Kaurane diterpene, kamebakaurin, inhibits NF-κB by directly targeting the DNA-binding activity of p50 and blocks the expression of antiapoptotic NF-κB target genes
Kaurane diterpene, kamebakaurin, inhibits NF-κB by directly targeting the DNA-binding activity of p50 and blocks the expression of antiapoptotic NF-κB target genes
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DOI：
10.1074/jbc.m201368200
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发表时间：
2002-05-24
影响因子：
4.8
通讯作者：
Lee, JJ
中科院分区：
文献类型：
--
作者：
Lee, JH;Koo, TH;Lee, JJ
贝壳杉烷二萜类化合物已从许多药用植物中鉴定出来，用于治疗炎症和癌症，但其分子作用机制尚不清楚。我们以前已经表明，kamebakaurin和其他三个贝壳杉烷二萜选择性抑制转录因子NF-κ B，细胞凋亡和免疫反应的中央介质的激活。我们在这里证明，kamebakaurin是一个有效的抑制剂NF-κ B激活直接靶向DNA结合活性的p50。Kamebakaurin阻止各种细胞类型中不同刺激物对NF-κ B的激活。Kamebakaurin没有阻止刺激诱导的IkappaB-α降解或NF-κ B核转位，但是，它显著干扰了细胞内和体外活化NF-κ B的DNA结合活性，并优先阻止p50介导的NF-κ B的DNA结合活性，而不是使用体外翻译的p50和RelA蛋白测量的RelA的DNA结合活性。此外，具有Cys-62 --> Ser突变的p50突变体不被kamebakaurin抑制，表明kamebakaurin的作用可能是由于其与p50中的半胱氨酸62相互作用。通过质谱分析进一步证明了kamebakaurin对p50的共价修饰，质谱分析显示kamebakaurin处理的p50的分子量增加，并且这种修饰不会通过添加二硫苏糖醇而逆转。这些结果表明，kamebakaurin通过直接共价修饰p50中的半胱氨酸62而表现出其抑制活性。此外，用kamebakaurin处理细胞可以防止肿瘤坏死因子-α的表达。（TNF-α）诱导的编码c-IAP 1（hiap-2）和c-IAP 2（hiap-1）的抗凋亡NF-κ B靶基因的表达，c-IAP 1和c-IAP 2（hiap-1）是凋亡抑制剂家族的成员，Bfl-1/A1是促存活Bcl-2同源物，并增强TNF-α诱导的caspase 8活性，从而导致MCF-7细胞对TNF-α诱导的凋亡敏感。综上所述，kamebakaurin是一种有价值的候选干预NF-κ B依赖的病理条件，如炎症和癌症。
Kaurane diterpenes have been identified from numerous medicinal plants, which have been used for treatment of inflammation and cancer, however, their molecular mechanism of action remains unclear. We have previously shown that kamebakaurin and other three kaurane diterpenes selectively inhibit activation of transcription factor NF-kappaB, a central mediator of apoptosis and immune responses. We here demonstrate that kamebakaurin is a potent inhibitor of NF-kappaB activation by directly targeting DNA-binding activity of p50. Kamebakaurin prevented the activation of NF-kappaB by different stimuli in various cell types. Kamebakaurin did not prevent either stimuli-induced degradation of IkappaB-alpha or nuclear translocation of NF-kappaB, however, it significantly interfered DNA binding activity of activated NF-kappaB in cell and in vitro and preferentially prevented p50-mediated DNA-binding activity of NF-kappaB rather than that of RelA as measured using in vitro translated p50 and RelA proteins. Moreover, a p50 mutant with a Cys-62 --> Ser mutation was not inhibited with kamebakaurin, indicating that the effect of kamebakaurin was probably due to its interaction with cysteine 62 in p50. The covalent modification of p50 by kamebakaurin was further demonstrated by mass spectrometry analysis that showed an increase in the molecular mass of kamebakaurin-treated p50, and this modification was not reverted by addition of dithiothreitol. These results suggested that kamebakaurin exhibited its inhibitory activity by a direct covalent modification of cysteine 62 in the p50. Also, treatment of cells with kamebakaurin prevented the tumor necrosis factor-alpha (TNF-alpha)-induced expression of antiapoptotic NF-kappaB target genes encoding c-IAP1 (hiap-2) and c-IAP2 (hiap-1), members of the inhibitor of apoptosis family, and Bfl-1/A1, a prosurvival Bcl-2 homologue, and augmented the TNF-alpha-induced caspase 8 activity, thereby resulting in sensitizing MCF-7 cells to TNF-alpha-induced apoptosis. Taken together, kamebakaurin is a valuable candidate for the intervention of NF-kappaB-dependent pathological conditions such as inflammation and cancer.