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The red clover (Trifolium pratense) isoflavone biochanin A inhibits aromatase activity and expression

Published online by Cambridge University Press:  01 February 2008

Yun Wang
Affiliation:
Department of Biochemistry, The Chinese University of Hong Kong, Shatin NT, Hong Kong
Wai Man Gho
Affiliation:
Department of Biochemistry, The Chinese University of Hong Kong, Shatin NT, Hong Kong
Franky L. Chan
Affiliation:
Department of Anatomy, The Chinese University of Hong Kong, Shatin NT, Hong Kong
Shiuan Chen
Affiliation:
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA
Lai K. Leung*
Affiliation:
Department of Biochemistry, The Chinese University of Hong Kong, Shatin NT, Hong Kong Food and Nutritional Sciences Programme, The Chinese University of Hong Kong, Shatin NT, Hong Kong
*
*Corresponding author: Dr Lai K. Leung, fax +852 26037732, email laikleung@cuhk.edu.hk
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Abstract

Biochanin A is an isoflavone isolated from red clover (Trifolium pratense), and is a commercially available nutraceutical for women suffering from postmenopausal symptoms. Isoflavones resemble the structure of oestrogen, and display agonistic and antagonistic interactions with the oestrogen receptor. Overexposure of oestrogen is a major contributing factor in the development of breast cancer, and cytochrome P450 (CYP) 19 enzyme, or aromatase, catalyses the reaction converting androgen to oestrogen. In the present study the effect of biochanin A on the gene regulation and enzyme activity of aromatase was investigated. By assaying MCF-7 cells stably transfected with CYP19, biochanin A inhibited aromatase activity and hampered cell growth attributing to the enzyme activity. In addition, 25 μm-biochanin A significantly reduced CYP19 mRNA abundance in the oestrogen receptor-negative breast cancer cells SK-BR-3. The transcriptional control of the CYP19 gene is exon-specific, and promoter regions I.3 and II have been shown to be responsible for CYP19 expression in SK-BR-3 cells. Luciferase reporter gene assays also revealed that biochanin A could repress the transcriptional control dictated by the promoter regulation. Interestingly, genistein did not inhibit aromatase but it might down regulate promoter I.3 and II transactivation. Since genistein is a major metabolite of biochanin A, it might contribute to biochanin A's suppressive effect on CYP19 expression. The present study illustrated that biochanin A inhibited CYP19 activity and gene expression.

Type
Full Papers
Copyright
Copyright © The Authors 2007

Epidemiological studies have shown that the use of exogenous oestrogen1, Reference Pike and Spicer2 or an augmented endogenous oestrogen concentrationReference Toniolo, Levitz, Zeleniuch-Jacquotte, Banerjee, Koenig, Shore, Strax and Pasternack3, Reference Adlercreutz, Gorbach, Goldin, Woods, Dwyer and Hamalainen4 is associated with increased breast cancer risk. In both cell and animal models a causal relationship between oestrogen exposure and breast cancer has also been establishedReference Colditz5.

The cancer-inducing mechanisms of oestrogen in the breast can be multifaceted, and may participate in either the initiation or promotion stage. Oestrogen can be metabolised into various catechol oestrogens, and oestrogen-2-hydroxylase and oestrogen-4-hydroxylase are cytochrome P450 (CYP) enzymes that hydroxylate oestrogen at the C-2 and C-4 positions, respectivelyReference Liehr6. These hydroxylated metabolites can further be converted into quinone and semiquinone structures, which have been shown to be carcinogenic in animal modelsReference Liehr, Fang, Sirbasku and Ari-Ulubelen7, Reference Li and Li8. In addition, free radicals generated by some of these metabolites may cause oxidative DNA damageReference Zhu and Conney9. These genotoxic effects of oestrogen have been demonstrated in MCF-7 cellsReference Yared, McMillan and Martin10 and rat mammary tissuesReference Zhang, Swanson, van Breemen, Liu, Yang, Gu and Bolton11.

The notion that oestrogen promotes breast cancer is reinforced in a transgenic mouse model that develops spontaneous mammary tumours. Treatment with oestrogen accelerates the development of neoplastic lesions and carcinomas in these miceReference Yoshidome, Shibata, Couldrey, Korach and Green12. Oestrogen-induced cell proliferation has been a major focus in breast cancer research. The pertained mechanisms lie in the regulation of cell-cycleReference Tsai and O'Malley13, Reference Dickson, Kasid, Huff, Bates, Knabbe, Bronzert, Gelmann and Lippman14 Bcl-2 family protein expressionReference Leung and Wang15, and the interaction with plasma membrane receptorsReference Watson, Norfleet, Pappas and Gametchu16.

Oestrogen is synthesised from cholesterol in several steps, and CYP19 (aromatase) catalyses the final rate-limiting reaction. Aromatase is encoded by a single-copy geneReference Means, Mahendroo, Corbin, Mathis, Powell, Mendelson and Simpson17, Reference Toda, Terashima and Kawamoto18. The promoter utilisation for CYP19 regulation varies in different tissues, which provides the basis for tissue-specific expressionReference Harada, Yoshimura and Honda19. Polymorphisms in the CYP19 gene have been associated with breast cancer riskReference Lee, Abel and Ko20. Many aromatase inhibitors have recently been developed, and some of them are promising agents for breast cancer prevention and therapyReference Cuzick21.

Some flavones have been documented to be aromatase inhibitors. The A and C rings of flavones may compete with the C and D rings of the androgen structure for binding to the active siteReference Jeong, Shin, Kim and Pezzuto22. Isoflavones are another class of flavonoids whose chemical structures highly resemble that of flavones. Nevertheless, biochanin A is the only aromatase-inhibitory isoflavone with reported 50 % inhibitory concentration (IC50) values varying from about 10 μm to 113 μmReference Jeong, Shin, Kim and Pezzuto22Reference Brueggemeier, Hackett and Diaz-Cruz25. We would like to examine and clarify the CYP19 inhibitory potential of the isoflavone and its effect on mRNA expression specifically driven by promoters I.3 and II in the present study. Since promoters I.3 and II are typically employed in breast cancerous tissuesReference Chen, Zhou, Okubo, Kao and Yang26, suppression on these promoters would halt oestrogen supply for their growth and development.

Materials and methods

Chemicals

Biochanin A was obtained from Sigma Chemicals (St Louis, MO, USA). All chemicals, if not stated, were purchased from Sigma Chemicals.

Cell culture

The breast cancer cell line SK-BR-3 was a generous gift from Dr Richard K. W. Choy (Obstetrics and Gynaecology Department, the Chinese University of Hong Kong, Kowloon, Hong Kong) and MCF-7 cells were obtained from the American Tissue Culture Collection (Rockville, MD, USA). MCF-7 cells stably transfected with human CYP19 (MCF-7aro) were prepared as previously describedReference Zhou, Pompon and Chen27.

The stably transfected MCF-7 cells were maintained in Eagle's minimum essential medium (Invitrogen, Grand Island, NY, USA) supplemented with 10 % fetal bovine serum (Invitrogen Life Technology, Rockville, MD, USA) and the selective antibiotic G418 (500 μg/ml; USB, Cleveland, OH, USA). SK-BR-3 cells were cultured in McCoy's 5A medium (Sigma Chemicals) with 10 % fetal bovine serum. Cells were incubated at 37°C and 5 % carbon dioxide, and were routinely sub-cultured when reaching 80 % of confluency. Biochanin A was administered in the solvent vehicle dimethyl sulfoxide, and the concentration was limited to 0·1 % (v/v). Cells were seeded uniformly at a density of 5 × 102 cells/mm2 in all experiments.

‘In-cell’ aromatase assays

The assays were performed as previously describedReference Grube, Eng, Kao, Kwon and Chen28. In brief, MCF-7aro cells were seeded and allowed 1 d for attachment. Assays were started by replacing the culture medium with serum-free medium containing [1β-3H]androstenedione and biochanin A. The final concentration of androstenedione was controlled at 25 nm, and the reaction was incubated at 37°C for 1 h. A sample of the medium was then mixed with an equal volume of chloroform, followed by a 10 000 g centrifugation at 4°C for 10 min. The aqueous phase was removed into a new tube containing 500 μl of 5 % activated charcoal suspension. After 30 min incubation, a sample of the supernatant fraction was taken out for scintillation counting. The protein content of the cells, on the other hand, was determined by using a BCA kit (Sigma Chemicals) after dissolving the cells in 0·5 m-NaOH.

A similar protocol was applied to assays performed on SK-BR-3 cells, except that the assays were designed to determine the level of expression as described previouslyReference Wang, Lee, Chan, Chen and Leung29. In brief, the cells were seeded in twelve-well plates at a density of 2 × 105 per well. Biochanin A was administered in the cell cultures and incubated for 24 h before adding the substrate [1β-3H(N)]androst-4-ene-3, 17-dione. The reaction was further incubated at 37°C for 3 h before the assay was performed.

For the enzyme inhibition assays performed on recombinant protein, 2 pmol Supersomes® was incubated with biochanin A and the substrate-containing assay buffer (25 nm-[1β-3H(N)]androst-4-ene-3, 17-dione, 3·3 mm-MgCl2, 100 mm-KH2PO4 (pH 7·4)). The reaction was initiated by the addition of 1·3 mm-NADPH and incubated at 37°C for 15 min.

Quantitative real-time polymerase chain reaction assay

In order to quantify the suppression of mRNA abundance, a cell line with reasonable amount of aromatase expression had to be used. Because aromatase mRNA was barely detectable in wild-type MCF-7 cells, we employed an aromatase-expressing cell line (SK-BR-3) for this assay. The real-time quantitative PCR was carried out as previously described by our laboratoryReference Wang, Lee, Chan, Chen and Leung29. In brief, CYP19 and β-actin cDNA fragments were amplified and cloned into pGEMT-Easy vector (Promega Corp., Madison, WI, USA) as templates for quantifying the absolute amount of mRNA expression. Plasmids containing the respective amplicon – pGEMT-CYP19 and pGEMT-β-actin – were sequenced and stored at − 20°C until use. SK-BR-3 cells were cultured and treated as described earlier. After 24 h 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 at 260 and 280 nm. Total RNA (3 μg), oligo-dT, and M-MLV Reverse Transcriptase (USB Corporation, Cleveland, OH, USA) were used for first strand synthesis. Target fragments were quantified by real-time PCR and an Opticon™ 2 system (MJ Research, Waltham, MA, USA). CYP19 copy number was determined by absolute quantification. A standard curve was constructed by 10-fold serial dilutions from 10 to 107 copies amplified from pGEMT-CYP19 or pGEMT-β-actin. Sample copy number was read from the standard curve. A SYBR green 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 200 nmol/l of forward and reverse primer, 2 μl cDNA and the final reaction volume was 20 μl. The reaction was initiated by preheating at 50°C for 2 min, followed by 95°C for 10 min. Subsequently, forty-five amplification cycles were then carried out with 15 s denaturation at 95°C and 1 min annealing and extension at 58°C. Copies of β-actin RNA were also determined and used for normalisation. The forward and reverse primers designed for CYP19 were 5′–ATC TCT GGA GAG GAA ACA CTC ATTA–3′ and 5′–CTG ACA GAG CTT TCA TAA AGA AGGG–3′; the forward and reverse primers for β-actin were 5′–CAC CAA CTG GGA CGA CAT–3′ and 5′–AGG CGT ACA GGG ATA GCA–3′. Dissociation curve and gel image analysis did not review non-specific amplifications generated from these primers.

Measurement of cell viability

Cell number was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetra-zolium bromide (MTT) staining as described by MosmannReference Mosmann30. Briefly, MCF-7aro cells were seeded in ninety-six-well plates and maintained in Eagle's minimal essential medium supplemented with 10 % charcoal dextran-treated serum (Hyclone, Logan, UT, USA). The cells were allowed 24 h for attachment and they were treated with testosterone and/or biochanin A for 48 h. At the end of the treatment, 50 μl of MTT (1 mg/ml) was added to the cells and incubated at 37°C for 4 h. Cell viability was assessed with respect to the absorbance at 544 nm.

Luciferase gene reporter assay

Construction of CYP19 promoter-driven reporter plasmid

A human CYP19 gene fragment ( − 446/+118) upstream to exon II was amplified from genomic DNA isolated from SK-BR-3 cells. The promoters I.3 and IIReference Chen, Zhou, Okubo, Kao and Yang26 have been reported to be associated with CYP19 expression in breast cancer cells. Primers were designed with the incorporation of KpnII and XhoI restriction sites. The amplified products were then digested and subcloned into a firefly luciferase reporter vector pGL3 basic (Promega Corp.), and the sequences were verified.

Dual luciferase assays

SK-BR-3 cells were plated in twenty-four-well dishes. After 24 h, the cells were transiently transfected with 0·25 μg of the CYP19 reporter plasmid and 2·0 ng of renilla luciferase control vector pRL (Promega Corp.) in LipofectAmine reagent (Invitrogen Life Technologies). After 1 d, the medium was removed and the cells were treated with biochanin A for 24 h. The cells were lysed and the activities of the luciferases were determined using the Dual-Luciferase Assay Kit (Promega Corp.). The luciferase bioluminescence was quantified by using a FLUOstar Galaxy plate reader (BMG Labtechnologies GmBH, Offenburg, Germany). The transactivation activities of the CYP19 promoter represented by firefly luciferase light units were then normalised with that of renilla luciferase.

Western analysis

Cells were washed once by PBS (pH 7·4) and harvested into a 1·5 ml microtube with 0·5 ml lysis buffer (PBS, 1 % NP40, 0·5 % sodium deoxycholate, 0·1 % SDS). The lysis buffer contained protease inhibitors (phenylmethylsulfonyl fluoride (40 mg/l), aprotinin (0·5 mg/l), leupeptin (0·5 mg/l), 1·1 mm-EDTA and pepstatin (0·7 mg/l)). The harvested cells were then lysed with a cell disruptor (Branson Ultrasonics Corp., Danbury, CT, USA) on ice for 30 s. The protein concentration of cell lysate was determined by the Dc protein assay (BioRad, Richmond, CA, USA). Lysate protein (50 μg) was separated on 10 % SDS-PAGE and transferred onto an Immobilon PVDF membrane (Millipore, Bedford, MA, USA). Anti-CYP19 (ABcam plc, Cambridge, UK), anti-actin primary (Sigma Chemicals) and secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were used for protein detection. An ECL Detection Kit (Amersham, Arlington Heights, IL, USA) provided the chemiluminescence substrate for horseradish peroxidase, and the targeted protein was visualised by autoradiography.

Statistical methods

A Prism® 3·0 (GraphPad Software, Inc., CA, USA) software package was utilised for statistical analysis. The results were analysed by one-way ANOVA followed by Bonferroni's multiple comparison test if significant differences (P < 0·05) were observed. The t test was employed for comparison of the means between biochanin A-treated and control cultures. Another software package, SigmaPlot (SPSS Inc., Chicago, IL, USA), was used for graphing the Lineweaver–Burk plots.

Results

Biochanin A inhibited aromatase enzymic activity

Enzyme inhibition assay performed on MCF-7aro cells and recombinant protein

A previous studyReference Wang, Chan, Chen and Leung31 has shown that MCF-7aro cells can be used for enzyme inhibition analysis. Biochanin A displayed an inhibitory effect with an IC50 value of about 8 μm in the MCF-7aro cells (Fig. 1 (A)). No significant drop in activity was observed in other isoflavones. The enzyme inhibition was further confirmed in the recombinant enzyme system (human CYP19 Supersomes®; BD Gentest, Woburn, MA, USA) and the IC50 value was determined to be 12·5 μm (Fig. 1 (B)).

Fig. 1 Inhibitory effect of isoflavones (equol (▾), genistein (♦), daidzein (●) and biochanin A (■)) on cytochrome P450 (CYP) 19 enzyme activity. MCF-7aro cells were maintained in Eagle's minimum essential medium and switched to a serum-free medium upon assay. [1β-3H]androstenedione and isoflavone were administered and incubated for 1 h. Biochanin A was able to inhibit the enzyme at the range of concentrations tested, and the 50 % inhibitory concentration (IC50) value was 8 μm (Fig. 1 (A)). Assays performed in CYP19 recombinant protein also displayed similar inhibition with an IC50 value of 12·5 μm (Fig. 1 (B)). Values are means (n 3), with their standard errors represeneted by vertical bars.

Enzyme kinetic assay

Five concentrations, i.e. 0, 6·25, 12·5, 25 and 50 μm-biochanin A, were selected for kinetic analysis. A Lineweaver–Burk plot showed that biochanin A had a mixed type of inhibition on CYP19 with a K i value of 10·8 μm in MCF-7aro cells (Fig. 2).

Fig. 2 Kinetic analysis of biochanin A inhibition on cytochrome P450 (CYP) 19. MCF-7aro cells were cultured and assayed for aromatase activity. Five concentrations of biochanin A (0 (●), 6·25 (○), 12·5 (▾), 25 (⋄) and 50 (■) μm) were co-administered to the cells for the enzyme kinetic assay (A). The Lineweaver–Burk plot (B) showed that biochanin A had a mixed type of inhibition on CYP19 with a i value of 10·8 μm.

Specific inhibition on testosterone-induced proliferation in MCF-7aro cells

Biochanin A was able to reduce the testosterone-induced proliferation of MCF-7aro cells through the inhibition of aromatase (Fig. 3). The administration of 10 nm-testosterone increased the cell number by 67 % as shown at 0 μm-biochanin A. At 12·5 μm, biochanin A could significantly (P < 0·05) reduce the cell proliferation. At 25 μm, biochanin A brought down the testosterone-induced cell growth to a level comparable with their testosterone-less counterparts.

Fig. 3 Effect of biochanin A in reducing testosterone-induced MCF-7aro cell proliferation. MCF-7aro cells were seeded in ninety-six-well plates and maintained in Eagle's minimal essential medium supplemented with 10 % charcoal dextran-treated serum. Cell number was quantified after 48 h under the influence of testosterone administration (10 nm; ■) or no testosterone (0 nm;□). Values are means (n 8), with their standard errors represeneted by vertical bars. Mean value is significantly higher than that of the cultures without testosterone treatment: **P < 0·01, ***P < 0·001. a,b,c Mean values with unlike letters are significantly different (P < 0·05) within the testostesone treatment group.

Biochanin A suppressed CYP19 promoter I.3 and II-driven transactivation

Effect of biochanin A on promoter I.3 and II activity of CYP19 in SK-BR-3 and MCF-7 cells

As the enzyme activity of CYP19 was reduced by biochanin A, we subsequently determined the transcriptional activity driven by promoter regions I.3 and II. We employed the breast cancer cell line SK-BR-3, which had been demonstrated using promoters I.3 and II for CYP19 regulationReference Chen, Zhou, Okubo, Kao and Yang26, for the assessment of promoter activity. At 50 μm, biochanin A was able to repress the promoter activity (Fig. 4 (A)) (P < 0·05). Similar suppression was observed in MCF-7 cells (Fig. 4 (B)), and the down regulation on CYP19 transactivity appeared to be universal for breast cells.

Fig. 4 Biochanin A suppression of cyp19 promoter I.3 and II-driven luciferase activity in SK-BR-3 (A) and MCF-7 cells (B). Cells were seeded in twenty-four-well plates. After 24 h, the cells were transiently transfected with 0·25 μg of the CYP19 reporter plasmid and 2·0 ng renilla luciferase control plasmid and the activities of the luciferases were determined in the cell lysate. Values are means (n 3), with their standard errors represeneted by vertical bars. *Mean value is significantly different from that of the control (P < 0·05).

Biochanin A reduced aromatase mRNA and protein expression in SK-BR-3 cells

Quantitative RT-PCR indicated that the mRNA abundance of aromatase was reduced by biochanin A. Cultures treated with 12·5, 25 and 100 μm-biochanin A revealed significant drops in aromatase expression, and 100 μm of the isoflavone could decrease the expression by more than 80 % (Fig. 5 (A)). Western analysis also revealed a similar pattern (Fig. 5 (B)).

Fig. 5 Messanger RNA (A) and protein expression of aromatase (B) in SK-BR-3 cells treated with biochanin A. SK-BR-3 cells were seeded in six-well plates and maintained in McCoy's 5A medium supplemented with 10 % charcoal dextran-treated serum. Biochanin A was administered to the cultures for 24 h. (A) CYP19 expression result determined by real-time RT-PCR. Values are means (n 3), with their standard errors represeneted by vertical bars. * Mean value is significantly different from that of the control cultures with no biochanin A treatment (P < 0·05).(B) Western analysis of aromatase. The image represents one of two blots with similar results. CYP, cytochrome P450.

Aromatase activity in SK-BR-3 cells treated with biochanin A

Since the mRNA abundance could be suppressed by biochanin A, we measured the aromatase activity as an indicator for reduced expression. After 24 h of treatment the aromatase activity was found to be significantly reduced by 25 μm-biochanin A (Fig. 6).

Fig. 6 Inhibitory effect of biochanin A on cytochrome P450 19 enzyme activity in SK-BR-3 cells. SK-BR-3 cells were seeded in six-well plates and maintained in McCoy's 5A medium supplemented with 10 % charcoal dextran-treated serum. Biochanin A was administered to the cultures for 24 h. The cultures were switched to serum-free medium upon assay. [1β-3H]androstenedione was administered and incubated for 1 h. Significant inhibition was seen at 25 μm and above. The 50 % inhibitory concentration value was determined to be 40 μm. Values are means (n 3), with their standard errors represeneted by vertical bars. *Mean value is significantly different from that of the control (P < 0·05).

A major metabolite of biochanin A – genistein – suppressed CYP19 promoter I.3 and II-driven transactivation

Effect of genistein on promoter I.3 and II activity of CYP19 in SK-BR-3 cells

Genistein is a major metabolite of biochanin A. Since biochanin A was shown to be active in suppressing CYP19 expression over a period of time, genistein might play some part at the transcriptional level. Genistein certainly suppressed promoter I.3 and II transactivity in SK-BR-3 cells, as depicted in Fig. 7 (A). Genistein treatment at 25 μm and above significantly suppressed the luciferase activity. The suppression was further supported by the respective enzyme activity (see Fig. 7 (B)).

Fig. 7 Suppressive effect of genistein on cytochrome P450 19 in SK-BR-3 cells. SK-BR-3 cells were seeded in six-well plates and maintained in McCoy's 5A medium supplemented with 10 % charcoal dextran-treated serum. Genistein was administered to the cultures for 24 h. (A) mRNA expression; (B) aromatase activity. Significant inhibition was seen in both mRNA expression and aromatase activity at 12·5 μm and above. Values are means (n 3), with their standard errors represeneted by vertical bars. *Mean value is significantly different from that of the control (P < 0·05).

Discussion

In the present study, we illustrated that biochanin A was the only aromatase inhibitor among the isoflavones tested. Enzyme kinetic analysis revealed that both competitive and non-competitive inhibitions were involved. Biochanin A could also suppress testosterone-induced MCF-7aro cell proliferation, which was attributed to the reduced aromatase activity. At the transcriptional level, the phytocompound also reduced the aromatase mRNA abundance in the breast cancer cell line SK-BR-3. The promoter utilisation of the human aromatase gene is tissue-specific and promoters I.3 and II have been identified to be responsible for the expression in breast cancer cells including SK-BR-3Reference Chen, Zhou, Okubo, Kao and Yang26. We further demonstrated that the transactivation activity of the gene fragment containing promoters I.3 and II was deactivated by biochanin A, and this suppression could be extended to MCF-7 cells. Genistein, which is a major metabolite of biochanin AReference Peterson, Coward, Kirk, Falany and Barnes32, Reference Heinonen, Wahala and Adlercreutz33, also blocked the transcriptional activity of promoters I.3 and II in SK-BR-3 cells. This implied that the metabolism of biochanin A could still be effective in suppressing CYP19 expression.

Biochanin A at 100 nm and 10 μm was found to be ineffective in inhibiting CYP19 at the enzyme and expression levels in human granulose-luteal cellsReference Rice, Mason and Whitehead34. The differences in treatment concentration and cell type could separate this and the present study. Genistein, on the other hand, displays a similar suppressive effect on CYP19 in the former study. Other phytochemicals have also been reported to be aromatase inhibitors. Extract of red wine inhibits aromatase activityReference Eng, Williams, Mandava, Kirma, Tekmal and Chen35, and reduces mammary hyperplasia in transgenic mice over-expressing CYP19. The active ingredients in the extract could be procyanidin B dimersReference Eng, Ye, Williams, Phung, Moore, Young, Gruntmanis, Braustein and Chen36 and resveratrolReference Wang, Lee, Chan, Chen and Leung29. Chalcones, which are a subclass of flavonoid, display inhibitory actions on aromatase in placental microsomes with IC50 values greater than or equal to 34·6 μmReference Le Bail, Pouget, Fagnere, Basly, Chulia and Habrioux37. Kao et al. Reference Kao, Zhou, Sherman, Laughton and Chen38 have shown that the flavonone naringinin is a stronger inhibitor than the chalcones. In the present study biochanin A was the only isoflavone demonstrated to inhibit the enzyme activity. Given the structural resemblance between biochanin A and genistein, the methyl ether group substitute at the 4′ C position may generate a significant steric hindrance in the active site of the enzyme.

At the transcriptional level, many factors have been described for the regulation of aromatase. Simpson et al. Reference Simpson, Zhao and Agarwal39 have reviewed that cyclic AMP, phorbol esters, dexamethasone, PG E2, transforming growth factor-β, and γ-interferon increase the transcriptional activity, whereas cyclo-oxygenase inhibitors suppress the mRNA expressionReference Diaz-Cruz, Shapiro and Brueggemeier40. Kinoshita & ChenReference Kinoshita and Chen41 have previously reported that mitogen-activated protein kinase inhibitor may reduce CYP19 transcription in breast cells, and biochanin A may inhibit mitogen-activated protein kinase in a different cell systemReference Vanden Berghe, Dijsselbloem, Vermeulen, Ndlovu, Boone and Haegeman42. This could also be a potential deactivating pathway in CYP19 transcription.

Many studies have documented biochanin A's chemopreventive effect on breast cancer. The isoflavone can protect against nitrosomethylurea-induced mammary carcinogenesis in ratsReference Gotoh, Yamada, Yin, Ito, Kataoka and Dohi43, and mammary tumour virus-induced spontaneous breast cancer in miceReference Mizunuma, Kanazawa, Ogura, Otsuka and Nagai44. In the context of drug or xenobiotic metabolism, biochanin A also inhibits CYP1Reference Chan, Wang and Leung45 and induces UDP-glucuronosyltransferaseReference Sun, Plouzek, Henry, Wang and Phang46 enzyme activities. The results of the present study provided a possible chemoprotective pathway for the isoflavone.

The biological relevance of biochanin A as a nutraceutical for preventing breast cancer has yet to be established. Like genistein, it exhibits biphasic effects on mammary cell proliferation. The phytochemical is growth inhibitory to human mammary epithelial cells and MCF-7 cells with IC50 values of about 20 μm after a 4 d incubation periodReference Peterson, Coward, Kirk, Falany and Barnes32, whereas it is growth stimulatory at a half maximal effective concentration (EC50) value of 9 nm after incubation for 6 dReference Van Meeuwen, Korthagen, de Jong, Piersma and van den Berg24. In the present study, biochanin A had no significant effect on MCF-7aro cell proliferation in the testosterone-less treatment group after a 24 h incubation period.

In human subjectsReference Heinonen, Wahala and Adlercreutz33 and the MCF-7 cell modelReference Peterson, Coward, Kirk, Falany and Barnes32, the phytocompound can be metabolised into genistein, biochanin A conjugates or hydroxy metabolites. Assuming the metabolites have an effect comparable with biochanin A, an oral dosage of 50 mg/kg could be able to sustain an aromatase-suppressing plasma concentration with respect to a pharmacokinetic study performed in ratsReference Moon, Sagawa, Frederick, Zhang and Morris47.

In summary, the present study suggested that biochanin A inhibited the enzyme activity and suppressed the transcriptional control of CYP19 in breast cancer cells.

References

1International Agency for Research on Cancer (1999) Hormonal Contraception and Post-menopausal Hormonal Therapy. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 72, Lyon, France: IARC Press.Google Scholar
2Pike, MC & Spicer, DV (1993) The chemoprevention of breast cancer by reducing sex steroid exposure: perspectives from epidemiology. J Cell Biochem Suppl 17G, 2636.CrossRefGoogle ScholarPubMed
3Toniolo, PG, Levitz, M, Zeleniuch-Jacquotte, A, Banerjee, S, Koenig, KL, Shore, RE, Strax, P & Pasternack, BS (1995) A prospective study of endogenous estrogens and breast cancer in postmenopausal women. J Natl Cancer Inst 87, 190197.Google Scholar
4Adlercreutz, H, Gorbach, SL, Goldin, BR, Woods, MN, Dwyer, JT & Hamalainen, E (1994) Estrogen metabolism and excretion in oriental and Caucasian women. J Natl Cancer Inst 86, 10761082.CrossRefGoogle ScholarPubMed
5Colditz, GA (1999) Hormones and breast cancer: evidence and implications for consideration of risks and benefits of hormone replacement therapy. J Womens Health 8, 347357.CrossRefGoogle ScholarPubMed
6Liehr, JG (2000) Is estradiol a genotoxic mutagenic carcinogen? Endocr Rev 21, 4054.Google ScholarPubMed
7Liehr, JG, Fang, WF, Sirbasku, DA & Ari-Ulubelen, A (1986) Carcinogenicity of catechol estrogens in Syrian hamsters. J Steroid Biochem 24, 353356.Google Scholar
8Li, JJ & Li, SA (1987) Estrogen carcinogenesis in Syrian hamster tissues: role of metabolism. Fed Proc 46, 18581863.Google ScholarPubMed
9Zhu, BT & Conney, AH (1998) Functional role of estrogen metabolism in target cells: review and perspectives. Carcinogenesis 19, 127.CrossRefGoogle ScholarPubMed
10Yared, E, McMillan, TJ & Martin, FL (2002) Genotoxic effects of oestrogens in breast cells detected by the micronucleus assay and the Comet assay. Mutagenesis 17, 345352.Google Scholar
11Zhang, F, Swanson, SM, van Breemen, RB, Liu, X, Yang, Y, Gu, C & Bolton, JL (2001) Equine estrogen metabolite 4-hydroxyequilenin induces DNA damage in the rat mammary tissues: formation of single-strand breaks, apurinic sites, stable adducts, and oxidized bases. Chem Res Toxicol 14, 16541659.CrossRefGoogle ScholarPubMed
12Yoshidome, K, Shibata, MA, Couldrey, C, Korach, KS & Green, JE (2000) Estrogen promotes mammary tumour development in C3(1)/SV40 large T-antigen transgenic mice: paradoxical loss of estrogen receptor (expression during tumour progression. Cancer Res 60, 69016910.Google Scholar
13Tsai, MJ & O'Malley, BW (1994) Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Ann Rev Biochem 63, 451486.CrossRefGoogle ScholarPubMed
14Dickson, RB, Kasid, A, Huff, KK, Bates, SE, Knabbe, C, Bronzert, D, Gelmann, EP & Lippman, ME (1987) Activation of growth factor secretion in tumorigenic states of breast cancer induced by 17 β-estradiol or v-Ha-ras oncogene. Proc Natl Acad Sci USA 84, 837841.Google Scholar
15Leung, LK & Wang, TT (1999) Paradoxical regulation of Bcl-2 family proteins by 17β-oestradiol in human breast cancer cells MCF-7. Br J Cancer 81, 387392.CrossRefGoogle ScholarPubMed
16Watson, CS, Norfleet, AM, Pappas, TC & Gametchu, B (1999) Rapid actions of estrogens in GH3/B6 pituitary tumour cells via a plasma membrane version of estrogen receptor-α. Steroids 64, 513.CrossRefGoogle Scholar
17Means, GD, Mahendroo, MS, Corbin, CJ, Mathis, JM, Powell, FE, Mendelson, CR & Simpson, ER (1989) Structural analysis of the gene encoding human aromatase cytochrome P-450, the enzyme responsible for estrogen biosynthesis. J Biol Chem 264, 1938519391.CrossRefGoogle ScholarPubMed
18Toda, K, Terashima, M, Kawamoto, T, et al. (1990) Structural and functional characterization of human aromatase P-450 gene. Eur J Biochem 193, 559565.CrossRefGoogle ScholarPubMed
19Harada, N, Yoshimura, N & Honda, S (2003) Unique regulation of expression of human aromatase in the placenta. J Steroid Biochem Mol Biol 86, 327334.Google Scholar
20Lee, KM, Abel, J, Ko, Y, et al. (2003) Genetic polymorphisms of cytochrome P450 19 and 1B1, alcohol use, and breast cancer risk in Korean women. Br J Cancer 88, 675678.CrossRefGoogle ScholarPubMed
21Cuzick, J (2003) Aromatase inhibitors in prevention – data from the ATAC (arimidex, tamoxifen alone or in combination) trial and the design of IBIS-II (the second International Breast Cancer Intervention Study). Recent Results Cancer Res 163, 96-103, 264266.Google Scholar
22Jeong, H-J, Shin, YG, Kim, L-H & Pezzuto, JM (1999) Inhibition of aromatase activity by flavonoids. Arch Pharm Res 22, 309312.Google Scholar
23Kao, YC, Zhou, C, Sherman, M, Laughton, CA & Chen, S (1998) Molecular basis of the inhibition of human aromatase (estrogen synthethase) by flavone and isoflavone phytoestrogens: a site-directed mutagenesis study. Environ Health Perspect 106, 8592.CrossRefGoogle Scholar
24Van Meeuwen, JA, Korthagen, N, de Jong, PC, Piersma, AH & van den Berg, M (2007) (Anti)estrogenic effects of phytochemicals on human primary mammary fibroblasts, MCF-7 cells and their co-culture. Toxicol Appl Pharmacol 221, 372383.CrossRefGoogle ScholarPubMed
25Brueggemeier, RW, Hackett, JC & Diaz-Cruz, ES (2005) Aromatase inhibitors in the treatment of breast cancer. Endocr Reviews 26, 331345.CrossRefGoogle ScholarPubMed
26Chen, S, Zhou, D, Okubo, T, Kao, Y-C & Yang, C (1999) Breast tumor aromatase: functional role and transcriptional regulation. Endocr Relat Cancer 6, 149156.CrossRefGoogle ScholarPubMed
27Zhou, DJ, Pompon, D & Chen, SA (1990) Stable expression of human aromatase complementary DNA in mammalian cells: a useful system for aromatase inhibitor screening. Cancer Res 50, 69496954.Google Scholar
28Grube, BJ, Eng, ET, Kao, YC, Kwon, A & Chen, S (2001) White button mushroom phytochemicals inhibit aromatase activity and breast cancer cell proliferation. J Nutr 131, 32883293.CrossRefGoogle ScholarPubMed
29Wang, Y, Lee, KW, Chan, FL, Chen, S & Leung, LK (2006) The red wine polyphenol resveratrol displays bi-level inhibition on aromatase in breast cancer cells. Toxicol Sci 92, 7177.CrossRefGoogle Scholar
30Mosmann, T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65, 5563.Google Scholar
31Wang, Y, Chan, FL, Chen, S & Leung, LK (2005) The plant polyphenol butein inhibits testosterone-induced proliferation in breast cancer cells expressing aromatase. Life Sci 77, 3951.Google Scholar
32Peterson, TG, Coward, L, Kirk, M, Falany, CN & Barnes, S (1996) The role of metabolism in mammary epithelial cell growth inhibition by the isoflavones genistein and biochanin A. Carcinogenesis 17, 18611869.CrossRefGoogle ScholarPubMed
33Heinonen, S-M, Wahala, K & Adlercreutz, H (2004) Identification of urinary metabolites of the red clover isoflavones formononetin and biochanin A in human subjects. J Agric Food Chem 52, 68026809.Google Scholar
34Rice, S, Mason, HD & Whitehead, SA (2006) Phytoestrogens and their low dose combinations inhibit mRNA expression and activity of aromatase in human granulose-luteal cells. J Steroid Biochem Mol Biol 101, 216225.Google Scholar
35Eng, ET, Williams, D, Mandava, U, Kirma, N, Tekmal, RR & Chen, S (2001) Suppression of aromatase (estrogen synthetase) by red wine phytochemicals. Breast Cancer Res Treat 67, 133146.Google Scholar
36Eng, ET, Ye, J, Williams, D, Phung, S, Moore, RE, Young, MK, Gruntmanis, U, Braustein, G & Chen, S (2003) Suppression of estrogen biosynthesis by procyanidin dimers in red wine and grape seeds. Cancer Res 63, 85168522.Google Scholar
37Le Bail, JC, Pouget, C, Fagnere, C, Basly, JP, Chulia, AJ & Habrioux, G (2001) Chalcones are potent inhibitors of aromatase and 17(-hydroxysteroid dehydrogenase activities. Life Sci 68, 751761.CrossRefGoogle ScholarPubMed
38Kao, YC, Zhou, C, Sherman, M, Laughton, CA & Chen, S (1998) Molecular basis of the inhibition of human aromatase (estrogen synthetase) by flavone and isoflavone phytoestrogens: a site-directed mutagenesis study. Environ Health Perspect 106, 8592.CrossRefGoogle ScholarPubMed
39Simpson, ER, Zhao, Y, Agarwal, VR, et al. (1997) Aromatase expression in health and disease. Recent Prog Horm Res 52, 185–213213–214.Google Scholar
40Diaz-Cruz, ES, Shapiro, CL & Brueggemeier, RW (2005) Cyclooxygenase inhibitors suppress aromatase expression and activity in breast cancer cells. J Clin Endocrinol Metab 90, 25632570.Google Scholar
41Kinoshita, Y & Chen, S (2003) Induction of aromatase (CYP19) expression in breast cancer cells through a nongenomic action of estrogen receptor α. Cancer Res 63, 35463555.Google Scholar
42Vanden Berghe, W, Dijsselbloem, N, Vermeulen, L, Ndlovu, N, Boone, E & Haegeman, G (2006) Attenuation of mitogen- and stress-activated protein kinase-1-driven nuclear factor-κB gene expression by soy isoflavones does not require estrogenic activity. Cancer Res 66, 48524862.CrossRefGoogle Scholar
43Gotoh, T, Yamada, K, Yin, H, Ito, A, Kataoka, T & Dohi, K (1998) Chemoprevention of N-nitroso-N-methylurea-induced rat mammary carcinogenesis by soy foods or biochanin A. Jpn J Cancer Res 89, 137142.Google Scholar
44Mizunuma, H, Kanazawa, K, Ogura, S, Otsuka, S & Nagai, H (2002) Anticarcinogenic effects of isoflavones may be mediated by genistein in mouse mammary tumor virus-induced breast cancer. Oncology 62, 7884.Google Scholar
45Chan, HY, Wang, H & Leung, LK (2003) The red clover (Trifolium pratense) isoflavone biochanin A modulates the biotransformation pathways of 7,12-dimethylbenz-[a]anthracene. Br J Nutr 90, 8792.Google Scholar
46Sun, XY, Plouzek, CA, Henry, JP, Wang, TT & Phang, JM (1998) Increased UDP-glucuronosyltransferase activity and decreased prostate specific antigen production by biochanin A in prostate cancer cells. Cancer Res 58, 23792384.Google ScholarPubMed
47Moon, YJ, Sagawa, K, Frederick, K, Zhang, S & Morris, ME (2006) Pharmacokinetics and bioavailability of the isoflavone biochanin A in rats. AAPS J 8, E433E442.Google Scholar
Figure 0

Fig. 1 Inhibitory effect of isoflavones (equol (▾), genistein (♦), daidzein (●) and biochanin A (■)) on cytochrome P450 (CYP) 19 enzyme activity. MCF-7aro cells were maintained in Eagle's minimum essential medium and switched to a serum-free medium upon assay. [1β-3H]androstenedione and isoflavone were administered and incubated for 1 h. Biochanin A was able to inhibit the enzyme at the range of concentrations tested, and the 50 % inhibitory concentration (IC50) value was 8 μm (Fig. 1 (A)). Assays performed in CYP19 recombinant protein also displayed similar inhibition with an IC50 value of 12·5 μm (Fig. 1 (B)). Values are means (n 3), with their standard errors represeneted by vertical bars.

Figure 1

Fig. 2 Kinetic analysis of biochanin A inhibition on cytochrome P450 (CYP) 19. MCF-7aro cells were cultured and assayed for aromatase activity. Five concentrations of biochanin A (0 (●), 6·25 (○), 12·5 (▾), 25 (⋄) and 50 (■) μm) were co-administered to the cells for the enzyme kinetic assay (A). The Lineweaver–Burk plot (B) showed that biochanin A had a mixed type of inhibition on CYP19 with a i value of 10·8 μm.

Figure 2

Fig. 3 Effect of biochanin A in reducing testosterone-induced MCF-7aro cell proliferation. MCF-7aro cells were seeded in ninety-six-well plates and maintained in Eagle's minimal essential medium supplemented with 10 % charcoal dextran-treated serum. Cell number was quantified after 48 h under the influence of testosterone administration (10 nm; ■) or no testosterone (0 nm;□). Values are means (n 8), with their standard errors represeneted by vertical bars. Mean value is significantly higher than that of the cultures without testosterone treatment: **P < 0·01, ***P < 0·001. a,b,c Mean values with unlike letters are significantly different (P < 0·05) within the testostesone treatment group.

Figure 3

Fig. 4 Biochanin A suppression of cyp19 promoter I.3 and II-driven luciferase activity in SK-BR-3 (A) and MCF-7 cells (B). Cells were seeded in twenty-four-well plates. After 24 h, the cells were transiently transfected with 0·25 μg of the CYP19 reporter plasmid and 2·0 ng renilla luciferase control plasmid and the activities of the luciferases were determined in the cell lysate. Values are means (n 3), with their standard errors represeneted by vertical bars. *Mean value is significantly different from that of the control (P < 0·05).

Figure 4

Fig. 5 Messanger RNA (A) and protein expression of aromatase (B) in SK-BR-3 cells treated with biochanin A. SK-BR-3 cells were seeded in six-well plates and maintained in McCoy's 5A medium supplemented with 10 % charcoal dextran-treated serum. Biochanin A was administered to the cultures for 24 h. (A) CYP19 expression result determined by real-time RT-PCR. Values are means (n 3), with their standard errors represeneted by vertical bars. * Mean value is significantly different from that of the control cultures with no biochanin A treatment (P < 0·05).(B) Western analysis of aromatase. The image represents one of two blots with similar results. CYP, cytochrome P450.

Figure 5

Fig. 6 Inhibitory effect of biochanin A on cytochrome P450 19 enzyme activity in SK-BR-3 cells. SK-BR-3 cells were seeded in six-well plates and maintained in McCoy's 5A medium supplemented with 10 % charcoal dextran-treated serum. Biochanin A was administered to the cultures for 24 h. The cultures were switched to serum-free medium upon assay. [1β-3H]androstenedione was administered and incubated for 1 h. Significant inhibition was seen at 25 μm and above. The 50 % inhibitory concentration value was determined to be 40 μm. Values are means (n 3), with their standard errors represeneted by vertical bars. *Mean value is significantly different from that of the control (P < 0·05).

Figure 6

Fig. 7 Suppressive effect of genistein on cytochrome P450 19 in SK-BR-3 cells. SK-BR-3 cells were seeded in six-well plates and maintained in McCoy's 5A medium supplemented with 10 % charcoal dextran-treated serum. Genistein was administered to the cultures for 24 h. (A) mRNA expression; (B) aromatase activity. Significant inhibition was seen in both mRNA expression and aromatase activity at 12·5 μm and above. Values are means (n 3), with their standard errors represeneted by vertical bars. *Mean value is significantly different from that of the control (P < 0·05).