Inhibition of transcription factor NF-kappaB signaling proteins IKKbeta and p65 through specific cysteine residues by epoxyquinone A monomer: correlation with its anti-cancer cell growth activity.
Transcription factor NF-kappaB is constitutively active in many human chronic inflammatory diseases and cancers. Epoxyquinone A monomer (EqM), a synthetic derivative of the natural product epoxyquinol A, has previously been shown to be a potent inhibitor of tumor necrosis factor-alpha (TNF-alpha)-induced activation of NF-kappaB, but the mechanism by which EqM inhibits NF-kappaB activation was not known. In this report, we show that EqM blocks activation of NF-kappaB by inhibiting two molecular targets: IkappaB kinase IKKbeta and NF-kappaB subunit p65. EqM inhibits TNF-alpha-induced IkappaBalpha phosphorylation and degradation by targeting IKKbeta, and an alanine substitution for Cys179 in the activation loop of IKKbeta makes it resistant to EqM-mediated inhibition. EqM also directly inhibits DNA binding by p65, but not p50; moreover, replacement of Cys38 in p65 with Ser abolishes EqM-mediated inhibition of DNA binding. Pretreatment of cells with reducing agent dithiothreitol dose-dependently reduces EqM-mediated inhibition of NF-kappaB, further suggesting that EqM directly modifies the thiol group of Cys residues in protein targets. Modifications of the exocyclic alkene of EqM substantially reduce EqM's ability to inhibit NF-kappaB activation. In the human SUDHL-4 lymphoma cell line, EqM inhibits both proliferation and NF-kappaB DNA binding, and activates caspase-3 activity. EqM also effectively inhibits the growth of human leukemia, kidney, and colon cancer cell lines in the NCI's tumor cell panel. Among six colon cancer cell lines, those with low amounts of constitutive NF-kappaB DNA-binding activity are generally more sensitive to growth inhibition by EqM. Taken together, these results suggest that EqM inhibits growth and induces cell death in tumor cells through a mechanism that involves inhibition of NF-kappaB activity at multiple steps in the signaling pathway.
Liang
Mei-Chih
MC
Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215, USA.
Bardhan
Sujata
S
Pace
Emily A
EA
Rosman
Diana
D
Beutler
John A
JA
Porco
John A
JA
Gilmore
Thomas D
TD
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects*; D002460:Cell Line; D003545:Cysteine / Q000187:drug effects*; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D020672:I-kappa B Proteins / Q000037:antagonists & inhibitors*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors*; D011809:Quinones / Q000494:pharmacology*; D051996:Transcription Factor RelA / Q000037:antagonists & inhibitors*
D000970:Antineoplastic Agents; D017931:DNA Primers; D020672:I-kappa B Proteins; C105627:NFKBIE protein, human; D011518:Proto-Oncogene Proteins; D011809:Quinones; D051996:Transcription Factor RelA; C508869:epoxyquinone A; D003545:Cysteine