b-sitosterol activates Fas signaling in human breast cancer cells

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1 ARTICLE IN PRESS Phytomeiine 14 (2007) Sitosterol tivtes Fs signling in humn rest ner ells A.B. Aw,, M. Chinnm, C.S. Fink, P.G. Brfor Deprtment of Exerise n Nutrition Sienes n Deprtment of Phrmology n Toxiology, Stte University of New York t Bufflo, Bufflo, NY 14214, USA Astrt -Sitosterol is the most unnt phytosterol. Phytosterols re enrihe in legumes, oil sees n unrefine plnt oils s foun in foos suh s penut utter, pisthios n sunflower sees. -Sitosterol inhiits the growth of severl speifi types of tumor ells in vitro n ereses the size n the extent of tumor metstses in vivo. The effets of - sitosterol on the extrinsi poptoti progrmme ell eth pthwy in humn rest MCF-7 n MDA-MB-231 enorinom ells were exmine, long with the extent of its inorportion into ellulr memrnes n its effets on ell growth, expression of Fs reeptor pthwy proteins, n spse-8 tivity. The results show tht -sitosterol exposure promotes its enrihment in trnsforme ell memrnes n signifintly inhiits tumor ell growth. Conurrently, Fs levels n spse-8 tivity re signifintly inrese. These tions re speifi, s expression of other proteins of the Fs reeptor pthwy, inluing Fs lign, FADD, p-fadd n spse-8, remin unhnge. These finings support the hypothesis tht -sitosterol is n effetive poptosis-promoting gent n tht inorportion of more phytosterols in the iet my serve preventive mesure for rest ner. r 2007 Elsevier GmH. All rights reserve. Keywors: Phytosterols; Brest ner; Extrinsi pthwy; Apoptosis; Signl trnsution Introution Aoring to the Worl Helth Orgniztion, more thn 1.2 million people worlwie will e ignose with rest ner this yer. However, the ourrene of rest ner vries wiely mong women from ifferent ountries n ultures, with higher inienes in Europen n North Amerin women s ompre to women in less evelope ountries n ountries relying more hevily on vegetrin iets (Amerin Cner Soiety, 2003). Dietry ftors, speifilly the proportion of niml versus plnt fts, my ply role in the evelopment of rest ner (Messin n Corresponing uthor. Tel.: x231; fx: E-mil ress: w@ufflo.eu (A.B. Aw). Brnes, 1991; Cho et l., 2003). Plnt foostuffs ontin speifi phytohemils whih my offer protetion from rest ner. Controlle ietry stuies with nimls suggest tht one lss of phytohemils, the phytosterols, my offer protetion from rest ner s well s protetion from other ners ommon to Western soieties, inluing ners of the olon n prostte (Aw et l., 2001, 2004; Aw n Fink, 2000). Phytosterols re holesterol-like hemils me exlusively y plnts. An ttrtive hypothesis whih my ount for the ner protetive tion of phytosterols is tht phytosterols inue poptosis or progrmme ell eth in highly prolifertive tumor ells. Apoptosis ours vi two min pthwys: the intrinsi or mitohonril pthwy n the extrinsi or eth reeptor-meite pthwy. The intrinsi pthwy /$ - see front mtter r 2007 Elsevier GmH. All rights reserve. oi: /j.phyme

2 748 ARTICLE IN PRESS A.B. Aw et l. / Phytomeiine 14 (2007) inues poptosis in response to internl signls. In this pthwy, internl mge to the ell is sense y the mitohonri n uses the relese of poptoti protese-tivting ftor-1 (Apf-1), ytohrome, n other pro-poptoti ftors from the intermitohonril memrne spe (Bunz, 2001; Zo rnig et l., 2001; Selens et l., 2004). These ftors ultimtely tivte the effetor protese, spse-3, riving progrmme ell eth (Drgovih et l., 1998; Fesik, 2005). The extrinsi pthwy of poptosis is triggere y externl signls tht tivte ell surfe eth reeptors, inluing Fs. Upon ining their ognte ligns, the intrellulr omins of eth reeptors reruit pter moleules suh s Fs-ssoite eth omin (FADD) n TNF reeptorssoite eth omin (TRADD) (Dniel et l., 2001), whih in turn reruit prospse-8, n inititor spse. Ativte spse-8 rives ownstrem effetor spses, inluing spse-3, ultimtely ulminting in ell eth. Resultnt poptoti oies re remove y phgoytosis. The ojetive of this stuy ws to ssess the effet of ellulr supplementtion with either -sitosterol or holesterol on the extrinsi spse-8 pthwy in the two rest ner ell lines, MCF-7 n MDA-MB-231. It is hypothesize tht -sitosterol my potentite Fseth omin signling, leing to spse-8 tivtion n ultimtely to poptosis. To test this hypothesis, the rest ner ell lines were trete with -sitosterol or with holesterol s ontrol n effets on ell growth, sterol inorportion in ell memrnes, expression of Fs-eth omin signling proteins, n spse-8 tivity were etermine. Mterils n methos Mterils Brest ner ell lines were otine from Amerin Type Culture Colletion (Mnsss, VA, USA). RPMI mei, trypsin-edta, ntiioti ntimyoti n fetl ovine serum were otine from Gio TM Invitrogen Corportion (Grn Isln, NY, USA). Cholesterol n -sitosterol were purhse from Sigm Chemil Compny (St. Louis, MO, USA). Protese inhiitor oktil ws otine from Rohe Dignostis (Mnnheim, Germny). Rey gels (12% Tris HCl, 50 ml, 10 wells), prestine SDS-PAGE stnrs n Bio-R DC protein ssy kit were otine from Bio- R Lortories (Herules, CA, USA). A-IETD-AFC (spse-8 sustrte) n A-IETD-CHO (spse-8 inhiitor) were otine from BIOMOL s Reserh Lortories, In. (Plymouth Meeting, PA, USA). Cyloextrin (2-hyroxypropyl et-yloextrin; CD) ws otine from Cerestr USA, In. (Hmmon, IN, USA). Antioies, hemiluminesene luminol regent n Cruz Mrker TM moleulr weight stnrs were otine from Snt Cruz Biotehnology, In. (Snt Cruz, CA, USA). Cell ulture MCF-7 n MDA-MB-231 ells, representing estrogen-epenent n estrogen-inepenent stges of humn rest rinom, were ulture t 37 1C in 5% CO 2 /95% ir s monolyers using RPMI 1640 growth meium supplemente with 2 g/l soium ironte, 5% fetl ovine serum n 1% ntiioti ntimyoti. Preprtion of sterol supplemente mei n mesurement of ell growth RPMI 1640 mei supplemente with holesterol or -sitosterol s CD omplexes were prepre s esrie (4) n e to growth meium to hieve rtio of 8 16 mm sterols: 5 mm CD. Control groups were trete with 5 mm CD vehile. For growth stuies, ells were seee t 5000 ells/m 2 into 24-well pltes n inute for 24 h. On y 1, mei were reple with tht ontining -sitosterol or holesterol in gre onentrtions or CD vehile. Mei were similrly hnge on ys 3 n 5. Cells were trypsinize n ounte on ys 2, 4 n 6 y Coulter Counter using the eletril sensing zone metho. Growth urves were generte from the Coulter Counter t. Mesurement of sterol ontent of ell memrnes y GLC Cells were seee into 6-well pltes, trete with sterols or vehile for 2, n then hrveste y srping. Cells were suspene, wshe 3 times n then frozen 80 1C in 350 ml 10 mm Tris 20 mm mnnitol uffer (ph 7.4) until further proessing. Smples were thwe n riefly sonite on ie. An liquot of eh smple ws use for protein nlysis. The smples were then sponifie t 80 1C in 95% ethnoli KOH in the presene of 5-holestne s n internl GLC stnr. After the ition of 2 ml of wter n 2 ml of hexne the upper orgni phse ontining the non-sponifile lipis (sterols) ws remove n rie uner nitrogen. The smples were reonstitute with ron isulfie n injete into gs liqui hromtogrph (Shimzu, moel GC-17A) fitte with 30 m DB-5M5, ID 0.25 mm olumn (JRW Sientifi, Folsom, CA, USA). The olumn temperture ws set t 265 1C n tht of injetion port n etetor t 290 1C. Sterol ontents were normlize to the internl stnr n expresse per mg protein.

3 ARTICLE IN PRESS A.B. Aw et l. / Phytomeiine 14 (2007) Determintion of spse-8 tivity Cells were seee in T-75 flsks t ensity of 10,000 ells/m 2 n llowe to tth for 24 h t whih time the mei were reple with sterol-supplemente or ontrol mei. After 3 tretment, ells were srpe, wshe in PBS (ph 7.4), n lyse y suspension on ie of 10 7 ells/200 ml in uffer ontining 10 mm HEPES, ph 7.4, 2 mm EDTA, 0.15 CHAPS, 0.1% Triton X-100 n 5 mm DTT. After 30 min, the lystes were spirte severl times through syringe fitte with 26-guge neele n then entrifuge t 10,000g for 30 min t 4 1C. The protein onentrtions of the superntnts (ell lystes) were nlyze y the Bio-R DC protein ssy moifie for thiol ontining smples. To quntify spse-8 tivity, ell lystes (100 mg were ssye with the speifi spse-8 sustrte, A-IETD-AFC, in the presene n sene of the speifi spse-8 inhiitor, A-IETD-CHO. Smples were inute with 50 mm A-IETD-CHO for 30 min t 37 1C. This inhiitor lnk ws use to orret eh smple for its own respetive spse-8 tivity. A-IETD-AFC ws then e to 50 mm n the smples inute t 37 1C. Reltive fluoresene ws monitore over time (1, 2, 3, 4, 19, n 20 h) t n exittion wvelength of 360 nm n n emission wvelength of 530 nm using fluoresene multi-well plte reer (CytoFluor TM II, PerSeptive Biosystems). Fluoresene vlues otine for smples trete with the speifi spse-8 inhiitor were sutrte from those tht i not reeive inhiitor. Western immunolot nlysis Western immunolot nlyses for Fs, FsL, FADD, p-fadd, spse 8 n -tin were performe on ell lystes (50 mg resolve y SDS-PAGE (12% polyrylmie gels) n eletrolotte onto PVDF memrnes. After loking with 5% nonft rie milk n 0.05% Tween20 Tris-uffere sline (TBS-T), lots were inute for 1 h with primry ntioy, wshe 3 times with TBS-T n inute with the seonry ntioy. Blots were trete with luminol regent for 1 min n expose to X-ry film. Intensities were nlyze y ensitometry (Bio-R Imging Densitometer, Moel GS-700) n quntifie using Quntity One (Version 4.2.3) softwre. Blots were strippe n re-proe for -tin retivity. Experimentl vlues were normlize to -tin retivity. Sttistil nlyses All experiments were performe in triplite. Vlues re expresse s mens n stnr errors of the mens. Dt were nlyze using one-wy nlysis of vrine (ANOVA). Differenes etween men vlues of the tretment groups were teste for signifine (po0.05) using the Stuent s Newmn Keuls post ho test. The sttistil softwre pkge use ws ProStt (Poly Softwre Int., Perl River, NY, USA). Results -Sitosterol supplementtion inhiits MCF-7 n MDA-MB-231 ell growth The effets of supplementtion with holesterol or - sitosterol on ell growth were stuie with the rest ner ell lines MCF-7 n MDA-MB-231. For MCF- 7 ells, holesterol supplementtion (8 n 16 mm) resulte in signifintly inrese ell growth when ompre to vehile ontrol fter 3 n 5 supplementtion (Fig. 1). There ws ose-epeneny of the effet, with 8 n 16 mm holesterol signifintly inresing ell growth y 29% n 60%, respetively. In ontrst, supplementtion with 8 or 16 mm - sitosterol for 1, 3, n 5 resulte in signifintly erese ell growth when ompre to vehile ontrol. At 5, the effet of 16 mm -sitosterol (81% erese) ws greter thn tht of 8 mm -sitosterol (63% erese) fter 5 supplementtion. The effet of sterol supplementtion on the ell growth of MDA-MB-231 ells ws lso exmine n Cell Density (ell # x 10 3 /m 2 ) Control Chol 8 um Chol 16 um Sit 8 um Sit 16 um 1 3 Dys of Tretment Fig. 1. Effet of sterol supplementtion on growth of MCF-7 ells. Cells were grown in RPMI-1640 growth meium supplemente with ifferent onentrtions of sterols: 8 or 16 mm holesterol; 8 or 16 mm -sitosterol. Control group reeive only 5 mm of the yloextrin vehile. Cell growth, s ssesse y ounter oulter, ws stuie up to 5 sterol supplementtion. Vlues (men7sem, n ¼ 3) with ifferent letters ( e) in one time point re signifintly ifferent (po0.05) [Control: 5 mm yloextrin (CD) vehile; Chol 8 mm: 8 mm holesterol in CD; Chol 16 mm: 16 mm holesterol in CD; Sit 8 mm: 8 mm -sitosterol in CD; Sit 16 mm: 16 mm - sitosterol in CD]. e 5

4 750 ARTICLE IN PRESS A.B. Aw et l. / Phytomeiine 14 (2007) oserve to e similr to the effet on MCF-7 ells (Fig. 2). Cholesterol supplementtion t 8 n 16 mm for 5 resulte in signifintly inrese ell growth ompre to vehile ontrol. After 3, the effet of 16 mm holesterol ws greter thn tht of 8 mm holesterol; however, there ws no ifferene in ell growth etween the two holesterol onentrtions fter 5 sterol supplementtion. Like the results seen with MCF-7 ells, supplementtion of MDA-MB-231 ells Cell Density (ell # x 10 3 /m 2 ) Control Chol 8 um Chol 16 um Sit 8 um Sit 16 um 1 3 Dys of Tretment Fig. 2. Effet of sterol supplementtion on growth of MDA- MB-231 ells. Cells were grown in RPMI-1640 growth meium supplemente with ifferent onentrtions of sterols: 8 or 16 mm holesterol; 8 or 16 mm -sitosterol. Control group reeive only 5 mm of yloextrin vehile. Cell growth, s ssesse y ounter oulter, ws stuie up to 5 sterol supplementtion. Vlues (men7sem, n ¼ 3) with ifferent letters ( ) re signifintly ifferent (po0.05) [Control: 5 mm yloextrin (CD) vehile; Chol 8 mm: 8 mm holesterol in CD; Chol 16 mm: 16 mm holesterol in CD; Sit 8 mm: 8 mm -sitosterol in CD; Sit 16 mm: 16 mm -sitosterol in CD]. 5 with either 8 or 16 mm -sitosterol signifintly erese ell growth ompre to vehile ontrol fter 1, 3 n 5 sterol supplementtion. However, there ws no signifint ifferene etween the two -sitosterol onentrtions on ny of the ys stuie. Sterol supplementtion lters omposition of MCF-7 n MDA-MB-231 ell memrnes Supplementtion of MCF-7 ells with 16 mm holesterol resulte in signifintly inrese holesterol ontent of the rue memrne preprtions ompre to CD vehile ontrol or -sitosterol groups (Tle 1). Likewise, supplementtion of MCF-7 ells with 16 mm -sitosterol signifintly inrese the -sitosterol ontent of rue ell memrnes. In the ontrol n holesterol supplementtion groups, no etetle levels of -sitosterol were oserve. However, totl sterol ontent of memrnes from -sitosterol-supplemente MCF-7 ells ws signifintly greter n out twie tht of the CD ontrol n holesterol groups, refleting the enrihment of memrnes with -sitosterol. With MDA-MB-231 ells, holesterol supplementtion resulte in signifint elevtion of totl memrne sterol ontent ompre to the CD vehile ontrol or - sitosterol groups (Tle 2). Likewise, when trete with -sitosterol, MDA-MB-231 ell memrnes ontine signifintly greter mounts of -sitosterol when ompre to ontrol or holesterol-trete groups. - Sitosterol ws not etete in either the ontrol or holesterol tretment group. -Sitosterol onstitute 61% of the totl sterol ontent of rue ell memrnes of MDA-MB-231 ells fter supplementtion with - Tle 1. Effet of 2 -sterol supplementtion on sterol ontent of MCF-7 ell memrnes Supplementtion Cholesterol (mg/mg -Sitosterol (mg/mg Totl sterol (mg/mg -Sitosterol (% totl sterol) CD vehile Cholesterol Sitosterol MCF-7 ells were trete for 2 with 16 mm sterol or vehile n sterol ontents of memrnes etermine y gs liqui hromtogrphy s esrie. Dt re mens7sem (n ¼ 3) n letters (, ) of vlues in eh olumn re signifintly ifferent (po0.05). Tle 2. Effet of 2 -sterol supplementtion on sterol ontent of MDA-MB-231 ell memrnes Supplementtion Cholesterol (mg/mg -Sitosterol (mg/mg Totl sterol (mg/mg -Sitosterol (% totl sterol) CD vehile Cholesterol Sitosterol MDA-MB-213 ells were trete for 2 with 16 mm sterol or vehile n sterol ontents of memrnes etermine y gs liqui hromtogrphy s esrie. Dt re mens7sem (n ¼ 3) n letters (, ) of vlues in eh olumn re signifintly ifferent (po0.05).

5 ARTICLE IN PRESS A.B. Aw et l. / Phytomeiine 14 (2007) sitosterol. The totl memrne sterol levels of the holesterol- n -sitosterol-supplemente groups were similr to eh other n oth signifintly greter thn the CD ontrol group. -Sitosterol inreses spse-8 tivity in MCF-7 n MDA-MB-231 ells Cspse-8 tivities in lystes from 3 sterol-supplemente MCF-7 n MDA-MB-231 ells were mesure kinetilly over 1 20 h of inution with the spse-8 sustrte, A-IETD-AFC. Ativities inrese linerly n in prllel for the 20 h inution perio in ll the tretment groups n results for only the 20 h time points re reporte. In -sitosterol-supplemente MCF- 7 ells, spse-8 tivity ws signifintly inrese Tle 3. Effet of 3 -sterol supplementtion on spse-8 tivity of MCF-7 n MDA-MB-231 ell lystes Supplementtion Cspse-8 tivity of MCF- 7 lystes Cspse-8 tivity of MDA-MB-231 lystes CD vehile Cholesterol Sitosterol Cells were trete for 3 with 16 mm sterol or vehile n spse- 8 tivities of ell lystes etermine. Dt re RFU/100 mg lyste protein n represent mens7sem (n ¼ 3). Letters (, ) of vlues in eh olumn re signifintly ifferent (po0.05). ompre to the CD vehile ontrol group (1.9-fol inrese) or the holesterol tretment group (2.9-fol inrese) (Tle 3). There ws no signifint ifferene in spse-8 tivity etween the CD vehile ontrol n holesterol groups t ny time point. Similrly, in - sitosterol-supplemente MDA-MB-231 ells, spse-8 tivity ws signifintly inrese when ompre to the CD vehile ontrol or holesterol tretment groups (Tle 3). There ws no signifint ifferene in spse- 8 tivity etween the vehile ontrol n the holesterol trete groups. Fs expression is seletively inue y -sitosterol The expression levels of the Fs-relte proteins Fs, FsL, FADD, phosphorylte FADD (p-fadd), n spse 8 were ssesse y quntittive immunolot. After 6 h of sterol supplementtion of MCF-7 ells, there were no etetle ifferenes in the mounts of Fs, FsL, FADD, p-fadd n spse 8 etween the two sterol tretment groups (t not shown). However, fter 24 h -sitosterol supplementtion of MCF-7 ells, there ws 30% inrese in Fs protein level ompre to MCF-7 ells supplemente with holesterol or CD vehile ontrol (Fig. 3). No ifferenes in ny other Fsrelte proteins were oserve t either time mong the three supplementtion groups. Similr t were otine from sterol-supplemente MDA-MB-231 ells; however, the -sitosterol epenent inrese in Fs protein levels, though signifint, ws only 10% ove ontrol fter 24 h (Fig. 4). CONT CHOL SIT Fs FsL FADD p-fadd Cspse-8 Atin Fig. 3. Effet of sterol supplementtion on expression levels of Fs-relte signling proteins in MCF-7 ells. MCF-7 ells were trete with sterols for 24 h s esrie. Expression of Fs-relte signling pthwy proteins ws quntifie y immunolot.

6 752 ARTICLE IN PRESS A.B. Aw et l. / Phytomeiine 14 (2007) CONT CHOL SIT Fs FsL FADD p-fadd Cspse-8 Atin Fig. 4. Effet of sterol supplementtion on expression levels of Fs-relte signling proteins in MDA-MB-231 ells. MDA-MB-231 ells were trete with sterols for 24 h s esrie. Expression of Fs-relte signling proteins ws quntifie y immunolot. Disussion Preliminry stuies showe tht phytosterol supplementtion of MDA-MB-231 humn rest ner ells inreses the tivities of spses-3, 8, n 9 (Aw et l., 2003). The purpose of the present stuy ws to expn this work y exmining the effets of -sitosterol on spse tivity in MCF-7 humn rest ner ells s well s to etermine the effets of -sitosterol on ell growth, memrne sterol ontent, n expression levels of Fs-relte poptoti proteins in oth ell lines. The MCF-7 ell line is estrogen reeptor-positive n onsiere moel of erly stge rest ner wheres the MDA-MB-231 ell line is estrogen reeptor-negtive, hormone-insensitive, n moel of lte or vne stge rest ner. The effet of sterols on the ell growth n the expression of the extrinsi poptoti pthwy in these ells hve not een stuie. -Sitosterol ws oserve to hve growth inhiitory effets on oth rest ner ell lines n these effets ourre over similr time (3 5 ) n onentrtion rnges (8 16 mm) in oth ell types. The growth inhiitory effet of phytosterols is t onentrtions relevnt to vegetrin iets similr to tht oserve with other tumor ell lines, inluing the humn prostte ner LNCP ell line (von Holtz et l., 1998) n the humn olon ner HT-29 ell line (Aw et l., 1998). These finings inite tht -sitosterol inhiits the growth of severl speifi ner ell lines representing ommon ners of Western soiety. The present finings re ontrry to previous report (Ju et l., 2004) in whih tretment of MCF-7 ells with - sitosterol t onentrtions of 150 mm inrese ell growth. Severl resons oul ount for this isrepny, perhps most signifint is tht -sitosterol is not reily solule t onentrtions in the hunre miromolr rnge. In ition to the negtive effet of -sitosterol on ell growth, -sitosterol tretment of tumor ells is ssoite with inrese poptosis (Aw et l., 2004). In the present stuy, potentil ellulr mehnisms unerlying phytosterol-inue poptosis were investigte. Speifi effets on spse-8, the inititor spse in the extrinsi poptoti pthwy, were ientifie. After 3, -sitosterol inue 1.9-fol inrese in spse-8 tivity in MCF-7 ells n 2.9-fol inrese in MDA- MB-231 ells. These effets were speifi n not mimike y holesterol. -Sitosterol is plnt sterol similr in struture to holesterol. Supplementtion of ells with -sitosterol ws hypothesize to le to its inorportion into ellulr memrnes. This tion might ffet signl trnsution pthwys leing to ell eth. Tretment of MCF-7 n MDA-MB-231 ells with -sitosterol n holesterol resulte in signifint inreses in the mounts of sterol inorportion in ellulr memrnes.

7 ARTICLE IN PRESS A.B. Aw et l. / Phytomeiine 14 (2007) Sitosterol tretment of MCF-7 ells i not lower the memrne holesterol ontent when ompre with the ontrol group ut rther enrihe the ellulr memrnes with -sitosterol, whih uner the speifi inution onitions, resulte in -sitosterol representing more thn hlf of the totl memrne sterols. The totl sterol ontent of memrnes from -sitosterol tretment group ws out two-fol tht of the ontrol or holesterol tretment groups. The holesterol-tretment ontrol proue 14% inrese in holesterol ontent of MCF-7 memrnes when ompre with the vehile ontrol. Similr results were otine in MDA- MB-231 ells, wherein -sitosterol onstitute 61% of totl sterols. Tretment of MDA-MB-231 ells with holesterol however resulte in out 49% inrese in holesterol ontent of ell memrnes. The totl sterol ontents in holesterol n -sitosterol tretment groups were similr n oth were signifintly greter thn tht of the ontrol group in this ell line. This enrihment of memrnes with sterols oul influene the ell signling mehnisms. These oservtions le to the suggestion tht the Fs reeptor-meite signl trnsution pthwy leing to tivtion of spse-8 might e influene y phytosterol-inue hnges in ellulr memrnes. Western lot evlution of series of Fs-relte poptoti proteins in -sitosterol-trete ells showe no effets on the expression levels of FsL, FADD, p- FADD n spse-8. However, there ws 30% inrese in Fs expression in MCF-7 ells fter 24 h - sitosterol tretment. This fining suggests speifi effet of -sitosterol supplementtion on Fs expression n tht this inrese might e responsile for the inrese tivity (1.9-fol) of the inititor spse, spse-8. The inrese expression of Fs, trnsmemrne protein, upon tretment with -sitosterol my e relte to the enrihment of ellulr memrnes with - sitosterol. The kinetis of these events eserves onsiertion. After 6 h tretment (t not shown) of MCF-7 ells with -sitosterol there were no etetle ifferenes in expression of Fs, FsL, FADD, p- FADD n spse-8 ross the tretment groups. However, t 24 h there ws -sitosterol-stimulte inrese in Fs levels n t 48 h (the only time mesure) there ws % inrese in memrne sterols (preominntly -sitosterol). During the first 24 h tretment spse-8 tivity ws inrese. It is possile tht the inrese in memrne sterol levels rives poptoti hnges n tht only lte in the proess o Fs levels hnge ppreily. The effets of -sitosterol on Fs levels in MDA-MB- 231 ells were not s rmti s those seen in MCF-7 ells: 24 h tretment resulte in only pproximtely 10% inrese s revele y Western immunolot. This inrese ws reprouile n signifint n my lso e onsequene of enrihment of -sitosterol into ellulr memrnes. Similr to MCF-7 ells, tretment of MDA-MB-231 ells with -sitosterol for 24 h i not show ny etetle hnges in the levels of other proteins of the extrinsi poptoti pthwy suh s FsL, FADD, p-fadd n spse-8 when ompre with the ontrol n holesterol tretment groups. In summry, the present stuy emonstrtes tht memrne enrihment with the phytosterol, -sitosterol, my ffet the mounts n tivity of omponents of the extrinsi poptoti pthwy in humn rest enorinom ells. This is signifint oservtion n investigtion of ny use n effet reltionship etween these oservtions is wrrnte. It is interesting to onsier tht the Fs reeptor hs een reporte to e lolize in holesterol- n sphingomyelin-rih memrne rfts (Hueer et l., 2002). Perhps ny isruption or hnge in the holesterol sphingomyelin rtio or sterol ontent y the ition of phytosterols my influene the tivities of lipi rfts n thus expression of n signling through Fs reeptors. Our previous oservtions inite tht -sitosterol inues reution in memrne sphingomyelin n n inrese the ermie levels in some tumor ells (von Holtz et l., 1998; Aw et l., 1998). Cermie oul ply role in the tivtion of the extrinsi pthwy s suggeste y oservtions of eth reeptor lustering in ermierih lipi rfts (Hueer, 2003; Sheel-Toellner et l., 2004). These finings suggest tht the effet of - sitosterol tretment to inrese spse-8 tivity n poptosis in these ells my e meite, t lest in prt, y hnges in memrne sterol ontent n effets on the Fs poptoti pthwy. Aknowlegment The support of The Penut Institute for this projet is ppreite. Referenes Amerin Cner Soiety In., Cner Fts n Figures, pp Aw, A.B., Fink, C.S., Phytosterols s ntiner ietry omponents: eviene n mehnism of tion. J. Nutr. 130, Aw, A.B., von Holtz, R.L., Cone, J.P., Fink, C.S., Chen, Y.C., Sitosterol inhiits growth of HT-29 humn olon ner ells y tivting the sphingomyelin yle. Antiner Res. 18, Aw, A.B., Fink, C.S., Willims, H., Kim, U., In vitro n in vivo (SCID mie) effets of phytosterols on the growth n issemintion of humn prostte ner PC-3 ells. Eur. J. Cner Prev. 10,

8 754 ARTICLE IN PRESS A.B. Aw et l. / Phytomeiine 14 (2007) Aw, A.B., Roy, R., Fink, C.S., Sitosterol, plnt sterol, inues poptosis n tivtes key spses in MDA-MB-231 humn rest ner ells. Onol. Rep. 10, Aw, A.B., Chinnm, M., Fink, C.S., Brfor, P.G., Trgeting ermie y ietry mens to stimulte poptosis in tumor ells. Curr. Top. Nutr. Res. 2, Bunz, F., Cell eth n ner therpy. Curr. Opin. Phrmol. 1, Cho, E., Spiegelmn, D., Hunter, D.J., Chen, W.Y., Stmpfer, M.J., Colitz, G., Willett, W.C., Premenopusl ft intke n risk of rest ner. J. Ntl. Cner Inst. 95, Dniel, P.T., Wieer, T., Sturm, I., Shulze-Osthoff, K., The kiss of eth: promises n filures of eth reeptors n ligns in ner therpy. Leukemi 15, Drgovih, T., Ruin, C.M., Thompson, C.B., Signl trnsution pthwys tht regulte ell survivl n ell eth. Onogene 17, Fesik, S.W., Promoting poptosis s strtegy for ner rug isovery. Nt. Rev. Cner 5, Hueer, A.O., Role of memrne miroomin rfts in TNFR-meite signl trnsution. Cell Deth Differ. 10, 7 9. Hueer, A.O., Bernr, A.M., Herino, Z., Couzinet, A., He, H.T., An essentil role for memrne rfts in the initition of Fs/CD95-triggere ell eth in mouse thymoytes. EMBO Rep. 3, Ju, Y.H., Clusen, L.M., Alre, K.F., Alm, A.L., Helferih, W.G., Sitosterol, -sitosterol gluosie n mixture of -sitosterol n -sitosterol gluosie moulte the growth of estrogen-responsive rest ner ells in vitro n in ovrietomize thymi mie. J. Nutr. 134, Messin, M., Brnes, S., The role of soy prouts in reuing the risk of ner. J. Ntl. Cner Inst. 83, Selens, X., Festjens, N., Wlle, L.V., Vn Gurp, M., vn Loo, G., Vner-Bele, P., Toxi proteins relese from mitohonri in ell eth. Onogene 23, Sheel-Toellner, D., Wng, K., Assi, L.K., We, P.R., Crok, R.M., Slmon, M., Lor, J.M., Clustering of eth reeptors in lipi rfts initites neutrophil spontneous poptosis. Biohem. So. Trns. 32, von Holtz, R.L., Fink, C.S., Aw, A.B., Sitostrol tivtes the sphingomyelin yle n inues poptosis in LNCP humn prostte ner ells. Nutr. Cner 32, Zo rnig, M., Heuer, A., Bum, W., Evn, G., Apoptosis regultors n their role in tumorigenesis. Biohim. Biophys. At 1151, F1 F37.