Apc‐driven colon carcinogenesis in pirc rat is strongly reduced by polyethylene glycol

Polyethylene glycol (PEG) is one of the most powerful agents in reducing chemically induced carcinogenesis in rat colon. However, contrasting results in Min mice dampened the enthusiasm on this potentially strong and virtually safe, cancer chemopreventing agent. Pirc (F344/NTac‐Apc ^am1137) rats carrying a germline heterozygous mutation in the Apc gene, spontaneously develop multiple tumours in the colon thus modelling both familial adenomatous polyposis (FAP) and sporadic colorectal cancer (CRC). Given this similarity, we thought that these rats could be appropriate to test the efficacy of PEG 8000 in reducing carcinogenesis. Pirc male rats aged one month were treated with 5% PEG in drinking water for 2 or 6 months. Precancerous lesions were dramatically reduced after 2 months of PEG treatment (Mucin depleted foci (MDF)/colon were 99 ±17 and 12± 8 in Controls and PEG‐treated rats, respectively; p < 0.001; mean ± SD). Similarly, colon tumors were significantly reduced after 6 months of treatment (tumors/rat were 8.1 ± 2.3 and 3.6 ± 2.2 in Controls and PEG‐treated rats, respectively; p < 0.05; mean ± SD). Colon proliferation, a parameter correlated to cancer risk, was also significantly lower in PEG‐treated rats than in Controls, while apoptosis was not significantly affected. In conclusion, PEG markedly reduces colon carcinogenesis in Pirc rats mutated in Apc; we thus suggest that PEG may be used as chemopreventive agent to reduce cancer risk in FAP and CRC patients.     

Dietary‐feeding of grape seed extract prevents azoxymethane‐induced colonic aberrant crypt foci formation in fischer 344 rats

Chemoprevention by dietary agents/supplements has emerged as a novel approach to control various malignancies, including colorectal cancer (CRC). This study assessed dietary grape seed extract (GSE) effectiveness in preventing azoxymethane (AOM)‐induced aberrant crypt foci (ACF) formation and associated mechanisms in Fischer 344 rats. Six‐week‐old rats were injected with AOM, and fed control diet or the one supplemented with 0.25% or 0.5% (w/w) GSE in pre‐ and post‐AOM or only post‐AOM experimental protocols. At 16 wk of age, rats were sacrificed and colons were evaluated for ACF formation followed by cell proliferation, apoptosis, and molecular analyses by immunohistochemistry. GSE‐feeding caused strong chemopreventive efficacy against AOM‐induced ACF formation in terms of up to 60% (P < 0.001) reduction in number of ACF and 66% (P < 0.001) reduction in crypt multiplicity. Mechanistic studies showed that GSE‐feeding inhibited AOM‐induced cell proliferation but enhanced apoptosis in colon including ACF, together with a strong decrease in cyclin D1, COX‐2, iNOS, and survivin levels. Additional studies showed that GSE‐feeding also decreased AOM‐caused increase in β‐catenin and NF‐κB levels in colon tissues. Compared to control animals, GSE alone treatment did not show any considerable change in these biological and molecular events in colon, and was nontoxic. Together, these findings show the chemopreventive efficacy of GSE against the early steps of colon carcinogenesis in rats via likely targeting of β‐catenin and NF‐κB signaling, and suggest its potential usefulness for the prevention of human CRC. © 2010 Wiley‐Liss, Inc.     

Calcium inhibits promotion by hot dog of 1,2‐dimethylhydrazine‐induced mucin‐depleted foci in rat colon

Epidemiology suggests that processed meat is associated with colorectal cancer risk, but few experimental studies support this association. We have shown that a model of cured meat made in a pilot workshop promotes preneoplastic lesions, mucin‐depleted foci (MDF) in the colon of rats. This study had two aims: to check if real store‐bought processed meats also promote MDF, and to test if calcium carbonate, which suppresses heme‐induced promotion, can suppress promotion by processed meat. A 14‐day study was done to test the effect of nine purchased cured meats on fecal and urinary biomarkers associated with heme‐induced carcinogenesis promotion. Fecal water from rats given hot dog or fermented raw dry sausage was particularly cytotoxic. These two cured meats were thus given to rats pretreated with 1,2‐dimethylhydrazine, to evaluate their effect on colorectal carcinogenesis. After a 100‐days feeding period, fecal apparent total N‐nitroso compounds (ATNC) were assayed and colons were scored for MDF. Hot dog diet increased fecal ATNC and the number of MDF per colon compared with the no‐meat control diet (3.0 ± 1.7 vs. 1.2 ± 1.4, p < 0.05). In a third study, addition of calcium carbonate (150 µmol/g) to the hot dog diet decreased the number of MDF/colon and fecal ATNC compared with the hot dog diet without calcium carbonate (1.2 ± 1.1 vs. 2.3 ± 1.4, respectively, p < 0.05). This is the first experimental evidence that a widely consumed processed meat promotes colon carcinogenesis in rats. It also shows that dietary prevention of this detrimental effect is possible.     

Antitumor activity of the MEK inhibitor trametinib on intestinal polyp formation in ApcΔ716 mice involves stromal COX‐2

Extracellular signal‐regulated kinase is an MAPK that is most closely associated with cell proliferation, and the MEK/ERK signaling pathway is implicated in various human cancers. Although epidermal growth factor receptor, KRAS, and BRAF are considered major targets for colon cancer treatment, the precise roles of the MEK/ERK pathway, one of their major downstream effectors, during colon cancer development remain to be determined. Using Apc^Δ716 mice, a mouse model of familial adenomatous polyposis and early‐stage sporadic colon cancer formation, we show that MEK/ERK signaling is activated not only in adenoma epithelial cells, but also in tumor stromal cells including fibroblasts and vascular endothelial cells. Eight‐week treatment of Apc^Δ716 mice with trametinib, a small‐molecule MEK inhibitor, significantly reduced the number of polyps in the large size class, accompanied by reduced angiogenesis and tumor cell proliferation. Trametinib treatment reduced the COX‐2 level in Apc^Δ716 tumors in vivo and in primary culture of intestinal fibroblasts in vitro. Antibody array analysis revealed that trametinib and the COX‐2 inhibitor rofecoxib both reduced the level of CCL2, a chemokine known to be essential for the growth of Apc mutant polyps, in intestinal fibroblasts in vitro. Consistently, trametinib treatment reduced the Ccl2 mRNA level in Apc^Δ716 tumors in vivo. These results suggest that MEK/ERK signaling plays key roles in intestinal adenoma formation in Apc^Δ716 mice, at least in part, through COX‐2 induction in tumor stromal cells.     

Long‐term treatment with Sitagliptin,a dipeptidyl peptidase‐4 inhibitor,reduces colon carcinogenesis and reactive oxygen species in 1,2‐dimethylhydrazine‐induced rats

Type 2 diabetes mellitus (T2DM) and insulin resistance (IR) increase colon cancer risk. Antidiabetic drugs stabilizing incretin hormones, such as inhibitors of dipeptidyl peptidase‐4 activity (DPP4i), may affect colon carcinogenesis; however, the data remain controversial. Therefore, the authors studied whether long‐term administration of the DPP4i Sitagliptin (SITA) affects 1,2‐dimethylhydrazine (DMH)‐induced colon carcinogenesis. Male F344 rats fed a high‐fat (HF) diet promoting colon carcinogenesis and IR, were induced with DMH (100 mg/kg × 2 times). One week later, the animals were allocated to two groups: one continuing with HF diet (controls; n = 8) and one receiving SITA (n = 8) mixed in the diet (260 ppm). Body weight, food consumption and glycemia were not affected by SITA. Fifteen weeks after DMH, the number of the precancerous lesions mucin‐depleted foci (MDF) was significantly lower in rats treated with SITA [MDF/colon: 9.5 ± 0.9 and 6.4 ± 0.9 in controls (n = 8) and SITA groups (n = 8), respectively; means ± SE, p < 0.05]. Reactive oxygen species in the blood were also significantly lower in the SITA group [6.75 ± 0.69 and 5.63 ± 0.75 (H₂O₂ in mM) in controls (n = 5) and SITA (n = 6), respectively; means ± SE, p < 0.05]. Rats treated with SITA had a lower DPP4 activity in the intestine but not in the plasma. Intestine growth morphometric parameters and colon proliferation, as proliferating cell nuclear antigen expression, were not affected by SITA. In conclusion, the results suggest a protective effect of DPP4i against colon carcinogenesis that could be exploited in chemoprevention trials.     

The secondary bile acid,deoxycholate accelerates intestinal adenoma–adenocarcinoma sequence in Apc min/+ mice through enhancing Wnt signaling

Colorectal cancer is one of the leading causes of cancer deaths. It correlates to a high fat diet, which causes an increase of the secondary bile acids including deoxycholate (DOC) in the intestine. We aimed to determine the effects of DOC on intestinal carcinogenesis in Apc ^min/+ mice, a model of spontaneous intestinal adenomas. Four-week old Apc ^min/+ mice were treated with 0.2 % DOC in drinking water for 12 weeks. The number and size of tumors were measured, and tissue sections were prepared for the evaluation of intestinal carcinogenesis, cell proliferation, and apoptosis. The activation of Wnt signaling was detected in the intestinal tumor cells of the Apc ^min/+ mice, and also in the human colon samples. DOC increased the number of intestine tumors by 165.1 % compared with that in untreated Apc ^min/+ mice mainly in the middle and distal segments of the small intestine and colon. The numbers of all sizes of tumors in the small intestine were increased. Intestinal carcinogenesis was confirmed in 75 % mice in DOC treated-Apc ^min/+ mice compared with 0 % in untreated mice. This was accompanied by promoting tumor cell proliferation and decreasing apoptosis, and increasing the percentage of β-catenin positive cells and its nuclear expression in intestinal tumor cells of Apc ^min/+ mice, and also up-regulating the expression of cyclin D1. In addition, the activation of Wnt signaling also played in modulating human colon carcinogenesis. Our studies suggest that DOC enhances the multiplicity of intestinal tumor, and accelerates intestinal adenoma–adenocarcinoma sequence in Apc ^min/+ mice mediated by stimulating tumor cell proliferation and decreasing apoptosis through enhancing Wnt signaling.     

The Ron receptor tyrosine kinase is not required for adenoma formation in ApcMin/+ mice

The Ron receptor tyrosine kinase is overexpressed in approximately half of all human colon cancers. Increased Ron expression positively correlates with tumor progression, and reduction of Ron levels in human colon adenocarcinoma cells reverses their tumorigenic properties. Nearly all colon tumors demonstrate loss of the adenomatous polyposis coli (APC) tumor suppressor, an early initiating event, subsequently leading to β‐catenin stabilization. To understand the role of Ron in early stage intestinal tumorigenesis, we generated Apc‐mutant (Apc^Min/+) mice with and without Ron signaling. Interestingly, we report here that significantly more Apc^Min/+ Ron‐deficient mice developed higher tumor burden than Apc^Min/+ mice with wild‐type Ron. Even though baseline levels of intestinal crypt proliferation were increased in the Apc^Min/+ Ron‐deficient mice, loss of Ron did not influence tumor size or histological appearance of the Apc^Min/+ adenomas, nor was β‐catenin localization changed compared to Apc^Min/+ mice with Ron. Together, these data suggest that Ron may be important in normal intestinal tissue homeostasis, but that the expression of this receptor is not required for the formation and growth of adenomas in Apc^Min/+ mice. © 2009 Wiley‐Liss, Inc.     

Altered expression of c-myc,p16 and p27 in rat colon tumors and its reversal by short-term treatment with chemopreventive agents

Modulation of gene expression in tumors has the potential of being a surrogate end-point biomarker for chemoprevention. Thus, we determined the modulation by chemopreventive agents of the protein and mRNA expression of genes in rat colon tumors. Male F344 rats were administered three weekly injections of 15 mg/kg azoxymethane. Forty-seven weeks later, they received aspirin (600), calcium chloride (50 000), 2-(carboxyphenyl) retinamide (2-CPR, 315), alpha-difluoromethylornithine (DFMO, 3000), piroxicam (200), quercetin (33 600), 9-cis retinoic acid (9-cis RA, 30), rutin (3000), or sulindac (280) in their diet at the indicated mg/kg concentration for 7 days and were then killed. In colon tumors relative to the mucosa, the protein and mRNA levels of c-myc were increased, while the levels of p16 and p27 were decreased. Calcium chloride, DFMO, piroxicam and sulindac administered for 7 days decreased the mitotic index and reduced the protein and mRNA levels of c-myc in colon tumors. Calcium chloride, DFMO and piroxicam increased the protein and mRNA levels of p16 and along with sulindac increased the protein level of p27, but not its mRNA. The other agents failed to modulate both the mitotic index and the expression of the genes. The ability of the chemopreventive agents to prevent colon tumors was determined. Male F344 rats were administered three weekly injections of 15 mg/kg azoxymethane and 8 weeks later they were administered aspirin, 2-CPR, DFMO, piroxicam, 9-cis RA and rutin in their diet. The rats were killed 26 weeks after they started to receive the chemopreventive agents. The multiplicity of colon tumors was reduced by DFMO and piroxicam, increased by rutin and not affected by the other agents. Hence, agents that prevented colon cancer decreased the mitotic index and altered the expression of c-myc, p16 and p27 suggesting that modulation in the expression of these genes are potential biomarkers for chemopreventive activity.     

Sulindac sulfide alters the expression of cyclin proteins in HT-29 colon adenocarcinoma cells

Sulindac sulfide (SS), the active metabolite of the colon cancer chemopreventive compound sulindac, inhibits the proliferation of HT-29 colon cancer cells mainly by inducing cell quiescence. We determined by bivariate flow-cytometric analysis both the DNA and cyclin protein content of individual cells. Thus, we assessed in detail the expression of several cyclins during the cell-cycle phases and demonstrated that SS (i) decreases the expression of cyclins B1 and E and (ii) increases the expression of cyclins D1, D2 and D3, particularly in the G₁ phase of the cell cycle. SS-induced apoptotic cells expressed both E- and D-type cyclins but not cyclin B1. The changes in cyclin expression combined with reduced catalytic activity of cyclin-dependent kinases could explain in molecular terms the anti-proliferative effect of SS on HT-29 colon cancer cells. These changes may contribute to the chemopreventive effect of sulindac. Int. J. Cancer 76:99–104, 1998.© 1998 Wiley-Liss, Inc.     

Fem1b,a proapoptotic protein,mediates proteasome inhibitor‐induced apoptosis of human colon cancer cells

In the treatment of colon cancer, the development of resistance to apoptosis is a major factor in resistance to therapy. New molecular approaches to overcome apoptosis resistance, such as selectively upregulating proapoptotic proteins, are needed in colon cancer therapy. In a mouse model with inactivation of the adenomatous polyposis coli (Apc) tumor suppressor gene, reflecting the pathogenesis of most human colon cancers, the gene encoding feminization‐1 homolog b (Fem1b) is upregulated in intestinal epithelium following Apc inactivation. Fem1b is a proapoptotic protein that interacts with apoptosis‐inducing proteins Fas, tumor necrosis factor receptor‐1 (TNFR1), and apoptotic protease activating factor‐1 (Apaf‐1). Increasing Fem1b expression induces apoptosis of cancer cells, but effects on colon cancer cells have not been reported. Fem1b is a homolog of feminization‐1 (FEM‐1), a protein in Caenorhabditis elegans that is regulated by proteasomal degradation, but whether Fem1b is likewise regulated by proteasomal degradation is unknown. Herein, we found that Fem1b protein is expressed in primary human colon cancer specimens, and in malignant SW620, HCT‐116, and DLD‐1 colon cancer cells. Increasing Fem1b expression, by transfection of a Fem1b expression construct, induced apoptosis of these cells. We found that proteasome inhibitor treatment of SW620, HCT‐116, and DLD‐1 cells caused upregulation of Fem1b protein levels, associated with induction of apoptosis. Blockade of Fem1b upregulation with morpholino antisense oligonucleotide suppressed the proteasome inhibitor‐induced apoptosis of these cells. In conclusion, the proapoptotic protein Fem1b is downregulated by the proteasome in malignant colon cancer cells and mediates proteasome inhibitor‐induced apoptosis of these cells. Therefore, Fem1b could represent a novel molecular target to overcome apoptosis resistance in therapy of colon cancer. © 2009 Wiley‐Liss, Inc.     

Randomized,double‐blind,placebo‐controlled trial of sulindac in individuals at risk for melanoma

BACKGROUND:

Reduced melanoma risk has been reported with regular use of nonsteroidal anti‐inflammatory drugs (NSAIDs). However, the ability of NSAIDs to reach melanocytes in vivo and modulate key biomarkers in preneoplastic lesions such as atypical nevi has not been evaluated.

METHODS:

This randomized, double‐blind, placebo‐controlled trial of sulindac was conducted in individuals with atypical nevi (AN) to determine bioavailability of sulindac and metabolites in nevi and effect on apoptosis and vascular endothelial growth factor A (VEGFA) expression in AN. Fifty subjects with AN ≥4 mm in size and 1 benign nevus (BN) were randomized to sulindac (150 mg twice a day) or placebo for 8 weeks. Two AN were randomized for baseline excision, and 2 AN and BN were excised after intervention.

RESULTS:

Postintervention sulindac, sulindac sulfone, and sulindac sulfide concentrations were 0.31 ± 0.36, 1.56 ± 1.35, and 2.25 ± 2.24 μg/mL in plasma, and 0.51 ± 1.05, 1.38 ± 2.86, and 0.12 ± 0.12 μg/g in BN, respectively. Sulindac intervention did not significantly change VEGFA expression but did increase expression of the apoptotic marker cleaved caspase‐3 in AN (increase of 3 ± 33 in sulindac vs decrease of 25 ± 45 in the placebo arm, P = .0056), although significance was attenuated (P = .1103) after adjusting for baseline expression.

CONCLUSIONS:

Eight weeks of sulindac intervention resulted in high concentrations of sulindac sulfone, a proapoptotic metabolite, in BN but did not effectively modulate VEGFA and cleaved caspase‐3 expression. Study limitations included limited exposure time to sulindac and the need to optimize a panel of biomarkers for NSAID intervention studies. Cancer 2012. © 2012 American Cancer Society.     

Distribution of preneoplastic lesions and tumors, and beta-catenin gene mutations in colon carcinomas induced by 1,2-dimethylhydrazine plus dextran sulfate sodium

Mucin-depleted foci (MDF) are considered as useful biomarkers in rat colon carcinogenesis. The purpose of the present study was to examine the mechanism(s) underlying rat colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) plus 1% Dextran Sulfate Sodium (DSS). Twelve male F344 rats were given subcutaneous injections (40mg/kg body) of DMH twice a week. They received DSS in the drinking water for 1 week after the first injection of DMH and then were maintained on tap water. The rats were sacrificed at 10 and 14 weeks after the first injection of DMH. Colon tissues were divided into 10 segments from anus to cecum (A/J) and stained with Alcian blue (AB) to identify MDF. We found that MDF and tumors were induced in the rat colon after treatment with DMH plus DSS and that the number of MDF in each segment of the colon was significantly correlated with that of tumors (p=0.006). In addition, we found that the beta-catenin protein was accumulated in cytoplasm and nuclei of MDF and the frequent beta-catenin gene mutations in the colon tumors. These results suggest that MDF is closely related to rat colon carcinogenesis induced by DMH plus DSS.     

Sustained proliferation and resistance to apoptosis after a cytotoxic insult are early alterations in rat colon carcinogenesis

To study the early alterations in carcinogenesis, we determined apoptosis and proliferation in rat mucin depleted foci (MDF), precancerous lesions in the colon under basal conditions and 24 h after treatment with 1,2-dimethylhydrazine (DMH), which induces apoptosis in the colon. Spontaneous apoptosis in MDF was higher than in normal mucosa (Apoptotic Index was 1.61 ± 0.30 and 0.21 ± 0.02 in MDF and normal mucosa, respectively, mean ± SE, p < 0.05). DMH (30 and 75 mg/kg) increased apoptosis in both normal mucosa and MDF (up to 20 times higher compared to basal levels in normal mucosa, but only two times in MDF). MDF had a higher and deregulated pattern of proliferation along the crypt compared to normal mucosa. After DMH, proliferation in normal mucosa was significantly depressed, but it did not vary in MDF. Survivin-Birc5 regulating apoptosis and proliferation was significantly over-expressed (RT-qPCR and immunohistochemistry experiments) in MDF vs. normal mucosa, but did not vary in response to DMH. The expression of the pro-apoptotic protein Bak did not vary in normal mucosa and MDF. Since inflammation is present in MDF, which may hamper apoptosis, we studied the effect of pre-treatment with aspirin (600 ppm in the diet for 10 days). No significant effects of aspirin were observed. In conclusion, MDF had a higher spontaneous apoptosis and proliferation coupled with a reduced response to apoptotic stimuli from cytotoxic compounds. Survivin over-expression in MDF indicates that this is an early event in colon carcinogenesis and suggests that down-regulation of Survivin may represent a strategy for cancer prevention.     

Chemopreventive efficacy of sulindac sulfone against colon cancer depends on time of administration during carcinogenic process.

Epidemiological and model studies with laboratory animals have provided evidence that nonsteroidal anti-inflammatory drugs reduce the risk of colon cancer. Sulindac, a nonsteroidal anti-inflammatory drug, has been shown to inhibit azoxymethane (AOM)-induced colon carcinogenesis in rats when administered continuously before, during, and after carcinogen treatment (initiation and postinitiation periods) or when given continuously beginning 14 weeks after carcinogen administration (promotion/ progression stage). The present study was designed to investigate the chemopreventive efficacy of sulindac sulfone (exisulind), the sulfone metabolite of sulindac, when administered during the promotion/progression stage of colon carcinogenesis in comparison to the effect during the initiation and postinitiation periods. We have also studied the modulating effect of exisulind on colonic tumor apoptosis. At 5 weeks of age, groups of male F344 rats were fed diets containing 0%, 0.06%, and 0.12% exisulind. At 7 weeks of age, groups of animals were injected s.c. with AOM (15 mg/kg body weight, once weekly for 2 weeks). Animals intended for the promotion/progression study and receiving 0% exisulind were switched to an experimental diet containing 0.12% exisulind at 14 weeks after the second AOM treatment. All rats remained on their respective dietary regimens until the termination of the study, 50 weeks after the second AOM injection. Colon tumors were evaluated histopathologically for tumor type. Administration of 0.06% and 0.12% exisulind during the initiation and postinitiation periods significantly inhibited the incidence and multiplicity of invasive and/or noninvasive adenocarcinomas of the colon. The inhibition of colon tumorigenesis by exisulind was associated with a significant retardation of body weight gain shortly after sulfone administration and increased apoptosis in the colon tumors. In contrast, administration of the higher dose (0.12%) of exisulind during the promotion/progression stage had only minimal effects on colon tumorigenesis and apoptosis in the colon tumors, suggesting that early administration, but not late administration, may be required for chemopreventive efficacy of this drug.     

Regional response leading to tumorigenesis after sulindac in small and large intestine of mice with Apc mutations

Sulindac and other NSAIDs have been widely studied as potential chemopreventive agents for colon cancer. Short-term studies have shown adenomatous polyps to regress in patients with familial adenomatous polyposis (FAP). In this study the effect of sulindac on cancer as an endpoint was evaluated in ApcMin mice, a preclinical model of FAP with an Apc mutation in codon 850 that leads to gastrointestinal adenomas and carcinomas. Three groups of mice were studied all of which were fed AIN-76A diet: one group was fed AIN-76A diet alone, a second group received sulindac 200 p.p.m. premixed in the diet and a third group received sulindac 180 p.p.m. added in drinking water. ApcMin mice were killed 9 weeks after feeding was initiated. Mice receiving sulindac developed fewer tumors in the intestine overall; the major decrease in tumor development after sulindac was seen in the small intestine regardless of route of administration. In the large intestine, however, sulindac significantly increased the incidence, multiplicity and volume of tumors in the colon of ApcMin mice, a regional response to sulindac differing from previous reports. Quantitative measurements of apoptosis, Bax and Bcl-xL protein expression in the ApcMin mice revealed the ratio of Bax/Bcl-xL expression and apoptosis increased in the small intestine but decreased in the cecum, consistent with the regional tumorigenesis observed after sulindac. These findings thus suggest involvement of Bax and apoptosis in tumors developing after sulindac treatment in this mouse model.     

Induction of Apoptosis by Sulindac in Azoxymethane-induced Possible Colonic Premalignant Lesions in Rats

We have reported that β-catenin-accumulated crypts (BCAC), which do not have the appearance of aberrant crypt foci (ACF) are possible colonic premalignant lesions in rats. Suppression of the occurrence and advancement of such lesions should have critical relevance to cancer prevention. This study examined whether sulindac, a chemopreventive nonsteroidal anti-inflammatory drug is able to induce apoptosis in such premalignant lesions. At 6 weeks of age, rats groups 1–3 were given azoxymethane (AOM) (15 mg/kg-body weight) once weekly for 3 weeks. Two groups were given sulindac in the diet (200 and 400 ppm), starting at 9 weeks of age. The rats were sacrificed at the termination, and the colons were carefully examined. The incidence and crypt multiplicity of BCAC and ACF were significantly less than those of the control group. The effect of sulindac on the expression of BCAC was greater than that on ACF. Exposure to sulindac significantly increased the apoptotic index (terminal deoxynucleotide transferase dUTP nick-end labeling (TUNEL)-positive cells) in BCAC. However, no significant increase of the index was found in the case of ACF. These results suggest that the chemopreventive effect of sulindac in rats is related to the induction of apoptosis in premalignant lesions. Our results also provide additional evidence that BCAC are premalignant lesions in colon carcinogenesis in rodents.     

Mechanism of lovastatin-induced apoptosis in intestinal epithelial cells

We earlier showed that lovastatin potentiated the chemopreventive effects of sulindac against colon neoplasia in a rodent model and augments apoptosis induced by 5-FU and cisplatin in human colon cancer cells. In the present study, we investigated effects of lovastatin in spontaneously immortalized rat intestinal epithelial cells, IEC-18 and their K-ras transformed clones. Lovastatin induced morphologic changes (cell rounding and detachment) and apoptosis that were not influenced by K-ras mutations, but were prevented by geranylgeranyl-pyrophosphate or by mevalonate. Clostridium difficile toxin B, which directly inactivates rho, induced similar morphologic changes and apoptosis. Cycloheximide prevented these effects of lovastatin, but not C. difficile toxin B. Lovastatin decreased the amounts of membrane bound rhoA and rhoB. Cycloheximide and geranylgeranyl-pyrophosphate prevented lovastatin induced morphologic changes and apoptosis but did not inhibit lovastatin-induced changes in membrane translocation of rho. Our data suggest that lovastatin induces morphologic changes and apoptosis by inhibiting geranylgeranylation of small GTPases of the rho family and thereby inactivating them. Restoration of membrane translocation of rho is not necessary for preventing lovastatin-induced morphologic changes or apoptosis.     

Loss of EphA2 receptor tyrosine kinase reduces Apcmin/+ tumorigenesis

The Eph receptor A2 (EphA2) is overexpressed in a range of human epithelial cancers, a phenotype that is associated with cancer cell proliferation, progression and angiogenesis. Mouse models of mammary neoplasia have confirmed the role of EphA2 as mice carrying a knockout allele of EphA2 were resistant to breast cancer, a phenotype that was associated with interactions between EphA2 and ErbB2. We investigated in vivo the role of EphA2 in GI cancer. To determine whether EphA2 influences intestinal tumorigenesis, we used qRT‐PCR to examine the mRNA expression levels of EphA2 in tumors from the small intestine and colon of Apc^Min/+ mice. We found that EphA2 was significantly up‐regulated in tumors from both regions when compared with normal control tissues. We then evaluated the spatial expression patterns of EphA2 protein using immunohistochemistry in both the small intestine and colon and found that in normal tissues EphA2 was robustly expressed in highly differentiated cells, such as cells of the villi, but that EphA2 expression was largely absent from the stem cell niche and proliferative zones of intestinal crypts. In contrast, in tumors EphA2 was broadly expressed. Finally, we created a strain of Apc^Min/+ mice carrying a genetic knockout of the EphA2 gene. These mice developed significantly fewer and smaller tumors in both the small and large intestine. Overall, our results indicate that EphA2 plays an oncogenic role in the mammalian intestine suggesting that strategies to target EphA2 activity may offer new therapeutic modalities for colorectal cancer. © 2008 Wiley‐Liss, Inc.     

Comparison of DNA‐adduct and tissue‐available dose levels of MeIQx in human and rodent colon following administration of a very low dose

[2‐¹⁴C]2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx) was administered orally (304 ng/kg body‐weight dose based upon an average 70‐kg‐body‐weight subject) to 5 human colon‐cancer patients (58 to 84 years old), as well as to F344 rats and B₆C₃F₁ mice. Colon tissue was collected from the human subjects at surgery and from the rodents 3.5 to 6 hr after administration. Colon DNA‐adduct levels and tissue available doses were measured by accelerator mass spectrometry (AMS). The mean levels of MeIQx in the histologically normal colon tissue were not different among the human (97 ± 26 pg MeIQx/g), rat (133 ± 15 pg/g) or mouse (78 ± 10 pg/g) tissues; and no difference existed between the levels detected in human normal and tumor tissue (101 ± 15 pg/g). Mean DNA‐adduct levels in normal human colon (26 ± 4 adducts/10¹² nucleotides) were significantly greater (p < 0.01) than in rats (17.1 ± 1 adduct/10¹² nucleotides) or mice (20.6 ± 0.9 adduct/10¹² nucleotides). No difference existed in adduct levels between normal and tumor tissue in humans. These results show that MeIQx forms DNA adducts in human colon at low dose, and that the human colon may be more sensitive to the effects of MeIQx than that of mice or rats. Int. J. Cancer 80:539–545, 1999. © 1999 Wiley‐Liss, Inc.     

Correlated downregulation of estrogen receptor beta and the circadian clock gene Per1 in human colorectal cancer

There is a growing body of evidence that disturbed circadian clock gene expression is associated with tumor development and tumor progression. Based on our initial experiments demonstrating decreased period 1 (Per1) expression in colon cancer, we evaluated clock gene and estrogen receptor (ER) alpha/beta expression in colon cancer cells of primary colorectal tumors and adjacent normal colon mucosa (NM) by real‐time RT‐PCR. Analysis of gene expression in G₂ and G₃ colorectal tumors revealed a decrease of Per1 mRNA compared with paired NM (G₂: 0.52‐fold; P = n.s. and G₃: 0.48‐fold; P = 0.03). A significant gender specific difference of Per1 expression was observed in G₂ tumors as compared with NM (female: 0.38‐fold; P = 0.004 vs. male: 0.73‐fold; P = n.s.). Expression of CLOCK was significantly elevated in G₂ tumors of male patients (1.63‐fold, P = 0.01). The expression of ER‐beta was significantly decreased in G₂ and G₃ tumors (G₂: 0.32‐fold; P = 0.003 and 0.27; P = 0.001). No significant gender specific differences of ER‐beta reduction in tumors were observed. A significant correlation between the decrease of Per1 and ER‐beta in colorectal tumors (r = 0.61; P < 0.001) was found. No changes in gene expression were detected for ER‐alpha and Per2. Our data demonstrate a correlated decrease of Per1 and ER‐beta in colorectal tumors, mediated probably by epigenetic mechanisms. The observed gender differences in the expression of CLOCK and Per1 in G₂ tumors might suggest a gender‐specific, distinctive role of the cellular clock in colorectal tumorigenesis. © 2009 Wiley‐Liss, Inc.