Human BGC823, SGC7901, MGC803, HGC-27, MKN45 and MKN74 GC cell lines were obtained from Shanghai Institute of Cell Biology (Shanghai, China) and cultured in RPMI-1640 medium, supplemented with 10% calf bovine serum, 50 U/ml penicillin and 50 U/ml streptomycin in an incubator at 37°C in 5% CO₂.

The DNA-PK inhibitor NU7441 (Tocris Bioscience, Bristol, UK) was dissolved in dimethyl sulfoxide (DMSO) as a 5 mmol/l stock solution and stored at −20°C. A casein kinase 2 (CK2) inhibitor, CX4945, was purchased from Selleck Chemicals (Houston, TX, USA). Cells were exposed to X-rays generated by a Rad Source RS2000 irradiator (Rad Source Technologies, Inc., Buford, GA, USA) operating at 25 mA with a 0.3 mm Al filter and effective photon energy of 160 kV. The dose rate at an irradiation distance of 48.6 cm was 1.31 Gy/min.

Cells were seeded into 6-well plates (2×10⁶) and treated with or without drug (0.1 µM NU7441 or 0.5 µM CX4945) following attachment for 4–6 h. The next day, the cells were exposed to different doses of IR. Cells were incubated for 10–14 days to form colonies, then fixed with 100% methyl alcohol for 5 min and stained with 1% crystal violet for 10 min at room temperature. Colonies containing >50 cells were counted. Survival fractions were normalized according to the non-irradiated subgroup to eliminate cytotoxicity generated by drug pretreatment. Cell survival curves were obtained using GraphPad Prism 7 software (GraphPad Software, Inc., La Jolla, CA, USA) according to the multitarget single hit model. The radiation-associated parameters, mean lethal dose (D0) and quasi-threshold dose (Dq), were calculated to evaluate the effect of drug pretreatment on the radiosensitivity of GC cells.

Determination of the IC₅₀ of NU7441 treatment of GC cells was performed using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Briefly, following treatment with increasing concentrations of NU7441 (5, 10, 15 and 20 µM) for 48 h, 10 µl CCK-8 solution was added to each well and incubated for 2 h. Subsequently, the cells were transferred into another 96-well plate and the absorbance was detected at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.) to reflect cell viability. The IC₅₀ was calculated using CompuSyn software version 1.0 (ComboSyn, Inc., Paramus, NJ, USA).

Cell extracts were prepared in radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and the concentration was measured using a Pierce BCA Protein Assay kit (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. Briefly, each sample (20 µg protein), prepared with loading buffer, was separated by SDS-PAGE on 8% gels and electrotransferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked in 5% non-fat milk for 1 h at room temperature and incubated with primary antibody at 4°C overnight. GAPDH primary antibody (cat. no. G8795; 1:5,000) was purchased from Sigma-Aldrich (Sigma-Aldrich; Merck KGaA), γ H2A histone family member X (γH2AX; cat. no. 33686; 1:1,000), poly [ADP-ribose] polymerase 1 (PARP1; cat. no. 31288; 1:1,000) and caspase3 (cat. no. 29034; 1:1,000) primary antibodies were from Signalway Antibody LLC (College Park, MD, USA). The membranes were then washed with three times with Tris-buffered saline and incubated with a horseradish peroxidase-conjugated secondary antibody (cat. no. 7074, 1:10,000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 2 h. Finally, the blots were visualized using enhanced chemiluminescence (EMD Millipore) and the semi-quantification of bands was performed using ImageJ (version 1.51; National Institutes of Health, Bethesda, MD, USA).

Cells were incubated with 3% H₂O₂ for 10 min, permeabilized with 0.3% Triton X-100 in PBS for 15 min and blocked with 10% goat serum for 1 h at room temperature. The sections were then incubated with primary antibody (γH2AX; cat. no. 33686; 1:100) at 4°C overnight. The next day, sections were washed, incubated with biotinylated goat anti-rabbit IgG (cat. no. BA-1000; 1:10,000; Vector Laboratories, Inc., Burlingame, CA, USA) in PBS for 1 h at room temperature and with VECTASTAIN (PK-6200; Vector Laboratories, Inc.) for 1 h. A horseradish peroxidase reaction product was visualized using an enhanced DAB peroxidase substrate kit (cat. no. SK-4100; Vector Laboratories, Inc.). Immunostaining signals were analyzed using the optical fractionator method with Microbrightfield Stereo-Investigator (MBF Bioscience, Williston, VT, USA). The total number of cells in four or five fields of view was counted per sample.

Data are presented as the mean ± standard deviation. Comparisons between two groups were evaluated by the Student's t-test, and comparisons among multiple groups were analyzed by analysis of variance followed by Newman-Keuls post-hoc analysis using GraphPad Prism 7 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

In the present study, six human GC cell lines, SGC7901, HGC-27, MKN45, MKN74, BGC823 and MGC803, were used, and the relative sensitivities to IR were determined by clonogenic survival assay. BGC823 and MGC803 cells were the most resistant to IR, with higher D0 and Dq values (Fig. 1A and Table I). In addition, the expression levels of key regulators involved in cell apoptosis and cell survival were detected in all cell lines treated with IR. The expression levels of cleaved caspase3 and γH2AX were relatively low in BGC823 and MGC803 cells compared with other cell lines following IR. PARP1, the substrate of active caspase3, was expressed at a relatively high level in BGC823 and MGC803 cells (Fig. 1B and C).

To further evaluate their response to IR, immunofluorescence was used to observe γH2AX expression following IR treatment. BGC823 cells exhibited weak fluorescence signals compared with MKN45 cells, indicating a mild double strand break induced by IR (Fig. 2A and B).

The expression of DNA-PKcs, whose expression predicts response to radiotherapy in various types of cancers, is responsible for DNA repair (12,19). Western blotting detection of DNA-PKcs and p-DNA-PKcs proteins indicated that DNA-PKcs was expressed more highly in BGC823 and MGC803 cells compared with other GC cell lines (Fig. 3A and B).

To determine whether DNA-PKcs were responsible for different sensitivities towards IR, NU7441 (a DNA-PKcs inhibitor) was used to inhibit DNA-PKcs activity. Cell viability assays revealed that the IC₅₀ was 3.724 µM and 4.704 µM for BGC823 and MGC803, respectively (Fig. 4A). At a very low concentration without significant growth inhibitory effect, 0.1 µM NU7441 induced a decreased survival fraction in response to IR compared with cells treated with DMSO in both BGC823 and MGC803 cells (Fig. 4B and C; Table II). In addition, NU7441 treatment increased γH2AX and cleaved-caspase3 protein expression levels in BGC823 and MGC803 cells treated with IR (Fig. 4D and E); 0.1 µM NU7441 significantly elevated IR induced cell apoptosis of BGC823 and MGC803 cells (Fig. 4F and G). Furthermore, immunofluorescence analysis confirmed an elevation of γH2AX expression in the nucleus in cells treated with NU7441 after IR in BGC823 (Fig. 5A and B). These data collectively demonstrated that DNA-PKcs was involved in radiotherapy sensitivity in GC.

A previous study indicated that CK2 inhibition could increase radiotherapy sensitivity via inhibition of X-ray repair cross complementing 1 (XRCC1) (20). In BGC823 and MGC803 cells, the combination of CK2 inhibitor (CX-4945) and DNA-PKcs inhibitor (NU7441), demonstrated a stronger enhancing effect in radiotherapy sensitivity compared with NU7441 alone (Fig. 6A and B; Table III). Furthermore, the combination of CX-4945 and NU7441 further increased γH2AX and cleaved-caspase3 protein expression levels in both cell lines (Fig. 6C and D); additionally, CX4945 further significantly elevated NU7441 induced cell apoptosis of GC823 cells and MGC803 cells (Fig. 6E and F). The elevation of γH2AX expression was validated in an immunofluorescence assay (Fig. 7A and B).