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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-0898
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 2 616-623
Copyright © 2007 by The Endocrine Society

A Novel Selective Progesterone Receptor Modulator Asoprisnil Activates Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Mediated Signaling Pathway in Cultured Human Uterine Leiomyoma Cells in the Absence of Comparable Effects on Myometrial Cells

Hiroko Sasaki, Noriyuki Ohara, Qin Xu, Jiayin Wang, Deborah A. DeManno, Kristof Chwalisz, Shigeki Yoshida and Takeshi Maruo

Department of Obstetrics and Gynecology (H.S., N.O., Q.X., J.W., S.Y., T.M.), Kobe University Graduate School of Medicine, Kobe 650-0017, Japan; and TAP Pharmaceutical Products Inc. (D.A.D., K.C.), Lake Forest, Illinois, 60045

Address all correspondence and requests for reprints to: Takeshi Maruo, M.D., Ph.D., Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, 7–5-1 Kusunoki-Cho, Chuo-Ku, Kobe 650-0017, Japan. E-mail: maruo{at}kobe-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: We previously demonstrated that asoprisnil, a selective progesterone receptor modulator, induces apoptosis of cultured uterine leiomyoma cells. This study was conducted to evaluate whether asoprisnil activates TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptotic pathway in cultured uterine leiomyoma and matching myometrial cells.

Objective and Methods: After subculture in phenol red-free DMEM supplemented with 10% fetal bovine serum for 120 h, cultured cells were stepped down to serum-free conditions for 24 h in the absence or presence of graded concentrations of asoprisnil. The levels of TRAIL signaling molecules and cellular inhibitors of apoptosis protein were assessed by Western blot analysis.

Results: TRAIL contents in untreated cultured leiomyoma cells were significantly (P < 0.01) lower compared with those in untreated cultured myometrial cells. There was no difference in death receptor (DR)4 and DR5 contents between the two types of cells. Asoprisnil treatment significantly (P < 0.05) increased TRAIL, DR4, and DR5 contents in cultured leiomyoma cells in a dose-dependent manner with a cleavage of caspase-8, -7, and -3, and decreased X-linked chromosome-linked inhibitor of apoptosis protein contents. In cultured myometrial cells, however, asoprisnil treatment did not affect either TRAIL signaling molecule or cellular inhibitors of apoptosis protein contents. The concomitant treatment with 100 ng/ml P4 significantly (P < 0.05) reversed asoprisnil-induced increase in DR4 and cleaved poly(adenosine 5'-diphosphate-ribose) polymerase contents in cultured leiomyoma cells.

Conclusions: These results suggest that asoprisnil induces apoptosis of cultured leiomyoma cells by activating TRAIL-mediated apoptotic pathway and down-regulating X-linked chromosome-linked inhibitor of apoptosis protein levels in the absence of comparable effects on myometrial cells.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEVERAL LINES OF evidence indicate that progesterone (P4) plays a critical role in regulating the growth of uterine leiomyomata (1, 2, 3). The diverse effects of P4 on target tissues are mediated by P4 receptor (PR), a nuclear receptor family of ligand-activated transcription factor (4). Asoprisnil (J867), a member of the novel class of 11ß-benzaldoxime-substituted selective progesterone receptor modulators, exerts tissue-selective P4 agonist, antagonist, or mixed agonist/antagonist effects on P4 target tissues in the in vivo studies (5, 6, 7). Recent clinical studies demonstrated that asoprisnil was effective in reducing the volume of leiomyoma, controlling menorrhagia, and improving pressure-related symptoms in patients with uterine leiomyomata (8, 9). We have recently reported that asoprisnil not only inhibits cell proliferation of cultured leiomyoma cells by down-regulating the expression of growth factors such as epidermal growth factor, IGF-I, and TGF-ß3, but also induces apoptosis of these cells by up-regulating cleaved caspase-3 and poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) and down-regulating Bcl-2 protein contents (10, 11).

Two major apoptotic signaling pathways are delineated to trigger apoptosis, including the intrinsic pathway mediated by the mitochondria and the extrinsic pathway mediated by death receptors on the cell surface (12). TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) is a type II transmembrane protein belonging to the TNF family of ligands that induce apoptosis of target cells through engagement of death receptors (DRs) (12, 13). TRAIL specifically exerts cytostatic or cytotoxic effects on various cancer cell lines while sparing most normal cells (14). TRAIL is believed to play a key role in suppressing tumor initiation and metastasis (15). The ligation of TRAIL to cell surface DR4 and DR5 results in trimerization of the receptors and clustering of a cytoplasmic death domain of receptors, leading to the formation of the death-inducing signaling complex. Trimerization of the death domains also leads to the recruitment of Fas-associated death domain, which in turn recruits and activates an initiator caspase-8 (12, 16). Activated caspase-8 cleaves and activates the downstream effector caspase-3, -6, and -7, and key death substrates such as PARP, thereby inducing apoptosis (12, 13). TRAIL gene was reported to be down-regulated in uterine leiomyomata tissues compared with normal myometrial tissues (17). However, it remains unknown whether asoprisnil activates TRAIL-mediated signaling pathway in cultured uterine leiomyoma cells.

In the present study, we investigated the effects of asoprisnil on the levels of TRAIL signaling molecules by Western blot analysis, including TRAIL, DR4, DR5, caspase-8, -7, and -3, and the inhibitors of apoptosis protein (IAPs) family such as X-linked chromosome-linked IAP (XIAP), cellular IAP-1 (c-IAP-1), and c-IAP-2 in cultured human uterine leiomyoma and matched myometrial cells. In addition, we examined the effects of P4 on the levels of TRAIL, DR4, DR5, and PARP in the absence or presence of asoprisnil in cultured leiomyoma cells.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue collection

After informed consent and approval by Kobe University Hospital Institutional Review Board was obtained, 16 independent uterine surgical specimens were collected from hysterectomies performed for leiomyomata in Japanese women with regular menstrual cycles. Uterine leiomyoma and the adjacent myometrial tissue samples were obtained from each hysterectomy specimen. The patients ranged in age from 35–45 yr, with a mean age of 39.4 yr, and had received no hormonal therapy for at least 6 months before surgery. Each uterine specimen was examined by a pathologist for histological evaluation. Samples were excluded from the study if accurate menstrual cycle dates could not be assigned or if unexpected pathology was found (e.g. adenomyosis). Endometrial tissues were obtained from the extirpated uterus, and the day of the menstrual cycle was determined by endometrial histological dating according to the method of Noyes et al. (18). Six samples were collected from the proliferative phase of the menstrual cycle, and 10 samples were from the secretory phase of the menstrual cycle.

Cell culture

Uterine leiomyoma tissues and the adjacent myometrium were obtained from the same individual uterus in the proliferative phase or secretory phase of the menstrual cycle. The central parts of leiomyoma tissues were collected with a careful removal of pseudo-capsules and fibrous septa materials. Tissues obtained were dissected from endometrial layers, cut into small pieces, and digested in 0.2% collagenase (wt/vol) at 37 C for 3 h (1). The collagenase treatment was shown to provide a pure population with smooth muscle cell characteristics without stromal or glandular epithelial cell contamination (1), and cultured leiomyoma cells were confirmed to be positive for the muscle-specific protein, desmin, and negative for cytoskeletal protein specific for epithelial cells, cytokeratin 19, by immunocytochemistry (1). The leiomyoma cells and myometrial cells were collected by centrifugation at 460 x g for 5 min and washed three times with phosphate-buffered saline containing 1% antibiotic solution, respectively. Cell viability was determined by trypan blue exclusion test. The isolated leiomyoma cells and myometrial cells were plated at densities of approximately 1 x 104/well in 96-well tissue culture plates. The isolated leiomyoma and myometrial cells in tissue culture plates were subcultured at 37 C for 120 h in a humidified atmosphere of 5% CO2 –95% air in phenol red-free DMEM supplemented with 10% fetal bovine serum (vol/vol; Invitrogen, Life Technologies, Inc., Grand Island, NY). Because the proliferating cell nuclear antigen-positive rate of leiomyoma cells was shown to be higher in the secretory phase than in the proliferative phase of the menstrual cycle (2), we subcultured isolated cells in phenol red-free DMEM supplemented with 10% fetal bovine serum to abrogate the menstrual cycle-dependent influence on the biological characteristics of cells, confirming that the subculture produced no differences in the proliferating cell nuclear antigen-positive rate of cultured leiomyoma cells obtained from the different menstrual phases (2). The monolayer cultures reaching approximately 70% confluence were stepped down to serum-free conditions for an additional 4, 8, and 24 h in the absence or presence of graded concentrations (10–8, 10–7, 10–6 M) of asoprisnil (TAP Pharmaceutical Products Inc., Lake Forest, IL). Asoprisnil was dissolved in absolute ethanol. Final concentration of ethanol in culture medium was less than 0.01%, and the same concentration of ethanol was used as a vehicle in control cultures.

Western blot analysis for TRAIL signaling molecules and c-IAPs

Western blot analysis was performed as described previously (2). At the termination of cultures, cultured leiomyoma and myometrial cells were incubated at 4 C for 5 min in the presence of lysis buffer consisting of 150 mM/liter NaCl, 2 mM phenylmethylsulfonyl fluoride, 1% Nonidet P-40, 0.5% deoxycholate, 1 mg/liter aprotinin, 0.1% sodium dodecyl sulfate, and 50 mM Tris-HCl (pH 7.5). Cells were subsequently scraped off the plates. The lysates were centrifuged at 13,000 x g for 5 min and the supernatants were collected. Protein quantity was evaluated by the Bradford assay. Each 60-µg aliquot of the proteins extracted from cultured cells was electrophoresed on a 10% SDS-PAGE under reducing conditions. The proteins were electrotransferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA).

For the detection of TRAIL signaling molecule proteins, blots were incubated with a goat polyclonal antibody against TRAIL (sc-6079; Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:200); goat polyclonal antibodies against DR4 (sc-6823, Santa Cruz Biotechnology; dilution 1:200), DR5 (sc-7191, Santa Cruz Biotechnology; dilution 1:200), and caspase-8 (sc-6135, Santa Cruz Biotechnology; dilution 1:100); a mouse monoclonal antibody against caspase-7 (MAB823, R&D systems, Minneapolis, MN; dilution 1:100); and a rabbit monoclonal antibody against caspase-3 (MAB835, R&D systems; diluted to 1.0 µg/ml). For the detection of the c-IAP family proteins, blots were incubated with a mouse monoclonal antibody against XIAP (MAB822, R&D Systems; diluted to 4.0 µg/ml), a rabbit polyclonal antibody against c-IAP-1 (sc-7943, Santa Cruz Biotechnology; dilution 1:100), and a goat polyclonal antibody against c-IAP-2 (sc-1957, Santa Cruz Biotechnology; dilution 1:100). In addition, blots were incubated with a rabbit polyclonal antibody against PARP (no. 9542; Cell Signaling Technology, Livermore, CA; dilution 1:500) and a mouse monoclonal antibody against pan-actin (Clone ACTN05, Lab Vision Corp, Fremont, CA; dilution 1:500). The membranes were incubated overnight with horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences, Arlington Heights, IL). Antigen-antibody complexes were detected with the ECL chemiluminescence detection system (Amersham Biosciences). Membranes were visualized by exposure to X-OMAT film (Eastman Kodak Co., Rochester, NY). The radioautograms were scanned and quantified with ChemiImager 4400 (Astec Co. Ltd, Osaka, Japan). The experiments were repeated with at least three independent cultured specimens and similar results were obtained; the reported results are representative.

Statistical analysis

The data were expressed as the mean ± SD from at least three independent experiments. Statistical significance was determined using Student’s t test and two-way ANOVA. A difference with a P < 0.05 was considered statistically significant.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The levels of TRAIL, DR4, and DR5 in untreated cultured leiomyoma and myometrial cells

We examined the basal TRAIL, DR4, and DR5 contents in cultured leiomyoma cells and myometrial cells by Western blot analysis after subculture of both cells in phenol red-free DMEM supplemented with 10% fetal bovine serum for 120 h (Fig. 1Go). There were no significant differences in TRAIL, DR4, and DR5 contents in untreated leiomyoma and myometrial cells between cultured cells obtained from the proliferative phase and those from the secretory phase of the menstrual cycle (data not shown). TRAIL contents in untreated cultured leiomyoma cells was significantly (P < 0.01) lower compared with those in untreated cultured myometrial cells. However, there were no significant differences in DR4 and DR5 contents between the two types of cells.


Figure 1
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  		FIG. 1. The levels of TRAIL, DR4, and DR5 in untreated cultured leiomyoma and myometrial cells. TRAIL contents in untreated cultured leiomyoma cells were significantly lower compared with those in untreated cultured myometrial cells, whereas no significant differences in DR4 and DR5 contents were noted between them. Densitometric analysis of TRAIL, DR4, and DR5 was performed as described in Materials and Methods. Pan-actin was used to ensure the even loading of each specimen. Results represent the mean ± SD of the fold increase over the control value of at least three independent experiments performed in triplicate. *, P < 0.01 vs. untreated cultured myometrial cells.

 
Effects of graded concentrations of asoprisnil on the levels of TRAIL, DR4, and DR5 in cultured leiomyoma and myometrial cells.

Two-way ANOVA of the indexes for TRAIL and DR4 assessed by Western blot analysis demonstrated significant effects of asoprisnil in cultured leiomyoma cells (P < 0.001, TRAIL; P < 0.01, DR4) (Fig. 2Go).


Figure 2
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  		FIG. 2. Effects of graded concentrations of asoprisnil on the levels of TRAIL, DR4, and DR5 in cultured leiomyoma and myometrial cells. In cultured leiomyoma cells, treatment with asoprisnil at concentrations greater than or equal to 10–7 M significantly increased TRAIL contents at 8 and 24 h compared with untreated control cultures (upper panel). Treatment with 10–6 M asoprisnil significantly increased DR4 and DR5 contents in these cells at 4 h, and treatment with asoprisnil at concentrations greater than or equal to 10–7 M significantly increased DR4 and DR5 contents at 8 and 24 h (upper panel). However, asoprisnil treatment did not affect TRAIL, DR4, and DR5 contents in cultured myometrial cells (lower panel). Densitometric analysis of TRAIL, DR4, and DR5 was performed as described in Materials and Methods. Pan-actin was used to ensure the even loading of each specimen. Results represent the mean ± SD of the fold increase over the control value of at least three independent experiments performed in triplicate. *, P < 0.05 vs. untreated control cultures at each experiment time.

 
TRAIL contents in cultured leiomyoma cells were significantly (P < 0.05) increased by the treatment with asoprisnil at concentrations greater than or equal to 10–7 M at 8 h and 24 h compared with untreated control cultures (Fig. 2Go, upper panel). DR4 and DR5 contents in cultured leiomyoma cells were also significantly (P < 0.05) increased by the treatment with 10–6 M asoprisnil at 4 h and by the treatment with asoprisnil at concentrations greater than or equal to 10–7 M at 8 h and 24 h compared with untreated control cultures (Fig. 2Go). In cultured myometrial cells, asoprisnil treatment did not affect TRAIL, DR4, and DR5 protein contents (Fig. 2Go, lower panel).

Effects of graded concentrations of asoprisnil on the levels of cleaved caspase-8, -7, and -3 in cultured leiomyoma and myometrial cells

Two-way ANOVA of the indexes for cleaved caspases assessed by Western blot analysis demonstrated significant effects of asoprisnil in cultured leiomyoma cells (P < 0.05, cleaved caspase-8; P < 0.001, cleaved caspase-7; P < 0.05, cleaved caspase-3) (Fig. 3Go).


Figure 3
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  		FIG. 3. Effects of graded concentrations of asoprisnil on the levels of cleaved caspase-8, -7, and -3 in cultured leiomyoma and myometrial cells. Compared with untreated control cultures, treatment with 10–6 M asoprisnil significantly increased cleaved caspase-8 contents in cultured leiomyoma cells at 4 and 8 h, and treatment with asoprisnil at concentrations greater than or equal to 10–7 M significantly increased cleaved caspase-8 contents in these cells at 24 h (upper panel). Treatment with asoprisnil at concentrations greater than or equal to 10–8 M significantly increased cleaved caspase-7 in cultured leiomyoma cells at 24 h, and treatment with 10–8 and 10–7 M asoprisnil significantly increased cleaved caspase-3 in these cells at 24 h (upper panel). In cultured myometrial cells, however, asoprisnil treatment had no apparent effects on the levels of cleaved caspases (lower panel). Densitometric analysis of cleaved caspase-8, -7, and -3 was performed as described in Materials and Methods. Pan-actin was used to ensure the even loading of each specimen. Results represent the mean ± SD of the fold increase over the control value of at least three independent experiments performed in triplicate. *, P < 0.05 vs. untreated control cultures at each experiment time.

 
Cleaved caspase-8 contents in cultured leiomyoma cells were significantly (P < 0.05) increased by the treatment with 10–6 M asoprisnil at 4 h and 8 h and by the treatment with asoprisnil at concentrations greater than or equal to 10–7 M at 24 h compared with untreated control cultures (Fig. 3Go, upper panel).

Furthermore, cleaved caspase-7 contents in cultured leiomyoma cells were also significantly (P < 0.05) increased by the treatment with asoprisnil at concentrations greater than or equal to 10–8 M at 24 h, whereas cleaved caspase-3 contents in cultured leiomyoma cells were significantly (P < 0.05) increased by the treatment with either 10–8 or 10–7 M asoprisnil at 24 h compared with untreated control cultures (Fig. 3Go). In cultured myometrial cells, asoprisnil treatment had no apparent effects on the levels of cleaved caspases (Fig. 3Go, lower panel).

P4 prevention of asoprisnil-induced TRAIL-mediated molecules and apoptosis in cultured leiomyoma cells

The effects of physiological tissue concentration of P4 (100 ng/ml) on TRAIL, DR4, and DR5 contents in cultured leiomyoma cells at 8-h treatment and cleaved PARP contents at 24-h treatment in the absence or presence of 10–7 M asoprisnil were assessed by Western blot analysis (Fig. 4Go). Asoprisnil treatment increased TRAIL, DR4, and DR5 contents in cultured leiomyoma cells compared with untreated control cultures, whereas the concomitant treatment with P4 significantly (P < 0.05) antagonized asoprisnil-induced up-regulation of DR4 and cleaved PARP contents in cultured leiomyoma cells. The concomitant treatment with P4 slightly reversed asoprisnil-induced increase in TRAIL and DR5 contents in cultured leiomyoma cells, but did not reach the statistical significance.


Figure 4
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  		FIG. 4. P4 prevention of asoprisnil-induced TRAIL-mediated molecules and apoptosis in cultured leiomyoma cells. The concomitant treatment with P4 significantly antagonized asoprisnil-induced up-regulation of DR4 contents at 8 h and cleaved PARP contents at 24 h in cultured leiomyoma cells. Densitometric analysis of TRAIL, DR4, DR5, and cleaved PARP was performed as described in Materials and Methods. Pan-actin was used to ensure the even loading of each specimen. Results represent the mean ± SD of the fold increase over the control value of at least three independent experiments performed in triplicate. *, P < 0.05 vs. leiomyoma cells treated with asoprisnil alone.

 
Effects of graded concentrations of asoprisnil on the levels of XIAP, c-IAP-1, and c-IAP-2 in cultured leiomyoma and myometrial cells

Compared with untreated control cultures, XIAP contents in cultured leiomyoma cells were significantly (P < 0.05) decreased at 4 h by the treatment with 10–6 M asoprisnil (Fig. 5Go, upper panel). Treatment with asoprisnil at concentrations greater than or equal to 10–7 M showed somewhat decreases in XIAP and c-IAP-1 contents in the cells at 8 h, but did not reach the statistical significance (Fig. 5Go). c-IAP-2 contents in cultured leiomyoma cells were not affected by the treatment with asoprisnil (Fig. 5Go). In cultured myometrial cells, asoprisnil treatment had no apparent effects on XIAP c-IAP-1, and c-IAP-2 contents (Fig. 5Go, lower panel).


Figure 5
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  		FIG. 5. Effects of graded concentrations of asoprisnil on the levels of XIAP, c-IAP-1, and c-IAP-2 in cultured leiomyoma and myometrial cells. In cultured leiomyoma cells, treatment with 10–6 M asoprisnil significantly decreased XIAP contents at 4 h (upper panel), but asoprisnil treatment did not affect c-IAP-1 and c-IAP-2 contents (upper panel). In cultured myometrial cells, asoprisnil treatment had no apparent effects on XIAP, c-IAP-1, and c-IAP-2 contents (lower panel). Densitometric analysis of XIAP, c-IAP-1, and c-IAP-2 was performed as described in Materials and Methods. Pan-actin was used to ensure the even loading of each specimen. Results represent the mean ± SD of the fold increase over the control value of at least three independent experiments performed in triplicate. *, P < 0.05 vs. untreated control cultures at each experiment time.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrated that TRAIL and DR proteins are expressed in untreated cultured leiomyoma and myometrial cells. TRAIL contents in untreated cultured leiomyoma cells were significantly lower compared with untreated cultured myometrial cells, but there were no significant differences in DR4 and DR5 contents between the two types of cells. This suggests that the two types of cells may differently respond to apoptotic stimuli. Compared with cultured myometrial cells, cultured leiomyoma cells may be more resistant to TRAIL-mediated apoptosis. However, treatment with asoprisnil induced a marked increase in TRAIL, DR4, and DR5 contents in cultured leiomyoma cells with the cleavage of caspase-8, -7, and -3, and key death substance, PARP. By contrast, asoprisnil treatment did not affect TRAIL, DR4, DR5, and cleaved caspase contents in cultured myometrial cells. Because TRAIL-mediated apoptosis is characterized by the activation of caspase-8, -7, -3, and -9 (19), and because PARP is a hallmark for the apoptotic paradigm (20), our results indicate that asoprisnil activates TRAIL-mediated signaling pathway and induce TRAIL-mediated apoptosis in cultured leiomyoma cells in a cell-type specific manner.

The concomitant treatment with P4 (100 ng/ml) counteracted an asoprisnil-induced increases in DR4 and cleaved PARP levels in cultured leiomyoma cells, suggesting that asoprisnil may exhibit a PR-antagonistic action. The concentrations of P4 used in the present study seem to be within the range of physiological tissue concentrations, because P4 levels in human myometrium and leiomyoma tissues were as high as 10–70 ng/g protein (21).

Several factors are involved in the regulation of DR expression in addition to TRAIL, which binds and activates DRs (13). Up-regulation of DR4 and DR5 was reported to enhance TRAIL-induced apoptosis in breast carcinoma treated with chemotherapeutic drugs in a p53-dependent manner (22, 23, 24, 25). In our previous study, however, P4 treatment did not affect p53 protein contents in cultured leiomyoma cells (26), suggesting that asoprisnil may modulate DR levels in these cells through a p53-independent manner. Multiple factors have been reported to up-regulate DR expression in a p53-independent pathway, including nuclear factor {kappa}B (NF{kappa}B) (27), activator protein 1 (28), a MAPK/ERK (MEK)-dependent pathway (29), and a c-Jun NH2-terminal kinase-mediated pathway (30). However, the precise mechanisms by which asoprisnil up-regulates DR levels in cultured leiomyoma cells still remain to be elucidated.

The c-IAPs are a family of antiapoptotic proteins characterized by baculoviral IAP repeat domains that bind and directly inhibit caspase-3, -7, and/or -9 (31). XIAP is the most potent apoptosis inhibitory protein among the c-IAPs (32). In the present study, asoprisnil treatment significantly decreased XIAP contents, but not c-IAP-1 or c-IAP-2 contents in cultured leiomyoma cells. Our results are in agreement with a report by Zhang et al. (33), who reported that XIAP was down-regulated during TRAIL-induced apoptosis in melanoma cell lines. Several studies demonstrated that the inhibition of XIAP expression by an antisense XIAP enhanced TRAIL potency in prostate cancer cells (34), whereas overexpression of XIAP attenuated TRAIL-induced apoptosis (35). Shi et al. (36) have reported that TRAIL promotes XIAP ubiquitination and proteasomal degradation in cancer cells, thereby resulting in the removal of the blockage on caspases and enhancement of apoptosis. Collectively, it seems reasonable to speculate that the down-regulation of XIAP contents in response to asoprisnil treatment may render cultured leiomyoma cells susceptible to TRAIL-mediated apoptosis.

The relative contribution of DR vs. mitochondrial pathway to apoptosis reflects the existence of two different cell types with respect to TRAIL signaling (12). In type I cells, activation of caspase-8 is sufficient for executing apoptosis, whereas in type II cells, amplication of the mitochondrial pathway is required through the cleavage of Bid by caspase-8 (16). The translocation of truncated Bid to the mitochondria promotes the release of cytochrome c and second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pl (Smac/DIABLO) from the mitochondria, thereby initiating a mitochondrial amplification loop (12). Smac/DIABLO promotes the activation of caspase-3 by eliminating the inhibitory effect of the c-IAPs on caspases (37), while Bcl-2 inhibits the release of Smac/DIABLO (38). We have demonstrated that P4 treatment augments Bcl-2 protein contents in cultured leiomyoma cells (1), whereas asoprisnil treatment down-regulates Bcl-2 protein contents in those cells (10). This suggests that leiomyoma cells may belong to the type II cells and that asoprisnil treatment may interfere with the antiapoptotic action of XIAP through interaction with the mitochondrial pathway. Although we have not examined yet Smac/DIABLO levels in cultured leiomyoma cells treated with asoprisnil, it cannot be excluded that an increased Smac/DIABLO release may suppress the inhibitory effects of XIAP on caspases in those cells.

TRAIL induces apoptosis in cancer cells without damaging most normal cells. However, the mechanisms underlying the differential effects of asoprisnil on the levels of TRAIL signaling molecules and XIAP between cultured leiomyoma and myometrial cells remain to be determined. Several factors have been described to influence TRAIL sensitivity in normal and malignant cells, including the relative number of decoy receptors (or DRs), mutations of caspase-8, FLICE-inhibitory protein (FLIP) levels, Akt activity, and NF{kappa}B activity (39). FLIP is an endogenous inhibitor of caspase-8 in TRAIL-induced apoptosis. c-FLIP splice variants include c-FLIPS and c-FLIPL. It has been speculated that c-FLIPS prevents an activation of caspase-8 (40), and that c-FLIPL inhibits DR-mediated apoptosis (41). Thus, asoprisnil might down-regulate c-FLIPS and/or c-FLIPL contents at the DISC in cultured leiomyoma cells, resulting in the promoted cleavage of caspase-8. Further study is necessary to elucidate the effects of asoprisnil on FLIP contents in cultured leiomyoma and myometrial cells.

The different responses of cultured leiomyoma cells and myometrial cells to asoprisnil might be due, at least in part, to the difference in PR isoform contents (10). PR exists as two isoforms, PR-A and PR-B (42), which have different transcriptional activities (43, 44, 45). PR-B functions as a transcriptional activator of P4-responsive genes, whereas PR-A functions as a ligand-dependent repressor of PR-B transcriptional activity (44). We have recently demonstrated that a PR modulator CDB-2914 increased PR-A contents, but decreased PR-B contents in cultured leiomyoma cells, whereas no changes in PR isoform contents were observed in cultured myometrial cells treated with CDB-2914 (46). Based on the similar effects of asoprisnil on apoptosis of cultured leiomyoma cells as observed with CDB2914, it seems likely that asoprisnil induces similar changes in PR isoform contents as CDB-2914 in cultured leiomyoma cells. Another possible explanation is the difference in the milieu of transcriptional cofactors between the two types of cells because the complex of liganded receptor and the balance of coactivators and corepressors impart cell-specific effects of selective progesterone receptor modulators (47). Coactivators enhance transcriptional activity, whereas corepressors elicit inhibitory effects on nuclear receptors (5). P4 agonists promote the interactions of nuclear receptors with coactivators, whereas P4 antagonist favors the interactions with corepressors or inhibits interactions with coactivators (5). The relative balance of coactivator and corepressor expression determines the relative agonist vs. antagonist activity of selective progesterone receptor modulators (47, 48). Giangrande et al. (49) demonstrated that antagonist-bound PR-A has a higher affinity for corepressors than antagonist-bound PR-B and that unlike PR-B, PR-A did not associate with coactivators. Given an antagonistic action of asoprisnil in cultured leiomyoma cells, asoprisnil treatment may interact with corepressors with an inhibition of the recruitment of coactivators, thereby suppressing the PR-mediated gene transcription. The differences in PR isoform and coregulator levels might contribute to the different responses to asoprisnil between the two types of cells. Taken together, asoprisnil activates TRAIL-mediated apoptotic pathway in cultured leiomyoma cells in a cell-type specific manner probably through exerting a PR-antagonistic action.

In conclusion, the present study demonstrates that asoprisnil triggers TRAIL-mediated apoptosis in cultured leiomyoma cells by up-regulating the levels of TRAIL, DR4, DR5, cleaved caspase-8, -7, and -3 levels, and by down-regulating XIAP levels in the absence of comparable effects on matching myometrial cells. These data suggest that asoprisnil may be useful in a novel strategy for the treatment of uterine leiomyomas.


   Footnotes
 
This work was supported by Grant-in-Aid for Scientific Research 1437053 from the Japanese Ministry of Education, Science and Culture (from July 2002 to March 2005) and by the Ogyaa-Donation Foundation of the Japan Association of Obstetricians and Gynecologists (from April 2004 to March 2006), and by TAP Pharmaceutical Products Inc. (from March 2005 to August 2006).

H.S., N.O., Q.X., J.W., and S.Y have nothing to declare. D.A.D. and K.C., who are employed by TAP Pharmaceutical Products Inc., supplied asoprisnil (J867) to T.M.

First Published Online November 14, 2006

Abbreviations: c-IAP, Cellular IAP; DR, death receptor; FLIP, FLICE-inhibitory protein; IAP, inhibitor of apoptosis protein; P4, progesterone; PR, P4 receptor; PARP, poly(adenosine 5'-diphosphate-ribose) polymerase; TRAIL, TNF-related apoptosis-inducing ligand; XIAP, X-linked chromosome-linked inhibitor of apoptosis protein.

Received April 26, 2006.

Accepted November 7, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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