NF-kappaB inhibition impairs the radioresponse of hypoxic EMT-6 tumour cells through downregulation of inducible nitric oxide synthase

Abstract

Hypoxic EMT-6 tumour cells displayed a high level of inducible nitric oxide synthase (iNOS) and an increased radiosensitivity after a 16 h exposure to lipopolysaccharide, a known activator of nuclear factor-kappaB (NF-kappaB). Both iNOS activation and radioresponse were impaired by the NF-kappaB inhibitors phenylarsine oxide and lactacystin. Contrasting to other studies, our data show that inhibition of NF-kappaB may impair the radioresponse of tumour cells through downregulation of iNOS.

Figure 1

Effect of lactacystin and PAO on nuclear NF-κB expression and binding activity in hypoxic EMT-6 cells. (A) Cultures were exposed to 0.1 μg ml⁻¹LPS in 1% oxygen for 0–90 min and afterwards analysed for the binding activity of NF-κB in nuclear extracts by EMSA. (B) An analysis of the NF-κB composition was performed using anti-p65 and anti-p50 antibodies. The specificity of NF-κB binding was confirmed by inclusion of unlabelled (cold) NF-κB consensus and its mutant in the binding reactions. (C, D) The effect of a 3 h pretreatment with lactacystin (LC) and a 10 min pretreatment with PAO on the expression and binding activity of NF-κB was evaluated after a 60 min exposure to 0.1 μg ml⁻¹ LPS. The figure is representative of three independent experiments.

Figure 2

Effect of lactacystin and PAO on iNOS expression in hypoxic EMT-6 cells. (A) EMT-6 cultures were exposed to 0.001–0.1 μg ml⁻¹ LPS in 1% oxygen and afterwards analysed for the expression of iNOS by Western blotting. The last lane illustrates the iNOS induction by LPS in 21% oxygen. (B, C) iNOS expression after a 3 h pretreatment with lactacystin (LC) and a 10 min pretreatment with PAO followed by a 16 h exposure to 0.1 μg ml⁻¹ LPS in 1% oxygen. The figure is representative of four independent experiments.

Figure 3

Effect of lactacystin and PAO on nitrite production by EMT-6 cells. (A) EMT-6 cultures were exposed to 0.0001–0.1 μg ml⁻¹ LPS for 16 h in 1% oxygen and afterwards analysed for the accumulation of nitrite, with or without 3 mM aminoguanidine. (B) Nitrite production after a 3 h pretreatment with lactacystin (LC) and a 10 min pretreatment with PAO followed by exposure to 0.1 μg ml⁻¹ LPS in 1% oxygen.

Figure 4

Radioresponse of hypoxic EMT-6 cells after exposure to different concentrations of LPS only (A) and pretreated with NF-κB inhibitors followed by LPS treatment (B). (A) Cells were exposed for 16 h to LPS at 0.001 μg ml⁻¹ (▪), 0.01 μg ml⁻¹ (▴) and 0.1 μg ml⁻¹ (•) in 1% oxygen prior to radiation. The survival curve for nontreated cells (○) and treated with 0.1 μg ml⁻¹ LPS in 21% oxygen (▾) are plotted for reference. (B) Cells were pretreated for 3 h with 40 μM lactacystin (□) for 10 min with 4 mM PAO (▵) or not pretreated (•) prior to exposure to 0.1 μg ml⁻¹ LPS in 1% oxygen. The survival curve for cells treated with 0.1 μg ml⁻¹ LPS and 3 mM aminoguanidine is plotted for reference (⧫).