BreastDefend enhances effect of tamoxifen in estrogen receptor-positive human breast cancer in vitro and in vivo

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1 Cheng et l. BMC Complementry nd Alterntive Medicine (217) 17:115 DOI /s RESEARCH ARTICLE BrestDefend enhnces effect of tmoxifen in estrogen receptor-positive humn rest cncer in vitro nd in vivo Shujie Cheng 1,6, Victor Cstillo 1, Mtt Welty 1, Mrk Alvrdo 1, Isc Eliz 2, Constnce J. Temm 3, George E. Sndusky 3 nd Dniel Sliv 1,4,5 Open Access Astrct Bckground: Tmoxifen (TAM) hs een widely used for the tretment of estrogen receptor (ER)-positive rest cncer nd its comintion with other therpies is eing ctively investigted s wy to increse efficcy nd decrese side effects. Here, we evlute the therpeutic potentil of co-tretment with TAM nd BrestDefend (BD), dietry supplement formul, in ER-positive humn rest cncer. Methods: Cell prolifertion nd poptosis were determined in ER-positive humn rest cncer cells MCF-7 y MTT ssy, quntittion of cytoplsmic histone-ssocited DNA frgments nd expression of cleved PARP, respectively. The moleculr mechnism ws identified using RNA microrry nlysis nd western lotting. Tumor tissues from xenogrft mouse model were nlyzed y immunohistochemistry. Results: Our dt clerly demonstrte tht comintion of 4-hydroxytmoxifen (4-OHT) with BD led to profound inhiition of cell prolifertion nd induction of poptosis in MCF-7 cells. This effect is consistent with the regultion of poptotic nd TAM resistnt genes t the trnscription nd trnsltion levels. Importntly, TAM nd BD co-tretment significntly enhnced poptosis, suppressed tumor growth nd reduced tumor weight in xenogrft model of humn ER-positive rest cncer. Conclusion: BD sensitized ER-positive humn rest cncer cells to 4-OHT/TAM tretment in vitro nd in vivo. BrestDefendcneusedinndjuvnttherpytoincresethetherpeuticeffectoftmoxifeninptients with ER-positive rest cncer. Keywords: Polyotnicl supplement, BrestDefend, Tmoxifen, Estrogen receptor, MCF-7, Xenogrft model, Apoptosis Bckground As the leding cuse of cncer deth in femles, rest cncer is heterogeneous disese tht cn e divided into three mjor sutypes: hormone (estrogen/progesterone) receptor-positive, HER2-positive, nd triple-negtive (estrogen, progesterone receptor nd HER2-negtive) [1, 2]. Among them, estrogen receptor (ER)-positive rest tumors comprise pproximtely 75%, depending on estrogen signling for growth nd survivl [3, 4]. Correspondence: dsliv@dstest-l.com 1 Cncer Reserch Lortory, Methodist Reserch Institute, Indin University Helth, Indinpolis, IN 4622, USA 4 Deprtment of Medicine, Indin University School of Medicine, Indinpolis, IN 4622, USA Full list of uthor informtion is ville t the end of the rticle Specific sutypes of rest cncer hve different responses to therpies nd tmoxifen (TAM) is the most commonly used endocrine therpy in tretment of ER-positive rest cncer. TAM is selective ER modultor nd its ctive metolite, 4-hydroxytmoxifen (4-OHT), cts s n estrogen ntgonist in rest cells tht inds ER nd locks its ctivity to hlt cell prolifertion nd induce poptosis [5 7]. Unfortuntely, de novo or cquired resistnce occurs in round 3% of ll ERpositive rest cncer nd tumor recurrence is oserved in mny ptients [8, 9]. Furthermore, long-term dministrtion of TAM my led to serious side effects, such s ftigue, pinful joints nd mood chnges [1, 11]. Therefore, in order to improve efficcy of the tretment nd The Author(s). 217 Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4. Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Cheng et l. BMC Complementry nd Alterntive Medicine (217) 17:115 Pge 2 of 11 increse the qulity of life, effective djuvnt therpies re urgently required. Numerous studies support tht nturl compounds or dietry gents, presented in vegetles, fruits nd mushrooms, cn ffect vrious moleculr trgets nd signling pthwys leding to their possile use in the comintion therpy of rest cncer [6, 12 14]. BrestDefend (BD) is dietry supplement formul, which contins extrcts from medicinl mushrooms (Gnoderm lucidum, Coriolus versicolor, Phellinus linteus), hers (Curcum long, Scutellri rt, Astrglus memrnceus), nd purified iologiclly ctive components (3, 3 -diindolylmethne, quercetin). These nturl gents in BD demonstrted nticncer ctivities ginst rest cncer through vrious mechnisms [15 23]. In ddition, BD lone or comined with PectSol-C modified citrus pectin (MCP) inhiits growth nd invsive ehvior of the highly metsttic triple-negtive humn rest cncer cells in vitro nd in vivo [24 26]. However, the effects of BD nd its comintion with TAM on ER-positive rest cncer hve never een evluted. Bsed on the dt descried ove, we investigted the sensitivity of ER-positive MCF-7 cells nd its tumor xenogrfts to BD, 4-OHT/TAM nd their comintion tretment. Here we show, for the first time, tht BD nd 4-OHT/TAM work synergisticlly ginst rest cncer y suppressing estrdiol-induced prolifertion of MCF-7 cells in vitro nd tumor growth in vivo, which relted to induced poptosis nd regultion of TAM resistnt proteins (p21/cdkn1a nd Bcl-2) expression. The findings revel novel potentil strtegy ginst ER-positive humn rest cncer using comintion tretment of tmoxifen with BD. Methods Cell culture A non-tumorigenic epithelil humn rest cell line MCF-1A, estrogen receptor (ER) -positive MCF-7 nd ER-negtive MDA-231 humn rest cncer cell lines were otined from ATCC (Mnsss, VA, USA). MCF- 1A were cultured in DMEM/F12 contining 1% horse serum, epiderml growth fctor (EGF, 2 ng/ml), hydrocortisone (.5 mg/ml). choler toxin (1 ng/ml), insulin (1 μg/ml) nd penicillin (5 U/ml), streptomycin (5 U/ml). MCF-7 nd MDA-231 cells were cultured in DMEM contining penicillin (5 U/ml), streptomycin (5 U/ml ) nd 1% fetl ovine serum (FBS). For in vitro cell culture ssys ssessing the effect of BD on the ER ctivity, MCF-7 cells were stripped of steroids for 3 dys efore seeding y culturing in steroid-free medium (SFM): phenol red-free DMEM, supplemented with 1% neworn clf serum (NCS), penicillin (5 U ml 1 ), streptomycin (5 U ml 1 ) nd 4 mm L-Glutmine. Medium, FBS, NCS nd culture supplements were otined from Gico BRL (Grnd Islnd, NY, USA). Chemicls nd regents 17β-estrdiol (E 2 ), 4-OHT, nstrozole, insulin, hydrocortisone, choler toxin, nd DMSO were purchsed from Sigm (St. Louis, MO). EGF ws purchsed from Peprotech (Rocky Hill, NJ), horse serum ws from Invitrogen (Crlsd, CA). TAM pellets (5 mg/pellet, 6-dy relese) nd E 2 pellets (.36 mg/pellet, 6-dy relese) were purchsed from Innovtive Reserch of Americ (Srsot, FL, USA). Mtrigel Mtrix Growth Fctor Reduced ws purchsed from BD Biosciences (Bedford, MA, USA). Anti-Rf-B, nti-p21, nti-bcl-2, nti-fironectin nd nti-β-ctin ntiodies were otined from Snt Cruz Biotechnology (Snt Cruz, CA, USA). BrestDefend (BD) ws supplied y EcoNugenics, Inc. (Snt Ros, CA, USA) nd dissolved in DMSO t concentrtion of 25 mg/ml then stored t 2 C. The chemicl composition of BD ws previously pulished [26]. All other chemicls nd regents were of nlyticl grde. Cell prolifertion ssy MCF-1A, MCF-7, MDA-231 cells were seeded into 96- well pltes (5 cell/well). After 24 h cells were treted with BD (1 5 μg/ml) for 3 dys. Steroid-depleted MCF- 7 cells were seeded into 96-well pltes (5 cell/well) in SFM. After 24 h, cells were treted with E 2 (1 nm) plus 4-OHT (1 μm), BD (1 5 μg/ml) or comintion of oth 4-OHT nd BD for 3 nd 6 dys, respectively. Alterntively, MCF-7 cells were seeded into 96-well pltes (5 cell/well) in DMEM, nd fter 24 h treted with nstrozole (4 μm), BD (1 μg/ml) or comintion of oth nstrozole nd BD for 48 h, respectively. Cell prolifertion ws determined s descried efore [27]. Dt points represent the men ± SD in one representtive experiment repeted t lest twice. Determintion of poptosis Steroid-depleted MCF-7 cells were seeded into 6-well pltes ( cell/well) in SFM. After 24 h, monolyers were treted with E 2 (1 nm) plus 4-OHT (1 μm), BD (1 μg/ml) or comintion of 4-OHT nd BD for 6 dys. Apoptosis induction ws ssessed y quntittion of cytoplsmic histone-ssocited DNA frgments using Cell Deth Detection ELISA PLUS Kit (Roche, Indinpolis, IN, USA). The mnufcturer s instructions were followed nd dt were expressed s the fold chnge vs. vehicletreted cells (set equl to 1). Dt points represent the men ± SD in three independent experiments. Western lotting for PARP clevge (c-parp) ws used to confirm the induction of poptosis.

3 Cheng et l. BMC Complementry nd Alterntive Medicine (217) 17:115 Pge 3 of 11 Microrry gene expression profiling Steroid-depleted MCF-7 cells were seeded into 6-well pltes t density of cell/well for 24 h nd treted with E 2 (1 nm) plus 4-OHT (1 μm), BD (1 μg/ml) or comintion of oth for 6 dys in SFM. Isoltion, quntifiction, reverse trnscription of RNA nd TqMn Arry Humn Moleculr Mechnisms of Cncer were performed s descried efore [28]. Reltive quntity (RQ) of gene expression ws normlized to GAPDH nd determined using the 2 -ΔΔCt method [29]. Dt were expressed s the fold chnge vs. vehicle-treted cells (set equl to 1) nd represent the men ± SD in three independent experiments. Western lot nlysis Steroid-depleted MCF-7 cells were seeded into 6-well pltes ( cell/well) for 24 h nd treted with E 2 (1 nm) plus 4-OHT (1 μm), BD (1 μg/ml) or comintion of oth for 6 dys in SFM. Whole protein extrcts isolted from cells were prepred nd western lot nlysis with nti-cleved PARP, nti-rf-b, nti-p21, nti-bcl-2, nti-fironectin nd nti-β-ctin ntiodies were performed s previously descried [27]. Western lots were quntified with HP-Scnjet 55c nd nlyzed y UN-SCAN-IT softwre (Silk Scientific, Orem, UT, USA). Quntittive dt composed of three independent experiments with sttisticl nlysis were expressed s the fold chnge vs. vehicle-treted cells (set equl to 1) nd dded elow or on the right of the representtive lot imges. Humn rest tumor xenogrft experiments Nu/Nu immune-compromised femle ovriectomized mice (4 5 weeks old) were otined from Hrln Lortories (Indinpolis, IN, USA) nd mintined under specific pthogen-free conditions with phytoestrogen-free d liitum food nd wter. After one week, MCF-7 cells (5 1 6 ) suspension in 5 μl sterilepbswsmixedwith5μl Mtrigel nd sucutneously implnted into oth sides of the inguinl mmmry ft pd. E 2 pellets (.36 mg/pellet, 6-dy relese) were implnted using 1-guge trochr into right ck etween the er nd shoulder of ll mice to permit tumors to form. Mice with plple tumors (~6 mm 3 ) were rndomly ssigned into four groups (n = 13): control, TAM, BD nd TAM + BD (numer of tumors per group). TAM pellets (5 mg/pellet, 6-dy relese) were implnted sucutneously into the left ck etween the er nd shoulder using 1-guge trochr. BD ws suspended in wter nd dministered y intrgstricl gvge 5 times per week with 1 mg kg 1 of ody weight for dditionl 4 weeks. During the tretment period, tumor sizes were mesured 3 times per week with microcliper nd ody weight ws recorded t the sme time. Tumor volumes were clculted with the stndrd formul: tumor volume (mm 3 )=L W 2.5, where L is the length nd W is the width of the tumor. At theendoftheexperiment(dy29),micewereeuthnized y CO 2 inhltion. Tumors were hrvested, weighed nd fixed in 1% neutrl-uffered formlin t 4 C for 24 h or snp frozen nd stored seprtely in liquid nitrogen. Animl experiments were conducted in ccordnce with the protocol pproved y the Animl Reserch Committee t the Indin University Helth Methodist Hospitl (protocol no ). Apoptosis mesurement in tumor xenogrft Formlin-fixed tumors were emedded in prffin within 48 h nd stined with hemtoxylin nd eosin (H&E). The slides were viewed using inverted microscope (Leic Microsystems, Wetzlr, Germny) nd poptosis in the vile tumor cell re ws quntified y counting poptotic odies in four fields of view (2 ) y two independent oservers (n =5-1). Immunohistochemistry Prffin-emedded tumor tissue sections were nlyzed y immunohistochemistry using primry ntiodies ginst B-rf or BRAF (Clone VE1, Spring Bioscience, Plesnton, CA, USA), Bcl-2 (Clone 124, Dko, Crpinteri, CA, USA), p21 (C-19, Snt Cruz Biotechnology, Snt Cruz, CA, USA) nd fironectin (H-3, Snt Cruz Biotechnology, Snt Cruz, CA, USA). Sections were de-prffinized, nd rehydrted. Het medited (2 min t 1 C, DAKO PT module) ntigen retrievl ws performed s follows for ech ntiody: DAKO high ph uffer for BRAF, Bcl-2 nd fironectin, while p21 ws in DAKO low ph uffer. Endogenous peroxidse ctivity ws locked y H 2 O 2 for 5 min nd slides were then incuted with BRAF nd Bcl-2 ntiodies for 2 min; fironectin nd p21 for 3 min. In negtive controls, the primry ntiody ws replced with PBS. Secondry ntiody (DAKO Flex system) for Bcl-2 nd BRAF ws dded to the sections for n incution time of 2 min; for p21 nd fironectin, incuted for 3 min with the Envision + R DAKO system. The stin ws developed using diminoenzidine (DAB) nd the sections counterstined with hemtoxylin. Locliztion nd intensity of immunorectivities ginst ll primry ntiodies used were exmined on slides y inverted microscope (Leic Microsystems, Wetzlr, Germny). For the immunohistochemicl quntifiction, rndomly selected imges were nlyzed in ech niml per group (n = 1) y ImgeJ [3]. Sttisticl nlysis All the sttisticl nlysis ws crried out using SigmPlot 11.2.(SysttSoftwreInc.,SnJose,CA,USA).Dtwere presented s men ± SD. Sttisticl comprisons etween mny groups of dt were crried out y ANOVA with the

4 Cheng et l. BMC Complementry nd Alterntive Medicine (217) 17:115 Pge 4 of 11 Prolifertion [%] Prolifertion [%] c Prolifertion [%] BD [ g/ml] BD [ g/ml] MCF-1A MCF-7 MDA-231 E2 E2 + 4-OHT BD [ g/ml] E2 E2 + 4-OHT Fig. 1 Effect of BD on the norml MCF-1A nd rest cncer MCF-7 nd MDA-231 cells. MCF-1A, MCF-7 nd MDA-231 cells were seeded nd treted with BD ( 5 μg/ml) for 3 dys., c MCF-7 cells were stripped of steroids for 3 dys efore seeding y culturing in steroid-free medium. After 24 h seeded into 96-well pltes, cells were treted with E 2 (1 nm) plus 4-OHT (1 μm), BD ( 5 μg/ml) or comintion of 4-OHT nd BD for 3dysndc 6 dys, respectively. Cell prolifertion ws determined y MTT ssy. Ech r represents the men ± SD of triplicte. Similr results were otined in three independent experiments. Sttisticl nlysis y ANOVA nd Holm-Sidk. P <.5 BD vs control ( μg/ml) for different cell lines., c P <.5 BD vs control ( μg/ml) in E 2 group, P <.5 BD vs control ( μg/ml) in E OHT group, different letters ove rs indicte significnt differences P <.5 for the sme concentrtion of BD ll pirwise multiple comprison procedures (Holm-Sidk method) t overll significnce level p <.5. Results Anti-prolifertive nd pro-poptotic effects of 4-OHT re ugmented y BD in ER-positive rest cncer cells MCF-7 To evlute the effect of BD on norml humn epithelil mmmry glnd cells nd ER-negtive MCF-7 nd ERpositive humn rest cncer cells we treted MCF-1A, MCF-7 nd MDA-231 cells with BD (1 5 μg/ml) for 3 dys. Here we show tht the low concentrtion of BD only slightly decresed prolifertion of norml rest cells MCF-1A, wheres BD strongly suppressed growth of rest cncer cells MCF-7 nd MDA-231 (Fig. 1). To evlute the effect of BD on the prolifertion of the estrogen-dependent ER-positive MCF-7 cells, we depleted these cells of estrogen nd treted MCF-7 cells with E 2, BD nd 4-OHT s descried in Mterils nd Methods. Our results show tht fter 3 nd 6 dys of tretment, 4- OHT (1 μm), ER receptor ntgonist, significntly inhiits E 2 -dependent prolifertion of MCF-7. Moreover, BD further enhnces nti-prolifertive effect of BD in dose- nd time-dependent mnner (Fig. 1-c). Thus, 1 μg/ml of BD in the comintion with 1 μm of4-ohtwsselectedfor future experiments in vitro. As shown in Fig. 1, E 2 -independent prolifertion of MCF-7 cells ws not sensitive to the inhiitory effects BD t lower concentrtions of BD. To determine whether the inhiition of cell prolifertion y 4-OHT nd BD re ssocited with poptosis, we evluted whether 4-OHT, BD nd their comintion induce nucler DNA frgmenttion [31]. As shown in Fig. 2, there were nerly 18 fold increses in the poptosis with 4-OHT (1 μm) nd BD (1 μg/ml) comintion compred to the vehicle-treted control fter 6 dys. Apoptosis induction in MCF-7 cells ws further confirmed y western lotting for the cleved frgment of PARP (c-parp) [32, 33], where comintion of 4-OHT nd BD distinctly incresed the mount of c-parp (Fig. 2-c).

5 Cheng et l. BMC Complementry nd Alterntive Medicine (217) 17:115 Pge 5 of c evlution of the comined effect of BD nd tmoxifen, we will ddress the comined effect of BD nd nstrozole in our future study. Reltive poptosis c Fold chnge (vs. vehicle treted group) c-parp ctin - 4-OHT - 4-OHT - - BD BD - 4-OHT - 4-OHT - - BD BD c. - 4-OHT - 4-OHT - - BD BD Fig. 2 Effect of 4-OHT nd BD on poptosis of MCF-7 humn rest cncer cells. MCF-7 cells were seeded nd treted s descried in Fig. 1c for 6 dys. Apoptosis ws evluted y Cell Deth Detection ELISA () nd western lotting for the expression of c-parp (). Representtive lots show expression of c-parp nd β-ctin ws used s loding control. Three independent experiments were done for the western lot studies nd quntittive dt with sttisticl nlysis were shown elow the representtive lot imge (c). Sttisticl nlysis y ANOVA nd Holm-Sidk. Different letters ove rs indicte significnt differences P <.5. The grphicl dt represent men +/ SD In ddition, we treted MCF-7 cells with BD (1 μg/ml) nd n romtse inhiitor nstrozole (4 μm) or their comintions for 48 h. Although nstrozole inhiited prolifertion of MCF-7 cells y 6% nd BD y 13%, their comintion suppressed prolifertion y 39%, suggesting possile synergistic effect (Additionl file 1: Figure S1). Since the mjor focus of the current study ws on the c 4-OHT nd BD comintion regultes multiple genes relted to poptosis nd TAM resistnce To further investigte moleculr mechnisms underlying enhnced poptosis induced y 4-OHT nd BD comintion, gene expression profiling with microrrys ws crried out using totl RNA from MCF-7 cells treted with 4-OHT nd BD. Ech set of four experiments ws done in triplicte to increse the precision of estimtion. The overlpping regulted genes with lrge recurring expression differences (t lest 1.6-fold chnged) compred to the vehicle-treted control fter tretment of 6 dys re summrized (T. 1). Bsed on the literture serch, severl genes ssocited to poptosis nd TAM resistnce were identified, such s upregultion of genes encoding v-rf murine srcom virl oncogene homolog B (BRAF), cspse 9 (CASP9), nd downregultion of genes encoding B-cell CLL/lymphom 2 (Bcl-2), fironectin 1 (FN1). Moreover, 4-OHT nd BD comintion showed mximl effects compring with individul tretment, which might e the reson of enhnced poptosis. Upon closer inspection, we found severl genes tht re regulted differently y BD versus 4-OHT. For exmple, CDKN1A, which encoding cyclin-dependent kinse inhiitor 1A (p21), is upregulted y oth BD nd comintion, ut not y 4-OHT. We evluted if 4-OHT nd BD comintion ffects expressions of genes, which involved in poptosis nd TAM resistnce, t the trnsltion level s well. Consistent with gene expression microrry dt otined t the mrna level fter tretment of 6 dys, induction of BRAF, p21, nd suppression of FN1, Bcl-2 in MCF-7 cells were detected y western lot nlysis (Fig. 3). Results of quntifiction indicted tht expression of BRAF is induced nerly 2.4 fold with BD, 1.8 fold with 4-OHT nd 2.8 fold with comintion; expression of Bcl-2/ FN1is suppressed round.6 fold/.7 fold with BD,.7 fold with 4-OHT nd.4 fold with comintion; expression of p21 is induced nerly 2.9 fold with BD nd 2.7 fold with comintion (Fig. 3). TAM nd BD co-tretment inhiits growth of tumor xenogrfts y induction of poptosis We hve recently demonstrted tht BD is not toxic in vivo. An intrgstric gvge of BD (1 mg/kg of ody weight for 4 weeks) did not ffect ody weight or ctivity in liver enzymes nd did not show ny sign of toxicity in liver, spleen, kidney, lung nd hert tissues in mice [26]. To determine the effect of TAM nd BD in vivo, the growth of ER-positive humn rest tumor xenogrfts ws monitored in ovriectomized nude mice sucutneously

6 Cheng et l. BMC Complementry nd Alterntive Medicine (217) 17:115 Pge 6 of 11 Tle 1 Comintion of 4-OHT with BD regultes expression of cncer progression relted genes Gene Description 4-OHT (RQ) BD (RQ) Comintion (RQ) BRAF serine/threonine-protein kinse B-Rf 2.91 ± ± ±.82 PTK2B protein tyrosine kinse 2 et 2.76 ± ± ± 1.41 CDKN1A cyclin-dependent kinse inhiitor 1A (p21) 1.6 ± ± ±.22 NFKBIA NF-kpp-B inhiitor 1.86 ± ± ±.16 FYN FYN oncogene relted to SRC, FGR, YES 1.81 ± ± ±.81 CASP9 cspse ± ± ±.67 BCL2 B-cell CLL/lymphom 2.13 ±.1.29 ±.23.8 ±.5 CCND2 cyclin D2.39 ±.8.12 ±.1.1 ±.1 FN1 fironectin 1.41 ±.4.3 ±.9.19 ±.11 ITGA2B integrin, lph 2.62 ± ±.2.41 ±.12 DNA-microrry nlysis ws performed on TqMn Arry Humn Moleculr Mechnisms of Cncer s descried in Mterils nd Methods. MCF-7 cells were stripped of steroids for 3 dys efore seeding y culturing in steroid-free medium. After 24 h seeded into 6-well pltes, cells were treted with E 2 (1 nm) plus 4-OHT (1 μm), BD (1 μg/ml) or comintion of oth for 6 dys in steroid-free medium. Dt re the mens ± SD of three independent experiments. Anlysis of the RQ gene expression dt ws performed using the 2 -ΔΔCT method. Sttisticl nlysis y ANOVA P <.5 injected with MCF-7 cells nd treted with TAM, BD or the comintion of TAM nd BD, s descried in Mterils nd Methods. The control group (E 2 lone) exhiited rpid growth of MCF-7 tumors from dy 7 nd tretments of BD or TAM resulted in the significnt inhiition of tumor growth when compred to control. In ddition, the comintion of TAM nd BD further enhnced inhiitory effect of TAM on tumor growth (Fig. 4). On dy 29, the verge tumor volume ± SD in the comintion group (TAM nd BD) ws pproximtely 139 ± 121 mm 3,which ws nerly 77% of inhiition compred with control (~68 ± 489 mm 3 ) (Fig. 4). Although the tretment of TAM or BD lso significntly suppressed tumor volume (TAM ~ 228 ± 216 mm 3, BD ~ 232 ± 18 mm 3 ), their comintion suppressed only slightly tumor volume on dy 29 (TAM nd BD ~139 ± 121 mm 3 ) (Fig. 4). In ddition, there were no significnt differences in ody weight etween control nd tretment groups (dt not shown). Interestingly, mice in the comintion group were more relx, ctive nd helthy thn mice in the TAM group (dt not shown). In ddition, verge tumor weight in the comintion group (TAM nd BD) (195 ± 141 mg) ws decresed y 67% when compred to the control group (587 ± 469 mg) t the end of tretment period on dy 29 (Fig. 4c). To determine if TAM nd BD comintion inhiits growth of ER-positive humn rest tumor y enhncing poptosis in vivo, we quntified the mounts of poptotic odies in tumor xenogrfts. As seen in Fig. 5, more poptotic odies were detected in the tumor tissues from TAM nd TAM nd BD comintion treted groups compred with the control group. Although there ws no sttisticl BRAF p21 Bcl-2 Fironectin -ctin Fold chnge (vs. vehicle-treted group) - 4-OHT - 4-OHT BRAF p21 Bcl-2 Fironectin - - BD BD Fig. 3 Comintion of 4-OHT with BD regultes expression of poptosis relted proteins. MCF-7 cells were treted for 6 dys s descried in Fig. 1. Whole protein extrcts isolted from cells were prepred nd western lot nlysis with nti-rf-b, nti-p21, nti-bcl-2, nti-fironectin nd nti-β-ctin ntiodies were performed s descried in Mterils nd Methods. β-ctin ws used s loding control nd representtive lots from three experiments were shown. Quntittive dt composed of ll the experiments in MCF-7 cells with sttisticl nlysis were on the right of the representtive lot imge. Sttisticl nlysis y ANOVA nd Holm-Sidk. Different letters ove rs indicte significnt differences P <.5. The grphicl dt represent men +/ SD c c c c Control 4-OHT BD 4-OHT + BD c

7 Cheng et l. BMC Complementry nd Alterntive Medicine (217) 17:115 Pge 7 of 11 Tumor volume [mm 3 ] Control TAM BD TAM + BD Time [dys] Fig. 4 Inhiition of humn rest tumor growth y TAM, BD, nd TAM nd BD comintion in vivo. Xenogrft experiments were performed s descried in Mterils nd Methods. During the tretment period, tumor sizes were mesured 3 times per week. Sttisticl nlysis y ANOVA nd Holm-Sidk. P <.5: control vs TAM, control vs. BD, control vs, BD + TAM. (n = tumors per group). Tumor sizes t the eginning (Dy1) nd the end (Dy 29) of the tretment. Sttisticl nlysis y ANOVA nd Holm-Sidk. Different letters ove rs indicte significnt differences P <.5. Box plots represent 5th/1th percentiles, men (white dotted line), horizontl rs represent medin vlues, whiskers indicte minimum to mximum vlues nd tringles represent outliers. c At the end of the experiment (Dy 29), tumors were hrvested nd weighed. Sttisticl nlysis y ANOVA nd Holm-Sidk. Different letters ove rs indicte significnt differences P<.5. Box plots represent 5th/1th percentiles, men (white dotted line), horizontl rs represent medin vlues, whiskers indicte minimum to mximum vlues nd tringles represent outliers 12 Tumor volume [mm 3 ] c c c Control BD c Tumor weigh [mg] TAM TAM+BD 2 Apoptotic odies TAM - TAM - - BD BD Fig. 5 Induction of poptosis in humn rest tumor xenogrfts. Representtive H&E stining of poptotic odies in MCF-7 humn rest tumors, quntifiction ws determined s descried in Mterils nd Methods. Sttisticl nlysis y ANOVA nd Holm-Sidk. Different letters ove rs indicte significnt differences P <.5. The grphicl dt represent men +/ SD. (n =5 1)

8 Cheng et l. BMC Complementry nd Alterntive Medicine (217) 17:115 Pge 8 of 11 difference etween TAM nd control group, TAM nd BD comintion showed mximl increse of poptotic odies (64%) compred with control, indicting tht suppression of tumor growth cn e ttriuted to the induction of poptosis in cncer cells (Fig. 2). Although the numer of poptotic odies in rest tumors is suggestive for the induction of poptosis it is necessry to confirm poptosis y other method, s is the expression of specific propoptotic protein Bcl-2 in tumors. Effect of BD nd TAM on the poptotic nd TAM resistnt proteins expression in tumors To ssess whether the medited tumor growth inhiition is ssocited with the expression of proteins involved in poptosis nd TAM resistnce, tumor tissues from TAM, BD nd TAM nd BD comintion treted mice were sujected to immunohistochemistry. As shown in Fig. 6, TAM nd BD comintion mrkedly induced expressions of BRAF nd p21, wheres expression of pro- poptotic Bcl-2 protein ws decresed compred to the vehicletreted control. Similr results were found in western lot nlysis of tumor tissues s well (dt not shown). These in vivo oservtions re in ccordnce with our in vitro dt, with MCF-7 cells treted with BD, 4-OHT or the comintion of BD nd 4-OHT. However, the expression of Fironectin ws not ffected in the rest cncer tumors. Discussion TAM hs een frontline tretment for oth erly nd dvnced ER-positive rest cncer in pre- nd postmenopusl women [34 36]. A new therpeutic strtegy is focus on the comintion with other gents tht increse efficcy nd decrese toxicity of TAM. Here, we evlute the therpeutic potentil of co-tretment of TAM with BD, nturl dietry supplement, in ER-positive humn rest cncer. Our results indicte tht the comintion of 4-OHT nd BD or the comintion of TAM 4 c BRAF BRAF positive cells [%] c c p21 p21 positive cells [%] Bcl-2 Bcl-2 positive cells [%] FN Fironectin positive cells [%] E 6 Fig. 6 Comintion of TAM with BD regultes expression of poptosis nd TAM resistnce relted proteins in humn rest tumors. Animl experiments were performed s descried in Fig. 3. Prffin-emedded tumor tissue sections were nlyzed y immunohistochemistry using ntiodies ginst BRAF, p21, Bcl-2 nd Fironectin (FN). Representtive locliztion nd intensity of immunorectivities ginst ll primry ntiodies re shown. Immunohistochemicl quntifiction of BRAF, p21, Bcl-2 nd FN were determined s descried in Mterils nd Methods. Sttisticl nlysis y ANOVA nd Holm-Sidk. Different letters ove rs indicte significnt differences P<.5. The grphicl dt represent men (white dotted line)+/ SD, tringles represent outliers. (n = 1)

9 Cheng et l. BMC Complementry nd Alterntive Medicine (217) 17:115 Pge 9 of 11 nd BD resulted in the suppression of cell nd tumor growth nd induction of poptosis in vitro nd in vivo, respectively. Microrry, western lot nd immunohistochemistry nlyses further demonstrte tht the comintion tretment regultes expression of proteins involved in the cncer growth nd cell deth. Importntly, TAM nd BD co-tretment significntly suppresses tumor growth in vivo. The emergence of TAM resistnce is lmost inevitle, which pose mjor clinicl prolem. Mechnisms my include chnges in the expression or function of ER, vrition in ER-ssocited trnscription fctor recruitment, ltered expression of specific micrornas, nd genetic polymorphisms involved in TAM metolic ctivity [37, 38]. Among of them, ER plys the mjor role in driving resistnce [39]. It hs een shown tht the enhnced cell prolifertion nd reduced susceptiility to cell deth medited y ER signling re in prt through the regultion of p21, key cell cycle rek, nd Bcl-2, the mjor nti-poptotic nd pro-survivl protein [4, 41]. Recently, Rh et l. estlished de novo nd cquired TAM-resistnt rest cncer models, which exhiit reduced p21 nd elevted Bcl-2 expression [37]. In clinicl studies, loss of p21 is ssocited with TAM growth-inducing phenotype nd incresed Bcl-2 expression is n importnt phenomenon in metsttic TAM-resistnt rest tumors [42, 43]. Our dt demonstrted tht 4-OHT/TAM lone hd no effect on the expression of p21, ut BD nd/or comintion with 4-OHT/TAM resulted in significnt upregultion of p21. Moreover, ddition of BD to 4-OHT/TAM leds to enhnced inhiition of Bcl-2. Altered expression of these key proteins my ttriute to quercetin, ioflvonoid presented in BD, which inhiits prolifertion nd induces poptosis in ER-positive rest cncer cells vi upregultion of p21 nd downregultion of Bcl-2 protein expression [44, 45]. In ddition, Oh et l. demonstrte tht quercetin suppresses ngiogenesis in TAM-resistnt rest cncer through inhiition of Pin1 [46]. Therefore, BD my reverse TAM resistnce y enhnced inhiition of Bcl-2 nd significnt induction of p21, which driving cells into poptosis. Although we found positive effects in the inhiition of prolifertion nd induction of poptosis which ws ssocited with the ltered gene expression in MCF-7 cells treted with BD nd 4-OH/TAM, these effects were determined in only one ER-positive humn rest cncer cells nd xenogrftmodel.therefore,itispossiletoexpecttht other ER-positive humn rest cncer cells would lso respond to this tretment. Nevertheless, since ech cncer cell type hs specific nd unique genetic mke-up, it is plusile tht other set of genes would e ssocited with the nticncer ctivity of BD nd 4-OH/TAM. We hve previously demonstrted tht therpeutic ctivity of BD itself ws ssocited with the expression of genes ssocited with prolifertion nd metstsis in highly invsive humn rest cncer cells MDA-MB-231 nd in n niml model of rest-to-lung cncer metstsis [24, 26]. Another crucil spect in gene trgeting is temporl gene expression. In our current study, we nlyzed gene expression t 6 dys ecuse t this time point we detected significnt response of BD nd 4-OH/TAM in the inhiition of prolifertion nd induction of poptosis in MCF-7 cells. Although it is importnt to evlute lso other time points, in vivo dt confirmed the originl cell culture dt, incresed expression of BRAF nd p21 nd decresed levels of Bcl-2 in tumors fter the comined tretment in mouse fter 29 dys. Indeed, temporl gene nlysis nd the use of other humn rest cncer cells is necessry for the evlution of specific moleculr trgets of BD nd their comintions with typicl rest cncer drugs. However, these nlyses re ehind the scope of the present mnuscript nd will e performed in future studies. Hormonl therpy using TAM results in menopusl symptoms nd serious symptoms not only gretly decrese the qulity of life, ut lso my led to discontinution of the tretment [47, 48]. Hence, non-prescription dietry supplements re often used to relieve TAM-induced side effects. 3,3 -diindolylmethne (DIM), nother purified components in BD, is the mjor product of indole- 3-crinol (I3C) in vivo nd hs promising ctivities ginst ER-positive rest cncer [49]. Ktchmrt et l. demonstrted significnt reduction in the N-oxygention of TAM ctlyzed y liver microsomes in rts fed with DIM, which my ctully decrese the toxicity of TAM. Bsed on the mrked shift in the metolic profiles of TAM, they hypothesize tht ptients tking TAM in concert with dministrtion of DIM dietry supplements could modulte the risk of developing toxic side effects if there is similr ltertion in humns [5]. Gnoderm lucidum, medicinl mushroom in BD, hs een used in Asin countries to improve helth nd promote longevity for centuries [15]. A pilot clinicl tril suggests tht spore powder of G. lucidum my hve eneficil effects on cncer-relted ftigue nd qulity of life in ER-positive rest cncer ptients undergoing endocrine therpy [51]. In our present study, mice in the comintion group exhiit less ftigue nd more energy compring mice in the TAM group. Definitely, more rigorous experiments re needed to confirm the findings nd clrify the specific mechnisms ehind them. Conclusions Our study is the first report descriing the comintion effects of TAM nd BD in ER-positive humn rest cncer. BD sensitizes rest cncer cells to 4-OHT/TAM tretment in vitro nd in vivo, promotes poptosis,

10 Cheng et l. BMC Complementry nd Alterntive Medicine (217) 17:115 Pge 1 of 11 interferes with multiple pthwys importnt for TAM resistnce nd hs potentil in decrese TAM-induced side effects. Therefore, BD could decrese future TAM resistnce in the comintion therpy in the originlly nti-estrogen responsive rest cncers. Thus, BD my e recommended s novel djuvnt polyotnicl preprtion for ptients with ER-positive rest cncer undergoing conventionl endocrine therpy. More ER-positive rest cncer cell models will e employed in the further study nd clinicl trils exploring efficiency of BD re required to support its use in rest cncer ptients. Additionl file Additionl file 1: Figure S1. Effect of BD nd nstrozole on MCF-7 rest cncer cells. (PPTX 39 k) Arevitions 4-OHT: 4-hydroxytmoxifen; BD: BrestDefend; ER: Estrogen receptor; TAM: Tmoxifen Acknowledgements We thnk to Dr. Jgdish Lognthn, Indin University Helth, for his ssistnce with niml experiments, to Dr. Zizheng Dong, Indin University School of Medicine, for his technicl ssistnce with the poptosis nlysis nd Dr. Dongsheng Gu, IU Simon Cncer Center, for his ssistnce with the nlysis of immunohistochemistry. Funding This study ws supported y reserch grnts from EcoNugenics, Inc., Snt Ros, CA, USA. The founder did not ply ny role in the design of the study nd collection, nlysis, nd interprettion of dt nd in writing the mnuscript. Avilility of dt nd mterils All dt generted or nlysed during this study re included in this pulished rticle. Authors contriutions SC, DS wrote the mnuscript, SC, VC, MT, MA performed experiments nd collected the dt, IE edited the mnuscript, SC, CJT, GES, DS nlyzed the dt, DS designed experiments, edited nd finlized the mnuscript. All uthors red nd pproved the finl mnuscript. Competing interests I.E. is the formultor nd owner of EcoNugenics, Inc., D.S. is CEO nd founder of DSTest lortories nd D.S. is consulting for EcoNugenics, Inc. The other uthors declre no conflict of interest. Consent for puliction Not pplicle. Ethics pprovl nd consent to prticipte Animl experiments were conducted in ccordnce with the protocol pproved y the Animl Reserch Committee t the Indin University Helth Methodist Hospitl (protocol no ). Author detils 1 Cncer Reserch Lortory, Methodist Reserch Institute, Indin University Helth, Indinpolis, IN 4622, USA. 2 Amith Medicl Clinic nd Heling Center, Snt Ros, CA 9541, USA. 3 Deprtment of Pthology, Indin University School of Medicine, Indinpolis, IN 4622, USA. 4 Deprtment of Medicine, Indin University School of Medicine, Indinpolis, IN 4622, USA. 5 DSTest Lortories, Purdue Reserch Prk, 5225 Explortion Drive, Indinpolis, IN 46241, USA. 6 Present ddress: Deprtment of Food Qulity nd Sfety, School of Engineering, Chin Phrmceuticl University, Nnjing, People s Repulic of Chin. Received: 22 August 216 Accepted: 2 Ferury 217 References 1. Torre LA, Bry F, Siegel RL, Ferly J, Lortet-Tieulent J, Jeml A. Glol cncer sttistics, 212. CA Cncer J Clin. 215;65: Sol M, Trnski D, Uzrevic Z, Ozretic P, Musni V, Rfj M, et l. Comintion of cyclopmine nd tmoxifen promotes survivl nd migrtion of mcf-7 rest cncer cells interction of Hedgehog-Gli nd estrogen receptor signling pthwys. 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Activtion nd potentition of interferon-gmm signling y 3,3 -diindolylmethne in MCF-7 rest cncer cells. Mol Phrmcol. 26;69: Ktchmrt S, Stresser DM, Dehl SS, Kupfer D, Willims DE. Concurrent flvin-contining monooxygense down-regultion nd cytochrome P-45 induction y dietry indoles in rt: implictions for drug-drug interction. Drug Met Dispos. 2;28: Zho H, Zhng Q, Zho L, Hung X, Wng J, Kng X. Spore powder of gnoderm lucidum improves cncer-relted ftigue in rest cncer ptients undergoing endocrine therpy: pilot clinicl tril. Evid Bsed Complement Alternt Med. 212;212: Sumit your next mnuscript to BioMed Centrl nd we will help you t every step: We ccept pre-sumission inquiries Our selector tool helps you to find the most relevnt journl We provide round the clock customer support Convenient online sumission Thorough peer review Inclusion in PuMed nd ll mjor indexing services Mximum visiility for your reserch Sumit your mnuscript t