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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-2379
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 4 1296-1304
Copyright © 2006 by The Endocrine Society

A Novel Selective Progesterone Receptor Modulator Asoprisnil (J867) Inhibits Proliferation and Induces Apoptosis in Cultured Human Uterine Leiomyoma Cells in the Absence of Comparable Effects on Myometrial Cells

Wei Chen, Noriyuki Ohara, Jiayin Wang, Qin Xu, Jin Liu, Akira Morikawa, Hiroko Sasaki, Shigeki Yoshida, Deborah A. Demanno, Kristof Chwalisz and Takeshi Maruo

Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine (W.C., N.O., J.W., Q.X., J.L., A.M., H.S., S.Y., T.M.), 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: Dr. Takeshi Maruo, 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: Asoprisnil, a selective progesterone (P4) receptor (PR) modulator (SPRM) with mixed P4 agonist/antagonist activities, reduces uterine leiomyoma volume in a dose-dependent manner in the presence of follicular phase estrogen concentrations. The evidence from clinical studies suggests that asoprisnil may directly target the uterine leiomyomata.

Objective and Methods: The present study evaluated the effects of asoprisnil on cell proliferation, the expression of apoptosis-related proteins, and apoptosis in cultured human uterine leiomyoma cells and matched normal myometrial cells. PR-A and PR-B expression in the two types of cells was comparatively evaluated. Cell proliferation, proliferating cell nuclear antigen (PCNA)-positive rate, and TUNEL-positive rate were assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, immunocytochemistry, and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) assay, respectively. The expression of apoptosis-related proteins and PR was assessed by Western blot analysis.

Results: Compared with untreated cultures, asoprisnil decreased the number of viable cultured cells, the PCNA-positive rate, and PCNA protein expression in cultured leiomyoma cells. Asoprisnil increased the TUNEL-positive rate, cleaved caspase-3, and cleaved poly(adenosine 5'-diphosphate-ribose) polymerase expression and decreased Bcl-2 protein expression in cultured leiomyoma cells. These effects were dose and time dependent. In cultured myometrial cells, however, asoprisnil did not affect cell proliferation and apoptosis. PR-B expression was elevated in cultured leiomyoma cells compared with cultured myometrial cells, whereas no differences in PR-A expression were noted between the two cell types.

Conclusions: These results show that asoprisnil inhibits proliferation and induces apoptosis in cultured uterine leiomyoma cells in the absence of comparable effects on cultured normal myometrial cells, suggesting a cell type-specific effect.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ACCUMULATING DATA SUPPORT the concept that progesterone (P4) plays a vital role in the growth of uterine leiomyomata (1). The effects of P4 on target tissues are mediated by the P4 receptor (PR), which belongs to the nuclear receptor family. PR functions as a ligand-activated transcription factor to regulate the expression of target genes (2). PR exists as two isoforms, PR-A and PR-B, which are transcribed from two promoters on a single gene (3). The two PR isoforms have distinct functions (4). PR-B functions as a transcriptional activator of P4-responsive genes, whereas PR-A may function as a transcriptional inhibitor (4, 5). Several studies have demonstrated that PR is up-regulated in uterine leiomyomata compared with adjacent normal myometrium (6, 7, 8) at mRNA and protein levels. Evidence from clinical studies suggests that synthetic progestins stimulate leiomyoma growth (9, 10, 11), whereas the P4 antagonist RU 486 has opposite effects (12, 13, 14). Collectively, these data suggest that PR may act to promote leiomyoma growth.

Asoprisnil (benzaldehyde, 4-[(11, 17)-17-methoxy-17-(methoxymethyl)-3-oxoestra-4,9-dien-11-yl]-1-oxime) belongs to a novel class of 11ß-benzaldoxime-substituted selective PR modulators (SPRMs). SPRMs are PR ligands that exert clinically relevant, tissue-selective P4 agonist, antagonist, partial, or mixed agonist/antagonist effects on various P4 target tissues in an in vivo situation depending on the biological action studied (15). Asoprisnil shows mixed P4 agonist/antagonist and tissue-selective effects in various animal models (16, 17). It has high PR specificity with no antiglucocorticoid and marginal androgenic effects in humans (15). Asoprisnil shows a high degree of selectivity in the endometrium of animals and humans (15, 16, 17). In premenopausal women, asoprisnil is reported to suppress menstruation and induce mixed P4 agonist/antagonist effects on the glandular epithelium, stroma, and clusters of thick-walled arterial vessels in the endometrium without showing the endometrial hyperplasia indicative of unopposed estrogen effects (18). Asoprisnil reached an advanced stage of clinical development for the treatment of uterine leiomyomata (19, 20) and endometriosis (21). Recent clinical studies with asoprisnil in patients with uterine leiomyomas demonstrated that asoprisnil reduces uterine volume and myoma volume in a dose- and time-dependent manner and improves leiomyoma pressure symptoms (19, 20). It also suppressed the duration and intensity of uterine bleeding. These effects were observed in the presence of follicular phase estrogen concentrations. Although the clinical evidence suggests that asoprisnil may exert a direct effect on uterine leiomyoma, the precise mechanism underlying the action of asoprisnil on uterine leiomyomata and normal myometrium remains to be elucidated. Recently, we demonstrated that the P4 antagonist CDB-2914 inhibits the proliferation of cultured human leiomyoma cells and induces apoptosis of those cells (22). However, SPRMs have not yet been studied in these cells.

In the present study we evaluated the effects of asoprisnil on proliferation and apoptosis in cultured human uterine leiomyoma cells and normal myometrial cells using different end points.


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

Twenty samples of uterine leiomyoma tissues and adjacent normal myometrium were obtained from Japanese women with regular menstrual cycles who underwent hysterectomy for uterine leiomyomas at Kobe University Hospital. Informed consent was obtained from each patient before surgery for the use of uterine leiomyoma and myometrial tissues for the present study. The institutional review board approved the use of uterine leiomyoma and myometrial tissues for culture experiments. The patients ranged in age from 33–45 yr, with a mean age of 39 yr, and had received no hormonal therapy for at least 6 months before surgery. The histological diagnosis of each uterine specimen was examined. Samples were excluded from the study if accurate menstrual cycle dates could not be assigned or if unexpected pathology was found (e.g. adenomyosis). Thirteen samples were collected from the proliferative phase of the menstrual cycle, and seven samples were obtained from the secretory phase of the menstrual cycle.

Cell culture

Uterine leiomyoma tissues and adjacent normal myometrium were obtained from the same individual uterus in the proliferative or secretory phase of the menstrual cycle, dissected from endometrial layers, cut into small pieces, and digested in 0.2% collagenase (wt/vol) at 37 C for 3–5 h (23). The collagenase treatment was shown to provide a pure population with smooth muscle cell characteristics without stromal or glandular epithelial cell contamination (23). Different types of primary cultures were used for different experiments. The leiomyoma cells and normal myometrial cells were collected by centrifugation at 460 x g for 5 min and washed three times with PBS containing 1% antibiotic solution, respectively. Cell viability was determined by trypan blue exclusion test. The isolated leiomyoma cells and normal myometrial cells were plated at densities of approximately 1 x 106 cells/dish in 10-cm2 culture dishes, 4 x 104 cells/well in two-well chamber glass slides, and 1 x 104/well in 96-well tissue culture plates. The isolated leiomyoma cells and normal myometrial cells in culture dishes and two-well chamber slides 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). The isolated leiomyoma cells and normal myometrial cells in 96-well tissue culture plates were subcultured for 72 h under the conditions described above. The monolayer cultures reaching approximately 70% confluence were treated with graded concentrations (10–8, 10–7, and 10–6 M) of asoprisnil (J867, TAP Pharmaceutical Products, Inc., Lake Forest, IL) in serum-free, phenol red-free DMEM for 24, 48, and 72 h. Asoprisnil was dissolved in absolute ethanol. The 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.

Cell proliferation assay

The cell viabilities of cultured leiomyoma cells and normal myometrial cells were colorimetrically determined using a cell proliferation assay kit (CellTiter 96 AQueous One Solution Cell Proliferation assay, Promega Corp., Madison, WI). After being treated in the absence or presence of graded concentrations (10–8, 10–7, and 10–6 M) of asoprisnil in serum-free DMEM for 24, 48, and 72 h in a 96-well tissue culture plate, 20 µl CellTiter 96 AQueous One Solution Reagent containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and phenazine ethosulfate was added to each well, and cultured cells were incubated at 37 C in a humidified, 5% CO2 atmosphere for 4 h. The absorbance of soluble formazan produced by cellular reduction of the MTS was measured at 490 nm using an MTP-120 ELISA plate reader (Corona Electric Co., Osaka, Japan). The MTS tetrazolium compound is bioreduced by cells into formazan product by reduced nicotinamide adenine dinucleotide phosphate or reduced nicotinamide adenine dinucleotide produced by dehydrogenase enzymes in metabolically active cells (24). The quantity of formazan product, as measured by the amount of 490-nm absorbance, is directly proportional to the number of living cells in culture. Experiments were performed in triplicate. Results were expressed as the OD at 490 nm.

Immunocytochemical staining for PCNA

The leiomyoma cells and normal myometrial cells cultured in two-well chamber slides were washed three times with PBS, fixed in methanol at 4 C for 20 min, and again washed with PBS three times. The fixed cells were subjected to immunostaining by the avidin/biotin immunoperoxidase method using a polyvalent immunoperoxidase kit (Omnitags, Lipshow, MI) according to the manufacturer’s instructions. A mouse monoclonal antibody to human proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used as the primary antibody at a dilution of 1:80. To assure the specificity of the immunological reaction, cultured cells were subjected to the same immunoperoxidase method, except that the primary antibody was replaced by nonimmune murine IgG (Miles, Erkhardt, IN) at the same dilution as the specific antibody. The replacement of the specific primary antibody with nonimmune murine IgG resulted in a lack of positive immunostaining for PCNA. Immunocytochemical staining was analyzed by two investigators in a blinded fashion without knowledge of the experimental group. The PCNA-positive rate was determined by observing more than 1000 nuclei for each experimental sample and was used for evaluating the proliferating activity of cultured leiomyoma cells and normal myometrial cells.

In situ terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) assay

In situ labeling of fragmented DNA in cultured leiomyoma cells and normal myometrial cells was performed with the TUNEL assay, using the ApopTag in situ apoptosis detection kit (Intergen Co., Purchase, NY) according to the manufacturer’s protocol for monolayer cultures. The leiomyoma cells and normal myometrial cells were subcultured in two-well glass chamber slides for 120 h, then cultured under serum deprivation conditions for 24 and 48 h in the absence or presence of graded concentrations of asoprisnil. At the termination of cultures, nucleotide-sized DNA fragments were tailed with digoxigenin-deoxy-UTP and then bound with peroxidase-conjugated antidigoxigenin antibodies. The nuclei were counterstained with hematoxylin (Zymed Laboratories, Inc., San Francisco, CA) for determining the TUNEL-positive rate of cultured leiomyoma cells and normal myometrial cells. The TUNEL-positive rate of cultured leiomyoma cells and normal myometrial cells was analyzed by two investigators in a blinded fashion without knowledge of the experimental group. All stained nuclei were scored as positive for TUNEL. The TUNEL-positive rate was determined by observing more than 1000 nuclei for each experimental sample.

Western blot analysis for PCNA, Bcl-2, cleaved caspase-3, and cleaved poly(adenosine 5'-diphosphate-ribose) polymerase (PARP)

Proteins were extracted from cultured leiomyoma cells and normal myometrial cells. At the termination of cultures, cells were lysed at 4 C for 20 min in the presence of a lysis buffer consisting of 150 mM NaCl, 2 mM phenylmethylsulfonylfluoride, 1% Nonidet P-40, 0.5% deoxycholate, 1 mg/liter aprotinin, 0.1% sodium dodecyl sulfate, and 50 mM Tris-HCl (pH 7.5). The lysates were subsequently centrifuged at 13,000 x g for 30 min at 4 C, and the supernatants were collected. Protein content in the supernatants was determined by the Bradford assay (25). Each 150-µg aliquot of the proteins extracted from cultured leiomyoma cells and normal myometrial cells was electrophoresed on 10% SDS-PAGE under reducing conditions. The proteins were then electrophoretically transferred from gels to nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA). The blots were exposed overnight to a mouse monoclonal antibody to PCNA (Santa Cruz Biotechnology, Inc.), a mouse monoclonal antibody to Bcl-2 (Santa Cruz Biotechnology, Inc.), a rabbit polyclonal antibody to caspase-3 that recognizes procaspase and active caspase (Cell Signaling Technology, Inc., Livermore, CA), a rabbit polyclonal antibody to PARP (Cell Signaling Technology, Inc.), and a mouse monoclonal antibody to PR (Santa Cruz Biotechnology, Inc.) at dilutions of 1:200, 1:200, 1:1000, 1:1000, and 1:500, respectively. The membranes were incubated for 1 h with horseradish peroxidase-conjugated antimouse or antirabbit secondary antibody (Amersham Biosciences, Arlington Heights, IL) that was diluted at 1:1000 with blocking buffer. The 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 then scanned and quantified with ChemiImager 4400 (Astec Co. Ltd., Osaka, Japan). The experiments were repeated with at least six different 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 six independent experiments. Statistical significance was determined using Student’s t test and one- or two-way ANOVA. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of graded concentrations of asoprisnil on the number of viable cultured leiomyoma cells and normal myometrial cells

Comparative effects of treatment with graded concentrations of asoprisnil on the number of viable cultured leiomyoma cells and normal myometrial cells were determined by MTS assay (Fig. 1Go). Compared with untreated control cultures, treatment with 10–6 M asoprisnil for 24 h and with asoprisnil at concentrations greater than or equal to 10–7 M for 48 and 72 h significantly decreased the number of viable cultured leiomyoma cells compared with untreated control cultures (P < 0.05, 10–6 M asoprisnil at 24 h; P < 0.01, 10–6 M asoprisnil at 48 and 72 h; P < 0.05, 10–7 M asoprisnil at 48 h; P < 0.01, 10–7 M asoprisnil at 72 h). However, asoprisnil had no effect on the number of viable cultured normal myometrial cells over the treatment period of 72 h.


Figure 1
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  		FIG. 1. Effects of graded concentrations of asoprisnil on the number of viable cultured leiomyoma cells and normal myometrial cells, as assessed by MTS assay. Compared with untreated control cultures, 24-h treatment with 10–6 M asoprisnil and either 48- or 72-h treatment with asoprisnil at concentrations greater than or equal to 10–7 M significantly decreased the number of viable cultured leiomyoma cells compared with untreated control cultures. In cultured normal myometrial cells, however, treatment with asoprisnil did not affect the number of viable cultured cells over the 72-h treatment period. Results represent the mean ± SD of at least six independent experiments performed in triplicate. *, P < 0.05; **, P < 0.01 (vs. untreated control cultures).

 
Effects of graded concentrations of asoprisnil on the PCNA-positive rate and PCNA protein expression in cultured leiomyoma cells and normal myometrial cells

The PCNA-positive rate of leiomyoma cells and normal myometrial cells cultured in the absence or presence of graded concentrations of asoprisnil for 24, 48, and 72 h was comparatively assessed by immunocytochemical analysis (Fig. 2Go, upper panel). Two-way ANOVA of the indexes for the PCNA-positive rate of cultured leiomyoma cells showed a significant interaction between time of culture and asoprisnil concentration (P < 0.001). Compared with untreated control cultures, treatment with 10–6 M asoprisnil significantly (P < 0.01) decreased the PCNA-positive rate of cultured leiomyoma cells at 24, 48, and 72 h. Treatment with either 10–8 or 10–7 M asoprisnil showed no significant effect at 24 h, but resulted in a significant (P < 0.01) decrease in the PCNA-positive rate of cultured leiomyoma cells at 48 and 72 h compared with untreated control cultures. In cultured normal myometrial cells, however, treatment with asoprisnil did not affect the PCNA-positive rate over the 72-h treatment period.


Figure 2
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  		FIG. 2. Effects of graded concentrations of asoprisnil on the PCNA-positive rate and PCNA protein expression in cultured leiomyoma cells and normal myometrial cells, as assessed by immunocytochemical analysis and Western blot analysis. Compared with untreated control cultures, treatment with 10–6 M asoprisnil significantly decreased the PCNA-positive rate of cultured leiomyoma cells at 24, 48, and 72 h, whereas treatment with either 10–8 or 10–7 M asoprisnil significantly decreased the PCNA-positive rate of cultured leiomyoma cells at 48 and 72 h (upper panel). In cultured normal myometrial cells, however, treatment with asoprisnil did not affect the PCNA-positive rate over the 72-h treatment period (upper panel). Results represent the mean ± SD of at least six independent experiments performed in triplicate. *, P < 0.01 vs. untreated control cultures at 24, 48, and 72 h. Western blot analysis revealed that compared with untreated control cultures, treatment with asoprisnil at concentrations greater than or equal to 10–7 M significantly decreased PCNA protein expression in cultured leiomyoma cells (lower panel). In cultured normal myometrial cells, however, treatment with asoprisnil did not affect PCNA protein expression. Densitometric analysis of PCNA was performed as described in Materials and Methods. ß-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 six independent experiments performed in triplicate. **, P < 0.01 vs. untreated control cultures.

 
Comparative effects of treatment with graded concentrations of asoprisnil on PCNA protein expression in leiomyoma cells and normal myometrial cells cultured for 48 h were assessed by Western blot analysis (Fig. 2Go, lower panel). One-way ANOVA of the indexes for PCNA protein expression in cultured leiomyoma cells showed significant effects of asoprisnil concentrations (P < 0.001). Compared with untreated control cultures, treatment with asoprisnil at concentrations greater than or equal to 10–7 M significantly (P < 0.01) decreased PCNA protein expression in cultured leiomyoma cells. There was a significant (P < 0.01) difference in PCNA protein levels between 10–7 and 10–6 M asoprisnil treatments. In cultured normal myometrial cells, however, treatment with asoprisnil did not affect PCNA protein expression over the 72-h treatment period.

Effects of graded concentrations of asoprisnil on the TUNEL-positive rate of cultured leiomyoma cells and normal myometrial cells

Figure 3Go shows the TUNEL-positive rate of leiomyoma cells and normal myometrial cells cultured for 24 and 48 h in the absence or presence of graded concentrations of asoprisnil. Two-way ANOVA of the indexes for the TUNEL-positive rate of cultured leiomyoma cells showed a significant interaction between time of culture and asoprisnil concentration (P < 0.001). Compared with untreated control cultures, treatment with 10–6 M asoprisnil significantly (P < 0.01) increased the TUNEL-positive rate of cultured leiomyoma cells at 24 and 48 h, whereas treatment with 10–7 M asoprisnil significantly (P < 0.01) augmented the TUNEL-positive rate at 48 h. In cultured normal myometrial cells, however, treatment with asoprisnil did not affect the TUNEL-positive rate.


Figure 3
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  		FIG. 3. Effects of graded concentrations of asoprisnil on the TUNEL-positive rate of cultured leiomyoma cells and normal myometrial cells, as assessed by TUNEL assay. Compared with untreated control cultures, treatment with 10–6 M asoprisnil significantly increased the TUNEL-positive rate of cultured leiomyoma cells at 24 and 48 h, whereas treatment with 10–7 M asoprisnil significantly augmented the TUNEL-positive rate at 48 h. In cultured normal myometrial cells, however, treatment with asoprisnil did not affect the TUNEL-positive rate. Results represent the mean ± SD of at least six independent experiments performed in triplicate. *, P < 0.01 vs. untreated control cultures at 24 and 48 h

 
Effects of graded concentrations of asoprisnil on Bcl-2 protein, cleaved caspase-3, and cleaved PARP expression in cultured leiomyoma cells and normal myometrial cells

Comparative effects of treatment with graded concentrations of asoprisnil on Bcl-2 protein, cleaved caspase-3, and cleaved PARP expression in cultured leiomyoma cells and normal myometrial cells were assessed by Western blot analysis at 48, 24, and 48 h, respectively (Fig. 4Go), because we have noted that cleaved caspase-3 expression reaches a peak at 24 h, followed by a peak of cleaved PARP expression at 48 h in cultured leiomyoma cells treated with the P4 antagonist CDB-2914 (22).


Figure 4
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  		FIG. 4. Effects of graded concentrations of asoprisnil on Bcl-2 protein, cleaved caspase-3, and cleaved PARP expression in cultured leiomyoma cells and normal myometrial cells, as assessed by Western blot analysis. Comparative effects of treatment with graded concentrations of asoprisnil on Bcl-2 protein, cleaved caspase-3, and cleaved PARP expression in cultured leiomyoma cells and normal myometrial cells were assessed by Western blot analysis at 48, 24, and 48 h, respectively. Treatment with asoprisnil at concentrations greater than or equal to 10–7 M significantly decreased Bcl-2 protein expression in leiomyoma cells cultured for 48 h compared with untreated control cultures (upper panel). In cultured myometrial cells, however, treatment with asoprisnil did not affect Bcl-2 protein expression (upper panel). Cleaved caspase-3 expression in leiomyoma cells cultured for 24 h was significantly augmented by treatment with asoprisnil at concentrations greater than or equal to 10–8 M compared with untreated control cultures (middle panel). In cultured normal myometrial cells, however, treatment with asoprisnil did not affect cleaved caspase-3 expression (middle panel). Cleaved PARP expression in leiomyoma cells cultured for 48 h was significantly increased by treatment with asoprisnil at concentrations greater than or equal to 10–8 M compared with untreated control cultures (lower panel). In cultured normal myometrial cells, however, treatment with asoprisnil did not affect cleaved PARP expression (lower panel). Densitometric analysis of Bcl-2 protein, cleaved caspase-3, and cleaved PARP was performed as described in Materials and Methods. ß-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 six independent experiments performed in triplicate. *, P < 0.01 vs. Bcl-2 protein, cleaved caspase-3, and cleaved PARP content in each untreated control culture.

 
One-way ANOVA of the indexes for Bcl-2 protein expression in cultured leiomyoma cells showed significant effects of asoprisnil concentrations (P < 0.001). Treatment with asoprisnil at concentrations greater than or equal to 10–7 M significantly (P < 0.01) decreased Bcl-2 protein expression in leiomyoma cells cultured for 48 h compared with untreated control cultures (Fig. 4Go, upper panel). In cultured normal myometrial cells, however, treatment with asoprisnil did not affect Bcl-2 protein expression (Fig. 4Go, upper panel).

One-way ANOVA of the indexes for cleaved caspase-3 expression in cultured leiomyoma cells showed significant effects of asoprisnil concentrations (P < 0.001). Cleaved caspase-3 expression in leiomyoma cells cultured for 24 h was significantly (P < 0.01) augmented by treatment with asoprisnil at concentrations greater than or equal to 10–8 M compared with untreated control cultures (Fig. 4Go, middle panel). There was a significant difference (P < 0.01) in cleaved caspase-3 expression between 10–8 and 10–7 M asoprisnil treatment. In cultured normal myometrial cells, however, treatment with asoprisnil did not affect cleaved caspase-3 expression (Fig. 4Go, middle panel).

One-way ANOVA of the indexes for cleaved PARP expression in cultured leiomyoma cells showed significant effects of the asoprisnil concentration (P < 0.001). Cleaved PARP expression in leiomyoma cells cultured for 48 h was significantly (P < 0.01) increased by treatment with asoprisnil at concentrations greater than or equal to 10–8 M compared with untreated control cultures (Fig. 4Go, lower panel). There was a significant difference (P < 0.01) in cleaved PARP expression between 10–7 and 10–6 M asoprisnil treatments. In cultured normal myometrial cells, however, treatment with asoprisnil did not affect cleaved PARP expression (Fig. 4Go, lower panel).

Expression of PR-A and PR-B in untreated cultured leiomyoma cells and untreated cultured normal myometrial cells

Figure 5Go shows the expression of PR-A and PR-B in untreated cultured leiomyoma cells and normal myometrial cells, as assessed by Western blot analysis. PR-B expression in untreated cultured leiomyoma cells was significantly (P < 0.05) higher than that in untreated cultured normal myometrial cells, but there were no differences in PR-A expression between the two cell types. The PR-A/PR-B ratio was significantly decreased (P < 0.05) in untreated cultured leiomyoma cells compared with that in untreated cultured normal myometrial cells.


Figure 5
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  		FIG. 5. The expression of PR-A and PR-B in untreated cultured leiomyoma cells and untreated cultured normal myometrial cells, as assessed by Western blot analysis. PR-B expression in untreated cultured leiomyoma cells was significantly higher than that in untreated cultured normal myometrial cells, but there were no differences in PR-A expression between the two types of cells. The PR-A/PR-B ratio in untreated cultured leiomyoma cells was significantly lower than that in untreated cultured normal myometrial cells. Densitometric analysis of PR was performed as described in Materials and Methods. ß-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 six independent experiments performed in triplicate. *, P < 0.05 vs. untreated cultured normal myometrial cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we demonstrate that asoprisnil exerts growth inhibitory effects on cultured leiomyoma cells by decreasing proliferation and increasing apoptosis of those cells without affecting the cell growth and survival of cultured normal myometrial cells obtained from the same uterus. Because the PCNA-positive rate of leiomyoma cells was shown to be higher in the secretory phase than in the proliferative phase of the menstrual cycle (26), we subcultured isolated cells in phenol red-free DMEM supplemented with 10% fetal bovine serum for 120 h to abrogate the menstrual cycle-dependent influence on the biological characteristics of cells. We confirmed that the 120-h subculture produced no differences in the PCNA-positive rate of cultured leiomyoma cells obtained from the different phases.

The effects of asoprisnil on leiomyoma cells were dose and time dependent. These findings are in accord with our previous data showing that P4 up-regulates PCNA protein expression in cultured leiomyoma cells, but not in cultured normal myometrial cells (26). Overall, these observations suggest that PR plays an important role in the regulation of both cell proliferation and programmed cell death in leiomyoma cells. Our results also suggest that the effects of asoprisnil on leiomyoma cells most likely represent the PR antagonistic activity of this compound in these particular cells. A partial PR antagonistic effect of asoprisnil was previously described in rabbit and guinea pig uterus (15, 16, 17). The exact mechanism of the antiproliferative effect of asoprisnil on leiomyoma cells remains to be evaluated. This effect may be due to the inhibition of P4-induced expression of local growth factors, such as epidermal growth factor (26). However, other potential mechanisms, such as PR-mediated effects on the cell cycle (27, 28), cannot be excluded.

Apoptosis is mediated through either the intrinsic mitochondrial pathway or the extrinsic pathway (29). In the intrinsic mitochondrial pathway, cytochrome c that is released from mitochondria into the cytosol forms the apoptosome together with apoptosis-protease activating factor-1 (29, 30), which recruits caspase-9 (31). Caspase-9 cleaves and activates the effector caspase, caspase-3 (29, 31). During the course of apoptosis, caspase-3 proteolytically cleaves the nuclear enzyme PARP (32), which is implicated in DNA replication, transcription, DNA repair, apoptosis, and genome stability (33). Thus, the cleavage of PARP is regarded as a hallmark event for the apoptotic paradigm (34). In contrast, Bcl-2 protein that resides in mitochondrial membranes acts to prevent the release of apoptogenic proteins from mitochondria (35).

In our study asoprisnil induced apoptosis of cultured leiomyoma cells in a dose- and time-dependent manner through up-regulating cleaved caspase-3 and cleaved PARP expression and down-regulating Bcl-2 protein expression. The proapoptotic effects of asoprisnil in cultured leiomyoma cells, but not in cultured normal myometrial cells, are in agreement with the data from our previous study showing that P4 up-regulates Bcl-2 protein expression in cultured leiomyoma cells, but not in cultured normal myometrial cells (23). Our results suggest that asoprisnil may act to trigger an intrinsic mitochondria-mediated apoptotic pathway by down-regulating Bcl-2 protein expression, subsequently activating caspase-3, which is the final executioner of apoptosis.

Unlike cultured leiomyoma cells, asoprisnil did not affect the growth and survival of cultured normal myometrial cells. This reinforces the cell type-selective action of asoprisnil. The relative expression levels of PR-A and PR-B are reported to determine the responsiveness of human myometrial cells to P4 (36). In the present study PR-B expression in untreated cultured leiomyoma cells was elevated compared with that in untreated cultured normal myometrial cells, whereas there were no differences in PR-A expression between the two types of cells. Consequently, the PR-A/PR-B ratio in untreated cultured leiomyoma cells was lower than that in untreated cultured normal myometrial cells. Our results were in agreement with previously reported data showing that PR-B protein expression is increased in leiomyoma tissues compared with adjacent normal myometrium (6), suggesting that cell culture may not affect the comparable expression patterns of PR isoforms between the two types of cells. Our observation suggests that differential expression of PR-B in leiomyoma vs. normal myometrial cells may play a role in the cell type-specific effects of asoprisnil.

Coactivators and corepressors are nuclear proteins that modulate the transcriptional activity of nuclear receptors. Coactivators enhance the transcriptional activity of nuclear receptors, whereas corepressors elicit inhibitory effects on nuclear receptors (15). P4 agonists promote the interactions of the nuclear receptor with coactivators, whereas P4 antagonist favors the interactions with corepressors (15). The differential responsiveness to asoprisnil of the two types of cells might be due to the difference in regulating the interactions with coactivators and corepressors. However, the precise mechanism underlying the differential effects of asoprisnil in cultured leiomyoma cells and normal myometrial cells remains to be determined.

In conclusion, we have demonstrated for the first time that asoprisnil selectively inhibits the proliferation of cultured human leiomyoma cells by down-regulating PCNA expression and induces apoptosis by up-regulating cleaved caspase-3 and cleaved PARP expression and down-regulating Bcl-2 protein expression without affecting the proliferation and apoptosis of cultured human normal myometrial cells. The results of the present study suggest that a progressive shrinkage of uterine leiomyomata during treatment with asoprisnil, as observed in clinical trials (19, 20), may be due at least in part to a direct, PR-mediated, and cell type-specific effect of asoprisnil on leiomyoma cells. Asoprisnil has the potential to become a novel treatment for symptomatic uterine leiomyomata.


   Footnotes
 
This work was supported by Grant-in-Aid for Scientific Research 1437053 from the Japanese Ministry of Education, Science, and Culture. the Ogyaa-Donation Foundation of the Japan Association of Obstetricians and Gynecologists, and TAP Pharmaceutical Products, Inc.

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

First Published Online February 7, 2006

Abbreviations: MTS, 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; P4, progesterone; PARP, poly(adenosine 5'-diphosphate-ribose) polymerase; PCNA, proliferating cell nuclear antigen; PR, progesterone receptor; SPRM, selective PR modulator; TUNEL, terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling.

Received November 1, 2005.

Accepted January 26, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Maruo T, Ohara N, Wang J, Matsuo H 2004 Sex steroidal regulation of uterine leiomyoma growth and apoptosis. Hum Reprod Update 10:207–220[Abstract/Free Full Text]
  2. Leonhardt SA, Edwards DP 2002 Mechanism of action of progesterone antagonists. Exp Biol Med 227:969–980[Abstract/Free Full Text]
  3. Kastner P, Krust A, Turcotte B, Stropp U, Tora L, Gronemeyer H, Chambon P 1990 Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B. EMBO J 9:1603–1614[Medline]
  4. Vegeto E, Shahbaz MM, Wen DX, Goldman ME, O’Malley BW, McDonnell DP 1993 Human progesterone receptor A form is a cell- and promoter-specific repressor of human progesterone receptor B function. Mol Endocrinol 7:1244–1255[Abstract]
  5. Wen DX, Xu Y-F, Mais DE, Goldman ME, McDonnell DP 1994 The A and B isoforms of the human progesterone receptor operate through distinct signaling pathways within target cells. Mol Cell Biol 14:8356–8364[Abstract/Free Full Text]
  6. Brandon DD, Bethea CL, Strawn EY, Novy MJ, Burry KA, Harrington MS, Erickson TE, Warner C, Keenan EJ, Clinton GM 1993 Progesterone receptor messenger ribonucleic acid and protein are overexpressed in human uterine leiomyomas. Am J Obstet Gynecol 169:78–85[Medline]
  7. Viville B, Charnock-Jones DS, Sharkey AM, Wetzka B, Smith SK 1997 Distribution of the A and B forms of the progesterone receptor messenger ribonucleic acid and protein in uterine leiomyomata and adjacent myometrium. Hum Reprod 12:815–822[Abstract/Free Full Text]
  8. Nisolle M, Gillerot S, Casanas-Roux F, Squifflet J, Berliere M, Donnez J 1999 Immunohistochemical study of the proliferation index, oestrogen receptors and progesterone receptors A and B in leiomyomata and normal myometrium during the menstrual cycle and under gonadotropin-releasing hormone agonist therapy. Hum Reprod 14:2844–2850[Abstract/Free Full Text]
  9. Tiltman AJ 1985 The effect of progestins on the mitotic activity of uterine fibromyomas. Int J Gynecol Pathol 4:89–96[Medline]
  10. Carr BR, Marshburn PB, Weatherall PT, Bradshaw KD, Breslau NA, Byrd W, Roark M, Steinkampf MP 1993 An evaluation of the effect of gonadotropin-releasing hormone analogs and medroxyprogesterone acetate on uterine leiomyomata volume by magnetic resonance imaging: a prospective, randomized, double blind, placebo-controlled, crossover trial. J Clin Endocrinol Metab 76:1217–1223[Abstract]
  11. Friedman AJ, Daly M, Juneau-Norcross M, Rein MS, Fine C, Gleason R, Leboff M 1993 A prospective, randomized trial of gonadotropin-releasing hormone agonist plus estrogen-progestin or progestin "add-back" regimens for women with leiomyomata uteri. J Clin Endocrinol Metab 76:1439–1445[Abstract]
  12. Kettel LM, Murphy AA, Morales AJ, Yen SSC 1994 Clinical efficacy of the antiprogesterone RU486 in the treatment of endometriosis and uterine fibroids. Hum Reprod 9(Suppl 1):116–120
  13. Murphy AA, Morales AJ, Kettel LM, Yen SSC 1995 Regression of uterine leiomyomata to the antiprogesterone RU486: dose-response effect. Fertil Steril 64:187–190[Medline]
  14. Eisinger SH, Meldrum S, Fiscella K, Le Roux HD, Guzick DS 2003 Low-dose mifepristone for uterine leiomyomata. Obstet Gynecol 101:243–250[Abstract/Free Full Text]
  15. Chwalisz K, Perez MC, DeManno D, Winkel C, Schubert G, Elger W 2005 Selective progesterone receptor modulator development and use in the treatment of leiomyomata and endometriosis. Endocr Rev [Erratum (2005) 26:703] 26:423–438
  16. DeManno D, Elger W, Garg R, Lee R, Schneider B, Hess-Stumpp H, Schubert G, Chwalisz K 2003 Asoprisnil (J867): a selective progesterone receptor modulator for gynecological therapy. Steroids 68:1019–1032[CrossRef][Medline]
  17. Schubert G, Elger W, Kaufmann G, Schneider B, Reddersen G, Chwalisz K 2005 Discovery, chemistry, and reproductive pharmacology of asoprisnil and related 11ß-benzaldoxime substituted selective progesterone receptor modulators (SPRMs). Semin Reprod Med 23:58–73[CrossRef][Medline]
  18. Chwalisz K, Elger W, Stickler T, Mattia-Goldberg C, Larsen L 2005 The effects of 1-month administration of asoprisnil (J867), a selective progesterone receptor modulator, in healthy premenopausal women. Hum Reprod 20:1090–1099[Abstract/Free Full Text]
  19. Chwalisz K, Parker RL, Williamson S, Larsen L, McCrary K, Elger W 2003 Treatment of uterine leiomyomas with the novel selective progesterone receptor modulator (SPRM) J867. J Soc Gynecol Invest 10(Suppl):301A (Abstract)
  20. Chwalisz K, Larsen L, McCrary K, Edmonds A 2004 Effects of the novel selective progesterone receptor modulator (SPRM) asoprisnil on bleeding patterns in subjects with leiomyomata. J Soc Gynecol Invest 11(Suppl):320A–321A (Abstract)
  21. Chwalisz K, Mattia-Goldberg K, Lee M, Elger W, Edmonds A 2004 Treatment of endometriosis with the novel selective progesterone receptor modulator (SPRM) asoprisnil. Fertil Steril 82:S83 (Abstract O-207)
  22. Xu Q, Takekida S, Ohara N, Chen W, Sitruk-Ware R, Johansson EDB, Maruo T 2005 Progesterone receptor modulator CDB-2914 down-regulates proliferative cell nuclear antigen and Bcl-2 protein expression and up-regulates caspase-3 and poly(adenosine 5'-diphosphate-ribose) polymerase expression in cultured human uterine leiomyoma cells. J Clin Endocrinol Metab 90:953–961[Abstract/Free Full Text]
  23. Matsuo H, Maruo T, Samoto T 1997 Increased expression of Bcl-2 protein in human uterine leiomyoma and its up-regulation by progesterone. J Clin Endocrinol Metab 82:293–299[Abstract/Free Full Text]
  24. Berridge MV, Tan AS 1993 Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Arch Biochem Biophys 303:474–482[CrossRef][Medline]
  25. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  26. Shimomura Y, Matsuo H, Samoto T, Maruo T 1998 Up-regulation by progesterone of proliferating cell nuclear antigen and epidermal growth factor expression in human uterine leiomyoma. J Clin Endocrinol Metab 83:2192–2198[Abstract/Free Full Text]
  27. Musgrove EA, Lee CSL, Cornish AL, Swarbrick A, Sutherland RL 1997 Antiprogestin inhibition of cell cycle progression in T-47D breast cancer cells is accompanied by induction of the cyclin-dependent kinase inhibitor p21. Mol Endocrinol 11:54–66[Abstract/Free Full Text]
  28. Gaddy VT, Barrett JT, Delk JN, Kallab AM, Porter AG, Schoenlein PV 2004 Mifepristone induces growth arrest, caspase activation, and apoptosis of estrogen receptor-expressing, antiestrogen-resistant breast cancer cells. Clin Cancer Res 10:5215–5225[Abstract/Free Full Text]
  29. Riedl SJ, Shi Y 2004 Molecular mechanisms of caspase regulation during apoptosis. Nat Rev Mol Cell Biol 5:897–907[CrossRef][Medline]
  30. Shiozaki EN, Shi Y 2004 Caspases, IAPs and Smac/DIABLO: mechanisms from structural biology. Trends Biochem Sci 39:486–494
  31. Shi Y 2004 Caspase activation, inhibition, and reactivation: a mechanistic view. Protein Sci 13:1979–1987[Abstract/Free Full Text]
  32. Cohen GM 1997 Caspases: the executioners of apoptosis. Biochem J 326:1–16[Medline]
  33. Bouchard VJ, Rouleau M, Poirier GG 2003 PARP-1, a determinant of cell survival in response to DNA damage. Exp Hematol 31:446–454[CrossRef][Medline]
  34. Pieper AA, Verma A, Zhang J, Snyder SH 1999 Poly(ADP-ribose) polymerase, nitric oxide and cell death. Trends Pharmacol Sci 20:171–181[CrossRef][Medline]
  35. Reed JC, Jurgensmeier JM, Matsuyama S 1998 Bcl-2 family proteins and mitochondria. Biochim Biophys Acta 1366:127–137[Medline]
  36. Pieber D, Allport VC, Hills F, Johnson M, Bennett PR 2001 Interactions between progesterone receptor isoforms in myometrial cells in human labour. Mol Hum Reprod 7:875–879[Abstract/Free Full Text]



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