Cancer Cells Cyclooxygenase Inhibitors Suppress Aromatase

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Cancer Cells Cyclooxygenase Inhibitors Suppress Aromatase Powered By Docstoc
					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

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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, 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,

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
          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.
    Ref. 36.
    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-
     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
     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
     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).

                                                                             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    
                                                                      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
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