Antibody against β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phosphorylated extracellular signal-regulated kinase (p-ERK), ERK, p-p38 and p38 were purchased from Cell Signaling Technology (Danver, MA, USA). Anti-γ H2A histone family, member X (γH2AX) antibody was purchased from Millipore (Billerica, MA, USA). The angiogenesis inhibitor SU5416 was synthesized at Sugen Inc. as previously described (8). For in vitro experiments, SU5416 was dissolved in DMSO as a 2 mmol/l stock solution that was stored at −20°C.

The human colorectal cancer cell lines HT29 and HCT116 were grown in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal bovine serum (FBS), glutamine, HEPES and antibiotics at 37°C in a 5% CO₂ humidified incubator.

Cells were plated in 60-mm dishes and incubated at 37°C in a 5% CO₂ humidified incubator, and grown to 70–80% confluency. 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.

SU5416 (2 μmol/l) was preincubated for 24 h before radiation exposure and then incubated for a total of 72 h. After 14–20 days, colonies were stained with 0.4% crystal violet (Sigma-Aldrich, St. Louis, MO, USA). The plating efficiency (PE) represents the percentage of seeded cells that grew into colonies under specific culture conditions for 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). To evaluate the radiosensitizing effects of SU5416, the enhancement ratio was calculated as the dose (Gy) for radiation alone divided by the dose for radiation plus SU5416 that yielded a surviving fraction of 50% (D₅₀).

After SU5416 exposure, the cells were treated with radiation for 48 h. Cells were washed with ice-cold phosphate-buffered saline (PBS), trypsinized and resuspended 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 the cell solution were mixed with 5 μl Annexin V-fluorescein isothiocyanate (FITC) (BD Pharmingen) and 10 μl propidium iodide (PI) stock solution (50 μg/ml in PBS) by gentle vortexing, followed by a 15-min incubation at room temperature in the dark. Cells were resuspended in 1X binding buffer (400 μl) and analyzed on a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). At least 10,000 cells were counted for each sample and data analysis was performed using CellQuest software (BD Biosciences).

The levels of intracellular ROS were assessed using the fluorescent probe 2′,7′-dichlorofluorescein diacetate (H₂DCF-DA). The cells were then incubated with 10 μM H₂DCF-DA for 30 min at 37°C, and the green fluorescence corresponding to the levels of intracellular ROS was detected through a 520-nm long-pass filter on an Olympus FV-1000 laser fluorescence microscope.

Colon cancer cells were washed twice in PBS, fixed at room temperature for 6–7 min in 2% formaldehyde/0.2% glutaraldehyde, then washed three times in PBS and incubated at 37°C with SA-β-gal staining solution [1 mg/m; 5-bromo-4-chloro-3-indolyl β-D-galactoside (Sigma-Aldrich)] in buffer containing 100 mM citric acid, 200 mM sodium phosphate, 5 mM potassium ferrocyanide, 5 mM potassium ferrocyanide, 150 mM NaCl and 2 mM MgCl₂ at pH 6.0. Staining was evident after 4–6 h. The cells were washed with PBS and then with distilled water before microscopic examination. Cells at passage 14 were used as a control.

Cells were seeded in 60-mm dishes with 60% confluency. After 24 h, cells were trypsinized, harvested, and fixed in 1 ml of 70% cold ethanol in test tubes and incubated at 4 ℃ for overnight. After incubation, cells were centrifuged at 2,000 rpm for 3 min, and the cell pellets were resuspended in 500 μl propidium iodine (10 μg/ml) containing 300 μg/ml RNase (Sigma, MO, USA). Cell cycle distribution was calculated from 10,000 cells with CellQuest software using FACScaliber (both from Becton Dickinson, CA, USA).

Immunohistochemistry was performed to determine the nuclear distribution of γ-H2AX in individual cells. Cells were grown on chamber slides for 1 day before irradiation or SU5416 treatment. For combination treatment, SU5416 was added to the cells before the radiation treatment. All treatments were performed while cells were attached to the slides. After treatment, the cells were fixed in 4% paraformaldehyde and permeabilized with 0.7% Triton X-100 in PBS. After blocking with 10% FBS/1% bovine serum albumin for 1 h, detection was performed using a 1:1,000 dilution of a FITC-labeled mouse monoclonal γ-H2AX antibody (Millipore).

Colon cancer cells were exposed to SU5416 and then treated with radiation for 1 and 24 h. The cells were lysed using radioimmunoprecipitation assay buffer, and equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 1% (v/v) non-fat dry milk in Tris-buffered saline with 0.05% Tween-20 and then incubated with the indicated antibodies. Blots were incubated with primary antibodies at 1:1,000 dilutions and with secondary antibodies at 1:5,000 dilutions. Immunoreactive protein bands were visualized by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA) and scanned.

Human colon cancer cells were seeded onto 6-well plates (Corning, Corning, NY, USA) at 2.5×10⁴/well in 3 ml RPMI-1640 medium supplemented with 10% FBS. At 2 days, the monolayers were disrupted mechanically using a sterile 200 μl tip. The assay was performed in duplicate. The wells were photographed every 24 h. Cells were then stained with 0.2% crystal violet. Cell migration was monitored using a Nikon Eclipse Ti microscope with a DS-Fi1 camera. The cells were counted using ImageJ software (United States National Institutes of Health, Bethesda, MD, USA).

The invasive ability of the cells in vitro was measured using a Transwell invasion assay kit (Chemicon, Millipore, Norcross, GA, USA) 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/ml in 150 μl serum-free RPMI medium and were left untreated or were treated with the indicated doses of SU5416, radiation, or a combination of the two. The medium in the lower chamber contained 10% FBS as a source of chemoattractants. After incubation for 24 h, the cells that passed through the Matrigel-coated membrane were stained with a cell stain solution containing crystal violet and photographed.

Statistical significance was determined by the Student's t-test. Differences were considered significant if the p-value was <0.05 or 0.001.

To investigate whether SU5416 treatment sensitizes colon cancer cells to ionizing radiation (IR)-induced cell killing, we conducted a clonogenic survival assay. Fig. 1A and B shows the radiation dose-response curves of radiosensitive HCT116 cells and radioresistant HT29 cells irradiated with γ-rays in the presence of SU5416. The survival rate decreased to a greater extent for cells treated with SU5416 before irradiation compared with cells irradiated without SU5416, suggesting that SU5416 acts as a potential radiosensitizer to increase the sensitivity of colon cancer cells to IR-induced cell killing. The SU5416 effect, expressed as the enhancement ratio compared with radiation alone was calculated at D₅₀.

To further explore the mechanisms by which SU5416 increases the radiation sensitivity of colon cancer cells, we investigated whether SU5416 promotes IR-induced senescence and apoptosis. We performed staining of the early apoptotic markers Annexin V and PI in colon cancer cell lines; radiation and SU5416 combination treatment for 72 h significantly increased the percentage of early apoptotic cells (Fig. 2A). To investigate the relationship between ROS production and enhancement of radiation-induced apoptosis by SU5416, we examined the effects of SU5416 on ROS production in colon cancer cells. ROS production was induced to a greater extent by the combination of SU5416 and radiation than by SU5416 or radiation alone (Fig. 2B). Recent studies have shown that IR induces premature senescence in cancer cells in a dose-dependent manner, suggesting that the induction of senescence plays an important role in IR-induced tumor suppression (9). We stained cells for SA-β-gal and found that the expression of SA-β-gal correlated with the loss of proliferation and was observed after approximately 5 days of exposure to various doses (Fig. 2C). The results showed that combined treatment with SU5416 and IR induced more SA-β-gal-positive senescent colon cancer cells than did either SU5416 or IR treatment alone (Fig. 2C). These results suggest that SU5416 may radiosensitize colon cancer cells by enhancing IR-induced premature senescence and apoptosis.

To further investigate the mechanisms underlying the cell cycle after treatment with SU5416, we performed flow cytometric analysis of PI-stained cells in the two colon cancer cell lines (Fig. 3A). As shown in Fig. 3B, the sub-G1 phase indicating the apoptotic population was not markedly altered after treatment with SU5416 alone; however, combined treatment caused slight accumulation of cells in the sub-G1 phase. Combination therapy caused a decrease in cells in the G2/M phase, suggesting efficient induction of cell cycle arrest at the G2/M phase in both colon cancer cell lines.

To analyze the effect of SU5416 on DNA double-strand break (DSB) processing, the level of phosphorylated H2AX (γ-H2AX), a marker of DSBs, was examined by immunohistochemistry and western blotting at 0, 1 and 24 h after treatment. As shown in Fig. 4, prolonged expression of γ-H2AX was observed 24 h after radiation exposure in the presence of SU5416 (Fig. 4A and B). Two MAPK members, ERK and p38, are involved in DNA damage responses (10). However, it has yet to be determined if SU5416 treatment affects the activities of ERK and p38 in colon cancer cells. To address this issue, western blot analyses were performed to assess the expression levels of p-ERK, ERK, p-p38 and p38 in the colon cancer cells. The results showed that SU5416 treatment significantly inhibited the expression levels of p-ERK and p-p38 in the irradiated colon cancer cells (Fig. 4C).

Cell migration and invasion play crucial roles in tumor metastasis (11). To further investigate the antimetastatic effect of SU5416, the ability of SU5416-treated colon cancer cell migration was assessed by scratch-wound and Transwell assays. The results from the scratch-wound assay showed that the wound healing of the combination treatment was gradually reduced compared with the wound healing ability of the cells treated with SU5416 or radiation alone (Fig. 5A and B). We next investigated the anti-invasive and anti-migratory activities of SU5416 on colon cancer cells using a Transwell system. As shown in Fig. 5, SU5416 in combination with radiation significantly inhibited the migration and invasion of the two colon cancer cell lines in the chamber Transwell assays (Fig. 5C–F). These results suggest that the combination of SU5416 and radiation affected the abilities of cell migration and invasion.