NSCLC cells demonstrate differential mode of cell death in response to the combined treatment of radiation and a DNA-PKcs inhibitor

Abstract

The current standard of care for lung cancer consists of concurrent chemotherapy and radiation. Several studies have shown that the DNA-PKcs inhibitor NU7441 is a highly potent radiosensitizer, however, the mechanism of NU7441's anti-proliferation effect has not been fully elucidated. In this study, the combined effect of NU7441 and ionizing radiation (IR) in a panel of non-small cell lung cancer cell lines (A549, H460 and H1299) has been investigated. We found that NU7441 significantly enhances the effect of IR in all cell lines. The notable findings in response to this combined treatment are (i) prolonged delay in IR-induced DNA DSB repair, (ii) induced robust G2/M checkpoint, (iii) increased aberrant mitosis followed by mitotic catastrophe specifically in H1299, (iv) dramatically induced autophagy in A549 and (v) IR-induced senescence specifically in H460. H1299 cells show greater G2 checkpoint adaptation after combined treatment, which can be attributed to higher expression level of Plk1 compared to A549 and H460. The enhanced autophagy after NU7441 treatment in A549 is possibly due to the higher endogenous expression of pS6K compared to H1299 and H460 cells. In conclusion, choice of cell death pathway is dependent on the mutation status and other genetic factors of the cells treated.

Figure 1. NU7441 sensitizes NSCLC cells to irradiation

(A–C) H460, A549 and H1299 cells were irradiated (10 Gy, 1 hour) with or without pretreatment with NU7441 (10 μM), autophosphorylation of DNA-PKcs was determined. (D–E) Clonogenic survival of NSCLC cells with or without NU7441 H460, A549, and H1299 cells were treated with NU7441 (1 and 2 μm, respectively) for 1 hour and treated with IR as indicated. Cells were trypsinized immediately and counted and colony formation was performed. (G–I) NU7441 in combination with IR led to significant tumor growth delay in H460, A549 and H1299 cells.

Figure 2. NU7441 enhanced cells sensitivity to IR correlates with deficient DSB repair

NSCLC cells were irradiated with 2 Gy with or without NU7441 (1 hour prior to IR) and samples were collected at the indicated time points after IR, immunostained for phospo-γH2AX (red) foci and counted (average, 50 nuclei). (A, C and E) The representative image of H460, A549 and H1299 cells. (B, D and F) Quantitative analysis of DNA repair kinetics in NSCLC cells.

Figure 3. NU7441 treatment results in a robust G2/M cell arrest in NSCLC cells and specifically leads to checkpoint adaptation in H1299 cells

(A–C) NSCLC cells were treated with IR (2 Gy), NU7441 (2 μM), and IR + NU7441 as indicated. Samples were collected at 0, 2, 6, and 24 hours post-treatment. Propidium iodide (PI) staining was used to detect the distribution of cells after various treatments. (D–F) The mitotic index was measured by flow cytometric analysis using PI staining for DNA content and anti–phospho-Histone H3 antibodies. Cells were irradiated at 2 Gy and samples were collected 24 h postradiation. Inset, the % of mitosis cells.

Figure 4. NU7441 + IR-induces mitotic catastrophe in H1299 cells

(A) H1299 cells were stained with anti-α-tubulin and anti-crest antibodies, the representative images show the normal and aberrant mitosis in H1299 cells. (B) The percentage of aberrant mitotic cells is determined by the morphology of the spindle formation 24 h after NU7441 and IR treatment. As shown in the figure the number of aberrant mitosis increased after NU7441 and IR treatment. (C) The phosphorylation of ATM and Chk2 and the expression of Plk1 were determined by Western blot analysis at the indicated time points.

Figure 5. Mitotic catastrophe-related apoptosis was dramatically increased in NU7441 + IR treatment in H1299 cells

(A) IR + NU7441-induced apoptosis was determined by cleaved-caspase 3 staining. Cells were treated with +/− NU7441 (5 μM) and IR (5 Gy) for 24 hours and the cells were stained with cleaved-caspase 3 and DAPI; the representative fluorescence images are shown. White arrows indicated the cleaved-caspase 3 positive apoptotic cells. (B) Quantitative analysis of apoptotic cells. The data are presented as the means ± SD of three independent experiments. (C) Analysis of PARP cleavage. Cells were lysed 24 hours after exposure to IR or IR + NU7441 and subjected to Western blot analysis.

Figure 6. NU7441 + IR-induces autophagic cell death in NSCLC cells

H460, A549 and H1299 cells were treated with NU744, IR, and IR + NU7441 for 72 hours and then stained with AO. (A) Fluorescent images of AVO-positive cells. (B) Flow cytometry analysis to assess autophagy. (C) Phosphorylation of mTOR and p70S6K were detected by Western blot analysis 24 hours after treatment.

Figure 7. NU7441 enhanced IR-induced senescence in NSCLC cells

H460, A549 and H1299 cells were treated with NU744, IR, and IR + NU7441 for 48 hours and then stained with X-Gal. (A) Representative image of SA-βGal activity after 48 hours treatment. (B) Quantitative analysis of senescent cells. The data are presented as the means ± SD of three independent experiments. (C) Cells were lysed 48 hours after exposure to IR or IR + NU7441 and subjected to Western blot analysis, the expression of p53 and p21 were determined.