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Nutrition and Cancer

Adenoma Growth Stimulation by the trans-10, cis-12 Isomer of Conjugated Linoleic Acid (CLA) Is Associated with Changes in Mucosal NF-B and Cyclin D1 Protein Levels in the Min Mouse

Johanna Rajakangas³, Samar Basu^*, Irma Salminen and Marja Mutanen

Department of Applied Chemistry and Microbiology, Division of Nutrition, University of Helsinki, Helsinki, Finland; ^* Section of Geriatrics and Clinical Nutrition Research, Faculty of Medicine, Uppsala University, Uppsala, Sweden; and Department of Nutrition, National Public Health Institute, Helsinki, Finland

³To whom correspondence should be addressed. E-mail: johanna.rajakangas{at}helsinki.fi.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Conjugated linoleic acid (CLA) is a term used to describe the different conjugated isomers of linoleic acid. CLA has been found to be anticarcinogenic in mammary cancer, but its effects on colon carcinogenesis are still inconclusive. In this study, the isomer-specific effects of the cis-9, trans-11 and trans-10, cis-12 CLA isomers were investigated in the Min mouse model for intestinal carcinogenesis. The Min mice (n = 10/group) were fed either an AIN-93G control diet or a diet containing 1 g/100 g cis-9, trans-11 or trans-10, cis-12 CLA for 8 wk. The number and size of adenomas were measured and the proteins from the small intestinal tissues extracted for immunoblotting analysis. The number of adenomas did not differ, but the size of the adenomas was greater in the distal part of the small intestine in mice fed the trans-10, cis-12 isomer than in controls (1.19 ± 0.16 vs. 0.94 ± 0.21 mm, mean ± SD, P < 0.01). The same isomer caused an increase in lipid peroxidation, measured as urinary 8-iso-prostaglandin (PG)F₂. Nuclear p65 protein of the mucosal tissue was not detectable in the trans-10, cis-12 group, which differed (P < 0.05) from the control group. Cyclin D1, a target for the nuclear factor (NF)-B pathway, was elevated in the trans-10, cis-12 group compared with the control group (P < 0.01), but cyclooxygenase-2 levels were not higher. There was no difference in ß-catenin protein levels between the groups. The results indicate that the trans-10, cis-12 isomer of CLA can act as a cancer promoter in colon carcinogenesis possibly through pathways affecting NF-B and cyclin D1.

KEY WORDS: • Min mouse • conjugated linoleic acid • colon cancer

The term conjugated linoleic acid (CLA) refers to the different conjugated isomers of linoleic acid (18:2). The two main forms are the cis-9, trans-11 and trans-10, cis-12 isomers; cis-9, trans-11 is the main isomer in milk and the meat of ruminants. A synthetic mixture of these two isomers has been used in the majority of the experimental and in vitro studies on CLA. Several studies have shown CLA to inhibit chemically induced tumor formation and growth (1–4) as well as the growth of cells in culture (5–7). The most convincing evidence of the anticarcinogenic effects of CLA was obtained from studies on the formation and growth of mammary tumors (2,3,8–11), but CLA has also been found to inhibit the formation of skin and forestomach neoplasia (1,4). Its effects on colon cancer, on the other hand, are still inconclusive. In rats, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine–induced mutations and aberrant crypt foci (ACF) formation were reduced by CLA in the colon, but increased in the cecum (12). When CLA was administered to rats after the initiation of cancer by azoxymethane, there was no inhibitory effect on ACF formation in the colon (13). Recently, a CLA mixture was studied in the multiple intestinal neoplasia (Min) mouse model (14,15). In these two experiments, CLA did not act as an anticarcinogenic substance; rather, CLA increased the size of adenomas developed in the intestine in our study (15). These results indicate that in contrast with mammary cancer, the actions of a mixture of CLA isomers may not be consistently favorable in intestinal cancer. It is quite possible, however, that the individual isomers influence the carcinogenic process differently. To study the effect of the main isomers on colon cancer formation and to elucidate possible mechanisms for adenoma growth, we fed the cis-9, trans-11 and trans-10, cis-12 isomers separately to Min mice, an animal model for intestinal carcinogenesis.

Min mice have a heterozygous mutation in the adenomatous polyposis coli (Apc) gene; as a consequence, they develop sporadic tumors in the intestine. The human APC gene is very similar to the murine Apc, and a mutation in the APC gene is found in up to 80% of colon cancer cases (16). A mutation in the Apc gene leads to a dysfunction of the degradation of ß-catenin by the Apc-GSK3ß complex (17,18). This leads to an accumulation of ß-catenin in the cell. In the nucleus, ß-catenin can bind to the Tcf-4 transcription factor and cause an overexpression of its target genes, which regulate cell proliferation (19–22). Dysregulated ß-catenin has been linked to the hyperproliferation of colonocytes (23) and the growth of colon cancer cells (24).

Previous research has shown that CLA increases in vivo lipid peroxidation in humans measured as urinary 8-iso-prostaglandin (PG)F₂ (25,26). This biomarker has been shown to be a reliable indicator of nonenzymatic lipid peroxidation (27,28). It has been suggested that lipid oxidation products would be toxic to the cancer cells and in that way have anticarcinogenic effects (29,30). On the other hand, it is possible that an increase in lipid peroxidation, as seen after CLA administration, could activate the nuclear factor (NF)-B pathway. NF-B is a nuclear transcription factor whose activation has been linked to the progression of cancer (31). In addition to oxidative stress, the pathway is activated by various other stress factors such as inflammation, mitogens, and UV-radiation [reviewed in (32)]. Activation of the NF-B pathway leads to the degradation of the inhibitory B (IB)-NF-B complex after which the active NF-B heterodimer is transferred into the nucleus. There it binds to its target genes such as cyclooxygenase (COX)-2 and cyclin D1 (33,34), both of which are important mediators of tumor growth (35–37). To test the hypothesis that increased lipid peroxidation induced by CLA is related to the NF-B pathway, the level of nuclear NF-B was measured as well as the level of its target gene products, cyclin D1 and COX-2. Because the ß-catenin pathway is dysregulated in Min mice, we also measured the levels of ß-catenin to determine whether this pathway is activated by CLA isomers and whether changes in cyclin D1 could be explained by increased ß-catenin.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The Laboratory Animal Ethics Committee of the Faculty of Agriculture and Forestry, University of Helsinki approved the study protocol. Male C57BL/6J Min mice (6 wk old) were obtained from Jackson Laboratories (Bar Harbor, ME). The mice were randomly divided into three experimental groups, n = 10. During the feeding period of 8 wk, the mice were kept in a temperature- and humidity-controlled animal facility with a 12-h light:dark cycle. The mice had free access to food and water throughout the experiment. The mice were weighed weekly to determine their growth rate.

The control mice were fed a low fat AIN-93G diet in which soybean oil was replaced by sunflower and rapeseed oils (38). The experimental diets were composed in accordance with the control diet (Table 1); 1 g/100 g cis-9, trans-11 (90.3% cis-9, trans-11 CLA, 4.5% oleic acid, 2.4% trans-10, cis-12 CLA, 1.3% trans-9, trans-11 and trans-10, trans-12 CLA and <1% cis-9, cis-11 and cis-10, cis-12 isomers) or trans-10, cis-12 CLA (92.5% trans-10, cis-12 CLA, 3.7% cis-9, trans-11 CLA, 1.7% trans-9, trans-11 and trans-10, trans-12 CLA and <1% cis-9, cis-11 and cis-10, cis-12 isomers) (Natural ASA, Hovdebygda, Norway) was added to the AIN-93G diet, and the equivalent amounts of sunflower and rapeseed oils were reduced to maintain the same proportions as those of the control diet.

Plasma total lipids were extracted and methylated as described by Folch et al. (40) and Stoffel et al. (41). The percentile distribution of methylated fatty acids was determined by gas chromatography (Hewlett-Packard 6890 using workstation software A.06.01, Palo Alto, CA) with a 60-m Supelco SP 2380 column (Supelco, Bellefonte, PA) and hydrogen as carrier gas. Fatty acid peaks from 14:0 to 22:6 were identified in a temperature-programmed run. The CLA isomers were identified with a standard mixture of methylated conjugated linoleate (Nu-Chek-Prep, Elysian, MN). The results were expressed as a normalized percentage fatty acid composition.

Unextracted urinary samples were analyzed for 8-iso-PGF₂ by a highly specific and validated RIA as described elsewhere (28). The cross-reactivity of the 8-iso-PGF₂ antibody with 15-keto-13,14-dihydro-8-iso-PGF₂, 8-iso-PGF_2ß, PGF₂, 15-keto-PGF₂, 15-keto-13,14-dihydro-PGF₂, thromboxane B₂, 11ß-PGF₂, 9ß-PGF₂ and 8-iso-PGF₃ was 1.7, 9.8, 1.1, 0.01, 0.01, 0.1, 0.03, 1.8 and 0.6%, respectively. The detection limit of the assay was 23 pmol/L. The urinary levels of 8-iso-PGF₂ were adjusted for creatinine concentration.

Total proteins of the adenoma tissue and the nuclear fraction of the mucosa were extracted from the collected tissue samples of the distal small intestine. The tissues were homogenized at 4°C in a 20 mmol/L TrisHCl, 10 mmol/L EGTA, 2 mmol/L EDTA, 1 mol/L saccharose buffer with added inhibitors. The mucosa was then centrifuged at 200 x g for 10 min so that cells and unhomogenized cell debris could be excluded from the sample. The supernatant was then further centrifuged at 1000 x g for 10 min. The pellet was resuspended in buffer containing 0.2% Triton X-100 and centrifuged at 15000 x g for 15 min at 4°C. The adenoma tissue was centrifuged once at 1000 x g for 10 min in a buffer containing Triton X-100. The supernatants containing the extracted nuclear proteins of the mucosa and the total proteins of the adenoma were then concentrated using Millipore ultrafree-4 concentration tubes (Millipore, Bedford, MA). Protein concentration of the samples was determined using the Bio Rad Protein Assay method (Bio Rad Laboratories, Hercules, CA). Sample buffer containing SDS was added and the samples were boiled to ensure denaturation of the proteins.

The protein was separated in a 10% acrylamide gel according to molecular weight by SDS-PAGE gel electrophoresis. NF-B and cyclin D1 were measured from the nuclear fraction of the mucosa, and ß-catenin and COX-2 were measured from the total protein of the adenoma. For the NF-B, cyclin D1 and COX-2 measurements, 20 µg of protein was loaded and a RAW-cell homogenate was used as a positive control. For ß-catenin measurements, 5 µg of protein was loaded onto the gel and rat brain homogenate was used as positive control. The protein was transferred to a nitrocellulose membrane (Hybond ECL, Amersham Pharmacia Biotech, Little Chalfont, UK) and blocked in a TBS buffer containing 0.1% Tween and 3.5% fat-free soy powder at 4°C overnight. The protein was detected by incubation in specific antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), washing with TBS-Tween and incubation with a horseradish peroxidase–conjugated secondary antibody. After incubation, the membranes were washed with TBS-Tween and the proteins were visualized using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). The membranes were analyzed using the Bio Rad GS-710 Calibrated Imaging Densitometer and the Quantity One program (Bio Rad Laboratories). Signal specificity was confirmed by using antibody-specific blocking peptides for NF-B, cyclin D1, COX-2 and ß-catenin (Santa Cruz Biotechnology) according to the manufacturer’s instructions. Lamin B (Santa Cruz Biotechnology) was used as a loading marker to ensure equal loading of nuclear proteins.

The data were analyzed statistically using the nonparametric Mann-Whitney U test for independent samples. SPSS (version 9.0, SPSS, Chicago, IL) statistical program was used in all analyses. Values presented are means ± SD. Differences were considered significant when P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The growth rate of the mice did not differ among the three groups. The initial weight of the mice was 21.4 ± 1.1 g. The final weights were 27.7 ± 2.0, 28.3 ± 2.1 and 27.6 ± 2.5 g in the control, cis-9, trans-11 and trans-10, cis-12 groups, respectively.

In the control mice, small amounts of the two CLA isomers were found in the plasma, whereas there was a significant increase in the plasma levels of the relevant isomer in the experimental groups. The cis-9, trans-11 group had 4.9 ± 0.7% of this isomer in the plasma, and small amounts of the trans-10, cis-12 isomer. The trans-10, cis-12 group had 3.9 ± 1.6% of this isomer in their plasma and only 0.39 ± 0.1% of the cis-9, trans-11 isomer (Table 2).

2

2

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View larger version (23K): [in this window] [in a new window]   FIGURE 1 Urinary levels of 8-iso-PGF2 in Min mice fed different isomers of conjugated linoleic acid (CLA) for 8 wk. For each group, the box represents the interquartile range, which contains 50% of values. The whiskers extend from the box to the highest and lowest values. Medians are indicated by lines across the boxes. n = 7 for control and cis-9, trans-11 groups; n = 4 for trans-10, cis-12 group. *Different from control, P < 0.01.

View this table: [in this window] [in a new window]   TABLE 4 Protein levels of nuclear factor (NF)-B, ß-catenin and cyclooxygenase (COX)-2 in the mucosa or adenomas of the distal small intestine of Min mice fed different isomers of conjugated linoleic acid (CLA)1

View larger version (17K): [in this window] [in a new window]   FIGURE 2 Representative Western blot for nuclear factor (NF)-B in the mucosal tissue of Min mice fed different isomers of conjugated linoleic acid (CLA) for 8 wk. The p65 subunit of NF-B was measured from the nuclear fraction of the mucosal tissue of Min mice fed a control diet (1), cis-9, trans-11 conjugated linoleic acid (CLA) (2) or trans-10, cis-12 CLA (3) for 8 wk. RAW-cell homogenate was used as an internal standard and lamin B as a loading control.

View larger version (25K): [in this window] [in a new window]   FIGURE 3 Nuclear cyclin D1 levels in the mucosal tissue of Min mice fed different isomers of conjugated linoleic acid (CLA) for 8 wk. Nuclear protein levels were measured by immunoblotting and results are represented as relative intensities. For each group, the box represents the interquartile range, which contains 50% of values. The whiskers extend from the box to the highest and lowest values. Medians are indicated by lines across the boxes. n = 7 for control and cis-9, trans-11 groups; n = 4 for trans-10, cis-12 group. *Different from control, P < 0.01.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

2

Earlier research shows that CLA increases lipid oxidation in vivo (25,26). We examined the effect of two CLA isomers on the peroxidation of PGF₂, and measured the amount of the peroxidation product, 8-iso-PGF₂ excreted in the urine. The peroxidation was significantly higher in the trans-10, cis-12 group than in the control group, although the oxidation in the cis-9, trans-11 group did not differ from that in the control group. The rise in peroxidation product was significant although only a few mice could be analyzed due to the small sample sizes. Lipid peroxidation has been proposed as one of the mechanisms by which CLA is toxic to cancer cells (29,30). Our study does not support this theory because the adenomas grew larger in the trans-10, cis-12 group, in which the peroxidation was higher than in the control group. Another possible effect of the increased oxidative stress may be the activation of the NF-B pathway. Inflammatory cytokines such as tumor necrosis factor- or interleukin-1, endotoxins, oxidative stress and other stress factors are all activators of NF-B. In the nucleus of the mucosal tissue in the control and cis-9, trans-11 groups, the protein levels of the p65 subunit of NF-B were high in some mice and undetectable in others. Interestingly, no NF-B appeared in the nucleus of the mucosa in the trans-10, cis-12 group, although this diet induced oxidative stress and growth of adenomas. One possible explanation for this surprising finding could be that this isomer of CLA does not activate this pathway. Nuclear levels of cyclin D1, however, were significantly higher in the trans-10, cis-12 group than in the control group. This could indicate that the pathway actually was activated, but active NF-B was already transported out from the nucleus at this stage in the carcinogenic process. Because we measured the protein levels of p65 in the nucleus, and not the transcriptional activity of the p65 subunit, it is also conceivable that the subunit is not transcriptionally active, although present in the nucleus.

Another possible explanation for adenoma growth with nuclear absence of NF-B is that in the mucosal cells of the Min mouse, the NF-B pathway activates apoptotic responses, and in situations in which the NF-B pathway is not activated, apoptosis is inhibited and cells proliferate. It is possible that the control and cis-9, trans-11 diets activate NF-B, and consequently also apoptosis by the NF-B targets Fas or through the tumor suppressor p53 (42–44). Another explanation is that the Apc mutation that causes the mucosal tissue to be cancer prone or the possible inflammation caused by the adenomas in the Min mouse is sufficient to activate the NF-B apoptotic responses. If trans-10, cis-12 CLA inhibits activation of NF-B, apoptosis is blocked, and cells can proliferate. In SW480 colon cancer cells, aspirin, a known inhibitor of tumor growth, in fact activated the NF-B pathway and caused apoptosis (45). In Caco-2 cells, tumorigenesis was accelerated by transfection with Ha-ras or PyMT oncogenes. In these cells, the tumorigenic progression was accompanied by decreased activity and expression of NF-B (46). Our results seem to indicate that in the mucosa of the Min mouse, the nuclear absence of NF-B is associated with the progression of cancer. Unfortunately, we were unable to measure the amounts of NF-B in the adenoma tissue, again due to small samples. The information from adenomas is necessary to draw further conclusions on the effect of the NF-B on adenoma development and growth in the Min mouse.

Possible activation of NF-B at an earlier stage of tumorigenesis is supported by the fact that in the mucosa, the trans-10, cis-12 isomer also elevated cyclin D1 expression. High levels of cyclin D1 have been found in human colon cancer tumors and in Min mouse adenomas, but not in their mucosa, as determined by immunohistochemistry (47). Our results indicate that cyclin D1 is found in the nuclear fraction of the mucosa of Min mice and that adenoma growth is accompanied by an increase in mucosal cyclin D1. Dysregulated ß-catenin seen in this mouse model could also have caused an increase in cyclin D1 expression. There was no difference, however, between the control and trans-10, cis-12 groups in ß-catenin protein levels. In our studies with the Min mouse, we have found that the ß-catenin in the adenoma tissue is a better indicator of adenoma growth; therefore, in this study, it was measured not in the mucosa, but the adenoma. Various other pathways including activator protein-1 activate cyclin D1, and it is fully possible that the increase in cyclin D1 is caused by the activation of another pathway (48).

In an attempt to clarify whether NF-B could have been activated earlier in the trans-10, cis-12 group, we measured the amount of COX-2 from the adenoma, because the NF-B pathway activates COX-2. In Min mouse adenomas, COX-2 expression is up-regulated (49), and activation of COX-2 has been shown to induce angiogenesis in colon carcinoma cells, and PGE₂, a product of COX-2 oxygenation, has been shown to inhibit apoptosis (35,36). There was an indication of a rise in COX-2 by the trans-10, cis-12 isomer (P = 0.13). It is possible that NF-B could have increased the expression of COX-2, but further studies with larger groups are required to verify these results. It cannot be ruled out, however, that a slightly higher level of COX-2, and, concomitantly, a higher level of its prostaglandin products, in the trans-10, cis-12 group was responsible for the growth-promoting effect of the adenomas.

Growth of the adenomas was clearly accompanied by an increase in nuclear cyclin D1 and with nuclear absence of NF-B in the mucosal tissue. The mucosa of the Min mouse is susceptible to cancer, and changes in mucosal signaling proteins can indicate changes that lead to the formation of tumors. Further studies are warranted to gain a better understanding of the mechanism of cancer modulation by CLA and of the involvement of the NF-B pathway in the carcinogenic process.

	ACKNOWLEDGMENTS

 
The authors thank Natural ASA, Norway for kindly providing the CLA isomers.

	FOOTNOTES

 
¹ Presented in part at the Conference on Cell Signaling, Transcription and Translation as Therapeutic Targets, February 2002, Luxembourg, Luxembourg [Rajakangas, J., Salminen, I., Kettunen, H., Basu, S. & Mutanen, M. (2002) The trans-10, cis-12 isomer of conjugated linoleic acid (CLA) enlarges the intestinal adenomas of the ApcMin mouse and causes lipid accumulation in the liver. Abs. VI (32) Abstract Book p.398].

² Supported in part by the Geriatrics Research Foundation, Sweden (S.B).

⁴ Abbreviations used: ACF, aberrant crypt foci; Apc, adenomatous polyposis coli; CLA, conjugated linoleic acid; COX-2, cyclooxygenase 2; Min, multiple intestinal neoplasia; NF-B, nuclear transcription factor B; PG, prostaglandin.

Manuscript received 7 January 2003. Initial review completed 24 January 2003. Revision accepted 18 February 2003.

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