Inhibition of Nuclear Factor κB by Direct Modification in Whole Cells—Mechanism of Action of Nordihydroguaiaritic Acid, Curcumin and Thiol Modifiers

This study was set up to investigate the mechanism of four inhibitors of interleukin-1(IL-1)-α and tumor necrosis factor-(TNF)α activated nuclear factor κB (NFκB) in whole cells. The compounds fall into two classes: the first comprised two chain-breaking antioxidants, curcumin (diferulolylmethane) and nordihydroguaiaritic acid. The second class were two thiol-modifying agents, N-ethylmaleimide (NEM) and 2-chloro-1,3-dinitrobenzene (CDNB). Both sets of compounds were found to inhibit NFκB in tumour necrosis factor-activated Jurkat T lymphoma cells and interleukin 1-activated EL4.NOB-1 thymoma cells as determined by electrophoretic mobility shift assay using a specific NFκB DNA probe. In unstimulated cells the compounds were found to modify NFκB prior to chemical dissociation with sodium deoxycholate. They also inhibited DNA binding by NFκB when added to nuclear extracts from stimulated cells. Both of these effects occurred over a concentration range comparable to that which inhibited cytokine-activated NFκB in intact cells. All four agents were found to react directly with the p50 subunit of NFκB. However, only the antioxidants, curcumin and nordihydroguaiaritic acid (NDGA) were found to inhibit IκBα degradation activated by tumour necrosis factor-α. These results suggest that NFκB itself is susceptible to direct inhibition by a range of pharmacological agents. Furthermore, curcumin and nordihydroguaiaritic acid inhibit NFκB by interfering with IκBα degradation and reacting with p50 in the NFκB complex. These findings are likely to be useful in the attempt to develop agents which inhibit NFκB-dependent gene transcription.