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Gonadotropin-releasing hormone type II (GnRH-II) agonist regulates the invasiveness of endometrial cancer… Wu, Hsien-Ming; Wang, Hsin-Shih; Huang, Hong-Yuan; Lai, Chyong-Huey; Lee, Chyi-Long; Soong, Yung-Kuei; Leung, Peter C Jun 20, 2013

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RESEARCH ARTICLE Open AccessGonadotropin-releasing hormone type II (GnRH-II)agonist regulates the invasiveness of endometrialcancer cells through the GnRH-I receptor andmitogen-activated protein kinase (MAPK)-dependentactivation of matrix metalloproteinase (MMP)-2Hsien-Ming Wu1*, Hsin-Shih Wang1*, Hong-Yuan Huang1, Chyong-Huey Lai1, Chyi-Long Lee1, Yung-Kuei Soong1and Peter CK Leung2AbstractBackground: More than 25% of patients diagnosed with endometrial carcinoma have an invasive primary canceraccompanied by metastases. Gonadotropin-releasing hormone (GnRH) plays an important role in reproduction. Inmammals, expression of GnRH-II is higher than GnRH-I in reproductive tissues. Here, we examined the effect of aGnRH-II agonist on the motility of endometrial cancer cells and its mechanism of action in endometrial cancertherapy.Methods: Immunoblotting and immunohistochemistry (IHC) were used to determine the expression of the GnRH-Ireceptor protein in human endometrial cancer. The activity of MMP-2 in the conditioned medium was determinedby gelatin zymography. Cell motility was assessed by invasion and migration assay. GnRH-I receptor si-RNA wasapplied to knockdown GnRH-I receptor.Results: The GnRH-I receptor was expressed in the endometrial cancer cells. The GnRH-II agonist promoted cellmotility in a dose-dependent manner. The GnRH-II agonist induced the phosphorylation of ERK1/2 and JNK, andthe phosphorylation was abolished by ERK1/2 inhibitor (U0126) and the JNK inhibitor (SP600125). Cell motilitypromoted by GnRH-II agonist was suppressed in cells that were pretreated with U0126 and SP600125. Moreover,U0126 and SP600125 abolished the GnRH-II agonist-induced activation of MMP-2. The inhibition of MMP-2 withMMP-2 inhibitor (OA-Hy) suppressed the increase in cell motility in response to the GnRH-II agonist. Enhanced cellmotility mediated by GnRH-II agonist was also suppressed by the knockdown of the endogenous GnRH-I receptorusing siRNA.Conclusion: Our study indicates that GnRH-II agonist promoted cell motility of endometrial cancer cells throughthe GnRH-I receptor via the phosphorylation of ERK1/2 and JNK, and the subsequent, MAPK-dependent activationof MMP-2. Our findings represent a new concept regarding the mechanism of GnRH-II-induced cell motility inendometrial cancer cells and suggest the possibility of exploring GnRH-II as a potential therapeutic target for thetreatment of human endometrial cancer.Keywords: GnRH-II agonist, Invasion, ERK1/2, JNK, MMP-2* Correspondence: danielwu@cgmh.org.tw; hswang@cgmh.org.tw1Department of Obstetrics and Gynecology, Chang Gung Memorial HospitalLinkou Medical Center, Chang Gung University School of Medicine,Taoyuan 333, TaiwanFull list of author information is available at the end of the article© 2013 Wu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Wu et al. BMC Cancer 2013, 13:300http://www.biomedcentral.com/1471-2407/13/300BackgroundEndometrial cancer is one of the most common gy-necological cancers in the world and accounts forapproximately 50,000 deaths worldwide each year [1].Patients with tumor confined to the uterus are treatedwith surgery and radiotherapy [2-4]. However, morethan 25% of patients diagnosed with endometrial car-cinoma have an invasive primary cancer accompaniedby metastases. Despite treatment with aggressive che-motherapeutic regimens, these patients have a 5-yearsurvival rate of less than 20% [1]. In fact, metastasisrepresents the main cause of death for patients withendometrial cancer, and the battle against this cancerwould greatly benefit from the identification of factorsinvolved in the metastatic process. Certain cases ofendometrial cancer with a particular morphology, ad-verse histopathological features or advanced stage arecharacterized by aggressive behavior and poor progno-sis [5]. The molecular pathogenesis of endometrial can-cer remains poorly understood, resulting in a limitedcure rate in the treatment of advanced cases. Thus, newtherapeutic approaches are needed for advanced or re-lapsed disease. The hypothalamic peptide GnRH playsan important role in the maintenance of intrauterinetissues and the development of endometrial cancer[6-9]. In mammals, GnRH-II is more widely present inperipheral tissues than GnRH-I, which suggests thatGnRH-II may have additional functions. GnRH-II hasbeen shown to have direct antiproliferative effects inthe growth of endometrial cancer cells [10]. These find-ings raise the possibility that GnRH-II could directlyregulate the tumor progression of endometrial cancercells. The role of GnRH-II in endometrial cancer cellinvasion is not known, and the mechanism by whichGnRH-II regulates the invasiveness of endometrial tu-mors has also not been established. The MAPKs areconsidered to be important components of GnRH-induced signaling pathways in various cell types[10-12]. We have previously demonstrated that theanti-proliferative effect of GnRH-II is mediated by theMAPKs signalings [10,13]. Different mechanisms havebeen suggested for MAPK activation through GPCRs[14,15]. MMPs are largely implicated in promotingangiogenesis and tumor metastasis [16,17]. Some evi-dence indicates an expanded role for GnRH in certainaspects of gynecologic tumor progression, such as me-tastasis, via the activation of MMPs and the subsequentincrease in cell migration and invasion [18]. In thepresent study, we examined the effect of a GnRH-IIagonist on the motility of endometrial cancer cells andthe mechanisms of the action involved. Our results sug-gest the possibility of exploring GnRH-II as a potentialtherapeutic target for the treatment of human endo-metrial cancer.ResultsGnRH-II stimulates migration and invasion of endometrialcancer cellsIn cancer invasion and metastasis, an imbalanced regula-tion of cell motility and proteolysis appears to be a criticalevent [19]. To study whether the expression of the GnRH-Ireceptor is associated with the metastasis of endometrialcancer cells, the effect of GnRH-II on cell migration and in-vasion was examined. Ishikawa and ECC-1 endometrialcancer cells, which express functional GnRH-I receptors[10], were treated with a GnRH-II agonist. The ability ofthe cells to migrate was assessed using a Transwell migra-tion assay. The GnRH-II agonist stimulated the migrationof endometrial cancer cells through the uncoated porousfilter in a dose-dependent manner at concentrations of 1nM to 1 μM with a maximal effect at 1 μM (Figure 1A).We also assessed the invasion of the cells in vitro inresponse to the GnRH-II agonist stimulus using Transwellswith filters coated with Matrigel. Our results indicated thatthe GnRH-II agonist induced endometrial cancer cell inva-sion in a dose-dependent manner at concentrations of 1nM to 1 μM with a maximal effect at 1 μM (Figure 1B).Expression of the GnRH-I receptor (GnRH-IR) inendometrial cancerTo examine the expression of the GnRH-I receptor,Ishikawa and ECC-1 endometrial cancer cells were lysed,and the expression of GnRH-I receptor was examined byimmunoblot analysis. As shown in Figure 2A, the GnRH-Ireceptor was detected in Ishikawa and ECC-1 endometrialcancer cells. Using immunohistochemical analysis, weconfirmed that the GnRH-I receptor was expressed in thehuman endometrial cancer tissue samples (Figure 2B).The GnRH-II-induced cell migration and invasion ismediated by GnRH-I receptors in endometrial cancer cellsIt is assumed that both GnRH-I and GnRH-II exert theirbiological effects by binding to a common GnRH-I re-ceptor [20]. To investigate whether the effects of GnRH-II on cell migration and invasion were mediated by theGnRH-I receptor, Ishikawa and ECC-1 endometrial can-cer cells were transfected with a GnRH-I receptor siRNAto knockdown the endogenous GnRH-I receptor expres-sion. The trnasfection efficiency of siRNA in bothIshikawa and ECC-1 was examined by using fluorescence-labeling siRNA, si-GLO. As shown in Figure 3A, both cellswere almost transfected after 24 hours si-GLO transfec-tion. Treatment with 50 nM GnRH-I receptor siRNAdown-regulated GnRH-I receptor expression in Ishikawaand ECC-1 endometrial cancer cells (Figure 3B). More-over, knockdown of the endogenous GnRH-I receptorsignificantly abolished the GnRH-II-mediated cell mi-gration (Figure 3C) and abolished the GnRH-II-pro-moted cell nvasion. Taken together, these results indicateWu et al. BMC Cancer 2013, 13:300 Page 2 of 11http://www.biomedcentral.com/1471-2407/13/300Figure 1 GnRH-II stimulates endometrial cancer cell migration and invasion. (A) Using the Transwell migration assay, endometrial cancercells were seeded. The GnRH-II agonist stimulated the migration of endometrial cancer cells through the uncoated porous filter in adose-dependent manner at concentrations of 1 nM to 1 μM with a maximal effect at 1 μM. (B) Endometrial cancer cells were seeded onto aMatrigel-precoated filter in the Transwell chambers in the presence or absence of increasing concentrations of GnRH-II agonist (1 nM to 1 μM, asindicated). After 24 (migration) and 48 (invasion) hours of incubation, cells in the upper side of the filter were removed and the migrated orinvaded cells were fixed, stained, and counted. Left, representative pictures. Columns, the mean number of migrated or invaded cells of five fieldsof triplicate wells from three independent experiments; bars, SD; *p<0.05, versus control.Wu et al. BMC Cancer 2013, 13:300 Page 3 of 11http://www.biomedcentral.com/1471-2407/13/300that the GnRH-II-induced cell migration and invasionin endometrial cancer cells are mediated by GnRH-Ireceptors.GnRH-II-induced cell migration and invasion are mediatedby ERK1/2 and JNK signaling in endometrial cancer cellsTo investigate the molecular mechanism of GnRH-II-induced cell migration and invasion in endometrialcancer cells, the activation of ERK1/2 and JNK signalingwere examined with immunoblot analysis. As shown inFigure 4A, GnRH-II activated ERK1/2 and JNK signalingin a time-dependent manner. The effects of GnRH-II onERK1/2 and JNK signaling activation were abolished bytransfecting the cells with GnRH-IR siRNA but not withcontrol siRNA (Figure 4B). To further evaluate the rolesof ERK1/2 and JNK signaling in GnRH-II-induced cellmigration and invasion, endometrial cancer cells weretreated with U0126 and SP600125 along with GnRH-II.As shown in Figure 4C, pretreatment of the cells withU0126 or SP600125 abolished the GnRH-II-stimulated cellmigration and invasion. These results suggest that GnRH-II induced the cell migration and invasion of endometrialcancer cells through the GnRH-I receptor and the activa-tion of the ERK1/2 and JNK signaling pathways.Effects of GnRH-II-induced MMP-2 expression on the cellmigration and invasion of endometrial cancer cellsMMP-2 is largely implicated in promoting angiogenesisand tumor metastasis. To determine whether MMP-2 is in-volved in GnRH-II-induced cell migration and invasion ofendometrial cancer cells, the cells were treated with GnRH-II, and the expression of MMP-2 was detected by immuno-blot analysis. As shown in Figure 5A, treatment with 1 nMto 1 μM GnRH-II obviously induced MMP-2 expression.Furthermore, MMP-2 enzymatic activity was measured bygelatin zymography using conditioned medium from endo-metrial cancer cells. The gelatin zymography indicatedstronger lytic zones at the molecular masses correspondingto the pro- and active-forms of MMP-2 (the 72-kDa and66-kDa forms, respectively) in the conditioned mediumFigure 2 Immunoblot and immunohistochemical analysis of GnRH-I receptor (GnRH-IR) protein expression in endometrial cancer.(A) GnRH-I receptor levels in Ishikawa and ECC-1 cells were monitored by immunoblot assays. (B) GnRH-I receptor was stained brown in thesecond of three columns depicting human endometrial cancer tissue sections. Sections were counterstained with hematoxylin to show thenuclei in column 1 of three columns depicting human endometrial cancer tissue sections. Sections were stained without the GnRH-I receptorantibody as a negative control in the third of three columns depicting human endometrial cancer tissue sections. Micrographs were takenwith a 40Χ objective lens. Scale bars represent 20 μm.Wu et al. BMC Cancer 2013, 13:300 Page 4 of 11http://www.biomedcentral.com/1471-2407/13/300Figure 3 (See legend on next page.)Wu et al. BMC Cancer 2013, 13:300 Page 5 of 11http://www.biomedcentral.com/1471-2407/13/300from cells treated with 1 nM to 1 μM GnRH-II comparedwith that from untreated cells (Figure 5B). A more import-ant observation was that the GnRH-II-induced cell migra-tion and invasion were abolished in cells pretreated withthe MMP-2 inhibitor, indicating that MMP-2 was criticalfor the effects of GnRH-II on the cell migration and inva-sion of endometrial cancer cells (Figure 5C).DiscussionThe GnRH pathway is important in the hypothalamus-pituitary-gonadal axis of reproduction [21]. Previous stud-ies have demonstrated the direct effects of GnRH analogsin human endometrial cancer cells [22,23]. Furthermore, ithas been demonstrated that GnRH-II has more potent ef-fects than GnRH-I in extra-pituitary tissues, such as endo-metrial tumors, suggesting that GnRH-II could beconsidered as a possible therapeutic target for endometrialcancers [22]. Metastasis represents the main cause ofdeath for patients with endometrial cancer, and the battleagainst this cancer would benefit greatly from the identifi-cation of factors involved in the metastatic process. How-ever, the underlying molecular mechanisms utilized byGnRH-II to regulate the cell migration and invasion ofendometrial cancer are not well known. The GnRH-Ireceptor is a member of the GPCR family. GPCRs arecharacterized by the presence of seven transmembranedomains and transfer their signals through multiple Gprotein subunits, often stimulating multiple signalingpathways [24]. Direct evidence showing the presence of afull-length, functional GnRH-II receptor mRNA in humantissues is insufficient, and the issue of whether the GnRH-I receptor mediates the effects of both GnRH-I andGnRH-II remains unresolved. In this study, we report forthe first time that GnRH-II may contribute to the migra-tion and invasion of endometrial cancer cells by inducingthe expression of MAPK-mediated MMP-2 through theGnRH-I receptor, providing an insight into the prospect ofdeveloping targeted therapy for endometrial cancer.In our previous study [10], the expression of GnRH-IIand its effects on cell growth were demonstrated inendometrial cancer. In the present study, the treatmentof Ishikawa and ECC-1 endometrial cancer cells withGnRH-II resulted in significant effects on cell migrationand invasion. These findings suggest that GnRH-IIdirectly induces the cell migration and invasion of endo-metrial cancer cells and provide in vitro confirmationthat GnRH-II induces cell motility in endometrial can-cer. These findings confirmed the previous studies[22,23,25-29] suggesting that GnRH-II may mediates thecell motility and anti-proliferation in gynecologic cancercell lines. Therefore, differences in levels of GnRH-Ireceptor, GnRH-II receptor and signaling differentiallyaffect the apoptotic and motile machinery within celllines and contribute to the cell type–specific effects ofGnRH analogues on cell growth and motility.In this study, GnRH-I receptor siRNA was used toselectively knock down the protein expression of GnRH-I receptors in Ishikawa and ECC-1 endometrial cancercells. Targeting GnRH-I receptors with siRNA abolishedthe GnRH-II-induced cell migration and invasion ofendometrial cancer cells, indicating that the effects ofGnRH-II on endometrial cancer cells is dependent uponGnRH-I receptors. This finding confirmed previous stud-ies [10,30-32] that suggested that the GnRH-I receptormay be a common receptor that mediates the effects ofboth GnRH-I and GnRH-II in gynecological cancer cells.In pituitary gonadotrope cells, MAPKs are consideredto be essential in GnRH-induced signaling pathways[12,33]. MAPKs contribute to signaling pathways thatmediate cellular responses to different extracellularstimuli and thereby determine the cell’s behavior. In thepresent study, we observed that GnRH-II (1 μM)resulted in the phosphorylation of ERK1/2 and JNK inIshikawa endometrial cancer cells, which is compatiblewith a previous study performed in COS-7 cells [34].Moreover, the activation of ERK1/2 and JNK was mark-edly attenuated by the specific inhibitors U0126 andSP600125 in Ishikawa endometrial cancer cells. Treat-ment with U0126 and SP600125 also attenuated theGnRH-II-induced cell migration and invasion, further in-dicating that the GnRH-II-induced activation of ERK1/2and JNK may have an important role in the regulation ofcell motility in Ishikawa endometrial cancer cells. Thepresent results indicate that the ERK1/2 and JNK path-ways might play an important role in mediating the motil-ity effects of GnRH-II in Ishikawa endometrial cancercells. Therefore, attempts to manipulate the ERK1/2 andJNK signaling that mediates the regulation of cell(See figure on previous page.)Figure 3 Effects of human GnRH-I receptor siRNA (si-GnRH-IR) transfection on endometrial cancer cells. (A) The transfection efficiency ofsiRNA in endometrial cancer cells. (B) GnRH-I receptor levels were monitored by immunoblot assays. The endometrial cancer cells weretransfected with human si-GnRH-IR or scrambled siRNA (si-Ctrl) for one day with Lipofectamine RNAiMAX. (C) The effects of si-GnRH-IRtransfection on the GnRH-II-induced cell migration. The cells were transfected with si-GnRH-IR and treated with GnRH-II (1 μM) for 24 h. The cellmotility was assessed with the migration assay. The results are expressed as the mean ± SEM of three independent experiments. (*p<0.05, versuscontrol; #p<0.05, versus GnRH-II). The effects of si-GnRH-IR transfection on GnRH-II-induced cell invasion. The cells were transfected with si-GnRH-IR and treated with GnRH-II (1 μM) for 48 h. The cell motility was assessed with the invasion assay. The results are expressed as the mean ± SEMof three independent experiments. (*p<0.05, versus control; #p<0.05, versus GnRH- II).Wu et al. BMC Cancer 2013, 13:300 Page 6 of 11http://www.biomedcentral.com/1471-2407/13/300Figure 4 The effects of ERK1/2 and JNK signaling in endometrial cancer cells. (A) The effects of GnRH-II on ERK1/2 and JNK signalingactivation. The cells were treated with GnRH-II (1 μM) at different time points. The phosphorylated ERK1/2 (p-ERK1/2), and phosphorylated JNK(p-JNK) levels were analyzed by immunoblot analysis, which indicated increases in the levels of p-ERK1/2 and p-JNK following 5 min ofstimulation. (B) The effects of human si-GnRH-IR transfection on the GnRH-II-induced activation of ERK1/2 and JNK. The activation of ERK1/2and JNK induced by GnRH-II (GII) was investigated after si-GnRH-IR transfection and showed significant decreases in the levels of p-ERK1/2 andp-JNK. (C) The effects of U0126 and SP600125 pretreatment on GnRH-II-induced ERK1/2 and JNK activation. Cells were pretreated individuallywith 1 μM U0126 or 1 μM SP600125 for 30 min followed by stimulation with 1 μM GnRH-II for 10 min. The control culture was treated withDMSO as a vehicle control. Pretreatment with 1 μM U0126 and 1 μM SP600125 individually attenuated the effects of GnRH-II on the inductionof cell migration and invasion. (Columns, mean from three independent experiments in three different passages of the cell line; bars, SE.,*p<0.05, versus control; #p<0.05, versus GnRH-II).Wu et al. BMC Cancer 2013, 13:300 Page 7 of 11http://www.biomedcentral.com/1471-2407/13/300migration and invasion may be an approach to explore theeffects of GnRH-II in endometrial cancer.Cancer cell metastasis is a complex process that in-volves proteolysis, increased cell motility, and decreasedcell adhesion. MMP-2 has been suggested to play a crit-ical role in cancer metastasis, and the up-regulation ofMMP-2 is associated with increased invasion and a poorprognosis in cancer [35-38]. In addition to theirenzymatic activities, MMPs can also promote cancer cellmigration by influencing cytoskeletal organization throughtheir association with different families of adhesion recep-tors [39]. In the present study, we demonstrated thatGnRH-II promotes the cell migration and invasion ofendometrial cancer cells through the increased expressionand proteolytic activity of MMP-2, which specificallydegrades the basement membrane. Pretreatment withFigure 5 The effects of GnRH-II on MMP-2 expression in endometrial cancer cells. (A) GnRH-II increased MMP-2 protein expression inendometrial cancer cells. (B) Conditioned medium from the treated cells was also collected and analyzed for MMP activity with gelatinzymography. Arrows, gelatinase activities corresponding to pro-MMP-2, and active-MMP-2. Lane 1, a human MMP-2 standard was used as apositive control. (C) The cells were pretreated with MMP-2 inhibitor. Pretreated cells were collected for the migration assay through uncoatedfilter (left) and the invasion assay through Matrigel (right) in the presence or absence of GnRH-II. The results are expressed as the mean ± SEM ofthree independent experiments. (*p<0.05, versus control; #p<0.05, versus GnRH-II).Wu et al. BMC Cancer 2013, 13:300 Page 8 of 11http://www.biomedcentral.com/1471-2407/13/300U0126 and SP600125 abolished the protein expression ofMMP-2 induced by GnRH-II, suggesting that the ERK1/2and JNK signaling pathways may play an important role inregulating MMP-2 expression. Taken together with theprevious results, the cell migration and invasion in endo-metrial cancer is regulated by the activation of the ERK1/2and JNK signaling pathways by GnRH-II and is accom-panied by the induction of MMP-2. This is one of thenovel findings in the present study. In aggregate, our datademonstrate that MMP-2 is closely associated with thepathways of the MAPKs involved in the GnRH-II-inducedcell migration and invasion of endometrial cancer cells.Targeting MMP-2 with an MMP-2 inhibitor blocked theGnRH-II-induced cell migration and invasion, indicatingthat the effects of GnRH-II in endometrial cancer cells arestrongly correlated with MMP-2 expression.ConclusionsIn conclusion, our findings suggest that the potentialrole of GnRH-II in promoting the cell migration andinvasion of endometrial cancer is through the binding ofGnRH-I receptors, the activation of the ERK1/2 andJNK pathways, and the subsequent induction of themetastasis-related proteinase MMP-2 activity. Thisinformation provides a mechanistic rationale for theobserved GnRH-I receptor expression in endometrialcancer. Our findings provide a new insight regarding themechanism of GnRH-II-induced cell motility in endo-metrial cancer and suggest the possibility of exploringGnRH-II as a potential therapeutic molecular target forthe treatment of human endometrial cancer.MethodsCell lines and cell cultureThe human endometrial cancer cell lines Ishikawa andECC-1 were utilized in this study. The human endomet-rial cancer cell line Ishikawa is a well-differentiatedendometrial adenocarcinoma cell line [40]. The ECC-1cell line, derived from a well-differentiated adenocarcin-oma of the endometrium [41], was obtained from theAmerican Type Culture Collection (US). The cells werecultured in Dulbecco’s minimum essential medium(DMEM) with 10% fetal bovine serum (FBS; HycloneLaboratories Inc., Logan, UT), 100 U/ml penicillin, and100 μg/ml streptomycin and incubated at 37°C in ahumidified incubator with 5% CO2. The cells were grownto 80% confluence and transferred to serum-free mediumfor 24 h prior to the treatment with the GnRH-II agonist.ReagentsThe GnRH-II agonist (D-Arg6, AzaGly10-GnRH II), asynthetic decapeptide, was purchased from Bachem (SanCarlos, CA). The MAPK/extracellular signal-regulatedprotein kinase (ERK) kinase (MEK) inhibitor U0126, theJNK inhibitor SP600125, and the MMP-2 inhibitor OA-Hy were purchased from Calbiochem (San Diego, CA).Immunoblot analysisThe cells were lysed in buffer containing 20 mM Tris,pH 7.4, 2 mM EGTA, 2 mM Na2VO3, 2 mM Na4P2O7,2% Triton X-100, 2% SDS, 1 μM aprotinin, 1 μMleupeptin and 1 mM PMSF. The protein concentrationwas determined with a protein assay kit using BSA stan-dards according to the manufacturer's instructions (Bio-Rad Laboratories, Hercules, CA). Equal amounts of celllysate were separated by SDS polyacrylamide gel electro-phoresis (PAGE) and transferred to a nitrocellulose mem-brane (Hybond-C, Amersham Pharmacia Biotech Inc.,Oakville, ON). Following blocking with Tris-bufferedsaline (TBS) containing 5% non-fat dry milk for 1 h, themembranes were incubated overnight at 4°C with anti-GnRH-I receptor (Neomarker, Fremont, CA), anti-phospho-ERK1/2 (Cell signaling), anti-ERK1/2 (Cellsignaling), anti-phospho-JNK (Cell signaling), anti-JNK(Cell signaling), or anti-MMP-2 (Calbiochem, San Diego,CA) antibody followed by incubation with HRP-conjugatedsecondary antibody. The immunoreactive bands weredetected with an enhanced chemiluminescence (ECL) kit.The membrane was then stripped with stripping buffer(62.5 mM Tris, 10 mM DTT, and 2% SDS, pH 6.7) at 50°Cfor 30 min and re-probed with anti-β-actin antibody (SantaCruz) as a loading control.Immunohistochemistry (IHC)To determine the expression of the GnRH-I receptorprotein in human endometrial cancer, IHC was per-formed on sections of human endometrial cancer tissueusing previously reported procedures [42]. The involve-ment of human subjects in this study was approved bythe Institutional Review Board of Chang Gung MemorialHospital (CGMH-IRB numbers 101-2187B and 100-3879C). Four-micrometer-thick formalin-fixed, paraffin-embedded (FFPE) tissue sections were deparaffinized inxylene and rehydrated with a graded series of ethanol so-lutions. The sections were then stained with an anti-human GnRH-I receptor polyclonal antibody (Neomarker;1:100) using an automated IHC stainer with the VentanaBasic DAB Detection kit (Tucson, AZ) according to themanufacturer’s protocol. Counterstaining was performedwith hematoxylin. Sections were stained without theGnRH-I receptor antibody as a negative control in thethird of three columns depicting the human endometrialcancer tissue sections.Small interfering RNA transfectionsiGENOME ON-TARGETplus SMARTpool human GnRH-I receptor siRNA and siCONTROL NON-TARGETINGpool siRNA were purchased from Dharmacon. The cellsWu et al. BMC Cancer 2013, 13:300 Page 9 of 11http://www.biomedcentral.com/1471-2407/13/300were transfected with siRNA (100 nM) using LipofectamineRNAiMAX. After a 24 h transfection, the medium wasremoved and changed to fresh serum-free medium. Toexamine the siRNA transfection, cells were transfected with100 nM si-GLO (Dharmacon) for 24 hr. The transfectionefficiency was examined by fluorescent microscopy.Invasion and migration assaysMigration and invasion assays were performed in Boy-den chambers with minor modifications. Cell cultureinserts (24-well, pore size 8 μm; BD Biosciences, Missis-sauga, ON) were seeded with 1x105 cells in 250 μL ofmedium with 0.1% FBS. Un-coated inserts were used formigration assays whereas inserts pre-coated with growthfactor reduced Matrigel (40 μl, 1 mg/ml; BD Biosciences)were used for invasion assays. Medium with 10% FBS(750 μl) was added to the lower chamber and served asa chemotactic agent. After 24 hr (migration) or 48 hr(invasion) incubation, non-migrating/invading cells werewiped from the upper side of the membrane and cellson the lower side were fixed in cold methanol (−20°C)and air dried. The cells that had not penetrated the filterwere removed by wiping, and the cells that had invadedthe lower surface of the filter were fixed with ice-coldmethanol and stained with 0.5% crystal violet.Gelatin zymographyThe activity of MMP-2 in the conditioned medium wasdetermined by gelatin zymography. The media were col-lected and clarified by centrifugation to remove cells anddebris. The samples were loaded under non-reducingconditions onto SDS-polyacrylamide gel polymerizedwith 1 mg/mL gelatin. Following electrophoresis, thegels were washed with 2.5% Triton X-100 to removeSDS and then incubated in a developing buffer overnightat 37°C. The gels were stained with 0.25% CoomassieBrilliant Blue R-250 and destained in the same solutionwithout dye. The gelatinase activity was visualized asclear bands against the blue-stained gelatin background.The molecular sizes were determined from mobilityusing gelatin zymography standards.Statistical analysisThe results are shown as the means ± SEM. Statisticalevaluation was conducted with the t-test for paired data.Multiple comparisons were first analyzed by one-wayANOVA, followed by Tukey’s multiple comparison test.A significant difference was defined as p<0.05.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsHMW, HSW, and PCKL performed the experiments, interpreted the resultsand prepared the manuscript. HMW, HSW, HYH, CHL, CLL, YKS, and PCKLcontributed to the scientific discussion and the manuscript editing. HSW andPCKL supervised in the design of the study and finalized the manuscript. Allauthors read and approved the final manuscript.AcknowledgementsThe authors acknowledge H.-L. Chen, J-E Yang, and A. Chao at theGynecological and Obstetrics Laboratory, Chang Gung Memorial Hospital,and JC Cheng at Department of Obstetrics and Gynecology, University ofBritish Columbia, Vancouver, British Columbia, Canada, for their technicalassistance in this study.FundingThis work was supported by Grant NSC-101-2314-B-182A-058-MY3 (to H.-M.W.)from the National Science Council, Taiwan and Grants CMRPG381671,CMRPG381672, CMRPG381673, and CMRPG3B1461 (to H.-M.W.). This work wasalso supported by Grants from the Canadian Institutes of Health Research to P.C.K.L. P.C.K.L. is the recipient of a Child & Family Research Institute DistinguishedInvestigator Award.Author details1Department of Obstetrics and Gynecology, Chang Gung Memorial HospitalLinkou Medical Center, Chang Gung University School of Medicine,Taoyuan 333, Taiwan. 2Department of Obstetrics and Gynecology,University of British Columbia, Vancouver, BC V6H3V5, Canada.Received: 5 February 2013 Accepted: 31 May 2013Published: 20 June 2013References1. 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