Regulation Of Nuclear Factor-kappa B Activity

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The IkappaB kinase (IKK) catalytic subunit, IKKbeta, plays a key role in cytokine-mediated increase in the pleiotropic responses of nuclear factor (NF)-kappaB signaling. Defects in IKKbeta activity inhibit NF-kappaB-mediated inflammation and oncogenesis, while promoting apoptosis. Manumycin is a natural epoxyquinoid compound that exhibits potent and selective inhibitory activity against farnesyltransferase, and it has been used for its antineoplastic action in a variety of experimental models. Our recent findings indicate that manumycin elicited a rapid and potent inhibition of TNF alpha-stimulated IKK activity in a number of cell lines and a primary culture of rat hepatocytes. Moreover, administration of manumycin to mice xenografted with murine B16F10 tumors caused potent IKK-suppressive effects (Bernier et al., 2006). Unexpectedly, other classes of farnesyltransferase inhibitors had no inhibitory effect under these experimental conditions. To identify the molecular mechanisms of manumycin action, we transfected cultured human HepG2 hepatoma cells with IKKalpha and IKKbeta constructs and demonstrated direct inhibition of IKK activity with concomittant formation of stable homotypic IKKbeta dimers in the presence of manumycin. A number of Cys-> Ala point mutants of IKKbeta were generated to investigate the possibility that IKKbeta covalent dimerization resulted from nucleophilic attack on the epoxyquinoid core of manumycin. Cells expressing IKKbeta mutated in the activation loop at Cys-179 exhibited similar dimer formation, whereas double substitution of Cys-662/716 in the carboxy-terminal domain of IKKbeta conferred protection against homotypic dimerization by manumycin. Dithiothreitol pre-treatment substantially blocked manumycin-mediated formation of covalent IKKbeta dimers in immunoprecipitates. Using mass spectrometry technique, we identified glutathione (GSH) as a molecule susceptible to modification via the epoxyquinoid function of manumycin, which suggests that the depletion of intracellular thiols could contribute, at least in part, to manumycin's antineoplastic properties. These results indicated that manumycin plays important regulatory function in IKK/NFkappaB signaling through pathways distinct from its role as farnesylation inhibitor. Despite these advances, it remains to be understood how manumycin affects the function of other specific elements of the IKK/NF-kappaB pathway. Biochemical and molecular biological studies are being carried out to characterize the inactivation of the IKK multimeric complex by manumycin and the pathways that it uses to induce apoptosis both in cultured cell lines and in cancer cells xenotransplanted into immunocompromised mice. Our data should provide novel insights into key aspects of NF-kappaB signaling, and may be of particular clinical relevance.