Activation of NF-kappaB is required for mediating proliferative and antiapoptotic effects of progastrin on proximal colonic crypts of mice, in vivo

Mice overexpressing progastrin (PG) in intestinal mucosa (fatty acid-binding protein (Fabp)-PG mice) are at an increased risk of proximal colon carcinogenesis in response to azoxymethane. Here, we report a significant increase in the length of proximal colonic crypts in Fabp-PG mice, associated with potent antiapoptotic effects of PG, which likely contributed to the previously reported increase in colon carcinogenesis in Fabp-PG mice. Phosphorylation of kinase of IkappaBalpha (IKKalpha/beta), inhibitor of kappaB (IkappaB)alpha and p65NF-kappaB was significantly elevated in proximal colonic crypts of Fabp-PG versus wild-type mice, which was associated with degradation of IkappaBalpha and nuclear translocation/activation of p65. Surprisingly, distal colonic crypt cells were not as responsive to elevated levels of PG in Fabp-PG mice. Annexin II, recently described as a high-affinity receptor for PG, strongly co-localized with PG intracellularly and on basolateral membranes of proximal crypt cells, providing evidence that annexin-II binds PG in situ in colonic crypt cells. Proliferative and antiapoptotic effects of PG on proximal crypts of Fabp-PG mice were attenuated to wild-type levels, on treatment with NEMO peptide (an inhibitor of nuclear factor-kappaB (NF-kappaB) activation), demonstrating for the first time a critical role of NF-kappaB in mediating hyperproliferative affects of PG on colonic crypts of Fabp-PG mice, in vivo. Thus, downregulation of NF-kappaB may significantly reduce the increased risk of colon carcinogenesis in response to PG.     

P53 gene mutation increases progastrin dependent colonic proliferation and colon cancer formation in mice

Transgenic mice overexpressing human progastrin (hGAS) show colonic crypt hyper-proliferation and elevated susceptibility to colon carcinogenesis. We aimed to investigate effects of p53 mutation on colon carcinogenesis in hGAS mice. We show that introducing a p53 gene mutation further increases progastrin dependent BrdU labeling and results in markedly elevated number of aberrant crypt foci (ACF) and colonic tumors. We demonstrate that hGAS/Lgr5-GFP mice have higher number of Lgr5+ colonic stem cells per crypt when compared to Lgr5-GFP mice indicating that progastrin changes crypt biology through increased stem cell numbers and additional p53 mutation leads to more aggressive phenotype in this murine colon cancer model.     

Transgenic expression of human MGMT protects against azoxymethane-induced aberrant crypt foci and G to A mutations in the K-ras oncogene of mouse colon

Transgenic mice over-expressing MGMT, which codes for the humanprotein O⁶-alkylguanine-DNA alkyltransferase, are protectedfrom methylating agent-induced thymic lymphomas. In this studywe evaluated the ability of transgenic overexpression of MGMTin the colon to protect mice from the development of azoxymethane(AOM)-inducedaberrant crypt foci (ACF) and mutations in K-ras. Colonic alkyltransferasein MGMT+ transgenic mice was > 5-fold higher than in nontransgenics:10.5 ± 1.1 vs 2.2 ± 1.1 fmol/µg DNA, P => 0.0001. Mice received 20 mg AOM/kg i.p. at 6 weeks or 15mg AOM/kg at 6 and 7 weeks of age, and 8 wks later colons wereexamined for ACF. A significant protective effect of MGMT wasseen in mice given single dose of 20 mg AOM/kg. The incidenceof ACF/colon was lower in MGMT+ mice (2.0 ± 1.2) thanin nontransgenic mice (3.9 ± 1.8, P = 0.02). G to A mutationsin codon 12 of K-ras were detected by PCR-RFLP in ACF and inrandom samples of normal appearing mucosa. The incidence ofACF with mutant K-ras in MGMT transgenic mice (0.6 ±0.7/colon) was significantly reduced compared to nontransgenicmice (2.3 ± 1.7/colon, P = 0.02). We propose that AOMinduces at least two overlapping but not identical premalignantlesions (aberrant crypt foci and K-ras mutations) which canbe prevented by over-expression of MGMT. Thus, MGMT may protectcolonic mucosa from carcinogenesis involving methylating agentssuch as AOM.     

Oncogenic K-ras promotes early carcinogenesis in the mouse proximal colon

Oncogenic K-ras mutations are frequently observed in colon cancers and contribute to transformed growth. Oncogenic K-ras is detected in aberrant crypt foci (ACF), precancerous colonic lesions, demonstrating that acquisition of a K-ras mutation is an early event in colon carcinogenesis. Here, we investigate the role of oncogenic K-ras in neoplastic initiation and progression. Transgenic mice in which an oncogenic K-ras(G12D) allele is activated in the colonic epithelium by sporadic recombination (K-rasLA2 mice) develop spontaneous ACF that are morphologically indistinguishable from those induced by the colon carcinogen azoxymethane (AOM). Similar neoplastic changes involving the entire colon are induced in transgenic mice constitutively expressing K-ras(G12D) throughout the colon (LSL-K-ras(G12D)/Villin-Cre mice). However, the biochemistry and fate of K-ras-induced lesions differ depending upon their location within the colon in these mice. In the proximal colon, K-ras(G12D) induces increased expression of procarcinogenic protein kinase C beta II (PKC beta II), activation of the MEK/ERK signaling axis and increased epithelial cell proliferation. In contrast, in the distal colon, K-ras(G12D) inhibits expression of procarcinogenic PKC beta II and induces apoptosis. Treatment of K-rasLA2 mice with AOM leads to neoplastic progression of small ACF to large, dysplastic microadenomas in the proximal, but not the distal colon. Thus, oncogenic K-ras functions differently in the proximal and distal colon of mice, inducing ACF capable of neoplastic progression in the proximal colon, and ACF with little or no potential for progression in the distal colon. Our data indicate that acquisition of a K-ras mutation is an initiating neoplastic event in proximal colon cancer development in mice.     

Differential effects of CYP2E1 status on the metabolic activation of the colon carcinogens azoxymethane and methylazoxymethanol.

Methylazoxymethanol (MAM) and its chemical and metabolic precursor, azoxymethane (AOM), both strong colon carcinogens in rodents, can be metabolically activated by CYP2E1 in vitro. Using CYP2E1-null mice, we found that CYP2E1 deficiency differentially affects the activation of AOM and MAM, as reflected in DNA guanine alkylation in the colon and in the formation of colonic aberrant crypt foci (ACF). Male and female inbred 129/SV wild-type (WT) and CYP2E1-null (null) mice were treated with 189 micromol/kg of either AOM or methylazoxymethyl acetate (MAMAc), and 7-methylguanine (7-MeG) and O(6)-methylguanine (O(6)-MeG) were measured in the DNAs of various organs. The levels of O(6)-MeG (as pmol/nmol guanine) in the liver, colon, kidney, and lung of male null mice treated with AOM were 87, 48, 70, and 43% lower, respectively, than in AOM-treated WT mice. In null mice treated with MAMAc, the DNA O(6)-MeG levels were lower by 38% in the liver but were higher by 368, 146, and 194% in the colon, kidney, and lung, respectively, compared with the same organs of WT mice treated in the same way. Determination of ACF revealed that although AOM-induced ACF formation was significantly lower in the null group than in the WT group, MAMAc-induced ACF formation was significantly higher in the null group than in the WT group. These results demonstrate an important role for CYP2E1 in the in vivo activation of AOM and MAM and suggest that agents that modify CYP2E1 activity at the tumor initiation stage might either enhance or inhibit colon carcinogenesis, depending on whether AOM or MAMAc is used as the carcinogen. The mechanism of this effect is discussed.     

Sequential and morphological analyses of aberrant crypt foci formation in mice of differing susceptibility to azoxymethane-induced colon carcinogenesis

Aberrant crypt foci (ACF), putative preneoplastic lesions, are early morphological changes induced by the colon carcinogen azoxymethane (AOM). Although inbred mice differ markedly in their susceptibility to AOM carcinogenesis, we have previously shown that ACF develop in both resistant and sensitive mouse strains after AOM treatment. The purpose of this study was to examine the sequential development and identify the morphological characteristics of ACF induced by AOM in the distal colon of sensitive and resistant mice. A/J (highly susceptible), SWR/J (relatively susceptible) and AKR/J (resistant) mice were treated with 10 mg/kg AOM or saline i.p. once a week for 6 weeks and were killed at 1, 2, 4, 6, 9 and 24 weeks after the last injection. The distal colons were stained with methylene blue and the numbers of ACF and tumors determined. Tumors were present as early as 4 weeks after AOM exposure in SWR/J and A/J mice and increased in frequency throughout the study in both strains. No tumors developed in the AKR/J mice. ACF, however, formed in all strains of mice. The greatest difference between susceptible and resistant strains was in the number of large ACF that developed at later time points. Furthermore, morphometric analysis revealed that A/J mice had the highest percentage of dysplastic ACF, followed by SWR/J mice. These data indicate that the difference in cancer risk from AOM may be due to the lack of progression of smaller ACF in the resistant mice and to the development of dysplasia in a higher percentage of ACF from susceptible strains.     

Tumor suppressor functions for the Cdk inhibitor p21 in the mouse colon

The Cdk inhibitor p21 regulates p53-mediated growth arrest following DNA damage. It is expressed during epithelial differentiation in a variety of organs including colon. We investigated susceptibility of p21-deficient mice to the colon carcinogen azoxymethane (AOM). After AOM injections, rodents develop putative premalignant lesions called aberrant crypt foci (ACF) that are localized to the distal three centimeters of the colon. p21-deficient mice developed significantly higher numbers of ACF than wild-type mice in response to AOM, and these were not restricted to distal colon. After AOM treatment, increased numbers of lymphoid aggregates were detected in p21-deficient colon. Proliferation was similar in wild type and p21-deficient colon before and after AOM injection, but AOM-induced apoptosis was detected only in wild-type crypt epithelial cells, and not in the p21-deficient colon. The proapoptotic function uncovered for p21 was unexpected, because p21 acts as an inhibitor of apoptosis in many systems, and is not required for p53-dependent apoptosis. Enhanced formation of ACF in p21-deficient mice supports a tumor suppressor function for p21 in the colon. Reduced apoptosis of colon epithelial cells with deleterious mutations may be an initiating event in the formation of ACF, with inflammatory cell cytokine expression contributing to their further expansion.     

Azoxymethane-induced colon tumors and aberrant crypt foci in mice of different genetic susceptibility

Azoxymethane (AOM) is an organotropic colon carcinogen that is commonly used to induce colon tumors in rodents. Unlike its parent compound, 1,2-dimethylhydrazine (DMH), a tumor susceptibility phenotype in inbred mice with respect to AOM has not been established. Thus, this study was undertaken to determine whether genetic susceptibility extends to this carcinogen. SWR/J, A/J (both susceptible to DMH carcinogenesis) and AKR/J (resistant) mice were treated with 10 mg/kg AOM i.p. once a week for 8 weeks. Twenty-five weeks after the initial injection, tumor yield was determined. With a single exception, only SWR/J and A/J mice developed tumors, with a distribution that was limited to the distal colon (16.3±1.1 and 36.4±2.4, respectively). The formation of aberrant crypt foci (ACF), putative preneoplastic lesions, was also assessed in whole-mount colons using Methylene Blue staining. Consistent with tumor multiplicity, the total number of ACF was highest in A/J mice, followed by SWR/J mice. In addition, A/J mice had a significantly greater number of large ACF (five or more crypts per foci) than the other strains. Despite the absence of colon tumors, however, AKR/J mice did develop a significant number of ACF. This finding suggests that ACF in resistant mice are persistent but do not progress to tumors.     

Deoxycholic acid promotes the growth of colonic aberrant crypt foci

AKR/J mice are resistant to the tumorigenic properties of the colon carcinogen, azoxymethane (AOM). Following AOM exposure, limited numbers of preneoplastic lesions, referred to as aberrant crypt foci (ACF), are formed in the colon, and their progression to tumors rarely occurs. To determine whether genetic resistance can be overcome by exposure to a dietary tumor promoter, AOM-exposed AKR/J mice were fed a diet containing 0.25% deoxycholic acid (DCA). DCA exposure was begun 1 wk prior to or 1 wk after tumor initiation with AOM. Mice placed on the DCA diet prior to AOM treatment developed a significantly higher multiplicity of ACF compared to AOM-exposed mice fed a control diet (15.50 +/- 0.96 vs. 6.17 +/- 0.48, respectively; P < 0.05). When DCA exposure was begun after AOM treatment (post-initiation), ACF formation was further enhanced (34.00 +/- 1.22). Interestingly, increased numbers of ACF were associated with the presence of nuclear beta-catenin, assessed by immunohistochemistry. While approximately 33% of ACF from mice exposed to DCA prior to AOM treatment contained positive nuclear beta-catenin staining, approximately 77% of ACF from mice fed DCA after AOM were positive. Accumulation of nuclear beta-catenin was not associated with a loss of E-cadherin from the plasma membrane, although loss of APC staining was a consistent feature of most AOM-induced ACF, regardless of DCA exposure. These results demonstrate that exposure to DCA, an important digestive component, is sufficient to sensitize the resistant AKR/J colon to formation of high-grade dysplasia, and that nuclear translocation of beta-catenin may play an important role in this process.     

Canola Oil Influence on Azoxymethane-induced Colon Carcinogenesis,Hypertriglyceridemia and Hyperglycemia in Kunming Mice

Azoxymethane (AOM) is a potent genotoxic carcinogen which specifically induces colon cancer. Hyperlipidemiaand diabetes have several influences on colon cancer development, with genetic and environmental exposureaspects. Here, we investigated plasma lipid and glucose concentrations in Kunming mice randomized into fourgroups; control (no AOM or oil exposure), AOM control, AOM + pork oil, and AOM + canola oil. Aberrantcrypt foci (ACF), plasma cholesterol, plasma triglyceride, plasma glucose and organ weight were examined 32weeks after AOM injection. Results revealed that AOM exposure significantly increased ACF number, plasmatriglyceride and glucose level. Further, male mice displayed a much higher plasma triglyceride level than femalemice in the AOM control group. Dietary fat significantly inhibited AOM-induced hypertriglyceridemia, andcanola oil had stronger inhibitory effect than pork oil. AOM-induced hyperglycemia had no sex-difference andwas not significantly modified by dietary fat. However, AOM itself not change plasma cholesterol level. AOMsignificantly increased liver and spleen weight in male mice, but decreased kidney weight in female mice. Onthe other hand, mice testis weight decreased when fed canola oil. AOM could induce colorectal carcinogenesis,hypertriglyceridemia and hyperglycemia in Kunming mice at the same time, with subsequent studies requiredto investigate their genome association.     

Tumor formation is correlated with expression of beta-catenin-accumulated crypts in azoxymethane-induced colon carcinogenesis in mice

We have reported that β-catenin-accumulated crypts (BCAC) are independent of aberrant crypt foci (ACF) in the colonic mucosa of rats exposed to colorectal carcinogens, and we suggested that they may be premalignant lesions. In the present study, we performed a comparative study on the formation of the two types of early-appearing lesions (BCAC and ACF), and tumors of the colon in two mouse strains with different susceptibility to azoxymethane (AOM). SWR/J mice are known to be relatively susceptible to AOM, whereas AKR/J mice are reported to be virtually resistant. Both AKR/J and SWR/J mice, 6 weeks old, received subcutaneous injections of AOM (15 mg/kg body weight) once a week for 3 weeks, and were sacrificed at 16 and 41 weeks of age. Colons of the animals sacrificed at 16 and 41 weeks of age were processed to examine expression of the early-appearing lesions and neoplasms. Although AKR/J mice had a lower incidence of colonic tumors than SWR/J mice did, AKR/J mice showed a similar frequency of ACF to that in SWR/J mice. In both strains, ACF were detected at high frequency in the proximal colon, whereas tumors developed mainly in the distal colon. Importantly, the incidence of BCAC in SWR/J mice was significantly higher than that in AKR/J mice, and the highest frequency was observed in the distal segments of the colon. These results support the idea that BCAC are a reliable surrogate endpoint for colon carcinogenesis in mice.     

Plasma membrane-associated sialidase (NEU3) promotes formation of colonic aberrant crypt foci in azoxymethane-treated transgenic mice

Human plasma membrane-associated sialidase (NEU3) specifically hydrolyzes gangliosides, and it is up-regulated in colon cancer and plays an essential role in the expression of malignant phenotypes. To clarify the role of NEU3 in tumorigenesis in vivo , we examined the susceptibility of NEU3 transgenic mice to induction of colonic aberrant crypt foci (ACF) by azoxymethane. Mice were injected with azoxymethane (i.p., 15 mg/kg/week) for 6 weeks, and 4 weeks later ACF had formed in the NEU3 transgenic mice significantly more than in the control wild-type mice. Enhanced phosphorylation of epidermal growth factor (EGF) receptor, Akt and ERK and up-regulation of Bcl-xL protein were observed in the transgenic colon mucosa, but no changes were found in cell proliferation, suggesting that the increased ACF formation is due to suppression of apoptosis. Immunohistological analysis with anti-cleaved caspase 3 antibody showed an actual reduction in apoptotic cells in the transgenic mucosa at 6 h after the first azoxymethane injection, when apoptosis in the colonic crypt occurs. Consistent with our previous observations of human colon cancer, thin-layer chromatography of the gangliosides from the transgenic colon mucosa revealed decreased GM3 and increased lactosylceramide as compared to those from the control mucosa, probably because of catalysis of gangliosides by NEU3. The results of this study provide the first evidence that NEU3 essentially increases azoxymethane-induced ACF formation in colon mucosa by suppression of apoptosis, possibly via activation of the EGF signaling pathway, and thus indicate that up-regulation of NEU3 is important to the promotion stage of colorectal carcinogenesis in vivo . ( Cancer Sci 2009; 100: 588–594)     

Qualitative and quantitative relationship between dysplastic aberrant crypt foci and tumorigenesis in the Min/+ mouse colon.

The multiple intestinal neoplasia (Min)/+ mouse, which harbors only one functional allele of the Apc gene, is susceptible to environmental factors that disrupt this gene and subsequently trigger Apc-driven tumorigenesis in the colon. Aberrant crypt foci (ACF) are assumed to be preneoplastic lesions in colon carcinogenesis. Recently, we reported the absence of "classical" ACF in the colon of untreated Min/+ mice. Instead we identified flat dysplastic lesions, which we denoted ACF(Min) (J. E. Paulsen et al., Scand. J. Gastroenterol., 35: 534-539, 2000). In contrast to the classical type, ACF(Min) are not elevated above the surrounding mucosa, and their detection is totally dependent on methylene blue staining and transillumination. In the present study, we treated Min/+ mice with 5 mg/kg body weight azoxymethane (AOM) at weeks 1 and 2 and demonstrated induction of both types of lesions. However, only ACF(Min) appeared to be associated with the development of adenomas. Monocryptal ACF(Min), large ACF(Min), and adenomas showed a uniform histopathological picture of dysplasia and cytoplasmic overexpression of beta-catenin, indicating a qualitative relationship between these lesions. Also a quantitative relationship was suggested because the dramatic decrease in ACF(Min) number from week 7 to 11 was paralleled by a reciprocal increase in tumor number, indicating fast-crypt multiplication of ACF(Min). In AOM-treated +/+ (wild-type) littermates, a low number of ACF(Min) and tumors with the same characteristics as in Min/+ mice was seen. In contrast to ACF(Min), histopathological and immunohistochemical examination of classical ACF showed normal or hyperplastic crypts with normal levels of beta-catenin expression. In AOM-treated Min/+ mice, the number of classical ACF was virtually constant from week 7 to 11, and only a modest increase of crypt multiplicity was observed. The number of AOM-induced classical ACF at week 11 was not different in Min/+ mice and +/+ mice. In conclusion, we identified two distinct populations of altered crypts in the colon of Min/+ mice after AOM treatment. The ACF(Min), which resemble the dysplastic ACF described previously, clearly showed a continuous development from the monocryptal stage to adenoma, and they were characterized by fast-growing crypts with altered control of beta-catenin. In contrast, the classical ACF, which resemble the hyperplastic ACF described previously, were characterized by slow-growing crypts with normal beta-catenin expression, and they were probably not related to tumorigenesis.     

Monosodium glutamate-induced diabetic mice are susceptible to azoxymethane-induced colon tumorigenesis

Obese people and diabetic patients are known to be high risk of colorectal cancer (CRC), suggesting need of a new preclinical animal model, by which to extensively study the diverse mechanisms, therapy and prevention. The present study aimed to determine whether experimental obese and diabetic mice produced by monosodium glutamate (MSG) treatment are susceptible to azoxymethane (AOM)-induced colon tumorigenesis using early biomarkers, aberrant crypts foci (ACF) and β-catenin-accumulated crypts (BCACs), of colorectal carcinogenesis. Male Crj:CD-1 (ICR) newborns were daily given four subcutaneous injections of MSG (2 mg/g body wt) to induce diabetes and obesity. They were then given four intraperitoneal injections of AOM (15 mg/kg body wt) or saline (0.1 ml saline/10 g body wt). Ten weeks after the last injection of AOM, the MSG-AOM mice had a significant increase in the multiplicity of BCAC (13.83 ± 7.44, P < 0.002), but not ACF (78.00 ± 11.20), when compare to the Saline-AOM mice (5.45 ± 1.86 of BCAC and 69.27 ± 8.06 of ACF). Serum biochemical profile of the MSG-treated mice with or without AOM showed hyperinsulinemia, hypercholesteremia and hyperglycemia. The mRNA expression of insulin-like growth factor-1 receptor (IGF-1R, P<0.01) was increased in the MSG-AOM mice, when compared with the mice given AOM alone. IGF-1R was immunohistochemically expressed in the BCAC, but not ACF, in the AOM-treated mice. Our findings suggest that the MSG mice are highly susceptible to AOM-induced colorectal carcinogenesis, suggesting potential utility of our MSG-AOM mice for further investigation of the possible underlying events that affect the positive association between obese/diabetes and CRC.     

Suppression of Azoxymethane-induced Rat Colon Aberrant Crypt Foci by Dietary Protocatechuic Acid

The modifying effect of dietary exposure to protocatechuic acid (PCA) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in male F344 rats. The effects of PCA feeding on the silver-stained nucleolar organizer regions protein (AgNORs) count in the colonic epithelial cells and on the ornithine decarboxylase (ODC) activity in the colonic mucosa were also estimated. Animals were given weekly s.c. injections of AOM (15 mg/kg body weight) for 3 weeks to induce ACF. These rats were fed diet containing 1000 or 2000 ppm PCA for 5 weeks, starting one week before the first dosing of AOM. All rats were killed 2 weeks after the last AOM injection, to measure the number of ACF, ODC activity, and AgNORs count per nucleus in the colon. In rats given AOM and PCA, the frequency of ACF/colon was significantly decreased compared with that in rats given AOM alone ( P < 0.005 at 1000 and P < 0.05 at 2000 ppm). ODC activity in the colon of rats given AOM and PCA at both doses was also significantly lower than that of rats treated with AOM alone ( P < 0.05). Similarly, the mean AgNORs count in rats fed PCA was significantly smaller than that of rats treated with AOM alone ( P < 0.0001). Treatment with PCA alone did not affect these three biomarkers. These results provide further evidence that PCA could be a chemopreventive agent against rat colon carcinogenesis.     

Identification of flat dysplastic aberrant crypt foci in the colon of azoxymethane-treated A/J mice

The role of aberrant crypt foci (ACF) as preneoplastic lesions in colon carcinogenesis is not clear. In Min/+ mice and their wild-type littermates treated with azoxymethane (AOM), we previously identified a subgroup of flat ACF that seem more immediate precursors of tumors than the classical elevated ACF. In the present study, we identified a similar subgroup of flat ACF in AOM-treated A/J mice and compared them with nascent tumors and classical elevated ACF. At week 1 and 2 after birth, A/J mice were injected subcutaneously with AOM (10 mg/kg bw/injection). At weeks 7-14, we examined the luminal surface of unsectioned colon preparations stained with methylene blue in the inverse light microscope. The lesions were also examined by histopathology and immunohistochemistry. Surface examination revealed flat ACF, classical elevated ACF and nascent tumors. Since flat ACF were not observed as elevated structures, their bright blue appearance and compressed pit pattern of crypt openings seen with transillumination were used as criteria for their identification. Flat ACF and nascent tumors displayed a uniform picture of severe dysplasia, compressed pit pattern, overexpression of cytoplasmic/nuclear beta-catenin and nuclear overexpression of cyclin D1. Apparently, flat ACF and tumors represented the same type of dysplastic lesions at different stages of crypt multiplication. In contrast, classical elevated ACF did not seem to be as clearly related to tumorigenesis. They infrequently (1/20) possessed severe dysplasia, overexpression of cytoplasmic/nuclear beta-catenin, or nuclear overexpression of cyclin D1, and they did not have compressed crypt openings. Furthermore, flat ACF grew significantly faster than classical elevated ACF. In conclusion, our data indicate a development from flat ACF to adenoma characterized by aberrant activation of the Wnt signaling pathway and fast crypt multiplication. Classical elevated ACF do not seem to be as closely related to tumorigenesis.     

Deguelin suppresses the formation of carcinogen-induced aberrant crypt foci in the colon of CF-1 mice

Deguelin [(7aS,BaS)-13,13a-dihydro-9,10-dimethoxy-3,3-dimethyl-3H-Bis[1]benzopyrano[3,4-b:6',5'-e]pyran-7(7aH)-one], a naturally occurring rotenone, has shown chemopreventive efficacy in several in vivo and in vitro models. In this report, the effectiveness of deguelin at inhibiting the development of AOM-induced colonic ACF was investigated in CF-1 mice. Loss of hex activity was assessed as a second biomarker. In an initial experiment, animals were given s.c. injections of AOM (10 mg/kg body weight) once a week for 2 weeks to induce ACF. Deguelin and vehicle (corn oil) were administered i.g. 7 days a week. Treatment was initiated 2 weeks prior to the first dose of carcinogen and continued for the duration of the study. The mean number of ACF for the control group was 29.0 +/- 4.3, whereas the mean numbers of ACF in the deguelin groups were 24.8 +/- 2.7, 7.2 +/- 1.5 and 4.6 +/- 1.4 at doses of 2.5, 5.0 and 10.0 mg/kg body weight, respectively. In a similar manner, treatment with deguelin significantly (p < 0.001) suppressed the appearance of hex(-) crypts in a dose-dependent manner. In a second study, the ability of deguelin to block the initiation and promotion stages of colon carcinogenesis was investigated. Greatest inhibition was observed when deguelin was administered during the promotional stage (73.3%, p < 0.001). These results demonstrate that deguelin is an efficacious chemopreventive agent against colon carcinogenesis.     

Resveratrol depresses the growth of colorectal aberrant crypt foci by affecting bax and p21(CIP) expression

We investigated whether resveratrol (RV) affects azoxymethane (AOM)-induced colon carcinogenesis, by administering RV (200 microg/kg/day in drinking water) to male F344 rats for 100 days, beginning 10 days before carcinogen treatment (two weekly doses of 15 mg/kg AOM). Aberrant crypt foci (ACF) were isolated and proliferation, apoptosis and expression of the cell cycle genes bax and p21 were determined. RV significantly reduced the number of ACF/colon [25.7 +/- 3.6 (mean +/- SEM) versus 39.4 +/- 3.3 in controls; P < 0.01] and their multiplicity (2.7 +/- 0.3 versus 4.9 +/- 0.6 in controls; P < 0.01), and also abolished large ACF. In RV-treated rats, bax expression was enhanced in ACF but not in the surrounding mucosa. In both controls and RV-treated rats, proliferation was higher in ACF than in normal mucosa. p21 was expressed in ACF of controls and of RV-treated rats and in normal mucosa of controls, but was lost in normal mucosa of RV-treated animals. In conclusion, the results suggest a protective role of RV in colon carcinogenesis with a mechanism involving changes in bax and p21 expression.     

Dietary Ziziphus jujuba Fruit Influence on Aberrant Crypt Formation and Blood Cells in Colitis-Associated Colorectal Cancer Mice

Ziziphus jujuba (ZJ) fruit is rich in bioactive functional components such as polysaccharides, triterpenoid acid, flavonoids and oleamide. It has been commonly used in the treatment of various diseases including diabetes, digestive disorders, diarrhea, skin infections, liver and urinary diseases. However, its dietary effect on chemoprevention of colon cancer has never been studied. The present study was to evaluate the protective effects of dietary ZJ on colitis-associated colon carcinogenesis in azoxymethane (AOM)-dextran sodium sulphate (DSS)-treated mice. AOM was injected (10 mg/kg b.wt., i.p.) and three cycles of 2% DSS in drinking water for 7 days with 14 days of normal drinking water in-between was administered to induce colitis-associated colon cancer. ZJ fruit was supplemented in feed as 5 and 10%. Dietary ZJ significantly attenuated aberrant crypt foci (ACF) formation thereby decreasing the progression of hyperplasia to dysplasia. In addition, it significantly reduced circulating white blood cells, lymphocytes, neutrophils, monocytes, eosinophils, basophils and platelets compared to colon cancer mice. We conclude that ZJ supplementation delayed the progression of colon cancer from hyperplasia to dysplasia and ultimately adenocarcinoma and cancer. In addition, it decreased circulating tumor-related leucocytes, main regulators of cancer inflammation. Therefore, dietary consumption of ZJ fruit attenuated the formation of ACF and delayed the progression of colon cancer.     

Formation of Early Lesions in 1,2-dimethylhydrazine-induced Colon Carcinogenesis in Rats

Aberrant crypt foci (ACF) are recognized as preneoplastic lesions for colon cancer, and ACF in rodents arewidely used as an intermediate biomarker to predict tumorigenicity in the colon. However, a lack of correlationsbetween the formation of ACF and the development of colonic tumors has been reported in several studies. Forexample, 2-(carboxyphenyl) retinamide (2-CPR) and genistein were reported to inhibit the carcinogen-inducedformation of ACF, whereas both of them were later found to enhance colon tumorigenesis in rats treated withazoxymethane (AOM). Recently, we have identified β-catenin-accumulated crypts (BCAC) in the colon of ratsshortly after administration of AOM, and provided evidence that these are independent early lesions of classicalACF, and BCAC might be direct precursors for colon cancers. In the present study, we performed a comparativeanalysis of the modifying effects of 2-CPR and genistein on 1,2-dimethylhydrazine (DMH)-induced BCAC andACF in male F344 rats. Dietary administration of 2-CPR (315 ppm) significantly reduced the total number,multiplicity and size of ACF in DMH-exposed colonic mucosa, while genistein (250 ppm) had no significant effectson DMH-induced ACF formation. In contrast, both of 2-CPR and genistein significantly enhanced the multiplicityand size of DMH-induced BCAC when compared with DMH alone group. In addition, both 2-CPR and genisteinsignificantly increased the proliferating cell nuclear antigen (PCNA) index preferentially in BCAC. Togetherwith previous findings that 2-CPR and genistein are tumor promoters in the colon, our results support the conceptthat BCAC are precursors of colon tumors and suggest that these lesions are more reliable short-term biomarkersfor colon carcinogenesis in rodents than ACF.