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Journal of Steroid Biochemistry & Molecular Biology 135 (2013) 51– 59

Contents lists available at SciVerse ScienceDirect

Journal of Steroid Biochemistry and Molecular Biology

jo u r n al hom epage: www.elsev ier .com/ locate / j sbmb

ffects of steroidal aromatase inhibitors on sensitive and resistant breast cancerells: Aromatase inhibition and autophagy

ristina Amarala,b, Carla Varelac, Margarida Azevedoa, Elisiário Tavares da Silvac,ernanda M.F. Roleirac, Shiuan Chend, Georgina Correia-da-Silvaa,b, Natércia Teixeiraa,b,∗

Laboratory of Biochemistry, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, no 228, 4050-313 Porto, PortugalInstitute for Molecular and Cell Biology (IBMC), University of Porto, Rua do Campo Alegre, 823, 4150-180 Porto, PortugalCEF, Center for Pharmaceutical Studies, Pharmaceutical Chemistry Group, Faculty of Pharmacy, University of Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra, PortugalDepartment of Cancer Biology, Beckman Research Institute of the City of Hope, Institute of the City of Hope, 1500 E. Duarte Road, Duarte, CA 91010, USA

r t i c l e i n f o

rticle history:eceived 25 July 2012eceived in revised form8 December 2012ccepted 29 December 2012

eywords:ormone-dependent breast cancerteroidal aromatase inhibitorsxemestaneromatase inhibitionndocrine resistance

a b s t r a c t

Several therapeutic approaches are used in estrogen receptor positive (ER+) breast cancers, being one ofthem the use of aromatase inhibitors (AIs). Although AIs demonstrate higher efficacy than tamoxifen, theycan also exhibit de novo or acquired resistance after prolonged treatment. Recently, we have described thesynthesis and biochemical evaluation of four steroidal AIs, 3�-hydroxyandrost-4-en-17-one (1), androst-4-en-17-one (12), 4�,5�-epoxyandrostan-17-one (13a) and 5�-androst-2-en-17-one (16), obtained frommodifications in the A-ring of the aromatase substrate, androstenedione. In this study, it was investigatedthe biological effects of these AIs in different breast cancer cell lines, an ER+ aromatase-overexpressinghuman breast cancer cell line (MCF-7aro cells), an estrogen-receptor negative (ER−) human breast can-cer cell line (SK-BR-3 cells), and a late stage of acquired resistance cell line (LTEDaro cells). The effectsof an autophagic inhibitor (3-methyladenine) plus AIs 1, 12, 13a or exemestane in LTEDaro cells werealso studied to understand the involvement of autophagy in AI acquired resistance. Our results showed

utophagy that these steroids inhibit aromatase of MCF-7aro cells and decrease cell viability in a dose- and time-dependent manner. The new AI 1 is the most potent inhibitor, although the AI 12 demonstrates to bethe most effective in decreasing cell viability. Besides, and in advantage over exemestane, AIs 12 and 13aalso reduced LTEDaro cells viability. The use of the autophagic inhibitor allowed AIs to diminish viabilityof LTEDaro cells, presenting a similar behavior to the sensitive cells. Thus, inhibition of autophagy maysensitize hormone-resistant cancer cells to anti-estrogen therapies.

. Introduction

Breast cancer is the leading cause of cancer death among

omen. Approximately 60% of pre-menopausal and 75% of post-enopausal patients have estrogen receptor positive (ER+) breast

ancer, which depend on estrogen for cancer cell proliferation and

Abbreviations: ER, estrogen receptor; ER+, estrogen receptor positive; ER-,strogen receptor negative; AIs, aromatase inhibitors; LTEDaro cells, long-erm estrogen deprivation human breast cancer cell line; MCF-7aro cells,R-positive aromatase-overexpressing human breast cancer cell line; SK-BR-3ells, ER-negative human breast cancer cell line; 3-MA, 3-methyladenine; PI3K,hosphatidylinositol 3-kinases; T, testosterone; E2, estradiol; MTT, tetrazolium salt-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; CFBS, charcoal fetal bovineerum; FBS, fetal bovine serum; LDH, lactate dehydrogenase.∗ Corresponding author at: Laboratory of Biochemistry, Department of Biological

ciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, no

28, 4050-313 Porto, Portugal. Tel.: +351 220428560; fax: +351 226093390.E-mail address: natercia@ff.up.pt (N. Teixeira).

960-0760/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.jsbmb.2012.12.017

© 2013 Elsevier Ltd. All rights reserved.

tumor growth [1,2]. Hormonal therapy in ER+ breast cancers canbe performed by selective ER modulators (SERMs), by selective ERdown-regulators (SERDs) that target the ER directly or block thebinding of estrogens to ER, and by aromatase inhibitors (AIs) thatinhibit the enzyme aromatase blocking the conversion of andro-gens to estrogens [1,3,4].

The third-generation of AIs are highly specific, potent andwith less adverse effects being two non-steroidal AIs, the anas-trozole and letrozole, and one steroidal, the exemestane [3,5,6].Steroidal AIs are derivative of androstenedione that compete withthe substrate for the binding to the active site and irreversiblyinactivate the enzyme [2–4,7]. Nowadays, these AIs, in post-menopausal women, are a successful alternative to tamoxifen asfirst-line therapy (anastrozole and letrozole) or second-line treat-ment (exemestane) for breast cancer [7].

Studies with AI-resistant cell lines, showed that not only dif-ferent resistance mechanisms exist, but also that the activationof ER� is critical for AI resistance [5,8,9]. Moreover, Masri et al.reported that besides the irreversible inactivation of aromatase

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2 C. Amaral et al. / Journal of Steroid Bioch

y the mechanism-based inhibitor exemestane, aromatase is stillunctional in AI-resistant cell lines [5,8], which suggests that AIesistance is not a consequence of alterations in aromatase expres-ion [5]. Furthermore, cells with a long-term estrogen deprivation,aused by prolonged AIs treatment, present acquired resistanceecause the cancer cells rely on other mechanisms or pathways torow [2,10–12]. The long-term estrogen deprivation human breastancer cell line, LTEDaro cells that over-express aromatase andimic the late stage of acquired resistance, presents a functional

R and has down-regulation of estrogen-responsive genes [8,9].oreover, the non-steroidal AI-resistant cells that are hormone-

ndependent contain an active ER� and an ER-dependent pathwayor cell proliferation and survival. However, LTEDaro cells thatre also hormone-independent and have active ER�, acquired aon-hormone-dependent pathway for survival [9]. On the otherand, unlike non-steroidal AI-resistant cells, exemestane-resistantells are hormone-dependent, since they activate the estrogen-esponsive gene AREG that drives EGFR-dependent signaling and,onsequently, cell growth [9]. Chen et al. demonstrated that resis-ance to letrozole and anastrozole is due to ER hypersensitivitynd growth-factor-ER crosstalk, whereas resistance to exemes-ane results from weak-like estrogen activity and the late stagef AI resistance results from growth-factor-ER crosstalk [5]. Thus,he third-generation AIs present different mechanisms of acquiredesistance for steroidal and non-steroidal AIs.

In the last years, several series of steroidal AIs have beenesigned and synthesized by our group [13–16] in an attempt toiscover AIs that might be more potent and specific, with less sideffects and with potential to overcome resistance to the AIs alreadyn clinical use. Recently, we have described the synthesis and bio-hemical evaluation of several potential AIs that were obtainedrom chemical modifications in the A- and D-ring of the aromataseubstrate, androstenedione [16].

In this work, the biochemical and biological effects of theost potent AIs, 3�-hydroxyandrost-4-en-17-one (1), androst-

-en-17-one (12), which was described by Numazawa [17,18],�,5�-epoxyandrostan-17-one (13a), and 5�-androst-2-en-17-ne (16) (Fig. 1), were investigated in three breast cancer cellines: the MCF-7aro cells, an ER+ aromatase-overexpressing humanreast cancer cell line; the SK-BR-3 cells, an ER− human breastancer cell line; and the LTEDaro cells, a late stage of acquiredesistant cell line. In addition, since it has been described thatutophagy may play a role in cancer cell survival and be involvedn acquired resistance to anti-cancer therapy [19–21], it was inves-igated the effect of the selected steroids and of exemestane on

CF-7aro and LTEDaro cells, in the presence of 3-methyladenine3-MA), an autophagic inhibitor that targets the phosphatidylin-sitol 3-kinases (PI3K) pathway.

. Materials and methods

.1. Materials

Eagles’s minimum essential medium (MEM), McCoy’s 5Aedium, fetal bovine serum (FBS), l-glutamine, antibiotic-

ntimycotic (10,000 units/ml penicillin G sodium, 10,000 mg/mltreptomycin sulphate and 25 mg/ml amphotericin B), GeneticinG418), sodium pyruvate and trypsin were supplied by Gibconvitrogen Co. (Paisley, Scotland, UK). Testosterone (T), estradiolE2), ethylenediaminetetracetic acid (EDTA), dimethylsulfox-de (DMSO), tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-

ifenyltetrazolium (MTT), 3-methyladenine (3-MA), charcoal andextran were from Sigma–Aldrich Co. (Saint Louis, USA). Cyto-ox 96 nonradioactive cytotoxity assay kit and Reporter Lysisuffer were from Promega Corporation (Madison, USA). [1�-3H]

y & Molecular Biology 135 (2013) 51– 59

androstenedione was obtained from Perkin-Elmer (Boston, MA,USA) and liquid scintillation cocktail Universol from ICN Radio-chemicals (Irvine, CA, USA). Bradford assay reagent was fromBio-Rad (Laboratories Melville, NY, USA). Exemestane (Aromasin)was from Sequoia Research Products Ltd. (Pangbourne, UK).

A detailed description of the synthesis, screening and IC50 inhuman placental microsomes of the studied steroids, 1, 12, 13a and16, was already published [16]. The stock solution of each steroidwas prepared in 100% DMSO and stored at −20 ◦C. The stock solu-tion of testosterone (T) and estradiol (E2) was prepared in absoluteethanol and stored at −20 ◦C. Appropriate dilutions were freshlyprepared with medium, just prior the assays and the final concen-tration of DMSO and ethanol in culture medium was less than 0.05%and 0.01%, respectively.

2.2. Cell culture

The ER− human breast cancer cell line, SK-BR-3, wasmaintained in McCoy’s 5A medium, supplemented with 1%penicillin–streptomycin–amphotericin B and 10% heat-inactivatedfetal bovine serum (FBS). The ER+ aromatase-overexpressinghuman breast cancer cell line, MCF-7aro, was prepared by sta-ble transfection with the human placental aromatase gene andGeneticin selection [22,23] and was maintained with Eagles’s mini-mum essential medium (MEM) with 1 mmol/L sodium pyruvate, 1%penicillin–streptomycin–amphotericin B, 700 ng/ml G418 and 10%heat-inactivated FBS. The long-term estrogen deprivation MCF-7aro cells, LTEDaro cells, were generated by prolonged culture ofparental MCF-7aro cells in steroid-depleted medium [8,9] and werecultured in the same conditions media as parental cells but withMEM without phenol red with 10% of pre-treated charcoal heat-inactivated fetal bovine serum (CFBS). Cells were regularly grownat 37 ◦C in 5% CO2 atmosphere and medium was changed everythree days.

To evaluate the biological effects of each compound in MCF-7aro cells, three days before starting the experiments and toavoid the interference of the steroids present in FBS and of theestrogenic effects of phenol-red [24], cells were cultured in anE2-free MEM medium without phenol-red, containing 5% heat-inactivated CFBS, 1 mmol/L sodium pyruvate, 2 mmol/L glutamineand 1% penicillin–streptomycin–amphotericin B. All the biologicalexperiments with MCF-7aro were performed according to theseconditions. MCF-7aro and LTEDaro cells were kindly provided byDr. Shiuan Chen from the Beckman Research Institute, City of Hope,Duarte, CA, U.S.A.

2.3. In cell aromatase assay

Aromatase activity and IC50 of each compound, 1, 12, 13a and16, in MCF-7aro cells were determined by a modification of themethod of Thompson and Siiteri [25] and Zhou et al. [22], using [1�-3H] androstenedione as substrate. Briefly, confluent MCF-7aro cellsplated in a 24-well plate, were cultured in serum-free medium con-taining the inhibitors at 10 �M, for aromatase activity screening,or at various concentrations (0.25–50 �M), for IC50 determination,with 50 nM of [1�-3H] androstenedione and 500 nM of proges-terone (that was used to suppress the 5�-reductase activity, whichalso use the androgen as substrate) and incubated at 37 ◦C during1 h.

The aromatase activity in disrupted MCF-7aro cells was alsoevaluated by a modified Thompson and Siiteri method [25]. Thepreparation of cell lysate was performed according the method

described by Kadohama et al. [26]. Confluent MCF-7aro cells regu-larly grown in 75 cm2 flasks were washed with PBS and collectedusing Reporter Lysis buffer 1×. After shaking for 3 h the cells werescraped, freezed at −80 ◦C and protein content was estimated by

C. Amaral et al. / Journal of Steroid Biochemistry & Molecular Biology 135 (2013) 51– 59 53

the st

tfifam

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bt

2

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Fw5ap

Fig. 1. Chemical structure of

he Bio-Rad Protein Assay. Briefly, the assay reaction was per-ormed with 8 �g of protein obtained from lysed cells incubatedn potassium phosphate 67 mM (pH 7.4) buffer, containing dif-erent concentrations (0–50 �M) of inhibitors, 50 nM of [1�-3H]ndrostenedione, 1 �M of progesterone, 0.1% of bovine serum albu-in and 300 �M of NADPH during 1 h in a water bath at 37 ◦C.Formestane at 1 �M and exemestane at 10 �M were used as

eference AIs. In order to compare with the new AIs, the IC50 ofxemestane in MCF-7aro cells was also evaluated.

The aromatase activity was evaluated as previously describedy our group [15]. All experiments were carried out in triplicate inhree independent experiments.

.4. Cell viability

To evaluate the effects of each steroid, 1, 12, 13a and 16, in MCF-aro, SK-BR-3 and LTEDaro cells viability, MTT and lactate dehydro-

enase (LDH) release assays were performed. Cells were culturedn 96-well plates at a cellular density of 2.5 × 104 cells/ml (for 2nd 3 days) and 1 × 104 cells/ml (for 6 days), with different con-entrations of each compound (5–50 �M). MCF-7aro cells cultured

AIsIC50 ( M) MCF-7aro

IC50 ( M) disrupted MCF-7a ro

1 0.6 - 12 5.0 0.7 13a 8.5 1.2 16 12.5 6.5 E 0.9 -

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ig. 2. Anti-aromatase activity of AIs 1, 12, 13a and 16. (A) Aromatase activity of AIs in plere determined by the in-cell aromatase assay in MCF-7aro cells (B) and in disrupted

0 nM of [1�-3H] androstenedione. (D) IC50 (�M) values for the steroids under study in is a reference AI. Data are presented as a percentage of the tritiated water control and cublished data in Varela et al. [16].

eroidal AIs 1, 12, 13a and 16.

in MEM without phenol-red containing CFBS were treated with1 nM of testosterone (T), the aromatase substrate and proliferationinducing agent, or with 1 nM of estradiol (E2), the product of aro-matase. MCF-7aro cells and LTEDaro cells treated with or withoutcompounds 1, 12 and 13a were also treated with 3-methyladenine(3-MA), at 1 mM for 3 and 6 days. Exemestane was used as controlat 5 and 10 �M.

After each incubation time, MTT (0.5 mg/ml) was added to eachwell and cells were incubated for 2 h and 30 min at 37 ◦C in 5% CO2.The formazan was quantified spectrophotometrically by addition ofDMSO:isopropanol mixture (3:1). LDH release was measured usingCytoTox 96 nonradioactive cytotoxity assay kit according to themanufacturer’s protocol.

All the assays were performed in triplicate in three indepen-dent experiments and results are expressed as a percentage of theuntreated control cells.

2.5. Statistical analysis

Statistical analysis of data was performed using analysisof variance (ANOVA) followed by Bonferroni test for multiple

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acental microsomes*, in intact and disrupted MCF-7aro cells. The IC50 (�M) valuesMCF-7aro cells (C), incubated with different concentrations (0–50 �M) of AIs andntact and disrupted MCF-7aro cells. Formestane (F) and exemestane (E) were usedorrespond to three independent experiments carried out in triplicate. * Previously

54 C. Amaral et al. / Journal of Steroid Biochemistry & Molecular Biology 135 (2013) 51– 59

Fig. 3. Effects of AIs 1 (A), 12 (B), 13a (C) and 16 (D) in cell viability of MCF-7aro cells, evaluated by MTT assay. (E) Comparison of the effects of all AIs. MCF-7aro cells werecultured with different concentrations of each AI (5–50 �M) and testosterone (T) at 1 nM during 2, 3 and 6 days. Cells cultured with T represent the maximum of cell viabilitya xperimw

csm

3

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nd were considered as control. Results are the mean ± SEM of three independent eith each AI are denoted by *p < 0.05, **p < 0.01 and ***p < 0.001.

omparisons and values of P < 0.05 were considered as statisticallyignificant. The data presented in this study are expressed as theean ± SEM.

. Results

.1. In cell aromatase assay

From a series of recently synthesized A- and D-ring modifiedteroidal AIs, [16], four of them which presented, in human placen-al microsomes, an anti-aromatase activity higher than 70% wereelected for studying their anti-aromatase activity in MCF-7aroells. The IC50 values of steroids 1, 12, 13a and 16, were evaluatedy a radiometric assay, using [1�-3H] androstenedione as substrate.he tritiated water released from [1�-3H] androstenedione into thencubation medium was used as an index of estrogen formation27]. The AIs 1, 12, 13a and 16 induced an aromatase inhibition, in

CF-7aro cells, of 93.21%, 62.59%, 52.18% and 45.39%, respectively.ormestane (1 �M) and exemestane (10 �M) presented an aro-atase inhibition of 96.63% and 97.58%, respectively. Compound

was the most potent inhibitor with an IC50 of 0.6 �M. Compound2, showed an IC50 of 5 �M, while compounds 13a and 16 had an

C50 of 8.5 �M and 12.5 �M, respectively (Fig. 2). The exemestaneresented an IC50 in MCF-7aro cells of 0.9 �M (Fig. 2).

ents, performed in triplicate. Significant differences between the control and cells

As the IC50 values were much higher in cells than in placentalmicrosomes for compounds 12, 13a and 16, the aromatase activityin disrupted MCF-7aro cells was also evaluated. In lysed cells theAIs 12, 13a and 16 presented an IC50 of 0.7 �M, 1.2 �M and 6.5 �M,respectively (Fig. 2).

3.2. Cell viability in MCF-7aro cells

The effects of steroids 1, 12, 13a and 16 (5–50 �M) in MCF-7arocells viability and cytotoxicity were studied by MTT and LDH releaseassays after 2, 3 and 6 days of treatment. Cells treated only with Twere considered as control. As observed in Fig. 3, compounds 1,12, 13a and 16 induced a decrease in cell viability in a dose- andtime-dependent manner. However, for the lowest concentration(5 �M) and after 2 days of treatment, compounds 1 (p < 0.001) and16 (p < 0.05) caused a significant increase in cell viability, suggest-ing an estrogenic effect. Nevertheless, all the compounds induced asignificant decrease (p < 0.01; p < 0.001) in cell viability for all timesof incubation and for the higher concentrations (10–50 �M). Com-pound 12 is the most efficient in decreasing cell viability and 16 isthe less potent (Fig. 3E). After 2 and 3 days of treatment no effects

were observed in LDH release for all compounds (data not shown).

To address the question whether reduction of cell viabilitywas due to aromatase inhibition, it was evaluated the effects ofcompounds on estradiol (E2)-treated MCF-7aro cells for the same

C. Amaral et al. / Journal of Steroid Biochemistry & Molecular Biology 135 (2013) 51– 59 55

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ell v

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Fig. 4. Effects of AIs 1 (A), 12 (B), 13a (C) and 16 (D) in cell viability of MCF-7aro cells cultured with T or E2 (E) and of SK-BR-3 cells (F). Cells were cultured with differentc threer ersusM < 0.05,

ptacTbe(

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t

oncentrations of each AI (5–50 �M) during 3 days. Results are the mean ± SEM ofeference AI. Significant differences between the E2-treated MCF-7aro cells with AI vCF-7aro cells with AI versus E2-treated MCF-7aro cells with AI are denoted by bp

eriods of time as for T-treated cells. As presented in Fig. 4, cellsreated with steroid 16 induced a similar reduction in both T-nd E2-treated cells viability, whereas compounds 1, 12, and 13aaused significant differences (p < 0.05; p < 0.01; p < 0.001) between- versus E2-treated cells. Moreover, the reduction on cell via-ility was more marked in T-treated cells. All the new AIs andxemestane induced a similar decrease on E2-treated cells viabilityFig. 4E).

.3. Cell viability in SK-BR-3 cells

To evaluate if the biological effects of the different steroids inCF-7aro cells were dependent on ER, it was also investigated

he effect of these compounds in an ER− human breast cancer celline, the SK-BR-3, during the same time periods. As observed inig. 4F, all the studied AIs induced a similar decrease on viabil-ty of SK-BR-3 cells in a dose-dependent manner. This effect was

ore marked on this cell line than on E2-treated MCF-7aro cells.urthermore, when comparing E2-treated MCF-7aro with SK-BR-

treated cells (Fig. 4), compounds 1 and 12 induced significantifferences (p < 0.01; p < 0.001) in cell viability for all times of treat-ent. Compound 13a caused a significant difference (p < 0.05) only

or the higher concentrations (25 and 50 �M), while compound 16aused also significant differences (p < 0.001), except for the higheroncentration (50 �M).

.4. Cell viability in LTEDaro cells

The biological effects of compounds 1, 12 and 13a were inves-igated in LTEDaro cells, as they represent a good model to

independent experiments, performed in triplicate. Exemestane (E) was used as a SK-BR-3 cells with AI are denoted by ap < 0.05, aap < 0.01 and aaap < 0.001; T-treatedbbp < 0.01 and bbbp < 0.001.

study AI acquired resistance [5,8,9]. Compound 16 was not stud-ied in LTEDaro cells as its effects in MCF-7aro cells appear tobe aromatase-independent. Except compound 12 (50 �M) thatpresented a significant decrease in cell viability (p < 0.01), theother AIs had no effect after 3 days of treatment (Fig. 5). How-ever, contrary to compound 1, steroids 12 and 13a presented asignificant (p < 0.001) reduction in cell viability after 6 days oftreatment (Fig. 5). Nevertheless, the reduction was always lessintense than in MCF-7aro cells viability (Fig. 6). Exemestane thatwas used as a reference control, at 5 and 10 �M, during 3 and 6days, did not affect LTEDaro cells viability (Fig. 5), as previouslyreported [8,9].

3.5. Effects of an inhibitor of autophagy in cell viability ofMCF-7aro cells and LTEDaro cells treated with compound 1, 12and 13a

As one of the mechanisms described for the AI-acquired resis-tance is due to autophagy, it was also evaluated, by MTT assay,the effects of the autophagic inhibitor 3-MA in AI-sensitive andAI-resistant breast cancer cell lines treated with compounds 1,12 and 13a, after 3 and 6 days. Cells treated with 3-MA wereconsidered as control, and presented a decrease in cell viabilityof only 9% and 10% in MCF-7aro and LTEDaro cells, respectively,when compared to cells without 3-MA. As presented in Fig. 6,when comparing AIs-treated MCF-7aro cells with or without

3-MA, no significant differences in cell viability were observed,which suggests that autophagy is not involved in the reduction ofcell viability. On the other hand, and as described previously byour group [28], exemestane, used as control in this study, induced

56 C. Amaral et al. / Journal of Steroid Biochemistry & Molecular Biology 135 (2013) 51– 59

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Fig. 5. Effects of AIs 1 (A), 12 (B), 13a (C) and exemestane (D), which was used as reference AI, on viability of LTEDaro cells treated with different concentrations ofA cellse AI-trb ted by

iv

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Is (5–50 �M) during 3 and 6 days with or without 3-MA. As controls it was usedxperiments, performed in triplicate. Significant differences between the control andetween the LTEDaro cells with AI versus LTEDaro cells with AI plus 3-MA are deno

n treated MCF-7aro cells plus 3-MA a significant decrease in celliability when comparing with cells without 3-MA.

Contrary to what was observed with these compounds inTEDaro cells, 3-MA plus compounds caused a significant (p < 0.01;

< 0.001) dose- and time-dependent decrease in cell viabilityFig. 5). Moreover, when comparing AIs treated LTEDaro cells withr without 3-MA, significant differences (p < 0.01; p < 0.001) werelso observed. As in the case of the new AIs, exemestane plus 3-A also induced a significant decrease (p < 0.001) in LTEDaro cells

iability. In addition, comparing these cells treated with or without-MA significant differences (p < 0.01; p < 0.001) were also observedFig. 5).

The effects of AIs plus 3-MA on LTEDaro cell viability were

imilar to the reduction of MCF-7aro cell viability with or with-ut 3-MA (no significant differences were observed between,TEDaro cells with 3-MA and MCF-7aro cells with or without 3-MA)Fig. 6).

treated with or without 3-MA. Results are the mean ± SEM of three independenteated LTEDaro cells with or without 3-MA are denoted by **p < 0.01 and ***p < 0.001;

ccp < 0.01 and cccp < 0.001.

4. Discussion

The compounds studied in this work, were obtained fromchemical modifications in the A-ring of the aromatase substrate,androstenedione, as previously described [16]. These competitiveAIs exhibit, in human placental microsomes, an IC50 of 0.18 �M forcompound 1, 0.14 �M for compound 12, 0.97 �M for compound13a and 1.73 �M for compound 16 [16]. In this study, the abovementioned steroids were further evaluated in MCF-7aro cells. Asexpected, these compounds were not as efficient inhibitors in thesecells as they were in placental microsomes. This is probably due tothe higher levels of aromatase expressed in that cell line and tothe accessibility of the inhibitors to aromatase (cell-free aromatase

system) in human placental microsomes. Hence, compound 1 pre-sented an IC50 of 0.6 �M, compound 12 of 5 �M, compound 13a of8.5 �M and compound 16 of 12.5 �M. Comparing our steroids withexemestane (IC50 of 0.9 �M), steroid 1 presented the lowest value

C. Amaral et al. / Journal of Steroid Biochemistry & Molecular Biology 135 (2013) 51– 59 57

Fig. 6. Comparison of the biological effects of AIs 1 (A), 12 (B) and 13a (C) (5–50 �M) on viability of MCF-7aro cells and LTEDaro cells with or without 3-MA, after 6 daysof treatment. As controls it was used cells treated with or without 3-MA. Results are the mean ± SEM of three independent experiments, performed in triplicate. Significantdifferences between the AI-treated LTEDaro cells versus AI-treated LTEDaro cells plus 3-MA or with AI-treated MCF-7aro cells or with AI-treated MCF-7aro plus 3-MA ared

oamdlitoba

cEtssmebcgaaba2dcliwtd

aM

enoted by ddp < 0.01 and dddp < 0.001.

f IC50. However, as the IC50 for compounds 12, 13a and 16 werepproximately 37, 8 and 7 times higher in cells than in placentalicrosomes, respectively, the IC50 values were also determined in

isrupted MCF-7aro cells. In this case, the IC50 values were muchower (0.7 �M for 12, 1.2 �M for 13a and 6.5 �M for 16) than inntact cells, which confirms that cell membrane interferes withhe uptake rate of these inhibitors. When analyzing all the databtained, we can conclude that steroid 1 is the most potent AI inoth systems and, unlike AIs 12, 13 and 16, cell membrane does notffect compound uptake.

In order to evaluate the consequences of these compounds uponell viability, it was investigated their effect in MCF-7aro cells, anR+ breast cancer cell line, stable transfected with aromatase gene,o express enough aromatase activity [29]. Our results demon-trated that, like exemestane [28], all the studied AIs induced aignificant decrease in cell viability in a dose- and time-dependentanner. However, compounds 1 and 16 induced also an estrogenic

ffect for the lower concentrations for 2 days of treatment. Thisehavior was already observed for exemestane [28] and for otherompounds formerly synthesised and biochemically studied by ourroup, specifically 5�-androst-3-en-17-one and 3�,4�-epoxy-5�-ndrostan-17-one [30]. Masri S. et al. also showed that exemestanet lower concentrations (1 �M) was able to drive proliferation ofreast cancer cells (MCF-7 and MCF-7aro) [31]. Other authors havelso demonstrated the estrogenic effect of other compounds, like-methoxyestradiol [32], genistein [33,34], resveratrol [35], 5�-ihydrotestosterone and 5�-androstane-3�,17�-diol [36] in breastancer cell lines. Comparing the studied AIs, compound 16 was theess efficient in decreasing cell viability of MCF-7aro cells, whichs consistent with its IC50 in these cells. Although, compound 12

as not considered the best anti-aromatase inhibitor in placen-al microsomes [16] and cells, it was the most potent steroid in

ecreasing cell viability.

To understand if these biological effects were dependent onromatase inhibition, it was also studied the effects of each AI inCF-7aro cells with E2, which is the product of the aromatization

reaction of testosterone by the enzyme aromatase. The AIs stud-ied, including exemestane, only induced on E2-treated MCF-7arocells a slight decrease in cell viability. This reduction was alwaysless marked than the one observed on T-treated MCF-7aro cells,but still significantly different for steroids 1, 12 and 13a. More-over, to evaluate if the decrease in cell viability was also due to theinteraction with estrogen receptor, SK-BR-3 cell line, was also used.Our results demonstrate that in SK-BR-3 cells, the new AIs presenta similar decrease in cell viability in a dose-dependent manner.Moreover, the compounds caused a significant decrease in SK-BR-3 cell viability that was more effective than in E2-treated MCF-7arocells, suggesting that the AIs induced their effects in cells in anER-independent manner. The SK-BR-3 cell line, besides express-ing all the enzymes required for estrogen synthesis, only expressesvery low levels of ER� and has no expression of ER�, indicatingthat this cell line represents a model for estrogen-independentbreast cancers. Moreover, estrogens may act through non-ER medi-ated pathway [37]. Therefore, our data indicates that compounds1, 12 and 13a induced a decrease on MCF-7aro cell viability inan aromatase-dependent but ER-independent manner, whereasfor compound 16 the reduction was in an aromatase- and ER-independent manner (Fig. 4). Nevertheless, we cannot excludethe hypothesis that other mechanisms, independent of ER or aro-matase, may also be involved.

In terms of structure–activity relationships (SAR), the presenceof a double bond in C-4/C-5 position, as in compounds 1 and 12, oran epoxide group, which has a similar geometry, as in compound13a, seems to be important for the reduction of cell viability bedependent on aromatase inhibition. Moreover, the presence of thedouble bond seems to contribute to this decrease in a more effec-tive manner than the epoxide group (compare 1 and 12 with 13a).Concerning compound 16, the farther is the double bond from the

A/B-ring junction in C-5, the lower is the anti-aromatase activityin MCF-7aro cells, being the anti-proliferative effects aromatase-independent. The SAR results obtained in MCF-7aro cells confirmthose obtained by our group in microsomes, which have been

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8 C. Amaral et al. / Journal of Steroid Bioch

ttributed to planarity requirement near the C-5 A/B-ring junction.his planarity can effectively be conferred by a double bond or anpoxide function in the C-4/C-5 position [13,14,16].

In order to evaluate the efficacy of our compounds in acquiredndocrine resistance, it was also studied the effect of compounds, 12 and 13a in LTEDaro cells, a good model of late stage acquiredesistance that does not respond to AI treatment [5,8,9]. Compound6 was not studied in LTEDaro cells once its effects in MCF-7aroells revealed to be aromatase-independent. Our data showed thatontrary to AIs 12 and 13a, the AI 1 had no effect on LTEDaroells. Moreover, as already described [8,9], the exemestane hado effect in LTEDaro cells viability. The exemestane resistant cell

ine [38] presents an ER that is estrogen-dependent or hormoneesponsive, unlike the non-steroidal AI-resistant cells [5,9]. Curi-usly, AI 1 like exemestane [28,31] has estrogenic-effects for loweroncentrations in MCF-7aro cells and has no significant inhibitionn cell growth of LTEDaro cells, which suggests that AI 1 mayave a resistance-mechanism similar to exemestane. Thus, unlikehe other AIs (12 and 13a) that induced a significant decrease iniability of LTEDaro cells, AI 1 may have a hormone-dependentesistant mechanism. Although further studies must be performedo understand the underlying mechanism, our data suggest thathe steroidal AIs under investigation have different mechanism ofcquired resistance, as already referred for steroidal (exemestane)nd non-steroidal AIs (letrozole and anastrozole) [5,39].

As it had been mentioned before, autophagy is involved inI acquired resistance. Therefore, the effects of the autophagic

nhibitor 3-MA, that targets class I and class III PI3K, were studiedn AI-treated LTEDaro and MCF-7aro cells. In autophagy, the class

PI3K leads to activation of AKt and mTOR, inhibiting autophagy,hereas the class III stimulate autophagic sequestration [40–44].esides the PI3K role in autophagy, survival and cell-cycle progres-ion, this pathway also interferes with ER directly or indirectly,romoting estrogen-dependent and -independent ER transcrip-ional activity [45,46]. Moreover, hyper-activation of PI3K pathwayas already been shown to reduce ER and ER-target gene expres-ion [12] that is associated to anti-estrogen resistance [46–48]. So,he inhibition of PI3K pathway reverses anti-estrogen resistance45–47].

Our results showed that no alterations in the reduction on celliability were observed when MCF-7aro cells were treated with AIs, 12 and 13a, in the absence or presence of 3-MA, which meanshat autophagy is not involved in this process. On the other hand,nd as described previously by our group [28], exemestane-treatedCF-7aro cells plus 3-MA presented a significant decrease in cell

iability when compared to cells without 3-MA. This suggests thatutophagy is involved and acted as a pro-survival mechanism [28].urthermore, and contrary to exemestane and AI 1, LTEDaro cellsere sensitive to the AIs 12 and 13a. However, the autophagic

nhibitor sensitizes this resistant cell line to exemestane and AI 1,hile for the other AIs caused a more pronounced decrease in cell

iability. For the new AIs, the behavior of the resistant cell line in theresence of 3-MA was similar to the sensitive cell line with or with-ut 3-MA, indicating that the autophagic inhibitor increases theensibility of resistant cells to AIs treatment. Moreover, in the casef exemestane, besides autophagy play a pro-survival role to pro-ect breast cancer cells from apoptosis [28], it is involved in acquiredesistance to exemestane treatment. In fact, Masri et al. showedhat LTEDaro cells have adapted a non-hormone-dependent path-ay for survival [9]. Thus, our data suggest that autophagy and PI3Kathway are involved in this type of resistance in cancer cells.

Autophagy is an evolutionary conserved catabolic process of

ubcellular degradation that may contribute to tumor cell sur-ival and may confer resistance to anti-cancer therapy, sinceome of the anti-hormonal therapies induce a protective and pro-urvival autophagy in tumor microenvironment [19–21]. Thus, the

y & Molecular Biology 135 (2013) 51– 59

inhibition of autophagy, by autophagic inhibitors, such as 3-MA, orby knockdown of autophagic related genes, may re-sensitize resis-tant cancer cells to radiation or chemotherapy [49–56] and lead toa decrease on tumor growth by increasing cell death [20]. There-fore, the inhibition of autophagy may effectively sensitize cancercells to therapeutic agents, improving in that way clinical benefitsin breast cancer treatment. These results are important to eluci-date the mechanisms, involved in anti-cancer drug resistance andsuggest that anti-estrogens and autophagic inhibitors that targetPI3K pathway may be a potential therapeutic strategy in cancer, tore-sensitize resistant cancer cells to anti-cancer therapy.

5. Conclusion

In summary, steroids, 1, 12, 13a and 16 inhibit aromatase inMCF-7aro cells causing a decrease in MCF-7aro cell viability in adose- and time-dependent manner. Compound 1 is the most potentaromatase inhibitor, being AI 12 the most effective in decreasingcell viability in an aromatase-dependent manner. This study alsoconfirms the importance of a double bond in C4, 5 position, forstrong anti-aromatase activity and for aromatase-dependent anti-proliferative effects. In addition, using LTEDaro cells as a modelof late stage of acquired resistance, we observed that betweensteroidal AIs there are different mechanisms of acquired resistance,since compounds 12 and 13a decreased LTEDaro cells viabilitywhile compound 1 and exemestane had no effect. Moreover, thecombination of the autophagic inhibitor 3-MA with compounds1, 12, 13a and exemestane exacerbated the reduction of LTEDarocell viability, being the behavior similar to the sensitive cells.Together, these results suggest that autophagy and PI3K pathwayare involved in resistance and inhibition of autophagy can sensitizethe resistant cancer cells to hormonal therapies.

Conflict of interest

The authors have no conflict of interest to declare.

Acknowledgements

The authors are grateful to Fundac ão para a Ciência e Tec-nologia (FCT) for the PhD grants attributed to Cristina Amaraland Carla Varela (SFRH/BD/48190/2008 and SFRH/BD/44872/2008,respectively). This work was funded by FEDER Funds through theOperational Competitiveness Program- COMPETE and by NationalFunds through FCT under the project FCOMP-01-0124-FEDER-020970 (PTDC/QUI-BIQ/120319/2010). We thank Dr. Shiuan Chenfor kindly supplying MCF-7aro and LTEDaro cells.

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