endometriosis, a common, benign, estrogen-dependent disease affecting 3–10% of women of reproductive age, is characterized by the ectopic growth of endometrial tissue, which is found primarily in the peritoneum, ovaries, and rectovaginal septum (21). Women suffering from endometriosis may present with chronic pelvic pain, dysmenorrhea, dyspareunia, and/or subfertility. The prevalence of endometriosis in women with pelvic pain and/or subfertility is estimated to be between 20–90%; thus, it is one of the most frequently encountered benign gynecological problems (9).

Various therapies have been used to treat endometriosis, including both surgical and medical strategies. Medical therapies historically have included contraceptive steroids, progestogens, and agonists of gonadotropin-releasing hormone (GnRH), as well as androgens and nonsteroidal anti-inflammatory agents (27). Current medical treatment aims to inhibit the growth of endometriotic implants by suppressing ovarian steroids and inducing a hypoestrogenic state (27). Of the medical agents mentioned above, GnRH agonists have gained predominance in the medical treatment of endometriosis. GnRH agonists suppress the release of follicle-stimulating hormone and luteinizing hormone from the pituitary gland and inhibit ovarian steroidogenesis, resulting in a hypoestrogenic state that is suitable for the remission of the endometriotic lesions (5). However, treatments for endometriosis that aim to lower circulating estradiol concentrations can be used only for a limited time due to unacceptable side effects. In addition, high recurrence rates of up to 45% after the completion of medical treatments remain a significant problem (4). Therefore, novel therapeutic strategies are necessary to improve the clinical management of patients with endometriosis.

We have demonstrated that endometriotic cells, but not normal endometrial cells, are resistant to cytokine-induced apoptosis (20). It has also been demonstrated that endometrial apoptosis in the eutopic endometrium is lower in women with endometriosis than controls and is further decreased in the ectopic endometrium (8, 10). Both the inability of endometriotic cells to transmit a “death” signal and the ability of the cells to avoid cell death have been associated with increased expression of anti-apoptotic factors [e.g., B-cell lymphoma/leukemia-2 (Bcl-2) and Bcl-X_L] and the decreased expression of preapoptotic factors (e.g., Bax) (15, 20). These findings encouraged the present authors to evaluate apoptosis-inducing agents for the treatment of endometriosis.

The pleiotropic transcription factor nuclear factor (NF)-κB has been identified as a critical component of several signal transduction pathways (2). One important function of NF-κB is its ability to protect cells from apoptosis by activating antiapoptotic genes (3, 31). Recent studies indicate that NF-κB is constitutively active in a number of malignant tumors, such as breast cancer and prostate cancer, and that it plays an antiapoptotic role in the survival of these tumor cells (17, 23, 31). Inhibition of NF-κB has been reported to enhance apoptosis and the sensitivity to anticancer drugs (14, 32). Wieser et al. (33) have demonstrated the constitutive activation of NF-κB in endometriotic cells, and it is suggested that NF-κB may play a significant role in the proliferation of endometriotic lesions (29).

Mitogen-activated protein kinase (MAPK) signal transduction pathways mediate intracellular signaling, which is initiated by a variety of extracellular stimuli. Phosphorylation of MAPKs leads to diverse cellular activities, which include cell proliferation, differentiation, and apoptosis (25). The human MAPK family includes three subgroups: c-Jun NH₂-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK). The ERK signaling pathway is often involved in the signal transduction in cell proliferation and differentiation. The JNK signaling pathway is often involved in the signal transduction in cell proliferation and differentiation. The JNK signaling pathway can be activated by a variety of stresses, including genotoxic stress, heat shock, osmotic shock, and metabolic stress, as well as proinflammatory cytokines (7). The persistent activation of the JNK pathway often mediates intracellular signaling that leads to cell death. The p38 MAPK signaling pathway is also stimulated by cellular stress and proinflammatory cytokines in a way that is similar to the JNK signaling pathway. ERK, JNK, and p38 MAPK are present in the endometriotic cells as well as in the endometrial cells (34). Significant activation of p38 MAPK has been demonstrated in endometriotic cells (34), and it is considered that MAPKs play pathophysiological roles in the development of endometriosis.

A synthetic compound, BAY 11-7085, has been shown to inhibit IκB phosphorylation and the expression of NF-κB-regulated gene products (26). In addition to its inhibition of IκB phosphorylation via IκB kinase (IKK) and the resultant decrease in the DNA-binding function of NF-κB (30), BAY 11-7085 has also been shown to activate JNK and p38 MAPK (26). It has been reported that BAY 11-7085 inhibits cell proliferation and induces apoptosis of colonic epithelial cells (30), keratinocytes (6), chondrocytes (28), and endothelial cells (26).

Molecular targeted treatment strategies are becoming an increasingly attractive option for the treatment of endometriosis, particularly because of the dismal clinical results of more traditional therapies. Our laboratory and others have focused on molecular strategies to alter the antiapoptotic response of endometriotic cells to both novel and traditional therapeutic agents (18, 20). In the present study, we evaluated the effects of BAY 11-7085, an inhibitor of IκB phosphorylation and activator of JNK and p38 MAPK, on the proliferation, cell cycle, and apoptosis of cultured endometriotic cells, using methods that were previously proposed for evaluating novel therapeutic agents for endometriosis (18, 20). We also discuss new therapeutic strategies for the treatment of endometriosis.

MATERIALS AND METHODS

Endometriotic cyst stromal cells and normal endometrial stromal cells: isolation procedure and cell culture conditions.

Endometriotic cyst stromal cells (ECSCs) were obtained from premenopausal patients who had undergone salpingo-oophorectomy or evisceration for ovarian endometriotic cysts (n = 10). Normal endometrial stromal cells (NESCs) were obtained from premenopausal patients who had undergone hysterectomy for subserosal or intramural leiomyoma (n = 9). The indication of hysterectomy was the size of the tumor or hypermenorrhea. None of the patients had received any hormonal treatments at least 2 yr prior to operation. All specimens were histologically diagnosed as being in the mid to late proliferative phase and were considered to be unaffected by the presence of leiomyoma on the basis of a standard histological examination of endometrial tissues. This study was approved by the Institutional Review Board of the Faculty of Medicine, Oita University, and written informed consent was obtained from all patients.

ECSCs were isolated from ovarian endometriotic tissues by enzymatic digestion, as previously described (19). Briefly, the tissues were minced in Hanks' balanced salt solution (GIBCO-BRL, Gaithersburg, MD) and digested with 0.5% collagenase (GIBCO-BRL) in Dulbecco's modified Eagle's medium (DMEM; GIBCO-BRL) at 37°C for 40 min. The dispersed cells were filtered through a 70-μm nylon mesh to remove undigested tissue pieces. The filtrated fraction was then further separated from the epithelial cell clumps by differential sedimentation at unit gravity as follows. The cells were resuspended in 2 ml of culture medium and layered slowly over 10 ml of the medium in a centrifuge tube. Sealed tubes were placed in an upright position at 37°C in 5% CO₂ in air for 30 min. After sedimentation, the top 8 ml of the medium were collected. Finally, the medium containing stromal cells was filtered through a 40-μm nylon mesh. Final purification was achieved by allowing stromal cells, which attach rapidly to plates, to adhere selectively to the culture dishes for 30 min at 37°C, followed by the removal of nonadhering epithelial cells. NESCs were also isolated from endometrial tissues by enzymatic digestion as previously described (19). Isolated NESCs were cultured in DMEM supplemented with 100 IU/ml penicillin, 50 mg/ml streptomycin, and 10% heat-inactivated fetal bovine serum (FBS) (all from GIBCO-BRL) at 37°C in 5% CO₂ in air.

ECSCs and NESCs in the monolayer culture after the third passage were >99% pure as determined by immunocytochemical staining with antibodies to vimentin, CD10, cytokeratin, factor VIII, and leukocyte common antigen and were used for the following experiments (18). Each experiment was performed in triplicate and repeated at least six times.

Assessment of cell proliferation and cell viability of ECSCs and NESCs.

The cell proliferation and cell viability of ECSCs and NESCs after BAY 11-7085 treatment were determined in 96-well plates by a modified methylthiazoletetrazolium (MTT) assay using WST-1 (Roche Diagnostics, Penzberg, Germany) following the manufacturer's protocol. We distributed 5 × 10⁴ cells in DMEM supplemented with 10% FBS into each well of a 96-well flat-bottomed microplate (Corning, New York, NY) and incubated them overnight. The medium was then removed, and the cells were incubated for 48 h with 200 μl of experimental medium containing various concentrations of BAY 11-7085 (0.01–10 μM; Nagara Science, Gifu, Japan). Thereafter, 20 μl of WST-1 dye were added to each well, and the cells were further incubated for 4 h. All experiments were performed in the presence of 10% FBS. Cell proliferation was evaluated by measuring absorbance at 540 nm. Data were calculated as the ratio of the values obtained for the BAY 11-7085-treated cells to those for the untreated controls.

Cell proliferation of ECSCs and NESCs after BAY 11-7085 treatment was determined by 5-bromo-2′-deoxyuridine (BrdU) incorporation using cell proliferation enzyme-linked immunosorbent assay (ELISA, Roche). We placed 1 × 10⁴ cells in DMEM supplemented with 10% FBS into each well of a 96-well flat-bottomed microplate and incubated them overnight. The medium was then removed, and the cells were incubated for 48 h with 100 μl of experimental medium containing various concentrations of BAY 11-7085 (0.01–10 μM). We then added 10 μl of BrdU (10 mM) to each well and incubated the samples for 2 h. BrdU incorporation was then evaluated according to the manufacturer's protocols. All experiments were performed in the presence of 10% FBS. Cell proliferation was evaluated by measuring absorbance at 450 nm. Data were calculated as the ratio of the values obtained for the BAY 11-7085-treated cells to those for the untreated controls.

Assessment of internucleosomal DNA fragmentation in ECSCs.

Internucleosomal DNA fragmentation in ECSCs after BAY 11-7085 treatment was evaluated using a Quick Apoptotic DNA Ladder Detection Kit (BioVision Research Products, Mountain View, CA), as previously described (20). Cells (1 × 10⁷) of ECSCs were plated onto 100-mm culture dishes (Corning) in 10 ml of DMEM supplemented with 10% heat-inactivated FBS and cultured overnight. The supernatant was then replaced with fresh culture medium (DMEM + 10% FBS) containing BAY 11-7085 (10 μM). Twenty-four hours after stimulation, the DNA was extracted from these cells according to the manufacturer's protocol. DNA fragmentation was analyzed by electrophoresis on an agarose gel (1.2%). The DNA bands were visualized by staining with ethidium bromide and were photographed under ultraviolet light using a transilluminator.

Assessment of BAY 11-7085-induced apoptosis in ECSCs and NESCs.

The BAY 11-7085-induced apoptosis of ECSCs and NESCs was quantified by direct determination of nucleosomal DNA fragmentation by Cell Death Detection ELISA (Roche) as previously described (20). The assay used specific monoclonal antibodies directed against histones from fragmented DNA, allowing the determination of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates. Briefly, 1 × 10⁶ cells were plated onto 24-well culture plates (Corning) in 1 ml of culture medium with 10% heat-inactivated FBS and cultured overnight. The supernatant was replaced with fresh culture medium (DMEM + 10% FBS) containing various amounts of BAY 11-7085 (0.01–10 μM). Twenty-four hours after stimulation, the cells were lysed according to the manufacturer's instructions, followed by centrifugation (200 g, 5 min). The mono- and oligonucleosomes contained in the supernatants were determined using an anti-histone-biotin antibody. The concentration of nucleosomes-antibody was determined photometrically at a wavelength of 405 nm using 2,2′-azino-di-(3-ethylbenzthiazoline-sulfonate) as a substrate. Data were calculated as the ratio of the values obtained for the BAY 11-7085-treated cells to those for the untreated controls.

Analysis of cell cycle by flow cytometry.

The cell cycle was analyzed by flow cytometry after 2 days of culture with or without BAY 11-7085, as previously described (18). Briefly, ECSCs were cultured in DMEM supplemented with 10% FBS at <60% confluence for 2 days with or without the presence of BAY 11-7085 (10 μM). They were then trypsinized, washed in phosphate-buffered saline (PBS), fixed in methanol, and incubated for 30 min at 4°C in the dark with a solution of 5 μg/ml propidium iodide, 1 mg/ml RNase (Sigma Chemical, St. Louis, MO), and 0.1% Nonidet P-40 (Sigma). Flow cytometric analysis of the cell cycle was performed immediately after staining using the CellFIT program (Becton-Dickinson, Sunnyvale, CA), in which the S-phase was calculated using an RFit model.

Assessment of the expression of apoptosis-related proteins in ECSCs.

The expression of apoptosis-related proteins (Bcl-2, Bcl-X_L, Bax, Fas, Fas ligand, caspase-3, caspase-8, and caspase-9) and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) in ECSCs was investigated by Western blotting analysis (20). Subconfluent ECSCs were cultured on 100-mm dishes for 24 h with or without the presence of BAY 11-7085 (10 μM). The cells were then washed with PBS, and whole cell extracts were prepared by lysing the cells in lysis buffer (50 mM Tris·HCl, 125 mM NaCl, 0.1% Nonidet P-40, 5 mM ethylenediamine tetraacetic acid, 50 mM NaF, and 0.1% phenylmethylsulfonyl fluoride). The suspension was centrifuged at 15,000 g for 15 min at 4°C, and the supernatant was collected. The total protein concentration was quantified using the Coomassie protein assay reagent (Pierce, Rockford, IL). The whole cell protein extract was resolved with sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a 10% polyacrylamide gel under reduced conditions. After transfer to an Immobilon-P transfer membrane (Millipore, Bedford, MA), the protein was stained with Ponceau S (Sigma) to verify uniform loading and transfer. The membranes were blocked with 5% skim milk (Becton-Dickinson) in Tris-buffered saline with Tween 20 (50 mM Tris·HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.4) (TBS-T) overnight and subsequently incubated with primary antibodies (Bcl-2, Bcl-X_L, Bax, Fas, and Fas ligand obtained from BD Biosciences, San Jose, CA; cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9 from Cell Signaling, Beverly, MA; and GAPDH from Ambion, Austin, TX) at appropriate dilutions for 1 h at room temperature. The membrane was washed three times with TBS-T and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Subsequently, the membrane was washed three times with TBS-T and analyzed by enhanced chemiluminescence (Amersham Pharmacia Biotech, Chicago, IL).

Statistical analysis.

Data were calculated as percentages relative to the untreated controls, presented as means (SD), and appropriately analyzed by the Bonferroni-Dunn test or Mann-Whitney U-test with StatView 4.5 (Abacus Concepts, Berkeley, CA). Values of P < 0.05 were considered to be statistically significant.

RESULTS

Effects of BAY 11-7085 on cell proliferation and cell viability of ECSCs and NESCs.

The effects of BAY 11-7085 on the cell proliferation and cell viability of ECSCs and NESCs were investigated by modified MTT assay. As shown in Fig. 1A, the number of viable ECSCs and NESCs was significantly decreased by the addition of increasing amounts of BAY 11-7085 (66.1 and 54.7% decreases, respectively, at a concentration of 10 μM). BAY 11-7085 showed a stronger inhibitory effect on the cell viability of ECSCs than on that of NESCs (Fig. 1B).

To further assess the effects of BAY 11-7085 on cell proliferation, the DNA synthesis of ECSCs and NESCs after BAY 11-7085 treatment was evaluated by BrdU incorporation assay. As shown in Fig. 2, BAY 11-7085 treatment resulted in a significant inhibition of the BrdU incorporation of ECSCs in a dose-dependent manner (53.2% decrease at a concentration of 10 μM), whereas BAY 11-7085 was associated with only a weak inhibitory effect on the BrdU incorporation of NESCs (38.2% decrease at a concentration of 10 μM).

Effects of BAY 11-7085 on apoptosis of ECSCs and NESCs.

The apoptotic effects of BAY 11-7085 on ECSCs and NESCs were assessed by evaluating the presence of internucleosomal DNA fragmentation. BAY 11-7085 induced the fragmentation of internucleosomal DNA in ECSCs (Figs. 3 and 4). A DNA ladder was detected by electrophoresis in BAY 11-7085-treated ECSCs, suggesting the presence of apoptotic cells (Fig. 3). The apoptosis of both ECSCs and NESCs was significantly induced by the addition of increasing amounts of BAY 11-7085 (725.1 and 368.2% increases, respectively, at a concentration of 10 μM; Fig. 4A). BAY 11-7085 showed a stronger stimulatory effect on the apoptosis of ECSCs than that of NESC (Fig. 4B).

Effects of BAY 11-7085 on the cell cycle of ECSCs.

The effect of BAY 11-7085 on the cell cycle of ECSCs was determined by flow cytometry. As shown in Fig. 5, the culturing of ECSCs cultured for 2 days in the presence of BAY 11-7085 (10 μM) resulted in an accumulation of these cells in the G0/G1 phase of the cell cycle, with a concomitant decrease in the proportion of those in the S-phase. Similar results were obtained in all repeated experiments.

Effects of BAY 11-7085 on expression of apoptosis-related proteins in ECSCs.

To analyze the underlying mechanisms of the above-mentioned findings, we evaluated the expression of apoptosis-related proteins in ECSCs. As shown in Fig. 6, BAY 11-7085 downregulated the expression of Bcl-2 and Bcl-X_L proteins and upregulated the expression of cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9 proteins in ECSCs. The levels of Bax, Fas, and Fas ligand protein in ECSCs remained unchanged. Similar results were obtained in all repeated experiments.

DISCUSSION

It has been suggested that decreased apoptosis in endometriotic cells plays the essential role in the development and progression of endometriosis (15, 20). Recently, we (20) demonstrated that ECSCs are resistant to cytokine-induced apoptosis compared with NESCs. Izawa et al. (13) have also demonstrated that ECSCs are resistant to staurosporine-induced apoptosis; enhanced expression of antiapoptotic molecules was considered to be involved in this phenomenon (20). We have also demonstrated (18) that bufalin, an apoptosis-inducing agent, can be a promising drug for the medical treatment of endometriosis. In the present study, we demonstrated for the first time that BAY 11-7085 inhibits cell proliferation by inducing apoptosis and the G0/G1 arrest of the cell cycle of ECSCs in vitro. Western blot analysis clearly showed the downregulation of the expression of Bcl-2 and Bcl-X_L in ECSCs after BAY 11-7085 treatments, suggesting that BAY 11-7085 induces the apoptosis of ECSCs by suppressing the antiapoptotic proteins. We also observed the activation of caspase-8 and caspase-9, initiator caspases closely coupled to proapoptotic signals, and caspase-3, a downstream effector caspase, after BAY 11-7085 treatments, suggesting that these caspase-mediated cascades are involved in the mechanism of BAY 11-7085-induced apoptosis (22). Interestingly, BAY 11-7085 showed only weak effects on the cell proliferation and apoptosis of NESCs compared with those of ECSCs, suggesting that BAY 11-7085 has cell-specific effects on ECSCs and that its effects could be weaker on the normal endometrium. These results indicate that BAY 11-7085 could be used as a novel therapeutic agent for the medical treatment of endometriosis.

It has been suggested that NF-κB, JNK, and p38 MAPK may play significant roles in the proliferation of endometriotic lesions (29, 34). Therefore, it would be reasonable to hypothesize that BAY 11-7085 might stimulate the expression of a specific proapoptotic gene that eventually triggers the downstream signaling pathway, leading ECSCs to apoptotic cell death, via NF-κB-, JNK-, and/or p38 MAPK-mediated pathways (26, 30). Interestingly, Bcl-2 and Bcl-X_L proteins that were downregulated by BAY 11-7085 are under the transcriptional regulation of NF-κB (16), JNK (24), and p38 MAPK (24). Treatment with BAY 11-7085 results in the complementation and activation of the mitochondrial pathway and activation of the downstream regions of caspase-9 and -3, resulting in apoptosis. Therefore, BAY 11-7085 could enhance apoptosis and diminish antiapoptotic signaling in ECSCs. We speculated that higher constitutive expression of Bcl-2 and Bcl-X_L proteins in ECSCs than in NESCs might be involved in this phenomenon (18, 20). Further investigations are necessary to fully elucidate the specific action mechanisms of BAY 11-7085 on ECSCs.

Endometriosis is considered to be a local inflammatory-like phenomenon (11, 19). Inflammatory responses are now thought to be mediated by cytokines and chemokines, which are activated by the transcription factor NF-κB (1). NF-κB can be activated by different stimuli, including proinflammatory cytokines, which are considered to be important in the progression of endometriosis (11, 12, 19, 29). The endometriotic cells themselves may be an important source of cytokines in the peritoneal cavity (19, 29). Therefore, in addition to the induction of cell growth arrest and apoptosis, the control of NF-κB activation seems to be a powerful therapeutic strategy for preventing pathological responses in endometriosis as a consequence of the release of excessive amounts of inflammatory mediators.

Current and standard medical treatments for endometriosis include GnRH agonists, contraceptive steroids, progestogens, and androgens (27), all of which aim to reduce circulating estradiol concentrations. Progesterone, which is commonly used in the clinical management of endometriosis, can also suppress NF-κB activity by stimulating the synthesis of IκB and/or by inducing the binding of progesterone receptor and NF-κB subunits (35). Sakamoto et al. (29) showed that GnRH agonist, currently the most effective drug for the treatment of endometriosis, also inhibits the NF-κB pathway in endometriotic cells. Such inhibition of NF-κB appears to suppress the expression of critical downstream effectors, including cytokines, growth factors, and apoptosis-regulatory proteins. It has been reported that N-tosyl-l-phenylalanine chloromethyl ketone, a synthetic NF-κB inhibitor, decreases the interleukin-8 protein production and cell proliferation of endometriotic cells (12). These findings further support our hypothesis that drugs targeting the NF-κB pathway may be beneficial in the treatment of endometriosis.

In summary, we have demonstrated that BAY 11-7085 is able to induce apoptosis and the G0/G1-phase cell cycle arrest of ECSCs. Downregulation of Bcl-2 and Bcl-X_L expression with simultaneous upregulation of cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9 expression was induced by BAY 11-7085 treatment, suggesting that BAY 11-7085 may be applicable for the medical treatment of endometriosis as an adjuvant approach in combination with current medical treatment for this disease. Further studies with other inhibitors of NF-κB, JNK, or p38 MAPK on the cell proliferation and apoptosis of endometriotic cells may contribute to the establishment of more effective and sophisticated treatment strategies for endometriosis.

GRANTS

This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 16591672 to K. Nasu).

FOOTNOTES

• The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.