The human lung cancer H1299 cell line was kindly provided by Professor Qinghua Shi (College of Biological Science, University of Science and Technology of China, Hefei, China). Cells were maintained in Dulbecco's modified Eagle medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), penicillin (100 U/ml) and streptomycin (100 µg/ml) (Sigma-Aldrich, St. Louis, MO, USA) at 37°C in an incubator containing a humid atmosphere of 95% air and 5% CO₂, and propagated according to the protocol supplied by the American Type Culture Collection (Manassas, VA, USA). UA was purchased from Nanjing Zelang Medical Technology Co., Ltd. (Nanjing, China), dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) at a stock concentration of 5 mmol/l and stored at −20°C.

The pcDNA3.0 vector with enhanced green fluorescence protein (EGFP) was kindly provided by Professor Qinghua Shi. HIF-1α complementary DNA (cDNA) for with three mutant motifs, including the prolines at the 402 and 564 sites in the oxygen-dependent degradation domain (ODDD) of HIF-1α, and the aspartic acid at the 803 site in the C-terminal transactivation domain (CTAD) of HIF-1α, was purchased from Beijing Zhongyuan Ltd. (Beijing, China). The M-HIF-1α cDNA was cloned into the pcDNA3.0-EGFP vector to construct the pcDNA3.0-EGFP-HIF-1α recombinant plasmid. The pcDNA3.0-EGFP empty vector was used as control. H1299 cells (5×10⁵) were transfected with 4 µg plasmid DNA using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The neomycin-resistant clones were selected in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 500 µg/ml G418 (Promega Corporation, Madison, WI, USA), and transferred into a 24-well culture plate with cloning discs (Sigma-Aldrich). The selected clones were expanded in medium containing 200 µg/ml G418, and identified by detecting the messenger RNA and protein expression of M-HIF-1α.

The influence of UA on cell growth was determined using the MTT (Sigma-Aldrich) assay. The parental H1299 cells and H1299 cells expressing the M-HIF-1α fragment were seeded in 96-well plates at a density of 5×10³ cells/well, and then treated with various concentrations of UA for 24 h. Next, the medium was replaced with fresh medium to allow cells to continuously grow for 72 h. MTT dye was then added to a final concentration of 50 mg/ml, and the cells were subsequently incubated for additional 4 h at 37°C. The medium containing residual MTT dye was carefully aspirated from each of the wells, and 200 µl dimethyl sulfoxide was added to each well to dissolve the reduced formazan dye. The survival rates of viable cells were calculated by comparing the optical absorbance of the culture exposed to UA treatment with that of the untreated control.

Irradiation was emitted using a 6 MV X-ray linear accelerator (Varian Inc., Palo Alto, CA, USA) at a dose rate of 250 cGy/min.

MN frequencies were tested with the cytokinesis-block technique as a biological end point for the response of mimetic hypoxia to irradiation (30). Briefly, the cells were exposed to 0.83 µg/ml cytochalasin B (Sigma-Aldrich) for 19–20 h, followed by 75 mM KCl hypotonic treatment for 1–3 min, and then fixed in situ with methanol:acetic acid (9:1 v/v) for 30 min. Air-dried cells were stained with 5% Giemsa for 10 min. MN were scored in binucleated cells, and the formation of binucleated cells was measured as the percentage of the total number of cells scored. For each sample, ≥1,000 binucleated cells were counted. The MN yield was calculated as the ratio of the number of MN to the number of binucleated cells scored.

Cells subjected to different treatments were scraped off from culture flasks and lysed in lysis buffer containing 10% glycerol, 10 mM Tris-HCl (pH 6.8), 1% sodium dodecyl sulfate (SDS), 5 mM dithiothreitol and 1X complete protease inhibitor cocktail (Sigma-Aldrich). The Bradford method was used to detect concentrations of protein in diverse samples. Protein concentration was measured using an automatic multifunctional microplate reader. Proteins (50 µg) were separated by 8% SDS-polyacrylamide gel electrophoresis. The separated proteins were electrophoretically transferred to polyvinylidene difluoride membranes, which were then blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 at room temperature for 1 h, and then incubated with mouse anti-HIF-1α antibody (catalog no. ab82832; Abcam, Cambridge, MA, USA) at a 1:500 dilution overnight at 4°C, followed by goat anti-mouse immunoglobulin G (catalog no. ab8226; Abcam) for 1 h at room temperature. Signals were detected with enhanced chemiluminescence (ECL Plus; GE Healthcare Life Sciences, Chalfont, UK). An antibody against the microtubule protein tubulin (anti-tubulin; Abcam) at a 1:1,000 dilution was used as an internal control to observe the changes in the HIF-1α bands.

Following the treatment of triplicate samples of 10⁶ cells with different reagents, the intracellular GSH content was measured with a glutathione reductase/5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) recycling assay kit obtained from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China), following the protocol recommended by the manufacturer. Briefly, GSH was determined using a reaction mixture containing 50 µl of cell lysates, 50 µl of 2.4 mM DTNB and 50 µl of 10.64 mU/µl glutathione reductase in the assay buffer (153 mM sodium phosphate and 8.4 mM ethylenediaminetetraacetic acid, pH 7.5). After 5 min incubation at 25°C, the reaction was started by the addition of 50 µl of reduced nicotinamide adenine dinucleotide phosphate (NADPH) solution (0.16 mg/ml) in the assay buffer. The standard and the sample cuvettes were placed into a dual-beam spectrophotometer, and the increases in absorbance at 412 nm were followed as a function of time.

Cell suspension from the different treatmentswas incubated with 10 µM of 2′,7′-dichloro-dihydro-fluorescein diacetate (DCFH-DA) for 30 min, and then washed three times with phosphate-buffered saline for removing excess DCFH probe. Upon counting the viable cells, the fluorescence intensities of the cells were observed under an inverted fluorescence microscope (Nikon Corporation, Tokyo, Japan), at excitation and emission wavelengths of 488 and 525 nm, respectively.

Data are reported as the mean ± standard error of the mean of three separate experiments unless stated otherwise. Statistical significance was measured by independent samples t-test and analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

The radiosensitivity of H1299 cells was regulated by transfecting recombinant pcDNA3.0-EGFP-HIF-1α plasmid. As shown in Fig. 2A and B, under aerobic conditions, overexpression of HIF-1α was detected in H1299 cells transfected with the H1299/M-HIF-1α recombinant plasmid, named H1299/M-HIF-1α cells. Both the parental H1299 cells and the H1299 cells transfected with empty vector (H1299/E cells) exhibited loss of HIF-1α expression. Upon exposure to 2 Gy irradiation, cellular viability was significantly upregulated in H1299/M-HIF-1α cells compared with that in H1299 and H1299/E cells (Fig. 2C). By contrast, no obvious difference was observed between H1299 cells and H1299/E cells. It was therefore demonstrated that H1299/M-HIF-1α cells had lower radiosensitivity than H1299 cells and H1299/E cells.

To evaluate the radiosensitizing effect of UA on NSCLC cells, the experimental concentration of UA was first selected. The result from the cytotoxicity test indicated that there were not significant changes in the survival rates of the three types of NSCLC cells pretreated with UA at 50 and 80 µmol/l, as shown in Fig. 3A. Alterations in the survival rates of the cells subjected to treatment with two different concentrations of UA for 24 h were detected upon irradiation at 2 Gy. It was observed that UA at two different concentrations could significantly increase the radiosensitivity of H1299/M-HIF-1α cells, leading to the reduction of survival rates of the irradiated cells. However, the radiosensitizing effects of UA on H1299 and H1299/E cells were only observed at high concentration (80 µmol/l) (Fig. 3B). Consequently, it was demonstrated that radioresistant NSCLC cells were the most sensitive to the UA treatment combined with irradiation.

The MN frequencies in irradiated cells were further measured for the assessment of DNA damage. As shown in Fig. 4, when NSCLC cells were not exposed to ionizing radiation, the intracellular MN frequencies were very low, even with UA pretreatment. Conversely, the MN ratio increased in irradiated cells. Subsequently, the addition of UA at 50 and 80 µmol/l could further increase the formation of MN in irradiated H1299/M-HIF-1α cells. Further elevation of MN frequencies, however, was not observed in H1299 or H1299/E cells subjected to the combination treatment of UA at 50 µmol/l concentration and radiation. It was thus obvious that UA effectively promoted the formation of MN in irradiated NSCLC cells, particularly in irradiated H1299/M-HIF-1α cells.

Due to the strong radioprotection of endogenous GSH, the intracellular GSH content was analyzed in NSCLC cells upon non-exposure or exposure to irradiation following UA pretreatment. The results revealed that UA could remarkably decrease the endogenous GSH content in H1299/M-HIF-1α cells not exposed to radiation but not in H1299 or H1299/E cells, as shown in Fig. 5A. Additionally, following NSCLC cells exposure to irradiation, the level of cellular GSH in H1299/M-HIF-1α cells was higher than that in H1299 cell and H1299/E cells, as shown in Fig. 5B. Furthermore, UA at high concentration (80 µmol/l) could effectively attenuate the intracellular GSH content of H1299 and H1299/E cells. The combination treatment of UA with radiation could decrease the GSH intracellular contents in H1299/M-HIF-1α cells, both at 50 and 80 µmol/l concentration of UA.

Under an inverted fluorescence microscope, there was fluorescence in H1299/E and H1299/M-HIF-1α cells without DCFH-DA probe treatment, due to the presence of EGFP in the transfected plasmid (Fig. 6A and E). The fluorescence intensities in the three groups of cells evaluated were weakly increased following DCFH-DA treatment. UA at different concentrations could enhance the levels of intracellular ROS and FR in these cells, particularly in H1299/M-HIF-1α cells (Fig. 6). The results shown in Fig. 7 further demonstrate that the combination of UA with radiation treatment significantly enhanced the generation of ROS and FR. It was revealed that UA with or without irradiation could promote an increase in cellular ROS and FR, particularly in H1299/M-HIF-1α cells.

Besides detection of intracellular GSH, the levels of HIF-1α in radioresistant cells were investigated upon UA pretreatment. It was observed that UA at different concentrations markedly reduced the protein expression levels of HIF-1α, as detected by western blotting (Fig. 8). The results also revealed that UA could suppress the expression of M-HIF-1α.