ABSTRACT. Cell proliferation and apoptosis in uterine leiomyoma were inves- tigated during therapy with GnRH agonist (GnRHa). Patients with uterine ...
0021-972X/98/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1998 by The Endocrine Society
Vol. 83, No. 4 Printed in U.S.A.
Suppression of Cell Proliferation and Induction of Apoptosis in Uterine Leiomyoma by GonadotropinReleasing Hormone Agonist (Leuprolide Acetate) TAKAHIRO MIZUTANI, AYAKO SUGIHARA, KARO NAKAMURO, NOBUYUKI TERADA
AND
Department of Obstetrics and Gynecology (T.M., K.N.), Osaka Prefectural Hospital, Sumiyoshi, Osaka 588 Japan; and Department of Pathology (A.S., N.T.), Hyogo College of Medicine, Hyogo 663 Japan ABSTRACT Cell proliferation and apoptosis in uterine leiomyoma were investigated during therapy with GnRH agonist (GnRHa). Patients with uterine leiomyomas were injected with 3.75 mg GnRHa (depot leuprolide acetate) at intervals of 4 weeks and underwent hysterectomy or myomectomy at the 2nd, 4th, 8th, 12th, or 16th week of GnRHa therapy. Tissue sections of leiomyomas from these patients and from control patients (control patients received no GnRHa therapy) were stained with the Ki-67 antibody or by an in situ DNA 39-end labeling method, and numbers of Ki-67 immunostained cells and DNA 39-endlabeled cells per cm2 were examined as indices of cell proliferation and apoptosis, respectively. The number of Ki-67 immunostained cells/
U
TERINE leiomyomas are benign tumors originated from uterine smooth muscle cells. Uterine leiomyomas have estrogen and progesterone receptors, and their growth and maintenance are sensitive to estrogen and/or progesterone; and depletion of these steroids induces their regression (1). GnRH agonist (GnRHa) inhibits gonadotropin secretion from the pituitary gland by causing pituitary desensitization by down-regulation of GnRH receptors, and GnRHa decreases estrogen and progesterone secretion from the ovary (2). Therefore, therapy of leiomyomas with GnRHa has been tried and has been shown to reduce leiomyoma volume (1, 3– 6). The reduction of tumor volume could be caused by a decrease in cell proliferation, an increase in cell loss by apoptosis or necrosis, and cellular atrophy. However, routine histological examinations of leiomyomas after GnRHa therapy have failed to demonstrate these changes, or their results about these changes contradict each other (7, 8). Moreover, there are no histological examinations in which time courses of these changes were examined. Therefore, the present study was intended to investigate time course effects of GnRHa therapy with depot leuprolide acetate on cell proliferation and apoptosis in uterine leiomyomas.
Received September 11, 1997. Revision received December 2, 1997. Accepted December 9, 1997. Address all correspondence and requests for reprints to: Takahiro Mizutani, Department of Obstetrics, Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 594 –1101, Japan.
cm2 in leiomyomas at the 2nd week of the GnRHa therapy was comparable with that of control patients. However, it decreased to a level less than one forth that of control patients at the 4th week, and it remained at similar low levels at the 8th, 12th, and 16th week. The number of DNA 39-end-labeled cells/cm2 in leiomyomas of control patients and in leiomyomas at the 2nd, 8th, 12th, and 16th weeks of GnRHa therapy were at low levels but, at the 4th week, was at an extremely high level (about 5 times more than that of control patients). The present results indicate that GnRHa therapy suppresses cell proliferation and causes a transient increase in apoptosis in uterine leiomyomas. (J Clin Endocrinol Metab 83: 1253–1255, 1998)
Materials and Methods Patients All procedures were carried out under a protocol approved by the Institutional Review Board of the Osaka Prefectural Hospital, and informed consent was obtained from all patients (61 patients, showing regular menstrual cycles). Their average age 6 sem was 43.6 6 1.0 (range: 30 – 49).
Treatment protocol Sixty-one patients, who were to undergo hysterectomy or myomectomy because of leiomyomas, were divided into 2 groups; a control group (18 patients) and a GnRHa-treatment group (43 patients). Patients in a GnRHa-treatment group were injected sc with 3.75 mg depot leuprolide acetate (Leuprine Depot, Takeda Pharmaceutical Co., Osaka, Japan) at intervals of 4 weeks. Five, 10, 13, 8, and 7 patients underwent hysterectomy or myomectomy at the 2nd, 4th, 8th, 12th, and 16th week of GnRHa treatment. Patients in a control group underwent hysterectomy without any medications. One intramural uterine leiomyoma obtained from each patient was fixed in phosphate (0.01 mol/L) buffered 10% paraformaldehyde, and tissue sections of a part of each leiomyoma were prepared. Blood was drawn from control patients, and patients who underwent hysterectomy or myomectomy at the 2nd and 4th week of GnRHa treatment, on the day of the operation, for assay of serum estradiol-17b (E2).
Immunohistochemistry of Ki-67 antigen Deparaffinized tissue sections (5-mm thickness), mounted on silanecoated glass slides, were treated with 3% H2O2 in 50% methanol for 30 min. Then, these sections were heated in 10 mmol/L sodium citrate buffer (pH 6.0) for 5 min in a microwave oven (Matsushita Electric Industries, Osaka, Japan) at 600 Watt, four times, to facilitate antigen retrieval. They were sequentially incubated with normal goat serum (1:50 dilution) for 30 min at room temperature, with the rabbit polyclonal antibody (1:100 dilution) for human Ki-67 antigen (Dako Japan, Kyoto, Japan) at 4 C overnight, with biotinylated goat antirabbit IgG (1:200
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dilution) (Vector, Peterborough, England) for 30 min at room temperature. The immunoreaction was visualized by an avidin-biotin peroxidase complex method using a Vectastain Elite ABC kit (Vector) with 3,39-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO). The sections were lightly counterstained with hematoxylin.
In situ DNA 39-end labeling In situ DNA 39-end labeling was carried out according to the method of Gavrieli et al. (9). Terminal deoxynucleotidyl transferase (Toyobo, Osaka, Japan) and biotin-16-deoxyuridine 59-triphosphate (Boehringer Mannheim Yamanouchi, Tokyo, Japan) were used at concentrations of 0.2 e.u./mL and 10 mmol/L, respectively. A Vectastain Elite ABC kit (Vector) and 3,39-diaminobenzidine tetrahydrochloride (Sigma) were used for the detection of biotinylated deoxyuridine 59-triphosphate incorporated into DNA.
Number of Ki-67 immunostained cells or apoptotic cells in leiomyomas A part of a tissue section of a leiomyoma on a slide glass was enclosed in ink, and the number of Ki-67-immunostained leiomyoma cells or in situ DNA 39-end-labeled cells in an enclosed area was counted. Then, an enclosed area was measured with an IBAS image analysis systems (Carl Zeiss, Oberkochen, Germany), and the number of these cells/cm2 was calculated. An average area was 0.975 cm2 (range: 0.756 –1.115 cm2).
FIG. 1. Ki-67 immunohistochemical staining (A) and in situ DNA 39-end labeling (B) of a leiomyoma during GnRHa therapy. Arrows show positively stained or labeled cells.
Assay of serum E2 level The serum E2 level was determined using an RIA kit (Diagnostic Products Co., Los Angeles, CA). The intra- and interassay coefficients of variation were 6.1% and 6.8%, respectively.
Statistics Values are presented as means 1 sem. Statistical significance was evaluated by one-way ANOVA (P , 0.01).
Results
Serum concentrations (means 6 sem) of E2 of control patients (receiving no GnRHa therapy) and patients at the 2nd and 4th weeks of GnRHa therapy were 109.5 6 30 (n 5 18), 145.5 6 35 (n 5 5), and 20.4 6 4 (n 5 10) pg/mL, respectively. A serum concentration of E2 did not decrease at the 2nd week, but it decreased markedly and significantly at the 4th week. Histologies of the uterine endometrium from these 18 control patients showed that 1 patient was at the proliferative phase and 17 patients at the secretory phase. Thinning of the endometrium, stromal fibrosis, and atrophy of the glands were found in the endometrium of the uteri at the 8th, 12th, and 16th week, but the appearance and extent of these changes in the endometrium at the 4th week varied among patients. Cell proliferation of leiomyomas during GnRHa therapy was evaluated by examining the number of Ki-67-immunostained cells/cm2 tissue section of a leiomyoma. Ki-67-immunostained cells were scattered in a tissue section (Fig. 1A), and an average number of Ki-67-immunostained cells/cm2 in leiomyomas of control patients was about 400 (Fig. 2). The number of Ki-67-immunostained cells/cm2 in leiomyomas at the 2nd week of GnRHa therapy was as much as that in leiomyomas of control patients. However, it decreased to a level less than one forth of the number in leiomyomas of control patients at the 4th week. The number of Ki-67-immunostained cells/cm2 at the 8th, 12th, and 16th week remained at low levels similar to that at the 4th week.
FIG. 2. Change of frequency of Ki-67 immunostained cells in leiomyomas during GnRHa therapy. Patients with leiomyomas were injected with depot leuprolide acetate (3.75 mg) at intervals of 4 weeks and underwent hysterectomy or myomectomy at the 2nd, 4th, 8th, 12th, or 16th week of the GnRHa therapy. Numbers of Ki-67-immunostained cells/cm2 of tissue sections of leiomyomas from these patients and control patients were estimated. The height of a bar represents a mean 1 SEM. The figure above the bar represents the number of leiomyomas examined. **, P , 0.01, significant difference from the value of a control group.
Apoptotic cells were recognized by detection of their DNA fragmentation by an in situ DNA 39-end-labeling method (Fig. 1B), and a frequency in a leiomyoma was evaluated by examining the number of apoptotic cells/cm2 tissue section of a leiomyoma (Fig. 3). Apoptotic cells were found in a cluster, rather than scattered. An average number of apoptotic cells/cm2 in leiomyomas of control patients was less than 100, and numbers of apoptotic cells/cm2 in leiomyomas at the 2nd, 8th, 12th, and 16th week of GnRHa therapy were at levels as low as that in leiomyomas of control patients. However, the number of apoptotic cells/cm2 at the 4th week was at an extremely high level, and an average number was
GnRHa THERAPY OF UTERINE LEIOMYOMAS
FIG. 3. Change of frequency of apoptotic cells in leiomyomas during GnRHa therapy. Tissue sections of leiomyomas described in the legend of Fig. 2 were stained by the in situ DNA 39-end-labeling method for the detection of apoptotic cells, and the number of apoptotic cells (labeled cells)/cm2 was estimated. The height of a bar represents a mean 1 SEM. The figure above the bar represents number of leiomyomas examined. **, P , 0.01, significant difference from the value of a control group, by one-way ANOVA.
about five times more than that in leiomyomas of control patients. Discussion
The Ki-67 antigen is a nuclear protein that presents in the G1, S, G2, and M phases but not in the G0 phase of the cell cycle, and it is used as a sensitive index of cell proliferation (10, 11). The present results showed that numbers of Ki-67stained cells in leiomyomas markedly decreased from the 4th week of GnRHa therapy, indicating that GnRHa reduces cell proliferation of leiomyomas. On the contrary, studies that examined cell proliferation by mitotic figures failed to detect an inhibitory effect of GnRHa on cell proliferation of leiomyomas (7, 8). The difference seems to be ascribed to the difference in sensitivity of the method for estimation of cell proliferation. Examination of apoptosis by the in situ DNA 39-end-labeling method revealed a transient increase in apoptosis of leiomyoma cells at the 4th week of GnRHa therapy, and low levels of apoptosis at all times other than this week. This transient increase in apoptosis seems, in part, to be responsible for the early reduction of leiomyoma volume within 1 month of GnRHa therapy (5, 6). A transient increase in apoptosis occurs in the rodent’s uterus after estrogen depletion, and this is responsible for its atrophy (12). On the other hand, the other mechanisms would be responsible for its late reduction after 1 month. Reduction of arterial blood flow of the uterus and leiomyomas caused by hypoestrogenism induced by GnRHa therapy would be one of these mechanisms, as suggested by several studies (1, 5, 6, 13, 14). A serum concentration of E2 decreased at the 4th week, when the number of Ki-67-immunostained cells per unit area decreased and the number of apoptotic cells per unit area increased. Therefore, the decrease in cell proliferation and the increase in apoptosis seem to be caused by the decrease
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in a serum concentration of E2. Chegini et al. (15) reported that uterine leiomyomas and myometrium had GnRH receptors, and that GnRHa directly inhibited DNA synthesis of cultured myometrial cells in the presence or absence of estrogen and/or progesterone, suggesting that GnRHa directly suppresses cell proliferation. However, this suggestion seems to be less possible because GnRHa did not suppress cell proliferation until the 4th week of GnRHa therapy. Hypoestrogenism induced by GnRHa therapy causes adverse effects, such as osteoporosis, vasomotor symptoms, and vaginal atrophy (1). Because of these adverse effects, the duration of GnRHa therapy is limited, and cessation of GnRHa therapy induces regrowth of leiomyomas (1). Therefore, to prevent these adverse effects, GnRHa/steroid addback therapy has been devised (1). In this therapy, estrogen and/or progesterone, at minimal doses (which do not affect the growth of leiomyomas) are administered to patients during GnRHa therapy. The present results indicate that GnRHa suppresses cell proliferation and induces apoptosis at the 4th week of GnRHa treatment. Therefore, we propose that estrogen should be administered after the 4th week of GnRHa therapy in GnRHa/steroid add-back therapy. References 1. Friedman AJ. 1993 Treatment of uterine myomas with GnRH agonists. Semin Reprod Endocrinol. 11:154 –161. 2. Monroe SE, Blumenfeld Z, Andreyko JL, Schriock E, Henzl MR, Jaffe RB. 1986 Dose-dependent inhibition of pituitary-ovarian function during administration of a gonadotropin-releasing hormone agonistic analog (nafarelin). J Clin Endocrinol Metab. 63:1334 –1341. 3. Filicori M, Hall DA, Loughlin JS, Rivier J, Vale W, Crowley WF. 1983 A conservative approach to the management of uterine leiomyoma: pituitary desensitization by a luteinizing hormone-releasing hormone analogue. Am J Obstet Gynecol. 147:726 –727. 4. Andreyko JL, Blumenfeld Z, Marshall LA, Monroe SE, Hricak H, Jaffe RB. 1988 Use of an agonistic analog of gonadotropin-releasing hormone (nafarelin) to treat leiomyomas: assessment by magnetic resonance imaging. Am J Obstet Gynecol. 158:903–910. 5. Shaw RW. 1989 Mechanism of LHRH analogue action in uterine fibroids. Horm Res. 32:150 –153. 6. Aleem FA, Predanic M. 1995 The hemodynamic effect of GnRH agonist therapy on uterine leiomyoma vascularity: a prospective study using transvaginal color Doppler sonography. Gynecol Endocrinol. 9:253–258. 7. Colgan TJ, Pendergast S, LeBlanc M. 1993 The histopathology of uterine leiomyomas following treatment with gonadotropin-releasing hormone analogues. Hum Pathol. 24:1073–1077. 8. Sreenan JJ, Prayson RA, Biscotti CV, Thornton MH, Easley KA, Hart WR. 1996 Histopathologic findings in 107 uterine leiomyomas treated with leuprolide acetate compared with 126 controls. Am J Surg Pathol. 20:427– 432. 9. Gavrieli Y, Sherman Y, Ben-Sasson SA. 1992 Identification of programmed cell death in situ via specific labeling nuclear DNA fragmentation. J Cell Biol. 119:493–501. 10. Gerdes J, Li L, Schlueter C, et al. 1991 Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67. Am J Pathol. 138:867– 873. 11. Cattoretti G, Becker MHG, Key G, et al. 1992 Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections. J Pathol. 168:357–363. 12. Finn CA, Publicover M. 1981 Hormonal control of cell death in the luminal epithelium of the mouse uterus. J Endocrinol. 91:335–340. 13. Matta WHM, Stabile I, Shaw RW, Campbell S. 1988 Doppler assessment of uterine blood flow changes in patients with fibroids receiving the gonadotropin-releasing hormone agonist Buserelin. Fertil Steril. 49:1083–1085. 14. Rutgers JL, Spong CY, Sinow R, Heiner J. 1995 Leuprolide acetate treatment and myoma arterial size. Obstet Gynecol. 86:386 –388. 15. Chegini N, Rong H, Dou Q, Kipersztok S, Williams S. 1996 Gonadotropinreleasing hormone (GnRH) and GnRH receptor gene expression in human myometrium and leiomyomata and the direct action of GnRH analogs on myometrial smooth muscle cells and interaction with ovarian steroids in vitro. J Clin Endocrinol Metab. 81:3215–3221.
