Cancer Research 09 AM Call for Abstracts
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lu, J.
Right arrow Articles by Kaminishi, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lu, J.
Right arrow Articles by Kaminishi, M.
[Cancer Research 65, 4769-4774, June 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Chemopreventive Effect of Peroxisome Proliferator–Activated Receptor {gamma} on Gastric Carcinogenesis in Mice

Jie Lu1,5, Kazuhiro Imamura1, Sachiyo Nomura1, Ken-ichi Mafune1, Atsushi Nakajima7, Takashi Kadowaki2, Naoto Kubota3, Yasuo Terauchi4, Genichiro Ishii6, Atsushi Ochiai6, Hiroyasu Esumi5 and Michio Kaminishi1

Departments of 1 Gastrointestinal Surgery and 2 Internal Medicine, Faculty of Medicine; 3 Department of Metabolic Diseases, Graduate School of Medicine; and 4 Department of Hematopoietic Factors, Institute of Medical Science, University of Tokyo, Tokyo, Japan; 5 Investigative Treatment Division and 6 Pathology Division, National Cancer Center Research Institute East, Kashiwa, Chiba, Japan; and 7 Gastroenterology Division, Yokohama City University Graduate School of Medicine, Yokohama, Japan

Requests for reprints: Jie Lu, Investigative Treatment Division, National Cancer Center Research Institute East, 6-5-1, Kashiwa, Chiba 277-8577, Japan. Phone: 81-471-34-6859; Fax: 81-471-34-6866; E-mail: jlu{at}east.ncc.go.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Peroxisome proliferator–activated receptor {gamma} (PPAR{gamma}) is known to be expressed in several cancers, and the treatment of these cancer cells with PPAR{gamma} ligands often induces cell differentiation and apoptosis. Recently, the chemopreventive potential of PPAR{gamma} ligands on colon carcinogenesis was reported, although the effect of PPAR{gamma} on colon carcinogenesis and the mechanism of the effect remain controversial. In this study, we attempted to elucidate the role of PPAR{gamma} in gastric carcinogenesis and explored the possible use of PPAR{gamma} ligand as a chemopreventive agent for gastric cancer. N-methyl-N-nitrosourea (MNU, 240 ppm) was given in drinking water for 10 weeks to induce gastric cancer in PPAR wild-type (+/+) and heterozygous-deficient (+/–) mice, followed by treatment with PPAR{gamma} ligand [troglitazone, 0.15% (w/w) in powder food] or the vehicle alone for 42 weeks. At the end of the experiment, PPAR{gamma} (+/–) mice were more susceptible to MNU-induced gastric cancer than wild-type (+/+) mice (89.5%/55.5%), and troglitazone significantly reduced the incidence of gastric cancer in PPAR{gamma} (+/+) mice (treatment 55.5%/vehicle 9%) but not in PPAR{gamma} (+/–) mice. The present study showed that (a) PPAR{gamma} suppresses gastric carcinogenesis, (b) the PPAR{gamma} ligand troglitazone is a potential chemopreventive agent for gastric carcinogenesis, and (c) troglitazone's chemopreventative effect is dependent on PPAR{gamma}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Peroxisome proliferator–activated receptor gamma (PPAR{gamma}) is a member of a superfamily of nuclear hormone receptors (1). PPAR{gamma} heterodimerizes with retinoid X receptor to bind to the PPAR response element, leading to the transcription of downstream genes (2). PPAR{gamma} is known to be expressed in various organs, including adipose tissue (3), mammary glands (4), small intestine (5), lung (6), colon (5), and stomach (7), and is also up-regulated in various types of cancer cells. Several specific ligands have been identified, such as the thiazolidinediones (including pioglitazone, rosiglitazone, and troglitazone), 15-deoxy-prostaglandin-J2, and certain polyunsaturated fatty acids. PPAR{gamma} ligands have been reported to induce cell differentiation and apoptosis in several cancers (8–12), suggesting a potential application as anticancer agents. Furthermore, some reports recently suggested that PPAR{gamma} ligands can be used as chemopreventive agents for colon, breast, and tongue carcinogenesis (13–16). However, the effect of PPAR{gamma} ligands on colon cancer is controversial (17, 18). On the other hand, some recent studies have reported that the biological effect of PPAR{gamma} ligand is independent of PPAR{gamma} (19–23).

Whereas gastric cancer mortality has markedly declined around the world, it remains the second leading cause of cancer death worldwide (24, 25). Increasing interest has been shown in the chemoprevention of gastric cancer because of the low curable rate and the poor relative survival rate (26–29). Although the anticancer effect of PPAR{gamma} ligands has been reported in several gastric cancer cell lines (30–34), no information is available on the role of PPAR{gamma} in gastric carcinogenesis or whether PPAR{gamma} ligands actually inhibit gastric carcinogenesis.

To address the above questions, N-methyl-N-nitrosourea (MNU) was used to induce gastric cancer in PPAR wild-type (+/+) and PPAR{gamma} heterozygous-deficient (+/–) mice, followed by treatment with a PPAR{gamma} ligand, troglitazone, for 1 year. Our results clearly showed that PPAR{gamma} plays a protective role in gastric carcinogenesis and that the chemopreventative effect of troglitazone is dependent on PPAR{gamma}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals. PPAR{gamma} knockout mice were generated as described previously (35, 36). Homozygous PPAR{gamma} knockout embryos (–/–) died because of placental dysfunction. We therefore used PPAR{gamma} heterozygous-deficient mice (+/–) in this study. To minimize the effect of the genetic background on carcinogenesis, one male and one female knockout mouse that had been generated by sister-brother mating for >10 generations were mated and all offspring were genotyped for the PPAR{gamma} gene. Then, all PPAR{gamma} heterozygous-deficient (+/–) offspring were mated again until the fourth generation mice were born. Both the wild-type and the heterozygous-deficient mice used in the present study were littermates. All mice were maintained in plastic cages with hardwood chip bedding in an air-conditioned room with a 12-hour light/12-hour dark cycle and given food (oriental CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) irradiated with 30 Gy of gamma rays and filtered tap water ad libitum.

Genetic typing. DNA was extracted from the ear of each mouse. Briefly, the ear was cut and the tissue was immersed into 400 µL of freshly prepared lysis buffer [80% saline sodium citrate, 2.5 mmol/L Tris-HCl (pH 8.0), 1 mmol/L EDTA (pH 8.0), 1% SDS, and 80 µg/mL proteinase K (Boehringer Mannheim GmbH, Indianapolis, IN)] and vortexed gently at 37°C overnight. Then 200 µL of phenol and 200 µL of chloroform were added to the lysate, and the mixture was rotated at room temperature for 1 hour, placed on ice for 5 minutes, and centrifuged at 15,000 rpm for 5 minutes. The aqueous phase was then transferred to a new tube. The above process was repeated a second time. Then, 400 µL of chloroform was added to the aqueous phase, and the solution was gently shaken by hand for 1 minute and centrifuged at 15,000 rpm for 5 minutes. DNA was recovered by the addition of 400 µL isopropanol, decanted, and rinsed with 70% ethanol thrice. The DNA was dissolved in 100 µL of TE buffer. The concentration and purity of the DNA was examined with a spectrophotometer DU 60 (Beckman, Fullerton, CA). The extracted DNA was then subjected to a PCR.

PCR. PCR was done in a TaKaRa PCR Thermal Cycler 480 (TaKaRa Biomedicals, Shiga, Japan) in a total volume of 50 µL containing 100 ng DNA, PCR buffer, 4 µL deoxynucleotide triphosphate (TaKaRa Biomedicals), 0.5 unit {gamma}Taq DNA polymerase (TaKaRa Biomedicals), 20 pmol of primer PPAR{gamma} sense, 10 pmol of primer PPAR{gamma} antisense, and 10 pmol of primer exon for 40 cycles of 30 seconds at 94°C for denaturation, 1 minute at 57°C for annealing, and 1 minute at 72°C for extension. The following primers were used: sense 5'-tctatgaggactgctctgcc-3', antisense 5'-ggtattcttggagcttcagg-3', and exon 5'-gccaccaaagaacggagccg-3'. The PCR product derived from the wild-type and knockout loci of PPAR{gamma} were easily differentiated by agarose gel electrophoresis. The 400-bp band corresponds to the knockout allele, and the 300-bp band corresponds to the wild-type allele (data not shown).

Chemicals. N-Methyl-N-nitrosourea (MNU; Sigma Chemical, St. Louis, MO) was dissolved in distilled water at a concentration of 240 ppm and freshly prepared thrice per week for administration in drinking water in light-shielded bottles ad libitum.

The PPAR{gamma}-specific ligand troglitazone was kindly provided by Sankyo Co., Ltd. (Tokyo, Japan). Troglitazone was mixed well in irradiated powder food (CE-2 M, Clea Japan, Inc., Tokyo, Japan) at a concentration of 0.15% (w/w) and freshly prepared thrice per week for administration.

Experimental design. The experimental design was approved by the Institutional Ethics Review Committee for animal experiments at the National Cancer Center. The experiment design is shown in Fig. 1. Sixty-five PPAR{gamma} (+/–) and 42 wild-type (+/+) mice, all male, age 4 to 7 weeks, were randomly divided into four groups, respectively. Animals in groups 2, 3, 6, and 7 were given drinking water containing 240 ppm MNU in light-shielded bottles on alternate weeks for a total of 10 weeks exposure, according to the protocol described in a previous report (37). Animals in groups 1, 4, 5, and 8 received only water. Several mice in each MNU-administered group died during the first 10 weeks and were omitted from the final analysis. At experimental week 11, all the drinking water was switched to autoclaved distilled water. The mice in groups 3, 4, 7, and 8 were then given powder food containing 0.15% troglitazone, whereas groups 1, 2, 5, and 6 received only the powder food for 42 weeks, until the end of the experiment. All mice were carefully autopsied at the time of their death, either after having been killed because they had become moribund or at the end of the experiment (week 52).



View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Experimental design. A total of 107 mice were used in the experiment. Groups 1 to 4 consisted of PPAR{gamma}-deficient (+/–) mice and groups 5 to 8 of wild-type (+/+) mice. Animals in groups 2, 3, 6, and 7 were given 240 ppm of MNU in drinking water on alternate weeks for a total of 10 weeks of exposure. Animals in groups 1, 4, 5, and 8 received only water. From the 11th week onward, all mice were given autoclaved distilled water, and the mice in groups 3, 4, 7, and 8 were given 0.15% (w/w) troglitazone mixed in powder food, whereas groups 1, 2, 5, and 6 received only the powder food for 42 weeks, until the end of the experiment. Black, MNU 240 ppm in drinking water; white, vehicle (distilled water and powder food); thatched, troglitazone (0.15% mixed in powder food).

 
Histopathologic analysis. At the end of the experiment, all mice were fasted for 24 hours and killed. Before sacrifice, blood samples were obtained directly from the heart. The stomachs and other organs were carefully examined macroscopically. The excised stomachs were fixed in 4% paraformaldehyde in PBS, cut into 1-mm-wide strips, embedded in paraffin, and cut into 4-µm-thick sections.

The 4-µm-thick sections were stained with H&E and carefully examined. Well-differentiated adenocarcinomas were characterized by excessive glandular proliferation with pronounced structural and cellular atypia invading at least the submucosa, moderately differentiated adenocarcinomas by cellular atypia and atypical glandular structures, and carcinoma in situ by glandular proliferation with marked structural and cellular atypia within the gastric mucosa. Other macroscopically abnormal organs were also examined histologically.

Western blot analysis of peroxisome proliferator–activated receptor {gamma}. Tissue samples obtained from mice were dissolved in lysis buffer, and the insoluble tissues were removed by centrifugation. Thirty milligrams of each solubilized lysate were then separated by gel electrophoresis on a polyacrylamide gel containing SDS and transferred to nylon membranes. PPAR{gamma} was detected with an anti-PPAR{gamma} (H-100) antibody (Santa Cruz Biotechnology, Santa Cruz, CA); visualization was accomplished using an enhanced chemiluminescence system (Amersham Pharmacia Biotech K.K., Buckinghamshire, United Kingdom).

Immunohistochemistry. The expression of PPAR{gamma} in the mouse stomach mucosa was examined by immunohistochemistry. The tissue sections were incubated at 4°C overnight with primary antibody for PPAR{gamma}, and biotinylated polyclonal anti-rabbit immunoglobulin G/horseradish peroxidase (BD Biosciences, San Jose, CA) was used as the secondary antibody. Then visualization was done. The specificity of the binding was confirmed by omitting the primary antibody, and this staining was used as a negative control.

Statistical analysis. The incidences of gastric cancer were analyzed using Fisher's exact test. Survival curves were drawn using the Kaplan-Meier method and analyzed using the log-rank test. P < 0.05 was regarded statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of peroxisome proliferator–activated receptor {gamma} in mouse gastric mucosa. The expression of PPAR{gamma} in PPAR{gamma} wild-type (+/+) and heterozygous-deficient (+/–) mice was examined immunohistochemically using a PPAR{gamma} antibody. As shown in Fig. 2A, the expression of PPAR{gamma} in wild-type (+/+) mice was significantly higher than that in heterozygous PPAR{gamma}-deficient (+/–) mice, whereas PPAR{gamma} (+/–) mice also exhibited a medial expression of PPAR{gamma}, compared with the negative control, because of the heterozygous deficiency of PPAR{gamma} in these mice.



View larger version (99K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. Expression of PPAR{gamma} in mouse stomach. A, representative section of mouse gastric mucosa. The expression of PPAR{gamma} in PPAR{gamma} (+/+) and (+/–) mice was examined immunohistochemically with PPAR{gamma} antibody. NC, negative control; WT, wild-type mouse (+/+); KO, heterozygous-deficient mice (+/–). B, Western blot analysis of PPAR{gamma} protein expression in the stomachs and colons of wild-type (+/+) and deficient (+/–) mouse. Thirty milligrams of tissue samples from heterozygous-deficient mice (+/–) (lane 1) and wild-type (+/+) (lane 2) were separated by SDS-PAGE electrophoresis and visualized using an anti-PPAR{gamma} antibody. C, colon; S, stomach; Actin, ß-actin.

 
To confirm that PPAR{gamma} is expressed at discernible levels in the mouse stomach, we also did Western blot analysis. Because PPAR{gamma} is well known to be strongly expressed in the colon, the colon was used as a positive control. As shown in Fig. 2B, a considerable level of PPAR expression was detected in the stomach of the wild-type mice, although lower than that in the colon, and a weaker level of expression was detected in the heterozygous PPAR{gamma}-deficient mice.

Loss of peroxisome proliferator–activated receptor {gamma} promotes gastric carcinogenesis. At the end of the experiment, all surviving mice were killed and their stomachs and other organs were carefully examined. The tumor incidences are summarized in Table 1.


View this table:
[in this window]
[in a new window]

  		Table 1. Incidence of stomach lesions in eight groups of mice at experiment week 52

 
None of the mice in groups 1, 4, 5, and 8 developed cancer. The carcinogen MNU induced gastric carcinoma in 17 of 19 PPAR{gamma} (+/–) mice (group 2, 89.5%): one carcinoma in situ, 15 well-differentiated adenocarcinomas, one moderately differentiated adenocarcinoma and even one lymphoma in group 2 (+/–/MNU, total of 19 mice). Gastric carcinoma was only induced in five of the nine wild-type mice that were examined (group 6, 55.5%; P < 0.05, compared with group 2; Table 1), all of which were well-differentiated gastric adenocarcinomas. These data suggest that PPAR{gamma} deficiency significantly sensitizes mice to MNU-induced gastric carcinogenesis.

Peroxisome proliferator–activated receptor {gamma} activation by troglitazone prevents the development of gastric carcinogenesis. Among the wild-type mice, five of nine mice treated with MNU were found to carry gastric carcinoma (55.5%), but the administration of 0.15% troglitazone for 42 weeks significantly reduced the incidence of gastric carcinogenesis to 1 of 11 mice (9%). However, troglitazone did not inhibit carcinogenesis in the heterozygous PPAR{gamma}-deficient mice (MNU 89.5%, MNU + Tro 80%; Table 1). Therefore, the preventative effect of troglitazone was considered dependent on PPAR{gamma}.

No appreciable histologic difference was observed in gastric carcinomas between wild-type and heterozygous peroxisome proliferator–activated receptor {gamma}–deficient mice. No significant macroscopic differences in the gastric tumors were observed between the wild-type and PPAR{gamma}-deficient mice. The microscopic morphology of the tumors was also carefully examined. Representative images of the macroscopic appearance of stomach adenocarcinoma (Fig. 3A) and the microscopic appearance of normal stomach mucosa (Fig. 3B), well-differentiated adenocarcinoma (Fig. 3C), and moderately differentiated adenocarcinoma (Fig. 3D), carcinoma in situ (Fig. 3E), and lymphoma in stomach (Fig. 3F) are shown. The gastric adenocarcinomas were mainly located in the pyloric mucosa and occasionally at the fundopyloric border. No appreciable histologic difference in the gastric carcinomas was observed between the wild-type and heterozygous PPAR{gamma}-deficient mice.



View larger version (142K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Gross and histologic appearance of representative stomach and its lesions. Stomach with tumor (A), the histologic appearance of normal stomach mucosa (B), well-differentiated adenocarcinoma (C), moderately differentiated adenocarcinoma (D), carcinoma in situ (E), and lymphoma in stomach (F). Inset, high power of the lymphoma cells.

 
Mice died after the 37th week because of carcinogen-induced gastric carcinoma. The survival curves for all eight mice groups are shown in Fig. 4. Before the end of the experiment (52nd week), 10 of 19 mice in group 2, 8 of 15 mice in group 3, 1 of 9 mice in group 6, and 1 of 11 mice in group 7 died. These data suggested that the increased gastric carcinoma burden reduced the survival time of PPAR{gamma} (+/–) mice, compared with that of wild-type mice (P < 0.01), but there was no difference between the control group and the troglitazone-treated group, either wild-type or PPAR{gamma} (+/–) mice. Because of the reported rare but severe hepatotoxicity of troglitazone, we examined the liver of mice treated with troglitazone. Neither degeneration nor necrosis was observed. Focal liver cell hyperplasia was found in one mouse in the group 3, but no dysplasia or cancer was observed. We did not find any abnormality in other organs.



View larger version (11K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. Survival curves of mice. The PPAR{gamma}-deficient (+/–) mice exhibited a significantly higher MNU-induced mortality, compared with the wild-type mice. **, P < 0.01 (log-rank test); 1-8, groups 1-8. Mice died after the 37th. Before the end of the experiment (52nd week), 10 of 19 mice in group 2, 8 of 15 mice in group 3, 1 of 9 mice in group 6, and 1 of 11 mice in group 7 died. No death occurred in the groups 1, 4, 5, and 8.

 
Peroxisome proliferator–activated receptor {gamma} expression was suppressed in cancerous gastric mucosa. The above observations suggest that PPAR{gamma} might exert a tumor suppressor activity in response to endogenous ligand(s). If this is the case, the gastric carcinomas that were induced might have lost their expression of PPAR{gamma} because of inhibitory selective pressure from either endogenous or exogenous ligands. To address this question, we examined the expression of PPAR{gamma} in cancerous and normal gastric mucosa from the same mouse using immunohistochemistry. The results showed that the expression of PPAR{gamma} in the malignant mucosa was much weaker than in the normal mucosa in both wild-type and heterozygous PPAR{gamma}-deficient mice, as shown in Fig. 5A, and the expression of PPAR{gamma} was also lower in the cancerous mucosa of the wild-type mouse treated with troglitazone that developed gastric carcinoma, although a stronger PPAR{gamma} expression was observed in the normal mucosa of mice treated with troglitazone. This result was confirmed by Western blot analysis using the protein extracted from either the normal or the cancerous gastric mucosa from wild-type mice (Fig. 5B).



View larger version (81K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Expression of PPAR{gamma} in cancerous gastric mucosa. A, the immunohistochemical expression of PPAR{gamma} in malignant gastric mucosa was significantly weaker than in normal mucosa. a, c, (+/+) mice; b, d, (+/–) mice; open arrow, normal mucosa; closed arrow, malignant mucosa. B, Western blot analysis for PPAR{gamma} expression in the gastric mucosa of wild-type (+/+) mouse. N, normal mucosa; C, cancerous mucosa.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recently, the potential of PPAR{gamma} as a target for the prevention and treatment of cancer has been widely explored (38). However, the therapeutic potential of PPAR{gamma} ligands has been questioned, based on the results of experiments using animal models for colon cancer, in which the PPAR{gamma} ligands increased the development of colon tumors (17). This contradictory result was supplemented by a recent report using transgenic mice that express a constitutive active form of PPAR{gamma} in mammary glands showing that PPAR{gamma} signaling accelerated tumor development in mammary glands (39). The actual role of PPAR{gamma} in cancer has been complicated by recent findings that PPAR{gamma} ligands affect cancer cells independent of PPAR{gamma} (21, 40–42). To date, no information on the role of PPAR{gamma} in gastric carcinogenesis is available.

The results of this study provide clear evidence of the critical importance of PPAR{gamma} in gastric carcinogenesis. The loss of one allele of PPAR{gamma} significantly enhanced carcinogen-induced gastric carcinogenesis and decreased the survival rate, compared with the wild-type (+/+) littermates. Our results are in good agreement with the report by Nicol et al. that PPAR{gamma} haploinsufficiency increased the susceptibility to carcinogen-induced breast carcinogenesis, suggesting that PPAR{gamma} acts as a tumor suppressor of skin, ovarian, and breast cancers (43). However, our results are inconsistent with those of one other study that suggested PPAR{gamma} acts as a tumor promoter in breast carcinogenesis, instead of a tumor suppressor (39). In that study, the loss of one allele of PPAR{gamma} did not influence breast tumorigenesis. This result cannot be easily explained. However, the histologic and pathologic differences between breast and stomach cancer, the method of inducing carcinogenesis (mouse mammary tumor virus/PyV transgenic mice were used to evaluate tumorigenesis in the other study, whereas the carcinogen MNU in drinking water was used to induce carcinogenesis in the present study), and the difference in the genetic backgrounds of the mice used in the two studies [the (C57BL/6 x DBA/2)F1(B6D2F1) mice were used in that study and the B6 x CBA x ICR mice in the present study] may be responsible for these inconsistencies, although further investigation is certainly needed.

The reduction in PPAR{gamma} expression observed among MNU-induced gastric adenocarcinomas from either PPAR{gamma} (+/–) or wild-type mice agreed well with the findings of several other reports showing a reduction in PPAR{gamma} protein expression in breast and colon cancers, where expression was highest in normal tissue and decreased from benign to malignant states of disease (44–46). Recently, Badawi et al. reported that PPAR{gamma} mRNA and protein levels were lower in MNU-induced rat mammary tumors than in normal tissues (47). Our present study showed the suppressed expression of PPAR{gamma} in MNU-induced carcinomas, showing a consistent pattern of PPAR{gamma} expression. These observations support the hypothesis that PPAR{gamma} can exert a tumor suppressing activity.

Furthermore, our results unambiguously showed the chemopreventive potential of a PPAR{gamma} ligand, troglitazone, in gastric carcinogenesis. The administration of troglitazone significantly suppressed the formation of MNU-induced gastric carcinoma in wild-type mice. No significant differences in the macroscopic and histologic features of the gastric adenocarcinomas were observed between the wild-type and PPAR{gamma}-deficient mice treated with or without troglitazone. Importantly, troglitazone suppressed gastric carcinogenesis without affecting the tumor pathology. In addition, troglitazone's preventive effect was only observed in wild-type mice but not in heterozygous PPAR{gamma}-deficient mice, and the reduction in PPAR{gamma} expression in the transformed mucosa clearly indicated that this effect was dependent of PPAR{gamma}. This result addresses the controversy regarding the dependence of troglitazone on PPAR{gamma}, at least in gastric carcinogenesis. However, in the present study, within 52 weeks, troglitazone did not alter the mortality rate. Only one mouse died in the wild-type mice group, either treated with or without troglitazone. It is necessary to extend the experimental period to evaluate troglitazone's effect on the mortality rate. Because troglitazone's preventive effect on gastric cancer seems dependent of PPAR{gamma}, as observed in the present study, the preventive effect of other ligands, such as pioglitazone, rosiglitazone and 15-prostaglandin-J2, on carcinogenesis should be investigated in the future. Recently, the preventive effect of PPAR{gamma} against acute gastric mucosal lesions associated with ischemia-reperfusion was reported (48). In that study, PPAR{gamma} ligands showed protection against acute gastric mucosal lesions formation induced by ischemia-reperfusion in mice in a dose-dependent manner, and the acute gastric mucosal lesions in PPAR{gamma} (+/–) mice was more severe than in wild-type mice. In addition, the inhibition of the up-regulation of tumor necrosis factor-{alpha}, intercellular adhesion molecule-1, inducible nitric oxide synthase, apoptosis, and nitrotyrosine formation in the stomach may be responsible for the preventive effect of PPAR{gamma}. This may provide us the speculation that the preventive effect of PPAR{gamma} against gastric carcinogenesis may be through inhibiting the nuclear factor {kappa}B–mediated transcription.

Our present results that PPAR{gamma} deficiency sensitizes mice to carcinogen-induced gastric carcinogenesis may provide a way of identifying certain populations susceptible to gastric cancer. In addition, the significant cancer-preventive effect of troglitazone has very important clinical implications. Provided that certain individuals at a higher risk of gastric cancer can be identified, these individuals might actually benefit from the use of PPAR{gamma} ligands as chemopreventive agents for gastric cancer.

Taken as a whole, the present study is the first report to show that (a) PPAR{gamma} (+/–) mice have an increased susceptibility to MNU-induced carcinogenesis, suggesting that PPAR{gamma} may function as a tumor suppressor; (b) the PPAR{gamma} ligand troglitazone is a potential chemopreventive agent for gastric cancer; and (c) troglitazone's chemopreventive effect is dependent on PPAR{gamma}.


   Acknowledgments
 
Grant support: Ministry of Education, Culture, Sports, Science, and Technology of Japan; Second-term Comprehensive Strategy for Cancer Control; Medical Frontier Program of the Ministry of Health, Labor, and Welfare of Japan; and Foundation for the Promotion of Cancer Research in Japan research resident fellowship (J. Lu).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/ 8/04. Revised 2/24/05. Accepted 3/ 8/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Green S, Tugwood JD, Issemann I. The molecular mechanism of peroxisome proliferator action: a model for species differences and mechanistic risk assessment. Toxicol Lett 1992;64 Spec:131–9.
  2. Bardot O, Aldridge TC, Latruffe N, Green S. PPAR-RXR heterodimer activates a peroxisome proliferator response element upstream of the bifunctional enzyme gene. Biochem Biophys Res Commun 1993;192:37–45.[CrossRef][Medline]
  3. Keller H, Mahfoudi A, Dreyer C, et al. Peroxisome proliferator-activated receptors and lipid metabolism. Ann N Y Acad Sci 1993;684:157–73.[Medline]
  4. Gimble JM, Pighetti GM, Lerner MR, et al. Expression of peroxisome proliferator activated receptor mRNA in normal and tumorigenic rodent mammary glands. Biochem Biophys Res Commun 1998;253:813–7.[CrossRef][Medline]
  5. Mansen A, Guardiola-Diaz H, Rafter J, Branting C, Gustafsson JA. Expression of the peroxisome proliferator-activated receptor (PPAR) in the mouse colonic mucosa. Biochem Biophys Res Commun 1996;222:844–51.[CrossRef][Medline]
  6. McGowan SE, Jackson SK, Doro MM, Olson PJ. Peroxisome proliferators alter lipid acquisition and elastin gene expression in neonatal rat lung fibroblasts. Am J Physiol 1997;273:1249–57.
  7. Huin C, Corriveau L, Bianchi A, et al. Differential expression of peroxisome proliferator-activated receptors (PPARs) in the developing human fetal digestive tract. J Histochem Cytochem 2000;48:603–11.[Abstract/Free Full Text]
  8. Elstner E, Muller C, Koshizuka K, et al. Ligands for peroxisome proliferator-activated receptorgamma and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice. Proc Natl Acad Sci U S A 1998;95:8806–11.[Abstract/Free Full Text]
  9. Tontonoz P, Singer S, Forman BM, et al. Terminal differentiation of human liposarcoma cells induced by ligands for peroxisome proliferator-activated receptor gamma and the retinoid X receptor. Proc Natl Acad Sci U S A 1997;94:237–41.[Abstract/Free Full Text]
  10. Rumi MA, Sato H, Ishihara S, et al. Peroxisome proliferator-activated receptor gamma ligand-induced growth inhibition of human hepatocellular carcinoma. Br J Cancer 2001;84:1640–7.[CrossRef][Medline]
  11. Heaney AP, Fernando M, Melmed S. PPAR-gamma receptor ligands: novel therapy for pituitary adenomas. J Clin Invest 2003;111:1381–8.[CrossRef][Medline]
  12. Ohta K, Endo T, Haraguchi K, Hershman JM, Onaya T. Ligands for peroxisome proliferator-activated receptor gamma inhibit growth and induce apoptosis of human papillary thyroid carcinoma cells. J Clin Endocrinol Metab 2001;86:2170–7.[Abstract/Free Full Text]
  13. Badawi AF, Badr MZ. Chemoprevention of breast cancer by targeting cyclooxygenase-2 and peroxisome proliferator-activated receptor-gamma (Review). Int J Oncol 2002;20:1109–22.[Medline]
  14. Brown PH, Lippman SM. Chemoprevention of breast cancer. Breast Cancer Res Treat 2000;62:1–17.[CrossRef][Medline]
  15. Kopelovich L, Fay JR, Glazer RI, Crowell JA. Peroxisome proliferator-activated receptor modulators as potential chemopreventive agents. Mol Cancer Ther 2002;1:357–63.[Abstract/Free Full Text]
  16. Li J, Lv Y, Dong X, Jin Z. Pioglitazone, a peroxisome proliferators-activated receptor gamma ligand, inhibits dimethylhydrazine (DMH) induced aberrant crypt foci in rats. Beijing Da Xue Xue Bao 2003;35:537–9.[Medline]
  17. Lefebvre AM, Chen I, Desreumaux P, et al. Activation of the peroxisome proliferator-activated receptor gamma promotes the development of colon tumors in C57BL/6J-APCMin/+ mice. Nat Med 1998;4:1053–7.[CrossRef][Medline]
  18. Saez E, Tontonoz P, Nelson MC, et al. Activators of the nuclear receptor PPAR{gamma} enhance colon polyp formation. Nat Med 1998;4:1058–61.[CrossRef][Medline]
  19. Wang M, Wise SC, Leff T, Su TZ. Troglitazone, an antidiabetic agent, inhibits cholesterol biosynthesis through a mechanism independent of peroxisome proliferator-activated receptor-gamma. Diabetes 1999;48:254–60.[Abstract]
  20. Castrillo A, Mojena M, Hortelano S, Bosca L. Peroxisome proliferator-activated receptor-gamma-independent inhibition of macrophage activation by the non-thiazolidinedione agonist L-796,449. Comparison with the effects of 15-deoxy-{delta}(12,14)-prostaglandin J(2). J Biol Chem 2001;276:34082–8.[Abstract/Free Full Text]
  21. Palakurthi SS, Aktas H, Grubissich LM, Mortensen RM, Halperin JA. Anticancer effects of thiazolidinediones are independent of peroxisome proliferator-activated receptor gamma and mediated by inhibition of translation initiation. Cancer Res 2001;61:6213–8.[Abstract/Free Full Text]
  22. Okano H, Shiraki K, Inoue H, et al. 15-deoxy-{delta}-12-14-PGJ2 regulates apoptosis induction and nuclear factor-{kappa}B activation via a peroxisome proliferator-activated receptor-gamma-independent mechanism in hepatocellular carcinoma. Lab Invest 2003;83:1529–39.[CrossRef][Medline]
  23. Weber SM, Scarim AL, Corbett JA. PPAR{{gamma}} is not required for the inhibitory actions of PGJ2 on cytokine signaling in pancreatic {ß}-cells. Am J Physiol Endocrinol Metab 2004;286:E329–36.[Abstract/Free Full Text]
  24. Roder DM. The epidemiology of gastric cancer. Gastric Cancer 2002;5 Suppl 1:5–11.[CrossRef][Medline]
  25. Parkin DM, Bray F, Ferlay J, Pisani P. Estimating the world cancer burden: Globocan 2000. Int J Cancer 2001;94:153–6.[CrossRef][Medline]
  26. Eguchi T, Fujii M, Takayama T. Mortality for gastric cancer in elderly patients. J Surg Oncol 2003;84:132–6.[CrossRef][Medline]
  27. Correa P. Chemoprevention of gastric cancer: has the time come? J Clin Oncol 2003;21:270–1.[CrossRef]
  28. Kakizoe T. Chemoprevention of cancer: focusing on clinical trials. Jpn J Clin Oncol 2003;33:421–2.[Abstract/Free Full Text]
  29. Wong JE, Ito Y, Correa P, et al. Therapeutic strategies in gastric cancer. J Clin Oncol 2003;21:267–9.
  30. Takahashi N, Okumura T, Motomura W, Fujimoto Y, Kawabata I, Kohgo Y. Activation of PPAR{gamma} inhibits cell growth and induces apoptosis in human gastric cancer cells. FEBS Lett 1999;455:135–9.[CrossRef][Medline]
  31. Sato H, Ishihara S, Kawashima K, et al. Expression of peroxisome proliferator-activated receptor (PPAR)gamma in gastric cancer and inhibitory effects of PPAR{gamma} agonists. Br J Cancer 2000;83:1394–400.[CrossRef][Medline]
  32. Takeuchi S, Okumura T, Motomura W, et al. Troglitazone induces G1 arrest by p27(Kip1)26 induction that is mediated by inhibition of proteasome in human gastric cancer cells. Jpn J Cancer Res 2002;93:774–82.[Medline]
  33. Chen YX, Zhong XY, Qin YF, Bing W, He LZ. 15d-PGJ2 inhibits cell growth and induces apoptosis of MCG-803 human gastric cancer cell line. World J Gastroenterol 2003;9:2149–53.[Medline]
  34. Nagamine M, Okumura T, Tanno S, et al. PPAR gamma ligand-induced apoptosis through a p53-dependent mechanism in human gastric cancer cells. Cancer Sci 2003;94:338–43.[CrossRef][Medline]
  35. Kubota N, Terauchi Y, Miki H, et al. PPAR gamma mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance. Mol Cell 1999;4:597–609.[CrossRef][Medline]
  36. Nakajima A, Wada K, Miki H, et al. Endogenous PPAR gamma mediates anti-inflammatory activity in murine ischemia-reperfusion injury. Gastroenterology 2001;120:460–9.[CrossRef][Medline]
  37. Yamachika T, Nakanishi H, Inada K, et al. N-methyl-N-nitrosourea concentration-dependent, rather than total intake-dependent, induction of adenocarcinomas in the glandular stomach of BALB/c mice. Jpn J Cancer Res 1998;89:385–91.[Medline]
  38. Sporn MB, Suh N, Mangelsdorf DJ. Prospects for prevention and treatment of cancer with selective PPAR{gamma} modulators (SPARMs). Trends Mol Med 2001;7:395–400.[CrossRef][Medline]
  39. Saez E, Rosenfeld J, Livolsi A, et al. PPAR gamma signaling exacerbates mammary gland tumor development. Genes Dev 2004;18:528–40.[Abstract/Free Full Text]
  40. Clay CE, Namen AM, Atsumi G, et al. Magnitude of peroxisome proliferator-activated receptor-gamma activation is associated with important and seemingly opposite biological responses in breast cancer cells. J Investig Med 2001;49:413–20.[Medline]
  41. Clay CE, Monjazeb A, Thorburn J, Chilton FH, High KP. 15-Deoxy-{delta}12,14-prostaglandin J2-induced apoptosis does not require PPAR{gamma} in breast cancer cells. J Lipid Res 2002;43:1818–28.[Abstract/Free Full Text]
  42. Place AE, Suh N, Williams CR, et al. The novel synthetic triterpenoid, CDDO-imidazolide, inhibits inflammatory response and tumor growth in vivo. Clin Cancer Res 2003;9:2798–806.[Abstract/Free Full Text]
  43. Christopher JN, Michung Y, Jerrold M, et al. PPAR gamma influences susceptibility to DMBA-induced mammary, ovarian and skin carcinogenesis. Carcinogenesis 2004;25:1747–55.[Abstract/Free Full Text]
  44. Mueller E, Sarraf P, Tontonoz P, et al. Terminal differentiation of human breast cancer through PPAR gamma. Mol Cell 1998;1:465–70.[CrossRef][Medline]
  45. Wang C, Pattabiraman N, Zhou JN, et al. Cyclin D1 repression of peroxisome proliferator-activated receptor gamma expression and transactivation. Mol Cell Biol 2003;23:6159–73.[Abstract/Free Full Text]
  46. Bogazzi F, Ultimieri F, Raggi F, et al. Changes in the expression of the peroxisome proliferator-activated receptor gamma gene in the colonic polyps and colonic mucosa of 27 acromegalic patients. J Clin Endocrinol Metab 2003;88:3938–42.[Abstract/Free Full Text]
  47. Badawi AF, Eldeen MB, Liu Y, Ross EA, Badr MZ. Inhibition of rat mammary gland carcinogenesis by simultaneous targeting of cyclooxygenase-2 and peroxisome proliferator-activated receptor gamma. Cancer Res 2004;64:1181–9.[Abstract/Free Full Text]
  48. Wada K, Nakajima A, Takahashi H, et al. Protective effect of endogenous PPAR{{gamma}} against acute gastric mucosal lesions associated with ischemia-reperfusion. Am J Physiol Gastrointest Liver Physiol 2004;287:452–8.



This article has been cited by other articles:


Home page
 Molecular Cancer TherapeuticsHome page
L. Qiao, Y. Dai, Q. Gu, K. W. Chan, B. Zou, J. Ma, J. Wang, H. Y. Lan, and B. C.Y. Wong
Down-regulation of X-linked inhibitor of apoptosis synergistically enhanced peroxisome proliferator-activated receptor {gamma} ligand-induced growth inhibition in colon cancer
Mol. Cancer Ther., July 1, 2008; 7(7): 2203 - 2211.
[Abstract] [Full Text] [PDF]

Home page
 Ann OncolHome page
K. N. Prasad, A. Saxena, U. C. Ghoshal, M. R. Bhagat, and N. Krishnani
Analysis of Pro12Ala PPAR gamma polymorphism and Helicobacter pylori infection in gastric adenocarcinoma and peptic ulcer disease
Ann. Onc., July 1, 2008; 19(7): 1299 - 1303.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol PatholHome page
T. Tsukamoto, T. Mizoshita, and M. Tatematsu
Animal Models of Stomach Carcinogenesis
Toxicol Pathol, August 1, 2007; 35(5): 636 - 648.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H. Tomita, Y. Yamada, T. Oyama, K. Hata, Y. Hirose, A. Hara, T. Kunisada, Y. Sugiyama, Y. Adachi, H. Linhart, et al.
Development of Gastric Tumors in ApcMin/+ Mice by the Activation of the {beta}-Catenin/Tcf Signaling Pathway
Cancer Res., May 1, 2007; 67(9): 4079 - 4087.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
Y. Yokoyama, B. Xin, T. Shigeto, M. Umemoto, A. Kasai-Sakamoto, M. Futagami, S. Tsuchida, F. Al-Mulla, and H. Mizunuma
Clofibric acid, a peroxisome proliferator-activated receptor {alpha} ligand, inhibits growth of human ovarian cancer
Mol. Cancer Ther., April 1, 2007; 6(4): 1379 - 1386.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Pathol.Home page
S. Stadlmann, U. Gueth, E. Wight, L. A Kunz-Schughart, A. Hartmann, and G. Singer
Expression of peroxisome proliferator activated receptor {gamma} and cyclo-oxygenase 2 in primary and recurrent ovarian carcinoma
J. Clin. Pathol., March 1, 2007; 60(3): 307 - 310.
[Abstract] [Full Text] [PDF]

Home page
 GutHome page
M Adachi, R Kurotani, K Morimura, Y Shah, M Sanford, B B Madison, D L Gumucio, H E Marin, J M Peters, H A Young, et al.
Peroxisome proliferator activated receptor {gamma} in colonic epithelial cells protects against experimental inflammatory bowel disease
Gut, August 1, 2006; 55(8): 1104 - 1113.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
M. A. Peraza, A. D. Burdick, H. E. Marin, F. J. Gonzalez, and J. M. Peters
The Toxicology of Ligands for Peroxisome Proliferator-Activated Receptors (PPAR)
Toxicol. Sci., April 1, 2006; 90(2): 269 - 295.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lu, J.
Right arrow Articles by Kaminishi, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lu, J.
Right arrow Articles by Kaminishi, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online