Vilariño Natalia, Nicolaou K C, Frederick Michael O, Vieytes Mercedes R, Botana Luis M
Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus Universitario, 27002 Lugo, Spain.
Biochem Pharmacol. 2007 Jul 15;74(2):327-35. doi: 10.1016/j.bcp.2007.04.004. Epub 2007 Apr 7.
Azaspiracid-1 (AZA-1) is a marine toxin discovered in 1995. Besides damage to several tissues in vivo, AZA-1 has been shown to cause cytotoxicity in a number of cell lines and alterations in actin cytoskeleton and cell morphology. We studied the reversibility of AZA-1-induced morphological changes in human neuroblastoma cells and their dependence on caspases and signaling pathways involved in cytoskeleton regulation. Morphological/cytoskeletal changes were clearly observed by confocal microscopy 24h after the addition of toxin, without recovery upon toxin removal. Interestingly, 2min of incubation with AZA-1 was enough for the cytoskeleton to be altered 24-48h later. The activation of caspases by AZA-1 was studied next using a fluorescent caspase inhibitor. A cell population with activated caspases was observed after 48h of exposure to the toxin, but not at 24h. Two fragments and a stereoisomer of AZA-1 were tested to analyze structure-activity relationship. Only ABCD-epi-AZA-1 was active with a similar effect to AZA-1. Additionally, regarding the involvement of apoptosis/cytoskeleton signaling in AZA-1-induced morphological effects, inhibition of caspases with Z-VAD-FMK did not affect AZA-1-induced cytoskeletal changes, suggesting, together with the activation kinetics, that caspases are not responsible for AZA-1-elicited morphological changes. Modulation of PKA, PKC, PI3K, Erk, p38MAPK, glutathione and microtubules with inhibitors/activators did not inhibit AZA-1-induced actin cytoskeleton rearrangement. The JNK inhibitor SP600125 seemed to slightly diminish AZA-1 effects, however due to the effects of the drug by itself the involvement of JNK in AZA-1 toxicity needs further investigation. The results suggest that AZA-1 binds irreversibly to its cellular target, needing moieties located in the ABCDE and FGHI rings of the molecule. Cytotoxicity of AZA-1 has been previously described without reference to the type of cell death, we report that AZA-1 induces the activation of caspases, commonly used as an early marker of apoptosis, and that these proteases are not responsible for AZA-1-induced cytoskeleton disarragement in human neuroblastoma cells.
azaspiracid - 1（AZA - 1）是1995年发现的一种海洋毒素。除了对体内多个组织造成损害外，AZA - 1已被证明可在多种细胞系中引起细胞毒性，并导致肌动蛋白细胞骨架和细胞形态的改变。我们研究了AZA - 1诱导的人神经母细胞瘤细胞形态变化的可逆性，以及它们对胱天蛋白酶和参与细胞骨架调节的信号通路的依赖性。在添加毒素24小时后，通过共聚焦显微镜清楚地观察到形态学/细胞骨架的变化，去除毒素后未恢复。有趣的是，与AZA - 1孵育2分钟就足以使细胞骨架在24 - 48小时后发生改变。接下来使用荧光胱天蛋白酶抑制剂研究AZA - 1对胱天蛋白酶的激活作用。在暴露于毒素48小时后观察到有激活的胱天蛋白酶的细胞群体，但在24小时时未观察到。测试了AZA - 1的两个片段和一个立体异构体以分析构效关系。只有ABCD - epi - AZA - 1具有活性，其效果与AZA - 1相似。此外，关于凋亡/细胞骨架信号传导在AZA - 1诱导的形态学效应中的作用，用Z - VAD - FMK抑制胱天蛋白酶并不影响AZA - 1诱导的细胞骨架变化，结合激活动力学表明，胱天蛋白酶与AZA - 1引起的形态学变化无关。用抑制剂/激活剂调节蛋白激酶A（PKA）、蛋白激酶C（PKC）、磷脂酰肌醇 - 3激酶（PI3K）、细胞外信号调节激酶（Erk）、p38丝裂原活化蛋白激酶（p38MAPK）、谷胱甘肽和微管，均不能抑制AZA - 1诱导的肌动蛋白细胞骨架重排。JNK抑制剂SP600125似乎略微减弱了AZA - 1的作用，然而由于该药物本身的作用，JNK在AZA - 1毒性中的作用需要进一步研究。结果表明，AZA - 1与其细胞靶点不可逆结合，需要分子中位于ABCDE环和FGHI环的部分。之前已描述了AZA - 1的细胞毒性，但未提及细胞死亡类型，我们报告AZA - 1诱导了胱天蛋白酶的激活，胱天蛋白酶通常用作凋亡的早期标志物，并且这些蛋白酶与AZA - 1诱导的人神经母细胞瘤细胞骨架解体无关。
