Section of Molecular Embryology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
Mol Cancer. 2011 Jul 31;10:95. doi: 10.1186/1476-4598-10-95.
Caspase-8 is a key upstream mediator in death receptor-mediated apoptosis and also participates in mitochondria-mediated apoptosis via cleavage of proapoptotic Bid. However, the role of caspase-8 in p53- and p73-dependent apoptosis induced by genotoxic drugs remains unclear. We recently reported that the reconstitution of procaspase-8 is sufficient for sensitizing cisplatin- but not etoposide-induced apoptosis, in chemoresistant and caspase-8 deficient HOC313 head and neck squamous cell carcinoma (HNSCC) cells.
We show that p53/p73-dependent caspase-8 activation is required for sensitizing etoposide-induced apoptosis by utilizing HOC313 cells carrying a temperature-sensitive p53G285K mutant. Restoration of wild-type p53 function under the permissive conditions, together with etoposide treatment, led to substantial transcriptional activation of proapoptotic Noxa and PUMA, but failed to induce apoptosis. In addition to p53 restoration, caspase-8 reconstitution was needed for sensitization to etoposide-induced apoptosis, mitochondria depolarization, and cleavage of the procaspases-3, and -9. In etoposide-sensitive Ca9-22 cells carrying a temperature-insensitive mutant p53, siRNA-based p73 knockdown blocked etoposide-induced apoptosis and procaspase-8 cleavage. However, induction of p73 protein and up-regulation of Noxa and PUMA, although observed in Ca9-22 cells, were hardly detected in etoposide-treated HOC313 cells under non-permissive conditions, suggesting a contribution of p73 reduction to etoposide resistance in HOC313 cells. Finally, the caspase-9 inhibitor Ac-LEHD-CHO or caspase-9 siRNA blocked etoposide-induced caspase-8 activation, Bid cleavage, and apoptosis in both cell lines, indicating that p53/p73-dependent caspase-8 activation lies downstream of mitochondria.
we conclude that p53 and p73 can act as upstream regulators of caspase-8, and that caspase-8 is an essential mediator of the p53/p73-dependent apoptosis induced by etoposide in HNSCC cells. Our data suggest the importance of caspase-8-mediated positive feedback amplification in the p53/p73-dependent apoptosis induced by etoposide in HNSCC cells.
Caspase-8 是死亡受体介导的细胞凋亡的关键上游介质，也通过切割促凋亡 Bid 参与线粒体介导的细胞凋亡。然而，Caspase-8 在基因毒性药物诱导的 p53 和 p73 依赖性细胞凋亡中的作用尚不清楚。我们最近报道，在化疗耐药和 Caspase-8 缺失的 HOC313 头颈部鳞状细胞癌（HNSCC）细胞中，重新组装 Caspase-8 足以增强顺铂而非依托泊苷诱导的细胞凋亡。
我们显示，利用携带温度敏感型 p53G285K 突变的 HOC313 细胞，p53/p73 依赖性 Caspase-8 激活对于增强依托泊苷诱导的细胞凋亡是必需的。在允许条件下恢复野生型 p53 功能，加上依托泊苷处理，导致促凋亡 Noxa 和 PUMA 的大量转录激活，但未能诱导细胞凋亡。除了 p53 的恢复，Caspase-8 的重建对于增强依托泊苷诱导的细胞凋亡、线粒体去极化和 Caspase-3 和 Caspase-9 的切割也是必需的。在携带温度不敏感突变 p53 的依托泊苷敏感的 Ca9-22 细胞中，基于 siRNA 的 p73 敲低阻断了依托泊苷诱导的细胞凋亡和 Caspase-8 的切割。然而，尽管在 Ca9-22 细胞中观察到 p73 蛋白的诱导和 Noxa 和 PUMA 的上调，但在非允许条件下用依托泊苷处理的 HOC313 细胞中几乎检测不到，这表明 p73 的减少有助于 HOC313 细胞对依托泊苷的耐药性。最后，Caspase-9 抑制剂 Ac-LEHD-CHO 或 Caspase-9 siRNA 阻断了两种细胞系中依托泊苷诱导的 Caspase-8 激活、Bid 切割和细胞凋亡，表明 p53/p73 依赖性 Caspase-8 激活位于线粒体下游。
我们得出结论，p53 和 p73 可以作为 Caspase-8 的上游调节剂，Caspase-8 是依托泊苷诱导的 HNSCC 细胞中 p53/p73 依赖性细胞凋亡的必需介质。我们的数据表明，在依托泊苷诱导的 HNSCC 细胞中，Caspase-8 介导的正反馈放大在 p53/p73 依赖性细胞凋亡中很重要。
