Human HT1080 fibrosarcoma cells were purchased from American Tissue Culture Collection (Rockville, IL, USA) and human A549 lung cancer cells and human SAS oral squamous cell carcinoma cells were purchased from the RIKEN Bio-Resource Center (Tsukuba, Japan). The HT1080 and SAS cells were maintained in RPMI-1640 (Gibco Life Technologies, Carlsbad, CA, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Japan Bioserum Co. Ltd., Fukuyama, Japan) in a 5% CO₂ incubator at 37°C. The A549 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% heat-inactivated FBS in 5% CO₂ at 37°C. All three cell lines were passaged at 75–90% confluence.

Culture dishes or microtiter plates containing the cells were exposed to X-rays from an X-ray machine (MBR-1520R-3; Hitachi, Tokyo, Japan) at 150 kV and 20 mA through a 0.5-mm aluminum and 0.3-mm copper filter, at a dose rate of 1.0 Gy/min. Prior to X-ray irradiation and/or treatment with AsA (Wako Pure Chemical Industries Ltd., Osaka, Japan), 10×10⁵ cells in 10 ml of medium were prepared in 10 cm dishes for 24 h. The dishes were divided into four groups: Control, AsA alone, X-ray irradiation alone and X-ray irradiation combined with AsA. The AsA was dissolved to a final concentration of 5 mM in medium following titration of the solution with NaOH (Wako Pure Chemical Industries Ltd.) to pH 7.4. The dishes treated with X-ray irradiation alone and X-ray irradiation combined with AsA, were irradiated 1 h after treatment with AsA.

HT1080 cells do not form colonies, and the cell survival rates of these cells as well as A549 and SAS cells were estimated without using colony assay in the present study. To remove all cells from the dishes following incubation, the cell monolayer was washed with phosphate buffered saline (PBS) and 0.1% trypsin/EDTA (1 ml per 25 cm² of surface area; Gibco Life Technologies, Carlsbad, CA, USA) was added to each well and incubated at 37°C for 2–10 min. When all the cells had detached, the number of viable cells were quantified using a trypan blue exclusion test (Wako Pure Chemical Industries Ltd.). The cells were stained with 0.2% trypan blue for 1 min and counted to estimate the number of viable cells in 1 ml medium.

The animals used in the present study were maintained, and the experiments were performed, according to the Principles of Laboratory Animal Care established by the NIH. All animal experiments described were previously approved by the ethics committee of Hirosaki University (Hirosaki, Japan) and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals in Hirosaki University. Female, 5-week-old BALB/cAJcl-nu/nu mice (CLEA Japan, Tokyo, Japan) were used in the present study. The mice were injected in the lateral neck with 200 µl PBS, containing HT1080 cells (5×10⁶ cells/ml) 1 week after arrival at Hirosaki University. The mice were divided into four groups: Control, AsA alone, X-ray irradiation alone and X-ray irradiation combined with AsA. The treatment consisted of X-ray irradiation and/or AsA injection 7 days and 9 days after implantation of the HT1080 cells (Fig. 2A). AsA was dissolved in PBS at a concentration of 5 mM and titrated with NaOH at pH 7.4. Each mouse in the AsA alone group and in the X-ray irradiation combined with AsA group were injected with 200 µl AsA solution. In the X-ray irradiation group and X-ray irradiation combined with AsA group, a field (40×40 mm) was made using a lead plate, which was placed centrally over the tumor. The mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg; Nembutor, Abbott Laboratories, Abbott Park, Il, USA) and were fixed in the prone position using synthetic rubber bands tied to each leg of the mouse. The mice were irradiated twice with 4 Gy with a total dose of 8 Gy. The irradiation was the same as that used for the cells treated in vitro, as described above. The mice in the X-ray irradiation and AsA combination groups were irradiated 15 min following injection with the AsA. The long diameter and short diameter of the tumors on the skin were measured daily. All mice were sacrificed by cervical dislocation following 5% halothane (Wako Pure Chemical Industries Ltd.) inhalation, 13 days after implantation of the HT1080 cells. The tumor mass was subsequently removed from the lateral neck of the mice and was weighed.

The extent of apoptosis was determined by annexin V-Fluorescein isothiocyanate (FITC; BioLegend, San Diego, CA, USA) and propidium iodide (PI; Sigma-Aldrich) staining, according to the manufacturer's instructions. Following X-ray irradiation and/or treatment with AsA, the cells were cultured for 48 h at 37°C. The cells were washed with PBS and suspended in 100 µl annexin V binding buffer (BioLegend). The annexin V-FITC (2.5 µg/ml) and PI solution (50 µg/ml) were added to the cell suspension and incubated for 15 min at room temperature in the dark. The number of apoptotic cells were determined using flow cytometry (Cytomics FC500; Beckman-Coulter, Fullerton, CA, USA). In the annexin V/PI quadrant gating, annexin V(−)/PI(−), annexin V(+)/PI(−) and annexin V(+)/PI(+) were used to identify the fraction of viable cells, early apoptotic cells and late apoptotic/necrotic cells, respectively (8).

An FITC-conjugated monoclonal active caspase 3 antibody kit for apoptosis (BD Biosciences, San Diego, CA, USA) was used to detect active caspase 3, according to the manufacturer's instructions. The cells were cultured for 48 h following X-ray irradiation and/or treatment with AsA. The cells were washed with PBS, suspended in 500 µl Cytofix/Cytoperm™ (BD Biosciences) and incubated for 20 min on ice. Following incubation, the cells were washed with Perm/Wash™ buffer (BD Biosciences) and resuspended in Perm/Wash™ buffer, containing 5% FITC-conjugated rabbit anti-active caspase 3 antibody (cat. no. 559341; BD Pharmingen, San Diego, CA, USA). Following 30 min incubation at room temperature in the dark, the cells were washed and then analyzed by flow cytometry (Cytomics FC500; Beckman-Coulter).

The HT1080 cells were seeded in 10 ml RPMI-1640 (1×10⁴ cells/ml) in 100 mm culture dishes (Iwaki, Tokyo, Japan) and incubated at 37°C for 24 h, in order to adhere to the dish. Following X-ray irradiation and/or treatment with AsA, the cells were cultured for a specific duration. The cells were fixed with 70% ethanol (Wako Pure Chemical Industries Ltd.) overnight at 4°C, washed with PBS and subsequently treated with RNase (200 µg/ml; Sigma-Aldrich) at 37°C for 30 min to hydrolyze the RNA. Following treatment, the cells were washed with PBS and stained with PI (25 µg/ml) for 30 min in the dark. A flow cytometer was used to analyze the cell cycle distribution.

Cell proliferation was measured using a cell proliferation ELISA, bromodexyuridine (BrdU) colorimetric kit (Roche, Basel, Switzerland). The cells (20,000/ml) in 200 µl of medium were placed into each well of a 96 well microtiter plate 24 h prior to treatment. Following X-ray irradiation and/or treatment with AsA, the cells were added to 20 µl BrdU (Roche) and cultured for a specific duration. The cells were incubated with 20 µl/well FixDenat solution (Roche) for 30 min and Anti-BrdU peroxidase (Roche) for 90 min at room temperature. The substrate reaction was measured using a spectrophotometer (Benchmark; Bio-Rad, Foster City, CA, USA) at 370 nm.

All the data are expressed as the mean ± standard deviation of at least three independent experiments. Statistical comparisons between the groups were performed using analysis of variance and Student's t-test. All statistical analyses were conducted using SPSS version 16.0 for Windows (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The number of viable cells were quantified 72 h after 4 Gy X-ray irradiation and/or treatment with 5 mM AsA. The percentage of viable cells compared with the control are shown in Fig. 1. The percentage of viable cells following treatment with AsA alone and X-ray irradiation alone significantly decreased in all three cell lines (P<0.05). In all three cell lines, the percentage of viable cells following X-ray irradiation combined with AsA treatment were significantly lower compared with X-ray irradiation alone (P<0.05).

The initial weight of the mice increased relative to natural growth, however, on the eighth day following transplantation of the HT-1080 cells, the weight of the mice decreased (Fig. 2A). No significant difference in weight was observed between the four groups. The results of the tumor sizes (long diameter x short diameter) are presented in Fig. 2B and demonstrate that, in the control mice, the tumor size increased daily. No significant difference in tumor size was observed between the control group and those subjected to treatment with AsA alone, although the tumor size did not increase following the second AsA treatment. The tumor size in the groups treated with X-ray irradiation alone and X-ray irradiation combined with AsA decreased following the second treatment. Furthermore, the tumor size in the X-ray irradiation combined with AsA group was lower compared with that in the X-ray irradiation alone group, with a significant difference between the two groups on the 13th day following transplantation of the HT-1080 cells (P<0.05). The weights of the tumor mass for each group on the 13^th day following the transplantation of HT-1080 cells is shown in Fig. 2C. The average tumor weight in the AsA alone group appeared lower compared with the control group, however, no significant difference was determined between the two groups. Statistical analysis revealed significant differences between the control group and the X-ray irradiation alone group, and between control group and X-ray irradiation combined AsA treatment group (P<0.05). The average weight of the tumor in the X-ray irradiation combined with AsA group was significantly lower than that in the X-ray irradiation alone group (P<0.05).

Annexin V/PI staining methods were used to analyze the induction of apoptosis. The percentage of early apoptotic annexin V(+)/PI(−) cells and annexin V(+)/PI(+) late apoptotic cells were significantly higher in the groups treated with X-ray irradiation and X-ray irradiation in combination with AsA, compared with the control and the group treated with AsA alone (P<0.05; Fig. 3A and C). However, no statistically significant difference was observed between the control cells and those treated with AsA alone. In addition, no statistically significant difference was observed between the cells treated with X-ray irradiation alone and X-ray irradiation combined with AsA. To further investigate the induction of apoptosis, the expression levels of activated caspase 3, an executioner of apoptosis, were analyzed. The activation of caspase 3 was higher in cells treated with X-ray irradiation and X-ray irradiation combined with AsA compared with the control and the group treated with AsA alone (Fig. 3B and D). However, no statistically significant difference was observed in the activation of caspase 3 between the control cells and those treated with AsA. In addition, no statistically significant difference was observed between the cells from the X-ray irradiation and X-ray combined AsA groups.

The effect of X-ray irradiation and/or treatment with AsA on the cell cycle profile of HT1080 cells was analyzed (Fig. 4). X-ray irradiation and X-ray irradiation combined with AsA increased the number of cells in the G2/M fraction following 12 h treatment, compared with the control and AsA alone groups (P<0.05; Fig. 4A). Treatment with X-ray irradiation alone and combined with AsA significantly increased the sub-G1 fraction following treatment for 72 h compared with the control and treatment with AsA alone (P<0.05; Fig. 4B). By contrast, no statistically significant differences were observed in the cell cycle profiles between the control cells and those treated with AsA alone, nor between the cells treated with X-ray irradiation alone and in combination with AsA. However, the proliferation of cells following treatment with AsA, with or without X-ray irradiation were lower compared with the control, and a significant difference was observed between the groups treated with X-ray irradiation alone and X-ray in combination with AsA treatment (P<0.05; Fig. 5).