The RPMI-1640 culture medium was purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd (Hangzhou, China). Dimethyl sulfoxide (DMSO), DFOM, Z-VAD-FMK, trypsin and trypan blue were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Primers for glutathione peroxidase 4 (GPX4), small interfering (si)RNA of GPX4, Lipofectamine^® 2000, TRIzol^®, OPTI-MEM I, MMLV reverse transcriptase, Taq DNA polymerase and Oligo dT primers were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). Erastin and olaparib were purchased from Selleck Chemicals (Houston, TX, USA). Protein molecular weight standards were purchased from Fermentas (Thermo Fisher Scientific, Inc.). Protein lysis buffer and the Bicinchoninic Acid (BCA) protein assay kit were purchased from the Beyotime Institute of Biotechnology (Wuhan, China). Protease inhibitors were purchased from Roche Diagnostics (Basel, Switzerland). Rabbit anti-GPX4 (catalog no. ab125066; 1:1,000) and anti-β-actin (catalog no. ab8226; 1:500) were purchased from Abcam (Cambridge, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (catalog no. TA130023; 1:3,000) was purchased from OriGene Technologies, Inc. (Beijing, China). All antibodies were diluted in bovine serum albumin. The Enhanced Chemiluminescence (ECL) chemiluminescence reagent was purchased from Thermo Fisher Scientific, Inc.

The human NSCLC cell lines A549 and H460 were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 IU penicillin and 100 µg/ml streptomycin, and incubated at 37°C in a 5% CO₂ humidified incubator.

Exponentially growing NSCLC cells A549 and H460 were irradiated with 5 does of 6 Gy. Irradiation was performed with 6-MV X-rays generated by a Siemens Primus H high-energy linear accelerator (Siemens Healthineers, Erlangen, Germany), as described previously (20,21). There was a 7–9 day break in between each irradiation (21). The radiation field was 10×10 cm, the distance from the source to target was 100 cm and the absorbed dose rate was 0.2 Gy/min. The surviving sublines (A549-R and H460-R) were then passaged for three months at 37°C and the radiosensitivity was determined by colony formation assays.

A single cell suspension was prepared from the A549, A549-R, H460 and H460-R cells in their logarithmic growth phase using 0.25% trypsin for digestion. Cells were seeded in 6-well plates and received single dose irradiation of 0, 2, 4, 6 and 8 Gy after a 12 h culture adherence at 37°C in a 5% CO₂ humidified incubator. Cell density was 200, 300, 600, 1,200 and 2,400 cells/well for each radiation dose. After 2 weeks, cells were fixed with 4% paraformaldehyde at 25°C for 20 min and were stained with Giemsa at 25°C for 15 min. Cell survival fraction (SF) was calculated based on the following formula: SF=colony numbers in experimental group/[plating number × plating efficiency (PE)]. PE=(colony numbers in the control group/plating number in control group) ×100%. A cell survival curve was obtained by the single-hit multi-target model [SF=l-(1-e^−D0/D)^N] using Sigma Plot 10.0 version (Systat Software, Inc, CA, USA). The parameters of cell survival fraction, a 2 Gy irradiation dose (survival fraction at 2 Gy irradiation, SF₂), mean lethal dose value (D₀), quasithreshold dose (D_q) and extrapolation number (N) were calculated. Higher values of SF₂, D₀ and Dq indicated an increased radioresistance (22).

A549-R and H460-R cells were divided into four groups for the following treatments: i) control group treated with DMSO; ii) treated with 4 µM erastin alone for 12 h at 37°C; iii) treated with 2 Gy IR alone and cultured in medium without erastin for 12 h at 37°C; and iv) treated with 2 Gy IR and then cultured in medium with 4 µM erastin for 12 h. Following the indicated treatments, the cells were trypsinized and stained with trypan blue for 3 min at 25°C, followed by counting with a hemocytometer using a standard protocol (21). Cells stained blue were considered as dead cells.

In addition, A549-R and H460-R cells were divided into eight groups for the following treatments: i) control group; ii) treated with 100 µM DFOM; iii) treated with 50 µM Z-VAD-FMK; iv) treated with 10 µM olaparib; v) treated with 4 µM erastin; vi) treated with 4 µM erastin and 100 µM DFOM; vii) treated with 4 µM erastin and 50 µM Z-VAD-FMK; and viii) treated with 4 µM erastin and 10 µM olaparib. All groups were incubated with an equal volume DMSO, including the control group. The eight groups of cells were treated for 12 h at 37°C. Following the indicated treatments, the cells were trypsinized and stained with trypan blue for 3 min at 25°C, followed by counting with a hemocytometer using the standard protocol (23). Cells stained blue were considered as dead cells.

A549, A549-R, H460 and H460-R cell lysates were prepared using the protein lysis buffer, according to the manufacturer's protocol. Following extraction, protein concentration was determined with a BCA assay. A total of 30 µg protein from each sample was loaded into a 10% SDS-PAGE, followed by a transfer to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% bovine serum albumin (catalog no. ST023-1000 g; Beyotime Institute of Biotechnology) for 2 h at 37°C. They were then incubated with rabbit anti-GPX4 (1:1,000) and anti-β-actin (1:500) antibodies at 4°C overnight. The polyvinylidene fluoride membrane was washed three times with TBS and Tween-20, 5 min each. Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit (1:3,000) antibodies for 1 h at 25°C. The ECL reagent then was added to the membranes and the optical density of the bands was analyzed using the Image J V1.8.0 software (National Institute of Health, Bethesda, MD, USA).

siGPX4 (catalog no. AM16708) and the control siRNA (siMock; catalog no. AM4611) were obtained from Invitrogen (Thermo Fisher Scientific, Inc.). A solution composed of 1 ml OPTI-MEM, 6 µl Lipofectamine 2000 and 2 µl siRNA (the final concentration was 2 µl/ml) was prepared and the mixture (1 ml/well) was incubated in a 6-well dish for 20 min at 37°C. Following incubation, 200,000 cells were suspended in 1 ml RPMI-1640 culture medium with 20% FBS (Thermo Fisher Scientific, Inc.). Subsequently, they were transferred to each well and incubated for 72 h. GPX4 expression was measured by western blot analysis, as aforementioned.

Statistical analysis was performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Data are presented as the mean ± standard error of the mean. Significant differences between the groups were determined by unpaired Student's t-test and by one-way analysis of variance followed by a Bonferroni post hoc test for multiple group comparisons (Figs. 3, 4 and 7). P<0.05 was considered to indicate a statistically significant difference.

To confirm that the radioresistant cell line was successfully established, a colony formation assay was performed to determine the radiosensitivity of A549-R and H460-R cells. The results demonstrated that A549-R and H460-R cells had increased survival fractions, compared with A549 and H460 cells, respectively. Cell survival curves were used to analyze the results. Therefore, the shoulder area under the survival curve was increased in H460-R and A549-R cells (Fig. 1). Compared with the A549 and H460 cells, A549-R and H460-R cells, respectively, had increased D₀, D_q, N and SF2 values (Table I), which represented increased radioresistance. Collectively, these results demonstrated that radioresistant (A549-R and H460-R) cells were successfully established.

A colony formation assay was performed to investigate the effect of erastin on radiosensitivity of A549-R and H460-R cells. The results demonstrated that erastin-treated groups had reduced survival fractions, compared with the control groups. Cell survival curves were used to analyze the results. Additionally, the shoulder area under the survival curves was reduced in the erastin-treated groups, compared with the control groups (Fig. 2). Compared with the control group cells, the erastin group cells had decreased D₀, D_q, N and SF₂ values (Table II), which indicated decreased radioresistance. The result demonstrated that erastin enhances the sensitivity to radiation therapy in the radioresistant NSCLC cells.

Following indicated treatment, cell death was measured. In A549-R cells, cell death fraction induced by erastin + IR was 60.2±2.7%. The cell death percentage induced by erastin alone was 32.5±1.9%, while the IR-induced cell death fraction was 19.1±2.5%. In H460-R cells, the cell death fraction in the erastin + IR group was significantly increased, compared with the erastin or IR groups (39.4±4.5% vs. 21.2±2.2% or 14.9±2.1%, respectively) (Fig. 3). These data revealed that erastin and IR exhibited a combined effect on killing cells.

DFOM is an iron chelator, which can inhibit ferroptosis (24). Z-VAD-FMK is a caspase-inhibitor, which can inhibit caspase-dependent apoptosis (25). Olaparib is a poly(ADP-ribose) polymerase 1 (PARP-1) inhibitor that can inhibit PARP-1-dependent apoptosis (26). Cell death was detected following treatment with DFOM (100 µM), Z-VAD-FMK (50 µM) and olaparib (10 µM) alone or with erastin (4 µM) for 12 h at 37°C. Treatment with erastin induced notable cell death. Treating cells with DFOM, Z-VAD-FMK and olaparib alone had no notable effect on cell death. When cells were treated with DFOM and erastin together, cell death was significantly decreased, compared with treated with erastin alone. However, when cells were treated with Z-VAD-FMK and erastin, cell death exhibited no significant difference, compared with treated with erastin alone. Additionally, olaparib had no significant effect on erastin-induced cell death (Fig. 4). The results indicated that DFOM partially rescued erastin-induced cell death, while Z-VAD-FMK and olaparib had no significant influence on it.

According to Yang et al (24), one of the mechanisms for erastin to induce cell death is that erastin inactivates GPX enzymes to trigger ferroptosis. GPX4 expression was detected in the radioresistant cells and GPX4 expression level was increased in A549-R and H460-R cells, compared with A549 and H460 cells, respectively (Fig. 5A). The radioresistant cells were treated with erastin and the expression of GPX4 was detected. The results demonstrated that GPX4 expression was suppressed by erastin in the A549-R and H460-R cells, compared with the control groups (Fig. 5B). Therefore, it was inferred that erastin increases radiosensitivity in NSCLC cells through inhibiting GPX4.

It was inferred that high levels of GPX4 may contribute to radioresistance in NSCLC cells. Therefore, siRNA was used to knockdown GPX4 expression in A549-R and H460-R cells. The radiosensitivity of the GPX4-knockdown cells (siGPX4) and control cells (siMock) was subsequently detected with a colony formation assay and the survival curves were calculated. In A549-R cells, siGPX4 cells exhibited decreased radioresistance, compared with siMock cells. In H460-R cells, similar results were achieved, demonstrating that knocking down the GPX4 expression resulted in decreased radioresistance (Fig. 6). The radiosensitivity parameters D₀, D_q, N and SF₂ are presented in Table III. Based on the results, it was concluded that knocking down the GPX4 expression resulted in decreased radioresistance.

Inhibiting GPX4 expression in A549-R and H460-R cells induced notable cell death. In A549-R cells, the cell death fraction was 56.03±3.63% in the siGPX4 group. In H460-R cells, the cell death fraction was 64.70±1.80% in the siGPX4 group. Treating siGPX4 group cells with DFOM decreased cell death fraction to 17.5±3.78% in A549-R cells. In H460-R cells, the cell death fraction was decreased to 16.00±3.05% following treatment with DFOM. However, treating cells with Z-VAD-FMK and olaparib did not significantly alter the cell death fraction (Fig. 7). Cell death fractions refer to the DMSO control. Collectively, these results indicated that the knockdown GPX4 decreases radioresistance in NSCLC cells by inducing ferroptosis.