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Cyclooxygenase Inhibitors Suppress Aromatase Expression and Activity in Breast Cancer Cells Edgar S. Díaz-Cruz, Charles L. Shapiro and Robert W. Brueggemeier J. Clin. Endocrinol. Metab. 2005 90:2563-2570 originally published online Feb 1, 2005; , doi: 10.1210/jc.2004-2029 To subscribe to Journal of Clinical Endocrinology & Metabolism or any of the other journals published by The Endocrine Society please go to: http://jcem.endojournals.org//subscriptions/ Copyright © The Endocrine Society. All rights reserved. Print ISSN: 0021-972X. Online 0021-972X/05/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 90(5):2563–2570 Printed in U.S.A. Copyright © 2005 by The Endocrine Society doi: 10.1210/jc.2004-2029 Cyclooxygenase Inhibitors Suppress Aromatase Expression and Activity in Breast Cancer Cells ´ Edgar S. Dıaz-Cruz, Charles L. Shapiro, and Robert W. Brueggemeier Division of Medicinal Chemistry and Pharmacognosy (E.S.D.-C., R.W.B.), College of Pharmacy; and Division of Hematology and Oncology (C.L.S.), Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, Ohio 43210 Estradiol is biosynthesized from androgens by the aromatase matase activity after treatment with all agents. Real-time PCR enzyme complex. Previous studies suggest a strong associa- analysis of aromatase gene expression showed a significant tion between aromatase (CYP19) gene expression and the ex- decrease in mRNA levels when compared with control for all pression of cyclooxygenase (COX) genes. Our hypothesis is agents. These results were consistent with enzyme activity that higher levels of COX-2 expression result in higher levels data, suggesting that the effect of COX inhibitors on aro- of prostaglandin E2, which, in turn, increases CYP19 expres- matase begins at the transcriptional level. Exon-specific real- sion through increases in intracellular cAMP levels. This bio- time PCR studies suggest that promoters I.3, I.4, and II are chemical mechanism may explain the beneficial effects of non- involved in this process. Thus, COX inhibitors decrease aro- steroidal antiinflammatory drugs on breast cancer. The matase mRNA expression and enzymatic activity in human effects of nonsteroidal antiinflammatory drugs, COX-1 and breast cancer cells in culture, suggesting that these agents COX-2 selective inhibitors on aromatase activity and expres- may be useful in suppressing local estrogen biosynthesis in sion were studied in human breast cancer cells. The data from the treatment of hormone-dependent breast cancer. (J Clin these experiments revealed dose-dependent decreases in aro- Endocrinol Metab 90: 2563–2570, 2005) A DENOCARCINOMA OF THE breast is the most com- mon cancer in women in the United States and ranks second only to lung cancer as a cause of cancer-related mor- the regulatory mechanism of aromatase expression from nor- mal breast tissue to cancerous tissue has been extensively investigated (4, 7, 8). Although promoter I.4 requires the tality. An estimated 215,990 new cases of invasive breast synergistic actions of glucocorticoids and class I cytokines cancer were expected to occur among women in the United (9), promoters I.3 and II are both transactivated by protein States during 2004, and an estimated 40,110 deaths were kinase A (PKA) cAMP-dependent signaling pathways (8, 10). anticipated from breast cancer in 2004 (1). Prostaglandin G/H endoperoxide synthase, also known as Approximately 60% of all breast cancer patients have hor- cyclooxygenase (COX), is a key enzyme that catalyzes the mone-dependent breast cancer, which contains estrogen re- conversion of arachidonic acid to prostaglandins. Two iso- ceptors and requires estrogen for tumor growth. Estrogens forms have been identified, COX-1 and COX-2 (11, 12). Al- are biosynthesized from androgens by aromatase, the prod- though COX-1 is present at a constant level in most cells and uct of the CYP19 gene and a member of the cytochrome P450 tissues and is believed to play a housekeeping role, some enzyme superfamily (2). The concentration of estradiol, the studies have shown that COX-1 activity and expression is most potent endogenous estrogen, is higher in the tumor elevated in human breast cancer tumors (13, 14). Most studies tissue than in the normal areas of the breast (3). Aromatase have shown that COX-2 is present in breast cancer tissue transcript expression (4) and activity (3, 5) in the tumor are samples but not in the normal breast tissue (14 –16). Prosta- greater than that in the normal breast tissue. Regulation of glandins produced by COX-2, predominantly prostaglandin aromatase expression in human tissues is quite complex, E2 (PGE2), induce inflammation and are potent mediators of involving alternative promoter sites that provide tissue-spe- a number of signal transduction pathways that are impli- cific control. In the normal breast cells, aromatase expression cated in cancer development. Concentrations of PGE2 in the is primarily derived by the tissue-specific promoter I.4 for normal tissue are lower than the levels detected in tumor and transcription, whereas expression from breast cancer pa- metastatic tissues (16, 17). tients switches from promoter I.4 to promoter I.3 and pro- Nonsteroidal antiinflammatory drugs (NSAIDs) are used moter II (6). These results suggest that promoters I.3 and II to decrease inflammation by inhibiting COX. A growing are the major promoters directing aromatase expression in body of experimental (18, 19) and epidemiological (19 –22) breast cancer and surrounding stromal cells. This switch in evidence suggests that the use of NSAIDs may decrease the risk of breast cancer. Aspirin (23) and flurbiprofen (24) were First Published Online February 1, 2005 shown to reduce mammary tumorigenesis. SC-560, cele- Abbreviations: COX, Cyclooxygenase(s); DMSO, dimethylsulfoxide; coxib, and indomethacin treatment resulted in statistically NSAIDs, nonsteroidal antiinflammatory drugs; PGE2, prostaglandin E2; PKA, protein kinase A; PKC, protein kinase C; RT, reverse transcription. significant inhibition of tumor size in comparison with ve- JCEM is published monthly by The Endocrine Society (http://www. hicle-treated control animals in a murine model of breast endo-society.org), the foremost professional society serving the en- cancer (25). Celecoxib and ibuprofen produced striking re- docrine community. ductions in the incidence of mammary cancer, tumor burden, 2563 2564 J Clin Endocrinol Metab, May 2005, 90(5):2563–2570 ´ Dıaz-Cruz et al. • Suppression of Aromatase by COX Inhibitors and tumor volume compared with those seen in the control After centrifugation, a 250- l aliquot containing the product was group in animal models (26). Genetic and pharmacological counted in 5 ml of liquid scintillation mixture. Results were corrected for blanks and for the cell contents of culture flasks, and results were evidence has shown that specific COX-2 inhibition is more expressed as picomoles of 3H2O formed per hour incubation time per effective than traditional NSAIDs in suppressing polyposis million live cells (pmol/h 106 cells). To determine the amount of live cells in the mouse (27). All of these findings indicate that COX are in each flask, the cells were trypsinized and analyzed using the diphe- involved in the promotion of this type of cancer. nylamine DNA assay adapted to a 96-well plate (29, 30). The relationship between COX and aromatase was exam- ined in a preliminary study of CYP19 gene expression with Enzyme immunoassay of PGE2 COX-1 and COX-2 gene expression in breast cancer patient To study PGE2 synthesis in cell culture media, experiments were specimens. Regression analysis using a bivariate model performed in 12-well plates. An aliquot of SK-BR-3 cells (150,000 cells) showed a strong linear association between the sum of was added to each well, and plates were incubated overnight to allow the cells to adhere to the plates. After this time, cells were serum starved COX-1 and COX-2 expression and CYP19 expression (15). in defined media for 24 h. This step was followed by replacement of Another study using immunohistochemistry staining for media with fresh media containing either vehicle (DMSO) or the indi- aromatase and COX-2 revealed a marked correlation be- cated concentration of agents. After 24-h incubation at 37 C, the media tween COX-2 and aromatase expression in tumor samples were collected, and the amount of PGE2 was determined by ELISA (28). (Cayman Chemical) according to the protocol provided by the manu- facturer. PGE2 concentration was normalized to total protein. Total The present study examines the activity and expression of proteins were extracted from adhered cells by 30-min treatment with 0.5 aromatase in breast cancer cell lines after the exposure to m NaOH at room temperature and shaking. Protein concentrations in nonselective and isozyme-selective COX inhibitors, to pro- these extracts were determined using a protein assay method (Bio-Rad vide additional information on the association between aro- Laboratories, Inc., Hercules, CA). matase, COX, and human breast cancer development. RNA extraction Materials and Methods Total RNA was isolated using the TRIzol reagent according to the Chemicals and reagents manufacturer’s protocol. Total RNA pellets were dissolved in nuclease- free water and quantitated using a spectrophotometer. The quality of Radiolabeled [1 -3H]androst-4-ene-3,17-dione was obtained from RNA samples was determined by electrophoresis through agarose gels NEN Life Science Products (Boston, MA). The following compounds and staining with ethidium bromide; the 18S and 28S rRNA bands were were purchased from Cayman Chemical (Ann Arbor, MI): niflumic acid, visualized under UV light. nimesulide, NS-398, SC-560, and SC-58125. The following compounds were purchased from Sigma (St. Louis, MO): ibuprofen, indomethacin, cDNA synthesis and piroxicam. Celecoxib was a gift from Dr. Ching-Shih Chen [The Ohio State University (OSU), College of Pharmacy, Columbus, OH]. Trypsin, Isolated total RNA (2 g) was treated with DNase I, amplification TRIzol, and all enzymes were obtained from Invitrogen (Carlsbad, CA). grade, according to the recommended protocol, to eliminate any DNA Radioactive samples were counted on a LS6800 liquid scintillation before reverse transcription (RT). Treated total RNA was denatured at counter (Beckman, Palo Alto, CA). Mixture 3a70B was obtained from 65 C for 5 min in the presence of 2.5 ng/ l random hexamers and 0.5 Research Prospect International Corp. (Mount Prospect, IL). mm deoxynucleotide triphosphate mix. The samples were snap-cooled on ice and centrifuged briefly. cDNA was synthesized using Superscript Cell culture II reverse transcriptase according to the recommended protocol. Briefly, the reactions were conducted in the presence of 1 first-strand buffer JAR, MCF-7, MDA-MB-231, and SK-BR-3 cell lines were obtained and 20 mm dithiothreitol at 42 C for 50 min and consequently inactivated from American Type Culture Collection (Rockville, MD). Cell cultures at 70 C for 15 min. The cDNA generated was used as a template in were maintained in phenol red-free custom media [MEM, Earle’s salts, real-time PCR. 1.5 amino acids, 2 nonessential amino acids, l-glutamine, and 1.5 vitamins (Life Technologies, Inc., Carlsbad, CA)] supplemented with Real-time PCR 10% fetal bovine serum, 2 mm l-glutamine, and 20 mg/liter gentamycin. Fetal calf serum was heat inactivated for 30 min in a 56 C water bath Real-time PCR was performed using the Opticon 2 system from MJ before use. Cell cultures were grown at 37 C in a humidified atmosphere Research (Waltham, MA). For the CYP19 total gene, the PCR mixture of 5% CO2 in a Hereaus CO2 incubator. For all experiments, cells were consisted of TaqMan Universal PCR Master Mix (Applied Biosystems, plated in either T-25 flasks or 100 mm plates and grown to subconflu- Foster City, CA), 600 nm of each primer (Invitrogen) (Table 1), 250 nm ency. Before treatment, the media was changed to a defined one con- TaqMan probe, 18S rRNA (Applied Biosystems), and 2.5 l of each RT taining DMEM/F12 media (Sigma) with 1.0 mg/ml human albumin sample in a final volume of 25 l. The TaqMan probe was designed to (OSU Hospital Pharmacy, Columbus, OH), 5.0 mg/liter human trans- anneal to a specific sequence of the aromatase gene between the forward ferrin, and 5.0 mg/liter bovine insulin. and the reverse primers (Table 1). Cycling conditions were 50 C for 2 min and 95 C for 10 min, followed by 50 cycles at 95 C for 15 sec and 60 C Tritiated water-release assay for 1 min. For the specific exon I promoter regions and TATA-box-binding Measurement of aromatase enzyme activity was based on the tritium protein, the PCR mixture consisted of DyNAmo Hot Start SYBR Green water-release assay (29). Cells in T-25 flasks or 100 mm plates were qPCR kit (MJ Research), 600 nm of each primer (Table 1), and 2.5 l of treated with 0.1% dimethylsulfoxide (DMSO; control), NSAIDs (ibu- each RT sample in a final volume of 20 l. SYBR Green uses a dye that profen, piroxicam, and indomethacin), COX-1 selective inhibitor SC-560, will bind to double-stranded DNA. In this methodology, the primers are and COX-2 selective inhibitors (SC-58125, NS-398, celecoxib, niflumic carefully designed to each of the promoter regions of aromatase exon I acid, and nimesulide) at the indicated concentrations. After 24 h, the cells (Table 1). Cycling conditions were 95 C for 15 min, followed by 50 cycles were incubated for 6 h with fresh media along with 50 nm androstenedi- at 94 C for 10 sec, 60 C for 25 sec, and 72 C for 30 sec. one including 2 Ci [1 -3H]androst-4-ene-3,17-dione. Subsequently, the reaction mixture was removed, and proteins were precipitated using Statistical analysis 10% trichloroacetic acid at 42 C for 20 min. After a brief centrifugation, the media was extracted three times with an equal amount of chloroform Statistical and graphical information were determined using Graph- to extract unused substrate and additional dextran-treated charcoal. Pad Prism software (GraphPad Software Inc., San Diego, CA) and Mi- ´ Dıaz-Cruz et al. • Suppression of Aromatase by COX Inhibitors J Clin Endocrinol Metab, May 2005, 90(5):2563–2570 2565 TABLE 1. Oligonucleotide primer and probe sequences for real-time PCR Gene Oligonucleotide Sequences (5 –3 ) CYP19 Primer (S) TGT CTC TTT GTT CTT CAT GCT ATT TCT C Primer (A) TCA CCA ATA ACA GTC TGG ATT TCC Probe 6FAM 5 -TGC AAA GCA CCC TAA TGT TGA AGA GGC AAT-3 TAMRA I.1a Primer (S) TGT GCT CGG GAT CTT CCA GAC Primer (A) GGT TCA GCA TTT CCA AAA CCA TC a I.3 Primer (S) GGG CTT CCT TGT TTT GAC TTG TAA Primer (A) AGA GGG GGC AAT TTA GAG TCT GTT I.4b Primer (S) AAC GTG ACC AAC TGG AGC CTG Primer (A) CAT CAC CAG CAT CGT GCC TG PIIb Primer (S) CTC TGA AGC AAC AGG AGC TAT AGA T Primer (A) CAT CAC CAG CAT CGT GCC TG TBPa Primer (S) TGC ACA GGA GCC AAG AGT GAA Primer (A) CAC ATC ACA GCT CCC CAC CA Total CYP19 gene was analyzed using TaqMan methodology and a sequence-specific fluorogenic probe. Exon I promoter-specific regions and TBP gene were analyzed using SYBR Green methodology. S, Sense; A, antisense. a Ref. 36. b Ref. 37. crosoft Excel (Microsoft Corp., Redmond, WA). Determination of IC50 in MCF-7 cells has also been reported by other groups (33), values was performed using nonlinear regression analysis. Statistically and the mechanism is not well understood. For these studies, significant differences were calculated with the two-tailed unpaired Student’s t test, and P values were reported at 95% confidence intervals. the SK-BR-3 cell line was selected as our breast cancer cell model because this cell line had the better ratio of aromatase Results activity and CYP19 expression than the other breast cancer Aromatase enzymatic activity and expression in human cell lines. breast cancer cell lines Decrease of aromatase enzymatic activity Aromatase activity was determined using the “in-cell” The effects of NSAIDs, COX-1, and COX-2 selective in- tritiated water-release assay and normalized to the number hibitors on aromatase activity were determined, and the of live cells in each flask. Different human breast cancer cell concentration producing a 50% decrease in activity (IC50) for lines (MCF-7, MDA-MD-231, and SK-BR-3) were compared each agent was calculated. All agents decreased aromatase for aromatase activity using the tritiated water-release assay activity under control (vehicle) levels in a dose-dependent (Table 2). Cell line SK-BR-3 showed the highest levels of manner. NSAIDs (nonselective COX inhibitors) decreased aromatase activity, followed by MCF-7 and MDA-MB-231. aromatase activity only at high micromolar concentrations The aromatase activity in the SK-BR-3 cell line is approxi- (Fig. 1). Indomethacin was the most potent NSAID in de- mately 20 times higher than that in MCF-7 cells and 35 times creasing aromatase activity, followed by piroxicam and ibu- higher than that in MDA-MB-231 cells. These results agree with previous studies from other researchers (31–33). Cell lines JAR, MCF-7, MDA-MB-231, and SK-BR-3 were used to compare CYP19 gene expression by real-time PCR (Table 2). CYP19 expression was normalized relative to 18s rRNA and compared with expression in JAR cells. The cho- riocarcinoma placental JAR cell line, a cell line that expresses high levels of CYP19, was selected as the calibrator. Results show that basal levels of CYP19 gene expression in MCF-7 cells are slightly higher than those in SK-BR-3 cells but con- siderably higher than those in MDA-MB-231 cells. The dis- crepancy in aromatase activity and CYP19 mRNA expression TABLE 2. Aromatase activitya and expressionb in cell lines Aromatase activity CYP19 expression relative to Cell line (pmol/h 106 cells) JAR cells (2 Ct ) JAR 6.31 2.17c 1.0 0.1 MCF-7 0.00015 0.00004 0.12 0.02 FIG. 1. Suppression of aromatase activity in SK-BR-3 breast cancer SK-BR-3 0.0028 0.0002 0.07 0.01 cells by NSAIDs and COX-1 selective inhibitor. SK-BR-3 cells were MDA-MB-231 0.00008 0.00001 0.004 0.001 treated with indomethacin (E), piroxicam (F), ibuprofen (f), or SC- a Values are expressed as pmol/h 106 cells and reported as mean 560 ( ), and aromatase activity was measured as described in Ma- SD (n 3). terials and Methods. Values are expressed as picomoles 3H2O formed b CYP19 expression was normalized relative to 18S rRNA. Values per hour incubation time per million live cells. The results were are expressed as CYP19 expression relative to a calibrator (JAR cells) normalized against a control treatment with vehicle. The value of and reported as mean SD (n 9). 100% is equal to 0.003 pmol/h 106 cells. Each data point represents the c Ref. 38. mean results of three independent determinations. 2566 J Clin Endocrinol Metab, May 2005, 90(5):2563–2570 ´ Dıaz-Cruz et al. • Suppression of Aromatase by COX Inhibitors profen, with IC50 values 157 5.7, 408 23, and 809 90 m, respectively. Treatment of SK-BR-3 cells for 24 h with SC-560, a COX-1 selective agent, resulted in a decrease of aromatase activity and an IC50 value of 5.8 1.2 m (Fig. 1). PGE2 is a powerful stimulator of aromatase activity and expression in human breast adipose stromal cells (29). Ad- ministration of exogenous PGE2 (1 m) resulted in a 1.4-fold increase in aromatase activity (0.0040 0.0005 pmol/h 106 cells). To examine the hypothesis that prostaglandin inhibi- tion could result in aromatase activity suppression, SK-BR-3 cells were treated for 24 h with COX-2 selective inhibitors. In fact, all COX-2 selective inhibitors decreased aromatase ac- tivity in SK-BR-3 cells in a dose-dependent manner (Fig. 2). NS-398 was the most potent agent in decreasing aromatase activity, with an IC50 value of 1.0 0.4 m. Celecoxib and SC-58125, a celecoxib-like agent, showed IC50 values of 37 4.5 and 24 1.8 m, respectively. Nimesulide and niflumic acid also decreased aromatase activity with IC50 values of 27 4.7 and 97 8.3 m, respectively. Aromatase activity was expressed as picomoles of 3H2O formed per hour incu- bation time per million live cells. Some agents (such as ibu- profen, indomethacin, and niflumic acid) showed some cell FIG. 3. Effect of cyclooxygenase inhibitors on PGE2 production of toxicity at very high concentrations, but aromatase activity SK-BR-3 cells. Cells were treated for 24 h with the indicated concen- was normalized by the number of live cells in each assay, to trations of agents. Results are expressed as means of the concentra- tion of PGE2 produced per microgram protein SEM. *, P 0.05 vs. assure that the loss of cells due to any toxicity effects by the control by unpaired t test (n 6). agents was taken into account. Levels of PGE2 production the production of PGE2, the experimental conditions re- quired the SK-BR-3 cells to use the endogenous substrate. The production of PGE2 was measured in cells treated with This assures that the conditions for the rest of the assays are NSAIDs, a COX-1 selective inhibitor, and COX-2 selective more consistent and comparable to one another. SK-BR-3 inhibitors (Fig. 3). The levels of COX activity in SK-BR-3 cells cells were treated for 24 h with the indicated concentration are low but detectable, consistent with other studies (34). of the agents. All agents resulted in a decrease in PGE2 Although exogenous arachidonic acid would have increased production in SK-BR-3 cells, but only ibuprofen, piroxicam, indomethacin, celecoxib, nimesulide, NS398, and SC-58125 resulted in significant reductions (P 0.05). CYP19 mRNA expression by real-time PCR Analysis of total CYP19 mRNA transcripts was performed using real-time PCR to determine whether the decrease in aromatase activity by COX inhibitors in SK-BR-3 cells was due to a down-regulation of aromatase expression at the transcriptional level. SK-BR-3 cells were treated with COX inhibitors for 24 h at concentrations at or near the IC50 value for each agent, to assure that the concentration was sufficient to suppress aromatase activity and not result in cell death. In fact, cell cytotoxicity assays showed that the concentrations used for this study were safe and not toxic for the cells. Total RNA was extracted at 24 h, and CYP19 transcript levels were compared with control (vehicle) treatment. NSAIDs and COX-2 selective inhibitors significantly decreased CYP19 gene expression in SK-BR-3 cells relative to the control (ve- hicle) treatment (Fig. 4A). The COX-1 selective inhibitor, FIG. 2. Suppression of aromatase activity in SK-BR-3 breast cancer cells by COX-2 selective inhibitors. SK-BR-3 cells were treated with SC-560, also resulted in a significant decrease in expression, NS-398 ( ), nimesulide (E), SC-58125 (f), celecoxib (F), or niflumic suggesting that COX-1 might also be involved in the mech- acid ( ), and aromatase activity was measured as described in Ma- anism. No effect on the expression level of the housekeeping terials and Methods. Values are expressed as picomoles 3H2O formed gene 18S rRNA was observed with any of the agents. These per hour incubation time per million live cells. The results were normalized against a control treatment with vehicle (DMSO). The results support the aromatase activity results, suggesting value of 100% is equal to 0.003 pmol/h 106 cells. Each data point that COX inhibitors have an effect on aromatase. To deter- represents the mean results of three independent determinations. mine whether similar results were observed in other cell ´ Dıaz-Cruz et al. • Suppression of Aromatase by COX Inhibitors J Clin Endocrinol Metab, May 2005, 90(5):2563–2570 2567 cells, EP1 and EP2. EP2 is coupled to stimulation of cAMP formation, whereas EP1 is coupled to protein kinase C (PKC) activation (35). This suggests that PGE2 is capable of acti- vating both PKA- and PKC-mediated signaling pathways. Exon-specific real-time PCR was performed to determine whether these agents specifically affected expression through promoters I.3 and II, which are the two aromatase promoters directly involved in PKA- and PKC-activation through cAMP. In a separate experiment, administration of exogenous PGE2 (1 m) resulted in elevated transcript levels of CYP19 mRNA through promoter II by approximately 1.5-fold when compared with control in SK-BR-3 cells. This confirms the results observed in the tritiated water-release assay when SK-BR-3 cells were treated with exogenous PGE2 (1 m). The NSAIDs, COX-1 and COX-2 selective inhibitors all demon- strated decreases in aromatase expression specific for pro- moter II (Fig. 5A). Aromatase expression is also driven through promoter I.3, the other promoter that is directly linked to the CYP19 switch in aromatase expression in breast tumors. The agents tested (COX-1 and COX-2 selective in- hibitors) showed significant decreases in aromatase expres- sion through this promoter (Fig. 5B). To study the possibility of other promoter regions being involved in the proposed mechanism, CYP19 promoter I.1- and I.4-specific mRNA ex- pression was analyzed. As expected, these agents had no effect on aromatase expression specific for I.1 (Fig. 5C). Sur- prisingly, on the other hand, these agents showed a decrease in aromatase expression specific for promoter I.4 (Fig. 5D). Discussion Aromatase is a key enzyme in the synthesis of estrogens (2) and plays an important role in the process of breast carcinogenesis of hormone-dependent breast cancers (3). COX has also been found to play a key role in this process (13, 14). Furthermore, PGE2 increases aromatase activity through increases in cAMP (29, 31, 35), and COX enzymes are involved in the synthesis of prostaglandins. To study a pos- sible mechanism explaining why NSAIDs exert chemopre- ventive and antitumor properties in human breast cancers, SK-BR-3 cells were treated with these agents and evaluated for aromatase activity and expression. Aromatase activity was decreased in SK-BR-3 cells by NSAIDs and COX selec- FIG. 4. Real-time RT-PCR analysis of CYP19 mRNA expression in tive inhibitors treatment in a dose-dependent manner. COX SK-BR-3 (A) and MCF-7 and MDA-MB-231 cells (B). Cells were selective agents are more effective in suppressing aromatase treated for 24 h with the indicated concentrations of agents, and total activity, with significantly lower IC50 concentrations than RNA was isolated. Results are expressed as means of CYP19 (nor- malized to 18S rRNA) SEM. *, P 0.05 vs. control by unpaired t test those required for NSAIDs (or nonselective COX inhibitors). (n 9). Treatment of SK-BR-3 cells with the various agents at concentrations at or near IC50 values resulted in a decreased production of PGE2, which supports our hypothesis that lines, MCF-7 and MDA-MB-231 cells were treated with se- PGE2 may be involved in CYP19 regulation. Real-time PCR lected COX inhibitors. Figure 4B shows similar decreases in analysis of aromatase gene expression demonstrated that CYP19 gene expression in both MCF-7 and MDA-MB-231 changes in mRNA expression were consistent with enzyme cells. activity data. The data from these experiments showed a significant decrease in mRNA levels when compare to con- CYP19 exon-specific mRNA expression by real-time PCR trol (vehicle) for all agents. The COX-1 selective inhibitor PGE2 is a powerful stimulator of aromatase activity and SC-560 resulted in a significant decrease in aromatase activ- expression in human breast adipose stromal cells (29, 31, 35). ity and expression in SK-BR-3 cells, suggesting that COX-1 PGE2 interacts with two receptor subtypes in adipose stromal may also produce prostaglandins that are involved in this 2568 J Clin Endocrinol Metab, May 2005, 90(5):2563–2570 ´ Dıaz-Cruz et al. • Suppression of Aromatase by COX Inhibitors FIG. 5. Real-time RT-PCR analysis of CYP19 exon-specific mRNA expression in SK-BR-3 cells. Promoter II (A), exon I.3 (B), exon I.1 (C), and exon I.4 (D). Cells were treated for 24 h with the indicated concentrations of agents, and total RNA was isolated. Results are expressed as means of CYP19 (normalized to TATA-box-binding protein) SEM. *, P 0.05 vs. control by unpaired t test (n 9). mechanism. Although COX-1 has also been implicated in through promoter II. NS-398 showed the most significant human breast cancers (13, 14), its levels are relatively con- effect followed by SC-58125, consistent with the enzyme stant. Aromatase expression in MCF-7 and MDA-MB-231 activity data. Promoter I.3 is the other promoter involved in cells is low compared with SK-BR-3 cells, and treatment of the promoter switch from normal breast to cancerous cells these cells with COX inhibitors also resulted in a decrease in (6). Decreases in aromatase expression through promoter I.3 aromatase expression. These results show that COX inhibi- were observed with the COX-2-specific inhibitors and the tors decrease aromatase mRNA expression and that these COX-1-specific inhibitor SC-560 as well, although the levels drugs may be potent therapeutic agents in the treatment of of promoter I.3 in SK-BR-3 cells are low. As expected, aro- breast cancer. matase expression through promoter I.1 was not affected by The effect of NSAIDs and COX selective inhibitors on the any of the agents studied. exon I-specific promoters for aromatase also was investi- Promoter I.4 was then studied, and, once again, all agents gated using real-time PCR. SK-BR-3 cells contain high abun- decreased aromatase expression through this promoter as dance of promoter I.1, followed by promoters II and I.4, and well. The effect was not as significant as promoter II; nev- relatively low abundance of promoter I.3. When SK-BR-3 ertheless, CYP19 expression through promoter I.4 was af- cells were treated with NSAIDs, COX-1 and COX-2 selective fected when treating cells with NSAIDs and COX-specific inhibitors resulted in decreases in aromatase expression inhibitors. Expression via promoter I.4 requires the action of ´ Dıaz-Cruz et al. • Suppression of Aromatase by COX Inhibitors J Clin Endocrinol Metab, May 2005, 90(5):2563–2570 2569 glucocorticoids and class I cytokines or TNF (9). Other NSAID or COX selective inhibitor, may increase efficacy studies have shown that PGE2 regulates TNF at the mRNA beyond the present treatments for postmenopausal hor- and protein level. The pleiotropic effect of prostaglandins, in mone-dependent breast cancer. general, may be playing a key role in this process. It is possible that the decrease in prostaglandin production by Acknowledgments COX-specific inhibitors results in alterations of other bio- Received October 13, 2004. Accepted January 20, 2005. chemical pathways within these cells affecting glucocorti- Address all correspondence and requests for reprints to: Robert W. coid and/or class I cytokine action. This would result in a Brueggemeier, College of Pharmacy, The Ohio State University, 500 decrease in CYP19 expression through promoter I.4. Addi- West 12th Avenue, Columbus, Ohio 43210. E-mail: Brueggemeier.1@ tional studies are underway to better understand this osu.edu. This work was supported by the National Institutes of Health (NIH) process. Grant R01 CA73698 (to R.W.B.), the NIH Chemistry and Biology Inter- These results suggest that PGE2 produced via COX may face Training Program Grant T32 GM08512 (to E.S.D.-C.), and The Ohio act locally in an autocrine fashion to increase the local bio- State University Comprehensive Cancer Center Breast Cancer Research synthesis of estrogen by the aromatase enzyme in hormone- Fund. dependent breast cancer development and lead to growth stimulation. In previous preliminary studies conducted in References human placental microsomes, NSAIDs and the COX-2-spe- 1. American Cancer Society 2004 Cancer facts and figures. Atlanta: American cific inhibitors NS-398 and celecoxib failed to directly inhibit Cancer Society 2. Simpson ER, Mahendroo MS, Means GD, Kilgore MW, Hinselwood MM, aromatase activity. These agents showed signs of aromatase Graham-Lorence S, Amarneh B, Ito Y, Fisher CR, Michael MD, Mendelson activity inhibition only at very high micromolar concentra- CR, Bulun SE 1994 Aromatase cytochrome P450, the enzyme responsible for tions. Thus, the effect of NSAIDs and the COX selective estrogen biosynthesis. Endocr Rev 15:342–355 3. 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