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Introduction
Uterine leiomyoma, a smooth muscle tumor arising from the uterus, occurs in about 20%_30% women over the age of 30,
and is the most frequently occurring tumor in
gynecology[1]. Although the vast majority of leiomyomas are benign, these
tumors could induce severe pain, excessive bleeding, infertility, repetitive pregnancy loss, and other pregnancy-related
complications. As a result, uterine leiomyomas are ranked as the major cause for hysterectomy, accounting for a third of all
hysterectomies[2,3]. Unfortunately, the underlying geneses of uterine leiomyoma are poorly understood. Estradiol benzoate
(E2) and progesterone (P) have generally been regarded as
the major factors responsible for the development of the
leiomyoma, since the growth of uterine leiomyoma is
associated with menstrual cycle and the fluctuation of steroid
hormone level[4,5]. Estradiol receptors (ER) and progesterone
receptors (PR), as members of a superfamily of nuclear
receptors that function as ligand-modulated transcriptional
regulators, are ligands for steroid hormone. ER and PR are
also regarded as predominant regulators in the promotion of
endometrium proliferation and uterine
leiomyoma[6]. In classical theory, steroid hormones act through the interaction of
their receptors with specific steroid responsive elements to
regulate the transcription of target gene networks. This
process causes hormone-dependent cell
proliferation[7,8]. There is recent evidence suggesting that the development of
leiomyomas may also be attributed to genetic and
environmental influences[6]. Several genes and/or growth factors,
such as transforming growth factor, epidermal growth factor,
and insulin-like growth factor have been demonstrated to be
differentially expressed in human tissues and
primary-cultured leiomyoma cells compared with normal
myometrium[9_12]
c-Src, belonging to Src family kinases, was first
recognized as a proto-oncogene with intrinsic protein tyrosine
kinase activity[13,14]. c-Src plays a vital role in the regulation
of both normal and oncogenic processes, including
prolifera-tion, differentiation, survival, motility, and
angiogenesis[15,16]. Elevated c-Src protein levels and kinase activity in several
human cancers have been reported since the early 1980s,
including tumors in the colon, breast, and ovaries. The
protein level of c-Src is significantly higher in carcinomas than
in normal matched tissues[17_21]. Recently, Migliaccio's
et al demonstrated that c-Src takes a critical role in hormone-
dependent tumor progression[22]. c-Src activity is stimulated
rapidly by estradiol, progestin, and androgens in breast and
prostate cancer cells and is involved in the nontranscriptional
action of estradiol[23,24]. Src is downstream of classical
sex-steroid receptors, such as ER, PR, and androgen receptors.
These sex steroid receptors can activate Src. Signaling via
Src plays an important role in ER activation. Estradiol
controls the expression of a cell-cycle regulator through the
activated signaling pathway, which triggers DNA synthesis
and cell proliferation[22,23]. The loss of the c-Src tyrosine
kinase is correlated with defects in ductal development as
well as in uterine and ovarian development. The mammary
gland, ovarian, and uterine tissues derived from the
c-Src-deficient animals displayed a dramatic development
delay[25]. This evidence indicates that c-Src may be a critical target
gene for steroid hormone action.
Since uterine leiomyoma is considered to be steroid-
hormone dependent, whether Src plays a role in the
development of uterine leiomyoma is questionable. It would help
to better understand the underlying causes of uterine
leiomyoma that lead to more targeted strategies to treat the
hormone-dependent tumor. Therefore, we investigated the
expression of c-Src kinase in a uterine leiomyoma of a guinea
pig model. To understand the association between the
activity of c-Src and the formation of the uterine leiomyoma,
the protein expression of phospho-416Src
autophosphoryla-tion site, Tyr416, and increases in c-Src activity were also
assayed.
Medical treatment of leiomyomas relies on drugs that
inhibit ovarian steroids and induce a hypoestradiolic state
that causes atrophy of the tumor at the same time. Gestrinone
is a synthetic trienic 19-norsteroid compound with potent
anti-estradiolic and antigonadotropic properties. The
compound also inhibits gonadotropin secretion and ovulation,
which prevents the occurrence of
menstruation[26]. Gestrinone was developed originally as a long-acting
contraceptive. Because of its marked inhibitory effects on ER and PR in the
endometrium and uterus, gestrinone has also been clinically
applied to treat endometriosis and uterine leiomyoma with
success[27,28]. Marca et al demonstrated that gestrinone
significantly increased blood flow impedance of the uterine
artery[29]. In the present study, we further investigated the
effect of gestrinone on the development of uterine
leio-myoma, and the expression of c-Src,
phospho-416Src, ER, and PR in the guinea pig model.
Materials and methods
Materials Gestrinone microparticle
(13β-ethyl-17α-
ethynyl-17β-hydroxy- gona-4,9,11-trien-3-one) is a patent of
Prof Qing-hua CHEN (Department of Pharmaceuticals,
Shanghai Institute of Pharmaceutical Industry, Shanghai, China)
and was generously gifted to us. The structure of gestrinone
is shown in Figure 1. E2 was purchased from Shanghai
General Pharmacy Company (Shanghai, China). The c-Src rabbit
polyclonal antibody and the anti-ER mouse monoclonal
antibody were purchased from Lab Vision Co (Lab Vision
Co, Fremont, CA, USA). The mouse anti-phospho-Src
(Tyr416) monoclonal antibody was from Upstate Co (Lake
Placid, NY, USA). The PR mouse monoclonal antibody was
from Novocastra Laboratories Ltd (Newcastle, UK). The
avidin-biotin-peroxidase complex (ABC) kit was the product
of Sino-American Biotechnology Co (Shanghai, China). Trizol
was purchased from Gibco-BRL (Grand Island, NY, USA).
An enhanced chemiluminescence (ECL) detection kit for
horseradish peroxidase (HRP) was purchased from Pierce
(Rockford, IL, USA). The magnetic average phase enzyme
immunoassay system (MGI) was the product of Serono Inc
(Geneva, Switzerland). Image processing was done with
Image-Pro Plus® Version 5.1.0 software (Media Cybernetics
Inc, Silver Spring, MD, USA).
Animals Dunkin-Hartley female guinea pigs (3 months
old, weighing 350_400 g) were purchased from the
Sino-British Experiment Animal Lab Co (Shanghai, China). The
animals were treated in accordance with protocols approved by
the Laboratory Animal Ethics Committee at the Shanghai
Institute of Planned Parenthood Research. All animals were
housed: 5 in 1 cage under a 12 h/12 h light/dark cycle with
free access to food and water. Each animal was weighed
once a week and fed 50 g cabbage twice a week for vitamin C
supplementation.
Model establishment and treatment Sixty-five female
guinea pigs were divided into 5 groups. Group 1 consisted
of normal control animals and group 2, castrated animals.
After bilateral oophorectomized, animals were bred as group
1; Group 3, model animals. After bilateral oophorectomy, the
animals were treated intramuscularly with
E2 100 µg/d twice per week from week 1 through to week 16. The bilateral
oophorectomized animals in groups 4 and 5 were treated
intramuscularly with E2 at 100 µg/d alone twice per week from
week 1 through to week 6, and then in combination with
subcutaneous administration of gestrinone microparticle at
2 or 4 mg/kg once biweekly from week 7 through to week 16.
Anesthetics were administered with 3% pentobarbital
sodium during oophorectomy. At the end of the sixteenth week,
blood was sampled from the animals' hearts. The animals
were killed by ether inhalation. Body weight was measured
before killing. After killing, the uteri were dissected, weighed,
and then fixed immediately in 10% neutral formalin or liquid
nitrogen. The collected blood was centrifuged, and sera
were frozen at -20 °C until the time of assay.
Histological examination The formalin-fixed tissues were
embedded in paraffin wax and sliced into 4 µm thick pieces
using a rotary microtome, and then stained with
hematoxylin-eosin (HE) for histological examination under light
microscope.
Serum E2 and P assays Serum estradiol and
progesterone levels were assayed using MGI with the Serono
magimu-zyme-III analyzer (Serono Inc, Geneva, Switzerland) at a
wavelength of 550 nm.
Immunohistochemical staining The paraffin-fixed
sections were baked overnight at 55 °C. Sections were
deparaf-finized with xylene and rehydrated with ethanol in gradient.
Endogenous peroxidase activity was blocked by incubating
the slides with 0.3% H2O2 in methanol for 10 min. Antigen
retrieval was performed with 10 mmol/L citric acid buffer
(pH 6.0) for a total of 10 min in a microwave oven.
Non-specific binding was blocked by incubating the specimens
in 0.1 mol/L Tris-HCl (pH 7.6) containing 10% goat serum for
1 h. The antigen was then detected using c-Src, anti-ER, or
anti-PR primary antibodies at a 1:100 dilution with blocking
buffer as diluents and incubated in a moist chamber at 37 °C
for 1 h. After washing with phosphate buffered saline (PBS),
the sections were incubated with biotinylated
goat-anti-mouse IgG followed by HRP conjugated streptavidin biotin
peroxidase complex (SABC) according to the manufacturer's
protocol. The resulted sections were then stained with
3,3'diaminobenzidine tetrahydrochloride (DAB). As a
negative control to assess potential nonspecific staining, 0.01
mol/L PBS was used instead of primary antibodies, and no
staining was observed. Finally, the sections were
dehydrated and sealed with mounting media for visualization by
light microscopy. The dark brown staining of nuclei or
cytoplasm was regarded as a positive reaction.
Immunohisto-chemistry was semiquantitatively evaluated using the
immunoreactive score (IRS). The IRS was obtained by the
product of intensity of immunostaining (none=0; weak=1;
moderate=2; and strong=3) and the percentage of positive
cells (none=0; <10%=1; 10%_50%=2; 51%_80%=3; and
>80%=4)[30,31]. The number of positive cells was recorded
with Image-Pro Plus® Version 5.1.0 software (Media
Cybernetics Inc). One section was examined for 1 animal.
Statistical analysis was performed on the average number of
positive cells of 10 sections per group.
Protein isolation and analysis Protein extracts were
prepared by grinding 100 mg of frozen tissue to powder with
liquid nitrogen in a mortar and pestle. The resulted proteins
were added with 1 mL extraction buffer (100 mmol/L Tris-HCl
pH 7.5, 0.5 mmol/L dithiothreitol, 2 mmol/L
phenylmethyl-sulfonyl fluoride, 5 mmol/L EDTA, 500 mmol/L NaCl, and
0.1% Triton X-100), and then centrifuged at 4 °C, 12
000×g for 15 min. The supernatant were collected and stored at
-70 °C. The protein concentration was measured using the
bicinchoninic acid (BCA) method (Pierce, Rockford, IL, USA).
Samples were diluted to 1 µg/µL with extraction buffer. An
aliquot of 20 µg was mixed with an equal volume of
SDS-PAGE loading buffer (50 mmol/L Tris-HCl, pH 6.8, 10%
glycerol, 2% SDS, 5% β-mercaptoethanol, 0.002%
bromophenol blue), and heated at 95 oC for 5 min before being loaded
onto 8% SDS-PAGE (30% acrylamide:bisacrylamide 29:1) for
electrophoresis in TBE buffer (Tris-Borate-EDTA). To
perform the Western blot analysis, the separated proteins were
electrophoretically transferred onto polyvinylidene difluoride
(PVDF) membranes. The membranes were incubated with
the primary antibody in 3% BSA (albumin bovin fraction
V)-PBS (1:600) at 37 °C for 2 h. After washing with 0.1% Tween
20-PBS, the membranes were incubated with
peroxidase-conjugated secondary antibody IgG in 3% BSA-PBS (1:2000) at
37 °C for 1 h. The blot was developed with ECL according to
the manual and was exposed to X-film. The relative levels of
protein were semiquantitatively determined with Image-Pro
Plus® Version 5.1.0 software (Media Cybernetics Inc). The
relative levels of protein were obtained by taking the ratio of
the band intensity of c-Src,
phospho-416Src, ER, or PR to β-actin. Statistical
analysis was performed on the average protein level of 5 animals per group.
Statistical analysis Data were presented as mean±SEM.
Various kinds of indexes between the control group and
administrated groups were analyzed by one-way ANOVA test
with SPSS 10.0 software (version 10.0 for Windows; SPSS,
Chicago, Illinois, USA). P<0.05 was considered statistically
significant.
Results
Weights and coefficients of uterine tissues
At the end of 16 weeks, there was no visible change in the guinea pigs'
body weight, the absolute and the relative uterine weights
between the control and the castrated group. Compared
with the control group, there was a significant decrease of
body weight in the model and the gestrinone treated group
(P<0.05). However, there was a marked increase of the
absolute and the relative uterine weights in the model group
(P<0.05). In the gestrinone-treated group, the
absolute and the relative weights of uterine tissues were lower than
that of the model group, and the organ coefficients in the 4
mg/kg gestrinone group were much lower than that of the 2
mg/kg gestrinone-treated group (P<0.05) (Table 1).
Morphological changes of uterine tissues
Necropsy and pathological analysis were performed to examine whether
there were leiomyoma features in the animals.
Visually, there was no node or cystitis tumor observed in the myometrium
of the control and the castrated animals. In the model animals,
the uteri became enlarged, proliferative, and had a thick
nodular or cystiform appearance. The nodules were generally
found on the uterine horn, the uterine body, or the
cervico-uterine junction. The general appearances of nodes were of
a solitary, firm, gray mass or cystiform full of fluid, appearing
as subserosa or intramural myometrium. In the 2 and 4
mg/kg gestrinone-treated animals, there were no nodes or severe
hyperplastic myometrium observed. The appearances were
similar to those of the control animals (Figure 2).
Histologically, proliferative features were demonstrated
both in the leiomyoma and the myometrium of model animals.
The majority of cell nuclei showed elongated, rod-like, or
oval-round shape with a deep nuclear stain and grew in
parallel arrays, exhibiting a typical, smooth,
muscle-like appearance. A few spindle-shaped cells were also observed.
The glandular cavity was enlarged. The muscle fiber showed
rarefaction and misalignment, exhibiting paliform or
fan-shape. In the castrated animals, the smooth muscle cells
lined up densely, and the glandular cavity was atrophic. In
the 2 and 4 mg/kg gestrinone-treated animals, the uterine
tissues were thin. The smooth muscle cells lined up in order
like those of the control animals (Figure 3).
Serum hormone concentrations In the
E2-treated model group, there was a visible increase in the serum
E2 level than that of the control and the castrated groups
(P<0.05). Compared with the model group, there was no significant
decrease in the serum E2 level in the 2 mg/kg gestrinone-treated
group (P>0.05), and there was an obvious decrease in the
serum E2 level in the 4 mg/kg gestrinone-treated group
(P<0.05). There was no significant fluctuation of serum P in the
control, castrated, model, and 2 and 4 mg/kg
gestrinone-treated groups (P>0.05) (Figure 4).
Immunohistochemical expression of ER, PR, and c-Src
The presence of ER, PR, and c-Src were confirmed by
immunohistochemical analysis in the myometrium of the control,
castrated, model, 2 and 4 mg/kg gestrinone-treated animals.
The expression of c-Src and ER distributed both in the
cytoplasm and nucleus. The staining of PR was located in the
nucleus. In the control and the castrated groups, the
coloration of c-Src, ER, and PR were stained light brown. By contrast,
the coloration of c-Src, ER, and PR in the model group were
dark brown and the positive staining areas were extended.
In the 2 and 4 mg/kg gestrinone-treated groups, the
coloration of c-Src, ER, and PR was lighter than that of the model
group. The number of positive uterine cells was less than
those in the model group (Table 2) (Figure 5).
Protein expressions of c-Src,
phospho-416Src, ER, and PR Western blots were performed to analyze the protein
level of c-Src, phospho-416Src, ER, and PR. The protein
expressions of c-Src, phospho-416Src, ER, and PR were
detectable in all of the groups. The
relative protein expressions of c-Src/β-actin,
phospho-416Src/β-actin, ER/β-actin, and
PR/β-actin were significantly higher in the model group than those
of the control group (P<0.05). Compared with the control
group, the relative protein expression of
c-Src/β-actin and phospho-416Src/β-actin was lower in the castrated group
(P<0.05), and there was no significant decrease in
ER/β-actin and PR/β-actin protein expression
(P>0.05). In the 2 and 4 mg/kg gestrinone-treated groups, the
relative protein expressions of c-Src/β-actin,
phospho-416Src/β-actin, ER/β-actin, and
PR/β-actin were significantly lower than those of the model group
(P<0.05), and there was no marked quantitative difference between the 2 and 4 mg/kg
gestrinone-treated group (Table 3) (Figure 6).
Discussion
Uterine leiomyoma is the most common benign tumor in
women. The cause of human uterine leiomyoma has not
been fully revealed. As a tumor developed from an estrogen
target organ (uterus), uterine leiomyoma is traditionally
considered an estrogen-associated
tumor[32]. In some clinical observations, surprisingly, there is no positive connection
found between E2 concentration and the genesis of
leiomyoma. Patients with uterine leiomyoma have similar
plasma E2 and P levels to those of normal
controls[6,33]. The development and growth of leiomyomas may involve a
multistep cascade[34]. In pharmacological research, nevertheless,
one of the effective methods for developing uterine
leiomyoma is to administer a guinea pig with
E2 for a long period of time. Studies on the guinea pig model of uterine
leiomyoma provide insights into unraveling molecular
mechanisms that regulate leiomyomas, and are widely used for
preclinical drug screening assays. Approximately 8% of aged
guinea pigs develop spontaneous leiomyomas, and
ovariectomy in young animals combined with high-dose estrogen
supplementation causes the development of uterine and
abdominal leiomyomas with high
frequency[35,36].
In this study, therefore, we induced uterine leiomyoma in
guinea pigs with an E2 injection twice a week for 4
months[35,37] with a modified
E2 injection frequency. During the experiment,
one animal in the model group died due to pneumonia and
the others lived healthily to the end of the experiment. At the
end of the 16 weeks, all of the animals underwent necropsy.
In the model group, the overall appearance of the myometrium
was enlarged and proliferative. Pronounced nodes were
observed in some model animals (8 of 14). The nodes were
located on the uterine horn, the uterine body, or the
cervico_uterine junction and exhibited intramural myoma (7 of 14),
subserosal myoma (1 of 14), and uterine necrosis (2 of 14).
There was a remarkable increase of the absolute and relative
uterine weights in the model group than that of the control
and castrated groups (Table l). Histologically, the
myometrium of all of the model animals appeared proliferative
and consisted of a mass of typical smooth muscle cells and
a few fibroblasts. It exhibited typical features of leiomyoma,
even if there was no pronounced nodule present. There was
no nodule found in the abdominal cavities of the model group.
According to Porter and Fujii et
al[35,37], however, an intramuscularly injection of
E2 3 times a week for 3 months would induce multiple nodules in the abdominal cavities, including
the uterine horn, peritoneum, spleen, pancreas, and omentum.
The marked disparity of the results may be attributed to the
different treatment frequency of E2, since we obtained a
similar result to that of Porter and Fujii et
al when following their protocol in a preliminary experiment.
In a preliminary experiment, we compared ovariectomized
animals with non-ovariectomized animals, both treated with
E2. The result showed that there was slight proliferation on
the myometrium and no nodes were observed in the
non-ovariectomized animal. The leiomyoma was better
developed in the ovariectomized animals. In order to equalize the
original hormones of the tested animals and induce uterine
leiomyoma successfully, we utilized oophorectomized guinea
pigs in the present study. In the castrated animals, the
serum E2 level significantly decreased when the ovary was
excised. There was a remarkable increase in the serum
E2 level of the model group after
E2 treatment. However, there was no appreciable fluctuation of serum P observed.
Furthermore, there was a higher expression of ER and PR in
the myometrium in the model group than the control group.
This evidence indicates that E2 could increase the level of
ER and PR in the myometrium, and that ER and PR play more
important roles during the formation of uterine leiomyoma.
Our observation is consistent with the previous reports that
high concentrations of ER and PR in the myometrium
contributes to the development of uterine
leiomyoma[6,38,39]. Compared with the control group, we demonstrated that there
was no marked decrease in immunoreactivity and protein
expression of ER and PR in the castrated group in the present
study. There was an overexpression of ER and PR in the
uterine leiomyoma of the guinea pig model.
Clinically, a gestrinone tablet or capsule is administered
orally at a dose of 2.5_5 mg twice weekly for at least 6_12
months for the treatment of leiomyoma. As an alternative for
those patients who developed gastric intolerance to steroids
and as a practical means to extend the action and bypass the
liver to increase the bioavailability of the drug, a gestrinone
microparticle was engineered by Prof CHEN. The
commercial product is expected to be available from Beijing Zizhu
Pharmaceutical Co Ltd (Beijing, China). In the present study,
we administered the guinea pig model with the gestrinone
microparticle once biweekly at 2 or 4 mg/kg for 10 weeks.
Visually, no node was observed in the 2 and 4 mg/kg
gestrin-one-treated groups. In a dose-dependent manner, the
gestrinone microparticle decreased the serum
E2 level, the absolute, and the relative uterine weights. Histologically,
the myometrium was thin and the smooth muscle cells lined
up in order, as that of the control animals. Gestrinone 2
mg/kg markedly reduced the immunoreactivity and
protein expression of ER and PR. There was no significant
difference in immunoreactivity and protein expression of ER
and PR between the 2 and 4 mg/kg gestrinone-treated groups.
The result indicated that gestrinone effectively reduced the
proliferation of uterine smooth muscles and suppressed the
formation of uterine leiomyoma in the guinea pig model.
Gestrinone 2 mg/kg is as effective as 4 mg/kg in inhibiting
the formation of uterine leiomyoma.
Recently, c-Src has been considered one of critical
factors in the genesis of breast and prostate
cancers[23,24]. Evidence shows that in
E2 responsive tissues such as breast and endothelia, the biological function of ER involves not
only the activation of estradiol-responsive elements, but also
induces rapid activation of various signaling molecules and
signal transduction pathways, which are regarded as
"nongenomic" or "non-transcriptional
signaling[40,41]. Migliaccio et al reported that in breast cancer cell lines, upon
activation by E2, ER stimulate a mitogenic signaling network,
such as Src/Ras/Erk and the PI3-K/AKT pathway, known to
be engaged by growth factors[24,42_45]. In these signaling
pathways, Src has been identified as a crucial molecule
downstream of ERα and mediates E2 rapid
action[22]. Activation of this signal pathway leads to S-phase entry of the cells and is
responsible for the proliferative effect against
apoptosis[42,23]. In the opinion of Migliaccio
et al, Src appears to be an initial target of sex steroid hormone action in breast and prostate
cancer[22].
In order to understand whether Src takes a role in the
development of uterine leiomyoma, we examined the
expression of c-Src in uterine leiomyoma in the guinea pig model.
An immunohistochemical assay demonstrated that c-Src was
expressed in the nucleus and cytoplasm of the myometrium.
Compared with the control group, we found that there was
lower immunoreactivity of c-Src in the castrated group, and
there was significant increase in immunoreactivity of c-Src
in the uterine leiomyoma of the guinea pig model induced by
E2 (Figure 5). In the Western blot analysis, the protein
expression of c-Src was weaker in the castrated group than
that the control group. The protein expression of c-Src was
stronger in the model group than that of the control and
castrated groups (Figure 6). These results indicate that
overexpressed c-Src plays an important role in the formation
of uterine leiomyoma. Furthermore, we also demonstrated
that gestrinone 2 and 4 mg/kg markedly downregulated cSrc
protein in the myometrium, which may be attributed to the
suppression of the formation of uterine leiomyoma in the
guinea pig model. There was no marked difference in
immunoreactivity and protein expression of c-Src between the 2
and 4 mg/kg gestrinone-treated groups.
Regulation of c-Src activity is complicated.
Phosphorylation of Tyr527, at the carboxy-terminal end of c-Src, results
in a decrease of c-Src kinase activity. In contrast,
phosphorylation of the autophosphorylation site, Tyr416, increases
c-Src activity[46,47]. To understand the relationship between
c-Src and the formation of uterine leiomyoma, we assayed
the protein level of phospho-416c-Src among the groups. The
results demonstrated that the level of
phospho-416c-Src was upregulated in the model group than the other groups.
Gestrinone 2 and 4 mg/kg significantly downregulated the
protein level of cSrc and
phospho-416c-Src. There was no marked difference between the 2 and 4 mg/kg
gestrinone-treated groups. The results indicated that the activity of
c-Src was enhanced in the model of uterine leiomyoma, and
gestrinone decreased the activity of c-Src.
On the basis of these findings, for the first time, we
propose that c-Src plays a significant role in the development of
uterine leiomyoma. The upregulation of Src and
phospho-416Src indicate that the activity of c-Src is augmented in the
leiomyoma model. c-Src is associated with the formation of
uterine leiomyoma in the guinea pig model. There is
significant downregulation of cSrc and
phospho-416Src protein after gestrinone 2 and 4 mg/kg treatment. Similar to ER and
PR, c-Src may be a vital factor contributing to the growth of
uterine leiomyoma. Gestrinone markedly suppressed the
growth of uterine leiomyoma induced by
E2 in the guinea pig model. Gestrinone inhibited not only the expression of ER
and PR, but also the protein expression of c-Src and
phospho-416Src in the leiomyoma model of guinea pigs. Although the
related mechanism is still to be elucidated, these results help
us to further understand the underlying causes of uterine
leiomyoma and provide a clue for a possible therapeutic
treatment with gestrinone in depressing the formation of uterine
leiomyoma via targeting the signaling pathway.
Acknowledgment
The authors greatly appreciate the help of Mrs Zhi-fang
ZHAO in carrying out the animal experiments.
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