Lin Yidan, Liu Xiangguo, Yue Ping, Benbrook Doris M, Berlin K Darrell, Khuri Fadlo R, Sun Shi-Yong
Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University School of Medicine, 1365-C Clifton Road Northeast, Atlanta, GA 30322, USA.
Mol Cancer Ther. 2008 Nov;7(11):3556-65. doi: 10.1158/1535-7163.MCT-08-0648.
The flexible heteroarotinoid, SHetA2, is a novel compound with apoptosis-inducing and anticancer activities in vitro and in vivo. Our previous research showed that up-regulation of death receptor 5 plays a critical role in the mechanism of SHetA2-induced apoptosis in human lung cancer cells. The hypothesis of this study was that the mechanism of SHetA2-induced apoptosis requires modulation of additional proteins critical for regulation of apoptosis, including cellular FLICE-inhibitory protein (c-FLIP), survivin, X-linked inhibitor of apoptosis, Bcl-2, Bcl-X(L), Bax, and Bim. Western blot analysis showed that c-FLIP and survivin were substantially reduced in all of the tested cell lines exposed to SHetA2 compared with other proteins that were reduced only in a subset of the cell lines tested. Strikingly, overexpression of c-FLIP, but not survivin, protected cells from SHetA2-induced apoptosis and enhancement of TRAIL-initiated apoptosis, although knockdown of endogenous survivin did slightly sensitize cells to SHetA2-induced apoptosis. Consistent with these results, small interfering RNA-mediated reduction of c-FLIP was more effective than survivin down-regulation in triggering apoptosis in these cell lines. SHetA2 increased ubiquitination of c-FLIP and the consequent degradation was abrogated by the proteasome inhibitor MG132. Although SHetA2 treatment led to increased c-Jun phosphorylation, the JNK inhibitor SP600125 did not prevent c-FLIP down-regulation by SHetA2. Thus, it appears that SHetA2 down-regulates c-FLIP levels by facilitating its ubiquitin/proteasome-mediated degradation independent of JNK activation. Collectively, the present study indicates that, in addition to death receptor 5 up-regulation, c-FLIP down-regulation is another important component of flexible heteroarotinoid (SHetA2)-induced apoptosis as well as enhancement of TRAIL-induced apoptosis.
柔性杂环维甲酸SHetA2是一种新型化合物，在体外和体内均具有诱导凋亡和抗癌活性。我们之前的研究表明，死亡受体5的上调在SHetA2诱导人肺癌细胞凋亡的机制中起关键作用。本研究的假设是，SHetA2诱导凋亡的机制需要调节对凋亡调节至关重要的其他蛋白质，包括细胞FLICE抑制蛋白（c-FLIP）、生存素、X连锁凋亡抑制蛋白、Bcl-2、Bcl-X(L)、Bax和Bim。蛋白质印迹分析表明，与仅在部分测试细胞系中减少的其他蛋白质相比，在所有暴露于SHetA2的测试细胞系中，c-FLIP和生存素均显著减少。引人注目的是，c-FLIP的过表达而非生存素的过表达可保护细胞免受SHetA2诱导的凋亡以及增强TRAIL引发的凋亡，尽管内源性生存素的敲低确实使细胞对SHetA2诱导的凋亡稍有敏感。与这些结果一致，在这些细胞系中，小干扰RNA介导的c-FLIP减少在触发凋亡方面比生存素下调更有效。SHetA2增加了c-FLIP的泛素化，蛋白酶体抑制剂MG132可消除随之而来的降解。尽管SHetA2处理导致c-Jun磷酸化增加，但JNK抑制剂SP600125并不能阻止SHetA2对c-FLIP的下调。因此，似乎SHetA2通过促进其泛素/蛋白酶体介导的降解来下调c-FLIP水平，而与JNK激活无关。总体而言，本研究表明，除了死亡受体5上调外，c-FLIP下调是柔性杂环维甲酸（SHetA2）诱导凋亡以及增强TRAIL诱导凋亡的另一个重要组成部分。
