Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Hôpital Notre-Dame and Institut du Cancer de Montréal, Montreal, QC H2L 4M1, Canada.
Int J Oncol. 2011 Feb;38(2):313-24. doi: 10.3892/ijo.2010.881. Epub 2010 Dec 20.
A lysosomal pathway, characterized by the partial rupture or labilization of lysosomal membranes (LLM) and cathepsin release into the cytosol, is evoked during the early events of 20-S-camptothecin lactone (CPT)-induced apoptosis in human cancer cells, including human histiocytic lymphoma U-937 cells. These lysosomal events begin rapidly and simultaneously with mitochondrial permeabilization and caspase activation within 3 h after drug treatment. Recently, in a comparative proteomics analysis performed on highly-enriched lysosomal extracts, we identified proteins whose translocation to lysosomes correlated with LLM induction after CPT treatment, including protein kinase C-δ (PKC-δ). In this study, we show that the PKC-δ translocation to lysosomes is required for LLM, as silencing its expression with RNA interference or suppressing its activity with the inhibitor, rottlerin, prevents CPT-induced LLM. PKC-δ translocation to lysosomes is associated with lysosomal acidic sphingomyelinase (ASM) phosphorylation and activation, which in turn leads to an increase in ceramide (CER) content in lysosomes. The accumulation of endogenous CER in lysosomes is a critical event for CPT-induced LLM as suppressing PKC-δ or ASM activity reduces both the CPT-mediated CER generation in lysosomes and CPT-induced LLM. These findings reveal a novel mechanism by which PKC-δ mediates ASM phosphorylation/activation and CER accumulation in lysosomes in CPT-induced LLM, rapidly activating the lysosomal pathway of apoptosis after CPT treatment.
溶酶体途径的特征是溶酶体膜的部分破裂或不稳定（LLM）和组织蛋白酶释放到细胞质中，在人癌细胞中，包括人组织细胞淋巴瘤 U-937 细胞中，20-S-喜树碱内酯（CPT）诱导凋亡的早期事件中会被引发。这些溶酶体事件在药物处理后 3 小时内迅速与线粒体通透性和半胱天冬酶激活同时开始。最近，在对高度富集的溶酶体提取物进行的比较蛋白质组学分析中，我们鉴定了与 CPT 处理后 LLM 诱导相关的转位到溶酶体的蛋白质，包括蛋白激酶 C-δ（PKC-δ）。在这项研究中，我们表明 PKC-δ向溶酶体的转位对于 LLM 是必需的，因为用 RNA 干扰沉默其表达或用抑制剂罗特林抑制其活性可防止 CPT 诱导的 LLM。PKC-δ向溶酶体的转位与溶酶体酸性鞘磷脂酶（ASM）的磷酸化和激活相关，这反过来又导致溶酶体中神经酰胺（CER）含量的增加。溶酶体中内源性 CER 的积累是 CPT 诱导的 LLM 的关键事件，因为抑制 PKC-δ或 ASM 活性会降低 CPT 介导的溶酶体中 CER 的产生和 CPT 诱导的 LLM。这些发现揭示了 PKC-δ介导 ASM 磷酸化/激活和 CER 在 CPT 诱导的 LLM 中在溶酶体中的积累的新机制，在 CPT 处理后迅速激活溶酶体凋亡途径。
