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Endocrinology, doi:10.1210/en.2007-1168
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Endocrinology Vol. 149, No. 3 1180-1189
Copyright © 2008 by The Endocrine Society

Cyclooxygenase-2 Regulates Survival, Migration, and Invasion of Human Endometriotic Cells through Multiple Mechanisms

S. K. Banu, J. Lee, V. O. Speights, Jr., A. Starzinski-Powitz and J. A. Arosh

Reproductive Endocrinology and Cell Signaling Laboratory (S.K.B., J.L., J.A.A.), Department of Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas 77843; Division of Anatomic Pathology (V.O.S.), Department of Pathology, Scott & White Memorial Hospital, Texas A&M University Health Science Center, Temple, Texas 76508; and Molekulare Zellbiologie und Humangenetik (A.S.-P.), Institut für Zellbiologie und Neurowissenschaft, Johann Wolfgang Goethe-Universität, 60323 Frankfurt am Main, Germany

Address all correspondence and requests for reprints to: Joe A. Arosh, Ph.D., Department of Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Mail Stop TAMU 4458, Texas A&M University, College Station, Texas 77843. E-mail: jarosh{at}cvm.tamu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Endometriosis is a debilitating disease characterized by the presence of functional endometrial glandular epithelium and stroma outside the uterine cavity that affects up to 20% of women of child-bearing age. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in the biosynthesis of prostaglandin E2 (PGE2), is highly expressed in endometriotic tissues and results in increased concentrations of peritoneal PGE2 in women. In this study, we determined the expression of COX-2 protein in ectopic and eutopic endometria in humans and the role of COX-2 in endometriotic cell survival, migration, and invasion in humans. Our results indicate that COX-2 protein is abundantly expressed in ectopic endometria compared with eutopic endometria. Comparatively, expression of COX-2 protein is higher in eutopic endometria from women with endometriosis compared with women without endometriosis. Inhibition of COX-2 decreases survival, migration, and invasion of endometriotic cells that are associated with decreased production of PGE2. Cell growth inhibitory effects of COX-2 inhibition/silencing are mediated through nuclear poly (ADP-ribose) polymerase-mediated apoptosis. Cell motility and invasion inhibitory effects of COX-2 inhibition/silencing are mediated through matrix metalloproteinase-2 and -9 activities. Interestingly, effects of COX-2 inhibition is more profound in endometriotic epithelial than in stromal cells. Furthermore, inhibition of COX-2 affects invasion rather than migration of endometriotic epithelial and stromal cells. It is the first evidence showing that inhibition of COX-2 decreases endometriotic epithelial and stromal cell survival, migration, and invasion in humans. Our results support the emerging concept that COX-2/PGE2 promotes the pathophysiology and pathogenesis of endometriosis in humans.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ENDOMETRIOSIS IS A DEBILITATING disease characterized by the presence of functional endometrial glandular epithelium and stroma outside the uterine cavity (1, 2, 3, 4). Endometriosis usually develops in the pelvic organs such as the peritoneum, ovaries, fallopian tubes, outside surface of the uterus, membranes and ligaments of the uterus and pelvis, and the pouch of Douglas (1, 2). Endometriosis affects up to 20% of women of childbearing age and is associated with dysmenorrhea, dyspareunia, noncyclic pelvic and abdominal pain, subfertility, infertility, and increased pelvic cancer susceptibility (1, 2, 5). The incidence of endometriosis is increased up to 40–60% higher in women with dysmenorrhea and 30–40% higher in women with subfertility (1, 2). At the time of clinical presentation, most women are experiencing advanced stages of endometriosis (1, 2). Laparoscopic examination is the only diagnostic method currently available. The available treatment options are also limited; drugs are targeted to inhibit ovarian activity and decrease ovarian estrogen production through the use of oral contraceptives, aromatase inhibitors, androgenic agents, and GnRH analogs or surgical removal of endometriotic tissues and uterus with or without ovary (1, 2, 6). All of these treatments compromise pregnancy. Nevertheless, the recurrence rate is up to 50–60% after cessation of therapy or surgery (1, 2). Lack of understanding of molecular behavior of endometriotic cells remains a major limiting factor for development of better therapeutic interventions in women.

The pathophysiology of endometriosis remains an enigma in reproductive medicine. The most widely accepted hypothesis first advanced by Sampson (7) is that the viable endometrial tissue fragments are refluxed through the fallopian tubes into the pelvic cavity during retrograde menstruation. The typical characteristics of endometriotic cells include 1) steroidogenic potential, 2) continuous proliferation and resistance to apoptosis, 3) promoting neoangiogenesis, 4) migrating and invading, and 5) modulating the local immune system (1, 4, 5, 8, 9). Endometriosis has been traditionally viewed as an estrogen-responsive disease (10). However, recent reports suggest that endometriosis is both an estrogen-responsive and a progesterone-unresponsive disease (5). A growing body of evidence indicates that growth factors, cytokines, and prostaglandins promote the establishment and maintenance of endometriosis (3, 9, 11, 12, 13).

Prostaglandins (PGs) are central mediators that play major roles in several physiological and pathological processes in humans and animals (14, 15, 16). Arachidonic acid (AA), an essential fatty acid stored in membrane phospholipids, is the primary precursor of PGs. Cytosolic phospholipase A2 (PLA2) liberates AA from phospholipids. Cyclooxygenases (COXs) COX-1 and COX-2, convert AA into PGH2, the common precursor for various PGs (14). PGH2 is then converted into PGE2, PGF2{alpha}, PGI2 (prostacyclin), PGD2, and thromboxane A2 by distinct PG synthases (14). COX-2 is an inducible enzyme under physiological conditions (14); however, it is constitutively expressed in a variety of pathological conditions including cancers. PGE2 exerts its biological effects through G protein-coupled receptors, designated EP1, EP2, EP3, and EP4, by activating multiple cell signaling pathways (15). Recent studies indicate that EP receptors transactivate MAPK, AKT, and Wnt signaling pathways intracellularly, thereby regulating cell proliferation, apoptosis, migration and invasion, and angiogenesis, immunomodulation, and pain (16, 17, 18, 19).

Concentrations of PGE2 in peritoneal fluid are higher in women suffering from endometriosis than in disease-free women (20). Much of the endometriotic pain is thought to be due to high levels of PGE2 (21). COX-2 is more abundantly expressed in ectopic endometria than in the eutopic endometria during similar phases of the menstrual cycle in women (22, 23). COX-2 is expressed in both glandular epithelium and stroma of endometriotic tissues in women (22, 23). Cytokines increase COX-2 expression in endometriotic stromal cells (3). PGE2 modulates expression of steroidogenic acute regulatory protein and aromatase in endometriotic stromal cells and thereby regulates estrogen metabolism (24), which is primarily mediated through the EP2 receptor (25). Moreover, inhibition of COX-2 prevents establishment of endometriosis (26, 27), decreases size and number of endometriotic tissues (13, 26, 27, 28), and prevents neoangiogenesis in endometriotic implants (13, 27) in different animal models for study of endometriosis. Inhibition of COX-2 induces apoptosis in endometriotic cells through caspase-3 pathways (13). We have recently shown that human immortalized endometriotic epithelial and stromal cells have migrating and invading potential that might be associated with increased PGE2 production and matrix metalloproteinase-2 (MMP2) and MMP9 activities (29).

Together, these results strongly suggest a requirement for COX-2 and PGE2 in the pathophysiology and pathogenesis of endometriosis in women (3). However, the underlying mechanisms are largely unknown. Our objectives were to 1) determine the expression of COX-2 protein in ectopic and eutopic endometria in humans and 2) determine the relationship between COX-2/PGE2 and endometriotic cell survival, migration, and invasion and underlying molecular and cellular mechanisms in humans. Our results indicate that inhibition of COX-2 decreases survival of endometriotic cell through poly-ADP-ribose polymerase (PARP)-mediated apoptosis and inhibits migration and invasion of endometriotic cells through MMP2- and MMP9-mediated mechanisms in humans.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The reagents used in this study were purchased from the following suppliers: prestained protein markers and Bio-Rad assay reagents and standards from Bio-Rad Laboratories (Hercules, CA), Protran BA83 nitrocellulose membrane from Whatman Inc. (Sanford, ME), Pierce ECL from Pierce (Rockford, IL), protease inhibitor from Roche Applied Biosciences (Indianapolis, IN), antibiotic-antimycotic, and trypsin-EDTA from Invitrogen Life Technologies Inc. (Carlsbad, CA), COX-2 inhibitor NS-398 and antihuman mouse β-actin monoclonal antibody from Sigma-Aldrich (St. Louis, MO), nuclear PARP antibody from Cell Signaling (Danvers, MA), MMP2 and MMP9 antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), goat antirabbit or antimouse IgG conjugated with horseradish peroxidase from Kirkegaard & Perry Laboratories (Gaithersburg, MA), Vectastain Elite ABC kit from Vector Laboratories Inc. (Burlingame, CA), Blue X-Ray film from Phenix Research Products (Hayward, CA), fetal bovine serum (FBS) from Hyclone (Logan, UT), and tissue culture dishes and plates from Corning Inc. (Corning, NY). The other chemicals used were molecular biological grade from Fisher (Pittsburgh, PA) or Sigma-Aldrich (St. Louis, MO). COX-2 antibody was a generous gift from Dr. Stacia Kargman, Merck Frost (Montreal, Quebec, Canada).

Endometriotic and endometrial tissues
The following tissues were collected from women presented at the Obstetrics and Gynecology Unit and processed at the Anatomic Pathology Laboratory for diagnostic purposes, Scott & White Memorial Hospital, Texas A&M University System Health Science Center. 1) Ectopic endometria (endometriotic tissue, n = 10) were collected from women with endometriosis and classified as stages I–IV based on criteria established by the American Society for Reproductive Medicine (30). 2) Normal eutopic endometrial tissues (n = 5) were collected from women undergoing hysterectomy for benign gynecological indications. 3) Eutopic endometria (n = 5) were collected from women with endometriosis. All these women reported regular menstrual cycles (25–40 d cycle length) and no hormonal medication in the last 3 months. All the endometriotic and endometrial tissues were collected from the proliferative phase of the menstrual cycle. For the present study, additional sections were cut from these archived paraffin-embedded tissues that were not needed for patient care. These studies were approved by the Institutional Review Board of the Texas A&M University System Health Science Center.

Cell culture
Immortalized human endometriotic epithelial cells (12-Z, 49-Z, and 11-Z) and stromal cells (22-B) (8) and immortalized human endometrial surface epithelial cells (HES) (31) and stromal cells (HESC) (32) were used. HES cells were a generous gift from Dr. Asgerally T. Fazleabas, University of Illinois at Chicago, Chicago, IL. HESC cells were a generous gift from Dr. Graciela Krikun, Yale University, New Haven, CT. The endometriotic epithelial and stromal cells and normal endometrial epithelial and stromal cells were cultured in DMEM/F12 without special steroid treatment (Sigma) containing 10% FBS (Hyclone) and penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin-B 2.5 µg/ml (Invitrogen) in a humidified atmosphere of 5% CO2 and 95% air at 37 C as we described previously (29).

COX-2 small interfering RNA (siRNA)
The cells (3.0 x 105 per well) were cultured in antibiotic-free DMEM/F12 with 10% FBS in six-well tissue culture plates. At 70–80% confluency, cells were used for COX-2 knockdown experiments using COX-2 SMART-pool siRNA duplex delivered by DharmaFect-1 per manufacturer’s instructions. As an internal control, siGLO RISC-Free siRNA or MOCK siRNA was used. COX-2 siRNA, siGLO RISC-free siRNA, and DharmaFect-1 were obtained from Dharmacon Inc. (Lafayette, CO). Briefly, siRNA duplexes (100 nM/well) and DharmaFect-1 (3 µl/well) were diluted in 50 µl antibiotic- and serum-free DMEM/F12 medium separately and mixed gently and incubated for 5 min at room temperature. Afterward, COX-2 siRNA and DharmaFect-1 were mixed (total volume 100 µl) and incubated at room temperature for 20 min. Then, 100 µl siRNA-DharmaFect-1 complex was diluted with 2 ml antibiotic-free medium with 10% FBS and added to the well. After 24 h, the medium was replaced with fresh DMEM/F12 with 10% FBS and incubated for 24 h. After 48 h of transfection, PGE2 levels were measured by ELISA (33), and endometriotic cell proliferation, apoptosis, migration and invasion were determined. Fluorescence-labeled siGLO RISC-free siRNA was transfected separately, and transfection efficiency was estimated using a fluorescence microscope. Efficiency of siRNA on COX-2 gene silencing was assessed by RT-PCR.

Cell proliferation assay
Endometriotic epithelial (12-Z, 49-Z, and 11-Z) and stromal (22-B) cells (10 x 104 per well) were cultured in DMEM/F12 with 10% FBS in six-well plates. At 70–80% of confluency, the cells were cultured in DMEM/F12 with 2% dextran charcoal-treated FBS and treated with NS-398 (100 µM) for 36 h. The concentrations for NS-398 were determined by dose-response experiments using 25, 50, and 100 µM (data not shown). At the end of experiments, adherent cells were washed with PBS and harvested using 0.1% trypsin-EDTA (Invitrogen). The cell pellets were resuspended in isotonic buffer (Val Tech Diagnostics Inc., Brackenridge, PA), and cell numbers were counted using a Coulter counter. Three experiments were conducted, and data are expressed as the mean ± SEM. To determine the effects of COX-2 siRNA on endometriotic cell proliferation, silencing of the COX-2 gene was performed as described above. After 24 h of transfection of COX-2 siRNA, the medium was replaced and the cells were cultured in 2% dextran charcoal-treated FBS, which was considered as time 0 h, and cell proliferation was estimated at 36 h as described above.

Terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) assay
Nonadherent and adherent cells were harvested, mixed together, and resuspended at the concentrations of 1 x 106 cells/ml. Nicks in the DNA were determined by terminal deoxynucleotidyl transferase and 5-bromo-2'-deoxyuridine (BrdU) 5'-triphosphate labeling using APO-BrdU TUNEL Assay Kit (Molecular Probes, Carlsbad, CA). Detection of BrdU incorporation at DNA break sites was achieved through Alexa Fluor 488 dye-labeled anti-BrdU antibody. The staining procedures were followed as recommended by the manufacturers. The number of apoptotic cells was analyzed by a flow cytometer (FACSCaliber; Becton Dickinson, San Jose, CA) using Cell Quest software. In addition, the presence of TUNEL-labeled DNA fragments and chromatin condensation was determined by fluorescence microscopy. Photos were captured using digital imaging and an image analysis workstation consisting of a Zeiss Axioplan 2 Research Microscope interfaced with a Zeiss Axiocam HR high resolution color CCD camera with Zeiss Axiovision (Carl Zeiss, Thornwood, NY).

Cell migration and invasion assays
In vitro migration and invasion assays were performed using uncoated or Matrigel-coated 24-well chambers/microfilters (8 µm pore size polycarbonate filters), respectively, according to the manufacturer’s instructions (BD Bioscience, Bedford, MA) and as described (29, 34, 35). Briefly, after rehydration of the chambers, the cells (5 x 104 cells per chamber) in 500 µl DMEM/F12 plus 10% FBS were seeded onto the upper chamber. In the lower chamber, 500 µl DMEM/F12 plus 10% FBS was placed. NS-398 (100 µM) was added onto both upper and lower chambers. Endometriotic cell motility/migration was measured as number of cells migrated from a defined area of the uncoated microfilter through micropores in the given time, 24 h. Invasion of endometriotic cells was measured as number of cells invaded from a defined area of the Matrigel-coated microfilter through micropores in the given time, 24 h. The micropore filters were stained with 4',6-diamidino-2-phenylindole (Vector), and cells migrated through filters were counted in eight representative areas per filter using a fluorescence microscope at x400 magnification and expressed as the mean ± SEM of three independent experiments. To determine the effects of COX-2 siRNA on endometriotic cell migration and invasion, silencing of the COX-2 gene was performed as described above. After 24 h of transfection of siRNA, the medium was replaced and the cells were cultured in DMEM/F12 with 10% FBS. Adherent cells were harvested by trypsinization at 48 h after transfection of COX-2 siRNA and placed onto the upper chamber in DMEM/F12 with 10% FBS at the concentrations of 5 x 104 cells per chamber. In the lower chamber, DMEM/F12 with 10% FBS was placed. The migration and invasion potential of endometriotic cells were determined as described above.

Gelatin zymography
MMP2 and MMP9 activity in the culture media was analyzed by zymography as we described recently (29). Proteins in culture medium (25 µl) were resolved on 7.5% SDS polyacrylamide gels (SDS-PAGE) (36) that were copolymerized with 0.1% (1 mg/ml) gelatin (29, 37, 38). Proteolytic activity was detected by gelatin lysis as evidenced as clear bands against the blue background of stained gelatin. The gels were analyzed using AlphaImager software (Alpha Innotech Corp., San Leandro, CA), and densitometry was performed. Numerical values are expressed as mean ± SEM of three independent experiments.

Protein extraction and immunoblotting
Total protein was isolated from tissues and cells, and immunoblotting/Western blotting was performed as previously described (36, 39). Protein concentration was determined using the Bradford method (40) and a Bio-Rad protein assay kit. Protein samples (50 µg) were resolved using 10% SDS-PAGE. Primary antibodies for COX-2 (1:3000), PARP (1:1000), MMP2 (1: 1000), MMP9 (1:1000), or β-actin (1:5000) were used. Goat antirabbit IgG conjugated with horseradish peroxidase was used as the secondary antibody (1:10,000). Chemiluminescent substrate was applied according to the manufacturer’s instructions. The blots were exposed to blue x-ray film, and densitometry of autoradiograms was performed using an Alpha Imager (Alpha Innotech).

Immunohistochemistry
Tissue sections were fixed in 4% buffered paraformaldehyde saline for 4 h at 4 C and processed using standard procedures (36). Paraffin sections (5 µm) were used for immunohistochemical localization of COX-2 protein using a Vectastain Elite ABC kit according to the manufacturer’s protocols. The tissue sections were incubated with the COX-2 antibody at the concentrations of 1:500 overnight at 4 C. The tissue sections were further incubated with the secondary antibody (goat antirabbit IgG biotinylated, 1:200) for 30 min at room temperature. For the negative control, rabbit serum at the respective dilution was used. Between each step, tissue sections were washed in PBS. Photos were captured with the use of an Axioplan 2 Research Microscope (Carl Zeiss).

ELISA
Commercially available PGE2 standards, antiserum, and acetylcholinesterase tracers were used (Cayman Chemicals, Ann Arbor, MI). ELISA was performed according to the manufacturers’ instructions and as described previously (33). Briefly, 50 µl of culture medium collected from different experiments was used. The reaction mixture was incubated overnight at room temperature. Concentrations of PGE2 were measured at 405 nm using a plate reader. A standard curve was developed with standards ranging from 50–5000 pg PGE2/ml.

Statistical analyses
Statistical analyses were performed using general linear models of Statistical Analysis System (SAS, Cary, NC). Effects of inhibition of COX-2 on its protein expression, PGE2 production, cell proliferation, apoptosis, migration, and invasion and expression and gelatinolytic activity of MMP2 and MMP9 in endometriotic cells were analyzed by one-way ANOVA followed by Tukey-Kramer honestly significant difference test. The relationship between PGE2 concentrations and endometriotic cell proliferation, apoptosis, migration, and invasion were analyzed by simple linear correlation. The numerical data are expressed as mean ± SEM. Statistical significance was considered as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of COX-2 protein in endometriotic tissues
Results (Fig. 1Go) indicated that COX-2 protein was expressed in glandular epithelia and stroma of ectopic endometria (endometriosis), eutopic endometria from women with endometriosis, and eutopic endometria from endometriosis-free women during the proliferative phase of the menstrual cycle. Interestingly, COX-2 protein was abundantly expressed in ectopic endometria compared with eutopic endometria. Furthermore, expression of COX-2 protein was relatively higher in eutopic endometria from women with endometriosis compared with women without endometriosis.


Figure 1
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  		FIG. 1. Immunohistochemical localization of COX-2 protein in ectopic and eutopic endometria in human. A, Negative control; B, ectopic endometrium (endometriosis); C, eutopic endometrium from women with endometriosis; D, eutopic endometrium from women without endometriosis. COX-2 protein was expressed in glandular epithelium (GLE) and stroma (STR). Ectopic endometria (n = 10), eutopic endometria with endometriosis (n = 5), and normal eutopic endometria (n = 5) were collected during the proliferative phase of the menstrual cycle. Representative immunohistochemistry pictures at x400 magnification are shown. Immunohistochemistry was performed using Vectastain Elite ABC kit according to the manufacturer’s protocols. Please see Materials and Methods for more details.

 
Regulation of COX-2 protein expression and PGE2 production in endometriotic cells
We have recently characterized human immortalized endometriotic epithelial (12-Z, 49-Z, and 11-Z) and stromal (22-B) cells (8, 29). We found that COX-2 gene was constitutively and abundantly expressed in these cell lines. In this study, we determined regulation of COX-2 protein and its functions. Results (Fig. 2Go) demonstrated that COX-2 protein was highly (P < 0.05) expressed in endometriotic epithelial (12-Z, 49-Z, and 11-Z) and stromal (22-B) cells compared with normal endometrial epithelial (HES) and stromal cells (HESC) under basal conditions. Endometriotic cells produced significant (P < 0.05) amounts of PGE2 compared with normal endometrial cells. The quantity of PGE2 production was associated with expression levels of COX-2 protein in these cells. Comparatively, endometriotic epithelial cells 12-Z and 11-Z produced (P < 0.05) a higher quantity of PGE2 than endometriotic stromal cells 22-B. Pharmacological inhibition of COX-2 by its selective functional inhibitor NS-398 partly decreased (P < 0.05) the expression of COX-2 protein (~2- to 3-fold) and inhibited (P < 0.05) PGE2 production up to 90% in both endometriotic epithelial and stromal cells.


Figure 2
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  		FIG. 2. Endometriotic epithelial cells 12-Z, 49-Z, and 11-Z; stromal cells 22-B; and normal endometrial epithelial cells (HES) and stromal cells (HESC) were cultured in DMEM/F12. At 70% confluency, the cells were treated with selective COX-2 functional inhibitor NS-398 (100 µM). Concentrations of PGE2 were measured using ELISA. A, Western blot analysis of COX-2 protein expression. As an internal control, β-actin protein was measured. B, Densitometry of COX-2 protein to β-actin ratio based on Western blot analysis. COX-2 protein was highly (P < 0.05) expressed in 12-Z, 49-Z, 11-Z, and 22-B cells but not expressed in HES and HESC cells under basal conditions. NS-398 decreased (P < 0.05) expression of COX-2 protein in 12-Z, 49-Z, 11-Z, and 22-B cells. C, Endometriotic epithelial and stromal cells produced large (P < 0.05) amounts of PGE2 compared with normal endometrial epithelial and stromal cells. NS-398 inhibited COX-2 activity and decreased (P < 0.05) PGE2 production up to 90% in 12-Z, 49-Z, 11-Z, and 22-B cells. Representative Western blots are shown. The lower band at 66 kDa represents the unglycosylated form of COX-2 protein (52 ). CONT, Control; IDV, integrated density value. Numerical data are expressed as mean ± SEM of three independent experiments. Please see Materials and Methods for more details.

 
Association between COX-2/PGE2 and endometriotic cell growth and proliferation
Inhibition of COX-2 by NS-398 decreased (P < 0.05) endometriotic epithelial and stromal cell proliferation (~60 and ~40%, respectively) within 36 h of treatment (Fig. 3AGo). However, the inhibitory effects of COX-2 were higher (~20%, P < 0.05) in endometriotic epithelial cells than in stromal cells. PGE2 levels were positively correlated (r = 0.99; r = 0.98) with proliferations of endometriotic epithelial and stromal cells, respectively.


Figure 3
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  		FIG. 3. A, Endometriotic cell proliferation. NS-398 (100 µm) decreased (P < 0.05) endometriotic epithelial and stromal cell proliferation. B, Western blot analysis of total and cleaved PARP protein in 12-Z and 22-B cells. As an internal control, β-actin protein was measured. C, Densitometry of cleaved PARP to total PARP protein ratio based on Western blot analysis. NS-398 cleaved (P < 0.05) PARP protein in 12-Z and 22-B cells. D, TUNEL assay using flow cytometry. DNA histogram shows the TUNEL-labeled cells (apoptotic cells) under M1 area in response to NS-398 in 12-Z and 22-B cells. E, Histogram showing the results of TUNEL assay in relative levels. NS-398 induced (P < 0.05) apoptosis in 12-Z and 22-B cells. F, TUNEL assays using fluorescence microscopy. DNA fragments and chromatin condensation were clearly detected by TUNEL labeling in 12-Z and 22-B cells. Representative Western blots, TUNEL assay DNA histogram, and fluorescence microscopy photomicrographs at x400 are shown. Cell proliferation was determined using a Coulter counter. Cell apoptosis was determined using APO-BrdU TUNEL Assay Kit. CONT, Control; IDV, integrated density value. Numerical data are expressed as mean ± SE of three independent experiments. Please see Materials and Methods for more details.

 
Next, we determined whether the growth inhibitory mechanisms of COX-2 inhibition in endometriotic cells were associated with increased apoptosis. Therefore, we examined cleavage of PARP protein, a well-accepted marker for cell apoptosis (41), in response to inhibition of COX-2 in endometriotic epithelial cells 12-Z and stromal cells 22-B. Results (Fig. 3Go, B and C) indicated that inhibition of COX-2 by NS-398 cleaved PARP protein within 24 h of treatment in both 12-Z and 22-B cells. Consistent with these findings, the TUNEL assay (Fig. 3Go, D and E) using flow cytometry indicated that inhibition of COX-2 by NS-398 increased (P < 0.05) apoptosis of both 12-Z and 22-B cells within 24 h. Interestingly, cell apoptosis was higher (200%, P < 0.05) in endometriotic epithelial cells 12-Z compared with stromal cells 22-B. TUNEL assay based on fluorescence microscopy clearly showed that inhibition of COX-2 induced DNA fragmentation and chromatin condensation in both 12-Z and 22-B cells within 24 h of treatment (Fig. 3FGo). PGE2 production levels were negatively correlated (r = –0.98; r = –0.97) with apoptosis of 12-Z and 22-B cells, respectively.

Association between COX-2/PGE2 and endometriotic cell migration and invasion
Results indicated that inhibition of COX-2 by NS-398 decreased (P < 0.05) endometriotic epithelial and stromal cell migration approximately 55 and 35%, respectively (Fig. 4Go, A and B). Inhibition of COX-2 decreased invasion of endometriotic epithelial and stromal cells approximately 65 and 45%, respectively (Fig. 4Go, C and D). Effects of COX-2 inhibition on migration and invasion potential were higher (~20%, P < 0.05) in endometriotic epithelial cells compared with stromal cells. Interestingly, effects of COX-2 inhibition on endometriotic cells invasion were higher (~10%, P < 0.05) than on migration. Concentrations of PGE2 were positively correlated with migration (r = 0.98) and invasion (r = 0.97) of endometriotic cells.


Figure 4
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  		FIG. 4. A, Migration/motility of endometriotic epithelial cells 12-Z, 49-Z, and 11-Z and stromal cells 22-B. NS-398 decreased (P < 0.05) migration of endometriotic epithelial and stromal cells. B, Representative photomicrographs at x400 on migration of 12-Z cells are shown. C, Invasion of endometriotic epithelial cells 12-Z, 49-Z, and 11-Z and stromal cells 22-B. NS-398 decreased (P < 0.05) invasion of endometriotic epithelial and stromal cells. D, Representative photomicrographs at x400 on invasion of 12-Z cells are shown. E, Western blot analysis on expression of total MMP9 and MMP2 proteins in 12-Z and 22-B cells. As an internal control, β-actin protein was measured. F, Densitometry of MMP9 or MMP2 and β-actin ratio based on Western blot analysis. NS-398 decreased (P < 0.05) expression of total MMP9 and MMP2 proteins in 12-Z and 22-B cells. G, Zymography showed the gelatinolytic activity of MMP9 and MMP2 in 12-Z and 22-B cells. (H) Densitometry of total gelatinolytic activity of MMP9 and MMP2 in 12-Z and 22-B. NS-398 decreased (P < 0.05) total gelatinolytic activity of MMP9 and MMP2 in 12-Z and 22-B cells. NS-398 decreased (P < 0.05) secretion of active forms of MMP9 and MMP2 proteins from the endometriotic cell into the culture medium, which is evident by gelatinolytic activity appearing at 82 and 66 kDa, respectively. Representative Western blots and gelatin zymography blots are shown. Numerical values are expressed as the mean ± SE of three independent experiments. Motility/migration and invasion assays were performed using 8-µm pore size uncoated or Matrigel-coated polycarbonate filters. The cells were treated with selective COX-2 functional inhibitor NS-398 (100 µm). CONT, Control; IDV, integrated density value. Please see Materials and Methods for more details.

 
Then, we examined whether the inhibitory effects of COX-2 on endometriotic cell migration and invasion were associated with metalloproteinases MMP9 and MMP2, the well-studied markers for cell migration and invasion (42). We used endometriotic epithelial cells 12-Z and stromal cells 22-B as representative cell lines. First, we examined regulation of total MMP9 and MMP2 proteins in whole-cell lysate using Western blot. Results (Fig. 4Go, E and F) indicated that inhibition of COX-2 with NS-398 decreased (P < 0.05) the expression of total MMP9 and MMP2 proteins (the antibodies used detect total MMP9 and MMP2 proteins including pro- and active forms). Next, we determined secretion and activity of MMP9 and MMP2 proteins. Culture media were harvested during the migration and invasion assay, and activities of MMP9 and MMP2 proteins were analyzed by gelatin zymography. The detection limits for pro- and active forms of MMP9 and MMP2 proteins and their gelatinolytic activities by zymography in these experiments were in agreement with the previous reports (37, 38). Results (Fig. 4Go, G and H) indicated that 12-Z and 22-B cells secreted and/or released MMP9 and MMP2 proteins into the culture medium. MMP2 protein was abundantly secreted by both 12-Z as well as 22-B cells; by contrast, MMP9 protein was primarily secreted (P < 0.05) by 12-Z compared with 22-B cells. Secretion of active forms of MMP9 and MMP2 proteins by endometriotic cells into the culture medium was clearly evident by gelatinolytic activity seen at 82 and 66 kDa, respectively. Inhibition of COX-2 decreased (P < 0.05) the total gelatinolytic activity of MMP9 and MMP2 approximately 32 and 30%, respectively, in 12-Z cells and 55 and 60%, respectively, in 22-B cells. The secretion and gelatinolytic activity of MMP2 and MMP9 were positively correlated (r = 0.97) with PGE2 production in endometriotic cells.

Effects of COX-2 siRNA on endometriotic cell survival, migration, and invasion
Finally, we confirmed the role of COX-2 in endometriotic cell proliferation, migration, and invasion by a genomics approach using siRNA. We used endometriotic epithelial cells 12-Z and stromal cells 22-B as representative cell lines. The success of siRNA transfection was 85–90%, and COX-2 gene silencingwas up to about 80% in 12-Z and 22-B cells. Results from COX-2 gene knockdown experiments indicated that silencing of COX-2 gene decreased (P < 0.05) PGE2 production about 85% in 12-Z and 22-B cells (Fig. 5AGo), proliferation of 12-Z cells about 53% and 22-B cells about 38% (Fig. 5BGo), migration of 12-Z and 22-B cells about 50 and 35%, respectively (Fig. 5CGo), and invasion of 12-Z and 22-B cells 60 and 40%, respectively (Fig. 5DGo). Concentrations of PGE2 were positively correlated with endometriotic cell proliferation (r = 0.99), migration (r = 0.98), and invasion (r = 0.99) and negatively correlated with apoptosis (r = 0.96). Effects of pharmacological inhibition of COX-2 protein activity were in agreement with the effects of genomic inhibition of COX-2 gene on endometriotic cell growth, migration, and invasion. Mock siRNA was used as an internal control, which did not have any detrimental effect on growth, migration, and invasion of 12-Z and 22-B cells compared with control.


Figure 5
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  		FIG. 5. Effects of COX-2 silencing on survival, migration, and invasion of endometriotic epithelial cells 12-Z and stromal cells 22-B. A, COX-2 silencing decreased (P < 0.05) PGE2 production in 12-Z and 22-B cells; B, COX-2 silencing decreased (P < 0.05) proliferation of 12-Z and 22-B cells; C, COX-2 silencing decreased (P < 0.05) migration of 12-Z and 22-B cells; D, COX-2 silencing decreased (P < 0.05) invasion of 12-Z and 22-B cells. Numerical values are expressed as the mean ± SE of three independent experiments. COX-2 knockdown was performed using COX-2 SMART-pool siRNA duplex delivered by DharmaFect-1. Please see Materials and Methods for more details.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we determined a role for COX-2 in endometriotic cell proliferation, survival, migration, and invasion using pharmacological and genomic approaches. It is the first evidence showing that inhibition of COX-2 1) decreased endometriotic epithelial and stromal cells survival by inducing PARP-mediated apoptosis and 2) inhibited endometriotic epithelial and stromal cells migration and invasion through MMP2- and MMP9-mediated mechanisms in human. Our results support the emerging concept that PGE2 promotes the pathophysiology and pathogenesis of endometriosis in human (3).

COX-2 protein is more abundantly expressed in ectopic endometria than in eutopic endometria, which is consistent with the previous reports (22, 23). In eutopic endometria, COX-2 protein is highly expressed in women with endometriosis compared with disease-free women. This result is in agreement with the emerging hypothesis that the presence of endometriotic lesions alters functions of eutopic endometrium (4). Increased expression of COX-2 protein in endometriosis suggests its important role in pathophysiology and pathogenesis of this disease.

PGE2 is an important mediator that regulates cell proliferation, apoptosis, migration, and invasion in several cell types (16). We used recently characterized immortalized human endometriotic epithelial and stromal cells (8, 29) to examine COX-2 functions and PGE2 biosynthesis. COX-2 protein is constitutively but very abundantly expressed in these cells, and they produce large amounts of PGE2. Comparatively, the level of PGE2 production is higher in epithelial than in stromal cells. Inhibition of COX-2 by its selective inhibitor NS-398 and siRNA significantly decrease PGE2 production in both endometriotic epithelial and stromal cells. These results suggest that COX-2 is the rate-limiting enzyme in PGE2 biosynthesis in endometriotic epithelial as well as stromal cells in human.

In the process of deciphering interactions between COX-2 and endometriosis, first we determined whether the increased COX-2 protein expression and PGE2 production in human endometriotic cells were associated with their survival. Results indicate that inhibition of COX-2 by NS-398 and siRNA significantly decreases PGE2 production and endometriotic epithelial and stromal cell proliferation. Nuclear PARP maintains the viability of the cells under normal physiological conditions. By contrast, in response to apoptotic stimuli, caspase-3 cleaves PARP protein, which in turn facilitates DNA repair and cellular disassembly and serves as the marker of cells undergoing apoptosis (41). Therefore, we determined a role for PARP protein in endometriotic cell apoptosis. Inhibition of COX-2 cleaves PARP protein, induces DNA fragmentation and chromatin condensation, and increases apoptosis of endometriotic epithelial and stromal cells. Together, these results suggest that PGE2 promotes survival and growth of human endometriotic epithelial and stromal cells. Available evidence clearly indicates that inhibition of COX-2 increases apoptosis by altering interactions between antiapoptotic and proapoptotic members and mitochondrial membrane integrity and release of cytochrome c and activation of caspase 3 and PARP proteins (43) and induces cell cycle arrest either in G0-G1 or G2-M phase by altering several cell cycle regulatory genes such as cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors p21 and p27 (44). Several cell cycle regulatory genes are expressed in these endometriotic epithelial and stromal cells (29). Further studies are required to identify and decipher mechanisms and interactions between COX-2 pathways and cell cycle and apoptotic machinery in these endometriotic cells.

Second, we determined whether the increased COX-2 expression and PGE2 production in human endometriotic cells were associated with their migration and invasion potential. Results indicate that endometriotic epithelial and stromal cells do have potential to migrate and invade, and endometriotic stromal cells possess more migrating and invading potential than epithelial cells, which is in consistent with our previous report (29). This difference might be due to the inherent nature of these two cell types. Surprisingly, inhibition of COX-2 by NS-398 and siRNA significantly decreases migration and invasion potential in both endometriotic epithelial and stromal cells; however, the effects are more profound in epithelial than in stromal cells, which are associated with quantity of PGE2 they produced. Furthermore, an interesting observation is that inhibition of COX-2 produces more detrimental effects on invasion compared with migration of endometriotic cells; however, the underlined molecular mechanisms for these selective effects are unknown. These results suggest that PGE2 promotes migration/motility and invasion of human endometriotic epithelial and stromal cells. Recent studies have reported that inhibition of COX-2 decreases invasiveness and establishment of endometriosis in rodent models (13, 26, 27, 28), and endometriotic/endometrial cells invade peritoneal mesothelial cells in vitro (9, 45), which might require activation of MMPs (46, 47).

MMPs are important enzymes in the degradation and reconstruction of the extracellular matrix (ECM), which is the prerequisite for the process of cell migration and invasion (42). MMP2 and MMP9 are most consistently detected in malignant tissues and are associated with tumor aggressiveness and metastatic potential (42). Therefore, we determined whether the inhibitory effects of COX-2 inhibition were mediated through MMP2 and MMP9 proteins. Results indicate that endometriotic epithelial and stromal cells secrete MMP2 and MMP9 proteins, which is consistent with our previous report (29). Inhibition of COX-2 by NS-398 decreases synthesis and gelatinolytic activities of MMP2 and MMP9 proteins, which is associated with decreased production of PGE2 in both endometriotic epithelial and stromal cell types. It suggests that PGE2 promotes the secretion/release of MMP2 and MMP9 proteins in endometriotic epithelial and stromal cells, and inhibitory effects of COX-2 on migration and invasion potential of endometriotic epithelial and stromal cell are partly mediated through MMP2 and MMP9. Our results are in agreement with previously published data for other cell types that COX-2 and PGE2 promote the secretion of MMP2 and MMP9 and pharmacological inhibition of COX-2 with NS-398 prevents the release of MMP2 and MMP9 (48), and inhibition COX-2 decreases expression and release of MMPs and associated cell migration and invasion (49).

It is well accepted that MMP2, MMP9, and other MMPs play an important role in cell migration and invasion (42). Synthesis and activation of MMPs exquisitely regulate degradation of ECM. The regulation of MMP synthesis and activation is both tissue/cell and MMP specific. In response to stimuli, specific MMP mRNA is synthesized, and this mRNA will be translated into a latent or pro form of the MMP protein. The majority of the pro-MMPs are secreted from the cells in a latent form that will be activated (active MMP) in the extracellular space by a variety of other proteases. This active MMP is capable of degrading ECM, and alternatively, this active MMP can be bound with its endogenous inhibitors such as tissue inhibitors of metalloproteinases (TIMPs) (50). The role of MMPs and TIMPs in endometrial biology has been extensively reviewed recently (50). A role for MMP3 and MMP7 in the pathophysiology of endometriosis has already been reported (51). The endometriotic cells used in the present study express MMP1, MMP3, MMP7, TIMP1, TIMP2, and TIMP3 genes at various levels (29). Further studies using these cell lines will determine interactions among MMPs, TIMPs, and PGE2 and their important roles in pathophysiology and pathogenesis of endometriosis in humans.

It is well known that COX-2-derived PGE2 is involved in the pathophysiology and pathogenesis of various cancers (16). Use of selective COX-2 inhibitors such as celecoxib, rofecoxib, and valdecoxib to inhibit PGE2 production has produced remarkable effects on remission of colon as well as other cancers (18). Unfortunately, therapeutic use of COX-2 inhibitors has been suspended because of undesirable cardiovascular side effects (18), which might be due to inhibition of other PGs such as PGI2 and thromboxane A2 but not due to inhibition of PGE2 (18). It challenges us to discover alternative targets downstream of COX-2 to inhibit selective biosynthesis, catabolism, and signaling of PGE2 (16, 18). In endometriotic women, peritoneal fluid concentrations of PGE2 are higher compared with disease-free women (20). Much of the endometriotic pain is thought to be due to high levels of PGE2 (21). Inhibition of COX-2 prevents the establishment of endometriosis (26, 27) and decreases the size and number of endometriotic tissues (13, 26, 27, 28). Taken together, the present results along with the findings from other laboratories (3, 13, 26, 27, 28) very strongly indicate that PGE2 has a profound role in pathophysiology and pathogenesis of endometriosis and also suggest that endometriosis is a PGE2-dependent disease. Ongoing studies determining a role for PGE synthases, PG dehydrogenases, and PGE2 receptors downstream of COX-2 may provide new information to control the selective production and action of PGE2 in endometriotic cells, and that may yield better therapeutic modalities for treatment of endometriosis in human.

In conclusion, results from the present study suggest that 1) COX-2 protein is highly expressed in endometriotic tissues; 2) increased expression of COX-2 and production of PGE2 regulate survival, migration, and invasion of endometriotic epithelial and stromal cells in human; and 3) inhibition of COX-2 by pharmacological and genomic approaches decreases growth, migration, and invasion of endometriotic epithelial and stromal cells in humans.


   Footnotes
 
This work was supported by Texas A&M University.

Disclosure Statement: The authors have nothing to disclose.

First Published Online November 26, 2007

Abbreviations: AA, Arachidonic acid; BrdU, 5-bromo-2'-deoxyuridine; COX, cyclooxygenase; ECM, extracellular matrix; FBS, fetal bovine serum; MMP, matrix metalloproteinase; PARP, poly-ADP-ribose polymerase; PG, prostaglandin; PLA2, phospholipase A2; siRNA, small interfering RNA; TIMP, tissue inhibitors of metalloproteinase; TUNEL, terminal deoxynucleotide transferase dUTP nick end labeling.

Received August 22, 2007.

Accepted for publication November 13, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
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