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1.  Enhanced induction of Mucin Depleted Foci in Estrogen Receptor β Knockout Mice 
The role of the estrogen receptor β (ERβ) in the colon has received considerable interest, yet in vivo models are needed to better define its protective actions. In the present study, wild-type (WT), ERα and ERβ knockout (αERKO and βERKO) mice were injected with azoxymethane (AOM), a colon chemical carcinogen. Fourteen weeks after AOM exposure the incidence of aberrant crypt foci (ACF) was assessed by methylene blue staining. βERKO mice showed significantly higher incidence (P < 0.001) of ACF (15.0 ± 2.5) as compared to αERKO (3.4 ± 1.0) and WT (4.6 ± 1.0) mice. The colons in several βERKO mice had increased thickness and loss of normal morphology. It has been reported that ERβ plays a role in maintenance of the colonic crypt architecture; this may explain the loss of crypt organization in the colonic epithelium of βERKO mice. The presence of mucin depleted foci (MDF) has been shown, both in humans and in rodents, as an early event in colon cancer. Therefore, in order to surpass the limitations with ACF scoring, we performed alcian blue-neutral red staining to assess the presence of MDF. This assay allowed the assessment of precancerous lesions on all the βERKO mice colons (38.3 ± 4.0; P < 0.001), comparing to WT and αERKO mice (6.6 ± 1.5, and 10.0 ± 1.9, respectively), and served to confirm the ACF results. Together these data support the use of MDF staining as a biomarker for precancerous lesions and the protective role of ERβ in colon carcinogenesis.
doi:10.1158/1940-6207.CAPR-10-0044
PMCID: PMC2933324  PMID: 20716634
Estrogen receptors; colon cancer; aberrant crypt foci; mucin depleted foci
2.  Dark Aberrant Crypt Foci with activated Wnt pathway are related to tumorigenesis in the colon of AOM-treated rat 
Background
To evaluate the relationship between Aberrant Crypt Foci (ACF) and tumorigenesis, we observed the sequential development from ACF to tumor in the colon of azoxymethane-exposed wistar rats.
Methods
Sixty wistar rats were sacrificed at different time points after exposure to azoxymethane and the colons were stained with methylene blue for stereomicroscopic analysis.
Results
We found two types of early lesions: classic ACF and dark ACF. Dark ACF were characterized by dark blue staining, mildly enlarged or small compressed crypts that are not elevated from the surrounding epithelium. Large dark ACF and nascent tumors displayed the same surface morphology. Furthermore, dark ACF grew significantly faster than classic ACF and showed dysplasia without hyperplasia. In contrast, classic ACF showed hyperplasia without dysplasia. Dark ACF has significant higher expression rate of β-catenin (100%) and MMP-7 (81.82%) compared with the expression of β-catenin and MMP-7 in classic ACF (4.84% and 7.87%, respectively).
Conclusion
Our data indicated that dark ACF is closely related to tumorigenesis while classic ACF is not. Furthermore, Wnt signal pathway was activated during the early period of dark ACF.
doi:10.1186/1756-9966-27-26
PMCID: PMC2529269  PMID: 18681964
3.  Aberrant crypt foci in patients with colorectal cancer. 
British Journal of Cancer  1998;77(12):2343-2348.
Aberrant crypt foci (ACF) are clusters of abnormally large colonic crypts identified on the mucosal surface of the human colon. They are thought to be preneoplastic lesions. The aim of the present study was to compare density (number of ACF per square cm of mucosal surface), crypt multiplicity (number of crypts per ACF) and histology of ACF in colonic resections of colorectal cancer patients resident in two Italian provinces with a twofold difference in colorectal cancer incidence rates. Thirty-two and 26 colonic resections were collected after operation in Ragusa (Southern Italy) and Modena (Northern Italy), respectively, and fixed in 10% formalin. Mucosal layers were observed under a light microscope at 25x after staining with methylene blue. Density of ACF was significantly higher in Modena (median 0.101 ACF cm(-2)) than in Ragusa (0.049, P = 0.001), whereas there was no difference in crypt multiplicity. ACF were classified into three groups according to histological features: ACF with mild alterations (hypertrophic ACF, 73%), ACF with hyperplasia (hyperplastic ACF, 17%) and ACF with dysplasia (microadenomas, 10%). The proportions of ACF in the three groups were similar in the two provinces. Density of ACF was higher and crypt multiplicity lower proceeding from proximal to distal large bowel. Microadenomas were observed only in the colon, whereas hyperplastic ACF were more frequent in the rectum. In conclusion, density of ACF correlates with colorectal cancer rates in two Italian provinces, and shows a positive gradient from proximal to distal large bowel. Histology of ACF suggests that they may be precursors of both hyperplastic and adenomatous polyps. These data provide further evidence of the role of ACF in human colorectal carcinogenesis.
Images
PMCID: PMC2150380  PMID: 9649156
4.  Characterisation of aberrant crypt foci in carcinogen-treated rats: association with intestinal carcinogenesis. 
British Journal of Cancer  1995;71(4):763-769.
Carcinogen-treated rats develop foci of aberrant crypts in the colon (ACFs) that have been interpreted as preneoplastic lesions. To characterise ACFs further, we studied in the unsectioned colon of rats the number, multiplicity, some morphological characteristics and the type of mucin production in ACFs. In ACFs observed 115 days after the administration of 50 mg kg-1 1,2-dimethylhydrazine (DMH), crypt multiplicity [number of aberrant crypts (AC) per focus] was positively correlated (P < 0.0001) with the reduction of goblet cells, and with luminal and nuclear alterations in the cells surrounding the lumen of the ACs. We studied mucin production in the unsectioned colon, demonstrating that ACFs producing sulphomucins (like the normal distal rat colon) were progressively reduced when ACF multiplicity increased, whereas ACFs containing sialomucins (correlated with an increased risk of colon cancer) or both sulphomucins and sialomucins increased with crypt multiplicity. We also studied ACFs in the colon and the occurrence of intestinal tumours in rats treated with azoxymethane (AOM; 64 mg kg-1). A significant association was found (P = 0.04) between tumours and the presence of 'large' ACFs (AC/ACF > 14 crypts) and a borderline significant association (P = 0.057) between the presence of tumours and sialomucin-producing ACFs. We found no association between the number of ACFs, ACF multiplicity and the presence of tumours.
Images
PMCID: PMC2033722  PMID: 7710942
5.  One Year Recurrence of Aberrant Crypt Foci 
Introduction
Aberrant crypt foci (ACF) are putative precursors of colorectal adenomas and have been postulated as a potential biomarker for colorectal cancer. Few studies have followed subjects after ACF removal to monitor recurrence.
Methods
Subjects enrolled in the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial were recruited for a study of ACF. A standardized protocol using magnified endoscopy and mucosal staining with methylene blue was implemented to detect rectal ACF. After removal of all baseline ACF, subjects returned one year later and recurrent ACF were observed and biopsied.
Results
A total of 434 of 505 (86%) subjects observed at baseline returned for the year 1 exam. The mean number of ACF at year 1 was strongly correlated with the number at baseline; subjects with 0, 1, 2–3, 4–6, and 7+ ACF at baseline had a mean of 1.2, 1.4, 1.7, 3.0 and 5.5 ACF, respectively, at year 1. ACF prevalence and mean count at year 1, 61% and 1.93, respectively, were only slightly lower than the corresponding values at year 0, 69% and 2.25. The locations of ACF at year 1 and baseline were significantly correlated. Of 96 ACF assessed for histology, 70 (73%) were hyperplastic and none dysplastic.
Conclusion
After removal of ACF at baseline, ACF counts one year later are only slightly reduced and are significantly correlated with the baseline ACF count. The results of this study do not support a role for ACF in clinical practice.
doi:10.1158/1940-6207.CAPR-09-0257
PMCID: PMC2900400  PMID: 20570885
6.  Sequential Changes in Aberrant Crypt Foci and Lectin Expression in the Early and Late Stages of DMH-Induced Colon Carcinogenesis in Rats 
Gut and Liver  2012;6(2):229-234.
Background/Aims
The purpose of this study was to investigate the malignant potential of aberrant crypt foci (ACF) by measuring the multiplicity of crypts and lectin expression in the early and late stages of 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis.
Methods
Six-week-old Wistar rats were injected subcutaneously with DMH for 27 weeks. We classified ACF according to the number of crypts per ACF as a few crypts (≤3 crypts, FC ACF) or numerous crypts (≥4 crypts, NC ACF). Immunohistochemistry was used to evaluate lectin expression.
Results
In the early stage, FC ACF (590/1,902, 31.0%) occurred more frequently than NC ACF (35/449, 7.8%); whereas in the late stage, NC ACF (176/449, 39.2%) occurred more frequently than FC ACF (324/1,902, 17.0%). The number of ACF peaked at 15 to 20 weeks. The ratio of NC/FC ACF increased gradually during carcinogenesis. The expression of both UEA1 and PNA was higher in NC ACF than FC ACF. Lectin expression increased in the late stage compared with the early stage.
Conclusions
The expression of lectin was higher in NC ACF and ACF in the late stage. Therefore, ACF with higher multiplicities in the late stage may have more malignant potential in DMH-induced colon carcinogenesis.
doi:10.5009/gnl.2012.6.2.229
PMCID: PMC3343162  PMID: 22570753
Aberrant crypt foci; Ulex europaeus agglutinin-I; Peanut agglutinin; Colon carcinogenesis
7.  Effects of hexahydrocurcumin in combination with 5-fluorouracil on dimethylhydrazine-induced colon cancer in rats 
AIM: To investigate the effects of hexahydrocurcumin (HHC), and its combination with 5-fluorouracil (5-FU) on dimethylhydrazine (DMH)-induced colon cancer in rats.
METHODS: Male Wistar rats weighing 100-120 g were used as subject models. Aberrant crypt foci (ACF), early preneoplastic lesions of colon cancer, were induced by subcutaneous injection of DHM (40 mg/kg) twice a week for two weeks. After the first DMH injection, rats were treated daily with vehicle (n = 12), curcumin (CUR) (50 mg/kg) (n = 12), HHC (50 mg/kg) orally (n = 12), and treated weekly with an intraperitoneal injection of 5-FU (50 mg/kg) (n = 12), or a combination of 5-FU plus CUR (n = 12) and HHC (n = 12) at the same dosage(s) for 16 wk. The total number of ACF and large ACF were assessed. Cyclooxygenase (COX)-1 and COX-2 expression were detected by immunohistochemistry in colon tissues. The quantitative data of both COX-1 and COX-2 expression were presented as the percentage of number of positive-stained cells to the total number of cells counted. Apoptotic cells in colon tissues were also visualized using the dUTP-biotin nick end labeling method. Apoptotic index (AI) was determined as the percentage of labeled nuclei with respect to the total number of nuclei counted.
RESULTS: The total number of ACF was highest in the DMH-vehicle group (1558.20 ± 17.37), however, the number of ACF was significantly reduced by all treatments, 5-FU (1231.20 ± 25.62 vs 1558.20 ± 17.37, P < 0.001), CUR (1284.20 ± 25.47 vs 1558.20 ± 17.37, P < 0.001), HHC (1086.80 ± 53.47 vs 1558.20 ± 17.37, P < 0.001), DMH-5-FU + CUR (880.20 ± 13.67 vs 1558.20 ± 17.37, P < 0.001) and DMH-5-FU + HHC (665.80 ± 16.64 vs 1558.20 ± 17.37, P < 0.001). Interestingly, the total number of ACF in the combined treatment groups, the DMH-5-FU + CUR group (880.20 ± 13.67 vs 1231.20 ± 25.62, P < 0.001; 880.20 ± 13.67 vs 1284.20 ± 25.47, P < 0.001) and the DMH-5-FU + HHC group (665.80 ± 16.64 vs 1231.20 ± 25.62, P < 0.001; 665.80 ± 16.64 vs 1086.80 ± 53.47, P < 0.001) were significantly reduced as compared to 5-FU or each treatment alone. Large ACF were also significantly reduced in all treatment groups, 5-FU (111.00 ± 7.88 vs 262.20 ± 10.18, P < 0.001), CUR (178.00 ± 7.33 vs 262.20 ± 10.18, P < 0.001), HHC (186.60 ± 21.51 vs 262.20 ± 10.18, P < 0.001), DMH-5-FU + CUR (122.00 ± 5.94 vs 262.20 ± 10.18, P < 0.001) and DMH-5-FU + HHC (119.00 ± 17.92 vs 262.20 ± 10.18, P < 0.001) when compared to the vehicle group. Furthermore, in the DMH-5-FU + CUR and DMH-5-FU + HHC groups the formation of large ACF was significantly reduced when compared to CUR (122.00 ± 5.94 vs 178.00 ± 7.33, P < 0.005) or HHC treatment alone (119.00 ± 17.92 vs 186.60 ± 21.51, P < 0.001), however, this reduction was not statistically different to 5-FU monotherapy (122.00 ± 5.94 vs 111.00 ± 7.88, P = 0.217; 119.00 ± 17.92 vs 111.00 ± 7.88, P = 0.619, respectively). The levels of COX-1 protein after all treatments were not different from normal rats. A marked increase in the expression of COX-2 protein was observed in the DMH-vehicle group. Over-expression of COX-2 was not significantly decreased by 5-FU treatment alone (95.79 ± 1.60 vs 100 ± 0.00, P = 0.198). However, over-expression of COX-2 was significantly suppressed by CUR (77.52 ± 1.68 vs 100 ± 0.00, P < 0.001), HHC (71.33 ± 3.01 vs 100 ± 0.00, P < 0.001), 5-FU + CUR (76.25 ± 3.32 vs 100 ± 0.00, P < 0.001) and 5-FU + HHC (68.48 ± 2.24 vs 100 ± 0.00, P < 0.001) in the treated groups compared to the vehicle group. Moreover, CUR (77.52 ± 1.68 vs 95.79 ± 1.60, P < 0.001), HHC (71.33 ± 3.01 vs 95.79 ± 1.60, P < 0.001), 5-FU + CUR treatments (76.25 ± 3.32 vs 95.79 ± 1.60, P < 0.001) and 5-FU + HHC (68.48 ± 2.24 vs 95.79 ± 1.60, P < 0.001) markedly decreased COX-2 protein expression more than 5-FU alone. Furthermore, the AI in all treated groups, 5-FU (38.86 ± 4.73 vs 23.56 ± 2.12, P = 0.038), CUR (41.78 ± 6.92 vs 23.56 ± 2.12, P < 0.001), HHC (41.06 ± 4.81 vs 23.56 ± 2.12, P < 0.001), 5-FU + CUR (49.05 ± 6.75 vs 23.56 ± 2.12, P < 0.001) and 5-FU + HHC (53.69 ± 8.59 vs 23.56 ± 2.12, P < 0.001) significantly increased when compared to the DMH-vehicle group. However, the AI in the combination treatments, 5-FU + CUR (49.05 ± 6.75 vs 41.78 ± 6.92, P = 0.192; 49.05 ± 6.75 vs 38.86 ± 4.73, P = 0.771) and 5-FU + HHC (53.69 ± 8.59 vs 41.06 ± 4.81, P = 0.379; 53.69 ± 8.59 vs 38.86 ± 4.73, P = 0.245) did not reach significant levels as compared with each treatment alone and 5-FU monotherapy, respectively.
CONCLUSION: The combined effects of HHC with 5-FU exhibit a synergistic inhibition by decreasing ACF formation mediated by down-regulation of COX-2 expression.
doi:10.3748/wjg.v18.i47.6951
PMCID: PMC3531679  PMID: 23322993
Hexahydrocurcumin; Curcumin analog; Colon cancer; Combination treatment; Cyclooxygenase-2; Apoptosis
8.  Suppressive effect of aspirin on aberrant crypt foci in patients with colorectal cancer 
Gut  2003;52(11):1598-1601.
Background and aims: Aspirin and other non-steroidal anti-inflammatory drugs have been shown to reduce the risk of colorectal cancer (CRC). Animal models have shown that aspirin is also effective in reducing the density of aberrant crypt foci (ACF). The aim of the study was to evaluate the effect of chronic administration of aspirin on the distribution pattern and histological characteristics of ACF in patients with CRC.
Methods: Our study compared the distribution patterns and histomorphological characteristics of ACF between a group of CRC patients treated with low dose aspirin (n=59) and a control group without aspirin (n=135). ACF were visualised on methylene blue stained macroscopically normal mucosa, microdissected, and serially cut.
Results: ACF were found in 75.8% of mucosal samples from the control group and in 36% of mucosal samples from the aspirin treated group, indicating a 47% decline in prevalence of ACF in colonic samples of patients treated with aspirin. A significant reduction from 92.5% to 40% (p<0.0001) was found in distal large bowel samples containing one or more ACF. Similarly, the aspirin treated group showed a reduction in ACF density of 64% and 82%, respectively, in both proximal and distal parts of the colon, indicating a significant reduction in ACF/cm2 in distal colon samples (p<0.01). The aspirin treated group displayed a 52% reduction in dysplastic ACF although this difference was not statistically significant.
Conclusions: Our study has provided evidence of the effective chemopreventive action of low dose aspirin on ACF in humans.
PMCID: PMC1773868  PMID: 14570729
aberrant crypt foci; colorectal carcinogenesis; aspirin; chemoprevention
9.  Cyclophilin C-associated protein (CyCAP) knock-out mice spontaneously develop colonic mucosal hyperplasia and exaggerated tumorigenesis after treatment with carcinogen azoxymethane1 
BMC Cancer  2009;9:251.
Background
The discovery of a "serrated neoplasia pathway" has highlighted the role of hyperplastic lesions of the colon as the significant precursor of colorectal adenocarcinoma. In mice, hyperplasia of the colonic mucosa is a regular phenomenon after a challenge with colonic carcinogens indicating that mucosal hyperproliferation and thickening, even without cytological dysplasia, represents an early pre-malignant change. Cyclophilin C-associated protein (CyCAP) has been described to down-modulate endotoxin signaling in colorectal murine mucosa and is a murine orthologue of the tumor-associated antigen 90 K (TAA90K)/mac-2-binding protein.
Methods
Female Balb/c wild-type (WT) and CyCAP knock-out (KO) mice (6–8 weeks old) were administered 2 or 6 weekly subcutaneous injections of azoxymethane. The animals were evaluated post-injection at six weeks for aberrant crypt foci (ACF) study and at five months for colon tumor measurement. The thickness of the colon crypts was measured in microns and the number of colonocytes per crypt was also determined in well-oriented crypts. Morphometric analyses of the colon mucosa were also performed in untreated 6–8 weeks old KO and WT animals. Formalin-fixed/paraffin-embedded colon sections were also studied by immunohistochemistry to determine the Ki-67 proliferation fraction of the colon mucosa, β-catenin cellular localization, cyclin D1, c-myc, and lysozyme in Paneth cells.
Results
Cyclophilin C-associated protein (CyCAP)-/- mice, spontaneously developed colonic mucosal hyperplasia early in life compared to wild-type mice (WT) (p < 0.0001, T-test) and crypts of colonic mucosa of the (CyCAP)-/- mice show higher proliferation rate (p = 0.039, Mann-Whitney Test) and larger number of cyclin D1-positive cells (p < 0.0001, Mann-Whitney Test). Proliferation fraction and cyclin D1 expression showed positive linear association (p = 0.019, Linear-by-Linear Association). The hyperplasia was even more pronounced in CyCAP-/- mice than in WT after challenge with azoxymethane (p = 0.005, T-test). The length of the crypts (r = 0.723, p = 0.018, Spearman Correlation) and the number of colonocytes per crypt (r = 0.863, p = 0.001, Spearman Correlation) in non-tumorous areas were positively associated with azoxymethane-induced number of tumors. CyCAP-/- developed larger numbers of tumors than WT animals (p = 0.003, T-Test) as well as overall larger tumor mass (p = 0.016, T-Test). Membranous β-catenin was focally overexpressed in KO mice including proliferative zone of the crypts.
Conclusion
CyCAP-/- represent the first described model of spontaneous colonic mucosal hyperplasia. We conclude that CyCAP-deficient mice spontaneously and after challenge with carcinogen develop significantly more colorectal mucosal hyperplasia, an early stage in murine colonic carcinogenesis.
doi:10.1186/1471-2407-9-251
PMCID: PMC2724547  PMID: 19627619
10.  Epidemiology of Colonic Aberrant Crypt Foci: Review and Analysis of Existing Studies 
Cancer letters  2006;252(2):171-183.
Since first described in a rodent model in 1987, aberrant crypt foci (ACF) in the colon have been shown to exhibit many of the molecular features of the more advanced colonic neoplasms including cancer. Therefore, they may be early lesions with potential for progression, and be valuable biomarkers for reduction of risk of colorectal cancer (CRC). For this review, we searched PubMed, and reference lists of recent publications, for studies which reported on associations of features of ACF in humans, such as number or size, with subject characteristics, such as age or family history of CRC. Over 150 papers have reported on ACF in humans. However, the vast majority of these publications are concerned with molecular and morphological features of biopsied lesions, and not their epidemiology. None of the epidemiological studies were of optimum design, primarily due to their absence of a well-defined subject sampling frame or method. Given their ‘first-generation’ nature, consistent findings were of increased ACF number with age and with synchronous advanced colonic neoplasia. One study reported a higher mean number of ACF in subjects with a family history of CRC than in those without. The strongest evidence on the ability of ACF to predict a diagnosis of CRC will be from prospective studies with baseline ACF assessment in a large sample of disease-free persons (many thousands) who are followed carefully for many years. In the interim, because ACF are asymptomatic, well-designed cross-sectional studies are feasible and will yield valuable information on the relation of ACF to the known risk factors for CRC. This information can then be used to improve the design of prospective studies, and of clinical intervention trials that use ACF as an intermediate endpoint.
doi:10.1016/j.canlet.2006.11.009
PMCID: PMC2017093  PMID: 17182176
11.  Colon Preneoplastic Lesions in Animal Models 
Journal of Toxicologic Pathology  2013;26(4):335-341.
The animal model is a powerful and fundamental tool in the field of biochemical research including toxicology, carcinogenesis, cancer therapeutics and prevention. In the carcinogenesis animal model system, numerous examples of preneoplastic lesions have been isolated and investigated from various perspectives. This may indicate that several options of endpoints to evaluate carcinogenesis effect or therapeutic outcome are presently available; however, classification of preneoplastic lesions has become complicated. For instance, these lesions include aberrant crypt foci (ACF), dysplastic ACF, flat ACF, β-catenin accumulated crypts, and mucin-depleted foci. These lesions have been induced by commonly used chemical carcinogens such as azoxymethane (AOM), 1,2-dimethylhydrazine (DMH), methylnitrosourea (MUN), or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Investigators can choose any procedures or methods to examine colonic preneoplastic lesions according to their interests and the objectives of their experiments. Based on topographical, histopathological, and biological features of colon cancer preneoplastic lesions in the animal model, we summarize and discuss the character and implications of these lesions.
doi:10.1293/tox.2013-0028
PMCID: PMC3921915  PMID: 24526805
preneoplastic lesion; colon carcinogenesis; animal model; topographic view
12.  Dietary-feeding of Grape Seed Extract Prevents Azoxymethane-induced Colonic Aberrant Crypt Foci Formation in Fischer 344 Rats 
Molecular carcinogenesis  2010;49(7):641-652.
Chemoprevention by dietary agents/supplements has emerged as a novel approach to control various malignancies, including colorectal cancer (CRC). This study assessed dietary grape seed extract (GSE) effectiveness in preventing azoxymethane (AOM)-induced aberrant crypt foci (ACF) formation and associated mechanisms in Fischer 344 rats. Six-week old rats were injected with AOM, and fed control diet or the one supplemented with 0.25% or 0.5% (w/w) GSE in pre- and post-AOM or only post-AOM experimental protocols. At 16 weeks of age, rats were sacrificed and colons were evaluated for ACF formation followed by cell proliferation, apoptosis and molecular analyses by immunohistochemistry. GSE-feeding caused strong chemopreventive efficacy against AOM-induced ACF formation in terms of upto 60% (P<0.001) reduction in number of ACF and 66% (P<0.001) reduction in crypt multiplicity. Mechanistic studies showed that GSE-feeding inhibited AOM-induced cell proliferation but enhanced apoptosis in colon including ACF, together with a strong decrease in cyclin D1, COX-2, iNOS and survivin levels. Additional studies showed that GSE-feeding also decreased AOM-caused increase in β-catenin and NF-κB levels in colon tissues. Compared to control animals, GSE alone treatment did not show any considerable change in these biological and molecular events in colon, and was non-toxic. Together, these findings show the chemopreventive efficacy of GSE against the early steps of colon carcinogenesis in rats via likely targeting of β-catenin and NF-κB signaling, and suggest its potential usefulness for the prevention of human CRC.
doi:10.1002/mc.20643
PMCID: PMC2892197  PMID: 20564341
Colorectal cancer; grape seed extract; aberrant crypt foci; chemoprevention
13.  Aberrant P-cadherin expression is an early event in hyperplastic and dysplastic transformation in the colon 
Gut  2002;50(4):513-519.
Background: Colorectal adenomatous and, probably, hyperplastic polyp development requires epithelial remodelling and stratification, with loss of E-cadherin expression implicated in adenoma formation. We have shown that P-cadherin, normally expressed in stratified epithelia and placenta, is aberrantly expressed in disturbed epithelial architecture associated with colitis.
Aims: (i) To investigate the role of P-cadherin in colonic polyp formation. (ii) To ascertain whether expression of P-cadherin is independent of or correlated with expression of its associated proteins— E-cadherin, β-catenin, and γ-catenin. (iii) To determine if P-cadherin is functional regarding catenin binding in polyps.
Methods: Expression and localisation of cadherins (E- and P-) and their associated catenins (β- and γ-) were determined in aberrant crypt foci (ACF), in polyps with hyperplastic morphology (hyperplastic polyps and serrated adenomas), and in adenomatous polyps by immunohistochemistry, western blotting, and mRNA in situ hybridisation. Assessment of cadherin-catenin binding was evaluated by co-immunoprecipitation. Adenomatous polyposis coli (APC) mutation was assessed in adenomatous polyps.
Results: P-cadherin was expressed from ACF through to hyperplastic and adenomatous polyps. Alterations in E-cadherin and catenin expression occurred later, with variant patterns in (i) ACF, (ii) hyperplastic polyps and serrated adenomas, and (iii) adenomatous polyps. P-cadherin present in adenomas was functional with regard to catenin binding, and its expression was independent of APC mutational status.
Conclusions: P-cadherin is aberrantly expressed from the earliest morphologically identifiable stage of colonocyte transformation, prior to changes in E-cadherin, catenin, and APC expression/mutation. P-cadherin expression alone does not predict tissue morphology, and such expression is independent of that of associated cadherins and catenins.
PMCID: PMC1773182  PMID: 11889072
cadherin; catenin; polyp; remodelling; colorectal cancer
14.  THE RELIABILITY AND ACCURACY OF ENDOSCOPIC APPEARANCE IN THE IDENTIFICATION OF ABERRANT CRYPT FOCI 
Gastrointestinal endoscopy  2009;70(2):322-330.
Background
Aberrant crypt foci (ACF) have emerged as a putative precursor to colorectal adenoma, with potential use as a biomarker of colorectal cancer (CRC). However, there are wide differences in ACF prevalence, dysplasia and histological confirmation rates across studies. These differences may in part be due to variability in identification of endoscopic criteria.
Objective
To systematically evaluate the accuracy and reliability of various endoscopic criteria used to identify ACF using magnification chromoscopic endoscopy (MCE).
Design
Images obtained via MCE were shown to participating endoscopists who diagnosed them as an ACF or not, and assessed them for the endoscopic characteristics used to identify ACF in the literature.
Main outcome measures
were the predictive ability of the endoscopic criteria (crypt number, staining, margin, crypt size, epithelial thickness, lumen shape) for histologic confirmation of an ACF, and their reliability across endoscopists. The accuracy of the examiners in identifying ACF that were histologically confirmed was also assessed.
Results
The inter rater agreement rate for all except one of the endoscopic criteria (crypt number) was low, and did not improve with training. None of the criteria could significantly predict histological confirmation of ACF. Despite training exercises, accuracy of endoscopists to correctly identify a histologically proven ACF remained low.
Limitations
Still images with 40X optical magnification were analyzed rather than real time endoscopy. All ACF samples were hyperplastic; none were dysplastic.
Conclusions
No endoscopic criteria evaluated by our study predicted histological confirmation of ACF. Magnification chromoendoscopy had low accuracy and poor reliability.
doi:10.1016/j.gie.2008.12.060
PMCID: PMC2727598  PMID: 19539919
15.  Relationship of human rectal aberrant crypt foci and formation of colorectal polyp: One-year following up after polypectomy 
AIM: To clarify the relationship of human rectal aberrant crypt foci and formation of colorectal polyp.
METHODS: Eighty-nine subjects were recruited from the population of Japanese individuals who underwent polypectomy at Yokohama City University Hospital. All patients had baseline adenomas removed at year 0 colonoscopy. Aberrant crypt foci (ACF) were defined as lesions in which the crypts were more darkly stained with methylene blue than normal crypts and had larger diameters, often with oval or slit-like lumens and a thicker epithelial lining.
RESULTS: A total of 366 ACFs were identified in 89 patients; all had baseline adenomas removed at the first examination (year 0) colonoscopy and returned for the second (year 1). ACF in the lower rectum were assessed at year 0 and study group were divided into two groups depend on ACF numbers, 0-3 or over 3. All participants were examined in the number and maximum size of adenoma. There was no statistical difference in number and maximum size of ACF at year 0, however, maximum size of adenoma was larger in over 3 group than 0-3 group at year 1.
CONCLUSION: The number of ACF may be a predictive factor of relatively large adenoma incidence in the pilot phase study.
doi:10.4253/wjge.v4.i12.561
PMCID: PMC3536853  PMID: 23293726
Aberrant crypt foci; Colorectal carcinogenesis; Visceral fat; Adiponectin
16.  Suppressive Effect of Zinc on the Formation of Colonic Preneoplastic Lesions in the Mouse Fed High Levels of Dietary Iron 
Toxicological Research  2012;28(1):39-49.
We investigated the effect of zinc on the formation of colonic aberrant crypt foci induced by azoxymethane (AOM) followed by dextran sodium sulfate (DSS) in mice with high iron diet (HFe; 450 ppm iron). Sixweek old ICR mice were fed on high iron diets with combination of three different levels of zinc in diets, low-zinc (LZn; 0.01 ppm), medium-zinc (MZn; 0.1 ppm), and high-zinc (HZn; 1 ppm) for 12 weeks. Animals were received weekly intraperitoneal injections of AOM (10 mg/kg B.W. in saline) for 3 weeks followed by 2% DSS (molecular weight 36,000~50,000) in the drinking water for a week. To confirm the iron storage in the body, the hepatic iron concentration has been determine chemically and compared with histological assessment visualized by Prussian blue reaction. Aberrant crypt (AC) and aberrant crypt foci (ACF) were analyzed in the colonic mucosa of mouse fed high dietary iron. Superoxide dismutase (SOD) activity and thiobarbituric acid-reactive substances (TBARS) level were also investigated. Apoptosis in the preneoplastic lesion was determined by terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling (TUNEL). In addition, immunohistochemistry of β-catenin was also performed on the mucous membrane of colon. The number of large ACF (≥ 4 AC/ACF), which possess greater tumorigenic potential, was significantly lower in MZn and HZn groups compared with LZn group. Cytosolic SOD activity in the liver was significantly higher in HZn group compared with LZn group. Hepatic MDA level was decreased significantly in HZn group compared with MZn and LZn groups. Apoptotic index was significantly higher in HZn group. Taken together, these findings indicate that dietary zinc might exert a protective effect against colonic preneoplastic lesion induced by AOM/DSS in ICR mice with high iron status, and suggest that dietary supplement of zinc might play a role in suppressing colon carcinogenesis in mice.
doi:10.5487/TR.2012.28.1.039
PMCID: PMC3834396  PMID: 24278588
Colon carcinogenesis; Zinc; Iron overload; Aberrant crypt foci (ACF); Mouse
17.  Novel diet-related mouse model of colon cancer parallels human colon cancer 
AIM: To investigate the close parallels between our novel diet-related mouse model of colon cancer and human colon cancer.
METHODS: Twenty-two wild-type female mice (ages 6-8 wk) were fed the standard control diet (AIN-93G) and an additional 22 female mice (ages 6-8 wk) were fed the control diet supplemented with 0.2% deoxycholic acid [diet + deoxycholic acid (DOC)] for 10 mo. Tumors occurred in the colons of mice fed diet + DOC and showed progression to colon cancer [adenocarcinoma (AC)]. This progression is through the stages of tubular adenoma (TA), TA with high grade dysplasia or adenoma with sessile serrated morphology, intramucosal AC, AC stage T1, and AC stage T2. The mouse tumors were compared to human tumors at the same stages by histopathological analysis. Sections of the small and large intestines of mice and humans were evaluated for glandular architecture, cellular and nuclear morphology including cellular orientation, cellular and nuclear atypia, pleomorphism, mitotic activity, frequency of goblet cells, crypt architecture, ulceration, penetration of crypts through the muscularis mucosa and presence of malignant crypts in the muscularis propria. In addition, preserved colonic tissues from genetically similar male mice, obtained from a prior experiment, were analyzed by immunohistochemistry. The male mice had been fed the control diet or diet + DOC. Four molecular markers were evaluated: 8-OH-dG, DNA repair protein ERCC1, autophagy protein beclin-1 and the nuclear location of beta-catenin in the stem cell region of crypts. Also, male mice fed diet + DOC plus 0.007% chlorogenic acid (diet + DOC + CGA) were evaluated for ERCC1, beclin-1 and nuclear location of beta-catenin.
RESULTS: Humans with high levels of diet-related DOC in their colons are at a substantially increased risk of developing colon cancer. The mice fed diet + DOC had levels of DOC in their colons comparable to that of humans on a high fat diet. The 22 mice without added DOC in their diet had no colonic tumors while 20 of the 22 mice (91%) fed diet + DOC developed colonic tumors. Furthermore, the tumors in 10 of these mice (45% of mice) included an adenocarcinoma. All mice were free of cancers of the small intestine. Histopathologically, the colonic tumor types in the mice were virtually identical to those in humans. In humans, characteristic aberrant changes in molecular markers can be detected both in field defects surrounding cancers (from which cancers arise) and within cancers. In the colonic tissues of mice fed diet + DOC similar changes in biomarkers appeared to occur. Thus, 8-OH-dG was increased, DNA repair protein ERCC1 was decreased, autophagy protein beclin-1 was increased and, in the stem cell region at the base of crypts there was substantial nuclear localization of beta-catenin as well as increased cytoplasmic beta-catenin. However, in mice fed diet + DOC + CGA (with reduced frequency of cancer) and evaluated for ERCC1, beclin-1, and beta-catenin in the stem cell region of crypts, mouse tissue showed amelioration of the aberrancies, suggesting that chlorogenic acid is protective at the molecular level against colon cancer. This is the first diet-related model of colon cancer that closely parallels human progression to colon cancer, both at the histomorphological level as well as in its molecular profile.
CONCLUSION: The diet-related mouse model of colon cancer parallels progression to colon cancer in humans, and should be uniquely useful in model studies of prevention and therapeutics.
doi:10.4251/wjgo.v6.i7.225
PMCID: PMC4092339  PMID: 25024814
Diet; Deoxycholate; Mouse model; Colon cancer; Histology; Chlorogenic acid; 8-OH-dG; Beclin 1; Beta-catenin
18.  Chemopreventive Efficacy of Andrographis paniculata on Azoxymethane-Induced Aberrant Colon Crypt Foci In Vivo 
PLoS ONE  2014;9(11):e111118.
Andrographis paniculata is a grass-shaped medicinal herb, traditionally used in Southeast Asia. The aim of this study was to evaluate the chemoprotective effects of A. paniculata on colorectal cancer. A. paniculata ethanol extract was tested on azoxymethane (AOM)-induced aberrant crypt foci (ACF) in vivo and in vitro. A. paniculata treated groups showed a significant reduction in the number of ACF of the treated rats. Microscopically, ACF showed remarkably elongated and stratified cells, and depletion of the submucosal glands of AOM group compared to the treated groups. Histologically, staining showed slightly elevated masses above the surrounding mucosa with oval or slit-like orifices. Immunohistochemically, expression of proliferating cell nuclear antigen (PCNA) and β-catenin protein were down-regulated in the A. paniculata treated groups compared to the AOM group. When colon tissue was homogenized, malondialdehyde (MDA) and nitric oxide (NO) levels were significantly decreased, whereas superoxide dismutase (SOD) activity was increased in the treated groups compared to the AOM group. A. paniculata ethanol extract showed antioxidant and free radical scavenging activity, as elucidated by the measure of oxidative stress markers. Further, the active fractions were assessed against cell lines of CCD841 and HT29 colon cancer cells.
doi:10.1371/journal.pone.0111118
PMCID: PMC4229078  PMID: 25390042
19.  The value of a study of the mucosubstances in rectal biopsies from patients with carcinoma of the rectum and lower sigmoid in the diagnosis of premalignant mucosa 
Journal of Clinical Pathology  1972;25(2):123-128.
One hundred and twenty-one rectal biopsies from 99 patients with carcinoma of the rectum or lower sigmoid colon were investigated using a high iron-diamine-Alcian blue technique for sulphated and non-sulphated acid mucins. It was found that in normal rectal mucosa sulphomucins are the main carbohydrate component of the goblet cell mucin. This normal `mucous pattern' changes in the `transitional' mucosa (histological normal mucosa adjacent to carcinoma) where there is an increase of non-sulphated acid mucins concomitantly with a decrease or absence of sulphated groups in 60 to 90% of the cases according to the type of tumour. The same type of changes in mucus were observed in the `transitional' mucosa surrounding adenomatous polyps and papillary adenomas; they were not marked in areas around carcinoma in situ and not observed in the metaplastic polyps. These changes seem to be in direct relationship to the grade of differentiation and invasiveness of the tumour.
The histochemical changes in the mucins seem to be in favour of a malignant potential in the so-called neoplastic polyps.
The high iron diamine-Alcian blue, because of its `specificity', consistent results, and easy technique is recommended for routine use together with haematoxylin and eosin staining in the diagnosis of premalignant changes.
Images
PMCID: PMC477240  PMID: 5017440
20.  Detection of sulfated glycoproteins in intestinal metaplasia: a comparison of traditional mucin staining with immunohistochemistry for the sulfo-Lewisa carbohydrate epitope 
Journal of Clinical Pathology  2003;56(9):703-708.
Background: Premalignant Barrett’s oesophagus (BO) and gastric intestinal metaplasia (IM) show phenotypic variability. Incompletely differentiated sulfomucin rich gastric IM (type III) may have increased malignant potential. The types of sulfated oligosaccharide structures present in IM, BO, and colon have not been fully characterised.
Aims: To compare sulfo-Lewisa epitope tissue distribution with high iron diamine (HID) positive sulfomucin in metaplastic, dysplastic, and neoplastic tissues from oesophagus and stomach.
Methods: Sections containing gastric IM or BO (some associated with dysplasia or adenocarcinoma) were stained by the HID/alcian blue (AB) method and immunohistochemically (antibody 91.9H) to detect sulfo-Lewisa. Based on HID/AB staining, IM was subtyped into type I (complete) or types II and III (incomplete).
Results: In total, 125 sections from 38 subjects were studied. Normal squamous oesophagus, normal gastric epithelium, and type I IM were negative for sulfomucin and sulfo-Lewisa. In type II IM, occasional goblet cells were HID and sulfo-Lewisa positive, but sialomucin secreting (AB positive) columnar cells were sulfo-Lewisa negative. Type III IM was always sulfo-Lewisa positive. Sulfomucin staining in dysplasia and cancer was variable, but HID positive areas were always sulfo-Lewisa positive.
Conclusions: Sulfo-Lea, which is expressed on colonic mucin, is invariably present on sulfomucins in gastric IM and BO. Its presence in incomplete variants of IM and its absence from type I IM emphasises the phenotypic differences between complete and incomplete forms of metaplasia. 91.9H immunostaining is useful in IM subtyping. Characterising the molecular basis of sulfo-Lewisa expression may help understand the process of aberrant differentiation.
PMCID: PMC1770063  PMID: 12944557
Barrett’s oesophagus; cancer; Lewisa intestinal metaplasia; sulfomucin
21.  Aberrant crypt focus and fragile histidine triad protein in sporadic colorectal carcinoma 
AIM: To characterize aberrant crypt focus (ACF) in adjoining mucosa in sporadic colorectal carcinoma and to evaluate fragile histidine triad (Fhit) protein and Ki67.
METHODS: ACF was identified grossly and classified histologically in 75 resected specimens. ACF was typed into hyperplastic ACF (HACF) and dysplastic ACF (DACF). Sections of ACF, carcinoma and normal colonic mucosa as control were studied for Fhit and Ki67 expressions by immunohistochemistry and were grouped according to staining intensity and the number of positive stained cells observed in different histological groups. Comparison was done between the different groups by Pearson’s χ2 test and γ test for the ordinal data. P value < 0.05 was considered as significant.
RESULTS: Age range was 40 to 86 years in males (mean = 43.36) and 45 to 70 years in females (mean = 56). HACF was identified in all cases studied in the non-tumorous colonic mucosa; ACF was observed as non-contiguous scattered foci, which supports the hypothesis of acquisition of single focus monoclonality by colonic epithelial cells in tumor generation. Twenty-four (32%) had DACF and were observed as closure to carcinoma foci. Intensity of Fhit expression: (1) HACF - 40% exhibited strong intensity, similar to normal, moderate in 36% and weak in 24%; (2) DACF - strong in 25%, moderate in 37.5% and weak in 37.5%; and (3) carcinoma - negative in 16%, strong in 43% and moderate and weak in 28.5% each. Significant difference was observed in intensity of the Fhit protein expressions by HACF and DACF (P < 0.05). Tumor in older patients showed a stronger Fhit intensity compared to younger patients (P = 0.036). Vegetarian diet intake and non-smokers showed stronger Fhit intensities. Advanced stage tumor, non-vegetarian diet and younger age was associated with loss of Fhit protein. Ki67 positivity was an extended crypt pattern in HACF and DACF showed extension up to the neck region of the crypts and surface epithelium. Carcinomas showed a marked increase in Ki67 expression (P < 0.05). Fhit protein had an inverse association with Ki67 expression.
CONCLUSION: Weaker Fhit intensity was associated with smoking, non-vegetarian diet intake and increasing Ki67 expression. Loss of Fhit protein expression is possibly influenced by environmental factors like smoking and non-vegetarian diet intake.
doi:10.4251/wjgo.v4.i12.250
PMCID: PMC3581850  PMID: 23443232
Aberrant crypt focus; Carcinogenesis, Colorectal carcinoma; Dysplasia; Fragile histidine triad protein; Ki67
22.  Vitamin D Receptor Deficiency Enhances Wnt/β-Catenin Signaling and Tumor Burden in Colon Cancer 
PLoS ONE  2011;6(8):e23524.
Aberrant activation of the Wnt/β-catenin pathway is critical for the initiation and progression of most colon cancers. This activation provokes the accumulation of nuclear β-catenin and the induction of its target genes. Apcmin/+ mice are the most commonly used model for colon cancer. They harbor a mutated Apc allele and develop intestinal adenomas and carcinomas during the first months of life. This phenotype is caused by the mutation of the second Apc allele and the consequent accumulation of nuclear β-catenin in the affected cells. Here we describe that vitamin D receptor (VDR) is a crucial modulator of nuclear β-catenin levels in colon cancer in vivo. By appropriate breeding of Apcmin/+ mice and Vdr+/− mice we have generated animals expressing a mutated Apc allele and two, one, or none Vdr wild type alleles. Lack of Vdr increased the number of colonic Aberrant Crypt Foci (ACF) but not that of adenomas or carcinomas in either small intestine or colon. Importantly, colon ACF and tumors of Apcmin/+Vdr-/- mice had increased nuclear β-catenin and the tumors reached a larger size than those of Apcmin/+Vdr+/+. Both ACF and carcinomas in Apcmin/+Vdr-/- mice showed higher expression of β-catenin/TCF target genes. In line with this, VDR knock-down in cultured human colon cancer cells enhanced β-catenin nuclear content and target gene expression. Consistently, VDR depletion abrogated the capacity of 1,25(OH)2D3 to promote the relocation of β-catenin from the nucleus to the plasma membrane and to inhibit β-catenin/TCF target genes. In conclusion, VDR controls the level of nuclear β-catenin in colon cancer cells and can therefore attenuate the impact of oncogenic mutations that activate the Wnt/β-catenin pathway.
doi:10.1371/journal.pone.0023524
PMCID: PMC3156234  PMID: 21858154
23.  Number of aberrant crypt foci associated with adiposity and IGF1 bioavailability 
Cancer causes & control : CCC  2008;20(5):653-661.
Background
Dysregulation of the insulin-like growth factor (IGF) system, a common consequence of adiposity-induced insulin resistance, may be a key underlying mechanism linking excess body weight with colon cancer. Evidence has been derived from studies of cancer and polyps. Supporting data about aberrant crypt foci (ACF), putative pre-polyp changes, have been generated only from animal studies to date.
Methods
We randomly selected 26 patients with sex-specific elevated waist-hip-ratio (WHR) and 26 with normal values from a series of 150 patients seeking routine colonoscopy at the University of Connecticut Health Center. Cross-sectional analyses were performed of ACF number (<5, ≥5) in relation to total IGF1, IGF-binding protein-3 (IGFBP3), insulin, body mass index (BMI), WHR and waist circumference (WC). Visualized ACF in the 20 cm of the distal colon were counted using advanced endoscopic imaging.
Results
Patients with ≥5 ACF had higher BMI, WHR, and WC compared with patients with >5 ACF (p = 0.04, p = 0.03, and p = 0.01, respectively). IGFBP3 was reduced (p = 0.02) and IGF1:IGFBP3 molar ratio was greater (p = 0.03) in patients with ≥5 ACF. We did not observe significant associations between ACF number and insulin or total IGF1.
Conclusions
Our study provides the first report in humans of a possible association of ACF prevalence and IGF1 bioavailability as characterized by IGF1:IGFBP3 molar ratio and IGFBP3 level. More research is needed to determine whether this relationship is varied by ACF features (e.g., size, dysplasia, molecular changes), synchronous cancer and polyps, and is modified by colon cancer risk factors.
doi:10.1007/s10552-008-9278-7
PMCID: PMC3099467  PMID: 19067190
Aberrant crypt foci; Obesity; Adiposity; Colon cancer; IGF1; IGFBP3; Insulin resistance; Metabolic syndrome
24.  Chemopreventive effects of Coltect, a novel dietary supplement, alone and in combination with 5-aminosalicylic acid in 1,2-dimethylhydrazine-induced colon cancer in rats 
Objectives: Coltect is a novel dietary supplement containing curcumin, green tea and selenomethionine. Previous reports have suggested that these agents can prevent colorectal cancer (CRC). The present study examined the chemopreventive effect of Coltect alone or combined with 5-aminosalicylic acid (5-ASA) using the 1,2-dimethylhydrazine (DMH) model in rats.
Methods: The effect of Coltect was examined on HT-29 CRC cells by growth inhibition assay. Apoptosis was determined by annexin V-FITC/PI staining. Male rats were injected with DMH in vivo and treated with Coltect 150 mg/kg, 5-ASA 50 mg/kg or their combination, by oral gavage. Aberrant crypt foci (ACF) were identified by methylene blue staining.
Results: HT-29 cells exhibited a dose-dependent response to Coltect. Part of the growth inhibition can be explained by the induction of mild-moderate apoptosis in cancer cells (28%) compared with the untreated cells (10%). In the in vivo model, the average number of ACF was divided into small (1–3 crypts) or large (≥4 crypts). The Coltect compound reduced the number of small and large ACF similarly to 5-ASA (40% reduction). This reduction was amplified by combining the two agents (70% reduction).
Conclusion: Coltect inhibits the growth of colon cancer cells, induces apoptosis and inhibits ACF development. Furthermore, it augments the growth inhibitory effect of 5-ASA in vivo. This may be clinically important since this safe dietary supplement-drug combination can be administered as a chemopreventive regimen for the treatment of CRC.
doi:10.1177/1756283X10379258
PMCID: PMC3002588  PMID: 21180609
aberrant crypt foci; 5-aminosalicylic acid; chemoprevention; colorectal cancer; Coltect
25.  Downregulation of prostaglandin E receptor subtype EP3 during colon cancer development 
Gut  2004;53(8):1151-1158.
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.
doi:10.1136/gut.2003.028787
PMCID: PMC1774140  PMID: 15247185
EP3 receptor; prostaglandin E2; colon cancer; colon carcinogenesis; gene expression

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