Vilariño Natalia, Nicolaou K C, Frederick Michael O, Cagide Eva, Ares Isabel R, Louzao M Carmen, Vieytes Mercedes R, Botana Luis M
Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain.
Chem Res Toxicol. 2006 Nov;19(11):1459-66. doi: 10.1021/tx060131z.
Azaspiracid-1 (AZA-1) is a marine toxin discovered 10 years ago. Since then, toxicologic studies have demonstrated that AZA-1 targets several organs in vivo, including the intestine, lymphoid tissues, lungs, and nervous system; however, the mechanism of action of AZA-1 remains unknown. Studies in vitro suggest that AZA-1 affects the actin cytoskeleton in nonadherent cells. We characterized the effects of AZA-1 on the cytoskeleton of adherent cells and on cell growth, an adhesion-dependent process in many cell types, and analyzed the structure dependency of this toxicity. Confocal and TIRF imaging of fluorescently labeled cytosketon showed that AZA-1 induced the rearrangement of stress fibers (actin filament bundles) and the loss of focal adhesion points in neuroblastoma and Caco-2 cells, without affecting the amount of polymerized actin. AZA-1 did not seem to alter the microtubule cytoskeleton, but it changed the cell shape and internal morphology observed by phase contrast imaging. Cell growth of lung carcinoma and neuroblastoma cells was inhibited by the toxin, as measured by a sulforhodamine B assay and BrdU incorporation to newly synthesized DNA. Fifteen different fragments and/or stereoisomers of AZA-1 were tested for cytoskeletal rearrangement and cell growth inhibition. Results showed that no fragment or stereoisomer had any activity, except for ABCD-epi-AZA-1, which conserved toxicity. AZA-1-induced reorganization of the actin cytoskeleton concurred with detachment and growth inhibition, three events that are probably related.
azaspiracid-1（AZA-1）是10年前发现的一种海洋毒素。从那时起，毒理学研究表明，AZA-1在体内靶向多个器官，包括肠道、淋巴组织、肺和神经系统；然而，AZA-1的作用机制仍然未知。体外研究表明，AZA-1会影响非贴壁细胞中的肌动蛋白细胞骨架。我们研究了AZA-1对贴壁细胞骨架以及细胞生长（许多细胞类型中依赖黏附的过程）的影响，并分析了这种毒性的结构依赖性。对荧光标记细胞骨架进行的共聚焦和全内反射荧光成像显示，AZA-1可诱导神经母细胞瘤和Caco-2细胞中的应力纤维（肌动蛋白丝束）重排以及粘着斑的丧失，而不影响聚合肌动蛋白的量。AZA-1似乎不会改变微管细胞骨架，但会改变相差成像观察到的细胞形状和内部形态。通过磺酰罗丹明B测定法和BrdU掺入新合成的DNA来测量，该毒素可抑制肺癌和神经母细胞瘤细胞的生长。对AZA-1的15种不同片段和/或立体异构体进行了细胞骨架重排和细胞生长抑制测试。结果表明，除保留毒性的ABCD-表-AZA-1外，没有片段或立体异构体具有任何活性。AZA-1诱导的肌动蛋白细胞骨架重排与脱离和生长抑制同时发生，这三个事件可能是相关的。
