Cell culture
Human liver cancer-derived HepG2 cells were grown in a monolayer in tissue culture flasks (Corning, NY, USA) in RPMI-1640 medium (Thermo Fisher, MA, USA) containing 10% fetal bovine serum (Thermo Fisher, MA, USA), 1% sodium pyruvate (Caisson Labs, UT, USA), 1% nonessential amino acids (Thermo Fisher, MA, USA), and 1 mM gentamicin (Thermo Fisher, MA, USA) at 37°C in a humidified incubator with 5% carbon dioxide. Cells were used in the logarithmic growth phase.
The chronic hypoxia condition was achieved as described previously. Briefly, cells were subjected repeatedly to hypoxia (1% O2, 5% CO2, 94% N2) for 4 h daily for 7 consecutive days. The following treatments and assays were performed under normoxic conditions.
Heat treatments
Confluent cells in 6-well culture plates containing 2 mL of medium per well were heated for 15 min or 30 min at 42°C, relative to controls (37°C), in temperature-controlled precision water baths (± 0.1°C) (Haake D8, Fisher Scientific, Montreal, QC). Cells were then cultured in fresh medium at 37°C in the presence (1 mM, working concentration) or absence of doxorubicin for various lengths of time until the following assays were performed.
Intracellular uptake of Doxorubicin
Cells were incubated in fresh culture medium in the presence or absence of doxorubicin (1 mM, working concentration) for 30 min at 37°C. After recovering in fresh medium without doxorubicin for 3 h, intracellular doxorubicin was measured by fluorescence intensity using flow cytometer (BD Biosciences, CA, USA) at 488 nm. Data were analyzed using FlowJo X software.
Cell viability assay
After culturing for 24 h, cell viability was assessed using the MTT assay (Sigma, USA) according to the manufacturer’s protocol. In brief, 10 μl of MTT solution was added to 200 μL of medium per well in 96-well plates, followed by incubation for 4 hours. The medium was discarded and 150 μl of DMSO per well was added. Absorbance at 568 nm was measured using a Thermomax microplate reader (Thermo, USA).
Cell cycle analysis
After treatment, the cells were collected for detection using a cell cycle analysis kit according to the manufacturer’s manual. In brief, cells were fixed in 70% ice-cold ethanol and stored at −20°C overnight. After washing with PBS, cells were incubated with 300 μl PBS, 125 μl RNase, and 25 μl PI (1 mg/ml) for 30 min at 37°C. A FACSCalibur flow cytometer (BD Bioscience, Heidelberg, Germany) was used to record 10,000 ungated events for each sample with excitation at 535 nm. Analysis of the events was performed using Modfit 3.2 software.
Apoptosis
Apoptosis assay was performed using a FITC Annexin V/Dead Cell Apoptosis Kit (Invitrogen) according to the manufacturer’s manual. In brief, cells were harvested and washed twice with cold PBS. The cells were then resuspended in 100 mL of 1 × Annexin V Binding Buffer, supplemented with 5 mL of Annexin V-FITC and 1 mL of propidium iodide, and incubated on ice in the dark for 15 min. Thereafter, 400 mL of 1 × Annexin V Binding Buffer was added and the stained cells were immediately analyzed by flow cytometer (BD Biosciences). Data were analyzed using FlowJo X software. Annexin V (+)/PI (-) cells were considered apoptotic and were analyzed as a percentage of the entire cell population.
Mitochondrial membrane potential (MMP)
MMP was detected using the TMRM mitochondrial membrane potential assay kit according to the manufacturer’s manual. After treatment, cells were labeled with 10 nmol/l tetramethylrhodamine methyl ester (TMRM), in serum free MEM medium for 20 min at 37°C. Samples were analyzed by flow cytometer. Data were analyzed using FlowJo X software.
Measurement of reactive oxygen species (ROS)
Cellular ROS levels of hydrogen peroxide were detected using an ROS assay kit according the manufacturer’s manual. Briefly, cells were incubated with DCFH-DA at a final concentration of 25 μM at 37°C for 30 min. To measure ROS generation, a FACSCalibur flow cytometer (BD Bioscience, Heidelberg, Germany) was used to detect the fluorescence intensity of dichlorofluorescein (DCF) at 535 nm. For each analysis, 10,000 events were recorded.
Total antioxidant capacity assay
Cellular total antioxidant capacity (TAC) was quantified using a TAC assay kit (Biovision, Mountain View, CA), according to the manufacturer's instructions. The concentration of TAC was calculated from the standard curves and the value was expressed as nmol/ng protein.
Statistical analysis
All of the experiments were repeated independently three times. Means ± SD of the three experiments are presented. ANOVA plus a multiple comparison test (Fisher’s protected least significant difference test, PLSD) was performed to determine group differences. P-values < 0.05 were regarded as significant for all of the statistical analyses.