Chemico-Biological Interactions AKR1C3 As a Potential Target for The

Chemico-Biological Interactions 178 (2009) 138–144 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint AKR1C3 as a potential target for the inhibitory effect of dietary flavonoids Lucie Skarydováˇ a, Lucie Zivnáˇ a, Guangming Xiong b, Edmund Maser b, Vladimír Wsól a,∗ a Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Heyrovského 1203, CZ-50005 Hradec Králové, Czech Republic b Institute of Toxicology and Pharmacology for Natural Scientists, Campus Kiel, University Medical School Schleswig-Holstein, Brunswiker Str. 10, D-24105 Kiel, Germany article info abstract Article history: AKR1C3 (also known as 17␤-hydroxysteroid dehydrogenase type 5 or 3␣-hydroxysteroid dehydroge- Available online 19 October 2008 nase type 2) functions as a 3-keto, 17-keto and 20-ketosteroid reductase and as a 3␣-, 17␤- and 20␣-hydroxysteroid oxidase. Relatively high mRNA expression of AKR1C3 was found in human prostate Keywords: and mammary gland where it is implicated in regulating ligand access to the androgen and estrogen Aldo-keto reductases (AKRs) receptor, respectively. AKR1C3 is an interesting target for the development of agents for treating hormone- Flavonoids dependent forms of cancer like prostate cancer, breast cancer, and endometrial cancer. However, only a few Oracin clinically promising and selective inhibitors have been reported so far. Very potent inhibitors of AKR1C3 Inhibition Selective inhibitor are the non-steroidal anti-inflammatory drugs, e.g. indomethacin or flufenamic acid. Also dietary phytoestrogens such as coumestrol, quercetin, and biochanin were reported to inhibit the enzyme in low micromolar concentrations. In this study, some dietary flavonoids and other phenolic compounds were tested for their ability to specifically inhibit AKR1C3. Carbonyl reduction of the anticancer drug oracin, which is a very good substrate for AKR1C3 and which could be well monitored by a sensitive HPLC system with fluorescence detection, was employed to determine the inhibitory potency of the compounds. Our results reveal that AKR1C3 could be potentially un-competitively inhibited by 2-hydroxyflavanone, whose IC50 value of 300 nM is clinically promising. Moreover, since the inhibition is selective towards AKR1C3, 2-hydroxyflavanone could be useful for treating or preventing hormone-dependent malignancies like prostate and breast cancer. © 2008 Elsevier Ireland Ltd. All rights reserved. 1. Introduction AKR1C1 acts preferentially as a 20␣-HSD by inactivation of pro- gesterone. It may therefore have a role in the development of breast Carbonyl reducing enzymes can be divided into two distinct and endometrial cancers [8]. AKR1C2 functions as peripheral 3␣- protein superfamilies, the short-chain dehydrogenases/reductases HSD by inactivation of 5␣-dihydrotestosterone [6]. (SDR) and the aldo-keto reductases (AKR) [1,2]. The AKR superfam- A number of enzymatic properties have been assigned to ily represents NAD(P)(H)-dependent oxidoreductases metabolizing AKR1C3 which has been referred to as both 3␣-hydroxysteroid a range of substrates including aliphatic aldehydes, monosaccha- dehydrogenase type 2 and 17␤-hydroxysteroid dehydrogenase type rides, steroids, prostaglandins, polycyclic aromatic hydrocarbons 5 [5]. AKR1C3 works as a 3-ketosteroid reductase (converting the and isoflavonoids [3]. Mammalian AKRs are found predominantly potent androgen 5␣-dihydrotestosterone into the weak andro- in the AKR1 and AKR7 families [4]. gen 3␣-androstanediol), and as a 17-ketoreductase (converting the The AKR1C subfamily members (hydroxysteroid dehydro- weak androgen 4-androstene-3,17-dione into the potent andro- genases, HSDs) function in endogenous steroid or eicosanoid gen testosterone and the weak estrogen estrone to the potent regulation and in xenobiotic metabolism [1,5]. They act predom- estrogen 17␤-estradiol) [7]. AKR1C3 has also been referred to as inantly as ketosteroid reductases by converting potent steroid prostaglandin F2 synthase [9]. hormones into their cognate inactive metabolites and vice versa Since AKR1C3 is predominantly expressed in the breast and have thus a role in the pre-receptor regulation of steroid hor- and prostate, and produces proliferative steroid hormones and mone action [5,6]. Whereas the human isoforms share at least 84% prostaglandins, it may contribute to the growth of prostate amino acid sequence identities, AKR1C1 and AKR1C2 differ by only and breast cancers. AKR1C3 is thus an interesting target for seven amino acids [7]. the development of agents for treating hormone-dependent forms of cancer. However, only a few inhibitors have been reported so far. Very potent inhibitors are indomethacin, N- ∗ Corresponding author. Tel.: +420 739488218. (4-chlorobenzoyl)-melatonin, flufenamic acid and some related E-mail address: [email protected] (V. Wsól). non-steroidal anti-inflammatory drugs [10]. The enzyme can be 0009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2008.10.015 L. Skarydováˇ et al. / Chemico-Biological Interactions 178 (2009) 138–144 139 inhibited also by dietary phytoestrogens (such as coumestrol, 2.2. Cloning, overexpression and purification of recombinant quercetin and biochanin) and the mycoestrogen zearalenone, as AKR1C3 well as by some other compounds like benzodiazepines, benzo- furanes and phenolphthalein derivatives [11]. The generation of recombinant AKR1C3 was performed accord- Phytoestrogens are plant-derived, non-steroidal compounds ing to standard techniques (Sammbrook, Maniatis). In brief, the which can act as agonists or antagonists of estrogen receptors and cDNA for human AKR1C3 was amplified from a commercially avail- they can modulate the activities of the key enzymes in estrogen able full length by PCR. The primers used were designated to anneal biosynthesis. They are structurally divided into four main groups: at the 5- and 3-ends of the coding region and had the following flavonoids, coumestans, stilbens and lignans, where the flavonoids sequences: 5-primer = 5-GGA ATT CCA TAT GGA TTC CAA ACA CCA are classified into at least 10 chemical groups [8,12]. G-3 (containing a NdeI restriction site) and 3-primer = 5-CGC GGA Flavonoids are found in almost all plant families in leaves, TCCTTAATATTCATCTGAATATG-3 (containing a BamHI restriction stems, roots, flowers and seeds. Flavanones, flavones, isoflavonoids, site). The PCR fragment obtained was cloned into pCR2.1-TOPO and flavans, anthocyanins and flavonols are especially common in the sequence confirmed by MWG. The fragment was then cloned the diet. Flavonols are the most abundant flavonoids in foods into the bacterial expression vector pET15b by use of the NdeI (quercetin, kaempferol and myricetin are the three most common and BamHI restriction sites. Overexpression of AKR1C3 was per- flavonols). Flavanones are mainly found in citrus fruits and flavones formed in E. coli strain BL21(DE3) by induction with 1 mM IPTG. For in celery. Catechins are present in large amounts in green and enzyme purification, cells were mechanically disrupted in a French black teas and in red wine. Anthocyanins are found in strawber- press and the cell debris were sedimented by ultracentrifuga- ries and other berries. Isoflavones are almost found in soy foods tion. The supernatant was directly applied to a Ni–agarose affinity [12]. chromatography column of an ÄKTA Purifier system (Pharmacia The biological roles of flavonoids in plants are not fully Biotech). The column was washed with 20 ml of starting buffer and understood. Some of them are thought to act as natural fungi- the His-tagged AKR1C3 was then eluted by using 500 mM imida- cides, UV-protectants and flower pigments [13]. As regular zole supplementation to the buffer. The successful overexpression constituents of the diet, they have some clinically relevant func- and purification was monitored by SDS–PAGE. Enzyme activity was tions: maintenance of capillary wall integrity, capillary resistance, checked with 5␣-androstanedione as the substrate. Active fractions antihypertensive, anti-arrhythmic, anti-inflammatory, anti-allergic containing the purified protein were pooled, supplemented with and hypocholesterolaemic activity, platelet and mast cell stabiliza- glycerol (final concentration 20%) and the protein concentrated to tion, anti-hepatotoxic, anti-fertility and anti-tumor properties [14]. 1.76 mg/ml in a 20 mM phosphate buffer, pH 7.4. Very little is known about the intake of flavonoids from food, as is their bioavailability and metabolism in humans. Their plasma 2.3. Purification of AKR1C1 and AKR1C2 concentration is depending on the diet. Individual flavonoid concentrations in subjects consuming diets similar to the habitual diet Enzyme purification was carried out as described by Breyer- of the general population range from nM to low ␮M. The bioavail- Pfaff and Nill [19]. Human liver samples were either from livers ability of flavonoids in tissues may be much more important than excluded from transplantation for medical reasons or excess nor- their plasma concentration, but these data are still very scarce, even mal tissue obtained on partial hepatectomy for tumor metastases. in animals [15,16]. Briefly, human liver cytosol corresponding to 5–7 g of liver was gel In the present study, we tested several dietary flavonoids and filtrated on a Sephadex G-100 column and active eluates were fur- phenolic compounds for their inhibitory effect towards AKR1C3. ther separated by ion exchange chromatography

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