Hypoxia-Inducible Factor 1α Determines Gastric Cancer Chemosensitivity via Modulation of p53 and NF-κB
Figure 5
HIF-1α-mediated activation of NF-κB limits the toxicity of 5-FU.
(A) Nuclear extracts of AGS KD and SCR cells were prepared at the indicated time points after treatment with 10 µg/ml 5-FU or TNFα as a positive control, and DNA-binding activity for NF-κB was examined by EMSA. For supershift experiments, nuclear extracts were incubated with an anti-p65 antibody. (B) Expression of NF-κB target genes cIAP1 and A20 mRNA in total RNA extracts from AGS KD and AGS SCR cells 48 h after treatment with 10 µg/ml 5-FU. Data were expressed relative to mRNA levels in untreated AGS SCR cells, set at 1.0 (*, P<0.05; **, P<0.01). (C) AGS KD cells were co-transfected with either pcDNA p65 or pcDNA 3.1 and treated with 5-FU 24 h post transfection. Cell numbers were after another 24 h and are presented as percent of untreated control cells (***, P<0.001). (D) DNA binding activity for NF-κB was examined by EMSA using nuclear extracts of MKN28 KD and SCR cells treated with 10 µg/ml 5-FU or TNFα for the indicated times. Antibody inhibition was performed with an anti-p65 antibody.
