Etoposide Induces Protein Kinase Cδ- and Caspase-3-Dependent Apoptosis in Neuroblastoma Cancer Cells
Etoposide Induces Protein Kinase Cδ- and Caspase-3-Dependent Apoptosis in Neuroblastoma Cancer Cells
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DOI：
10.1124/mol.109.054999
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发表时间：
2009-09-01
影响因子：
3.6
通讯作者：
Safa, Ahmad R.
中科院分区：
文献类型：
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作者：
Day, Travis W.;Wu, Ching-Huang;Safa, Ahmad R.
在本报告中，我们揭示依托泊苷可抑制 SK-N-AS 神经母细胞瘤癌细胞的增殖，并促进蛋白激酶 C δ (PKC δ) 和 caspase 依赖性细胞凋亡。依托泊苷诱导 PKC δ 依赖 caspase-3 裂解为其活性 p40 片段，而活性 PKC delta 通过正反馈机制触发 caspase-3 的加工。用 caspase-3 特异性抑制剂 N-苄氧羰基-Asp-Glu-Val-Asp-氟甲基酮或 caspase-3 特异性小相互作用 RNA (siRNA) 处理细胞，可防止依托泊苷诱导的 caspase-8 激活并抑制细胞凋亡。使用 siRNA 沉默 caspase-2 或 caspase-8 基因并不影响依托泊苷诱导的 caspase-3 加工，表明这些 caspase 在此信号通路中位于 caspase-3 的下游。此外，依托泊苷诱导的 caspase-2 加工需要 caspase-8 的表达，而依托泊苷介导的 caspase-8 加工需要 caspase-2 的表达，表明这两种 caspase 在依托泊苷处理后相互激活。我们还观察到，用 caspase-6 特异性抑制剂 benzyloxycarbonyl-Val-Glu(OMe)-Ile-Asp-(OMe)-氟甲基酮处理细胞，可以减少依托泊苷介导的细胞凋亡，并且 caspase-6 通过 caspase-8 依赖性机制被激活。最后，我们发现 Rottlerin 通过抑制 PKC δ 介导的 caspase-3 激活和降解 caspase-2（从而阻止 caspase-8 激活）来阻断依托泊苷诱导的细胞凋亡。我们的结果为依托泊苷如何介导细胞凋亡信号传导以及靶向这些途径如何导致神经母细胞瘤治疗新疗法的开发提供了重要见解。
In this report, we reveal that etoposide inhibits the proliferation of SK-N-AS neuroblastoma cancer cells and promotes protein kinase C delta (PKC delta)- and caspase-dependent apoptosis. Etoposide induces the caspase-3-dependent cleavage of PKC delta to its active p40 fragment, and active PKC delta triggers the processing of caspase-3 by a positive-feedback mechanism. Treatment of cells with the caspase-3-specific inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone or caspase-3-specific small interacting RNA (siRNA) prevented the etoposide-induced activation of caspase-8 and inhibited apoptosis. The silencing of the caspase-2 or caspase-8 genes using siRNAs did not affect the etoposide-induced processing of caspase-3, indicating that these caspases lie downstream of caspase-3 in this signaling pathway. Furthermore, the etoposide-induced processing of caspase-2 required the expression of caspase-8, and the etoposide-mediated processing of caspase-8 required the expression of caspase-2, indicating that these two caspases activate each other after etoposide treatment. We also observed that etoposide-mediated apoptosis was decreased by treating the cells with the caspase-6-specific inhibitor benzyloxycarbonyl-Val-Glu(OMe)-Ile-Asp-(OMe)-fluoromethyl ketone and that caspase-6 was activated by a caspase-8-dependent mechanism. Finally, we show that rottlerin blocks etoposide-induced apoptosis by inhibiting the PKC delta-mediated activation of caspase-3 and by degrading caspase-2, which prevents caspase-8 activation. Our results add important insights into how etoposide mediates apoptotic signaling and how targeting these pathways may lead to the development of novel therapeutics for the treatment of neuroblastomas.