Samples and chemicals
Hot air-dried broccoli sprout powder was purchased from Damaonherb Co. (Yeongcheon, South Korea). The 11 standard compounds for high-performance liquid chromatography (HPLC) such as gallic acid, caffeic acid, p-coumaric acid, chlorogenic acid, 4-hydroxybenzoic acid, sinapic acid, catechin hydrate, rutin hydrate, myricetin, quercetin, and epicatechin and the HPLC-grade solvents such as methanol and acetic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Solvent fractionation
Broccoli sprout powder (100 g) was extracted with 2 L of 80% methanol, sonicated three times, and filtered using Whatman No. 2 filter paper. The extract was evaporated at 40 °C in a vacuum rotary evaporator and lyophilized to generate crude ME. Dried ME powder (25 g) was suspended in 500 mL of distilled water and fractionated by n-hexane, chloroform, ethyl acetate, n-butanol, and water (1:1) in a stepwise manner. Each fraction was evaporated at 40 °C and lyophilized to generate the hexane fraction (HF), chloroform fraction (CF), ethyl acetate fraction (EF), butanol fraction (BF), and residual water fraction (WF). Each extract was dissolved in dimethyl sulfoxide (DMSO) to 200 mg/mL for use in subsequent experiments.
Measurement of total polyphenol content
Distilled water (1.375 mL) and the fractions (125 μL) were added to 500 μL of Folin–Ciocalteu phenol reagent and incubated for 3 min. Then, 10% Na2CO3 (1 mL) was added, and the mixture was allowed to react in the dark for 30 min. The absorbance at 700 nm was measured using a microplate reader. Results are expressed as gallic acid equivalent (mg GAE/g).
Measurement of total flavonoid content
Distilled water (80 μL) was added to 40 μL of the fractions and 6 μL of 5% NaNO2. After incubation for 5 min, 10% AlCl3 (12 μL) was added and the mixture was incubated for 6 min. NaOH (40 μL; 1 N) and distilled water (42 μL) were then added, and the absorbance at 510 nm was determined using a microplate reader. Results are expressed as rutin equivalent (mg RE/g).
DPPH radical scavenging assay
DPPH radical scavenging activity was assayed as described previously, with some modifications [15, 16]. Each fraction in DMSO (40 µL) was added to 160 μL of 200 μM DPPH radical solution and incubated at 37 °C for 30 min. Scavenging activity was quantified using catechin as the positive control. EC50 values were calculated using Prism 7.0 software (GraphPad Software, Inc., La Jolla, CA, USA).
ABTS radical scavenging activity
ABTS radical scavenging activity was assayed as described previously, with some modifications [17]. ABTS solution (900 µL) was added to 100 µL of extract in DMSO and incubated for 2 min, and absorbance at 734 nm was measured using a UV1800 spectrophotometer (Shimadzu, Kyoto, Japan). Scavenging activity was calculated using α-tocopherol as the positive control.
Alkyl radical scavenging activity based on electron spin resonance
Alkyl radical scavenging activity was assayed as described previously [15] using a JES-FA200 electron spin resonance (ESR) spectrometer (JEOL, Tokyo, Japan). Signal intensities were compared using a magnetic ESR standard (Mn2+ marker), and results are expressed as relative height ratios. Catechin was used as the positive control.
High-performance liquid chromatography
Phenolic compounds were quantitatively analyzed using a slightly modified method [18], HPLC-ultraviolet detector (HPLC-UVD) (CBM-20A; Shimadzu, Tokyo, Japan) and Shim-pack ODS 5 μm column (Shimadzu). The mobile phase consisted of solvent A: water: methanol: acetic acid (95%, 2.5%, 2.5%; v/v/v), and solvent B: water: methanol; acetic acid (5%, 92.5%, 2.5%; v/v/v). The binary gradient was as follows: 0 min (100% A; 0% B), 10 min (90% A; 10% B), 48 min (20% A; 80% B), and 58 min (100% A; % B). The column and flow rate were maintained at 30 °C and 1.0 mL/min, respectively, and the injection volume and detection wavelength were 20 μL and 280 nm, respectively. The concentrations of phenolic compounds were calculated using a standard curve in triplicate.
Cell culture
Human breast cancer MCF-7 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). The BCSC line MCF-7/SCs were sorted from MCF-7 cells based on CD44+ and CD24−, and characterized as described previously [19]. MCF-7 cells and MCF-7/SCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and Roswell Park Memorial Institute (RPMI) medium containing 10% heat-activated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were maintained at 37 °C in an atmosphere containing 5% CO2.
Cell viability assay
MCF-7 cells and MCF-7/SCs were seeded (5000/well) in 96-well plates and incubated for 24 h. Cells were treated with the fractions for 24 h and exposed to 100 μL of 1 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 37 °C for 2 h. Next, 150 μL of DMSO was added to each well to solubilize formazan, and the plates were shaken for 30 min in the dark. Absorbance at 570 nm was evaluated using a microplate reader. IC50 values were calculated using GraphPad Prism 7.0 software.
Colony formation assay
MCF-7/SCs (400/mL) were seeded for 24 h and treated with CF for 10 days. Colonies were washed twice with PBS, fixed with 4% paraformaldehyde, and stained with 2% crystal violet for 30 min.
Flow cytometry
Flow cytometry was performed using a FACSCalibur flow cytometer (BD Biosciences, Franklin, NJ, USA), as described previously [20]. Identical numbers of cells (1 × 105/dish) were seeded for 24 h and treated with the CF for 24 h. To assay ALDH activity, the ALDEFLUOR Assay Kit (Stem Cell Technologies, Vancouver, BC, Canada) was used according to the manufacturer’s instructions. Diethylaminobenzaldehyde (DEAB), an inhibitor of ALDH, was used as the negative control. PE-conjugated anti-human CD24 and FITC-conjugated anti-human CD44 antibodies (BD Pharmingen, San Diego, CA, USA) were added to 100 μL of immunofluorescence staining buffer and incubated for 10 min at 4 °C. The cells were washed with PBS and the CD44+/CD24− cell population was analyzed. To identify apoptotic cells, the Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen, San Diego, CA, USA) was used following the supplier’s instructions. Briefly, cells were suspended in annexin V-FITC (1:20 dilution in 1 × binding buffer) and propidium (PI; 1:50) and analyzed within 30 min.
Western blot assay
MCF-7/SCs were prepared at 4 × 105 in 100 mm dishes. After incubation for 24 h and treatment with the solvent fractions, cells were lysed with RIPA lysis buffer and protein concentrations in the lysates were quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The lysates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Except for β-actin (1:10,000), the primary antibodies were used at 1:1000 dilutions, and secondary antibodies (anti-rabbit and -mouse IgG) were used at 1:5000 dilutions. Protein bands were developed using the ECL Plus Kit (Biosesang, Seongnam, South Korea).
Wound healing assay
MCF-7/SCs (2 × 105/mL) were seeded in six-well plates until 95% confluence, and a scratch wound was made in each well using a sterile pipette tip. Next, the cells were treated with the CF and incubated. Wounds were visualized using an inverted phase-contrast microscope at 4 × magnification.
Invasion assay
The upper chamber of 24-well Transwell plates (Corning, Corning, NY, USA) was filled with Matrigel and normal culture medium. After the gel had solidified, each well was loaded with 2.5 × 105 cells in serum-free medium, with or without CF, and the lower chamber was loaded with culture medium. After 24 h, the cells were fixed with 4% paraformaldehyde and stained with 2% crystal violet. The cells were observed under a phase-contrast microscope.
Hoechst 33342 staining
MCF-7/SCs were seeded into wells (1 × 104/well) and incubated for 24 h. Next, cells were treated with the CF for 24 h, stained with Hoechst 33,342 solution (10 μg/mL) for 10 min in the dark, and visualized using a fluorescence microscope (× 100) (IX73; Olympus Corporation, Tokyo, Japan).
Gas chromatography-mass spectrometry
Gas chromatography-mass spectrometry (GC–MS) of CF was carried out using the Shimadzu GCMS-QP-2010 Plus instrument in the Bio-Health Materials Core-Facility of Jeju National University, with a DB-5MS GC column (30 m length, 0.25 mm internal diameter, 0.25 µm film thickness). The injection volume of 1 µL (100 µg/mL dissolved in methanol) was delivered in splitless mode. Helium was used as the carrier gas at a constant flow rate of 1 mL min−1. The temperature ranged from 80 to 300 °C (80 °C hold for 5 min; 80–280 °C at 5 °C/min for 10 min; and 280–300 °C at 10 °C/min for 10 min). The total run time was 67 min, and mass spectra were detected using W9N08 Wiley library ver. 9.0 at a similarity cut-off of 90%.
Statistical analysis
Groups were compared using GraphPad Prism 7.0 software, Student’s t-test, and one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. Data are expressed as means ± standard deviation (SD) of three replicates and statistical significance was set at P < 0.05. Pearson’s rank correlation was performed using the ‘correlation’ tool in Prism 7.0 software.