Anti-cyclin B, anti-cyclin A, anti-β-actin and anti-matrix metalloproteinase-9 (MMP-9) were purchased from Santa Cruz Biotechnology. Anti-cleaved poly(ADP-ribose) polymerase-1 (PARP1) antibody, anti-cleaved caspase 3, anti-pDNA-dependent protein kinase (DNA-PK; S2059), anti-PERK and anti-pAkt were purchased from Cell Signaling Technology (Danvers, MA, USA), and anti-phosphorylated H2AX (γH2AX) was obtained from Millipore (Billerica, MA, USA). Sorafenib was purchased from Selleckchem. For in vitro experiments, sorafenib was dissolved in dimethyl sulfoxide to make a 16 mmol/l stock solution and was stored at 4°C.

The human colorectal cancer cell lines HT29, HCT116 and SW480 were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), glutamine, HEPES and antibiotics at 37°C in a 5% CO₂ humidified incubator. Human umbilical vein endothelial cells (HUVECs) were maintained in endothelial cell basal medium (EGM-2; Cambrex) containing EGM-2 SingleQuot growth supplements (Cambrex) and maintained for no more than 8 culture passages.

Cells were plated in 60-mm diameter dishes and incubated at 37°C under humidified conditions and 5% CO₂ until they reached 70–80% confluence. Cells were irradiated with a ¹³⁷Cs γ-ray source (Atomic Energy of Canada, Ltd., Ontario, Canada) at a dose rate of 3.81 Gy/min.

Sorafenib was added to cells 1 h after radiation exposure to a final concentration of 16 μmol/l, and the cells were then incubated for 72 h. After 14–20 days, colonies were stained with 0.4% crystal violet (Sigma, St. Louis, MO, USA). The plating efficiency (PE) represents the percentage of seeded cells that grew into colonies under the specific culture conditions of a given cell line. The survival fraction, expressed as a function of irradiation, was calculated as follows: Survival fraction = colonies counted/(cells seeded × PE/100). PEs of HT29, HCT116 and SW480 were 0.52±0.18, 0.50±0.15, and 0.48±0.10, respectively. To evaluate the radiosensitizing effects of sorafenib, the ratio for radiation alone and radiation plus sorafenib was calculated as the dose (Gy) for radiation alone divided by the dose for radiation plus sorafenib at a surviving fraction of 50% 50% of cells.

Cells were cultured, harvested at the indicated times, stained with propidium iodide (PI; 1 μg/ml, Sigma), according to the manufacturer’s protocol, and then analyzed using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). A minimum of 10,000 cells was counted for each sample, and data analysis was performed with the use of CellQuest software (BD Biosciences).

After the exposure to radiation, sorafenib was added to the cells, which were then incubated for a further 48 h. Cells were washed with ice-cold phosphate-buffered saline (PBS), trypsinized and re-suspended in 1X binding buffer [10 mm HEPES/NaOH (pH 7.4), 140 mm NaCl and 2.5 mm CaCl₂] at 1×10⁶ cells/ml. Aliquots (100 μl) of cell solution were mixed with 5 μl Annexin V FITC (Pharmingen) and 10 μl PI stock solution (50 μg/ml in PBS) by gentle vortexing, followed by 15 min of incubation at room temperature in the dark. Buffer (400 μl, 1X) was added to each sample and analyzed on a FACScan flow cytometer (Becton Dickinson). A minimum of 10,000 cells was counted for each sample, and data analysis was performed using CellQuest software (BD Biosciences).

The fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA) was used for the assessment of intracellular reactive oxygen species (ROS). For fluorocytometrical analysis, cells were plated in 60-mm diameter dishes (1×10⁵ cells/dish) and loaded for 30 min at room temperature with 10 μM DCFH-DA in 5 ml PBS. Unincorporated DCFH-DA was removed with 2 washes in PBS. DCFH-DA-loaded cells were treated with sorafenib or radiation alone, or in combination. Fluorescence was measured using a flow cytometer (Becton Dickinson).

Immunohistochemistry was performed to determine the nuclear distribution of γ-H2AX in individual cells. Cells were grown on chambered slides 1 day prior to irradiation or sorafenib treatment. After radiation exposure, sorafenib was added to the cells, and the cells were treated for various lengths of time. All treatments were performed while cells remained attached to the slides, followed by fixing with 4% paraformaldehyde and permeabilization with 0.2% Triton X-100 in PBS. Detection was performed after blocking the slides in 10% FBS/1% bovine serum albumin for 1 h at a 1:1,000 dilution of FITC-labeled mouse monoclonal antibody against γ-H2AX (Millipore) in the background-reducing antibody diluent (DAKO plus S3022; Millipore).

Following irradiation, sorafenib was added to the colon cancer cells, which were then incubated for 1 or 24 h. The cells were then lysed with RIPA buffer. Proteins were separated by sodium-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 1% (v/v) non-fat dried milk in Tris-buffered saline with 0.05% Tween-20 and incubated with the required antibodies. Primary antibodies were used at a 1:1,000 dilution and secondary antibodies at a 1:5,000 dilution. Immunoreactive protein bands were visualized by enhanced chemiluminescence (Amersham Biosciences) and scanned.

The cell motility assay was conducted in 6-well plates. A fine scratch in the form of a groove was made using a sterile pipette tip in a layer of cells at ~90% confluency. Cells were then treated with sorafenib, radiation or a combination of both. The migration of cells was monitored using a Nikon Eclipse Ti microscope with a DS-Fi1 camera.

The invasive ability was measured in vitro using Transwell chambers, according to the manufacturer’s protocol. Briefly, cells were seeded onto the membrane of the upper chamber of the Transwell at a concentration of 4×10⁵ cells/ml in 150 μl of RPMI medium and were left untreated or treated with the indicated doses of sorafenib, radiation, or a combination of both for 24 h. The medium in the upper chamber was serum-free, while the medium in the lower chamber contained 10% FBS as a source of chemo-attractants. Cells that passed through the Matrigel-coated membrane were stained with Cell Stain Solution containing crystal violet supplied in the Transwell invasion assay (Chemicon, Millipore) and images were captured after 24 h of incubation.

Endothelial cell tube formation was assessed using Matrigel-coated chamber slides as previously described (12). The results of each assay were photographed (Nikon Eclipse Ti microscope with DS-Fi1 camera) at ×40 magnification. Tube formation was quantified by counting the number of connected cells in randomly selected fields at ×400 magnification with a microscope, and dividing that number by the total number of cells in the same field.

All data were plotted as the mean ± standard error of the mean (SEM). Results of colony forming assays were analyzed using a paired t-test with SPSS 18.0 software (SPSS, Chicago, IL, USA). All other data were analyzed by parametric repeated measure one-way analysis of variance (ANOVA), followed by Tukey’s honestly significant difference test (SPSS 18.0). Statistical significance was set at P<0.05.

In order to evaluate the effects of sorafenib on radiation-induced cytotoxicity, a clonogenic survival assay was performed using HT29, HCT116 and SW480 cells. We used a sorafenib concentration resulting in a 25% growth inhibition after a 48-h exposure in each experiment (data not shown), and added this drug post-radiation as this was previously shown to be more efficacious than pre-radiation treatment (13). Dose-response curves of the 3 colon cancer cell lines irradiated in the presence or absence of sorafenib are shown in Fig. 1. Combined radiation and sorafenib treatment was more effective than radiation alone in all cases. The effect of sorafenib on radiation-mediated cell killing was expressed as an enhancement ratio (D₅₀).

To investigate whether sorafenib and radiation induce apoptosis, we assessed early apoptosis by Annexin V and PI staining. In colon cancer cell lines, combined 48-h sorafenib treatment and radiation exposure significantly increased the proportion of cells in early apoptosis in all 3 colon cancer cell lines (Fig. 2A). We next investigated whether sorafenib-enhanced radiation cytotoxicity resulted from the increased activation of caspase, resulting in enhanced apoptotic cell death. We observed increased activation of caspase-3 and increased PARP cleavage in response to combined radiation and sorafenib treatment compared to sorafenib alone (Fig. 2B). We also investigated the relationship between ROS production and enhancement of radiation-induced apoptosis by sorafenib. The production of ROS was synergistically induced by the combined treatment of sorafenib and radiation in colon cancer cell lines (Fig. 2C), indicating that ROS generated by the combined treatment increases intracellular caspase signaling and, thus, apoptosis.

We next investigated whether combined sorafenib/radiation-induced cytotoxicity resulted from differences in cell cycle regulation by analyzing cell cycle progression through flow cytometry. Sorafenib treatment combined with radiation significantly increased the proportion of cells in G2 to M phase for all 3 cell lines, compared to radiation alone (Fig. 3A). We also examined the expression of cell cycle regulators after combined sorafenib and radiation treatment. Western blotting revealed that radiation alone resulted in a significant accumulation of cyclin A and cyclin B, a key cell cycle regulator involved in the G2/M transition, whereas sorafenib alone and combined treatment reduced expression of cyclin A and cyclin B levels (Fig. 3B).

To analyze the effect of sorafenib on double-strand break (DSB) repair, the level of γH2AX, a marker for DSB, was examined by immunofluorescence and western blotting 0, 1 and 24 h after treatment. Prolonged expression of γH2AX was observed after 24-h radiation exposure in the presence of sorafenib (Fig. 4A). All 3 colon cancer cell lines treated with combined sorafenib and irradiation exhibited damaged DNA foci, which appeared 1 h after treatment and were still present 24 h after exposure (Fig 4A). In addition, cell lysates were immunoblotted with anti-γH2AX antibody (Fig. 4B), which confirmed the results of the immunofluorescence assay, and the levels of phosphorylated DNA-PK were slightly reduced after combined treatment (Fig. 4C).

We next evaluated the effects of sorafenib and radiation on the invasive and migratory capacities of colon cancer cells using Matrigel and scratch assays. In the latter, combined sorafenib and radiation treatment significantly inhibited cell migration compared with sorafenib or radiation alone (Fig. 5A and B). In the Matrigel invasion assay, combined sorafenib and radiation treatment was highly effective in inhibiting the invasive behavior of all 3 colon cancer cell lines, by 10, 15 and 8% (Fig. 5C and D). We also evaluated the extent to which MMP-9 expression is altered in colon cancer cells treated with sorafenib and radiation. Notably, sorafenib suppressed the upregulation of MMP-9 caused by irradiation (Fig. 5E).

We next examined whether combined sorafenib and radiation treatment blocks angiogenesis by inhibiting VEGF. Combined sorafenib and radiation completely inhibited VEGF-mediated endothelial tube formation in HUVECs, whereas either treatment alone inhibited tube formation by only 33 and 36%, respectively (Fig. 6A and B). VEGF predominantly mediates angiogenesis via the PI3K/Akt and MAPK signaling cascades (5), and combination treatment significantly inhibited Akt and ERK1/2 activation compared with sorafenib or radiation alone, as shown by western blotting (Fig. 6C).