Cell lines and Khz-cp treatment
The BEAS-2B (normal immortalized), 1799 (non-transformed), 1198 (transformed but non-tumorigenic), and 1170-I (tumorigenic) cell lines that compose the in vivo lung carcinogenesis model used in this study have been previously described
[30, 31]. The human gastric cancer cell line SNU-1 was maintained in RPMI 1640 media supplemented with 10% fetal bovine serum, 100 U/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, and 0.25 μg/ml amphotericin B. Unless otherwise indicated, all the cells were treated with Khz-cp diluted 1:100 in the media.
Extraction of Khz-cp (crude polysaccharide extract obtained from the fusion of G. lucidumand P. umbellatusmycelia)
First, 1 kg of powder was added to 8.5 L of clean water, heated to 115°C, and extraction was performed for 60 min under pressure. This was followed by a 60-min maturation period. Next, the remaining water from the first extraction was added to 7.5 L of clean water and heated to 115°C; extraction was performed under pressure for 60 min, followed by maturation for a further 60 min. The first and second extracts were then mixed, boiled, and placed in bottles after 5 min.
We purified Khz-cp from Khz by using the Sevag method for deproteinization. The Sevag reagent, which is a 4:1 mixture of chloroform and n-butanol, was added with shaking; the volume of the reagent added was one-fourth that of the sample solution. After the mixture was allowed to stand and separate, the water layer and the solvent layer at the junction of the denatured protein were removed. This step was repeated several times until the denatured protein content was minimized. Preliminary experiments showed that a fourfold excess of 95% ethanol with respect to the sample volume was appropriate for precipitation. Therefore, after the removal of the denatured protein, 95% ethanol (4 times the sample volume) was added slowly until additional precipitation did not occur. The mixture was centrifuged and the supernatant was removed. The sediment collected was a brown precipitate and represented the total bacterial crude polysaccharide fraction. The polysaccharides were then dissolved and filtered by membrane ultrafiltration (molecular weight cutoff: 100,000 Da). Khz-cp was obtained from BrainGroup (Seoul, South Korea).
Reagents and antibodies
Mitochondrion-targeted ubiquinone (MitoQ) is an ubiquinol antioxidant attached to a lipophilic triphenylphosphonium (TPP) cation
. MitoQ and TPP were kind gifts from Dr. Michael P. Murphy (Medical Research Council Dunn Human Nutrition Unit, UK). SB203580, apocynin, and cyclosporin A (CsA) were purchased from Calbiochem (San Diego, CA, USA), and N-acetyl cysteine (NAC) and ethylene glycol tetraacetic acid (EGTA) were purchased from Sigma (St. Louis, MO, USA). z-VAD-fmk was obtained from R&D Systems (Minneapolis, MN, USA), diphenylene iodonium (DPI) was from Cayman Chemical (Ann Arbor, MI, USA), and BAPTA-AM was from Invitrogen (Eugene, OR, USA).
Antibodies against p38 (sc-7972), p47phox (sc-14015), p67phox (sc-15342), caspase 3 (sc-7148), PARP (sc-7150), and cytochrome c (sc-13561) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p-P38 (9255) antibodies were obtained from Cell Signaling (Danvers, MA, USA), and a COX IV antibody (A21347) was obtained from Invitrogen.
Western blot analysis
Cells were lysed in an extraction buffer (31.25 mM Tris–HCl [pH 6.8], 1% sodium dodecyl sulfate [SDS], 10% glycerol, and 2.5% mercaptoethanol), and the whole cell lysate was subjected to 10% SDS-polyacrylamide gel electrophoresis. Size-fractionated proteins on the gel were transferred onto a nitrocellulose membrane. The membrane was blocked in 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 and was incubated with a primary antibody. After washing, the membrane was incubated with the peroxidase-conjugated secondary antibody. The protein band of interest was detected using enhanced chemiluminescence reagents (Amersham).
Cells treated with Khz-cp were washed twice in cold phosphate-buffered saline (PBS) and stained with annexin-V-FITC (A13199; Invitrogen) and propidium iodide (PI) according to the manufacturer’s instructions. Briefly, annexin-V-FITC (5 μL) was added to the cells, which were resuspended in 100 μL of binding buffer (10 mM HEPES, 140 mM NaCl, and 2 mM CaCl2; pH 7.4). The cells were then incubated at room temperature for 15 min, and PI was added before flow cytometry or fluorescence microscopy analysis. Apoptosis were determined using a FACS calibur (Becton and Dickson) and analysed using Cell Quest pro software. Images were analyzed using NIS Elements software (Nikon).
Assessment of cytoplasmic and mitochondrial ROS levels
The levels of cytoplasmic ROS were estimated using the oxidation-sensitive fluorescent dye H2DCF-DA (2′,7′-dichlorodihydrofluorescein diacetate; Invitrogen) or the Amplex Red hydrogen peroxide assay kit (Invitrogen). For DCF staining, the cells were loaded with H2DCF-DA (100 nM) for 1 h at 37°C and washed once with PBS. After treatment with Khz-cp, ROS levels were analyzed using a flow cytometer (FACSCalibur; Becton Dickinson, San Jose, CA, USA) or a fluorescence microscope (Eclipse 80i; Nikon, Tokyo, Japan). The Amplex Red hydrogen peroxide assay was performed according to the manufacturer’s protocol. In brief, the cells were lysed in 50 μM Amplex Red solution supplemented with 0.1 U/mL horseradish peroxidase and incubated in the dark for 30 min. Fluorescence was measured using a plate reader (Victor 2; Perkin-Elmer Life Sciences, Boston, MA, USA) with an excitation wavelength of 540 nm and an emission wavelength of 590 nm.
Mitochondrial superoxide anion levels were analyzed by staining with MitoSOX™ Red (Invitrogen). Cells were loaded with MitoSOX Red (5 μM) for 30 min at 37°C and then treated with Khz-cp. The fluorescence was then analyzed by flow cytometry or fluorescence microscopy.
Preparation of subcellular fractions
To prepare the mitochondrial and cytosolic fractions, the cells (1 × 107) were washed once in PBS and disrupted by passing them through a glass homogenizer 80 times in ice-cold isolation buffer (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, and 0.1 mM PMSF). Nuclei and non-disrupted cells were removed by centrifugation at 750 × g for 20 min at 4°C. The supernatant was further centrifuged at 10,000 × g for 15 min at 4°C to obtain a mitochondrion-enriched pellet and a cytoplasm-enriched supernatant.
Membrane and cytosolic fractions were prepared using the Compartmental Protein Extraction kit (Millipore, Temecula, CA, USA) according to the manufacturer’s instructions.
Digital imaging of the intracellular free Ca2+ was performed using the fura-2 AM dye (Invitrogen). When fura-2 binds to Ca2+, its maximal absorption wavelength shifts from 363 to 335 nm. SNU-1 cells (1 × 104) were cultured in 35-mm glass-bottomed dishes and loaded with fura-2 AM (2 μM) for 30 min at 37°C. Fluorescence images of fura-2 were digitally captured at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm with an IX70 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a digital cooled charge-coupled device camera. Paired 340/380 ratiometric images were analyzed using the Metafluor software (Molecular Devices, Sunnyvale, CA, USA). Confocal images of intracellular free Ca2+ were obtained using the fluo-4 AM Ca2+-sensitive fluorescent dye (Invitrogen). The cells were loaded with fluo-4 AM (1 μM) for 30 min at 37°C, and Ca2+ imaging was performed using a confocal laser scanning microscope (LSM510; Carl Zeiss, Jena, Germany).
Transfection of siRNA and plasmids
SNU-1 cells were transfected with siRNA using Lipofectamine 2000 (Invitrogen), as described previously. The coding strand sequences of the siRNA were as follows: 5′-CUG GUA UGA UCC UUC UGA AdTdT-3′ (P381), 5′-GAG GUA UAC ACA UAC UGA dTdT-3′ (Nox2), 5′-CUG UUG UGG ACC CAA UUC AdTdT-3′ (Nox4), and 5′-GUU CAG CGU GUC CGG CGA GdTdT-3′ (GFP). Bcl-2 cDNA was transfected into cells using the Lipofectamine-PLUS reagent (Invitrogen) according to the manufacturer’s instructions. Stably transfected cells were selected using G418 (3 mg/mL).