HeLa cells obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cell cultures were stored at 37°C in an atmosphere containing 5% CO₂ in a humidified incubator. Cells used for each experiment were grouped according to the dose of irradiation (0, 2, 4, 6, 8, 10, 12.5, 15 and 20 Gy).

X-irradiation experiments were performed at the Irradiation Facility of Zhongnan Hospital of Wuhan University (Wuhan, China). The medium was replaced prior to irradiation in a horizontal position. Cells were divided into different groups and exposed under (0, 2, 4, 6, 8, 10, 12.5, 15 and 20 Gy) X-rays separately with a Siemens Primus Accelerator machine (6 Mv; Siemens AG, Munich, Germany).

CCK-8 reagent (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used to determine the proliferation rate of cells, the cells was grouped according to the dose of irradiation (0, 2, 4, 6, 8, 10, 12.5, 15 and 20 Gy). The seeded (1×10⁴ cells/well in 96-well plates) serum-starved cells were exposed to different doses of radiation (0, 2, 4, 6, 8, 10, 12.5, 15 and 20 Gy). Cells were washed using PBS prior to detection, then CCK-8 solution was added (10 µl CCK-8 and 90 µl Dulbecco's modified Eagle's medium without any supplements). At 3 h following the addition of CCK-8, the absorbance at 450 nm was measured at 24, 48, 72, 96 and 120 h followimg irradiation of cells in each group (0, 2, 4, 6, 8, 10, 12.5, 15 and 20 Gy) by micro-tablet reader (Microplate Reader, Sunrise, Tecan). The cell proliferation rates were calculated and the optical density value from the first day was used for normalization.

Cells were plated and grown on coverslips (5×10⁴ cells/well), 12 h after cells were seeded, a series of dose irradiation was performed as aforementioned (0, 2, 4, 6, 8, 10, 12.5, 15 and 20 Gy). At 20 min following irradiation, cells were fixed in 5% paraformaldehyde (Merck KGaA, Darmstadt, Germany) for 30 min at room temperature, then washed with PBS and permeabilized in 0.5% Triton (Sigma-Aldrich; Merck KGaA) for 10 min at room temperature. The cells were treated with the blocking fluid (5% bovine serum albumin, cat. no. SBJ-1239; NanJing SenBeiJia Biotechnology Co. Ltd., Nanjing, China) for 1 h at room temperature, then washed with PBS. The cells were subsequently probed with mouse anti-γ-H2AX antibody (ser139) (1:200 dilution; cat. no. 05-636-AF488; EMD Millipore, Billerica, MA, USA) and incubated overnight at 4°C. The cells were then washed with PBS, stained with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit secondary antibody (1:500 dilution; cat. no. 31635; Invitrogen; Thermo Fisher Scientific, Inc.) for 2 h at room temperature. All antibody dilutions were prepared in 5% bovine serum albumin (cat. no. SBJ-1239; NanJing SenBeiJia Biotechnology Co. Ltd.). Subsequently, three washing steps were performed with PBS, following which a cover glass was incubated with propidium iodide (PI) for 20 min at room temperature. Finally, images were captured with a fluorescence microscope (Nikon E-600; Nikon Corporation, Tokyo Japan) using a 1.3 aperture plan fluor ×40 numerical aperture oil objective.

Cells were collected at 0, 6, 12, 18, 24, 30, 36, 42 and 48 h after irradiation by trypsinization purification at room temperature. Considering that there may be adherent cells, the supernatants and PBS used in washing steps were retained. Samples were fixed in cold 70% EtOH solution at 4°C for 24 h. Cells were then washed with PBS two times, then stained with 500 µl PI solution (50 µg/ml PI and 1% RNase A; Sigma-Aldrich; Merck KGaA) for 50 min at 37°C. Samples were measured immediately by flow cytometry (Accuri C6, BD Biosciences, San Jose, CA, USA). PI fluorescence of a minimum of 1×10⁴ cells was measured. Cells in the G0/G1, S and G2/M phases were determined followed filtering for doublets and aggregates. Doublets were filtered based on a FSC-A vs. FSC-H dot plot with BD Accuri C6 software version 1.0.202.1 (BD Biosciences).

During apoptotic cell detection, 1×10⁵ cells were inoculated in the 6-well culture plate and cultured for 12 h prior to irradiation, at 37°C in an atmosphere containing 5% CO₂ in a humidified incubator. Cells were harvested at 48 and 72 h after irradiation at room temperature, then the cells were re-suspended in 100 µl staining buffer (PBS, cat. no. SH30256.01B; GE Healthcare Life Sciences, Hyclone, Logan, UT, USA) with 5 µl Annexin V-FITC (BD Pharmingen; BD Biosciences) and stained at room temperature for 15 min, followed by a quick staining with 1 µg/ml PI (cat. no. P4864; Sigma-Aldrich; Merck KGaA) at room temperature for 10 min. The samples were analyzed on a LSRII flow cytometer (BD Biosciences). The data was then analyzed using Flow Jo software version 9.6.2 (FlowJo LLC, Ashland, OR, USA).

The JC-1 kit (Beyotime Institute of Biotechnology, Haimen, China) was used to detect the mitochondrial membrane potentials. Cell suspensions at 1×10⁵ cells/ml were cultured on glass cover-slips for 12 h at 37°C in an atmosphere containing 5% CO₂ in a humidified incubator. At 6 h after irradiation, the cells were incubated with Dulbecco's PBS (DPBS) which was provided in the JC-1 kit and 1 µl JC-1 reagent at 37°C for 15 min. Subsequently, the cells were washed with DPBS three times. The mitochondrial membrane potential was detected under a fluorescence microscope (×100 magnifications, Nikon E-600). The reduction in red fluorescence and the increase in green fluorescence indicated a decrease in the mitochondrial membrane potential.

With the use of a 12-well plate, cells were plated at a density of 5×10⁴ cells/ml were incubated in Dulbecco's modified Eagle's medium containing 20 µM DHE (Vigorous Biotechnology Beijing Co., Ltd., Beijing, China; http://www.bioon.com.cn/show/reagent_88470_c0_p2.html.) at 37°C for 10 min. Following loading DHE into the cells, the plate was mounted on a fluorescence microscope (×100 magnification, Nikon E-600), and fluorescence images were captured with a digital charge-coupled device camera (Roper Scientific RTE/CCD-1300-Y/HS) controlled by MetaMorph Image-analysis software version 4.01 (Universal Imaging, Inc., Bedford Hills, NY, USA). The Eth-DNA fluorescence was monitored at 490 nm excitation and 610 nm emission.

Cells were washed with ice-cold PBS twice. Lysis buffer (25 Mm Tris-HCl, Ph 7.4, 25 Mm NaCl, 0.5 Mm EDTA, 1 Mm sodium orthovanadate, 10 Mm NaF, 25 Mm β-glycerophosphate, 10 Mm sodium pyrophosphate, 0.2 Mm sodium molybdate, 10 mg/ml aprotinin, 2 Mm phenylmethylsulfonyl fluoride and 1% Triton X-100) was added to the cells, which were subjected to lysis by sonication for 60 sec on ice. The lysates were treated by centrifugation for 10 min at 12,000 × g at 4°C, and the protein concentration was determined with a BCA protein concentration assay (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of protein (30 µg) were separated on 10–12.5% SDS-PAGE gels, electrophoretically transferred to polyvinylidene difluoride membranes with transfer buffer (25 mM Tris, 250 mM glycine and 10% methanol), the membranes were blocked with Western Blocking Buffer (cat. no. SW3010; Solarbio Life Sciences, China) at room temperature for 1 h. Probed with primary antibodies at 4°C for 8 h, then horseradish peroxidase-conjugated secondary antibodies (Goat anti-rabbit IgG, HRP-linked Antibody, 1:1,000 dilution; cat. no. 7074; Cell Signaling Technology Inc., Danvers, MA, USA) at room temperature for 1 h. Immunoblots were detected using an ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences, Little Chalfont, UK), according to the manufacturer's protocol, autoradiography and recorded on film. Quantification was performed using ImageJ software version 1.45S (National institutes of Health, Bethesda, MD, USA). The primary antibodies used included: Rabbit anti-β-actin (1:5,000 dilution; cat. no. A2066; Sigma-Aldrich; Merck KGaA), γ-H2AX (ser139) (cat. no. 05-636-AF488; EMD Millipore) (1:2,000 dilution), cyclin dependent kinase 1 (CDK1; 1:5,000 dilution; cat. no. PLA0287; Sigma-Aldrich; Merck KGaA), checkpoint kinase 1 (CHK1; 1:500; cat. no. SAB4500207; Sigma-Aldrich; Merck KGaA), RAD51 recombinase (Rad51; dilution 1:500; cat. no. SAB2101936; Sigma-Aldrich; Merck KGaA), Ku70 (1:1,000 dilution; cat. no. 4104; Cell Signaling Technology Inc., Danvers, MA, USA) and Ku80 (1:1,000 dilution; cat. no. 2753; Cell Signaling Technology Inc.), B-cell lymphoma (Bcl-2; 1:5,000 dilution; cat. no. SAB4500003; Sigma-Aldrich; Merck KGaA), Bcl-2-associated X (Bax; 1:500 dilution; cat. no. SAB4502546; Sigma-Aldrich; Merck KGaA), BH3 interacting domain death agonist (Bid; 1:500 dilution; cat. no. SAB3500353; Sigma-Aldrich; Merck KGaA) and Caspase-9 (1:500 dilution; cat. no. 9502; Cell Signaling Technology Inc.), hypoxia inducible factor-1α (HIF-1α; 1:2,000 dilution; cat. no. SAB1405933; Sigma-Aldrich; Merck KGaA), glucose transporter 1 (GLUT1; 1:1,000 dilution; cat. no. SAB4502803; Sigma-Aldrich; Merck KGaA), c-Myc (1:1,000 dilution; cat. no. 13987; Cell Signaling Technology Inc.) and pyruvate kinase M2 (PKM2; 1:1,000; cat. no. 4053; Cell Signaling Technology Inc.).

All the experiments were repeated ≥3 times. Statistical analysis was performed with GraphPad Prism 5.0 Software (GraphPad Software, Inc., La Jolla, CA, USA). Data are presented as the mean ± SEM. P≤0.05 was considered to indicate a statistically significant difference. Cell cycle data were analyzed by two-way analysis of variance with dose and time point as independent variables, with Tukey's honestly significant difference (Tukey's HSD) as post-hoc test.

Cells were incubated with DHE for 10 min after incremental doses of irradiation were administered. Red fluorescence produced by the formation and binding of ethidium (Eth) from DHE represents ROS production. The typical fluorescence microscopic images recorded at 10 min after incubation of DHE with different irradiation doses present maximal ROS-induced Eth-DNA red fluorescence. Red fluorescence of cells following irradiation, indicating production of ROS, was increased with a greater X-ray irradiation dose. As illustrated in Fig. 1, the ROS production in cells following irradiation was increased, and increased at increased doses. However, the fluorescence intensity demonstrated that there was no significant difference in ROS production between the 2 and 4 Gy groups.

DNA double-strand breaks (DSBs) are induced directly by ionizing energy or indirectly by secondary radicals mediated by ROS (11). Phosphorylation of H2AX at Ser 139 (γ-H2AX) is the most sensitive marker used to detect DNA damage (12,13). In the present experiment, γ-H2AX was used as a marker of radiation-induced DNA DSBs.

To examine the dose-effect of radiation, DNA DSBs were visualized using immunofluorescent staining for γ-H2AX foci at 15 min after irradiation. Representative images of the γ-H2AX foci in HeLa cells were depicted in Fig. 2A. γ-H2AX was stained with green fluorescence (FITC), indicating that DSBs merged with the nucleus stained with red fluorescence (PI). A clear dose-dependent induction of the γ-H2AX foci was observed and was associated with the presence of DSBs. The expression of γ-H2AX at the protein level was detected by western blot analysis. The results of western blot analysis demonstrated that the expression of γ-H2AX increased following irradiation (Fig. 2B).

Radiation-induced cell cycle changes were analyzed by flow cytometry at 0, 6, 12, 18, 24, 30, 36, 42 and 48 h after X-irradiation using PI staining. The primary results are depicted in Fig. 3A. The change trend of S-phase under different doses of irradiation was similar, within the 24 h following irradiation, the S-phase of cells with different dose were decreased gradually; and 30 h following irradiation, the change of S-phase exhibited a gradual increase. Following irradiation, a decrease trend of G1-phase was exhibited in HeLa cells, the higher the irradiation dose received, the greater the tendency of G1 phase reduction. Correspondingly, a significant increase in the number of HeLa cells in the G2/M-phase was observed following irradiation, indicative of DNA damage repair following irradiation (14,15). An increased dose of irradiation resulted in a greater G2/M delay. Cell cycling distribution returned to normal within 24 h after 2 Gy irradiation. The cells exposed to 4, 6 and 8 Gy returned to a normal cell cycle within 36 h, and cells exposed to 10 Gy returned to a normal cell cycle within 48 h. The cell cycle did not return to normal levels within 48 h when the radiation dose was >10 Gy (Fig. 3A). The expression of cell cycle-associated protein CDK1 markedly decreased following various doses of irradiation and the expression of CHK1 increased following irradiation. Variations in CDK1 and CHK1 were consistent with the accumulation of G2/M-phase cells (Fig. 3B).

Cell proliferation was detected by CCK-8 assays. The results represent the survival curves of HeLa cells exposed to the different doses of X-irradiation (Fig. 3C). The proliferation ability of cells irradiated with X-rays decreased with increasing doses.

Decrease in the mitochondrial membrane potential is an important signal of apoptosis in its early stage (16). The decrease in red fluorescence combined with the increase in green fluorescence indicated a loss of mitochondrial membrane potential, as depicted in Fig. 4. Compared with the cells without irradiation (0 Gy), the mitochondrial membrane potential of HeLa cells decreased following irradiation, and decreased at increased doses. The change of fluorescence also demonstrated that there was similar change of mitochondrial membrane potential between the 4 and 2 Gy groups. A dose-dependent decrease in mitochondrial membrane potential (MMP) was observed in cells irradiated at doses >4 Gy.

Further investigation of the dose response of apoptosis was performed by harvesting cells at 48 and 72 h after irradiation, staining with Annexin V-fluorescein isothiocyanate and PI, and analysis using a flow cytometer. It was determined that the proportion of apoptotic cells at 72 h after irradiation was increased, compared with the same dose at 48 h following irradiation (Fig. 5). Additionally, 48 and 72 h after irradiation have the identical change tendencies of apoptotic cells with different irradiation doses, a dose-dependent increase in the percentage of apoptotic cells was observed 48 and 72 h following irradiation. The results of the apoptosis assay indicated an increase in the percentage of apoptotic cells following irradiation (Fig. 5) The radio of apoptotic cells of two groups with adjacent doses were compared with each other. Except 2 and 4 Gy groups, the difference in the percentage of apoptotic cells between other adjacent two groups were significant; however, the difference in the percentage of apoptotic cells between the 2 and 4 Gy groups was not significant.

The expression of DNA damage-associated proteins was detected by western blot analysis (Fig. 6A). The expression of Rad51, which participates in homologous recombination (HR) repair (17), was markedly increased following irradiation, and the expression in the 2, 8 and 10 Gy groups was reduced, compared with the other groups. Generally, the expression of Ku70 and Ku80, which serve a notable role in Non-Homologous Endjoining (NHEJ) (18), were also increased following irradiation. Furthermore, levels of Ku70 in the 2, 8, 10, 12.5, 15 and 20 Gy groups, and Ku80 in the 6 and 8 Gy groups were markedly reduced, compared with the other groups. As reported, HR repair occurs in the S and G2-phases; however, NHEJ occurs throughout all phases of the cell cycle (19,20). Combined with the results of these two primary repair pathways, it may be concluded that for HeLa cells cultured in vitro, the DNA damage repair capability in the groups of 2, 6, 8 and 10 Gy were reduced, compared with the other groups, following irradiation.

Expression of apoptosis-associated proteins was detected by western blot analysis (Fig. 6B). Compared with the 0 Gy group, the expression of the Bcl-2 apoptosis suppression protein was markedly decreased following different doses of irradiation. However, pro-apoptosis protein Bax increased following irradiation, but the expression of Bax in the 6, 8, 10 and 12.5 Gy groups was notably increased, compared with the other groups. Additionally, the apoptosis-associated proteins Bid was elevated following irradiation, compared with the control group (0 Gy). The expression of Bid in the 6, 8 and 10 Gy groups were increased, compared with the other groups. The expression of procaspase-9 and cleaved Caspase-9 was increased in the 2, 4, 6 and 8 groups, however, a downward trend was observed following 6 Gy. Generally, increased expression levels of apoptosis-associated proteins were determined in the 2, 6, 8 and 10 Gy groups.

Given that radiation may alter tumor glucose metabolism through the aerobic glycolysis metabolic pathway (21,22), the present study detected changes associated with aerobic glycolysis following irradiation that may alter the energy of cancer cells.

Compared with 0 Gy, the results of the western blot analysis (Fig. 6C) demonstrated an increasing trend in the expression of HIF-1α following irradiation in a dose-dependent manner, and a similar trend was observed in the expression of c-Myc; however, the expression of HIF-1α and c-Myc in the 4, 6, 12.5 and 20 Gy were higher than the other groups. GLUT1 is one of most important glucose transporters in cancer cells (23). The expression level of GLUT1 was markedly increased following irradiation, but was reduced in the 12.5, 15 and 20 Gy groups. HIF-1α, c-Myc and GLUT1 serve an important role in promoting glycolysis and glucose uptake (24). The expression of PKM2 in the groups with irradiation doses from 2–10 Gy were reduced, compared with the other groups, indicating that the levels of glycolysis in these groups may be reduced, compared with the other groups.