Seija I. Oikarinen

Disruption of estrogen receptor signaling enhances intestinal neoplasia in ApcMin/+ mice

Estrogen receptors (ERs) [ERalpha (Esr1) and ERbeta (Esr2)] are expressed in the human colon, but during the multistep process of colorectal carcinogenesis, expression of both ERalpha and ERbeta is lost, suggesting that loss of ER function might promote colorectal carcinogenesis. Through crosses between an ERalpha knockout and Apc(Min) mouse strains, we demonstrate that ERalpha deficiency is associated with a significant increase in intestinal tumor multiplicity, size and burden in Apc(Min/+) mice. Within the normal intestinal epithelium of Apc(Min/+) mice, ERalpha deficiency is associated with an accumulation of nuclear beta-catenin, an indicator of activation of the Wnt-beta-catenin-signaling pathway, which is known to play a critical role in intestinal cancers. Consistent with the hypothesis that ERalpha deficiency is associated with activation of Wnt-beta-catenin signaling, ERalpha deficiency in the intestinal epithelium of Apc(Min/+) mice also correlated with increased expression of Wnt-beta-catenin target genes. Through crosses between an ERbeta knockout and Apc(Min) mouse strains, we observed some evidence that ERbeta deficiency is associated with an increased incidence of colon tumors in Apc(Min/+) mice. This effect of ERbeta deficiency does not involve modulation of Wnt-beta-catenin signaling. Our studies suggest that ERalpha and ERbeta signaling modulate colorectal carcinogenesis, and ERalpha does so, at least in part, by regulating the activity of the Wnt-beta-catenin pathway.

Effects of a flaxseed mixture and plant oils rich in α-linolenic acid on the adenoma formation in multiple intestinal neoplasia (Min) mice

Flaxseed is a dietary source of possible chemopreventive compounds such as lignans and alpha-linolenic acid (ALA). To study the effects of a flaxseed mixture on adenoma formation in multiple intestinal neoplasia mice, the mice were fed a diet containing 2.7 % flaxseed, 4.5 % fibre and 3.7 % ALA. To elucidate the effect of oils of the mixture we also composed a diet without flaxseed but with the same oil composition. The median number of adenomas in the small intestine was fifty-four for the control group, and thirty-seven (P=0.023) and forty-two (P=0.095) for flaxseed and oil groups, respectively. Compared with controls (1.2 mm), the adenoma size was smaller in the flaxseed (0.9 mm; P=0.002) and oil (1.0 mm; P=0.012) groups. Both diets changed the proportions of n-3 and n-6 fatty acids in the colonic mucosa. Membrane beta-catenin and protein kinase C (PKC)-zeta levels were reduced in the adenoma v. mucosa (P<0.05), and an inverse association was found between the membrane PKC-zeta in the mucosa and the adenoma number (r -0.460, P=0.008, n 32). Only the flaxseed diet increased lignan levels in the caecum (P=0.002) and in plasma (P=0.002) but they were not associated with tumour formation. The results suggest that the preventive effect of flaxseed on colon carcinogenesis may be due to the oil part of flaxseed, and the loss of beta-catenin and PKC-zeta from the membranes of the mucosal tissue may play a permissive role in intestinal tumour development.

Chemopreventive activity of crude hydroxsymatairesinol (HMR) extract in ApcMin mice

We studied the effects of a lignan, hydroxymatairesinol (HMR), and rye bran on intestinal tumor development in adenomatous polyposis colimultiple intestinal neoplasia (Apc)(Min) mice. HMR showed a strong chemopreventive effect in this animal model. The mean number of adenomas in the small intestine was significantly lower (26. 6+/-11.0, P<0.05) in mice fed the inulin and HMR when compared with the inulin and inulin/rye bran fed mice (39.6+/-8.9 and 36.0+/-7.4, respectively). HMR resulted in normalization of beta-catenin levels in adenoma tissue, indicating that HMR mediates its chemopreventive effect through the Apc-beta-catenin pathway. In the cytosolic fraction, beta-catenin level in adenoma tissue was significantly elevated (P=0.008-0.013) in all the diet groups as compared with that of the surrounding mucosa. In the nuclear fraction, beta-catenin in the inulin (3.15+/-2.9 relative units) and inulin/rye (5.17+/-6.94 relative units) groups was also significantly higher (P=0.003-0.009) in the adenoma tissue when compared with the surrounding mucosa (0.5+/-0.5 and 0.35+/-0.39 relative units). However, HMR was able to restore nuclear beta-catenin level of the adenoma tissue (0.41+/-0.25 relative units) to the level found in the surrounding mucosa (0.36+/-0.28 relative units).

Diets enriched with cereal brans or inulin modulate protein kinase C activity and isozyme expression in rat colonic mucosa.

The role of dietary fibres in colon carcinogenesis is controversial. To elucidate the mechanisms by which different dietary fibre sources may affect colonic tumour development, we studied the effects of diets enriched with cereal brans or inulin on protein kinase C (PKC) activity and isozyme expression in rat colon. Male Wistar rats (twelve per group) were fed one of the following AIN-93G-based diets (Reeves et al. 1993) for 4 weeks: a non-fibre high-fat diet or one of the four high-fat diets supplemented with either rye, oat or wheat bran or inulin at 100 g/kg diet. The fat concentration (20 g/100 g) and fatty acid composition of the non-fibre high-fat diet was designed to approximate that in a typical Western-type diet. In the proximal colon, rats fed the inulin diet had a significantly higher membrane PKC activity and a higher membrane PKC d level than rats fed the non-fibre dietOP , 0·05U: In the distal colon, rats fed the inulin and oat bran diets had a higher total PKC activity and a higher membrane PKC b2 level than rats fed the wheat-bran diet. Rats in the non-fibre and wheat-bran groups had the lowest concentrations of luminal diacylglycerol. In conclusion, feeding of wheat bran resulted in low distal PKC activity and expression of PKC b2, a PKC isozyme related to colonic cell proliferation and increased susceptibility for colon carcinogenesis, which may explain in part the protective effect of wheat bran against tumour development in a number of experimental colon cancer studies. The increase in PKC activity and PKC b2 expression by feeding inulin may be a drawback of inulin as a functional food. Cereal brans: Inulin: Protein kinase C isozymes: Colon

Plasma enterolactone or intestinal Bifidobacterium levels do not explain adenoma formation in multiple intestinal neoplasia (Min) mice fed with two different types of rye-bran fractions

The study was designed to evaluate whether two types of rye-bran fractions result in distinct bifidogenic effect or enterolactone production in multiple intestinal neoplasia (Min) mice and whether these parameters are associated with intestinal tumorigenesis in this animal model. The experimental diets were a non-fibre diet (control), a rye-bran diet, and diets containing either the soluble extract or the insoluble fraction prepared from rye bran. The main result on adenoma formation in these experiments was the observation that the soluble extract increased number (P=0.012) and size (P=0.008) of adenomas in the distal small intestine when compared with the non-fibre group. All rye-supplemented diets supported similarly the in vivo growth of Bifidobacterium (10(8)-10(9) colony forming units/g) in Min mice, whereas the non-fibre diet lowered intestinal Bifidobacterium below the level of detection. The results show that water solubility does not affect the bifidogenicity of rye bran. Mean plasma enterolactone concentration was highest in the rye-bran group (30.0 nmol/l; P=0.002), which along with the soluble-extract group (16.2 nmol/l; P=0.024) differed significantly from the non-fibre diet group (7.5 nmol/l). Thus, the mice fed with the rye bran were the best enterolactone producers. In conclusion, rye bran and rye fractions influence adenoma formation in Min mice to a varying degree but plasma enterolactone levels or the production of bifidogenic bacteria do not mediate the effect.

Genetic mapping of Mom5, a novel modifier of ApcMin-induced intestinal tumorigenesis

The initial purpose of this study was to assess the role of estrogen receptor beta (ERbeta) in intestinal tumorigenesis by examining the effects of an ERbeta knockout (ERbeta(-/-)) on Apc(Min) mice. In order to accomplish this goal on a uniform genetic background, we were required to backcross the ERbeta knockout from the 129P2 genetic background to the B6 genetic background for 10 generations. Midway through this process, we performed a test cross in which mice from the N(5) backcross generation of the ERbeta knockout strain were intercrossed with Apc(Min/+) mice to obtain Apc(Min/+) ERbeta(+/+), Apc(Min/+) ERbeta(+/-) and Apc(Min/+) ERbeta(-/-) mice. Intestinal tumorigenesis in the N(5)F(2) mice was evaluated at 14 weeks of age. The analysis of the impact of ERbeta in the N(5) cross was complicated by segregating 129P2-derived alleles that affected tumor number and were unlinked to ERbeta. Genetic linkage analysis of this cross permitted the localization of a single genetic modifier of tumor number in Apc(Min/+) mice. This locus, Modifier of Min 5 (Mom5), maps to proximal mouse chromosome 5; the 129P2 allele of this locus is associated with a 50% reduction in mean intestinal tumor number. Through in silico analysis and confirmatory sequencing, we have identified the Rad50-interacting protein-1 gene as a strong candidate for Mom5.

A high-beef diet alters protein kinase C isozyme expression in rat colonic mucosa.

We recently reported that a red meat (beef) diet relative to a casein-based diet increases protein kinase C (PKC) activity in rat colonic mucosa. The purpose of this study was to further elucidate the effects of a high-beef diet on colonic intracellular signal transduction by analyzing steady-state protein levels of different PKC isozymes as well as activities of the three types of sphingomyelinases. Male Wistar rats (n = 12/group) were fed AIN93G-based diets either high in beef or casein for 4 weeks. Rats fed the beef diet had significantly (P < 0.05) higher cytosolic PKC alpha and lower membrane PKC delta protein levels than rats fed the casein diet. The beef-fed rats also had alterations in subfractions of PKC zeta/lambda so that they had a significantly (P = 0.001) lower level of membrane 70 & 75 kDa fraction and a higher (P = 0.001) level of cytosolic 40 & 43 kDa fraction than rats fed the casein diet. Because protein levels analyzed with a PKC zeta-specific antibody were similar, these differences in PKC zeta/lambda were probably due to changes in PKC lambda expression. PKC beta2 levels did not differ between the dietary groups. Diet had no significant effect on the activity of acid, neutral, or alkaline sphingomyelinase. This study demonstrated that consumption of a high-beef diet is capable of modulating PKC isozyme levels in rat colon, which might be one of the mechanisms whereby red meat affects colon carcinogenesis.

Tumour formation in multiple intestinal neoplasia (ApcMin/+) mice fed with filtered or unfiltered coffee.

Background The aetiology of colorectal cancer has strong dietary links, and there may be an association between coffee and colorectal cancer risk. Objective To study the effects of filtered (low levels of kahweol/cafestol) and unfiltered (high levels of kahweol/cafestol) coffee on tumour formation in multiple intestinal neoplasia (Apc Min/+) mice. Design Apc Min/+ mice (n=11 per group) were fed for 9 weeks with 10% w/w of these two types of coffee. Coffee was served as a dietary ingredient mixed with a semi-synthetic AIN-93G-based diet. Plasma levels of caffeine and paraxanthine were used as compliance markers. At the end of the feeding period intestinal tumour number and size were determined. The levels of β-catenin and cyclin D1, two cell-signalling proteins important to the progression of neoplasia, were also analysed in the tumour tissue. Results Plasma caffeine and paraxanthine concentrations were 3.2±1.4 and 1.7±0.4 µmol l−1 in the filtered coffee group and 3.6±2.3 and 1.6±0.6 µmol l−1 in the unfiltered coffee group. The level of plasma xanthines was below detection in the control group. The total number of tumours was equal between the dietary groups: 29 for the control, 30 (p =0.767) for the filtered coffee and 29 (p=0.430) for the unfiltered coffee groups. The levels of β-catenin and cyclin D1 in the nuclear fraction of the tumour tissue were also the same between the groups. Conclusions Filtered or unfiltered coffee (10% w/w) does not exert antitumorigenic activity in Apc Min/+ mice or change β-catenin and cyclin D1 signalling in the adenoma tissues. The results suggest that coffee does not change neoplasia progression in this animal model.

Green Leafy Vegetables in Cancer Prevention

Green leafy vegetables contain a wealth of potential chemopreventive compounds. Chlorophyll and its derivatives can trap aflatoxin and other mutagens by complex formation and appear protective against carcinogens in various animal and human models. They also have antioxidative and immunomodulatory properties. Folate is essential in DNA synthesis and methylation, and is required especially by rapidly proliferating tissues. For cancer prevention, dietary folate may be preferable to the much more stable folic acid used in fortification. Of the various green vegetables, spinach and perilla have been widely studied. Spinach has high antioxidant content, and its glycolipid fractions inhibit cancer cell proliferation and suppress tumours in murine models. Luteolin, rosmarinic acid and triterpenes extracted from perilla leaves are potent antitumourigenic and anti-inflammatory agents.