Elsevier

Biochemical Pharmacology

Volume 55, Issue 7, 1 April 1998, Pages 965-973
Biochemical Pharmacology

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

https://doi.org/10.1016/S0006-2952(97)00535-2Get rights and content

Abstract

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.

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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.

References (40)

  • AG Bowie et al.

    Lipid peroxidation is involved in the activation of NFκB by TNF but not IL1 in the human endothelial cell line ECV304

    J Biol Chem

    (1997)
  • S Baldwin

    The NFκB and the IκB proteins: New discoveries and insights

    Annu Rev Immunol

    (1996)
  • DW Ballard et al.

    HTLV-I tax induces cellular proteins that activate the κB element in the IL-2 receptor α gene

    Science

    (1988)
  • MI Cybulsky et al.

    Gene structure, chromosomal location, and basis for alternative mRNA splicing of the human VCAM1 gene

    Proc Natl Acad Sci USA

    (1991)
  • MR Edbrooke et al.

    Identification of cis-acting sequences responsible for phorbol ester induction of human serum amyloid A gene expression via a nuclear factor κB-like transcription factor

    Mol Cell Biol

    (1989)
  • B Hoyos et al.

    Kappa B-specific DNA binding proteinsrole in the regulation of human interleukin-2 gene expression

    Science

    (1989)
  • G Nabel et al.

    An inducible transcription factor activates expression of human immunodeficiency virus in T cells

    Nature

    (1987)
  • PA Baeuerle et al.

    Function and activation of NFκB in the immune system

    Annu Rev Immunol

    (1994)
  • AA Beg et al.

    Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of IκBαa mechanism for NFκB activation

    Mol Cell Biol

    (1993)
  • T Henkel et al.

    Rapid proteolysis of IκBα is necessary for activation of transcription factor NFκB

    Nature

    (1993)
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