Erastin decreases radioresistance of NSCLC cells partially by inducing GPX4-mediated ferroptosis

Abstract

The aim of the present study was to examine whether erastin influences radioresistance in non-small cell lung cancer (NSCLC) cells and produce a preliminary investigation into its mechanism of action. The radioresistant subtype of NSCLC cells, A549-R and H460-R, were induced by high-dose hypofractionated irradiation. Erastin was used to treat the radioresistant cells and radiosensitivity was examined by colony formation assays. Cell death was determined after the cells were treated with erastin, irradiation (IR) or erastin together with IR. The expression of glutathione peroxidase 4 (GPX4) expression in the parental cells and radioresistance cells was detected by western blotting. GPX4 expression in the radioresistance cells was subsequently inhibited, radiosensitivity and cell death was measured, and erastin enhanced radiosensitivity in A549-R and H460-R cells. Erastin and IR exhibited a combined effect on killing cells, as co-treatment with erastin and IR demonstrated a higher effect on killing cells compared with erastin or IR alone. GPX4 expression was inhibited by erastin in the radioresistant cells. Inhibiting GPX4 expression also radiosensitized NSCLC cells to radiation in the radioresistant cell lines. Erastin-induced and GPX4-inhibition-induced cell death could partially be rescued by deferoxamine, but not Z-VAD-FMK and olaparib, which indicated that erastin and GPX4-inhibition induced ferroptosis in the radioresistant cells. Erastin decreased radioresistance of NSCLC cells partially by inducing GPX4-mediated ferroptosis.

Keywords: erastin; ferroptosis; glutathione peroxidase 4; non-small cell lung cancer; radioresistance.

Figure 1.

Clone formation assay determining 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. The shoulder area under the survival curve was increased in H460-R and A549-R cells. (A) Radiosensitivity of A549 and A549-R cells. (B) Radiosensitivity of H460 and H460-R cells. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001 A549-R vs. A549 or H460-R vs. H460. R, radioresistant; IR, irradiation.

Figure 2.

Colony formation assay investigating the effect of erastin on radiosensitivity of A549-R and H460-R. The results demonstrated that groups treated with erastin had reduced survival fractions, compared with the control groups. The shoulder area under the survival curves was reduced in erastin groups. (A) Radiosensitivity of A549-R cells. (B) Radiosensitivity of H460-R cells. Data are presented as the mean ± standard deviation. ***P<0.001 vs. control. R, radioresistant; IR, irradiation.

Figure 3.

Cell death measurements following the administration of different treatments. The cell death fraction in the erastin + IR group was significantly increased, compared with the erastin or IR groups. (A) Cell death fraction in A549-R cells. (B) Cell death fraction in H460-R cells. Data are presented as the mean ± standard deviation. ***P<0.001. R, radioresistant; IR, irradiation; DMSO, dimethyl sulfoxide.

Figure 4.

Treatment with erastin induces notable cell death. Treating cells with DFOM, Z-VAD-FMK and olaparib alone exhibited no significant effect on cell death. When cells were treated with DFOM and erastin together, cell death was decreased, compared with treatment with erastin alone. However, when cells were treated with Z-VAD-FMK and erastin, cell death exhibited no significance difference to the group treated with erastin alone. Additionally, olaparib had no significant effect on erastin-induced cell death. (A) Cell death fraction in A549-R cells. (B) Cell death fraction in H460-R cells. Data are presented as the mean ± standard deviation. ***P<0.001. R, radioresistant; DFOM, deferoxamine.

Figure 5.

GPX4 expression detection in the radioresistant cells. GPX4 protein expression level was increased in A549-R and H460-R cells. Following treatment with erastin, GPX4 expression was markedly decreased. (A) GPX4 expression in the parental and radioresistant cells. (B) GPX4 expression in the control group and erastin group cells. R, radioresistant; GPX4, glutathione peroxidase 4.

Figure 6.

siRNA was used to knockdown GPX4 expression in radioresistant cells, and the radiosensitivity of the GPX4-knockdown cells (siGPX4) and control cells (siMock) was determined with a colony formation assay. siGPX4 cells exhibited decreased radioresistance, compared with siMock cells. (A) GPX4 expression in A549-R and H460-R cells. (B) Radiosensitivity of A549-R cells. (C) Radiosensitivity of H460-R cells. Data are presented as the mean ± standard deviation. **P<0.01 and ***P<0.001. siRNA, small interfering RNA; GPX4, glutathione peroxidase 4; R, radioresistant; IR, irradiation.

Figure 7.

siRNA inhibition of GPX4 expression and cell death examination. Inhibiting GPX4 expression induced significant cell death. Treating siGPX4 group cells with DFOM significantly decreased cell death. However, treating cells with Z-VAD-FMK and olaparib did not significantly change the cell death fraction. (A) Cell death fraction in A549-R cells. (B) Cell death fraction in H460-R cells. Data are presented as the mean ± standard deviation. ***P<0.001. siRNA, small interfering RNA; GPX4, glutathione peroxidase 4; R, radioresistant; DFOM, deferoxamine; DMSO, dimethyl sulfoxide.