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

Abstract

Cellular hypoxia, detectable in up to 80% of non-small cell lung carcinoma (NSCLC) tumors, is a known cause of radioresistance. High linear energy transfer (LET) particle radiation might be effective in the treatment of hypoxic solid tumors, including NSCLC. Cellular hypoxia can activate nuclear factor κB (NF-κB), which can modulate radioresistance by influencing cancer cell survival. The effect of high-LET radiation on NF-κB activation in hypoxic NSCLC cells is unclear. Therefore, we compared the effect of low (X-rays)- and high (¹²C)-LET radiation on NF-κB responsive genes' upregulation, as well as its target cytokines' synthesis in normoxic and hypoxic A549 NSCLC cells. The cells were incubated under normoxia (20% O₂) or hypoxia (1% O₂) for 48 h, followed by irradiation with 8 Gy X-rays or ¹²C ions, maintaining the oxygen conditions until fixation or lysis. Regulation of NF-κB responsive genes was evaluated by mRNA sequencing. Secretion of NF-κB target cytokines, IL-6 and IL-8, was quantified by ELISA. A greater fold change increase in expression of NF-κB target genes in A549 cells following exposure to ¹²C ions compared to X-rays was observed, regardless of oxygenation status. These genes regulate cell migration, cell cycle, and cell survival. A greater number of NF-κB target genes was activated under hypoxia, regardless of irradiation status. These genes regulate cell migration, survival, proliferation, and inflammation. X-ray exposure under hypoxia additionally upregulated NF-κB target genes modulating immunosurveillance and epithelial-mesenchymal transition (EMT). Increased IL-6 and IL-8 secretion under hypoxia confirmed NF-κB-mediated expression of pro-inflammatory genes. Therefore, radiotherapy, particularly with X-rays, may increase tumor invasiveness in surviving hypoxic A549 cells.

Keywords: 12C ions; A549; IL-6/IL-8 secretion; NF-κB; high-LET radiation; hypoxia-induced radioresistance; p65 (RelA) nuclear localization; tumor hypoxia.

Figure 1

Venn diagrams of differentially upregulated NF-κB target genes in A549 cells after exposure to X-rays or ¹²C ions; (a) effect of hypoxia in irradiated and unirradiated cells; (b) effect of irradiation in hypoxic and normoxic cells. * H0, A549 cells exposed to 0 Gy X-rays or ¹²C ions under hypoxia; N0, A549 cells exposed to 0 Gy X-rays or ¹²C ions under normoxia; H8, A549 cells exposed to 8 Gy X-rays or ¹²C ions under hypoxia; N8, A549 cells exposed to 8 Gy X-rays or ¹²C ions under normoxia. n = 4.

Figure 2

IL-6 and IL-8 content of cell culture supernatants of A549 cells after exposure to X-rays or carbon ions under normoxia and hypoxia; (a) IL-6; 6 h (b) IL-8; 6 h (c) IL-6; 24 h (d) IL-8; 24 h. Mean (filled circle) ± SE of cytokine secreted within 6 h or 24 h by one million cells are shown for n = 3–6. Significance was tested using two-way ANOVA and Sidak multiple comparison tests with ** p < 0.01, *** p < 0.001, **** p < 0.0001.