The rat glioma C6 and human lung cancer A549 cell lines (Cell Bank, Chinese Academy of Sciences) were cultured in RPMI-1640 medium (BAL Gibco, Grand Island, NY, USA) supplemented with 10% FBS (Gibco), penicillin (100 U/ml), streptomycin (100 μg/ml) (Sigma-Aldrich) 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. The cells in the exponential phase of growth were incubated in culture media with 100 μm CoCl₂ (Sigma-Aldrich), a common mimetic hypoxia reagent. OA was purchased from Nanjing Zelang Medical Technology Co., Ltd., (Jiangsu, China) and was dissolved in dimethyl sulfoxide (DMSO; Sigma) at a stock concentration of 250 μg/ml and stored at −20°C. The cells were treated with OA at different concentrations for 24 h prior to exposure to irradiation.

The influence of OA on cell growth was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) method. C6 and A549 cells were seeded in 96-well plates at a density of 5×10³ cells/well. They were then treated with OA at different concentrations for 24 h. Furthermore, the medium was replaced with fresh medium allowing cells to undergo continuous growth up to 72 h. MTT dye was added to a final concentration of 50 mg/ml, and cells were subsequently incubated for another 4 h at 37°C. The medium containing residual MTT dye was carefully aspirated from each of the wells, and 200 μl DMSO (Sigma-Aldrich) was added to each well to dissolve the reduced formazan dye. The fraction of viable cells was calculated by comparing the optical absorbance of the culture exposed to OA treatment with that of the untreated control.

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

The radiosensitivity of tumor cells was determined using the clonogenic assay. Both tumor cell lines were seeded and cultured overnight at an appropriate density in T25 flasks, and subsequently the drugs at different concentrations were added into the medium for 24 h. After being pretreated with control and OA, cells were subjected to 0, 1, 2, 3, 5 or 7 Gy X-ray irradiation. The medium was then replaced with fresh medium allowing cells to continuously grow for colony formation for 9 to 12 days. Cell colonies were fixed by absolute methanol and stained with Giemsa (Sigma-Aldrich) for counting. The clonogenic survival fraction (SF) was calculated as the number of colonies/(the number of seeded cells × plating efficiency). Plating efficiency was defined as the number of colonies/the number of seeded cells of the untreated control. Survival curve was fitted with the single target multi-model of an equation: S=1–1(1−e^−D/D0)^N. The oxygen enhancement ratio (OER) was calculated accordingly, comparing the hypoxic D₀ with the corresponding aerobic D₀.

Micronucleus (MN) frequencies were tested with the cytokinesis-block technique as a biological end point for the response of cells under mimetic hypoxia to irradiation. 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. Micronuclei 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, at least 1,000 binucleated cells were counted. The MN yield, Y_MN, is the ratio of the number of micronuclei to the number of binucleated cells scored.

After triplicate samples of 10⁶ cells were treated with different reagents, the intracellular GSH content was measured with the glutathione reductase/5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) recycling assay kit (Beyotime Institute of Biotechnology, Shanghai, China) following the methods 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 (pH 7.5) containing 153 mM sodium phosphate and 8.4 mM EDTA. After a 5-min incubation at 25°C, the reaction was started by the addition of 50 μl NADPH solution (0.16 mg/ml) in assay buffer. The standard sample and checking sample cuvettes were placed into a dual-beam spectrophotometer, and the increases in absorbance at 412 nm were followed as a function of time.

Cells (10⁶) were homogenized in 50 mM potassium phosphate (pH 7.5) containing TES/SB buffer (20 mM Tris, 1 mM EDTA, 250 mM sucrose, 20 mM sodium borate, 2 mM serine) for the α-GCS assay. Homogenates were centrifuged at 12,000 rpm (15 min, 4°C), and the supernatants were maintained on ice for determination of enzyme activity. The protein concentration of the cell supernatants was measured using the Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Hertfordshire UK) and enzyme activity was reported as units/mg protein, where a unit of activity is the amount of enzyme required to convert 1 μmole of substrate to product per minute at 25°C. The α-GCS assay is an adaptation of the method previously described, in which α-GCS in cell extracts synthesizes α-glutamylcysteine which is then reacted with 2,3-naphthalenedicarboxaldehyde (NDA) to form a highly fluorescent product that can be measured fluorimetrically at 520 nm (29).

Tumor cells were plated in 60-mm culture dishes at a density of 10⁶ cells/dish then divided into different groups for various methods of pretreatment. The intracellular GSS content was measured using the GSS assay kit (Hefei Lanxu Biotech Co., Ltd., Hefei, China). Briefly, the cells were subjected to repeated freeze-thaw cycles to lyse these cells for release of intracellular components. Cell lysates were centrifuged at 3,000 rpm (20 min, 4°C), and the supernatants were maintained on ice for determination of enzyme activity. All procedures were performed according to the protocol of the kit. After the reaction was terminated, the absorbance was measured at 450 nm on an ELISA reader. The activity of GSS in the sample was then determined by comparing the OD of the samples to the standard curve.

The cells in the different treatment groups were scraped off from the culture flasks and lysed in lysis buffer containing 10% glycerol, 10 mM Tris-HCl (pH 6.8), 1% SDS, 5 mM dithiothreitol (DTT) and 1X complete protease inhibitor cocktail (Sigma, St. Louis, MO, USA). The method of Bradford was used to detect the concentrations of protein in the diverse samples. The protein concentration was measured using an auto multifunction microplate reader. Fifty micrograms of proteins was separated by 8% polyacrylamide-SDS in consecutive gel electrophoresis. The separated proteins were electrophoretically transferred to a polyvinylidene difluoride membrane. Membranes were blocked with 5% skim milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 at room temperature for 1 h and then incubated with mouse HIF-1α antibody (Abcam, Cambridge, MA, USA) at a 1:500 dilution overnight at 4°C, followed by goat anti-mouse IgG for 1 h at room temperature. Signals were detected with enhanced chemiluminescence (ECL Plus; Amersham, Pittsburgh, PA, USA). Microtubule protein (Tubulin; Abcam) at a 1:1,000 dilution was used as an internal control to observe the changes in the HIF-1α bands.

Data are reported as the means ± SEM of three separate experiments. Statistical significance was measured by the independent sample t-test and analysis of variance. A value of P<0.05 was considered to indicate a statistically significant result.

The cytotoxicity test showed that the half maximal inhibitory concentrations (IC₅₀) of OA in the hypoxic C6 and A549 cells were 80 and 81 μg/ml, respectively (Fig. 1). The concentrations of OA at 16 μg/ml (20% IC₅₀) and 24 μg/ml (30% IC₅₀) were used to pretreat the cells in order to observe the alteration of radiosensitivity in both hypoxic tumor cell lines.

No statistical differences were observed in the numbers of colonies formed in the C6 and A549 cells with CoCl₂ treatment and those without CoCl₂ treatment. However, after the cells were exposed to radiation, both cell lines in the mimetic hypoxic microenvironment had a higher resistance to irradiation. The OER values of the C6 and A549 cells were 1.52 and 1.31, respectively (Fig. 2A and B). Subsequently, we observed the alteration in radiosensitivity of the C6 and A549 cells following treatment with OA at the different concentrations. After the hypoxic cells were exposed to irradiation, the SF of the cells treated with OA was lower than that of the cells without OA treatment. Following calculation of the sensitive enhancement ratio (SER), the SER of the irradiated cells was elevated concomitant with the increase in OA concentrations. The SERs of the hypoxic C6 and A549 cells treated with OA at 30% IC₅₀ were 2.09 and 1.71, respectively (Fig. 2C and D).

MN assay showed that there was no obvious influence of OA on the frequencies of MN in both hypoxic cell lines unexposed to irradiation. Subsequently, the numbers of intracellular MN were significantly increased concomitant with the irradiation doses. When both irradiated cell lines were pretreated with OA at different concentrations, further enhancement in the numbers of intracellular MN was noted. Compared with the irradiated cells without OA treatment, the irradiated cells pretreated with 16 and 24 μg/ml OA displayed a statistically significant increase in intracellular MN frequencies (Fig. 3).

To further observe the mechanism of the influence of OA on the radiosensitivity of hypoxic cells, intracellular GSH levels were measured following treatment of OA at different concentrations for 24 h. Significant decreases in the GSH levels of hypoxic C6 cells were noted in the presence of OA, when compared with levels in the absence of OA. Moreover, a similar phenomenon occurred in hypoxic A549 cells, when intracellular GSH levels showed a gradually declining tendency concomitant with increases in OA concentrations (Fig. 4A). Since α-GCS is the key limiting-enzyme in the synthesis of intracellular GSH, its activity was further measured. As shown in Fig. 4B, the different concentrations of OA significantly decreased α-GCS activity in both hypoxic cell lines. Notably, the activity of GSS, another synthetic enzyme, did not exhibit a statistically significant change in the tumor cells with the same treatments (Fig. 4C).

High expression of intracellular HIF-1α was induced by CoCl₂ treatment. The hypoxic cells exposed to irradiation still exhibited high levels of HIF-1α. Meanwhile, HIF-1α expression in the hypoxic cells without irradiation did not exhibit a statistically significant change following OA treatment. However, the combination of OA treatment with irradiation suppressed the high levels of HIF-1α expression in the hypoxic cells (Fig. 5).