Involvement of ERK-Nrf-2 Signaling in Ionizing Radiation Induced Cell Death in Normal and Tumor Cells

<div><p>Prolonged oxidative stress favors tumorigenic environment and inflammation. Oxidative stress may trigger redox adaptation mechanism(s) in tumor cells but not normal cells. This may increase levels of intracellular antioxidants and establish a new redox homeostasis. Nrf-2, a master regulator of battery of antioxidant genes is constitutively activated in many tumor cells. Here we show that, murine T cell lymphoma EL-4 cells show constitutive and inducible radioresistance via activation of Nrf-2/ERK pathway. EL-4 cells contained lower levels of ROS than their normal counterpart murine splenic lymphocytes. In response to radiation, the thiol redox circuits, GSH and thioredoxin were modified in EL-4 cells. Pharmacological inhibitors of ERK and Nrf-2 significantly enhanced radiosensitivity and reduced clonogenic potential of EL-4 cells. Unirradiated lymphoma cells showed nuclear accumulation of Nrf-2, upregulation of its dependent genes and protein levels. Interestingly, MEK inhibitor abrogated its nuclear translocation suggesting role of ERK in basal and radiation induced Nrf-2 activation in tumor cells. Double knockdown of ERK and Nrf-2 resulted in higher sensitivity to radiation induced cell death as compared to individual knockdown cells. Importantly, NF-kB which is reported to be constitutively active in many tumors was not present at basal levels in EL-4 cells and its inhibition did not influence radiosensitivity of EL-4 cells. Thus our results reveal that, tumor cells which are subjected to heightened oxidative stress employ master regulator cellular redox homeostasis Nrf-2 for prevention of radiation induced cell death. Our study reveals the molecular basis of tumor radioresistance and highlights role of Nrf-2 and ERK.</p></div

EL-4 cells were more resistant to ionizing radiation induced cell death as compared to normal murine lymphocytes.

<p>(A) Flow cytometric profile of propidium iodide stained normal murine splenic lymphocytes cultured for 24 h post 4 Gy irradiation. Pre-G1 peak (gate RN1) represents apoptotic population. (B) Bar graph represents percent apoptotic cells. Data points represent mean±S.E.M. from nine replicates from three independent experiments. *p<0.01, as compared to irradiated lymphocytes. (C) ROS levels in normal splenic lymphocytes and EL-4 cells were estimated by staining with DCF-DA (20 µM), DHE or DHR123 (5 µM each) 30 min at 37°C followed by fluorescence emission measured at their respective wavelength. Bar graph shows relative fluorescence units indicating ROS levels, mean±S.E.M. from nine replicates from three independent experiments. *p<0.01, as compared to normal lymphocytes. (D&E) One million splenic lymphocytes stimulated with anti-CD3/anti-CD28 antibodies for 72 h in 24-well plate or EL-4 cells were exposed to ionizing radiation (4 Gy) and cultured for 24 h. Cells were stained with propidium iodide and acquired on flowcytometer. Data points represent mean±S.E.M. from nine replicates from three independent experiments. *p<0.01, as compared to activated and irradiated lymphocytes.</p

Murine T cell lymphoma showed active redox circuits as compared to normal lymphocytes.

<p>(A) Intracellular GSH levels were measured by conventional enzyme cycling method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065929#pone.0065929-Rahman1" target="_blank">[41]</a> at 2, 6, 12 or 24 h post irradiation (4 Gy) in normal lymphocytes and EL-4 lymphoma cells. Bar graph represents GSH/GSSG ratio in normal murine splenic lymphocytes and EL-4 cells. Data points represent mean±S.E.M. from nine replicates from three independent experiments. *p<0.05 as compared to untreated lymphoma cells (B) Thioredoxin activity in wild type EL-4 cells at 2, 6, 12 & 24 h post irradiation (4 Gy) was measured. Bar diagram shows thioredoxin activity in EL-4 cells. Each bar shows mean±S.E.M from nine replicates from three independent experiments. *p<0.01 as compared to untreated lymphoma cells.</p

Schematic representation showing the involvement of ERK-Nrf-2 signaling pathway in radioresistance of EL-4 cells.

<p>Schematic representation showing the involvement of ERK-Nrf-2 signaling pathway in radioresistance of EL-4 cells.</p

Inhibitors of ERK or Nrf-2 significantly enhanced radiosensitivity of tumor cells.

<p>(A) EL-4 cells were incubated with different concentration of pharmacological inhibitors of ERK (10 µM for 2 h) or JNK (10 µM for 2 h) or P38 (10 µM for 2 h) or NF-kB inhibitory peptide (10 µM for 2 h) or Nrf-2 (ATRA) (5 µM for 2 h) or HO-1 (SnPP) (5 µM for 2 h) or TrxRd1 (auranofin) (25 nM for 2 h) or Ras (FTA) (10 µM for 2 h) and were cultured for 48 h in 5% CO₂ at 37°C with or without exposure to 4 Gy ionizing radiation. Cell death was analyzed by propidium iodide staining and flow cytometry. Representative flow cytometric histograms show apoptotic population as pre-G1 cells. (B) Bar graph represent radiation induced apoptosis in EL-4 cells incubated in the presence of different inhibitors. Data points represent mean±S.E.M. from nine replicates from three independent experiments. *p<0.05, as compared to untreated cells and ^#p<0.05, as compared to irradiated cells (C) EL-4 cells incubated in the presence of different pharmacological inhibitors were assessed by clonogenic assay for clonogenicity. Bar graph shows number of colonies in different treatment groups. Each bar shows mean±S.E.M from nine replicates from three independent experiments. *p<0.01, as compared to untreated cells and ^#p<0.01, as compared to irradiated cells.</p

ERK or Nrf-2 knockdown EL-4 cells are significantly more radiosensitive than wild type cells.

<p>(A) EL-4 cells transfected with scrambled shRNA or ERK or Nrf-2 shRNA plasmid were exposed to ionizing radiation (4 Gy) and cultured for 48 h. Apoptosis was estimated by propidium iodide staining and flow cytometry. (B) The frequency of apoptotic cells (gate RN1) is shown in the bar graph. Data points represent mean±S.E.M. from nine replicates from three independent experiments. *p<0.05, as compared to unirradiated cells and ^#p<0.05, as compared to irradiated cells. (C) Genomic DNA from wild type and knock down EL-4 cells cultured for 48 h post radiation exposure or alone was resolved on agarose gel and stained with ethidium bromide. DNA ladder indicates cells undergoing apoptosis.</p