Chinese Journal of Biochemistry and Molecular Biology

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology 21 (5) : Sp1,,,, 3, Charles Y F YOUNG 2) 3 ( ; 2) Department of Urology, Mayo Clinical College of Medicine, Mayo Clinic, Rochester, MN 55905,USA) (androgen receptor, AR) AR AR (quercetin), LNCaP, AR AR GC box AR, Sp1, Sp1 AR, AR Sp1 AR Western, Sp1, c2jun, c2jun Sp1 AR c2jun Sp1 Sp1 AR Sp1 c2jun AR, c2jun Sp1 Sp1 AR,Sp1, Q5,Q7 Inhibitory Effects of Quercetin on Proliferation of Prostate Cancer Cells and Activity of Transcription Factor Sp1 YUAN Hui2Qing, YANGLing2Ling, J IANG An2Li, KONG Feng, ZHANGJian2Ye 3, Charles Y F YOUNG 2) 3 ( Department of Biochemistry and Molecular Biology, School of Medicine, Shandong University, Jinan , China ; 2) Department of Urology, Mayo Clinical College of Medicine, Mayo Clinic, Rochester, MN 55905, USA) Abstract Androgen receptor ( AR) plays an important role in prostate cancer etiology The aberrant regulation of AR is implicated in the formation and progression of the prostate cancers Quercetin, one of the main polyphenols from vegetables and fruits, could inhibit proliferation of the human prostate cancer cell LNCaP in a dose2dependent manner as demostrated by MTS analysis Since ubiquitous transcription factor Sp1 is the one of factors that facilitates the AR expression Luciferase activity analysis showed that ectopic transfection of Sp1 increased AR promoter activity and transcriptional activity of the AR, while quercetin repressed the Sp12mediated stimulation on the AR However, quercetin has no detectable effect on Sp1 expression In addition, overexpression of c2j un induced by quercetin could reverse the stimulation of Sp1 on : : (No Y2004C24) 3 Tel : , E2mail : edu cn ;Tel : , E2mail : young edu Received :December 3, 2004 ; Accepted :March 15, 2005 Supported by Shandong Natural Science Foundation(No Y2004C24) 3 Corresponding author Tel : , E2mail : edu cn ; Tel : , E2mail : young edu China Academic Journal Electronic Publishing House All rights reserved

2 the AR, and interaction between c2j un and Sp1 was further detected by co2immunoprecipitation These data might lead us to conclude that inhibitory effect of quercetin on proliferation of LNCaP cells may result from protein2protein interactions of transcriptional factors or coregulators in addition of decreasing the AR expression Key words quercetin, cell proliferation, Sp1, prostate cancer cells, inhibitory effect (androgen receptor, AR) 100 mm 60 mm, 5 % RPMI ,5 %CO 2, 3 4 d AR 24 h, AR,, AR AR 5 % 1 (prostate cancers,pcs) nmolπl (mibolerone,mib) [1,2 ] 1 nmolπl Mib Mib New England Nuclear (hormone refractory prostate cancers, HRPCs) LKT DMSO AR [3 5 ] 1 2 MTSΠPMS PCs LNCaP h 24 h, PCs 5 % 10 nmolπl (quercetin) (DHT) 4 d, 4 MTS, ( 32( 4, 52dimethylthiaol222yl ) 252( 32carboxy2methoxyp2, : ( henyl)222( 42sulfophenyl )22H2tetrazolium inner salt ) NADPH c PMS(phenazine methosulfate) Promega, l, [6 ] ; (2) 20 l, 37 5 %CO 2 B1 (cyclin B,cdc2 1 2 h, 490 nm [7 ] ; (3) 1 3 [8 ] ; (4) 60 mm C PI, PIP LNCaP GeneFECTOR ( Venn IP3 Nova, LLC) (luciferase) [9,10 ] ;, AR2kb p GL3 (p GL32 c2myc, ras p53 ( AR2kb) 215 gπ,psa6kb p GL3 ), (p GL32PSA6kb),215 gπ prsv2c2jun [11 MCF27 ] pcdna2sp1 p GL3 basic CMV ( 2gal),014 gπ h 24 h 2gal 3 LNCaP China Academic Journal Electronic Publishing House All rights reserved

3 5 : Sp1 675 (WE) :LNCaP 100 mm, Mib Mib 24 h, (phosphate buffered saline, PBS) [12 ] MTS Fig11, (015 mmolπl ( PMSF), 50 gπml aprotinin, 1 mmolπl, 10 mmolπl LNCaP 10 mmolπl 2 ), - 80 (NE) (CE) : Mib Mib + 24 h LNCaP 2, Clontech Lab TransFactor Extraction,, - 80 : Bradford (Bio2Rad, Hercules, CA, USA) SDS2PAGE : SDS2PAGE(4 % 12 %) (Bio2Rad) Western : analysis,, 5 % TBST (20 mmolπl Tris2HCl, 137 mmolπl NaCl, and 011 % Tween 20, ph 810) 1 h Sp1 (Santa Cruze Biotechnology Inc ) 2 2 Sp1 AR [3,4 ] 1 h, AR AR 1 h, ECL ( ECL, Amersham Corporation, Arlington Heights, IL) Fig 1 [13,Photo image ] mrna 1 5 AR AR (WE) IgG Sp1 GC box, GC box A2agarose beads ( Santa Cruz Biotechnology, Inc ) [14,15 AR ], 500 g 2 g 4 Sp1 AR (20 mmolπl HEPES, ph 719, 20 %, 150 Sp1 Fig 2, mmolπl KCl, 012 mmolπl EDTA, 015 mmolπl AR Sp1, 015 mmolπl PMSF, 50 gπml aprotinin, 1 EDTA) 4, Western 2 (DHT), Fig 1 Antiproliferative effect of quercetin on LNCaP cells by MTS Cells were incubated with the indicated agents for 4 days Viable cells were measured by MTS method and expressed in A490 nm (absorbance at 490 nm) 3 3 P < 0105, 3 P < 0101 when compared with control,sp1 mmolπl, mmolπl 2,Sp1 AR, ) A2agarose beads 4 2 h 2 3 Sp1 AR (50 mmolπl Tris2HCl, ph 715, 015 % IGEPAL CA2630, 150 mmolπl NaCl, 012 mmolπl (prostate2specific antigen, PSA), China Academic Journal Electronic Publishing House All rights reserved

4 Fig 2 Inhibition of Sp12mediated transactivation on the AR promoter by quercetin in LNCaP Cells LNCaP cells were co2transfected with pgl32ar promoter2luciferase reporter plasmid and increasing amounts of pcdna2sp1 expression plasmid as indicated The cells were treated with 1 nmolπl Mib or 1 nmolπl Mib molπl quercetin for additional 24 hours after transfection 2 4 Sp1, Sp1 AR Sp1, Sp1 AR AR? Western, Sp1, Fig 4A Sp1 Mib Sp1 ( 1 2,3), Sp1 ( 2 3) Sp1, Sp1 Fig 4B,Sp1 ( 1,2,3 4,5,6) Sp1, Fig 4A Sp1 (androgen response elements, AREs) AR,AR PSA [16,17 AREs PSA ] AR,Sp1? PSA AR PSA Sp1 Fig 3,Sp1 AR PSA, PSA, Sp1 AR, Fig 4 Western blot analysis of Sp1 expression level in LNCaP cells The Sp1 protein expression in whole cell lysates ( WE), cytosolic (CE) and nuclear extracts (NE) from LNCaP cells treated with or without the above chemical was analyzed by western blot Ponceau Fig 3 Function synergy of Sp1 on the AR transcription activity of androgen2response genes was repressed by quercetin LNCaP cells were cotransfected with p GL32PSA6kb promoter luciferase reporter, the pcdna2sp1 expression plasmid (1 g) and the pcmv2 2galatosidase The pgl3 basic vector was included as a control The cells were incubated with 1 nmolπl Mib or 1 nmolπl plus indicated amounts of quercetin for 24 hours after transfection P < 0101, 3 P < when compared with control S2stained protein bands were served to monitor protein loading and transferring efficiency 2 5 c2jun Sp1 c2jun [18 ] [19 21 ],Sp1 c2jun 2 Sp1 AR, China Academic Journal Electronic Publishing House All rights reserved

5 5 : Sp1 677 Sp1 AR, 3, Sp1 c2jun Sp1, AR? LNCaP AR PSA AR, PSA [13 ] Fig 5A,c2Jun PSA, AR,Sp1 c2jun [3,4 ], Sp1 Sp1 c2jun AR, c2jun Sp1 Fig 5B Sp1 AR, Mib,c2Jun Sp1, c2jun Sp1, Sp1 PSA Fig 5 Sp12mediated stimulation on the transcription activity of the AR was inhibited by c2jun (A) LNCaP cells were cotransfected with pgl32psa6kb luciferase reporter in the absence (2) or presence different amounts of expression vectors for pcdna2sp1 andπor c2jun in the indicated combinations The empty pgl3 basic vector was included as a control The normalized, relative luciferase activities (mean S E) of at least three independent experiments performed in duplicate were shown 3 3 P < 0105, P < 0101, 3 P < when compared with the control ; (B) Co2immunoprecipitation of Sp1 and c2jun c2jun Sp1 PSA AR [14,15 ],Sp1 AR, PSA Sp1 AR ( Sp1 DNA ) mrna AR AR, Sp1 Western, Sp1, Sp1 AR c2jun, c2jun AR [18,22 ] Fig 5 c2jun Sp1 2 Sp1,Sp1 AR Sp1Πc2Jun Sp1 Sp1Πc2J un, p21 WAF1ΠCip1 Sp1 c2jun, c2jun Sp1 Sp1 [19 ], EGF c2jun, c2jun Sp1 12 (S)2 [20 lipoxygenase ],Wang [21 ],Sp1Πc2Jun p21 WAF1ΠCip1,Sp1Πc2Jun 2,, c2jun c2j unπsp1 2 Sp1, AR, AR China Academic Journal Electronic Publishing House All rights reserved

6 AR Sp1 c2jun ( References) inhibits expression and function of the androgen receptor in LNCaP 1 Koivisto P Androgen receptor gene amplification, a possible molecular prostate cancer cells Carcinogenesis, 2001, 22 : mechanism for androgen deprivation therapy failure in prostate cancer 14 Faber P W, Rooij H C Van, Schipper H J, Brinkmann A O, Trapman Cancer Res, 1997, 57 (2) : J Two different, overlapping pathways of transcription initiation are 2 Culig Z, Hobisch A, Bartsch G, Klocker H Androgen receptor an active on the TATA2less human androgen receptor promoter The role of update of mechanism of action in prostate cancer Urol Res, 2000, 28 : Sp1 J Biol Chem, 1993, 268 : Faber, P W, van Rooij, H C, van der Korput, H A, Baarends, W M, 3 Eder I E, Hoffmann J, Rogatsch H, Schgfer G, Zopf D, Bartsch G, Brinkmann A O, Grootegoed J A, Trapman J Characterization of the Klocker H Inhibition of LNCaP prostate tumor growth in vivo by an human androgen receptor transcription unit J Biol Chem, 1991, 266, antisense oligonucleotide directed against the human androgen receptor Cancer Gene Ther,2002,9 : Brinkmann A O, Blok L J, de Ruiter P E, Doesburg P, Steketee K, 4 Zegarra2Moro O L,Schmidt L J,Huang H J,Tindall D J Disruption of Berrevoets C A, Trapman J Mechanisms of androgen receptor activation androgen receptor function inhibits proliferation of androgen2refractory and function J Steroid Biochem Mol Biol, 1999, 69 : prostate cancer cells Cancer Res,2002,62 : Baumann C T, Lim C S, Hager G L Intracellular localization and 5 DNA PSA ( Zhang Peng2Ju, Zhang Jian2Ye, Chen Wei2Wen,Jiang An2Li,Zhang Lian2Ying, Guo Qiang Inhibition of androgen responsive induced by quercetin and resveratrol inhibits the expression and function element decoy DNA on the promoter of prostate specific antigen Chin J of the androgen receptor in human prostate cancer cells Cancer Lett, Biochem Mol Biol),2003,19(5) : , 213 : Rodgers E H, Grant M H The effect of the falvonoids, quercetin, 19 Kardassis D, Papakosta P, Pardalis K, Moustakas A c2jun myricetin and epicatechin on the growth and enzyme activities of MCF7 transactivates the promoter of the human p21 WAF1ΠCip1 gene by acting as a human breast cancer cells Chem Biol Interact, 1998, 116(3) : 213 superactivator of the ubiquitous transcription factor Sp1 J Biol Chem, , 274 : Choi J A, Kim J Y, Lee J Y, Kang C M, Kwon H J, Yoo Y D, Kim 20 Chen B K, Chang W C Functional interaction between c2jun and T W, Lee Y S, Lee S J Induction of cell cycle arrest and apoptosis in promoter factor Sp1 in epidermal growth factor2induced gene expression human breast cancer cells by quercetin Inter J oncol, 2001, 19 : 837 of human 12 ( S)2lipoxygenase Proc Natl Acad Sci USA, 2000, 97 : Wei Y Q, Zhao X, Kariya Y, Fukata H, Teshigawara K, Uchida A 21 Wang C H, Tsao Y P, Chen H J, Wang H W, Chen S L Induction of apoptosis by quercetin : involvement of heat shock protein Transcriptional repression of p21 WAF1ΠCip1 gene by c2jun through Sp1 Cancer Res, 1994, 54 : site Biochem Biophys Res Commun, 2000, 270 : Csokay B, prajda N, Weber G, Olah E Molecular mechanisms in the 22 Sato N, Sadar M D, Bruchovsky N, Saatcioglu F, Rennie P S, Sato S, antiproliferative action of quercetin Life Sci, 1997, 60 : Lange P H, Gleave M E Androgenic induction of prostate2specific 10 C (Liu Wen, Song Zhi2Juan,Liang Nian2Ci, She Ji,Mo Li2Er Effects of sodium quercetin sulfate on cytosolic calcium concentrion and protein kinase C in pig platelets Chin J Biochem Mol Biol),1999,15 (2) : Miodini P, Fioravanti L, Di Fronzo G, Cappelletti V The two phyto2 oestrogens genistein and quercetin exert different effects on oestrogen receptor function Br J Cancer, 1999, 80 : Jiang S W, Eberhardt N L A micro2scale method to isolate DNA2 binding proteins suitable for quantitative comparison of expression in levels from transfection cells 3608 Nucleic Acids Res, 1995, 23 : Xing N Z, Chen Y, Mitchell S H, Young Charles Y F Quercetin trafficking of steroid receptors Cell Biochem Biophys, 1999, 31 : Yuan H Q, Pan Y Q, Young Charles Y F Overexpression of c2jun antigen gene is repressed by protein2protein interaction between the androgen receptor and AP21Πc2Jun in the human prostate cancer cell line LNCaP J Biol Chem, 1997, 272 : China Academic Journal Electronic Publishing House All rights reserved