MINIREVIEW Marine toxins and the cytoskeleton: pectenotoxins, unusual macrolides that disrupt actin Begon ˜ a Espin ˜ a 1 and Juan A. Rubiolo 1,2 1 Departamento de Farmacologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain 2 Departamento de Fisiologia Animal, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain The pectenotoxins (PTXs), macrolactones with multi- ple polyether ring units that have been shown to con- taminate shellfish in various parts of the world (Fig. 1) [1–7], were first isolated from the Japanese scallop Patinopecten yessoensis [8]. PTX-2 is produced by many species of the dinofla- gellate genus Dinophysis, and it was initially detected in Dinophysis fortii [9]. Later, this toxin was isolated from Dinophysis acuminate, Dinophysis norvegica, Din- ophysis rotundata and Dinophysis acuta [1–3,6,10–13]. After consumption of the algae by the shellfish, PTX-2 can be metabolized to other PTX derivatives. In the digestive gland of the scallop P. yessoensis, the C43 methyl group in PTX-2 is oxidized to the alcohol (PTX-1), aldehyde (PTX-3) and carboxylic acid (PTX- 6) forms [7,13]. Also, PTX-4 and PTX-7 have been isolated from the digestive glands of scallops collected in Japan, and these are stereoisomers of PTX-1 and PTX-6, respectively. After acid treatment of PTX-4 and PTX-7, two isomers, named PTX-8 and PTX-9 respectively, were formed. These last two toxins are not naturally occurring compounds, but artificial toxins generated during isolation or acid treatment [14]. On the other hand, in most bivalve species, PTX-2 is metabolized to PTX-2 seco acid (PTX-2 SA), in which the lactone ring of PTX-2 is hydrolyzed to Keywords actin cytoskeleton; biotoxins; cell line; dinoflagellates; hepatotoxicity; macrolide; mouse bioassay; pectenotoxin; primary culture; red tide Correspondence B. Espin˜ a, Departamento de Farmacologı ´ a, Facultad de Veterinaria, 27002 Lugo, Spain Fax: +34 982 252 242 Tel: +34 982 252 242 E-mail: begona.espina@usc.es (Received 7 July 2008, revised 4 September 2008, accepted 8 September 2008) doi:10.1111/j.1742-4658.2008.06714.x In recent years, many natural macrolactones have been found that display toxicity against the actin cytoskeleton. Pectenotoxins are macrolactones produced by species of the dinoflagellate genus Dinophysis. They were ini- tially classified within the diarrheic shellfish poisoning group of toxins, because of their co-occurrence and biological origin, but mice toxicity assays demonstrated that pectenotoxins do not induce diarrheic symptoms. Intraperitoneal injection of pectenotoxins into mice produces high hepato- toxicity as the principal symptom, so the liver seems to be their target organ. Up to now, 15 pectenotoxin analogs have been discovered, with dif- ferent toxicological potencies that are related to their structures. Now, it is generally accepted that the actin cytoskeleton is the principal molecular target of pectenotoxins. Although recent studies have demonstrated that pectenotoxins induce actin filament disruption by a capping effect, other kinds of activity, such as sequestration of actin, cannot be ruled out. All of the active analogs tested triggered disruption of the actin cytoskeleton and displayed potencies that correlated with their toxicity in mice. Moreover, pectenotoxins induce apoptosis to a higher degree in tumor cells than in normal cells of the same tissue. This fact opens the prospect of studying new chemotherapy agents and actin cytoskeleton dynamics with potential clinical applications. Abbreviations F-actin, filamentous actin; G-actin, globular actin; OA, okadaic acid; PTX, pectenotoxin; SA, seco acid. 6082 FEBS Journal 275 (2008) 6082–6088 ª 2008 The Authors Journal compilation ª 2008 FEBS the seco acid form. Epimerization of PTX-2 SA yields the thermodynamically more stable 7-epi-PTX-2 SA, and both have been detected in shellfish from Portugal [6], Ireland [2], New Zealand [4] and Croatia [3]. Besides PTX-2, four other oxidized forms of this toxin, PTX-11, PTX-12, PTX-13 and PTX-14, have been isolated from samples of Dinophysis [12,15–17]. PTX-11 is the 34S-hydroxy PTX-2 and is more resis- tant to enzymatic hydrolysis than PTX-2 when exposed to mussel hepatopancreas [17]. The PTX- 12 SA ⁄ PTX-12 ratio in naturally contaminated mussel samples is markedly lower than the PTX-2 SA ⁄ PTX-2 ratio [12], suggesting that PTX-12 is more resistant to hydrolysis by blue mussel enzymes than PTX-2 but not as resistant as PTX-11 [17]. PTX-13 is the 32a-hydroxy-PTX-2 whereas PTX-14 is a dehydro analog of PTX-13 derived by elimination of a water molecule between the 32- and 36-hydroxyl groups to form a C32–C36 ether linkage [15]. When these oxidized derivatives accumulate in scallops instead of PTX-2 SA, it is an indication that these shellfish lack the enzymes necessary to hydrolyze PTXs to their SAs, a process that is considered to be a detoxification mechanism (Fig. 1). PTXs were originally classified within the diarrheic shellfish poisoning group of toxins [8,18]. Earlier stud- ies indicated that PTX-2 produced severe diarrhea in mice [19] and that it was toxic after oral administra- tion [20]. It was also suggested that PTX-2 SA and 7-epi-PTX-2 SA were responsible for outbreaks of severe diarrheic illness in Australia [21], but it was later shown that the SAs employed in the dosing Fig. 1. Chemical structures of pectenotoxins. B. Espin˜ a and J. A. Rubiolo Actin cytoskeleton disruption by pectenotoxins FEBS Journal 275 (2008) 6082–6088 ª 2008 The Authors Journal compilation ª 2008 FEBS 6083 experiment were contaminated with okadaic acid esters, and the latter were considered to be responsible for the observed effects on the intestine [22]. Recent oral dosing studies failed to induce diarrhea or toxic- ity, even at doses as high as 5 mgÆkg )1 [17,23]. They also showed that PTXs are much less toxic via the oral route than via the intraperitoneal route. It is now accepted that the PTXs do not induce diarrhea [24,25]. PTXs are toxic to the liver when administered intra- peritoneally in mice, PTX-2 being the most potent. Oxidation of this toxin to PTX-1, PTX-3 and PTX-6 is accompanied by a decrease in toxicity [7]. PTX-11 has been reported to be as toxic as PTX-2, producing the same symptoms of intoxication in mice [17]. Ito et al. reported that the liver injuries produced by PTX-6 are different from those produced by PTX-2; whereas PTX-2 produced congestion under the liver capsule as a result of circulatory disorder, PTX-6 caused severe bleeding in the liver [26]. The first to establish the hepatotoxicity of these toxins were Terao et al., who showed that PTX-1 produced liver damage after intraperitoneal injection into mice, inducing necrosis of hepatocytes, principally in the periportal regions of the hepatic lobules [25]. Hepatocyte death was also observed by Fladmark et al. [27] in freshly isolated rat and salmon hepato- cytes, but in this case PTX-1 induced apoptosis rather than necrosis according to the chromatin hyperconden- sation, cell shrinkage and lack of Trypan blue uptake observed [27]. It has been shown that PTX-1, PTX-2, PTX-6 and PTX-11 disrupt the filamentous actin (F-actin) cytoskeleton in NRK-52E cells, rabbit enterocytes and neuroblastoma cells [28–31], and it is proposed that PTXs exert their toxicity by this mechanism. PTX structure–activity relationship Fifteen PTX analogs have been discovered and described up to now (Fig. 1), although only a few of them could be included in simultaneous comparative studies because of their scarcity. There are two routes of PTX transformation that are of particular interest, due to their natural occur- rence and implications: oxidation at C43, and lactone ring rupture. PTX-2, considered to be the parental compound, oxidizes progressively at C43 to PTX-1, PTX-3 and PTX-6 in the Japanese scallop P. yessoensis [7,13]. These chemical reactions may constitute a detoxifica- tion route, because toxicity in a mouse bioassay decreases in the order PTX-2 > PTX-1 > PTX-3 > PTX-6 [8]. However, oxidation at C34 does not change intraperitoneal toxicity: PTX-11 has the same LD 50 for mice as PTX-2 [17]. On the other hand, rupture of the lactone ring inac- tivates the PTX molecule [23,28]. Although preliminary studies indicated toxicological and pathological effects in mice after oral administration of PTX-2 SA and 7-epi-PTX-2 SA at high doses (up to 875 lgÆkg )1 ) [32], it was suggested that other contaminants from shellfish were responsible for this toxicity [22]. In fact, more recent investigations with highly purified PTX-2 SAs isolated from algae showed these compounds to be nontoxic to mice by the oral and intraperitoneal routes at 5000 lgÆkg )1 [23]. Toxicological studies indicated evident differences between PTX potencies that can be related to their particular structures (Fig. 1). Analogs isomerized so as to contain a six-membered B-ring, PTX-8 and PTX-9, are more than one order of magnitude less toxic than those containing a five-membered B-ring. They are believed to be artefacts produced during the purifica- tion process [14]. PTX-3 and PTX-6, the 7R-epimers of PTX-4 and PTX-7, are significantly less toxic than their corresponding 7S-epimers [7]. Only two in vitro studies have compared the effects of diverse PTXs on cells, and both agree with the above cited in vivo toxicological assays. Daiguji et al. reported that PTX-2 SA was not toxic to KB cells even at 1.8 lgÆmL )1 , whereas PTX-2 was cytotoxic at 0.05 lgÆmL )1 [10]. Ares et al. found that PTX-1, PTX-2 and PTX-11 modified the actin cytoskeleton and morphology of BE(2)-M17 neuroblastoma cells. However, PTX-2 SA did not show any effect on these cells. Moreover, PTX-2 and PTX-11 were more potent than PTX-1 (Fig. 2) [28]. This supports the idea that lactone ring integrity is essential for the action of PTXs; oxidation on C43 decrease the toxic- ity of the molecule, and oxidation on C34 does not affect the potency of PTXs. Unusual macrolides – antiactin drugs PTXs are unusual macrolides found in recent years that, to varying degrees, mimic the activity of endoge- nous actin-binding proteins [31]. Typically, a macro- cyclic lactone, a cetonic group and a glycosidic linked amino-sugar constitute the macrolide structure. Recent studies have demonstrated that the globular actin (G-actin)-binding and actin-filament-severing activity of some typical macrolides, such as kabiramide-C, reside in the glycosidic amino-sugar region of the molecule [33]. However, PTXs and the macrolactones described in the next paragraph lack this amino-sugar chain. Actin cytoskeleton disruption by pectenotoxins B. Espin˜ a and J. A. Rubiolo 6084 FEBS Journal 275 (2008) 6082–6088 ª 2008 The Authors Journal compilation ª 2008 FEBS Although they share a common structure and target, these antiactin drugs display diverse mechanisms of action. Latrunculins specifically sequester monomeric actin, mimicking proteins such as b-thymosins and inhibiting polymerization of G-actin and promoting depolymerization of F-actin [31,34]. Mycalolide-B and aplyronine-A inhibit polymerization of purified actin, apparently by sequestration of monomeric actin and actin severing because of the rapid depolymerization that they induce [31,34]. However, jasplakinolides induce actin polymerization, stabilizing actin filaments and actin nucleation [35]. Another group of macrolides, the halichondramides, exhibit barbed-end capping and F-actin-severing activity. Moreover, unusual dimeric macrolides, swinholide-A and misakinolide-A, were, surprisingly, found to display different effects on actin dynamics; swinkolide-A severs actin, whereas misakino- lide-A rapidly caps barbed ends of filaments [31]. The actin cytoskeleton – molecular target of PTXs Initial studies performed to elucidate the mechanism of action of PTXs and their in vitro biochemical effects demonstrated that PTX-2 inhibited actin polymeriza- tion in a concentration-dependent manner [36]. PTX-2 also inhibited contractions induced by KCl in the isolated rat aorta, and formed a 1 : 4 complex with G-actin [36,37]. Spector et al. reported that PTX-2 sequestered monomeric actin with a K d of 20 nm, but did not exhibit severing or capping activity. Moreover, PTX-2 disrupted the organization of actin in several cell types in a time- and concentration-dependent manner [31]. In a recent study, Allingham et al. obtained the X-ray structure of the PTX-2–actin complex. In con- trast to the results of Hori et al., they described a 1 : 1 stoichiometry in a novel site between subdomains 1 and 3 of the actin molecule [38]. In cells, capping pro- teins such as gelsolin play an important role; capping of old filaments funnels actin polymerization to sites where new barbed ends are present, and the generation of new barbed ends relies on the nucleation mechanism [39]. But PTXs could also sequester actin monomers binding to G-actin [36] in a similar way to latrunculin- A [28,31], avoiding new actin polymerization or nucle- ation. On the basis of models of the actin filament, PTX binding would disrupt key lateral contacts between the PTX-bound actin monomer and the lower lateral actin monomer within the filament, thereby cap- ping the barbed end without inducing filament sever- ing, even though, on the basis of the previous work of Spector et al., an actin-sequestering effect could not be ruled out as another possible action (Fig. 3). Fig. 3. Simplified hypothetical PTX mechanism of action. Two dif- ferent activities on the actin filaments can be attributed to PTXs: capping of the growing (+) barbed end of F-actin by binding of the PTX molecule to the lower lateral actin monomer within the filament, and G-actin monomer sequestration. Fig. 2. Effects of diverse PTX analogs on the actin cytoskeleton of BE(2)-M17 human neuroblastoma cells. Full projection of Z-ser- ies captured with confocal microscopy of cells in which F-actin was labeled with Ore- gon Green 514 Phalloidin. Control cells (A) and cells incubated for 4 h with 200 n M PTX-11 (B), PTX-2 (C), PTX-1 (D) or PTX-2 SA (E). Figure modified from [28]. Scale bar: 50 lm. B. Espin˜ a and J. A. Rubiolo Actin cytoskeleton disruption by pectenotoxins FEBS Journal 275 (2008) 6082–6088 ª 2008 The Authors Journal compilation ª 2008 FEBS 6085 Cytological implications of disruption of the actin cytoskeleton Although, according to the histopathological studies, the liver seems to be the target organ of PTXs, many cellular models have shown substantial effects of PTX treatments in the nanomolar range [27,40–42]. The induction of apoptotic cell death rather than necrosis by PTX-1 and PTX-2 is well established, on the basis of morphological changes, reduction of mitochondrial membrane potential, increases in cytoplasmic cyto- chrome c and Smac ⁄ DIABLO, and caspase-3 and caspase-9 activation. The inhibition of PTX-induced cell death by caspase inhibitors supports the apoptotic pathway [27,40]. Actin polymerization and dynamics are required for essential cell processes such as motility, endocytosis, cytokinesis, and establishing cell–cell and cell–substrate contacts. In vitro and in vivo studies showed that some macrolide–actin complexes reduced free G-actin below the critical concentration and blocked extension of the (+)-end of the filament, while the free macrolide also bound to and severed actin filaments [43]. In living cells, the combined effects of these interactions leads to a cessation of new filament growth and a disruption of existing filaments, and is accompanied by a loss of motility, breakdown of adherens junctions, polyploidy and, ultimately, apoptosis [43]. Very few studies have compared the effects of PTXs in diverse cellular models. Chae et al. found that PTX-2 induced apoptosis in p53-deficient cells [40], showing enhanced cytotoxicity as compared with normal cells. Recently, Espin ˜ a et al. tested the effect of PTXs on actin cytoskeleton and cell viability in two hepatic cellu- lar models [44]. Interesting results point to different effects of PTXs in Clone 9 cells, a rat hepatocyte cell line used previously in metabolic and cancer studies [45], and primary cultured rat hepatocytes (Fig. 4). Although primary rat hepatocytes, as well as Clone 9 cells, showed a marked change in the pattern of distri- bution and a decrease in polymerized actin after the treatment with PTX-2, no morphological effects could be observed in primary hepatocytes, whereas Clone 9 cells were contracted and rounded. The higher sensitiv- ity of Clone 9 cells to PTXs in comparison to normal hepatocytes could be related to the fact that the cell lines share cytoskeletal and morphological characteris- tics with cancerous cells. In accordance with this fact, the tumor cells of the study of Chae et al. were more sensitive to PTX-2 than normal cells [44]. Fig. 4. Effect of PTX-2 on the actin cyto- skeleton of two hepatic cellular models. Clone 9 cells (A, B) and primary cultured rat hepatocytes (C, D) were incubated for 3 h with 200 n M PTX-2 (B, D) or with the toxin vehicle (control; A, C). Then, they were stained for F-actin and G-actin with Oregon Green 514 Phalloidin and Texas Red DNAse I, and visualized with a confocal microscope. Scale bar: 50 lm. Actin cytoskeleton disruption by pectenotoxins B. Espin˜ a and J. A. Rubiolo 6086 FEBS Journal 275 (2008) 6082–6088 ª 2008 The Authors Journal compilation ª 2008 FEBS Perspectives The importance of the actin cytoskeleton in pathogenic cellular process such as angiogenesis, cell adhesion, cytokinesis and metastasis has made it an attractive target for the development of anticancer drugs. Drugs that block the regulation of actin filament dynamics within tumor cells or in cells infected with pathogens could be useful in treating cancer and other diseases. In addition, differential actions of antiactin com- pounds can reveal interesting data about actin cyto- skeleton dynamics. Although the presence in the food chain of PTXs is being regulated, together with that of diarrhetic shell- fish poisoning toxins, toxicological tests by oral admin- istration point to these compounds as nonhazardous molecules for human health with regard to shellfish consumption. However, the selective cytotoxicity of PTXs against certain tumor cell lines and their ability to induce apoptosis of p53-deficient cell lines, which are often resistant to other chemotherapeutic agents, makes them interesting new candidates for cancer therapy. References 1 Draisci R, Lucentini L, Giannetti L, Boria P & Poletti R (1996) First report of pectenotoxin-2 (PTX-2) in algae (Dinophysis fortii) related to seafood poisoning in Europe. Toxicon 34, 923–935. 2 James KJ, Bishop AG, Draisci R, Palleschi L, Marchiaf- ava C, Ferretti E, Satake M & Yasumoto T (1999) Liquid chromatographic methods for the isolation and identifi- cation of new pectenotoxin-2 analogues from marine phytoplankton and shellfish. J Chromatogr A 844, 53–65. 3 Pavela-Vrancic M, Mestrovic V, Marasovic I, Gillman M, Furey A & James KK (2001) The occurrence of 7-epi-pectenotoxin-2 seco acid in the coastal waters of the central Adriatic (Kastela Bay). Toxicon 39, 771–779. 4 Suzuki T, Mackenzie L, Stirling D & Adamson J (2001) Pectenotoxin-2 seco acid: a toxin converted from pecte- notoxin-2 by the New Zealand Greenshell mussel, Perna canaliculus. Toxicon 39, 507–514. 5 Suzuki T, MacKenzie L, Stirling D & Adamson J (2001) Conversion of pectenotoxin-2 to pectenotoxin-2 seco acid in the New Zealand scallop, Pecten novaeze- landiae. Fish Sci 67, 506–510. 6 Vale P & Sampayo MA de M (2002) Pectenotoxin-2 seco acid, 7-epi-pectenotoxin-2 seco acid and pectenotoxin-2 in shellfish and plankton from Portugal. Toxicon 40, 979–987. 7 Yasumoto T, Murata M & Lee J (1989) Polyether toxins produced by dinoflagellates. In Mycotoxins and Phycotoxins ‘88 (Natori S, Hashimoto K & Ueno Y, eds), pp. 375–382. Elsevier, Amsterdam. 8 Yasumoto T, Murata M, Oshima Y, Sano M, Matsum- oto Gk & Clardy J (1985) Diarrheic shellfish toxins. Tetrahedron 41, 1019–1025. 9 Lee JS, Yanagi Y, Kenma R & Yasumoto T (1987) Fluorometric determination of diarrhetic shellfish toxins by high performance liquid chromatography. Agricul Biol Chem 51, 877–881. 10 Daiguji M, Satake M, James KJ, Bishop AG, MacKen- zie L, Naoki H & Yasumoto T (1998) Structures of new pectenotoxin analogs, pectenotoxin-2 seco acid and 7-epi-2 seco acid, isolated from a dinoflagelate and greenshell mussels. Chem Lett 7, 653–654. 11 MacKenzie L, Holland P, McNabb P, Beuzenberg V, Selwood A & Suzuki T (2002) Complex toxin profiles in phytoplankton and Greenshell mussels (Perna canalicu- lus), revealed by LC-MS ⁄ MS analysis. Toxicon 40, 1321–1330. 12 Miles CO, Wilkins AL, Samdal IA, Sandvik M, Petersen D, Quilliam MA, Naustvoll LJ, Rundberget T, Torgersen T, Hovgaard P et al. (2004) A novel pecteno- toxin, PTX-12, in Dinophysis spp. and shellfish from Norway. Chem Res Toxicol 17, 1423–1433. 13 Suzuki T, Mitsuya T, Matsubara H & Yamasaki M (1998) Determination of pectenotoxin-2 after solid- phase extraction from seawater and from the dinoflagel- late Dinophysis fortii by liquid chromatography with electrospray mass spectrometry and ultraviolet detec- tion. Evidence of oxidation of pectenotoxin-2 to pecte- notoxin-6 in scallops. J Chromatogr A 815, 155–160. 14 Sasaki K, Wright JL & Yasumoto T (1998) Identifica- tion and characterization of pectenotoxin (PTX) 4 and PTX7 as spiroketal stereoisomers of two previously reported pectenotoxins. J Org Chem 63, 2475–2480. 15 Miles CO, Wilkins AL, Hawkes AD, Jensen DJ, Selwood AI, Beuzenberg V, Mackenzie AL, Cooney JM & Holland PT (2006) Isolation and identification of pectenotoxins-13 and -14 from Dinophysis acuta in New Zealand. Toxicon 48, 152–159. 16 Suzuki T, Beuzenberg V, Mackenzie L & Quilliam MA (2003) Liquid chromatography–mass spectrometry of spiroketal stereoisomers of pectenotoxins and the analy- sis of novel pectenotoxin isomers in the toxic dinoflagel- late Dinophysis acuta from New Zealand. J Chromatogr A 992, 141–150. 17 Suzuki T, Walter JA, LeBlanc P, MacKinnon S, Miles CO, Wilkins AL, Munday R, Beuzenberg V, MacKenzie AL, Jensen DJ et al. (2006) Identification of pecteno- toxin-11 as 34S-hydroxypectenotoxin-2, a new pecteno- toxin analogue in the toxic dinoflagellate Dinophysis acuta from New Zealand. Chem Res Toxicol 19, 310–318. 18 Yasumoto T & Murata M (1993) Marine toxins. Chem Rev 93, 1897–1909. 19 Ishige M, Satoh N & Yasumoto T (1988) Pathological studies on the mice administration with the causative agent of diarrhetic shellfish poisoning (okadaic acid and B. Espin˜ a and J. A. Rubiolo Actin cytoskeleton disruption by pectenotoxins FEBS Journal 275 (2008) 6082–6088 ª 2008 The Authors Journal compilation ª 2008 FEBS 6087 pectenotoxin-2). Hokkaidoritsu Eisei Kenkyushoho 38, 15–18. 20 Ogino H, Kumagai M & Yasumoto T (1997) Toxico- logic evaluation of yessotoxin. Nat Toxins 5, 255–259. 21 Burgess V & Shaw G (2001) Pectenotoxins – an issue for public health: a review of their comparative toxicol- ogy and metabolism. Environ Int 27, 275–283. 22 Burgess V & Shaw G (2003) Investigations into the tox- icology of pectenotoxin-2-seco acid and 7-epi pecteno- toxin-2 seco acid to aid in a health risk assessment for the consumption of shellfish contaminated with these shellfish toxins in Australia. Report on Project No. 2001/258. National Research Centre for Environmental Toxicology, Archerfield, Australia. 23 Miles CO, Wilkins AL, Munday R, Dines MH, Hawkes AD, Briggs LR, Sandvik M, Jensen DJ, Cooney JM, Holland PT et al. (2004) Isolation of pectenotoxin-2 from Dinophysis acuta and its conversion to pecteno- toxin-2 seco acid, and preliminary assessment of their acute toxicities. Toxicon 43, 1–9. 24 Hamano Y, Kinoshita Y & Yasumoto T (1985) Suck- ling mice assay for diarrhetic shellfish toxins. In Toxic Dinoflagellates (Anderson DM, White AW & Baden DB, eds), pp. 383–388. Elsevier, Amsterdam. 25 Terao K, Ito E, Yanagi T & Yasumoto T (1986) Histo- pathological studies on experimental marine toxin poi- soning. I. Ultrastructural changes in the small intestine and liver of suckling mice induced by dinophysistoxin-1 and pectenotoxin-1. Toxicon 24, 1141–1151. 26 Ito E, Suzuki T, Oshima Y & Yasumoto T (2008) Stud- ies of diarrhetic activity on pectenotoxin-6 in the mouse and rat. Toxicon 51, 707–716. 27 Fladmark KE, Serres MH, Larsen NL, Yasumoto T, Aune T & Doskeland SO (1998) Sensitive detection of apoptogenic toxins in suspension cultures of rat and salmon hepatocytes. Toxicon 36 , 1101–1114. 28 Ares IR, Louzao MC, Espina B, Vieytes MR, Miles CO, Yasumoto T & Botana LM (2007) Lactone ring of pecte- notoxins: a key factor for their activity on cytoskeletal dynamics. Cell Physiol Biochem 19, 283–292. 29 Ares IR, Louzao MC, Vieytes MR, Yasumoto T & Botana LM (2005) Actin cytoskeleton of rabbit intes- tinal cells is a target for potent marine phycotoxins. J Exp Biol 208, 4345–4354. 30 Leira F, Cabado AG, Vieytes MR, Roman Y, Alfonso A, Botana LM, Yasumoto T, Malaguti C & Rossini GP (2002) Characterization of F-actin depolymerization as a major toxic event induced by pectenotoxin-6 in neuroblastoma cells. Biochem Pharmacol 63, 1979–1988. 31 Spector I, Braet F, Shochet NR & Bubb MR (1999) New anti-actin drugs in the study of the organization and function of the actin cytoskeleton. Microsc Res Tech 47, 18–37. 32 Burgess VA, Seawright A, Eaglesham G, Shaw G & Moore M (2002) The acute oral toxicity of the shellfish toxins pectenotoxin-2 seco acid and 7-epi-pectenotoxin- 2 seco acid in mice. 4th International Conference on Molluscan Shellfish Safety. Santiago de Compostela, Spain. 33 Perrins RD, Cecere G, Peterson I & Marriot G (2008) Synthetic mimetics of actin-binding macrolides: rational design of actin-targeted drugs. Chem Biol 15, 287–294. 34 Yarmola EG, Somasundaram T, Boring TA, Spector I & Bubb MR (2000) Actin–latrunculin A structure and function. Differential modulation of actin-binding protein function by latrunculin A. J Biol Chem 275, 28120–28127. 35 Bubb MR, Spector I, Beyer BB & Fosen KM (2000) Effects of jasplakinolide on the kinetics of actin polymerization. An explanation for certain in vivo observations. J Biol Chem 275, 5163–5170. 36 Hori M, Matsuura Y, Yoshimoto R, Ozaki H, Yasumoto T & Karaki H (1999) Actin depolymerizing action by marine toxin, pectenotoxin-2. Nippon Yakurigaku Zasshi 114(Suppl. 1), 225P–229P. 37 Karaki H, Matsuura Y, Hori M, Yoshimoto R, Ozaki H & Yasumoto T (1999) Pectenotoxin-2, a new actin depolymerizing compound isolated from scallop Patino- pecten yessoensis. Jpn J Pharmacol 79, 268. 38 Allingham JS, Miles CO & Rayment I (2007) A struc- tural basis for regulation of actin polymerization by pectenotoxins. J Mol Biol 371, 959–970. 39 Giganti A & Friederich E (2003) The actin cytoskeleton as a therapeutic target: state of the art and future direc- tions. Prog Cell Cycle Res 5, 511–525. 40 Chae HD, Choi TS, Kim BM, Jung JH, Bang YJ & Shin DY (2005) Oocyte-based screening of cytokinesis inhibitors and identification of pectenotoxin-2 that induces Bim ⁄ Bax-mediated apoptosis in p53-deficient tumors. Oncogene 24, 4813–4819. 41 Jung JH, Sim CJ & Lee CO (1995) Cytotoxic com- pounds from a two-sponge association. J Nat Prod 58, 1722–1726. 42 Zhou ZH, Komiyama M, Terao K & Shimada Y (1994) Effects of pectenotoxin-1 on liver cells in vitro. Nat Toxins 2, 132–135. 43 Tanaka J, Yan Y, Choi J, Bai J, Klenchin VA, Rayment I & Marriott G (2003) Biomolecular mimicry in the actin cytoskeleton: mechanisms underlying the cytotoxicity of kabiramide C and related macrolides. Proc Natl Acad Sci USA 100, 13851–13856. 44 Espin ˜ a B, Louzao MC, Ares IR, Cagide E, Vieytes MR, Vega FV, Rubiolo JA, Miles CO, Suzuki T, Yasumoto T et al. (2008) Cytoskeletal toxicity of pectenotoxins in hepatic cells. Br J Pharmacol, doi: 10.1038/bjp.2008.323. 45 Louzao MC, Espina B, Vieytes MR, Vega FV, Rubiolo JA, Baba O, Terashima T & Botana LM (2007) ‘Fluo- rescent glycogen’ formation with sensibility for in vivo and in vitro detection. Glycoconj J 25, 503–510. Actin cytoskeleton disruption by pectenotoxins B. Espin˜ a and J. A. Rubiolo 6088 FEBS Journal 275 (2008) 6082–6088 ª 2008 The Authors Journal compilation ª 2008 FEBS . MINIREVIEW Marine toxins and the cytoskeleton: pectenotoxins, unusual macrolides that disrupt actin Begon ˜ a Espin ˜ a 1 and Juan A. Rubiolo 1,2 1. not affect the potency of PTXs. Unusual macrolides – antiactin drugs PTXs are unusual macrolides found in recent years that, to varying degrees, mimic the activity