Hypoxia Modulates Radiosensitivity and Response to Different Radiation Qualities in A549 Non-Small Cell Lung Cancer (NSCLC) Cells

Abstract

Hypoxia-induced radioresistance reduces the efficacy of radiotherapy for solid malignancies, including non-small cell lung cancer (NSCLC). Cellular hypoxia can confer radioresistance through cellular and tumor micro-environment adaptations. Until recently, studies evaluating radioresistance secondary to hypoxia were designed to maintain cellular hypoxia only before and during irradiation, while any handling of post-irradiated cells was carried out in standard oxic conditions due to the unavailability of hypoxia workstations. This limited the possibility of simulating in vivo or clinical conditions in vitro. The presence of molecular oxygen is more important for the radiotoxicity of low-linear energy transfer (LET) radiation (e.g., X-rays) than that of high-LET carbon (12C) ions. The mechanisms responsible for 12C ions’ potential to overcome hypoxia-induced radioresistance are currently not fully understood. Therefore, the radioresistance of hypoxic A549 NSCLC cells following exposure to X-rays or 12C ions was investigated along with cell cycle progression and gene expression by maintaining hypoxia before, during and after irradiation. A549 cells were incubated under normoxia (20% O2) or hypoxia (1% O2) for 48 h and then irradiated with X-rays (200 kV) or 12C ions (35 MeV/n, LET ~75 keV/µm). Cell survival was evaluated using colony-forming ability (CFA) assays immediately or 24 h after irradiation (late plating). DNA double-strand breaks (DSBs) were analyzed using γH2AX immunofluorescence microscopy. Cell cycle progression was determined by flow cytometry of 4′,6-diamidino-2-phenylindole-stained cells. The global transcription profile post-irradiation was evaluated by RNA sequencing. When hypoxia was maintained before, during and after irradiation, hypoxia-induced radioresistance was observed only in late plating CFA experiments. The killing efficiency of 12C ions was much higher than that of X-rays. Cell survival under hypoxia was affected more strongly by the timepoint of plating in the case of X-rays compared to 12C ions. Cell cycle arrest following irradiation under hypoxia was less pronounced but more prolonged. DSB induction and resolution following irradiation were not significantly different under normoxia and hypoxia. Gene expression response to irradiation primarily comprised cell cycle regulation for both radiation qualities and oxygen conditions. Several PI3K target genes involved in cell migration and cell motility were differentially upregulated in hypoxic cells. Hypoxia-induced radioresistance may be linked to altered cell cycle response to irradiation and PI3K-mediated changes in cell motility and migration in A549 cells rather than less DNA damage or faster repair.

NF-κB in the Radiation Response of A549 Non-Small Cell Lung Cancer Cells to X-rays and Carbon Ions under Hypoxia

“…While cytokine-mediated inflammation is established as an important component of cellular responses to both irradiation and cellular hypoxia in normal and tumor cells [112][113][114][115][116], there was no direct comparison of NF-κB-induced cytokine secretion by cancer cells under normoxia and hypoxia following exposure to high-and low-LET radiation to the best of our knowledge up to now. In a holistic view of the tumor response to the different radiation qualities, the impact on survival has to be considered in addition to radio-induced inflammation, which is higher for carbon ions compared to X-rays, with relative biological effectiveness (RBE) above 2.3 in normoxic and hypoxic A549 cells, as we recently described [97]. Such a higher killing efficiency of carbon ions could counteract the tumor-supporting effects of the hypoxic milieu.…”

“…Recently, we correlated pro-EMT gene expression signature in hypoxic A549 cells following X-ray exposure in comparison to carbon ion irradiation with greater radioresistance to X-rays in this cell line [97]. To establish whether X-rays exposure may lead to greater cell survival in NSCLC cells than 12 C ions irradiation via EMT enhancement and even immune escape requires functional (e.g., migration) tests and animal experiments.…”

“…The remaining range of the ions in water was 2550 µm, indicating that the cells were exposed in the plateau region of the Bragg curve. The fluence for heavy ions (P/cm 2 ) was used to calculate radiation dose (Gy) [97].…”