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. Author manuscript; available in PMC: 2009 Sep 29.
Published in final edited form as: Free Radic Biol Med. 2007 May 16;43(4):568–580. doi: 10.1016/j.freeradbiomed.2007.05.009

FIGURE 1. Glutathione abrogates curcumin-mediated inhibition of NF-κB.

FIGURE 1

A, Curcumin inhibited TNF-induced activation of NF-κB and GSH blocked the effect of curcumin in a dose-dependent manner. KBM-5 cells were incubated with indicated concentrations of GSH for 2 h, treated with 50 μM curcumin for 4 h. Then cells were activated with 0.1 nM of TNF for 30 min, and then subjected to EMSA to assay for NF-κB activation. B, The direct effect of curcumin on the NF-κB complex was investigated. Nuclear extracts were prepared from untreated cells or cells treated with 0.1 nM TNF and incubated for 30 min with the indicated concentrations of curcumin. They were then assayed for NF-κB activation by EMSA. C, NF-κB induced by TNF is composed of p65 and p50 subunits. Nuclear extracts from untreated or TNF-treated cells were incubated with the indicated antibodies, unlabeled NF-κB oligo-probe, or mutant oligo-probe and then assayed for NF-κB activation by EMSA. D, Glutathione inhibited curcumin-mediated suppression of IKK activity. KBM-5 cells were preincubated with indicated concentrations of GSH for 2 h, treated with 50 μM concentration of curcumin for 4 h. Then cells were activated with 1 nM TNF for 15 min. Whole-cell extracts were immunoprecipitated with antibody against IKK-α and analyzed by an immune complex kinase assay. To examine the effect of curcumin ad GSH on level of IKK proteins, whole-cell extracts were fractionated on SDS-PAGE and examined by Western blot analysis using anti-IKK-α and anti-IKK-β antibodies. E, Curcumin suppressed TNF-mediated phosphorylation of AKT, as shown by Western blotting; GSH significantly blocked the effect of curcumin. KBM-5 cells were preincubated with indicated concentrations of GSH for 2 h, treated with 50 μM concentration of curcumin for 4 h. Then cells were activated with 1 nM TNF for 10 min. Whole cell extracts were prepared, separated on 10% SDS-PAGE, and electrotransferred to nitrocellulose membrane. Western blot analysis was performed using phospho AKT antibody. Anti-AKT was used as a loading control.