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

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 Modulates Radiosensitivity and Response to Different Radiation Qualities in A549 Non-Small Cell Lung Cancer (NSCLC) 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 Changes Energy Metabolism and Growth Rate in Non-Small Cell Lung Cancer Cells.

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.

Unraveling astrocyte behavior in the space brain: Radiation response of primary astrocytes.

Introduction: Exposure to space conditions during crewed long-term exploration missions can cause several health risks for astronauts. Space radiation, isolation and microgravity are major limiting factors. The role of astrocytes in cognitive disturbances by space radiation is unknown. Astrocytes' response toward low linear energy transfer (LET) X-rays and high-LET carbon (12C) and iron (56Fe) ions was compared to reveal possible effects of space-relevant high-LET radiation. Since astronauts are exposed to ionizing radiation and microgravity during space missions, the effect of simulated microgravity on DNA damage induction and repair was investigated. Methods: Primary murine cortical astrocytes were irradiated with different doses of X-rays, 12C and 56Fe ions at the heavy ion accelerator GSI. DNA damage and repair (γH2AX, 53BP1), cell proliferation (Ki-67), astrocytes' reactivity (GFAP) and NF-κB pathway activation (p65) were analyzed by immunofluorescence microscopy. Cell cycle progression was investigated by flow cytometry of DNA content. Gene expression changes after exposure to X- rays were investigated by mRNA-sequencing. RT-qPCR for several genes of interest was performed with RNA from X-rays- and heavy-ion-irradiated astrocytes: Cdkn1a, Cdkn2a, Gfap, Tnf, Il1ß, Il6, and Tgfß1. Levels of the pro inflammatory cytokine IL-6 were determined using ELISA. DNA damage response was investigated after exposure to X-rays followed by incubation on a 2D clinostat to simulate the conditions of microgravity. Results: Astrocytes showed distinct responses toward the three different radiation qualities. Induction of radiation-induced DNA double strand breaks (DSBs) and the respective repair was dose-, LET- and time-dependent. Simulated microgravity had no significant influence on DNA DSB repair. Proliferation and cell cycle progression was not affected by radiation qualities examined in this study. Astrocytes expressed IL-6 and GFAP with constitutive NF-κB activity independent of radiation exposure. mRNA sequencing of X-irradiated astrocytes revealed downregulation of 66 genes involved in DNA damage response and repair, mitosis, proliferation and cell cycle regulation. Discussion: In conclusion, primary murine astrocytes are DNA repair proficient irrespective of radiation quality. Only minor gene expression changes were observed after X-ray exposure and reactivity was not induced. Co-culture of astrocytes with microglial cells, brain organoids or organotypic brain slice culture experiments might reveal whether astrocytes show a more pronounced radiation response in more complex network architectures in the presence of other neuronal cell types.

Diacylglycerol lipase alpha in astrocytes is involved in maternal care and affective behaviors.

Genetic deletion of cannabinoid CB1 receptors or diacylglycerol lipase alpha (DAGLa), the main enzyme involved in the synthesis of the endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG), produced profound phenotypes in animal models of depression-related behaviors. Furthermore, clinical studies have shown that antagonists of CB1 can increase the incidence and severity of major depressive episodes. However, the underlying pathomechanisms are largely unknown. In this study, we have focused on the possible involvement of astrocytes. Using the highly sensitive RNAscope technology, we show for the first time that a subpopulation of astrocytes in the adult mouse brain expresses Dagla, albeit at low levels. Targeted lipidomics revealed that astrocytic DAGLa only accounts for a minor percentage of the steady-state brain 2-AG levels and other arachidonic acid derived lipids like prostaglandins. Nevertheless, the deletion of Dagla in adult mouse astrocytes had profound behavioral consequences with significantly increased depressive-like behavioral responses and striking effects on maternal behavior, corresponding with increased levels of serum progesterone and estradiol. Our findings therefore indicate that lipids from the DAGLa metabolic axis in astrocytes play a key regulatory role in affective behaviors.