Plant material and extraction
The aerial parts of A. pilosa were purchased from Kyungdong Market (Seoul, Korea) in dried form and identified by the Classification and Identification Committee of the Korea Institute of Oriental Medicine (KIOM). A voucher specimen (KIOM109-122Aa) has been deposited at the herbarium of the Department of Herbal Resources Research of the KIOM. Each of the dried components was extracted twice with 10 volumes of water at 80°C for 3 h. The extracts were filtered through filter paper (Whatman, Maidstone, UK) and were concentrated under reduced pressure by a rotary evaporator (EYELA, Tokyo, Japan) at 40°C. The water filtrates were frozen and lyophilized. The lyophilized extracts were stored at −20°C until use.
Analysis of A. pilosa aqueous extract composition
The chemical composition of the A. pilosa aqueous extract was determined using liquid chromatography-mass spectrometry (LC-MS). Briefly, aqueous extracts of A. pilosa were subjected to ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) analysis. LC-MS was performed using a LTQ Orbitrap XL linear ion trap mass spectrometer system (Thermo Fisher Scientific Co., Waltham, MA) equipped with an electrospray ionization source. The UHPLC separations were performed with an Accela UHPLC system (Thermo Fisher Scientific) by using an Acquity BEH C18 column (1.7 μm, 100 × 2.1 mm; Waters Corp., Milford, MA). Mobile phase A was water and phase B was acetonitrile, where both phases contained 0.1% formic acid. The gradient elution, at a flow rate of 0.3 mL/min, was performed as follows: 0–1 min, 1% B (isocratic); 1–15 min, 1–30% B (linear gradient); 15–20 min, 30–60% B (linear gradient); 20–25 min, 60–100% B (linear gradient); and 25–27 min 100% B (isocratic). Full-scan mass spectra were obtained using the negative-ion mode at with an m/z range of 100–1000. Data-dependent tandem mass spectrometry (MSn) experiments were controlled by menu-driven software provided with the Xcalibur system (Thermo Fisher Scientific).
ERα- and ERβ-binding assays
Estrogen receptor-binding ability was examined using an estrogen receptor (ER) competitive binding assay kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The relative affinity of the test material for ER was determined by the change in polarization value (Molecular Devices Inc., Sunnyvale, CA) in the presence of a test plant.
MCF-7, an ER-positive human breast cancer cell line, was purchased from the Korean Cell Line Bank (Seoul, Korea) and cultured in RPMI-1640 containing 10% fetal bovine serum and penicillin-streptomycin solution (100 units/mL penicillin and 100 μg/mL streptomycin; Hyclone Laboratories, Inc., South Logan, UT). The cells were grown at 37°C in a humidified atmosphere of 95% air/5% CO2. The medium was renewed 2–3 times per week, and before reaching confluence, the cells were subcultured every 3–4 days in a 1: 4 ratio.
Proliferation assay of MCF-7 cells (E-screen assay)
Confluent MCF-7 cells were washed twice with phosphate-buffered saline (PBS) (Hyclone Laboratories) and 0.05% trypsin-EDTA solution (Invitrogen) was added for 1 min. After trypsin-EDTA was removed, the culture was left at room temperature (~20°C) for 5–10 min; subsequently, the cells were detached, resuspended in RPMI-1640 medium, counted, and seeded into 24-well plates at a density of 2 × 104 cells/well in normal growth medium. After 24 h, the cells were completely attached to the well bottom; the medium was then aspirated and estrogen-free medium containing both phenol-red-free RPMI (Invitrogen) and 5% charcoal-dextran-stripped human serum (Hyclone Laboratories) was added. MCF-7 cells were treated with different concentrations of test material and were cultured for 144 h. In addition, test material were added to the medium at some concentrations where they showed estrogenic activity, either with or without the ER-antagonist ICI 182,780 (Tocris, Bristol, UK). 17β-Estradiol and PBS were used as the positive and negative controls, respectively.
MTT proliferation assay
Cell proliferation was assessed after 7 d in culture, using the MTT proliferation assay. After the incubation period, cells were added with 100 μL of 5 mg/mL thiazolyl blue tetrazolium bromide (Sigma, St. Louis, MO) solution/well and were incubated further for 4 h in a humidified atmosphere (37°C in 5% CO2). The medium was replaced with 1 mL dimethyl sulfoxide (DMSO). The absorbance was measured at 540 nm in a microplate reader (Molecular Devices Inc., Sunnyvale, CA). Cell proliferation was expressed as percentage values compared with the negative PBS control, which was considered to represent 100% cell proliferation.
MCF-7 cells were seeded in 75-cm2 culture flasks at a density of 2 × 104 cells/cm2 in RPMI-1640 medium and incubated at 37°C at 5% CO2. On the following day, the medium was shifted to estrogen-free medium containing phenol-red-free RPMI (Invitrogen) and 5% charcoal-dextran-stripped human serum (Hyclone Laboratories) with controls or test material. After the cells were incubated for 24 h at 37°C and 5% CO2, they were washed twice with PBS, and total RNA was extracted from cells using the RNeasy Plus Mini Kit (Qiagen, Venlo, Netherlands), according to the manufacturer’s protocol. The quantity and purity of the total RNA obtained were determined on the basis of the absorbance at 260 and 280 nm. RNA quality was determined by gel electrophoresis on 2% agarose gels stained with ethidium bromide (0.5 μg/mL). RNA samples were stored at −20°C until use.
The expression levels of estrogen-dependent genes were determined by a real-time one-step RT-PCR performed using the SYBR Green PCR master mix (Qiagen) and a thermal cycler Rotor-Gene 3000 (Corbett Research, Mortlake, Australia), in accordance with the manufacturer’s protocol. The primer sequences for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pS2, progesterone receptor (PR), and cathepsin D are as follows (forward and reverse, respectively):
GAPDH: 5′-CCATGGAGAAGGCTGGGG-3′, 5′-CAAAGTTGTCATGGATGACC-3′; pS2: 5′-CATCGACGTCCCTCCAGAAGAG-3′, 5′-CTCTGGGACTAATCACCGTGCTG-3′; PR: 5′-CGCGCTACCCTGCACTC-3′, 5′-TGAATCCGGCCTCAGGTAGTT-3′; and cathepsin D: 5′-CTGAGCAGGGACCCAGATG-3′, 5′-CAGGTGGACCTGCCAGTAG-3′.
One step RT-PCR was performed as follows: (1) reverse transcription at 50°C for 30 min; (2) initial denaturation at 95°C for 15 min; and (3) 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and elongation at 72°C for 30 s. Melting-curve analysis was performed at 72–95°C to verify the specificity of amplification and was supplemented with 2% agarose gel electrophoresis of randomly selected samples. The relative quantitation values were calculated by analyzing the changes in SYBR Green fluorescence during PCR, according to the manufacturer’s instructions. Ct values were defined as the threshold cycles at which a statistically significant increase occurred in the intensity of SYBR Green emission. Using the 2-ΔΔCt method, the changes (in orders of magnitude) relative to the control were calculated. Ct values were normalized to those for the housekeeping gene, GAPDH and the ΔCt values of the A. pilosa-treated cells were normalized to the mean ΔCt values of the controls.
Data was expressed as mean ± SD values. Cell proliferation was expressed as percentage values compared with that for the negative PBS control, which was taken to represent 100% cell proliferation. Duncan’s multiple range tests were used to detect differences between groups when analysis of variance was significant at p < 0.05. Student’s t-test was used to establish significant differences between any 2 groups.