Irreversible cytoskeletal disarrangement is independent of caspase activation during in vitro azaspiracid toxicity in human neuroblastoma cells

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 cytotoxicit)y in a number of cell lines and alterations in actin cyl:oskeleton 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 24 h after the addition of toxin, without recovery upon toxin removal. Interestingly, 2 min of incubation with AZA-1 was enough for the cytoskeleton to be altered 24-48 h 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 48 h of exposure to the toxin, but not at 24 h. Two fragments and a stereoisomer of AZA-1 were tested to analyze structure-activity relationship. Only ABCD-epi-AZA- I 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 inhuman neuroblastoma. cells. (C) 2007 Elsevier Inc. All rights reserved.