AKR1C3 as a potential target for the inhibitory effect of dietary flavonoids

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Abstract

AKR1C3 (also known as 17β-hydroxysteroid dehydrogenase type 5 or 3α-hydroxysteroid dehydrogenase type 2) functions as a 3-keto, 17-keto and 20-ketosteroid reductase and as a 3α-, 17β- and 20α-hydroxysteroid oxidase. Relatively high mRNA expression of AKR1C3 was found in human prostate and mammary gland where it is implicated in regulating ligand access to the androgen and estrogen receptor, respectively. AKR1C3 is an interesting target for the development of agents for treating hormone-dependent forms of cancer like prostate cancer, breast cancer, and endometrial cancer. However, only a few clinically promising and selective inhibitors have been reported so far. Very potent inhibitors of AKR1C3 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.

Introduction

Carbonyl reducing enzymes can be divided into two distinct protein superfamilies, the short-chain dehydrogenases/reductases (SDR) and the aldo-keto reductases (AKR) [1], [2]. The AKR superfamily represents NAD(P)(H)-dependent oxidoreductases metabolizing a range of substrates including aliphatic aldehydes, monosaccharides, steroids, prostaglandins, polycyclic aromatic hydrocarbons and isoflavonoids [3]. Mammalian AKRs are found predominantly in the AKR1 and AKR7 families [4].

The AKR1C subfamily members (hydroxysteroid dehydrogenases, HSDs) function in endogenous steroid or eicosanoid regulation and in xenobiotic metabolism [1], [5]. They act predominantly as ketosteroid reductases by converting potent steroid hormones into their cognate inactive metabolites and vice versa and have thus a role in the pre-receptor regulation of steroid hormone action [5], [6]. Whereas the human isoforms share at least 84% amino acid sequence identities, AKR1C1 and AKR1C2 differ by only seven amino acids [7].

AKR1C1 acts preferentially as a 20α-HSD by inactivation of progesterone. It may therefore have a role in the development of breast and endometrial cancers [8]. AKR1C2 functions as peripheral 3α-HSD by inactivation of 5α-dihydrotestosterone [6].

A number of enzymatic properties have been assigned to AKR1C3 which has been referred to as both 3α-hydroxysteroid dehydrogenase type 2 and 17β-hydroxysteroid dehydrogenase type 5 [5]. AKR1C3 works as a 3-ketosteroid reductase (converting the potent androgen 5α-dihydrotestosterone into the weak androgen 3α-androstanediol), and as a 17-ketoreductase (converting the weak androgen Δ4-androstene-3,17-dione into the potent androgen testosterone and the weak estrogen estrone to the potent estrogen 17β-estradiol) [7]. AKR1C3 has also been referred to as prostaglandin F2 synthase [9].

Since AKR1C3 is predominantly expressed in the breast and prostate, and produces proliferative steroid hormones and prostaglandins, it may contribute to the growth of prostate and breast cancers. AKR1C3 is thus an interesting target for 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-(4-chlorobenzoyl)-melatonin, flufenamic acid and some related non-steroidal anti-inflammatory drugs [10]. The enzyme can be inhibited also by dietary phytoestrogens (such as coumestrol, quercetin and biochanin) and the mycoestrogen zearalenone, as well as by some other compounds like benzodiazepines, benzofuranes and phenolphthalein derivatives [11].

Phytoestrogens are plant-derived, non-steroidal compounds which can act as agonists or antagonists of estrogen receptors and they can modulate the activities of the key enzymes in estrogen biosynthesis. They are structurally divided into four main groups: flavonoids, coumestans, stilbens and lignans, where the flavonoids are classified into at least 10 chemical groups [8], [12].

Flavonoids are found in almost all plant families in leaves, stems, roots, flowers and seeds. Flavanones, flavones, isoflavonoids, flavans, anthocyanins and flavonols are especially common in the diet. Flavonols are the most abundant flavonoids in foods (quercetin, kaempferol and myricetin are the three most common flavonols). Flavanones are mainly found in citrus fruits and flavones in celery. Catechins are present in large amounts in green and black teas and in red wine. Anthocyanins are found in strawberries and other berries. Isoflavones are almost found in soy foods [12].

The biological roles of flavonoids in plants are not fully understood. Some of them are thought to act as natural fungicides, UV-protectants and flower pigments [13]. As regular constituents of the diet, they have some clinically relevant functions: maintenance of capillary wall integrity, capillary resistance, antihypertensive, anti-arrhythmic, anti-inflammatory, anti-allergic and hypocholesterolaemic activity, platelet and mast cell stabilization, anti-hepatotoxic, anti-fertility and anti-tumor properties [14]. Very little is known about the intake of flavonoids from food, as is their bioavailability and metabolism in humans. Their plasma concentration is depending on the diet. Individual flavonoid concentrations in subjects consuming diets similar to the habitual diet of the general population range from nM to low μM. The bioavailability of flavonoids in tissues may be much more important than their plasma concentration, but these data are still very scarce, even in animals [15], [16].

In the present study, we tested several dietary flavonoids and phenolic compounds for their inhibitory effect towards AKR1C3. For evaluating their inhibitory potencies, we measured the carbonyl reduction of the anticancer drug oracin as a specific substrate [17], in combination with a very sensitive HPLC method and fluorescence detection [18]. 2′-Hydroxyflavanone was found to be a strong inhibitor of AKR1C3. The low IC50 value together with its selective inhibition of AKR1C3, in comparison to AKR1C1 and AKR1C2, predetermine 2′-hydroxyflavanone as a potential drug for clinical use.

Section snippets

Chemicals

Flavonoids and phenolic compounds (vitexin, isovitexin, 2′-hydroxyflavanone, 4′-hydroxyflavanone, 3-hydroxyflavone, 5-hydroxyflavone, 7-hydroxyflavone, quercetin, caffeic acid, naringenin, chlorogenic acid, rutin, 4-hydroxybenzoic acid, quercitrin, epigallocatechin gallate, cyanin chloride, luteolin, taxifolin, silibinin and apigenin) were obtained from Fluka (Prague, Czech Republic) and Sigma–Aldrich (Prague, Czech Republic). Oracin and DHO (11-dihydrooracin) were provided by the Research

Results

In the present study, several flavonoids and phenolic compounds were tested for their ability to effectively and selectively inhibit human AKR1C3. We chose compounds commonly occurring in the diet: vitexin, isovitexin, 2′-hydroxyflavanone, 4′-hydroxyflavanone, 3-hydroxyflavone, 5-hydroxyflavone, 7-hydroxyflavone, quercetin, caffeic acid, naringenin, chlorogenic acid, rutin, 4-hydroxybenzoic acid, quercitrin, epigallocatechin gallate, cyanin chloride, luteolin, taxifolin, silibinin and apigenin.

Discussion

The human AKR1C isozymes are involved in the pre-receptor regulation of steroid hormone action by regulating the concentration of active and inactive androgens, estrogens and progestins in target tissues [22], [23], [24]. AKR1C isozymes catalyze the reduction of ketosteroids at the C3, C17 or C20 positions [7]. Selective inhibitors of AKR1C isoforms provide tissue-specific effects of steroid hormones [6].

AKR1C3 reduces the weak androgen androstenedione to the potent androgen testosterone and

Acknowledgements

The authors are grateful to Prof. U. Breyer-Pfaff for providing isolated human liver cytosolic enzymes AKR1C1 and AKR1C2. This project was supported by the Grant Agency of Charles University, Grant No. 108/2006/C and the Deutsche Forschungsgemeinschaft (MA 1704/5-1).

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