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Gut. 2004 Aug; 53(8): 1151–1158.
PMCID: PMC1774140

Downregulation of prostaglandin E receptor subtype EP3 during colon cancer development

Abstract

Background and aims: Involvement of prostaglandin E2 (PGE2) receptors EP1, EP2, and EP4 in the formation of aberrant crypt foci (ACF) and/or intestinal polyps has been suggested. In contrast, EP3 appears to have no influence on the early stages of colon carcinogenesis. In the present study, we examined expression of PGE2 receptor subtypes EP1, EP2, EP3, and EP4 in normal colon mucosa and colon cancers, and assessed the contribution of EP3 to colon cancer development.

Methods: mRNA expression of PGE2 receptor subtypes EP1, EP2, EP3, and EP4 in normal colon mucosa and colon cancers in azoxymethane (AOM) treated mice and rats, and in humans, were examined by reverse transcription-polymerase chain reaction (RT-PCR), quantitative real time RT-PCR, and immunohistochemical analyses. Evaluation of the role of EP3 was performed by intraperitoneal injection of AOM, using EP3 receptor knockout mice. Effects of EP3 receptor activation on cell growth of human colon cancer cell lines were examined using ONO-AE-248, an EP3 selective agonist. Moreover, EP3 expression in colon cancer cell lines was analysed with or without 5-aza-2′-deoxycytidine (5-aza-dC) treatment.

Results: Expression levels of EP1 and EP2 mRNA were increased in cancer tissues. EP4 mRNA was constantly expressed in normal mucosa and cancers. In contrast, expression of EP3 mRNA was markedly decreased in colon cancer tissues, being 5% in mice, 9% in rats, and 28% in humans compared with normal colon mucosa, analysed by quantitative real time RT-PCR. Immunohistochemical staining demonstrated the rat EP3 receptor protein to be expressed in epithelial cells of normal mucosa and some parts of small carcinomas but hardly detectable in large carcinomas of the colon. Colon cancer development induced by AOM in EP3 receptor knockout mice was enhanced compared with wild-type mice, with a higher incidence of colon tumours (78% v 57%) and mean number of tumours per mouse (2.17 (0.51) v 0.75 (0.15); p<0.05). Expression of EP3 mRNA was detected in only one of 11 human colon cancer cell lines tested. Treatment with 5 μM of an EP3 selective agonist, ONO-AE-248, resulted in a 30% decrease in viable cell numbers in the HCA-7 human colon cancer cell line in which EP3 was expressed. Treatment with 5-aza-dC restored EP3 expression in CACO-2, CW-2, and DLD-1 cells but not in WiDr cells, suggesting involvement of hypermethylation in the downregulation of EP3 to some extent.

Conclusion: The PGE2 receptor subtype EP3 plays an important role in suppression of cell growth and its downregulation enhances colon carcinogenesis at a later stage. Hypermethylation of the EP3 receptor gene could occur and may contribute towards downregulating EP3 expression to some extent in colon cancers.

Keywords: EP3 receptor, prostaglandin E2, colon cancer, colon carcinogenesis, gene expression

Clear benefits have been reported in epidemiological studies with non-steroidal anti-inflammatory drugs (NSAIDs) as chemopreventive agents against colon cancers, one of the most common malignancies in humans.1 Chemically induced colon carcinogenesis in rodents is also suppressed by administration of NSAIDs.2–4 Moreover, intestinal polyp formation in familial adenomatous polyposis coli patients is markedly reduced after application of agents such as sulindac or indomethacin.5–8 The common mechanism of action of NSAIDs is inhibition of cyclooxygenase (COX) activity, two distinct isoforms of which have been reported: a constitutive enzyme, COX-1, and an inducible enzyme, COX-2.9 COX-1 and COX-2 are rate limiting enzymes in the synthesis of prostanoids which affect cell proliferation, tumour growth, apoptosis, and immune responsiveness, and both COX isoforms have been reported to be involved in colon carcinogenesis.1,4,10

Prostanoids such as prostaglandin (PG)E2, PGD2, PGF, PGI2, and TXA2 exert their biological actions through binding to nine specific receptors with seven transmembrane domains: the four subtypes EP1–EP4 for PGE2, DP and CRTH2 for PGD2, FP for PGF, IP for PGI2, and TP for TXA2.11,12 Several reports have demonstrated increased levels of PGE2 in human colon cancer tissues compared with surrounding normal mucosa.13–15 Signal transduction pathways of PGE2 receptors have been studied by examining agonist induced changes in the levels of second messengers such as cAMP and free Ca2+ and by identifying G protein coupling by various methods.11 The EP1 receptor is known to mediate PGE2 induced elevation of free Ca2+ concentration although the species of G protein to which EP1 receptor is coupled remains unidentified. EP2 and EP4 receptors are coupled to Gs and stimulate cAMP production by adenylate cyclase. In contrast, the major signalling pathway for the EP3 receptor is inhibition of adenylate cyclase via Gi. In addition, another function has been suggested for this receptor type in which cell phenotype is regulated through activation of Rho via G proteins other than Gi.16

Establishment of mice lacking the genes encoding prostanoid receptors has promoted understanding of the involvement of prostanoids11 and their receptors in the development of colon cancer.16–18 In previous studies, we demonstrated that deficiency of either EP1 or EP4 receptor decreases formation of azoxymethane (AOM) induced aberrant crypt foci (ACF), putative preneoplastic lesions in the colon.17,18 Moreover, antagonists of EP1 and EP4 receptors suppress formation of AOM induced ACF in the colon of mice and intestinal polyp formation in Apc gene deficient Min mice.17,18 Recently, it was also reported that homozygous deletion of the gene encoding the EP2 receptor resulted in a decrease in intestinal polyp formation in Apc knockout mice.19 As already mentioned, EP2 and EP4 stimulate adenylate cyclase whereas EP3 exerts an inhibitory influence, suggesting a possible suppressive role against colon carcinogenesis. However, deficiency of EP3 did not affect AOM induced ACF formation in our previous study.17

In the present study, we hypothesised that EP3 might act at a later stage in colon carcinogenesis. Examination of mRNA expression for EP1, EP2, EP3, and EP4 in colon carcinomas of mice, rats, and humans demonstrated that levels of EP3 were markedly decreased compared with normal mucosa. An increase in colon carcinoma formation induced by AOM was also demonstrated in EP3 receptor knockout mice. Furthermore, activation of the EP3 receptor showed a suppressive effect on cell growth in a colon cancer cell line in which EP3 was expressed. In most human colon cancer cell lines tested, EP3 expression was not detected but treatment with 5-aza-2′-deoxycytidine (5-aza-dC) restored EP3 expression in some cell lines. On the basis of the results obtained, the role of the EP3 receptor in colon carcinogenesis is discussed.

MATERIALS AND METHODS

Animals

The mouse gene encoding the PGE2 receptor EP3 was disrupted by a gene knockout method using homologous recombination, as reported previously.17 The generated chimeric mice were backcrossed with C57BL/6Cr mice, and the resulting homozygous mutant mice of these F2 progeny were backcrossed into the C57BL/6Cr background for 10 generations. EP3 receptor deficient male mice were used at six weeks of age. Genotypes of the knockout mice were confirmed by polymerase chain reaction (PCR) according to the method described previously.17 Animals were housed in plastic cages at 24±2°C and 55% relative humidity with a 12 h/12 h light/dark cycle. Water and basal diet (AIN-76A; Bio-Serv, Frenchtown, New Jersey, USA) were given ad libitum. Body weights and food intake were measured weekly.

Colon tumour samples and cell lines

Mouse colon tumours and normal colon mucosa tissues were obtained from C57BL/6J male mice treated with AOM, as previously reported.18 Rat colon tumours and normal colon mucosa tissues were obtained from eight F344 male rats treated with AOM, as previously reported.20 Frozen samples of mouse and rat tissues were used for reverse transcription (RT)-PCR analyses, and formalin fixed, paraffin embedded rat tissue samples were employed for immunohistochemical staining.

Surgical specimens of human colon cancer and adjacent normal colon mucosa tissues were taken from eight Japanese patients who had undergone surgical operations for colorectal cancers at the National Cancer Center Hospital, Tokyo, and samples were immediately frozen in liquid nitrogen.

Eleven human colon cancer cell lines were subjected to RT-PCR analysis. HCA-7 colony 29, a human colon adenocarcinoma cell line, was kindly provided by Dr Susan Kirkland, Imperial College of Science, Technology, and Medicine (London, UK).21 HCA-7 cells were maintained in Dulbecco’s minimum essential medium supplemented with 5% heat inactivated fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah, USA) and antibiotics (100 μg/ml of streptomycin and 100 units/ml of penicillin) at 37°C in 5% CO2. Colo 201, DLD-1, HCT-116, SW48, SW480, SW620, WiDr (Dainippon Pharmaceutical Co., Ltd, Osaka, Japan), CACO-2, Colo 320, and CW-2 (Riken Cell Bank, Tsukuba, Japan) were purchased and cultured according to the manufacturer’s instructions.

Analysis of EP receptor expression in colon cancers by RT-PCR

Total RNA was extracted from tissues and cultured cells by direct homogenisation in Isogen (Nippon Gene Co., Tokyo, Japan), and spectrophotometry was used for quantification. Aliquots (3 μg) of total RNA were subjected to the RT reaction with oligo-dT primer using an Omniscript Reverse Transcriptase kit (Qiagen, Hilden, Germany). After reverse transcription, PCR was carried out with Hotstartaq (Qiagen), according to the manufacturer’s instructions. To test cDNA integrity, the β-actin gene was amplified for each sample. Primers were designed using the computer program OLIGO 4.0-s (National Biosciences, Maryland, USA) and were based on published sequences in Genbank. Primers were designed to cross an exon-exon boundary or insertion of intron to ensure that genomic DNA was not being amplified. BLAST searches confirmed that the primers were specific for the target gene. Primers for the β-actin and EP receptor genes are listed in table 1. PCR amplifications were performed in a thermocycler (Gene Amp PCR System 9600; Perkin-Elmer Applied Biosystems, Foster City, California, USA), with 18–40 cycles of 94°C for 20 seconds, 60°C for 30 seconds, and 72°C for one min using the specific primer sets. PCR products were then analysed by electrophoresis on 2% agarose gel.

Table 1
 List of primers used for reverse transcription-polymerase chain reaction

Quantitative real time RT-PCR analysis

Quantitative real time RT-PCR analysis was performed using the Smart Cycler system with the Ex Taq R-PCR version 2 kit and SYBR Green (Takara Shuzo Co., Shiga, Japan) according to the manufacturer’s instructions. Primers for the β-actin and EP3 genes, and cycle conditions for PCR, are listed in table 2. To assess the specificity of each primer set, amplicons generated from the PCR reaction were analysed by their melting point curves and additionally run on 2% agarose gels to confirm the correct sizes of the PCR products. Each PCR product was subcloned into the TA cloning plasmid vector pGEN-T easy vector (Promega Co., Madison, Wisconsin, USA) and used as a positive control for real time PCR analyses. The number of molecules of specific gene products in each sample was determined using a standard curve generated by amplification of 102–108 copies of the control plasmid. Each sample was analysed in triplicate.

Table 2
 List of primers used for real time reverse transcription-polymerase chain reaction

Immunohistochemical staining

Immunohistochemical analyses of colon tumours and normal mucosa samples from F344 male rats treated with AOM were performed with the avidin-biotin complex immunoperoxidase technique, as previously reported.20 As the primary antibody, a polyclonal rabbit anti-EP3 antibody raised against rat EP3 receptors was used at a 50× dilution.22 As the secondary antibody, biotinylated antirabbit IgG (H+L) raised in a goat, affinity purified, and absorbed with rat serum (Vector Laboratories, Inc., Burlingame, California, USA) was used at a 200× dilution. Staining was performed using avidin-biotin reagents (Vectastain ABC reagents; Vector Laboratories.), 3,3′-diaminobenzidine, and hydrogen peroxide. Sections were counterstained with haematoxylin. As a negative control, the primary antibody was preincubated with a 16-fold (molar ratio) excess amount of the fusion protein used as the immunogen for one hour at room temperature prior to incubation of the sections.22

AOM induced colon tumour development in EP3 receptor knockout mice

Male EP3 receptor deficient homozygous mice (EP3−/−) and wild-type mice received AOM at a dose of 10 mg/kg body weight intraperitoneally once a week for six weeks. At 56 weeks of age, mice were sacrificed under ether euthanasia and complete autopsy was performed. After laparotomy, the entire intestines were resected and opened longitudinally, and the contents were flushed with normal saline. Using a dissection microscope, colon tumours were noted grossly for their location, number, and diameter, measured with callipers. All tumours from AOM treated mice were subjected to histological examination after routine processing and haematoxylin and eosin staining. The experimental protocol was according to the guidelines for Animal Experiments in the National Cancer Center.

Effects of ONO-AE-248 on growth of colon cancer cells

The EP3 receptor selective agonist 16-(3-methoxymethyl)phenyl-ω-tetranor-3,7-dithiaPGE1 (ONO-AE-248) was chemically synthesised at Ono Pharmaceutical Co. Ltd.23 DLD-1 and HCA-7 cells were seeded in plastic 96 well plates at a density of 2×103 cells per well, and grown for 24 hours with media containing 5% FBS. The EP3 receptor selective agonist ONO-AE-248 was added daily on days 0–4, and then numbers of viable cells on day 1, 3, and 5 were measured by colorimetric assay using the cell proliferation assay reagent WST-1 (Wako Chemicals, Osaka, Japan) with a microplate reader (Bio Rad, Hercules, California, USA) at a reference wavelength of 655 nm and a test wavelength of 450 nm. Cell viability was determined as per cent of control values. Experiments were repeated three times and data were measured six times (n = 6).

5′-Aza-2′-deoxycytidine treatment

CACO-2, CW-2, DLD-1, HCA-7, and WiDr cells were seeded at a density of 5×104 cells/10 cm dish on day 0 and treated with 1 and 2 μM 5-aza-dC (Sigma, St Louis, Missouri, USA) on days 1, 3, and 5. After each treatment, cells were placed in fresh media and harvested on day 6, and total cellular RNA was prepared using Isogen on day 7.

Statistical analysis

The significance of differences in the incidences of tumours was analysed using the χ2 test and other differences using the Student’s t test. Differences were considered statistically significant at p<0.05.

RESULTS

Different expression of PGE2 receptors EP1, EP2, EP3, and EP4 in normal colon mucosa and colon tumours

Expression of PGE2 receptors EP1, EP2, EP3, and EP4 in normal colon mucosa and colon tumours of AOM treated mice and rats, and in human tissues, were examined by RT-PCR (figs 1, 2). In the three mouse colon adenocarcinomas tested, expression of EP1 and EP2 receptor mRNAs was increased compared with levels in normal mucosa. EP4 mRNA was equally expressed in carcinomas and normal mucosa. In contrast, expression of EP3 mRNA was markedly decreased in all carcinoma samples compared with normal colon mucosa (fig 1A). Expression patterns of EP1, EP2, EP3, and EP4 receptors in eight pairs of samples of adenocarcinoma and normal mucosa from AOM treated rats were similar to those in mice. Patterns for EP1, EP2, EP3, and EP4 receptors in four typical pairs of samples are shown in fig 1B. In the case of human colon tissues, EP3 receptor mRNA was markedly decreased in seven of eight samples for adenocarcinomas compared with adjacent normal mucosa of the colon. Expression levels of EP2 receptor mRNA were increased in seven of eight human colon adenocarcinomas compared with levels in normal mucosa, but expression of EP1 receptor was not clearly increased in human colon carcinoma. EP4 mRNA was equally expressed in carcinomas and normal mucosa in all cases. Figure 2A shows expression of EP1, EP2, EP3, and EP4 receptors of colon carcinoma and normal mucosa in four typical pairs of samples.

Figure 1
 Analyses of prostaglandin E2 (PGE2) receptors EP1, EP2, EP3, and EP4 mRNA expression. (A) Azoxymethane (AOM) treated mouse normal colon mucosa and colon carcinomas. Two pairs of samples (lanes ...
Figure 2
 Analyses of prostaglandin E2 (PGE2) receptors EP1, EP2, EP3, and EP4 mRNA expression in human colon tissues. (A) Reverse transcription-polymerase chain reaction (RT-PCR) analysis patterns in four ...

Furthermore, downregulation of EP3 was confirmed by quantitative real time RT-PCR (figs 1C, 1D, 2B, 2C). Expression of EP3 receptor mRNA was significantly downregulated in tumours, being 5% in mice (fig 1C), 9% in rats (fig 1D), and 28% in humans (fig 2C) of the average value of that in the respective normal colon mucosa.

Localisation of EP3 receptor protein in rat colon tumours

Immunohistochemical analysis of paraffin embedded specimens of eight colon tumours and normal colon mucosa in rats treated with AOM was performed. Slight background staining was widely detected in both negative controls, those stained without antirat EP3 receptor antibody (fig 3A, B) and those stained with anti-EP3 receptor antibody preabsorbed with fusion EP3 receptor protein (fig 3C, D). Moreover, slight non-specific staining was detected in red blood cells. In normal colon mucosa tissues, EP3 receptor expression was prominent in epithelial cells (fig 3E), and the muscular coat was also positively stained. Similarly, positive staining of EP3 receptors was observed in hyperplastic ACF of the colon (data not shown). In contrast, staining was very faint, minimal, or absent in epithelial cells of colon adenocarcinomas (fig 3F), being totally lacking in seven cases, sized 3–9 mm in diameter. Only one carcinoma sample was weakly stained, and its size was 2 mm.

Figure 3
 Immunohistochemical staining for the rat prostaglandin E2 receptor subtype EP3 of normal colon mucosa (A, C, and E) and colon adenocarcinoma (B, D, and F). Non-specific staining of some red blood cells and weak ...

Colon tumour development in EP3 receptor knockout mice

To assess the role of EP3 receptors in colon tumour development, EP3 receptor knockout mice were used in an in vivo model. Data for the incidence (percentage of mice with tumours) and multiplicity (number of tumours per mouse) of colon tumours induced by AOM are summarised in table 3. Tumour incidence was increased to 78% in EP3 receptor knockout mice compared with 57% in wild-type mice. Regarding tumour multiplicity, values were 2.17 (0.51) for EP3 receptor knockout mice and 0.75 (0.15) for wild-type mice (p<0.05). Histopathological examination revealed 20 colon tumours to be adenocarcinomas in wild-type, and 50 colon tumours to be three adenomas and 47 adenocarcinomas in EP3 receptor knockout mice. Figure 4 shows the size distribution, demonstrating a significant increase in tumours measuring ⩾2.0 mm in diameter in EP3 receptor knockout mice (2.00 (0.48) v 0.50 (0.11); p<0.01) but not in those measuring <2.0 mm in diameter (0.17 (0.08) v 0.25 (0.11)).

Figure 4
 Size distribution of colon tumours induced by azoxymethane in wild-type and prostaglandin E2 receptor subtype EP3 knockout (KO) mice. The number of tumours/mouse in each size class is expressed as mean (SEM). **Significantly ...
Table 3
 Colon tumour development in EP3 receptor knockout mice

Expressions of PGE2 receptors in colon cancer cell lines, and effects of the EP3 selective agonist on growth of colon cancer cells

Expression of PGE2 receptors in 11 human colon cell lines was examined by RT-PCR. EP1, EP2, and EP4 were widely detected in the human colon cancer cell lines (in 10 of 11 for EP1, nine of 11 for EP2, and nine of 11 for EP4) but EP3 was only detected in HCA-7 (fig 5A).

Figure 5
 Effect of ONO-AE-248 treatment on cell growth of DLD-1 and HCA-7 cells. (A) Expression of prostaglandin E2 (PGE2) receptors EP1, EP2, EP3, and EP4 was analysed by reverse transcription-polymerase chain reaction ...

To evaluate the physiological functions of the EP3 receptor, the effect of an EP3 receptor selective agonist ONO-AE-248 on viable cell numbers of DLD-1 and HCA-7 in monolayer cultures was examined. In the HCA-7 human colon adenocarcinoma cell line, expression of the EP3 receptor and other PGE2 receptors (EP1, EP2, and EP4) were detected by RT-PCR analysis (fig 5A). As shown in fig 5B, HCA-7 cell numbers were significantly decreased dose dependently by addition of ONO-AE-248, with 8%, 17%, and 30% decreases (p<0.05, p<0.01, and p<0.01) in the presence of 1, 3, and 5 μM ONO-AE-248 on day 5, respectively. On the other hand, treatment with ONO-AE-248 did not affect growth of DLD-1 cells which were not expressing EP3 mRNA. The experiments were repeated three times and similar results were obtained.

Effect of 5-aza-dC on EP3 expression

To determine whether silencing by DNA methylation could be involved in reduced expression of EP3 receptor in colon tumours, we tested the effects of 5-aza-dC, a demethylating agent, on EP3 receptor expression in colon cancer cell lines. Human colon cancer cell lines CACO-2, CW-2, DLD-1, HCA-7, and WiDr were treated with 5-aza-dC, and expression levels of EP3 receptor were analysed by RT-PCR. Without 5-aza-dC treatment, expression of EP3 receptor was detected in HCA-7, but not in CACO-2, CW-2, DLD-1, or WiDr (fig 5A). After 5-aza-dC treatment, expression was restored in CACO-2, CW-2, and DLD-1, but not in WiDr (fig 6).

Figure 6
 5-Aza-2′-deoxycytidine (5-aza-dC) treatment of CACO-2, CW-2, DLD-1, HCA-7, and WiDr colon cancer cell lines. Each cell line was treated with 1 and 2 μM 5-aza-dC three times. EP3 receptor expression was analysed ...

DISCUSSION

In the present study, examination of mRNA expression levels for EP1, EP2, EP3, and EP4 receptors in colon tissues in mice, rats, and humans by RT-PCR and quantitative RT-PCR provided evidence of a marked reduction in EP3 receptors in colon cancers, in clear contrast with the increase observed for EP1 and EP2. Additionally, results of mRNA expression of EP receptors in 11 human colon cancer cell lines support the above findings and further indicate the events may occur in colon cancer cells. Recently, we reported enhancement of AOM induced colon tumours with exogenous administration of PGE2 in male F344 rats, and that colon tumours exhibited similar expression patterns in EP receptors as those observed in the present study.24 Sonoshita et al reported that mRNA expression of EP2 was strongly increased and EP3 was weakly decreased in colon polyps compared with normal colon in APCΔ716mice.19 These reports support our data that downregulation of EP3 is a common feature in colon cancer of mice, rats, and humans. It has been reported that expression of the EP3 receptor is widely distributed throughout the body, and its mRNA has been identified in almost all tissues in mice and rats, as well as in humans.25–27 Northern blot analysis revealed that expression of EP3 receptor mRNA was mainly localised in the muscle layer in the rat gastrointestinal tract,27 and the present immunohistochemical analysis indicated that EP3 receptors were detectable in rat normal colon epithelial cells and the muscular coat, but not in rat colon adenocarcinomas. In our previous study, we demonstrated that deficiency of EP1 or EP4 receptor reduced formation of AOM induced ACF while EP3 receptors had no effect, using eight types of EP receptor knockout mice.17,18 However, long term in vivo examination of AOM induced colon tumour development using EP3 receptor knockout mice, conducted here in the present study, demonstrated enhancement of tumour incidence and multiplicity. Moreover, the size of the tumours was significantly increased. Thus based on our present and previous results, we suggest that the EP3 receptor does not influence the early stage of colon carcinogenesis, including ACF formation, but its downregulation could be important to cancer development at a later stage.

In our present study, PCR primers of mouse, rat, and human EP3 receptors targeted a common sequence in each species. PCR products would be expected to be derived from the entire range of splice variants (figs 1A–B, 2A, 5A). It is noteworthy that there are three splice variants of the EP3 receptor in mice and rats, and nine in humans, coupled to different G protein signalling pathways.28–33 These variants are different in the carboxy terminal tail, and the amino acid sequence has an important role in G protein coupling specificity.30,31 Two of the three variants of the mouse EP3 receptors are EP and EP, which are coupled to Gi and cause inhibition of adenylate cyclase.30 The mouse EP receptor, in contrast, is coupled to Gs, in addition to Gi, and evokes pertussis toxin insensitive cAMP production.31 Preliminarily, we examined expression of three splice variants of mouse EP3 receptors by RT-PCR using specific primers for each variant, and found EP to be the major form in mouse normal mucosa (data not shown). These observations support the conclusion that the major splice variants of EP3 receptors are coupled to Gi and act to inhibit adenylate cyclase in normal colon mucosa in mice. On the other hand, EP2 and EP4 receptors are coupled to Gs and stimulate cAMP production by this enzyme. Increased cAMP levels result in activation of cAMP dependent protein kinase (PKA) and transcriptional factors that bind to cAMP responsive elements to transactivate the transcription of specific primary response genes that initiate cell proliferation.34 In our previous study,18 the EP4 receptor selective agonist ONO-AE1-329 was shown to enhance colony formation by the HCA-7 human colon adenocarcinoma cell line. The EP3 receptor selective agonist ONO-AE-248 was demonstrated to suppress cell growth in HCA-7 in the present study. It has been reported that ONO-AE-248 attenuates the rise in intracellular cAMP induced by forskolin, an activator of adenylate cyclase, in CHO cells transfected with EP receptor.23 Therefore, the EP3 receptor pathway may play an important role in counteracting the effects of EP2 and EP4 receptors, and its downregulation in later stages of colon carcinogenesis may enhance cancer development. Additional studies are needed to investigate interactions between the EP3 receptor signalling pathway and others linked to EP receptors.

Hypermethylation of CpG islands in promoter regions is known to cause silencing of genes in various human cancers,35,36 and silencing of COX-2 and APC genes by hypermethylation has been reported in human colon cancer.37,38 Although hypermethylation of the prostaglandin receptor gene has not been reported,37,38 DNA sequences in the promoter region and exon 1 of the human EP3 gene are GC rich (Genbank AL031429). Therefore, in the present study, we examined the effects of demethylation of DNA with 5-aza-dC on EP3 expression in human colon cancer cell lines. Demethylation of five cell lines by 5-aza-dC treatment resulted in restoration of EP3 receptor expression in three cell lines. These findings suggest that the DNA sequence of the EP3 receptor may be methylated but further studies are needed to clarify whether hypermethylation of the EP3 receptor gene occurs and regulates EP3 expression in colon cancers.

In conclusion, data obtained in our present and previous studies suggest that the PGE2 receptor subtype EP3 plays an important role in suppression of cell growth and that its downregulation enhances colon carcinogenesis at a later stage. The underlying mechanisms clearly warrant further investigation.

Acknowledgments

This work was supported in part by Grants-in-Aid for Cancer Research, for the Second-Term Comprehensive 10-Year Strategy for Cancer Control, and for the Research on Advanced Medical Technology from the Ministry of Health, Labor and Welfare of Japan.

Abbreviations

  • PGE2, prostaglandin E2
  • ACF, aberrant crypt foci
  • AOM, azoxymethane
  • COX, cyclooxygenase
  • NSAIDs, non-steroidal anti-inflammatory drugs
  • RT-PCR, reverse transcription-polymerase chain reaction
  • 5-aza-dC, 5-aza-2′-deoxycytidine
  • FBS, fetal bovine serum

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