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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 18, 2002 as doi:10.1096/fj.01-0720fje. |
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Unit of Surgical Oncology, Aberdeen University Medical School, Foresterhill, Aberdeen AB25 9ZD, Scotland;
* Kunsan National University, Kunsan City, South Korea; and
Rowett Research Institute, Bucksburn, Aberdeen, AB21 9SB, Scotland, UK
2Correspondence: Lipid and Cell Biology Unit, Rowett ReSEarch Institute, Bucksburn, Aberdeen, AB21 9SB, Scotland, UK. E-mail: kwjw{at}rri.sari.ac.uk
SPECIFIC AIMS
Conjugated linoleic acid (CLA) inhibits mammary tumorigenesis in animals and attenuates animal and human cancer cell proliferation. The aim of this study was a detailed determination of the modulatory effect of CLA on the expression (mRNA and protein) of major proto-oncogenes that regulate cell proliferation and apoptosis in benign (MCF-10A) and malignant (estrogen receptor positive MCF-7 and negative, MDA-MB-231) mammary tumor cells.
PRINCIPAL FINDINGS
1. CLA mix differentially regulates expression of p53 and p21WAF1/CIP1 and bcl-2 mRNA in MCF-7, MBA-MD-231, and MCF-10A cells
The three cell lines were grown to subconfluence under standard culture conditions in 75 cm2 flasks and treated with varying concentrations of CLA (a 50:50 mix of 9-cis, trans-11, and trans-10, cis-12 isomers) for 24 h. Total RNA and protein was extracted and subjected to Northern, Western, and ELISA using standard procedures to determine changes in gene expression (mRNA and protein) of the above-mentioned oncogenes.
Northern blot analysis of mRNA from the three different cell types clearly showed that CLAs differentially regulated the expression of proapoptotic/antiproliferative p53 and p21WAF1/CIP1 and the anti-apoptotic bcl-2 genes (Fig. 1
A–C). p53 expression was increased significantly with 12.5–100 µM CLA in MCF-7 and MCF-10A cells, but in MDA-MB-231 cells a trend to an increase at 12.5 and 25 µM was not significant (Fig. 1A
). p21WAF1/CIP1 expression was increased with 12.5–100 µM CLA in MCF-7, 25 µM in MDA-MB-231, and was unaffected in MCF-10A cells (Fig. 1B
). In contrast, anti-apoptotic bcl-2 expression was increased in MDA-MB-231 cells with 12.5–100 µM CLA whereas MCF-7 and MCF-10A cells were unresponsive (Fig. 1C
). These observations in MCF-7 and MCF-10A cells support the reported anti-tumorigenic role of CLA in animals and clearly indicate that their mechanism of action is through regulation of major genes involved in apoptosis. Increased bcl-2 expression in MDA-MB-231 cells suggested an anti-apoptotic effect of CLA in these cells, but this was contrary to the induction of apoptosis/DNA damage observed with CLA in these cells (results not shown). Clearly, other genes were involved that counteracted the increase in bcl-2 to induce cell death (see below).
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2. CLA differentially regulates p53 wild-type, p53 mutant, p21 WAF1/CIP1 and Bcl-2 protein expression in the three cell types
Significantly increased expression of wild-type p53 protein in MCF-7 cells was observed at 50 and 100 µM CLA using specific ELISAs (Fig. 2
A). Results with Western blot analysis were similar (not shown). CLA had no effect on wild-type p53 expression in MCF-10A cells but completely suppressed the expression of the mutant p53 in MDA-MB-231 cells (Fig. 2A
). This is the first report of CLA completely reversing or suppressing the translational expression of a mutant gene and suggests new possibilities for regulation of mutated gene-products by dietary means.
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Basal expression of p21WAF1/CIP1 protein was greater in MCF-7 than the other cells (Fig. 2B
). The CLA-induced increase in expression was similar to that of wild-type p53 in MCF-7 cells, being increased at 50 and 100 µM CLA (Fig. 2B
). Increases were also observed with MDA-MB-231 and MCF-10A cells, although in absolute terms values were much lower than in MCF-7 cells (Fig. 2B
). High Bcl-2 protein expression in MCF-7 cells was significantly reduced and the low basal expression in MDA-MB-231 cells was significantly increased when assessed by specific ELISA (Fig. 2C
). The low basal expression of Bcl-2 in MCF10A cells was reduced further by CLA treatment at 12.5 and 25 µM concentrations (Fig. 2C
). These findings generally mirrored the mRNA results (see above) and all but the increased Bcl-2 in MDA-MB-231 cells suggested a strong apoptotic effect of the CLA. The increased Bcl-2 protein expression in MDA-MB-231 cells reflected the increased mRNA. This was paradoxical since both increases suggested anti-apoptosis, but CLA had induced apoptosis in this cell line. This paradox led us to investigation the possibility that other proapoptotic oncogenes were being affected by CLA. bcl-2 has numerous homologous genes such as bcl-Xs, bax, bad, and bcl-XL that have pro- and anti-apoptotic regulatory effects (see Fig. 3
).
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3. CLA increases the expression of Bax and Bcl-Xs protein in MDA-MB-231 cells
We investigated the paradoxical increase in Bcl-2 protein expression in MDA-MB-231 cells by assessing the CLA-induced expression of Bax and Bcl-Xs, two proapoptotic members of the bcl-2 family of proto-oncogenes. These proteins can oppose the effects of Bcl-2, and the ratio of Bax-Bcl-Xs to Bcl-2 is regarded as a more important regulator of apoptosis than the absolute amounts of the individual proteins. Incubation of these estrogen receptor negative cells with CLA elicited a significant increase in the expression of Bax (r=0.963; P<0.03) and Bcl-Xs (r=0.947; P<0.05) as assessed by Western blot. The proportions of these proteins greatly increased the Bax-Bcl-Xs to Bcl-2 ratio by 2–3 to 1 (results not shown).
DISCUSSION AND CONCLUSIONS
CLAs are dienoic acids derived from linoleic acid by reducing or oxidizing conditions and comprise a range of positional and geometric isomers, the principal of which are the cis-9, trans-11–18:2 and trans-10, cis-12–18:2 forms. The major source of these fatty acids in the human food chain is ruminant food products, particularly dairy products, but also meat products. Linoleic acid is partially hydrogenated by rumen microbes to produce trans-vaccenic acid (trans-11–18:1) and CLAs. Trans-vaccenic acid can be further desaturated by ruminant tissue, particularly mammary tissue, by delta-9 desaturase to give the cis-9, trans-11CLA. This is postulated to be a major source of CLAs in cow’s milk. Murine and human tissues are also capable of desaturating trans-vaccenic acid to CLA.
CLAs are attributed with health benefits including anti-obesity, anti-diabetic, anti-atherogenic, and proimmune functions. These effects have been observed mainly in animals where the most potent findings have been marked anti-tumorigenic effects in animal models of cancer. CLAs also elicit inhibition of cell proliferation and induction of apoptosis in animal and human cancer cells and animal tissues. Previous reports indicated that bcl-2 expression in rat mammary tissue and c-myc expression in human mammary cancer cells is reduced by these fatty acids. Despite these earlier findings, no detailed investigation of the cellular mechanisms influenced by CLAs at the level of oncogene expression, particularly those involved in apoptosis, has to our knowledge been undertaken in human cells.
We found that a 50:50 mixture of the major isoforms, cis-9, trans-11 and trans-10, cis-12 CLAs, inhibited cell proliferation and induced apoptosis in the cell lines studied that supported previous published findings on the anti-cancer effects of these fatty acids. Linoleic acid, the precursor of CLA, was without effect on cell proliferation/cell cytotoxicity, and the CLA mix was more effective than individual isomers (not shown here; see full-length paper on-line).
Positive up-regulation by CLAs of proapoptoic p53 and p21WAF1/CIP1 and negative down-regulation of anti-apoptotic bcl-2 oncogenes at the level of mRNA and protein expression observed in the MCF-7 and MCF-10A cells clearly indicated that CLAs could elicit anti-tumorigenic effects through classical p53-dependent signal mechanisms (see Fig. 3
). MDA-MB-231 cells express the mutant form of p53 protein that is no longer proapoptotic and is believed to impair the function of any remaining wild-type p53, thereby inhibiting apoptosis. Its complete suppression by CLA in our studies was surprising and a novel finding. To our knowledge, this is the first indication that deleterious mutations can be regulated by fatty acids commonly found in the human diet. Unfortunately, the level of expression of wild-type p53 in MDA-MB-231 cells after CLA treatment was not assessed. It was also apparent that in these estrogen-negative cells, CLA induction of apoptosis was through a p53-independent mechanism (see Fig. 3
). Increased expression of the wild-type protein in MCF-7 cells with CLA further emphasized the selective, differential regulatory effects of these fatty acids that were dependent on cell type.
Induction of bcl-2 expression in MDA-MB-231 cells was also surprising since overexpression of this oncogene is known to be anti-apoptotic. However, it is the relative proportions of anti-apoptotic Bcl-2 protein to the proapoptotic analogs Bax and Bcl-Xs that determine apoptosis (see Fig. 3
). The concomitant induction of expression of these analogs of Bcl-2 by CLA observed in the MDA-MB-231 cells resulted in a higher ratio of Bax-Bcl-Xs to Bcl-2, which could explain the apoptosis elicited by CLAs in these cells.
Our findings clearly show that the CLA mix can regulate the expression of some of the major oncogenes involved in cell survival and programmed cell death signaling in human mammary tumor cells. Observations that p53-dependent and -independent pathways are induced by CLA mix in different cell types and that mutant p53 is suppressed in MDA-MB-231 cells clearly support earlier suggestions in animal studies that these fatty acids may exert anti-cancer effects through induction of apoptosis.
We did not investigate the effects of individual isomers on oncogene expression in breast cancer cells in the present study because the CLA mix was a more effective anti-proliferative agent than LA and individual isomers. However, in a recent, parallel study from our laboratory (see paper on-line) using androgen-insensitive prostate cancer cells (PC3), we observed similar proapoptotic effects of the CLA mix on cell proliferation, cytotoxicity, and oncogene expression (both mRNA and protein). Furthermore, these effects appeared to be due mainly, but possibly not entirely, to the presence of the trans-10, cis-12 isomer as the cis-9, trans-11 isomer was without significant effect. A synergism between individual isomers cannot be excluded as the mix was the most effective agent throughout. The trans 10, cis-12 CLA isomer also appears to be the most effective component of the CLA mix in some reported cell responses.
These findings also have implications for nutritional therapies in cancer prevention.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0720fje; to cite this article, use FASEB J. (July 18, 2002) 10.1096/fj.01-0720fje 
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