Food and Chemical Toxicology 66 (2014) 158–165

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a-Mangostin-induced apoptosis is mediated by estrogen receptor a in human breast cancer cells Yeong-Seon Won a,b,1, Ju-Hye Lee a,1, Soon-Jae Kwon c, Jae-Yong Kim d, Ki-Hun Park e, Mi-Kyung Lee a, Kwon-Il Seo a,⇑ a

Department of Food and Nutrition, Sunchon National University, Suncheon 540-950, Republic of Korea Department of Bioscience and Biotechnology, Kyushu University, Fukuoka 812-8581, Japan School of Food Science and Biotechnology, Kyungpook National University, Daegu 702-701, Republic of Korea d Jeonnam Institute of National Resources Research, Jangheung 529-851, Republic of Korea e Division of Applied Life Science, IALS, Graduate School of Gyeongsang National University, Jinju 660-701, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 1 November 2013 Accepted 22 January 2014 Available online 28 January 2014 Keywords: a-Mangostin Apoptosis Human breast cancer cells Caspase ERa

a b s t r a c t In this study, we evaluated the effects of a-mangostin on cell growth inhibition and induction of apoptosis in MCF-7 ERa-positive human breast cancer cells. Our results showed that a-mangostin inhibited MCF-7 cell proliferation whereas ERa-negative MDA-MB-231 cells were less sensitive to the agent. Additionally, a-mangostin effectively induced apoptosis as evidenced by the appearance of apoptotic nuclei observed with Hoechst 33258 staining and evaluation of sub-G1 DNA contents by flow cytometry. aMangostin also activated caspases-8, -9, and -7; increased the protein levels of Bax, p53, and cytosolic cytochrome c; and induced PARP cleavage while reducing Bid and Bcl-2 protein expression. In addition, apoptosis-inducing factor (AIF) was transported from mitochondria to the cytosol after a-mangostin treatment. a-mangostin also induced apoptosis in 17-b-estradiol (E2)-stimulated MCF-7 cells in parallel with the non-stimulated cells. Moreover, treatment with 10 lM a-mangostin for 48 h specifically decreased the expression of ERa and pS2, an estrogen-responsive gene, in MCF-7 cells. Furthermore, knockdown of ERa expression in MCF-7 cells with siRNA attenuated a-mangostin-induced cell growth inhibition and caspase-7 activation. These results suggest that ERa is required for a-mangostin-induced growth inhibition and apoptosis in human breast cancer cells. Therefore, a-mangostin may be used to prevent and treat of ER-positive breast cancer. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Breast cancer is the most prevalent cancer in women worldwide (Parkin et al., 2005). Most types of breast cancer cells express high levels of estrogen receptor (ER), and exhibit estrogen-dependent development and proliferation (Ijichi et al., 2011). Estrogen induces cell proliferation and suppresses apoptosis through ERa, which is essential for the survival of both normal and cancerous

Abbreviations: ERa, estrogen receptor a; PARP, poly (ADP-ribose) polymerase; AIF, apoptosis inducing factor; FBS, fetal bovine serum; PI, propidium iodide; CC, column chromatography; SRB, sulforhodamine B; BSA, bovine serum albumin; PMSF, phenylmethylsulfonyl fluoride. ⇑ Corresponding author. Address: Department of Food and Nutrition, Sunchon National University, 315 Maegok, Sunchon, Jeonnam 540-742, Republic of Korea. Tel.: +82 61 750 3655; fax: +82 61 752 3657. E-mail address: [email protected] (K.-I. Seo). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.fct.2014.01.040 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.

cells. Furthermore, ERa serves as a prognostic factor for cases of breast cancer (Głuszko et al., 2011). Previously, several natural substances were found to inhibit the growth of breast cancer cells by down-regulating ERa expression. Celastrol, apicidin, furanodienone, and pomegranate extract induce apoptosis and inhibit the growth of MCF-7 breast cancer cells by reducing ERa levels (Im et al., 2008; Jang et al., 2011; Li et al., 2011; Sreeja et al., 2012). Thus, the induction of apoptosis via down-regulation of ERa expression by natural substances can contribute to the treatment of breast cancer. Mangosteen, Garcinia mangostana Linn., is a tropical plant found in India, Malaysia, Vietnam, and Thai-land. It known as ‘‘queen of fruits’’ is white color with soft texture whereas the rind is firm and dark purple color. Southeast Asians and Chinese have used the pericarp of mangosteen as a medicinal agent for centuries to treat tuberculosis, inflammation, fever, dysentery, diarrhea, eczema, and amoebic dysentery (Pedraza-Chaverri et al., 2008).

Y.-S. Won et al. / Food and Chemical Toxicology 66 (2014) 158–165

a-Mangostin is a major xanthone isolated from mangosteen pericarp and has been extensively studied due to its antioxidant, anti-cancer, anti-inflammatory, anti-bacterial, anti-allergic, and antiviral properties (Chae et al., 2012; Gan and Latiff, 2011; Kosem et al., 2013; Pothitirat et al., 2009; Wang et al., 2011). a-Mangostin has recently been shown to induce cell cycle arrest and apoptosis in various cancer cell lines (Krajarng et al., 2012; Nakagawa et al., 2007). Apoptosis is a form of programmed cell death involving caspases, (specialized proteases found in animal cells as inactive proenzymes) and is characterized by specific morphological features. Apoptosis is considered to be a vital component of various processes including the hormone-dependent pathway signaling (Elmore, 2007; Spencer and Sorger, 2011). a-Mangostin has been shown to induce the down-regulation of Bcl-2 expression and up-regulate Bax and p53 production in head and neck squamous cell carcinoma (HNSCC) cell lines (Kaomongkolgit et al., 2011). In addition, this compound induces caspase-3-dependent apoptosis in leukemia cell lines (Matsumoto et al., 2004), activates caspases-8, -9, and -3 in breast cancer cell lines; and increases the production of cytosolic cytochrome c during mitochondrial-mediated apoptosis (Moongkarndi et al., 2004; Shibata et al., 2011). However, the precise mechanism underlying ERa-mediated apoptotic cell death triggered by a-mangostin remains unclear. Therefore, the purpose of the present study was to investigate ERa-mediated molecular apoptotic mechanisms initiated by a-mangostin in breast cancer cells. 2. Materials and methods 2.1. Chemicals RPMI 1640 medium, Dulbecco’s modified Eagle medium (DMEM), phenol redfree DMEM, fetal bovine serum (FBS), trypsin–EDTA, penicillin, streptomycin, and an antimycotic were purchased from Gibco BRL Co. (Carlsbad, CA, USA). Propidium iodide (PI), RNase, bis-benzomide, and Lipofectamine were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). The general caspase inhibitor z-VAD-fmk was obtained from R&D Systems (Minneapolis, MN, USA). Anti-ERa, anti-Bid, anti-Bax, anti-Bcl-2, anti-caspase-7, anti-caspase-8, anti-caspase-9, anti-p53, anti-cytochrome c, anti-AIF, anti-poly (ADP ribose) polymerase (PARP), anti-pS2, and anti-b-actin antibodies as well as ERa-specific and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). An enhanced chemiluminesence (ECL) kit was purchased from Amersham Life Science (Amersham, UK). A mitochondria isolation kit was purchased from Thermo Scientific (Rockford, IL, USA).

2.2. Extraction and isolation of a-mangostin The dried seedcases of G. mangostana (0.5 kg) underwent extraction two times with ethanol (30 L in total) for 3 d with continuous stirring at room temperature. The solvent was filtered and evaporation under reduced pressure, yielding the ethanol extract (65.5 g) that was applied to a silica gel column (10  30 cm, 230–400 mesh, 700 g) and eluted using hexane/acetone solutions [100:1 (1.5 L), 50:1 (1.5 L), 30:1 (1.5 L), 20:1 (1.5 L), 10:1 (3 L), 6:1 (2.5 L), 4:1 (2 L), 2:1 (2 L), 1:1 (1 L)] and acetone alone (2 L) to recover eight pooled fractions (F1–F8) based on thin layer chromatography (TLC) profiles. Fraction F3 (24.1 g) was subjected to flash column chromatography (CC) using a gradient of hexane to acetone to produce 32 subfractions (F3.1–F3.32). Subfractions F3.12–F3.23 were combined (4.3 g), further purified by reverse-phase CC (ODS-A, 12 nm, S-150 lM), and eluted with CH3OH:H2O (4:1) to obtain compound 1 (453 mg). The isolated compound was identified based on spectroscopic data. a-Mangostin has a molecular formula of C24H26O6, and with a molecular weight of 410.45. The recovered a-mangostin was dissolved in DMSO and stored at 20 °C.

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2.4. Sulforhodamine B (SRB) assay Cell proliferation was measured with a sulforhodamine B (SRB; Sigma–Aldrich, Co.) assay. MCF-7 and MDA-MB-231 cells were seeded at a concentration of 5  104 cells/mL and incubated with a-mangostin for 48 h with or without 10 nM 17-bestradiol (E2; Sigma–Aldrich, Co.) in a humidified atmosphere of 5% CO2 at 37 °C. The cells were then fixed with 12% trichloroacetic acid. After 1 h of incubation at 4 °C, the cells were washed three times with water. The cells were stained with 0.4% SRB at room temperature for 1 h and then washed three times with 1% acetic acid. Bound SRB was dissolved with 10 mM Tris buffer after which absorbance of the 96-well plate was measured using an enzyme-linked immunosorbent assay (ELISA) reader at 540 nm as previously described (Moon et al., 1998). 2.5. Cell cycle analysis MCF-7 and MDA-MB-231 cells were seeded at a density of 1  106 cells/mL in 6well plates and cultured for 24 h in RPMI 1640 medium. After culturing, the cells were treated with a-mangostin for 48 h with or without E2 in a humidified atmosphere of 5% CO2 at 37 °C. For growth inhibition analysis and measurement of subG1 DNA contents, the cells were collected, fixed in ice-cold 70% ethanol in media, and stored at 4 °C overnight. After resuspended, the cells were washed three times with PBS and incubated with 1 lL of RNase (1 mg/mL), 20 lL of PI (1 mg/mL), and 500 lL of PBS at 37 °C for 30 min. After staining, flow cytometry was used to analyze the sub-G1 DNA contents as previously described (Park et al., 2005). 2.6. Hoechst staining Characteristic apoptotic morphological changes were assessed by fluorescent microscopy using bis-benzimide (Hoechst 33258) staining. MCF-7 and MDA-MB231 cells were seeded at a density of 5  105 cells/mL in 6-well plates, and treated with a-mangostin for 48 h in a humidified atmosphere of 5% CO2 at 37 °C. After harvested, the cells were washed twice with PBS and then stained with 100 lL of bisbenzimide (1 lg/mL) for 20 min at room temperature. Ten lL of the cell suspension was placed on a glass slide and covered with a cover slip. The cells were examined with a fluorescence microscope in order to evaluate nuclei fragmentation and chromatin condensation as previously described (Ricote et al., 2006). 2.7. Caspase inhibitor activity MCF-7 cells were seeded at a density of 1  106 cells/mL in 6-well plates and cultured for 24 h in RPMI 1640 medium. The cells were than incubated with 5 lM z-VAD-fmk for 2 h and treated with 10 lM a-mangostin for 48 h in a humidified atmosphere of 5% CO2 at 37 °C. Next, the cells were fixed with 12% TCA for 1 h at 4 °C before the plate was washed three times with distilled water. The cells were stained with 0.4% SRB at room temperature for 1 h and then washed three times with 1% acetic acid. Finally, bound SRB was dissolved with 10 mM Tris buffer, after which absorbance of the 96-well plate was read using an ELISA reader (540 nm) as previously described (Kuo et al., 2005). 2.8. Western blotting MCF-7 cells were seeded at a density of 1  106 cells/mL in a 100 mm dish and cultured for 24 h in RPMI 1640 medium in a humidified atmosphere of 5% CO2 at 37 °C. After culturing, the cells were treated with a-mangostin for 48 h, and collected by centrifugation. Cells in the pellets were lysed in lysis buffer (50 mM Tris–Cl, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 30 mM Na4P2O7, 1 mM PMSF, 2 lg/mL of aprotinin) for 30 min on ice. To examine the subcellular localization of cytochrome c and AIF, cytosolic extracts were prepared with a mitochondria isolation kit according to manufacturer’s instructions (Thermo Scientific). Protein content of the supernatant was measured using a BCA protein assay. Protein samples (10 lg of protein/lane) were separated with 12% SDS–PAGE at 100 V of constant voltage/slab for 1.5 h. The proteins were then transferred onto nitrocellulose membranes. After blocking with 2.5% and 5% bovine serum albumin (BSA) for 1 h at 37 °C, the membranes were incubated with primary antibody (anti-ER a, anti-caspase-7, anti-caspase-8, anti-caspase-9, anti-Bid, anti-Bax, anti-Bcl 2, anti-cytochrome c, anti-AIF, and anti-PARP antibody) overnight at 4 °C. Next, the membranes washed with T-TBS, incubated with horseradish peroxidase-coupled secondary antibodies for 1 h at 4 °C, and washed again with T-TBS. Antibody binding was detected using an ECL kit as previously described (Wan et al., 2006). 2.9. ERa knockdown analysis

2.3. Cell culture MCF-7 and MDA-MB-231 cells were purchased from the Korea Cell Line Bank of Seoul National University (Korea). The cells were cultured in RPMI 1640 medium, DMEM, or phenol red-free DMEM supplemented with 1%, 2.5%, or 10% FBS, penicillin (100 IU/mL), and streptomycin (100 lg/mL) in a humidified atmosphere of 5% CO2 at 37 °C.

MCF-7 cells were seeded at a density of 1  105 cells/mL in 6-well plates and cultured for 48 h in RPMI 1640 medium in a humidified atmosphere of 5% CO2 at 37 °C. RNA interference assay involving the transfection of siRNA was performed according to the manufacturer’s protocol. The cells were transfected with siRNA using Lipofectamine. After transient transfection with ERa-specific siRNA, the cells were treated with 10 lM a-mangostin for 48 h and evaluated by both an SRB assay and Western blotting (Li et al., 2011).

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2.10. Statistical analysis Data were presented as the mean ± SD. Statistical significance was analyzed by one-way ANOVA followed by Dunnett’s test. P-values