Chemico-Biological Interactions xxx (2014) xxx–xxx Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint Important roles of the AKR1C2 and SRD5A1 enzymes in progesterone metabolism in endometrial cancer model cell lines ⇑ Maša Sinreih a, Maja Anko a, Sven Zukunft b, Jerzy Adamski b,c,d, Tea Lanišnik Rizˇner a, a Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia b Institute of Experimental Genetics, Genome Analysis Centre, Helmholtz Zentrum München, München, Germany c Lehrstuhl für Experimentelle Genetik, Technische Universität München, 85356 Freising-Weihenstephan, Germany d German Centre for Diabetes Research, 85764 Neuherberg, Germany article info abstract Article history: Endometrial cancer is the most frequently diagnosed gynecological malignancy. It is associated with Available online xxxx prolonged exposure to estrogens that is unopposed by progesterone, whereby enhanced metabolism of progesterone may decrease its protective effects, as it can deprive progesterone receptors of their active Keywords: ligand. Furthermore, the 5a-pregnane metabolites formed can stimulate proliferation and may thus 3-Keto/20-keto-reductases contribute to carcinogenesis. The aims of our study were to: (1) identify and quantify progesterone 5a-Reductases metabolites formed in the HEC-1A and Ishikawa model cell lines of endometrial cancer; and (2) pinpoint Pre-receptor metabolism the enzymes involved in progesterone metabolism, and delineate their roles. Progesterone metabolism 5a-Pregnanes studies combined with liquid chromatography–tandem mass spectrometry enabled identification and 4-Pregnenes quantification of the metabolites formed in these cells. Further quantitative PCR analysis and small-inter- fering-RNA-mediated gene silencing identified individual progesterone metabolizing enzymes and their relevant roles. In Ishikawa and HEC-1A cells, progesterone was metabolized mainly to 20a-hydroxy- pregn-4-ene-3-one, 20a-hydroxy-5a-pregnane-3-one, and 5a-pregnane-3a/b,20a-diol. The major difference between these cell lines was rate of progesterone metabolism, which was faster in HEC-1A cells. In the Ishikawa and HEC-1A cells, expression of AKR1C2 was 110-fold and 6800-fold greater, respectively, than expression of AKR1C1, which suggests that 20-ketosteroid reduction of 5a-pregnanes and 4-pregnenes is catalyzed mainly by AKR1C2. AKR1C1/AKR1C2 gene silencing showed decreased pro- gesterone metabolism in both cell lines, thus further supporting the significant role of AKR1C2. SRD5A1 was also expressed in these cells, and its silencing confirmed that 5a-reduction is catalyzed by 5a-reduc- tase type 1. Silencing of SRD5A1 also had the most pronounced effects, with decreased rate of progester- one metabolism, and consequently higher concentrations of unmetabolized progesterone. Our data confirm that in model cell lines of endometrial cancer, AKR1C2 and SRD5A1 have crucial roles in progesterone metabolism, and may represent novel targets for treatment. Ó 2014 Elsevier Ireland Ltd. All rights reserved. 1. Introduction The majority of cases are diagnosed in post-menopausal women, while 20% of EC patients are pre-menopausal and 5% are younger Endometrial cancer (EC) is the most frequently diagnosed than 40 years of age [4]. EC patients are usually treated surgically, gynecological malignancy [1]. Worldwide, it is the sixth most com- but with patients who wish to preserve fertility, alternative mon cancer in women, with 320,000 new cases diagnosed in 2012 treatments with progestins are used [5,6]. [2]. Both incidence and mortality from EC appear to be rising [3]. From a histological and molecular pathology perspective, the majority of EC cases can be divided into two groups. About 80% Abbreviations: AKR1C1/2/3, aldo–keto reductase 1C1/1C2/1C3; EC, endometrial of diagnosed cases are endometrioid type (type 1). These tumors cancer; HSD17B2, 17b-hydroxysteroid dehydrogenase type 2; SRD5A1/2, 5a- are well differentiated and estrogen-dependent, and they have reductase types 1/2. good prognosis. Most type 1 cases originate from endometrial ⇑ Corresponding author at: Institute of Biochemistry, Faculty of Medicine, hyperplasia, and they occur in premenopausal and postmeno- University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia. Tel.: +386 1 pausal patients and are less aggressive. Type 2 tumors are 5437657; fax: +386 1 5437641. E-mail address: [email protected] (T.L. Rizˇner). diagnosed mainly in postmenopausal patients and are considered http://dx.doi.org/10.1016/j.cbi.2014.11.012 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved. Please cite this article in press as: M. Sinreih et al., Important roles of the AKR1C2 and SRD5A1 enzymes in progesterone metabolism in endometrial cancer model cell lines, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.11.012 2 M. Sinreih et al. / Chemico-Biological Interactions xxx (2014) xxx–xxx as estrogen independent. These are more aggressive forms of this 2. Materials and methods cancer that arise spontaneously. Histologically, type 2 tumors are serous, clear-cell, and adenosquamous adenocarcinomas [3]. 2.1. Reagents and chemicals The risk factors for the development of EC include nulliparity, early age at menarche, late age at menopause, obesity, diabetes, All chemicals and solvents were of the highest purity available. tamoxifen treatment, and long-term use of estrogen-only hormone The commercial standards of testosterone, 3a-hydroxy-pregn-4- replacement therapy. Many of these risk factors support the ene-20-one, 3b-hydroxy-pregn-4-ene-20-one, 20a-hydroxy- hypothesis that chronic exposure to estrogens that is unopposed pregn-4-ene-3-one ((20S)-20-hydroxy-pregn-4-ene-3-one), 20b- by progesterone or progestins can lead to increased mitotic activity hydroxy-pregn-4-ene-3-one ((20R)-20-hydroxy-pregn-4-ene-3- of the endometrial cells, and increased DNA replication errors and one), 5a-pregnane-3,20-dione, 3a-hydroxy-5a-pregnane-20-one, somatic mutations, which can result in a malignant phenotype 3b-hydroxy-5a-pregnane-20-one, 20a-hydroxy-5a-pregnane-3- [3,7]. one ((20S)-20-hydroxy-5a-pregnane-3-one), 20b-hydroxy-5a- Progesterone has protective effects on the endometrium, as it pregnane-3-one ((20R)-20-hydroxy-5a-pregnane-3-one), 5a-preg- has anti-proliferative actions and can stimulate differentiation nane-3a,20a-diol ((20S)-5a-pregnane-3a,20-diol), 5a-pregnane- [8]. In pre-menopausal women, progesterone is synthesized 3b,20a-diol ((20S)-5a-pregnane-3b,20-diol), and 5a-pregnane- mainly in the corpus luteum, and its plasma concentrations change 3a,20b-diol ((20R)-5a-pregnane-3a,20-diol) were from Steraloids according to the menstrual cycle. After menopause, the concentra- (Newport, RI, USA). Progesterone, formic acid, Eagle’s minimal tion of progesterone in the blood decreases as the ovaries cease to essential medium, McCOY 5A, fetal bovine serum, and trypsin– function. However, progesterone can still be formed in the adrenal EDTA solution were from Sigma–Aldrich Chemie GmbH (Deisenho- gland and in some other peripheral tissues, including cancerous fen, Germany). OptiMEM was from Life Technologies (Carlsbad, CA, endometrium [9–11]. Progesterone that is formed locally de novo USA). from cholesterol can act in the same cell (intracrine action) or on Solvents were obtained from Carlo Erba Reagents (Rodano, neighboring cells (paracrine action) via progesterone receptors Italy), Carl Roth GmbH (Karlsruhe, Germany), Sigma–Aldrich Che- [9], or it can be further metabolized. mie GmbH (Deisenhofen, Germany), Thermo Fisher Scientific (Geel, The metabolism of progesterone in the endometrium proceeds Belgium) and Alfa Aesar (Heysham, UK). via the actions of 20-ketosteroid reductases (to form 20a- The following steroids were synthesized by the group of Dr. hydroxy-pregn-4-ene-3-one), 3-ketosteroid reductases (to form Gobec from the Faculty of Pharmacy, University of Ljubljana 3a/b-hydroxy-pregn-4-ene-20-one) and 5a-reductases (to form (Ljubljana, Slovenia) (unpublished data): pregn-4-ene-3a,20a-diol 5a-pregnane-3,20-dione). The reduction of the 20-keto group of ((20S)-pregn-4-ene-3a,20-diol), pregn-4-ene-3a,20b-diol ((20R)- progesterone is catalyzed by the aldo–keto reductases AKR1C1– pregn-4-ene-3a,20-diol), pregn-4-ene-3b,20a-diol ((20S)-pregn- AKR1C3, which act as 3-keto, 17-keto and 20-ketosteroid 4-ene-3b,20-diol), and pregn-4-ene-3b,20b-diol ((20R)-pregn-4- reductases, with varying catalytic efficiencies [12–15]. The pro- ene-3b,20-diol). The reduction of progesterone was performed as gesterone metabolite 20a-hydroxy-pregn-4-ene-3-one thus described earlier by Wiebe et al., with some modifications [21]. formed can be oxidized back to progesterone by the 20a- hydroxysteroid dehydrogenase activity of 17b-hydroxysteroid 2.2. Cell culture dehydrogenase type 2 (HSD17B2) [16]. The AKR1C enzymes do not reduce the 3-keto group of 4-pregnenes [17], and thus the The Ishikawa cell line was obtained from The European Collec- conversion of progesterone to 3a/b-hydroxy-pregn-4-ene-20-one tion of Cell Cultures (ECACC) and the HEC-1A cell line was obtained is catalyzed by as-yet-unidentified ketosteroid reductases. The from American Type Culture Collection (ATCC). The cell lines were formation of 5a-pregnane-3,20-dione from progesterone is cata- cultured according to the ECACC and ATCC recommendations. The lyzed by 5a-reductases types 1 and 2 (SRD5A1, SRD5A2). 5a- Ishikawa cells were cultured in Eagle’s minimal essential medium Pregnane-3,20-dione can be further metabolized to mono- and containing 5% fetal bovine serum, and the HEC-1A cells were cul- di-hydroxy-5a-pregnanes by the actions of the AKR1C1–AKR1C3 tured in McCOY 5A medium containing 10% fetal bovine serum. enzymes (Fig. 1) [15,18]. Both of these cell lines were grown without any antibiotics, in a Enhanced metabolism of progesterone may decrease its protec- humidified atmosphere of 5% CO and 95% air, at 37 °C.