Ostreocin-D Impact on Globular Actin of Intact Cells - American

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Articles Ostreocin-D Impact on Globular Actin of Intact Cells Isabel R. Ares,† Eva Cagide,† M. Carmen Louzao,† Begonã Espinã,† Mercedes R. Vieytes,‡ Takeshi Yasumoto,§ and Luis M. Botana*,† Departamento de Farmacologıá and Departamento de Fisiologıá Animal, Facultad de Veterinaria, UniVersidad de Santiago de Compostela, Campus de Lugo, 27002 Lugo, Spain, and Tama Laboratory, Japan Food Research Laboratories, Tokyo 206-0025, Japan ReceiVed July 29, 2008

Ostreocin-D, discovered in the past decade, is a marine toxin produced by dinoflagellates. It shares structure with palytoxin, a toxic compound responsible for the seafood intoxication named clupeotoxism. At the cellular level, the action sites and pharmacological effects for ostreocin-D are still almost unknown. Previously, we demonstrated that these toxins change the filamentous actin cytoskeleton, which is essential for multiple cellular functions. However, nothing has yet been reported about what happens with the unpolymerized actin pool. Here (i) the effects induced by ostreocin-D on unpolymerized actin, (ii) the Ca2+ role in such a process, and (iii) the cytotoxic activity of ostreocin-D on the human neuroblastoma BE(2)-M17 cell line are shown for the first time. Fluorescently labeled DNase I was used for staining of monomeric actin prior to detection with both laser-scanning cytometry and confocal microscopy techniques. Cellular viability was tested through a microplate metabolic activity assay. Ostreocin-D elicited a rearrangement of monomeric actin toward the nuclear region. This event was not accompanied by changes in its content. In addition, the presence or absence of external Ca2+ did not change these results. This toxin was also found to cause a decrease in the viability of neuroblastoma cells, which was inhibited by the specific blocker of Na+/K+-ATPase, ouabain. All these responses were comparable to those obtained with palytoxin under identical conditions. The data suggest that ostreocin-D modulates the unassembled actin pool, activating signal transduction pathways not related to Ca2+ influx in the same way as palytoxin. Introduction Ostreocin-D is a very toxic compound (LD50 ) 750 ng/kg ip in mice) produced by the dinoflagellate Ostreopsis siamensis (1). This molecule is a structural analogue of palytoxin (2, 3) (Figure 1), one of the most active and complex marine toxins related to seafood. Palytoxin induces a highly fatal intoxication named clupeotoxism, mainly associated with consumption of tropical fishes belonging to the Clupeidae and Engraulidae families (4, 5). Very recently, the first report revealing contamination of shellfish by palytoxins in European coastal waters was published (6). A new palytoxin analogue, named ovatoxina, has been identified from samples of the dinoflagellate Ostreopsis oVata collected during an outbreak on the Ligurian coast (Italy) (7). This suggests that the potential risk of seafood contaminated with palytoxin compounds is not restricted only to tropical areas, but is a global problem. Moreover, the real hazard of intoxication is not exclusive from palytoxin, but also it could be associated with its analogues (8-10). The alarming increase in the presence of palytoxin or palytoxin-like compounds makes it a priority to know in depth their toxic machinery. * To whom correspondence should be addressed. Phone and fax: +34 982 252 242. E-mail: [email protected]. † Departamento de Farmacologıá, Universidad de Santiago de Compostela. ‡ Departamento de Fisiologıá Animal, Universidad de Santiago de Compostela. § Japan Food Research Laboratories.

Contrary to the scarce available information about the biological activity of ostreocin-D, several aspects of the action mode of palytoxin have been described. In this way, it is well established that Na+/K+-ATPase is a high-affinity cellular action site for palytoxin. This molecule binds to Na+/K+-ATPase, and the pump is locked into a conformation that allows simultaneous opening of both gates, giving rise to characteristic channel activity (11-14). Ouabain, a cardiac glycoside and specific inhibitor of the sodium pump, blocks palytoxin action; conversely, palytoxin inhibits ouabain binding (15, 16). By altering the cell permeability for monovalent cations, palytoxin induces depolarization as a primary event in different systems (17-20). Intracellular Ca2+ movements are another of the well-known palytoxin-induced effects (21-24). It has been reported that the actin cytoskeleton undergoes constant remodeling in response to a variety of stimuli that include Ca2+ signals (25-28). Modifications of this complex structure as a consequence of such stimulations are possible owing to a large pool of unpolymerized actin that coexists in the cell with polymerized actin (29-31). Some conditions such as polyamine depletion or extracellular ATP as well as exposure to marine toxins such as pectenotoxin-2 or swinholide lead to alterations in both filamentous actin (F-actin)1 and globular actin (G-actin) in cells (32-34). Recently, we have demonstrated that ostreocin-D, and also palytoxin, changes quantitatively and 1 Abbreviations: AB, alamar blue; F-actin, filamentous actin; G-actin, globular actin; LSC, laser-scanning cytometry; LAT A, latrunculin A.

10.1021/tx800273f CCC: $40.75  2009 American Chemical Society Published on Web 01/20/2009

Ostreocin-D Impact on Globular Actin

qualitatively the polymeric actin of the human BE(2)-M17 cell line in a process partially dependent on Ca2+ entry (35). Currently, their effect on G-actin remains unknown. The present study was designed for characterizing, on the same cell line, the consequences of ostreocin-D action on the monomeric actin pool in comparison to that of palytoxin. The connection of these events to signaling pathways involving Ca2+ influx and the cytotoxic potential of both marine toxins in neuroblastoma cells were also determined.

Experimental Procedures Materials. Dr. Yasumoto (Japan Food Research Laboratories) kindly provided ostreocin-D and also a purified extract of the dinoflagellate O. oVata. This extract was obtained from a sample collected during an outbreak of this dinoflagellate that occurred from December 2001 to January 2002 on the Brazilian coast (36). Compounds from O. oVata were purified in a method similar to the those previously described for other Ostreopsis species (1, 3). Afterward, this extract was stored at -80 °C until analysis. Palytoxin from Palythoa caribaeorum was obtained from SigmaAldrich (Madrid, Spain). Deoxyribonuclease I-Texas red conjugate (Texas red-DNase I) used for labeling G-actin was from Molecular Probes (Leiden, The Netherlands). Latrunculin A (LAT A) was acquired from Sigma-Aldrich. Alamar Blue (AB) was from Biosource (Nivelles, Belgium). The culture medium, nonessential amino acids, gentamicin, and amphotericin B were purchased from Biochrom AG (Berlin, Germany). All other chemicals were reagent grade and purchased from Sigma-Aldrich, Panreac (Barcelona, Spain), or Merck (Darmstadt, Germany). Tissue culture flasks were from Nunc (Roskilde, Denmark) and 96-multi-well plates from Corning (Schiphol-Rijk, The Netherlands). Cellular Culture and Solutions. The human neuroblastoma cell line BE(2)-M17 was grown in EMEM/Ham’s F12 (1:1) supplemented with glutamine, 1% nonessential amino acids, 10% heatinactivated fetal bovine serum, 50 µg/mL gentamicin, and 50 ng/ mL amphotericin B. They were maintained and subcultured as described previously (35, 37, 38). The standard salt solution used for assessing G-actin contained 150 mM NaCl, 6 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose (pH 7.4). When the experiments were performed in nominally Ca2+-free solution, CaCl2 was not added. Fluorescent Labeling for G-Actin. When treatment with the toxins was complete, neuroblastoma cells on glass supports were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 for 5 min. After a brief washing with PBS, the preparations were blocked with 1% BSA/PBS solution for 30 min prior to the G-actin labeling with 9 µg/mL Texas red-DNase I (596 and 615 nm excitation and emission wavelengths, respectively) for 20 min in the dark. Finally, after washing with PBS twice, the samples were mounted on slides with a mixture of PBS/glycerol and their margins sealed. Measurement of the G-Actin Level with Laser-Scanning Cytometry (LSC). To quantify the fluorescence associated with G-actin, the slides were scanned using a laser-scanning cytometer (CompuCyte, Cambridge, MA) attached to a BX50 Olympus microscope. The LSC settings were optimized to avoid the loss of fluorescence information. Around 2000-3000 cells were examined per condition. For all events considered during scanning, the area and maximum red pixel fluorescence were recorded using WinCyte software. The diagrams displayed in the Results were directly extracted from LSC and are shown as representative experiments of each assay performed. Confocal Imaging Analysis. Fluorescence images of neuroblastoma cells fixed and stained with Texas red-DNase I were recorded using a 60× oil immersion objective of a Nikon Eclipse TE2000-E inverted microscope attached to a C1 laser confocal system (EZC1 V.2.20 software, Nikon Instruments Europe B.V., The Netherlands). They were captured as optical sections of a z-series at 0.5 µm

Chem. Res. Toxicol., Vol. 22, No. 2, 2009 375 intervals and collected in a mode format of 512 × 512 pixels. Simultaneous transmitted light was also collected in a separate channel. Metabolic Activity Assay. Neuroblastoma cells were cultured on 96-well plates (40 000 cells/well) 24 h prior to the experiment initiation, enough time for cell attachment to the bottom of the microplate. The cellular metabolic inhibition was directly assessed in the wells as a cytotoxic effect using the fluorescent probe AB. After addition of toxins and vehicle (triplicate assays), a dilution of 1:10 of AB was aseptically incorporated into the culture medium of each well. The fluorescence was measured at different toxin exposure times using an FL600 fluorescence plate reader (Bio-Tek, Vermont) at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Statistical Analysis. The statistical significance of the results was determined using Student’s t test. A P value of 0.05, Figure 4). This means that the quantity of unpolymerized actin within neuroblastoma cells remained unaltered in the presence of ostreocin-D or palytoxin. To verify our ability to detect changes in the G-actin quantity, LAT A was introduced as a positive control. This is a known drug that binds actin monomers and as a consequence moves the F-actin/ G-actin equilibrium in cells toward G-actin (34, 39-41). We analyzed the state of F-actin and G-actin after treatment with LAT A to test its anticytoskeletal effects on neuroblastoma cells. F-actin assays were performed utilizing fluorescently labeled phalloidin for labeling assembled actin and LSC for its detection, as previously described (35, 37). It was found that 500 nM LAT A caused F-actin depolymerization, with neuroblastoma cells losing 42 ( 2% of their actin cytoskeletons (data not shown). In addition, as was expected, this actin-disrupting drug induced a significant elevation of the G-actin content in neuroblastoma cells by 24 ( 8% above the control level (Figure 4). In confocal images, the cells appeared condensed; however, monomeric

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Figure 1. Molecular structure of palytoxin and ostreocin-D.

Figure 2. Images displaying G-actin staining patterns. Volume render projections corresponding to the fluorescence of control cells (A) and neuroblastoma cells incubated with ostreocin-D (B) or with palytoxin (C). For each case, to examine the regions where monomeric actin was located, transmissions of the same cells are shown as (D), (E), and (F). Scale bar 20 µm.

actin was homogeneously disposed through the whole cell, as occurred in the controls (Figure 4). Several lines of evidence have suggested that changes in intracellular Ca2+ can lead to alterations in the cytoskeleton (27, 42, 43). Interestingly, we have previously reported that ostreocin-D and palytoxin modify the cytosolic Ca2+ level of BE-M(17) human neuroblastoma cells, but only when external Ca2+ is present in the medium (35). Thus, our next step in the

present study was to verify whether the G-actin changes and Ca2+ influx evoked by these toxins could be linked. For this purpose, we incubated the cells with the toxins in a nominally Ca2+-free solution. After treatment of neuroblastoma cells with ostreocin-D or palytoxin in these conditions, confocal microscopy revealed that localization of their monomeric actin was almost indistinguishable from that exhibited in a Ca2+-containing medium (Figure 5). Also no significant modifications in the


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Figure 3. LSC detection of Texas red-DNase I bound to G-actin of human neuroblastoma cells. Scattergrams of red fluorescent signal recorded in control cells (A, D) and cells treated with 75 nM ostreocin-D (B) or with 75 nM palytoxin (E) for 4 h. Profiles of histograms derived from the area selected in the scattergrams compare the staining intensity of cells without and with ostreocin-D (C) or palytoxin (F).

Figure 4. (A) G-actin state in cells incubated with toxins or 500 nM LAT A. Percentage of fluorescent signal registered by LSC in neuroblastoma treated cells versus controls ()100%) (mean ( SEM of three assays). The asterisk indicates the result is significantly different from that of the control cells (P < 0.05). (B) The histogram extracted from LSC displays profiles reflecting the increase of the fluorescence intensity of LAT A-treated cells in comparison to untreated cells. Right (top): confocal pictures exhibit the increase in cellular staining after LAT A treatment. Transmission images of the same cells in each case are displayed in the bottom panels. Scale bar 20 µm.

quantity of G-actin detected by LSC were found compared to the controls (Figure 6). On the basis of these results, unpolymerized actin reorganization caused by ostreocin-D or palytoxin seems to be independent of Ca2+ mobilization. Although it is known that palytoxin causes metabolic alterations and decreases cellular viability in different models (20, 44-46), there is almost no information about the cytotoxic action of ostreocin-D. In Figure 7, the effect induced by

ostreocin-D (Figure 7A) and palytoxin (Figure 7B) on neuroblastoma cells measured with the metabolic activity dye AB is comparatively shown. The fall in the fluorescence intensity is related to a decrease in cellular viability. Therefore, we registered the effect of 75 and 15 nM concentrations of both toxins for 24 h, and as can be observed in Figure 7, ostreocin-D evoked a high toxicity comparable to that of palytoxin. In addition, since it is widely accepted that palytoxin acts on Na+/

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Figure 5. Visualization of G-actin of cells labeled with Texas red-DNase I in nominally Ca2+-free medium. Fluorescence (volume render projections) of control cells (A) and neuroblastoma cells incubated with ostreocin-D (B) or with palytoxin (C) for 4 h. For each case, transmissions of the same cells are shown as (D), (E), and (F). Scale bar 20 µm.

Figure 6. Analysis of G-actin in nominally Ca2+-free solution using LSC. The overlap of the profiles indicates similar fluorescence intensity between the controls and cells incubated with ostreocin-D (A) or palytoxin (B) for 4 h. (C) displays the quantification of fluorescence associated with G-actin in toxin-treated cells versus controls ()100%) (mean ( SEM of three assays).

Figure 7. Dose-dependent effect of ostreocin-D (A, filled symbols) and palytoxin (B, filled symbols) on human neuroblastoma viability and response inhibition induced by 400 µM ouabain (A, B, empty symbols). Neuroblastoma cells were incubated with the toxins and AB for 24 h. The results are expressed as the percentage of fluorescence versus controls ()100%). Mean ( SEM of experiments performed in triplicate.

K+-ATPase and ouabain is a specific inhibitor of this pump, we combined it with palytoxin, as well as with ostreocin-D, by preincubating the cells with oubain for 30 min and then adding the toxins. Figure 7A shows that ouabain inhibited ostreocin-D and palytoxin cytotoxicity in the same way (Figure 7B).

Finally, we also present the results of a preliminary assay using a purified extract of O. oVata on Clone 9 cells, a rat hepatic cell line widely used in metabolic studies (47). In a recent study by Espinã et al. (48), it was found that Clone 9 hepatocytes exhibited a high sensitivity, even greater than that of neuro-


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Table 1. Percentage of Cellular Viability Exhibited by Clone 9 Rat Hepatocytes after Incubation with an O. oWata Extracta time (h)

1:100 dilution

1:1000 dilution

1:10000 dilution

3 6 8 12 24

4.4 3 2.3 2 2

9.3 4.9 4.9 3.2 3.1

97 76 67.2 44.8 39

a Dilutions of 1:100, 1:1000, and 1:10000 of this sample were incubated with cells and AB for 3, 6, 8, 12, and 24 h. The results are expressed as the percentage of fluorescence versus controls ()100%). The decrease in the percentage of fluorescence reflects the cytotoxic effect of the extract.

blastoma cells, to the effect on cellular viability exerted by palytoxin and palytoxin-like compounds contained in Ostreopsis extracts. In this way, three different dilutions of our O. oVata extract were tested with AB for cytotoxicity on the Clone 9 cell line cultivated as previously specified (47). The experimental procedure was analogous to that utilized for neuroblastoma cells. As displayed in Table 1, all dilutions induced a clear reduction in the metabolic rate of Clone 9 cells after 24 h of incubation. The loss of cellular viability was shown to be progressive when the smallest quantity of sample was checked and almost whole in the case of 1:100 and 1:1000 dilutions after the first time of incubation, which seems to suggest that the extract action is time-and dose-dependent. Additional experiments were carried out to observe whether this cytotoxicity could also be inhibited by ouabain. Only 30 min of preincubation with ouabain was enough to reduce by 20% the inhibition elicited by 1:1000 and 1:10000 dilutions on the viability of Clone 9 hepatocytes (data not shown).

Discussion According to the specificity and sensitivity demonstrated by fluorescent conjugates of DNase I, they have become a valuable analysis tool for detecting and measuring unpolymerized actin in cells (49-51). Using this probe, we investigated the outcome of ostreocin-D (75 nM) on monomeric actin of living cells comparatively with palytoxin (75 nM). It was found that the stimulus evoked by these marine toxins on neuroblastoma cells led to modifications in their G-actin localization, similarly with both compounds and not associated with Ca2+ influx. Such alterations are condensation of unpolymerized actin in the nuclear area, mainly in the form of intense spots, which were present on all confocal z-sections (data not shown). This last observation could indicate that G-actin is arranged as either vertical rods or highly compacted actin aggregates. This phenomenon has already been considered in other studies with mast cells and mouse fibroblasts, where actin appeared to form complexes with cofilin, an actin-binding protein (52, 53). The past few years have brought evidence suggesting a regulated nuclear import and export of actin (54, 55). Some types of stress, such as heat shock and DMSO treatment or adenine nucleotides, are known to induce nuclear translocation of actin in various eukaryotic cells (33, 52, 56). The hypothesis of translocation fits well with our results, mainly bearing in mind that unpolymerized actin reorganization is not related to changes in its quantity. In fact, this could explain the sharp fall in cytoplasmic G-actin staining observed at the same time that condensation was detected in the nucleus of toxin-treated cells. In the transport from the cytoplasm to the nucleus of monomeric actin, cofilin seems to play a relevant role (53, 57). It is interesting to observe that ostreocin-D and palytoxin induce

translocation and compaction of G-actin in the nucleus and that cofilin has been reported to be implicated in both processes. Although, of course, our data do not probe a relation between these observations, they could be the starting point for new studies in this way. The assembly and disassembly of actin filaments is a dynamic process with continual dependent reversible noncovalent selfassociation of G-actin to the polymer at the barbed end and net release from the pointed end, which allows actin filaments to turn over and respond to appropriate stimuli (58, 59). In a previous report, we have determined that 4 h of treatment with ostreocin-D or palytoxin induced on the BE(2)-M17 cell line a sharp reduction (around 50%) in its polymerized actin (35). Then the loss of filaments triggered by both marine toxins would be expected to be associated with a corresponding increase in G-actin. Instead, and independently of the incubation conditions (with or without external Ca2+), our present study has revealed no substantial changes in unassembled actin. We wondered whether the failure to observe an elevation in this case could be related to artifacts during preparative steps to perform our experiments, such as loss of the monomers from the cell. However, the confirmation that G-actin molecules were stably retained in cells during such procedures was obtained with LAT A. This macrolide compound, which sequesters actin monomers and inhibits their assembly (39, 40), raised efficiently the level of nonassembled actin in neuroblastoma cells as expected. Therefore, a different explanation should support our findings. An unchanged G-actin content in the face of a filamentous actin decrease was found in cellular models exposed to bacterial toxins such as Clostridium botulinum exoenzyme C3, Clostridium difficile toxin B, and also Clostridium noVyi R-toxin (60-62). Another compound with similar effects is lovastatin (61), a fungal secondary metabolite, utilized as an anticholesterolemic drug (63). It has been suggested that, after treatment with these compounds, depolymerized actin could form oligomers too small to be detected as polymeric actin and too large to be recognized as G-actin by the DNase I probe; this would explain the lack of increase in the levels of monomeric actin. However, this phenomenon could also occur if the compound affects the degradation or synthesis of actin subunits. This last possibility is thought to be the most probable one in the case of cadmium, a toxic metal which also distorts the actin filament system without modifying the state of the G-actin pool (64). In any case, the activity of ostreocin-D and palytoxin on cells led to redistribution of unpolymerized actin to the nuclear area, even though for elucidating in which way they affect the G-actin levels more detailed pharmacological studies are required. The drop in cell viability observed in cells treated with ostreocin-D indicates that this toxin was toxic for neuroblastoma cells, with a potency similar to that of palytoxin. Cytotoxic activity was also detected for the extract obtained from O. oVata, which contains a putative palytoxin-like compound. On the other hand, 75 nM ostreocin-D induced a lower cytotoxicity than 75 nM palytoxin, pointing out that ostreocin-D could be slightly less potent. This lower potency agrees with the reported data about the toxicity after intraperitoneal injection in mammals, where palytoxin has a higher LD50 value (450 ng/kg) than ostreocin-D (750 ng/kg) (5). Na+/K+-ATPase is the specific target of heart glycosides and palytoxin (65-69). Palytoxin disturbs the function of this pump by transforming it in a permanently open ion channel and therefore varying the cellular homeostasis (11, 13, 15, 70). Ouabain is a heart glycoside that binds to Na+/K+-ATPase and inhibits the action of palytoxin. We found that incubation with ouabain prevented the cytotoxic

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effects induced by ostreocin-D in the same way as by palytoxin. From these results, and the high mortality rate evoked by both toxins, we observed that ostreocin-D exhibits a functional behavior similar to that of palytoxin. Actually, the inhibition induced by ouabain verifies the interaction with Na+/K+ATPase. Hence, this work confirms that ostreocin-D is a palytoxin analogue not only in a structural way, but also in a functional way. Acknowledgment. This work was funded with the following grants: contract grant sponsor Ministerio de Ciencia y Tecnologıá, Spain, Contract Grant Numbers AGL2006-08439/ ALI, AGL2005-23689-E, AGL2005-23687-E, AGL200408268-C02-02/ALI, AGL2007-60946/ALI, and REN200306598-C02-01; contract grant sponsor Xunta de Galicia, Spain, Contract Grant Numbers GRC 30/2006 PGIDIT05PXIC26101PM, PGIDIT05PXIC26102PM, PGIDIT05PXIC26102PN, PGIDIT04TAL261005PR, PGIDIT 07MMA006261PR, and PGIDT07CSA012261PR; EU VIth Frame Program, Grant Numbers IP FOOD-CT-2004-06988 (BIOCOP), STREP FOOD-CT-2004-514055 (DETECTOX), and CRP 030270-2 (SPIES-DETOX).

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Ostreocin-D Impact on Globular Actin of Intact Cells - American

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