Isoquinoline alkaloids as a novel type of AKR1C3 inhibitors

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SBMB 4186 1–9

Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx

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Isoquinoline alkaloids as a novel type of AKR1C3 inhibitors

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Lucie Skarydova a , Jakub Hofman a , Jakub Chlebek b , Jana Havrankova a , Katerina Kosanova a , Adam Skarka a , Anna Hostalkova b , Tomas Plucha a , Lucie Cahlikova b , Vladimir Wsol a,∗ a Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic b ADINACO Research Group, Department of Pharmaceutical Botany and Ecology, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic

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Article history: Received 2 February 2014 Received in revised form 10 April 2014 Accepted 12 April 2014 Available online xxx

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Keywords: AKR1C3 Inhibitor Alkaloids Natural Isoquinoline Cancer

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1. Introduction

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AKR1C3 is an important human enzyme that participates in the reduction of steroids and prostaglandins, which leads to proliferative signalling. In addition, this enzyme also participates in the biotransformation of xenobiotics, such as drugs and procarcinogens. AKR1C3 is involved in the development of both hormone-dependent and hormone-independent cancers and was recently demonstrated to confer cell resistance to anthracyclines. Because AKR1C3 is frequently upregulated in various cancers, this enzyme has been suggested as a therapeutic target for the treatment of these pathological conditions. In this study, nineteen isoquinoline alkaloids were examined for their ability to inhibit a recombinant AKR1C3 enzyme. As a result, stylopine was demonstrated to be the most potent inhibitor among the tested compounds and exhibited moderate selectivity towards AKR1C3. In the follow-up cellular studies, stylopine significantly inhibited the AKR1C3-mediated reduction of daunorubicin in intact cells without considerable cytotoxic effects. This inhibitor could therefore be used as a model AKR1C3 inhibitor in research or evaluated as a possible therapeutic anticancer drug. Furthermore, based on our results, stylopine can serve as a model compound for the design and future development of structurally related AKR1C3 inhibitors. © 2014 Published by Elsevier Ltd.

AKR1C3 (also known as 17␤-hydroxysteroid dehydrogenase type 5) is an NADPH-dependent carbonyl-reducing enzyme that participates in the metabolism of many important eobiotics and xenobiotics. This enzyme is responsible for the pre-receptor regulation of steroid hormone action because the interconversion of a ketone and hydroxyl groups dramatically changes their affinity to appropriate receptors. Major reactions that are catalysed by AKR1C3 are described in Fig. 1. A reduction of estrone and 4androstene-3,17-dione (Adion) leads to the formation of potent

Abbreviations: Adion, 4-androstene-3,17-dione; DHO, dihydrooracin; DHO assay, reduction of oracin to DHO assay; DMSO, dimethyl sulfoxide; HPLC, high-performance liquid chromatography; MTT, 3-(4,5-dimethylthiazoyl-2yl)2,5diphenyl tetrazolium bromide; NADPH, nicotinamide adenine dinucleotide phosphate; PBS, phosphate buffered saline; T assay, reduction of Adion to testosterone assay; UHPLC, ultra high-performance liquid chromatography. ∗ Corresponding author at: Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 50005 Hradec Kralove, Czech Republic. Q2 Tel.: +420 739488218; fax: +420 495067168. E-mail addresses: [email protected], [email protected] (V. Wsol).

hormones that stimulate cell proliferation, whereas a reduction of progesterone produces weak hormone 20␣-hydroxyprogesterone. Moreover, AKR1C3 also catalyses some minor reactions, such as the conversion of dihydroepiandrosterone to 5-androstene-3␤,17␤diol or 5␣-androstane-3,17-dione to dihydrotestosterone [1–3]. Other recognised substrates of AKR1C3 are prostaglandins. AKR1C3 catalyses the reduction of PGH2 to PGF2␣ and PGD2 to 9␣,11␤-PGF2 ; both products activate signalling pathways that stimulate cell proliferation [4,5]. The previously described role of AKR1C3 in hormone-dependent (e.g., breast, prostate and endometrium) [1,6–10] and hormoneindependent (e.g., lung, brain, and kidney) cancers [11–13] is related to the above noticed activities or to the activation of carcinogens (e.g., polycyclic aromatic hydrocarbons in lung [13]). The expression of AKR1C3 is frequently upregulated in these types of cancer and in cancer cell lines and is believed to be one of the mediators promoting their development [6,8,9]. AKR1C3 seems to be a principal enzyme responsible for the excessive formation of active androgens (mainly testosterone) causing the over-activation of androgen receptors and subsequent cell proliferation in prostate cancer, primarily in its castrate-resistant subtype [6,10]. The most important reaction catalysed by AKR1C3 in breast

http://dx.doi.org/10.1016/j.jsbmb.2014.04.005 0960-0760/© 2014 Published by Elsevier Ltd.

Please cite this article in press as: L. Skarydova, et al., Isoquinoline alkaloids as a novel type of AKR1C3 inhibitors, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.005

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Fig. 1. Steroid reactions that can be catalysed by AKR1C3 in cells. Products of AKR1C3 reduction are potent steroids (testosterone, 17␤-estradiol) that interact with appropriate receptors. The reduced metabolite of progesterone (20␣hydroxyprogesterone) is a weak steroid; this reaction leads to the deactivation of the progesterone effect.

2. Materials and methods

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2.1. Chemicals

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4-Androstene-3,17-dione, testosterone, daunorubicin, and 3-(4,5-dimethylthiazoyl-2-yl)2,5diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Prague, Czech Republic), whereas daunorubicinol was obtained from Toronto Research Chemicals (Toronto, Canada). Oracin and 11-dihydrooracin (DHO) were obtained from the Research Institute for Pharmacy and Biochemistry (Prague, Czech Republic). Enzymatic tests were performed with NADP+ , glucose-6-phosphate (Sigma–Aldrich, Prague, Czech Republic), and with glucose-6-phosphate dehydrogenase (Roche Diagnostics, Mannheim, Germany). jetPRIME, a transfection reagent, was purchased from Polyplus Transfection (Illkirch, France). Cell culture reagents were supplied by Lonza (Walkersville, MD, USA) and by PAA Laboratories (Pasching, Austria). All other chemicals and reagents were of the highest purity commercially available. 2.2. Cell cultures

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and endometrial cancer is most likely identical to that in prostate cancer (Adion→testosterone); formed testosterone is a substrate for aromatase, which produces active oestrogen, 17␤-estradiol. In addition, AKR1C3 is able to directly reduce estrone to 17␤estradiol. The main enzyme responsible for the direct formation of 17␤-estradiol in breast cancer is most likely 17␤-HSD1; however, in cells with low expression of 17␤-HSD1, the role of AKR1C3 in this reaction may increase [14]. Moreover, AKR1C3 contributes to weakening the protective role of progesterone [15,16]. In addition to its involvement in cancer development, AKR1C3 constitutes an important enzyme that participates in the biotransformation of anticancer drugs (e.g., anthracyclines). Over-expressed AKR1C3 in cancer cells can potentiate the reduction of a parent drug to its less active metabolite, which, in turn, limits the efficacy of anthracycline treatment. AKR1C3 has been described as one of the biotransformation enzymes responsible for the development of anthracycline resistance [17,18]. Moreover, AKR1C3 play a role in the resistance against a cisplatin derivate that is based on a diverse mechanism because this substance is not the substrate of AKR1C3 [19]. Considering the important role of AKR1C3 in carcinogenesis and in drug metabolism, the modulation of this enzyme is of great clinical interest; considerable effort has been devoted to the development of potent and specific AKR1C3 inhibitors because such molecules could attenuate the pro-proliferative signalling or drug resistance induced by the activity of AKR1C3 in cancer cells. Several AKR1C3 inhibitors from different classes have been described thus far–non-steroidal anti-inflammatory drugs and their derivatives [5,7,20–22], natural compounds (phytoestrogens, flavonoids and related compound) [23–26], steroidal compounds [20] and other chemical substances [27,28]. Although some of these inhibitors are potent and selective, no AKR1C3 inhibitors have reached the clinical practice, although their utilisation appears to be advantageous in comparison to the recently approved CYP17A1 inhibitor Abiraterone, which is used for the treatment of castration-resistant prostate cancer. In addition to inhibiting CYP17A1, this compound also blocks the production of corticoids, which leads to a serious adverse effect [6]. The aim of this study was to elucidate the possible inhibitory effect of diverse types of isoquinoline alkaloids isolated from plant sources on a recombinant form of AKR1C3. In addition, the selectivity of strong inhibitors using the same method with recombinant AKR1C1, AKR1C2, AKR1C4 and CBR1 was demonstrated. Finally, toxicity and functionality on the cellular level were determined for stylopine, the strongest inhibitor.

Human breast adenocarcinoma MCF7 cells were purchased from the European Collection of Cell Cultures. The HCT 116 (human colorectal carcinoma) cell line was obtained from the American Type Culture Collection. Cells were cultured in complete Dulbecco’s modified Eagle’s medium, which was supplemented with 10% foetal bovine serum, at standard conditions (37 ◦ C, 5% CO2 ). Routine cultivation and all experiments were performed in antibioticfree medium. Cell lines were periodically tested for mycoplasma contamination. Cells from passages 10 to 20 were used in all experiments. Dimethyl sulfoxide (DMSO) was applied as a stylopine solvent in concentrations not exceeding 0.5%; no effect on tested parameters was observed in control experiments. 2.3. Isoquinoline alkaloids (−)-Isocorydine was purchased from Sigma–Aldrich, Czech Republic. (−)-Californidine iodide, (−)-escholtzine, (−) -caryachine, (−)-argemonine, (−)-O-methylcaryachine and (+)N-methyllaurotetanine were isolated from aerial parts and roots of Eschscholzia californica [29]. Protopine, allocryptopine and (−)-stylopine were isolated from aerial parts and roots of Chelidonium majus [30]. Cryptopine, (+)-bulbocapnine, (+)-corydine, (−)-corypalmine, (+)-tetrahydropalmatine, (−)-scoulerine and (+)-corydaline were isolated from tubers of Corydalis cava [31]. (−)-Tetrahydrocolumbamine was isolated from tubers (200 g) of Corydalis yanhusuo, and (−)-canadine was isolated from the rhizome (150 g) of Hydrastis canadensis. The alkaloidal extracts were prepared as described in a previous study [30]. The extracts (1.5 g and 1.2 g, respectively) were subjected to preparative TLC (toluene:diethylamine 95:5) to give (−)-tetrahydrocolumbamine (145 mg) and (−)-canadine (95 mg). The structures of isolated alkaloids were identified by comparison with published data [32,33]. Stock solutions (10 mM) of particular alkaloids were prepared in DMSO. Working solutions for the screening and determination of inhibition constants were also prepared in DMSO. 2.4. Preparation of a recombinant form of AKR1C3 A recombinant form of human AKR1C3 was prepared in an Escherichia coli expression system and purified to homogeneity as described previously [24]. AKR1C1, AKR1C2, AKR1C4 and CBR1 were prepared similarly in the same expression system [34].

Please cite this article in press as: L. Skarydova, et al., Isoquinoline alkaloids as a novel type of AKR1C3 inhibitors, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.005

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2.5. Determination of kinetic parameters of Adion reduction by AKR1C3

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The kinetic parameters of Adion reduction by AKR1C3 were measured by a similar procedure as in the Section 2.6. There were no tested substances in the incubation mixtures and, the Adion concentration range of 10–120 ␮M was utilised. The kinetic parameters were determined using the software GraphPad Prism 5.02 (GraphPad Software Inc.), and Vmax was expressed as a specific activity (nmol/mg/min).

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2.6. Testosterone and DHO assays

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concentration of 1 mg/mL was added to cells, and cells were subsequently incubated at standard conditions for 45 min. The medium was aspirated, and cells were lysed with DMSO on an automatic shaker for 15 min. The absorbance was measured at 570 and 690 nm using a microplate reader Infinite M200 (Tecan, Salzburg, Austria); background values of 690 nm were subtracted from those absorbances obtained at 570 nm. For the reproduction of results from these experiments, the normalisation of raw data was performed using the software GraphPad Prism 5.02. 2.10. Transient transfections

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Incubation mixtures, which contained 1.5 ␮g of pure recombinant AKR1C3, an NADPH-generation system (13 mM NADP+ , 96 mM glucose-6-phosphate, 3.5 U glucose-6-phosphate dehydrogenase, 50 mM MgCl2 ), 20 ␮M (for screening purposes) or 2–200 ␮M (for inhibition constant determination) tested substance and 0.1 M Na-phosphate buffer, pH 7.4, were incubated 15 min on ice and subsequently preincubated for 5 min at 37 ◦ C. The enzymatic reaction was initiated by the addition of Adion or oracin in a final concentration of 12 or 500 ␮M, respectively. The reaction mixture in a volume of 100 ␮L was incubated at 37 ◦ C for 30 min and then stopped by the addition of 40 ␮L of 25% NH4 OH and by cooling to 0 ◦ C. After 10 min on ice, metabolites (testosterone or DHO) were extracted with 1 mL of ethyl acetate by shaking for 10 or 15 min, respectively. Each sample was then centrifuged for 2 min at 13,000 rpm. The organic phases were transferred to new Eppendorf microtubes and evaporated to dryness under vacuum at 30 ◦ C. Residues were dissolved in 300 ␮L of mobile phase and subjected to high-performance liquid chromatography (HPLC) analysis (Section 2.12). Control samples of identical composition containing only DMSO without tested compounds were prepared in identical manner. Similar procedure as in the case of tested substances was used for model inhibitors of AKR1C3, indomethacin and 2 -hydroxyflavanone.

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2.7. Determination of selectivity of chosen inhibitors

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Particular related carbonyl-reducing enzymes, AKR1C1, AKR1C2, AKR1C4 and CBR1, in the amount of 1.5 ␮g were incubated in the presence of 20–200 ␮M stylopine or canadine with 0.5 mM oracin identical to the method described in Section 2.6. The amount of formed DHO was determined by the HPLC method, as described in Section 2.12.

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2.8. Determination of mode of AKR1C3 inhibition by stylopine

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Mode of AKR1C3 inhibition by stylopine was determined using both assays (T and DHO) described in Section 2.6. Concentrations of stylopine were chosen according to IC50 values. Concentrations of stylopine were 8 and 20 ␮M in T assay when Adion concentrations ranging from 5 to 120 ␮M were utilised. DHO assay was performed with 25–500 ␮M oracin concentration range and two stylopine concentrations, 1 and 3 ␮M. Amounts of formed metabolites were subsequently determined by the HPLC/UHPLC method, as described in Section 2.12. Ki values were determined using Cheng–Prussof equation [35].

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2.9. Cytotoxicity of stylopine

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In brief, HCT 116 (3.0 × 104 ) and MCF7 (1.6 × 104 ) cells were seeded on 96-well Plates 24 h before the experiment; the medium was then replaced with a fresh medium containing stylopine in an 8-point concentration scale (1–50 ␮M). Cell viability was determined after 72 h of incubation at standard conditions (37 ◦ C, 5% CO2 ). MTT solution in phosphate buffered saline (PBS) at a final

HCT 116 (30 × 104 ) and MCF7 (18 × 104 ) cells were seeded on 24-well Plates 24 h before transfection. In total, 0.75 ␮g of jetPRIME transfection reagent was mixed with 0.25 ␮g of pCI AKR1C3 [18] or with the empty pCI plasmid, and the mixture was incubated for 10 min at room temperature. The growth medium was changed before the transfection began by the drop-wise addition of polyplexes into the wells. After 6 h, polyplexes were aspirated, and 2 mM sodium butyrate in medium was added to the cells that were transfected with pCI AKR1C3 to boost AKR1C3 expression. To maintain identical conditions, sodium butyrate was also added to the transfections with the empty pCI vector. After an additional 18 h of incubation at standard conditions (37 ◦ C, 5% CO2 ), transfected cells were used for follow-up inhibition experiments. The expression of AKR1C3 was verified using qRT-PCR. The uniformity of transfection was monitored by the concomitant transfection of the pmaxGFP vector at identical conditions to those conditions that were used for the transfections with pCI AKR1C3 and pCI. Green fluorescent protein was quantified as described previously [36]. Differences up to 10% in the reporter gene expression between experiments were accepted; in the case of higher differences, the experiment was terminated. 2.11. Inhibition of AKR1C3 by stylopine in intact cells At time 0, the medium from transfected cells was aspirated, and a fresh medium with 500 nM daunorubicin with or without stylopine was added. Samples were collected after 4 and 8 h of incubation at standard conditions (37 ◦ C, 5% CO2 ); the medium was harvested, and cells were lysed in lysis buffer (25 mM Tris, 150 mM NaCl, 1% Triton X-100, pH 7.8) for 15 min at room temperature. The harvested medium with the cell lysate was extracted twice with 1 mL of ethyl acetate by shaking on an automatic shaker for 15 min, which was followed by centrifugation for 2 min at 13,000 rpm. Organic phases were evaporated under vacuum, and then residues were dissolved in a mobile phase and subjected to ultra high-performance chromatography (UHPLC) analysis (Section 2.12). 2.12. HPLC/UHPLC analysis The testosterone formed after the enzymatic conversion of Adion by recombinant AKR1C3 was determined using an HPLC Agilent 1100 Series system (Santa Clara, CA, USA), which was equipped with a chromatography column BDS Hypersil C18 column (250 × 4.6 mm, 5 ␮m) equipped with BDS 10 × 4 mm i.d. 5 ␮m guard column (Thermo Electron Corporation, UK). A mobile phase consisting of methanol:water 70:30 (v/v) at a flow rate 0.6 mL/min was used, and testosterone was detected using a diode array detector at 240 nm. The total amount of DHO after the enzymatic conversion of oracin by recombinant AKR1C3 in the presence of tested substances was determined by a modified method according Wsól et al., 1996 [37]. An UHPLC Agilent 1290 Series chromatographic system, which was equipped with a chromatography column Zorbax C18 Eclipse Plus column (50 × 2.1 mm, 1.8 ␮m) with a 1290 Infinity inline filter

Please cite this article in press as: L. Skarydova, et al., Isoquinoline alkaloids as a novel type of AKR1C3 inhibitors, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.005

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(Agilent, Santa Clara, CA, USA), and the mobile phase, which consisted of 10 mM Na-hexanesulphonate with 0.04 mM triethylamine (pH 3.27) and acetonitrile in a ratio of 78:22 (v/v), at flow rate 1 mL/min were utilised for the detection of DHO using a fluorescence detector at 340/418 nm. The daunorubicinol formed in experiments with intact cells was detected using a UHPLC Agilent 1290 Series chromatographic system, which was equipped with a Zorbax C18 Eclipse Plus (50 × 2.1 mm, 1.8 ␮m) column with a 1290 Infinity inline filter (Agilent, Santa Clara, CA, USA). The HPLC method that was employed in our previous study [38] was adapted to the UHPLC system: isocratic elution of 1.0 mL/min by 0.1% formic acid in water and acetonitrile in a ratio of 74:26, with the column compartment thermostated to 40 ◦ C and fluorescence detection (480/560 nm).

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3. Results

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3.1. Kinetic parameters of AKR1C3-catalysed reactions

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The kinetic parameters for the AKR1C3-catalysed reduction of Adion were determined using incubations with a pure recombinant AKR1C3. The obtained Michaelis constant Km = 3.82 ± 0.94 ␮M, Vmax expressed as a specific activity 3.96 ± 0.12 nmol/mg/min and kcat = 0.15 min−1 correlate well with values that have been reported previously [39]. A 12 ␮M concentration (∼3 × Km ) of Adion was chosen for the inhibitor screening and under such conditions; the time dependence of testosterone formation has been tested. The amount of testosterone produced by AKR1C3 linearly increased up to 35 min; thus, the incubation time was determined to be 30 min. Similar measurements for the second substrate, oracin, were previously performed by our workgroup, and a Michaelis constant of 110 ± 11 ␮M and a Vmax of 169 ±6 nmol/mg/min were reported [40]. Identical to our previous study [24], a 500 ␮M concentration (∼4 × Km ) of oracin was used for the inhibitor screening.

Table 1 Comparison of inhibitory potencies of studied alkaloids and well-known inhibitors of AKR1C3 (20 ␮M) under conditions of this study. × – not determined because of interference. Values are given as means ± S.D. from n = 3 experiments. Inhibitor

T assay (% of inhibition)

DHO assay (% of inhibition)

Stylopine Canadine Indomethacin 2 -Hydroxyflavanone

54.7 ± 3.4 27.2 ± 3.4 67.7 ± 4.4 ×

90.6 59.0 94.7 98.6

± ± ± ±

0.6 0.6 0.1 0.4

is a model anticancer drug that is also extensively metabolised by related carbonyl-reducing enzymes. The percentage of inhibition at 20 ␮M concentration of the tested substances in the presence of the saturation amount of a particular substrate was determined; the results (together with subgroup of isoquinoline alkaloids) are shown in Fig. 2. Clearly, members of pavinane, aporphine and protopine types are poor inhibitors of AKR1C3. Their inhibitory efficiency is lower than 20% for both reactions at defined conditions. Higher inhibitory potency to AKR1C3 was proven for tetrahydrocolumbamine, canadine and stylopine from the protoberberine group. Because the inhibitory potency can vary considerably depending on the assay conditions, well-known inhibitors of AKR1C3 (indomethacin, 2 -hydroxyflavanone [5,24]) were added to this study for a better comparison of our results with other studies. The results of this comparison are shown in Table 1. Our data show that stylopine possesses almost an identical inhibitory effect on AKR1C3 as indomethacin and a slightly lower effect in comparison with 2 -hydroxyflavanone. Canadine also significantly inhibited AKR1C3 catalytic function; however, this interaction was weaker than in the case of stylopine. The inhibitory effect of 2 -hydroxyflavanone and of some tested alkaloids (all are signed × in Fig. 1 and Table 1) was not possible to detect by T assay because of their interference with the HPLC method used. 3.3. Selectivity of inhibitors

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3.2. Screening of AKR1C3-inhibitory potency of isoquinoline alkaloids In total, nineteen isoquinoline alkaloids were examined for their ability to inhibit the recombinant AKR1C3. Based on their structural similarity, these substances may be divided into several groups – protoberberine, pavinane, apomorphine and protopine types. Their inhibitory effect was tested on two different reactions – the reduction of Adion (12 ␮M) to testosterone (T assay), which is a physiological reaction catalysed by AKR1C3 in the cell, and the reduction of oracin (500 ␮M) to dihydrooracin (DHO assay), which

In addition to significant inhibitory potency towards the target enzyme, selectivity is another important feature of an inhibitor, particularly when thinking concerning its possible introduction into clinical practice. The most related enzymes from the AKR1C family and the well-known carbonyl-reducing enzyme CBR1 were chosen to verify the selectivity of the most promising alkaloids by DHO assay because all mentioned enzymes participate in the biotransformation of oracin [41]. Two stylopine and canadine concentrations of 20 and 200 ␮M were evaluated in this experiment (Fig. 3).

Fig. 2. Inhibitory effect of tested substances at 20 ␮M concentration on recombinant AKR1C3. Two different assays were used–T assay utilised the physiological reaction mediated by AKR1C3, the reduction of Adion (12 ␮M) to testosterone and DHO assay employed one of the biotransformation reactions catalysed by AKR1C3, the reduction of anticancer drug oracin (500 ␮M) to DHO. Tested compounds from isoquinoline alkaloids are grouped according to structural types. × – not determined because of interference. Values are given as means ± S.D. from n = 3 experiments.

Please cite this article in press as: L. Skarydova, et al., Isoquinoline alkaloids as a novel type of AKR1C3 inhibitors, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.005

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Fig. 3. Inhibitory effect of selected, strongest inhibitors of AKR1C3, stylopine and canadine, towards related enzymes from the AKR1C family and towards another carbonylreducing enzyme, CBR1. Stylopine and canadine were tested at concentrations of 20 and 200 ␮M. Values are given as means ± S.D. from n = 3 experiments.

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Both tested substances at 20 ␮M concentration do not interact at all with the AKR1C1 enzyme; however, so strong preference for AKR1C3 in the case of the other tested enzymes was not observed. Nevertheless, good selectivity towards AKR1C3 was still proven when testing AKR1C4; stylopine and canadine are ∼45-, ∼37-times stronger inhibitors of AKR1C3 than AKR1C4, respectively. The relatively weak preference of AKR1C3 over AKR1C2 and CBR1 was recorded for both tested substances; however, both compounds are significantly stronger inhibitors of AKR1C3 than AKR1C2 (∼5 times for stylopine, ∼3 times for canadine) and CBR1 (∼7 times for stylopine, ∼10 times for canadine). When compared with 20 ␮M alkaloid concentration, inhibitory potencies of stylopine and canadine at concentration of 200 ␮M are significantly higher only in the case of AKR1C1 and AKR1C4, but still less than 50%. Due to the limited solubility of tested substances, it was not possible to determine IC50 values for all tested enzymes. Nevertheless, in regard to obtained data, it can be presumed that this value would exceed 150 ␮M. Taken together, the tested alkaloids can be ranked as moderately selective inhibitors of AKR1C3.

3.4. IC50 determination for the strongest inhibitors IC50 values for the strongest inhibitors from the group of alkaloids have been determined to fully characterise these inhibitors of AKR1C3. Both reduction reactions were used for these measurements (see Table 2). Because inhibitory potency and IC50 values strongly depend on substrate and reaction conditions, it is logical that the values obtained from the reduction of Adion and oracin differ from each other.

3.5. Mode of AKR1C3 inhibition by stylopine

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Evaluation of type of AKR1C3 inhibition by stylopine was determined for both AKR1C3 model substrates using two stylopine concentrations (≈IC50 and ≈3 × IC50 ). Inhibition of Adion reduction to testosterone best fitted in the competitive model (Fig. 4), value of Ki determined by Cheng–Prusoff equation for classic mode of inhibition is 1.86 ␮M. Conversion of oracin to dihydrooracin was inhibited by stylopine in a noncompetitive manner (Fig. 4), value of Ki determined by Cheng–Prusoff equation for classic mode of inhibition is 0.9 ␮M. 3.6. Effect of stylopine on cell viability

Table 2 IC50 values for the strongest inhibitors of AKR1C3 from the group of isoquinoline alkaloids and two well-known inhibitors (indomethacin and 2 -hydroxyflavanone) as determined by two diverse reactions – the reduction of Adion to testosterone (T assay) and oracin to DHO (DHO assay). × – not determined because of interference. Values are given as mean ± S.D. from n = 3 experiments.

Stylopine Canadine Indomethacin 2 -Hydroxyflavanone

IC50 (␮M) T assay

DHO assay

7.7 29.0

0.9 12.2

3.7 ×

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The MTT proliferation test was used to investigate the possible cytotoxic influence of stylopine, a most potent candidate, on HCT 116 and MCF7 cell viability. Stylopine showed a highly favourable cytotoxic profile, reducing HCT 116 and MCF7 cell viability only by 46% and 29% at the highest concentration tested (50 ␮M), respectively (Fig. 5). The data from this experiment were used for the

Inhibitor

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0.46 0.3 [23]

Fig. 4. Lineweaver–Burk double reciprocal plots demonstrating mode of AKR1C3 inhibition by stylopine for reduction of Adion (T assay) and oracin (DHO assay) catalysed by AKR1C3. Concentrations of stylopine were 0 ␮M (䊉), 1 resp. 8 ␮M () and 3 resp. 20 ␮M (). Values are given as means ± S.D. from n = 3 experiments.

Please cite this article in press as: L. Skarydova, et al., Isoquinoline alkaloids as a novel type of AKR1C3 inhibitors, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.005

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Fig. 5. Cell viability of HCT 116 (A) and MCF7 (B) cells following 72 h exposure to stylopine as determined by MTT assay. It was impossible to obtain a complete dose-response curve and to calculate IC50 values because stylopine precipitated in the medium in concentrations higher than 50 ␮M. For the reproduction of results from these experiments, the normalisation of raw data was performed using the software GraphPad Prism 5.02 (GraphPad Software Inc.). For the normalisation of absorbance values, absorbance from cells where only medium was added, was considered as 100% viability. Absorbance equal to 0% viability was obtained from cells incubated in 10% DMSO. The data are expressed as the means ± S.D. of three independent experiments.

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design of the stylopine concentration for the follow-up functional study described below. 3.7. Effect of stylopine on the AKR1C3-mediated metabolism of daunorubicin in intact cells

10 and 20 ␮M) significantly blocked the acceleration of daunorubicinol generation by 54, 65 and 79%, respectively (Fig. 6B). Taken together, these data demonstrate the capability of stylopine to effectively inhibit AKR1C3 at intracellular conditions. 4. Discussion

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To evaluate the ability of stylopine to inhibit AKR1C3 on the cellular level, a study employing human cancer cells transfected with pCI AKR1C3 (encoding AKR1C3) and an empty pCI vector was performed. HCT116 and MCF7 cells were chosen for this experiments because these cells endogenously express negligible and low levels of AKR1C3, respectively [18]. In our concurrent study, we further observed daunorubicin to be a good substrate of AKR1C3 [18]; therefore, this anthracycline was applied as an appropriate model substrate for this study. Based on the results of the cell viability assessment, three non-cytotoxic concentrations of stylopine (5, 10 and 20 ␮M) were used. The production of daunorubicinol was markedly accelerated in cells transfected with the pCI AKR1C3 vector in comparison with those cells transfected with the empty pCI vector. HCT 116 and MCF7 cells ectopically overexpressing AKR1C3 generated 22.3- and 3.1-fold higher amounts of daunorubicinol than non-overexpressing cells, respectively. In HCT116 cells, both 5 and 10 ␮M stylopine blocked the AKR1C3-mediated production of daunorubicinol by 39%, but without statistical significance. In contrast, 20 ␮M concentration of stylopine was sufficient to significantly inhibit the overproduction of daunorubicinol by 62% in this cell line (Fig. 6A). In MCF7 cells, all three tested concentrations (5,

Alkaloids constitute a highly diverse group of compounds that contain a ring structure with a nitrogen heteroatom. These secondary metabolites are abundant in plants (approximately 12,000 different alkaloids) [42] and carry a variety of pharmacologically important properties, such as anti-inflammatory, antimicrobial, antihypertensive, anticancer etc. Alkaloids are often responsible for the effects of traditional herbal medicine, and many clinically used drugs belong to plant alkaloids or to their derivatives, e.g., the analgesic codeine and the antimicrobial agent sanguarine. Furthermore, camptothecin, paclitaxel and vinblastine have been successfully introduced into clinical practice as efficient chemotherapeutics for the treatment of various types of cancers [43]. Presently, alkaloids are intensively tested for their interaction with nucleic acids, protein, receptors and entire regulatory pathways. The inhibition of pharmacodynamically significant enzymes has been recognised as one possible alkaloid mode of action. Galanthamine is a potent, inhibitor of acetylcholine esterase clinically used for the treatment of Alzheimer’s disease, and camptothecin is an inhibitor of topoisomerase I whose derivatives are used as anticancer agents [43]. Many natural compounds, including alkaloids, have been tested as potential inhibitors of aldose reductase (AKR1B1), which has been

Fig. 6. Effect of stylopine on the AKR1C3-mediated intracellular metabolism of daunorubicin in HCT 116 (A) and MCF7 (B) cells. Cells were transiently transfected with pCI AKR1C3 or with the empty pCI vector and exposed to 500 nM daunorubicin with or without stylopine. Medium with cells was harvested at indicated time intervals; the metabolite was then extracted with ethyl acetate and assessed via UHPLC. 䊉, cells transfected with the pCI AKR1C3 vector without stylopine; , cells transfected with the pCI AKR1C3 vector with 5 ␮M stylopine; , cells transfected with the pCI AKR1C3 vector with 10 ␮M stylopine; , cells transfected with the pCI AKR1C3 vector with 20 ␮M stylopine; , cells transfected with the empty pCI vector without stylopine. The values from the end point were subjected to the statistical analysis using a one-way ANOVA followed by Bonferroni’s test (GraphPad Prism 5.02, GraphPad Software Inc.). The values are expressed as the means ± S.D. of three independent experiments. *p < 0.05; **p < 0.01.

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implicated in the development of diabetic complications, but without a significant effect [44,45]. This enzyme shares 47% homology with the studied AKR1C3. To our knowledge, AKR1B1 is the only one carbonyl-reducing enzyme that has been tested for an inhibitory effect of alkaloids thus far. In this study, the inhibitory effect of 19 different isoquinoline alkaloids of four structural types on AKR1C3 was tested by two assays; the first assay utilises a physiological reaction catalysed by AKR1C3 in cells, and the second assay employs a biotransformation reaction of a model compound. From these assays, two significant inhibitors were found: stylopine and canadine, which are members of the protoberberine group of isoquinoline alkaloids. The specific members of this group that were tested in this study are in Fig. 7; these members are ordered in the direction of decreasing inhibitory effect. Benzodioxole groups appear to be important structural moieties required for the inhibitory effect of these compounds because substances without this structural feature have only limited potency to inhibit AKR1C3. However, several other tested substances of different structural alkaloid types contain this/these benzodioxole group/s (Fig. 8); however, this/these group/s are connected to other types of heterocycles. In contrast, stylopine, which exhibits high AKR1C3 inhibitory potency, is derived from the quinolisidine heterocycle, protopine, which also bears two benzodioxole groups identical to stylopine, contains azecine heterocycle and possesses no inhibitory effect to AKR1C3. From this point of view, it is clear that, other than benzodioxole group/s, the structure of the heterocycle is also an important moiety for AKR1C3 inhibition. The inhibitory potency of the new AKR1C3 inhibitor, stylopine, is almost identical (Table 2) to that of the well-known AKR1C3 inhibitor, indomethacin [4]. Although some more potent inhibitors have been described (e.g., 2 -hydroxyflavanone, baccharin, and N(4-chlorobenzoyl)-melatonin [5,24,26]), there is a new structural type of AKR1C3 inhibitors that can be used for the further testing of natural compounds derived from (S)-reticuline, similar to stylopine (e.g., cheilanthiofoline, chelirubine, and macarpine [46,47]) and also for the synthesis of more potent analogues, as in the case of natural cinnamic acids [25,48]. To evaluate stylopine mode of action, the type of AKR1C3 inhibition was determined. The type of AKR1C3 inhibition by stylopine was demonstrated to be substrate dependent; reduction of Adion was inhibited in a competitive manner whereas inhibition of oracin reduction was shown to exhibit noncompetitive type of inhibition. This phenomenon is probably due to the diverse reaction mechanism and reaction intermediates. Similarly, Byrns et al. observed that biotransformation of 9,10-phenanthrenequinone is inhibited noncompetitively by indomethacin whereas reduction of Adion is inhibited competitively by the same inhibitor [5]. Apart from inhibitory potency and type of inhibition, selectivity towards the target protein is another important feature of a valuable inhibitor, and therefore, this property was tested for both new inhibitors. Closely related carbonyl-reducing enzymes (Fig. 3) were chosen for this experiment because these enzymes share the highest structural homology with AKR1C3. The inhibition of AKR1C2 is not desirable because this enzyme, which acts inversely to AKR1C3 (produces a weak androgen 5-androstane-3␣,17␤-diol), is expressed in the same tissues as AKR1C3. The inhibition of AKR1C2 might outweigh the effect of AKR1C3 inhibition, which could lead to the promotion of proliferative signalling in the prostate [28]. In addition, several papers have reported that stylopine is an inhibitor of other important enzymes (e.g., COX-1 and CYP P450 [49,50]); however, it is impossible to compare the potency of such interactions with the level of AKR1C3 inhibition described in this study. It is apparent that stylopine and canadine possess relatively high selectivity towards AKR1C3. In regards to the possible serious side-effects caused by

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Fig. 7. Structure of tested isoquinoline alkaloids from the protoberberine group. The strongest AKR1C3 inhibitor is stylopine; other compounds are ordered according to their decreasing AKR1C3 inhibitory potency.

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Fig. 8. Structures of tested isoquinoline alkaloids from pavinane (1–5), aporphine (6–8) and protopine (9–12) groups.

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insufficient selectivity, this property should be carefully evaluated in the future search of AKR1C3 inhibitors from the group of related substances. The screening of inhibitory potency at the level of pure isolated recombinant enzymes is important; however, this type of investigation does not ensure that the examined compound is equally active at the cellular level or even at the whole organism level. Some substances may be potent inhibitors in an isolated system; however, their biological activity in cells may be limited by their inability to enter the target cell or by their high toxicity. From published results, it is supposed that stylopine (and other members of protoberberine alkaloids) is able to penetrate cells with more effective penetration in a non-cancer cell line (NIH/3T3) in comparison with cancer cell lines (MCF7 and B16F10) [51], which, thus, rationalises our decision to determine the effect (toxicity functionality) of stylopine on the cellular level. The cytotoxicity of stylopine was tested on two cancer cell lines (Fig. 5), and this substance was not toxic for these cells at reasonable concentrations. Our results correspond with the published results of stylopine toxicity on Abelson murine leukaemia cells [49] and also with the results of a study that investigated the effect of a combination of five alkaloids, including stylopine, on the MCF7 cell line [51]. The participation of AKR1C3 in cancer cell resistance to daunorubicin treatment was recently found by our work group [18]. Model cell lines with overexpressed AKR1C3, which were previously established in that study, were used for the evaluation of the inhibitory effect of stylopine on AKR1C3 function. Significant inhibition of AKR1C3 at intracellular conditions (without considerable effect on cell viability) was found (Fig. 6). Differences in the extent of the inhibition of daunorubicinol formation in diverse cell lines can depend on various factors – the expression of other enzymes that participate in daunorubicin metabolism (e.g., CBR1 and AKR1A1 [52,53]), differences in the permeability of cells for stylopine [51], etc. However, based on our present results, we hypothesise that stylopine could, at least partially, restore the sensitivity of AKR1C3-overexpressing cancer cells to the antiproliferative effect of daunorubicin. The inhibition of AKR1C3 has been proposed as a possible therapeutic approach to attenuate its pro-proliferating potential or to overcome anthracycline resistance mediated by this enzyme [28].

In conclusion, two natural alkaloids were described as novel and potent AKR1C3 inhibitors with moderate selectivity towards this enzyme. Stylopine, the strongest AKR1C3 inhibitor from isoquinoline alkaloids, significantly inhibits AKR1C3 in intact cells without a considerable cytotoxic effect. Therefore, this inhibitor could be used as a model AKR1C3 inhibitor in research or evaluated as a possible therapeutic anticancer drug. Moreover, stylopine constitutes a novel structural type in the family of AKR1C3 inhibitors; thus, stylopine can be used as a model substance for the further development of related compounds with better characteristics in terms of inhibition potency and selectivity by molecular docking, site-directed mutagenesis and organic synthesis. Acknowledgments This work was co-financed by the European Social Fund and by the state budget of the Czech Republic (project no. Q3 CZ.1.07/2.3.00/30.0061) and supported by Charles University in Prague (UNCE 204026/2012). The publication was also co-financed by the European Social Fund and by the state budget of the Czech Republic, project No. CZ.1.07/2.3.00/20.0235, the title of the project: TEAB. References [1] D.R. Bauman, S. Steckelbroeck, T.M. Penning, The roles of aldo-keto reductases in steroid hormone action, Drug News Perspect. 17 (2004) 563–578. [2] T.M. Penning, Hydroxysteroid dehydrogenases and pre-receptor regulation of steroid hormone action, Hum. Reprod. Update 9 (2003) 193–205. [3] T.L. Rizner, T.M. Penning, The role of aldo-keto reductase family 1 (AKR1) enzymes in human steroid metabolism, Steroids 79 (2014) 49–63. [4] T.M. Penning, M.C. Byrns, Steroid hormone transforming aldo-keto reductases and cancer, Ann. N. Y. Acad. Sci. 1155 (2009) 33–42. [5] M.C. Byrns, S. Steckelbroeck, T.M. Penning, An indomethacin analogue, N-(4chlorobenzoyl)-melatonin, is a selective inhibitor of aldo-keto reductase 1C3 (type 2 3alpha-HSD, type 5 17beta-HSD, and prostaglandin F synthase), a potential target for the treatment of hormone dependent and hormone independent malignancies, Biochem. Pharmacol. 75 (2008) 484–493. [6] A.R.A.H. Hamid, M.F. Pfeiffer, A. Dudek, M. Voller, G.W. Verhaegh, F.P. Smit, et al., AKR1C3 – a potential marker and therapeutic target in castration resistance prostate cancer, Eur. Urol. Suppl. 11 (2012) E239. [7] M.C. Byrns, T.M. Penning, Type 5 17beta-hydroxysteroid dehydrogenase/prostaglandin F synthase (AKR1C3): role in breast cancer and inhibition by

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[25]

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[27]

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[28]

664 665 666 667 668

[29]

non-steroidal anti-inflammatory drug analogs, Chem. Biol. Interact. 178 (2009) 221–227. T.M. Penning, S. Steckelbroeck, D.R. Bauman, M.W. Miller, Y. Jin, D.M. Peehl, et al., Aldo-keto reductase (AKR) 1C3: role in prostate disease and the development of specific inhibitors, Mol. Cell. Endocrinol. 248 (2006) 182–191. T.L. Rizner, T. Smuc, R. Rupreht, J. Sinkovec, T.M. Penning, AKR1C1, AKR1C3 may determine progesterone and estrogen ratios in endometrial cancer, Mol. Cell. Endocrinol. 248 (2006) 126–135. A.O. Adeniji, M. Chen, T.M. Penning, AKR1C3 as a target in castrate resistant prostate cancer, J. Steroid Biochem. Mol. Biol. 137 (2013) 136–149. V.L. Miller, H.K. Lin, P. Murugan, M. Fan, T.M. Penning, L.S. Brame, et al., Aldoketo reductase family 1 member C3 (AKR1C3) is expressed in adenocarcinoma and squamous cell carcinoma but not small cell carcinoma, Int. J Clin. Exp. Pathol. 5 (2012) 278–289. J.T. Azzarello, H.K. Lin, A. Gherezghiher, V. Zakharov, Z. Yu, B.P. Kropp, et al., Expression of AKR1C3 in renal cell carcinoma, papillary urothelial carcinoma, and Wilms’ tumor, Int. J. Clin. Exp. Pathol. 3 (2009) 147–155. A.L. Park, H.K. Lin, Q. Yang, C.W. Sing, M. Fan, T.B. Mapstone, et al., Differential expression of type 2 3alpha/type 5 17beta-hydroxysteroid dehydrogenase (AKR1C3) in tumors of the central nervous system, Int. J. Clin. Exp. Pathol. 3 (2010) 743–754. M.C. Byrns, L. Duan, S.H. Lee, I.A. Blair, T.M. Penning, Aldo-keto reductase 1C3 expression in MCF-7 cells reveals roles in steroid hormone and prostaglandin metabolism that may explain its over-expression in breast cancer, J. Steroid Biochem. Mol. Biol. 118 (2010) 177–187. K.K. Sharma, A. Lindquist, X.J. Zhou, R.J. Auchus, T.M. Penning, S. Andersson, Deoxycorticosterone inactivation by AKR1C3 in human mineralocorticoid target tissues, Mol. Cell. Endocrinol. 248 (2006) 79–86. N. Beranic, S. Gobec, T.L. Rizner, Progestins as inhibitors of the human 20ketosteroid reductases, AKR1C1 and AKR1C3, Chem. Biol. Interact. 191 (2011) 227–234. A.D. Heibein, B. Guo, J.A. Sprowl, D.A. Maclean, A.M. Parissenti, Role of aldoketo reductases and other doxorubicin pharmacokinetic genes in doxorubicin resistance, DNA binding, and subcellular localization, BMC. Cancer 12 (2012) 381. J. Hofman, B. Malcekova, A. Skarka, E. Novotna, V. Wsol, Anthracycline resistance mediated by reductive metabolism in cancer cells: the role of aldo-keto reductase 1C3, Toxicol. Appl. Pharm. (2014) (unpublished result). T. Matsunaga, A. Hojo, Y. Yamane, S. Endo, O. El-Kabbani, A. Hara, Pathophysiological roles of aldo-keto reductases (AKR1C1 and AKR1C3) in development of cisplatin resistance in human colon cancers, Chem. Biol. Interact. 202 (2013) 234–242. D.R. Bauman, S.I. Rudnick, L.M. Szewczuk, Y. Jin, S. Gopishetty, T.M. Penning, Development of nonsteroidal anti-inflammatory drug analogs and steroid carboxylates selective for human aldo-keto reductase isoforms: potential antineoplastic agents that work independently of cyclooxygenase isozymes, Mol. Pharmacol. 67 (2005) 60–68. S. Gobec, P. Brozic, T.L. Rizner, Nonsteroidal anti-inflammatory drugs and their analogues as inhibitors of aldo-keto reductase AKR1C3: new lead compounds for the development of anticancer agents, Bioorg. Med. Chem. Lett. 15 (2005) 5170–5175. A.J. Liedtke, A.O. Adeniji, M. Chen, M.C. Byrns, Y. Jin, D.W. Christianson, et al., Development of potent and selective indomethacin analogues for the inhibition of AKR1C3 (Type 5 17beta-hydroxysteroid dehydrogenase/prostaglandin F synthase) in castrate-resistant prostate cancer, J. Med. Chem. 56 (2013) 2429–2446. A. Krazeisen, R. Breitling, G. Moller, J. Adamski, Phytoestrogens inhibit human 17beta-hydroxysteroid dehydrogenase type 5, Mol. Cell. Endocrinol. 171 (2001) 151–162. L. Skarydova, L. Zivna, G. Xiong, E. Maser, V. Wsol, AKR1C3 as a potential target for the inhibitory effect of dietary flavonoids, Chem. Biol. Interact. 178 (2009) 138–144. P. Brozic, B. Golob, N. Gomboc, T.L. Rizner, S. Gobec, Cinnamic acids as new inhibitors of 17beta-hydroxysteroid dehydrogenase type 5 (AKR1C3), Mol. Cell. Endocrinol. 248 (2006) 233–235. S. Endo, T. Matsunaga, A. Kanamori, Y. Otsuji, H. Nagai, K. Sundaram, et al., Selective inhibition of human type-5 17beta-hydroxysteroid dehydrogenase (AKR1C3) by baccharin, a component of Brazilian propolis, J. Nat. Prod. 75 (2012) 716–721. D.M. Heinrich, J.U. Flanagan, S.M. Jamieson, S. Silva, L.J. Rigoreau, E. Trivier, et al., Synthesis and structure-activity relationships for 1-(4-(piperidin-1ylsulfonyl)phenyl)pyrrolidin-2-ones as novel non-carboxylate inhibitors of the aldo-keto reductase enzyme AKR1C3, Eur. J. Med. Chem. 62 (2013) 738–744. M.C. Byrns, Y. Jin, T.M. Penning, Inhibitors of type 5 17beta-hydroxysteroid dehydrogenase (AKR1C3): overview and structural insights, J. Steroid Biochem. Mol. Biol. 125 (2011) 95–104. L. Cahlikova, K. Macakova, J. Kunes, M. Kurfurst, L. Opletal, J. Cvacka, et al., Acetylcholinesterase and butyrylcholinesterase inhibitory compounds from Eschscholzia californica (Papaveraceae), Nat. Prod. Commun. 5 (2010) 1035–1038.

9

[30] L. Cahlikova, L. Opletal, M. Kurfurst, K. Macakova, A. Kulhankova, A. Hostalkova, Acetylcholinesterase and butyrylcholinesterase inhibitory compounds from Chelidonium majus (Papaveraceae), Nat. Prod. Commun. 5 (2010) 1751–1754. [31] J. Chlebek, K. Macakova, L. Cahlikovi, M. Kurfurst, J. Kunes, L. Opletal, Acetylcholinesterase and butyrylcholinesterase inhibitory compounds from Corydalis cava (Fumariaceae), Nat. Prod. Commun. 6 (2011) 607–610. [32] E.K. Jeong, S.Y. Lee, S.M. Yu, N.H. Park, H.S. Lee, Y.H. Yim, et al., Identification of structurally diverse alkaloids in Corydalis species by liquid chromatography/electrospray ionization tandem mass spectrometry, Rapid Commun. Mass Spectrom. 26 (2012) 1661–1674. [33] R. Marek, O. Humpa, J. Dostal, J. Slavik, V. Sklenar, N-15 NMR study of isoquinoline alkaloids, Magn. Reson. Chem. 37 (1999) 195–202. [34] L. Skarydova, R. Tomanova, L. Havlikova, H. Stambergova, P. Solich, V. Wsol, Deeper insight into the reducing biotransformation of bupropion in human liver, Drug Metab. Pharmacokinet. 29 (2014) 177–184. [35] R.Z. Cer, U. Munuduri, R. Stephens, F.J. Lebeda, IC50 to Ki: web based tool for converting of IC50 to Ki for inhibitors of enzyme activity and ligand binding, Nucleic Acids Res. 37 (2009) 441–445. [36] J. Hofman, M. Buncek, R. Haluza, L. Streinz, M. Ledvina, P. Cigler, In vitro transfection mediated by dendrigraft poly(l-lysines): the effect of structure and molecule size, Macromol. Biosci. 13 (2013) 167–176. [37] V. Wsol, E. Kvasnickova, B. Szotakova, I.M. Hais, High-performance liquid chromatographic assay for the separation and characterization of metabolites of the potential cytostatic drug oracine, J. Chromatogr. B: Biomed. Appl. 681 (1996) 169–175. [38] A. Skarka, L. Skarydova, H. Stambergova, V. Wsol, Anthracyclines and their metabolism in human liver microsomes and the participation of the new microsomal carbonyl reductase, Chem. Biol. Interact. 191 (2011) 66–74. [39] T.M. Penning, M.E. Burczynski, J.M. Jez, C.F. Hung, H.K. Lin, H. Ma, et al., Human 3alpha-hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C4) of the aldo-keto reductase superfamily: functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones, Biochem. J. 351 (2000) 67–77. [40] R. Novotna, V. Wsol, G. Xiong, E. Maser, Inactivation of the anticancer drugs doxorubicin and oracin by aldo-keto reductase (AKR) 1C3, Toxicol. Lett. 181 (2008) 1–6. [41] V. Wsol, B. Szotakova, H.J. Martin, E. Maser, Aldo-keto reductases (AKR) from the AKR1C subfamily catalyze the carbonyl reduction of the novel anticancer drug oracin in man, Toxicology 238 (2007) 111–118. [42] T. Takemura, N. Ikezawa, K. Iwasa, F. Sato, Metabolic diversification of benzylisoquinoline alkaloid biosynthesis through the introduction of a branch pathway in Eschscholzia californica, Plant Cell Physiol. 51 (2010) 949–959. [43] J.J. Lu, J.L. Bao, X.P. Chen, M. Huang, Y.T. Wang, Alkaloids isolated from natural herbs as the anticancer agents, Evid. Based Complement Alternat. Med. 2012 (2012) 485042. [44] S. Suzen, E. Buyukbingol, Recent studies of aldose reductase enzyme inhibition for diabetic complications, Curr. Med. Chem. 10 (2003) 1329–1352. [45] M. Kubo, H. Matsuda, K. Tokuoka, Y. Kobayashi, S. Ma, T. Tanaka, Studies of anti-cataract drugs from natural sources. I. Effects of a methanolic extract and the alkaloidal components from Corydalis tuber on in vitro aldose reductase activity, Biol. Pharm. Bull. 17 (1994) 458–459. [46] N. Ikezawa, K. Iwasa, F. Sato, Molecular cloning and characterization of methylenedioxy bridge-forming enzymes involved in stylopine biosynthesis in Eschscholzia californica, FEBS J. 274 (2007) 1019–1035. [47] N.V. Khodorova, A.L. Shavarda, M. Lequart-Pillon, J.C. Laberche, O.V. Voitsekhovskaja, M. Boitel-Conti, Biosynthesis of benzylisoquinoline alkaloids in Corydalis bracteata: compartmentation and seasonal dynamics, Phytochemistry 92 (2013) 60–70. [48] M. Gazvoda, N. Beranic, S. Turk, B. Burja, M. Kocevar, T.L. Rizner, et al., 2,3-Diarylpropenoic acids as selective non-steroidal inhibitors of type-5 17beta-hydroxysteroid dehydrogenase (AKR1C3), Eur. J. Med. Chem. 62 (2013) 89–97. [49] S.I. Jang, B.H. Kim, W.Y. Lee, S.J. An, H.G. Choi, B.H. Jeon, et al., Stylopine from Chelidonium majus inhibits LPS-induced inflammatory mediators in RAW 264.7 cells, Arch. Pharm. Res. 27 (2004) 923–929. [50] K.A. Salminen, A. Meyer, L. Jerabkova, L.E. Korhonen, M. Rahnasto, R.O. Juvonen, et al., Inhibition of human drug metabolizing cytochrome P450 enzymes by plant isoquinoline alkaloids, Phytomedicine 18 (2011) 533–538. [51] M. Kulp, O. Bragina, Capillary electrophoretic study of the synergistic biological effects of alkaloids from Chelidonium majus L. in normal and cancer cells, Anal. Bioanal. Chem. 405 (2013) 3391–3397. [52] O.S. Bains, M.J. Karkling, T.A. Grigliatti, R.E. Reid, K.W. Riggs, Two nonsynonymous single nucleotide polymorphisms of human carbonyl reductase 1 demonstrate reduced in vitro metabolism of daunorubicin and doxorubicin, Drug Metab. Dispos. 37 (2009) 1107–1114. [53] O.S. Bains, T.A. Grigliatti, R.E. Reid, K.W. Riggs, Naturally occurring variants of human aldo-keto reductases with reduced in vitro metabolism of daunorubicin and doxorubicin, J. Pharmacol. Exp. Ther. 335 (2010) 533–545.

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