Bikash Konda

Hypoxia-induced radioresistance in non-small cell lung cancer (NSCLC) hinders radiotherapy efficacy. Fractionated schedules exploit reoxygenation between fractions to reverse this resistance, but the effects of post-irradiation reoxygenation remain unclear and may depend on radiation quality. We investigated survival, cell cycle progression, cytokine secretion, and gene expression in hypoxic (1 % O₂) and reoxygenated A549 cells irradiated with X-rays or carbon ions. Colony-forming assays revealed an Oxygen Enhancement Ratio (OER) > 1 for both hypoxic and reoxygenated cells after X-rays, indicating persistent radioresistance; carbon ion OER ≈ 1 reflected oxygen-independent cytotoxicity. Hypoxia weakened radiation-induced G₂ arrest, and this was unaffected by reoxygenation. IL-6 secretion increased after X-rays and IL-8 after carbon ions exposure; both were enhanced under hypoxia and reoxygenation. RNA sequencing revealed that hypoxia induced a pro-survival, epithelial-to-mesenchymal transition (EMT)-promoting, and immune-evasive transcriptional program, which was largely reversed by reoxygenation but without increased clonogenic killing. These findings indicate that short-term reoxygenation after irradiation can normalize hypoxia-driven transcriptional changes yet does not restore radiosensitivity, supporting the advantage of high-linear energy transfer (LET) carbon ions for targeting resistant hypoxic NSCLC cells.

Hypoxia-induced radioresistance limits therapeutic success in cancer. In addition, p53 mutations are widespread in tumors including non-small cell lung carcinomas (NSCLCs), and they might modify the radiation response of hypoxic tumor cells. We therefore analyzed the DNA damage and inflammatory response in chronically hypoxic (1% O₂, 48 h) p53 null H358 NSCLC cells after X-ray exposure. We used the colony-forming ability assay to determine cell survival, γH2AX immunofluorescence microscopy to quantify DNA double-strand breaks (DSBs), flow cytometry of DAPI-stained cells to measure cell cycle distribution, ELISAs to quantify IL-6 and IL-8 secretion in cell culture supernatants, and RNA sequencing to determine gene expression. Chronic hypoxia increased the colony-forming ability and radioresistance of H358 cells. It did not affect the formation or resolution of X-ray-induced DSBs. It reduced the fraction of cells undergoing G2 arrest after X-ray exposure and delayed the onset of G2 arrest. Hypoxia led to an earlier enhancement in cytokines secretion rate after X-irradiation compared to normoxic controls. Gene expression changes were most pronounced after the combined exposure to hypoxia and X-rays and pertained to senescence and different cell death pathways. In conclusion, hypoxia-induced radioresistance is present despite the absence of functional p53. This resistance is related to differences in clonogenicity, cell cycle regulation, cytokine secretion, and gene expression under chronic hypoxia, but not to differences in DNA DSB repair kinetics.

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.

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.

Hypoxia occurs in 80% of non-small cell lung carcinoma (NSCLC) cases, leading to treatment resistance. Hypoxia’s effects on NSCLC energetics are not well-characterized. We evaluated changes in glucose uptake and lactate production in two NSCLC cell lines under hypoxia in conjunction with growth rate and cell cycle phase distribution. The cell lines A549 (p53 wt) and H358 (p53 null) were incubated under hypoxia (0.1% and 1% O₂) or normoxia (20% O₂). Glucose and lactate concentrations in supernatants were measured using luminescence assays. Growth kinetics were followed over seven days. Cell nuclei were stained with DAPI and nuclear DNA content was determined by flow cytometry to determine cell cycle phase. Gene expression under hypoxia was determined by RNA sequencing. Glucose uptake and lactate production under hypoxia were greater than under normoxia. They were also significantly greater in A549 compared to H358 cells. Faster energy metabolism in A549 cells was associated with a higher growth rate in comparison to H358 cells under both normoxia and hypoxia. In both cell lines, hypoxia significantly slowed down the growth rate compared to proliferation under normoxic conditions. Hypoxia led to redistribution of cells in the different cycle phases: cells in G1 increased and the G2 population decreased. Glucose uptake and lactate production increase under hypoxia in NSCLC cells indicated greater shunting of glucose into glycolysis rather than into oxidative phosphorylation compared to normoxia, making adenosine triphosphate (ATP) production less efficient. This may explain the redistribution of hypoxic cells in the G1 cell cycle phase and the time increase for cell doubling. Energy metabolism changes were more prominent in faster-growing A549 cells compared to slower-growing H358 cells, indicating possible roles for the p53 status and inherent growth rate of different cancer cells. In both cell lines, genes associated with cell motility, locomotion and migration were upregulated under chronic hypoxia, indicating a strong stimulus to escape hypoxic conditions.

Nuclear factor κB (NF-κB) activation might be central to heavy ion-induced detrimental processes such as cancer promotion and progression and sustained inflammatory responses. A sensitive detection system is crucial to better understand its involvement in these processes. Therefore, a DD-tdTomato fluorescent protein-based reporter system was previously constructed with human embryonic kidney (HEK) cells expressing DD-tdTomato as a reporter under the control of a promoter containing NF-κB binding sites (HEK-pNFκB-DD-tdTomato-C8). Using this reporter cell line, NF-κB activation after exposure to different energetic heavy ions (¹⁶O, 95 MeV/n, linear energy transfer—LET 51 keV/µm; ¹²C, 95 MeV/n, LET 73 keV/μm; ³⁶Ar, 95 MeV/n, LET 272 keV/µm) was quantified considering the dose and number of heavy ions hits per cell nucleus that double NF-κB-dependent DD-tdTomato expression. Approximately 44 hits of ¹⁶O ions and ≈45 hits of ¹²C ions per cell nucleus were required to double the NF-κB-dependent DD-tdTomato expression, whereas only ≈3 hits of ³⁶Ar ions were sufficient. In the presence of Shield-1, a synthetic molecule that stabilizes DD-tdTomato, even a single particle hit of ³⁶Ar ions doubled NF-κB-dependent DD-tdTomato expression. In conclusion, stabilization of the reporter protein can increase the sensitivity for NF-κB activation detection by a factor of three, allowing the detection of single particle hits’ effects.

Astronauts are exposed to considerable doses of space radiation during long-term space missions. As complete shielding of the highly energetic particles is impracticable, the cellular response to space-relevant radiation qualities has to be understood in order to develop countermeasures and to reduce radiation risk uncertainties. The transcription factor Nuclear Factor κB (NF-κB) plays a fundamental role in the immune response and in the pathogenesis of many diseases. We have previously shown that heavy ions with a linear energy transfer (LET) of 100–300 keV/µm have a nine times higher potential to activate NF-κB compared to low-LET X-rays. Here, chemical inhibitor studies using human embryonic kidney cells (HEK) showed that the DNA damage sensor Ataxia telangiectasia mutated (ATM) and the proteasome were essential for NF-κB activation in response to X-rays and heavy ions. NF-κB’s role in cellular radiation response was determined by stable knock-down of the NF-κB subunit RelA. Transfection of a RelA short-hairpin RNA plasmid resulted in higher sensitivity towards X-rays, but not towards heavy ions. Reverse Transcriptase real-time quantitative PCR (RT-qPCR) showed that after exposure to X-rays and heavy ions, NF-κB predominantly upregulates genes involved in intercellular communication processes. This process is strictly NF-κB dependent as the response is completely absent in RelA knock-down cells. NF-κB’s role in the cellular radiation response depends on the radiation quality.