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* Laboratory of Mammary Gland Biology, Department of Nutritional Sciences, and Department of Cell Biology and Anatomy, Arizona Cancer Center, The University of Arizona, Tucson, AZ 85721
1 To whom correspondence should be addressed. E-mail: donato{at}u.arizona.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • conjugated linoleic acid • cyclooxygenase-2 • activator protein-1 • breast cancer
Cyclooxygenases (COX)2 catalyze the conversion of arachidonic acid (AA) to prostaglandins (PG). The 2 isoforms of COX are differentially regulated and expressed. COX-1 is generally considered to be a housekeeping enzyme that functions to maintain cellular homeostasis, whereas COX-2 overexpression is rapidly induced in response to growth factors, oncogenes, tumor promoters, cytokines, and endotoxins [reviewed in (1)]. The involvement of COX-2 in carcinogenesis is supported by several lines of evidence. COX-2 was reported to be overexpressed in a variety of human malignancies including breast cancer (2). Studies with transgenic mice demonstrated that overexpression of COX-2 was sufficient to induce tumorigenesis in the mammary gland (3). Moreover, the use of nonsteroidal anti-inflammatory drugs that target cyclooxygenases was correlated with reduced incidence of several cancers including colorectal and breast (4,5). In addition, treatment with COX-2 selective inhibitors was shown to reduce tumorigenesis in animal models [reviewed in (6)].
The expression of COX-2 is regulated at the transcriptional, post-transcriptional, and post-translational levels. Regulatory elements in the 5'-flanking region of the COX-2 gene include a cAMP response element (CRE, –59 to –53, 5'-TTCGTCA-3') (7). The CRE recognition sequence is essential for both constitutive and induced COX-2 expression in most cells (8,9) and shares sequence homology with the consensus 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive element (TRE = 5'-TGACTCA-3') site found in the collagenase-1 promoter (10). The TRE is a consensus site for the transcription factor activator protein-1 (AP-1) (11). Binding of AP-1 proteins to the CRE and TRE elements is strongly induced by the tumor promoter and proinflammatory agent TPA (9,11). Binding of CREB proteins to the CRE element is induced by UV irradiation (8). In contrast, UVA induces COX-2 expression through stabilization of the COX-2 mRNA (12).
Conjugated linoleic acid (CLA) has garnered attention as a dietary adjuvant for its anticarcinogenic properties as well as other biological activities including modulation of lipid metabolism, atherogenesis, diabetes, and immune functions [reviewed in (13)]. CLA is referred to as a mixture of positional and geometric isomers of conjugated dienoic linoleic acid (18:2); they originate in ruminants from the conversion of linoleic acid (LA) by bacterial isomerases. The c9, t11-CLA is the predominant isomer found in milk fat and meats from ruminant animals (14,15). However, other isomers are also present, including t10, c12-CLA, which was shown to possess significant biological activity (16). Several studies reported that CLA reduced the synthesis of PG, including PGE2, in a variety of cell culture and animal models (17–21). CLA may exert its protective effects by decreasing AA concentration in the phospholipid membranes, thus reducing PG production (17), as well as inhibiting COX-2 expression (22,23). Nevertheless, the mechanisms through which CLA modulates COX-2 expression remain largely unknown.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The pGL3-COX-2 luciferase reporter construct (pCOX-2) containing a putative peroxisome proliferator-activated receptor (PPAR) response element (PPRE) sequence (–3721 to –3707, AGGCGACAGGTCA) and the pGL3-3.5-kb luciferase reporter construct (p3.5COX-2) lacking the PPRE were a gift of Dr. Tom McIntyre (University of Utah, Salt Lake City, UT), and were described previously (24,25). The pCMV-c-Jun plasmid (pcJun) was a gift from Dr. Andrei Bakin (Roswell Park Cancer Institute, Buffalo, NY). Details concerning the collagenase-1 promoter (–73 to +63) luciferase constructs are described elsewhere (8).
Semiquantitative RT-PCR. Total cellular RNA was extracted using a guanidinium thiocyanate procedure (26). RT was performed using total RNA incubated with random hexamer primers, Moloney murine leukemia virus reverse transcriptase, RNase inhibitor, and RT buffer at 42°C for 1 h. Oligonucleotides used to amplify COX-2 were as follows: (forward) 5'-TTCACGCATCAGTTTTTCAA-3' and (reverse) 5'-ACAGCAAACCGTAGATGCT C-3'. The COX-2 PCR product was of the expected size (248 bp) and the authenticity to the COX-2 sequence deposited in the GenBank (Accession number M90100) was verified by direct sequencing. The 18S ribosomal RNA amplification product (488 bp) was used as an internal standard for equal loading and monitoring of PCR conditions. For amplification of 18S RNA, we used the competimer oligonucleotide module from Ambion using a ratio of 1.75:1 18S competimer primers:18S primers. For amplification of COX-2 and 18S, we ascertained linear accumulation of the PCR products during 36 PCR cycle amplification reactions (data not shown). Relative expression levels of COX-2 were estimated by Alpha Imager analysis (Alpha Innotech) and corrected for 18S control (COX-2/18S).
Prostaglandin assay. MCF-7 cells were cultured in DMEM with 0.5% FBS in the presence or absence of TPA (0.1 µmol/L). Determination of PG levels in conditioned media was carried out using the Prostaglandin Screening EIA Kit (514012) with a limit of detection of 40 mg/L, according to manufacturer's instructions (Cayman Chemical).
Western blot analysis. Western blot analysis was performed as previously described (27). Equal amounts of proteins were subjected to SDS-PAGE analysis and subsequent immunoblotting was carried out with antibodies raised against COX-2 protein (Cayman Chemical) and cJun (Cell Signaling). For cytoplasmic extracts, immunoblotting was performed using antibodies that recognize the phosphorylated forms of p44/42 and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) from Cell Signaling. Levels of immunocomplexes for ß-actin (Oncogene Research Products) were used as an internal standard for equal loading. The immunocomplexes were detected by enhanced chemiluminescence (Amersham).
Transient transfections and luciferase assay. Cells (1 x 105 in 2 mL DMEM, 10% FBS/well) were plated in 24-well Costar tissue culture plates. Transient transfections were performed using the Lipofectamine-Plus procedure according to the manufacturer's instructions (Life Technologies). Briefly, 24 h after cells were plated, each well was cotransfected with 1.5 µg of COX-2 plasmid and 0.8 µg of the internal control plasmid pRL-TK (renilla luciferase gene). Cells were incubated with the DNA-liposome complex for 3 h at 37°C in 5% CO2. After transfection, cells were maintained in DMEM (10% FBS) and allowed to recover for 48 h. Cells were then treated in DMEM (0.5% FBS) containing either control (ethanol vehicle) or various experimental compounds for the times indicated.
Electrophoretic mobility shift assay (EMSA).
Cells (5 x 105 in 2 mL DMEM, 10% FBS/well) were plated in 6-well Costar tissue culture plates. After 24 h, cells were treated for 4.5 h then subsequently harvested. Briefly, cells were trypsinized, then washed with ice-cold DPBS. Cells were resuspended in ice-cold 25 mmol/L Hepes buffer at pH 7.6 containing 1.5 mmol/L EDTA, 1 mmol/L dithiothreitol (DTT), 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF), and 5 g/L aprotinin and placed on ice for 10 min. Cells were pelleted and resuspended in 1 mL ice-cold 25 mmol/L Hepes buffer containing 1.5 mmol/L EDTA, 10% (v:v) glycerol, 1 mmol/L DTT, 0.5 mmol/L PMSF, and 5 g/L aprotinin. The cell suspension was transferred to a mortar for drilling with a Teflon pestle until >90% of the cells in a 2-µL aliquot were unable to exclude trypan blue. After centrifugation (1600 x g; 10 min), cell pellets were resuspended in 150 µL ice-cold 25 mmol/L Hepes buffer containing 1.5 mmol/L EDTA, 10% (v:v) glycerol, 0.5 mol/L KCl, 1 mmol/L DTT, 0.5 mmol/L PMSF, and 5 g/L aprotinin and placed on ice with intermittent mixing on a vortex. Cell debris was removed by centrifugation (26,000 x g; 60 min). Supernatants containing nuclear protein were stored at –70°C. Nuclear protein concentration was determined using the bicinchoninic acid protein assay (Pierce Chemical). Oligonucleotides used for the binding assay were: COX-2 CRE, 5'-AAACAGTCATTTCGTCA CATGGGCTTG-3' (sense) and 5'-CAAGCCCATGTGACGAAATGACTGTTT-3' (antisense); and consensus-TRE, 5'-TCGCTTGATGACTCAGCCGGA-3' (sense) and 5'-TCCGGCTGAGTCATCAAGCGA-3' (antisense) from the collagenase-1 promoter (10). The complementary oligonucleotides were annealed, then phosphorylated at the 5'-end with [
-32 P]ATP and T4 polynucleotide kinase. Unincorporated nucleotides were removed using the TE-10 spin columns (Clontech). Binding assays were performed by incubating 5 µg of nuclear protein in the binding buffer and then incubated with the labeled oligonucleotides for 20 min. For cold competition, a 100-fold excess of the respective unlabeled oligonucleotides was added to the binding reaction 10 min before the addition of the labeled oligonucleotides. Samples were electrophoresed through a 5% nondenaturing polyacrylamide gel at 200 V for 90 min. Finally, the gel was dried and exposed to a phosphor screen, and digital phosphorimages were retrieved using the Storm system (Molecular Dynamics).
DNA-protein binding assay. The binding assay was performed as previously described (28). Biotinylated oligonucleotides were synthesized by Sigma Genosys using the sequences containing the COX-2 CRE and collagenase-1 TRE utilized in the EMSA experiments. Nuclear extracts were harvested from cells that were pretreated for 2 h with 80 µmol/L of each fatty acid (CLA mix, c9, t11-CLA, t10, c12-CLA, and LA) then cotreated for 6 h with fatty acids + TPA (0.1µmol/L). The binding assay was performed by incubating 200 µg of the nuclear extracts, 2 µg biotin-labeled double-stranded DNA oligonucleotides, and 20 µL of 4% beaded-agarose conjugated with streptavidin in 600 µL PBSI buffer (PBS buffer containing multiple protease inhibitors: 1 mmol/L Na3VO4, 10 mmol/L NaF, 25 mmol/L ß-glycerophosphate, 0.1 mmol/L PMSF, 0.06 g/L aprotinin, 1 g/L leupeptin, 0.5 mmol/L DTT) for 2 h with shaking at room temperature. Beads were pelleted by centrifugation at 550 x g for 1 min and then washed 3 times with cold PBSI. Nuclear proteins were dissociated by incubating the mixture at 95°C for 5 min. The binding proteins were separated on a 4–12% SDS-PAGE and subsequently subjected to Western blot analysis with an antibody that detects total cJun protein regardless of phosphorylation state (Cell Signaling).
Statistical analysis.
Statview, the SAS Institute statistical analysis software was used for ANOVA, with multiple comparisons by Fisher's protected least significant different test. For ANOVA and multiple comparisons, P-values 
0.05 were considered significant. Data are presented as means ± SE.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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46 and 54 kDa) (Fig. 7B). However, the cotreatment with CLA caused a dose-dependent (80 > 40 µmol/L) shift in the profile of phosphorylated SAPK/JNK isoforms as shown by the increased intensity of the faster migrating band. TPA treatment also induced the phosphorylation of p44/42 [extracellular signal-regulated kinase (ERK)1/2]. However, the CLA cotreatment did not have an effect of TPA-induced activation of p44/42. These results suggested that the CLA cotreatment altered the TPA-induced activation of SAPK/JNK and subsequent phosphorylation of cJun, but did not influence TPA-induced phosphorylation of p44/42. |
DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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in RAW264.7 mouse macrophage cells. More recently, Cheng et al. (23) reported that CLA (20–200 µmol/L) reduced COX-2 mRNA and protein levels in RAW 264.7 cells after treatment with lipopolysaccharide. Although these cumulative studies provided important clues concerning the protective effects of CLA, the mechanisms of action of CLA on COX-2 expression activity remain elusive. The objective of this study was to examine whether CLA modulated COX-2 transcription activity. To induce the expression of COX-2, we used the proinflammatory agent TPA, which in the current study effectively induced COX-2 mRNA, protein, and PG production in MCF-7 cells. The CLA mix reduced COX-2 protein expression induced by TPA. In breast cancer MCF-7 cells transiently transfected with a COX-2 reporter construct, treatment with TPA stimulated the activity of the COX-2 promoter. Conversely, cotreatment with the CLA mix or selected isomers (t10, c12-CLA; c9, t11-CLA) attenuated TPA-induced COX-2 transcription in a dose-dependent manner. At a dose of 20 µmol/L, which is within the normal physiological range of 10–70 µmol/L found in humans (34), the t10, c12-CLA and c9, t11-CLA isomers were more effective than the CLA mix in reducing TPA-induced COX-2 promoter activity. These results were somewhat expected because the CLA mix used in this study contained other isomers in addition to those tested. The treatment of MCF-7 cells with LA also reduced COX-2 promoter activity; however, it was not as effective as the CLA mix or isomers at 40 and 80 µmol/L. Previous reports documented that LA reduced COX-2 mRNA (22) and promoter activity (35). In contrast, other studies reported that LA-enriched diets increased COX-2 mRNA in the rat mammary gland (36), and hepatoma cells (37). Therefore, it is feasible that the hampering effects of LA on COX-2 transcription observed in this study may be due to a feedback loop in which AA and/or PGE2 downregulates COX-2 transcription rather than a direct effect of LA on the COX-2 promoter (38). Nonetheless, our data indicated that CLA attenuated TPA-induced COX-2 transcription, and further studies were designed to investigate the mechanism of inhibition of COX-2 transcription by CLA.
Studies documented that treatment with TPA induced the recruitment of AP-1 dimers to cis-acting TRE on promoter regions inducing transcription of genes involved in cellular metabolism, cell proliferation, and metastasis (39). CLA inhibited the reporter activity of the collagenase-1 promoter, suggesting that CLA may modulate transcription through an anti-AP-1 mechanism. Previous investigations reported that binding of the AP-1 transcription factor to the CRE in the COX-2 promoter was strongly induced by the tumor-promoting agent TPA. Conversely, deletion or mutation of the CRE site resulted in the loss of TPA-induced transcription, suggesting that the CRE element mediated the stimulatory effects of AP-1 (9). Therefore, we formulated the hypothesis that CLA may interfere with the recruitment of AP-1 at the CRE site. In the current study, EMSA revealed that TPA induced the binding of nuclear proteins to oligonucleotides comprising the COX-2 CRE or the consensus TRE. The latter is a putative sequence in the collagenase-1 promoter that is known to bind AP-1 transcription factors (10,11). The cotreatment with CLA cleared the complex recruited at the CRE and TRE after treatment with TPA in a dose-dependent fashion. Conversely, the overexpression of cJun in MCF-7 cells relieved the suppressive effects of CLA on COX-2 promoter activity, as well as on binding of nuclear proteins to the COX-2 CRE and collagenase-1 TRE oligonucleotides. In binding studies, we observed that the t10, c12-CLA isomer was more effective than the CLA mix and c9, t11-CLA in reducing TPA-induced recruitment of the cJun protein to the oligonucleotides containing the COX-2 CRE or the collagenase-1 TRE. In contrast, the treatment with LA increased cJun binding to the COX-2 CRE and collagenase-1 TRE. The latter results were in agreement with previous observations documenting that AP-1 binding was induced by LA (40). These cumulative data support a model in which CLA antagonizes COX-2 transcription by preventing the recruitment of an AP-1 complex at the CRE element harbored in the proximal COX-2 promoter.
The anti-AP-1 properties of CLA may be exerted through 2, possibly not mutually exclusive, mechanisms. First, CLA may directly repress signal transduction pathways required for activation of AP-1, thus preventing the formation of a productive AP-1 complex at the CRE. The induction of COX-2 expression by TPA was shown to be regulated at least in part by upstream signal transduction cascades such as the protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) pathways (11,41). Previous studies investigated the effects of CLA on PKC and MAPK signaling pathways. In human prostate cancer cells, CLA treatment modulated the abundance of PKC isoforms in cytosol and membrane protein fractions (42). In human adipocytes, treatment with t10, c12 CLA resulted in the activation of MAPK signaling (16). However, in HT-29 colon cancer cells, treatment with a mix of CLA isomers resulted in decreased levels of both phosphorylated and total p44/42 (ERK1/2) (43). In the current investigation, CLA cotreatment did not reduce TPA-induced phosphorylation of p44/42 in MCF-7 cells, suggesting that the effects of CLA may be cell and tissue specific.
The activation of SAPK/JNK proteins is required for phosphorylation of transcription factors including cJun (33). In the current investigation, CLA cotreatment decreased TPA-induced phosphorylation of cJun and altered the profile of phosphorylated SAPK/JNK isoforms. One possible interpretation of these results is that CLA may interfere with activation of SAPK/JNK and consequently repress the levels of phosphorylated cJun. The effects of CLA on signal transduction pathways are complex and should be the target of future investigations.
Second, CLA may repress COX-2 transcription by activating PPAR , a member of the superfamily of nuclear hormone ligand-dependent transcription factors. CLA is a potential ligand of PPAR (13,22). Interestingly, previous investigations documented that PPAR ligands downregulated COX-2 in breast cancer cells by sequestering transcription factors such as the cAMP responsive element binding protein and p300 from the COX-2 CRE site (29). The COX-2 promoter contains an upstream PPRE (–3721/–3707) (24,25). Therefore, one possibility is that CLA may repress COX-2 transcription by interfering with activation of COX-2 at the PPRE. However, in transfection studies, we found that CLA repressed TPA-induced transcription of a 3.5-kb COX-2 promoter fragment lacking the PPRE (data not shown). These results suggest that the repressive effects of CLA on COX-2 transcription may not be mediated by the upstream PPRE.
Overall, the results of the present study document that CLA antagonizes AP-1–dependent induction of COX-2 transcription. Through this mechanism, CLA may reduce COX-2 levels and prostaglandin biosynthesis, thereby contributing to its anticarcinogenic and anti-inflammatory properties.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received 3 May 2005. Initial review completed 2 June 2005. Revision accepted 10 November 2005.
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