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Department of Obstetrics and Gynecology, School of Medicine, Kanazawa University, Kanazawa 920-0934, Japan
Address all correspondence and requests for reprints to: Makio Shozu, M.D., Ph.D., Department of Obstetrics and Gynecology, School of Medicine, Kanazawa University, Kanazawa 920-0934, Japan. E-mail: shozu{at}med.kanazawa-u.ac.jp
Abstract
We have shown that in situ estrogen synthesized in leiomyoma of the uterus plays a possible role in the promotion of leiomyoma cell growth via an autocrine/paracrine mechanism. In the present study, we demonstrated that leuprorelin acetate, a GnRH agonist widely used for treatment of uterine leiomyoma by down-regulation of pituitary-ovarian function, suppressed the expression of aromatase P450 (an estrogen synthetase) in leiomyoma cells. Given the role of in situ estrogen in leiomyoma cell growth, the inhibition of in situ estrogen synthesis may play a role in GnRH agonist-induced rapid regression of leiomyomas. Quantitative RT-PCR revealed that in women receiving no medication uterine leiomyomas express aromatase P450 mRNA at levels 20 times higher than that in the surrounding myometrium. Leuprorelin acetate treatment (1.88 mg every 4 wk, sc injection) for 12–24 wk reduced the expression of aromatase P450 mRNA in leiomyoma tissue as well as in the myometrium, to approximately one tenth of that in the myometrium of untreated women. Suppression of aromatase P450 expression was also demonstrated by Western blot analysis and aromatase activity assay of microsomal fractions prepared from leiomyomas. On the other hand, no differences in the levels of activity and mRNA of aromatase P450 were observed between leiomyoma cells obtained from women treated with and without leuprorelin acetate injections when cells were cultured ex vivo and stimulated by various combinations of stimulants such as dexamethasone + IL-1ß. The addition of various concentrations of E2 did not affect the aromatase activity of leiomyoma cells, suggesting that deprivation of circulating (ovarian) estrogen is not a cause of decreased expression of aromatase during leuprorelin acetate therapy. On the other hand, 8-d treatment with leuprorelin acetate (100 nmol/liter) reduced dexamethasone + IL-1ß-induced activity and a mRNA level of aromatase by 28% and 42%, respectively. These results indicated that leuprorelin acetate inhibits the expression of aromatase P450 in leiomyoma cells, which contributes to the rapid regression of leiomyoma during leuprorelin acetate therapy.
GnRH is a decapeptide that is produced in the hypothalamus and secreted into the portal vein to stimulate gonadotropin release from the pituitary. GnRH is now recognized as a central regulator of the hypothalamus-pituitary-gonadal axis. Recently, the expression of GnRH as well as its receptor was demonstrated in a variety of healthy and diseased extra-pituitary tissues, suggesting autocrine/paracrine roles for GnRH and its receptor at sites distal to the pituitary (1, 2). These extra-pituitary tissues are sex steroid-dependent tissues and their tumors, from such sites as the breast (3), ovary (4, 5), prostate (6), endometrium (7), and placenta (8). Cell lines that established from these tissues also express GnRH, GnRH receptors, or both (2). The physiological role of GnRH and GnRH receptor expression in these extra-pituitary tissue and cells remain to be elucidated. It is, however, widely accepted that GnRH agonists exhibit antiproliferative effects on certain human tumors of the reproductive organs and cells (9, 10, 11, 12). In vitro experiments using cell lines derived from these organs demonstrate that some of these growth inhibitory effects are due to the direct action of the GnRH agonist (7, 13, 14). GnRH agonists have already been applied to the treatment of ovarian cancers, particularly in refractory cases (15, 16, 17, 18, 19, 20, 21), and significant reduction in tumor size has been observed in one third of cases treated (10, 22).
GnRH agonists are now widely used for the treatment of leiomyoma of the uterus. In most cases, GnRH agonists decrease the size of uterine leiomyomas by 30% or more within 3–6 months of therapy (23, 24). The underlying rationale of this therapy is that GnRH agonists desensitize pituitary gonadotrophs and reduce ovarian steroids, which in turn induce a reduction in the steroid hormone-dependent growth of leiomyomas. In addition to this indirect mechanism, GnRH agonists may also exhibit direct suppression of cellular growth of leiomyomas, as in the case of malignant tumors described above. Recently, other researchers and we have provided evidence that leiomyomas overexpress aromatase P450, an estrogen synthetase, and actually synthesize sufficiently significant amounts of estrogen to promote their own cell growth (25, 26). In situ estrogen may play a role in the rapid growth of a leiomyoma over the surrounding myometrium. More recently, we found that preoperative treatment with leuprorelin acetate (LA), a GnRH agonist, markedly reduced the expression of aromatase P450 in leiomyomas. This raises the possibility that GnRH agonists inhibit aromatase P450 in leiomyoma cells to reduce production of in situ estrogen, which, in turn, reduces the growth of leiomyoma cells. Thus, suppression of in situ estrogen can be an additional mechanism of GnRH agonist-induced growth retardation of leiomyoma cells. This may also explain why leiomyomas shrink rapidly in women treated with GnRH agonist while the same tumor shows relatively slow regression after natural menopause (27).
In the present study, we demonstrate the inhibitory action of GnRH agonist on the aromatase P450 of leiomyoma cells. We also discuss the role of in situ estrogen on the cellular growth of leiomyoma cells during LA therapy as compared with that after natural menopause.
Materials and Methods
Tissue acquisition
Myometrial tissue was obtained from women at hysterectomy for uterine leiomyoma following approval from the Medical Ethics Committee of Kanazawa University (no. 067). Informed consent was obtained from all 25 patients who were randomly selected and enrolled in this study. Women with evidence of adenomyosis and/or endometriosis at the time of laparotomy were excluded. Leiomyoma specimens were taken from the leiomyomatous tissue just beneath the capsule of the nodule that showed ordinary leiomyomatous histology, with no cellular, epithelioid, bizarre, or plexiform variants. In the case of multiple nodules, leiomyoma specimens were taken from the largest nodule. LA (1.88 mg) was administered by sc injection every 4 wk for 12–24 wk before surgery. Although preoperative LA therapy (LA group) was offered to all patients, 8 of 25 patients rejected LA therapy and were, thus, assigned to the "no LA group." Consequently, differences in the size of the largest nodule (maximum diameter measured before the beginning of LA therapy) and age at laparotomy were not significant between the two groups: 7.4 ± 0.6 cm in the LA group vs. 7.3 ± 0.7 cm in the no LA group and 44.1 ± 0.9 yr old in the LA group vs. 44.9 ± 1.4 yr old in the no LA group, respectively. Durations from the initial injection to the surgery and from the last injection to surgery in the LA group were 15.4 ± 1.4 wk and 12.5 ± 1.6 d, respectively. Tissue samples were dissected immediately after surgery, snap-frozen in liquid nitrogen, and then stored at -76 C. Surrounding myometrium was taken more than 2 cm away from the leiomyoma capsule and treated and stored similarly. Because the level of aromatase P450 mRNA expression did not statistically differ between leiomyomas obtained at the proliferative phase and those at the secretory phase, all patients without LA therapy were included in one group irrespective of the menstrual cycle.
Cell culture
Isolation and culture of smooth muscle-like cells from leiomyoma
nodules (leiomyoma cells) and morphological validation of smooth
muscle-like cells were performed as described previously
(26). Briefly, leiomyoma tissue was minced and digested by
collagenase type II (1 mg/ml; Roche, Mannheim, Germany)
and deoxyribonuclease I (0.15 mg/ml; Sigma, St. Louis, MO)
for 3 h at 37 C with vigorous shaking. Digests were filtrated
through three layers of sterile gauze and then cultured in DMEM/F-12
medium supplemented with 10% FBS (Sigma), 100 IU/ml
penicillin, 100 µg/ml streptomycin, and 100 µg/ml kanamycin
(Life Technologies, Inc., Gaithersburg, MD). It took 3–8
d for the freshly isolated cells to reach confluency. The culture was
maintained for no more than 4 wk. Cells in culture were confirmed to
have the characteristic features of uterine muscle cells: a fusiform
shape, expression of smooth muscle-specific 
-actin, and estrogen
responsiveness. More than 95% of cells stained positively with smooth
muscle-specific -actin (-smooth muscle actin immunohistology kit;
Sigma). All experiments on leiomyoma cells were conducted
on first or second passage cells.
RT-PCR
Total RNA was extracted from snap-frozen tissue samples using an Ultraspec RNA isolation kit (Biotecx, Houston, TX) according to the manufacturer’s instructions. RNA concentration was determined spectrophotometrically. Competitive RT-PCR to quantify the aromatase P450 coding sequence was conducted as described previously (26). Briefly, 1 µg total RNA was combined with a known amount of internal-standard RNA that had 107 bp of wild-type sequence deleted between exon II and III and then reverse-transcribed. The synthesized cDNA representing 25 ng total RNA was PCR-amplified using the primers Arom205 (5'- CTCCTCACTGGCCTTTTTCTC-3') and Arom203 (5'-GCCGAATCGAGAGCTGTAAT-3'). The resultant PCR products were resolved on a 1.5% agarose gel. After staining with ethidium bromide, the gel was photographed and the intensity of the signal was analyzed using the NIH Image program (version 1.61). Densitometric values were normalized to the length of the band. The ratios of the densitometric values of the target to that of the internal standard was used to calculate the amount of aromatase mRNA in a given sample. The standard curves (plots of the logarithmically transformed ratios of the target intensity to the competitor intensity against the log amount of initially added target) showed a significant linear regression over the three-log scale (0.01–24 attomol). Calculated intra-assay and interassay variabilities were 7% and 17%, respectively. Intra-assay variability was calculated as a mean of variability from four different samples assayed in triplicates, and interassay variability was calculated from the results of four independent RT-PCR assay of the same RNA aliquot. Similarly, mRNA of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was quantitated to monitor the quality of total RNA samples as described elsewhere (26). The PCR primers used were G3PDH1 (5'-CTGAGAACGGGAAGCTTGTCATCAATGG-3') and G3PDH2 (5'-TGTGGTCATGAGTCCTTCCACG- ATACCA-3').
Assay for aromatase activity
The aromatase activity of primary cells was assayed by detecting
the formation of tritiated water from
[1ß-3H]-androstenedione (NEN Life Science Products, Boston, MA) as described (26, 28, 29). A preliminary experiment showed that aromatase activity
measured by this technique increased linearly from a 4- to 12-h
incubation with [1ß-3H]-androstenedione. We
used a 6-h incubation for the experiment shown in Fig. 5
and a 12-h
incubation for those shown in Figs. 3 and 4 to evaluate the basal level
of expression as precisely as possible. Aromatase activity was
expressed as the rate of incorporation of tritium into water per
milligram of protein per 6 or 12 h of incubation. The LA used for
cell culture experiment was a kind gift from Takeda Pharmaceutical
Company (Osaka, Japan).
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1 g) were
homogenized in 5 ml buffer containing 10 mmol/liter PBS (pH 7.4), 150
mmol/liter potassium chloride, 1 mmol/liter EDTA, 2 mmol/liter
magnesium chloride, 1 mmol/liter dithiothreitol, 1 mmol/liter
phenylmethylsulfonyl fluoride, 1 mmol/liter pepstatin, and 1x
Complete (Roche) using a Polytron homonenizer
(PT10–35; Kinematica AG, Littau/Luzern, Switzerland). Homogenates were
centrifuged at 1,000 x g for 10 min, and the
supernatant was then centrifuged for 60 min at 105,000 x
g. Resultant pellets were washed twice and resusupended in
200 µl buffer containing 50 mmol/liter PBS (pH 7.4), 20% glycerol, 1
mmol/liter EDTA, and 1 mmol/liter dithiothreitol. A 500-µg sample of
microsomal protein was incubated with 1 ml assay buffer containing 50
mmol/liter PBS, 10 mmol/liter NADPH, 4 mmol/liter magnesium chloride,
100 nmol/liter cold androstenedione, and 50 nmol/liter
[1ß-3H]-androstenedione. The reaction was
initiated by the addition of the mixture of cold and radiolabeled
androstenedione at 37 C and stopped after 30 min by the addition of 1
ml 20% trichloroacetic acid. Unused
[1ß-3H]-androstenedione was removed from the
mixture by chloroform extraction, followed by dextran-coated charcoal
adsorption as described for cell samples. Radioactivities of the water
phase were measured, and aromatase activity was expressed as the rate
of incorporation of tritium into water per milligram of protein per 30
min. Western blotting
Western blotting and detection of signals were conducted as described (26). Antibody for aromatase was a kind gift from Dr. N. Harada (Fujita Health University, Nagoya, Japan).
Protein quantitation
Cells in 12-well plates were lysed in 1 ml PBS containing 0.5% SDS. Protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL). The protein concentrations of microsomal fractions were determined from 50-fold dilution of samples.
Statistics
All data are expressed as the mean ± SEM. Differences between two groups were evaluated using the Mann-Whitney U test for unpaired data and the Wilcoxon signed rank test for paired data (StatView 4.0 software; SAS Institute, Inc., Cary, NC). The effects of LA treatment on aromatase P450 mRNA levels were assessed using a 2 x 2 ANOVA test. Statistical significance was established at the P less than 0.05 level.
Results
Suppression of aromatase P450 expression in tissue samples by preoperative LA therapy
RT-PCR analysis of tissue samples. The level of aromatase P450
mRNA was quantified by competitive RT-PCR (Fig. 1A). In women receiving no preoperative
LA therapy (no LA group), leiomyomas expressed 20 times more aromatase
P450 mRNA than the homologous myometrium did, as seen in our previous
study (Fig. 1A, two left columns) (26). LA
therapy for 12–24 wk (LA group) significantly reduced the expression
of aromatase P450 mRNA in both leiomyoma and myometrium tissue (Fig. 1A, two right columns), to approximately one tenth of that
found in the myometrium of untreated women (Fig. 1A, left).
After LA therapy, there was no longer any difference in the levels of
expression between leiomyoma and the corresponding myometrium
samples (Fig. 1A, two right columns).
|
, all three leiomyoma samples from LA
group showed lower protein levels as well as activity than the three
samples obtained from the no LA group.
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RT-PCR analysis of leiomyoma cells in culture. To examine
whether LA-induced suppression of aromatase P450 is maintained in
vitro, the level of aromatase P450 mRNA was quantified in cultured
smooth muscle cells obtained from leiomyoma nodules (leiomyoma cells).
Leiomyoma cells obtained from the LA group and those from the no LA
group were incubated in the presence or absence of dexamethasone (DEX)
+ IL-1ß for 24 h, and then aromatase activity was measured. As
shown in Fig. 1B
, there was no difference in either the basal level of
aromatase P450 expression or in the DEX + IL-1ß-induced level
of aromatase P450 expression between the LA group and the no LA group.
Thus, preoperative LA therapy suppressed the level of expression in
tissue in vivo but had no similar prolonged effect once the
cells were cultured ex vivo. Interestingly, the basal level
of expression, as low as that observed in myometrial and leiomyomatous
tissue of the LA group (shown in Fig. 1A, right), increased
close to the tissue level of expression in one day of culture (Fig. 1A, left). This finding is compatible with the notion that
leiomyoma cells maintain their ability to express aromatase P450 in
response to suitable stimulants that exist in the tissues and decrease
the basal level of expression when cultured ex vivo because
of the removal of stimulants.
Regulation of aromatase activity in
leiomyoma cells in culture.The expression of aromatase P
450 is regulated both in the myometrium and leiomyomas by various
stimulants, such as phorbol myristyl acetate (PMA), IL-1ß,
DEX, and PGE2 (26). To examine whether preoperative LA
therapy alter the regulation of aromatase P450 in leiomyoma cells in
culture, aromatase activity was determined in the presence of these
stimulants. As shown in Fig. 3, the basal level of aromatase activity
did not differ between the LA group and the no LA group. Among the
stimulants tested, the combination of DEX + IL-1ß increased aromatase
activity most effectively, followed by PMA alone and PGE2 + IL-1ß.
Neither increases in the response to each stimulant or the basal level
of expression differed between the LA group and the no LA group. Thus,
aromatase activity of leiomyoma cells in vitro was not
altered by preoperative LA therapy.
Deprivation of ovarian estrogen does not cause suppression of aromatase P450 in leiomyoma cells during LA therapy
Aromatase P450 might be up regulated by its product, E2 (30, 31). In other words, the lower the level of circulating
(ovarian) estrogen, the lesser the amount of aromatase P450 transcripts
in extragonadal cells is. If this is true, we could assume that GnRH
agonist-induced suppression of circulating (ovarian) estrogen is a
causal factor of decreased aromatase P450 expression in leiomyoma cells
during GnRH agonist therapy. To test this possibility, we examined the
effects of estrogen on the expression of aromatase P450 in leiomyoma
cells. Leiomyoma cells obtained from the no LA group were treated with
various concentrations of E2 for 24 h, and aromatase activity then
was measured. As shown in Fig. 4, treatment with 100 pmol/liter to 10 nmol/liter E2 did not affect
aromatase activity. This result does not support the notion that a
decrease in circulating (ovarian) estrogen causes suppression of
aromatase P450 expression in leiomyomas.
In vitro suppression of aromatase P450 expression in leiomyoma cells by prolonged treatment with LA
To investigate the direct actions of LA on leiomyoma cells, we
treated leiomyoma cells with LA (100 nmol/liter) for 1–8 d.
Twenty-four hours before the end of incubation, leiomyoma cells were
treated with DEX (25 nmol/liter) + IL-1ß (1 ng/ml), and then
aromatase activity was measured. The basal (nonstimulated) level of
aromatase activity gradually increased by approximately 100% over
8 d of incubation (Fig. 5A).
Conversely, the DEX + IL-1ß-induced level of aromatase activity was
suppressed by preincubation with LA and the decrease reached a
significant level at the eighth day of incubation in comparison to
nonpreincubated control. This decrease in the DEX + IL-1ß-induced
level was statistically significant even when compared with the control
that were similarly preincubated for the same period (8 d) in the
absence of LA. There was no significant change in cellular protein
levels during the incubation period (Fig. 5B), suggesting that the
decrease in response to the stimulants was not the result of
nonspecific cell damage during culture. The decrease in aromatase
activity was attributable to a reduction in the mRNA level (Fig. 5C).
Discussion
In our previous report, we demonstrated that leiomyoma cells express a high level of aromatase P450 and are able to synthesize estrogen to promote their own cell growth (26). In that experiment, we estimated the conversion of androgen into E in leiomyoma cells in culture and found that a physiological level of androstenedione (10 nmol/liter) promoted cell growth with a slight increase in E2 concentration in the media (10 pmol/liter), which is one order lower than that the minimum concentration of E2 necessary to induce cell growth (100 pmol/liter). Thus, in situ estrogen synthesized in leiomyoma cells appears to act in a paracrine/autocrine/intracrine manner rather than in "an endocrine manner." In premenopausal women, the amount of circulating E2 (100 pmol/liter or higher) secreted from the ovary is considerable in comparison with estrogen synthesized in situ. Thus, in situ estrogen is not likely important for the cell growth of leiomyoma cells. On the other hand, after cessation of follicular growth, in situ estrogen possibly becomes the sole source of estrogen available for leiomyoma cells in postmenopausal women. As the plasma level of androstenedione (5–10 nmol/liter) in women at early menopause is maintained at the level of or at a slightly higher level of the apparent Km of aromatase P450 to androstenedione (3 nmol/liter), a physiologically significant amount of estrogen might be synthesized in situ (26). Presumably, in situ estrogen plays a role in preventing leiomyoma cells from rapid regression, which would be viewed as the result of the complete depletion of ovarian estrogen. Actually, more than 85% of leiomyomas do not show detectable regression in the first 6 months of natural menopause in comparison with the rapid decrease in plasma E2 levels (27). In contrast to this slow regression seen in natural menopause, most leiomyomas shrink rapidly during LA therapy, loosing 30% or more volume in the first 12 wk of LA treatment (23, 24). The regression rate of leiomyoma nodules is estimated to be three times faster in women treated with GnRH agonist than that in women of the early stage of menopause (27). The present study demonstrated that therapeutic levels of LA reduced the expression of aromatase P450 in leiomyoma cells less than one twentieth, indicating that there is virtually no in situ estrogen available to prevent rapid regression during LA therapy. LA reduces the amount of both ovarian estrogen and in situ estrogen available, causing leiomyomas to show rapid and profound shrinkage. There is further evidence to support the importance of this role of in situ estrogen in leiomyoma shrinkage. Some women who show significant regression of leiomyoma during LA therapy still maintain a higher level of plasma estradiol (50–100 pmol/liter) than women at menopause do (32). Even women who continue to have significant circulating estrogen levels (150 pmol/liter or higher) and experience repetitive genital bleeding during treatment with another GnRH agonist, buserelin, show significant shrinkage of leiomyomas (33). Therefore, complete down-regulation of the ovary and hypoestrogenemia is not necessary for successful shrinkage of leiomyomas during GnRH agonist therapy. Moreover, low-dose estrogen add-back did not induce regrowth of leiomyomas during GnRH agonist therapy (34). Given these findings, the role of in situ E would be important in borderline-hypoestrogenemic conditions, and, therefore, inhibition of in situ aromatase may be beneficial for successful shrinkage during LA administration. Further study is needed to confirm this hypothesis.
In the present study, we used enzymatically isolated leiomyoma cells as
a model to study the action of LA. More than 95% of cells that we used
were positive for smooth muscle specific -actin (26).
However, it has been noted that myometrial cells in primary culture
undergo a transformation from contractile to synthetic phenotype,
losing some morphological characteristics for smooth muscle cells
(35). We used only the first and the second passage cells
to minimize transition from the contractile phenotype. So far, we have
confirmed that the leiomyoma cells used expressed a higher level of ER
mRNA and proliferated in response to E2, whereas myometrial smooth
muscle cells cultivated from the normal myometrium did not show any
response to E2 (26). Thus, the leiomyoma cells we used
possess some features characteristic to leiomyoma cells in terms of the
functional ER. We also confirmed that LA activated MAPK in these
leiomyoma cells, indicating the presence of functional receptors for a
GnRH agonist (data not shown). Moreover, these leiomyoma cells
maintained the potential to express aromatase P450 in response to
suitable stimulants. Given these features, we used the enzymatically
isolated cells to study the regulation of aromatase in
vitro.
The present study clearly demonstrated that preoperative LA administration reduced the expression of aromatase P450 in leiomyoma tissues. Once leiomyoma cells were isolated and cultured ex vivo in the absence of LA, there was no difference in the expression of aromatase P450 between cells obtained from the LA group and the no LA group. Conversely, isolated leiomyoma cells decreased such expression when they were cultured in the presence of LA in culture media. Although we cannot exclude the possibility that leiomyoma cells change their in vivo nature during culture, thus making our in vitro model unsuitable in the event, the most likely explanation of our findings at this time is that LA directly acts on leiomyoma cells to reduce the expression of aromatase P450, and this process is reversible. Our in vivo model may be useful to study LA action. The magnitude of suppression in the expression level was, however, smaller in the in vitro model (approximately 30% reduction) than in vivo tissues samples (more than 90% reduction). Duration of treatment with LA may explain the difference. Further study is needed to define the precise mechanism of LA action on the expression of aromatase P450.
There are at least seven different promoters for a single copy gene of aromatase P450 (CYP19) and these promoters were alternatively used in a tissue- and stimulant-specific manner. For example, the promoters PII, I.4, 1f, and I.1 of aromatase P450 were mainly used for the ovary and prostate, adipose tissues and skin fibroblasts, brain, and placenta, respectively. Each promoter possesses different regulatory cis-elements upstream and is regulated differently through these regulatory cis-elements. Thus, it is necessary to identify the promoter use of aromatase P450 in leiomyoma cells to define the possible action of LA on the transcriptional regulation of aromatase P450. Bulun et al. (25) have reported that leiomyoma tissue primarily uses promoter PII and partly promoter I.4. Judging from the many cell types that have been analyzed, to date, cAMP and PGE2 are the most potent stimulators for PII-induced transcription and the combination of DEX + IL-1ß or DEX + another cytokines for promoter I.4-induced transcription. We tested these combinations in the present study and found that DEX + IL-1ß was the most potent and that PGE2 alone was ineffective. This indicates that the promoter I.4 is the most active promoter in these leiomyoma cells. Precise promoter use in leiomyomas is currently under investigation.
Besides the deprivation of estrogen, several different mechanisms have been proposed for LA-induced regression of leiomyomas. Leiomyoma tissue and its receptors express a higher level of TGFß1 than unaffected myometrium, and women who received LA therapy had a substantially lower level of expression than untreated controls (36). Given that TGFß1 is an autocrine growth factor of leiomyomas, suppression of TGFß1 may contribute to LA-induced growth retardation of leiomyomas (37). As secretion of active form of TGFß1 from leiomyoma cells is up-regulated by estrogen, LA-induced suppression of TGFß1 may result from estrogen suppression (36, 37). Kobayashi et al. (14) proposed another mechanism, in which excessive treatment of leiomyoma cells with buserelin reduces the expression of cyclin estrogen and p33cdk2, the G1-related genes necessary for G1 to S progression and, thus, inhibits cell proliferation. In some cancer cell lines such as EFO-21 (ovarian cancer), EFO-27 (ovarian cancer), and HEC-1A (endometrial cancer), GnRH agonists activate phosphotyrosine phosphatase, which then abolishes intracellular signaling of growth factors through phosphorylation (38). In the Caov-3 cell line, another ovarian cancer cell line, prolonged activation of MAPK induced by GnRH agonist treatment causes hypophosphorylation of pRB and then suppresses G1-S transition, although the precise mechanism of hypophosphorylation of pRB is unknown (13). These possibilities remain to be tested in leiomyoma cells.
In conclusion, the present study demonstrated that LA abolished overexpression of aromatase P450 in situ probably through direct action on leiomyoma cells. As discussed above, GnRH agonist (LA) therapy would render suppression of in situ aromatase a key determinant in achieving a maximum shrinkage of leiomyomas. The importance of in situ estrogen on leiomyoma growth may justify estrogen add-back therapy, in which small amounts of estrogen are administered during GnRH agonist therapy in an attempt to prevent adverse effects of long-term deprivation of estrogen, such as hot flashes and loss of bone mass, without nullifying their therapeutic effects (34, 39, 40). Further study is required to substantiate the clinical significance of in situ estrogen on leiomyoma cell growth to validate such add-back therapy. The mechanism by which LA represses the transcription of aromatase P450 also remains to be elucidated.
Footnotes
This work was supported by Grants-in-Aid for Scientific Research B12557136, B13470348, and C11676102 from the Ministry of Education, Science, Sports, and Culture, Japan.
Abbreviations: DEX, Dexamethasone; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; LA, leuprorelin acetate; PMA, phorbol myristyl acetate.
Received October 25, 2000.
Accepted April 26, 2001.
References
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  S. E. Bulun, Z. Lin, G. Imir, S. Amin, M. Demura, B. Yilmaz, R. Martin, H. Utsunomiya, S. Thung, B. Gurates, et al. Regulation of Aromatase Expression in Estrogen-Responsive Breast and Uterine Disease: From Bench to Treatment Pharmacol. Rev., September 1, 2005; 57(3): 359 - 383. [Abstract] [Full Text] [PDF]
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, P <
0.05 vs. DEX + IL-1ß-stimulated levels in the group
that was not pretreated with LA; 
, P < 0.05
vs. DEX + IL-1ß-stimulated levels of the group that
were pretreated with vehicle for 8 d. C, Leiomyoma cells obtained
from the no LA group were plated on 9-cm wells and were treated with LA
for 8 d. At the last 24 h of incubation, DEX (25 nmol/liter)
+ IL-1ß (1ng/ml) was added to the media as described in A and B.
Cells were lysed in 1 ml Ultraspec, and total RNA was extracted. Levels
of mRNA containing aromatase-coding sequence were measured by
competitive RT-PCR, as described in Materials and
Methods. Data from groups treated with LA for 0, 1, and 5 d
were not available. Data represent the mean ± SEM of
three independent experiments. *, P < 0.05
vs. the control that was cultured with LA for 8 d
and was not stimulated with DEX + IL-1ß before aromatase assay; 
