Rats have mostly been employed for animal colorectal carcinogenesis models, and AOM, methylazoxymethanol (MAM) acetate, and 1,2-dimethylhydrazine (DMH) have been widely used as colorectal carcinogens [15]. About 30 weeks are required for development of CRC in about half of rats that are initiated with these colonic carcinogens. On the other hand, in experiments and studies using mice, multiple administrations of the colorectal carcinogens are required and it takes a long-term of 40 weeks or longer to develop CRC [31]. Therefore, I have developed a novel mouse model that would produce CRC in a short-term in the inflamed colorectum [18]. AOM-DSS, here called the TANAKA model [15,17,18], is a well-characterized experimental model for UC-associated colorectal carcinogenesis (Figure 1a–e) [18,32,33,34,35,36]. Mice that received a single injection of a low dose of the classic colon carcinogen AOM prior to administration of DSS in drinking water develop inflammation and mucosal ulcer (Figure 1b) [18,37,38] as well as dysplasia (Figure 1c), adenoma (AD) (Figure 1d) and ADC (Figure 1e) with pathologic features that resemble those of human UC-associated neoplasia [18,33,34,36]. The extent of neoplastic lesions depends on several factors like strain susceptibility as well as duration, dosage, and schedule of cyclic DSS application [39,40,41].

To settle the issue of the influence of peroxisome proliferator-activated receptor (PPAR) agonists on colorectal carcinogenesis, which has been a topic since 1998 [42,43,44], we confirmed that colitis induced by DSS, which is a non-genotoxic carcinogen [45,46], using aberrant crypt foci (ACF) as a biomarker [16,47,48,49], had tumor promoter activity to enhance development of ACF in rats and hypothesized that a combination of DSS and AOM would induce CRC in a short-term period in mice as well [50].

Instead of AOM, experiments with DMH [51] or a heterocyclic amine, PhIP [52] as an initiator (colon carcinogen) and followed by DSS treatment resulted in rapid development of colorectal neoplasms. Histopathologically, ADC induced by DMH/DSS showed severer atypia and more aggressive biological nature than that induced by AOM/DSS. As noticed in the cancers induced by AOM/DSS, the ADC cells developed in the inflamed colon of mice that received DMH and DSS were positive for cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), and β-catenin. Mutation patterns of the β-catenin gene slightly differ among the ADCs that were induced by the different treatment regimens: AOM/DSS, codon 32–34, 37, and 41; DMH/DSS, codon 32, 34, 37, and 41; and PhIP/DSS, codon 32 and 34. However, these mutations was limited to the codon 32–34, 37, 41, and 45 that played an important role in degradation of β-catenin protein.

In Apc^Min/+ mice, known as an animal model for familial adenomatous polyposis (FAP), multiple tumors (tubular ADs) develop in the small intestine, instead of the large intestine in human FAP, and markedly few tumors develop in the large bowel. However, dysplastic crypts are observed in the colonic mucosa of Apc^Min/+ mice [53,54]. Therefore, DSS possibly enhances the growth of dysplastic crypts, and finally the lesions progress to ADCs. To investigate whether DSS-induced inflammation in the colonic mucosa would accelerate the growth of dysplastic crypts, Apc^Min/+ mice were given drinking water containing 2% DSS for one week without the initiation (carcinogen) treatment [53]. Surprisingly, multiple colorectal tumors, which were histopathologically tubular ADs and ADCs, developed four weeks after the end of DSS treatment. Immunohistochemistry showed that the developed colorectal ADCs were positive against β-catenin, COX-2, iNOS, and p53 antibodies, suggesting that these factors were involved in the development of colorectal neoplasms in the Apc^Min/+ mice by the DSS treatment, in addition to oxidative stress and nitrosative stress. The findings suggested that DSS-induced inflammation in the large bowel of Apc^Min/+ mice exert powerful tumor-promotion and/or progression effects on the growth of dysplastic crypts, which had already existed after the birth [53,54].

The mouse inflammation-associated colorectal carcinogenesis model was named the TANAKA model. With this model it was possible to induce colorectal tumors in a short-term period in rats as well as by similar treatment regimens (AOM/DSS and DMH/DSS) [55,56]. The administration dose of colon carcinogens for initiation is too low to induce colon tumors, but it can initiate or induce DNA modification [18]. Treatment with DSS followed by AOM did not produce colonic neoplasms [18]. The TANAKA model will help advance the research on elucidation of the mechanisms of inflammation-associated colorectal carcinogenesis, inhibition of carcinogenesis, and clarification of the mechanisms of the tumor-promotion ability of DSS. In particular, development of challenging research using the Kyoto Apc Delta (KAD) rats (Figure 2a–e) [57] and gpt delta rats [58] will give new insight in the pathogenesis of CRC development in the inflamed colon [57]. Interestingly, atypical and neoplastic cells of dysplastic crypts, AD, and ADC in the KAD rats that received AOM and DSS contain Paneth’s granules (Figure 2f), which consist of several anti-microbial compounds and other compounds that are known to be important in immunity and host-defense, in their cytoplasm. We have thus confirmed that in rats with or without genetically alterations colonic tumors are rapidly produced as observed in mice and the rat AOM/DSS and DMH/DSS models can be applied to determine the chemopreventive ability of target compounds.

We could identify initiators and promoters of colorectal carcinogenesis by modifying the regimens of AOM/DSS and DMH/DSS. When applied test compounds to the initiation treatment instead of AOM or DMH and followed by DSS treatment, we could evaluate initiation activity of test compounds [59]. When test chemicals are applied after initiation treatment with AOM or DMH, we could determine tumor promotion activity of test compounds (unpublished data and [60]). Thus, modification of two regimens in the TANAKA model will determine environmental carcinogens and tumor promoters in the colorectum.

A flavonol, morin (3,5,7,2',4'-pentahydroxyflavone) found in almonds, mill, fig, mulberry, and other Moraceae, acts as a potent antioxidant, inhibitor of xanthine oxidase, protein kinase C and proliferation, apoptosis inducer and modulator of lipoxygenase and cyclooxygenase activities. This flavone has been reported to inhibit the growth of COLO205 cells in nude mice [66], exhibit intestinal anti-inflammatory activity in the acute phase of rat colitis induced by trinitrobenzenesulfonic acid [67,68]. We have previously reported that morin inhibits AOM-induced putative precursor lesions, ACF, in rats [69] and inhibit chemically-induced rat tongue carcinogenesis [70]. Morin exerts anti-inflammatory effects on septic shock induced by lipopolysaccharide [71]. This study aimed to determine possible inhibitory potential of morin in colitis-associated colon carcinogenesis initiated with AOM and promoted by DSS in male F344 rats.

Materials and methods: A total of 66 male rats (5-week-old) were initiated with a single s.c. injection of AOM (20 mg/kg bw), and then they were given promotion stimuli by the treatment with 1.5% DSS in their drinking water for seven days. They were then given a basal diet containing 50, 250 and 1,000 ppm of morin for 17 weeks. Experimental groups included the AOM/1.5% DSS (n = 14), AOM/1.5% DSS/50 ppm morin (n = 10), AOM/1.5% DSS/250 ppm morin (n = 11), AOM/1.5% DSS/1,000 ppm morin (n = 11), AOM alone (n = 5), 1.5% DSS alone (n = 5), 500 ppm morin alone (n = 5), and untreated (n = 5) groups. At the end (week 20) of the study histopathological analysis of colorectum was performed on hematoxylin and eosin (H&E)-stained histologic sections (3 μm thickness). Proliferation activity of colonic ADCs was determined by immunofluorescence technique using anti-Mcm2 antibody (BD Biosciences PharMingen, Tokyo, Japan). Apoptotic cells were detected by fluorescein in situ tunnel method, TACS TdT kit (R&D Systems, Inc., Minneapolis, MN, USA). Polyamine levels [72] and mRNA expression of nuclear factor-kappaB (NF-κB), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, Stat3, and hypoxia-inducible factor (HIF)-1α [73] in colonic mucosa were determined in mice randomly selected from each group. All measurements were statistically analyzed using either the Tukey multiple comparison post test or Fisher’s extract probability test. Differences were considered to be statistically significant at p < 0.05.

Results: At week 20, the treatment with morin inhibited colonic mucosal ulcer (Figure 3) and dysplastic crypts (p < 0.05 at 1,000 ppm, Figure 3). The incidence (p < 0.005) and multiplicity (p < 0.01) of colonic AD were significantly reduced by feeding with 1000 ppm morin (Figure 4). Also, dietary administration with 1,000 ppm morin significantly inhibited the incidence (p < 0.02) and multiplicity (p < 0.05) of colonic ADC (Figure 4), when compared to the AOM/DSS group (93% incidence and 2.36 ± 1.95 multiplicity). Feeding with 50 ppm and 250 ppm morin lowered the incidence and multiplicity of ADC, but the inhibition rates did not reach statistically significance. The treatments also modulated proliferation and apoptosis in ADCs. Mcm2 positive rates (%) of ADCs in rats fed the diets containing 50 ppm morin (n = 6, 78.7 ± 6.8), 250 ppm morin (n = 7, 63.4 ± 6.3, p < 0.001), and 1,000 ppm morin (n = 5, 55.6 ± 12.5, p < 0.001) were lower than that of rats given AOM and DSS (n = 13, 83.4 ± 8.8). When compared with the AOM and DSS group (n = 13, 8.23 ± 1.24), apoptotic index (%) of ADCs was increased by feeding with morin: 50 ppm morin (n = 6, 10.83 ± 3.66), 250 ppm morin (n = 7, 11.29 ± 3.95), and 1,000 ppm morin (n = 5, 13.00 ± 2.74, p < 0.05).

Growth inhibition and apoptosis induction by morin in CRC might be caused by activation of caspase 3 and increase of p21 protein [66]. Suppression of NF-κB-regulated gene products and enhancement of apoptosis induced by TNF [74] also contribute to inhibition of colitis-related colorectal carcinogenesis by morin. Our findings suggest that dietary morin is able to inhibit colitis-related colon carcinogenesis in rats and a flavonol morin is one of the candidates for clinical application of chemoprevention against CRC development in patients with ulcerative colitis.

Because morin can inhibit inflammation, inhibit tumor promotion, suppress tumor growth, and down-regulate the expression of certain genes regulated by NF-κB, it may be possible that morin modulates the activation of NF-κB and NF-κB-regulated gene expression induced by carcinogens, inflammatory agents, and immune modulators. In fact, Manna et al. [74] have recently reported morin suppresses the activation of NF-κB and NF-κB-regulated gene expression that leads to enhancement of apoptosis.

CRC is one of the leading forms of malignancy in the developed countries. Epidemiologic and animal studies have suggested that risk factors for coronary artery disease like insulin resistance and dyslipidemia are probably related to the development of colon cancer [75,76,77]. In particular, nuclear peroxisome proliferator-activated receptors (PPARs), mainly PPARα and PPARγ, which play a central role in lipid and glucose metabolism, had been hypothesized as being involved in colon carcinogenesis [50,78,79]. Furthermore, synthetic PPAR ligands (glitazones and bezafibrate) with proven beneficial effects on insulin resistance and triglyceride levels had been proposed to be candidates as tumor preventive agents [50,63,79]. This study aimed to determine inhibitory potential of bezafibrate in colitis-associated colon carcinogenesis initiated with AOM and promoted by DSS in mice.

Materials and methods: A total of 100 male ICR mice (5-week old) initiated with a single s.c. injection of AOM (10 mg/kg bw) were promoted by 1.5% DSS in their drinking water for seven days. They were then given a basal diet containing 50, 100 and 500 ppm of bezafibrate for 17 weeks. Mice were divided into 8 groups: AOM/1.5% DSS (n = 20), AOM/1.5% DSS/50 ppm bezafibrate (n = 20), AOM/1.5% DSS/100 ppm bezafibrate (n = 20), AOM/1.5% DSS/500 ppm bezafibrate (n = 20), AOM alone (n = 5), 1.5% DSS alone (n = 5), 500 ppm bezafibrate alone (n = 5), and untreated (n = 5) groups. At the end (week 20) of the study histopathological analysis of colorectum was performed on H&E-stained histological sections of 3 μm in thickness. Immunofluorescence technique using anti-Mcm2 antibody (BD Biosciences PharMingen) and fluorescein in situ tunnel method, TACS TdT kit (R&D Systems, Inc.) were used for determination of proliferation activity and apoptosis index of colonic ADCs, respectively. Polyamine levels [72] and mRNA expression of NF-κB, TNF-α, IL-1β, Stat3, and HIF-1α [73] in colonic mucosa were assayed in some mice of each group. Measurements were statistically analyzed using either the Tukey multiple comparison post-test or Fisher’s extract probability test. Differences were considered to be statistically significant at p < 0.05.

Results: At week 20, the bezafibrate feeding inhibited the occurrence of mucosal ulcer (the incidence at 500 ppm, p < 0.02; and the multiplicity at 50, 100 and 500 ppm, p < 0.01 or p < 0.001, Figure 5) and dysplastic crypts (the multiplicity at 500 ppm, p < 0.05, Figure 5). As illustrated in Figure 6, he development of colonic ADC was significantly inhibited by feeding with 500 ppm bezafibrate (incidence: 73% reduction, p < 0.01; and multiplicity: 92%, p < 0.05), when compared to the AOM/DSS group (73% incidence and 2.53 ± 3.14 multiplicity). Feeding with 50 ppm (47% incidence with a multiplicity of 1.40 ± 1.92) and 100 ppm bezafibrate (67% incidence with a multiplicity of 1.13 ± 1.19) also lowered the incidence and multiplicity of ADC, but the inhibition was not statistically significant when compared to the AOM/DSS group (Figure 6). Although statistically insignificant, feeding with 50 and 100 ppm bezafibrate increased the incidence and multiplicity of ADs, but 500 ppm bezafibrate decreased the development of ADs (Figure 6). These findings may suggest the threshold of chemopreventive ability of bezafibrate. Feeding with bezafibrate lowered Mcm2 positive rates (%) of ADCs, when compared with the AOM and DSS group (n = 10, 72.9 ± 10.1): 50 ppm bezafibrate (n = 7, 54.3 ± 9.5), 100 ppm bezafibrate (n = 4, 38.8 ± 7.4, p < 0.01), and 500 ppm bezafibrate (n = 3, 22.7 ± 3.2, p < 0.001) were lower than that of rats given AOM and DSS (n = 13, 83.4 ± 8.8). When compared with the AOM and DSS group (n = 10, 8.18 ± 1.17), apoptotic index (%) of ADCs was increased by feeding with bezafibrate: 50 ppm bezafibrate (n = 7, 10.71 ± 3.35), 100 ppm bezafibrate (n = 4, 13.20 ± 2.49, p < 0.05), and 500 ppm bezafibrate (n = 3, 13.25 ± 3.10, p < 0.05).

Anti-angiogenic effects and anti-inflammatory activity of PPARα agonist [80] are considered to contribute to inhibition of CRC growth. Down-regulation of the anti-apoptotic gene Mcl-1 by PPARα agonist [81] causes apoptosis. The findings suggest that dietary bezafibrate is able to inhibit colitis-related colon carcinogenesis in mice and a hypolipidemic drug bezafibrate is one of the candidates for clinical application of chemoprevention against CRC development in patients with ulcerative colitis.

Accumulating animal experimental, human laboratory and epidemiologic data [50,63,79,82,83] support the hypothesis linking triglyceride levels and insulin resistance to the development of colon cancer. These facts emphasize the potential for this cancer to become a preventable disease not only via screening and removal of polyps but through relevant lifestyle changes and pharmacological interventions which can provide even more avenues for prevention [83,84,85].

The incidence of insulin resistance has been increasing in the Western world where colon cancer is the second leading cause of cancer death. This suggests the interrelationship of these conditions. The biological role of PPARs in various diseases, including inflammation and cancer, has been highlighted recently [50,63,78,85,86,87]. PPARs are members of the nuclear hormone receptor family of ligand-activated transcription factors that play a prominent role in the regulation of many metabolic processes. The PPAR isoformes α and γ are important regulators in lipid and glucose metabolism, cell differentiation and inflammatory response [87,88]. These data propose that PPAR may be associated with many aspects of colon cancer development including insulin- and inflammation-related mechanisms. The fibric acid derivative bezafibrate is the pan PPAR (α, β/δ, and γ) activator with predominantly PPARα (as all fibrates) and β/δ effects but also with perceptible PPARγ properties [88,89,90]. The use of bezafibrate is associated with triglyceride-lowering and high density-cholesterol raising effects resulting in decreased systemic availability of fatty acid, diminished of fatty acid uptake by muscle and improvement of insulin sensitization [91,92]. These direct and indirect effects may have contributed to the suppression of the development of colonic tumors in rodents by bezafibrate [50,63,79]. Recently, Tenenbaum et al. [93] have reported possible preventive effects of bezafibrate on the development of CRC from patients with coronary artery disease.

Epigenetic modification plays an important role in tumorigenesis. Affecting epigenetic and tumorigenic alterations is a promising strategy for anticancer targeted therapy [94,95,96]. Among the key chromatin modifying enzymes which influence gene expression, histone acetyltransferases (HATs) and histone deacetylases (HDACs) have attracted interest because of their impact on tumor development and progression. Histone deacetylase inhibitors (HDACIs) represent a new and promising class of antitumor drugs that influence gene expression by enhancing acetylation of histones in specific chromatin domains. HDACIs also exert potent anticancer activities inducing cell cycle arrest and apoptosis. Moreover, HDACIs down-regulate genes involved in tumor progression, invasion and angiogenesis. Based on the ability of HDACIs to regulate many signaling pathways, co-treatment of these compounds that are currently under clinical investigation with molecular targeted drugs is a promising strategy against many types of tumors.

VPA (2-propylpentanoic acid) [97] is a well-established drug for the therapy of epilepsy. It is teratogenic when administered during early pregnancy and can induce birth defects such as neural tube closure defects and other malformations. This well-tolerated antiepileptic drug was found to be a powerful HDAC-1 inhibitor [97]. VPA induces differentiation of carcinoma cells, transformed hematopoietic progenitor cells and leukemic blasts from acute myeloid leukemia patients [98]. Our microarray analysis during the AOM-DSS carcinogenesis revealed alteration of Wif-1 expression [34]. VPA has been reported to modify the Wif-1 expression [99]. These findings may suggest possible modifying effects of VPA on AOM-DSS colorectal carcinogenesis. In this study, we determined whether VPA is able to inhibit colitis-associated colon carcinogenesis in mice.

Materials and methods: A total of 85 mice aged five weeks was used and they were divided into 8 groups: AOM/2% DSS (n = 19), AOM/2% DSS/50 ppm VPA (n = 15), AOM/2% DSS/250 ppm VPA (n = 15), AOM/2% DSS/1,000 ppm VPA (n = 16), AOM alone (n = 5), 2% DSS alone (n = 5), 1,000 ppm VPA alone (n = 5), and untreated (n = 5) groups. Mice were initiated with a single s.c. injection of AOM (10 mg/kg bw) were promoted by 2% DSS in their drinking water for seven days. They were then given a basal diet containing 50, 250 or 1,000 ppm of VPA for 17 weeks. At the end (week 20) of the study histopathological examination of large bowel was performed on H&E-stained histological sections (3 μm in thickness). Immunofluorescence technique using anti-Mcm2 antibody (BD Biosciences PharMingen) for evaluating proliferating activity and fluorescein in situ tunnel method, TACS TdT kit (R&D Systems, Inc.) for detecting apoptosis cells were applied on histological sections of colonic ADCs. Polyamine levels [72] and mRNA expression of NF-κB, TNF-α, IL-1β, Stat3, and HIF-1α [73] in colonic mucosa were assayed in some mice of each group.At the end of the study (week 20), Measurements were statistically analyzed using either the Tukey multiple comparison post test or Fisher’s extract probability test. Differences were considered to be statistically significant at p < 0.05.

Results: VPA feeding inhibited the development of mucosal ulcer (Figure 7) and dysplastic crypts (the incidence at 1,000 ppm VPA, p < 0.05; and the multiplicity at 50 ppm, p < 0.05 and at 1,000 ppm, p < 0.01, Figure 7).

The development of colonic AD and ADC was lowered by feeding with VPA, but the reduction rates were statistically insignificant (Figure 8). When fed with VPA-containing diet, Mcm2 positive rates (%) of ADCs were lower than that of the AOM and DSS group (n = 9, 80.1 ± 6.8): 125 ppm VPA (n = 7, 73.7 ± 8.0), 250 ppm VPA (n = 6, 64.2 ± 2.5, p < 0.01), and 1,000 ppm VPA (n = 5, 61.6 ± 9.6, p < 0.001). As to apoptotic index (%) of ADCs, the values of mice fed with 125 ppm VPA (n = 7, 10.67 ± 3.44), 250 ppm VPA (n = 6, 12.33 ± 1.75, p < 0.05), and 1,000 ppm VPA (n = 5, 12.80 ± 2.39, p < 0.05) were higher thab the AOM and DSS group (n = 9, 8.44 ± 1.33).

Our findings suggest slight chemopreventive effects of VPA on colitis-related colon carcinogenesis in mice, suggesting that single use of a HDAC inhibitor VPA is not practical for inhibiting CRC development in inflamed colon. VPA combined with other known chemopreventive or chemotherapeutic agent(s) may exert to inhibit CRC that develop in colitic mucosa.

VPA was studied in combination with all-trans retinoid acid in patients with acute myeloid leukemia who were not candidates for intensive chemotherapy [100]. Using human hepatocellular carcinoma (HCC) cells, HepG2, combination treatment with acyclic retinoid and VPA is demonstrated to be an effective regimen for the chemoprevention and chemotherapy of HCC [101]. Acyclic retinoid and VPA cooperatively increase the expression of retinoid X receptor (RXR)-β and p21 (CIP1), while inhibiting the phosphorylation of RXRα, and these effects were associated with induction of apoptosis and the inhibition of cell growth in HepG2 cells. Several HDACIs seem to exert an antitumor effect in a synergistic manner with different anticancer compounds and to overcome the resistance induced by conventional chemotherapeutic drugs [102,103,104,105]. A phase I trial of single agent VPA was reported in patients with newly diagnosed cervical cancer [106]. Twelve patients were included. VPA doses ranged from 20 mg/kg to 40 mg/kg daily for five days. Tumor HDAC activity decreased in 8 patients. Many lines of evidence suggest that tumor cells are characterized by histone hypoacetylation and that over-expression of HDACs is involved in tumorigenesis of various human malignancies [107,108]. Recently, a population-based case-control study with long-term users of VPA has not supported HDAC inhibition by VPA as a pharmacologic principle for general chemoprevention [109]. However, VPA doses (0.35–0.70 mmol/L) used in clinical practice could be too low to achieve cancer preventive effects in contrast the doses (0.50–3.0 mmol/L) that inhibit HDAC. Since VPA is reported to suppress progression of urological malignancies [110,111], it is worthy to evaluate the modifying effects of VPA on different stage of carcinogenesis.