BACKGROUND Cell death triggered by unmitigated endoplasmic reticulum (ER) stress plays an important role in physiology and disease, but the death-inducing signaling mechanisms are incompletely understood. To gain more insight into these mechanisms, the ER stressor thapsigargin (Tg) is an instrumental experimental tool. Additionally, Tg forms the basis for analog prodrugs designed for cell killing in targeted cancer therapy. Tg induces apoptosis via the unfolded protein response (UPR), but how apoptosis is initiated, and how individual effects of the various UPR components are integrated, is unclear. Furthermore, the role of autophagy and autophagy-related (ATG) proteins remains elusive. METHODS To systematically address these key questions, we analyzed the effects of Tg and therapeutically relevant Tg analogs in two human cancer cell lines of different origin (LNCaP prostate- and HCT116 colon cancer cells), using RNAi and inhibitory drugs to target death receptors, UPR components and ATG proteins, in combination with measurements of cell death by fluorescence imaging and propidium iodide staining, as well as real-time RT-PCR and western blotting to monitor caspase activity, expression of ATG proteins, UPR components, and downstream ER stress signaling. RESULTS In both cell lines, Tg-induced cell death depended on death receptor 5 and caspase-8. Optimal cytotoxicity involved a non-autophagic function of MAP1LC3B upstream of procaspase-8 cleavage. PERK, ATF4 and CHOP were required for Tg-induced cell death, but surprisingly acted in parallel rather than as a linear pathway; ATF4 and CHOP were independently required for Tg-mediated upregulation of death receptor 5 and MAP1LC3B proteins, whereas PERK acted via other pathways. Interestingly, IRE1 contributed to Tg-induced cell death in a cell type-specific manner. This was linked to an XBP1-dependent activation of c-Jun N-terminal kinase, which was pro-apoptotic in LNCaP but not HCT116 cells. Molecular requirements for cell death induction by therapy-relevant Tg analogs were identical to those observed with Tg. CONCLUSIONS Together, our results provide a new, integrated understanding of UPR signaling mechanisms and downstream mediators that induce cell death upon Tg-triggered, unmitigated ER stress. Video Abstract.

中文翻译：

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ER 应激源毒胡萝卜素诱导的细胞死亡涉及死亡受体 5、MAP1LC3B 的非自噬功能以及未折叠蛋白反应成分的独特贡献。


背景技术由未缓解的内质网（ER）应激引发的细胞死亡在生理学和疾病中发挥着重要作用，但诱导死亡的信号传导机制尚不完全清楚。为了更深入地了解这些机制，内质网应激源毒胡萝卜素 (Tg) 是一种实用的实验工具。此外，Tg 构成了设计用于靶向癌症治疗中细胞杀伤的类似前药的基础。 Tg 通过未折叠蛋白反应 (UPR) 诱导细胞凋亡，但细胞凋亡是如何启动的，以及各个 UPR 成分的个体效应如何整合尚不清楚。此外，自噬和自噬相关（ATG）蛋白的作用仍然难以捉摸。方法 为了系统地解决这些关键问题，我们使用 RNAi 和抑制药物来靶向死亡受体 UPR，分析了 Tg 和治疗相关的 Tg 类似物在两种不同来源的人类癌细胞系（LNCaP 前列腺癌细胞和 HCT116 结肠癌细胞）中的作用。成分和 ATG 蛋白，结合通过荧光成像和碘化丙啶染色测量细胞死亡，以及实时 RT-PCR 和蛋白质印迹来监测 caspase 活性、ATG 蛋白、UPR 成分和下游 ER 应激信号的表达。结果在两种细胞系中，Tg 诱导的细胞死亡依赖于死亡受体 5 和 caspase-8。最佳细胞毒性涉及 MAP1LC3B 上游的 procaspase-8 裂解的非自噬功能。 PERK、ATF4 和 CHOP 是 Tg 诱导的细胞死亡所必需的，但令人惊讶的是它们是并行作用的，而不是作为线性途径； ATF4 和 CHOP 是 Tg 介导的死亡受体 5 和 MAP1LC3B 蛋白上调所独立需要的，而 PERK 通过其他途径发挥作用。 有趣的是，IRE1 以细胞类型特异性的方式促进 Tg 诱导的细胞死亡。这与 c-Jun N 末端激酶的 XBP1 依赖性激活有关，该激酶在 LNCaP 细胞中促凋亡，但在 HCT116 细胞中则不然。治疗相关 Tg 类似物诱导细胞死亡的分子要求与用 Tg 观察到的相同。结论 总之，我们的结果提供了对 UPR 信号传导机制和下游介质的新的、综合的理解，这些下游介质在 Tg 触发的、未缓解的 ER 应激下诱导细胞死亡。视频摘要。