. In short,
total RNA was used as a template for DNA synthesis using a superscript preamplification
system (GIBCO-BRL) according to the manufacturer instructions. PCR was
performed with each cDNA; PPAR-α, -β, and -γ; or G3PDH
primer and Taq DNA polymerase (NIPPON GENE, Toyama, Japan). The synthetic
oligonucleotides were obtained from Nippon Flour Mills (Kanazawa, Japan). We
used G3PDH mRNA as a control.The primers used were as follows:(a)
PPAR-α: sense; 5′-CCAGTATTTAGGACGCTGTCC-3′  and
antisense 5′-AAGTTCTTCAAGTAGGCCAGC-3′;(b)
PPAR-β: sense; 5′-AACTGCAGATGGGCTGTAAC-3′  and antisense 5′-GTCTCGATGTCGTGGATCAC-3′;(c)
PPAR-γ: sense; 5′-TCTCTCCGTAATGGAAGACC-3′  and antisense
5′-GCATTATGAGACATCCCCAC-3′;(d)
human G3PDH: sense;5′-CCACCCATGGCAAATT CCATGGCA-3′ and
antisense; 5′-TCTAGAGGGCAG  GTCAGGTCCACC-3′.The primer sets yield PCR products of 492, 484, 474,
and 598 base pair for PPAR-α, -β, and -γ or G3PDH, respectively. Reactions were
incubated in an automatic heat-block for 30 cycles of denaturation 40 seconds,
94°C ; annealing for
50 seconds, 50°C; extension for
50 seconds, 72°C [19]. PCR products were run on 2% agarose gel
in TAE buffer (40 mM Tris acetate, 1 mM EDTA) and visualized by ethidium
bromide staining.


## 2.4. Immunohistochemistry

Tissues sections (4μm thick) were incubated with
anti-PPAR-α, -β, and -γ antibody (2 μg/mL) or purified normal goat IgG (2 μg/mL)
in a humid chamber for 24 hours, and further incubated with biotinylated rabbit
antigoat IgG (Vector Laboratories, Inc. Burlingame, Calif, USA) for 30 minutes.
After washing with PBS, the sections were incubated with the vectastatin
avidin-biotin peroxidase complex kit (Vector, Burlingame, Calif, USA) [20] for 45 minutes. Color was developed by immersing the
sections in a solutions of 0.05% wt/vol 3,3′-diaminobenzidine
tetrahydrochloride (Sigma-Aldrich, St. Louis, Mo, USA). The sections were
counterstained with hematoxylin (Sigma-Aldrich, St. Louis, Mo, USA).


## 2.5. Statistical Analysis

The extent and intensity of staining with PPAR-α, -β,
and -γ antibodies were graded on a scale of 0 to 4 (+) by two blind observers
on two separate occasions using coded slides, and an average score was
calculated [21]. Staining was
classified into 5 grades from 0 to 4 (+) according to the intensity of staining
and the number of positive cells. The observers assessed all tissues on the
slides to assign the score. A 4 (+) grade implies that all staining was
maximally intense throughout the specimen, whereas 0 implies that staining was
absent throughout the specimen. The microanatomical sites of staining were also
recorded. To quantify the expressions of PPAR-α, -β, and -γ, the same two
pathologists made assessments throughout the study, staining control specimens
simultaneously. This method, therefore, increases the credibility of data. In
addition, all specimens were reassessed, which also contributed to the exclusion
of any subjective variability.


## 2.6. Cell Cultures

The human PC cell lines (LNCaP, PC3, DU-145) and TC
cell line (NEC-8) were obtained from Health Science Research Resources Bank
(Osaka, Japan). Cells were grown in culture flask (Nunc, Roskilde, Denmark) in
RPMI 1640 supplemented with 10% FBS, 100 U/mL of penicillin and 100μg/mL of
streptomycin in a humidified 5% CO2 atmosphere at 37°C. The media were
changed every 3 days and the cells were separated via trypsinization, using
trypsin/EDTA when they reached subconfluence.


## 2.7. Cell Proliferative Studies

Troglitazone (thiazolidinedione compounds) was
obtained from Sankyo Pharmaceuticals (Tokyo, Japan). 15-deoxy-Δ12,14-prostaglandin
J2 (15-d-PGJ2) was purchased from Cayman Chemical Company
(St. Louis, Mo, USA). GW9662 was purchased from BIOMOL Research Laboratories
Inc. (Pa, USA). Approximately 1.0×104 cells (all PC and TC cell lines) placed onto 8×8 mm diameter multichamber slides
(Nunc, Copenhagen, Denmark) were treated with troglitazone and 15-d-PGJ2 (5–40 μM) dissolved in ethanol. The final concentration of ethanol was <0.05%.
Cell viability was measured after 48 hours by a microplate reader using a
modified 3-[4,5-dimethylthiazol-2-thiazolyl]-2,5-diphenyltetrazolium bromide
(MTT) assay (WST-1 assay; Dojindo, Kumamoto, Japan) and presented as the
percentage of control-culture conditions (N=6).


## 2.8. Flow Cytometry (Annexin V and Propidium Iodide Staining)

The effects of PPAR-γ ligands on PC (PC3) and TC
(NEC-8) cell lines were determined by dual staining with Annexin V-FITC and
propidium iodide using Annexin V-FITC Apoptosis Detection Kit I (Biosciences Pharmingen, Calif, USA). Annexin
V-FITC and propidium iodide (PI) were added to the cellular suspension as in
the manufacturer instruction, and sample fluorescence of 1.0×104 cells was analyzed
flow cytometry. Flow cytometry was with FACScan (Becton Dickinson, Heidelberg, Germany). Cell which were Annexin V-FITC positive and PI
negative were identified as early apoptosis. Cell which were Annexin V-FITC
positive and PI positive were identified as late apoptosis or necrosis.


## 2.9. Flow Cytometry (Identification of  DNA Fragmentation)

The assay was performed by TdT-mediated dUTP Nick End
Labelling (TUNEL) method using APO-DIRECT kit (Becton Dickinson). Following the
experiments, PC (PC3) and TC (NEC-8) cell lines in suspension (1×106/mL)
were fixed with 1% PBS, washed in PBS, and suspended in 70% (v/v) ice-cold
ethanol. The cells were stored in ethanol at −20°C until use. The positive and negative controls and the
sample were stained with FITC-dUTP by incubation in terminal deoxynucleotidyl
transferase buffer as in the manufacturer instruction, and sample fluorescence
of 1×104 cells was analyzed by flow cytometry (Becton Dickinson). Results are given as
% of TUNEL-positive cells.


## 2.10. Detection of Apoptosis by Hoechst Staining

DNA chromatin morphology was assessed using Hoechst
staining. PC (PC3) and TC (NEC-8) cell (5×105  cells) were incubated
with 20 μM PPAR-γ ligands for 24 hours. Cells were washed by RPMI-1640 and
labeled with 8 mg/mL of hoechest 33342 (Sigma-Aldrich Japan K.K. Tokyo, Japan) for 10 minutes; PI (Sigma-Aldrich Japan K.K. Tokyo, Japan) was added (10 mg/mL final concentration), and the cells
were examined by fluorescence microscopy.


## 2.11. Statistical Analysis

All results are presented as the mean± SD. Analysis
of data was performed using the analysis of variance (ANOVA) [22].


## 3. Results

### 3.1. Tumor Specimens

#### 3.1.1. PC Tissue Sample

The
156 patients with PC were male aged 59–78 years (mean age67±5.3 years).
We used Gleason score to evaluate PC. Gleason score is given to
PC based upon its microscopic appearance. Gleason score is important because
higher Gleason scores are associated with worse prognosis. This is because
higher Gleason scores are given to cancer which is more aggressive. Gleason
score ranges from 2 to 10. Gleason score of 2 is associated with the best
prognosis and a score of 10 with the worst.  The final score is a combination of
two different scores which each range from 1 to 5. Gleason score is as follows: low group: Gleason score, 2, 3, 4, 5, middle group:
Gleason score, 6, 7, 8, high group: Gleason score, 9, 10. In clinical PC, Gleason
score is almost over 5.The 50 patients were in the low group, 54 were in the
middle group, and 52 were in the high group. The 15 patients with PIN averaged64±5.9 (52–73) years. The 20 patients with BPH averaged 68±4.7 years (59–75),
and all had nodular hyperplasia.  The 12 patients with NP averaged 52±7.6
(44–62) years.


#### 3.1.2. TC Tissue Sample

The 72 TC patients were a mean age of31.0±12.3 years.
Tumors of single
histologic types were found in 58 patients and more than two histological types
in 14 patients. Seminoma occurred in 31 patients, embryonal carcinoma in 8
patients, yolk sac tumor in 7 patients, choriocarcinoma in 7 patients, and
teratoma in 5 patients. Tumors having more than two histologic types included
embryonal carcinoma and teratoma in 4 patients, choriocarcinoma and other types
in 3 patients, and other combinations in 7 patients. The average age of 20
patients NT tissues was 61.4±8.6 years.


### 3.2. RT-PCR

To check PPAR-α, -β, and -γ mRNA variation, RT-PCR was
performed with total RNA extracted from all specimens. Using specific primers
for PPAR-α, -β, and -γ and G3PDH, the
amplification predicted, respectively, fragments of 492, 484, 474, and 598 base
pair (bp) in length.


#### 3.2.1. PC Tissue Sample

The PPAR-α and -β mRNA were detected in PC, PIN, BPH,
and NP samples. However, we detected a specific band of PPAR-γ mRNA in the
samples from PIN and PC, and we also detected a very weak specific band of
PPAR-γ mRNA in the sample from BPH, whereas sample from NP displayed no band of
PPAR-γ mRNA (see Figure 1).RT-PCR analysis of PPAR-α, -β, and -γ in prostate tissue samples from the patients with PC, PIN,
BPH and NP. A slight, but clear, band of PPAR-α and -β m-RNA was
detected in all samples. However, the specific band of PPAR-γ mRNA in the
samples from prostate cancer (PC) and prostatic intraepithelial neoplasia (PIN)
was detected, while samples from benign prostatic hyperplasia (BPH) displayed a
very weak band, and in a sample from normal prostate (NP) no clear band was
detected.


#### 3.2.2. TC Tissue Sample

The PPAR-α and -β mRNA were
detected in all TC and NT samples.
However, we detected a specific band of PPAR-γ mRNA in all TC groups,
whereas sample from NT displayed no band of PPAR-γ mRNA.


### 3.3. Immnohistostaining of PPAR-α, -β, and -γ

To assess the tissue distribution of PPAR-α, -β, and -γ
polypeptides, we stained paraffin-embedded samples with the affinity-purified
PPAR-α, -β, and -γ antibodies that recognize specifically PPAR-α, -β, and -γ.
The specificity of this antibody was proved by the previous experiments [23].


#### 3.3.1. PC Tissue Sample

PPAR-α, and -β were expressed
in PC, PIN, BPH, and NP tissues. Although very weak expression of PPAR-γ was
found in BPH and NP tissues, PPAR-γ was strongly expressed in all PC groups and PIN (see Figure 2).Representative immunostaining for PPAR-γ in prostate tissues samples. A significant strong PPAR-γ expression in all prostate cancer (PC) group tissues and prostatic intraepithelial neoplasia (PIN) tissue was
detected, whereas PPAR-γ expression is very weak in benign prostatic hyperplasia (BPH) tissues and normal prostate  (NP) tissue.
(a)(b)(c)(d)(e)(f)


#### 3.3.2. TC Tissue Sample

PPAR-α, and -β were expressed
in all TC and NT tissues. Although no expression of PPAR-γ was found in NT tissues, PPAR-γ was strongly expressed
in all TC groups (see Figure 3).Representative immunostaining for PPAR-γ in testicular tissues samples. A significant strong PPAR-γ expression in all testicular cancer (TC) tissues was detected, whereas PPAR-γ expression is very weak in normal testis (NT) tissues.
(a)
Seminoma(b)
Embryonal carcinoma(c)
Yolk sac tumors(d)
Choriocarcinoma(e)
Teratoma(f)
Nomal testis


### 3.4. Statistical Analysis of PPAR-α, -β, and -γ Immunostaining

To extent and intensity
of staining with PPAR-α, -β, and -γ, antibody was
graded 0 to 4 (+) by 2 blind observers.


#### 3.4.1. PC Tissue Sample

PPAR-α, -β immunostaining were significantly intense
in all cases.  There were no differences among PC, PIN, BPH, and NP.  There was
no significant difference of the intensity of PPAR-α, -β staining between PC,
PIN, BPH, and NP. However, PPAR-γ immunostaining was significantly more
extensive and intense in tumor cells (mean: low group; 2.6±0.7, middle
group; 2.7±0.9, high group; 3.3±1.0, P<.01)
and in PIN (mean: 2.5±0.8, P<.01) than in tissue of BPH (mean:
0.8±0.6).
PPAR-γ staining was also high in blood vessels and stromal
tissues of prostate cancer and PIN, with no significant difference between them
(1.8–2.0). However, the expression of the PPAR-γ in the blood vessels and stromal tissues from BPH and
NP was at the basic level (0.5–0.7) (see Table 1).Table 1
Statistical analysis of PPAR-α, -β, and -γ immunostaining.
TumorBlood vesselStromal tissuePPAR-αPC (N=156)Low groupN=50)2.2±0.62.0±0.92.0±0.9Middle groupN=54)2.4±0.72.3±0.71.9±0.6High groupN=52)2.3±0.71.9±0.62.0±0.8PIN (N=15)1.9±0.61.8±0.61.7±0.7BPH (N=20)2.1±0.61.7±0.51.7±0.5NP (N=12)Not present2.1±0.72.2±1.1PPAR-βPC (N=156)Low groupN=50)2.0±1.01.7±0.71.9±0.6Middle groupN=54)2.3±1.21.6±0.71.8±0.7High groupN=52)2.1±1.01.7±0.61.8±0.9PIN (N=15)2.2±0.81.8±0.91.8±0.7BPH (N=20)2.0±0.71.9±0.81.6±0.7NP (N=12)Not present1.9±0.71.9±0.8PPAR-γPC (N=156)Low groupN=50)2.6±0.7*1.8±0.9*1.8±0.7*Middle groupN=54)2.7±0.9*1.8±0.8*1.9±0.9*High groupN=52)3.3±1.0*2.0±0.8*1.7±0.9*PIN (N=15)2.5±0.8*1.9±0.8*1.7±0.9*BPH (N=20)0.8±0.60.6±0.50.7±0.5NP (N=12)Not present0.5±0.40.5±0.3* Graded 0–4 on the coded sections by two blind observers. 0, no staining; 4+, maximum intensity. Statistical analysis was performed using the ANOVA (p-value). PPAR-α and -β immunostaining were significantly intense in all cases. There were no differnces among PC, PIN, BPH and NP. PPAR-γ immunostainings of tumor were significantly more extensive and intense in PC and in PIN than in tissue of BPH or NP. PPAR-γ staining was high in blood vessels and stromal tissues of PC and PIN, with no significant difference between them. However, the expressions of the PPAR-γ in the blood vessels and stromal tissues from BPH and NP were at the basic level. (*P<.01).


#### 3.4.2. TC Tissue Sample

PPAR-α, -β immunostaining were
significantly intense in all TC groups and NT. However, PPAR-γ immunostaining was significantly more extensive and
intense in tumor cells and blood vessels of the TC groups than in NT. There was
no significant differences occurred between all TC group in tumor cells and
blood vessels (see Table 2).Table 2
Statistical analysis of PPAR-α, -β, and -γ immunostaining.
Av.± SDTumor typeEpitheliumBlood vesselPPAR-αSeminoma (N=34)2.2±0.81.7±0.9Embryonal carcinoma (N=15)2.4±1.12.1±1.0Yolk sac tumor (N=11)1.8±1.01.6±0.7Choriocarcinoma (N=10)2.5±1.22.0±0.9Teratoma (N=12)1.8±0.91.6±0.9Normal testis (N=20)2.5±1.12.1±0.9PPAR-βSeminoma (N=34)2.4±0.92.2±1.1Embryonal carcinoma (N=15)2.6±1.42.3±1.2Yolk sac tumor (N=11)2.5±1.42.1±0.6Choriocarcinoma (N=10)2.2±1.01.9±0.9Teratoma (N=12)2.4±0.92.2±1.3Normal testis (N=20)2.5±1.12.3±1.0PPAR-γSeminoma (N=34)2.2±0.8*1.9±0.9*Embryonal carcinoma (N=15)2.8±1.1*2.5±1.0*Yolk sac tumor (N=11)2.2±0.9*2.1±1.1*Choriocarcinoma (N=10)2.9±1.0*2.4±1.0*Teratoma (N=12)2.0±1.3*1.9±1.1*Normal testis (N=20)0.7±0.60.6±0.4* Graded 0 to 4 on the coded sections by two blind observers. 0, no staining; 4+, maximum intensity. Statistical analysis was performed using the analysis of variance (P value; ANOVA). PPAR-α, and -β immunostaining were significanty apparent in all TC and NT tissues. PPAR-γ immunostaining of tumor was significantly more extensive and intense in all TC groups than in NT tissue. PPAR-γ staining was high in blood vesseld of TC, with no significant difference between them. However, the expression of PPAR-γ in blood vessels from NT was at the basic level. P<.01.


### 3.5. PPAR-γ Ligands Induced Growth Inhibition in PC and TC Cell Lines by MTT Assay

To investigate the effects of PPAR-γ ligands on all PC
(LNCaP, PC3, DU-145) and TC cell (NEC-8) lines proliferation, we analyzed cell
viability in vitro by modified MTT assay.


#### 3.5.1. PC Cell Line

PPAR-γ ligands induced the reduction of cell viability with the
half-maximal concentration of growth inhibition of all PC cell lines (LNCaP,
PC3, DU-145) in the range of 5–40 μM (see Table 3). PPAR-γ ligands stopped the growth of all PC cell
lines.Table 3
Effects of troglitazone, 15-d-PGJ2 and GW9662 in viabity of human PC and TC cell lines.
5μM10μM20μM40μMTroglitazonePC cell linesLNCaP72.4%25.7%12.6%8.4%PC348.6%15.5%14.7%6.5%DU-14560.1%35.1%7.6%7.7%TC cell lineNEC-838.7%35.3%36.6%38.1%15-d-PGJ2PC cell linesLNCaP78.9%63.7%22.4%5.6%PC369.7%59.0%34.1%6.8%DU-14573.8%59.3%5.8%5.8%TC cell lineNEC-875.1%66.7%52.3%46.8%GW9662PC cell linesLNCaP106.8%112.4%103.7%106.2%PC3116.8%118.6%119.4%120.2%DU-145122.6%119.4%117.8%115.6%TC cell lineNEC-8108.4%115.5%110.6%112.3%The dose-response analysis of viability in human cancer cells treated with troglitazone, 15-d-PGJ2 and GW9662 (5–40μM, 48 hr) was measured by the MTT assay and expressed as % of control culture conditions (N=6).


#### 3.5.2. TC Cell Line

Similar to PC cell lines, PPAR-γ ligands induced the reduction of
cell viability with the half-maximal concentration of growth inhibition of TC
cell line (NEC-8) in the range of 5–40 μM (see Table 3). PPAR-γ ligands stopped the growth of TC cell line
(NEC-8).


### 3.6. PPAR-γ Ligands Induced Apoptosis by Flow Cytometry

To evaluate whether or not cell death induced by PPAR-γ
ligands was through apoptosis, we evaluated using flow cytometry. The higher
left quadrants represent early apoptosis (Annexin V-FITC-positive cells and
PI-negative cells). The higher right quadrants represent late apoptosis or
necrosis (Annexin V-FITC-positive cells and PI-positive cells).


#### 3.6.1. PC Cell Line

PC cell line (PC3) with
treatment of 25μM PPAR-γ ligand
(15-d-PGJ2) could induce early apoptosis, not late apoptosis or
necrosis (see Figure 4) and DNA fragmentation (see Figure 5). Diagrams of
FITC-Annexin V/PI flow cytometry and typical flow cytometry analysis histogram
are presented.Effects of PPAR-γ ligands on apoptosis by flow cytometry in PC and TC cell lines. PC cells (PC3) and TC cell (NEC-8) lines with treatment of 25 μM 15-d-PGJ2 could induce early apoptosis not late apoptosis or necrosis. The higher left quadrants represent early apoptosis (Annexin V-FITC-positive cells and
PI-negative cells). The higher right quadrants represent late apoptosis or necrosis (Annexin V-FITC-positive cells and PI-positive cells). Diagrams of FITC-Annexin V/PI flow cytometry are presented.
(a)(b)(c)(d)(e)(f)PPAR-γ ligands induce DNA fragmentation in PC and TC cell lines. 15-d-PGJ2 (2 μM) could induce DNA fragmentation in PC cell (PC3) and TC cell line (NEC-8). Typical flow cytometry analysis histograms are presented.
(a)(b)(c)(d)(e)(f)


#### 3.6.2. TC Cell Line

TC cell line (NEC-8) with treatment of 25μM PPAR-γ ligands
(15-d-PGJ2) could induce early apoptosis not late apoptosis or
necrosis (see Figure 4) and DNA fragmentation (see Figure 5). Diagrams of
FITC-Annexin V/PI flow cytometry and typical flow cytometry analysis histogram
are presented.


### 3.7. Effect of PPAR-γ Ligands in Induction of Apoptosis on PC and TC Cell Lines

To evaluate whether or not cell death induced by PPAR-γ
ligands was through apoptosis, we evaluated the chromatin morphology of PC (PC3) cell and TC cell (NEC-8) lines using hoechst
staining.


#### 3.7.1. PC Cell Line

PC cell line (PC3) treated with
PPAR-γ ligands showed significant chromatin condensation, cellular shrinkage,
small membrane-bound bodies (apoptotic bodies), and cytoplasmic condensation.
These cellular changes were typically redundant characteristics of apoptosis.
PC cell lines (PC3) without PPAR-γ ligands maintained normal chromatin patterns
and cell size (see Figure 6). Typical photographs are presented in Figure 6.Effects of PPAR-γ ligands in induction of apoptosis on human PC cell line. PC cell line (PC3) treated
with PPAR-γ ligands ((b); 20 μM Troglitazone, (c); 20 μM 15-d-PGJ2) showed significant chromatin
condensation, cellular shrinkage, small membrane-bound bodies (apoptotic bodies), and cytoplasmic condensation. These cellular changes were typically redundant characteristics of apoptosis. PC cells without PPAR-γ ligands maintained normal chromatin patterns and cell size (a). Typical photographs are presented.


#### 3.7.2. TC Cell Line

Similar to PC cell line, TC cell line (NEC-8) treated
with PPAR-γ ligands showed significant chromatin condensation, cellular
shrinkage, small membrane-bound bodies (apoptotic bodies), and cytoplasmic
condensation. These cellular changes were typically redundant characteristics
of apoptosis. TC cell line without PPAR-γ ligands maintained normal chromatin
patterns and cell size.


## 3.1. Tumor Specimens

### 3.1.1. PC Tissue Sample

The
156 patients with PC were male aged 59–78 years (mean age67±5.3 years).
We used Gleason score to evaluate PC. Gleason score is given to
PC based upon its microscopic appearance. Gleason score is important because
higher Gleason scores are associated with worse prognosis. This is because
higher Gleason scores are given to cancer which is more aggressive. Gleason
score ranges from 2 to 10. Gleason score of 2 is associated with the best
prognosis and a score of 10 with the worst.  The final score is a combination of
two different scores which each range from 1 to 5. Gleason score is as follows: low group: Gleason score, 2, 3, 4, 5, middle group:
Gleason score, 6, 7, 8, high group: Gleason score, 9, 10. In clinical PC, Gleason
score is almost over 5.The 50 patients were in the low group, 54 were in the
middle group, and 52 were in the high group. The 15 patients with PIN averaged64±5.9 (52–73) years. The 20 patients with BPH averaged 68±4.7 years (59–75),
and all had nodular hyperplasia.  The 12 patients with NP averaged 52±7.6
(44–62) years.


### 3.1.2. TC Tissue Sample

The 72 TC patients were a mean age of31.0±12.3 years.
Tumors of single
histologic types were found in 58 patients and more than two histological types
in 14 patients. Seminoma occurred in 31 patients, embryonal carcinoma in 8
patients, yolk sac tumor in 7 patients, choriocarcinoma in 7 patients, and
teratoma in 5 patients. Tumors having more than two histologic types included
embryonal carcinoma and teratoma in 4 patients, choriocarcinoma and other types
in 3 patients, and other combinations in 7 patients. The average age of 20
patients NT tissues was 61.4±8.6 years.


## 3.1.1. PC Tissue Sample

The
156 patients with PC were male aged 59–78 years (mean age67±5.3 years).
We used Gleason score to evaluate PC. Gleason score is given to
PC based upon its microscopic appearance. Gleason score is important because
higher Gleason scores are associated with worse prognosis. This is because
higher Gleason scores are given to cancer which is more aggressive. Gleason
score ranges from 2 to 10. Gleason score of 2 is associated with the best
prognosis and a score of 10 with the worst.  The final score is a combination of
two different scores which each range from 1 to 5. Gleason score is as follows: low group: Gleason score, 2, 3, 4, 5, middle group:
Gleason score, 6, 7, 8, high group: Gleason score, 9, 10. In clinical PC, Gleason
score is almost over 5.The 50 patients were in the low group, 54 were in the
middle group, and 52 were in the high group. The 15 patients with PIN averaged64±5.9 (52–73) years. The 20 patients with BPH averaged 68±4.7 years (59–75),
and all had nodular hyperplasia.  The 12 patients with NP averaged 52±7.6
(44–62) years.


## 3.1.2. TC Tissue Sample

The 72 TC patients were a mean age of31.0±12.3 years.
Tumors of single
histologic types were found in 58 patients and more than two histological types
in 14 patients. Seminoma occurred in 31 patients, embryonal carcinoma in 8
patients, yolk sac tumor in 7 patients, choriocarcinoma in 7 patients, and
teratoma in 5 patients. Tumors having more than two histologic types included
embryonal carcinoma and teratoma in 4 patients, choriocarcinoma and other types
in 3 patients, and other combinations in 7 patients. The average age of 20
patients NT tissues was 61.4±8.6 years.


## 3.2. RT-PCR

To check PPAR-α, -β, and -γ mRNA variation, RT-PCR was
performed with total RNA extracted from all specimens. Using specific primers
for PPAR-α, -β, and -γ and G3PDH, the
amplification predicted, respectively, fragments of 492, 484, 474, and 598 base
pair (bp) in length.


### 3.2.1. PC Tissue Sample

The PPAR-α and -β mRNA were detected in PC, PIN, BPH,
and NP samples. However, we detected a specific band of PPAR-γ mRNA in the
samples from PIN and PC, and we also detected a very weak specific band of
PPAR-γ mRNA in the sample from BPH, whereas sample from NP displayed no band of
PPAR-γ mRNA (see Figure 1).RT-PCR analysis of PPAR-α, -β, and -γ in prostate tissue samples from the patients with PC, PIN,
BPH and NP. A slight, but clear, band of PPAR-α and -β m-RNA was
detected in all samples. However, the specific band of PPAR-γ mRNA in the
samples from prostate cancer (PC) and prostatic intraepithelial neoplasia (PIN)
was detected, while samples from benign prostatic hyperplasia (BPH) displayed a
very weak band, and in a sample from normal prostate (NP) no clear band was
detected.


### 3.2.2. TC Tissue Sample

The PPAR-α and -β mRNA were
detected in all TC and NT samples.
However, we detected a specific band of PPAR-γ mRNA in all TC groups,
whereas sample from NT displayed no band of PPAR-γ mRNA.


## 3.2.1. PC Tissue Sample

The PPAR-α and -β mRNA were detected in PC, PIN, BPH,
and NP samples. However, we detected a specific band of PPAR-γ mRNA in the
samples from PIN and PC, and we also detected a very weak specific band of
PPAR-γ mRNA in the sample from BPH, whereas sample from NP displayed no band of
PPAR-γ mRNA (see Figure 1).RT-PCR analysis of PPAR-α, -β, and -γ in prostate tissue samples from the patients with PC, PIN,
BPH and NP. A slight, but clear, band of PPAR-α and -β m-RNA was
detected in all samples. However, the specific band of PPAR-γ mRNA in the
samples from prostate cancer (PC) and prostatic intraepithelial neoplasia (PIN)
was detected, while samples from benign prostatic hyperplasia (BPH) displayed a
very weak band, and in a sample from normal prostate (NP) no clear band was
detected.


## 3.2.2. TC Tissue Sample

The PPAR-α and -β mRNA were
detected in all TC and NT samples.
However, we detected a specific band of PPAR-γ mRNA in all TC groups,
whereas sample from NT displayed no band of PPAR-γ mRNA.


## 3.3. Immnohistostaining of PPAR-α, -β, and -γ

To assess the tissue distribution of PPAR-α, -β, and -γ
polypeptides, we stained paraffin-embedded samples with the affinity-purified
PPAR-α, -β, and -γ antibodies that recognize specifically PPAR-α, -β, and -γ.
The specificity of this antibody was proved by the previous experiments [23].


### 3.3.1. PC Tissue Sample

PPAR-α, and -β were expressed
in PC, PIN, BPH, and NP tissues. Although very weak expression of PPAR-γ was
found in BPH and NP tissues, PPAR-γ was strongly expressed in all PC groups and PIN (see Figure 2).Representative immunostaining for PPAR-γ in prostate tissues samples. A significant strong PPAR-γ expression in all prostate cancer (PC) group tissues and prostatic intraepithelial neoplasia (PIN) tissue was
detected, whereas PPAR-γ expression is very weak in benign prostatic hyperplasia (BPH) tissues and normal prostate  (NP) tissue.
(a)(b)(c)(d)(e)(f)


### 3.3.2. TC Tissue Sample

PPAR-α, and -β were expressed
in all TC and NT tissues. Although no expression of PPAR-γ was found in NT tissues, PPAR-γ was strongly expressed
in all TC groups (see Figure 3).Representative immunostaining for PPAR-γ in testicular tissues samples. A significant strong PPAR-γ expression in all testicular cancer (TC) tissues was detected, whereas PPAR-γ expression is very weak in normal testis (NT) tissues.
(a)
Seminoma(b)
Embryonal carcinoma(c)
Yolk sac tumors(d)
Choriocarcinoma(e)
Teratoma(f)
Nomal testis


## 3.3.1. PC Tissue Sample

PPAR-α, and -β were expressed
in PC, PIN, BPH, and NP tissues. Although very weak expression of PPAR-γ was
found in BPH and NP tissues, PPAR-γ was strongly expressed in all PC groups and PIN (see Figure 2).Representative immunostaining for PPAR-γ in prostate tissues samples. A significant strong PPAR-γ expression in all prostate cancer (PC) group tissues and prostatic intraepithelial neoplasia (PIN) tissue was
detected, whereas PPAR-γ expression is very weak in benign prostatic hyperplasia (BPH) tissues and normal prostate  (NP) tissue.
(a)(b)(c)(d)(e)(f)


## 3.3.2. TC Tissue Sample

PPAR-α, and -β were expressed
in all TC and NT tissues. Although no expression of PPAR-γ was found in NT tissues, PPAR-γ was strongly expressed
in all TC groups (see Figure 3).Representative immunostaining for PPAR-γ in testicular tissues samples. A significant strong PPAR-γ expression in all testicular cancer (TC) tissues was detected, whereas PPAR-γ expression is very weak in normal testis (NT) tissues.
(a)
Seminoma(b)
Embryonal carcinoma(c)
Yolk sac tumors(d)
Choriocarcinoma(e)
Teratoma(f)
Nomal testis


## 3.4. Statistical Analysis of PPAR-α, -β, and -γ Immunostaining

To extent and intensity
of staining with PPAR-α, -β, and -γ, antibody was
graded 0 to 4 (+) by 2 blind observers.


### 3.4.1. PC Tissue Sample

PPAR-α, -β immunostaining were significantly intense
in all cases.  There were no differences among PC, PIN, BPH, and NP.  There was
no significant difference of the intensity of PPAR-α, -β staining between PC,
PIN, BPH, and NP. However, PPAR-γ immunostaining was significantly more
extensive and intense in tumor cells (mean: low group; 2.6±0.7, middle
group; 2.7±0.9, high group; 3.3±1.0, P<.01)
and in PIN (mean: 2.5±0.8, P<.01) than in tissue of BPH (mean:
0.8±0.6).
PPAR-γ staining was also high in blood vessels and stromal
tissues of prostate cancer and PIN, with no significant difference between them
(1.8–2.0). However, the expression of the PPAR-γ in the blood vessels and stromal tissues from BPH and
NP was at the basic level (0.5–0.7) (see Table 1).Table 1
Statistical analysis of PPAR-α, -β, and -γ immunostaining.
TumorBlood vesselStromal tissuePPAR-αPC (N=156)Low groupN=50)2.2±0.62.0±0.92.0±0.9Middle groupN=54)2.4±0.72.3±0.71.9±0.6High groupN=52)2.3±0.71.9±0.62.0±0.8PIN (N=15)1.9±0.61.8±0.61.7±0.7BPH (N=20)2.1±0.61.7±0.51.7±0.5NP (N=12)Not present2.1±0.72.2±1.1PPAR-βPC (N=156)Low groupN=50)2.0±1.01.7±0.71.9±0.6Middle groupN=54)2.3±1.21.6±0.71.8±0.7High groupN=52)2.1±1.01.7±0.61.8±0.9PIN (N=15)2.2±0.81.8±0.91.8±0.7BPH (N=20)2.0±0.71.9±0.81.6±0.7NP (N=12)Not present1.9±0.71.9±0.8PPAR-γPC (N=156)Low groupN=50)2.6±0.7*1.8±0.9*1.8±0.7*Middle groupN=54)2.7±0.9*1.8±0.8*1.9±0.9*High groupN=52)3.3±1.0*2.0±0.8*1.7±0.9*PIN (N=15)2.5±0.8*1.9±0.8*1.7±0.9*BPH (N=20)0.8±0.60.6±0.50.7±0.5NP (N=12)Not present0.5±0.40.5±0.3* Graded 0–4 on the coded sections by two blind observers. 0, no staining; 4+, maximum intensity. Statistical analysis was performed using the ANOVA (p-value). PPAR-α and -β immunostaining were significantly intense in all cases. There were no differnces among PC, PIN, BPH and NP. PPAR-γ immunostainings of tumor were significantly more extensive and intense in PC and in PIN than in tissue of BPH or NP. PPAR-γ staining was high in blood vessels and stromal tissues of PC and PIN, with no significant difference between them. However, the expressions of the PPAR-γ in the blood vessels and stromal tissues from BPH and NP were at the basic level. (*P<.01).


### 3.4.2. TC Tissue Sample

PPAR-α, -β immunostaining were
significantly intense in all TC groups and NT. However, PPAR-γ immunostaining was significantly more extensive and
intense in tumor cells and blood vessels of the TC groups than in NT. There was
no significant differences occurred between all TC group in tumor cells and
blood vessels (see Table 2).Table 2
Statistical analysis of PPAR-α, -β, and -γ immunostaining.
Av.± SDTumor typeEpitheliumBlood vesselPPAR-αSeminoma (N=34)2.2±0.81.7±0.9Embryonal carcinoma (N=15)2.4±1.12.1±1.0Yolk sac tumor (N=11)1.8±1.01.6±0.7Choriocarcinoma (N=10)2.5±1.22.0±0.9Teratoma (N=12)1.8±0.91.6±0.9Normal testis (N=20)2.5±1.12.1±0.9PPAR-βSeminoma (N=34)2.4±0.92.2±1.1Embryonal carcinoma (N=15)2.6±1.42.3±1.2Yolk sac tumor (N=11)2.5±1.42.1±0.6Choriocarcinoma (N=10)2.2±1.01.9±0.9Teratoma (N=12)2.4±0.92.2±1.3Normal testis (N=20)2.5±1.12.3±1.0PPAR-γSeminoma (N=34)2.2±0.8*1.9±0.9*Embryonal carcinoma (N=15)2.8±1.1*2.5±1.0*Yolk sac tumor (N=11)2.2±0.9*2.1±1.1*Choriocarcinoma (N=10)2.9±1.0*2.4±1.0*Teratoma (N=12)2.0±1.3*1.9±1.1*Normal testis (N=20)0.7±0.60.6±0.4* Graded 0 to 4 on the coded sections by two blind observers. 0, no staining; 4+, maximum intensity. Statistical analysis was performed using the analysis of variance (P value; ANOVA). PPAR-α, and -β immunostaining were significanty apparent in all TC and NT tissues. PPAR-γ immunostaining of tumor was significantly more extensive and intense in all TC groups than in NT tissue. PPAR-γ staining was high in blood vesseld of TC, with no significant difference between them. However, the expression of PPAR-γ in blood vessels from NT was at the basic level. P<.01.


## 3.4.1. PC Tissue Sample

PPAR-α, -β immunostaining were significantly intense
in all cases.  There were no differences among PC, PIN, BPH, and NP.  There was
no significant difference of the intensity of PPAR-α, -β staining between PC,
PIN, BPH, and NP. However, PPAR-γ immunostaining was significantly more
extensive and intense in tumor cells (mean: low group; 2.6±0.7, middle
group; 2.7±0.9, high group; 3.3±1.0, P<.01)
and in PIN (mean: 2.5±0.8, P<.01) than in tissue of BPH (mean:
0.8±0.6).
PPAR-γ staining was also high in blood vessels and stromal
tissues of prostate cancer and PIN, with no significant difference between them
(1.8–2.0). However, the expression of the PPAR-γ in the blood vessels and stromal tissues from BPH and
NP was at the basic level (0.5–0.7) (see Table 1).Table 1
Statistical analysis of PPAR-α, -β, and -γ immunostaining.
TumorBlood vesselStromal tissuePPAR-αPC (N=156)Low groupN=50)2.2±0.62.0±0.92.0±0.9Middle groupN=54)2.4±0.72.3±0.71.9±0.6High groupN=52)2.3±0.71.9±0.62.0±0.8PIN (N=15)1.9±0.61.8±0.61.7±0.7BPH (N=20)2.1±0.61.7±0.51.7±0.5NP (N=12)Not present2.1±0.72.2±1.1PPAR-βPC (N=156)Low groupN=50)2.0±1.01.7±0.71.9±0.6Middle groupN=54)2.3±1.21.6±0.71.8±0.7High groupN=52)2.1±1.01.7±0.61.8±0.9PIN (N=15)2.2±0.81.8±0.91.8±0.7BPH (N=20)2.0±0.71.9±0.81.6±0.7NP (N=12)Not present1.9±0.71.9±0.8PPAR-γPC (N=156)Low groupN=50)2.6±0.7*1.8±0.9*1.8±0.7*Middle groupN=54)2.7±0.9*1.8±0.8*1.9±0.9*High groupN=52)3.3±1.0*2.0±0.8*1.7±0.9*PIN (N=15)2.5±0.8*1.9±0.8*1.7±0.9*BPH (N=20)0.8±0.60.6±0.50.7±0.5NP (N=12)Not present0.5±0.40.5±0.3* Graded 0–4 on the coded sections by two blind observers. 0, no staining; 4+, maximum intensity. Statistical analysis was performed using the ANOVA (p-value). PPAR-α and -β immunostaining were significantly intense in all cases. There were no differnces among PC, PIN, BPH and NP. PPAR-γ immunostainings of tumor were significantly more extensive and intense in PC and in PIN than in tissue of BPH or NP. PPAR-γ staining was high in blood vessels and stromal tissues of PC and PIN, with no significant difference between them. However, the expressions of the PPAR-γ in the blood vessels and stromal tissues from BPH and NP were at the basic level. (*P<.01).


## 3.4.2. TC Tissue Sample

PPAR-α, -β immunostaining were
significantly intense in all TC groups and NT. However, PPAR-γ immunostaining was significantly more extensive and
intense in tumor cells and blood vessels of the TC groups than in NT. There was
no significant differences occurred between all TC group in tumor cells and
blood vessels (see Table 2).Table 2
Statistical analysis of PPAR-α, -β, and -γ immunostaining.
Av.± SDTumor typeEpitheliumBlood vesselPPAR-αSeminoma (N=34)2.2±0.81.7±0.9Embryonal carcinoma (N=15)2.4±1.12.1±1.0Yolk sac tumor (N=11)1.8±1.01.6±0.7Choriocarcinoma (N=10)2.5±1.22.0±0.9Teratoma (N=12)1.8±0.91.6±0.9Normal testis (N=20)2.5±1.12.1±0.9PPAR-βSeminoma (N=34)2.4±0.92.2±1.1Embryonal carcinoma (N=15)2.6±1.42.3±1.2Yolk sac tumor (N=11)2.5±1.42.1±0.6Choriocarcinoma (N=10)2.2±1.01.9±0.9Teratoma (N=12)2.4±0.92.2±1.3Normal testis (N=20)2.5±1.12.3±1.0PPAR-γSeminoma (N=34)2.2±0.8*1.9±0.9*Embryonal carcinoma (N=15)2.8±1.1*2.5±1.0*Yolk sac tumor (N=11)2.2±0.9*2.1±1.1*Choriocarcinoma (N=10)2.9±1.0*2.4±1.0*Teratoma (N=12)2.0±1.3*1.9±1.1*Normal testis (N=20)0.7±0.60.6±0.4* Graded 0 to 4 on the coded sections by two blind observers. 0, no staining; 4+, maximum intensity. Statistical analysis was performed using the analysis of variance (P value; ANOVA). PPAR-α, and -β immunostaining were significanty apparent in all TC and NT tissues. PPAR-γ immunostaining of tumor was significantly more extensive and intense in all TC groups than in NT tissue. PPAR-γ staining was high in blood vesseld of TC, with no significant difference between them. However, the expression of PPAR-γ in blood vessels from NT was at the basic level. P<.01.


## 3.5. PPAR-γ Ligands Induced Growth Inhibition in PC and TC Cell Lines by MTT Assay

To investigate the effects of PPAR-γ ligands on all PC
(LNCaP, PC3, DU-145) and TC cell (NEC-8) lines proliferation, we analyzed cell
viability in vitro by modified MTT assay.


### 3.5.1. PC Cell Line

PPAR-γ ligands induced the reduction of cell viability with the
half-maximal concentration of growth inhibition of all PC cell lines (LNCaP,
PC3, DU-145) in the range of 5–40 μM (see Table 3). PPAR-γ ligands stopped the growth of all PC cell
lines.Table 3
Effects of troglitazone, 15-d-PGJ2 and GW9662 in viabity of human PC and TC cell lines.
5μM10μM20μM40μMTroglitazonePC cell linesLNCaP72.4%25.7%12.6%8.4%PC348.6%15.5%14.7%6.5%DU-14560.1%35.1%7.6%7.7%TC cell lineNEC-838.7%35.3%36.6%38.1%15-d-PGJ2PC cell linesLNCaP78.9%63.7%22.4%5.6%PC369.7%59.0%34.1%6.8%DU-14573.8%59.3%5.8%5.8%TC cell lineNEC-875.1%66.7%52.3%46.8%GW9662PC cell linesLNCaP106.8%112.4%103.7%106.2%PC3116.8%118.6%119.4%120.2%DU-145122.6%119.4%117.8%115.6%TC cell lineNEC-8108.4%115.5%110.6%112.3%The dose-response analysis of viability in human cancer cells treated with troglitazone, 15-d-PGJ2 and GW9662 (5–40μM, 48 hr) was measured by the MTT assay and expressed as % of control culture conditions (N=6).


### 3.5.2. TC Cell Line

Similar to PC cell lines, PPAR-γ ligands induced the reduction of
cell viability with the half-maximal concentration of growth inhibition of TC
cell line (NEC-8) in the range of 5–40 μM (see Table 3). PPAR-γ ligands stopped the growth of TC cell line
(NEC-8).


## 3.5.1. PC Cell Line

PPAR-γ ligands induced the reduction of cell viability with the
half-maximal concentration of growth inhibition of all PC cell lines (LNCaP,
PC3, DU-145) in the range of 5–40 μM (see Table 3). PPAR-γ ligands stopped the growth of all PC cell
lines.Table 3
Effects of troglitazone, 15-d-PGJ2 and GW9662 in viabity of human PC and TC cell lines.
5μM10μM20μM40μMTroglitazonePC cell linesLNCaP72.4%25.7%12.6%8.4%PC348.6%15.5%14.7%6.5%DU-14560.1%35.1%7.6%7.7%TC cell lineNEC-838.7%35.3%36.6%38.1%15-d-PGJ2PC cell linesLNCaP78.9%63.7%22.4%5.6%PC369.7%59.0%34.1%6.8%DU-14573.8%59.3%5.8%5.8%TC cell lineNEC-875.1%66.7%52.3%46.8%GW9662PC cell linesLNCaP106.8%112.4%103.7%106.2%PC3116.8%118.6%119.4%120.2%DU-145122.6%119.4%117.8%115.6%TC cell lineNEC-8108.4%115.5%110.6%112.3%The dose-response analysis of viability in human cancer cells treated with troglitazone, 15-d-PGJ2 and GW9662 (5–40μM, 48 hr) was measured by the MTT assay and expressed as % of control culture conditions (N=6).


## 3.5.2. TC Cell Line

Similar to PC cell lines, PPAR-γ ligands induced the reduction of
cell viability with the half-maximal concentration of growth inhibition of TC
cell line (NEC-8) in the range of 5–40 μM (see Table 3). PPAR-γ ligands stopped the growth of TC cell line
(NEC-8).


## 3.6. PPAR-γ Ligands Induced Apoptosis by Flow Cytometry

To evaluate whether or not cell death induced by PPAR-γ
ligands was through apoptosis, we evaluated using flow cytometry. The higher
left quadrants represent early apoptosis (Annexin V-FITC-positive cells and
PI-negative cells). The higher right quadrants represent late apoptosis or
necrosis (Annexin V-FITC-positive cells and PI-positive cells).


### 3.6.1. PC Cell Line

PC cell line (PC3) with
treatment of 25μM PPAR-γ ligand
(15-d-PGJ2) could induce early apoptosis, not late apoptosis or
necrosis (see Figure 4) and DNA fragmentation (see Figure 5). Diagrams of
FITC-Annexin V/PI flow cytometry and typical flow cytometry analysis histogram
are presented.Effects of PPAR-γ ligands on apoptosis by flow cytometry in PC and TC cell lines. PC cells (PC3) and TC cell (NEC-8) lines with treatment of 25 μM 15-d-PGJ2 could induce early apoptosis not late apoptosis or necrosis. The higher left quadrants represent early apoptosis (Annexin V-FITC-positive cells and
PI-negative cells). The higher right quadrants represent late apoptosis or necrosis (Annexin V-FITC-positive cells and PI-positive cells). Diagrams of FITC-Annexin V/PI flow cytometry are presented.
(a)(b)(c)(d)(e)(f)PPAR-γ ligands induce DNA fragmentation in PC and TC cell lines. 15-d-PGJ2 (2 μM) could induce DNA fragmentation in PC cell (PC3) and TC cell line (NEC-8). Typical flow cytometry analysis histograms are presented.
(a)(b)(c)(d)(e)(f)


### 3.6.2. TC Cell Line

TC cell line (NEC-8) with treatment of 25μM PPAR-γ ligands
(15-d-PGJ2) could induce early apoptosis not late apoptosis or
necrosis (see Figure 4) and DNA fragmentation (see Figure 5). Diagrams of
FITC-Annexin V/PI flow cytometry and typical flow cytometry analysis histogram
are presented.


## 3.6.1. PC Cell Line

PC cell line (PC3) with
treatment of 25μM PPAR-γ ligand
(15-d-PGJ2) could induce early apoptosis, not late apoptosis or
necrosis (see Figure 4) and DNA fragmentation (see Figure 5). Diagrams of
FITC-Annexin V/PI flow cytometry and typical flow cytometry analysis histogram
are presented.Effects of PPAR-γ ligands on apoptosis by flow cytometry in PC and TC cell lines. PC cells (PC3) and TC cell (NEC-8) lines with treatment of 25 μM 15-d-PGJ2 could induce early apoptosis not late apoptosis or necrosis. The higher left quadrants represent early apoptosis (Annexin V-FITC-positive cells and
PI-negative cells). The higher right quadrants represent late apoptosis or necrosis (Annexin V-FITC-positive cells and PI-positive cells). Diagrams of FITC-Annexin V/PI flow cytometry are presented.
(a)(b)(c)(d)(e)(f)PPAR-γ ligands induce DNA fragmentation in PC and TC cell lines. 15-d-PGJ2 (2 μM) could induce DNA fragmentation in PC cell (PC3) and TC cell line (NEC-8). Typical flow cytometry analysis histograms are presented.
(a)(b)(c)(d)(e)(f)


## 3.6.2. TC Cell Line

TC cell line (NEC-8) with treatment of 25μM PPAR-γ ligands
(15-d-PGJ2) could induce early apoptosis not late apoptosis or
necrosis (see Figure 4) and DNA fragmentation (see Figure 5). Diagrams of
FITC-Annexin V/PI flow cytometry and typical flow cytometry analysis histogram
are presented.


## 3.7. Effect of PPAR-γ Ligands in Induction of Apoptosis on PC and TC Cell Lines

To evaluate whether or not cell death induced by PPAR-γ
ligands was through apoptosis, we evaluated the chromatin morphology of PC (PC3) cell and TC cell (NEC-8) lines using hoechst
staining.


### 3.7.1. PC Cell Line

PC cell line (PC3) treated with
PPAR-γ ligands showed significant chromatin condensation, cellular shrinkage,
small membrane-bound bodies (apoptotic bodies), and cytoplasmic condensation.
These cellular changes were typically redundant characteristics of apoptosis.
PC cell lines (PC3) without PPAR-γ ligands maintained normal chromatin patterns
and cell size (see Figure 6). Typical photographs are presented in Figure 6.Effects of PPAR-γ ligands in induction of apoptosis on human PC cell line. PC cell line (PC3) treated
with PPAR-γ ligands ((b); 20 μM Troglitazone, (c); 20 μM 15-d-PGJ2) showed significant chromatin
condensation, cellular shrinkage, small membrane-bound bodies (apoptotic bodies), and cytoplasmic condensation. These cellular changes were typically redundant characteristics of apoptosis. PC cells without PPAR-γ ligands maintained normal chromatin patterns and cell size (a). Typical photographs are presented.


### 3.7.2. TC Cell Line

Similar to PC cell line, TC cell line (NEC-8) treated
with PPAR-γ ligands showed significant chromatin condensation, cellular
shrinkage, small membrane-bound bodies (apoptotic bodies), and cytoplasmic
condensation. These cellular changes were typically redundant characteristics
of apoptosis. TC cell line without PPAR-γ ligands maintained normal chromatin
patterns and cell size.


## 3.7.1. PC Cell Line

PC cell line (PC3) treated with
PPAR-γ ligands showed significant chromatin condensation, cellular shrinkage,
small membrane-bound bodies (apoptotic bodies), and cytoplasmic condensation.
These cellular changes were typically redundant characteristics of apoptosis.
PC cell lines (PC3) without PPAR-γ ligands maintained normal chromatin patterns
and cell size (see Figure 6). Typical photographs are presented in Figure 6.Effects of PPAR-γ ligands in induction of apoptosis on human PC cell line. PC cell line (PC3) treated
with PPAR-γ ligands ((b); 20 μM Troglitazone, (c); 20 μM 15-d-PGJ2) showed significant chromatin
condensation, cellular shrinkage, small membrane-bound bodies (apoptotic bodies), and cytoplasmic condensation. These cellular changes were typically redundant characteristics of apoptosis. PC cells without PPAR-γ ligands maintained normal chromatin patterns and cell size (a). Typical photographs are presented.


## 3.7.2. TC Cell Line

Similar to PC cell line, TC cell line (NEC-8) treated
with PPAR-γ ligands showed significant chromatin condensation, cellular
shrinkage, small membrane-bound bodies (apoptotic bodies), and cytoplasmic
condensation. These cellular changes were typically redundant characteristics
of apoptosis. TC cell line without PPAR-γ ligands maintained normal chromatin
patterns and cell size.


## 4. Discussion

PPAR-α is highly expressed in the liver, heart,
kidney, muscle, brown adipose tissue, and gut, which exhibit high carbolic rates
of fatty acid. PPAR-β may be expressed ubiquitously and its function is
relatively unknown. Recent studies suggest that PPAR-β may be a target for nonsteroidal
antiinflammatory drugs (NSAIDs)-induced tumor suppression in colorectal tumors.
PPAR-γ is expressed at high level in adipose tissue and is a critical regulator
of adipocyte differentiation. In addition, PPAR-α, and -γ have been considered
important immunomodulatory factors. PPAR-α knockout mice exhibit exacerbated
inflammatory responses, and leukotriene B4, a chemotractic mediator,
appears to regulate the clearance of itself as an agonist of PPAR-α. However, PPAR-γ
is also expressed in the immune system, in the spleen monocytes, bone-marrow precursors, and
helper T-cell clones. PPAR-γ is also expressed in chondrocytes as well as in synovial
and bone tissues. Recent data have shown that PPAR-γ ligands lead to inhibition
of phorbol ester-induced nitric oxide and macropharge-derived cytokines such as
tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6
(IL-6), chemokines, and adhesion molecules, in part by antagonizing the
activities of transcriptional factors [7].Recently, it has been evidenced that
thiazolidinedione, a new class of antidiabetic as a specific ligand for PPAR-γ,
and retinoic receptor agonists can regulate differentiation of cancer cells [24],
and that nuclear-acting
prostanoids, including 15-d-PGJ2, are potent activators of the PPAR-γ
receptor isoform [25, 26]. In fact, 15-d-PDJ2 induces apoptosis in macropharge, endothelial cell, choriocarcinoma cell [3, 10, 27], as well as thiazolidinediones-induced fibroblast
apoptosis [9]. PPAR-γ ligands
also inhibit vascular endothelial cell growth factor-induced angiogenesis in
vivo [28]. Angiogenesis is
important for tumorigenesis. Antiangiogenetic therapy is highly promising since
it does not induce
aquired anticancer drug resistance [29, 30].
Drevs et al. demonstrated the effect of PTK787/ZK 222584, a specific inhibitor
of vascular endothelial growth factor receptor tyrosine kinases, on primary
tumor, metastasis, vessel density, and blood flow in an animal model of renal
cell carcinoma [31]. PPAR-γ agonists
induce apoptosis in endothelial cells and inhibit vascular endothelial growth
factor-induced angiogenesis in rats. Therefore, PPAR-γ ligands may have anticancer
effects through inhibition of cell proliferation and angiogenesis.In this time, concerning about PC, we demonstrated a stronger
expression of PPAR-γ in PC and PIN tissues than in BPH or NP tissues by
immunohistochemical staining and RT-PCR. We classified 3 categories (epithelial
cells, blood vessels, and
stromal tissues) in PC, PIN, BPH, and NP tissues, and examined the intensity of
PPAR-α, -β, and -γ expressions in all tissue categories. There were no
significant differences in the intensity of PPAR-α and -β in PC, PIN, BPH, and
NP tissues. However, in all categories, PPAR-γ expression was significantly
more extensive and intense in PC and PIN tissues than in BPH and NP tissues,
and PPAR-γ expression was higher in G3 cancer than in G1 cancer. Paltoo et al.
demonstrated that there
were no significant differences between PPAR-γ expression in grades and stages [16].
Using competitive PCR, these differences may be demonstrated in the near
future.Next, we demonstrated that PPAR-γ ligands induced reduction of the
viability in PC cells in the range of 5–40 μM by using MTT assay. Furthermore,
we also demonstrated that PC cells treated with PPAR-γ ligands could induce
early apoptosis and DNA fragmentation in PC cells. Subbarayan et al. have also
demonstrated similar results [17].  Several reports support the efficacy of PPAR-γ ligands in PC [16, 18]. We expect that additional research will be
progressed.Concerning about TC,  we demonstrate stronger
expression of PPAR-γ in all tissue types of TC than in normal testicular
tissues by immunohistochemical staining and RT-PCR. There were no significant
differences among 5 histopathologic groups. We classified 2 categories
(epithelial cells and blood vessels) in TC and NT tissues, and examined the
intensity of PPAR-α, -β, and-γ expression. There were no significant
differences in the intensity of PPAR-α, -β expression between all categories of
TC and NT tissues. However, PPAR-γ expression was significantly more extensive
and intense in all categories of TC than in NT tissues. Next, we demonstrated that PPAR-γ ligands
induced the reduction of viability in TC cells in the range of 5–40 μM by MTT
assay. Furthermore, we also demonstrated that TC cells treated with PPAR-γ ligands
could induce early apoptosis and DNA fragmentation in TC cells. However, no further data on TC
and PPAR-γ have been documented in other reports. We
expect additional research will be progressed.In summary, PPAR-γ expression was significantly more
extensive and intense in malignant tissues than in normal tissue, and PPAR-γ
expression was higher in G3 cancer than in G1 cancer. Furthermore, PPAR-γ
ligands induced the reduction of malignant cell viability through apoptosis in
vitro. These results indicate that PPAR-γ participates in initiation and
promotion of tumorigenesis.These results raise the possibility that PPAR-γ may
play role in the pathogenesis and progression of PC and TC. While it is
difficult at this time to use PPAR-γ ligands at a clinical dose (relatively
nontoxic therapeutic approach) as suppressive cancer therapy,  we strongly
suggest that further research may confirm PPAR-γ ligands as a novel approach to
the treatment of PC and TC.



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*Source: 249849-2008-02-19.xml*