title: Irreversible cytoskeletal disarrangement is independent of caspase activation during in vitro azaspiracid toxicity in human neuroblastoma cells.
abstract: 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.
authors.affiliation: Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus Universitario, 27002 Lugo, Spain.
authors.forename: Natalia
authors.initials: N
authors.lastname: Vilariño
authors.forename: K C
authors.initials: KC
authors.lastname: Nicolaou
authors.forename: Michael O
authors.initials: MO
authors.lastname: Frederick
authors.forename: Mercedes R
authors.initials: MR
authors.lastname: Vieytes
authors.forename: Luis M
authors.initials: LM
authors.lastname: Botana
mesh_terms: D020169:Caspases / Q000502:physiology*; D045744:Cell Line, Tumor; D003599:Cytoskeleton / Q000187:drug effects*; D004789:Enzyme Activation; D006801:Humans; D008387:Marine Toxins / Q000633:toxicity*; D009447:Neuroblastoma / Q000473:pathology; D015398:Signal Transduction / Q000187:drug effects; D013141:Spiro Compounds / Q000633:toxicity*; D000095702:Polyether Toxins
chemical_list: D020169:Caspases; D008387:Marine Toxins; D013141:Spiro Compounds; C406592:azaspiracid; D000095702:Polyether Toxins