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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-1591
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 2 884-892
Copyright © 2005 by The Endocrine Society

Gonadotropin-Releasing Hormone Antagonist Cetrorelix Down-Regulates Proliferating Cell Nuclear Antigen and Epidermal Growth Factor Expression and Up-Regulates Apoptosis in Association with Enhanced Poly(Adenosine 5'-Diphosphate-Ribose) Polymerase Expression in Cultured Human Leiomyoma Cells

Wei Chen, Shigeki Yoshida, Noriyuki Ohara, Hiroya Matsuo, Mayumi Morizane and Takeshi Maruo

Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, Kobe, 650-0017, Japan

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
 
The objective of this study was to elucidate the effects of GnRH antagonist Cetrorelix on proliferation and apoptosis in human leiomyoma cells cultured in vitro. Isolated leiomyoma cells were subcultured in phenol red-free DMEM supplemented with 10% fetal bovine serum for 120 h and then stepped down to serum-free conditions in the presence or absence of graded concentrations of Cetrorelix (10–5 to 10–8 mol/liter) for 6 d. Cultured leiomyoma cells were used for semiquantitative RT-PCR, immunocytochemistry, Western blot analysis, and terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling assay. RT-PCR analysis revealed the presence of mRNAs encoding for GnRH receptor and epidermal growth factor (EGF) in cultured leiomyoma cells. The number of viable cultured leiomyoma cells was significantly (P < 0.01) decreased by treatment with Cetrorelix compared with untreated control cultures. Immunocytochemical examination demonstrated that treatment with Cetrorelix attenuated the expression of proliferating cell nuclear antigen (PCNA) and EGF in cultured leiomyoma cells. Western blot analysis revealed that treatment with 10–5 mol/liter Cetrorelix significantly (P < 0.01) decreased PCNA expression. In addition, treatment with 10–5 mol/liter Cetrorelix remarkably increased the terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling-positive rate and poly(ADP-ribose) polymerase expression at 24 h of treatment compared with untreated control cultures (P < 0.01). Furthermore, treatment with 10–5 mol/liter Cetrorelix decreased immunoreactive EGF protein and EGF mRNA expression in cultured leiomyoma cells at 4 d of treatment. GnRH antagonist Cetrorelix may directly inhibit leiomyoma cell growth by down-regulating proliferation in association with a decrease in EGF mRNA expression and by up-regulating apoptosis in those cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
UTERINE LEIOMYOMA IS the most common smooth muscle tumor that occurs in as many as 30% of women over 35 yr of age (1). Leiomyoma is believed to be a sex steroid hormone-dependent neoplasm because it grows during the reproductive years and regresses after menopause. Treatment with GnRH agonists is a well-established therapeutic approach in the management of premenopausal women with leiomyomas (2). GnRH agonists are known to inhibit the hypophyseal-gonadal axis through down-regulation of pituitary GnRH receptors, desensitization of the pituitary gonadotrophs, and suppression of circulating levels of gonadotropins and sex steroids (3). Recently, clinical trials have demonstrated that GnRH antagonists cause a rapid reduction in the size of leiomyoma without any flare-up effect, reinforcing the usefulness of this new treatment modality (4, 5, 6, 7). Unlike GnRH agonists, GnRH antagonists cause an immediate inhibition of the release of gonadotropins and sex steroids without an initial flare-up phenomenon seen with GnRH agonists due to a competitive blockade of GnRH and competitive receptor occupancy of pituitary GnRH receptors without a marked down-regulation of pituitary GnRH receptors in a clinical setting (3, 8, 9).

The regression in leiomyoma size after administration of GnRH analogs has been attributable to the deprivation of circulating sex steroid hormones. However, the direct action of GnRH analogs has been speculated because GnRH receptors exist in the extrapituitary tissues. An increasing body of evidence has demonstrated the presence of GnRH and GnRH receptors in leiomyomas (10, 11, 12, 13) and a variety of cancers, including breast, endometrial, ovarian, and prostate cancer (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24). The presence of high-affinity binding sites and the expression of GnRH and GnRH receptor mRNAs suggest an autocrine/paracrine role for GnRH in the extrapituitary tissues. Actually, the agonistic and antagonistic GnRH analogs are shown to exert a direct antiproliferative effect on breast, endometrial, and ovarian cancer cells through GnRH receptor coupling (25, 26, 27). Our previous study has also demonstrated that GnRH agonist directly inhibits the growth of cultured leiomyoma cells by suppressing cell proliferation and inducing apoptosis (13). However, the precise mechanism of action of GnRH antagonists on leiomyoma growth and apoptosis remains unknown.

Accumulating data have demonstrated that growth factors are involved in the regulation of leiomyoma growth (28). The mRNAs encoding for epidermal growth factor (EGF) and its receptor are expressed in leiomyoma (29, 30). EGF plays a crucial role in the autocrine/paracrine regulation of leiomyoma growth (29, 30) and stimulates the proliferation of leiomyoma cells in synergy with sex steroid hormones (31). The antiproliferative effect of GnRH analogs is reported to be mediated through interaction with the mitogenic signal transduction pathways of EGF receptor in breast, endometrial, ovarian, and prostate cancer (32, 33, 34, 35, 36). However, no information is available regarding the effect of GnRH antagonist on EGF expression in leiomyoma cells.

In the present study, we have examined the presence of GnRH receptor mRNA expression in cultured human uterine leiomyoma cells and determined the effects of GnRH antagonist Cetrorelix (Zentaris GmbH, Frankfurt, Germany) on the expression of proliferating cell nuclear antigen (PCNA), immunoreactive EGF protein and EGF mRNA, and poly(ADP-ribose) polymerase (PARP) and on apoptosis in cultured uterine leiomyoma cells by semiquantitative RT-PCR analysis, immunocytochemistry, Western blot analysis, and terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay.


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

Phenol red-free DMEM and antibiotics (1 x 105 U/liter penicillin and 50 mg/liter streptomycin) were purchased from Life Technologies (Grand Island, NY). Fetal bovine serum was obtained from Sigma Chemical Co. (St. Louis, MO). Collagenase was obtained from Wako Pure Biochemical Industries (Osaka, Japan). Mouse monoclonal antibodies to human PCNA (PCNA; Ab-1, PC10) and EGF were purchased from Oncogene Science, Inc. (Cambridge, MA). A mouse monoclonal antibody against PARP was purchased from Cell Signaling Technology Inc. (Livermore, CA). GnRH antagonist Cetrorelix, [Ac-D-Nal (2)1, D-Phe(4Cl)2, D-Pal (3)3, D-Cit6, D-Ala10]GnRH, was obtained from Zentaris GmbH.

Tissue collection, cell culture, and cell counting

Leiomyoma tissues were obtained from premenopausal women with regular menstrual cycles who underwent hysterectomy or myomectomy for uterine leiomyomas at Kobe University Hospital (Kobe, Japan). The institutional review board approved the study protocol for the collection of surgical specimens, and informed consent was obtained from each patient before surgery for the use of uterine tissues for the present study. Patients received no hormonal therapy at least for 6 months before surgery. The age of patients ranged from 29–46 yr, with a mean age of 36.9 yr. Eight samples were collected from the proliferative phase of the menstrual cycle, and 10 samples were from the secretory phase of the menstrual cycle.

Leiomyoma tissues were dissected from endometrial cell layers, washed in PBS, cut into small pieces, and digested in 0.2% collagenase at 37 C for 3–5 h. Leiomyoma cells were collected by centrifugation at 460 x g for 5 min and washed three times with PBS containing 1% antibiotic solution. The cell viability was determined by trypan blue exclusion test. Isolated leiomyoma 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 2 x 103/well in six-well chamber glass slides. Isolated leiomyoma cells 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% FBS. The monolayer cultures that were grown to 70–80% confluence were used for experiments. For the cell-counting assay, immunocytochemistry, Western blot analysis, and semiquantitative RT-PCR analysis after 5 d of subculture, the cells were treated in the presence or absence of graded concentrations of Cetrorelix (10–8, 10–7, 10–6, and 10–5 mol/liter) in serum-free, phenol red-free DMEM for 2, 4, and 6 d. We used 10% dimethylsulfoxide (DMSO) to dissolve Cetrorelix, resulting in a final concentration of DMSO of 0.1% in culture media. We used 0.1% DMSO as a vehicle in control cultures. For the analysis of apoptosis after 5 d of subculture, the cells were treated in the same conditions as mentioned earlier for 12, 24, and 36 h,

The number of cultured leiomyoma cells per dish was determined using a hemocytometer after treatment with graded concentrations of Cetrorelix at 2, 4, and 6 d. The cell counting of cultured leiomyoma cells was estimated by trypsinizing cells and making triplicate determinations of three aliquots of the cell suspension using a Coulter counter (Coulter, Hialeah, FL).

Semiquantitative RT-PCR

Total RNA was obtained from leiomyoma cells cultured under serum-free conditions for 2 and 4 d using RNeasy Mini Kit (Qiagen, Inc., Chatsworth, CA). First-strand cDNA was synthesized from 2 µg total RNA using an Omniscript RT Kit (Qiagen, Inc.). PCR was performed using 1 µl cDNA as template, 6.25 pmol/liter of each primer, 2.5 mmol/liter deoxynucleotide triphosphates, 0.125 UTaq DNA polymerase (Roche Diagnostics Inc., Mannheim, Germany), 1x reaction buffer containing 10 mmol/liter Tris-HCl (pH 8.3), 50 mmol/liter KCl, 1.5 mmol/liter MgCl2, and 0.01% gelatin in 25-µl reaction volume. The amplification procedure, which was performed on a Gene Amp PCR System 9600-R (PerkinElmer Corp., Norwalk, CT), was as follows: initial denaturation step at 94 C for 5 min, denaturation step at 94 C for 30 sec, annealing step at 55 C for 30 sec, and extension step at 72 C for 30 sec. The reactions were subjected to 34 cycles using human-specific PCR primers for GnRH receptor (sense: 5'-TCTAGCAGACAGCTCTGGACA-3'; and antisense: 5'-GAGTCTTCAGCCGTGCTCTT-3') (37). We designed the oligonucleotide primers for EGF from the 330-bp mRNA derived from EGF repeat transmembrane protein (sense: 5'-ACATCAAATATCCTCAATGG-3'; and antisense: 5'-GTGGCATCAAGACCGGGCTGC-3'). In addition, tubes containing all PCR components except the reverse transcriptase reaction mixture were also amplified, which served as a negative control to check for the presence of DNA that may have been carried over from a previous reaction. RT-PCR of RNA extracted from human pituitary and human ovarian cancer cell line SK-OV-3 (European Collection of Cell Cultures, Porton Down, UK) was used as the positive control for GnRH receptor and EGF, respectively. PCR for ß-actin (sense: 5'-CTTCTACAATGAGCTGCGTG-3'; and antisense: 5'-TCATGAGGTAGTCAGTCAGG-3') was performed on all the samples to test for the possibility of RNA degradation or RNA transcription default. The PCR products specific for EGF and ß-actin were visualized under UV light after gel electrophoresis on a 3% agarose gel stained with ethidium bromide and then photographed with POLAROID MP-4 Camera (Polaroid, Waltham, MA). The bands were scanned with GT-9700F (Epson Co., Tokyo, Japan) and qualified with NIH Image version 1.60 (National Institutes of Health, Bethesda, MD). The experiments were repeated with at least three different cultured specimens with similar results, and the reported results are representative. The amount of mRNA was expressed relative to the abundance of ß-actin mRNA. Data are presented as the fold increase over the control value and the mean ± SD. The PCR products were cloned, and sequence analysis revealed their specificity.

Immunocytochemistry

Leiomyoma cells were subcultured in two-well glass chamber slides for 120 h and then cultured under serum-deprivation conditions for 2, 4, and 6 d in the presence or absence of graded concentrations of Cetrorelix. After removal of the culture medium, the cells were washed with PBS twice, fixed in methanol at 4 C for 20 min, and 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). Mouse monoclonal antibodies to human PCNA and EGF were used as the primary antibodies at the dilution of 1:80 and 1:50, respectively. To ensure the specificity of the immunological reaction, the cultured cells were subjected to the same immunoperoxidase method, except that the primary antibodies were replaced by nonimmune murine IgG (Miles, Erkhardt, IN) at the same dilution as the specific antibodies. The replacement of the specific primary antibodies with nonimmune murine IgG resulted in a lack of positive immunostaining for PCNA and EGF.

Immunostained cells were 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 of the experimental samples and used for evaluating the proliferating activity of cultured leiomyoma cells.

Western blot analysis

Proteins were extracted from cultured leiomyoma cells as described previously (31). At the termination of cultures, cultured leiomyoma cells were incubated at 4 C for 20 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 30 min, and the supernatants were collected. Protein contents in the supernatants were determined by the Bradford assay (38). Each 60-µg aliquot of the proteins extracted from cultured leiomyoma cells was electrophoresed on a 10% SDS-PAGE under a reducing condition and transferred to polyvinylidene difluoride membrane. The bolts were exposed to monoclonal antibodies to PCNA and PARP at the same dilution of 1:1000 in TBS. The antigen-antibody complexes were detected with the secondary antibodies using an ECL chemiluminescence detection system (Amersham Pharmacia Biotech, Uppsala, Sweden). 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 three different cultured specimens with the similar results, and the reported results are representative.

TUNEL assay

ApopTag In Situ Apoptosis Detection Kit (Intergen Co., Purchase, NY) was used to identify TUNEL-positive nuclei in cultured leiomyoma cells. Leiomyoma cells were subcultured in two-well glass chamber slides for 120 h and then cultured under serum-deprivation conditions for 12, 24, and 36 h in the presence or absence of graded concentrations of Cetrorelix. After removal of the culture medium, the cells were washed with PBS twice and fixed. Then, 3'-hydroxyl-DNA strand breaks in cultured cells were enzymatically labeled with digoxigenin-nucleotide by terminal deoxynucleotidyl transferase and subsequently exposed to horseradish peroxidase-conjugated antidigoxigenin antibody. Staining was developed in diaminobenzidine, and slides were counterstained with hematoxylin. TUNEL assay of cultured leiomyoma cells was performed by two investigators in a blinded fashion without knowledge of the experimental group. All stained nuclei were scored to be positive for TUNEL assay. The TUNEL-positive rate was determined by observing more than 2000 nuclei for each of the experimental samples.

Statistical analysis

The data were expressed as the mean ± SD from at least three independent experiments. Statistical significance was evaluated using one-way ANOVA. A difference with a P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GnRH receptor mRNA expression in cultured leiomyoma cells

The expression of GnRH receptor mRNA was examined using semiquantitative RT-PCR in leiomyoma cells cultured for 2 d under no hormonal treatment. RT-PCR analysis revealed an anticipated 251-bp fragment of GnRH receptor mRNA in cultured leiomyoma cells (Fig. 1Go, lane 1).



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  		FIG. 1. The presence of GnRH receptor mRNA in leiomyoma cells cultured for 2 d under no hormonal treatment. Semiquantitative RT-PCR analysis revealed the presence of a 251-bp fragment of GnRH receptor mRNA in cultured leiomyoma cells. Lane 1, Untreated leiomyoma cells cultured for 2 d; lane 2, ß-actin; lane N, negative control; lane P, positive control (pituitary cDNA); lane M: marker. The experiments were repeated with at least three different cultured specimens with similar results, and the reported results are representative.

 
Effects of Cetrorelix on the number of viable cultured leiomyoma cells

Compared with untreated control cultures, treatment with 10–5 mol/liter Cetrorelix significantly (P < 0.01) decreased the number of viable leiomyoma cells at 2 d of treatment, and this decrease persisted up to 6 d of treatment (Fig. 2Go). Treatment with either 10–6 or 10–7 mol/liter Cetrorelix did not affect the number of viable cells at 2 d of treatment but resulted in a significant decrease (P < 0.05) in the number of viable cells at 4 d of treatment (Fig. 2Go). By contrast, treatment with 10–8 mol/liter Cetrorelix did not affect the number of viable cells throughout the treatment period for 6 d (Fig. 2Go).



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  		FIG. 2. Effects of Cetrorelix on the number of viable cultured leiomyoma cells, as assessed by cell-counting assay. The number of viable cells cultured with graded concentrations of Cetrorelix was counted at 2, 4, and 6 d of treatment. Treatment with 10–8 mol/liter Cetrorelix did not affect the number of viable cultured leiomyoma cells. Treatment with either 10–7 or 10–6 mol/liter Cetrorelix had no effect on the number of viable cultured leiomyoma cells at 2 d of treatment but significantly decreased the number of those cells at 4 and 6 d of treatment. Treatment with 10–5 mol/liter Cetrorelix remarkably decreased the number of viable cultured leiomyoma cells even at 2 d of treatment, and this effect persisted up to 6 d of treatment. Data are expressed as the mean ± SD of at least six independent experiments. *, P < 0.05 vs. untreated control cultures; **, P < 0.01 vs. untreated control cultures.

 
Effects of Cetrorelix on PCNA expression in cultured leiomyoma cells

Compared with untreated control cultures for 2 d (Fig. 3BGo), 4 d (Fig. 3DGo), and 6 d (Fig. 3FGo), PCNA-positive nuclei in cultured leiomyoma cells treated with 10–5 mol/liter Cetrorelix for 2 d (Fig. 3AGo), 4 d (Fig. 3CGo), and 6 d (Fig. 3EGo) were apparent less abundantly. Treatment with either 10–7 or 10–6 mol/liter Cetrorelix resulted in an apparent decrease in PCNA-positive nuclei only at 4 d of treatment compared with untreated control cultures (data not shown). Replacement of the primary antibody with nonimmune murine IgG showed a lack of positive immunostaining in the cultured leiomyoma cell nuclei (Fig. 3GGo).



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  		FIG. 3. Effects of Cetrorelix on immunoreactive PCNA expression in cultured leiomyoma cells, as assessed by immunocytochemistry. PCNA expression was apparent less abundant at 2 d (A), 4 d (C), and 6 d (E) of treatment with 10–5 mol/liter Cetrorelix compared with untreated control cultures for 2 d (B), 4 d (D), and 6 d (F). Replacement of the primary antibody with nonimmune murine IgG resulted in a lack of immunostaining (G). Bars, 5 µm. Original magnification, x200.

 
Figure 4Go shows the PCNA-positive rate of cultured leiomyoma cells treated with graded concentrations of Cetrorelix at indicated treatment periods. Compared with untreated control cultures, treatment with 10–5 mol/liter Cetrorelix significantly (P < 0.01) decreased the PCNA-positive rate of cultured leiomyoma cells at 2 d of treatment. Treatment with either 10–7 or 10–6 mol/liter Cetrorelix showed no significant effect on the PCNA-positive rate at 2 d of treatment but resulted in a significant decrease (P < 0.01) in the PCNA-positive rate at 4 d of treatment compared with untreated control cultures.



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  		FIG. 4. Effects of Cetrorelix on the PCNA-positive rate in cultured leiomyoma cells. Treatment with either 10–7 or 10–6 mol/liter Cetrorelix had no effect at 2 d of treatment but decreased the PCNA-positive rate at both 4 and 6 d of treatment. Treatment with 10–5 mol/liter Cetrorelix significantly decreased the PCNA-positive rate even at 2 d of treatment, and this effect persisted up to 6 d of treatment. Data are expressed as the mean ± SD of at least six independent experiments. *, P < 0.01 vs. untreated control cultures.

 
Western blot analysis of proteins extracted from leiomyoma cells cultured for 2 d in the presence or absence of graded concentrations of Cetrorelix revealed that cultured leiomyoma cells contained immunoreactive PCNA protein with an approximate molecular mass of 36 kDa (Fig. 5Go). The PCNA protein content in cultured leiomyoma cells was significantly (P < 0.01) decreased when treated with 10–5 mol/liter Cetrorelix compared with untreated control cultures (Fig. 5Go). Treatment with either 10–7 or 10–6 mol/liter Cetrorelix for 2 d did not affect the PCNA protein content in cultured leiomyoma cells (Fig. 5Go).



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  		FIG. 5. Effects of Cetrorelix on PCNA protein expression in cultured leiomyoma cells, as assessed by Western blot analysis. Isolated leiomyoma cells were cultured for 2 d in serum-free DMEM in the presence or absence of graded concentrations of Cetrorelix. Each 60-µg aliquot of proteins extracted from cultured leiomyoma cells was subjected to Western blot analysis with a mouse monoclonal antibody to PCNA. The 36-kDa PCNA protein expression observed in untreated leiomyoma cells was decreased by treatment with 10–5 mol/liter Cetrorelix, whereas treatment with either 10–7 or 10–6 mol/liter Cetrorelix for 2 d did not affect the PCNA protein expression in cultured leiomyoma cells. Experiments were repeated at least three times with similar results. Densitometric analysis of PCNA protein levels in cultured leiomyoma cells was performed as described in Materials and Methods. Data were presented as the fold increase over the control value and as the mean ± SD of at least three independent experiments. *, P < 0.01 vs. untreated control cultures.

 
Effects of Cetrorelix on EGF expression in cultured leiomyoma cells

Compared with untreated control cultures for 2 d (Fig. 6BGo), 4 d (Fig. 6DGo), and 6 d (Fig. 6FGo), immunoreactive EGF protein expression in cultured leiomyoma cells treated with 10–5 mol/liter Cetrorelix for 2 d (Fig. 6AGo), 4 d (Fig. 6CGo), and 6 d (Fig. 6EGo) was apparent less abundantly. Treatment with either 10–7 or 10–6 mol/liter Cetrorelix decreased immunoreactive EGF protein expression in cultured leiomyoma cells at 4 d of treatment, although no effect was observed at 2 d of treatment compared with untreated control cultures (data not shown). Replacement of the primary antibody with nonimmune murine IgG showed a lack of positive immunostaining in cultured leiomyoma cells (Fig. 6GGo).



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  		FIG. 6. Effects of Cetrorelix on immunoreactive EGF expression in cultured leiomyoma cells, as assessed by immunocytochemistry. Compared with untreated control cultures for 2 d (B), 4 d (D), and 6 d (F), immunoreactive EGF protein expression in cultured leiomyoma cells treated with 10–5 mol/liter Cetrorelix for 2 d (A), 4 d (C), and 6 d (E) was apparent less abundantly. Replacement of the primary antibody with nonimmune murine IgG resulted in a lack of immunostaining (G). Bars, 5 µm. Original magnification, x200.

 
Semiquantitative RT-PCR analysis demonstrated the presence of a 330-bp fragment of EGF mRNA in cultured leiomyoma cells (Fig. 7Go). The bottom panel illustrates the fold increase of EGF mRNA normalized to the respective ß-actin when treated with 10–5 mol/liter Cetrorelix for 2 and 4 d. Although treatment with 10–5 mol/liter Cetrorelix for 2 d (lane 3) did not affect EGF mRNA expression in cultured leiomyoma cells, the treatment with 10–5 mol/liter Cetrorelix for 4 d (lane 4) resulted in a significant decrease (P < 0.05) in EGF mRNA expression in those cells compared with untreated control cultures for 4 d (lane 2).



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  		FIG. 7. Effects of Cetrorelix on EGF mRNA expression in cultured leiomyoma cells. Semiquantitative RT-PCR analysis revealed the presence of a 330-bp transcript corresponding to EGF mRNA in cultured leiomyoma cells. Experiments were repeated three times with similar results for each. M, Marker; lane 1, untreated leiomyoma cells cultured for 2 d; lane 2, untreated leiomyoma cells cultured for 4 d; lane 3, leiomyoma cells treated with 10–5 mol/liter Cetrorelix for 2 d; lane 4, leiomyoma cells treated with 10–5 mol/liter Cetrorelix for 4 d; lane N, negative control; lane P, positive control (human ovarian cancer cell line SK-OV-3). The bottom panel illustrates the fold increase of EGF mRNA normalized to the respective ß-actin when treated with 10–5 mol/liter Cetrorelix for 2 and 4 d. Although treatment with 10–5 mol/liter Cetrorelix for 2 d (lane 3) did not affect EGF mRNA expression in cultured leiomyoma cells, treatment with 10–5 mol/liter Cetrorelix for 4 d (lane 4) resulted in a significant decrease in EGF mRNA expression in those cells compared with untreated control cultures for 4 d (lane 2). 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 for 4 d; NS, not significant vs. untreated control cultures for 2 d.

 
Effects of Cetrorelix on the TUNEL-positive rate of cultured leiomyoma cells

No apparent effect of graded concentrations of Cetrorelix on the TUNEL-positive nuclei was attained at 12 h of treatment (data not shown). However, TUNEL-positive nuclei were apparent more abundantly when treated with 10–5 mol/liter Cetrorelix for 24 h (Fig. 8AGo) and 36 h (Fig. 8CGo) compared with untreated control cultures for 24 h (Fig. 8BGo) and 36 h (Fig. 8DGo), respectively.



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  		FIG. 8. Effects of Cetrorelix on the appearance of TUNEL-positive nuclei in cultured leiomyoma cells, as assessed by TUNEL assay. TUNEL-positive nuclei were apparent more abundantly when treated with 10–5 mol/liter Cetrorelix for either 24 h (A) or 36 h (C) compared with untreated control cultures for 24 h (B) and 36 h (D). Bars, 5 µm. Original magnification, x200.

 
Figure 9Go shows the TUNEL-positive rate of cultured leiomyoma cells when treated with graded concentrations of Cetrorelix. Compared with untreated control cultures, treatment with 10–5 mol/liter Cetrorelix resulted in a significant increase (P < 0.01) in the TUNEL-positive rate of cultured leiomyoma cells at 24 and 36 h of treatment but not at 12 h of treatment. Treatment with either 10–6 or 10–7 mol/liter Cetrorelix caused a significant increase (P < 0.01) in the TUNEL-positive rate only at 36 h of treatment.



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  		FIG. 9. Effects of Cetrorelix on the TUNEL-positive rate in cultured leiomyoma cells. Treatment with 10–5 mol/liter Cetrorelix increased the TUNEL-positive rate in cultured leiomyoma cells at 24 h of treatment, whereas treatment with either 10–6 or 10–7 mol/liter Cetrorelix resulted in a significant increase in the TUNEL-positive rate only at 36 h of treatment. Data are expressed as the mean ± SD of at least six independent experiments. *, P < 0.01 vs. untreated control cultures.

 
Effects of Cetrorelix on PARP expression in cultured leiomyoma cells

Western blot analysis of proteins extracted from leiomyoma cells cultured for 24 h demonstrated that cultured leiomyoma cells contained immunoreactive cleaved PARP with a molecular mass of 89 kDa (Fig. 10Go). Compared with untreated control cultures, treatment with 10–5 mol/liter Cetrorelix for 24 h significantly (P < 0.01) increased cleaved PARP expression in cultured leiomyoma cells (Fig. 10Go). Treatment with either 10–6 or 10–7 mol/liter Cetrorelix for 24 h did not affect cleaved PARP expression in cultured leiomyoma cells (Fig. 10Go).



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  		FIG. 10. Effects of Cetrorelix on cleaved PARP expression in cultured leiomyoma cells, as assessed by Western blot analysis. Isolated leiomyoma cells were cultured for 24 h in serum-free DMEM in the presence or absence of graded concentrations of Cetrorelix. Each 60-µg aliquot of proteins extracted from cultured leiomyoma cells was subjected to Western blot analysis with a mouse monoclonal antibody to PARP. Western blot analysis revealed the presence of 89-kDa cleaved PARP in untreated leiomyoma cells. Treatment with 10–5 mol/liter Cetrorelix increased cleaved PARP expression in cultured leiomyoma cells, whereas treatment with either 10–6 or 10–7 mol/liter Cetrorelix did not affect cleaved PARP expression in those cells. Experiments were repeated at least three times with similar results. Densitometric analysis was performed as described in Materials and Methods. Data are presented as the fold increase over the control value and as the mean ± SD of at least three independent experiments. *, P < 0.01 vs. untreated control cultures.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The presence of GnRH receptors with two types (10) has been identified in leiomyoma (10, 11, 12, 13). One is a low-affinity binding site, and the other is high-affinity binding site for GnRH, and the latter is comparable to the pituitary GnRH receptor (10). We have demonstrated GnRH receptor mRNA expression in cultured leiomyoma cells using semiquantitative RT-PCR, suggesting a role for GnRH in the regulation of leiomyoma growth. In the present study, the GnRH antagonist Cetrorelix inhibited the proliferation of cultured leiomyoma cells in a dose- and time-dependent manner. Treatment with Cetrorelix decreased the number of viable cultured leiomyoma cells and PCNA protein expression in those cells in a dose- and time-dependent manner. Our results are in accord with the reports showing that GnRH antagonist inhibited the growth of a variety of human cancer cell lines, including breast, endometrial, ovarian, and prostate cancer (25, 26, 27).

It has been reported that the GnRH receptor signal transduction mechanisms that operate in the pituitary are not involved in the mediation of the antiproliferative effects of GnRH analogs in cancer cells (27). GnRH receptors in the pituitary couple to G protein {alpha}q and activate phospholipase C and protein kinase C (39), whereas GnRH receptors in reproductive tumors couple to pertussis toxin-sensitive G proteins of the Gi family and activate phosphotyrosine phosphatase (40, 41). The difference in the signal transduction pathway linked to the GnRH receptor between the pituitary and peripheral tumors may explain the antagonistic action of GnRH antagonist in the pituitary and agonistic action of GnRH antagonist in cancer cells. However, it has been speculated that the dichotomy of GnRH agonists and antagonists does not exist in tumor cells because the proliferation of cancer cell lines expressing GnRH receptors is inhibited by both agonistic and antagonistic analogs of GnRH (34).

In our study, treatment with Cetrorelix down-regulated immunoreactive EGF protein and EGF mRNA expression in cultured leiomyoma cells in a dose- and time-dependent manner. These results suggest a role of the cross-link between Cetrorelix and EGF in the regulation of leiomyoma growth. Several studies have reported that chronic treatment with GnRH antagonist reduces the EGF concentrations in rat prostate cancer (42) and that GnRH agonist counteracts the mitogenic action of EGF in canine mammary tumor cells (43). The antiproliferative activity of GnRH agonist and antagonist has been speculated to be mediated through interaction with the mitogenic signal transduction pathway of EGF receptor in breast, endometrial, ovarian, prostate, and pancreatic cancer (32, 33, 34, 35, 36). GnRH antagonist is shown to down-regulate EGF receptors in prostate, ovarian, and pancreatic cancer (17, 33, 35). GnRH agonist and antagonist reduce EGF-induced c-fos mRNA and c-Fos protein expression in breast, endometrial, and ovarian cancer cell lines expressing GnRH receptor (34). Moreover, GnRH agonist inhibits EGF-induced tyrosine autophosphorylation of EGF receptor (32, 36) and EGF-induced activation of MAPK (44). Gründker et al. (36) suggest that the antiproliferative signal transduction by GnRH agonist is mediated through the pertussis toxin-sensitive G protein {alpha}i and that GnRH receptor activates a phosphotyrosine phosphatase that counteracts EGF receptor tyrosine kinase activity in endometrial and ovarian cancer cells, resulting in down-regulation of mitogenic signal transduction and cell proliferation. Taken together, it seems likely that GnRH antagonist may inhibit the growth of leiomyoma cells through inhibiting the autocrine/paracrine mitogenic activity of EGF and autophosphorylation of EGF receptors.

The present study also demonstrated that treatment with Cetrorelix induced apoptosis in cultured leiomyoma cells in a dose- and time-dependent manner. Treatment with 10–5 mol/liter Cetrorelix increased the TUNEL-positive rate by as early as 24 h of treatment compared with untreated control cultures. Western blot analysis revealed that treatment with Cetrorelix augmented cleaved PARP expression in cultured leiomyoma cells in a dose-dependent manner. Treatment with either 10–6 or 10–7 mol/liter Cetrorelix did not affect cleaved PARP expression in cultured leiomyoma cells, whereas 10–5 mol/liter Cetrorelix significantly increased cleaved PARP expression at 24 h of treatment compared with untreated control cultures. These results indicate that Cetrorelix exhibits the proapoptotic activity in cultured leiomyoma cells. Likewise, GnRH antagonist has been reported to induce apoptosis in prostate cancer xenografted into nude mice (45) and ovarian cancer cells (21). However, little is known about the molecular mechanism underlying GnRH antagonist action on the induction of apoptosis of leiomyoma cells. Nonetheless, we have previously demonstrated that treatment with GnRH agonist can induce apoptosis in cultured leiomyoma cells by increasing Fas expression and by inducing Fas ligand, suggesting that the Fas/Fas ligand system may participate in GnRH agonist-induced apoptosis in cultured leiomyoma cells (13). More recently, Bifulco et al. (46) have provided molecular evidence that GnRH agonist-induced reduction in leiomyoma volume is mediated by a decreased activation of the phosphatidyl inositol 3-kinase (PI3k)/protein kinase B (PKB) survival pathway and by the suppression of antiapoptotic proteins such as Fas-associated death domain-like IL-1ß-converting enzyme (FLICE)-like inhibitory protein (FLIP) and phosphoprotein enriched in astrocytes of 15 kDa (PED/PEA15).

During a process of apoptosis, caspase-3 is shown to cause the proteolytic cleavage and inactivation of PARP (47, 48, 49), producing an 89-kDa C-terminal fragment and a 24-kDa N-terminal fragment (49). PARP is a Zn-finger nuclear protein activated by DNA breaks. PARP is implicated in DNA replication, transcription, DNA repair, apoptosis, and genome stability (50). Thus, PARP cleavage has been used as a prominent biochemical hallmark of apoptosis (48, 49). In the present study, of particular importance was the enhanced expression of cleaved PARP in cultured leiomyoma cells treated with Cetrorelix. The fact that treatment with Cetrorelix augmented the 89-kDa cleaved PARP expression in cultured leiomyoma cells suggests that Cetrorelix can induce apoptosis in cultured leiomyoma cells at least in part through activation of the caspase cascade.

Clinical use of GnRH antagonists in most patients with uterine leiomyomas is safe and highly effective (4, 5, 6, 7). Mean plasma Cetrorelix concentrations in patients with leiomyoma when treated with 60 mg im Cetrorelix were reported to be 18.6 ng/ml within 1 h after the first dose of depot Cetrorelix and fell to 5.61 ng/ml within 6 d (6). Compared with plasma Cetrorelix concentrations in a clinical setting, the concentrations of Cetrorelix used in the present study were extremely high. However, the plasma Cetrorelix concentrations may not necessarily be the same as those in culture media. The optimal tissue Cetrorelix concentrations to induce regression of leiomyoma size remain to be elucidated. This elucidation will contribute to the development of an efficient clinical use of Cetrorelix by providing the optimal tissue concentrations in leiomyomas.

In conclusion, we have demonstrated that GnRH antagonist Cetrorelix exerts antiproliferative effects by down-regulating PCNA and EGF expression and proapoptotic effects in cultured leiomyoma cells by inducing apoptosis through the proteolysis of PARP. Further studies on the molecular mechanism underlying the action of GnRH antagonist on leiomyoma growth and apoptosis will open up a new avenue for the potential clinical application of GnRH antagonist in the treatment of uterine leiomyomas.


   Acknowledgments
 
We thank Zentaris GmbH (Frankfurt, Germany) for providing us with Cetrorelix for the present study.


   Footnotes
 
This work was supported in part by Grants Aid for Scientific Research No. 10470346 from the Japanese Ministry of Education, Science and Culture and by the Ogyaa-Donation Foundation of the Japan Association of Maternal Welfare.

First Published Online November 9, 2004

Abbreviations: DMSO, Dimethylsulfoxide; EGF, epidermal growth factor; PARP, poly(ADP-ribose) polymerase; PCNA, proliferating cell nuclear antigen; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling.

Received August 10, 2004.

Accepted October 29, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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