Distinct biological effects of low-dose radiation on normal and cancerous human lung cells are mediated by ATM signaling

Abstract

Low-dose radiation (LDR) induces hormesis and adaptive response in normal cells but not in cancer cells, suggesting its potential protection of normal tissue against damage induced by conventional radiotherapy. However, the underlying mechanisms are not well established. We addressed this in the present study by examining the role of the ataxia telangiectasia mutated (ATM) signaling pathway in response to LDR using A549 human lung adenocarcinoma cells and HBE135-E6E7 (HBE) normal lung epithelial cells. We found that LDR-activated ATM was the initiating event in hormesis and adaptive response to LDR in HBE cells. ATM activation increased the expression of CDK4/CDK6/cyclin D1 by activating the AKT/glycogen synthase kinase (GSK)-3β signaling pathway, which stimulated HBE cell proliferation. Activation of ATM/AKT/GSK-3β signaling also increased nuclear accumulation of nuclear factor erythroid 2-related factor 2, leading to increased expression of antioxidants, which mitigated cellular damage from excessive reactive oxygen species production induced by high-dose radiation. However, these effects were not observed in A549 cells. Thus, the failure to activate these pathways in A549 cells likely explains the difference between normal and cancer cells in terms of hormesis and adaptive response to LDR.

Keywords: ATM; biological effects; low-dose radiation; lung cancer cells; normal lung cells.

Figure 1. LDR stimulates cell proliferation and cell cycle progression of HBE cells but not A549 cells

HBE (5 × 10³) and A549 (3 × 10³) cells were seeded in 96-well plates and irradiated with 20, 50, 75,100, 200, 1000, and 3000 mGy of X-ray. After irradiation, cells were transferred to an incubator and cultured for another 24 h. (A) Cell viability at indicated doses was determined with the WST-1 assay. (B) Cell viability was assessed at different times after exposure to 75 mGy X-ray with the WST-1 assay. (C) Representative images of colony formation of cells after exposure to 75 mGy X-ray. (D) Statistical analysis of colonies numbers in HBE and A549 cells 10 days after treated. (E) Cell cycle distribution after exposure to 75 mGy X-ray was analyzed 24 h post-LDR by flow cytometry. (F) Quantitative analysis of cells in each phase of the cell cycle. Data are presented as mean ± standard deviation of three separate experiments, with six replicates in each experiment. *P < 0.05 vs. control; **P < 0.01 vs.control.

Figure 2. LDR induces ATM and AKT phosphorylation in HBE cells but not in A549 cells

ATM inhibition blocked LDR-induced phosphorylation of ATM and AKT, and proliferation of HBE cells but not A549 cells. Cells were treated with 75 mGy X-ray, then transferred to an incubator and cultured for 24 h. For ATM inhibition, cells were pretreated with 5 mM caffeine or 25 nM ATM siRNA and then irradiated with 75mGy X-ray, then cultured for an additional 24 h before analysis. (A, B, C) ATM and AKT phosphorylation was assessed by western blotting and quantitative analysis in both HBE and A549 cell lines after LDR with or without pretreated with caffeine. (D, E, F) ATM and AKT phosphorylation were assessed by western blotting and quantitative analysis in both HBE and A549 cell lines after LDR with or without pretreated with ATM siRNA. (G) Cell viability was assessed with the WST-1 assay in both HBE and A549 cell lines after LDR with or without pretreated with caffeine. (H) Cell viability was assessed with the WST-1 assay in both HBE and A549 cell lines after LDR with or without pretreated with ATM siRNA. Data are presented as mean ± standard deviation of three separate experiments, with at least two of each sample per experiment. *P < 0.05.

Figure 3. LDR increases GSK-3β phosphorylation and CDK4/CDK6/cyclin D1 expression in HBE cells but not in A549 cells

For AKT inhibition, cells were pretreated with 40 μM LY294002 for 2 h, then irradiated at 75 mGy and cultured for an additional 24 h before analysis. (A–F) GSK-3β phosphorylation and CDK4/CDK6/cyclin D1 level were determined 24 h after irradiation with 75 mGy X-ray by western blotting and quantitative analysis in HBE and A549 cells with or without pretreated with LY294002. Data are presented as mean ± standard deviation of three separate experiments, with at least two of each sample per experiment. *P < 0.05.

Figure 4. LDR increases transcript levels of Nrf2-dependent antioxidants via ATM-mediated signaling in HBE cells but not in A549 cells

Cells were treated with 75 mGy X-ray, then transferred to an incubator and cultured for 24 h. For ATM inhibition, cells were pretreated with caffeine or ATM siRNA. (A–D) mRNA levels of NQO1 and HO-1 were assessed by real-time qPCR and normalized to that of β-actin, respectively. Data are presented as mean ± standard deviation of three separate experiments, with at least two of each sample per experiment. *P < 0.05.

Figure 5. ATM inhibition with caffeine abolished LDR-induced phosphorylation of AKT and GSK-3β, nuclear accumulation of Nrf2, and expression of antioxidants in HBE cells but not in A549 cells

Cells were pretreated with 5 mM caffeine for 2 h, then irradiated with 75 mGy X-ray and cultured for 24 h before analysis. (A–G) ATM, AKT, and GSK-3β phosphorylation, nuclear Nrf2, NQO1, and HO-1 level were determined by western blotting and quantitative analysis in HBE and A549 cells, respectively. Data are presented as mean ± standard deviation of three separate experiments, with at least two of each sample per experiment. *P < 0.05.

Figure 6. Knockdown of ATM with siRNA blocks LDR-induced phosphorylation of AKT and GSK-3β, nuclear accumulation of Nrf2, and expression of antioxidants in HBE cells but not in A549 cells

For knockdown of ATM, cells were pretreated with 25 nM ATM siRNA. Twenty-four hours after transfection, cells were irradiated with 75 mGy X-ray and cultured for 24 h before analysis. (A–G) ATM, AKT, and GSK-3β phosphorylation nuclear Nrf2, NQO1 and HO-1 level were determined by western blotting and quantitative analysis in HBE and A549 cells, respectively. Data are presented as mean ± standard deviation of three separate experiments, with at least two of each sample per experiment. *P < 0.05.

Figure 7. Knockdown of ATM with siRNA attenuated the protective effect of LDR in HBE cells but not in A549 cells

Cells were pretreated with 25 nM ATM siRNA. Twenty-four hours after transfection, cells were exposure to LDR (75 mGy X-ray), cultured for another 24 h, and then treated by HDR (5 Gy X-ray) before analysis. Statistical analysis of the data from WST-1 assay showed that cellular viabilities of both HBE and A549 cells decreased drastically at 24 h after these cells were treated with HDR. Pretreatment with LDR protected HBE cells but not A549 cells against decrease in cell viability induced by HDR. However, knockdown of ATM attenuated significantly the protective effect of LDR against the cell viability decrease induced by HDR in HBE cells. Data are presented as mean ± standard deviation of three separate experiments, with at least two of each sample per experiment. *P < 0.05.

Figure 8. LDR pretreatment protects HBE cells but not A549 cells against increases in intracellular ROS induced by HDR

Cells were divided into four groups: control (sham-irradiated), D1 (75 mGy), D2 (5 Gy), and D1 + D2 (75 mGy + 5 Gy). The interval between D1 and D2 was 24 h. Cells were analyzed after an additional 24 h of culture. (A) Intracellular ROS levels were analyzed with the oxidation-sensitive DCFH-DA probe and detected by flow cytometry. (B) Flow cytometry in combination with JC-1 staining showed that the mitochondrial decline caused by HDR in D2 group of HBE cells was significantly inhibited by pretreatment with LDR in D1 + D2 group. (C) The apoptosis rate of the four groups by flow cytometry showed that the increased apoptosis rate caused by HDR in D2 group of HBE cells was obviously attenuated by LDR pretreatment in D1 + D2 group. Data are presented as mean ± standard deviation of three separate experiments, with at least two of each sample per experiment. *P < 0.05.

Figure 9. Model of LDR-induced hormesis and adaptive response in normal cells but not in cancer cells

DSB, double-strand break; p, phosphorylated; ROS, reactive oxygen species; SSB, single-strand break.