Experimental Cell Research 309 (2005) 329 – 339 www.elsevier.com/locate/yexcr

Research Article

Monomeric and oligomeric flavanols are agonists of membrane androgen receptors Artemissia-Phoebe Nifli a, Antoine Bosson-Kouame´ c, Natalia Papadopoulou b, Christina Kogia a, Marilena Kampa a, Chantal Castagnino c, Christos Stournaras b, Joseph Vercauteren c, Elias Castanas a,* a

Laboratory of Experimental Endocrinology, University of Crete School of Medicine, P.O. Box 2208, Heraklion GR-71003, Greece b Laboratory of Biochemistry, University of Crete School of Medicine, Heraklion, Greece c Laboratory of Pharmacognosy, University of Montpellier 1 School of Pharmacy, Montpellier, France Received 4 April 2005, revised version received 18 June 2005, accepted 21 June 2005 Available online 20 July 2005

Abstract The present work reports a new mode of action of the naturally occurring flavanols catechin and epicatechin and their dimers B2 and B5, in the breast cancer T47D cell line, namely, their interaction with membrane androgen receptors. We show that monomeric and dimeric flavanols are complete (B2) or partial displacers of radiolabeled testosterone bound on T47D membranes, with affinities ranging from 1.7 (B5) to 82.2 nM (B2). In addition, they trigger the phosphorylation of the same signaling molecules (FAK, PI3K) as testosterone-BSA, minutes after binding to membrane receptors, leading to actin cytoskeleton polymerization and redistribution, with formation of filopodia and lamellipodia. The PI3K inhibitor wortmannin reverts the effect of polyphenols and testosterone-BSA, providing additional evidence about activation of a similar signaling cascade. Incubation of T47D cells for more than 2 h with polyphenols or testosterone-BSA induces apoptosis, which follows the same time-dependent pattern. We conclude that flavanols (monomers or dimers) are agonists of membrane androgen receptors and could be used as testosterone – protein conjugates for the management of tumors, in which, application of testosterone-BSA induces regression, providing additional data about the mechanism of their antiproliferative action. D 2005 Elsevier Inc. All rights reserved. Keywords: Flavanol (catechin, epicatechin); Flavanol oligomers (B2, B5); Androgen receptor (membrane); Breast cancer cell (T47D); Actin cytoskeleton; Apoptosis

Introduction A number of epidemiological and animal studies have shown that dietary micronutrients could have a positive impact on cancer prevention, incidence, and mortality [1–6]. Among these, polyphenols, a class of naturally plant phenolic compounds, play a major role [7]. Flavonoids form a distinct subclass of polyphenols, comprising about 8000 members. They are produced through the mixed shikimate and polyketide pathway (Fig. 1A), all sharing a C15-molecular scaffold. Flavanols represent a restricted group of flavonoids, featuring the 2-phenylchromanol skeleton. In this series, the * Corresponding author. Fax: +30 2810 394581. E-mail address: [email protected] (E. Castanas). 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.06.011

two main monomers, (+)-catechin and ( )-epicatechin, may form oligomers, also known as condensed tannins or procyanidolic oligomers (OPC). The monomers may equally display hydroxylation of phenyl rings, methoxylation, esterification, and/or glycosylation, increasing the diversity of naturally occurring molecules (Fig. 1B). Fruits (grapes, apples, cocoa beans, red fruits, etc.), vegetables (salads), or some beverages (e.g., coffee, tea, wine) are particularly rich in flavanol monomers and oligomers [7]. Catechin and epicatechin are potent inhibitors of cancer cell proliferation [8–10], interacting with different cellular mechanisms, including cell growth arrest, cell cycle modulation, and inhibition of Nitric Oxide Synthase (NOS) activity, leading ultimately to apoptosis (see [11] for review). In addition, catechin derivatives were found to act specifically on

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Fig. 1. (A) Biogenetic relationships in the flavonoid series. (B) Molecular structure of monomeric flavanols and the epicatechin dimers B2 and B5.

cancer cells than on their non-cancer cell counterparts [12– 15]. In addition, catechin has also been shown to increase the effect of cancer drugs in vitro, through enhancement of their intracellular concentration or bioavailability [16–18]. Finally, condensed tannins showed excellent protection against oxidative stress and free radical-mediated tissue injury [19], while a polyphenolic fraction from grape seeds resulted in the inhibition of TPA-induced tumor promotion [20].

Actin cytoskeleton rearrangement, modifying cell-substratum adhesion, controls many cell functions such as motility, division, and secretion and is involved in a large number of human diseases, including cancer [21–23], while its dissolution results in cell apoptosis [24–27]. However, the disassembly of stress fibers and focal adhesions is also implicated in the stimulation of cell motility and the promotion of invasion [28]. In a recent work, Khan et al. [29] reported that red wine

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extract, rich in condensed tannins, induces changes in actin cytoskeleton organization in bovine aortic endothelial cells, comparable with the one found in a number of cancer cells after application of testosterone-BSA, the prototype agonist of membrane androgen receptors [30–32]. As the redistribution of actin cytoskeleton was reported to be a major mechanism inducing the pro-apoptotic effect of testosterone through membrane androgen receptors [30,32], we have investigated whether condensed tannins are competitors of these sites. Membrane androgen receptors are a new class of membrane binding sites present preferentially on cancer cells. Our previous results show that these sites are found in prostate cell lines [30,31,33], and are expressed preferentially on cancer rather than in non-neoplastic cells [34]. Activation of these sites induced actin cytoskeleton polymerization, redistribution of microfilaments [31,32], and apoptosis [30,33]. Membrane androgen receptors were also found on T47D breast cancer cells [33], being responsible for the testosteroneBSA-induced apoptosis. It is interesting to note that membrane androgen receptors may be different from intracellular androgen receptors, as they are not recognized by antibodies against the latter [31], and they are not inhibited by a series of commonly used anti-androgens in vitro [32] or in vivo [30]. In the present study, we report that both the prototype flavanol natural molecules (catechin, epicatechin) as well as their dimers (B2 and B5) compete with testosterone for binding on membrane androgen receptors, and induce potent actin cytoskeleton reorganization, regulated by a similar signaling cascade as described for testosterone, leading ultimately to cell apoptosis. The above findings provide new evidences for the reported antiproliferative and proapoptotic action of polyphenols.

Materials and methods Cell cultures T47D were obtained from the European Collection of Cell Cultures (Salisbury, UK). They were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), in a humidified atmosphere of 5% CO2 in air. All culture media and serum were from Gibco BRL (Life Technologies, Paisley, UK). The serum batches used were assayed, prior to use, for the presence of polyphenol oxidase (seruloplasmin) and transferrin, by conventional nefelometric techniques. In no case, measurable levels of either substance were found.

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with unnatural stereochemistry. Therefore, optical purities of both isomers were verified by measuring their specific rotation values in acetone solutions (c = 1) at k = 589 nm. Dimers B2 and B5 were isolated with ethanol:water (70:30) from cocoa beans [36]. Xanthine bases (theobromine and caffeine) were removed by liquid – liquid separation in chloroform. Residual water was removed by successive CPC using the same solvent system as above and further preparative reverse C18 High-Performance Liquid Chromatography (Prep-HPLC) using a 15 –100% methanol:water gradient. Structural identification of both molecules was confirmed by comparison with references after co-injection on analytical HPLC system and by recording of 2D NMR long-range heteronuclear 13C – 1H spectra [37]. Detection of membrane androgen receptors Cultured T47D cells were washed with phosphatebuffered saline (PBS), removed by scrapping and centrifuged at 1500 rpm. Pelleted cells were homogenized by sonication in 50 mM Tris base pH 7.4, containing freshly added protease inhibitors (10 Ag/ml PMSF and 1 Ag/ml aprotinin). Unbroken cells were removed by centrifugation at 2500  g for 15 min. Membranes were collected by centrifugation at 45,000  g for 1 h, then acidified with one volume of 50 mM glycine pH 3 for 3 min, in order to dissociate any intracellular loosely bound or adsorbed androgen receptor [31,38,39], and resuspended in ten volumes Tris base buffer. After an additional centrifugation at 45,000  g for 1 h, protein concentration was measured by the method of Bradford [40]. Binding experiments were performed in a final volume of 0.1 ml, containing T47D cell membranes (2 mg/ml) and 5 nM of [3H]testosterone (specific activity 95 Ci/mmol, Amersham-Pharmacia, Buckinghamshire, UK) in the absence or in the presence of different concentrations of dihydrotestosterone (DHT) or polyphenols, ranging from 10 12 to 10 6 M. Non-specific binding was estimated in the presence of 5 AM DHT. After overnight incubation at 4-C, bound radioactivity was separated by filtration under reduced pressure, through GF/B filters, pre-soaked in 0.5% polyethylenimine (PEI) in water and rinsed three times with ice-cold 50 mM Tris base buffer pH 7.4. Filters were mixed with 3 ml scintillation cocktail and the bound radioactivity was counted in a scintillation counter (Perkin Elmer, Foster City, CA) with 60% efficiency for Tritium. Determination of the monomeric and polymerized actin

Polyphenol isolation (+)-Catechin and ( )-epicatechin monomers were extracted from grape seeds with acetone:water (70:30), and subsequent Centrifugal Partition Chromatography (CPC) in Hexane/Ethyl acetate/Ethanol/Water (1:8:2:7; v/ v/v/v) [35]. During isolation, epimerization at the C2 stereocenter could occur, resulting in the formation of monomers

For measurements of the monomeric (Triton X-100 soluble) and polymerized (Triton X-100 insoluble) actin, T47D cells, seeded in 6-well plates, were incubated for 3 h in serum-free medium. Thereafter, they were incubated in the same medium for the indicated time periods with or without testosterone-BSA or polyphenols (10 7 M). Thereafter, 500 Al of Triton-extraction buffer was added (0.3% Triton X-100, 5

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mM Tris, pH 7.4, 2 mM EGTA, 300 mM sucrose, 2 AM phalloidin, 1 mM PMSF, 10 Ag/ml leupeptin, 20 Ag/ml aprotinin, 1 mM sodium orthovanadate, 50 mM NaF), and the mixture was incubated for 5 min on ice. Soluble proteins in the collected supernatants were precipitated with equal volumes of 6% PCA. The Triton-insoluble fraction remaining on the plate was precipitated with 1 ml of 3% PCA and collected with scrapping. Samples were centrifuged for 5 min at 12,000 rpm and pellets were resuspended in 0.2 ml NaOH 0.1 N. Equal volumes of each fraction were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The resulting protein bands were transferred onto nitrocellulose membrane, and the membrane was blocked with 5% nonfat dry milk in TBS-T (20 mM Tris pH 7.6, 137 mM NaCl, 0.05% Tween-20) for 1 h at room temperature. Antibody solutions (in TBS-T) were added for 1 h at room temperature [monoclonal mouse antiactin first antibody (Amersham-Pharmacia, Buckinghamshire, UK) and second horseradish peroxidase-coupled antibody (Chemicon, Temecula, CA)]. Blots were developed using the ECL system (Amersham-Pharmacia) and the band intensities were quantitated by PC-based image analysis (Image Analysis Inc., Ontario, Canada) [41,42]. Confocal laser scanning microscopy of the actin cytoskeleton Cytoskeleton was visualized with direct fluorescence staining of actin microfilaments by rhodamine – phalloidin (Molecular Probes, Leiden, NL). T47D cells (3  105) were seeded at poly-l-lysine-coated cover slips, placed in 6-well plates. One day after seeding, cells were preincubated with serum-free medium for 3 h and subsequently treated with testosterone-BSA or polyphenols (10 7 M) for 1 h. In the case of wortmannin treatment, the agent (obtained from Sigma) was introduced at a concentration of 100 nM 1 h prior to the application of testosterone-BSA or polyphenols. After fixation with 3.7% formaldehyde and ice-cold acetone, cells were incubated for 40 min at room temperature with rhodamine – phalloidin (1 unit/well) to stain the filamentous actin. Slides were mounted with Mowiol antifading medium. The cover slips were analyzed using a confocal laser scanning module (Leica Lasertechnik, Heidelberg, Germany) equipped with an Ar – Kr laser [42,43].

For immunoblot analysis, the cell lysates or the immunoprecipitates were suspended in Laemmli’s sample buffer and separated by SDS-PAGE. Proteins were transferred onto nitrocellulose membrane, and blocked with 5% nonfat dry milk in TBS-T (20 mM Tris pH 7.6, 137 mM NaCl, 0.05% Tween-20) for 1 h at room temperature. Antibody solutions (in TBS-T containing 5% nonfat dry milk) were added overnight at 4-C (first antibody) and for 1 h (second horseradish peroxidase-coupled antibody). Blots were developed using the ECL system and the band intensities were quantitated by PC-based image analysis (Image Analysis Inc., Ontario, Canada). Anti-phosphotyrosine (PY20) as well as polyclonal antibody for FAK (rabbit) were from Santa Cruz Biotechnology Inc. Rabbit polyclonal anti-PI-3 kinase (p85) antibody was purchased from Upstate Biotechnology Inc. [32]. Apoptosis assay T47D cells were cultured in RPMI1640 medium, 10% FBS (Gibco BRL, Life Technologies, Paisley, UK), in gelatin pre-coated 96-well plates. Testosterone-BSA and polyphenols were added in culture medium 24 h later. After the indicated time periods, medium was discarded, and apoptosis was assayed using the APOPercentage Apoptosis Assay kit (Biocolor Ltd., Belfast, N. Ireland), as previously described [44]. The assay uses a dye which is imported by cells undergoing apoptosis, when the ‘‘flip–flop’’ mechanism translocates phosphatidylserine to the outer membrane leaflet. The dye has a purple–red color. Thereafter, the dye from labeled cells is released in the supplier’s buffer and the concentration of intracellular dye is measured at 540 nm with a reference filter at 620 nm, in a microplate colorimeter (Dynatech MicroElisa reader Chantilly, VA). Previous data have shown that results obtained with this method correlate well with oligo-nucleosome determination or apoptosis assays based on Annexin V/PI staining and flow cytometry [39]. Analysis of the results Data were analyzed using the Origin V5 (Microcal Software, Northampton, MA) and Systat V10 (SPSS Inc., Chicago, IL) microcomputer-based programs.

Immunoprecipitation and immunoblotting analysis Results Testosterone-BSA- or polyphenol-treated cells (10 7 M, for the indicated time periods), as well as untreated (control) cells were washed three times with ice-cold PBS and suspended in cold lysis buffer containing 1% Nonidet P-40, 20 mM Tris pH 7.4, and 137 mM NaCl, supplemented with protease and phosphatase inhibitors. Cleared lysates were preadsorbed with protein A-Sepharose for 1 h at 4-C, centrifuged and the supernatants (equal amounts of protein) were subjected to immunoprecipitation using the indicated antibodies and the protein A-Sepharose beads.

Monomeric and dimeric flavanols compete for membrane androgen receptor binding on T47D cells Fig. 2 presents the results of competition experiments on membrane androgen receptors of T47D cells. Dihydrotestosterone (DHT) competes, as expected, for radiolabeled testosterone binding, with an apparent IC50 of 4.7 nM, compatible with the affinity of DHT for membrane androgen receptors in the same [33] or different human cell systems

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Fig. 2. Competition for binding to membrane androgen receptors of catechin, epicatechin, and the B2 and B5 dimers. Isolated T47D membranes, acidified briefly, in order to dissociate any bound intracellular receptor or steroid, were incubated with a fixed concentration of [3H] testosterone (5 nM), and varying concentrations of the indicated monomeric (left panel) or dimeric flavanols (right panel), at concentrations ranging from 10 12 to 10 6 M. Figure presents the specific binding in each case. Results (mean T SEM) of three independent experiments performed in triplicate.

[31,45]. Catechin and epicatechin are partial competitors on this site. They displace about 30% of the specific testosterone binding with an apparent IC50 of 21.4 and 19.2 nM, respectively. On the other hand, the B2 epicatechin dimer is a complete displacer of [3H]testosterone binding with an apparent IC50 of 82.2 nM, while B5 displaces 60% of the specifically bound radioligand with an apparent IC50 of 1.7 nM (Table 1). These results indicate that monomeric and especially dimeric flavanols are partial or complete competitors of membrane androgen receptors. As all tested polyphenols compete at concentrations