research article estrogen induces metastatic potential of...

Hindawi Publishing CorporationInternational Journal of EndocrinologyVolume 2013 Article ID 941568 6 pageshttpdxdoiorg1011552013941568

Research ArticleEstrogen Induces Metastatic Potential of Papillary ThyroidCancer Cells through Estrogen Receptor 120572 and 120573

Wenwu Dong12 Hao Zhang1 Jing Li2 Haixia Guan2 Liang He1 Zhihong Wang1

Zhongyan Shan2 and Weiping Teng2

1 Department of General Surgery The First Affiliated Hospital of China Medical University No 155 Nanjing Bei StreetHeping District Shenyang Liaoning 110001 China

2Department of Endocrinology and Metabolism Institute of Endocrinology Liaoning Provincial Key Laboratory ofEndocrine Diseases The First Affiliated Hospital of China Medical University No 155 Nanjing Bei Street Heping DistrictShenyang Liaoning 110001 China

Correspondence should be addressed to Hao Zhang haozhangmailcmueducn and Jing Li lijingendocrine126com

Received 6 July 2013 Revised 18 September 2013 Accepted 18 September 2013

Academic Editor Małgorzata Kotula-Balak

Copyright copy 2013 Wenwu Dong et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Estradiol (E2) promotes metastatic propensity However the detailed mechanism remains largely unknown E-cadherin vimentinand MMP-9 play a dominant role in the metastatic process We aimed to investigate the effects of E2 on metastatic potential ofPTC cell line BCPAP and on E-cadherin vimentin and MMP-9 protein expression PTC cell line BCPAP was evaluated for thepresence of estrogen receptor (ER) by western blot analysis The effects of E2 PPT (a potent ER120572-selective agonist) and DPN (apotent ER120573-selective agonist) on modulation of metastatic phenotype were determined by using in vitro scratch wound assay andinvasion assay In addition the effects on E-cadherin vimentin and matrix metalloproteinase-9 (MMP-9) protein expression wereevaluated byWestern blot analysis We found that BCPAP cells expressed ER120572 and ER120573 E2 and PPT enhanced but DPN inhibitedthe migration and invasion of BCPAP cells in an in vitro experimental model system that is modulated by E-cadherin vimentinand MMP-9 These findings indicate that E2 induces the metastatic potential of BCPAP cells through ER120572 and ER120573 The two ERsubtypes play differential roles in modulation of BCPAP cell metastasis and the related molecule expressions including E-cadherinvimentin and MMP-9

1 Introduction

Thyroid cancer is the most common cancer of the endocrinesystem and the sixth most common cancer in women inthe United States [1] Thyroid cancers are classified into 3main histologic types as differentiated (including papillaryfollicular andHurthle)medullary and anaplastic (aggressiveundifferentiated tumor) with papillary thyroid cancer (PTC)accounting for 80 of all cases Differentiated thyroid cancer(DTC) has a faveorable long-term prognosis with a 10-yearrelative survival rate of more than 90 However up to10 of DTC invades through the outer border of the glandand grows directly into surrounding tissues increasing bothmorbidity and mortality Recurrence rates are 2 times higherwith locally invasive tumors and as many as 33 of patientswith such tumors die of cancer within a decade [2 3] DTC

can metastasize to the lymph nodes of the neck in 20ndash90of the patients [4] Distant metastasis which occurs in 5ndash23 of patients with known DTC is the most common causeof death in these patients [5] Therefore great efforts shouldbe made to improve the prognosis of these patients withaggressive tumor

Clinical and epidemiological studies have shown thatthe female predominance in thyroid cancer is the greatestduring reproductive age and the incidence decreases aftermenopause [6 7] The use of oral contraceptives appears toresult in a moderately increased risk of developing thyroidcancer and an elevated risk was also reported in womenwho used estrogens for gynecological problems but not forlow-dose estrogen replacement therapy in postmenopausalwomenThere are also experimental lines of evidence that E2could affect tumor progression by increasing cell proliferation

2 International Journal of Endocrinology

as well as promoting invasion or cell mobility in thyroid can-cer [8ndash10] However the underlyingmolecular mechanism ofE2 on promoting PTC progression is still unclear

The regulation of gene expression by E2 is amultifactorialprocess involving both genomic and nongenomic actionsthat converge at certain response elements located in thepromoters of target genes [11] In the genomic pathwayestrogens bind to two estrogen receptor (ER) subtypes ER120572and ER120573 inducing an activating conformational changewithin the ER promoting dimerization and high-affinitybinding to specific estrogen response elements (EREs) locatedwithin the regulatory regions of target genes ER120572 and ER120573show significant overall sequence homology but the differentsizes of the binding pockets and sequences of their activationfunction domains indicate that ER120572 and ER120573 should havedifferent specificities for ligands and biological responses[12] Thus ER120572 and ER120573 selective agonists are promisingnew approach for treating specific conditions associated withendocrine-related disease Besides that estrogens are alsoable to exert nongenomic events mediated by a novel trans-membrane ER G protein-coupled receptor 30 (GPR30) [13]

The metastatic process that is the dissemination ofcancer cells throughout the body to seed secondary tumors atdistant sites requires cancer cells to leave the primary tumorand to acquire migratory and invasive capabilities Manydifferent processes are involved in tumor cell invasion andmetastasis such as an epithelial to mesenchymal transition(EMT) adhesion molecules downregulation and matrixmetalloproteinases (MMPs) upregulation in cancer cellsLoss of epithelial protein marker E-cadherin the concurrentupregulation of mesenchymal protein markers vimentin andthe upregulation of MMP-9 play a dominant role in themetastatic process and could be regulated by E2 in variouscancers including breast ovarian colon and lung cancer[14ndash17] However whether and how the effects of E2 onmigration and invasion of thyroid cancer may be involved inthe regulation of E-cadherin vimentin andMMP-9 is poorlyunderstood

In this study we explore the effects of E2 PPT and DPNon metastatic potential of PTC cell line BCPAP and evaluatethe roles of ER120572 and ER120573 in metastasis of BCPAP cells Wefurther determine the metastasis-related proteins by whichE2 affects BCPAP cell metastasis

2 Materials and Methods

21 Cell Culture The human PTC cell line BCPAP waspurchased from DSMZ (Braunschh Germany) The humanbreast cancer cell line MCF-7 was obtained from the CellBank of the Chinese Academy of Sciences (Shanghai China)The cells were maintained in RPMI1640 medium supple-mented with 10 fetal bovine serum 100UmL penicillinand 100 120583gmL streptomycin in a humidified atmosphere of5 CO

2at 37∘C

22 Treatments Cells were stripped from endogenoussteroids by changing the medium to phenol red-freeRPMI1640 containing 10 charcoal-dextran stripped fetal

ER120572

ER120573

120573-actin

BCPAP MCF-7

Figure 1 BCPAP cells express ER120572 and ER120573Whole cell protein wasresolved by SDS-PAGE followed by western blot analysis for ER120572ER120573 and 120573-actinMCF-7 and ER-positive cells were used as positivecontrol

bovine serum (FBS) (Biowest Nuaille France) 48 h beforetreatment Cells were then incubated in fresh medium withvehicle (DMSO) alone 10minus8M E2 (Sigma St Louis MO)10minus6MPPT (an ER120572-selective agonist) (Tocris BallwinMO) or 10minus6MDPN (an ER120573-selective agonist) (TocrisBallwin MO) for 24 h Concentrations of E2 PPT and DPNare used according to the report by Zeng et al Controlcultures received the same volume of DMSO

23 Western Blot Analysis Total protein was extracted usingTotal Protein Extraction Kit (KeyGEN Nanjing China)according to themanufacturerrsquos instructions Protein concen-tration was determined using a Bradford assay with bovineserum albumin as a standard Protein was resolved with10 SDS-PAGE and then electrophoretically transferred topolyvinylidene difluoride (PVDF) membranes (MilliporeCA USA) The membranes were blocked with 5 nonfatdry milk at room temperature for 1 h and incubated at 4∘Covernight with antibodies directed against ER120572 (AbcamUSA 1 500) ER120573 (Abcam USA 1 1000) E-cadherin (SantaCruz USA 1 1000) vimentin (Boster China 1 500) MMP-9 (Santa Cruz USA 1 1000) and 120573-actin (Santa Cruz USA1 1000) After washing three times with TBS containing 01Tween 20 the membranes were incubated with an HRP-conjugated goat anti-rabbit or goat anti-mouse secondaryantibody (Zhongshan Golden Bridge China 1 5000) andvisualized using SuperSignal West Pico ChemiluminescentSubstrate (Pierce Rockford IL USA)

24 Scratch Wound Assay Migratory ability of BCPAP cellswas assessed by a scratch wound assay Cells were platedin a six-well plate and grow to confluent cell monolayersSubsequently three vertical scratches were made gently withsterile pipette tip across the diameter of the well and rinsedwith PBS to remove debris The wounded cell monolayerwas then incubated in fresh complete medium with vehicle(DMSO) alone 10minus8M E2 10minus6MPPT or 10minus6MDPN Foreach well at least five pictures were taken microscopicallyat a magnification of 10x at 0 and 24 h after scratch

International Journal of Endocrinology 3

Control E2 PPT DPN

0h

24h

180

160

140

120

100

80

60

40

20

0

Control E2 PPT DPN

lowastlowast

lowast

Cell

mig

ratio

n (

of c

ontro

l)

Figure 2 E2 enhances migration of BCPAP cells Confluent monolayers of BCPAP cells were wounded with a uniform scratch washed toremove cell debris and cultured in the presence of vehicle (DMSO) alone 10minus8M E2 10minus6MPPT or 10minus6MDPN for 24 h Images of cellcultures were captured at 0 and 24 h after scratching representative pictures are shown in upper panel The amount of wound repair wasexpressed as uncovered area at the indicated time compared with initial uncovered area of vehicle-treated control at time zero (lower panel)Values are the mean plusmn SD of three separate experiments (normalized to the untreated control) lowast119875 lt 005 compared with control

The percentage of nonrecovered wound area was calculatedby dividing the nonrecovered area after treatments by theinitial wound area at time zero

25 Invasion Assay Invasion assay was carried out usingtranswell invasion chambers (8120583m pore size) in 24-wellplates (Corning Life Sciences Corning NY USA) Tran-swell was coated with Matrigel (BD Biosciences Two OakPark Bedford MA) overnight Cells were starved for 48hours using the starvation medium (phenol-red-free RPMImedium supplemented with 10 charcoal-dextran strippedFBS) before plating to the upper chamber After Matrigelinvasion chambers were rehydrated using the phenol-red-free and serum-free RPMI medium at 37∘C cells (20 times 104cells per well) in the phenol-red-free RPMI medium (100 120583L)with 1 charcoal-dextran stripped FBS containing vehicle(DMSO) alone 10minus8M E2 10minus6MPPT or 10minus6MDPN wereloaded onto the upper chamber and 500120583L of phenol-red-free RPMI medium with 10 charcoal-dextran stripped FBSwas loaded onto the bottom chamber as a chemoattractantAfter 24 hours of incubation the noninvading cells on theupper chambers were removed with a cotton-tipped swabCells were fixed with 4 paraformaldehyde and stained with1 crystal violet The invaded cells were counted microscop-ically in five random fields of view at 200x magnification andexpressed as the mean number of cells per field of view

3 Statistical Analysis

Data were presented as mean plusmn SD for at least three indepen-dent replicates Differences between groups were examinedusing Studentrsquos t-test Differences with 119875 lt 005 wereconsidered to be statistically significant

4 Results

41 BCPAP Cells Express ER120572 and ER120573 Thyroid cancercells are not known to act as traditional estrogen-responsivetissues such as breast cancer In order to determine the statusof the ERs in BCPAP cells western blots were performedMCF-7 a classical ER expressing breast cancer cell line wasused as a positive control for detection of ER120572 and ER120573 Weobserved that both the thyroid and breast cancer cell linesassayed expressed both ER isoforms ER120572 and ER120573 (Figure 1)This suggests that these cells are presumably responsive to theE2-ER-mediated signaling pathway

42 Effect of E2 PPT and DPN on Migration of BCPAP CellsThe migratory potential of BCPAP cells following E2 PPTand DPN exposure was assayed by performing the scratchwound assay The wounded area of BCPAP cell monolayershealed slowly in vehicle-treated cells The closure of thewounded gap was significantly accelerated in the presence


Control E2 PPT DPN

160

140

120

100

80

60

40

20

0

Control E2 PPT DPN

lowast

lowast

lowast

Cell

inva

sion

( o

f con

trol)

Figure 3 E2 enhances invasion of BCPAP cells Cells (20times 104 cells perwell) in the phenol-red-free RPMImedium (100 120583L)with 1 charcoal-dextran stripped FBS containing vehicle (DMSO) alone 10minus8M E2 10minus6MPPT or 10minus6MDPN were loaded onto the upper chamber and500 120583L of phenol-red-free RPMImediumwith 10 charcoal-dextran stripped FBSwas loaded onto the bottom chamber as a chemoattractantAfter 24 h the invaded cells were counted microscopically in five random fields of view at 200x magnification and expressed as the meannumber of cells per field of view Values are the mean plusmn SD of three separate experiments (normalized to the untreated control) lowast119875 lt 005compared with control

of E2 and PPT but significantly decelerated in the presenceof DPN at 24 h (Figure 2) Compared with control cells thissignificant increase inmigration was approximately 516 forE2-treated cells and 436 for PPT-treated cells in contrastthe significant decrease in migration was 489 for DPN-treated cells (normalized to 100 and 119875 lt 005) (Figure 2)This result indicates that E2 enhanced migration of BCPAPcells ER120572 and ER120573 had opposite functions in migration ofBCPAP cells

43 Effects of E2 PPT and DPN on Invasion of BCPAP CellsThe invasive potential of BCPAP cells following E2 PPTand DPN exposure was assayed by the invasion assay Weobserved that cell invasion increased in the presence of E2and PPT but decreased in the presence of DPN (Figure 3)Compared with control cells this increase in invasion wasapproximately 462 for E2-treated cells and 322 for PPT-treated cells in contrast this decrease in invasion was 273for DPN-treated cells (normalized to 100 and 119875 lt 005)(Figure 3) This result indicates that E2 enhanced invasionof BCPAP cells ER120572 and ER120573 had opposite functions ininvasion of BCPAP cells

44 E-Cadherin Vimentin and MMP-9 Expression followingE2 PPT andDPNExposure To ascertain themolecular basisof E2 PPT and DPN on modulation of metastatic potentialof BCPAP cells we analyzed expression of E-cadherinvimentin and MMP-9 protein by western blot analysis inBCPAP cells treated with vehicle (DMSO) alone 10minus8ME2 10minus6MPPT or 10minus6MDPN The 24-hour treatment of

BCPAP with E2 and PPT caused a significant decreasein expression of E-cadherin but a significant increase inexpression of vimentin and MMP-9 In contrast the 24-hourtreatment of BCPAP with DPN caused a significant increasein expression of E-cadherin but a significant decrease inexpression of vimentin and MMP-9 (Figure 4) These obser-vations are suggestive of a possible role of E-cadherinvimentin and MMP-9 in migration and invasion of BCPAPcells regulated by E2 PPT and DPN

5 Discussion

In the present study we confirmed that both ER120572 and ER120573were expressed in the PTC cell line BCPAP and E2 couldinduce metastatic potential of BCPAP This is consistentwith the results of previous studies that ER120572 and ER120573 wereexpressed in the PTC line KAT 5 NPA 87 and BCPAP and E2could enhance adhesion migration and invasion of BCPAPcells [8 9] Moreover we further found that the two ERsubtypes play differential roles in modulation of BCPAP cellmetastasis and the related molecule expressions including E-cadherin vimentin and MMP-9

Given the importance of E2 demonstrated by previousstudies understanding the mechanistic basis through whichit controls thyroid carcinogenesis will have profound bio-logical and medical implications In fact the proliferativeeffects of E2 in thyroid cancer were found to be mediatedthrough regulation of genes involved in growth control suchas bcl-2 Bax and c-fos [10 18] In contrast to the rolein regulating cell proliferation very little is known about


ControlE2

PPTDPN

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

25

2

15

1

05

0

E-cadherin

Vimentin

MMP-9

120573-actin

Expr

essio

n le

velr

elat

ive c

ontro

l

Control E2 PPT DPN

lowast

E-cadherin Vimentin MMP-9

Figure 4 E2 induces metastasis via downregulation of E-cadherinand upregulation of vimentin andMMP-9 BCPAP cells were treatedwith vehicle (DMSO) alone 10minus8M E2 10minus6MPPT or 10minus6MDPNWhole cell lysates were extracted and E-cadherin vimentin andMMP-9 protein were detected by western blot analysis 120573-actin wasused as a loading control lowast119875 lt 005 compared with control

whether and how E2 promotes thyroid cancer cell metastasisRajoria et al reported that E2 could enhance adhesionmigration and invasion of BCPAP cells through 120573-cateninandMMP-9 [8 19]The studies on other types of cancer haveindicated that E2 may promote tumor metastasis throughactivation of genes linked to cell metastasis E2 has beenfound to increase themetastatic potential of human epithelialovarian cancer cell through upregulation of MMP-2 anddownregulation of E-cadherin [14 20] E2 enhanced breastcancer cell motility and invasion via extranuclear activationof actin-binding protein ezrin [16] E2 promoted lung cancercell migration through downregulation of E-cadherin and 120573-catenin and upregulation of fibronectin and vimentin [17]In this regard our data implied that E2-induced metastaticpotential of BCPAP cells was at least in part mediatedthrough downregulation of E-cadherin and upregulation ofvimentin and MMP-9

In our recent study we found that ER120572 and ER120573 proteinexpression was lost in 528 and 19 respectively of the106 PTC lesions (unpublished data) This implicates that ERsubtypesmay play the distinct roles in thyroid carcinogenesisHowever their functions and the molecular mechanismsin thyroid carcinogenesis have just begun to be unveiled

Zeng et al reported that ER120572 promoted cell proliferationbut ER120573 induced cell apoptosis in thyroid cancer [10] Parket al also found that E2 triggered the metastatic behaviorsexclusively through an ER120572-dependent pathway but ER120573 hadan opposing action on ER120572 in ovarian cancer [14] To dissectwhich ER subtype plays a dominant role in the prometastaticeffect of E2 we treated BCPAP with the preferential ER120572agonist PPT or with the ER120573 agonist DPN We found thatPPT enhanced but DPN inhibited migration and invasionof BCPAP cells through the differential regulation of E-cadherin vimentin and MMP-9

The findings in this study have been limited to thetypical PTC cell linemdashBCPAPWewould verify them in otherPTC cell lines and through other methods such as ER geneknockdown technique in the future study

6 Conclusions

As a whole our data suggest a strong link between E2 andBCPAP cell metastasis as evidenced by its effect on in vitromigration and invasion We found that E2 exposure alsoled to downregulation of E-cadherin and upregulation ofvimentin and MMP-9 Furthermore ER120572 and ER120573 playthe distinct roles in BCPAP cell metastasis through thedifferential regulation of E-cadherin vimentin and MMP-9It carries clinical implications for selective targeting of theERs in therapeutic and prevention strategies against thyroidcancer

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Hao Zhang and Jing Li contributed to this paper equally

Acknowledgments

This research was supported by the Key Scientific andTechnological Project from Liaoning Province (JH2) (no2008225007) Program for Liaoning Excellent Talents inUniversity (LNET) (no LJQ 2011080) the Innovation TeamProject of Education Department of Liaoning Province (noLT2010102) and the Liaoning Baiqianwan Talents Project(no 2010921070) The authors thank Medjaden BioscienceLimited for editing and proofreading the paper

References

[1] G C Kabat M Y Kim J Wactawski-Wende D Lane SWassertheil-Smoller and T E Rohan ldquoMenstrual and repro-ductive factors exogenous hormone use and risk of thyroidcarcinoma in postmenopausal womenrdquo Cancer Causes andControl vol 23 no 12 pp 2031ndash2040 2012

[2] E L Mazzaferri and S M Jhiang ldquoLong-term impact of initialsurgical and medical therapy on papillary and follicular thyroidcancerrdquoTheAmerican Journal ofMedicine vol 97 no 5 pp 418ndash428 1994


[3] H Salvesen P R Njolstad L A Akslen G Albrektsen OSoreide and J E Varhaug ldquoPapillary thyroid carcinoma a mul-tivariate analysis of prognostic factors including an evaluationof the p-TNM staging systemrdquoThe European Journal of Surgeryvol 158 no 11-12 pp 583ndash589 1992

[4] L Rotstein ldquoThe role of lymphadenectomy in the managementof papillary carcinoma of the thyroidrdquo Journal of SurgicalOncology vol 99 no 4 pp 186ndash188 2009

[5] I C Huang F F Chou R T Liu et al ldquoLong-term outcomesof distant metastasis from differentiated thyroid carcinomardquoClinical Endocrinology vol 76 no 3 pp 439ndash447 2012

[6] R Rahbari L Zhang and E Kebebew ldquoThyroid cancer genderdisparityrdquo Future Oncology vol 6 no 11 pp 1771ndash1779 2010

[7] R Siegel DNaishadham andA Jemal ldquoCancer statistics 2013rdquoCAACancer Journal for Clinicians vol 63 no 1 pp 11ndash30 2013

[8] S Rajoria R Suriano A Shanmugam et al ldquoMetastatic pheno-type is regulated by estrogen in thyroid cellsrdquo Thyroid vol 20no 1 pp 33ndash41 2010

[9] AKumar CMKlinge andR EGoldstein ldquoEstradiol-inducedproliferation of papillary and follicular thyroid cancer cells ismediated by estrogen receptors 120572 and 120573rdquo International Journalof Oncology vol 36 no 5 pp 1067ndash1080 2010

[10] Q Zeng G G Chen A C Vlantis and C A van HasseltldquoOestrogen mediates the growth of human thyroid carcinomacells via an oestrogen receptor-ERK pathwayrdquo Cell Proliferationvol 40 no 6 pp 921ndash935 2007

[11] M Marino P Galluzzo and P Ascenzi ldquoEstrogen signal-ing multiple pathways to impact gene transcriptionrdquo CurrentGenomics vol 7 no 8 pp 497ndash508 2006

[12] D C Leitman S Paruthiyil O I Vivar et al ldquoRegulationof specific target genes and biological responses by estrogenreceptor subtype agonistsrdquo Current Opinion in Pharmacologyvol 10 no 6 pp 629ndash636 2010

[13] A P Santin and T W Furlanetto ldquoRole of estrogen in thyroidfunction and growth regulationrdquo Journal of Thyroid Researchvol 2011 Article ID 875125 7 pages 2011

[14] SH Park LWCheungA SWong andPC Leung ldquoEstrogenregulates snail and slug in the down-regulation of E-cadherinand inducesmetastatic potential of ovarian cancer cells throughestrogen receptor 120572rdquoMolecular Endocrinology vol 22 no 9 pp2085ndash2098 2008

[15] H H Hsu C J Liu C Y Shen et al ldquop38120572 MAPK mediates17120573-Estradiol inhibition of MMP-2 and -9 expression and cellmigration in human lovo colon cancer cellsrdquo Journal of CellularPhysiology vol 227 no 11 pp 3648ndash3660 2012

[16] S Zheng J Huang K Zhou et al ldquo17120573-Estradiol enhancesbreast cancer cell motility and invasion via extra-nuclear acti-vation of actin-binding protein ezrinrdquo PLoS ONE vol 6 no 7Article ID e22439 2011

[17] G Zhao Y Nie M Lv L He T Wang and Y Hou ldquoER120573-mediated Estradiol enhances epithelial mesenchymal transitionof lung adenocarcinoma through increasing transcription ofmidkinerdquoMolecular Endocrinology vol 26 no 8 pp 1304ndash13152012

[18] A Vivacqua D Bonofiglio L Albanito et al ldquo17120573-Estradiolgenistein and 4-hydroxytamoxifen induce the proliferation ofthyroid cancer cells through the g protein-coupled receptorGPR30rdquoMolecular Pharmacology vol 70 no 4 pp 1414ndash14232006

[19] S Rajoria R Suriano A George et al ldquoEstrogen inducedmetastatic modulators MMP-2 and MMP-9 are targets of 331015840-diindolylmethane in thyroid cancerrdquo PLoS ONE vol 6 no 1Article ID e15879 2011

[20] J X Ding Y J Feng L Q Yao M Yu H Y Jin and L HYin ldquoThe reinforcement of invasion in epithelial ovarian cancercells by 17120573-Estradiol is associated with up-regulation of snailrdquoGynecologic Oncology vol 103 no 2 pp 623ndash630 2006

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


as well as promoting invasion or cell mobility in thyroid can-cer [8ndash10] However the underlyingmolecular mechanism ofE2 on promoting PTC progression is still unclear

The regulation of gene expression by E2 is amultifactorialprocess involving both genomic and nongenomic actionsthat converge at certain response elements located in thepromoters of target genes [11] In the genomic pathwayestrogens bind to two estrogen receptor (ER) subtypes ER120572and ER120573 inducing an activating conformational changewithin the ER promoting dimerization and high-affinitybinding to specific estrogen response elements (EREs) locatedwithin the regulatory regions of target genes ER120572 and ER120573show significant overall sequence homology but the differentsizes of the binding pockets and sequences of their activationfunction domains indicate that ER120572 and ER120573 should havedifferent specificities for ligands and biological responses[12] Thus ER120572 and ER120573 selective agonists are promisingnew approach for treating specific conditions associated withendocrine-related disease Besides that estrogens are alsoable to exert nongenomic events mediated by a novel trans-membrane ER G protein-coupled receptor 30 (GPR30) [13]

The metastatic process that is the dissemination ofcancer cells throughout the body to seed secondary tumors atdistant sites requires cancer cells to leave the primary tumorand to acquire migratory and invasive capabilities Manydifferent processes are involved in tumor cell invasion andmetastasis such as an epithelial to mesenchymal transition(EMT) adhesion molecules downregulation and matrixmetalloproteinases (MMPs) upregulation in cancer cellsLoss of epithelial protein marker E-cadherin the concurrentupregulation of mesenchymal protein markers vimentin andthe upregulation of MMP-9 play a dominant role in themetastatic process and could be regulated by E2 in variouscancers including breast ovarian colon and lung cancer[14ndash17] However whether and how the effects of E2 onmigration and invasion of thyroid cancer may be involved inthe regulation of E-cadherin vimentin andMMP-9 is poorlyunderstood

In this study we explore the effects of E2 PPT and DPNon metastatic potential of PTC cell line BCPAP and evaluatethe roles of ER120572 and ER120573 in metastasis of BCPAP cells Wefurther determine the metastasis-related proteins by whichE2 affects BCPAP cell metastasis

2 Materials and Methods

21 Cell Culture The human PTC cell line BCPAP waspurchased from DSMZ (Braunschh Germany) The humanbreast cancer cell line MCF-7 was obtained from the CellBank of the Chinese Academy of Sciences (Shanghai China)The cells were maintained in RPMI1640 medium supple-mented with 10 fetal bovine serum 100UmL penicillinand 100 120583gmL streptomycin in a humidified atmosphere of5 CO

2at 37∘C

22 Treatments Cells were stripped from endogenoussteroids by changing the medium to phenol red-freeRPMI1640 containing 10 charcoal-dextran stripped fetal

ER120572

ER120573

120573-actin

BCPAP MCF-7

Figure 1 BCPAP cells express ER120572 and ER120573Whole cell protein wasresolved by SDS-PAGE followed by western blot analysis for ER120572ER120573 and 120573-actinMCF-7 and ER-positive cells were used as positivecontrol

bovine serum (FBS) (Biowest Nuaille France) 48 h beforetreatment Cells were then incubated in fresh medium withvehicle (DMSO) alone 10minus8M E2 (Sigma St Louis MO)10minus6MPPT (an ER120572-selective agonist) (Tocris BallwinMO) or 10minus6MDPN (an ER120573-selective agonist) (TocrisBallwin MO) for 24 h Concentrations of E2 PPT and DPNare used according to the report by Zeng et al Controlcultures received the same volume of DMSO

23 Western Blot Analysis Total protein was extracted usingTotal Protein Extraction Kit (KeyGEN Nanjing China)according to themanufacturerrsquos instructions Protein concen-tration was determined using a Bradford assay with bovineserum albumin as a standard Protein was resolved with10 SDS-PAGE and then electrophoretically transferred topolyvinylidene difluoride (PVDF) membranes (MilliporeCA USA) The membranes were blocked with 5 nonfatdry milk at room temperature for 1 h and incubated at 4∘Covernight with antibodies directed against ER120572 (AbcamUSA 1 500) ER120573 (Abcam USA 1 1000) E-cadherin (SantaCruz USA 1 1000) vimentin (Boster China 1 500) MMP-9 (Santa Cruz USA 1 1000) and 120573-actin (Santa Cruz USA1 1000) After washing three times with TBS containing 01Tween 20 the membranes were incubated with an HRP-conjugated goat anti-rabbit or goat anti-mouse secondaryantibody (Zhongshan Golden Bridge China 1 5000) andvisualized using SuperSignal West Pico ChemiluminescentSubstrate (Pierce Rockford IL USA)

24 Scratch Wound Assay Migratory ability of BCPAP cellswas assessed by a scratch wound assay Cells were platedin a six-well plate and grow to confluent cell monolayersSubsequently three vertical scratches were made gently withsterile pipette tip across the diameter of the well and rinsedwith PBS to remove debris The wounded cell monolayerwas then incubated in fresh complete medium with vehicle(DMSO) alone 10minus8M E2 10minus6MPPT or 10minus6MDPN Foreach well at least five pictures were taken microscopicallyat a magnification of 10x at 0 and 24 h after scratch


Control E2 PPT DPN

0h

24h

180

160

140

120

100

80

60

40

20

0

Control E2 PPT DPN

lowastlowast

lowast

Cell

mig

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n (

of c

ontro

l)






4 Results




Control E2 PPT DPN

160

140

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100

80

60

40

20

0

Control E2 PPT DPN

lowast

lowast

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Cell

inva

sion

( o

f con

trol)






5 Discussion




ControlE2

PPTDPN

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

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2

15

1

05

0

E-cadherin

Vimentin

MMP-9

120573-actin

Expr

essio

n le

velr

elat

ive c

ontro

l

Control E2 PPT DPN

lowast







6 Conclusions






Acknowledgments


References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control E2 PPT DPN

0h

24h

180

160

140

120

100

80

60

40

20

0

Control E2 PPT DPN

lowastlowast

lowast

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mig

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n (

of c

ontro

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4 Results




Control E2 PPT DPN

160

140

120

100

80

60

40

20

0

Control E2 PPT DPN

lowast

lowast

lowast

Cell

inva

sion

( o

f con

trol)






5 Discussion




ControlE2

PPTDPN

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

25

2

15

1

05

0

E-cadherin

Vimentin

MMP-9

120573-actin

Expr

essio

n le

velr

elat

ive c

ontro

l

Control E2 PPT DPN

lowast







6 Conclusions






Acknowledgments


References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control E2 PPT DPN

160

140

120

100

80

60

40

20

0

Control E2 PPT DPN

lowast

lowast

lowast

Cell

inva

sion

( o

f con

trol)






5 Discussion




ControlE2

PPTDPN

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

25

2

15

1

05

0

E-cadherin

Vimentin

MMP-9

120573-actin

Expr

essio

n le

velr

elat

ive c

ontro

l

Control E2 PPT DPN

lowast







6 Conclusions






Acknowledgments


References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ControlE2

PPTDPN

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

25

2

15

1

05

0

E-cadherin

Vimentin

MMP-9

120573-actin

Expr

essio

n le

velr

elat

ive c

ontro

l

Control E2 PPT DPN

lowast







6 Conclusions






Acknowledgments


References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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