Anne-Maria Pajari

Promotion of intestinal tumor formation by inulin is associated with an accumulation of cytosolic β‐catenin in Min mice

Inulin, polydisperse β (2‐1) fructan, has been suggested to protect against colon carcinogenesis and is currently used in a number of food applications. However, the data regarding the role of inulin in intestinal carcinogenesis remains controversial since the results of our previous study suggested that inulin promotes intestinal tumor formation in Min mice, an animal model for intestinal cancer with a mutation in the Apc tumor suppressor gene (Carcinogenesis 2000;21:1167–73). In our present study, we further examined the effects of inulin on intestinal tumor formation in Min mice by carefully analyzing β‐catenin expression and cellular localization at 3 different time points during the tumorigenic process. Min mice were fed a high‐fat inulin‐enriched (10% w/w) diet or the high‐fat diet without any added fiber from the age of 6 weeks to the ages of 9, 12 or 15 weeks. The results showed that inulin significantly increased the number (by 20%) and especially the size (by 44%) of adenomas in the small intestine. At week 15, the promotion of tumor development was accompanied by an accumulation of cytosolic β‐catenin in the adenoma tissue. In the normal appearing mucosa, levels of membrane β‐catenin and PCNA were reduced in the inulin‐fed mice, possibly indicating impaired enterocyte migration. These data do not support the earlier suggestions on the cancer preventive effects of inulin and emphasize the need for further research and evaluation where health claims for inulin are concerned.

Fibers with different solubility characteristics alter similarly the metabolic activity of intestinal microbiota in rats fed cereal brans and inulin

We compared the effects of different sources of fiber (rye-bran, oat-bran, wheat-bran) and inulin on the activity of fecal bacterial enzymes and concentration of short chain fatty acids (SCFA) in the colonic contents of rats. A diet without fiber was used as a control. The cereal brans and inulin had similar, increasing effect on the activities of β-glucuronidase and urease (P < 0.05). All bran diets also increased β-glucosidase activity while the increase by inulin was non-significant. Concentration of total SCFA was higher in the inulin and rye-bran groups compared to non-fiber and wheat-bran groups (P < 0.05). Concentration of butyrate was higher in rye-bran and inulin groups compared to the non-fiber group (P < 0.05), and that of propionate higher in the inulin group compared to non-fiber and wheat-bran groups (P < 0.05). The SCFA profile in the oat-bran group did not differ significantly from SCFA profile in the other groups. These findings indicate that the source of the fiber does not markedly affect the enzyme activities of the fecal bacterial microbiota, but may cause differences in the profile of SCFA in the rat colon.

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.

Ellagic acid and natural sources of ellagitannins as possible chemopreventive agents against intestinal tumorigenesis in the Min mouse.

Abstract: Ellagic acid has been shown to have chemopreventive effects in various experimental cancer models. We wanted to see whether pure ellagic acid and natural ellagitannins from cloudberry (Rubus chamaemorus) seed and pulp have any effect on adenoma formation in Apc-mutated Min mice. From the age of 5 wk, the mice were fed either a control diet, a diet containing pure ellagic acid at 1,564 mg/kg, or diets containing 4.7% (wt/wt) cloudberry seeds or 5.3% cloudberry pulp. The concentrations of ellagitannins and free ellagic acid in the seed diet were 807 and 42 mg/kg and in the pulp diet 820 and 34 mg/kg, respectively. After the 10-wk feeding period, ellagic acid had no effect on the number or size of adenomas in the distal or total small intestine, but it increased adenoma size in the duodenum when compared with the control diet (1.50 ± 0.29 vs. 1.16 ± 0.31 mm; P = 0.029). Neither cloudberry seed nor pulp diets had any effect on the adenoma formation. Chemopreventive effects and mechanisms of whole cloudberry and other similar sources of phenolic compounds should, however, be studied, further taking into account food matrix and interactions with other dietary constituents that may be involved in the bioavailability and metabolism of ellagitannins.

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

Role of red meat and arachidonic acid in protein kinase C activation in rat colonic mucosa.

Two studies were conducted to investigate the role of meat and arachidonic acid in colonic signal transduction, particularly protein kinase C (PKC) activation. In Study 1, 26 male Wistar rats were fed a casein- or a beef-based diet for four weeks. PKC activity was measured from the proximal and distal colonic mucosa and diacylglycerol concentration from fecal samples. The beef diet significantly increased membrane PKC activity in the proximal and distal colon and cytosolic PKC in the distal colon. No differences were found in fecal diacylglycerol concentration for the rats maintained on the two diets. In Study 2, 57 male Wistar rats were divided into three dietary treatment groups: a control group, a group supplemented with arachidonic acid at 8 mg/day (an amount equivalent to that available from the beef diet in Study 1), and a group supplemented with fish oil at 166 mg/day. After a four-week supplementation period, 6 rats per group were used for colonic phospholipid fatty acid analysis and 13 rats per group were used for analysis of colonic prostaglandin E2 concentration, sphingomyelinase, and PKC activities. Supplementation of dietary arachidonic acid resulted in incorporation of arachidonic acid into colonic phosphatidylcholine, which was associated with an increase in mucosal prostaglandin E2 concentration compared with the fish oil group. However, arachidonate supplementation had no effect on sphingomyelinase or PKC activities. These data indicate that meat significantly increases colonic PKC activity, but this effect is probably not due to the arachidonic acid content of meat.

Protein kinase C activation in rat colonic mucosa after diets differing in their fatty acid composition

We studied the effects of different types of dietary fats on fatty acid composition and activity of protein kinase C (PKC) in rat colonic mucosa. Activation of PKC, a key enzyme in signal transduction and growth regulation, provides a mechanism by which dietary components could be involved in colon carcinogenesis. Male Wistar rats (n = 12/group) were fed a semisynthetic high fat diet (43% of energy) containing either sunflower oil, rapeseed oil, or butter for 4 weeks ad libitum. The control group received a low fat sunflower oil diet (10% of energy). The butter diet increased membrane-associated PKC activity in rat colonic mucosa compared with the low fat control diet (1237 vs. 917 pmol/min per mg prot.; P = 0.028). Mucosal fatty acids reflected dietary fatty acid composition even though there was no clear association between the amount of mucosal fatty acids and PKC activity. More research is needed to elucidate how dietary fatty acids regulate colonic PKC activity.

Phospholipid fatty acid composition and protein kinase C activity in the large intestine of rats fed on butter and coconut-oil diets

Protein kinase C (PKC) has been proposed to play an important role in the aetiology of colon cancer. Therefore, we investigated whether the amount and type of saturated fat could affect colonic PKC activity by modifying either mucosal phospholipid fatty acid composition or faecal diacylglycerol production. Male Wistar rats (n 13 per group) were fed on diets containing butter or coconut oil at energy levels of 10% and 43% for 4 weeks. The control group received a low-fat diet providing 10% of energy from sunflowerseed oil. PKC activity was higher in the distal than the proximal colon but the quantity or type of fat did not alter PKC activity in either region of the colon. Saturated fats caused moderate changes in the fatty acid composition of caecal phospholipids, which were more obvious in the phosphatidylethanolamine than in the phosphatidylcholine fraction. A significant correlation was found between fatty acid composition of phosphatidylcholine and membrane PKC activity. In particular, there was a positive correlation between the proportion of saturated 14:0 and 18:0 and increased PKC activity while unsaturated 18:2n-6, 20:4n-6 and 16:1n-7 were inversely correlated with PKC activity. No relationship was found between phosphatidylethanolamine fatty acids and PKC activity. Concentration of faecal diacylglycerol was not affected by the diet. Overall the data suggest that diets high in saturated fat may not alter colonic PKC activity to a significant extent.

Inulin results in increased levels of beta-catenin and cyclin D1 as the adenomas increase in size from small to large in the Min/+ mouse.

The mechanism that drives the growth of some colonic adenomas towards malignancy, while permitting others to remain for decades in quiescence, remains unknown. Diets can alter the growth rate of intestinal tumours but it is still unknown whether diets are able to alter the molecular biology of these adenomas in a way that predicts further outcome. To address this issue we fed Min/+ mice with two diets known to lead to different adenoma outcomes: a high-fat control diet (n 15) or a high-fat inulin-enriched (10 % w/w) diet (n 13). To study the effect of diet on cell signalling during adenoma growth, the adenomas of each Min/+ mouse were divided into three size-categories, and the levels of beta-catenin, E-cadherin, cyclin D1 and matrix metalloproteinase-9, which are known to be involved in colon tumorigenesis, were determined. The growth-promoting inulin diet resulted in more large adenomas than the control feeding (P = 0.003) and doubled the total area of the adenomas (P = 0.008). The inulin diet increased the expression of nuclear beta-catenin (P = 0.004) and its target cyclin D1 (P = 0.017) as the adenomas increased in size from small to large, indicating the presence of an accelerated cancerous process. Neither phenomenon was seen in the control group during adenoma growth. Our results suggest that in addition to the number, size, and growth rate of adenomatous polyps, the signalling pattern of the adenomas should also be considered when evaluating preventive dietary strategies.