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* Division of Research, Department of Gynecology and Obstetrics, Emory University School of Medicine, Atlanta, GA 30322; and
Accelerated Pharmaceuticals, Augusta, GA 30903
3To whom correspondence should be addressed. E-mail: Nsidell{at}emory.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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expression at both mRNA and protein levels and decreased binding activity of nuclear protein to a canonical estrogen response element (EREv). Using a reporter gene construct (EREv-tk-Luc) that was transiently transfected into MCF-7 cells, we also demonstrated inhibition of promoter activity by CLA that was directly mediated by blockage of activity through the ERE. The results indicated that the order of potency of the CLA isomers for inhibiting activation of EREv was similar to that demonstrated for their antiproliferative activity on MCF-7 cells. Taken together, these findings demonstrate that CLA compounds possess potent antiestrogenic properties that may at least partly account for their antitumor activity on breast cancer cells.
KEY WORDS: • conjugated linoleic acid • breast cancer • estrogen • isomers • anticarcinogenic
Conjugated linoleic acid (CLA),4 a natural derivative of linoleic acid, is found in dairy products and meat from ruminants (1). CLA is a collective term used to describe a mixture of positional and geometric isomers of linoleic acid, which are synthesized in vivo by rumen bacteria and may also be synthesized during heat processing of animal-derived foods. At least 8 different CLA isomers have been identified. Of these, the 9cis,11trans form is likely the most common natural form of CLA and it has demonstrated biological effects on lipid metabolism (2). In recent years, biological activity has been proposed for other forms, especially the 10trans,12cis isomer.
Pariza and colleagues first demonstrated that CLA has anticarcinogenic properties against epidermal cancer cells (3–5). Later reports by a number of investigators showed that these compounds possess potent antitumor activity against breast cancer. The mechanism of this effect has not yet been elucidated although some studies have suggested involvement of the estrogen signaling pathway (6). To this end, CLA was reported to have direct oncostatic, cell cycle–inhibitory effects on estrogen receptor–positive (ER+), but not ER-, breast cancer cells in vitro (6). In contrast, linoleic acid was without activity (6,7) or even had opposite (i.e., growth stimulatory) effects (8,9). In vivo studies showed that CLA possesses substantial activity in inhibiting mammary carcinogenesis in animal models relating to the induction of estrogen-dependent tumors (10). Majumder et al. (11) reported the ability of CLA to alter the expression of key apoptotic genes such as p53, p21 WAF1/CIP1, and Bcl-2 in human breast cancer cells. A possible link between the regulation of these genes by CLA and its effects on estrogen signaling was not addressed. Other proposed mechanisms of CLA in various cell types include activation of nuclear peroxisome proliferatior-activated receptors (12) and alteration of phospholipid sensitivity to oxidative stress (13). In this study, we investigated whether CLA has direct antiestrogenic activity or can otherwise interfere with estrogen signaling in ER+ breast cancer cells.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The ER+ breast cancer cell line MCF-7 and the ER- cell line MDA-MB-231 were obtained from the American Type Culture Collection. Cells were grown and maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 50 kIU/L penicillin, 50 mg/L streptomycin, and 1 mg/L fungizone (complete medium).
Cell growth. The effects of a mixture of CLA and 5 different purified isoforms were assessed on cell growth by the sulforhodamine B (SRB) assay as previously described (14). Briefly, cells were plated in 12-well plates and treated for 2 d at the indicated concentrations of a CLA preparation containing a mixture of isomers or 5 different purified isomers or solvent control. After 2 d of culturing, the cells were fixed with 10% trichloroacetic acid (TCA) and incubated at 4°C for 1 h. Then the cells were washed 5 times with deionized water and the plates dried at room temperature. The TCA-fixed cells were stained with 0.4% SRB (Sigma Chemical) for 30 min. The excess SRB was removed by quick rinsing 4–5 times with 1% acetic acid and the plates were dried again. The dye was then solubilized with 10 mmol/L unbuffered Tris base (pH 10.5) for 5 min on a gyrotory shaker. Optical density was measured at 564 nm.
RNA isolation and RT-PCR.
Total RNA was isolated from cells using TRI-reagent (Sigma Aldrich) following the provider’s protocol. Total RNA extracts were frozen at -80°C until analysis. To amplify the cDNA fragment, the sequences of PCR primers used for detection of ER were 5'-TGTGCAATGACTATGCTTCA-3' and 5'-GCTCTTCCTCCTGTTTTTA-3', resulting in a 150-bp product. The samples were first denatured at 95°C for 3 min before 40 PCR cycles, each with temperature variations as follows: (a) 95°C for 1 min; (b) 60°C for 1 min; and (c) 72°C for 1 min. After the last cycle, an additional extension incubation of 9 min at 72°C was performed. Analysis of amplicons was visualized on 1% agarose gel containing 0.2 g/L ethidium bromide. Negative controls were parallel experiments performed in the absence of RT enzyme. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR product (200-bp) was ethidium bromide stained to confirm comparable levels of cDNA template added to the PCR. A 100-bp ladder (Promega) was used as a size standard.
Western blot analysis.
Western blot analysis was performed on whole cell extracts obtained by direct dissolution of cell pellets in sample buffer (1 mmol/L NaHCO3; 0.2 mol/L phenylmethylsulfonyl fluoride; 0.1 mol/L NaVO4; 1 mol/L NaF), followed by protein determination using a bicinchoninic acid (BCA) protein assay kit (Sigma Chemical). Protein (35 µg) from cells treated with medium (control) or CLA (200 µmol/L) was loaded on the 10% SDS-PAGE gel and then transferred to nitrocellulose and blocked with 5% skim milk in PBS. Blots were probed with primary anti-ER mouse monoclonal antibody (Neomarker) and then incubated with the secondary antibody linked to horseradish peroxidase. The immunoreactive bands were visualized using the enhanced chemiluminescence system (Amersham). Blots were washed, reprobed with an anti-ß-actin antibody, and developed in an identical manner for assessing ß-actin protein levels to ensure even loading.
Electrophoretic mobility shift assay (EMSA).
Nuclear extracts from MCF-7 cells were isolated (15). The protein concentration of the nuclear extract was determined using the BCA protein assay kit. A total of 5 µg of nuclear extract was incubated in a final volume of 20 µL containing 10 mmol/L Tris-HCl (pH 7.9), 50 mmol/L NaCl, 1 mmol/L EDTA (pH 8.0), 19% glycerol, 1 mmol/L dithiothreitol, 1 µg polydeoxyinosinic-deoxycytidylic acid at room temperature for 10 min. A total of 10 fmol of 32P-radiolabeled oligonucleotide corresponding to the vitellogenin A2-estrogen response element (EREv) [>2 x 104 counts per minute (CPM)] was added (16) and incubated for an additional 30 min. Competition was performed with the addition of oligonucleotides at a 100-fold molar excess and a mutant oligonucleotide with "GT"-"TA" substitution in the ER binding site. The reaction mixture was loaded directly onto a 5% polyacrylamide gel in 0.5X Tris-borate-EDTA buffer and analyzed at 200 V for 2 h, and the gel was dried and autoradiographed at -80°C with an intensifying screen.
Plasmid constructs and transient transfection.
The EREV-tk-luciferase (Luc) (firefly) reporter construct contains the vitellogenin A2 estrogen response element sequence from –336 to –310 (17) inserted upstream of the HSV thymidine kinase promoter (–105 to +54) (18). The peroxisome proliferator response element (PPRE)3-tk-Luc reporter construct was provided by Dr. Mitchell Lazar [University of Pennsylvania (19)]. The GAPDH-tk-Luc (renilla) reporter was used as a control for transfection efficiency and cell number. Transfection was performed with the reagent FuGene 6 method according to the manufacturer’s instruction (Boehringer-Mannheim) and cells were immediately treated with solvent control or CLA at the indicated concentrations. In some experiments, the cells were additionally cotransfected with an ER expression plasmid HEO (14) as indicated. After 48 h, cells were harvested and a dual-luciferase reporter assay system (Promega) was performed according to the manufacturer’s instructions to measure simultaneous activity of both luciferase constructs. Results were calculated as the ratio of the optical densities of luciferase activities (firefly/renilla).
Statistical analysis.
Values presented are means ± SEM of at least 3 measurements. The data in some figures are from a representative experiment, which was qualitatively similar in the replicate experiments. Statistical significance (P 
0.05) was determined using Student’s t test (2-tailed) between an experimental group and the corresponding control conditions set as 100% ("1-sample" t test).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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least potent): 9cis,11cis > 10trans,12cis > 9trans,11trans > 11cis,13trans 
9cis,11trans).
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CLA suppresses ER expression and ERE binding activity.
The ability of CLA to inhibit the growth of ER+ MCF-7 cells but not the ER- MDA-MB-231 cell line suggests the possibility of some form of interaction or "cross-talk" between the compounds and the estrogen signaling pathways. This hypothesis is consistent with studies showing the inhibitory effects of CLA in a variety of rodent models of chemically induced mammary carcinogenesis. To investigate whether CLA possesses direct antiestrogenic activity in MCF-7 cells, we tested for its effects on ER expression levels and the ability of this receptor to bind to EREv. Treatment of MCF-7 cells with 25–100 µmol/L CLA resulted in a reduction in ER mRNA of up to 70% (Fig. 3A). Western blot analysis revealed that this effect was accompanied by decreased ER protein levels (Fig. 3B).
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levels in CLA-treated cells was accompanied by reduced nuclear protein binding to EREv, EMSA was performed. Nuclear extracts prepared from untreated MCF-7 cells produced a prominent band that corresponded in size to an ER-ERE binding complex (Fig. 4, lane 3) whereas a weaker band was detected using extract from CLA-treated cells (lane 4). The presence of ER within the complex was confirmed by the addition of ER-specific antibody, which resulted in the appearance of a supershifted complex and a reduction in the intensity of the ER-ERE band (lane 2). The specificity of binding was further demonstrated by the disappearance of the band with excess unlabeled oligonucleotide corresponding to EREv (lane 1) or when using a 32P-labeled mutant EREv as probe (not shown).
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levels and nuclear protein binding activity of EREv, we were interested in whether this activity resulted in interference with promoter activation through EREv. We tested the ability of CLA to alter the production of luciferase in MCF-7 cells that were transiently transfected with EREv-tk-Luc (firefly), a reporter construct containing 2 vitellogenin EREs directly upstream of tk-Luc. GAPDH-tk-Luc (renilla) was used as control vector for standardization purposes. After 48 h of treatment with different concentrations of CLA (mixture) or the various isomers, the cells were harvested and the dual-luciferase assay was performed. CLA at 25–200 µmol/L inhibited luciferase activity by 38–84% (Fig. 5A). The order of inhibitory potency of the CLA isomers was (most least potent) 9cis,11cis > 10trans,12cis > 9trans,11trans 11cis,13trans > 9cis,11trans (Fig. 5B), basically the same as that demonstrated for the antiproliferative effects of the isomers on MCF-7 cells (see above).
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protein levels, we assessed the ability of CLA to alter EREv-tk-Luc reporter gene activity in MCF-7 cells when the cells were cotransfected with the HEO ER expression plasmid. Transfection of this expression vector resulted in levels of ER protein >threefold higher than the endogenous level (Fig. 6A). In the presence of 200 µmol/L CLA (mixture), there was approximately a 60% reduction in the level of endogenous ER protein. Reduced levels of protein were also observed in the HEO-transfected cells, which could basically be accounted for by the marked reduction in the endogenous component of the total ER expressed. Nevertheless, the level of ER in CLA-treated transfectants was consistently greater than the endogenous protein in untreated cells. CLA markedly inhibited EREv-tk-Luc activity in cultures cotransfected with HEO (Fig. 6B); inhibition of activity was >90% at 200 µmol/L CLA compared to control cultures. These results indicate that reduction of endogenous ER by CLA cannot, by itself, account for its inhibition of EREv-dependent transcriptional activation of reporter gene activity.
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agonists (14,29–31). To address this possible association between CLA and MCF-7 cells, we assessed the transactivating potential on the PPRE-tk-Luc reporter of the most potent CLA isomer we tested for antiestrogenic activity (9cis,11cis) versus the least potent isomer tested (9cis,11trans). The relative abilities of these isomers to transactivate PPRE-tk-Luc in MCF-7 cells are shown (Fig. 7). At all concentrations tested, the 9cis,11cis isomer induced greater levels of luciferase activity than 9cis,11trans.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The present work was designed to investigate whether CLA can directly interfere with estrogen signaling in MCF-7 cells. To address this question, we assessed the effects of CLA on a number of variables that play obligatory roles in this pathway; that is, ER mRNA and protein levels, nuclear protein binding activity to a canonical ERE, and transcriptional activation mediated by the ERE. The data presented herein are consistent with CLA acting as an antiestrogenic agent: (1) CLA was shown to inhibit the growth of ER+ MCF-7, but had little effect on ER- MB-MDA-231 cells. Our results have extended the observations of Durgam and Fernandes (6) to specific CLA isomers; (2) In MCF-7 cells, CLA inhibited ER expression at both the mRNA and protein levels; (3) EMSA demonstrated that nuclear extracts from CLA-treated cells exhibited decreased binding activity to the canonical estrogen response element, EREv; and (4) CLA inhibited transactivation through EREv as evidenced by a reduction in the activity of the EREv-tk-Luc reporter construct.
Previous studies demonstrated that CLA can function as a ligand for PPAR in cells of the macrophage/monocyte lineage and adipocytes (33,34). Our results showing activation of a PPRE construct by CLA represent the first report that these compounds can serve a similar function in breast cancer cells. This property of CLA has been suggested to account for at least part of its biologic activity (12). A recent study by Qin et al. (35) demonstrated that PPAR activation decreases ER protein levels in MCF-7 cells through proteasome-dependent degradation involving enhanced protein ubiquitination. As such, the ability of CLA to activate PPAR in these cells may at least partially explain its downregulation of ER protein levels. Because the ubiquitin system has been implicated in various aspects of transcriptional regulation (36), it is possible that PPAR activation by CLA is also involved in the suppression of ER mRNA levels.
Our data show that CLA inhibits EREv-tk-Luc activation, a finding that is consistent with its ability to alter the level or function of ER or other transactivating proteins that are able to bind to EREv. Thus, downregulation by CLA of endogenous ER expression may serve to decrease ER-dependent transcriptional activation of the EREv reporter plasmid. On the other hand, an argument against this possibility was provided by experiments demonstrating that cotransfection of an ER expression vector that increased the protein level of ER >threefold compared to endogenous levels failed to abolish CLA inhibition of EREv-tk-Luc. Another possible mechanism of action is that CLA, like tamoxifen and other clinically utilized antiestrogens, competes with estrogen for interaction with the ER. However, based upon the known structural requirement of steroid ring components for compounds to bind to these hormone receptors (37), direct binding of CLA to ER would seem unlikely. In addition to binding and activating PPREs, Keller et al. (30) provided evidence that liganded PPAR/RXR heterodimers are also able to bind some ERE sequences (including EREv), but, depending upon promoter context, may not mediate gene transcription. Thus, it is possible that PPAR/RXR complexes could act as roadblocks, competing with activated ER for binding to the ERE. In this way, it would be possible for CLA to interfere with the stimulatory effects of estrogen on at least some target genes. Our results showing that the enhanced ability of 9cis11cis versus 9cis11trans CLA to stimulate a PPRE reporter construct correlated with a greater potency of the former versus the latter to inhibit EREv activation are consistent with this PPAR-mediated hypothesis. Although structural differences between the CLA isomers that affect their efficacy to serve as receptor ligands (e.g., PPAR ligands) might account for their divergent potencies, it is also possible that their biological impact could be related to differences in their cellular uptake and incorporation. In this regard, recent studies have demonstrated a threefold higher incorporation of 10trans,12cis versus 9cis,11cis CLA into platelet lipids and differences in their ability to be esterified into umbilical vein endothelial cells (38).
The possibility that CLA induces proteins to interact with ERs in such a way as to inhibit their subsequent binding with or transactivation of EREv must also be considered. Such protein-protein interactions were demonstrated between, for example, nuclear retinoic acid receptors and AP-1 proteins (39,40) and glucocorticoid receptors and subunits of the transcription factor NF-
B (41–44). Of special relevance to us are the recent studies by Hu et al. (45) showing that the TR2 orphan receptor, another member of the nuclear hormone receptor superfamily which includes ERs and PPARs, can interfere with ER DNA binding via direct protein-protein interaction that disrupts ER dimerization. Future investigations will determine whether inhibitory ER-protein complexes containing CLA-liganded PPAR can also form in the absence of high-affinity DNA binding.
Our findings provide the first evidence that CLA possesses potent antiestrogenic properties and that its mechanism of action is at the level of the ERE. Our study demonstrated that these compounds have antiproliferative action on ER+ breast cancer cells and that their antiestrogenic properties can at least partly account for their antitumor activity on this cell type. These findings provide the impetus for future studies into a novel mechanism of action that might prove valuable in antiestrogen therapy or dietary intervention of certain cancers by way of inhibiting estrogen signaling in the affected tissue.
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FOOTNOTES |
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2 The first 2 authors contributed equally to this work.
4 Abbreviations used: BCA, bicinchoninic acid; CLA, conjugated linoleic acid; EMSA, electrophoretic mobility shift assay; ER, estrogen receptor; ERE, estrogen response element; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Luc, luciferase; PPRE, peroxisome proliferator response element; SRB, sulforhodamine B; TCA, trichloroacetic acid.
Manuscript received 23 October 2003. Initial review completed 4 November 2003. Revision accepted 15 December 2003.
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S. C. Degner, M. Q. Kemp, G. T. Bowden, and D. F. Romagnolo Conjugated Linoleic Acid Attenuates Cyclooxygenase-2 Transcriptional Activity via an Anti-AP-1 Mechanism in MCF-7 Breast Cancer Cells J. Nutr., February 1, 2006; 136(2): 421 - 427. [Abstract] [Full Text] [PDF]
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) breast cancer cells by CLA. Cells were treated for 2 d with the indicated concentration of CLA (mixture) or solvent control. Values are means ± SEM, n = 3, and are expressed as a percentage of control cultures as assessed by the SRB protein assay. *Different from the vehicle control, P < 0.05.
