Biomedicine & Pharmacotherapy 66 (2012) 569–577

Original article

Gestrinone inhibits growth of human uterine leiomyoma may relate to activityregulation of ERa, Src and P38 MAPK

Yan Zhu a, Tingting Zhang b,*, Shuwu Xie a, Ruiqin Tu c, Yang Cao b, Xiangjie Guo a, Jieyun Zhou a,Xianying Zhou a, Lin Cao a

a Department of Reproductive Pharmacology, NPFPC Key Laboratory of Contraceptives and Devices, Shanghai Institute of Planned Parenthood Research, Shanghai 200032, Chinab Department of Obstetrics and Gynecology, Shanghai Yueyang Hospital, Shanghai 200437, Chinac Department of Obstetrics and Gynecology, Shanghai Zhongshan Hospital, Fudan University, Shanghai 200032, China

A R T I C L E I N F O

Article history:

Received 29 December 2011

Accepted 29 February 2012

Keywords:

Gestrinone

Uterine leiomyoma

c-Src

ERap38 MAPK

A B S T R A C T

The study was to investigate the effect of gestrinone on the growth of human uterine leiomyoma cells

and on the levels and activity of p38, Src and estrogen receptor alpha (ERa). Human uterine leiomyoma

cells were cultured and treated with dimethylsulfoxide (DMSO) or a gestrinone concentration gradient.

Morphological changes were observed and apoptosis was evaluated. Levels of p38 and phosphorylated-

p38 (pp38) were assayed by enzyme-linked immunosorbent assay (ELISA). Levels of ERa and Src were

analyzed using real-time RT-PCR and Western blotting. The result showed that gestrinone significantly

inhibited the growth of cultured human uterine leiomyoma cells in a concentration- and time-

dependent manner, with a 50% inhibitory concentration (IC50) value and corresponding 95% confidence

intervals (CI) of 43.67 (23.46�81.32), 27.78 (12.51�61.68) and 15.25 (7.17�32.43) mmol/L at 20, 40 and

60 h, respectively. Compared with control-treated leiomyoma cells, gestrinone significantly reduced

both the expression of ERa (P < 0.05) and the levels of phospho-Ser167-ERa (P < 0.05). Gestrinone also

markedly suppressed the level of phospho-Tyr416-Src (P < 0.05). Moreover, gestrinone significantly

increased the ratio of phospho-p38/p38 mitogen-activated protein kinase (MAPK) (P < 0.05). However,

no significant increase in apoptosis or cell cycle arrest was observed (P > 0.05) in response to the tested

concentrations of 0.1 to 3.0 mmol/L. As a conclusion, gestrinone suppresses the proliferation of uterine

leiomyoma cells mainly by regulating the activity of ERa/Src/p38 MAPK in a concentration-dependent

manner at a low concentration of 0.1�3.0 mM, but not significantly regulating apoptosis. Gestrinone

opposes the growth of uterine leiomyoma through multiple genes.

� 2012 Published by Elsevier Masson SAS.

Available online at

www.sciencedirect.com

1. Introduction

Uterine leiomyoma (fibroid or myoma) is a common benigntumor and affects more than 70% of women of reproductive age[1,2]. Uterine leiomyomas are hyper-responsive to estrogen.Evidence has also shown that cytokines and growth factors,including platelet-derived growth factor (PDGF), epidermalgrowth factor (EGF) and insulin-like growth factor (IGF)-I and -IIand their receptors participate in the uterine leiomyoma develop-ment [1,2]. Some genes, such as HMGA2 and Tsc-2, have also beenfound to be frequently aberrantly expressed in leiomyomas [2].

* Corresponding authors. Department of Obstetrics and Gynecology, Yueyang

Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai

University of Traditional Chinese Medicine, Shanghai 200437, China. Tel.: +86 21

6516 1782 3307; fax: +86 21 6516 4621, tel: +86 21 6422 9919;

fax: +86 21 6422 9919.

E-mail addresses: tingting185@yahoo.com.cn, zhuyan@sippr.org (T. Zhang).

0753-3322/$ – see front matter � 2012 Published by Elsevier Masson SAS.

doi:10.1016/j.biopha.2012.02.003

These findings suggest that multiple genes are involved inleiomyoma formation.

Recently, the up-regulation of mitogen-activated protein kinase(MAPK) signaling pathway has been described in uterineleiomyomas [3,4]. The activated p38 MAPK pathway engagespathways that can block proliferation or promote apoptosis [5].P38 MAPK was involved in regulating endothelin-1-mediatedsurvival in ELT3 uterine leiomyoma cells [6]. Increased p38 MAPKlevels are associated with reduced leiomyoma volume [7].Evidence has also demonstrated that estrogen receptor (ER)signaling is enhanced by activated MAPK. ERa is involved in thetransient activation of MAPK, leading to hyper-responsiveness ofuterine leiomyoma cells, which promotes leiomyoma prolifera-tion. Hypo-estrogen therapy significantly down-regulates theactivity of this signaling pathway and contributes to growthsuppression in uterine leiomyomas [8].

c-Src, a non-receptor protein tyrosine kinase, has also beenshown to associate with the development of uterine leiomyoma. c-Src over-expression and aberrant activation have been reported in

Y. Zhu et al. / Biomedicine & Pharmacotherapy 66 (2012) 569–577570

the development of hormone-related tumors [4]. We havepreviously found that Src was hyperactive in a guinea pig modelof uterine leiomyoma induced by estradiol [9] and in culturedleiomyoma cells [10,11]. Through the MAPK pathway, activated c-Src stimulates diverse cellular processes, including cell prolifera-tion, cellular adhesion and differentiation [12]. For instance, in theH9c2 and C2C12 cells lines, suppression of c-Src activity stimulatesmuscle differentiation by unidirectionally activating p38 MAPK[13]. The activation of the IGF-IR signaling pathway through Srchomology/collagen(Shc)/Grb2/MAPK is important for mediatinguterine leiomyoma growth [4]. Reducing the activity of Src helps tosuppress leiomyoma development [11].

Gestrinone, acts as an anti-estrogen, anti-progestin, androgenicand weak estrogen-like activity (Markiewicz and Gurpide, 1994),was initially tested as a contraceptive, and then widely used as atherapy for endometriosis and uterine leiomyoma; Usually, it takes6�12 months to suppress endometriosis and reduce uterinevolume [14,15]. To our knowledge, there was little laboratoryevidence to clarify the mechanism of action of gestrinone.Therefore, in this study, we investigated the effect of gestrinoneon the growth of human uterine leiomyoma cells and on theactivity of several signaling molecules, including ERa, Src and p38MAPK, which are involved in the growth of uterine leiomyomas.

2. Materials and methods

2.1. Chemicals and reagents

Gestrinone (13-ethyl-17-hydroxy-18,19-dinor-17a-pregna-4,9,ll-trien-20-yn-3-one) and C21H24O2 (molecular weight 308.41, puritygreater than 98%) were kind gifts from Beijing ZiZhu pharmaceutical(Beijing, China). The structure of gestrinone is shown in Fig. 1. SB-203580 was purchased from Calbiochem (EMD Biosciences, Inc.,Merck KGaA, Darmstadt, Germany). Dextran-coated charcoal-treatedfetal calf serum (D-FCS) was obtained from Biological Industries Ltd.(Kibbutz Beit Haemek, Israel). DMEM-F12 (1:1), Trizol and HEPESwere purchased from Gibco, Invitrogen Co. (Carlsbad, CA, USA).Dulbecco’s Modified Eagle’s Medium (DMEM) and Hank’s BalancedSalt Solution (HBSS), DMSO and type II collagenase were obtainedfrom Sigma Chemical Co. (St Louis, MO, USA). The CCK-8 kit wasobtained from Dojindo Molecular Technologies (Kumamoto, Japan).The FragEL DNA fragmentation detection kit was obtained fromCalbiochem (EMD Biosciences, Inc., Merck KGaA, Darmstadt,Germany). The mouse anti-ERa monoclonal antibody (65 kD), rabbitc-Src polyclonal antibody (60 kD), mouse anti-phospho-Tyr416-Srcmonoclonal antibody (60 kD) and mouse anti-phospho-Tyr527-Srcmonoclonal antibody (60 kD) were purchased from Cell SignalingTechnologies (Beverly, MA, USA). The anti-phospho-ERa (Ser167)antibody (66 kD) was obtained from Upstate Cell Signaling Solutions(Lake Placid, NY, USA). The enhanced chemiluminescence (ECL)

Fig. 1. Structure of gestrinone.

detection kit and bicinchoninic acid (BCA) kit were purchased fromPierce (Rockford, IL, USA). The Taq polymerase was purchased fromPromega (Shanghai Promega Biological Products, Ltd. Shanghai,China), and SYBR Green I was purchased from Invitrogen (InvitrogenCo, San Diego, CA, USA). The p38 pathway activation enzyme-linkedimmunosorbent assay (ELISA) kit was purchased from Kangchen Bio-tech (Shanghai, China).

2.2. Tissue collection

The leiomyoma tissue samples were obtained from premen-opausal women (38 to 45 years old) with regular menstrualcycles who were undergoing hysterectomies. None of thepatients had received any hormonal therapy before surgery.The patients underwent surgery in 2006 to 2009 at ShanghaiZhongshan Hospital. Informed consent for the use of the tissuewas obtained from each patient before surgery. The study wasapproved by the Ethical Committee for Clinical Research of theHospital and the Shanghai Institute of Planned ParenthoodResearch. The histological diagnosis of each uterine specimenwas examined. Samples were collected from the scheduledsurgeries at the patient’s convenience during the proliferativephase of the menstrual cycle.

2.3. Cell culture and treatment

The culture of leiomyoma cells was performed according to Zhuet al. [11]. Briefly, the leiomyoma tissue samples were rinsed inCa2+- and Mg2+-free HBSS, cut into small pieces, and digested inpre-warmed HBSS containing 2.0% type II collagenase (wt/vol) and25 mmol/L HEPES at 37 8C for 4 to 5 h. The cells were collected bycentrifugation at 460 � g for 5 min and washed several times withHBSS. The isolated leiomyoma cells were then transferred to a 75-cm2 flask at a density of 1 � 106 cells/mL and cultured in phenol-red free DMEM-F12 (1:1) with 10% (v/v) D-FCS, 25 mmol/L HEPES(pH 7.4), 2 mmol/L glutamine, 1 mmol/mL sodium pyruvate,10 mmol/mL non-essential amino acids and 1 mmol/mL insulin-transferrin-selenium-G supplement. The cells were cultured in ahumidified atmosphere of 5% CO2/95% O2. The culture medium wasreplaced every 3 to 4 d until the cells reached confluence (10�12d). The cultured cells were then digested with 0.25% trypsin andsubcultured under the same conditions as the primary cultures.The primary and subcultured smooth muscle cells were confirmedby immunohistochemical staining for the muscle-specific proteinsdesmin and a-smooth muscle actin.

At approximately 70% confluence, the leiomyoma cell cultureswere treated with gestrinone or DMSO alone as a control.Gestrinone was dissolved in DMSO and diluted to the desiredconcentration. The final concentration of DMSO in the culturemedia was 0.5% (v/v).

2.4. Cell viability assay

The viabilities of the cultured leiomyoma cells were determinedby the WST-8[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophen-yl)-2H-tetrazolium, monosodium salt] assayusing cell-counting kit-8 (CCK-8). The cells were cultured in 96-well plates and treated with DMSO or graded concentrations ofgestrinone (0.1, 0.5, 1.0, 5.0, 10, 50 or 100 mmol/L) for 20, 40 and60 h. Using a spectrophotometric plate reader (Bio-Tek ELX-800,USA), the absorbance (OD) at 450 nm was read to determine thecell viability in each well. Results are presented as the percentageof cell growth inhibition rate (%) and the 50% inhibitoryconcentration (IC50) and the corresponding 95% confidenceintervals (CI). The cell growth inhibition rate (%) was calculatedaccording to the following equation: [(1-OD of gestrinone-treated

Y. Zhu et al. / Biomedicine & Pharmacotherapy 66 (2012) 569–577 571

wells/OD of control wells) � 100%]. The IC50 and its corresponding95% CI were calculated from the dose response using the Blissmethod. The experiments were performed using three differentsamples.

2.5. Transmission electron microscopy

Morphological changes in the leiomyoma cells were visualizedby electron microscopy. Briefly, the cells treated with DMSO orgraded concentrations (0.1, 1.0 and 3.0 mmol/L) of gestrinone for60 h were collected by digesting with 0.25% trypsin andsequentially treat with 2.5% glutaraldehyde, 1% osmium tetroxide,propylene oxide and 1:1 Epon-propylene oxide mix. Ultrathinsections were cut and stained with 1% uranyl acetate and leadcitrate and examined with a transmission electron microscope(JEM-1230, JEOL, Japan) at an accelerating voltage of 80 kV with a40-mm objective aperture.

2.6. Cell cycle assay

The leiomyoma cells treated with DMSO or graded concentra-tions (0.1, 1.0 and 3.0 mmol/L) of gestrinone for 60 h were collectedand sequentially treated with cold ethanol, 0.1% Triton X-100 andRNase A (50 mg/mL). Next, the cell pellet was stained withpropidium iodide (50 mg/mL). The DNA content of the cells wasthen analyzed by flow cytometry using a FACSCalibur flowcytometer (Becton Dickinson, San Jose, CA, USA). At least 8000cells were analyzed in each sample. The presented values are theaverage ratio from three different samples.

2.7. Terminal deoxynucleotidyl transferase-mediated digoxigenin-

dUTP nick-end labeling assay (TUNEL)

Cells treated with DMSO or graded concentrations (0.1, 1.0 and3.0 mmol/L) of gestrinone for 60 h were collected and stained bytransferase-mediated digoxigenin-dUTP nick-end labeling assay(TUNEL) using the TdT-FragEL DNA fragmentation detection kit.Briefly, the adherent cells were cultured and fixed in a 96-wellNunc plate. Then, we performed the experiment according to themanufacturer’s instructions. The cells were stained with DAB, andthe numbers of positively stained cells were determined using alight microscope. For the negative control, dH2O was substitutedfor the TdT in the reaction mixture during the labeling step. At least1000 cells were analyzed in each sample. The presented values arethe average ratio of the number of positive cells out of the totalcells in three different samples.

2.8. P38 activity assay

ELISAs were used to evaluate the levels and activity of p38. Cellstreated with DMSO or graded concentrations (0.1, 1.0 and3.0 mmol/L) of gestrinone for 60 h were collected. The ELISA wasperformed according to manufacturer’s instructions. Briefly, celllysates were prepared by extracting proteins in a buffer containing1% (v/v) NP-40, 10% (v/v) glycerol, 137 mmol/L NaCl, 20 mmol/LTris-HCl (pH 7.4), 1% (v/v) Triton X-100, and protease inhibitors.The protein concentration was determined using the bicinchoninicacid (BCA) method according to the manufacturer’s instructions.The cell lysates were diluted to a concentration of 0.5 mg/mL. Thelysates were pipetted into wells that had been pre-coated withmonoclonal antibodiesagainst p38, pp38, or GAPDH and incubatedat 37 8C for 2 h. The unbound cell lysates were removed, and thewells were incubated with the detection antibody at 37 8C for 1 h,followed by secondary antibody at 37 8C for 30 min. Finally, thetetramethylbenzidine (TMB) substrate was added and incubated at37 8C for 10 min, and the absorbance at 450 nm was read

immediately. The presented values are the average ratio ofphosphorylated-p38 (pp38)/p38 in three different samples.

2.9. Quantitative real-time RT-PCR

Real-time RT-PCR was used to evaluate the mRNA levels of ERaand c-Src. The leiomyoma cells treated with DMSO or gradedconcentrations (0.1, 1.0 and 3.0 mmol/L) of gestrinone for 60 h werecollected. Total RNA was extracted using Trizol reagent and reverse-transcribed to form cDNA according to the routine method. b-actinwas used as internal control gene. The specific primer sequences usedin the real-time PCR are: b-actin forward: 5-’CCTGTACGCCAACA-CAGTGC-3’, reverse: 50-ATACTCCTGCTTGCTGATCC-30; Src forward:50-TCCTCGTGCGAGAAAGTGAG-30, reverse: 5-’CAGCTTGCGGATC-TTGTAGTG-30; ERa forward: 50-AAGGAGACTCGCTACTGTGC-30, re-verse: 50-CCACCTTTCATCATTCCCAC-30; The PCR products of b-actin,Src and ERa were 211, 107 and 224 bp, respectively. The purities ofthe amplified products were determined as a single peak in thedissociation curve. The final results are presented as the ratios of therelative amount of the target gene (c-Src and ERa) to the control gene(b-actin) using the 2�

DCT equation, where –DCT = CT, target gene –CT, internal control gene, according to the method described by Livaket al. [16] and Fujisawa et al. [17] The real-time PCR reaction wasperformed using a Rotor-Gene 3000 Real-time PCR device, and theresults were analyzed with Rotor-Gene 6.0 software (CorbettResearch Pty Ltd, Mortlake Australia). The results were comparablewith those normalized to b-actin, and the statistical analysis wasperformed using the average relative mRNA levels from threeindependent samples.

2.10. Western blotting

Western blotting was used to evaluate the levels and activity ofERa and Src according to the standard procedure. Cells treatedwith DMSO or graded concentrations (0.1, 1.0 and 3.0 mmol/L) ofgestrinone were collected and treated with RIPA solution contain-ing Tris-HCl (50 mM, pH 7.4), NP-40 (1%), NaCl (150 mM), EDTA(1 mM), PMSF (1 mM), aprotinin (25 mM), leupeptin (25 mM), andpepstatin (25 mM). The protein concentrations were determinedusing the bicinchoninic acid (BCA) method according to themanufacturer’s instructions. The primary antibodies against ERa,Src, phospho-167-ER, phospho-416-Src and phospho-527-Srcwere diluted 1:3000. The primary b-actin antibody was diluted1:10,000. The relative levels of protein were semi-quantitativelydetermined using Image-Pro Plus1 Version 5.1.0 software (MediaCybernetics Inc.) by taking the ratio of the band intensities of thetarget proteins over b-actin. Statistical analyses were performedon the average protein level of three independent samples.

2.11. Statistical analysis

The data are presented as means � SD. The statistical signifi-cance of the inhibition rate at various times was determined with therepeated measures general linear model using SPSS 11.5 software(version 11.5 for Windows; SPSS, Chicago, Illinois, USA). Thestatistical significance among three or more groups was determinedby one-way ANOVA with post hoc multiple comparisons using SPSS11.5 software. P < 0.05 was considered statistically significant.

3. Results

3.1. Effects of gestrinone on the growth of cultured uterine leiomyoma

cells

The effect of the gestrinone concentration gradient on thenumber of viable cultured leiomyoma cells was determined. The

Fig. 2. Inhibitory effects of graded concentrations of gestrinone on the growth of

cultured leiomyoma cells at 20, 40 and 60 h, as assessed by CCK-8. The experiments

were performed in triplicate with three different cultured samples. Leiomyoma

cells were treated with graded concentrations of gestrinone (0.1�100 mM). Data are

presented as means � SD of three independent samples. aP < 0.05 vs. cells treated for

20 h; bP < 0.05 vs. cells treated for 40 h.

Y. Zhu et al. / Biomedicine & Pharmacotherapy 66 (2012) 569–577572

IC50 of gestrinone and the corresponding 95% CI were 43.67(23.46�81.32), 27.78 (12.51�61.68) and 15.25 (7.17�32.43)mmol/L at 20, 40 and 60 h, respectively. There was a significantdifference between the degree of cell viability inhibition detectedat 20, 40 and 60 h (P < 0.05, Fig. 2). This suggested that gestrinoneexhibited stronger inhibitory effects on the growth of leiomyomacells at 60 h than that at 20 and 40 h.

3.2. Effects of gestrinone on the morphology of cultured uterine

leiomyoma cells

The morphologies of the leiomyoma cells treated withgestrinone were visualized by optical and electron microscopy.The control cells appeared robust, manifested an elongated shapeand abundant cytoplasm, were well aligned and more dense andexhibited a whirlpool or fan-shape. After treatment with thegraded concentrations of gestrinone for 60 h, morphologicalalterations were observed. If the concentration of gestrinonewas less than 10 mM, no pronounced morphological changes wereobserved; however, when the concentration of gestrinone wasabove 10 mM, the cells appeared less dense, the cytoplasm wasatrophic, the intercellular connections dwindled and nuclearaggregations were observed. When the concentration of gestri-none reached 100 mM, the cells appeared swollen and dead (Fig. 3).

Fig. 3. Effects of gestrinone on the morphology of cultured uterine leiomyoma cells as as

concentrations of gestrinone (at 1.0, 3.0 10.0 and 100 mmol/L) for 60 h. (A) Leiomyoma ce

gestrinone, respectively. (D) Leiomyoma cells treated with 10.0 mM gestrinone. (E) Lei

Under the electron microscope, a series of morphologicalchanges were further observed after treatment with the gradedconcentrations of gestrinone. At concentrations of 0.1�3.0 mMgestrinone, the cells showed slight signs of shrinkage, nucleardeformation, and chromatin aggregation. At a gestrinone concen-tration of 10 mM, some of the cells exhibited damaged nuclei, butthe mitochondria remained intact (Fig. 4).

3.3. Effects of gestrinone on the cell cycle and apoptosis in the cultured

uterine leiomyoma cells

The DNA content and cell cycle analysis in the leiomyoma cellswas performed by flow cytometry using a FACSCalibur instrument.Compared to the control, in the presence of 0.1�3.0 mmol/Lgestrinone, there was a subtle increase in the DNA content in theG0-G1 phase and a decrease in the S phase. In addition, gestrinoneinduced a slight increase in the sub-G1 peak in a concentration-dependent manner. However, no pronounced differences weredetected (P > 0.05, Fig. 5).

Apoptosis was further assessed by TUNEL staining. The non-apoptotic cells were predominantly round and appeared counter-stained, and the apoptotic cells appeared dark brown and exhibiteda variety of distinctive morphological changes, including pyknosis,dark brown encircling and densely stained oval, crescent or ring-shaped nuclei. In the presence of 0.01�3.0 mmol/L gestrinone,more dark brown-stained apoptotic leiomyoma cells wereobserved compared to in the control cells. However, there wasno statistically significant increase in the ratio of apoptotic cellsdetected after the treatment with graded gestrinone (P > 0.05,Fig. 6).

3.4. Effects of gestrinone on the levels of estrogen receptor alpha

(ERa) and phospho-Ser167-ERa in uterine leiomyoma cells

The effect of gestrinone on the mRNA levels of ERa was analyzedby real-time RT-PCR. The results showed that gestrinone treatmentreduced the relative mRNA levels of ERa in a concentration-dependent manner at concentrations of 0.1�3.0 mmol/L comparedto the control (Fig. 7A).

The effect of gestrinone on the protein levels of ERa wasanalyzed by Western blotting. Similar to the decreased mRNAlevels, gestrinone reduced the protein levels of ERa (expressed as aratio vs. b-actin) in the leiomyoma cells in a concentration-dependent manner at concentrations of 0.1�3.0 mmol/L, and therewas a pronounced decline in the ratio of ERa/b-actin in thepresence of 1.0 and 3.0 mmol/L gestrinone compared to in thecontrol cells (P < 0.05, Fig. 7B).

Phospho-Ser167-ERa is an active form of ERa that isphosphorylated at serine-167; therefore, the effect of gestrinone

sessed by optical microscopy. The cells were cultured in DMSO (control) or graded

lls treated with DMSO alone. (B and C) Leiomyoma cells treated with 1.0 and 3.0 mM

omyoma cells treated with 100 mM gestrinone.

Fig. 4. Effects of gestrinone on the morphology of cultured uterine leiomyoma cells, as assessed by electron transmission microscopy. The cells were cultured in DMSO

(control) or graded concentrations of gestrinone (0.1, 1.0, 3.0 and 10.0 mmol/L) for 60 h. (A) Leiomyoma cells treated with DMSO alone. (B) Leiomyoma cells treated with

0.1 mM gestrinone. (C) Leiomyoma cells treated with 1.0 mM gestrinone. (D) Leiomyoma cells treated with 3.0 mM gestrinone. (E) Leiomyoma cells treated with 10.0 mM

gestrinone.

Fig. 6. Effects of gestrinone on apoptosis of cultured uterine leiomyoma cells, as assesse

method. The cells were cultured in DMSO (control) or graded concentrations of gestrinon

0.01 (Ges-0.01), 0.1 (Ges-0.1) 1.0 (Ges-1.0) and 3.0 (Ges-3.0) mM (� 200). Data are pre

Fig. 5. Effects of gestrinone on the DNA content and cell cycle in cultured uterine

leiomyoma cells, as assessed by flow cytometry. The cells were cultured in DMSO

(control) or graded concentrations of gestrinone (0.1, 1.0, and 3.0 mmol/L) for 60 h.

Y. Zhu et al. / Biomedicine & Pharmacotherapy 66 (2012) 569–577 573

on the level of phospho-Ser167-ERa was also analyzed. The resultsindicated that there was a concentration-dependent decrease inphospho-Ser167-ERa/b-actin in the presence of gestrinone, and apronounced difference was also detected at concentrations of 1.0and 3.0 mmol/L in the treated leiomyoma cells compared to thecontrol cells (P < 0.05, Fig. 7C).

3.5. Effects of gestrinone on the levels of c-Src, phospho-Tyr416-Src

and phospho-Tyr527-Src in leiomyoma cells

Because the function of ER is related to the activity of c-Src, theeffect of gestrinone on the mRNA levels of c-Src was also analyzedby real-time RT-PCR. The results showed that gestrinone treatmentincreased the relative mRNA levels of Src/b-actin in a concentra-tion-dependent manner at concentrations of 0.1�3.0 mmol/Lcompared with the control (Fig. 8A).

The effect of gestrinone on the protein level of c-Src wasanalyzed by western blotting. Compared with the control, therewas an obvious reduction in the protein levels of Src/b-actin in thecells treated with 0.1 mmol/L gestrinone (P < 0.05). Similar to the

d by the transferase-mediated digoxigenin-dUTP nick-end labeling assay (TUNEL)

e (0.01, 0.1, 1.0, and 3.0 mmol/L) for 60 h. Leiomyoma cells treated with 0 (control),

sented as means � SD of three independent samples.

Fig. 7. Effects of gestrinone on the levels of estrogen receptor alpha (ERa) and Phospho-Ser167-ERa in cultured uterine leiomyoma cells, as assessed by real-time RT-PCR or

Western blotting. The cells were cultured in DMSO (control) or graded concentrations of gestrinone (0.1, 1.0, and 3.0 mmol/L) for 60 h. Data are presented as means � SD of

three independent samples. cP < 0.05 vs. the control cells. (A) The effect of gestrinone on the mRNA levels of ERa. (B) The effect of gestrinone on the protein levels of ERa. (C) The

effect of gestrinone on the levels of phospho-Ser167-ERa.

Fig. 8. Effects of gestrinone on the levels of c-Src, phospho-Tyr416-Src and phospho-Tyr527-Src in cultured leiomyoma cells, as assessed by real-time RT-PCR or Western

blotting. The cells were cultured in DMSO (control) or graded concentrations of gestrinone (0.1, 1.0, and 3.0 mmol/L) for 60 h. Data are presented as means � SD of three

independent samples. cP < 0.05 vs. control cells; aP < 0.05 vs. cells treated with 0.1 mmol/L gestrinone; dP < 0.05 vs. cells treated with 3.0 mmol/L gestrinone. (A) The effect of

gestrinone on Src mRNA levels. (B) The effect of gestrinone on the protein levels of Src. (C) The effect of gestrinone on the levels of phospho-Tyr416-Src. (D) The effect of gestrinone on

the level of phospho-Tyr527-Src.

Y. Zhu et al. / Biomedicine & Pharmacotherapy 66 (2012) 569–577574

Y. Zhu et al. / Biomedicine & Pharmacotherapy 66 (2012) 569–577 575

alteration in mRNA levels, there was a concentration-dependentincrease in the protein levels of c-Src/b-actin after treatment withgestrinone at concentrations of 0.1�3.0 mmol/L (Fig. 8B).

The levels of phospho-Tyr416-Src and phospho-Tyr527-Src inthe leiomyoma cells were also analyzed. The results indicated thatthere was a concentration-dependent change in the levels ofphospho-Tyr416-Src and phospho-Tyr527-Src in the leiomyomacells after treatment with 0.1–3.0 mmol/L gestrinone. Compared tothe control, there was a concentration-dependent decrease in thelevels of phospho-Tyr416-Src, and a pronounced decline wasobserved at 3.0 mmol/L gestrinone (P < 0.05, Fig. 8C). Furthermore,the results also indicated that there was a slight concentration-dependent increase in the expression of phospho-Tyr527-Src aftertreatment with 1.0�3.0 mmol/L gestrinone, but no pronouncedincrease was observed (P > 0.05, Fig. 8D).

3.6. Effects of gestrinone on the activity of p38 mitogen-activated

protein kinase (MAPK) in uterine leiomyoma

We investigated the activity of p38 MAPK to further analyze themechanism of action of gestrinone. As an inhibitor of p38 MAPK,SB203580 suppressed the ratio of pp38 to p38 MAPK in theleiomyoma cells. Compared with the control, gestrinone increasedthe relative ratio of pp38/p38 MAPK in a concentration-dependentmanner, and there was a pronounced increase in cells treated with3.0 mmol/L gestrinone (P < 0.05, Fig. 9). This implies thatgestrinone increases the activity of p38 MAPK.

4. Discussion

In this study, we provide evidence supporting the effectivenessof gestrinone inhibiting the growth of human uterine leiomyomacells. At the tested concentrations, the mechanism of action ofgestrinone involves regulating the activity of ERa/Src/p38/MAPK,but does not obviously involve promoting apoptosis.

In a preliminary test, we investigated the inhibitory effects ofgestrinone on the growth of leiomyoma and myometrial cells. Wefound that gestrinone exerts an inhibitory effect on the growth ofboth leiomyoma and myometrial cells, but there was a differencebetween the two type of cells. In the myometrial cells, the IC50

values of gestrinone and the corresponding 95% CI were 30.55(18.39�50.73), 22.61 (12.33�41.44) and 25.46 (14.05�46.14)mmol/L at 20, 40 and 60 h, respectively. There was no obviousdifference in the inhibitory rates among these time points. In the

Fig. 9. Effects of gestrinone on the activity of p38 mitogen-activated protein kinase

(MAPK) in cultured leiomyoma cells, as assessed by enzyme-linked immunosorbent

assay (ELISA). The leiomyoma cells were cultured in DMSO (control), SB203580 or

graded concentrations of gestrinone (0.1, 1.0 and 3.0 mmol/L) for 60 h. ‘‘Leio’’ stands

for leiomyoma cells; ‘‘SB’’ stands for SB203580. Data are presented as means � SD of

three independent samples. cP < 0.05 vs. control leiomyoma cells; aP < 0.05 vs.

2.6 mmol/L SB203580-treated leiomyoma cells; bP < 0.05 vs. 0.1 mmol/L gestrinone

(G-0.1)-treated leiomyoma cells; dP < 0.05 vs. 1.0 mmol/L gestrinone (G-1.0)-treated

leiomyoma cells; eP < 0.05 vs. 3.0 mmol/L gestrinone (G-3.0)-treated leiomyoma cells.

leiomyoma cells, however, gestrinone caused a markedly strongerinhibition on the growth of leiomyoma cells at 60 h compared to at20 and 40 h. This result suggests that gestrinone exerts a strongerinhibition on the growth of leiomyoma cells vs. myometrial cells at60 h. This indicates that gestrinone may exhibit weaker inhibitoryeffects and fewer adverse effects on the growth of myometrial cellsthan leiomyoma cells in a long-term of clinical treatment.Therefore, we focused on investigating the mechanism of actionof gestrinone on the leiomyoma cells.

It has been confirmed that ERa plays a vital role in thedevelopment of uterine leiomyoma. Clinically, reducing theexpression of ERa is a vital mechanism of action for treatmentof uterine leiomyoma. Herein, we found that gestrinone decreasesthe RNA and protein levels of ERa in a concentration-dependentmanner. The result was in accordance with our previous reportusing the guinea pig model of uterine leiomyoma [9]. Thefunctional regulation of ER is mediated by phosphorylation ofkey residues, including Ser118 and Ser167, which influences bothERa-DNA binding and the recruitment of cofactor molecules [18].Therefore, the phosphorylation of these sites plays an importantrole in regulating ERa activity. For instance, the activation of ERaby phosphorylation at Ser-167 may confer tamoxifen resistance inbreast cancer patients [20,21]. In this study, we found thatgestrinone significantly decreased the levels of phospho-Ser167-ERa in a concentration-dependent manner. The findings suggestthat gestrinone not only decreased the expression of ERa but alsoablated the activity of ERa. Because the phosphorylation of amino-terminal sites on ERa increases the transcriptional activity of ERa[22], the inhibitory effect of gestrinone on the activity of ERa willreduce the transcriptional activity of ERa, which will contribute toabating the resistance of leiomyoma cells to the estrogen responseand inhibit leiomyoma proliferation.

The function of ER is related to c-Src activity. As a tyrosinekinase, c-Src has recently received attention because of theprevalence of activated Src in hormone-dependent tumor progres-sion and metastasis [23]. Src is downstream of classical sex steroidreceptors, including ER, PR, and androgen receptors. There is adirect association between ER and Src, and this association isprevented by a pure anti-estrogen agent [24]. Src activity can berapidly stimulated by estrodiol; in turn, activated c-Src canphosphorylate the ER and drive expression of certain ERa-targetgenes [19]. This process is involved in the non-transcriptionalaction of estradiol [24]. There is a feed-forward signaling loopinvolving estrogen, ERa and Src. On one hand, crosstalk betweenERa and c-Src improves the transcriptional activity of ERa; on theother hand, the crosstalk also activates ERa proteolysis. IncreasedSrc activity correlates with reduced ERa t1/2, and the inhibition ofc-Src impairs ERa ubiquitylation and ERa loss, leading to elevatedlevels of ERa [19]. In this study, we found that there was a cleardecline in the protein levels of Src/b-actin in cells treated with0.1 mmol/L gestrinone. These results suggest that gestrinonedecreases Src levels at the low concentration of 0.1 mmol/L. Asthe concentration increased, there was a concentration-dependentincrease in the mRNA and protein levels of c-Src/b-actin;meanwhile, gestrinone decreased the level of ERa in a concentra-tion-dependent manner at concentrations of 1.0 and 3.0 mmol/L.This result is consistent with the findings of Chu et al. [19].According to these results, we presume that gestrinone acts on thefeed-forward signaling loop between Src and ERa. Becausegestrinone exhibited a clear inhibitory effect on the levels ofERa, we suppose that the elevated expression of c-Src is a result ofERa inhibition. Gestrinone primarily functions as an inhibitor ofhormone receptors but is not an inhibitor of Src tyrosine kinase.

Furthermore, we found that gestrinone reduced the level ofphospho-Tyr416-Src and increased the level of phospho-Tyr527-Src in a concentration-dependent manner. The various Src

Y. Zhu et al. / Biomedicine & Pharmacotherapy 66 (2012) 569–577576

phosphorylation sites are responsible for the activity or inactivityof Src. Phosphorylation of a conserved tyrosine in the activationloop (Tyr-416) enhances the autophosphorylation of Src andincreases the kinase activity of Src [25]. The phosphorylation ofanother conserved tyrosine (Tyr527) inactivates this activity andkeeps Src in an inactive state [26]. Our results have demonstratedthat gestrinone clearly down-regulates the autophosphorylation ofSrc and slightly increases the inactive state of Src in a concentra-tion-dependent manner. These results suggest that gestrinonesuppresses the growth of cultured leiomyoma cells by abating Srcactivity, contributing to decreased ERa activity and a reducedresponse of the leiomyoma to estrogen. Taken together, we believethat gestrinone functions much more as an active regulator ratherthan as an inhibitor of Src at the tested concentrations.

However, our results were not consistent with our previousfindings in an animal model of guinea pig leiomyoma [9]. In oneprevious in vivo study, we found that gestrinone (at doses of 2 and4 mg/kg, administered orally to the guinea pigs) simultaneouslydecreased the levels of Src and ER in a dose-dependent manner; inthe present in vitro study, however, gestrinone increased the levelsof Src in a concentration-dependent manner. We presumed thatthe contradiction may stem from the different experimentalsystems and tested dosages. The different impact of gestrinone onthe expression of Src may relate to the doses administered, theconcentration of estrogen, and the type of estrogen receptor, butmore research is required.

Moreover, recent evidence shows that there is crosstalkbetween c-Src and p38 MAPK [27]. Src acts upstream of p38MAPK [13]. Suppression of c-Src activity stimulates muscledifferentiation by activating p38 MAPK unidirectionally [13].Kim et al. found that inhibition of c-Src attenuated p38 MAPKactivation and enhanced radiation-induced cell death in humancervical cancer cell lines [28]. These findings suggest that there isan inverse correlation between the activities of c-Src and pp38MAPK [13]; and a positive association between c-Src and pp38MAPK. Because gestrinone demonstrated a concentration-depen-dent increasing in the levels of c-Src/b-actin and reducing the levelof phospho-Tyr416-Src, we further investigated the effect ofgestrinone on the activity of p38 MAPK. We found that gestrinonemarkedly enhanced pp38 MAPK in a concentration-dependentmanner while increasing the expression of Src and abating theactivity of Src. These results are consistent with Lim et al. [13] andKim et al. [28]. According to Lim et al., we presume that theenhancement in active p38 MAPK is a result of reduced Src activity.However, more research is required to prove this. Our result wasalso similar to that of Xu et al., who reported that the progestinlevonorgestrel (LNG) inhibited the proliferation of uterineleiomyoma cells and significantly increased p38 phosphorylation[7]. Similar to LNG, we believe that gestrinone inhibits theproliferation of uterine leiomyoma cells in a mechanism related tothe increased activity of p38 MAPK.

In addition, we found that there was no pronounced increase inthe apoptosis rate or DNA content in the cell cycle in the presenceof 0.1�3.0 mmol/L gestrinone compared to the control. Theleiomyoma cells exhibited obvious features of apoptosis onlywhen the concentration of gestrinone reached 100 mM. Therefore,at concentrations of 3.0 mmol/L, we believe that gestrinonesuppresses the proliferation of uterine leiomyoma cells mainlyby regulating the activity of ERa/Src/p38 MAPK and not by directlyinducing significant levels of apoptosis. We expect that this mightbe one of the reasons why the long-term administration ofgestrinone is required to reduce the volume of uterine leiomyoma.

Our results suggest that gestrinone acts on multiple genesrather than a single gene while impeding abnormal proliferation ofuterine leiomyoma. In addition, although gestrinone exerts aweaker inhibitory effect on myometrial cells compared to on

leiomyoma cells, it is possible that a similar mechanism of ERa/Src/p38 MAPK activity modulation occurs in the myometrial cells,and more research is required.

Disclosure of interest

The authors declare that they have no conflicts of interestconcerning this article.

Acknowledgements

This work was supported by a grant from the Shanghai LeadingAcademic Discipline Project (grant No. S30303).

References

[1] Walker CL. Role of hormonal and reproductive factors in the etiology andtreatment of uterine leiomyoma. Recent Prog Horm Res 2002;57:277–94.

[2] Walker CL, Stewart EA. Uterine fibroids: the elephant in the room. Science2005;308:1589–92.

[3] Chen HW, Liu JC, Chen JJ, Lee YM, Hwang JL, Tzeng CR. Combined differentialgene expression profile and pathway enrichment analyses to elucidate themolecular mechanisms of uterine leiomyoma after gonadotropin-releasinghormone treatment. Fertil Steril 2008;90:1219–25.

[4] Yu L, Saile K, Swartz CD, He H, Zheng X, Kissling GE, et al. Differentialexpression of receptor tyrosine kinases (RTKs) and IGF-I pathway activationin human uterine leiomyomas. Mol Med 2008;14:264–75.

[5] Bulavin DV, Fornace Jr AJ. p38 MAP kinase’s emerging role as a tumorsuppressor. Adv Cancer Res 2004;92:95–118.

[6] Oyeniran C, Tanfin Z. MAPK14 cooperates with MAPK3/1 to regulate endothe-lin-1-mediated prostaglandin synthase 2 induction and survival in leiomyomabut not in normal myometrial cells. Biol Reprod 2011;84:495–504.

[7] Xu Q, Qiu L, Zhu L, Luo L, Xu C. Levonorgestrel inhibits proliferation and inducesapoptosis in uterine leiomyoma cells. Contraception 2010;82:301–8.

[8] Di X, Yu L, Moore AB, Castro L, Zheng X, Hermon T, et al. A low concentration ofgenistein induces estrogen receptor alpha and insulin-like growth factor-Ireceptor interactions and proliferation in uterine leiomyoma cells. HumReprod 2008;23:1873–83.

[9] Zhu Y, Qiu XY, Wang L, Wu JH, Liu GM, He GL, et al. Effects of gestrinone onuterine leiomyoma and expression of c-Src in a guinea pig model. ActaPharmacol Sin 2007;28:685–94.

[10] Strizzi L, Bianco C, Hirota M, Watanabe K, Mancino M, Hamada S, et al.Development of leiomyosarcoma of the uterus in MMTV-CR-1 transgenicmice. J Pathol 2007;211:36–44.

[11] Zhu Y, Xie SW, Zhang JF, Zhang TT, Zhou JY, Cao Y, et al. Involvement of Bcl-2,Src, and ERalpha in gossypol-mediated growth inhibition and apoptosis inhuman uterine leiomyoma and myometrial cells. Acta Pharmacol Sin2010;31:1593–603.

[12] Biscardi JS, Tice DA, Parsons SJ. c-Src, receptor tyrosine kinases, and humancancer. Adv Cancer Res 1999;76:61–119.

[13] Lim MJ, Seo YH, Choi KJ, Cho CH, Kim BS, Kim YH, et al. Suppression of c-Srcactivity stimulates muscle differentiation via p38 MAPK activation. ArchBiochem Biophys 2007;465:197–208.

[14] Coutinho EM. Treatment of large fibroids with high doses of gestrinone.Gynecol Obstet Invest 1990;30:44–7.

[15] La Marca A, Giulini S, Vito G, Orvieto R, Volpe A, Jasonni VM. Gestrinone in thetreatment of uterine leiomyomata: effects on uterine blood supply. Fertil Steril2004;82:1694–6.

[16] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods2001;25:402–8.

[17] Fujisawa T, Velichko S, Thai P, Hung LY, Huang F, Wu R. Regulation of airwayMUC5AC expression by IL-1beta and IL-17A; the NF-kappaB paradigm. JImmunol 2009;183:6236–43.

[18] Lannigan DA. Estrogen receptor phosphorylation. Steroids 2003;68:1–9.[19] Chu I, Arnaout A, Loiseau S, Sun J, Seth A, McMahon C, et al. Src promotes

estrogen-dependent estrogen receptor alpha proteolysis in human breastcancer. J Clin Invest 2007;117:2205–15.

[20] Campbell RA, Bhat-Nakshatri P, Patel NM, Constantinidou D, Ali S, Nakshatri H.Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptoralpha: a new model for anti-estrogen resistance. J Biol Chem 2001;276:9817–24.

[21] Guo JP, Shu SK, Esposito NN, Coppola D, Koomen JM, Cheng JQ. IKKepsilonphosphorylation of estrogen receptor alpha Ser-167 and contribution totamoxifen resistance in breast cancer. J Biol Chem 2010;285:3676–84.

[22] Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, et al. Activa-tion of the estrogen receptor through phosphorylation by mitogen-activatedprotein kinase. Science 1995;270:1491–4.

[23] Migliaccio A, Castoria G, Di Domenico M, De Falco A, Bilancio A, Auricchio F. Srcis an initial target of sex steroid hormone action. Ann N Y Acad Sci2002;963:185–90.

Y. Zhu et al. / Biomedicine & Pharmacotherapy 66 (2012) 569–577 577

[24] Migliaccio A, Castoria G, Di Domenico M, de Falco A, Bilancio A, Lombardi M,et al. Sex steroid hormones act as growth factors. J Steroid Biochem Mol Biol2002;83:31–5.

[25] Boulet I, Fagard R, Fischer S. Correlation between phosphorylation and kinaseactivity of a tyrosine protein kinase: p56lck. Biochem Biophys Res Commun1987;149:56–64.

[26] Thomas JE, Soriano P, Brugge JS. Phosphorylation of c-Src on tyrosine 527 byanother protein tyrosine kinase. Science 1991;254:568–71.

[27] Lee J, Hong F, Kwon S, Kim SS, Kim DO, Kang HS, et al. Activation of p38MAPK induces cell cycle arrest via inhibition of Raf/ERK pathwayduring muscle differentiation. Biochem Biophys Res Commun 2002;298:765–71.

[28] Kim MJ, Byun JY, Yun CH, Park IC, Lee KH, Lee SJ. c-Src-p38 mitogen-activatedprotein kinase signaling is required for Akt activation in response to ionizingradiation. Mol Cancer Res 2008;6:1872–80.