Activation of insulin-like growth factor I receptor-mediated pathway by ginsenoside Rg1

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1 British Journl of Phrmology (6) 147, & 6 Nture Pulishing Group All rights reserved /6 $3. Ativtion of insulin-like growth ftor I reeptor-medited pthwy y ginsenoside 1, Wen-Fng Chen, 1,3 Wi-Sum Lu, 1 Pik-Yuen Cheung, 4 De-An Guo &,1,3 Mn-Su Wong 1 Centrl Lortory of the Institute of Moleulr Tehnology for Drug Disovery nd Synthesis, Deprtment of Applied Biology nd Chemil Tehnology, The ong Kong Polytehni University, ung om, Kowloon, ong Kong SAR, People s Repuli of Chin; Deprtment of Physiology, Medil College of Qingdo University, Qingdo 661, People s Repuli of Chin; 3 Stte Key Lortory of Chinese Mediine nd Moleulr Phrmology, Shenzhen 51857, People s Repuli of Chin nd 4 Shnghi Reserh Center for TCM Moderniztion, Shnghi Institute of Mteri Medi, Shnghi Institutes for Biologil Sienes, Chinese Ademy of Sienes, Shnghi 13, People s Repuli of Chin Keywords: Arevitions: 1 Ginsenoside, n tive ingredient in ginseng, ws previously shown to e novel lss of potent phytoestrogen. The present study ims t investigting the moleulr mehnisms involved in mediting its tions in humn rest ner (MCF-7) ells. (1 pm) stimultes ell prolifertion (Po.1) nd estrogen-responsive ps mrna expression (Po.5) without ltertion of estrogen reeptor lph (ER) protein or mrna expression in MCF-7 ells. In ddition, M of does not demonstrte speifi inding to ER. 3 We hypothesize tht my exert its tions in MCF-7 ell vi the tivtion of rosstlk etween ER- nd insulin growth ftor I reeptor ()-dependent pthwys. 4 The results indite tht signifintly inreses expression nd promoter tivity in MCF-7 ells (Po.5). Cotretment of MCF-7 ells with 1 mm of estrogen ntgonist ICI 18,78 ompletely olishes the effets of on expression. 5 Furthermore, enhnes tyrosine phosphoryltion of IRS-1 in MCF-7 ells upon IGF-I stimultion nd the tivtion of IRS-1 phosphoryltion is lso ER-dependent. 6 Tken together, our results suggest tht not only inreses expression ut lso enhnes -medited signling pthwys in MCF-7 ells. The stimultion of expression y in MCF-7 ells ppers to require ER, nd its tions might involve lignd-independent tivtion of ER. British Journl of Phrmology (6) 147, doi:1.138/sj.jp.7664; pulished online 16 Jnury 6 Ginsenoside ; estrogen; insulin-like growth ftor I reeptor; humn rest ner; phytoestrogen CX, yloheximide; DMEM, Duleo s modified Egle s medium;,17-estrdiol; ER, estrogen reeptor; FBS, fetl ovine serum;, glyerldehyde-3-phosphte dehydrogense; IGF, insulin-like growth ftor;, insulin-like growth ftor I reeptor; IRS-1, insulin reeptor sustrte 1; PBS, phosphte-uffered sline; RT PCR, reverse trnsriptse polymerse hin retion; Sh, Sr homology/ollgen; sfbs, hrol-stripped fetl ovine serum Introdution Ginsenosides, the prinipl tive omponents of ginseng (Liu & Xio, 199), hve een shown to possess vriety of enefiil effets on humn helth, inluding nti-inflmmtory, ntioxidnt nd ntiner effets (Mohizuki et l., 1995; Kim et l., 1998; Wkyshi et l., 1998). There re two mjor lsses of ginsenosides, nmely protopnxtriol (, Rg, Re, Rf, nd Rh1) nd protopnxdiol (R1, R, R, Rd, Rg3, Rh, nd Rh3). Numerous studies hve demonstrted tht ginseng extrts (Punnonen & Lukol, 198; Dud et l., 1996; Liu et l., 1; Amto et l., ) nd its onstituents (Cui et l., 1; Chn et l., ; Lee et l., 3, ) possess estrogen-like tivities in oth in vitro Author for orrespondene t: Centrl Lortory of the Institute of Moleulr Tehnology for Drug Disovery nd Synthesis, Deprtment of Applied Biology nd Chemil Tehnology, The ong Kong Polytehni University, ung om, Kowloon, ong Kong SAR, People s Repuli of Chin; E-mil: [email protected] nd in vivo models. Ginseng extrts n stimulte the growth of estrogen reeptor (ER)-positive ells nd the indution of estrogen-dependent ps gene expression (Dud et l., 1996; Liu et l., 1; Amto et l., ). Our previous study demonstrted tht ginsenoside, steroidl sponin of high undne in ginseng, ould medite its tion vi the ER in humn rest ner ells (Chn et l., ). Susequent studies y others hve reported tht ginsenoside R1 (Lee et l., 3; Cho et l., 4) nd Rh1 (Lee et l., 3) ould tivte ER in humn rest ner ells, wheres ginsenoside R nd Re n stimulte MCF-7 ell growth s well s indue -Fos expression independent of ER tivtion (Lee et l., 3). Among these ginsenosides, ppers to e of the highest poteny s its EC5 for tivtion of humn rest ner ell prolifertion is.5 pm (Chn et l., ), while the reported tivities of R1 nd Rh1 re in miro-molr rnge. Similrly, it is y fr the most potent phytoestrogens eing reported s most of

2 W.-F. Chen et l enhnes IGF-I reeptor-medited pthwys 543 the other nturlly ourring ompounds (nonsteriodl gents like flvonoids, oumestn derivtives nd lignns; polyphenoli ompounds) demonstrted wek estrogeni tivity t miro-molr rnge (Murkies et l., 1998; Thm et l., 1998; Anter et l., 5; Klinge et l., 5). Most importntly, the inding ffinities of different ginsenosides for ER re found to e different. Both (Chn et l., ) nd R1 (Cho et l., 4) did not show speifi inding to ER, while Rh1 ould disple 3 -leled 17-estrdiol ( ) inding to ER (Cho et l., 4). The demonstrtion of estrogeni tivities of ginsenosides in the sene of diret intertion with ER suggests tht ginsenosides my tivte ER vi ligndindependent pthwy. Reent studies indited tht ER n e tivted in lignd-independent mnner y vriety of stimuli, inluding insulin-like growth ftor I (IGF-I) (Lee et l., 1997, ), epiderml growth ftor (EGF) (Kto et l., 1995), serum (Krs et l., 1998), nd, reently, leptin (Ctlno et l., 4). Moreover, rosstlk etween ER nd growth ftor hs een oserved in ER-positive rest ner ells (Kto et l., ). n indue the expression of severl memers of the IGF fmily in humn rest ner ells, inluding IGF-I reeptor (), IGF-II reeptor (IGF-IIR), IGF inding proteins (IGFBPs), insulin reeptor sustrte 1 (IRS-1), nd insulin reeptor sustrte (IRS-). The upregultion of these proteins y provides the potentil mehnisms for its utorine nd prrine ontrol of rest ner ell mitogenesis (Stewrt et l., 199; Lee et l., 1999). Reiprolly, IGF-I ws found to modulte tion y ting vi the tyrosine kinse of. Lignds for oth the EGF reeptor nd n regulte ER gene expression nd ER-medited trnsription in n estrogen-independent mnner (Kto et l., 1995; Lee et l., 1997, ). In the present study, we systemtilly evluted the moleulr tions of ginsenoside in ER-positive rest ner (MCF-7) ell line. We hypothesize tht might exert its tion vi the rosstlk etween ER- nd dependent pthwys. We hve studied the effet of on expression nd promoter tivity, s well s on tyrosine phosphoryltion of IRS-1, downstrem signling moleule in -medited pthwys, in MCF-7 ells. The results indite tht IGF-I signling pthwy is indeed involved in mediting the tion of in humn rest ner ells, nd provide new evidene to support our hypothesis for the tivtion of ER nd rosstlk y in MCF-7 ells. Methods Purifition of ginsenoside from Pnx notoginseng The method of ginsenoside purifition used ws s desried previously (Chn et l., ). Briefly, the powder of roots of P. notoginseng ws extrted using 7% ethnol three times. The extrt ws evported to dryness t less thn 51C under redued pressure, nd the residue ws dissolved in wter nd extrted y queous utnol three times. The utnol extrt ws evported to dryness t less thn 51C under redued pressure nd ws then sujeted to hromtogrphy on D11 resin, whih ws eluted y using 1% grdient of wter ethnol mixture. After removl of solvent, the 5% Figure 1 C O O O O O O O C 3 C 3 O 3 C C 3 C O O O O O O ethnol extrt ws sujeted to hromtogrphy on sili gel. ws eluted from the olumn using CCl 3 MeO O (5 : 1 : 1). The purity of ws determined y PLC nd ws found to e more thn 99% pure. The struture of is shown in Figure 1. Culture of humn rest ner ell line (MCF-7) nd humn emryoni kidney ell line (EK93) MCF-7 ells (ATCC No. TB-) nd EK93 ells (ATCC No. CRL-1573, humn emryoni kidney ell line) were routinely ultured in Duleo s modified Egle s medium (DMEM) supplemented with 5% fetl ovine serum (FBS), peniillin 1 IU ml 1, nd streptomyin 1 mgml 1 (Invitrogen, Crlsd, CA, U.S.A.) t 371C in humidified tmosphere of 95% ir nd 5% CO. Cells were trnsferred to phenol-red free DMEM supplemented with 1% hrolstripped fetl ovine serum (sfbs), peniillin 1 IU ml 1, nd streptomyin 1 mgml 1 y stndrd methods of trypsiniztion, plted in six-well dishes for 5 dys nd llowed to replite to 8% onfluene. Cells were then treted either with (1 pm) or (1 nm) (Sigm, St Louis, MO, U.S.A.) for 6, 4, 48, nd 7 h. For dose response tretment, MCF-7 ells were exposed to.1 pm 1 mm of for 48 h. The medium nd test ompounds were replenished t 4 h. For ntiestrogen or yloheximide (CX) tretment, MCF-7 ells were exposed to or in the presene or sene of estrogen ntgonist ICI 18,78 (1 mm) (Toris, Bristol, U.K.) or CX (5 mgml 1 ) (Sigm, St Louis, MO, U.S.A.) for 48 h. For immunopreipittion experiment, ells were then stimulted with either IGF-1 (5 min) or (48 h) seprtely, or sequentilly ( for 48 h followed y IGF-1 for 5 min). The ell lystes were used for immunopreipittion. Cell prolifertive ssys C 3 C C C C C C 3 C 3 Chemil struture of ginsenoside. C 3 For growth study, MCF-7 ells were seeded in 96-well pltes (3 1 3 ells per well) in phenol-red free DMEM supplemented with 1% sfbs for 4 dys nd then treted with vrious onentrtions (1 6,1 8,1 1,1 1, nd 1 14 M) of or for 48 h. As n indiret mesure of growth, the 3-(4,5- dimethylthizol--yl),5-diphenyltetrzolium romide (MTT) British Journl of Phrmology vol 147 (5)

3 544 W.-F. Chen et l enhnes IGF-I reeptor-medited pthwys ssy ws used s desried previously (Denizot & Lnd, 1986). Briefly, the medium ws removed nd repled with 1 ml of tetrzolium (MTT, 5 mg ml 1, Sigm, St Louis, MO, U.S.A.) in PBS. The pltes were inuted for 4 h t 371C nd followed y ddition of 1 ml lysis uffer (.4 N Cl in propn--ol). The multiwell pltes re shken for 1 h nd red on miroplte reder t wvelength of 595 nm. ER ompetition ssy The ER ompetition ssy ws performed using the humn reominnt Estrogen Reeptor Competitor Assy Beon Kit ording to the mnufturer s protool (Invitrogen, Crlsd, CA, U.S.A.). In this ssy kit, n ER/fluoresent estrogen lignd (ES ) omplex with high polriztion vlue is dded to the test ompound. If the ompound ompetes with estrogen for ER inding, it will disple ES from the ER/ES omplex nd use redution in the polriztion vlue. Non- ER-inding ompetitors will not disple ES from the ER/ES omplex, nd the polriztion vlue remins high. The shift in polriztion in the presene of test ompound is used to determine the reltive ffinity of the ompound for ER tht ws lulted from the resulting urve using Prism 3.3 softwre (GrphPd Softwre, Sn Diego, CA, U.S.A.). Reverse trnsriptse polymerse hin retion (RT PCR) for ps, ER nd expression Totl RNA ws isolted from ells y using Trizol regent ording to the stndrd protool. Totl RNA ( mg) ws used to generte DNA in eh smple using SuperSript II reverse trnsriptse with oligo(dt) 1 18 primers (Invitrogen, Crlsd, CA, U.S.A.). The evlution of ps, ER, nd IGF- IR expression ws performed y semiquntittive RT PCR s desried previously (Chen & Wong, 4). For ps, ER,, nd the housekeeping gene glyerldehyde-3-phosphte dehydrogense (), the primers used were 5 - ATGGCCACCATGGAGAACAAGG-3 (ps forwrd) nd 5 -CATAAATTCACACTCCTCTTCTGG-3 (ps reverse), 5 -AAGTTCAGGCACAATTGGATG-3 (ER forwrd) nd 5 -CCCTGCATGACACTGATTACA-3 (ER reverse), 5 - ACTATGCCGGTGTCTGTGTG-3 ( forwrd) nd 5 -TGCAAGTTCTGGTTGTCGAG-3 ( reverse), nd 5 -ACCACAGTCCATGCCTACAC-3 (forwrd) nd 5 -TTCACCACCCTGTTGCTGTA-3 (reverse) to yield produts of 5, 5, 5, nd 4 p, with 3, 3, 3, nd PCR yles, respetively. PCR mplifition ws performed on GeneAmp 96 PCR system (Perkin-Elmer, Foster City, CA, U.S.A.). The PCR produts were nlyzed using grose gel eletrophoresis. Optil densities of ethidium romide-stined DNA nds were quntified using Lumi- Imger (Rohe Mnnheim, Germny) nd the mrna expression levels were normlized to the expression of. promoter luiferse ssy The effet of on promoter tivity ws studied y trnsient trnsfetion ssys using genomi DNA frgment extending from nuleotides 35 to þ 64 (nuleotide orresponds to the trnsription site of rt gene) or promoterless luiferse onstrut (polu) tht ws kindly provided y Dr Derek LeRoith (Dietes Brnh, Bethesd, MD, U.S.A.). MCF-7 ells were seeded in 1-well pltes for 1 dy in DMEM medium supplement with 5% FBS; ells were then trnsferred to phenol-red free DMEM supplement with 1% sfbs for nother dys. MCF-7 ells were trnsfeted with.8 mg of reporter plsmid long with.4 mg of the ontrol reporter plsmids prl-tk using the Lipofetmine regent ording to the mnufturer s instrutions (Invitrogen, Crlsd, CA, U.S.A.). At 5 h fter trnsfetion, ells were treted with or for nother 4 h nd then hrvested. Luiferse tivity enoded y experimentl nd internl ontrol plsmid ws mesured sequentilly using TD- / Luminometer (Turner Design, Sunnyvle, CA, U.S.A.) nd the DLR ssy regents ording to the supplier s reommendtion (Promeg, Mdison, WI, U.S.A.). The IGF- IR promoter tivity ws expressed s firefly luiferse vlues normlized y prl-tk renill luiferse vlues. Immunolotting nd immunopreipittion For Western lotting, protein ws isolted from ells using Trizol regent ording to the stndrd protool. Protein onentrtions were nlyzed y the method of Brdford (Bio- Rd, erules, CA, U.S.A.) (Brdford, 1976). Equl mount of proteins (5 mg) were seprted y SDS PAGE on 1% reduing gels t onstnt voltge (15 V) for 1 h s desried previously (Sriussdporn et l., 1995), nd trnslotted onto PVDF memrnes (Immoilin-P, Millipore Corp., MA, U.S.A.). Immunodetetion ws performed fter loking nonspeifi inding sites on the memrne with 5% skimmed milk. The lots were proed with polylonl rit ntihumn, IRS-1 (1 : ; Snt Cruz Biotehnology, In., Snt Cruz, CA, U.S.A.) or ER (1 : 3; Sigm, St Louis, MO, U.S.A.) s the primry ntiody, whih ws followed y inution with got nti rit ntiody onjugted with horserdish peroxidse (1 : ; Snt Cruz Biotehnology, In., Snt Cruz, CA, U.S.A.) s the seondry ntiody for 1 h. The ntigen ntiody omplexes were then deteted with enhned hemiluminesene (ECL) regent (Mttson & Bellehumeur, 1996) nd visulized y the Lumi- Imger using Lumi Anlyst version 3.1 softwre (Rohe, Mnnheim, Germny). For immunopreipittion, ells were lysed with Nonidet P-4 uffer ( mm Tris-Cl, p 7.5, 15 mm NCl, 1 mm CCl, 1mM MgCl, 1% glyerol, 1% Nonidet P-4) ontining protese inhiitors (protinin mgml 1, leupeptin mgml 1, 1mM PMSF) nd phosphtse inhiitors (1 mm sodium orthovndte, 1 mm NF). Lystes were entrifuged t 14, g for 3 min t 41C nd superntnt protein onentrtions were nlyzed y the method of Brdford. Phosphoryltion of IRS-1 ws determined s follows: 5 mgof protein lyste ws preipitted with 6.5 mg of the orresponding ntiodies t 41C on roker pltform for h, followed y the ddition of 1 ml protein A Sephrose slurry, nd inuted for h t 41C. After three sequentil wshes using Nonidet P-4 uffer, the resulted pellets were resuspended in eletrophoresis smple uffer nd oiled for 5 min nd susequently deteted y immunodetetion with ntiphosphotyrosine monolonl ntiody (P-Tyr-1, 1 : 1; Cell Signling Tehnology, ithin, erts, U.K.) s the primry ntiody nd got nti-mouse-onjugted horserdish peroxidse s the seondry ntiody (1 : 1). The smple ws British Journl of Phrmology vol 147 (5)

4 W.-F. Chen et l enhnes IGF-I reeptor-medited pthwys 545 then deteted y ECL kit (Piere, IL, U.S.A.), nd nlyzed y Lumi-Anlyst 3.1 softwre (Rohe, Mnnheim, Germny). Sttistil nlysis Dt re reported s the men7s.e.m. Signifine of differene etween group mens ws determined y one-wy nlysis of vrine (ANOVA). The independent Student s t test ws used to lulte sttistil signifine etween the ontrol group nd eh tretment group in MTT ssy nd tyrosine Phosphoryltion of IRS-1. A P-vlue of o.5 ws onsidered sttistilly signifint. Asorne, reltive to ontrol C Conentrtion (M) Results Ginsenoside stimultes ell prolifertion nd ps mrna expression in humn rest ner (MCF-7) ells h 6h 4h 48h 7h PS As shown in Figure, inresed ell prolifertion of MCF-7 ells in dose-dependent mnner. The stimultion of ell prolifertion y different onentrtions of ppered to e iphsi. The mximl stimultion of MCF-7 t low onentrtions of (from 1 14 to 1 8 M) ourred t 1 pm, nd 1 mm of ppered to inrese MCF-7 ell prolifertion to n extent higher thn tht hieved y 1 pm of. Tretment of MCF-7 ells with 1 pm nd 1 mm of for 48 h resulted in nd 5-fold inrese in ell numer, respetively (Po.5 vs vehile-treted ells). lso inresed ell prolifertion of MCF-7 ells in dose-dependent mnner; the ell numer inresed s the onentrtion of inresed. To determine if mimis nd indues ER-dependent gene trnsription, the ility of to indue estrogenregulted gene ps ws determined. The ps gene ws originlly identified s n estrogen-induile trnsript in MCF-7 ells nd enoded for seretry protein from MCF-7 ells (Msikowski et l., 198). ps-gene expression is frequently used s mrker for ssessing the estrogeniity of vrious ompounds (Soto et l., 1997). Figure lerly shows tht 1 pm inresed ps gene expression in time-dependent fshion. The signifint inrese ws found t 7 h tretment. In the se of (1 nm), the indution of ps gene expression ourred s erly s 4 h nd throughout the 7 h of tretment. These results indite tht indeed ehves s potent phytoestrogen t onentrtion s low s 1 pm nd its tivtion pttern in MCF-7 ells is not ompletely similr to estrogen. Role of ER in mediting the tion of ginsenoside The inding of or to humn reominnt ER ws determined y ER ompetitive inding ssy. In the se of, speifi inding of ES with reominnt ER ws displed y inresing onentrtion of unleled nd the polriztion vlue deresed in dose-dependent mnner (Figure 3). In ontrst, (.1 pm 1 mm) filed to disple the speifi inding of ES from its reeptor (Figure 3), onfirming our previous finding (Chn et l., ) tht no diret intertion exists etween ER nd t onentrtion s high s 1 mm. ER is previously demonstrted to e n essentil meditor of tion; we therefore determined if exerts -like tivity y regulting the level of ER expression in MCF-7 ps/, % of ontrol PS ells. As ER, ut not ER, is the mjor isoform expressed in MCF-7 ell line, its expressions in response to were eing studied in the present study. As shown in Figure 4, the dose response effets of on ER protein nd mrna expressions were determined. Our results showed tht ( M) did not signifintly lter ER protein expression, lthough n upwrd trend of stimultion ws oserved when treted with M of (Figure 4 nd ). Similrly, the expressions of ER mrna expression in MCF-7 ells were not ltered in response to tretment with M of (Figure 4 nd d). The time-ourse responses of ER expression to tretment with nd in MCF-7 ells were lso determined. As shown in Figure 4e nd f, 1 nm of signifintly redued ER protein nd mrna expressions in MCF-7 ells throughout the durtion of tretment (Po.5). In ontrst, did not h 6h 4h 48h 7h Figure Ginsenoside stimultion of ell prolifertion nd ps mrna expression in humn rest ner (MCF-7) ells. () MCF- 7 ells were treted with inresing doses of nd 17- estrdiol ( ) for 48 h. Cell numer ws then determined y MTT ssy. The result is representtive of three independent experiments expressed s men7s.e.m. Po.5 vs ontrol (), n ¼ 6. () MCF-7 ells were treted with either 1 pm or 1 nm with inresing time (6, 4, 48 nd 7 h). Totl RNA ws isolted nd ps nd mrna expression were sujeted to semiquntittive RT PCR nlysis. The mrna expression level ws expressed s rtio to the expression of. Grphi results shown re representtive of three independent experiments nd expressed s men7s.e.m. Po.5 vs ontrol, n ¼ 3. British Journl of Phrmology vol 147 (5)

5 546 W.-F. Chen et l enhnes IGF-I reeptor-medited pthwys Polriztion (mp) Competitor onentrtion (nm) Figure 3 ER ompetition ssy. Seril dilutions of or mixed with 5 ml of omplex were inuted in the drk t room temperture for h nd the polriztion vlues were determined. A smple ontining 5 ml ofes sreening uffer nd 5 ml of omplex ws used s negtive ontrol, whih represents % ompetition. Results were otined from three independent experiments nd expressed s men7s.e.m. suppress oth ER protein nd mrna expressions in MCF-7 ells, nd it, though insignifintly, ppered to rise ER protein expression in time-dependent mnner. Thus, it ppers tht ehves differently from nd does not suppress the expression of ER in MCF-7 ells. Ginsenoside stimultes insulin-like growth ftor I reeptor () expression in MCF-7 ells The ft tht exerts its -like tivity without diret intertion with ER suggests tht other signling pthwys might e involved in mediting its tions. A reent report (Kto et l., ) indited tht rosstlk exists etween ER nd signling in humn rest ner ells. We, therefore, studied if ould lter protein expressions in MCF-7 ells in response to for time ourse rnging from 6 to 7 h. As shown in Figure 5 nd, the expression of protein inresed signifintly in timedependent mnner nd rehed 1.8-fold y 7 h in response to tretment with 1 pm (Po.5). Suh hnge mimiked the effet of 1 nm on protein expression in MCF-7 ells (Figure 5 nd ). We then determined if would lso mimi the effets of on mrna expression. As shown in Figure 5 nd d, oth (1 pm) nd (1 nm) indued mrna expression of mrna in MCF-7 ells in time-dependent mnner, nd rehed 1.8- nd.1-fold, respetively, y 48 h of tretment. Thus, our results suggest tht the -like tions of might e medited through the rosstlk etween ER nd -dependent signling pthwys vi the upregultion of expression in MCF-7 ells. Mehnisms involved in ginsenoside indution of expression in MCF-7 ells Our previous study (Chn et l., ) reported tht the prolifertive tions of in MCF-7 ells were ERdependent. To determine if the oserved effets of on expression re lso ER-dependent, ICI 18,78, n estrogen ntgonist tht olishes the tivtion funtions (oth AF-1 domin nd AF- domin) of ER, ws used. Coinution of MCF-7 ells with ICI 18,78 (1 mm) ompletely prevented the upregultion of protein (Figure 6 nd ) nd mrna (Figure 6 nd d) expression y (1 pm) nd (1 nm). These results suggest tht the indution of expression y requires the funtionl tivity of ER. To further investigte the potentil mehnism involved in upregultion of expression y, MCF-7 ells treted with either or were oinuted with or without CX (5 mgml 1 ), de novo protein synthesis inhiitor. The expressions of indued y nd were ompletely loked y CX t oth protein (Figure 7 nd ) nd mrna level (Figure 7 nd d), inditing tht de novo protein synthesis is indeed required for the tion of nd on regultion. Effet of ginsenoside on promoter tivity To determine if the inrese in expression y ginsenoside ws medited y the upregultion of promoter tivities, trnsient trnsfetion ssy ws performed. MCF-7 ells were trnsiently trnsfeted with IGF- IR luiferse promoter p(35/ þ 64)Lu or promoterless luiferse onstrut (polu) for 5 h, followed y tretment with (1 pm) or (1 nm) for nother 4 h. As shown in Figure 8, nd signifintly inresed the promoter luiferse tivity nd their indution ould e olished y otretment with ntiestrogen ICI 18,78, while no luiferse tivity ws determined in the ells whih were trnsfeted with promoterless luiferse onstrut. These results suggest tht nd ould upregulte gene expression y their stimultory effets on promoter tivities, nd these effets re lso ER dependent. Ginsenoside potentites the effet of IGF-I on signling sde in MCF-7 ells To investigte if n lter signling sde, we ssessed the responses of tyrosine phosphoryltion of IRS-1 in MCF-7 ells to IGF-I (5 ng ml 1 ) in the presene or sene of 1 pm of. MCF-7 ells were stimulted with either IGF-1 (5 min) or (48 h) seprtely, or sequentilly ( for 48 h followed y IGF-1 for 5 min). Protein expression level s well s tyrosine phosphoryltion of IRS-1 were then determined (Figure 9). The results indite tht the sl tyrosine phosphoryltion levels of IRS-1 were low nd undetetle in MCF-7 ells (Figure 9, lne 1). lone did not use n indution of tyrosine phosphoryltion of IRS-1 (Figure 9, lne ). Tretment with IGF-I for 5 min lone resulted in n inrese in tyrosine phosphoryltion level of IRS-1 (Figure 9, lne 3). Most importntly, IGF-I stimultion of MCF-7 ells pretreted with resulted in enhned tyrosine phosphoryltion of the IRS-1 (Figure 9, lne 4). Figure 9 indites tht the oserved effets on tyrosine phosphoryltion of IRS-1 were not due to unequl loding of proteins for immunopreipittion s well s Western lotting. These results indite tht not only stimultes expression ut lso enhnes tyrosine phosphoryltion of downstrem signling proteins, suh s IRS-1, in MCF-7 ells. In order to investigte if the -medited enhnement of IRS-1 phosphoryltion y IGF-I is ER-dependent, n British Journl of Phrmology vol 147 (5)

6 W.-F. Chen et l enhnes IGF-I reeptor-medited pthwys 547 C R14 R1 R1 R8 R6 ERα C R14 R1 R1 R8 R6 ERα β-tin ER/et tin, % of ontrol d ER/, % of ontrol C R14 R1 R1 R8 R6 C R14 R1 R1 R8 R e h 6h 4h 48h 7h ERα f h 6h 4h 48h 7h ER β-tin ERα E ER β-tin g ER/et tin, % of ontrol h ER/, % of ontrol h 6h 4h 48h 7h h 6h 4h 48h 7h Figure 4 Effet of on the ER protein nd ER mrna expression. MCF-7 ells were treted with.1 pm (R14), 1 pm (R1), 1 pm (R1), 1 nm (R8), 1 mm (R6) for 48 h (, ) nd treted with 1 nm nd 1 pm (R1) with inresing time (6, 4, 48 nd 7 h) (e, f). Protein nd totl RNA were isolted nd sujeted to Western lotting nd RT PCR nlysis. () The dose response of on ER nd -tin protein expression. () The dose response of on ER nd mrna expression. (, d) The grphi representtion of the ER protein nd mrna expression levels, whih were expressed s rtio to the expression of -tin nd, respetively. (e) ER protein nd mrna expression treted with nd. (f) Semiquntittive RT PCR nlysis of ER nd mrna expression treted with nd. (g, h) Grphi representtion of the ER protein nd mrna expression levels, whih were expressed s rtio to the expression of -tin nd, respetively. Results were otined from three independent experiments nd expressed s men7s.e.m. Po.5 vs ontrol, n ¼ 3. ER-negtive humn emryoni kidney (EK93) ell ws used insted of MCF-7 ells. As shown in Figure 9, tretment with IGF-I for 5 min lone resulted in n inrese in tyrosine phosphoryltion level of IRS-1 (Figure 9, lne 7). owever, pretretment of EK93 ells with did not enhne IGF- I-indued tyrosine phosphoryltion of the IRS-1 (Figure 9, lne 8). Thus, the result strongly suggests tht the enhnement of IGF-I-indued phosphoryltion of IRS-1 y requires the presene of ER. Disussion Ginsenoside is steroid sponin tht shres similr struturl fetures nd trget orgns with steroid hormone, inluding the rin nd rdiovsulr system (Attele et l., 1999). Ginsenosides re mphiphili in nture nd hve the ility to interlte into the plsm memrne (Bnthorpe, 1994). There is evidene suggesting tht ginsenosides intert diretly with speifi memrne proteins (Kimur et l., 1994; Brnn et l., 1995). Moreover, like steroid hormones, hs previously een shown to intert with gluoortioid reeptor nd initite genomi effets (Lee et l., 1997, ). Our previous study hs shown tht hs estrogen-like properties nd exerts its tion vi the tivtion of, ut without diret inding to, ER in humn rest ner MCF-7 ells (Chn et l., ). The present study further demonstrtes tht in MCF-7 ells ginsenoside : (1) mimis the effets of nd indues ell prolifertion, ER-dependent trnsription of ps, protein, nd mrna expression, s well s British Journl of Phrmology vol 147 (5)

7 548 W.-F. Chen et l enhnes IGF-I reeptor-medited pthwys h 6h 4h 48h 7h h 6h 4h 48h 7h β-tin β-tin /et tin, % of ontorl d 3 E h 6h 4h 48h 7h h 6h 4h 48h 7h Figure 5 Western lotting nd RT PCR nlysis of the expression of in MCF-7 ells treted with. MCF-7 ells were ultured nd treted with 1 pm or 1 nm for 6, 4, 48 nd 7 h. () nd -tin protein expression. () Semiquntittive RT PCR nlysis of nd mrna expression. (, d) Grphil representtion of protein nd mrna expression, respetively. Results were otined from three independent experiments nd expressed s men7s.e.m. Po.5 nd Po.1 vs ontrol, n ¼ 3. /, % of ontorl β-tin ICI ICI /et tin, % of ontorl d C R R+I E E+I ICI C R R+I E E+I ICI /, % of ontorl Figure 6 Effet of ICI 18,78 on the regultion of expression y or. MCF-7 ells were ultured nd treted with 1pM (R) or 1 nm (E) in the presene nd sene of 1 mm ICI 18,78 (ICI) for 48 h. () nd -tin protein expression. () Semiquntittive RT PCR nlysis of nd mrna expression. (, d) Grphil representtion of protein nd mrna expression, respetively. Results shown were otined from three independent experiments. Columns with different letters re signifintly different from one other (Po.5, n ¼ 3). promoter tivity; () does not signifintly lter ER expression; nd (3) enhnes IGF-I-indued tyrosine phosphoryltion of IRS-1, ut not. It is pprent tht ER is essentil for mediting the tions of in MCF-7 ells. In our previous study, stimultion of MCF-7 ell prolifertion nd estrogen response element (ERE)-dependent tivtion of luiferse reporter gene tivity ould e ompletely olished y otretment with pure ER ntgonist, ICI 18,78 (Chn et l., ). In the present study, our results lerly demonstrte tht upregultion of expression nd indution of promoter tivity y ould lso e ompletely loked y otretment with ICI 18,78. In ddition, our results provide further support tht ehves s phytoestrogen nd stimultes estrogenregulted ps gene expression in time-dependent mnner. owever, unlike nd other phytoestrogen suh s genistein British Journl of Phrmology vol 147 (5)

8 W.-F. Chen et l enhnes IGF-I reeptor-medited pthwys 549 IGF- IR CX CX /et tin, % of ontorl d C R R+C E E+C CX C R R+C E E+C CX /, % of ontorl Figure 7 Effet of CX on the regultion of expression y or. MCF-7 ells were ultured nd treted with 1 pm (R) or 1 nm (E) in the presene nd sene of 1 mm CX for 48 h. () nd -tin protein expression. () Semiquntittive RT PCR nlysis of nd mrna expression. (, d) Grphil representtion of protein nd mrna expression, respetively. Results shown were otined from three independent experiments. Columns with different letters re signifintly different from one other (Po.5, n ¼ 3). promoter tivity, % of ontrol C E E+I R R+I ICI Figure 8 promoter luiferse ssy. Ativities of luiferses enoded y experimentl nd internl ontrol plsmid were mesured sequentilly with the DLR ssy regents. The promoter firefly luiferse tivities were normlized with prl-tk renill luiferse vlues. The promter luiferse tivity of ontrol ws defined s 1%. Results were otined from three independent experiments nd expressed s men7s.e.m. Columns with different letters re signifintly different from one other (Po.5, n ¼ 3). IP: IRS-1A WB: IRS-1A A MC F-7 EK IRS-1 PY B IGF Figure 9 Effets of on tyrosine phosphoryltion of IRS-1 in MCF-7 nd EK93 ells. MCF-7 nd EK93 ells were stimulted with IGF-1 (5 ng ml 1 ) for 5 min, (1 pm) for 48 h, or with oth gents. Cell lystes were immunopreipitted with nti- IRS-1 (A, B) ntiodies. After SDS PAGE, lots were inuted with nti-irs-1 (A) or ntiphosphotyrosine of IRS-1 (B). IP stnds for immunopreipittion; WB stnds for Western lotting nd PY stnds for tyrosine phosphoryltion. Results shown were otined from three independent experiments; n ¼ 3. (Chen & Wong, 4), did not ind to ER nor suppress its expression level in MCF-7 ells. Thus, the inrese in -like tivity y in MCF-7 ells ppers to tivte ER through distint moleulr mehnisms. The present study lerly demonstrtes the involvement of the rosstlk etween ER--dependent signling pthwys in mediting the tions of in MCF-7 ells. n stimulte expression in MCF-7 ells in mnner similr to. Suh mehnism ws originlly proposed to serve s mens for to exert utorine nd prrine ontrol of rest ner ells mitogenesis (Stewrt et l., 199; Lee et l., 1997, ; Kto et l., ). In ddition, n enhne IGF-I-indued tyrosine phosphoryltion of IRS-1 in MCF-7 ells, suggesting tht n enhne medited signling pthwys in this ell type. Our results using ER-negtive EK93 ells showed tht filed to inrese tyrosine phosphoryltion of IRS-1 in the sene of ER, suggesting tht ER is required for enhnement of IGF- IR-medited signling pthwys. Thus, our results suggest tht the moleulr mehnism of in MCF-7 ells involves: (1) the ER-dependent enhnement of signling pthwy; () the ER-dependent inrese in protein nd mrna expression. Though -like tivities of ginseng or its onstituents hve een reported repetedly, this study is the first to report tht nother signling pthwy, nmely -medited pthwy, is involved in the -like tion of ginsenosides in humn rest ner MCF-7 ells. This newly disovered mehnism of tion of provides new insight to understnd the phrmologil effets of ginseng in other physiologil systems. Aumulting evidene support the ft tht rosstlk etween ER nd pthwys plys n importnt role in British Journl of Phrmology vol 147 (5)

9 55 W.-F. Chen et l enhnes IGF-I reeptor-medited pthwys regulting neurl funtions, inluding the promotion of neuronl differentition, survivl, nd neuroprotetion (Duens et l., 1996; Gri-Segur et l., ; Grdon- Gomez et l., ). Tretment of dult ovrietomized rts with resulted in dose-dependent inrese in the phosphoryltion of ERK in rin tissues (Grdon-Gomez et l., ), suggesting tht n tivte growth ftor reeptormedited pthwys in rin tissues. Previous studies hve reported-tht ply mjor role in modulting neurotrnsmission nd prevent sopolmine-indued memory defiits y inresing holinergi tivity (Ymguhi et l., 1996). Reent in vivo nd in vitro experiments lso showed tht ginsenoside hs neurotropi nd neuroprotetive effets (Chen et l., 3; Rdd et l., 4). Thus, it is possile tht the effet of in rin tissue might e medited through the tivtion of rosstlk etween ER- nd -medited pthwys. Further study will e needed to determine the role of -medited pthwy in mediting the moleulr tions of in rin tissue. In summry, the present study provides new prdigm for hrteriztion of the moleulr tions of newly identified phytoestrogen. In ontrst to other well-hrterized phytoestrogens suh s genistein (Chen & Wong, 4), the -like tivities of ginsenoside re not medited y diret inding intertion with ER. Most importntly, our results showed tht ER is essentil for mediting the tions of in stimultion of ell prolifertion nd expression, s well s in enhnement of growth ftor reeptor-medited signling pthwys in humn rest ner ells. owever, it should e noted tht there re mny unresolved issues relting to the mehnism of tion of ginsenoside. For exmple, it is not ler whether ER, low-undne isoform of ER in MCF-7 ells, plys role in mediting the tions of nd whether indeed tivtes ER in lignd-independently mnner in MCF-7 ells. It is lso intriguing to determine the role of these signling pthwys in mediting the tion of or other ginsenosides in different ER-positive ell types suh s those found in rin nd one tissues. Future study will e needed to delinete the detiled mehnism of tion of ginsenosides in oth MCF-7 ells nd other ER-positive ell types, s well s to provide insights for understnding the omplex tions of ginseng extrt in humn ody. We thnk Dr LeRoith D. for the promoter onstrut nd the support provided y the Stte Key Lortory of Chinese Mediine nd Moleulr Phrmology. This work ws supported y the Ares of Exellene Sheme Estlished under the University Grnts Committee of the ong Kong Speil Administrtive Region, Chin (AOE/P-1/1), the Are of Strtegi Development Grnt of the ong Kong Polytehni University (A14), the Centrl llotion grnt from the Reserh Committee of the ong Kong Polytehni University (G-W15, G-YC81), nd the Ntionl Nturl Siene Foundtion of Chin (357573). Referenes AMATO, P., CRISTOPE, S. & MELLON, P.L. (). Estrogeni tivity of hers ommonly used s remedies for menopusl symptoms. Menopuse, 9, ANTER, E., CEN, K., SAPIRA, O.M., KARAS, R.. & KEANEY JR, J.F. 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