Sunitinib (S1042; Selleck Chemicals, Houston, TX, USA) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) as a concentrated stock solution of 10 mg/ml. The human malignant esophageal cancer cell line ECA109 was obtained from the Shanghai Institute of Cell Biology (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) and 1% penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were kept under conditions of 5% CO₂ in an incubator at 37°C.

Hypoxia was induced by incubating cells in an hypoxia chamber [a glass chamber maintaining 0.5–1.0% partial pressure of oxygen (pO₂)]. IR was performed at 566 cGy/min using an X-ray medical linear accelerator (Elekta AB, Stockholm, Sweden). Cells were irradiated at a single dose at room temperature.

Cell cytotoxicity effect was measured by MTT assay. ECA109 cells were seeded into 96-well plates at a concentration of 5–6×10³ cells/well and allowed to adhere. Next, the cells were treated with increasing sunitinib doses (0, 1, 2.5, 5, 10, 15, 20 and 25 µM). After 24 or 48 h of exposure to sunitinib, 10 µl of 5 mg/ml MTT reagent was added to each well. After incubation for 4 h, the supernatants were removed, and 150 µl of DMSO was added to dissolve the MTT crystals (formazan). The absorbance of the plates was measured at a wavelength of 490 nm using a microplate reader (ELx800; BioTek Instruments, Inc., Winooski, VT, USA). The half maximal inhibitory concentration (IC₅₀) values were calculated using the SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Each experiment was performed thrice.

ECA109 cells were seeded into 96-well plates. The cells were incubated overnight and then treated with the indicated concentrations of sunitinib (1 or 2.5 µM) under normoxic or hypoxic conditions for 24 h, and then subjected to X-rays at 8 Gy. After 24 h, cell proliferation was assessed by MTT assay. The percentage cell growth inhibition for each group was calculated by adjusting the control group to 100%. Each experiment was performed thrice.

ECA109 cells were seeded into 6-well plates. The cells were incubated overnight and then treated with the indicated concentrations of sunitinib (1 or 2.5 µM) or DMSO (control) under normoxic or hypoxic conditions for 24 h, and then subjected to X-rays at 2, 4, 6 or 8 Gy. Subsequently, the cells were incubated at 37°C for 10–14 days under normoxic conditions, fixed with methanol and stained with Giemsa for 30 min. Finally, the plates were examined under the microscope, and the number of colonies with ≥50 cells was counted. The cell survival curves were fitted according to a single-hit multi-target model, and the survival enhancement ratio (SER) was calculated as the ratio of the mean inactivation dose in control cells divided by the mean inactivation dose in sunitinib-treated cells. Each experiment was performed thrice.

Annexin-V/fluorescein isothiocyanate (FITC) and propidium iodide (PI) dual staining was performed to determine the percentage of apoptotic cells. The cells were seeded into 6-well plates and treated with or without sunitinib (1 or 2.5 µM) under normoxic or hypoxic conditions for 24 h. Subsequently, the cells were subjected to X-ray IR (8 Gy). The cells were collected 48 h after IR and analyzed with BD Pharmingen™ FITC Annexin V Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA) by flow cytometry. Each experiment was performed thrice.

ECA109 cells were incubated in 6-well plates (1×10⁶ cells/well) and then divided into the following groups: Normoxia (Norm), hypoxia (Hypo), sunitinib 1 µM (SU 1 µM) and sunitinib 2.5 µM (SU 2.5 µM). The groups of SU 1 µM and SU 2.5 µM were pretreated with 1 µM or 2.5 µM sunitinib. After 24 h, all cells were collected and washed with cold 1X PBS, and then resuspended in 70% ethanol at 4°C overnight. The cells were incubated with 6 µl of 1 g/l RNase A, 1 ml of 1 mg/ml PI and 400 µl of PBS at room temperature for 15 min. The cell cycle distribution was analyzed using flow cytometry. Each experiment was performed thrice.

Total proteins were extracted from the cells using SDS Lysis Buffer (Sigma-Aldrich; Merck Millipore) at 24 h after the last sunitinib treatment under normoxic or hypoxic conditions. The protein concentrations of the supernatants were determined by bicinchoninic acid assay. Equal amounts of protein were loaded into each lane, and proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk, incubated with primary antibodies against HIF-1α (14179; dilution, 1:250; Cell Signaling Technology, Inc., Danvers, MA, USA), VEGF (sc-507; dilution, 1:250) and GAPDH (sc-25778; dilution, 1:1,000) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight, and then incubated with horseradish peroxidase-conjugated secondary antibodies (BS13278; dilution, 1:1,000; Bioworld Technology, Inc., St. Louis Park, MN, USA) for 1 h at room temperature. The immunoblotted proteins were visualized with enhanced chemiluminescence reagents (EMD Millipore), and the signals were detected using the ChemiDoc™ XRS+ imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and analyzed with Quantity One quantitation software (Bio-Rad Laboratories, Inc.).

All data are expressed as means ± standard deviation. Data were analyzed using SPSS 17.0 software (SPSS, Inc.). Survival curves were fitted using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Student's t test was applied to compare the groups. P<0.05 was considered to indicate a statistically significant difference.

MTT assay was performed at 24 and 48 h following sunitinib administration at various concentrations (≤25 µM) to determine the sensitivity of human ESCC cells to sunitinib as a single agent. The IC₅₀ value for ECA109 cells at 24 h was 7.07 µM. Fig. 1A demonstrates that sunitinib produced a cytotoxic effect in a dose-dependent manner. The survival rates in 1 or 2.5 µM sunitinib-treated ECA109 cells for 24 h were 89.23 and 78.29%, respectively. These results indicated a low cytotoxic effect on the growth of ESCC cells. Thus, these low-cytotoxic concentrations (1 and 2.5 µM) were selected for the following assays.

MTT assay was also performed to assess the effect of sunitinib on hypoxic ECA109 cells. ECA109 cells under hypoxic conditions (0.8–1.0% pO₂) exhibited a significant resistance to IR, while sunitinib sensitized hypoxic cancer cells to IR. The cell growth inhibition with sunitinib treatment was significantly lower than that without sunitinib treatment (Fig. 1B).

Annexin-V/PI staining was performed to quantify the apoptosis of hypoxic ESCC cells exposed to IR after sunitinib treatment for 48 h (Fig. 2A and B). The results revealed that the apoptosis rate was significantly lower in the hypoxic group than in the normoxic group (P<0.05). Following treatment with sunitinib at 1 or 2.5 µM, the apoptosis rate was higher compared with that of hypoxia alone (P<0.05).

The percentage of cells in each phase of the cell cycle in the different groups are summarized in Fig. 2C and D. Compared with the normoxia group, no significant accumulation of ECA109 cells in the G0/G1 or G2/M phases was noted in the hypoxia alone group or in the hypoxia group treated with sunitinib at 1 or 2.5 µM.

Clonogenic survival assays were performed to assess the potential radiosensitization activity of sunitinib. Cells under hypoxic conditions (0.8–1.0% pO₂) exhibited a significant increase in their ability to form colonies following IR, indicating their resistance to IR. However, sunitinib sensitized hypoxic cancer cells to IR significantly. The dose-survival curves are shown in Fig. 3A. The surviving fraction (SF) data were fitted into the following single-hit multi-target model formula: SF=1-(1-e^−D/D0)ⁿ, where D₀ is the mean lethal dose and D_q is the quasi-threshold dose. The results revealed that the SFs at 2 Gy (SF2)s were 0.48 and 0.67 for ECA109 cells under normoxic and hypoxic conditions, respectively. Upon treatment with sunitinib at 1 or 2.5 µM, the SF2 of hypoxic cells increased to 0.61 or 0.58 in ECA109 cells, respectively. The survival enhancement ratio (SER) was calculated according to the clonogenic results: SER = D₀ (test group)/D₀ (hypo group). The SERs of hypoxic cells were 1.31 or 1.59 in ECA109 cells treated with sunitinib at 1 or 2.5 µM, respectively, compared with the hypoxic condition alone (Table I). These data demonstrated that treating hypoxic ESCC cells with sunitinib resulted in a significant radiosensitization effect.

Western blot analysis was performed to confirm the effect of sunitinib on the VEGF and HIF-1α expression induced by hypoxia. ECA109 cells were treated with 1 or 2.5 µM sunitinib for 24 h. It was observed that hypoxia increased the expression of VEGF and HIF-1α. However, sunitinib could inhibit the expression of VEGF and HIF-1α, particularly at high doses (Fig. 3B).