research article inhibition of androgen receptor...

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 519397 8 pageshttpdxdoiorg1011552013519397

Research ArticleInhibition of Androgen Receptor Expression with SmallInterfering RNA Enhances Cancer Cell Apoptosis by SuppressingSurvival Factors in Androgen Insensitive Late Stage LNCaP Cells

Sang Soo Kim1 Hee Joo Cho2 Jung Yoon Kang2 Hee Kyu Kang3 and Tag Keun Yoo1 2

1 Eulji Medi-Bio Research Institute Seoul Republic of Korea2Department of Urology College of Medicine Eulji-University Hangeulbiseok-gil Hagye-dong Nowon-ku Seoul Republic of Korea3 Department of Biomedical Laboratory Science College of Health Sciences Eulji University Gyeonggi-DoSeongnam 461-713 Republic of Korea

Correspondence should be addressed to Tag Keun Yoo ytk5202euljiackr

Received 14 December 2012 Accepted 10 January 2013

Academic Editors T Esen and A Tefekli

Copyright copy 2013 Sang Soo Kim 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

IntroductionThe aim was to evaluate the changes of androgen receptor (AR) expression quantitatively and to identify influence ofAR on cancer related survival markers in LNCap cell line Materials and Methods We compared expressions of AR heat shockprotein 27 (HSP27) clusterin (CLU) glucose-related protein 78 (GRP78) and cellular FLICE-like inhibitory protein (c-FLIP)and their genes between es-LNCaP (less than 33 times subcultured L-33) ls-LNCaP (over 81 times subcultured H-81) and si-LNCaP (AR siRNA transfected ls-LNCaP) by Western blotting and RT-PCR Results The expressions of AR HSP27 CLU GRP78and c-FLIP were increased in ls-LNCaP compared with es-LNCaP (AR 157 HSP27 132 CLU 146 GRP78 138 c-FLIP152) However in si-LNCaP cell line protein expressions were reversed to the level of es-LNCaP cell lines (25 102 109 98 and101) and gene expressions on real-time PCR were also reversed to the expression level of es-LNCaP (ls-LNCaP 179 156 133 123and 167 si-LNCaP 22 93 103 112 and 107) Conclusions This finding suggests that androgen receptor can be related to theincreased expression of cancer related survival markers such as HSP27 GRP78 CLU and c-FLIP in late stage prostate cancer andalso inhibition of AR gene can be a therapeutic target in this stage of cancer

1 Introduction

Nearly 29 of patients with newly diagnosed cancer inUnited States were diagnosed with prostate cancer which isthe second most common cause of cancer death (11 22720patients) [1 2]

At early stage the prostate cancer is influenced markedlyby androgen acting through the androgen receptor (AR) andclinically could be treated with surgical castration radiationor antiandrogen therapy However after initial response tothese treatments most androgendependent prostate cancercells commonly progress to a highly aggressive metastaticandrogenindependent state

Androgen receptor (AR) is a 110 kDa phosphoprotein andone of the nuclear receptor superfamily of ligand activated

transcription factors which elicits the biological response ofandrogens [3ndash5] The AR is expressed in nearly all prostatecancer cells [6ndash8] Growth and development of aggressiveprostate cancer depend on androgen induced AR function[9ndash11]

Androgen independent prostate cancer development canbe explained by the following five theories (1) the AR hyper-sensitivity under chemical castrated state induced by andro-gen ablation treatment more AR is produced or the AR hasenhanced sensitivity to androgen (2) The promiscuous ARhypothesis factors other than testosterone (ie estrogensprogestins and antiandrogens) acts as a mutated AR ago-nists due to broaden specificity of AR (3) The outlaw ARhypothesis hormone independent prostate cancer growth orprogress of AR independently through PTEN mutation and

2 The Scientific World Journal

activation of AR independent pathways such as PI3K andMAPK (4) The bypass AR hypothesis in androgen depriva-tion state other antiapoptotic signal pathways through Bcl-2overexpression and oncogene activation induce progressionto HRPC (5) The lurker cell hypothesis androgen indepen-dent prostate cancer cells basically exist among epithelialstem cells even at androgen dependent state After androgendeprivation androgen independent malignant stem cells areselected to be activated [12]

LNCaP cell line is androgen sensitive human prostatecancer cells derived from the lymph node metastasis [1314] Igawa et al (2002) suggested the hormone sensitiveLNCaP models changed to hormone independent cancercells through long-term subcultures [15]

We focused on the factors related to the development ofandrogen independent prostate cancer In previous studiesusing proteomic analysis we confirmed that high passagesubcultured LNCaP cells that acquired androgen indepen-dent property and the silencing of AR with small interferingRNA (siRNA) transfection resulted in the reversion of pro-teomic profile to level of es-LNCap cell line [16] The aimof the present study was to evaluate changes of androgenreceptor (AR) expression quantitatively and to identify influ-ences of AR on cancer related proteins in LNCap cell line bycomparing es-LNCaP and ls-LNCaP

2 Materials and Methods

21 Cell Culture and Experimental Groups LNCaP cellsobtained from American Type Culture Collection (BethesdaMD) were maintained in RPMI 1640 medium and madetwo LNCaP clones described in previous study [16] Allclones of LNCaP human prostate cancer cells were originatedfrom the same source cell The es-LNCaP cell was derivedfrom low (less than 33) passage subculture and the ls-LNCaP androgenindependent LNCaP derived from high(more than 81) passage subculture The si-ls-LNCaP sublinewas established by stably transfecting the ls-LNCaP cells withsiRNA sequence As control to silencing with siRNA thescrambled siRNA scr-ls-LNCaP was used

22 Doxazosin and siRNA Treatment Doxazosin (SigmaAldrich Korea Seoul Korea) was prepared as describedin previous study [17] Cells were refed with fresh mediaat 80 confluence and treated with doxazosin or serum-free media containing 025 DMSO as control The mRNAtarget sequences to AR (GeneBank Accession NumberNM000044) were designed using a siRNA template designtool (Ambion Austin TX) and siRNA was prepared witha Silencer siRNA construction kit (Ambion) Three oligonu-cleotides AR-1 (51015840-GAC CUA CCG AGG AGC UUU CdTT-31015840) AR-2 (51015840-UCG AGG CCC UGU AAC UUG-31015840) andAR-3 (51015840-CAG UAG UUC GGA CAA ACG AAG A-31015840)were designed based on the publicly released AR DNAsequenceThe siRNAswere transfected into LNCaP cells withLipofectamine 2000 (Invitrogen) employing 50 nM in 250120583LOptiMEMmedium60mmculture dishThe transfected cellswere allowed to grow for 24 48 and 72 h at 37∘C in a 5CO2incubator and harvested for RT-PCR and immunoblot

analysis We performed immunohistochemical staining toconfirm the expressions of AR in various LNCaP cells

23 Total RNA Extraction Conventional RT-PCR and Real-Time RT-PCR Total RNA was extracted using the TRIzolmethod (Invitrogen Carlsbad CA) Cells (50 times 105) weremixed in a test tubewith 1mLTRIzol solution Prepared RNAwas denaturated at 65∘C for 15min in a volume of 30 120583L andcooled on ice for at least 1min 20120583g of denatured RNAwere then annealed by addition of reaction mixture to a totalvolume of 20 120583L (40 120583L of 5 times RT buffer 10 pmol of primers20 120583L of 25mM MgCl

2 20120583L of 10mM dNTPs and 02 120583L

of 1M DTT in nuclease-free water) and incubated at 42∘Cfor 70min The reaction was terminated at 95∘C for 5minchilled on ice for 5min and collected by brief centrifugationTo remove RNA 1 120583L of RNase H were added to each tubefollowed by incubation at 37∘C for 20min 1 120583L of cDNAwereused for each PCR reaction

Amplifications of cDNAs by PCR using specific primerpairs for AR were performed in 20 120583L reaction volumescontaining 10mM Tris-HCl pH 83 50mM KCl 15mMMgCl

2 0001 gelatin 02120583M each dNTP 02 120583M of each

primer 1 unit of Taq DNA polymerase (Invitrogen CA) and10 120583L cDNA as template

Real-time PCR was performed with an SLAN real-timePCR detection system (LG Life science Korea) and SYBRGreen reagents (Invitrogen Carlsbad CA) Specific primersfor human GAPDH AR HSP27 CLU GRP78 and c-FLIPwere designed to work in the same cycling conditions (50∘Cfor 2min to permit uracil N-glycosylase cleavage 95∘C for10min followed by 40 cycles of 95∘C for 15 s and 60∘C for1min) We used 10 120583L of the reverse transcriptase productfor PCR in a final volume of 25 120583L

24 Western Blot Preparation of total cell lysate and the pro-cedures for Western blot analyses were performed essentiallyas described previously [16]The antibodies againstGRP78 c-FLIP andARwere purchased fromSantaCruz Biotechnology(Santa Cruz CA) Antibody for HSP27 purchased from Mil-lipore (Millipore MA) The quantity of the applied proteinwas normalized with anti-actin polyclonal antibody (SigmaAldrich Korea Seoul Korea)

Samples with equal amounts of protein (20120583g) fromlysates of cultured LNCaP cells were subjected to SDS-PAGE and then transferred to a PVDF filter The filterswere blocked in TBS containing 5 nonfat milk powderat 4∘C overnight and then incubated for 1 h with a dilutedeach primary antibodies (Actin 1 10000 AR HSP27 CLUGRP78 1 1000 c-FLIP 1 2000 Santa Cruz CA)

25 Immunocytochemical Analysis and TUNEL StainingCells on coverslips were rinsed 1 times phosphate-buffered saline(PBS) and then fixed with ice-coldmethanol for 15min Sam-ples were further permeabilized with PBS containing 0025Triton-X detergent (1timesPBS-TX) for 10min and blocked with3BSA in 1timesPBS for 30min Cells were reactedwith primaryantibodies (AR HSP27 CLU GRP78 1 100 c-FLIP 1 50Santa Cruz CA) for 1 hour at room temperature Cells werewashed 3 times for 5minwith 1timesPBS-TX and then incubated

The Scientific World Journal 3

Phase contrast FITC

(a)

Control NC 24 48 72AR-1

AR-2

AR-3

GAPDH

(b)

Figure 1 Effective silencing of the androgen receptor (AR) gene expression in ls-LNCap cells after small interfereing RNA (siRNA) treatment(a) Transfection efficiency shown by fluorescence microscopy (b) RT-PCR band of AR from si-ls-LNCap cells after siRNA CNTL untreatedsiRNA NC treated scrambled siRNA

with horseradish peroxidase (HRP) conjugated secondaryantibodies (goat anti-mouse IgG and goat anti-rabbit IgGSanta Cruz CA) Diaminobenzidine (DAB) was used asthe chromogen and counterstaining was done with Mayershematoxylin Following three 5 min washes cellscoverslipswere mounted on slides with coverslips

For TUNEL assays fixed cells were incubated with anequilibrium buffer for 5min using the in situ apoptosisdetection kit Fluorescein (Apoptag Roche BMS) and thentreated in reaction buffer with 10 units of terminal deoxynu-cleotidyl transferase and 1 unit of deoxyuridine triphosphatedigoxigenin at 37∘C for 1 hour The reaction was terminatedby adding stopwash buffer and then washed twice withTris buffer Anti-digoxigenin-FITC was added and reacted at37∘C for 30min After washing with distilled water nucleiwere counterstained withHoechst 33258 (Sigma Chemical StLouis MO) and apoptosis in the cells was observed under afluorescent microscope Cells with green fluorescent (FITC)colored nuclei were considered apoptotic For quantifyingapoptotic cells apoptotic and total cells were counted in 5random fields scoring between 300 and 500 cells and thenumbers of apoptotic cells were expressed as percentagesof the total cell population Immunocytochemical stainingslides and TUNEL staining slides were observed with micro-scope (TE-300 Nikon Japan)

26 Statistics Analysis Results were analyzed using a two-tailed Studentrsquos 119905-test to assess statistical significance Valuesof 119875 lt 005 were considered statistically significant

3 Results

31 Androgen Receptor siRNA Transfection Efficiency Toexplore the feasibility of siRNA transfection efficiency inknocking down AR expression in prostate cancer cells thatharbor the AR gene fluorescent oligo staining with BLOCK-iT was used and the green staining of more than 95 of cellswas confirmed in siRNA transfected cells under fluorescencemicroscopy (Figure 1(a)) We designed and synthesized threesiRNAs AR-1 AR-2 andAR-3 against humanARgene After48 hours transfection with three sequence-specific siRNAsone relatively potent siRNA AR-1 was identified in knockingdown AR expression compared with others by checkingthe mRNA with RT-PCR This knocking down effect wassequence-specific event because a negative control siRNAwith a scrambled sequence had no effect on AR expressionlevel (Figure 1(b))

32 Immunocytochemistry of Markers Among the fourexperimental cell lines (es-LNCaP ls-LNCaP scr-ls-LNCaPand si-ls-LNCaP) we confirmed the expression level of fiveprostate cancer related proteins (AR HSP27 CLU GRP78and c-FLIP) with immunocytochemical staining (Figure 2)Positive staining of AR protein was more intensive in ls-LNCaP than es-LNCaP but the expression level of AR proteinin si-ls-LNCaP was almost disappeared (Figure 2) Theseresults showed AR gene expression was almost inhibited bysiRNA transfection Moreover the knocking down effect onthe other cancer related proteins was also verified similarly


AR HSP27 CLU GRP78

es-LNCaP

ls-LNCaP

si-ls-LNCaP

scr-ls-LNCaP

c-FLIP

Figure 2 Immunocytochemical analysis of HSP27 clusterin GRP78 and c-FLIP at the es-LNCap ls-LNCap scr-ls-LNCap and si-ls-LNCaPcells

Actin

AR

HSP27

es ls

CLU

c-FLIP

si-ls

(a)

0

100

200

300

400

AR HSP27 CLU c-FLIP

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

es-LNCaPls-LNCaPsi-ls-LNCaP

Relat

iver

atio

of

targ

eta

ctin

(b)

Figure 3 Electrophoretogram of immunoblot for androgen receptor HSP27 c-FLIP and clusterin expression at the es- ls- and the si-ls-LNCap cells

The positive staining to HSP27 CLU GRP78 and c-FLIPproteins in ls-LNCaP cells was more intensive than in es-LNCaP cells and was dramatically decreased in si-ls-LNCaPcells

33 Gene and Protein Expressions of Markers The expressionof five prostate cancer related proteins (AR HSP27 CLUGRP78 and c-FLIP) increased in ls-LNCaP compared withes-LNCaP (AR 157 HSP27 132 CLU 146 GRP78 138and c-FLIP 152 Figure 3) But in si-ls-LNCaP cell lineprotein expressions were decreased to level of es-LNCaPcell lines (25 102 109 98 and 101 Figure 3) and geneexpressions on real-time PCR were decreased similarly (ls-LNCaP 179 156 133 123 and 167 si-ls-LNCaP 22 93 103112 and 107 Figure 4)

34 TUNEL Assay TUNEL assay was performed to see howdoxazosin induced apoptosis was affected by the inhibitionof AR gene (Figure 5) The number of TUNEL positivecells appeared less in ls-LNCaP cells compared to es-LNCaPcounterpart But after AR was silenced the number ofTUNEL positive cells increased significantly

4 Discussion

Prostate cancer cells are basically androgen dependent andandrogen deprivation therapy (ADT) consistently causesprostate apoptosis and involution in first diagnosed prostatecancer But when prostate cancer advance further it pro-gresses into a more aggressive form of castration resistantprostate cancer (CRPC) refractory to all kinds of ADT


es ls si-ls

LNCap cells

ARHSP27

CLUGRP78c-FLIP

GAPDH

(a)

AR HSP27 CLU GRP78 c-FLIP0

100

200

300

Mar

kers

GA

PDH

Relat

ivei

nten

sity

()


(b)


10050

200150


(c)

Figure 4 Electrophoretogram and its densitogram of conventional RT-PCRreal-time PCR product for androgen receptor HSP27 CLUGRP78 and c-FLIP expression at the es- ls- and the si-ls-LNCaP cells

Hoechst TUNEL Hoechst TUNEL

Untreated Doxazosin treated (25 120583M)

es-LNCaP

ls-LNCaP

si-ls-LNCap

Figure 5 In situ detection of apoptotic cells in AR siRNA transfected cells at 48 hours after doxazosin treatment (25 120583M) In situ detectionof apoptotic cells in LNCaP cells was performed by 31015840-end labeling with digoxigenin-dUTP using terminal transferase

Treatment of CRPC is very difficult and not that satisfactoryso far Docetaxel based chemotherapy is one of the mosteffective ways of treatments [18ndash20] but the overall survivalbenefit is only 2-3 months compared to conventional meth-ods [21]

Diverse pathways have been discussed regarding progres-sion toCRPC fromandrogen dependent counterpart Amongthem AR is considered having one of the most importantroles with the possible mechanisms of hypersensitive AR ormutation of AR gene [12] LNCaP cell lines are well known as


their androgen sensitive characteristics but in our previousstudy we showed the changes of cellular characteristics ofearly stage LNCaP cells (L-33) into androgen independentmanner after long-term subculture (H-81) [16] And we alsofound that there were significant differences in the expressionof important survival antiapoptotic factors such as TimGRP78 and HSP27 using proteomics technique We thinkthat early and late stage LNCaP cell line model can help usto understand the mechanism of progression into the form ofCRPC to some extent In the present study we showed higherlevel of AR mRNA and protein expression in H-81 comparedto L-33 and this implicates that AR is closely related to theprogression into H-81 characteristics via direct or indirectways

Among the possiblemechanisms of chemotherapy failureof CRPC several antiapoptotic factors such as c-FLIP [22]HSP27 [23] clusterin [17] and GRP78 [24] have been dis-cussed These factors have protective roles against apoptosisinducing stimuli They are upregulated in various types ofcancer cells and the degree of upregulation is proportionalto the cancer aggressiveness It also has been speculatedthat these survival factors help cancer cells to resist againstvarious forms of anticancer treatment such as radiotherapyand cytotoxic chemotherapy

Hsp27 suppresses apoptosis and probably has a criticalrole in progression toCRPC [25ndash29] It has been reported thatandrogen insensitive LNCaP cells showed upregulation ofHSP27 against androgen withdrawal and antiacancer drugssuch as paclitaxel [30]

GRP78 is a key member of the molecular chaperone heatshock protein (HSP) 70 family [31ndash33] GRP78 expression isincreased when AR expression is upregulated in LNCaP cellstreated with DHT [34]This is consistent with our findings inthis study showing further upregulation ofGRP78 expressionin H-81 cells

Clusterin acts as an antiapoptotic factor and plays animportant role in resistance to chemotherapeutic drugs [35]When clusterin is overexpressed using vector transfection inrat prostate cell lines transfected cells survived with blockingTNF-120572 induced apoptosis

c-FLIP is also involved in apoptosis pathway regardingFas signal transduction [22] It is generally considered tohave antiapoptotic roles in the prostate cancer [36] c-FLIPexpression is highly upregulated in the prostate cancer tissuewhen compared to normal tissue It seems that maintaininghigh level of c-FLIP is essential and important in overcomingTNF related apoptosis in the prostate cancer [37] It has alsobeen known that transcription of c-FLIP is affected by AR[38]

Our study showed increased expression of clusterinHSP27 GRP78 and c-FLIP in H-81 compared to L-33mRNAs and proteins of these factors are downregulatedbelow the levels of L-33 afterARknock-out using siRNA tech-nique Furthermore the same concentration of doxazosincould induce more significant apoptosis after AR silencingThese observations suggest that Clusterin HSP27 GRP78and c-FLIP take part in the progression into H-81 in LNCaPmodel and AR is closely related to the upregulated expressionand suppression of these survival factors Our study also

helps us to speculate that while prostate cancer cells becomemore aggressive with change of AR and overproduction ofsurvival factors under the extremely stressful circumstanceslike androgen deprivation these cells can be reverted intotreatment sensitive traits when successful AR blocking causessuppression of various survival factors It also supports theidea that therapeutic approach targeting AR can enhancethe efficacy of anticancer treatment in the patients withmetastatic CRPC resisting against all forms of treatment

Suppression of gene expression using siRNA is a simpli-fied experimental technique which can regulate functions ofspecific factors at the gene level AR silencing in the genelevel is essential in the study of AR block because AR isa transcription factor related to synthesis regulation andsecretion of various kinds of proteins In our present studyAR silencing successfully showedover 80efficiencyWe alsoshowed higher level of discrimination using real time RT-PCR which enables us to find more significant differencesbetween cell groups

It can be summarized that in this LNCaP model weobserved that c-FLIP HSP27 clusterin and GRP78 take partin the progression into androgen insensitive status and theexistence and overexpression of AR are closely related inthis process We think that these findings can be appliedin the understanding of CRPC progression and treatmentresistance of metastatic CRPC New therapeutic approachestargeting AR regulation could be an effective solution againstmetastatic CRPC currently incurable and therefore variousscientific efforts should be focused

Conflict of Interests

None of the contributing authors have any conflict of inter-ests including specific financial interests and relationshipsand affiliations relevant to the subject matter materials ormethods discussed in the paper

Acknowledgment

This study was supported by grants from Eulji Medi-BioResearch Institute (EMBRI-2010-SN-02)

References

[1] R Siegel E Ward O Brawley and A Jemal ldquoCancer statistics2011 the impact of eliminating socioeconomic and racial dis-parities on premature cancer deathsrdquo CA Cancer Journal forClinicians vol 61 no 4 pp 212ndash236 2011

[2] A Jemal F Bray M M Center J Ferlay E Ward and D For-man ldquoGlobal cancer statisticsrdquoCACancer Journal for Cliniciansvol 61 no 2 pp 69ndash90 2011

[3] D J Mangelsdorf and RM Evans ldquoThe RXR heterodimers andorphan receptorsrdquo Cell vol 83 no 6 pp 841ndash850 1995

[4] D J Lamb N L Weigel and M Marcell ldquoAndrogen receptorsand their biologyrdquoVitamins andHormones vol 62 pp 199ndash2302001

[5] S M Dehm and D J Tindall ldquoMolecular regulation of andro-gen action in prostate cancerrdquo Journal of Cellular Biochemistryvol 99 no 2 pp 333ndash344 2006


[6] J A R De Winter P J A Janssen H M E B Sleddens et alldquoAndrogen receptor status in localized and locally progressivehormone refractory human prostate cancerrdquo American Journalof Pathology vol 144 no 4 pp 735ndash746 1994

[7] G W Chodak D M Kranc L A Puy H Takeda K Johnsonand C Chang ldquoNuclear localization of androgen receptor inheterogeneous samples of normal hyperplastic and neoplastichuman prostaterdquo Journal of Urology vol 147 no 3 pp 798ndash8031992

[8] M V Sadi P CWalsh and E R Barrack ldquoImmunohistochem-ical study of androgen receptors in metastatic prostate cancercomparison of receptor content and response to hormonal ther-apyrdquo Cancer vol 67 no 12 pp 3057ndash3064 1991

[9] C W Gregory B He R T Johnson et al ldquoA mechanism forandrogen receptor-mediated prostate cancer recurrence afterandrogen deprivation therapyrdquo Cancer Research vol 61 no 11pp 4315ndash4319 2001

[10] H I Scher and C L Sawyers ldquoBiology of progressive castra-tion-resistant prostate cancer directed therapies targeting theandrogen-receptor signaling axisrdquo Journal of Clinical Oncologyvol 23 no 32 pp 8253ndash8261 2005

[11] M E Taplin and S P Balk ldquoAndrogen receptor a key moleculein the progression of prostate cancer to hormone indepen-dencerdquo Journal of Cellular Biochemistry vol 91 no 3 pp 483ndash490 2004

[12] B J Feldman and D Feldman ldquoThe development of androgen-independent prostate cancerrdquoNature Reviews Cancer vol 1 no1 pp 34ndash45 2001

[13] T Otsuka K Iguchi K Fukami et al ldquoAndrogen receptorW741C and T877A mutations in AIDL cells an androgen-independent subline of prostate cancer LNCaP cellsrdquo TumourBiology vol 32 no 6 pp 1097ndash1102 2011

[14] F Thomas S Patel J M P Holly R Persad A Bahl and CM Perks ldquoDihydrotestosterone SEnsitises LNCaP cells to deathinduced by epigallocatechin-3-Gallate (EGCG) or an IGF-Ireceptor inhibitorrdquo Prostate vol 69 no 2 pp 219ndash224 2009

[15] T Igawa F F Lin M S Lee D Karan S K Batra andM F LinldquoEstablishment and characterization of androgen-independenthuman prostate cancer LNCaP cell modelrdquo Prostate vol 50 no4 pp 222ndash235 2002

[16] H Y Yun S Kim Y B Young and K Y Tag ldquoProteomic analy-sis of androgen-independent growth in low and high passagehuman LNCaP prostatic adenocarcinoma cellsrdquo Journal ofBiochemistry and Molecular Biology vol 41 no 10 pp 722ndash7272008

[17] Y H Youm H Yang Y D Yoon D Y Kim C Lee and T KYoo ldquoDoxazosin-induced clusterin expression and apoptosis inprostate cancer cellsrdquoUrologic Oncology vol 25 no 6 pp 483ndash488 2007

[18] I Marech A Vacca G Ranieri A Gnoni and F DammaccoldquoNovel strategies in the treatment of castration-resistantprostate cancer (Review)rdquo International Journal of Oncologyvol 40 no 5 pp 1313ndash1320 2012

[19] C J Logothetis E Basch A Molina et al ldquoEffect of abirateroneacetate and prednisone compared with placebo and pred-nisone on pain control and skeletal-related events in patientswithmetastatic castration-resistant prostate cancer exploratoryanalysis of data from the COU-AA-301 randomised trialrdquo TheLancet Oncology vol 13 no 12 pp 1210ndash1217 2012

[20] D Keizman N Maimon and M Gottfried ldquoMetastatic hor-mone refractory prostate cancer recent advances in standard

treatment paradigm and future directionsrdquo American Journalof Clinical Oncology 2012

[21] J Ansari S A Hussain A Zarkar J S Tanguay J Bliss andJ Glaholm ldquoDocetaxel chemotherapy for metastatic hormonerefractory prostate cancer as first-line palliative chemotherapyand subsequent re-treatment birmingham experiencerdquo Oncol-ogy Reports vol 20 no 4 pp 891ndash896 2008

[22] H Ye Y Li J Melamed et al ldquoStromal anti-apoptotic androgenreceptor target gene c-FLIP in prostate cancerrdquo Journal ofUrology vol 181 no 2 pp 872ndash877 2009

[23] C Garrido M Brunet C Didelot Y Zermati E Schmitt andG Kroemer ldquoHeat shock proteins 27 and 70 anti-apoptoticproteins with tumorigenic propertiesrdquo Cell Cycle vol 5 no 22pp 2592ndash2601 2006

[24] H Miyake I Hara S Arakawa and S Kamidono ldquoStress pro-tein GRP78 prevents apoptosis induced by calcium ionophoreionomycin but not by glycosylation inhibitor tunicamycin inhuman prostate cancer cellsrdquo Journal of Cellular Biochemistryvol 77 no 3 pp 396ndash408 2000

[25] S W Lee E K Kim S S Kim H S Uh K S Cha and T KYoo ldquoExpression of heat shock protein 27 according to Gleasonscore and pathologic stage of prostate cancerrdquoKorean Journal ofUrology vol 50 no 6 pp 547ndash552 2009

[26] P Rocchi A So S Kojima et al ldquoHeat shock protein 27increases after androgen ablation and plays a cytoprotective rolein hormone-refractory prostate cancerrdquo Cancer Research vol64 no 18 pp 6595ndash6602 2004

[27] S A Thomas ldquoDetection and distribution of heat shockproteins 27 and 90 in human benign and malignant prostatictissuerdquo British Journal of Urology vol 77 no 3 pp 367ndash3721996

[28] D G Bostwick L Klotz and M Garnick ldquoImmunohisto-chemical changes in prostate cancer after androgen deprivationtherapyrdquoMolecular Urology vol 4 no 3 pp 101ndash107 2000

[29] A Valeri R Azzouzi E Drelon et al ldquoHeat-shock proteinsinhibit induction of prostate cancer cell apoptosisrdquoProstate vol45 no 1 pp 58ndash65 2000

[30] C Andrieu D Taieb V Baylot et al ldquoHeat shock protein 27confers resistance to androgen ablation and chemotherapy inprostate cancer cells through eIF4Erdquo Oncogene vol 29 no 13pp 1883ndash1896 2010

[31] J Zhang Y Jiang Z Jia et al ldquoAssociation of elevated GRP78expression with increased lymph node metastasis and poorprognosis in patients with gastric cancerrdquo Clinical and Exper-imental Metastasis vol 23 no 7-8 pp 401ndash410 2006

[32] M ShudaNKondohN Imazeki et al ldquoActivation of theATF6XBP1 and grp78 genes in human hepatocellular carcinoma apossible involvement of the ER stress pathway in hepatocarcino-genesisrdquo Journal of Hepatology vol 38 no 5 pp 605ndash614 2003

[33] G Gazit J Lu and A S Lee ldquoDe-regulation of GRP stressprotein expression in human breast cancer cell linesrdquo BreastCancer Research and Treatment vol 54 no 2 pp 135ndash146 1999

[34] S S Tan I Ahmad H L Bennett et al ldquoGRP78 up-regulationis associated with androgen receptor status Hsp70-Hsp90client proteins and castrate-resistant prostate cancerrdquo Journal ofPathology vol 223 no 1 pp 81ndash87 2011

[35] H Miyake C Nelson P S Rennie and M E Gleave ldquoAcqui-sition of chemoresistant phenotype by overexpression of theantiapoptotic gene Testosterone-repressed prostate message-2in prostate cancer xenograft modelsrdquo Cancer Research vol 60no 9 pp 2547ndash2554 2000


[36] S Gao P Lee H Wang et al ldquoThe androgen receptordirectly targets the cellular FasFasL-associated death domainprotein-like inhibitory protein gene to promote the androgen-independent growth of prostate cancer cellsrdquo MolecularEndocrinology vol 19 no 7 pp 1792ndash1802 2005

[37] J Zhang N Gao S Kasper K Reid C Nelson and R JMatusik ldquoAn androgen-dependent upstream enhancer is essen-tial for high levels of probasin gene expressionrdquo Endocrinologyvol 145 no 1 pp 134ndash148 2004

[38] D B Longley T R Wilson M McEwan et al ldquoc-FLIP inhibitschemotherapy-induced colorectal cancer cell deathrdquoOncogenevol 25 no 6 pp 838ndash848 2006

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


activation of AR independent pathways such as PI3K andMAPK (4) The bypass AR hypothesis in androgen depriva-tion state other antiapoptotic signal pathways through Bcl-2overexpression and oncogene activation induce progressionto HRPC (5) The lurker cell hypothesis androgen indepen-dent prostate cancer cells basically exist among epithelialstem cells even at androgen dependent state After androgendeprivation androgen independent malignant stem cells areselected to be activated [12]

LNCaP cell line is androgen sensitive human prostatecancer cells derived from the lymph node metastasis [1314] Igawa et al (2002) suggested the hormone sensitiveLNCaP models changed to hormone independent cancercells through long-term subcultures [15]

We focused on the factors related to the development ofandrogen independent prostate cancer In previous studiesusing proteomic analysis we confirmed that high passagesubcultured LNCaP cells that acquired androgen indepen-dent property and the silencing of AR with small interferingRNA (siRNA) transfection resulted in the reversion of pro-teomic profile to level of es-LNCap cell line [16] The aimof the present study was to evaluate changes of androgenreceptor (AR) expression quantitatively and to identify influ-ences of AR on cancer related proteins in LNCap cell line bycomparing es-LNCaP and ls-LNCaP

2 Materials and Methods

21 Cell Culture and Experimental Groups LNCaP cellsobtained from American Type Culture Collection (BethesdaMD) were maintained in RPMI 1640 medium and madetwo LNCaP clones described in previous study [16] Allclones of LNCaP human prostate cancer cells were originatedfrom the same source cell The es-LNCaP cell was derivedfrom low (less than 33) passage subculture and the ls-LNCaP androgenindependent LNCaP derived from high(more than 81) passage subculture The si-ls-LNCaP sublinewas established by stably transfecting the ls-LNCaP cells withsiRNA sequence As control to silencing with siRNA thescrambled siRNA scr-ls-LNCaP was used

22 Doxazosin and siRNA Treatment Doxazosin (SigmaAldrich Korea Seoul Korea) was prepared as describedin previous study [17] Cells were refed with fresh mediaat 80 confluence and treated with doxazosin or serum-free media containing 025 DMSO as control The mRNAtarget sequences to AR (GeneBank Accession NumberNM000044) were designed using a siRNA template designtool (Ambion Austin TX) and siRNA was prepared witha Silencer siRNA construction kit (Ambion) Three oligonu-cleotides AR-1 (51015840-GAC CUA CCG AGG AGC UUU CdTT-31015840) AR-2 (51015840-UCG AGG CCC UGU AAC UUG-31015840) andAR-3 (51015840-CAG UAG UUC GGA CAA ACG AAG A-31015840)were designed based on the publicly released AR DNAsequenceThe siRNAswere transfected into LNCaP cells withLipofectamine 2000 (Invitrogen) employing 50 nM in 250120583LOptiMEMmedium60mmculture dishThe transfected cellswere allowed to grow for 24 48 and 72 h at 37∘C in a 5CO2incubator and harvested for RT-PCR and immunoblot

analysis We performed immunohistochemical staining toconfirm the expressions of AR in various LNCaP cells

23 Total RNA Extraction Conventional RT-PCR and Real-Time RT-PCR Total RNA was extracted using the TRIzolmethod (Invitrogen Carlsbad CA) Cells (50 times 105) weremixed in a test tubewith 1mLTRIzol solution Prepared RNAwas denaturated at 65∘C for 15min in a volume of 30 120583L andcooled on ice for at least 1min 20120583g of denatured RNAwere then annealed by addition of reaction mixture to a totalvolume of 20 120583L (40 120583L of 5 times RT buffer 10 pmol of primers20 120583L of 25mM MgCl

2 20120583L of 10mM dNTPs and 02 120583L

of 1M DTT in nuclease-free water) and incubated at 42∘Cfor 70min The reaction was terminated at 95∘C for 5minchilled on ice for 5min and collected by brief centrifugationTo remove RNA 1 120583L of RNase H were added to each tubefollowed by incubation at 37∘C for 20min 1 120583L of cDNAwereused for each PCR reaction

Amplifications of cDNAs by PCR using specific primerpairs for AR were performed in 20 120583L reaction volumescontaining 10mM Tris-HCl pH 83 50mM KCl 15mMMgCl

2 0001 gelatin 02120583M each dNTP 02 120583M of each

primer 1 unit of Taq DNA polymerase (Invitrogen CA) and10 120583L cDNA as template

Real-time PCR was performed with an SLAN real-timePCR detection system (LG Life science Korea) and SYBRGreen reagents (Invitrogen Carlsbad CA) Specific primersfor human GAPDH AR HSP27 CLU GRP78 and c-FLIPwere designed to work in the same cycling conditions (50∘Cfor 2min to permit uracil N-glycosylase cleavage 95∘C for10min followed by 40 cycles of 95∘C for 15 s and 60∘C for1min) We used 10 120583L of the reverse transcriptase productfor PCR in a final volume of 25 120583L

24 Western Blot Preparation of total cell lysate and the pro-cedures for Western blot analyses were performed essentiallyas described previously [16]The antibodies againstGRP78 c-FLIP andARwere purchased fromSantaCruz Biotechnology(Santa Cruz CA) Antibody for HSP27 purchased from Mil-lipore (Millipore MA) The quantity of the applied proteinwas normalized with anti-actin polyclonal antibody (SigmaAldrich Korea Seoul Korea)

Samples with equal amounts of protein (20120583g) fromlysates of cultured LNCaP cells were subjected to SDS-PAGE and then transferred to a PVDF filter The filterswere blocked in TBS containing 5 nonfat milk powderat 4∘C overnight and then incubated for 1 h with a dilutedeach primary antibodies (Actin 1 10000 AR HSP27 CLUGRP78 1 1000 c-FLIP 1 2000 Santa Cruz CA)

25 Immunocytochemical Analysis and TUNEL StainingCells on coverslips were rinsed 1 times phosphate-buffered saline(PBS) and then fixed with ice-coldmethanol for 15min Sam-ples were further permeabilized with PBS containing 0025Triton-X detergent (1timesPBS-TX) for 10min and blocked with3BSA in 1timesPBS for 30min Cells were reactedwith primaryantibodies (AR HSP27 CLU GRP78 1 100 c-FLIP 1 50Santa Cruz CA) for 1 hour at room temperature Cells werewashed 3 times for 5minwith 1timesPBS-TX and then incubated


Phase contrast FITC

(a)


AR-2

AR-3

GAPDH

(b)





3 Results




AR HSP27 CLU GRP78

es-LNCaP

ls-LNCaP

si-ls-LNCaP

scr-ls-LNCaP

c-FLIP


Actin

AR

HSP27

es ls

CLU

c-FLIP

si-ls

(a)

0

100

200

300

400

AR HSP27 CLU c-FLIP

lowast

lowast

lowastlowast

lowastlowast

lowastlowast


Relat

iver

atio

of

targ

eta

ctin

(b)





4 Discussion



es ls si-ls

LNCap cells

ARHSP27

CLUGRP78c-FLIP

GAPDH

(a)


100

200

300

Mar

kers

GA

PDH

Relat

ivei

nten

sity

()


(b)


10050

200150


(c)




es-LNCaP

ls-LNCaP

si-ls-LNCap

















Acknowledgment


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Phase contrast FITC

(a)


AR-2

AR-3

GAPDH

(b)





3 Results




AR HSP27 CLU GRP78

es-LNCaP

ls-LNCaP

si-ls-LNCaP

scr-ls-LNCaP

c-FLIP


Actin

AR

HSP27

es ls

CLU

c-FLIP

si-ls

(a)

0

100

200

300

400

AR HSP27 CLU c-FLIP

lowast

lowast

lowastlowast

lowastlowast

lowastlowast


Relat

iver

atio

of

targ

eta

ctin

(b)





4 Discussion



es ls si-ls

LNCap cells

ARHSP27

CLUGRP78c-FLIP

GAPDH

(a)


100

200

300

Mar

kers

GA

PDH

Relat

ivei

nten

sity

()


(b)


10050

200150


(c)




es-LNCaP

ls-LNCaP

si-ls-LNCap

















Acknowledgment


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















AR HSP27 CLU GRP78

es-LNCaP

ls-LNCaP

si-ls-LNCaP

scr-ls-LNCaP

c-FLIP


Actin

AR

HSP27

es ls

CLU

c-FLIP

si-ls

(a)

0

100

200

300

400

AR HSP27 CLU c-FLIP

lowast

lowast

lowastlowast

lowastlowast

lowastlowast


Relat

iver

atio

of

targ

eta

ctin

(b)





4 Discussion



es ls si-ls

LNCap cells

ARHSP27

CLUGRP78c-FLIP

GAPDH

(a)


100

200

300

Mar

kers

GA

PDH

Relat

ivei

nten

sity

()


(b)


10050

200150


(c)




es-LNCaP

ls-LNCaP

si-ls-LNCap

















Acknowledgment


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















es ls si-ls

LNCap cells

ARHSP27

CLUGRP78c-FLIP

GAPDH

(a)


100

200

300

Mar

kers

GA

PDH

Relat

ivei

nten

sity

()


(b)


10050

200150


(c)




es-LNCaP

ls-LNCaP

si-ls-LNCap

















Acknowledgment


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























Acknowledgment


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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