Cell lines and agents
Human hepatocellular carcinoma cell lines, SMMC-7721 and Bel-7402, were obtained from Nanfang Hospital of Southern Medical University, Guangzhou, Guangdong, China , and were cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Hyclone, Logan City, Utah) at 37°C in a humidified atmosphere containing 5% CO2. Sorafenib (Bayer, Leverkusen, Germany) was dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 25 mmol/L and stored at -20°C.
The 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2 H-terazolium (MTT) assays
The MTT (3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2 H-terazolium) assays (Promega, Madison, WI) were performed as instructed by the manufacturer to assess cell viability. Briefly, SMMC-7721 (3 Χ 103 cells/well) and BEL-7402 cells (4 ×103) were seeded into 96-well plates in quadruplicate. After incubation for 1 d, cells were treated with sorafenib 30 min before (pre-irradiation sorafenib) or 24 h following irradiation (post-irradiation sorafenib). Cells were irradiated at the indicated doses using a 60Co irradiator. Cell viability was measured on d0 to d6 after irradiation. Absorbance values were shown as the percentage of the treated samples relative to the controls which received neither irradiation nor sorafenib. Inhibition of cell growth was measured as the percentage of viable cells relative to the controls, which was calculated as follows: % of viable cells = ODT/ODC x 100%, where ODT is the average OD value of the treatment samples, and ODC is the average OD value of the control samples. Results were analyzed using the CalcuSyn software program (Biosoft, Cambridge, UK). Combination indices (CI) were used to assess the interaction between the two treatment modalities.
Apoptotic study and cell cycle analysis
SMMC-7721 and BEL-7402 cells were irradiated, treated with sorafenib for 30 min followed by irradiation (pre-irradiation sorafenib), or irradiated and treated 24 h later with sorafenib (post-irradiation sorafenib). Apoptosis was detected in cells washed with phosphate buffered saline (PBS) at 48 h post-irradiation (irradiated controls, pre-irradiation sorafenib) or 72 h post-irradiation (post-irradiation sorafenib) by staining with annexin V and propidium iodide as instructed by the manufacturer (BD Biosciences, Franklin Lake, NJ). Stained cells were analyzed by flow cytometry with a FACSCalibur flow cytometer (BD Biosciences). For cell cycle analysis, treated cells were washed once with PBS, trypsinized, washed in PBS with 2% FBS, fixed in ice-cold ethanol for at least 1 h, washed, stained with propidium iodide (30 μg/mL), and treated with RNase (0.6 mg/ml) in PBS plus 0.5% (v/v) Tween 20 and 2% FBS. Stained cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences) by using the CellQuest software. Mod-Fit program (Verity Software House Inc., Topsham, ME) was used to analyze the cell-cycle profiles.
Colony formation assays
This procedure was performed as previously described . Briefly, cells were irradiated at a dose of 0, 2, 4, and 8 Gy alone or in combination with sorafenib administered 30 min prior to (pre-irradiation sorafenib) or 24 h following irradiation (post-irradiation sorafenib). After incubation of 12 d (SMMC-7721) or 14 d (BEL-7402), cells were stained with 0.5% crystal violet in absolute ethanol, and colonies containing more than 50 cells were counted under a dissection microscope. Clonogenic survival curves were constructed by fitting the average survival levels. Subsequent experiments utilized a radiation dose of 6 Gy because the percentage of cells remaining after 8 Gy (SMMC-7721: 0.9-4%; BEL-7402: 2-5%) was too low for analysis. SMMC-7721 and BEL-7402 cells in subsequent experiments received one of the four treatments: (a) none (control), (b) 6 Gy radiation, (c) 15 μM sorafenib 30 min before 6 Gy radiation, or (d) 6 Gy radiation followed 24 h later with 15 μM sorafenib.
DNA damage immunofluorescence microscopy
Immunofluorescence microscopy was done as previously described . Rabbit anti-γ-H2AX antibody (serine 139; Abcam, Cambridge, MA), and secondary antibodies Alex Fluor 488 goat anti-rabbit IgG (Invitrogen, Carlsbad, CA) were used. Nuclear staining was done by using 4’, 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, USA). A cell containing more than 10 γ-H2AX foci was considered to be positive for damages to DNA.
Cell cycle G2/M distribution assay
After the indicated time period, cells were rinsed with PBS, fixed with 70% ethanol, and incubated overnight at -20°C. Fixed cells were washed and suspended in 500 μl of staining solution (50mcg/ml of propidium iodide, 100mcg/ml RNAase and 0.2% Triton X-100) for 30 min. The fluorescence associated with PI-bound DNA was measured by flow cytometry (Beckman Coulter, cytomics FC 500, CA). Cell cycle profiles of G2/M phase were calculated using MultiCycle software.
Cell proliferation assays
SMMC-7721 and BEL-7402 cells were plated at 1 x 103 cells per well in collagen-coated 96-well plates. Cell proliferation assays were performed by using the Cell Counting Kit-8 (CCK8) (Dojindo, Kumamoto, Japan) according to the manufacturer's protocol. Briefly, a 10 μL of CCK-8 solution was added to each well and incubated at 37°C for 2 h in a humidified CO2 incubator. Optical density (OD) was measured at 450 nm using a Microplate Reader (Bio-Tek Instruments, Winooski, VT) and the proliferation index was calculated as the experimental OD value/control OD value. Each experiment was done in quadruplicate and at least three times independently.
After incubation for 0 h, 24 h, or 48 h after sorafenib treatment, cells were harvested, rinsed, and stained with Annexin V-FITC and propidium iodide, as previously described .
Normally distributed continuous variables were compared by one-way analysis of variance (ANOVA). When a significant difference between groups was apparent, multiple comparisons of means were performed using the Dunnett test. Data are presented as mean ± standard deviation (SD). All statistical assessments were two-sided and evaluated at the 0.05 level of significant difference. Statistical analyses were performed using SPSS 15.0 statistics software (SPSS Inc, Chicago, IL).