Dietary flavones and flavonones display differential effects on aromatase (CYP19) transcription in

Dietary flavones and flavonones display differential effects on aromatase (CYP19)transcription in the breast cancer cells MCF-7Fengjuan Li b ,1,Lan Ye b ,1,Shu-mei Lin c ,Lai K.Leung a ,b ,⇑aFood and Nutritional Sciences Programme,School of Life Sciences,Faculty of Science,The Chinese University of Hong Kong,Shatin,N.T.,Hong Kong bBiochemistry Programme,School of Life Sciences,Faculty of Science,The Chinese University of Hong Kong,Shatin,N.T.,Hong Kong cDepartment of Food Science,National Chiayi University,Chiayi City,Taiwana r t i c l e i n f o Article history:Received 29March 2011Received in revised form 9June 2011Accepted 24June 2011Available online 30June 2011Keywords:Luteolin Hesperetin AromataseBreast cancer cellsa b s t r a c tAromatase or cytochrome P450(CYP19)enzyme catalyzes the rate-determining reaction in estrogen syn-thesis.Inhibiting aromatase is a major strategy in treating breast cancer patients.However,suppression on the transcriptional activity may be equally important in controlling aromatase.Dietary flavones and flavo-nones have been previously demonstrated to be the most potent aromatase-inhibitory flavonoids.In the present study we examined their effects on the transcription regulation of CYP19in MCF-7cells.Real-time PCR results indicated that luteolin suppressed CYP19mRNA expression while hesperetin increased it.Reporter gene assays were employed to look into the transactivity of CYP19driven by promoters I.3and II,and the result was consistent with the observation in mRNA expression.Further investigation using truncation reporter gene and electrophoretic mobility shift assays suggested that luteolin and hesperetin differentially influenced AP-1-and C/EBP-binding on the CYP19promoter.Western blot analysis indicated that signaling transduction pathways involving JNK and ERK could be the underlying mechanisms for their actions.The present study showed that dietary flavones and flavonones might differentially regulate aro-matase transcription in breast cells in addition to the inhibition at the enzyme level.Ó2011Elsevier Ireland Ltd.All rights reserved.1.IntroductionOver-exposure to estrogen has been linked to a higher risk for breast carcinogenesis (Colditz,1999;Kaaks,2001).This causal ef-fect is also validated by a transgenic animal model (Yoshidome et al.,2000).Cytochrome P450(CYP19)or aromatase catalyzes the rate-determining reaction in estrogen synthesis.Polymor-phisms in CYP19gene with increased estrogen production are associated with increased risk of breast cancer (Lee et al.,2003;Hirose et al.,2004).Estrogen is generally considered as non-geno-toxic,but its 4-hydroxyl metabolite retains the cell proliferative properties and can be converted into a DNA-attacking moiety (Liehr et al.,1986;Li and Li,1987;Zhu and Conney,1998).Estro-gen may interact with estrogen receptor and induce proliferation of breast cancer cells.The antiestrogen tamoxifen is commonlyadministered as an adjuvant therapy for estrogen-responsive breast cancer patients.Alternatively,a contemporary treatment for such cancer is in the inhibition of CYP19(aromatase).The administration of aromatase inhibitor is claimed to be more effec-tive in preventing breast cancer development than tamoxifen (Kudachadkar and O’Regan,2005).Breast cancer tissue has a higher expression of aromatase than normal breast tissue,and in-creased plasma estrogen concentration is seen in breast cancer patients’circulation (Miller and Dixon,2001).Studies have shown that estrogen synthesized in the cancerous vicinity is crucial in promoting tumor growth (Santner et al.,1993;Yue et al.,1998).This may explain why aromatase inhibition is a better option in treating breast cancer.The promoter utilization of human aromatase gene is tissue-specific and promoters I.3and II dictate the expression regulation in breast cancer cells (Chen et al.,1999).Many substances or fac-tors have been described affecting the transcription of aromatase.Cyclic AMP,phorbol esters,dexamethasone,prostaglandin E2,transforming growth factor-b ,and c -interferon increase its tran-scriptional activity (Simpson et al.,1997).In contrast,cyclooxygen-ase inhibitors suppress its expression (Diaz-Cruz et al.,2005).Luteolin or 30,40,50,70-tetrahydroxyflavone is a common flavo-noid which can be isolated from vegetables such as celery and green peppers.It may protect against bioactivation of benzo[a]pyr-0303-7207/$-see front matter Ó2011Elsevier Ireland Ltd.All rights reserved.doi:10.1016/j.mce.2011.06.024Abbreviations:CYP,cytochrome P450;AP-1,activated protein-1;C/EBP,CCAAT/enhancer binding proteins;EMSA,electrophoretic mobility shift assay;DMSO,dimethyl sulphoxide.⇑Corresponding author.Address:Food and Nutritional Sciences Programme,School of Life Sciences,The Chinese University of Hong Kong,Rm.507C MMW Bldg,Shatin,N.T.,Hong Kong.Tel.:+852********;fax:+852********.E-mail address:laikleung@.hk (L.K.Leung).1Thsese authors contributed equally for this work.ene by inhibiting CYP1family enzymes(Kim et al.,2005;Miller and Ramos,2001).Hesperetin or30,5,7-trihydroxy-4-methoxyflav-anone,is aflavonone found abundantly in citrus fruits and has po-tential anticarcinogenic activity.It prevents against1,2-dimethyl hydrazine-induced colon carcinogenesis in rats(Aranganathan et al.,2008),and inhibits cell proliferation of breast cancer cells through G1-phase arrest(Choi,2007).Dietaryflavones andflavonones have previously been shown to be the most potent aromatase-inhibitoryflavonoids(Jeong et al., 1999;van Meeuwen et al.,2008),and their structures are highly resembled to each other(Fig.1).However,their effects on the tran-2.2.Cell cultureThe breast cancer cell line MCF-7was purchased from A.T.C.C., Rockville,MD.The cells were maintained in MEM medium(Invit-rogen,Grand Island,NY,USA)supplemented with10%fetal bovine serum(Invitrogen Life Technology,Rockville,MD).Cells were incu-bated at37°C,5%carbon dioxide and routinely sub-cultured when reaching80%of confluency.Three days before experiments,the cells were incubated in phenol-red free RPMI medium(Invitrogen Life Technology)instead of MEM.Flavonoid was administered in the solvent vehicle dimethyl sulphoxide(DMSO),and the concen-Fig.1.Structures of dietaryflavones andflavonones with aromatase-inhibitory property.52 F.Li et al./Molecular and Cellular Endocrinology344(2011)51–58containing500l l of5%activated charcoal suspension.After 30min incubation,an aliquot of the supernatant fraction was re-moved for scintillation counting.For the assay performed on MCF-7cells,the protocol was previ-ously described(Grube et al.,2001).In brief,MCF-7cells were seeded and allowed1day for attachment.Assays were started by replacing the culture medium with serum-free medium containing [1b-3H(N)]-androst-4-ene-3,17-dione andflavonoid.Thefinal con-centration of andro-stenedione was controlled at25nM,and the reaction was incubated at37°C for1h.The tritiated water release was then processed as abovementioned.The protein content of the cells,on the other hand,was determined by using a BCA kit(Sigma Chemicals)after dissolving the cells in0.5mol/l NaOH.2.4.Quantitative real time RT-PCR assay on aromatase expressionThe protocol for real-time quantitative PCR was carried out as previously described by our lab(Wang et al.,2008).Sequences of the forward and reverse primers were50-GGA GAA TTC ATG CGA GTC TGG AT-30and50-GGA ACA TAC TTG AGG ACT TGC TGA T-30, respectively.The mRNA species were quantified by tailor-made TaqmanÒMGB probes and normalized by GAPDH expression (Assay-on-DemandÒ,Applied Biosystems,Foster City,CA,USA). MCF-7cells were cultured and treated as described above.After 24h of treatment,total RNA was extracted from the cells using TRIzol reagent(Invitrogen,Carlsbad,CA USA).The concentration and purity of the isolated RNA were determined by the absorbance reading observed at260and280nm.3l g of total RNA,oligo-dT, and M-MLV Reverse Transcriptase(USB Corporation,Cleveland, Ohio,USA)were used forfirst strand synthesis.Target fragments were quantified by real-time PCR and an Opticon™2system(MJ Research,Waltham,MA,USA).Real-time PCR Master Mix Reagent kit was obtained from Applied Biosystems and PCR reactions were set up as described in the manual.A typical reaction contained 200nmol/l of forward and reverse primer,2l l cDNA and thefinal reaction volume was20l l.The reaction was initiated by preheat-ing at50°C for2min,followed by heating at95°C for10min.Sub-sequently,45amplification cycles were then carried out with15s denaturation at95°C and1min annealing and extension at58°C.2.5.Preparation of CYP19promoter-driven reporter plasmidsA human CYP19gene fragment(556/+10)upstream to exon II encompassing promoters I.3and II was amplified from genomic DNA and subcloned into afirefly luciferase reporter vector pGL3 basic(Promega Corp,Madison,WI,USA).Truncation plasmids were constructed with this CYP19gene fragment as previously described (Ye et al.,2009).2.6.Luciferase gene reporter assayMCF-7cells were plated in24-well dishes.After allowing24h for attachment followed by6h serum-free incubation,the cells were transiently transfected with0.25l g of the CYP19reporter plasmid and2.0ng of renilla luciferase control vector pRL(Prome-ga,Madison,WI,USA)in LipofectAmine reagent(Invitrogen Life Technologies).After1day,the medium was removed and the cells were treated with luteolin or hesperetin for24h.The cells were lysed and the activities of the luciferases were determined using Dual-Luciferase Assay Kit(Promega).The luciferase biolumines-cence was quantified by using a FLUOstar Galaxy plate reader (BMG Labtechnologies GmBH,Offenburg,Germany).The transac-tivation activities of the CYP19promoter represented byfirefly luciferase light units were then normalized with that of renilla luciferase.2.7.Electrophoretic mobility shift assay(EMSA)After the truncation reporter gene analysis,C/EBP and CRE/AP1 sites were identified to be potential transcriptional factors in-volved in the regulation using the computer software MATCH™(Biological Databases GmbH,Wolfenbuttel,Germany).EMSA was employed to examine the DNA binding.Nuclear protein extract was isolated by using NucBuster™protein extraction kit(Nova-genÒ,EMD Biosciences,Inc.,La Jolla,CA,USA.).In brief,cells were washed,trypsinised,and packed at500g at4°C.Reagent1was added to the packed cells.Nuclear extract was isolated from the cell suspension by vortexing and centrifugation.The nuclear protein was stored atÀ80°C until it was assayed.Two oligonucle-otides encompassing promoter PII(À66/À37)and(À92/À57)were synthesized.The sequences were shown as below,and were labeled by DIG Gel Shift Kit,2nd Generation(Roche Diagnostics GmbH).C/EBP(-66/-37):50-CATAAATTAGTCTTGCCTAAATGTCTGATCACAT-30 AP1(-92/-57):50-TGGGCTTCCTTGTTTTGACTTGTAACCATAAATTA-30 The nuclear protein was thawed and incubated with the labeled probe,sonicated salmon sperm DNA,poly(dI-dC),and binding buf-fer(400mm KCl,80mm HEPES,2mm DTT,0Á8mm EDTA,80% glycerol,pH8)provided in electrophoretic mobility shift assay accessory kit(Novagen)for30min at room temperature.For testing the band specificity,antibodies against C/EBP or c-Jun (Santa Cruz Biotechnology)was co-incubated with the reaction. The reaction mix was then separated on a4–6%non-denaturing gel in0Á5ÂTris-borate EDTA at100V.The labeled oligonucleotide was electro-transferred to a Nylon membrane,fixed by uv light, blocked and washed.The shifted oligonucleotide was detected by anti-Digoxigenin-AP conjugate and the chemiluminescent sub-strate CSPDÒprovided in the kit.2.8.Western analysisCells were washed once by PBS(pH7.4)and harvested into a 1.5ml microtube with0.5ml lysis buffer(PBS,1%NP40,0.5% sodium deoxycholate,0.1%SDS).The lysis buffer contained protease inhibitors(40mg/L PMSF,0.5mg/L aprotinin,0.5mg/L leupeptin, 1.1mmol/L EDTA and0.7mg/L pepstatin)and PhosSTOP phosphatase inhibitor cocktail tablet(Roche Diagnos-tics GmbH,Penzberg,Germany)was added.The harvested cells were then lysed with a cell disruptor(Branson Ultrasonics Corp., Danbury,CT,USA)on ice for30s.The protein concentration of cell lysate was determined by Dc protein assay(BioRad, Richmond,CA,USA).50l g of lysate protein was separated on 10%SDS–PAGE and transferred onto an Immobilon PVDF mem-brane(Millipore,Bedford,MA,USA).Anti-phospho-ERK-1/2, anti-phospho-JNK,anti-phospho-MEK-1/2,anti-phospho-P38 (Santa Cruz Biotechnology,Santa Cruz,CA,USA),anti-actin primary(Sigma Chem)and secondary antibodies conjugated with horseradish peroxidase(Santa Cruz Biotechnology)were used for protein detection.An ECL Detection Kit(Amersham, Arlington Heights,IL,USA)provided the chemiluminescence substrate for HRP,and the targeted protein was visualized by autoradiography.2.9.Statistical analysisThe data were analyzed by the software package Prizm5Ò(GraphPad Software,Inc.,San Diego,CA,USA).One Way ANOVA with Tukey’s Multiple Comparison Test was used for comparison.F.Li et al./Molecular and Cellular Endocrinology344(2011)51–585354 F.Li et al./Molecular and Cellular Endocrinology344(2011)51–583.Results3.1.Enzyme inhibition assay performed on recombinant proteinApigenin,luteolin,hesperetin and naringenin inhibited aroma-tase with IC50values ranging from0.7to4l M(Fig.2).Apigenin appeared to the most potent among theflavones andflavonones tested.3.2.Differential effects on aromatase mRNA expression in MCF-7cellsMessenger RNA abundance of CYP19was quantified under the treatment of theflavones andflavonones.Luteolin at or above 1l M significantly(P<0.05)reduced the transcript numbers, whereas hesperetin increased(P<0.05)the expression at or above 10l M.Bothflavonoids displayed dose-dependent relationship.No changes were observed in apigenin or naringenin-treated cultures (Fig.3).3.3.Enzyme activity assay in MCF-7cellsSince luteolin and hesperetin might affect aromatase expression as shown above,we proceeded to assay the related enzyme activ-ities.Consistent to the mRNA expression results,hesperetin in-creased the aromatase activity(Fig.4A)and the reverse was true in luteolin-treated cultures after48-h incubation(Fig.4B).3.4.Promoter I.3/II-driven CYP19transactivity in MCF-7cellsFollowing up with the mRNA results,we subsequently deter-mined the transcriptional activity driven by promoters I.3and II. 20l M hesperetin induced the transactivation(Fig.5A)while lute-olin suppressed the same at P5l M(Fig.5B).These results agreed to the mRNA expression patterns.3.5.Truncation CYP19gene reporter assayContrasting to the signal in cells transfected with immediate shorter construct(0/+10),the transactivity in cells transfected with (À56/+10)-driven reporter plasmids was induced by hesperetin (Fig.6A)or suppressed by luteolin(Fig.6B).This indicated that the transcriptional factor binding within(À56/0)might be acti-vated or deactivated upon theflavonoid treatment.The increased or reduced transactivity could explain the induction or suppression on the mRNA expression of CYP19.3.6.Effects of hesperetin and luteolin on C/EBP and AP-1binding in Promoter IIFrom the result of truncation reporter gene assay described above,we scanned through(À56/0)of promoter II of CYP19using the software MATCH™,one C/EBP binding site and one AP-1bind-ing site were located in the segment(À56/0).EMSA assay wasF.Li et al./Molecular and Cellular Endocrinology344(2011)51–5855performed to verify the functionality of the binding.Nuclear ex-tracts isolated from cultures treated with hesperetin displayed an increasing trend of CCAAT/enhancer binding proteins(C/EBP)bind-ing(Fig.7A)with null effect on AP-1binding(data not shown).On the other hand,luteolin-treated nuclear extracts exhibited a declining trend of Activated protein-1(AP-1)binding(Fig.7B). No such effect was observed in C/EBP binding(data not shown). The band specificity was demonstrated by the weakened intensity upon incubating with the antibody of C/EBP or c-Jun.3.7.Protein kinase signalingSince the transcriptional factors C/EBP and AP-1could be regu-lated by phosphorylation,we investigated the activation states of the related signaling protein kinases by western blot analysis.Hesperetin appeared to increase pERK-1and-2(Fig.8A),while luteolin reduced p-JNK(Fig.8B).These were the potential signaling pathways involved in the induction and suppression of CYP19tran-scriptional activities.4.DiscussionFormer studies have demonstrated thatflavones andflavonones are inhibitors to aromatase at the enzyme level with comparable potencies.The effect on aromatase activity in cells can occur at the enzyme level and at the transcriptional level.In the present study,we verified the inhibition of aromatase activity of these flavonoids at the enzyme level by using recombinant aromatase. We also demonstrated that at least two within theflavonoiddifferentially shifted the PI.3/II oligomers containing AP-1and C/EBP.MCF-7cells were cultured in T-75luteolin.Nuclear extracts were isolated and used for electromobility shift assays.Fig.7A illustrates the band incubating with DIG-labeled PII(-66/-37)nes0,0.5,1,5,10,20are the respective concentrations(treated with20l M.Fig.7B is an illustration for luteolin incubating with DIG-labelled PII(À92/À57)oligonucleotide.0+AB,c-Jun antibody and extract from control cells.The autoradiograph is a representation of three independentthe supershift band.56 F.Li et al./Molecular and Cellular Endocrinology344(2011)51–58subclasses could differentially regulate aromatase at the transcrip-tional level.Luteolin suppressed while hesperetin induced the transcription.The 30-OH group separates the structure between luteolin and apigenin.Given the differential results observed be-tween these two flavones,the hydroxyl group should be solely contributing to the downregulation of aromatase expression.On the other hand,attachment position of the B-ring may also matter.With the B-ring attached to C3instead of C2position as in apigenin,genistein could suppress the expression at around 12.5l M (Wang et al.,2008).Hesperetin,on the other hand,has two structural differences when compared to naringenin.Hesperetin has a 30-OH group and a methoxyl group instead of hydroxyl group at the 40position.An analogous structure disparity at the 40position is seen in bioch-anin A and genistein.Our lab has previouisly shown that biochanin A has similar potency to genistein in suppressing CYP19expression (Wang et al.,2008).It is still unclear that either one of the 30and 40structures or a combination of the two is responsible for the induc-tion of expression.Alternatively,the transcription might be regu-lated by metabolites of these flavonoids.Human aromatase gene is regulated by alternate splicing and is tissue-specific.Previous study has identified that the transcription of CYP19in breast cancer cells is controlled by Promoters I.3and II (Chen et al.,1999).Hesperetin induced the transactivity of these promoters,while luteolin suppressed the transactivation in MCF-7cells.This attenuation of CYP19expression is shared by resveratrol (Wang et al.,2006)biochanin A (Wang et al.,2008)and isoliquiritigenin (Ye et al.,2009).Luteolin appears to be more potent than resveratrol and biochanin A,whereas isoliquiritigenin could suppress the expression with a comparable strength.Isoli-quiritigenin blocks the transcription by reducing ERK phosphoryla-tion.In contrast,luteolin might suppress the same by way of JNK inactivation in the present study.These might provide alternative strategies for targeting aromatase in breast cells.This project isolated two transcriptional factors that were important in the CYP19regulation,i.e.C/EBP and AP-1.As reviewed by Grimm and Rosen (Grimm and Rosen,2003),C/EBP isoforms can be crucial in the development of breast cancer.Previous study has shown that C/EBP d can stimulate PI.3/II-driven transcription of aromatase in breast cancer cells (Kijima et al.,2008).In the present study,we identified that C/EBP binding in the PII segment was in-duced by hesperetin.This activation might be introduced through pERK,since mitogen-activated protein kinases are able to phos-phorylate and activate C/EBP (Li et al.,2007).Yet,no alterationwas seen in the upstream protein kinase pMEK.The increased pERK could be the result of augmented expression.AP-1is also a transcription factor that has been intensively studied and Shaulian (Shaulian,2010)has reviewed its role in cancer.Luteolin,on the other hand,suppressed the aromatase expression through AP-1.The signaling pathway appeared to be executed by way of JNK.In view of the differential effects on aromatase expression,lute-olin seems to be the most potent suppressor among the com-pounds tested.For the signal transduction events,Choi and Lee,2010have also demonstrated that p38MAPK and JNK are inacti-vated by luteolin in interleukin-1-induced synovial sarcoma cells SW982.Regarding its action on breast cancer,luteolin has been re-ported to be sensitizing MCF-7cells to doxorubicin treatment by reversing the drug resistance at hypoxic condition (Du et al.,2008).Administration of luteolin in combination of cyclophospha-mide can reduce the tumor size and number in 7,12-dimethyl-benz[a]anthracene-initiated mammary carcinogenesis in rats (Samy et al.,2006).After oral dosing with 50and 15mg/kg of the respective flavo-noids,the maximum plasma concentrations of hesperetin and lute-olin are 0.6l M (Matsumoto et al.,2004)and 3l M (Shimoi et al.,1998).In the present study,luteolin could initiate its effect starting as low as 1l M.Given the physiologically relevant concentration in inhibiting the enzyme activity and suppressing the transcription of aromatase,luteolin could be one of the most promising flavonoids in targeting aromatase.Further study using an animal model for testing is warranted.In contrast,the induction of CYP19expression observed in hesperetin would less likely to occur in vivo.In summary,the present study illustrated that dietary flavones and flavonones could regulate aromatase expression differently in breast cancer cells MCF-7.Effect on the transcription level might be important when targeting aromatase in therapeutic application.Conflict of interest statement None declared.AcknowledgmentThis work was supported by Chinese University of Hong Kong Direct Grant Project No.2030430.aromatase and signaling protein kinases in MCF-7cells treated with luteolin and hesperetin.MCF-7cells were seeded in 6-well hesperetin (Fig.8A)or luteolin (Fig.8B)for 24h.Phosphorylated forms of ERK-1/2,P38and JNK in cell lysates were determined by western hesperetin-treated lysates because ERK was found activated.There were triplicates of samples in each experiment.The image is experiments with similar 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