Liang Mei-Chih, Bardhan Sujata, Pace Emily A, Rosman Diana, Beutler John A, Porco John A, Gilmore Thomas D
Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215, USA.
Biochem Pharmacol. 2006 Feb 28;71(5):634-45. doi: 10.1016/j.bcp.2005.11.013. Epub 2005 Dec 19.
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.
转录因子核因子-κB（NF-κB）在许多人类慢性炎症性疾病和癌症中呈组成性激活。环氧醌A单体（EqM）是天然产物环氧喹诺醇A的合成衍生物，先前已被证明是肿瘤坏死因子-α（TNF-α）诱导的NF-κB激活的有效抑制剂，但EqM抑制NF-κB激活的机制尚不清楚。在本报告中，我们表明EqM通过抑制两个分子靶点来阻断NF-κB的激活：IκB激酶IKKβ和NF-κB亚基p65。EqM通过靶向IKKβ抑制TNF-α诱导的IκBα磷酸化和降解，并且在IKKβ激活环中用丙氨酸替代半胱氨酸179使其对EqM介导的抑制具有抗性。EqM还直接抑制p65与DNA的结合，但不抑制p50；此外，用丝氨酸替代p65中的半胱氨酸38可消除EqM介导的对DNA结合的抑制。用还原剂二硫苏糖醇对细胞进行预处理可剂量依赖性地降低EqM介导的对NF-κB的抑制，进一步表明EqM直接修饰蛋白质靶点中半胱氨酸残基的巯基。EqM外环烯烃的修饰显著降低了EqM抑制NF-κB激活的能力。在人SUDHL-4淋巴瘤细胞系中，EqM抑制增殖和NF-κB与DNA的结合，并激活caspase-3活性。EqM还能有效抑制美国国立癌症研究所（NCI）肿瘤细胞库中的人白血病、肾和结肠癌细胞系的生长。在六种结肠癌细胞系中，组成性NF-κB DNA结合活性较低的细胞系通常对EqM介导的生长抑制更敏感。综上所述，这些结果表明EqM通过一种涉及在信号通路多个步骤抑制NF-κB活性的机制来抑制肿瘤细胞的生长并诱导细胞死亡。
