Figure 4

5-FU induces upregulation of NF-κB, which is downregulated by curcumin, and inactivation of NF-κB inhibits the synergism. (a) Dose dependence of 5-FU-mediated DNA binding activity of NF-κB in MDA-MB-231 cells. Nuclear extracts prepared from MDA-MB-231 cells exposed to different concentrations of 5-FU (0–100 μM) assayed for NF-κB activation by electrophoretic mobility shift assay (EMSA). (b) Kinetics of 5-FU-induced activation of NF-κB in MDA-MB-231 cells. Nuclear extracts were prepared after exposing the cells to 10 μM 5-FU for different time intervals (0–12 h) and NF-κB status was assessed by EMSA. (c) Individual and combined effects of 5-FU and curcumin on NF-κB activation in MDA-MB-231 cells compared with untreated controls. NF-κB activation was assayed by EMSA as described earlier. (d) Supershift analysis, using anti-p50 and p65 antibodies to indicate band specificity, is carried out as described in Materials and Methods. (e) Kinetics of IκBα degradation corresponding to nuclear translocation of NF-κB. Cytoplasmic extract collected after exposing the cells to 10 μM 5-FU for different time periods were subjected to western blotting using antibody against IκBα. (f) Inhibition of 5-FU-induced IκBα degradation by curcumin. Cytosolic extract prepared from MDA-MB-231 cells after treating with 5-FU and curcumin, either alone or in combination, for 1 h was subjected to western blotting using anti-IκBα antibody. (g) Effect of curcumin on 5-FU-induced phosphorylation of IκB kinase (IKK). Cytosolic lysates from MDA-MB-231 cells after treatment with 5-FU and/curcumin were subjected to western blotting using anti-phospho-IKK antibody. (h) Effect of 5-FU and curcumin, alone or in combination, on control and SN-50-pretreated MDA-MB-231 cells. Cell viability was checked using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described earlier. Data represent three independent sets of experiments and results are shown as the mean±S.D. ***, **, and ^Φ represents P-values ⩽0.0001, ⩽0.001 and >0.05, respectively. (i) Effect of 5-FU and curcumin, alone or in combination, on empty vector (pcDNA3) or pcDNA3-IκB-α DM plasmid-transfected MDA-MB-231 cells. Cell viability was checked using MTT assay as described earlier. Data represent three independent sets of experiments and results are shown as the mean±S.D. ***, **, and ^Φ represents P-values ⩽0.0001, ⩽0.001 and >0.05 respectively