Dulbecco’s Modified Eagle’s Medium, an RPMI 1640 medium, fetal bovine serum, penicillin/streptomycin, and medium supplements were purchased from Life Technologies (Gibco, Grand Island, NY). Monoclonal antibodies and a peroxidase-conjugated secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). DOX, GS, and other agents were obtained from Sigma Chemical (St. Louis, MO).
All rat cardiac H9C2 myocardial cells, spontaneously immortalized ventricular rat embryo myoblasts, and DLD-1 cells (human colon adenocarcinoma) were purchased from the Food Industry Research and Development Institute, Taiwan (BCRC). The H9C2 cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C in 5% CO2, and DLD-1 cells were cultured in the RPMI 1640 medium. The media were changed every 2–3 d.
Sub-confluent cells were trypsinized and seeded onto 96-well plates at a density of 1.5 × 105 cells/ml and incubated for 24 h before treatment. Thereafter, the cells were exposed to DOX 1 μM for 24 h and then incubated in a fresh medium with Z-GS at various concentrations for an additional 24 h. The effects of GS on DOX-induced cytotoxicity were assessed using the MTT assay, as previously described
. The unwashed dye was eluted and quantified spectrophotometrically at 550 nm using a microplate reader. Cell viability was determined as the percentage of surviving cells compared with that of the DOX-treated control.
Lactate dehydrogenase (LDH) release assay for cytotoxicity
GS-induced cytotoxicity leading to plasma membrane damage was measured using the LDH Cytotoxicity Detection Kit (Boehringer Mannheim, Mannheim, Germany). The LDH release assay has been widely used in cytotoxicity studies
. The detailed assay was performed as previously described
Intracellular ROS induced by DOX was measured using 2’7’-dichlorodihydrofluorescein diacetate (DCFH-DA) as a fluorescent probe
. H9C2 cells were loaded with DCFH-DA (20 μ) for 10 min, followed by 2 washes with HBSS. Dichlorodihydrofluorescein (DCF) fluorescence was detected using a fluorescence spectrophotometer with an excitation of 485 nm and an emission of 520 nm, and the fluorescence image was visualized using a fluorescence microscope.
Lipid peroxidation was assayed using the thiobarbituric acid (TBA) reaction. The amount of thiobarbituric acid reactive substance (TBARS) was determined using a standard curve of 1,1,3,3-tetramethoxypropane. The detailed assay procedure was performed as previously described
Preparation of total cell lysates and nuclear and cytosolic extracts
H9C2 cells (5 × 105 cells/well) or DLD-1 cells in 6-well plates were incubated with or without concentrations of GS and DOX (1 μM) for 24 h. The total cell lysates were obtained using a lysis buffer (250 mM Tris–HCl (pH 6.8), 1% Triton-100, 0.1% SDS, 1 mM Na3VO4, 1 mM EDTA, 5 mM sodium fluoride, 1 mM PMSF, and 1 mg/ml leupeptin), and cell debris was removed using a centrifuge at 10 000 × g for 10 min at 4°C and stored at −80°C until required. The protein content of the cell lysates was determined using the Bradford assay
Western blot analysis
Equal amounts of cell lysates (30 μg) were electroblotted onto a nitrocellulose membrane (Millipore, MA), following separation using 8%-12% SDS-polyacrylamide gel electrophoresis. The blot was probed using a primary antibody against poly (ADP-ribose) polymerase (PARP), caspase-3, bcl-2, bax, cytochrome C, and β-actin (Santa Cruz Biochemicals, Santa Cruz, CA). The intensity of each band was quantified using density analysis software (MetaMorph Imaging System, Meta Imaging Series 4.5), and the density ratio represented the relative intensity of each band against controls in each experiment.
Hoechst 33258 staining
Cells were rinsed twice in 4°C PBS and fixed in 4% formaldehyde at 4°C for 10 min. After washing, the cells were incubated using Hoechst 33258 (5 μg/ml) staining at room temperature for 10 min in the dark. The cells were then observed and imaged using a laser scanning confocal microscope (Bio-Rad MRC-1000, American Laboratory, USA) with an excitation of 350 nm and an emission of 460 nm
Caspase-3 activity assay
Caspase-3 activity was determined using the ApoAlert Caspase Colorimetric Assay kit (Clontech Laboratories, Inc. USA), according to manufacturer’s protocol.
Data and statistical analysis
The results of all the experiments were expressed as the mean ± standard error (SE) obtained from the number of replicate treatments. Data were analyzed using analysis of variance (ANOVA), followed by Dunn’s post hoc test for comparison, and P values of < 0.05 were considered statistically significant.