一种 Smac 模拟物通过调节 IAP 和 NF-κB 使前列腺癌细胞对 TRAIL 诱导的细胞凋亡敏感。
A Smac-mimetic sensitizes prostate cancer cells to TRAIL-induced apoptosis via modulating both IAPs and NF-kappaB.
机构信息
Department of Radiation Oncology, University of Michigan, Ann Arbor, MI 48109, USA.
出版信息
BMC Cancer. 2009 Nov 6;9:392. doi: 10.1186/1471-2407-9-392.
BACKGROUND
Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for human cancer therapy, prostate cancer still remains resistant to TRAIL. Both X-linked inhibitor of apoptosis (XIAP) and nuclear factor-kappaB function as key negative regulators of TRAIL signaling. In this study, we evaluated the effect of SH122, a small molecule mimetic of the second mitochondria-derived activator of caspases (Smac), on TRAIL-induced apoptosis in prostate cancer cells.
METHODS
The potential of Smac-mimetics to bind XIAP or cIAP-1 was examined by pull-down assay. Cytotoxicity of TRAIL and/or Smac-mimetics was determined by a standard cell growth assay. Silencing of XIAP or cIAP-1 was achieved by transient transfection of short hairpin RNA. Apoptosis was detected by Annexin V-PI staining followed by flow cytometry and by Western Blot analysis of caspases, PARP and Bid. NF-kappaB activation was determined by subcellular fractionation, real time RT-PCR and reporter assay.
RESULTS
SH122, but not its inactive analog, binds to XIAP and cIAP-1. SH122 significantly sensitized prostate cancer cells to TRAIL-mediated cell death. Moreover, SH122 enhanced TRAIL-induced apoptosis via both the death receptor and the mitochondrial pathway. Knockdown of both XIAP and cIAP-1 sensitized cellular response to TRAIL. XIAP-knockdown attenuated sensitivity of SH122 to TRAIL-induced cytotoxicity, confirming that XIAP is an important target for IAP-inhibitor-mediated TRAIL sensitization. SH122 also suppressed TRAIL-induced NF-kappaB activation by preventing cytosolic IkappaB-alpha degradation and RelA nuclear translocation, as well as by suppressing NF-kappaB target gene expression.
CONCLUSION
These results demonstrate that SH122 sensitizes human prostate cancer cells to TRAIL-induced apoptosis by mimicking Smac and blocking both IAPs and NF-kappaB. Modulating IAPs may represent a promising approach to overcoming TRAIL-resistance in human prostate cancer with constitutively active NF-kappaB signaling.
背景
尽管肿瘤坏死因子相关凋亡诱导配体（TRAIL）是人类癌症治疗的一种很有前途的药物，但前列腺癌仍然对 TRAIL 具有抗性。X 连锁凋亡抑制因子（XIAP）和核因子-κB 均作为 TRAIL 信号的关键负调控因子发挥作用。在这项研究中，我们评估了小分子模拟物第二线粒体衍生的半胱天冬酶激活剂（Smac）SH122 对前列腺癌细胞中 TRAIL 诱导的细胞凋亡的影响。
方法
通过下拉实验检测 Smac 模拟物与 XIAP 或 cIAP-1 的结合潜力。通过标准细胞生长测定法确定 TRAIL 和/或 Smac 模拟物的细胞毒性。通过瞬时转染短发夹 RNA 实现 XIAP 或 cIAP-1 的沉默。通过 Annexin V-PI 染色结合流式细胞术和 caspase、PARP 和 Bid 的 Western Blot 分析检测细胞凋亡。通过亚细胞分离、实时 RT-PCR 和报告基因测定法确定 NF-κB 激活。
结果
SH122，但不是其无活性类似物，与 XIAP 和 cIAP-1 结合。SH122 显著增强了前列腺癌细胞对 TRAIL 介导的细胞死亡的敏感性。此外，SH122 通过死亡受体和线粒体途径增强 TRAIL 诱导的细胞凋亡。XIAP 和 cIAP-1 的敲低均增强了细胞对 TRAIL 的反应敏感性。XIAP 敲低减弱了 SH122 对 TRAIL 诱导的细胞毒性的敏感性，证实 XIAP 是 IAP 抑制剂介导的 TRAIL 增敏的重要靶标。SH122 还通过阻止细胞质 IkappaB-α降解和 RelA 核易位以及抑制 NF-κB 靶基因表达来抑制 TRAIL 诱导的 NF-κB 激活。
结论
这些结果表明，SH122 通过模拟 Smac 并阻断两种 IAP 和 NF-κB 来增强人前列腺癌细胞对 TRAIL 诱导的细胞凋亡的敏感性。调节 IAP 可能是克服具有组成性 NF-κB 信号的人类前列腺癌对 TRAIL 耐药性的一种很有前途的方法。
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