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

De Ridder M, Van den Berge DL, Verovski VN, Monsaert C, Wauters N, Storme GA - Br. J. Cancer (2003)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Oncology Center, Cancer Research Unit, Academic Hospital Free University Brussels (A.Z.-V.U.B.), Laarbeeklaan 101, B 1090, Brussels, Belgium. Mark.De.Ridder@vub.ac.be

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.

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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.
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fig1: 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.

Mentions: EMT-6 cultures were exposed to 0.1 μg ml−1 LPS in 1% oxygen to model the reduced oxygenation in solid tumours. Afterwards, the binding activity of NF-κB in nuclear extracts was examined by EMSA. The activation of NF-κB was maximal after a 60 min exposure (Figure 1AFigure 1


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

De Ridder M, Van den Berge DL, Verovski VN, Monsaert C, Wauters N, Storme GA - Br. J. Cancer (2003)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2376775&req=5

fig1: 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.
Mentions: EMT-6 cultures were exposed to 0.1 μg ml−1 LPS in 1% oxygen to model the reduced oxygenation in solid tumours. Afterwards, the binding activity of NF-κB in nuclear extracts was examined by EMSA. The activation of NF-κB was maximal after a 60 min exposure (Figure 1AFigure 1

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Oncology Center, Cancer Research Unit, Academic Hospital Free University Brussels (A.Z.-V.U.B.), Laarbeeklaan 101, B 1090, Brussels, Belgium. Mark.De.Ridder@vub.ac.be

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.

Show MeSH
Related in: MedlinePlus