Nontoxic concentration of DNA-PK inhibitor NU7441 radio-sensitizes lung tumor cells with little effect on double strand break repair

Abstract

High-linear energy transfer (LET) heavy ions have been increasingly employed as a useful alternative to conventional photon radiotherapy. As recent studies suggested that high LET radiation mainly affects the nonhomologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair, we further investigated this concept by evaluating the combined effect of an NHEJ inhibitor (NU7441) at a non-toxic concentration and carbon ions. NU7441-treated non-small cell lung cancer (NSCLC) A549 and H1299 cells were irradiated with X-rays and carbon ions (290 MeV/n, 50 keV/μm). Cell survival was measured by clonogenic assay. DNA DSB repair, cell cycle distribution, DNA fragmentation and cellular senescence induction were studied using a flow cytometer. Senescence-associated protein p21 was detected by western blotting. In the present study, 0.3 μM of NU7441, nontoxic to both normal and tumor cells, caused a significant radio-sensitization in tumor cells exposed to X-rays and carbon ions. This concentration did not seem to cause inhibition of DNA DSB repair but induced a significant G2/M arrest, which was particularly emphasized in p53-null H1299 cells treated with NU7441 and carbon ions. In addition, the combined treatment induced more DNA fragmentation and a higher degree of senescence in H1299 cells than in A549 cells, indicating that DNA-PK inhibitor contributes to various modes of cell death in a p53-dependent manner. In summary, NSCLC cells irradiated with carbon ions were radio-sensitized by a low concentration of DNA-PK inhibitor NU7441 through a strong G2/M cell cycle arrest. Our findings may contribute to further effective radiotherapy using heavy ions.

Keywords: DNAPK protein; G2 cell cycle arrest; heavy ions; non-small cell lung cancer; p53 genes.

Figure 1

Both X‐rays and carbon ions induce radio‐sensitization in non‐small cell lung cancer (NSCLC) cells with nontoxic conce ntrations of NU7441 treatment. The cellular toxicities of NU7441 in HFL1 cells (a) and NSCLC cells (b) were determined by clonogenic survival assay. The survival fraction in A549 (c) and H1299 (d) cells after indicated treatments were determined by clonogenic survival assay. Colonies containing more than 50 cells were scored. Data represent mean ± SD from three independent experiments.

Figure 2

Low concentration of NU7441 does not seem to show double strand break (DSB) repair inhibition in X‐ray‐irradiated and carbon‐irradiated non‐small cell lung cancer (NSCLC) cells. The quantification of DSB was analyzed by γ‐H2AX signal using flow cytometry 1, 4 and 24 h after 4 Gy of irradiation. The fold change of the γ‐H2AX level at the indicated time after irradiation was calculated with respect to control (without irradiation and NU7441). DSB repair kinetics with 0.3 and 3 μM NU7441 were analyzed in A549 cells irradiated with X‐rays (a) and carbon ions (c), and in H1299 cells irradiated with X‐rays (b) and carbon ions (d). Data represent mean ± SD from at least two independent experiments.

Figure 3

Non‐small cell lung cancer (NSCLC) cells treated with NU7441 (0.3 μM) and radiation show significant G2/M arrest. Cell cycle distribution after indicated treatments was analyzed 24 h after 4 Gy of irradiation using flow cytometry. The figures show the representative histogram and the stacked chart of cell cycle distribution in A549 cells (a, c) and in H1299 cells (b, d). Data represent mean ± SD from at least three independent experiments.

Figure 4

NU7441 (0.3 μM) causes a remarkable increase of DNA fragmentation in irradiated H1299 cells. DNA fragmentation after indicated treatments was determined by analyzing the sub‐G1 population in A549 cells (a) and H1299 cells (b) 1, 2 and 3 days after 4 Gy of irradiation using flow cytometry. Data represent mean ± SD from at least three independent experiments.

Figure 5

NU7441 (0.3 μM) enhances cellular senescence in irradiated H1299 cells. Cellular senescence after indicated treatments was determined by analyzing SA‐βGal using flow cytometry in A549 cells (a) and H1299 cells (b) and p21 expression in both cells (c) using western blotting 3 days after 4 Gy of irradiation. Data represent mean ± SD from at least three independent experiments.