Apc deficiency is associated with increased Egfr activity in the intestinal enterocytes and adenomas of C57BL/6J-Min/+ mice

Overexpression of the epidermal growth factor receptor (EGFR) and its increased tyrosine kinase activity are implicated in colorectal cancer (CRC) development and malignant progression. The C57BL/6J-Min/+ (Min/+) mouse is a model for CRC and develops numerous intestinal adenomas. We analyzed the normal mucosa of Min/+ and Apc+/+ (WT) littermate mice together with Apc-null adenomas to gain insight into the roles of Egfr in these intestinal tissues. Protein analyses showed that Egfr activity was highest in the tumors, and also up-regulated in Min/+ relative to WT enterocytes. Expression of ubiquitylated Egfr (Egfr-Ub) was increased in Min/+ enterocytes and tumors. Tumors exhibited increased association of Egfr with clathrin heavy chain (CHC), Gab1, and p85alpha, the regulatory subunit of phosphoinositide 3-kinase (PI3K), and tumors also overexpressed c-Src, PDK1, and Akt. Immunohistochemistry for Akt-p-Ser473 revealed a low level of this active kinase in Min/+ and WT enterocytes and its strong presence in tumors. Prostaglandin E2 (PGE2) is a product of cyclooxygenase-2 (Cox-2) activity that is up-regulated in Min/+ tumors and transactivates Egfr. PGE2 expression was significantly higher in untreated Min/+ tumors and reduced by treatment with the Cox-2 inhibitor, celecoxib. Dietary administration of this NSAID also inhibited Egfr activity in tumors. Increased activation of the EGFR-PI3K-Akt signaling pathway in tumors relative to Apc+/+ and ApcMin/+ enterocytes provides potential opportunities for therapeutic interventions to differentially suppress tumor formation, promotion, progression, and/or recurrence.     

Inhibition of intestinal polyposis with reduced angiogenesis in ApcMin/+ mice due to decreases in c-Myc expression

The c-myc oncogene plays an important role in tumorigenesis and is frequently deregulated in many human cancers, including gastrointestinal cancers. In humans, mutations of the adenomatous polyposis coli (Apc) tumor suppressor gene occur in most colorectal cancers. Mutation of Apc leads to stabilization of beta-catenin and increases in beta-catenin target gene expression (c-myc and cyclin D1), whose precise functional significance has not been examined using genetic approaches. Apc(Min/+) mice are a model of familial adenomatous polyposis and are heterozygous for an Apc truncation mutation. We have developed a model for examining the role of c-Myc in Apc-mediated tumorigenesis. We crossed c-myc(+/-) mice to Apc(Min/+) to generate Apc(Min/+) c-myc(+/-) animals. The compound Apc(Min/+) c-myc(+/-) mice were used to evaluate the effect of c-myc haploinsufficiency on the Apc(Min/+) phenotype. We observed a significant reduction in tumor numbers in the small intestine of Apc(Min/+) c-myc(+/-) mice compared with control Apc(Min/+) c-myc(+/+) mice. In addition, we observed one to three polyps per colon in Apc(Min/+) c-myc(+/+) mice, whereas only two lesions were observed in the colons of Apc(Min/+) mice that were haploinsufficient for c-myc. Moreover, reduction in c-myc levels resulted in a significant increase in the survival of these animals. Finally, we observed marked decreases in vascular endothelial growth factor, EphA2, and ephrin-B2 expression as well as marked decreases in angiogenesis in intestinal polyps in Apc(Min/+) c-myc(+/-) mice. This study shows that c-Myc is critical for Apc-dependent intestinal tumorigenesis in mice and provides a potential therapeutic target in the treatment of colorectal cancer.     

Promotion of adenoma growth by dietary inulin is associated with increase in cyclin D1 and decrease in adhesion proteins in Min/+ mice mucosa

We have earlier shown that dietary fructo-oligosaccharide inulin enhances adenoma growth in multiple intestinal neoplasia (Min/+) mice. To further explore inulin-induced early biochemical changes in the normal-appearing mucosa, Min/+ mice were fed from the age of 5 weeks to the ages of 8 and 15 weeks a control diet or an inulin-enriched diet (10% w/w). In addition, the wild-type littermates were fed with the same diets until the age of 8 weeks, in order to determine whether similar changes happen both in the wild-type and Min/+ mice. The mucosa without adenomas was collected and fractionated to nuclear, cytosolic and membrane pools. The protein levels of beta-catenin, cyclin D1 and E-cadherin were determined by Western blotting at both time points, and immunohistochemical stainings were done for 8-week-old mice. The promotion of adenoma growth by inulin (week 15, 1.3-fold increase, P=.0004) was associated with accumulation of cytosolic and nuclear beta-catenin, and increased amount of cytosolic cyclin D1 (1.5-fold increase, P=.003) in the normal-appearing mucosa of the Min/+ mice. Furthermore, inulin feeding reduced the membranous pools of beta-catenin and E-cadherin. Also in the wild-type mice the drop in membranous beta-catenin was clear (P=.015), and, moreover, a subset of crypts had enhanced nuclear beta-catenin staining. These data indicate that dietary inulin can already activate in the normal-appearing mucosa beta-catenin signaling, which in the presence of Apc mutation induces adenoma growth and even in the wild-type mice direction of the changes is similar.     

Deficient E-cadherin adhesion in C57BL/6J-Min/+ mice is associated with increased tyrosine kinase activity and RhoA-dependent actomyosin contractility

The Min/+ mouse is a model for APC-dependent colorectal cancer (CRC). We showed that tumorigenesis in this animal was associated with decreased E-cadherin adhesion and increased epidermal growth factor receptor (Egfr) activity in the non-tumor intestinal mucosa. Here, we tested whether these abnormalities correlated with changes in the actin cytoskeleton due to increased Rho-ROCK signaling. We treated Apc+/+ (WT) littermate small intestine with EGTA, an inhibitor of E-cadherin, and with LPA, an RhoA activator; both induced effects on adhesion and kinase activity that mimicked the Min/+ phenotype. GTP-bound Rho was increased in Min/+ enterocytes relative to WT. Since RhoA activity is associated with actomyosin contractility, markers of this signaling cascade were assessed including phosphorylated myosin light chain (MLC), cofilin, Pyk2, Src, and MAPK kinases. The increased actomyosin contractility characterizing Min/+ intestinal tissue was suppressed by the ROCK inhibitor, Y27632, but was inducible in the WT by EGTA or LPA. Finally, ultrastructural imaging revealed changes consistent with actomyosin contractility in Min/+ enterocytes. Thus, the positive regulation of E-cadherin adhesion provided by Apc+ in vivo allows proper negative regulation of Egfr, Src, Pyk2, and MAPK, as well as RhoA activities.     

Mammary tumorigenesis in ApcMin/+ mice is enhanced by X irradiation with a characteristic age dependence

The Apc(Min/+) (Min) mouse is genetically predisposed to both intestinal and mammary tumorigenesis. We investigated age-related changes in the susceptibility of mice (before, during and after puberty) to radiation-induced mammary tumorigenesis using this model. Female Min and wild-type mice having the C57BL/6J background were irradiated with 2 Gy of X rays at 2, 5, 7 and 10 weeks and killed humanely at 18 weeks of age. Min mice irradiated at 7-10 weeks of age (after puberty) developed mammary tumors with squamous metaplasia, whereas their wild-type littermates did not. Interestingly, irradiation of Min mice at 2-5 weeks (before and during puberty, respectively) did not induce mammary tumors but rather cystic nodules with metaplasia. The mammary tumors exhibited increased nuclear beta-catenin protein and loss of the wild-type Apc allele. Our results show that susceptibility to radiation-induced mammary tumorigenesis increases after puberty in Min mice, suggesting that the tumorigenic effect of ionizing radiation targets the lobular-alveolar progenitor cells, which increase in number with age and are controlled by beta-catenin signaling.     

Bortezomib and sphingosine kinase inhibitor interact synergistically to induces apoptosis in BCR/ABl+ cells sensitive and resistant to STI571 through down-regulation Mcl-1

IL-17 plays an important role in gut homeostasis. However, the role of IL-17F in intestinal tumorigenesis has not been addressed. Here we demonstrate that ablation of IL-17F significantly inhibits spontaneous intestinal tumorigenesis in the small intestine of Apc(Min/+) mice. IL-17F ablation decreased IL-1β and Cox-2 expression as well as IL-17 receptor C (IL-17RC) expression, which were increased in tumors from Apc(Min/+) mice. Lack of IL-17F did not reverse the splenomegaly but partially restored thymic atrophy, suggesting a local effect of IL-17F in the intestine. IL-17F deficient Apc(Min/+) mice showed a significant decrease in immune cell infiltration in the lamina propria. Interestingly, the expression of IL-17A from CD4 T cells in the lamina propria remains unchanged in the absence of IL-17F. Collectively, our results suggest the proinflammatory and essential role of IL-17F to develop spontaneous intestinal tumorigenesis in Apc(Min/+) mice in the presence of IL-17A.     

Effect of exercise on biological pathways in ApcMin/+ mouse intestinal polyps

Many epidemiological studies have demonstrated that level of exercise is associated with reduced colorectal cancer risk. Treadmill training can decrease Apc(Min/+) mouse intestinal polyp number and size, but the mechanisms remain unclear. Understanding the molecular changes in the tumor following exercise training may provide insight on the mechanism by which exercise decreases Apc(Min/+) mouse polyp formation and growth. The purpose of this study was to determine if exercise can modulate Apc(Min/+) mouse intestinal polyp cellular signaling related to tumor formation and growth. Male Apc(Min/+) mice were randomly assigned to control (n = 20) or exercise (n = 20) treatment groups. Exercised mice ran on a treadmill at a moderate intensity (18 m/min, 60 min, 6 days/wk, 5% grade) for 9 wk. Polyps from Apc(Min/+) mice were used to quantify markers of polyp inflammation, apoptosis, and beta-catenin signaling. Exercise decreased the number of macrophages in polyps by 35%. Related to apoptosis, exercise decreased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells by 73% in all polyps. Bax protein expression in polyps was decreased 43% by exercise. beta-Catenin phosphorylation was elevated 3.3-fold in polyps from exercised mice. Moderate-intensity exercise training alters cellular pathways in Apc(Min/+) mouse polyps, and these changes may be related to the exercise-induced reduction in polyp formation and growth.     

Intracellular role for sphingosine kinase 1 in intestinal adenoma cell proliferation

Sphingosine kinase (Sphk) enzymes are important in intracellular sphingolipid metabolism as well as in the biosynthesis of sphingosine 1-phosphate (S1P), an extracellular lipid mediator. Here, we show that Sphk1 is expressed and is required for small intestinal tumor cell proliferation in Apc Min/+ mice. Adenoma size but not incidence was dramatically reduced in Apc Min/+ Sphk(-/-) mice. Concomitantly, epithelial cell proliferation in the polyps was significantly attenuated, suggesting that Sphk1 regulates adenoma progression. Although the S1P receptors (S1P1R, S1P2R, and S1P3R) are expressed, polyp incidence or size was unaltered in Apc Min/+ S1p2r(-/-), Apc Min/+ S1p3r(-/-), and Apc Min/+ S1p1r(+/-) bigenic mice. These data suggest that extracellular S1P signaling via its receptors is not involved in adenoma cell proliferation. Interestingly, tissue sphingosine content was elevated in the adenomas of Apc Min/+ Sphk1(-/-) mice, whereas S1P levels were not significantly altered. Concomitantly, epithelial cell proliferation and the expression of the G1/S cell cycle regulator CDK4 and c-myc were diminished in the polyps of Apc Min/+ Sphk1(-/-) mice. In rat intestinal epithelial (RIE) cells in vitro, Sphk1 overexpression enhanced cell cycle traverse at the G1/S boundary. In addition, RIE cells treated with sphingosine but not C6-ceramide exhibited reduced cell proliferation, reduced retinoblastoma protein phosphorylation, and cyclin-dependent kinase 4 (Cdk4) expression. Our findings suggest that Sphk1 plays a critical role in intestinal tumor cell proliferation and that inhibitors of Sphk1 may be useful in the control of intestinal cancer.     

Adenomatous polyposis coli truncation mutations in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced intestinal tumours of multiple intestinal neoplasia mice

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces intestinal tumours in C57BL/6J-multiple intestinal neoplasia (Min)/+ mice. The main mechanism for PhIP-induced tumour induction in Min/+ mice is loss of the wild-type adenomatous polyposis coli (Apc) allele, i.e. loss of heterozygosity (LOH). In this study, single injections of either 10, 17.5 or 25 mg/kg PhIP on days 3-6 after birth all increased the mean number of small intestinal tumours two to three-fold, from 37.7 in controls to 124.8 in the PhIP-treated Min/+ mice. In total, we analysed 292 small intestinal tumours and 253 of these had LOH. The frequency of LOH in the Apc gene was 88, 93, 83 and 84% in tumours of 0, 10, 17.5 and 25 mg/kg PhIP-treated mice, respectively. Therefore, these lower doses of PhIP did not reduce the frequency of LOH, as found in our previous study with a single injection of 50 mg/kg PhIP (Mutat. Res. 1-2 (2002) 157). In the second part of this study, we wanted to characterise Apc truncation mutations from tumour samples apparently retaining the Apc wild-type allele from this and two previous experiments with PhIP-exposed Min/+ mice. In the first half of exon 15 in Apc, we verified 25 mutations from 804 tumour samples of PhIP-treated mice. Of these were 60% G-->T transversions, and 16% G deletions, indicating that these are the predominant types of PhIP-induced truncation mutations in the Apc gene in Min/+ mice. Most of the mutations were located between codon 989 and 1156 corresponding to the first part of the beta-catenin binding region. We also identified two Apc truncation mutations from 606 spontaneously formed intestinal tumours from untreated Min/+ mice, one C-->T transition and one T insertion, which were different from those induced by PhIP.     

Interleukin-6 and cachexia in ApcMin/+ mice

The Apc(Min/+) mouse has a mutation in the Apc tumor suppressor gene and develops intestinal polyps, beginning at 4 wk of age. This mouse develops cachexia by 6 mo, characterized by significant loss of muscle and fat tissue. The purpose of the present study was to determine the role of circulating interleukin-6 (IL-6) and the polyp burden for the development of cachexia in Apc(Min/+) mice. At 26 wk of age, mice exhibiting severe cachectic symptoms had a 61% decrease in gastrocnemius muscle weight, complete loss of epididymal fat, a 10-fold increase in circulating IL-6 levels, and an 89% increase in intestinal polyps compared with mildly cachectic animals. Apc(Min/+)/IL-6(-/-) mice did not lose gastrocnemius muscle mass or epididymal fat pad mass while overall polyp number decreased by 32% compared with Apc(Min/+) mice. Plasmid-based IL-6 overexpression in Apc(Min/+)/IL-6(-/-) mice led to a decrease in gastrocnemius muscle mass and epididymal fat pad mass and increased intestinal polyp burden. IL-6 overexpression did not induce cachexia in non-tumor-bearing mice. These data demonstrate that IL-6 is necessary for the onset of adipose and skeletal muscle wasting in the Apc(Min/+) mouse and that circulating IL-6 can regulate Apc(Min/+) mouse tumor burden.     

The Min (multiple intestinal neoplasia) mutation: its effect on gut epithelial cell differentiation and interaction with a modifier system

Min is a fully penetrant dominant mutation that leads to the development of multiple intestinal adenomas throughout the duodenal-to-colonic axis. Min/+ C57BL6/J mice have an average life-span of 120 d. Multi-label immunocytochemical studies of these lesions demonstrate patches of differentiated enterocytes, and scattered enteroendocrine, goblet and Paneth cells. Expression of endogenous marker genes within these differentiated cells can be directly correlated with the position occupied by the adenoma along the duodenal-to-colonic axis and mirrors the regional differentiation of the normal gut epithelium. The presence of multiple lineages in adenomas together with their retention of spatial information suggests that tumorigenesis in Min/+ mice may be initiated in a multipotent stem cell normally located at the base of intestinal crypts. To study the time-dependent properties of these tumors, genetic conditions were sought in which Min/+ animals could survive for up to 300 d. Min is fully penetrant in hybrids with either AKR/J or MA/MyJ. However, the hybrids demonstrate a reduction in the number of intestinal adenomas. Preliminary backcross analysis is consistent with a single major modifier locus unlinked to Min in both the AKR/J and MA/MyJ strains. The increased lifespan of the hybrid animals is also associated with the development of invasive tumors. New tumors do not arise continuously over the lifespan of these animals; instead all adenomas appear to be established by 100 d of age or sooner. These studies indicate that the Min/+ mouse is a powerful model system for analyzing the mechanisms that establish and maintain a balance between proliferation and differentiation in the continuously renewing gut epithelium and for an assessment of the multi-step hypothesis of intestinal neoplasia.     

Discordant effect of aspirin and indomethacin on intestinal tumor burden in Apc(Min/+)mice

Epidemiologic and animal studies indicate that sustained use of non-steroidal anti-inflammatory drugs (NSAIDs) have a chemopreventive effect against the incidence of colorectal neoplasia and subsequent mortality. We previously demonstrated that sulindac significantly reduces intestinal tumor load in Apc(Min/+)mice and the tumor regression was not necessarily correlated with prostaglandin biosynthesis. In the present study, we further investigate the relationship of NSAID treatment and tumorigenesis in the Apc(Min/+)mouse model. We demonstrate that indomethacin (9 ppm) is a very potent chemopreventive agent, reducing tumor load by 85% and significantly inhibiting basal and ex vivo prostaglandin formation (P< 0.006 and P< 0.0001, respectively). Aspirin (400 ppm) has a similar impact on reducing prostaglandin levels, but in contrast to indomethacin, is uneffective in reducing the tumor load. The data indicate a discordance between the impact of different NSAIDs on tumorigenesis in Apc(Min/+)mice.     

Analysis of reciprocal congenic lines reveals the C3H/HeJ genome to be highly resistant to ApcMin intestinal tumorigenesis

Genetic background affects polyp development in the Multiple intestinal neoplasia (Apc(Min)) mouse model. The Modifier of Min 1 (Mom1) locus accounts for approximately 50% of the variation in polyp multiplicity. We generated reciprocal congenic lines, such that the recipient C57BL/6J (B6) strain carries a donor C3H/HeJ (C3H) Mom1 allele, and the recipient C3H strain carries a donor B6 Mom1 allele. Hybrid progeny from congenic females mated to B6 Apc(Min/+) males were analyzed. A single C3H Mom1 locus on the B6 background reduced small intestinal polyp numbers by 50% and colon polyp incidence by 66% compared to their susceptible B6 Mom1(S/S)Apc(Min/+) siblings. These findings show that the C3H genome contains a resistant Mom1(R) locus. The reciprocal congenic line, which carries the susceptible B6 Mom1(S) locus on the C3H background, reduced small intestinal polyp numbers by 80% and colon polyp incidence by 95% compared to B6 Mom1(S/S)Apc(Min/+) mice. These data demonstrate that unidentified modifiers in the C3H strain can suppress intestinal polyp multiplicity in Apc(Min/+) mice, and act in the absence of a resistant Mom1(R) locus.     

Toll‐like receptor 4 regulates spontaneous intestinal tumorigenesis by up‐regulating IL‐6 and GM‐CSF

Inflammation is as an important component of intestinal tumorigenesis. The activation of Toll‐like receptor 4 (TLR4) signalling promotes inflammation in colitis of mice, but the role of TLR4 in intestinal tumorigenesis is not yet clear. About 80%–90% of colorectal tumours contain inactivating mutations in the adenomatous polyposis coli (Apc) tumour suppressor, and intestinal adenoma carcinogenesis in familial adenomatous polyposis (FAP) is also closely related to the germline mutations in Apc. The Apc^Min/+ (multiple intestinal neoplasia) model mouse is a well‐utilized model of FAP, an inherited form of intestinal cancer. In this study, Apc^Min/+ intestinal adenoma mice were generated on TLR4‐sufficient and TLR4‐deficient backgrounds to investigate the carcinogenic effect of TLR4 in mouse gut by comparing mice survival, peripheral blood cells, bone marrow haematopoietic precursor cells and numbers of polyps in the guts of Apc^Min/+ WT and Apc^Min/+ TLR4^?/? mice. The results revealed that TLR4 had a critical role in promoting spontaneous intestinal tumorigenesis. Significant differential genes were screened out by the high‐throughput RNA‐Seq method. After combining these results with KEGG enrichment data, it was determined that TLR4 might promote intestinal tumorigenesis by activating cytokine‐cytokine receptor interaction and pathways in cancer signalling pathways. After a series of validation experiments for the concerned genes, it was found that IL6, GM‐CSF (CSF2), IL11, CCL3, S100A8 and S100A9 were significantly decreased in gut tumours of Apc^Min/+ TLR4^?/? mice compared with Apc^Min/+ WT mice. In the functional study of core down‐regulation factors, it was found that IL6, GM‐CSF, IL11, CCL3 and S100A8/9 increased the viability of colon cancer cell lines and decreased the apoptosis rate of colon cancer cells with irradiation and chemical treatment.     

One dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) induces tumours in Min/+ mice by truncation mutations or LOH in the Apc gene

The C57BL/6J-Min/+ (multiple intestinal neoplasia) mouse has a heterozygous nonsense Apc(Min) (adenomatous polyposis coli) mutation, and numerous adenomas spontaneously develop in the intestine. Neonatal exposure of Min/+ mice to the food carcinogens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) (one injection of 50mg/kg) increased the number of small intestinal tumours about three- and two-fold, respectively. The number of colonic tumours was only increased in males. We examined whether the wild-type Apc allele was affected in intestinal tumours induced by either PhIP or IQ. In spontaneously formed and in IQ-induced small intestinal and colonic tumours from these mice, the main mechanism for tumour induction was loss of wild-type Apc allele, i.e. loss of heterozygosity (LOH). In contrast to the IQ-induced (84% LOH) and spontaneously (88% LOH) formed tumours, only 55% of the PhIP-induced small intestinal tumours from males showed LOH. Tumours that apparently had retained the wild-type Apc allele were further analysed for the presence of truncated Apc proteins by the in vitro synthesised protein (IVSP) assay. Truncated Apc proteins, indicating truncation mutations in exon 15 of the Apc gene, were detected in two of the 12 PhIP-induced tumours in segment 2 (codons 686-1217), and two of five IQ-induced tumours, one in segment 2 and the other in segment 3 (codons 1099-1693). Three of these four mutations, all in segment 2 of the Apc gene, were confirmed by sequencing. The PhIP-induced mutations were detected at codon 1125 (C deletion) and 1130 (G-T transversion), and the IQ-induced mutation was at codon 956 (C-T transition). Importantly, no truncated proteins were detected in tumours from unexposed mice with apparently retained wild-type Apc allele. These results show that one injection of either PhIP or IQ induces intestinal tumours in the Min/+ mice by inactivation of the wild-type Apc allele either by causing LOH or truncation mutations.     

Apc mutation enhances PyMT-induced mammary tumorigenesis

The Adenomatous Polyposis Coli (APC) tumor suppressor gene is silenced by hypermethylation or mutated in up to 70% of human breast cancers. In mouse models, Apc mutation disrupts normal mammary development and predisposes to mammary tumor formation; however, the cooperation between APC and other mutations in breast tumorigenesis has not been studied. To test the hypothesis that loss of one copy of APC promotes oncogene-mediated mammary tumorigenesis, Apc(Min/+) mice were crossed with the mouse mammary tumor virus (MMTV)-Polyoma virus middle T antigen (PyMT) or MMTV-c-Neu transgenic mice. In the PyMT tumor model, the Apc(Min/+) mutation significantly decreased survival and tumor latency, promoted a squamous adenocarcinoma phenotype, and enhanced tumor cell proliferation. In tumor-derived cell lines, the proliferative advantage was a result of increased FAK, Src and JNK signaling. These effects were specific to the PyMT model, as no changes were observed in MMTV-c-Neu mice carrying the Apc(Min/+) mutation. Our data indicate that heterozygosity of Apc enhances tumor development in an oncogene-specific manner, providing evidence that APC-dependent pathways may be valuable therapeutic targets in breast cancer. Moreover, these preclinical model systems offer a platform for dissection of the molecular mechanisms by which APC mutation enhances breast carcinogenesis, such as altered FAK/Src/JNK signaling.     

NO-donating aspirin inhibits intestinal carcinogenesis in Min (APC(Min/+)) mice

The chemopreventive effect of nitric oxide-releasing aspirin (NO-ASA) against gastrointestinal tumorigenesis was evaluated in Min (APC(Min/+)) mice. NO-ASA consists of a traditional ASA that bears covalently attached to it an NO-releasing moiety. Four groups (N=10) of six-week-old female C57BL/6J APC(Min/+) and the corresponding C57BL/6J(+/+) wild type mice were treated either with vehicle or NO-ASA 100 mg/kg/day intrarectally for 21 days. There were no signs of overt toxicity including gastrointestinal toxicity from NO-ASA. Vehicle treated Min mice had 24.7 +/- 3.8 tumors (mean +/- SEM) and NO-ASA treated Min mice had 10.1 +/- 1.4 tumors (59% reduction; P<0.001). Wild type mice showed no tumors. NO-ASA did not affect cell proliferation in small intestinal mucosa, determined by immunohistochemical staining for PCNA. Our findings establish the strong inhibitory effect of NO-ASA in intestinal carcinogenesis in the Min mouse and suggest that this agent merits further evaluation as a chemopreventive agent against colon cancer.     

Intestinal tumorigenesis is not affected by progesterone signaling in rodent models

Clinical data suggest that progestins have chemopreventive properties in the development of colorectal cancer. We set out to examine a potential protective effect of progestins and progesterone signaling on colon cancer development. In normal and neoplastic intestinal tissue, we found that the progesterone receptor (PR) is not expressed. Expression was confined to sporadic mesenchymal cells. To analyze the influence of systemic progesterone receptor signaling, we crossed mice that lacked the progesterone receptor (PRKO) to the Apc(Min/+) mouse, a model for spontaneous intestinal polyposis. PRKO-Apc(Min/+) mice exhibited no change in polyp number, size or localization compared to Apc(Min/+). To examine effects of progestins on the intestinal epithelium that are independent of the PR, we treated mice with MPA. We found no effects of either progesterone or MPA on gross intestinal morphology or epithelial proliferation. Also, in rats treated with MPA, injection with the carcinogen azoxymethane did not result in a difference in the number or size of aberrant crypt foci, a surrogate end-point for adenoma development. We conclude that expression of the progesterone receptor is limited to cells in the intestinal mesenchyme. We did not observe any effect of progesterone receptor signaling or of progestin treatment in rodent models of intestinal tumorigenesis.