Abstract
Objective: This study aimed to investigate the effect of ataxia telangiectasia mutated (ATM)–mediated autophagy on the radiosensitivity of lung cancer cells under low-dose radiation and to further investigate the role of ATM and its specific mechanism in the transition from hyper-radiosensitivity (HRS) to induced radioresistance (IRR).Methods: The changes in the HRS/IRR phenomenon in A549 and H460 cells were verified by colony formation assay. Changes to ATM phosphorylation and cell autophagy in A549 and H460 cells under different low doses of radiation were examined by western blot, polymerase chain reaction (PCR), and electron microscopy. ATM expression was knocked down by short interfering RNA (siRNA) transfection, and ATM-regulated molecules related to autophagy pathways were screened by transcriptome sequencing analysis. The detection results were verified by PCR and western blot. The differential metabolites were screened by transcriptome sequencing and verified by colony formation assay and western blot. The nude mouse xenograft model was used to verify the results of the cell experiments.Results: (1) A549 cells with high expression of ATM showed positive HRS/IRR, whereas H460 cells with low expression of ATM showed negative HRS/IRR. After the expression of ATM decreased, the HRS phenomenon in A549 cells increased, and the radiosensitivity of H460 cells also increased. This phenomenon was associated with the increase in the autophagy-related molecules phosphorylated c-Jun N-terminal kinase (p-JNK) and autophagy/Beclin 1 regulator 1 (AMBRA1). (2) DL-Norvaline, a product of carbon metabolism in cells, inhibited autophagy in A549 cells under low-dose radiation. DL-Norvaline increased the expression levels of ATM, JNK, and AMBRA1 in A549 cells. (3) Mouse experiments confirmed the regulatory role of ATM in autophagy and metabolism and its function in HRS/IRR.Conclusion: ATM may influence autophagy through p-JNK and AMBRA1 to participate in the regulation of the HRS/IRR phenomenon. Autophagy interacts with the cellular carbon metabolite DL-Norvaline to participate in regulating the low-dose radiosensitivity of cells.
Highlights
Radiotherapy is an important treatment method for lung cancer
Radioresistance was observed in A549 cells at 0.3–0.5 Gy, which is consistent with the HRS/induced radioresistance (IRR) phenomenon
The results showed that DL-Norvaline increased the autophagy level of A549 cells (Figures 6J,K), which further confirmed the interaction within Ataxia telangiectasia mutated (ATM), DL-Norvaline and autophagy
Summary
Radiotherapy is an important treatment method for lung cancer. Radiotherapy resistance and damage to surrounding normal tissue during radiotherapy limit its application in clinical practice. Due to the presence of the HRS/IRR phenomenon, at the same total dose, compared with conventionally fractionated radiotherapy, lowdose fractionated radiotherapy can significantly increase the radiosensitivity of tumor cells, increase the tumor control rate, and protect normal tissues (Valentini et al, 2010; Gupta et al, 2011; Dilworth et al, 2013; Schoenherr et al, 2013; Zhang et al, 2015). Słonina et al (2018) showed that in the fibroblasts of patients with HRS-positive tumors, the HRS phenomenon was closely related to the number of focal points of phosphorylated (p)-ATM. The above experimental results all suggest that ATM plays an important role in the transition from HRS to IRR under low-dose radiation
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