Reagents and standards
All reagents used for total Se, Se speciation, and sulforaphane analysis were of analytical grade and were purchased from Wako Pure Chemical (Tokyo, Japan) unless stated otherwise. For the preparation of solutions and sample treatment, Ultrapure Milli-Q water (Millipore, Tokyo, Japan) was used. Cyclohexane and 2,3-diaminonaphthalene, used for total Se determination, were purchased from Dojindo Laboratories (Tokyo, Japan). Se standards, sodium selenate, selenomethionine, and Se-methylselenocysteine were purchased from Wako Pure Chemical (Tokyo, Japan); sodium selenite was purchased from Sigma Chemical (MO, USA) and selenocystine was purchased from Acros Organic (Geel, Belgium). Sulforaphane standard was purchased from LKT Laboratories (MN, USA).
Broccoli sprout extract preparation
SeSp was prepared using the method described by Sugihara et al. . Briefly, broccoli sprout seeds (Tohoku Co., Ltd., Ibaraki, Japan) were germinated in 10 ppm of a sodium selenite solution to produce SeSp or in deionized water to produce CSp. The total germination period was 7 days. The sprouts were then harvested and kept at -80°C until extraction.
For Se speciation and cell culture treatment, 500 mg of broccoli sprouts (wet weight) was extracted in 1 ml of deionized water by sonication (Branson Sonifier 450, CT, USA) for 1 minute on ice. After centrifugation at 2,500 rpm for 10 minutes, the supernatant was filtered through a 0.45- μm membrane filter (Toyo Roshi, Tokyo, Japan). The extract was prepared on the same day as the Se speciation analysis or cell culture treatment.
Determination of total Se concentration
The total Se content of broccoli sprouts was measured by a method described previously  using a Twinkle LB970 spectrofluorometer (Berthold Technologies, Bad Wildbad, Germany) at an excitation wavelength of 378 nm and an emission wavelength of 525 nm. The accuracy of this analysis was monitored by the measurement of bovine liver SRM 1577b as the reference material (National Institute of Standards and Technology, MD, USA).
Se speciation analysis by HPLC/ICP-MS
Se speciation was measured using high-performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICP-MS). Fifty microliters of extract was injected into an Asahipak GS-320 HQ size exclusion column (Showa Denko, Tokyo, Japan) and detected by an ICP-MS (Perkin-Elmer Sciex ELAN 6100, Ontario, Canada) connected to a GemTip™ cross-flow nebulizer with a Scott-type spray chamber. All ICP-MS operating parameters were adjusted to obtain maximum signal intensity. The instrumental operating conditions are given in Additional File 1.
Sulforaphane analysis by HPLC
Sulforaphane extraction and analysis were performed by slight modification of the method described by Liang et al. . Briefly, 500 mg of broccoli sprouts (wet weight) was homogenized by sonication in 1 ml of de-ionized water for 1 minute on ice. After incubation at room temperature for 30 minutes, the homogenate was extracted three times with 5 ml of methylene chloride. The methylene chloride fractions were combined and salted with 1 g of anhydrous sodium sulfate. The methylene chloride fraction was then dried at 30°C using a rotary evaporator (Eyela Rotary Vacuum Evaporator N-N series, Tokyo, Japan). The residue was dissolved in acetonitrile and stored at -80°C until analysis.
Analysis of sulforaphane was performed via the standard addition method. Sulforaphane standards of 10, 20, and 40 μg/ml in acetonitrile were mixed with the sulforaphane extract at a ratio of 1:1 (v:v). Fifty microliters of this mixed sample was then applied to an HPLC system (Waters, MA, USA). Separation was performed by an Inertsil ODS-3 column 4.6 × 250 mm (GL Sciences, Tokyo, Japan) with 70% acetonitrile as the mobile phase at a flow rate of 1 ml/min. Sulforaphane was detected at a UV absorbance of 254 nm.
Cell culture and treatment
Three human prostate cancer cell lines (LNCaP, PC-3 and DU-145) were purchased from Dainippon Pharmaceutical (Tokyo, Japan). One non-cancerous cell line (CHEK-1, an immortalized human esophageal cell line) was provided by Dr. H. Matsubara. CHEK-1 was established by transduction of human papillomavirus type 16 E6/E7 into primary cultures of esophageal keratinocytes . The cell lines were cultured in RPMI-1640 medium (Sigma, MO, USA) supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin).
For cell treatments, various concentrations of CSp and SeSp extracts were added to the cell culture medium, followed by the addition of L-Methioninase (Wako, VA, USA) in order for SeSp to generate methylselenol. After 24 hours, cells were released from the CSp or SeSp treatment, the medium was replaced, and cells were harvested at the indicated time.
Drug sensitivity assay
Cell proliferation analysis was performed with cells in the presence of various concentrations of broccoli sprout extracts by a colorimetric methyl thiazolyl tetrazolium (MTT) assay, as described previously . Briefly, cells (2 × 104 in 50 μl/well) were plated in 96-well plates. After the initial cell seeding, different concentrations of broccoli sprout extracts were added and incubated for 24 hours. Ten microliters of WST-8 assay cell-counting solution (Dojindo Lab., Tokyo, Japan) was added to each well and incubated at 37°C for 3 hours. After the addition of 100 μl/well of 1 N HCl, the cell proliferation rate was then determined by measuring the absorbance at a wavelength of 450 nm with a reference wavelength of 650 nm. The absorbance was read using a microtiter plate reader (Becton-Dickinson, NJ, USA). Results were derived from triplicate experiments.
Prostate-Specific Antigen (PSA) analysis
At approximately 80% confluence, the cells were treated with the IC50 of each sprout extract. After 24 hours, the cell medium was collected, dead cells and debris were removed by centrifugation, and the clear supernatant fraction was stored at -80°C until analysis of PSA secretion. PSA values were determined at the Gunma University Hospital Laboratory by an immunoenzymatic assay using an ST AIA-PACK PSA II kit (Tosoh Bioscience, Tokyo, Japan), following the method described by the manufacturer. PSA values were normalized to the total protein concentration of viable cells, as described elsewhere , using a ND-1000 Spectrophotometer (NanoDrop Technology, DE, USA).
Flow cytometric analysis
Cell sample preparation and propidium iodide staining were performed as described previously . At around 80% confluency, the cells were treated with the IC50 of each sprout extract. For cell cycle distribution experiments, living cells were harvested at 0 (untreated), 24, 48, and 72 hours and analyzed using a FACScan Coulter EPICS XL Flow Cytometer (Beckman Coulter, CA, USA).
Cells were cultured and treated with CSp and SeSp as described above. Morphological changes were examined at the time indicated and photographed using a regular phase-contrast microscope.
Cell extraction and Western blot analysis
Protein concentrations were determined using a BCA Protein Assay Kit (Pierce, IL, USA). Protein (40 μg) was electrophoresed on 5-20% Tris-Tricine ReadyGel (Bio-Rad, Tokyo, Japan), and electro-transferred to a hybond-enhanced chemiluminescence membrane (Amersham, Buckinghamshire, UK). Apoptosis-related proteins were analyzed using caspase-3, caspase-8, caspase-9, and PARP antibodies in 1:1000 dilutions (Cell Signaling Technology, MA, USA). Survival-related proteins were analyzed using antibodies against total Akt, phospho-Akt (p-Akt), total mTOR, and phospho-mTOR (p-mTOR) at 1:1000 dilutions (Cell Signaling Technology, MA, USA). Protein bands were detected using an enhanced chemiluminescence detection system (Amersham, Buckinghamshire, UK). β-actin (Sigma) served as the loading control. Scanning densitometry was performed with Adobe Photoshop (Apple, Inc., CA, USA), and analyzed with Quantity One (Bio-Rad, Tokyo, Japan).
Results of the total Se and sulforaphane concentrations were compared using paired t-tests. A two-way ANOVA was also used to analyze the difference of the effect of CSp and SeSp to PARP, p-Akt and p-mTOR protein expression. All statistical analyses were performed by R statistical software .