Dietary flavonoids suppress azoxymethane-induced colonic preneoplastic lesions in male C57BL/KsJ-db/db mice

Obesity is known to be a risk factor for colon carcinogenesis. Although there are several reports on the chemopreventive abilities of dietary flavonoids in chemically induced colon carcinogenesis, those have not been addressed in an obesity-associated carcinogenesis model. In the present study, the effects of 3 flavonoids (chrysin, quercetin and nobiletin) on modulation of the occurrence of putative preneoplastic lesions, aberrant crypt foci (ACF), and β-catenin-accumulated crypts (BCACs) in the development of colon cancer were determined in male db/db mice with obesity and diabetic phenotypes. Male db/db mice were given 3 weekly intraperitoneal injections of azoxymethane (AOM) to induce the ACF and BCAC. Each flavonoid (100 ppm), given in the diet throughout the experimental period, significantly reduced the numbers of ACF by 68–91% and BCAC by 64–71%, as well as proliferation activity in the lesions. Clinical chemistry results revealed that the serum levels of leptin and insulin in mice treated with AOM were greater than those in the untreated group. Interestingly, the most pronounced suppression of development of preneoplastic lesions and their proliferation were observed in the quercetin-fed group, in which the serum leptin level was lowered. Furthermore, quercetin-feeding decreased leptin mRNA expression and secretion in differentiated 3T3-L1 mouse adipocytes. These results suggest that the present dietary flavonoids are able to suppress the early phase of colon carcinogenesis in obese mice, partly through inhibition of proliferation activity caused by serum growth factors. Furthermore, they indicate that certain flavonoids may be useful for prevention of colon carcinogenesis in obese humans.     

A computer model of oxygen dynamics in human colon mucosa: implications in normal physiology and early tumor development

Based on the geometry of the colon mucosa, we built a model to compute the oxygen supply, the oxygen diffusion across the interstitial matrix, and the oxygen consumption by cryptal and stromal cells. By using an iterative algorithm, we have been able to solve a set of discretized (time and space) oxygen balance equations and determine the three-dimensional distribution of pO₂ in the mucosa. Although significant longitudinal and radial pO₂ variations were found, cells appeared to operate at their maximum respiratory capacity, regardless of their location in the tissue. The estimated oxygen extraction fraction was 47%, while the capillary oxygen permeability was 1.57×10⁻⁵ cm m s⁻¹. We concluded that cellular metabolism in normal colon mucosa is not limited by oxygen supply, thus prompting the idea that oxygenation does not determine the characteristic microenvironments occurring along the normal Lieberkhün crypts. In an extended model, simulation of an aberrant crypt focus (ACF)—the earliest stage in the adenomatous polyp-carcinoma sequence—showed instead that respiratory activity decreased when the capillary array symmetry is disrupted due to the ACF growth. A unified explanation about the alternative of a hypoxic-independent and/or a hypoxic-dependent early angiogenic response associated to the development of ACF is proposed.     

Colon Cryptogenesis: Asymmetric Budding

The process of crypt formation and the roles of Wnt and cell-cell adhesion signaling in cryptogenesis are not well described; but are important to the understanding of both normal and cancer colon crypt biology. A quantitative 3D-microscopy and image analysis technique is used to study the frequency, morphology and molecular topography associated with crypt formation. Measurements along the colon reveal the details of crypt formation and some key underlying biochemical signals regulating normal colon biology. Our measurements revealed an asymmetrical crypt budding process, contrary to the previously reported symmetrical fission of crypts. 3D immunofluorescence analyses reveals heterogeneity in the subcellular distribution of E-cadherin and β-catenin in distinct crypt populations. This heterogeneity was also found in asymmetrical budding crypts. Singular crypt formation (i.e. no multiple new crypts forming from one parent crypt) were observed in crypts isolated from the normal colon mucosa, suggestive of a singular constraint mechanism to prevent aberrant crypt production. The technique presented improves our understanding of cryptogenesis and suggests that excess colon crypt formation occurs when Wnt signaling is perturbed (e.g. by truncation of adenomatous polyposis coli, APC protein) in most colon cancers.     

Significance and role of early-lesions in experimental colorectal carcinogenesis

Preneoplastic or precancerous lesions in the large bowel have attracted much attention, and aberrant crypt foci (ACF) topographically identified in the colonic mucosa have found application as effective endpoint lesions for detection of chemopreventive agents as well as carcinogenic risk assessment of environmental agents. While many ACF are regarded as hyperplastic in nature, lacking the potential lesion to give rise to neoplasia, a subset termed dysplastic ACF, or newly identified "mucin depleted foci (MDF)", and "beta-catenin accumulated crypts (BCAC)" are suggested to be more reliably related to colorectal tumorigenesis in rodents. ACF and MDF can be visualized on the surface of colonic mucosa and BCAC were recently identified by our laboratory in rodents en face in cross sections. In particular, BCAC having a similar pattern of beta-catenin gene mutation to that observed in colonic carcinomas appear to be direct precursors. This review provides a review and discussion of the relevant literature relative to early lesions in colorectal carcinogenesis.     

Aberrant crypt foci in colorectal carcinogenesis. Cell and crypt dynamics

Aberrant crypt foci (ACF) have been identified on the colonic mucosal surface of rodents treated with colon carcinogens and of humans after methylene-blue staining and observation under a light microscope. Several lines of evidence strongly suggest that ACF with certain morphological, histological, cell kinetics, and genetic features are precursor lesions of colon cancer both in rodents and in humans. Thus, ACF represent the earliest step in colorectal carcinogenesis. This paper has the main purpose of reviewing the evidence supporting this view, with particular emphasis on cell and crypt dynamics in ACF. ACF have been used as intermediate biomarkers of cancer development in animal studies aimed at the identification of colon carcinogens and chemopreventive agents. Recently, evidence has also shown that ACF can be effectively employed in chemopreventive studies also in humans.     

Influence of colonizing micro-flora on the mucin histochemistry of the neonatal mouse colon

Summary Mucin histochemistry on sections of colon from germ-free and conventional mouse pups showed that all goblet cell mucins were sulphated at birth. During the first two weeks of post natal development, the pattern of mucin production in the ascending colon changed to a distribution of non-sulphated mucins towards the apical zone of the crypts and sulphated sialomucins basally. In conventional animals during the third postnatal week when the complex micro-flora of the colon was becoming established, the typical adult mucin distribution pattern developed, with sulphated mucins now confined to the upper third of the crypt. However, in the absence of a colonizing micro-flora crypt mucins become more and more sulphated until at weaning, most goblet cells of the ascending colon were producing fully or partially sulphated mucins, except for one or two cells at the very base of the crypt.     

Expression of Human Lactoferrin in Bacteroides uniformis and its Effect on Azoxymethane-induced Aberrant Crypt Focus Formation in the Rat Colon

To express a human lactoferrin (hLF) gene in Bacteroides uniformis, a member of the anaerobic microflora in the colon, we constructed a recombinant plasmid, pVLFK, by subcloning hLF cDNA to an Escherichia coli–Bacteroides shuttle vector, pVAL-1. The plasmid pVLFKwas transferred to B. uniformis strain BU1001 by the filter mating procedure creating strain TCTK101. The lactoferrin protein in B. uniformis strain TCTK101 was detected by Western blot analysis with an anti-human lactoferrin monoclonal antibody. A culture of strain TCTK101 inhibited the growth ofE. coli strain HB101 in vitro compared to a culture of strain TCTK11, which is a B. uniformis strain-carrying plasmid pVAL-1, suggesting that the lactoferrin produced from strain TCTK101 possesses biological activity. To determine the effect of lactoferrin-producing B. uniformis on the formation of azoxymethane-induced aberrant crypt foci (ACF), putative neoplastic lesions, overnight cultures of strains TCTK11 and TCTK101 were given to rats as drinking water. The numbers of ACF and ACF having more than three crypts per focus five weeks after the beginning of the experiment significantly increased in the group treated by a culture of strain TCTK11 compared with those in the non-treated water group. However, rats treated with a culture of strain TCTK101 carrying plasmid pVLFK showed a significantly lower number of ACF than rats with a culture of strain TCTK11 (34% reduction), suggesting that hLF which is produced in a prokaryotic expression system prevents formation of ACF in the rat colon.     

Consumption evaluation of one apple flesh a day in the initial phases prior to adenoma/adenocarcinoma in an azoxymethane rat colon carcinogenesis model

Colorectal cancer (CRC) is the fourth cancer with the most new cases reported in 2018 worldwide. Consumption of fruit and vegetables is a protective factor against the risk of CRC. Beyond this, flavonoids could orchestrate these healthy effects. Apart from containing the typical apple flavonoids, red-fleshed apples also contain anthocyanins, mainly cyanidin-3-O-galactoside (Cy3Gal). Through an azoxymethane rat carcinogenesis model, a study was carried out in order to assess the possible protective effects of apple polyphenols, with special attention to anthocyanins. In addition, apart from negative and positive controls, a group with chemotherapy with 5-fluorouracil (5FU) was included to compare their performance against the output collected from the animal treatments with white-fleshed apple (WF), red-fleshed apple (RF) and Cy3Gal (AE).Although the 5FU group presented the best performance towards aberrant crypt foci (ACF) inhibition (70.1%), rats fed with white-fleshed apples ('Golden Smoothee') were able to achieve 41.3% ACF inhibition, while none of the challenged treatments (WF, RF and AE) suffered mucin depletion in their colonocytes. Expression changes of 17 genes related to CRC were assessed. In detail, the ACF inhibition phenotype detected in 5FU and WF groups could be explained through the expression changes detected in the apoptosis-related genes of Aurka, p53 and Cox2. Moreover, in the apple consumption groups (WF and RF), a reduced protein expression of matrix metalloproteinases with gelatinase activity (MMP-2 and 9) was detected.Overall, our study suggests an effect of apple polyphenols and apple anthocyanin Cy3Gal against colon carcinogenesis, retarding/diminishing the appearance of the precancerous markers studied.     

Cell population kinetics in the epithelium of the colon of the male rat.

The present study was undertaken in order to try to define some of the kinetic parameters in the colonic mucosa of normal Wistar rats. Preliminary observations showed considerable morphological differences in the mucosa from site to site along the length of the colon. In particular the height of the crypts (measured in cells) was variable. In addition labelling index studies demonstrated dramatic variations in the distribution of labelling along the length of the crypts from site to site in the bowel. A single site in the descending colon was selected for more detailed study using a stathmokinetic agent, vincristine, and the continous labelling technique with tritiated thymidine. The results of these investigations suggest that there exists at the base of the crypt a subpopulation of cells cycling more slowly than the cells in the rest of the proliferative compartment. Growth fraction appears to fall with rising cell positions within the crypt.     

Aberrant crypt foci and beta-catenin accumulated crypts; significance and roles for colorectal carcinogenesis

Preneoplastic or precancerous lesions in the large bowel have been characterized in terms of morphology and histochemical phenotype. However, the detailed histogenesis and relation of particular lesions to malignancies has not yet to be unequivocally clarified. Aberrant crypt foci (ACF), identified in whole-mount preparations of colonic mucosa in rodents and also recognized in human colon, are now frequently used as effective surrogate biomarkers for experimentally detection of chemopreventive agents against colorectal cancers, but the preneoplastic or precancerous nature of ACF in rodents and humans still remains inconclusive. Relatively recently, early appearing beta-catenin accumulated crypts (BCAC) have been described in en face preparations of colonic mucosa in rodents which differ from ACF in many features. BCAC are suggested to be premalignant rather than preneoplastic. The pathological significance of both lesions, including their advantages and disadvantages as surrogate end points for large bowel neoplasms, and roles in colorectal carcinogenesis are discussed here.     

A Method for Serial Tissue Processing and Parallel Analysis of Aberrant Crypt Morphology,Mucin Depletion,and Beta-Catenin Staining in an Experimental Model of Colon Carcinogenesis

The use of architectural and morphological characteristics of cells for establishing prognostic indicators by which individual pathologies are assigned grade and stage is a well-accepted practice. Advances in automated micro- and macroscopic image acquisition and digital image analysis have created new opportunities in the field of prognostic assessment; but, one area in experimental pathology, animal models for colon cancer, has not taken advantage of these opportunities. This situation is primarily due to the methods available to evaluate the colon of the rodent for the presence of premalignant and malignant pathologies. We report a new method for the excision and processing of the entire colon of the rat and illustrate how this procedure permitted the quantitative assessment of aberrant crypt foci (ACF), a premalignant colon pathology, for characteristics consistent with progression to malignancy. ACF were detected by methylene blue staining and subjected to quantitative morphometric analysis. Colons were then restained with high iron diamine–alcian blue for assessment of mucin depletion using an image overlay to associate morphometric data with mucin depletion. The subsequent evaluation of ACF for beta-catenin staining is also demonstrated. The methods described are particularly relevant to the screening of compounds for cancer chemopreventive activity.     

Novel mucilage fraction of Sinapis alba L. (mustard) reduces azoxymethane-induced colonic aberrant crypt foci formation in F344 and Zucker obese rats

Seeds of Sinapis alba Linn. (commonly called yellow or white mustard) and their components have been reported to possess anticancer properties. In this study, we evaluated the efficacy of a novel mucilaginous fraction of mustard seeds in inhibiting colonic preneoplastic changes in animal models of sporadic and obesity-associated colon cancer. In two separate studies, male Sprague-Dawley or female Zucker obese rats, injected with azoxymethane (15 or 10 mg/kg body wt. once a week for 2 weeks, respectively), were fed AIN-93G diets with or without 5% mustard mucilage (MM) (w/w) for 8 weeks. Our aim was to measure the ability to modulate the number of aberrant crypt foci (ACF), putative preneoplastic lesions of the colon. The data were classified into total numbers of ACF and large ACF (crypt multiplicity of 4 or more). We report here that 5% MM significantly (p<0.05) decreased the number of total (approximately 21% inhibition) and large (approximately 50% inhibition) ACF in the colons of Sprague-Dawley rats compared to that in untreated controls. In addition, 5% MM supplemented diet significantly lowered (p<0.05) the number of total (approximately 63% inhibition) and large (approximately 60% inhibition) colonic ACF in Zucker obese rats compared to untreated obese rats, and had no effect on fasting plasma cholesterol or triglyceride levels. These results demonstrate the possible role of MM as a functional food against sporadic and obesity-associated colon cancer, and provide impetus to conduct research to understand the underlying mechanism(s) of action.     

Biased competition between Lgr5 intestinal stem cells driven by oncogenic mutation induces clonal expansion

The concept of ‘field cancerization’ describes the clonal expansion of genetically altered, but morphologically normal cells that predisposes a tissue to cancer development. Here, we demonstrate that biased stem cell competition in the mouse small intestine can initiate the expansion of such clones. We quantitatively analyze how the activation of oncogenic K-ras in individual Lgr5⁺ stem cells accelerates their cell division rate and creates a biased drift towards crypt clonality. K-ras mutant crypts then clonally expand within the epithelium through enhanced crypt fission, which distributes the existing Paneth cell niche over the two new crypts. Thus, an unequal competition between wild-type and mutant intestinal stem cells initiates a biased drift that leads to the clonal expansion of crypts carrying oncogenic mutations.     

Human colon tissue in organ culture: preservation of normal and neoplastic characteristics

Normal and neoplastic human colon tissue obtained at surgery was used to establish conditions for organ culture. Optimal conditions included an atmosphere of 5% CO₂ and 95% O₂; tissue partially submerged with mucosa at the gas interface; and serum-free medium with 1.5 mM Ca²⁺ and a number of growth supplements. Histological, histochemical, and immunohistochemical features that distinguish normal and neoplastic tissue were preserved over a 2-d period. With normal tissue, this included the presence of elongated crypts with small, densely packed cells at the crypt base and mucin-containing goblet cells in the upper portion. Ki67 staining, for proliferating cells, was confined to the lower third of the crypt, while expression of extracellular calcium-sensing receptor was seen in the upper third and surface epithelium. E-cadherin and β-catenin were expressed throughout the epithelium and confined to the cell surface. In tumor tissue, the same disorganized, abnormal glandular structures seen at time zero were present after 2 d. The majority of cells in these structures were mucin-poor, but occasional goblet cells were seen and mucin staining was present. Ki67 staining was seen throughout the abnormal epithelium and calcium-sensing receptor expression was weak and variable. E-cadherin was seen at the cell surface (similar to normal tissue), but in some places, there was diffuse cytoplasmic staining. Finally, intense cytoplasmic and nuclear β-catenin staining was observed in cultured neoplastic tissue.     

DNA damage and aberrant crypt foci as putative biomarkers to evaluate the chemopreventive effect of annatto (Bixa orellana L.) in rat colon carcinogenesis

Chemoprevention opens new perspectives in the prevention of cancer and other degenerative diseases. Use of target-organ biological models at the histological and genetic levels can markedly facilitate the identification of such potential chemopreventive agents. Colon cancer is one of the highest incidence rates throughout the world and some evidences have indicated carotenoids as possible agents that decrease the risk of colorectal cancer. In the present study, we evaluate the activity of annatto (Bixa orellana L.), a natural food colorant rich in carotenoid, on the formation of aberrant crypt foci (ACF) induced by dimethylhydrazine (DMH) in rat colon. Further, we investigate, the effect of annatto on DMH-induced DNA damage, by the comet assay. Male Wistar rats were given s.c. injections of DMH (40 mg/kg body wt.) twice a week for 2 weeks to induce ACF. They also received experimental diets containing annatto at 20, 200 or 1000 ppm for five 5 weeks before (pre-treatment), or 10 weeks after (post-treatment) DMH treatment. In both protocols the rats were sacrificed on week 15th. For the comet assay, the animals were fed with the same experimental diets for 2 weeks. Four hours before the sacrifice, the animals received an s.c. injection of DMH (40 mg/kg body wt.). Under such conditions, dietary administration of 1000 ppm annatto neither induce DNA damage in blood and colon cells nor aberrant crypt foci in rat distal colon. Conversely, annatto was successful in inhibiting the number of crypts/colon (animal), but not in the incidence of DMH-induced ACF, mainly when administered after DMH. However, no antigenotoxic effect was observed in colon cells. These findings suggest possible chemopreventive effects of annatto through their modulation of the cryptal cell proliferation but not at the initiation stage of colon carcinogenesis.     

Long-term feeding of various fat diets modulates azoxymethane-induced colon carcinogenesis through Wnt/beta-catenin signaling in rats

The Wnt signaling pathway plays an essential role in carcinogenesis, and the amount of fat intake and composition of dietary fatty acids are crucial factors for colon carcinogenesis. We investigated whether various dietary fats affected the Wnt signaling pathway of colon tumorigenesis in azoxymethane (AOM)-treated rats. Male Sprague-Dawley rats were given intraperitoneal injections of AOM and supplemented with 10% corn, olive, beef, and fish oil for 44 wk. Aberrant crypt foci (ACF) and tumors were examined at 12 and 44 wk. Normal appearing colon mucosal proliferation and apoptosis were evaluated by 5-bromo-2'-deoxyuridine (BrdU) incorporation and percentages of fragmented DNA, respectively. Expressions of beta-catenin, cyclin D(1), Wnt2, Wnt3, and Wnt5a of normal appearing colon mucosa were analyzed by Western blot analysis. Long-term dietary corn oil and beef tallow increased ACF, tumor incidence, and tumor numbers in AOM-treated rats. In contrast, both olive and fish oil inhibited them. Dietary corn oil and beef tallow increased BrdU incorporation and the expression of cytosolic beta-catenin and cyclin D(1) and decreased apoptosis in the colon mucosa. Expressions of Wnt2 and Wnt3 in rats fed with beef tallow and Wnt5a in rats fed with corn oil increased with or without AOM-treatment. BrdU-incorporated cells were often observed at the tops of crypts in rats fed with beef tallow, whereas this was not observed in rats fed with the other diet. Long-term high intake of corn oil and beef tallow enhanced cell proliferation through Wnt signaling and modulated the distribution of proliferating cells, which might contribute to promoting effects in colon tumorigenesis.     

Epithelial cell kinetics in the descending colon of the rat

The influence of experimental bypass on the epithelial cell kinetics in the rat descending colon was studied. It was found that the number of cells per crypt was markedly reduced at 6 weeks after bypass. The percentage of labelled crypt cells, 1 h after 3HTdR, and the distribution of labelled cells in the crypt was normal. Also the life span of the epithelial cells was the same in control and bypassed colon. The response of crypt cell proliferation to ischaemia-induced cell loss in the bypassed descending colon was similar to the one previously described for normal descending colon. This indicates that the absence of the normal luminal contents does not result in a different response of colonic crypts to induced cell loss. Furthermore, it was found that the number of cells per crypt and the proliferative activity did not change in the transverse colon after temporary ischaemia of the bypassed descending colon. This indicates that the increase in crypt cell proliferation after ischaemia-induced cell loss is a local response.     

Effect of common Brassica vegetables (Brussels sprouts and red cabbage) on the development of preneoplastic lesions induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in liver and colon of Fischer 344 rats

Aim of the present study was the investigation of effects of juices from commonly consumed Brassica vegetables (two cultivars of Brussels sprouts and two cultivars of red cabbage) on formation and development of preneoplastic lesions in colons (aberrant crypt foci, ACF) and livers (glutathione-S-transferase placental form, GST-P+) in male F344 rats. The foci were induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a widespread carcinogenic heterocyclic aromatic amine which is found in fried meats. Recently, we reported on pronounced protective effects in the two-organ foci model when the vegetable juices were given during the carcinogen treatment but several findings by other groups indicated that breakdown products of glucosinolates contained in Brassica vegetables cause tumour promotion in various organs of laboratory rodents. In the present study, the animals received the juices in the drinking water (5%) over a period of 20 days after treatment with IQ (100 mg/kg bw on 10 alternate days). To increase the foci yield (which facilitates the detection of modifying effects), the animals were fed with a modified (high fat, fibre free) AIN-76 diet. With exception of the sprout variety "Cyrus", all juices lowered the number of GST-P+ foci as well as the foci area in the liver, but none of these effects was statistically significant. In the colon, none of the juices had an impact on crypt multiplicity (number of crypts/focus), whereas the number of ACF was decreased; only with the sprout variety Maximus the protective effect was significant (reduction 49%). The present findings show that administration of vegetable juices to the animals after the carcinogen does not increase the number and size of IQ-induced preneoplastic lesions in liver and colon.