Japanese Journal of Clinical Oncology 32:238-243 (2002)
© 2002 Foundation for Promotion of Cancer Research
Decreased Peroxisome Proliferator-activated Receptor Gamma Gene Expression is Correlated with Poor Prognosis in Patients with Esophageal Cancer
Department of Surgery II, Nagoya City University Medical School, Nagoya, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Background: Peroxisome proliferator-activated receptor gamma (PPAR
) induces apoptosis by ligand stimulation in various tumor cell lines. In esophageal cancer cell lines, PPAR activation also has suppressed the proliferation.
Methods: In 55 primary esophageal squamous cell carcinomas (ESCCs) we examined the correlation between the expression of PPAR mRNA with prognosis of esophageal cancer patients. The expression of PPAR mRNA was quantified by real-time reverse transcription polymerase chain reaction using LightCycler. Immunohistochemistry was used to study the expression of PPAR protein.
Results: The expression of PPAR mRNA was significantly decreased in esophageal cancer cells compared with normal esophageal mucosa (P = 0.0084). Among the clinical factors, PPAR mRNA expression was lower in the tumors with extensive lymph node metastasis (n4) than those with less extensive lymph node metastasis (n0–3) (P = 0.0059). Patients with low PPAR mRNA expression had significantly shorter postoperative survival time than those with high PPAR mRNA expression (P = 0.0191). In immunohistochemistry, PPAR protein was expressed in the nuclei of cells in some cases and expressed in the nuclei and cytoplasm in others. The expression of PPAR protein is decreased in esophageal cancer tissue compared with normal esophageal squamous epithelium. However, we could not deduce the apparent relation for the expression between PPAR mRNA and PPAR proteins in immunohistochemistry (P = 0.284).
Conclusions: In esophageal cancer tissues, the expression of PPAR was decreased compared with normal esophageal epithelium. The mRNA expression level of PPAR may be a marker of prognosis after operation in esophageal cancer patients.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Peroxisome proliferator-activated receptor gamma (PPAR
) is a member of the nuclear receptor superfamily (1). It is expressed in multiple organs including breast, colon, stomach, lung and ovary (2–5). It binds to specific recognition sites on DNA and transactivates the target genes as a heterodimer with the retinoid X receptor (5). PPAR has been reported to play a role in differentiation of adipocytes and monocytes (6–7). Recent studies have shown that PPAR is associated with differentiation and apoptosis in various human cancers (5,8,9). PPAR is capable of being activated by 15-deoxy-
-prostaglandin J2 and thiazolidinedione (5) and activated PPAR has been reported to inhibit cancer cell growth. In lung, prostate, colon, thyroid, gastric and pancreatic cancers, PPAR activation inhibited cell growth in a dose-dependent manner (4,8,10–17). On the other hand, PPAR leads to G1 cell cycle arrest in colon, bladder, gastric and pancreatic cancer cells (4,12,14,18). However, the mechanism by which PPAR affects cell proliferation or differentiation remains unclear. In this study, we investigated mRNA expression of PPAR in 55 primary esophageal squamous cell carcinomas (ESCCs) and their paired normal esophageal mucosa and its correlation with clinicopathological factors and prognosis of ESCC patients. We quantified the PPAR mRNA expression level by real-time reverse transcription polymerase chain reaction (RT-PCR) using LightCycler (19). We also investigated the expression and localization of PPAR protein using immunohistochemistry. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Tissue Samples
Samples were obtained from 55 esophageal cancer patients who had undergone operations at the Department of Surgery II, Nagoya City University Medical School, between 1996 and 2000. They were classified according to the guidelines for the clinical and pathological studies on carcinoma of the esophagus (20). All samples for RT-PCR were frozen immediately in liquid nitrogen and stored at –80°C until analysis. All tissues for immunohistochemistry were fixed in formalin and embedded in paraffin.
RNA Extraction and RT-PCR Analysis
Total RNA was extracted from the esophageal cancer tissues, normal esophageal mucosa taken from as far away from the tumor as possible and esophageal cancer cell lines, using an Isogen kit (Nippon Gene, Tokyo, Japan). The concentration of the total RNA was adjusted to 200 ng/ml, using a spectrophotometer. Reverse transcriptional reaction was carried out at 42°C for 90 min and 95°C for 5 min followed by incubation at 72°C for 15 min, using 1 µg of total RNA, 0.5 µg of oligo(dT) primer and Superscript II enzyme (Gibco BRL, Gaithersburg, MD). To confirm the exactitude of RNA extraction and RT-PCR, all samples were subjected into PCR amplification, using a LightCycler-FastStart DNA Master SYBR Green I kit (Roche Molecular Biochemicals, Mannheim, Germany). We used the following set of primers: forward primer PPAR-F, 5-TCTCTCCGTAATGGAAGACC-3, and reverse primer PPAR-R, 5-GCATTATGAGACATCCCCAC-3 (474 bp). The PCR protocol was as follows: initial denature at 95°C for 10 min, followed by 60 cycles at 94°C for 15 s, annealing at 63°C for 5 s, extension at 72°C for 19 s. The PCR product was quantified via the intensity of SYBR Green I at 83°C.
Immunohistochemistry
Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded primary 45 human esophageal cancer tissues of 55 ESCCs with RT-PCR analysis, using an affinity-purified, rabbit polyclonal anti-PPAR antibody, H-100 (Santa Cruz Biotechnology, Santa Cruz, CA). This antibody recognizes the epitope of 6–105 sequence mapping at the N-terminus of PPAR. Paraffin-embedded sections of tumor were deparaffinized, rehydrated and heat-treated for citrate microwave antigen retrieval in 10 mM citrate buffer for 15 min and then cooled to room temperature. Sections were then blocked for peroxidases in 0.3% H2O2 in methanol for 30 min, blocked with non-specific goat serum for 10 min and incubated with primary antibody H-100 overnight at room temperature in a humidity chamber. For detection, a DAKO ENVISION System HRP with DAB staining and hematoxylin counterstaining was used.
Statistical Analysis
Data are expressed as means ± SD. Statistical analysis was done using the Stat-View software package (Abacus Concepts). The Mann–Whitney U-test was used to evaluate the significance of the expression of PPAR mRNA. The survival of ESCC patients after operation was examined by the Kaplan–Meier method. The result was considered significant when the P-value was <0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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First, we examined PPAR
mRNA expression in esophageal cancer cell lines (TE5, TE7, TE8, TE9, TE10) by RT-PCR using LightCycler. PPAR mRNA was detectable in all the cell lines studied (Fig. 1a). Next, 55 primary ESCC samples and their paired normal esophageal tissues were examined. PPAR mRNA expression was detectable in the majority of ESCC tissues and all the normal esophageal mucosa (Fig. 1b). PPAR mRNA expression level was analyzed with reference to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The PPAR mRNA expression level was significantly decreased in ESCC tissues (0.722 ± 0.637), compared with normal esophageal mucosa (1.105 ± 1.007) (P = 0.0084). (Fig. 2).
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In clinicopathological studies, the PPAR
mRNA expression level of ESCCs with extensive lymph node metastasis (n4) (0.264 ± 0.321, n = 9) was significantly decreased compared with those with less extensive lymph node metastasis (n0–3) (0.812 ± 0.647, n = 46, P = 0.0059) (Fig. 3). Other factors such as differentiation status of tumor cells, tumor size, stage and gender of the patients, were not correlated with the PPAR mRNA expression of the tumors (Table 1).
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We investigated the correlation between PPAR
mRNA expression level and survival time of ESCC patients after operation with a median follow-up of 14.6 months. Patients with low PPAR mRNA expression (PPAR/GAPDH <0.866) had a significantly shorter survival time after the operation than those with high PPAR mRNA expression (PPAR/GAPDH >0.866, P = 0.0191; log-rank test, P = 0.0089, Breslow–Gehan–Wilcoxson test, median follow-up time 14.6 months) (Fig. 4). We decided on a borderline of a PPAR/GAPDH ratio of 0.866 for the most significant difference in prognosis between low and high expression of PPAR from ESCC.
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To examine the expression and localization of PPAR
, we performed immunohistochemistry. PPAR protein was recognized in the tumor cells and normal squamous epithelial cells. In normal epithelial cells, PPAR was stained in the nuclei of the cells at the basal layer (Fig. 5). Of 45 ESCC tissues studied, 29 samples expressed PPAR stain (64.4%) and 16 were negative for PPAR (35.6%). Of 29 samples positive for PPAR stain, 10 showed positive stain only in the nuclei (34.5%). Seven samples showed positive staining only in the cytoplasm (24.1%). Twelve samples showed positive staining in both nuclei and cytoplasm (41.4%) (Fig. 6). We could not find a significant correlation between the level of PPAR mRNA expression by RT-PCR and PPAR protein expression by immunohistochemistry (P = 0.284, Mann–Whitney U-test).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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It has been reported that PPAR
is expressed in multiple normal tissues and cancers. In colon cancer and pancreatic cancer, its expression level was shown to be equal to or higher than that in the normal tissue (15,21). In this study, we have shown that PPAR mRNA was significantly decreased in ESCC compared with normal esophageal mucosa. In a recent study, we found that PPAR mRNA expression was down-regulated in lung cancer (22). Hence, in human cancers, PPAR expression level does not seem to show a consistent tendency, suggesting tissue-specific expression of PPAR.
PPAR seems to play an important role in the proliferation of cancer cells. PPAR activation by its ligand leads to up-regulation of p21 or p27Kip1 and inhibits cell growth in neuroblastoma, hepatoma and lung, thyroid, bladder, pancreatic cancer cell lines (8,15,16,19,23,24). A recent study has reported that treatment by the ligand for PPAR led to growth inhibition in esophageal cancer cell lines (9); the down-regulation of PPAR might contribute to the progression of esophageal cancer.
In this study, we found a significant difference between the PPAR mRNA expression in n0–3 ESCCs and that in n4 ESCCs. It is not known how the decreased PPAR is related to the tumor invasiveness. Matrix metalloproteinase-2 (MMP-2) is related to invasion and lymph node metastasis of cancer. It has been reported that activated PPAR led to reduced activity of MMP-2 in lung cancer (25). The down-regulated PPAR in n4 ESCCs as reported in this study may have favored the invasion of the esophageal cancer by sustaining the level of MMP-2.
PPAR has been reported to affect differentiation of various cells, e.g. adipocyte, monocyte and several cancer cells. In breast, lung and bladder cancer, PPAR is up-regulated during differentiation of cancer cells (3,8,18) and PPAR induces differentiation in colon cancer, pancreatic cancer and liposarcoma cells (3,14,26). In our study, we could not find a significant relationship between PPAR expression and the differentiation of ESCC cells.
Prognosis of ESCC patients is generally poor and it is important to identify patients with potentially poor prognosis. Previous studies have shown that reduction of cyclin D1 and expression of E-cadherin were significant prognostic factors in ESCC patients (27,28). In bladder cancer, reduction of cyclin D1 by activated PPAR has been reported (18). In esophageal cancer, the low expression of PPAR might result in the reduction of cyclin D1 and in the progression of esophageal cancer. These effects might be the cause our result where we have shown that the PPAR mRNA expression level was significantly correlated with the prognosis of ESCC patients (P = 0.0191). Moreover, activated PPAR has been reported to induce apoptosis in several tumors (8,9), and the reduction of PPAR may also result in suppression of apoptosis in esophageal cancer.
In this study, PPAR protein varied and was found in both the nuclei and cytoplasm of tumor cells. We also failed to detect a significant correlation between the expression of mRNA and protein of PPAR. This may be because we used different parts of the tumor to study the mRNA and protein. PPAR is not known to possess a nuclear localization sequence and further study is needed to confirm and analyze the significance of localization of PPAR protein.
In conclusion, the expression level of PPAR is decreased in ESCC compared with normal esophageal epithelium. PPAR mRNA expression could be a prognostic marker of ESCC patients. Further study and longer follow-up of the patients are required to define its exact role in esophageal cancer invasion and patient prognosis.
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Acknowledgment |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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The authors thank Ms Shinobu Makino for her excellent technical assistance.
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FOOTNOTES |
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+ For reprints and all correspondence: Yoshitaka Fujii, Department of Surgery II, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. E-mail: yosfujii@med.nagoya-cu.ac.jp
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Received December 18, 2001; accepted April 19, 2002
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