Chemoprevention of colon and small intestinal tumorigenesis in APC(Min/+) mice by licofelone, a novel dual 5-LOX/COX inhibitor: potential implications for human colon cancer prevention

Preclinical and clinical studies suggest that 5-lipoxygenase (5-LOX), such as COX-2, is a potential target for colon cancer inhibition and, in part, contributes to cardiovascular side effects associated with COX-2 inhibitors. Experiments were designed to assess the chemopreventive effects of a novel dual 5-LOX/COX inhibitor, licofelone {[6-(4-chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-1H-pyrrolizin-5-yl] acetic acid}, in APC(Min/+) mouse intestinal tumorigenesis. Six-week-old male and female APC(Min/+) mice (n = 10 per group) were fed with control American Institute of Nutrition-76A diet or diets containing 150 or 300 ppm licofelone for 14 weeks (～100 days), and intestinal tumors were evaluated for tumor multiplicity and size. Licofelone significantly inhibited total intestinal tumor multiplicity and size in a dose-dependent manner (P < 0.0001; mean tumors for 0, 150, and 300 ppm: 48.8, 17, and 8, respectively, in male mice; and 34.3, 8.8, and 5.5, respectively, in female mice). Licofelone at high dose showed more than 83% (P < 0.0001) tumor inhibition in both genders of mice. One hundred and fifty and 300 ppm licofelone resulted in 86% to 97% inhibition of polyps having size greater than 2 mm. One hundred and fifty and 300 ppm licofelone caused more than 72% and 100% inhibition of colonic tumors, respectively. Importantly, in mice fed with licofelone, tumors showed significantly reduced proliferating cell nuclear antigen expression (70%, P < 0.0001), increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells (75%, P < 0.0001), and there was dose-dependent suppression of serum triglycerides (71%-83%, P < 0.0001), decreased inflammatory cytokines; and decreased COX and 5-LOX activities (57%-64%, P < 0.0001). Also, compared with 300 ppm celecoxib, 300 ppm licofelone provided better efficacy in suppressing tumor growth. These observations show that a novel dual 5-LOX/COX inhibitor dramatically suppresses small intestinal and colonic tumor formation in APC(Min/+) mice.     

Chemoprevention of familial adenomatous polyposis by low doses of atorvastatin and celecoxib given individually and in combination to APCMin mice

Preclinical and clinical studies have established evidence that cyclooxygenase-2 (COX-2) inhibitors and statins [hydroxy-3-methylglutaryl CoA reductase (HMGR) inhibitors] inhibit colon carcinogenesis. Chronic use of high doses of COX-2 inhibitors may induce side effects, and combining the low doses of agents may be an effective way to increase their efficacy and minimize the side effects. We assessed the chemopreventive efficacy of atorvastatin (Lipitor) and celecoxib individually or in combination in an animal model of familial adenomatous polyposis. Six-week-old male C57BL/6J-APCmin/+ mice were either fed diets containing 0 or 100 ppm atorvastatin or 300 ppm celecoxib, or a combination of both for approximately 80 days. Mice were sacrificed, and their intestines were scored for tumors. Normal-seeming mucosa and intestinal tumors were harvested and assayed for apoptosis (terminal deoxynucleotidyl transferase-mediated nick-end labeling) and HMGR and COX-2 protein expression and activity. We observed that 100 ppm atorvastatin significantly (P < 0.002) suppressed intestinal polyp formation. As anticipated, 300 ppm celecoxib decreased the rate of formation of intestinal polyps by approximately 70% (P < 0.0001). Importantly, the combination of 100 ppm atorvastatin and 300 ppm celecoxib in the diet suppressed the colon polyps completely and small intestinal polyps by >86% (P < 0.0001) compared with the control group. The inhibition of tumor formation by the atorvastatin and celecoxib combination was significant (P < 0.005) when compared with tumor inhibition by celecoxib alone. In addition, increased rates of apoptosis in intestinal tumors (P < 0.01-0.0001) were observed in animals fed with atorvastatin and celecoxib and more so with the combinations. Tumors of animals fed atorvastatin showed a significant decrease in HMGR-R activity. Similarly, tumors of mice exposed to celecoxib showed significantly lower levels of COX-2 activity. These observations show that atorvastatin inhibits intestinal tumorigenesis and that, importantly, when given together with low doses of celecoxib, it significantly increases the chemopreventive efficacy in an APC(min) mice.     

Beef induces and rye bran prevents the formation of intestinal polyps in Apc(Min) mice: relation to beta-catenin and PKC isozymes

Epidemiological studies suggest that high consumption of red meat and saturated fat and low consumption of fiber are associated with an increased risk of colon cancer. Therefore, we studied whether diets high in red meat or high in different grain fibers as well as inulin, polydisperse beta(2-->1) fructan, could affect the formation of intestinal polyps in Apc(Min) mice. Min mice were fed the following high-fat (40% of energy) diets for 5-6 weeks; a high-beef diet and a casein-based diet without added fiber or casein-based diet with 10% (w/w) oat, rye or wheat bran, or 2.5% (w/w) inulin. One group had a normal low-fat AIN93-G diet. The mice fed the rye-bran diet had the lowest number of polyps in the distal small intestine [15.4 +/- 8.7 (mean +/- SD)], and in the entire intestine (26.4 +/- 12.1). The rye-bran group differed significantly (P = 0. 001-0.004) from the beef group (36.6 +/- 9.4 and 52.8 +/- 13.2). In addition, the beef group differed significantly from the AIN93-G group (P = 0.009) and also from the wheat-bran group (21.0 +/- 6.1 and 35.0 +/- 8.2; P = 0.02) in the distal small intestine. The inulin group (32.9 +/- 14.3 and 49.3 +/- 16.3), on the other hand, was close to the beef group and it differed significantly from the rye-bran group in the distal small intestine. The number of animals bearing tumors in the colon + caecum was only 33% in the rye-bran group when compared with 89% in the beef and 100% in the inulin groups. The mice fed the rye-bran and beef diets had the lowest levels of cytosolic beta-catenin (0.60 +/- 0.42 and 0.67 +/- 0.26) and they differed significantly (P = 0.040 and 0.062) from the mice fed the oat-bran diet (1.46 +/- 0.43). No differences between groups in expression of protein kinase C (PKC) alpha, betaII, delta and zeta were found. The four PKC isozymes were positively correlated with cytosolic beta-catenin levels (r = 0.62-0.68; P < 0.0001).     

Chemoprevention of colon cancer by a glutathione conjugate of 1,4-phenylenebis(methylene)selenocyanate, a novel organoselenium compound with low toxicity

We have consistently shown that several synthetic Organoselenium compounds are superior cancer chemopreventive agents and less toxic than selenite or certain naturally occurring selenoamino acids. 1,4-Phenylenebis(methylene)selenocyanate (p-XSC) is the lead Organoselenium compound in that it has been shown to be the most effective and the least toxic agent in several experimental cancer models. It is not known whether p-XSC or one of its metabolites is responsible for its chemopreventive efficacy. As an initial step, we synthesized one of its putative metabolites, i.e., the glutathione conjugate of p-XSC (p-XSe-SG), and determined its stability in the pH range from 2 to 8 and in the diet under normal feeding conditions. We also assessed its maximum tolerated dose and examined its chemopreventive efficacy against azoxymethane (AOM)-induced colon carcinogenesis in male F344 rats. p-XSe-SG proved to be very stable over the pH range tested. The maximum tolerated dose of p-XSe-SG determined in a 6-week subchronic toxicity study was found to be >210 ppm (>40 ppm selenium) when the compound was added to AIN-76A high-fat diet. To assess the efficacy of this agent in the postinitiation period of colon carcinogenesis, male F344 rats 6 weeks of age were fed the high-fat diet, and at beginning of weeks 7 and 8, all of the rats intended for carcinogen treatment were given AOM at a dose of 15 mg/kg body weight by s.c. injection. Two days after the carcinogen treatment, the groups of rats consuming the high-fat control diet began their respective high-fat experimental diet regimens with 0, 56, or 84 ppm p-XSe-SG (0.1, 10, and 15 ppm of selenium) supplementation. All animals continued on their respective diets for 38 weeks after the AOM-treatment and were then killed. Colon tumors were evaluated histologically using routine procedures and were also analyzed for cyclooxygenase (COX)-1 and COX-2 expression and enzymatic activities. The results indicate that p-XSeSG administered during the post-initiation stage significantly inhibited both the incidence (P < 0.05-0.01) and the multiplicity (P < 0.05-0.005) of AOM-induced colon adenocarcinomas. This agent also greatly suppressed the multiplicity (P < 0.01-0.001) of AOM-induced exophytic adenocarcinomas in a dose-dependent manner. Feeding of 56 or 84 ppm p-XSe-SG in the diet significantly suppressed total COX activity (P < 0.02 to -0.01) and COX-2 specific activity (P < 0.005-0.0005) but had minimal effect on the protein expression levels of COX-1 and COX-2. These results suggest that the newly developed synthetic Organoselenium compound, p-XSe-SG, is stable in the diet and at wide pH ranges, inhibits colon carcinogenesis when administered during the postinitiation stage, and inhibits COX activity. Compared with previous efficacy studies and considering the toxicity associated with selenium, p-XSe-SG seems to be the least toxic Organoselenium chemopreventive agent thus far tested in the experimental colon carcinogenesis. Studies are in progress to delineate whether p-XSe-SG is also effective when administered during the progression stage of colon carcinogenesis.     

Combined effects of cyclooxygenase-1 and cyclooxygenase-2 selective inhibitors on intestinal tumorigenesis in adenomatous polyposis coli gene knockout mice

As with cyclooxygenase (COX)-2, genetic disruption of COX-1 gene or pharmacologic inhibition of its activity has been shown to decrease the number of intestinal polyps in Apc gene-deficient mice. The present study was designed to investigate the combined effects of COX-1 and COX-2 selective inhibitors on spontaneous polyp formation in APC1309 female mice. The animals were treated with 300 or 600 ppm mofezolac (a COX-1 selective inhibitor) alone, 200 or 400 ppm nimesulide (a COX-2 selective inhibitor) alone, 300 ppm mofezolac plus 200 ppm nimesulide, 600 ppm mofezolac plus 400 ppm nimesulide, or 10 ppm indomethacin (a dual-COX inhibitor) in the diet from 7 weeks of age for 4 weeks. Percentage inhibition of polyp area in the intestine was 17% with 600 ppm mofezolac alone and 25% with 400 ppm nimesulide alone, their sum of 42% being almost equal to the 37% observed for the combination treatment. Administration of 300 ppm mofezolac plus 200 ppm nimesulide also significantly decreased polyp area in the intestine by 30%. Moreover, the numbers of polyps more than 2.5 mm in diameter were markedly decreased by combined treatment of both COX inhibitors. With 10 ppm indomethacin, the dual inhibitor, polyp area was also clearly reduced by 46%. Our results indicate that COX-1 and COX-2 may to some extent contribute to polyp formation independently and inhibitor combination treatment thus has particular potential for chemoprevention of colon carcinogenesis.     

Inhibitory effects of mofezolac,a cyclooxygenase-1 selective inhibitor,on intestinal carcinogenesis

Cyclooxygenase (COX)-2, one enzyme isoform responsible for producing prostanoids from arachidonic acid, contributes to colon carcinogenesis. Recently, genetic disruption of COX-1, the other isoform, was shown to decrease the number of intestinal polyps and prostaglandin E(2) levels in intestinal mucosa, like the case with COX-2 gene disruption, in Min mice. We therefore investigated whether a COX-1 selective inhibitor, mofezolac, suppresses intestinal carcinogenesis in rodents. F344 male rats, receiving azoxymethane (AOM, 15 mg/kg body wt) s.c. injections at 5 and 6 weeks of age, were fed a diet containing 600 or 1200 p.p.m. mofezolac for 4 weeks. The number of aberrant crypt foci (ACFs) per rat and the bromodeoxyuridine labeling index of the crypt epithelium were dose-dependently decreased by administration of mofezolac, the value for the former at 1200 p.p.m. being 60% of control value. When Apc gene knockout mice (APC1309 mice) were given 600 or 1200 p.p.m. mofezolac in their diet for 8 weeks, the numbers of intestinal polyps were also dose-dependently decreased, with reduction to 59% of that in the control diet group at the higher dose. Nimesulide, a COX-2 selective inhibitor used as positive control, showed similar suppressive effects on the development of ACFs in AOM-treated rats and polyps in Apc gene knockout mice. The data indicate that both COX-1 and COX-2 can contribute to intestinal tumorigenesis.     

Suppression of intestinal polyp development by low-fat and high-fiber diet in Apc(delta716) knockout mice

Most epidemiological and animal studies show a positive correlation of the dietary intake of fat with the incidence of colon cancer, whereas an inverse correlation of the dietary intake of fiber. In rats fed a diet low in fat and high in wheat bran fiber and calcium, a significant decrease was reported in the number of azoxymethane-induced aberrant crypt foci compared with those fed a high-fat, low-fiber and low- calcium diet. Mutations in the human APC gene play a key role, not only in familial adenomatous polyposis, but also in many sporadic cancers of the entire digestive tract. We previously constructed a mouse strain Apc(delta716), carrying a truncation mutation at codon 716 of the Apc gene, the homolog of human APC (10). The heterozygous mice developed numerous intestinal polyps, and all microadenomas dissected from the earliest polyps had already lost the wild-type allele, indicating the loss of heterozygosity. Using these Apc(delta716) knockout mice, we have investigated the effect of a low-fat and high-fiber diet (LRD for 'low-risk' diet) on intestinal polyposis, and compared it with that of a high-fat and low-fiber diet (HRD for 'high-risk' diet). The mice were fed either diet for 7 weeks, and the number and size of intestinal polyps were scored. The LRD-fed mice had fewer polyps than the HRD-fed mice, by 36% in the small intestine and by 64% in the colon. As for the polyp size distribution, there was no significant difference between the HRD- and LRD-fed mice. These results indicate that LRD can suppress intestinal polyposis compared with HRD which does not, and suggest that its suppression is at the initiation of polyp formation. This is likely to be due to a decreased frequency of loss of heterozygosity, rather than a retarded growth of the polyp adenomas.      

Suppression of intestinal polyps in Msh2-deficient and non-Msh2-deficient multiple intestinal neoplasia mice by a specific cyclooxygenase-2 inhibitor and by a dual cyclooxygenase-1/2 inhibitor

Epidemiological studies suggest that nonsteroidal anti-inflammatory agents decrease the risk of colorectal cancer. This is believed to be mediated, at least in part, by inhibition of cyclooxygenase (COX) activity. There are two COX isoenzymes, namely the constitutively expressed COX-1 and the inducible COX-2. COX-2 is overexpressed in adenomas and colorectal cancers, and COX-2-specific inhibitors have been shown to inhibit intestinal polyps in Apc(Delta716) mice more effectively than dual COX-1/COX-2 inhibitors such as sulindac. Various Apc knockout mice, including the multiple intestinal neoplasia (Min) mouse and the Apc(Delta716) mouse, are limited by their lack of large numbers of colonic adenomas and aberrant crypt foci, the putative precursors of large-bowel polyps and cancers. Our DNA mismatch-repair-deficient Min mouse model (Apc+/-Msh2-/-) has genetic features of both familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer, and most importantly, rapidly develops numerous small- and large-bowel adenomas, as well as colonic aberrant crypt foci. The purpose of this study was to determine the effects of COX inhibitors on intestinal adenomas and colonic aberrant crypt foci in this accelerated polyposis, mismatch-repair-deficient Min mouse model, in addition to a standard Min mouse model. Weanling Apc+/-Msh2-/- and Min mice were fed diets containing no drug, sulindac, or a specific COX-2 inhibitor (MF-tricyclic). Apc+/-Msh2-/- and Min mice were sacrificed after 4 weeks and 5 months on diet, respectively. Apc+/-Msh2-/- mice treated with MF-tricyclic had significantly fewer small-bowel polyps (mean +/- SD, 178 +/- 29) compared with mice on sulindac (278 +/- 80), or control diet (341 +/- 43; P < 0.001). There was no difference in numbers of large-bowel polyps or aberrant crypt foci in mice in the three groups. MF-tricyclic was also effective in reducing both small- and large-bowel polyps in Min mice. Western analysis demonstrated COX-2 expression in both large- and small-bowel polyps from mice of both genotypes. This study demonstrates that a specific COX-2 inhibitor is effective in preventing small-bowel polyps in mismatch-repair-deficient Min mice and both small- and large-bowel polyps in standard Min mice. Therefore, specific COX-2 inhibitors may be useful as chemopreventive and therapeutic agents in humans at risk for colorectal neoplasia.     

The role of NAG-1/GDF15 in the inhibition of intestinal polyps in APC/Min mice by sulindac

The antitumor effects of nonsteroidal anti-inflammatory drugs (NSAID) are assumed to be due to the inhibition of COX activity, but COX-independent mechanisms may also play an important role. NSAID-activated gene (NAG-1/GDF15) is induced by NSAIDs and has antitumorigenic activities. To determine the contribution of COX-2 inhibition and NAG-1/GDF15 expression to the prevention of colon carcinogenesis by NSAIDs, we evaluated several sulindac derivatives [des-methyl (DM)-sulindac sulfide and its prodrug DM-sulindac] that do not inhibit COX-2 activity. Sulindac sulfide and DM-sulindac induced the expression of NAG-1/GDF15 in HCT116 cells as determined by quantitative real-time PCR and Western blot. We fed APC/Min mice with 320 ppm of sulindac and doses of DM-sulindac. Only sulindac significantly inhibited tumor formation inAPC/Min mice. To determine the pharmacokinetic properties of sulindac and DM-sulindac in vivo, wild-type C57/B6 mice were fed with sulindac and DM-sulindac at 80, 160, and 320 ppm. High-performance liquid chromatography analysis revealed that the conversion of DM-sulindac to DM-sulindac sulfide (active form) was less efficient than the conversion of sulindac to sulindac sulfide (active form) in the mice. Lower levels of DM-sulindac sulfide accumulated in intestinal and colon tissues in comparison with sulindac sulfide. In addition, NAG-1/GDF15 was induced in the liver of sulindac-fed mice but not in the DM-sulindac-fed mice. Collectively, our results suggest that the tumor-inhibitory effects of sulindac in APC/Min mice may be due to, in part, NAG-1/GDF15 induction in the liver. Our study also suggests that pharmacologic properties should be carefully evaluated when developing drug candidates.     

Microadenomatous lesions involving loss of Apc heterozygosity in the colon of adult Apc(Min/+) mice

Mutations in the human adenomatous polyposis coli (APC) gene are causative for familial adenomatous polyposis (FAP), a rare condition in which numerous colonic polyps arise during puberty and, if left untreated, lead to colon cancer. Mouse model for human FAP, Apc(Min/+) mouse, contains a truncating mutation in the Apc gene and spontaneously develops intestinal adenomas. However, the distribution of tumors along the intestine found in Apc(Min/+) mice is different from that in human FAP. A majority of intestinal polyps in the Apc(Min/+) mice is known to be located in the small intestine but rarely detected in the colon. We report here that adult Apc(Min/+) mice develop dozens of microadenomatous lesions in the colon (>20 lesions/colon). Surprisingly, the vast majority of such adenomatous lesions consisting of colonic crypts were <300 microm in their greatest dimension, whereas lesions in the small intestine showed various sizes. The allelic loss analysis revealed that these adenomatous crypts in the colon have already lost the remaining allele of Apc. Such findings suggest that, in contrast to tumorigenesis in the small intestine, loss of heterozygosity of the Apc gene is not sufficient for tumor development in the colon of Apc(Min/+) mice. Our results may give an account for the low incidence of colonic tumors in Apc(Min/+) mice.     

Chemoprevention of familial adenomatous polyposis by bromo-noscapine (EM011) in the Apc(Min/+) mouse model

Germline mutation of the tumor suppressor gene, adenomatous polyposis coli (APC), is responsible for familial adenomatous polyposis (FAP) with nearly 100% risk for colon cancer at an early age. Although FAP is involved in only 1% of all colon cancer cases, over 80% of sporadic cancers harbor somatic mutations of APC. We show here that bromo-noscapine (EM011), a rationally designed synthetic derivative of a natural nontoxic tubulin-binding alkaloid-noscapine, that reduces the dynamics of microtubules, causes a reversible G(2) /M arrest in wild type murine embryonic fibroblasts (MEFs), but an aberrant exit from a brief mitotic block, followed by apoptosis in MEFs after APC deletion with small interfering RNA. Furthermore, both β-catenin levels and activity fell to half the original levels with a concomitant reduction of cell proliferation-inducing cyclin D1, c-Myc, and induction of cytostatic protein p21 before caspase-3 activation. Additionally, we show a statistically significant reduction in the number of newly emerging intestinal polyps (to 35% compared with untreated mice) as well as the mean size of polyps (to 42% compared with untreated mice) in EM011-treated Apc(Min/+) mice as compared to their sham-treated control littermates. The remaining polyps in the EM011 treated group of Apc(Min/+) mice showed evidence of elevated apoptosis as revealed by immunohistochemistry. We failed to detect any evidence of histopathological and hematological toxicities following EM011 treatment. Taken together, our data are persuasive that a clinical trial of EM011 is possible for the prevention/amelioration of polyposis in FAP patients.     

Expression of beta-catenin and full-length APC protein in normal and neoplastic colonic tissues

Mutations of the APC gene are thought to be early events in the process of colorectal carcinogenesis. Although the complete function(s) of the APC gene product is not known, it has been shown that the APC protein interacts with beta-catenin in a multi-protein complex to regulate the level of expression of beta-catenin. Loss of normal APC protein function can lead to an accumulation of beta-catenin in the cytosol and the nucleus. Immunohistochemical methods were used to determine the relationship between APC and beta-catenin protein expression in human colonic tissues (150 normal, 9 hyperplastic, 58 adenomas and 83 carcinomas) and 12 paired samples of normal and cancer tissue in mouse colon. In all samples of normal human and mouse colonic mucosa and in human hyperplastic polyps both APC and beta-catenin immunoreactivity were present in colonocytes. APC expression was cytoplasmic, with maximal immunoreactivity in the goblet cells. beta-Catenin expression was predominantly localized to the plasma membrane, with no nuclear immunoreactivity. APC immunoreactivity was absent in all of the mouse adenocarcinomas and 83% of the human colon cancers. All of the human and mouse carcinomas had nuclear and cytoplasmic beta-catenin expression. In contrast, only 29% of the 58 colonic adenomas were completely negative for APC immunoreactivity. Regardless of the presence or absence of APC, all of the adenomas had cytoplasmic and nuclear beta-catenin immunoreactivity. Many colonic adenomas retain expression of full-length APC protein whereas it is usually lost in colorectal cancers. Regardless of the status of APC protein expression, beta-catenin protein was found in the cytoplasm and nucleus of all neoplastic colonic mucosa. The dissociation between loss of expression of APC and accumulation of beta-catenin in the nucleus suggests that inactivation of both alleles of the APC gene may not be required for beta-catenin nuclear accumulation in colonic adenomas.     

Frequent alterations of the beta-catenin and TCF-4 genes, but not of the APC gene, in colon cancers with high-frequency microsatellite instability

High-frequency microsatellite instability (MSI-H) due to defective DNA mismatch repair (MMR) is a characteristic of the majority of tumors from kindreds with hereditary nonpolyposis colorectal cancer (HNPCC) and a subset of sporadic cancers. To better understand the molecular characteristics of colon cancers with MSI-H, we analyzed these cancers for alterations of genes, such as APC, beta-catenin, and TCF-4 genes, involved in the Wnt signaling pathway. Following the National Cancer Institute (NCI) criteria, 385 unselected colon cancers were classified as follows: 50 (13%) MSI-H tumors, 36 (9%) low-frequency MSI (MSI-L) tumors, and 299 (78%) microsatellite stable (MSS) tumors. The frequency of APC mutations was significantly lower in MSI-H tumors (9 out of 50) than in MSI-L (12 out of 20) and MSS (66 out of 100) tumors (P = 0.0005 and P < 0.0001, respectively). In contrast, the frequency of exon 3 mutations in the beta-catenin gene was higher in MSI-H tumors (10 out of 50) than in MSI-L tumors (0 out of 30; P = 0.0110) and MSS tumors (3 out of 100; P = 0.0010). Frameshift mutations in a (A)9 tract of the TCF-4 gene were detected in 44% (22 out of 50) of MSI-H tumors, but not in any of the 20 MSI-L tumors or 40 MSS tumors. In total, 78% of MSI-H tumors and 84% of the remaining tumors had at least one alteration in APC, beta-catenin, or the TCF-4 genes. Although further analysis is needed to functionally characterize the consequences of each of these alterations on beta-catenin/TCF target gene expression, our results suggest that the activation of the Wnt signaling pathway plays a pivotal role in colon tumorigenesis, irrespective of MSI status.     

Suppression of tumorigenesis in the Apc(min) mouse: down-regulation of beta-catenin signaling by a combination of tea plus sulindac

Epidemiological and animal studies suggest that tea may be protective towards cancers of the GI tract. White tea, the least processed form of tea, contains high levels of polyphenols and, like green tea, is chemopreventive towards heterocyclic amine-initiated colonic aberrant crypt formation in male F344 rats. We examined for the first time the relative effectiveness of white and green tea in suppressing intestinal tumorigenesis in C57BL/6J-Apc(Min/+) (Apc(min)) mice. Each tea was also compared with sulindac, a non-steroidal anti-inflammatory drug known to be highly effective in Apc(min) mice. Male C57BL/6J (+/+) (wild-type) and Apc(min) mice were treated in the drinking water with white tea or green tea (1.5% w/v, 2 min brew-time), 80 p.p.m. sulindac, a combination of 80 p.p.m. sulindac in 1.5% white tea, or pH buffered water. After 12 weeks of treatment, Apc(min) mice given white tea, green tea, or sulindac had significantly fewer tumors than controls (P < 0.05). The protection provided by 1.5% green or white tea was comparable to that provided by 80 p.p.m. sulindac. Mice treated with a combination of white tea plus sulindac had significantly fewer tumors than either treatment alone (P < 0.05). beta-catenin and beta-catenin/Tcf-4 regulated proteins Cyclin D(1) and c-Jun were readily detected in polyps, but markedly reduced in normal-looking intestines of mice treated with both tea and sulindac. This research provides evidence that teas, particularly when administered in combination with sulindac, are highly effective at inhibiting intestinal neoplasia in male Apc(min) mice via direct or indirect effects on the beta-catenin/APC pathway.     

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.     

Chemoprevention of colon carcinogenesis by the synthetic organoselenium compound 1,4-phenylenebis(methylene)selenocyanate.

The chemopreventive effect of 40% and 80% maximum tolerated dose (MTD) levels of 1,4-phenylenebis(methylene)selenocyanate (p-XSC) administered in the diet during the initiation phase (2 weeks before, during, and up to 3 days after carcinogen administration) and the post-initiation phase (3 days after carcinogen treatment until termination) of azoxymethane (AOM)-induced colon carcinogenesis was studied in male F344 rats. The MTD of p-XSC was determined in male F344 rats and found to be 50 ppm. Beginning at 5 weeks of age, all animals were divided into various experimental groups (42 rats/group) and fed the high-fat semipurified diet or diets containing 20 (40% MTD) and 40 (80% MTD) ppm p-XSC. At 7 weeks of age, all animals (30 rats/group) except the vehicle-treated groups (12 rats/group) were administered s.c. injections of AOM (15 mg/kg body weight/week for 2 weeks). Three days after the second injection of AOM or vehicle (normal saline), groups of animals fed the p-XSC diets and control diet were transferred, respectively, to control diet and p-XSC diets and continued on these diets until the termination of the study. All animals were necropsied during the 36th week after AOM treatment. Colonic mucosal prostaglandin E2 and selenium-dependent glutathione peroxidase were measured in animals fed the control and p-XSC diets at the termination of the study. The results indicate that 40 ppm p-XSC administered during the initiation phase significantly inhibited the colon tumor incidence (percentage of animals with tumors). Dietary p-XSC administered at 20 and 40 ppm levels during the initiation phase significantly inhibited colon tumor multiplicity (tumors/animal and tumors/tumor-bearing animal). Colon tumor incidence and multiplicity were significantly reduced in groups fed 20 and 40 ppm p-XSC diets at the postinitiation phase of carcinogenesis. Colonic mucosal selenium-dependent glutathione peroxidase activity was increased, and prostaglandin E2 was reduced in animals fed the p-XSC diet compared to animals fed the control diet. Whereas the precise mechanisms of p-XSC-induced inhibition of colon carcinogenesis remain to be elucidated, it is likely that the effect during the initiation and postinitiation phases may be due to alteration in carcinogen metabolism and to modulation of prostaglandin synthesis and selenium-dependent glutathione peroxidase activity.