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Carcinogenesis Advance Access originally published online on January 12, 2008
Carcinogenesis 2008 29(4):688-695; doi:10.1093/carcin/bgm299
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

15-Deoxy-{Delta}12,14-prostaglandin J2 induces COX-2 expression through Akt-driven AP-1 activation in human breast cancer cells: a potential role of ROS

Eun-Hee Kim1, Hye-Kyung Na1, Do-Hee Kim1, Sin-Aye Park1, Ha-Na Kim1, Na-Young Song1 and Young-Joon Surh1,2,*

1 National Research Laboratory of Molecular Carcinogenesis and Chemoprevention, College of Pharmacy, Seoul National University, Shinlim-Dong, Kwanak-Gu, Seoul 151-742, South Korea
2 Cancer Research Institute, Seoul National University, Seoul 110-799, South Korea

* To whom correspondence should be addressed. Tel: +82 2 880 7845; Fax: +82 2 874 9775; Email: surh{at}plaza.snu.ac.kr


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Recent studies suggest that inflammation is causally linked to carcinogenesis. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in the biosynthesis of prostaglandins, is inappropriately expressed in various cancers and hence recognized as one of the hallmarks of chronic inflammation-associated malignancies. However, the mechanistic role of COX-2 as a link between inflammation and cancer remains undefined. Here, we report that 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2), one of the final products of COX-mediated arachidonic acid metabolism, upregulates the expression of COX-2 in the human breast cancer MCF-7 cell line. 15d-PGJ2-induced COX-2 expression was mediated by activation of Akt and subsequently activator protein-1 (AP-1). Furthermore, 15d-PGJ2 formed reactive oxygen species, which led to increased phosphorylation of Akt, DNA binding of AP-1 and expression of COX-2. In contrast to 15d-PGJ2, 9,10-dihydro-15d-PGJ2 did not elicit any of effects induced by 15d-PGJ2 in this study, suggesting that an electrophilic carbon center present in 15d-PGJ2 is critical for COX-2 expression as well activation of upstream signal transduction induced by this cyclopentenone prostaglandin. Taken together, these observations suggest that 15d-PGJ2 produced by COX-2 overexpression may function as a positive regulator of COX-2 in human breast cancer MCF-7 cells.

Abbreviations: AP-1, activator protein-1; COX-2, cyclooxygenase-2; CRE, cyclic AMP response element; 15d-PGJ2, 15-deoxy-{Delta}12,14-prostaglandin J2; GSH, reduced glutathione; KD, kinase-dead; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAC, N-acetyl-L-cysteine; PBS, phosphate-buffered saline; PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; PTEN, phosphatase and tensin homologue deleted on chromosome 10; ROS, reactive oxygen species


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
15-Deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2), an endogenous ligand of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), has potent antiproliferative properties in several types of cancerous or transformed cells as evidenced by its inhibition of malignant cell growth or induction of apoptosis (1,2). 15d-PGJ2 induces apoptosis through modulation of genes associated with cell cycle and cell survival/death in various types of cancer cells. The antitumorigenic effects of 15d-PGJ2 are also manifested by its inhibition of invasiveness and angiogenesis (3). Moreover, 15d-PGJ2 exerts anti-inflammatory effects that are associated with interruption of nuclear factor-{kappa}B and subsequent blockade of expression of some pro-inflammatory genes, such as cyclooxygenase-2 (COX-2) (reviewed in ref. 3). However, there are some reports demonstrating the opposite effects of 15d-PGJ2 on the development of tumors (1,2). Thus, 15d-PGJ2 significantly enhanced the rate of formation, the size and vascularization of the papillomas when topically applied onto mouse skin (4). 15d-PGJ2 induced the proliferation of COX-2-depleted colorectal cancer (HCA-7) cells (5). However, precise mechanisms responsible for proliferative effects of 15d-PGJ2 remain incompletely clarified.

It is noticeable that 15d-PGJ2 is one of the major terminal products of COX-2. COX-2 has been shown to contribute to carcinogenesis by promoting cell proliferation and angiogenesis as well as by protecting cells from apoptosis (reviewed in ref. 6). Since abnormal overexpression of COX-2 is implicated in the pathogenesis of various human cancers, it is reasonable to assume that increased 15d-PGJ2 synthesis as a consequence of COX-2 overexpression can facilitate carcinogenesis and tumor cell growth. Recently, Vichai et al. (7) reported the positive feedback regulation by 15d-PGJ2 of COX-2 expression in mouse lung fibroblasts. Other studies also demonstrate the similar effect of 15d-PGJ2 on COX-2 production (8). However, molecular mechanisms by which 15d-PGJ2 modulates COX-2 expression remain unresolved. In an attempt to elucidate the molecular mechanisms underlying 15d-PGJ2-mediated COX-2 upregulation, we explored the intracellular signaling pathways that can be possibly activated by this cyclopentenone prostaglandin in relation to its oncogenic potential in human breast cancer cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Materials
15d-PGJ2, 9,10-dihydro-15d-PGJ2 and GW9662 were purchased from Cayman Chemical Co. (Ann Arbor, MI). RPMI 1640 medium and fetal bovine serum were obtained from Gibco BRL (Grand Island, NY). Primary antibodies for Akt and pAkt were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against COX-2 and actin were obtained from Lab Vision Corporation (Fremont, CA) and Sigma-Aldrich (St Louis, MO). LY294002, SB203580, SP600125 and U0126 were obtained from Tocris (Ellisville, MO). The anti-rabbit and anti-mouse horseradish peroxidase-conjugated secondary antibodies were purchased from Zymed Laboratories (San Francisco, CA). Oligonucleotide probes containing activator protein-1 (AP-1) consensus sequences located in the mouse COX-2 promoter region were obtained from Promega (Madison, WI). The enhanced chemiluminescence and [{gamma}-32P]ATP were supplied from Amersham Pharmacia Biotech (Buckinghamshire, UK).

Plasmid and expression vector
A series of human COX-2 promoter deletion constructs ligated to luciferase gene were described previously (9). Hemaglutinin-tagged full-length Akt and Akt with a K179M mutation [kinase-dead (KD)-Akt] cloned into pCMV5 were kindly provided by Dr An-Sik Chung (Korea Advanced Institute of Science and Technology, Daejeon, South Korea) (10). For analysis of AP-1, transcriptional activity and determination of the effects KD-Akt (AktK179M) on the expression of COX-2, MCF-7 cells were transfected with mammalian expression vectors. Appropriate control plasmids (pGL2 for COX-2 promoter deletion plasmids and AP-1-luciferase; hemaglutinin-tagged full-length Akt for KD-Akt) were used to properly assess the plasmid transfection effect.

Cell culture
MCF-7 and MDA-MB-231 cells were maintained routinely in RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 ng/ml penicillin–streptomycin–fungizone mixture at 37°C in a humidified atmosphere of 5% CO2/95% air.

MTT reduction assay.
Cells were plated at a density of 3 x 104 cells/300 µl in 48-well plates, and the cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. After incubation, cells were treated with the MTT solution (final concentration, 1 mg/ml) for 2 h. The dark blue formazan crystals formed in intact cells were dissolved with dimethyl sulfoxide and the absorbance at 570 nm was read using a microplate reader. Results were expressed as the percentage of MTT reduction obtained in the treated cells, assuming that the absorbance of control cells was 100%.

Thymidine incorporation.
Cells were cultured in six-well dish and cotreated with [methyl-3H]thymidine at a final concentration of 1 mCi/ml and dimethyl sulfoxide or 15d-PGJ2. After 24 h incubation, cells were trypsinized, washed with phosphate-buffered saline (PBS) and centrifuged. The supernatant was removed, and ice-cold 10% trichloroacetic acid solution (20 ml) was added to the pellet, followed by vortex mixing. The mixture was centrifuged, and the resulting pellet was mixed with 400 ml of 0.2 N NaOH (with 0.5% sodium dodecyl sulfate) and further incubated at 37°C for 30 min. After incubation, samples were mixed with 1 N HCl (68 ml) and 1 ml cocktail solution. Radioactivity in trichloroacetic acid-precipitable material was measured by liquid scintillation counting.

Western blot analysis
MCF-7 cells (2 x 105 cells/ml) were plated in a 60 mm dish and treated with 15d-PGJ2 under specified conditions. After rinse with PBS, the cells were exposed to the lysis buffer [20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid disodium, 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin and protease inhibitors] (Cell Signaling Technology) in the ice for 15 min. After centrifugation at 12,000g for 15 min, supernatant was separated and stored at –70°C until use. The protein concentration was determined by using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Protein samples were electrophoresed on a 12% sodium dodecyl sulfate–polyacrylamide gel and transferred to polyvinylidene difluoride membrane at 300 mA for 3 h. Blots were incubated in fresh blocking buffer (0.1% Tween 20 in PBS containing 5% non-fat dry milk, pH 7.4) for 1 h followed by incubation with primary antibodies for COX-2, Akt, phospho-Akt/protein kinase B in PBS with 3% non-fat dry milk. After washing with 0.1% Tween 20 in PBS three times, blots were incubated with horseradish peroxidase-conjugated secondary antibody in PBS with 3% non-fat dry milk for 1 h at room temperature. Blots were washed again three times in 0.1% Tween 20 in PBS buffer, and transferred proteins were detected with an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech).

Preparation of nuclear proteins
After treatment with 15d-PGJ2, cells (3 x 106 cells/10 ml in 100 mm dish) were washed with PBS, centrifuged and resuspended in ice-cold isotonic buffer A (10 mM 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol and 0.2 mM phenylmethanesulfonyl fluoride). After the incubation in ice bath for 10 min, cells were centrifuged again and resuspended in ice-cold buffer C containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (pH 7.9), 20% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol and 0.2 mM phenylmethanesulfonyl fluoride followed by incubation at 0°C for 20 min. After vortex mixing, the resulting suspension was centrifuged, and the supernatant was stored at –70°C after the determination of protein concentrations.

Electrophoretic mobility shift assay
Briefly, the AP-1 oligonucleotide probe (5'-CGCTTGATGAGTCAGCCGGAAC-3') was labeled with [{gamma}-32P]ATP by T4 polynucleotide kinase and purified on a Nick column (Amersham Pharmacia Biotech). The binding reaction was carried out in a total volume of 25 µl containing 10 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 4% glycerol, 0.1 mg/ml sonicated salmon sperm DNA, 10 µg of nuclear extracts and 100,000 c.p.m. of the labeled probe. A 100-fold excess of unlabeled oligonucleotide (competitor) was added where necessary. After 50 min incubation at room temperature, 2 µl of 0.1% bromophenol blue was added and samples were electrophoresed through a 6% non-denaturating polyacrylamide gel at 150 V for 2 h. Finally, the gel was dried and exposed to X-ray film.

Transient transfection and the luciferase reporter gene assay
MCF-7 cells were plated at a confluence of 60% in six-well plate and grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2/95% air. Transient transfections were performed using the N-[1-(2,3-dioleolloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) liposomal transfection reagents according to the instructions supplied by the manufacturer (Roche, Basel, Switzerland). After 8–12 h transfection, cells were treated with 15d-PGJ2 for additional 12 h, and the cell lysis was carried out with the reporter lysis buffer. After mixing the cell extract with a luciferase substrate (Promega), the luciferase activity was measured by the luminometer (AntoLumat LB953, EG and G Berthold, Bad Widbad, Germany). The β-galactosidase assay was done according to the supplier’s instructions (Promega β-Galactosidase Enzyme Assay System) for normalizing the luciferase activity.

Preparation of c-fos and c-jun antisense
c-jun and c-fos antisense oligonucleotides were purchased from Bionics (Seoul, South Korea). The oligonucleotide sequences are as follows: c-jun, 5'-TGCAGTCATAGAAC-3'; c-fos, 5'-GAAGCCCGAGAACATCAT-3' and control, 5'-ATGAGTTTCTCGGGCTTG-3'. The cells were incubated with the c-jun and c-fos antisense oligonucleotides at different concentrations (1–10 µM) for 4 h followed by stimulation with 15d-PGJ2.

Measurement of intracellular reactive oxygen species accumulation
To monitor net intracellular accumulation of reactive oxygen species (ROS), the fluorescent probe 2',7'-dichlorofluorescein diacetate was used. Following treatment, cells were rinsed with Krebs ringer solution and 10 µM 2',7'-dichlorofluorescein diacetate was loaded. After 15 min incubation at 37°C, cells were examined under a confocal microscope equipped with an argon laser (488 nm, 200 mW).

Statistical analysis
When necessary, data were expressed as means ± SDs of at least three independent experiments, and statistical analysis for single comparison was performed using the Student’s t-test. The criterion for statistical significance was P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
15d-PGJ2 induces the expression of COX-2 in human breast cancer cells
A kinetic study revealed that COX-2 protein expression was induced by 15d-PGJ2 in a time-dependent manner (Figure 1A). Treatment with 15d-PGJ2 (0, 3, 10 and 30 µM) for 24 h resulted in the induction of COX-2 in a concentration-dependent manner (Figure 1B). MCF-7 cells express estrogen receptor-{alpha}. We also examined the effect of 15d-PGJ2 on COX-2 expression in an estrogen receptor-{alpha}-negative cell line, MDA-MB-231. As shown in Figure 1B, upregulation of COX-2 was observed in both cell lines. When both cells were treated with 30 µM 15d-PGJ2 up to 24 h, ~80% of treated cells were viable (Figure 2). Although the prostaglandin exhibited some cytotoxicity at this time point, the COX-2 upregulation still continued (Figure 1A). Since 15d-PGJ2 is known to provoke diverse effects by acting as an endogenous ligand of PPAR{gamma} (11), we examined the effect of a PPAR{gamma} antagonist GW9662 on COX-2 upregulation by 15d-PGJ2 in MCF-7 cells. As illustrated in Figure 1C, 15d-PGJ2-induced COX-2 expression was not affected by GW9662 treatment. These findings indicate that COX-2 expression by 15d-PGJ2 is independent of estrogen receptor and PPAR{gamma}.


Figure 1
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  		Fig. 1. 15d-PGJ2-induced expression of COX-2. (A) Western blot analysis of the total cell extract was performed with COX-2 antibody. MCF-7 cells were treated with 30 µM 15d-PGJ2 for 0, 3, 6, 12 or 24 h. (B) Human breast cancer cell lines, MCF-7 and MDA-MB-231, as well as human breast epithelial cells, MCF-10A were treated with 0, 3, 10 or 30 µM of 15d-PGJ2 for 24 h. Actin was used as an equal loading control for normalization. (C) MCF-7 cells were treated with 30 µM of 15d-PGJ2 in the absence or presence of GW9662 (25 µM) for 24 h at 37°C. GW9662 was added to the media 30 min before the 15d-PGJ2 treatment.

 


Figure 2
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  		Fig. 2. Effect of 15d-PGJ2 on viability and proliferation of human breast cancer cell lines. MCF-7 (A and B) and MDA-MB-231 (C) cells were treated with indicated concentrations of 15d-PGJ2 for 12 or 24 h, and their viability and proliferation were determined by the MTT assay (A and C) and the thymidine incorporation assay (B), respectively as described in Materials and Methods. The concentration of 15d-PGJ2 was 30 µM for the experiment conducted for (B).

 
15d-PGJ2 upregulates the expression of COX-2 through AP-1 activation
The regulation of COX-2 synthesis occurs mainly at the transcriptional level, although messenger RNA stabilization is also involved in response to specific signals. Transcription factors involved in the induction of COX-2 gene expression are diversified and cell type and/or stimulus specific. Several cis-acting elements are found in the COX-2 promoter, such as nuclear factor-{kappa}B, nuclear factor-interleukin-6, cyclic AMP response element (CRE) and E-box (12–14). To determine which transcription factors are preferentially involved in 15d-PGJ2-induced COX-2 expression, cells were transfected with human COX-2 promoter-luciferase constructs (–327/+59) (Figure 3A) and challenged with 15d-PGJ2 for 12 h. Treatment with 15d-PGJ2 resulted in a significant increase in the COX-2 promoter (–327/+59) activity. To elucidate the critical region of the COX-2 promoter responsible for COX-2 expression by 15d-PGJ2, we utilized a series of COX-2 deletion constructs (–327/+59, –220/+59, –124/+59 and –52/+59). The COX-2 promoter activity was most prominent when the –327/+59 promoter construct was used (Figure 3B). As the promoter length was shortened, COX-2 activities became gradually diminished. It is noticeable that the –52/+59 construct exhibited an almost complete loss of the COX-2 promoter activity compared with the –327/+59 construct. CRE is present between nucleotides –59 and –53, suggesting that this element might be responsible for mediating the COX-2-inducing effects of 15d-PGJ2. To precisely define which of these cis-acting elements are involved in 15d-PGJ2-induced COX-2 promoter activity, MCF-7 cells were transiently transfected with site-specific mutant COX-2 promoter constructs. As shown in Figure 3C, CRM (–327/+59 construct with mutation at the CRE site) significantly lowered the COX-2 promoter activity in 15d-PGJ2-treated cells. These results suggest that CRE plays an important role in mediating the induction of COX-2 gene expression by 15d-PGJ2 in MCF-7 cells.


Figure 3
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  		Fig. 3. COX-2 promoter activity induced by 15d-PGJ2. (A) A schematic representation of the human COX-2 promoter. (B) Determination of cis-acting elements of COX-2 promoter. MCF-7 cells were transfected with 2.5 µg of a series of human COX-2 promoter deletion constructs (–327/+59, –220/+59, –124/+59 and –52/+59) ligated to luciferase. (C) Identification of the regions responsible for 15d-PGJ2-induced promoter activity of the human COX-2 gene. MCF-7 cells were transfected with 2.5 µg of a series of human COX-2 promoter-luciferase constructs (–327/+59, ILM, CRM). ILM and CRM represent the –327/+59 COX-2 promoter constructs with mutations in binding sites for nuclear factor-interleukin-6 (NF-IL6) and CRE, respectively. For the experiments related to (B) and (C), MCF-7 cells were transiently cotransfected with pCOX-2 promoter and pCMV-β-galactosidase (0.5 µg) for 24 h by using N-[1-(2,3-dioleolloxy)propyl]-N,N,N-trimethylammonium methylsulfate liposomal transfection reagent according to the manufacturer’s instructions. Transfectant cells were treated with 15d-PGJ2 (30 µM) for 12 h and the cells were lysed with reporter lysis buffer for the measurement of luciferase activity. Fold inductions in the luciferase activity were normalized to β-galactosidase activity. Data are means ± SDs (n = 4). *Significantly different (P < 0.05).

 
Based on these findings, we examined whether 15d-PGJ2 could activate AP-1, a representative transcription factor that preferentially binds to CRE. The AP-1 proteins are homo- or heterodimers composed of basic region-leucine zipper proteins such as Jun, Fos and Jun dimerization partners and the closely related activating transcription factor subfamily proteins (ATF2, LRF1/ATF3 and B-ATF) (15,16). When MCF-7 cells were treated with 15d-PGJ2, the increased DNA-binding activity of AP-1 was observed up to 12 h (Figure 4A). Furthermore, 15d-PGJ2 also enhanced the luciferase reporter activity of AP-1 (Figure 4B). To verify the role of AP-1 in regulating 15d-PGJ2-induced COX-2 expression, we designed antisense oligonucleotides for c-jun and c-fos that encode major AP-1 components as well as the control oligonucleotide containing the equal number of base pairs (17). When cells were treated with each of the antisense oligonucleotides (1 or 10 µM) for 4 h prior to 15d-PGJ2 treatment, both AP-1 DNA binding (Figure 4C) and COX-2 expression (Figure 4D) were reduced in a concentration-dependent fashion. The inhibitory effect of c-fos antisense on COX-2 expression as well as AP-1 DNA binding appeared to be stronger than that of c-jun antisense. These results support that 15d-PGJ2-induced upregulation of COX-2 expression is mediated at least in part via activation of AP-1 signaling.


Figure 4
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  		Fig. 4. Involvement of AP-1 in the induction of COX-2 by 15d-PGJ2 in MCF-7 cells. (A) MCF-7 cells were treated with 15d-PGJ2 (30 µM) for indicated time periods. The DNA-binding activity of AP-1 in MCF-7 cells stimulated with 15d-PGJ2 was measured by electrophoretic mobility shift assay. The nuclear extract isolated from 15d-PGJ2-treated cells was used for electrophoretic mobility shift assay as described under Materials and Methods. (B) The 15d-PGJ2-mediated increase in the transcriptional activation of AP-1 was measured by the luciferase reporter gene assay. After overnight transfection, cells were exposed to 30 µM 15d-PGJ2 for 12 h and treated with reporter lysis buffer for the measurement of the luciferase activity. *Significantly different from the value obtained with the blank vector (P < 0.005). (C) Antisense c-fos and c-jun oligonucleotides inhibit the 15d-PGJ2-induced DNA-binding activity of AP-1 in MCF-7 cells. Cells were treated with the c-fos and c-jun antisense oligonucleotides (1 and 10 µM) for 4 h followed by stimulation with 15d-PGJ2 (30 µM) for additional 12 h. Quantitative data on the AP-1 DNA-binding activity are provided on the right. *Significantly different from the value obtained with the dimethyl sulfoxide-treated group (P < 0.005). **Significantly different from the value obtained with the 15d-PGJ2-treated group (P < 0.05). (D) Antisense c-fos and c-jun oligonucleotides inhibit 15d-PGJ2-induced upregulation of COX-2 in MCF-7 cells. Cells were either left untreated or treated with 15d-PGJ2 (30 µM) for 24 h in the presence or absence of c-fos or c-jun antisense oligonucleotides (1 and 10 µM). COX-2 protein levels were determined by Western blot analysis as described under Materials and Methods. Quantification of COX-2 immunoblot was normalized to that of actin followed by statistical analysis of relative image density. *Significantly different from the value obtained with the dimethyl sulfoxide-treated group (P < 0.005). **Significantly different from the value obtained with the 15d-PGJ2-treated group (P < 0.005).

 
Both AP-1 binding and COX-2 expression were not completely blocked in treatment of MCF-7 cells with c-jun or/and c-fos antisense. It is not clear whether the expression of COX-2 by 15d-PGJ2 in MCF-7 cells is solely regulated by c-Jun and/or c-Fos. It is probable that AP-1 activation by 15d-PGJ2 in MCF-7 is modulated by factors other than c-Jun and c-Fos. Some other factors may still act as functional components of AP-1 even if c-jun and c-fos are inhibited by their corresponding antisense oligonucleotides. Our results show that c-Fos is a more critical AP-1 protein involved in regulating COX-2 expression by 15d-PGJ2 in MCF-7 cells. As a member of the AP-1 transcription factor complex, c-Fos is an essential modulator of cell proliferation, differentiation and transformation (18). Lu et al. (19) observed that c-fos antisense more effectively suppresses breast cancer cell growth than does c-jun antisense. From these studies, they conclude that c-Fos functions as a critical mediator of AP-1-regulated breast cancer cell growth. Our current results are consistent with those from these previous studies.

The expression of COX-2 is regulated via Akt activation
In an attempt to identify the upstream signaling pathways responsible for AP-1 activation and COX-2 induction by 15d-PGJ2, we initially examined its effect on the activation of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase, p38 and c-Jun N-terminal kinase, and also on the activation of the survival signaling represented by Akt. Akt is a serine/threonine kinase that regulates both growth and survival mechanisms by phosphorylating a large number of substrates (20). Recent reports have shown that Akt regulates COX-2 overexpression in various cell cultures and in in vivo studies (21–23). In line with this notion, 15d-PGJ2 transiently activated Akt via phosphorylation (Figure 5A). In a subsequent experiment, MCF-7 cells were transfected with the plasmid carrying the KD-Akt with K179M mutation and then stimulated with 15d-PGJ2. MCF-7 cells transfected with functionally inactive Akt (KD) were much less responsive to 15d-PGJ2 in terms of induced expression of COX-2 (Figure 5B) when compared with the cells transfected with wild-type Akt. To further verify the involvement of the phosphatidylinositol-3 kinase–Akt pathway in AP-1-mediated induction of COX-2 expression, cells were preincubated for 30 min with LY294002 (a phosphatidylinositol-3 kinase–Akt inhibitor) followed by treatment with 15d-PGJ2 for additional 6 h. The pharmacologic inhibition of Akt with LY294002 abrogated 15d-PGJ2-induced COX-2 expression in a concentration-dependent manner (Figure 5C). Likewise, AP-1 DNA binding induced by 15d-PGJ2 was attenuated by genetic and pharmacologic inhibition of phosphatidylinositol-3 kinase activity (Figure 6). These findings suggest that 15d-PGJ2 induces AP-1 activation and subsequently COX-2 expression via Akt activation. However, the pharmacologic inhibition of the other MAPKs (extracellular signal-regulated kinase, p38 and c-Jun N-terminal kinase) barely affected COX-2 induction by 15d-PGJ2 (Figure 5D), suggesting that the MAPKs pathway are unlikely to be involved in 15d-PGJ2-induced COX-2 upregulation in MCF-7 cells.


Figure 5
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  		Fig. 5. 15d-PGJ2-induced expression of COX-2 through activation of Akt signaling. (A) The time-related activation of Akt was assessed by measuring the respective phosphorylated form. MCF-7 cells were stimulated with 30 µM 15d-PGJ2 for the indicated time periods followed by immunoblot analysis with specific antibodies that recognize different kinases and its phosphorylation state. (B) The Akt inhibition suppresses the expression of COX-2 induced by 15d-PGJ2. MCF-7 cells were transfected transiently with hemaglutinin-tagged full-length Akt or KD-Akt. The expression of COX-2 protein was monitored by Western blot analysis. Total protein was isolated after treatment with 30 µM 15d-PGJ2 for 24 h. (C) The effect of the phosphatidylinositol-3 kinase inhibitor on the induction of COX-2 was assessed by Western blot analysis in MCF-7 cells exposed to 30 µM 15d-PGJ2 for 24 h in the presence of 0, 1, 5 or 20 µM LY294002. (D) The effect of the inhibitors of extracellular signal-regulated kinase, c-Jun N-terminal kinase and p38 on the induction of COX-2 was assessed by Western blot analysis in MCF-7 cells exposed to 30 µM 15d-PGJ2 for 24 h in the presence of U0126 (1 and 5 µM), SP600125 (5 and 25 µM) or SB203580 (5 and 25 µM).

 


Figure 6
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  		Fig. 6. Role of Akt signaling in 15d-PGJ2-induced activation of AP-1. The effect of the genetic (A) and pharmacologic inhibition (B) on the DNA-binding activity of AP-1 was assessed by the electrophoretic mobility shift assay in MCF-7 cells exposed to 30 µM 15d-PGJ2 for 12 h. (A) After transient transfection with hemaglutinin-tagged full-length Akt and KD-Akt, the DNA-binding activity of AP-1 was measured by the electrophoretic mobility shift assay in MCF-7 cells. (B) The effect of the Akt inhibitor on the AP-1 binding in nucleus was assessed by the gel shift assay in MCF-7 cells exposed to 30 µM 15d-PGJ2 for 12 h with or without LY294002 (20 µM).

 
15d-PGJ2 generates ROS that induces COX-2 expression
15d-PGJ2 and other cyclopentenone prostaglandins have been shown to induce the production of ROS in diverse cell types, which could mediate their effects on various cellular events (24–27). Therefore, we attempted to determine whether ROS could mediate the 15d-PGJ2-induced Akt activation and downstream signaling. Treatment of MCF-7 cells with 15d-PGJ2 resulted in ROS production which was significantly suppressed by the addition of the ROS scavenger N-acetyl-L-cysteine (NAC) (Figure 7A). Pretreatment of MCF-7 cells with NAC significantly reduced 15d-PGJ2-induced phosphorylation of Akt (Figure 7B). Similarly, the increased DNA-binding activity of AP-1 caused by 15d-PGJ2 was attenuated in the presence of NAC (Figure 7C). Furthermore, the 15d-PGJ2-induced COX-2 expression was also abolished by pretreatment with NAC (Figure 7D). These results suggest that ROS generated by 15d-PGJ2 activate Akt and AP-1 signaling in sequence, which consequently induces expression of COX-2 in MCF-7 cells.


Figure 7
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  		Fig. 7. ROS involvement in the activation of AP-1 and Akt and COX-2 expression induced by 15d-PGJ2. (A) 15d-PGJ2 stimulates ROS production in MCF-7 cells. MCF-7 cells were treated with 30 µM 15d-PGJ2 for 3 h with or without NAC (5 mM) pretreatment. When necessary, the cells were incubated for 1 h with NAC before the 15d-PGJ2 treatment. ROS production was determinated by the 2',7'-dichlorofluorescein diacetate assay as described in Materials and Methods. (B) 15d-PGJ2-induced phosphorylation of Akt was abolished by NAC. MCF-7 cells were preincubated for 1 h with NAC (5 mM) followed by treatment with 15d-PGJ2 (30 µM) for additional 6 h. Whole-cell lysate was prepared to detect phosphorylated Akt and Akt by immunoblot analysis. (C) Nuclear protein (5 µg) was prepared and incubated with the [{gamma}-32P]-labeled oligonucleotides containing the AP-1 consensus sequence for the electrophoretic mobility shift assay. The electrophoretic mobility shift assay was performed in triplicate and a representative result is shown. MCF-7 cells were preincubated for 1 h with NAC (5 mM) followed by treatment with 15d-PGJ2 (30 µM) for additional 12 h. Lane 1, free probe only (no nuclear extract); lane 2, dimethyl sulfoxide control; lane 3, 15d-PGJ2 alone; lane 4, NAC (5 mM) plus 15d-PGJ2; lane 5, NAC (5 mM) only; lane 6, 15d-PGJ2-treated sample plus 100-fold of excess unlabeled oligonucleotide. (D) 15d-PGJ2-induced COX-2 expression was blocked by NAC. MCF-7 cells were treated with 15d-PGJ2 (30 µM) in the presence of NAC (1, 5 and 10 mM) for 24 h. The COX-2 level in the cell lysates was determined by Western blot analysis. Actin was used as a control for equal loading.

 
The {alpha},β-unsaturated carbonyl moiety present in the cyclopentenone ring of 15d-PGJ2 is crucial to induce ROS production and COX-2 expression
The presence of the {alpha},β-unsaturated carbonyl group in the cyclopentenone ring of 15d-PGJ2 has been suggested to be prerequisite for inducing the alteration of cellular redox status and/or the modulation of target protein functions (3). This prompted us to determine whether the ROS production and activation of intracellular signaling for COX-2 induction by 15d-PGJ2 is attributed to its characteristic {alpha},β-unsaturated carbonyl functional group. To validate such structural requirement, MCF-7 cells were exposed to 15d-PGJ2 and its analog 9,10-dihydro-15d-PGJ2 that lacks the cyclopentenone structure (Figure 8A), and induction of ROS production and COX-2 expression were compared. As shown in Figure 8B, while the intracellular ROS production in MCF-7 cells was induced by 15d-PGJ2, 9,10-dihydro-15d-PGJ2 failed to induce ROS production. Moreover, 9,10-dihydro-15d-PGJ2 was much less effective than 15d-PGJ2 in inducing Akt activation and COX-2 expression (Figure 8C). Thus, elimination of the the {alpha},β-unsaturated carbonyl moiety by catalytic hydrogenation of the double bond in the cyclopentenone ring of 15d-PGJ2 virtually abolished the pro-oxidant and pro-inflammatory effects of 15d-PGJ2, indicating that these biological activities can be attributed to the electrophilic carbon at the position 9 of 15d-PGJ2.


Figure 8
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  		Fig. 8. Comparative effects of 15d-PGJ2 and 9,10-dihydro-15d-PGJ2 on ROS production and COX-2 expression. (A) The chemical structures of 15d-PGJ2 (upper) and 9,10-dihydro-15d-PGJ2 (lower). Asterisks depict electrophilic carbons (position 9 and 13). (B) Intracellular ROS production in MCF-7 cells treated with 15d-PGJ2 or 9,10-dihydro-15d-PGJ2 (30 µM each) for 3 h was examined in MCF-7 cells as described in Materials and Methods. (C) MCF-7 cells were exposed to 15d-PGJ2 or 9,10-dihydro-15d-PGJ2 (each 30 µM) for 6 h (for pAkt) or 24 h (for COX-2). Whole-cell extracts were analyzed for Akt phosphorylation or COX-2 expression by immunoblotting.

 

   Discussion
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 Materials and methods
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Funding
 References
 
Abnormally elevated COX-2 expression has been frequently, although not all, observed in human breast cancer tissues (28–31). In addition, COX-2 upregulation has been shown to correlate with distant metastases in breast cancer (32,33). Although some studies failed to find a significant correlation between aspirin use and breast cancer risk, there is now enough evidence for the involvement of COX-2 in breast cancer and for the beneficial effects of COX-2 inhibitors (34). While human breast tumor specimens exhibit elevated COX-2 levels, compared with surrounding tissues, there was relatively low extent of constitutive COX-2 expression in breast cancer cell lines that we examined. However, this is not surprising as augmented COX-2 expression as well as other pro-inflammatory enzymes/cytokines in human tumor tissues is influenced by the tumor microenvironment that is composed of surrounding host stromal tissue and infiltrated inflammatory/immune cells as well as tumors tissues. Such microenvironment is not anticipated to exist in cultured cells. However, when MCF-7 cells were treated with 15d-PGJ2 in human breast cancer cell lines, COX-2 induction started as early as in 6 h and continued up to 24 h.

15d-PGJ2 has been reported to inhibit AP-1 DNA-binding activity in interleukin-1β-treated mesangial cells (35) and human chondrocytes (36,37), which were PPAR{gamma} dependent (37) or independent (35,36). Simonin et al. (38) demonstrated that 15d-PGJ2 inhibited lipopolysaccharide-induced DNA-binding activity of AP-1 and nuclear factor-{kappa}B in a PPAR{gamma}-independent manner. Jozkowicz et al. (39) reported that 15d-PGJ2 inhibited AP-1 DNA-binding activity via PPAR{gamma}-independent mechanisms in human umbilical vein endothelial cells. In particular, the antioxidant NAC blocked the inhibitory effect of 15d-PGJ2 on AP-1 DNA binding, indicative of the involvement of ROS in 15d-PGJ2-mediated AP-1 inactivation (40). The direct interaction of 15d-PGJ2 with AP-1 proteins was revealed by Perez-Sala et al. (41). However, our present study clearly demonstrates that COX-2 expression induced by 15d-PGJ2 is mediated through DNA binding and transcriptional activity of AP-1, a representative transcription factor harboring the CRE-binding site. We found that a mutation at the CRE-binding site completely abolished the COX-2 promoter activity, suggesting that this site located in the COX-2 promoter plays a crucial role in regulating COX-2 transcription under the control of AP-1. Treatment with c-jun and c-fos antisense oligonucleotides abrogated the AP-1-driven COX-2 expression in 15d-PGJ2-stimulated MCF-7 cells. Considering the important role of AP-1 in tumor promotion, the activation of this transcription factor is likely to contribute to the oncogenic effects of 15d-PGJ2 on human breast carcinogenesis. Classical regulation of cellular AP-1 activity occurs via two mechanisms: one is an increase in the transcription of c-fos and c-jun and the other is the phosphorylation of c-Fos and c-Jun proteins. The AP-1 activity is also regulated via redox-dependent mechanisms (reviewed in ref. 42). The pretreatment of MCF-7 cells with LY294002 and transient transfection of the cells with functionally inactive KD-Akt diminished COX-2 expression as well as AP-1 DNA binding. However, MAPK inhibitors were not effective in blocking the same processes. Therefore, Akt appears to be a major upstream target for pro-inflammatory and plausible tumor-promoting activity of 15d-PGJ2.

ROS are potent biological messenger molecules and have been implicated in the induction of gene expression through regulating several distinct intracellular signaling cascades (43). A variety of cellular enzyme systems are potential sources of ROS production. These include NAD(P)H oxidase, xanthine oxidase, uncoupled endothelial NO synthase, arachidonic acid xenobiotic-metabolizing enzymes including cytochrome P-450, lipoxygenase, COX and enzymes in the mitochondrial respiratory chain (44). ROS-induced oxidative stress is considered to be a general mechanism for AP-1 activation and COX-2 expression by a variety of agents (45,46). In support of this notion, the antioxidant NAC suppressed not only 15d-PGJ2-induced ROS production but also AP-1 DNA binding and COX-2 expression. Furthermore, NAC blocked the phosphorylation of Akt induced by 15d-PGJ2. These findings suggest that Akt can function as a downstream effector of ROS in transducing 15d-PGJ2-initiated signals via AP-1 to induce COX-2 expression in MCF-7 cells. However, the precise mechanism underlying association between ROS and Akt in 15d-PGJ2-induced signaling cascades remains to be clarified. One possibility for the activation of Akt is the interaction of 15d-PGJ2 with phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a negative regulator of the Akt pathway. A recent report has revealed that PTEN can be inactivated by formation of a disulfide bond between the active site Cys124 and Cys71 as a consequence of oxidation of these cysteine residues in NIH3T3 and HeLa cells treated with H2O2 (47). A preliminary data from our laboratory also revealed that 15d-PGJ2 treatment inhibited the expression of PTEN in MCF-7 cells. Moreover, by utilizing biotinylated 15d-PGJ2, we were able to demonstrate the direct interaction of 15d-PGJ2 with PTEN (E.-H.Kim and Y.-J.Surh, in preparation). Consequently, the inactivation of PTEN by 15d-PGJ2 appears to contribute to the activation of Akt, leading to DNA binding of AP-1 and the expression of COX-2.

Several lines of evidence suggest ROS generation by exogenously added 15d-PGJ2. While molecular mechanisms of ROS generation by 15d-PGJ2 remain to be defined, one plausible mechanism may involve conjugation of 15d-PGJ2 with reduced glutathione (GSH), an important component of the cellular antioxidant defense system. Paumi et al. (48) demonstrated that 15d-PGJ2 conjugated with GSH at carbon 9, whereas 9,10-dihydro-15d-PGJ2 that lacks the electrophilic carbon center at position 9 failed to form such conjugate. It is well known that reduction of the cellular GSH results in the generation of ROS. In this regard, the formation of 15d-PGJ2–GSH conjugates may, at least in part, contribute to ROS production by this cyclopentenone prostaglandin. Another possibility of ROS production by 15d-PGJ2 may involve disruption of mitochondrial respiratory electron flow. 15d-PGJ2 increased ROS production in MCF-7 cells, which was attenuated by the mitochondrial complex I inhibitor rotenone (25). Similar increases in ROS formation have also been reported in isolated mitochondria treated with 15d-PGJ2, and it is noteworthy that an electrophile-responsive proteome in the mitochondria is composed of proteins with reactive thiols (49–51). The cyclopentenone moiety of 15d-PGJ2 has been proposed as a critical structural feature responsible for some of its biological effects (3). 9,10-Dihydro-15d-PGJ2, which differs from 15d-PGJ2 only in the absence of the endocyclic double bond, was unable to generate ROS in MCF-7 cells, lending support that the electrophilic carbon center at position 9 of 15d-PGJ2 is required for ROS formation. Consistent with this finding, 9,10-dihydro-15d-PGJ2 did not mimic the marked inducible effect of 15d-PGJ2 on the expression of COX-2 as well as phosphorylation of Akt. Taken together, these observations suggest that the unique cyclopentenone structure of 15d-PGJ2 is a critical determinant in its induction of COX-2 in MCF-7 cells.

Chronic inflammation is a well-known risk factor for cancer development. It is noteworthy that many malignant cells produce ROS to a greater extent than normal cells for their growth (43). Just as inflammation can promote cancer development, a tumor often induces an inflammatory response that can be often mediated by ROS. In this context, 15d-PGJ2 formed as a consequence of COX-2 induction may represent a potential molecular link between increased ROS production and chronic inflammation-induced cancer. Our present study does show that 15d-PGJ2 functions as a positive regulator of COX-2 in human breast cancer cells via the Akt-AP-1 signaling pathway (Figure 9), which may provide these cells with survival advantage. As the expression level of COX-2 as well as the ability to respond to 15d-PGJ2 is variable in different cell lines, our results obtained from a single cell line cannot be extrapolated to other cell lines. In order to explore the role of endogenous 15d-PGJ2 in mammary carcinogenesis, our present study should be expanded to use of alternative cell lines and also tumor tissues, which have high expression level of COX-2.


Figure 9
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  		Fig. 9. Schematic representation of 15d-PGJ2-induced upregulation of COX-2 expression via activation of Akt and AP-1. The mechanisms involve at least two possibilities; oxidation or direct modification of cysteine residues of PTEN and conjugation of 15d-PGJ2 with reduced glutathione (GSH) with concomitant generation of ROS. Both events will lead to increased phosphorylation of Akt, DNA binding of AP-1 and subsequently expression of COX-2.

 

   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Korea Science and Engineering Foundation through the National Research Laboratory Program (M10400000366-06J0000-36610); Innovative Drug Research Center through the Ministry of Science and Technology.


   Acknowledgments
 
The authors thank Dr H. Inoue, Nara Women's University, for generous supply of COX-2 promoter constructs.

Conflict of Interest statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received June 12, 2007; revised November 15, 2007; accepted December 20, 2007.


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