Hum. Reprod. Advance Access published online on August 20, 2008
Human Reproduction, doi:10.1093/humrep/den315
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Effects of a selective cyclooxygenase-2 inhibitor on endometrial epithelial cells from patients with endometriosis
1 Instituto de Biología y Medicina Experimental (IBYME-CONICET), Vuelta de Obligado 2490 (C1428ADN), Ciudad Autónoma de Buenos Aires, Argentina 2 Hospital de Clínicas Jose de San Martín, Av. Córdoba 2351 (C1120AAR), Ciudad Autónoma de Buenos Aires, Argentina 3 Centro de Ginecología y Reproducción (CEGYR), Viamonte 1432 (C1055ABB), Ciudad Autónoma de Buenos Aires, Argentina
4 Correspondence address. Tel: +54-11-47832869; Fax: +54-11-47862564; E-mail: meresman{at}dna.uba.ar
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Abstract |
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BACKGROUND: Celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, also has anti-proliferative properties and pro-apoptotic effects on different in vivo and in vitro models, two actions that may be efficacious in therapy for endometriosis. We evaluated the effects of celecoxib on apoptosis and proliferation, and vascular endothelial growth factor (VEGF) production and COX-2 expression and activity in endometrial epithelial cells (EECs).
METHODS AND RESULTS: Thirty-two endometriosis and 13 control women were included in the study. EECs from eutopic endometrium and control biopsies were cultured with different doses of celecoxib. Celecoxib at 50, 75 and 100 µM (versus vehicle control) inhibited EEC proliferation in cultures from controls (P < 0.05, P < 0.01 and P < 0.01, respectively) and patients with endometriosis (P < 0.05, P < 0.01 and P < 0.01), as assessed by 3H-thymidine uptake. Celecoxib at 50, 75 and 100 µM induced apoptosis in EEC from controls (P < 0.05, P < 0.001 and P < 0.001) and patients with endometriosis (P < 0.001, P < 0.001 and P < 0.01), as revealed by the Acridine Orange–Ethidium Bromide technique. Western blot analysis showed that celecoxib was effective at increasing COX-2 protein at 100 µM in EEC from endometriosis patients (P < 0.05). In EEC from endometriosis patients, celecoxib at 25, 50 and 100 µM was also effective in reducing COX-2 activity, reflected in the reduction of prostaglandin E2 (PGE2) synthesis (P < 0.001), and VEGF secretion (P < 0.001; P < 0.05 and P < 0.001), assessed by enzyme-linked immunosorbent assay. Exogenous PGE2 did not reverse celecoxib-induced growth inhibition.
CONCLUSIONS: This study suggests a direct effect of celecoxib on reduction of endometrial growth and supports further research on selective COX-2 inhibition as a novel therapeutic modality in endometriosis.
Key words: endometriosis/cyclooxygenase-2 inhibitor/eutopic endometrium/apoptosis/cell proliferation
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Introduction |
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Endometriosis is one of the most common benign gynaecological diseases in women of reproductive age and is defined as the presence and proliferation of endometrial glands and stroma outside the uterine cavity. This disease is estrogen-dependent and its medical treatment is principally aimed at down-regulating the ovarian estrogen production by the application of GnRH agonists, progestins, androgenic agents or oral contraceptives either in a continuous or cyclic fashion. Unfortunately, these treatments are often associated with side effects and high recurrence rates (Valle and Sciarra, 2003
; Mihalyi et al., 2006). There is a real need for more effective approaches to the prevention and treatment of endometriosis. Cyclooxygenase (COX)-2 inhibitors show great promise in this respect (Ebert et al., 2005).
COX-1 and COX-2 are isoenzymes that execute the synthesis of prostaglandins (PGs). While COX-1 is constitutively expressed in tissue, COX-2 is inducible and up-regulated in pathways of pain and inflammation (Vane et al., 1994; Ebert et al., 2005). Non-steroidal anti-inflammatory drugs (NSAIDs) are the most widely used therapeutic agents in the treatment of pain and inflammation. Unlike regular NSAIDs, COX-2 selective inhibitors do not inhibit COX-1 and thus show promise as drugs that spare the gastrointestinal system. Celecoxib is a potent COX-2 inhibitor being developed for the treatment of rheumatoid arthritis and osteoarthritis (Chun and Surh, 2004).
Consistent data suggest that COX-2 inhibitors can reduce cancer risk in patients, can prevent tumorigenesis and suppress established tumour growth in animals (Bundred and Barnes, 2005). In addition, COX-2-specific inhibitors have the ability to block cell growth and induce apoptosis and cell cycle arrest in various tumour cell lines (Kundu et al., 2002; Basu et al., 2005). Also, it has been shown that COX-2 inhibitors such as celecoxib are potent inhibitors of angiogenesis both in vitro and in vivo (Gately and Li, 2004; Basu et al., 2005).
COX-2 overexpression was detected in eutopic and ectopic endometrium from patients with endometriosis (Ota et al., 2001; Matsuzaki et al., 2004b). Furthermore, large amounts of PGs have been found in peritoneal fluid from endometriosis patients and endometriotic lesions (De Leon et al., 1986). It has been shown that PGE2 overproduction leads to excessive estradiol formation and that estradiol and cytokines (interleukin-1β and tumour necrosis factor-
), which are increased in endometriosis, induce COX-2 (Attar and Bulun, 2006). In addition, the COX-2/vascular endothelial growth factor (VEGF) association has been shown in human samples of non-small cell lung cancer (Gately and Li, 2004). PGE2 increases VEGF production (Gately and Li, 2004) and then VEGF stimulates COX-2 expression in endothelial cells (Tamura et al., 2006). This pathway establishes a positive feedback loop in favour of high concentrations of estrogen, PGE2 and VEGF in endometriosis (Bulun et al., 2002). Based on these data, we can speculate that inhibition of COX-2 would be a promising therapy toward controlling the growth of the endometriotic lesion.
In endometriosis research, recent animal studies demonstrated that treatment with COX-2 inhibitors prevents the implantation of endometrium to ectopic sites (Matsuzaki et al., 2004a) and induces the regression of endometrial explants in rats (Dogan et al., 2004) and mice (Ozawa et al., 2006). However, the mechanisms underlying this regression remain unclear.
To examine the potential application of celecoxib, a potent COX-2 inhibitor, in the treatment of endometriosis, we investigated the effects of this drug on cell proliferation and apoptosis of cultured human endometrial epithelial cells (EECs). In addition, our study evaluated the effect of celecoxib on in vitro VEGF and PGE2 secretion, as well as on COX-2 expression in EECs from patients with endometriosis.
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Materials and Methods |
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Subjects
A total of 45 patients who underwent diagnostic laparoscopy for infertility participated in this study: 32 with untreated endometriosis (Stages I and II) and 13 controls. Control subjects were infertile women without endometriosis, with tubal factor infertility or unexplained infertility, undergoing diagnostic laparoscopy. Male factor infertility was discarded. To avoid false negatives, only patients who did not complain of pelvic pain were considered for the control group. All patients were infertile, showed regular menstrual cycles and had not received any hormonal medical treatment for the last 6 months. Staging of the disease was performed according to the Revised American Society for Reproductive Medicine Classification (ASRM, 1997
). Further confirmation of the disease was performed by histologic documentation. Biopsy specimens of eutopic endometrium were obtained from all subjects in the proliferative phase, as described previously (Meresman et al., 2000). Our experience indicates that there is a more efficient recovery of epithelial cells in the proliferative phase than in the secretory phase of the cycle and other authors have observed the same (Bongso et al., 1988). The biopsies were taken from the anterior uterine wall toward the fundal area. This study was approved by the Ethics and Research Committee of the Biology and Experimental Medicine Institute of Buenos Aires, Argentina, and all subjects included in the study signed informed consents.
Isolation and culture of EECs
EECs were obtained from eutopic endometrial biopsies of endometriosis patients and controls. The tissue was immediately placed into culture medium and processed within 60 min of collection. Epithelial cells were enzymatically separated and isolated by successive centrifugation, and primary cultures were established for in vitro studies using the method described previously (Meresman et al., 2003a,b). Briefly, the explant was minced, washed and placed in basic medium (Dulbecco’s modified Eagle’s medium F-12, Gibco, Paisley, UK) containing 100 IU/ml penicillin, 100 µg/ml streptomycin and 25 µg/ml amphotericin B (Gibco) with 0.5 mg/ml collagenase (type I, Gibco). After a 2-h incubation at 37°C in an atmosphere of 5% CO2, the resulting suspension was centrifuged at 100 x g for 5 min. The pellet containing glands was re-suspended in nutrient medium and spun again at 100 x g for 5 min. The final pellet mainly contained epithelial cells. Any stromal cells remaining with the glands were further separated by selective adherence to plastic tissue culture dishes for 2 h. The enriched epithelial cells were cultured with media enriched with 10% fetal bovine serum (FBS; Gibco) and were grown to sub-confluence (70–80%) at 37°C for 48 h before the experiments. In all cases, each biopsy was utilized to run one or more experiments and there was no pooling of biopsies.
It has been previously shown that this method guarantees purity of epithelial cells in culture (Meresman et al., 2003a,b). Briefly, after 48-h culture, EECs were fixed in 100% methanol for 20 min and stained by indirect immunofluorescence using mouse monoclonal anti-cytokeratin 56 kDa (reacts with cytokeratin polypeptides 1, 2, 5, 6, 7, 8, 11, 14, 16, 17, 18; Serotec Ltd, Oxford, UK) followed by anti-immunoglobulin(Ig)-fluorescein isothiocyanate (Dako Ltd, Cambridge, UK) as secondary antibody. Cultures incubated without the primary antibody were included as controls in all experiments. Slides were viewed on a microscope equipped with fluorescence and differential interference contrast optics. Identical evaluation was also done on endometrial stromal cells.
The broad-spectrum cytokeratin antibody produced clear labelling in the epithelial cells. No staining of stromal cells was observed with this anti-cytokeratin antibody and no fluorescent labelling was observed in control cultures incubated with the secondary antibody alone. The purity of culture was 85–90% for epithelial cells as determined by immunofluorescent staining.
Cell proliferation assay
Fifty thousand EECs per well were plated in 96-microwell plates and incubated with 10% FBS medium. After a 48-h incubation, the cells were washed and celecoxib (Pfizer, Caguas, USA) 10, 20, 25, 40, 50, 75 and 100 µM was added to supplemented 2.5% FBS medium. The cells were incubated with the agent for 48 additional hours, at which time the cells had reached 70% confluence. Vehicle control cells consisted of EECs incubated with 2.5% FBS medium with 1 µl dimethylsulphoxide (D5879, Sigma, St Louis, MO, USA) per ml of medium, the vehicle in which celecoxib was reconstituted.
Twenty-four hours before harvesting, 1 µCi 3H-thymidine (Nen, Dupont, Boston, MA, USA) was added to each microwell and DNA synthesis was assessed by 3H-thymidine incorporation using a liquid scintillation counter (Meresman et al., 2003a).
In order to determine whether added PGE2 could counteract the growth inhibitory effect of celecoxib, EECs were treated with celecoxib (50 µM) and 200–2000 pg/ml PGE2 and incubated for 48 h before determining 3H-thymidine incorporation, as mentioned above. The dose of celecoxib used in this assay was chosen based on the lowest concentration that had a significant effect on inhibiting cell proliferation.
Apoptosis assay
A total of 120 000 EECs per well were plated in Lab-Tek 8-well culture chambers (Nalge Nunc, Naperville, IL, USA). After 48-h incubation with 10% FBS medium, the cells were washed and cultured with 2.5% FBS medium. Cells were incubated with the addition of celecoxib at 10, 20, 25, 40, 50, 75 and 100 µM or vehicle control. The concentrations in the study were based on our titrations assessing apoptosis with celecoxib for the EECs and from several published studies on other cells (Jendrossek et al., 2003; Basu et al., 2005). The cells were incubated for 24 additional hours and the percentage of apoptotic cells was assessed by the Acridine Orange–Ethidium Bromide (AO–EB) technique. AO is taken up by both viable and non-viable cells and emits green fluorescence if intercalated into double-stranded nucleic acid (DNA) or red fluorescence if bound to single-stranded nucleic acid (RNA). EB is taken up only by non-viable cells and emits red fluorescence by intercalation into DNA (Baskic et al., 2006). It is specific for apoptotic forms of cell death and does not significantly label cells undergoing necrotic death provoked by injury (Abrams et al., 1993; Ribble et al., 2005). After addition of the AO (4 µg/ml)–EB (4 µg/ml) mix, the cells were viewed using a fluorescence microscope by two observers. There was no significant difference in the results obtained between the two observers (P = 0.66).
Detection of COX-2 by immunocytochemistry
A total of 120 000 EECs per well were plated in Lab-Tek 8-well culture chambers (Nalge Nunc). After a 48-h incubation with 10% FBS medium, the cells were washed and incubated for an additional 24-h period with celecoxib at 25, 40, 50, 75 and 100 µM in medium supplemented with 2.5% FBS or vehicle control. Following treatments, chamber slides were fixed and standard procedures for immunocytochemistry were performed. Cells were incubated overnight with rabbit polyclonal anti-human COX-2 antibody (1:25, sc-1747, Santa Cruz, Santa Cruz Biotechnology Inc., CA, USA) at 4°C. After that, cells were treated for 60 min with the corresponding biotinylated secondary antibody and later with streptavidine conjugated to horseradish peroxidase (LSAB+System, Dako).
Binding was visualized by incubating slides with diaminobenzidine and lightly counterstained with haematoxylin. As a negative control, Ig of the same Ig class and concentration as the primary antibody was used. The negative control showed absence of specific staining. COX-2-positive cells were identified by the presence of brown cellular reactivity.
Evaluation of COX-2 expression by western blot
A total of 106 EECs per well were plated in 6-well plates. After incubation for 48 h with 10% FBS medium, the cells were washed and incubated for an additional 24-h period with celecoxib at 25, 40, 50, 75 and 100 µM in medium supplemented with 2.5% FBS or vehicle control. Following treatment, cells were lysed by scraping in chilled lysis buffer supplemented with protease inhibitors (P8340, Sigma). After freeze thawing, the lysate was centrifuged at 15 000 x g for 10 min at 4°C and protein concentrations in the supernatant were measured by the Bradford assay (Bradford, 1976). Equal protein samples (20 µg) were electrophoresed through a 10% sodium dodecyl sulphate–polyacrylamide gel. The separated proteins were transferred to nitrocellulose membranes, blocked for 1 h in 5% low-fat powdered milk and incubated with the rabbit-polyclonal anti-human COX-2 antibody (1:200, sc-1747, Santa Cruz) at 4°C overnight. After overnight incubation, membranes were incubated with a goat peroxidase-conjugated anti-rabbit IgG secondary antibody (1:1000, A4914, Sigma) at room temperature for 1 h. Protein bands were visualized by incubating the membranes with an enhanced chemiluminescence reagent (Perkin Elmer Life Science, Boston, MA, USA) followed by exposure to Kodak X-Omat AR films. The levels of protein were compared and analysed by densitometric studies using Scion Image for Windows (Scion Corporation, Worman’s Mill, CT, USA). Consistency of protein loading was validated with β-actin loaded as control, using a mouse-monoclonal anti-human β-actin antibody (1:2000, Abcam, Cambridge, UK) as primary antibody and an anti-mouse IgG as secondary antibody (1:1000, HAF007, R&D Systems, Minneapolis, MN, USA).
Measurement of VEGF and PGE2 by ELISA
EEC cultures were left undisturbed at 37°C for 48 h. Subsequently, the cells were washed and incubated with basic medium supplemented with 2.5% FBS for an additional 24 h after the addition of celecoxib at 25, 50 and 100 µM or vehicle control. Cells were prepared for assays as described above. The cell culture supernatant was removed from each well and centrifuged for 5 min at 2000 x g to pellet any floating cells. Supernatants not assayed immediately were frozen at –80°C. The conditioned medium was collected and assayed for VEGF and PGE2 using commercial enzyme-linked immunosorbent assay (ELISA) kits (Cytelisa, Human VEGF kit, EL-V, Cytimmune Sciences Inc., MD, USA, and Correlate-EIA, PGE2 Enzyme Inmunoassay Kit, Assay Designs, Ann Arbor, MI, USA). The sensitivity level for the VEGF ELISA was 26.6 pg/ml and for the PGE2 ELISA was 13.4 pg/ml. The intra-assay variability for VEGF and PGE2 was ±7.3 and 8.9%, respectively, while the inter-assay variability was ±10.5 and 3.0% for VEGF and PGE2, respectively. All samples were assessed in duplicate. Results were expressed as percentage of vehicle control cells ± SEM.
Statistics
Statistical comparisons were performed by Kruskal–Wallis non-parametric analysis of variance test, followed by Dunn’s multiple comparison test. Regardless of the statistical test, only a P-value 
0.05 was considered significant.
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Effects of celecoxib on EEC proliferation
The effects of different concentrations of celecoxib on EEC proliferation are displayed in Fig. 1. We found that in EEC cultures from controls, celecoxib at low concentrations (10, 20, 25 and 40 µM) had no effect on basal DNA synthesis. In contrast, after exposure to 50, 75 and 100 µM of celecoxib, the EECs showed a significantly lower degree of cell proliferation (P < 0.05, P < 0.01 and P < 0.001, respectively, versus vehicle control cell proliferation).
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In EEC cultures from endometriosis patients, 3H-thymidine uptake was significantly down-regulated by celecoxib at high concentrations: 50, 75 and 100 µM (P < 0.05, P < 0.01 and P < 0.01, respectively, versus vehicle control cell proliferation). Lower concentrations of celecoxib (10, 20, 25 and 40 µM) had no significant effect on basal cell proliferation.
Effects of celecoxib on EEC apoptosis
The effects of different concentrations of celecoxib on EEC apoptosis are displayed in Fig. 2. Celecoxib 50, 75 and 100 µM showed a significant effect on endometrial growth, enhancing apoptosis in endometrial cell cultures from controls (P < 0.05, P < 0.001 and P < 0.001, respectively, versus vehicle control apoptosis). In contrast, celecoxib 10, 20, 25 and 40 µM had no effect on basal apoptosis.
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As well, in EECs from patients with endometriosis, celecoxib 50, 75 and 100 µM showed a significant effect enhancing the apoptosis levels (P < 0.001, P < 0.001 and P < 0.01, respectively, versus vehicle control apoptosis). In contrast, celecoxib 10, 20, 25 and 40 µM had no significant effect on basal apoptosis.
In this study and in previous works performed in our laboratory (Meresman et al., 2003a; Bilotas et al., 2007), we found no significant differences in the degrees of cell proliferation and in the percentage of apoptotic cells between endometrial cell cultures from endometriosis patients and controls. Taking all these results into account, we decided to utilize only cultures from patients with endometriosis to evaluate the experiments that follow.
Effect of celecoxib on COX-2 expression
The effect of different doses of celecoxib on the expression of COX-2 in EECs in culture from patients with endometriosis was studied by immunocytochemistry. COX-2 expression was observed in the cytoplasm of EECs treated with celecoxib (25 and 50 µM) as well as in vehicle control cells (Fig. 3A).
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To assess whether there were variations in the level of COX-2 protein after exposure to different doses of celecoxib (25, 40, 50, 75 and 100 µM), COX-2 expression was analysed by western blot. The pattern observed was similar to the one seen by immunocytochemistry. COX-2 protein was observed in celecoxib-treated EECs and in vehicle control cells, showing a significant increase after treatment with 100 µM of celecoxib (P < 0.05) (Fig. 3B).
Effect of celecoxib on PGE2 synthesis
COX-2 activity was evaluated by measuring the levels of PGE2 released into the conditioned media by EECs in culture treated with different doses of celecoxib for 24 h. Fig. 4 shows that the concentrations of celecoxib tested (25, 50 and 100 µM) significantly reduced the levels of PGE2 in supernatants (P < 0.001 versus vehicle control).
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Effect of celecoxib on VEGF secretion
Fig. 5 shows the effect of celecoxib on the secretion of the pro-angiogenic factor VEGF by EECs in culture treated with different doses of celecoxib for 24 h. The concentrations of celecoxib tested (25, 50 and 100 µM) induced a significant reduction in VEGF secretion compared with vehicle control (P < 0.001, P < 0.05 and P < 0.001, respectively).
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Effect of the addition of celecoxib and PGE2 on EEC proliferation
Since celecoxib caused growth inhibition and inhibited PGE2 secretion, we speculated that the addition of exogenous PGE2 to celecoxib-stimulated EECs in culture might abrogate the effect of celecoxib on cell proliferation. Therefore, to determine whether celecoxib-induced growth inhibition could be reversed by exogenous PGE2, EECs from patients with endometriosis were stimulated simultaneously with 50 µM celecoxib and PGE2 in different doses. Growth inhibition induced by 50 µM celecoxib could not be restored by addition of exogenous PGE2 (P > 0.05 versus EECs stimulated with celecoxib alone), thereby suggesting that celecoxib-induced growth inhibition in these cells may be independent of PGE2 levels. Results are displayed in Fig. 6.
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Discussion |
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Current medical therapies for endometriosis, which are usually aimed at reducing the endogenous steroid production, are not effective in at least half of these patients, and their adverse effect profiles constitute a significant problem (Rice, 2002
; Mihalyi et al., 2006). Given the inefficiency of the available treatments, developing novel therapeutic strategies may be useful to improve our ability to eliminate endometriotic lesions and to prevent the recurrence of the disease.
In this study, epithelial cell cultures of eutopic human endometrium from endometriosis patients were used in a model to evaluate cell proliferation, apoptosis, COX-2 expression and VEGF and PGE2 secretion in response to the addition of the COX-2 inhibitor celecoxib. Although the cells employed in this investigation were not derived from endometriotic implants and their in vitro response may not be identical, the rationale for the use of eutopic endometrial cells in short-term culture as a model for endometriosis research has been documented previously (Surrey and Halme, 1990). Furthermore, the epithelial fraction was the one studied since it has been seen that COX-2 is preferentially expressed in this cell type rather than in stromal cells (Ota et al., 2001).
Endometriosis is associated with increased COX-2 expression in eutopic and ectopic endometrium (Ota et al., 2001; Matsuzaki et al., 2004b). It has also been shown that PG levels are elevated in serum and peritoneal fluid from endometriosis patients (De Leon et al., 1986; Gazvani and Templeton, 2002). Inhibition of COX by conventional NSAIDs or selective COX-2 inhibitors may therefore lead to relief of pain associated with endometriosis (Nasir and Bope, 2004).
Recently, multiple studies have suggested that the administration of a COX-2 inhibitor reduces tumour development by suppressing proliferation and angiogenesis as well as by increasing apoptosis (Elder et al., 2000; Leahy et al., 2002; Basu et al., 2005; Grosch et al., 2006). However, the effect of COX-2 inhibitors on the endometrial cell growth in endometriosis is still unknown. The present study clearly demonstrates that in EECs from patients with endometriosis and controls, treatment with the selective COX-2 inhibitor, celecoxib, effectively reduces cell proliferation and induces cell death. We observed that celecoxib-induced apoptosis in EECs in culture revealed by the AO–EB technique. Staining of apoptotic cells with fluorescent dyes such as AO and EB is considered a correct method for evaluating the changed nuclear morphology. Recently, Baskic et al. compared several methods for detecting apoptosis and necrosis in human leukocytes. The authors conclude that both fluorescence methods, AO and EB, provided reliable and reproducible results and distinguished clearly between subpopulations of apoptotic cells (Baskic et al., 2006).
Celecoxib-induced apoptosis has previously been reported by other authors. Basu et al. (2005) and Jendrossek et al. (2003) described that treatment with celecoxib enhanced apoptosis in human breast cancer cells and Jurkat T cells, respectively. There is evidence that the intrinsic pathway is implicated in the activation of apoptosis after celecoxib treatment (Jendrossek et al., 2003; Dandekar et al., 2005), yet other authors have reported activation of the extrinsic pathway (Kim et al., 2004; Liu et al., 2004). Furthermore, the doses of celecoxib that significantly induced apoptosis in the present study are clinically relevant since studies conducted by other authors reveal that the concentration of COX-2 inhibitors in serum of treated patients varies from 20 to 100 µM (Munkarah et al., 2003; Basu et al., 2005). Nevertheless, there are discrepancies in this regard. Other authors state that the plasma concentration corresponds to 3 µM when patients were treated with daily doses of 400 mg of celecoxib (Maier et al., 2004; Grosch et al., 2006). According to these investigators, the differences found in COX-2 inhibitor concentration needed to observe anti-proliferative effects relies on the fact that, in order to induce regression of tumour growth in patients, several weeks of treatment are needed while, in vitro, the anti-proliferative effects are seen in a few hours (Maier et al., 2005).
Our findings are consistent with previous results from other authors who found that COX-2 inhibitors were successful at inhibiting tumour growth in different cell lines (Wu et al., 2004; Basu et al., 2005). Nevertheless, provision of exogenous PGE2 did not reverse celecoxib-induced growth inhibition in EECs. This suggests that celecoxib-induced growth inhibition in EECs is independent of PGE2. Corroborating our findings are previous reports that conclude that the addition of PGE2 had no effect on growth inhibition induced by COX-2 inhibitors (Elder et al., 2000; Basu et al., 2005).
Additionally, celecoxib treatment reduces PGE2 secretion by EECs from patients with endometriosis. PGE2 is the major PG produced by EECs, and the production of secreted PGE2 is therefore an appropriate measure of COX activity. A concentration of 25 µM of celecoxib was effective at inhibiting PGE2 secretion, however, under these conditions, growth inhibition was not observed. The reduction of PGE2 production induced by 25 µM celecoxib in EECs without the induction of growth inhibition supports the proposal that the growth arrest induced by higher concentrations (50–100 µM) of celecoxib is not simply due to reduced PGE2 secretion. Similar results were obtained by Grosch et al. (2001) and Han et al. (2004) who speculated that the mechanisms involved in the inhibition of cell proliferation due to celecoxib treatment are independent of COX-2 activity and expression, since the concentrations needed to induce cell cycle arrest and inhibit proliferation are higher than those needed to inhibit COX-2 activity. In agreement, it has been reported that celecoxib induces apoptosis through mechanisms independent of COX-2 in cancer cell lines (Jendrossek et al., 2003; Lai et al., 2003; Grosch et al., 2006), and it can also induce apoptosis in cells deficient in COX-2 (Grosch et al., 2001; Pang et al., 2007). Further studies are needed to confirm whether the inhibitory effect triggered by celecoxib is independent of COX-2 activity.
In addition to growth inhibition and COX-2 activity reduction, we observed that COX-2 protein is significantly increased in EECs from endometriosis patients treated with 100 µM celecoxib. Also, the western blot analysis for COX-2 protein showed a clear but not statistically significant increase after treatment with 25–75 µM celecoxib (Fig. 3B). The mechanism for this is not known, however, it has been suggested that COX-2-dependent products may inhibit COX-2 expression in a negative feedback loop: here, celecoxib treatment inhibited COX-2 activity, shown by reduced PGE2 secretion, perhaps resulting in removal of negative feedback and COX-2 induction. There are similar data on COX-2 inhibitor treatment leading to strong up-regulation of COX-2 protein expression in several reports (Elder et al., 2000; Levitt et al., 2004; Basu et al., 2005).
The establishment, maintenance and growth of endometriosis are dependent in part on the development of new blood vessels and it is known that the VEGF family is critical in the process of angiogenesis. Activity of COX-2 results in an increased VEGF expression (Li et al., 2002). In addition, it has been shown that patients with endometriosis have a greater concentration of VEGF in peritoneal fluid compared with controls (Mahnke et al., 2000). Treating rats with rofecoxib induced a decrease in the explant size accompanied by a decrease in peritoneal fluid VEGF levels (Dogan et al., 2004). In our study, VEGF levels were significantly lower in the celecoxib-treated EECs in comparison to vehicle control cells. Down-regulation of eicosanoid production by COX-2 inhibitors is reported widely as a mechanism underlying their antiangiogenic activity (Gately and Li, 2004).
To the best of our knowledge, this is the first in vitro study suggesting a direct effect of celecoxib COX-2 inhibitor on the reduction of endometrial growth. Nevertheless, in animal models for endometriosis, it was shown that celecoxib prevented the establishment of new endometriotic lesions and the growth of already established ones (Matsuzaki et al., 2004a).
With the development of COX-2-specific inhibitors, better-tolerated therapeutic options are available. In humans, only one report has been published and suggested that the use of COX-2-specific inhibitors was effective in the management of pelvic pain associated with endometriosis (Cobellis et al., 2004).
The role of COX-2 in endometrial growth inhibition continues to be under investigation, and the data in our study support further research on selective COX-2 inhibition as a novel therapeutic modality in endometriosis.
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This study was supported by ANPCYT (BID 1728/OC-AR PICT 2004 5-26047) and CONICET (PIP 5471), Buenos Aires, Argentina.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingReferences |
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Abrams JM, White K, Fessler LI, Steller H. Programmed cell death during Drosophila embryogenesis. Development (1993) 117:29–43.
ASRM. Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertil Steril (1997) 67:817–821.[CrossRef][Web of Science][Medline]
Attar E, Bulun SE. Aromatase and other steroidogenic genes in endometriosis: translational aspects. Hum Reprod Update (2006) 12:49–56.
Baskic D, Popovic S, Ristic P, Arsenijevic NN. Analysis of cycloheximide-induced apoptosis in human leukocytes: fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide. Cell Biol Int (2006) 30:924–932.[CrossRef][Web of Science][Medline]
Basu GD, Pathangey LB, Tinder TL, Gendler SJ, Mukherjee P. Mechanisms underlying the growth inhibitory effects of the cyclo-oxygenase-2 inhibitor celecoxib in human breast cancer cells. Breast Cancer Res (2005) 7:R422–R435.[CrossRef][Web of Science][Medline]
Bilotas M, Baranao RI, Buquet R, Sueldo C, Tesone M, Meresman G. Effect of GnRH analogues on apoptosis and expression of Bcl-2, Bax, Fas and FasL proteins in endometrial epithelial cell cultures from patients with endometriosis and controls. Hum Reprod (2007) 22:644–653.
Bongso A, Gajra B, Lian NP, Wong PC, Soon-Chye N, Ratnam S. Establishment of human endometrial cell cultures. Hum Reprod (1988) 3:705–713.
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem (1976) 72:248–254.[CrossRef][Web of Science][Medline]
Bulun SE, Gurates B, Fang Z, Tamura M, Sebastian S, Zhou J, Amin S, Yang S. Mechanisms of excessive estrogen formation in endometriosis. J Reprod Immunol (2002) 55:21–33.[CrossRef][Web of Science][Medline]
Bundred NJ, Barnes NL. Potential use of COX-2-aromatase inhibitor combinations in breast cancer. Br J Cancer (2005) 93(Suppl 1):S10–S15.[CrossRef][Web of Science][Medline]
Chun KS, Surh YJ. Signal transduction pathways regulating cyclooxygenase-2 expression: potential molecular targets for chemoprevention. Biochem Pharmacol (2004) 68:1089–1100.[CrossRef][Web of Science][Medline]
Cobellis L, Razzi S, De Simone S, Sartini A, Fava A, Danero S, Gioffre W, Mazzini M, Petraglia F. The treatment with a COX-2 specific inhibitor is effective in the management of pain related to endometriosis. Eur J Obstet Gynecol Reprod Biol (2004) 116:100–102.[CrossRef][Web of Science][Medline]
Dandekar DS, Lopez M, Carey RI, Lokeshwar BL. Cyclooxygenase-2 inhibitor celecoxib augments chemotherapeutic drug-induced apoptosis by enhancing activation of caspase-3 and -9 in prostate cancer cells. Int J Cancer (2005) 115:484–492.[CrossRef][Web of Science][Medline]
De Leon FD, Vijayakumar R, Brown M, Rao CV, Yussman MA, Schultz G. Peritoneal fluid volume, estrogen, progesterone, prostaglandin, and epidermal growth factor concentrations in patients with and without endometriosis. Obstet Gynecol (1986) 68:189–194.[Web of Science][Medline]
Dogan E, Saygili U, Posaci C, Tuna B, Caliskan S, Altunyurt S, Saatli B. Regression of endometrial explants in rats treated with the cyclooxygenase-2 inhibitor rofecoxib. Fertil Steril (2004) 82(Suppl 3):1115–1120.[CrossRef][Web of Science][Medline]
Ebert AD, Bartley J, David M. Aromatase inhibitors and cyclooxygenase-2 (COX-2) inhibitors in endometriosis: new questions–old answers? Eur J Obstet Gynecol Reprod Biol (2005) 122:144–150.[CrossRef][Web of Science][Medline]
Elder DJ, Halton DE, Crew TE, Paraskeva C. Apoptosis induction and cyclooxygenase-2 regulation in human colorectal adenoma and carcinoma cell lines by the cyclooxygenase-2-selective non-steroidal anti-inflammatory drug NS-398. Int J Cancer (2000) 86:553–560.[CrossRef][Web of Science][Medline]
Gately S, Li WW. Multiple roles of COX-2 in tumor angiogenesis: a target for antiangiogenic therapy. Semin Oncol (2004) 31:2–11.[Web of Science][Medline]
Gazvani R, Templeton A. Peritoneal environment, cytokines and angiogenesis in the pathophysiology of endometriosis. Reproduction (2002) 123:217–226.[Abstract]
Grosch S, Tegeder I, Niederberger E, Brautigam L, Geisslinger G. COX-2 independent induction of cell cycle arrest and apoptosis in colon cancer cells by the selective COX-2 inhibitor celecoxib. FASEB J (2001) 15:2742–2744.
Grosch S, Maier TJ, Schiffmann S, Geisslinger G. Cyclooxygenase-2 (COX-2)-independent anticarcinogenic effects of selective COX-2 inhibitors. J Natl Cancer Inst (2006) 98:736–747.
Han C, Leng J, Demetris AJ, Wu T. Cyclooxygenase-2 promotes human cholangiocarcinoma growth: evidence for cyclooxygenase-2-independent mechanism in celecoxib-mediated induction of p21waf1/cip1 and p27kip1 and cell cycle arrest. Cancer Res (2004) 64:1369–1376.
Jendrossek V, Handrick R, Belka C. Celecoxib activates a novel mitochondrial apoptosis signaling pathway. FASEB J (2003) 17:1547–1549.
Kim SH, Song SH, Kim SG, Chun KS, Lim SY, Na HK, Kim JW, Surh YJ, Bang YJ, Song YS. Celecoxib induces apoptosis in cervical cancer cells independent of cyclooxygenase using NF-kappaB as a possible target. J Cancer Res Clin Oncol (2004) 130:551–560.[Web of Science][Medline]
Kundu N, Smyth MJ, Samsel L, Fulton AM. Cyclooxygenase inhibitors block cell growth, increase ceramide and inhibit cell cycle. Breast Cancer Res Treat (2002) 76:57–64.[CrossRef][Web of Science][Medline]
Lai GH, Zhang Z, Sirica AE. Celecoxib acts in a cyclooxygenase-2-independent manner and in synergy with emodin to suppress rat cholangiocarcinoma growth in vitro through a mechanism involving enhanced Akt inactivation and increased activation of caspases-9 and -3. Mol Cancer Ther (2003) 2:265–271.
Leahy KM, Ornberg RL, Wang Y, Zweifel BS, Koki AT, Masferrer JL. Cyclooxygenase-2 inhibition by celecoxib reduces proliferation and induces apoptosis in angiogenic endothelial cells in vivo. Cancer Res (2002) 62:625–631.
Levitt RJ, Buckley J, Blouin MJ, Schaub B, Triche TJ, Pollak M. Growth inhibition of breast epithelial cells by celecoxib is associated with upregulation of insulin-like growth factor binding protein-3 expression. Biochem Biophys Res Commun (2004) 316:421–428.[CrossRef][Web of Science][Medline]
Li G, Yang T, Yan J. Cyclooxygenase-2 increased the angiogenic and metastatic potential of tumor cells. Biochem Biophys Res Commun (2002) 299:886–890.[CrossRef][Web of Science][Medline]
Liu X, Yue P, Zhou Z, Khuri FR, Sun SY. Death receptor regulation and celecoxib-induced apoptosis in human lung cancer cells. J Natl Cancer Inst (2004) 96:1769–1780.
Mahnke JL, Dawood MY, Huang JC. Vascular endothelial growth factor and interleukin-6 in peritoneal fluid of women with endometriosis. Fertil Steril (2000) 73:166–170.[CrossRef][Web of Science][Medline]
Maier TJ, Schilling K, Schmidt R, Geisslinger G, Grosch S. Cyclooxygenase-2 (COX-2)-dependent and -independent anticarcinogenic effects of celecoxib in human colon carcinoma cells. Biochem Pharmacol (2004) 67:1469–1478.[CrossRef][Web of Science][Medline]
Maier TJ, Janssen A, Schmidt R, Geisslinger G, Grosch S. Targeting the beta-catenin/APC pathway: a novel mechanism to explain the cyclooxygenase-2-independent anticarcinogenic effects of celecoxib in human colon carcinoma cells. FASEB J (2005) 19:1353–1355.
Matsuzaki S, Canis M, Darcha C, Dallel R, Okamura K, Mage G. Cyclooxygenase-2 selective inhibitor prevents implantation of eutopic endometrium to ectopic sites in rats. Fertil Steril (2004) a 82:1609–1615.[CrossRef][Web of Science][Medline]
Matsuzaki S, Canis M, Pouly JL, Wattiez A, Okamura K, Mage G. Cyclooxygenase-2 expression in deep endometriosis and matched eutopic endometrium. Fertil Steril (2004) b 82:1309–1315.[CrossRef][Web of Science][Medline]
Meresman GF, Vighi S, Buquet RA, Contreras-Ortiz O, Tesone M, Rumi LS. Apoptosis and expression of Bcl-2 and Bax in eutopic endometrium from women with endometriosis. Fertil Steril (2000) 74:760–766.[CrossRef][Web of Science][Medline]
Meresman GF, Bilotas M, Buquet RA, Baranao RI, Sueldo C, Tesone M. Gonadotropin-releasing hormone agonist induces apoptosis and reduces cell proliferation in eutopic endometrial cultures from women with endometriosis. Fertil Steril (2003) a 80(Suppl 2):702–707.[CrossRef][Web of Science][Medline]
Meresman GF, Bilotas MA, Lombardi E, Tesone M, Sueldo C, Baranao RI. Effect of GnRH analogues on apoptosis and release of interleukin-1beta and vascular endothelial growth factor in endometrial cell cultures from patients with endometriosis. Hum Reprod (2003) b 18:1767–1771.
Mihalyi A, Simsa P, Mutinda KC, Meuleman C, Mwenda JM, D’Hooghe TM. Emerging drugs in endometriosis. Expert Opin Emerg Drugs (2006) 11:503–524.[CrossRef][Medline]
Munkarah AR, Genhai Z, Morris R, Baker VV, Deppe G, Diamond MP, Saed GM. Inhibition of paclitaxel-induced apoptosis by the specific COX-2 inhibitor, NS398, in epithelial ovarian cancer cells. Gynecol Oncol (2003) 88:429–433.[CrossRef][Web of Science][Medline]
Nasir L, Bope ET. Management of pelvic pain from dysmenorrhea or endometriosis. J Am Board Fam Pract (2004) 17(Suppl):S43–S47.
Ota H, Igarashi S, Sasaki M, Tanaka T. Distribution of cyclooxygenase-2 in eutopic and ectopic endometrium in endometriosis and adenomyosis. Hum Reprod (2001) 16:561–566.
Ozawa Y, Murakami T, Tamura M, Terada Y, Yaegashi N, Okamura K. A selective cyclooxygenase-2 inhibitor suppresses the growth of endometriosis xenografts via antiangiogenic activity in severe combined immunodeficiency mice. Fertil Steril (2006) 86(Suppl 4):1146–1151.[CrossRef][Web of Science][Medline]
Pang RP, Zhou JG, Zeng ZR, Li XY, Chen W, Chen MH, Hu PJ. Celecoxib induces apoptosis in COX-2 deficient human gastric cancer cells through Akt/GSK3beta/NAG-1 pathway. Cancer Lett (2007) 251:268–277.[CrossRef][Web of Science][Medline]
Ribble D, Goldstein NB, Norris DA, Shellman YG. A simple technique for quantifying apoptosis in 96-well plates. BMC Biotechnol (2005) 5:12.[CrossRef][Medline]
Rice VM. Conventional medical therapies for endometriosis. Ann N Y Acad Sci (2002) 955:343–352.[Web of Science][Medline]
Surrey ES, Halme J. Effect of peritoneal fluid from endometriosis patients on endometrial stromal cell proliferation in vitro. Obstet Gynecol (1990) 76:792–797.[Web of Science][Medline]
Tamura K, Sakurai T, Kogo H. Relationship between prostaglandin E2 and vascular endothelial growth factor (VEGF) in angiogenesis in human vascular endothelial cells. Vascul Pharmacol (2006) 44:411–416.[CrossRef][Web of Science][Medline]
Valle RF, Sciarra JJ. Endometriosis: treatment strategies. Ann N Y Acad Sci (2003) 997:229–239.[CrossRef][Web of Science][Medline]
Vane JR, Mitchell JA, Appleton I, Tomlinson A, Bishop-Bailey D, Croxtall J, Willoughby DA. Inducible isoforms of cyclooxygenase and nitric-oxide synthase in inflammation. Proc Natl Acad Sci USA (1994) 91:2046–2050.
Wu T, Leng J, Han C, Demetris AJ. The cyclooxygenase-2 inhibitor celecoxib blocks phosphorylation of Akt and induces apoptosis in human cholangiocarcinoma cells. Mol Cancer Ther (2004) 3:299–307.
Submitted on April 25, 2008; resubmitted on July 16, 2008; accepted on July 21, 2008.
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