Original ArticlesInhibition of Nuclear Factor κB by Direct Modification in Whole Cells—Mechanism of Action of Nordihydroguaiaritic Acid, Curcumin and Thiol Modifiers
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Materials
The murine thymoma cell line EL4.NOB-1 and the human lymphoblast Jurkat E6.1 were obtained from the European Collection of Animal Cell Cultures. RPMI 1640 and FCS were from Greiner GmbH. Penicillin-streptomycin was purchased from Life Technologies. Human recombinant TNFα was a gift from Dr. Steve Foster, Zeneca Pharmaceuticals. IL-1α was a gift from Dr. J. Saklatvala (Kennedy Institute for Rheumatology-Sunley Division). Poly (dI.dC) was from Pharmacia Biosystems. T4 polynucleotide kinase, the
Curcumin and NDGA Inhibit NFκB Activated by IL-1α or TNFα
Two T cell lines, the murine thymoma EL4.NOB-1 and the human lymphoma Jurkat E6.1 were tested for inhibition of NFκB by curcumin and NDGA. We have previously shown EL4 to respond strongly to the cytokine IL-1α [18]while Jurkat are commonly used in TNFα studies. The NFκB complexes have been characterised in both cell types 18, 25. Both cell types were pre-incubated with the agents for 30 min prior to cytokine stimulation for 1 hr. Cells were harvested and washed once in PBS. Nuclear extracts
Discussion
This study has shown that the antioxidants NDGA and curcumin inhibit NFκB by direct modification. Modification can occur in the cytosol of intact cells, where NFκB was complexed to IκB, implying that the site of modification was not obscured by IκB. We reached this conclusion from experiments with deoxycholate, in which we found that in cytosolic extracts prepared from drug-treated cells, DNA binding by NFκB as revealed by deoxycholate treatment in vitro was inhibited. It is also possible,
Acknowledgements
This work was supported by grants from the Irish Health Research Board, Forbairt, and BioResearch Ireland. We thank Dr. Tim Mantle for useful discussions.
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