L-Sulforaphane (purity ≥ 98%) (Figure 1), urea, thiourea, 3-[(3-cholamidopropy) dimethyammonio]-1-propanesulfonate (CHAPS), dithiothreitol (DTT), benzamidine, Bradford solution, acrylamide, iodoacetamide, bis-acrylamide, sodium dodecyl sulphate (SDS), acetonitrile, trifluoroacetic acid and α-cyano-4-hydroxycinnamic acid were purchased from Sigma-Aldrich Co. (St. Louis, MO). Pharmalyte (pH 3.5-10), IPG DryStrips (pH 4-10 NL, 24 cm) and Modified porcine trypsin (sequencing grade) were from Amersham Biosciences, Genomine Inc. and Promega (Maison, WI), respectively.
LNCaP cells were purchased from the American Type Culture Collection (ATCC) (Manassas, VA) and maintained in RPMI 1640 supplemented with 10% fetal bovine serum (Welgene, Korea), 2 mmol/L L-glutamine, 10 mmol/L HEPES, 1 mmol/L sodium pyruvate and 45 g/L glucose with antibiotics at 37°C in a humidified atmosphere containing 5% CO2.
The cytotoxicity of SFN was assessed by XTT assay as described previously [43
]. Cells were seeded onto 96-well microplates at a density of 1 × 104
cells per well in 100 μl of RPMI 1640 medium and grown for 24 h. Then the cells were exposed to various concentrations (0, 25, 50 or 100 μM) of SFN in serum-free medium for 24 h and 50 μl of XTT (1 mg/ml in phosphate buffered saline (PBS)) mixture containing phenazine methosulfate (PMS) (1.53 mg/ml in PBS) at the ratio of 100:1 was added to the cells. Cells were incubated at 37°C for 2 h and the optical density was measured using microplate reader (Molecular Devices Co.) at 450 nm. Cell viability was calculated as a percentage of viable cells in SFN-treated group versus untreated control using the following equation:
Two-dimensional polyacrylamide gel electrophoresis (2DE-PAGE)
Cells were treated with or without SFN (80 μM) for 24 h, homogenized using mortor-driven homogenizer (PowerGen125, Fisher Scientific) in lysis buffer (7 M urea, 2 M thiourea containing 4% CHAPS, 1% DTT and 2% pharmalyte, and 1 mM benzamidine) and incubated for 1 h at room temperature. After centrifugation at 15,000× g for 1 h at 15°C, soluble fraction was collected and used for two-dimensional gel electrophoresis. Protein concentration was measured by the Bradford assay.
The first-dimensional isoelectric focusing (IEF) was carried out on Pharmacia Immobiline IPG DryStrip system (Uppsala, Sweden). For the first dimension of electrophoresis, the samples containing 100 μg protein for analysis gels were diluted to 350 μL with a rehydration solution (7 M urea, 2% w/v CHAPS, 50 mM DTT, 0.5% v/v IPG buffer (pH 3-10 nonlinear and pH 4-7 linear), and trace bromophenol blue) before loading onto 17 cm IPG strips (pH 3-10 nonlinear and pH 4-7 linear). IEF was then performed using IPG electrophoresis unit according to the manufacturer's instructions. Thereafter, the strips were equilibrated with a solution (6 M urea, 30% v/v glycerol, 2% w/v SDS, and 50 mM Tris-HCl, pH 8.8), reduced with 1% w/v DTT for 15 min, and alkylated with 2.5% w/v iodoacetamide for 15 min. Strips were then rinsed in electrophoresis buffer (25 mM Tris base, 192 mM glycine, and 0.1% w/v SDS), applied to 11% acrylamide gels, and sealed with melted agarose (0.5% w/v agarose in electrophoresis buffer containing a trace of bromophenol blue). SDS-PAGE was carried out using Hoefer SE 600 vertical chambers and a Tris-glycine buffer (25 mM Tris and 192 mM glycine) containing 0.1% w/v SDS, with initial separation at a constant 10 mA/gel for 30 min followed by 20 mA/gel. The second-dimensional SDS-PAGE was developed until the bromophenol blue dye marker had reached the bottom of the gel. The total run time was typically 4 to 4.5 hours. Gels were fixed in 10% v/v acetic acid, 40% v/v ethanol before sensitization for 30 min in a buffer containing 30% v/v ethanol, 0.2% w/v sodium thiosulphate, and 0.83 M sodium acetate. This was followed by three 15 min washes in deionised water. The proteins were then stained with 0.1% w/v silver nitrate for 20 min, washed twice in deionised water for 1 min, and developed in 2.5% w/v sodium carbonate containing 0.04% v/v formaldehyde (37% solution). The development was stopped with 1% v/v acetic acid, and the gels were washed three times in water.
Quantitative analysis of digitized images was carried out using the PDQuest (version 7.0, BioRad) software according to the protocols provided by the manufacturer. Quantity of each spot was normalized by total valid spot intensity. Protein spots with intensity changed over two folds compared with control samples were selected for further studies.
Enzymatic digestion of protein in-gel
Protein spots were enzymatically digested in-gel similar to that described by Shevchenko et al  but modified using porcine trypsin. Gel pieces were washed with 50% acetonitrile to remove SDS, salt and stain, dried to remove solvent and then rehydrated in Trypsin (8-10 ng/μl) Digest Solution and incubated 8-10 h at 37°C. The proteolytic reaction was terminated by addition of 5 μl 0.5% trifluoroacetic acid. Tryptic peptides were recovered by combining the aqueous phase from several extractions of gel pieces with 50% aqueous acetonitrile. After concentration the peptide mixture was desalted using C18ZipTips (Millipore), and peptides eluted in 1 to 5 μl of acetonitrile. An aliquot of this solution was mixed with an equal volume of a saturated solution of a-cyano-4-hydroxycinnamic acid in 50% aqueous acetonitrile, and 1 μl of mixture spotted onto a target plate.
MALDI-TOF analysis and database search
Protein analysis was performed using an Ettan MALDI-TOF (Amersham Biosciences). Peptides were evaporated with a N2 laser at 337 nm, and using a delayed extraction approach. They were accelerated with 20 Kv injection pulse for time of flight analysis. Each spectrum was the cumulative average of 300 laser shots. The search program ProFound, developed by The Rockefeller University http://126.96.36.199/profound_bin/WebProFound.exe was used for protein identification by peptide mass fingerprinting. Spectra were calibrated with trypsin auto-digestion ion peak m/z (842.510, 2211.1046) as internal standards.
Cell lysates were prepared by using lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 1× protease inhibitor cocktail). The extracts were incubated on ice, spun at 14,000× g for 20 min at 4°C and the supernatants were collected. Protein concentrations were determined by Bradford assay (Bio-Rad), and the protein (50-100 μg) was separated by electrophoresis on 4-12% NuPAGE Bis-Tris gels (Novex). Proteins were then transferred to Hybond ECL transfer membranes to evaluate their expression using PGM3 (Santa Cruz Biotechnologies) and β-actin (Sigma) antibodies.
Apoptotic cell death was examined by using DeadEnd™fluorometric TUNEL assay kit as described in the manufacturer's instructions. Briefly, cells were plated onto the poly-L-lysine-coated slides, fixed with 4% paraformaldehyde for 15 min and incubated in TdT enzyme buffer containing fluorescein-12-dUTP for 1 h at 37°C. After mounting in medium containing DAPI (Vectashield, Vector Labs), cells were visualized under a Carl Zeiss LSM5 confocal microscope.
Cells were transiently transfected with PGM3 or control siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) at 50 nM of final concentration by using INTERFERin siRNA transfection reagent (Polyplus transfection) for 72 h.
Total RNA was prepared using the Trizol reagent (Invitorgen) following the manufacturer's instructions and reverse transcribed to cDNA using oligo-dT and random primers. The cDNA was amplified by PCR using the following specific primers:
PGM3: forward 5'-ACACGCCAAGCCCAATGGACT- 3',
GAPDH: forward 5'-TCACCATCTTCCAGGAGCGA-3'
PCR conditions were as follows: 92°C for 2 min; 94°C for 30 sec, 59°C for 30 sec, 72°C for 30 sec (30 cycles); and 72°C for 5 min. The amplified products were separated on 2% agarose gels.
All data were expressed as means ± SD. The statistically significant differences between control and SFN treated group were calculated by the Student's t- test.