Cell Growth Inhibition and Actin Cytoskeleton ... - ACS Publications

Chem. Res. Toxicol. 2006, 19, 1459-1466

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Cell Growth Inhibition and Actin Cytoskeleton Disorganization Induced by Azaspiracid-1 Structure-Activity Studies Natalia Vilarinõ,† K. C. Nicolaou,‡,§ Michael O. Frederick,‡ Eva Cagide,† Isabel R. Ares,† M. Carmen Louzao,† Mercedes R. Vieytes,| and Luis M. Botana*,† Departamento de Farmacologıá, Facultad de Veterinaria, UniVersidad de Santiago de Compostela, 27002 Lugo, Spain, Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, Department of Chemistry and Biochemistry, UniVersity of California, San Diego, 9500 Gilman DriVe, La Jolla, California 92093, and Departamento de Fisiologıá, Facultad de Veterinaria, UniVersidad de Santiago de Compostela, 27002 Lugo, Spain ReceiVed June 19, 2006

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. Introduction In 1995, an outbreak of poisoning in Killary Harbour, Ireland, related to the consumption of mussels (Mytilus edulis) was reported (1, 2). The poisoning would become known as azaspiracid poisoning (AZP) with symptoms similar to diarrhetic shellfish poisoning (DSP), including diarrhea, headache, nausea, vomiting, and stomach cramps. Toxicity studies in mice revealed several organs affected by azaspiracid intoxication, including injury to the intestine, with atrophic lamina propria and shortening and erosion of villi, necrosis of lymphoid tissues and fatty liver after oral administration, and lung tumors after chronic administration (3, 4). Additionally, mice developed neural symptoms such as spasm and paralysis to the limbs after intraperitoneal administration (5). The causative toxins are known as the azaspiracids, and the structure of azaspiracid-1 (AZA-1)1 would first be proposed by Satake. A total synthesis of the originally proposed structure by the Nicolaou group, however, would prove the structure incorrect (6, 7). Through degradative and synthetic studies, the * Corresponding author: Tel: 34-982 252 242. Fax: 34-982 252 242. E-mail: [email protected]. † Departamento de Farmacologıá, Universidad de Santiago de Compostela. ‡ The Scripps Research Institute. § University of California, San Diego. | Departamento de Fisiologıá, Universidad de Santiago de Compostela. 1 Abbreviations: AZA-1, azaspiracid-1; EMEM, Eagle’s minimum essential medium; HBSS, Hank’s balanced salt solution; LSC, laser scanner cytometry.

Nicolaou group would prove the correct structure of AZA-1 to be that seen in Table 1 (8-12). In total, 11 azaspiracids have been isolated, with each differing by the presence or absence of methyl and hydroxyl groups through the backbone of AZA-1 (2, 13-16). The mechanism of action of the azaspiracids is not known yet, and although symptoms are similar to DSP toxins, AZA-1 does not inhibit protein phospatase 2A, a well-known target of okadaic acid and its analogs (17). An involvement of AZA-1 in the regulation of the microfilament cytoskeleton has been suggested because of an AZA-1-induced reduction in the number of pseudopodia (plasmatic membrane protrusions) in Jurkat T cells (17). Additionally, high doses of AZA-1 seemed to reduce the amount of polymerized actin in neuroblastoma cells (18). AZA-1 also has cytotoxic effects in several cell lines at doses in the nanomolar range (17); however, variability is prominent among cell lines. Regarding signaling events, the AZAs have been shown to modify calcium signaling and cAMP levels in human lymphocytes (18-20). Many natural toxins alter actin cytoskeleton organization in different ways, probably due to the involvement of microfilaments in multiple aspects of cell physiology (21-24). Microfilaments provide structural support to the cell body but also participate in cell movement, contraction, organelle trafficking, and attachment to the matrix. Because of the neurological and intestinal toxicity observed in ViVo with AZA-1, we chose to study the effects AZA-1 has on the cytoskeleton of two adherent cell lines, neuroblastoma BE(2)-M17 and Caco-2 intestinal cells. Because the induction of lung tumors in mice suggests that AZA-1 may be tumorigenic, we also analyzed the effects of

10.1021/tx060131z CCC: $33.50 © 2006 American Chemical Society Published on Web 10/17/2006

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Table 1. Chemical Structure of AZA-1 and 15 Molecules Tested for Cytoskeletal/morphological Changes and Cytotoxicitya

a Chemical structures are shown together with effects on neuroblastoma BE(2)-M17 cytoskeleton and lung carcinoma NCI-H460 maximal growth inhibition. Morphological/ cytoskeletal effects were tested at 50 nM and 200 nM concentrations of all molecules, and data are displayed as Yes if they had an effect and No for negative results. Maximal growth inhibition values were from a dose-response curve for AZA-1 (see Figure 7) and ABCD-epi-AZA-1. The rest of the compounds did not inhibit cell growth to allow for a full dose-response experiment; therefore, data are shown for 10 µM concentrations. The Emax of the cisplatin control when fragments of AZA-1 were tested was 61 ( 9% inhibition.

Effects of Azaspiracid-1 on Actin Cytoskeleton

AZA-1 on proliferation, which in many cellular models is regulated by cell adhesion. Effects on proliferation were also tested in two cell lines, lung carcinoma and neuroblastoma, in order to evaluate if the AZA-1 target could be ubiquitous or if on the contrary the toxin acted in a different way in different cell models. Additionally, we studied the toxic activity in cell cultures of a number of isomers and fragments of AZA-1 (Table 1) to probe the structure-activity relationship and to learn more about the mechanism of action, which might lead to a new therapeutic chemical strategy.

Materials and Methods Materials. Eagle’s minimum essential medium (EMEM), Ham’s F12, glutamine, nonessential amino acids, gentamycin, amphotericine B, and penicillin/streptomycin were purchased from Biochrom AG (Berlin, Germany). Oregon Green 514 Phalloidin, BrdU, Alexa Fluor 488 anti-BrdU, Texas red DNAse I, and TubulinTrackerGreen Reagent were obtained from Molecular Probes (Eugene, Oregon). Fetal bovine serum (FBS), paraformaldehyde, Triton X-100, sulforhodamine B, DMSO, trichloroacetic acid, and glycerol were obtained from Sigma (St. Louis, MO). BSA was obtained from ICN Biomedicals (Aurora, Ohio). Hank’s balanced salt solution (without phenol red) (HBSS) was 1.2 mM CaCl2, 5.4 mM KCl, 0.4 mM KH2PO4, 0.5 mM MgCl2, 0.4 mM MgSO4, 137 mM NaCl, 4.2 mM NaHCO3, 0.3 mM Na2HPO4, 1 g/L d-glucose at pH 7.4. HCl/Triton X-100 denaturing solution was 2 M HCl and 0.5 g/100 mL Triton X-100. PBS was 137 mM NaCl, 8.2 mM Na2HPO4, 3.2 mM KCl, and 1.5 mM KH2PO4 at pH 7.4. AZA-1 and Enantiomer/Fragment Synthesis. AZA-1, AZA-1 isomers, and AZA-1 fragments were synthesized by Nicolaou and co-workers (6-12). Cell Line Culture. The neuroblastoma BE(2)-M17 cell line (European Collection of Cell Cultures) was cultured in EMEM/ Ham’s F12 (1:1) supplemented with 2 mM glutamine, 1% nonessential amino acids, 10% FBS, 50 mg/L gentamycin, and 50 µg/L amphotericine B. The CACO-2 cell line (ECCC) was cultured in EMEM supplemented with 2 mM glutamine, 1% nonessential amino acids, 10% fetal bovine serum, and 50 µg/mL penicillin/50 µg/mL streptomycin. For imaging assays, neuroblastoma cells were plated on glass coverslips at a density of 2.5-5 × 104 cells/well and used after 5-7 days, CACO-2 cells were plated and allowed to grow and differentiate for 22-26 days. Human lung carcinoma NCIH460 (ATCC) cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum. Cell cultures were kept at 37 °C in 5% CO2. Cytoskeleton Imaging. The actin cytoskeleton was stained with Oregon Green 514 Phalloidin that binds specifically to F-actin. Cells previously incubated with the toxin or carrier were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. After blocking for 30 min with 1% BSA/PBS, the cells were incubated with 165 nM Oregon Green Phalloidin in 1% BSA/PBS for 20 min and washed three times with PBS. The coverslips were then mounted in 50% glycerol/PBS and sealed with nail polish. Microtubules were fluorescently labeled using TubulinTracker Green, a specific probe for live-cell polymerized tubulin. After toxin or carrier treatment, cells were incubated in HBSS for 30 min at 37 °C/5% CO2 in the presence of 250 nM TubulinTracker Green. Toxin or carrier concentration was maintained during this 30 min incubation. The live-cell preparation was washed three times with prewarmed HBSS, and fluorescence was recorded immediately after staining. Images were captured with a Nikon Eclipse TE2000-E confocal microscope using an argon laser light source with excitation wavelength 488 nm and a Nikon 60× Plan Apo Tirf objective. Emission was captured at 510 nm. Phase contrast images were taken using a Nikon 40×/1.3 oil Plan Fluor objective. Confocal images were recorded with C1 normal 2.20 software (Nikon) as sections in the Z-axis at 0.5-µm steps and, unless specified, shown as volume rendered with the maximum projection method. Tirf images were

Chem. Res. Toxicol., Vol. 19, No. 11, 2006 1461 captured in the same microscope using a Hamamatsu ORCA-ER camera and Metamorph version 6.1 software (Universal Imaging Corporation). Quantification of polymerized actin and tubulin was done in duplicate for each experiment, fluorescence was quantified for individual cells at a middle-basal level by a single exposure of the preparation to the laser beam, and then fluorescence values of 20-30 cells per recording were averaged. Cytotoxicity Assays. The effect of AZA-1 on cell growth was evaluated using the sulforhodamine B assay. Briefly, cells were grown in 96 well plates at 150,000 cells/well for 24 h and treated for 48 h with toxin or carrier (DMSO). Then, cells were fixed with 10% TCA (trichloroacetic acid) for 60 min at 4 °C. After washing with H2O, cells were marked with 0.4% sulforodamine B/1% acetic acid by incubating for 10 min with constant shaking. Cells previously washed with 0.1% acetic acid were left in 10 mM Trizma for 15 min at room temperature with constant shaking. Absorbance at 515 nm was measured in a Tecan Ultra Evolution spectrometer. The experiments were done in triplicate, and cell growth was estimated versus that of the control cells cultured in a medium without growth factors. DMSO induced a 2-3% inhibition of cell growth. The percentage of cell growth inhibition was calculated as 100 - ((AO × 100)/AT), where AO is the absorbance in wells containing toxin, and AT is the absorbance in wells containing DMSO. Percentage of inhibition was plotted versus concentration, and the curve was adjusted to the equation y ) Emax/(1+(IC50/x)n) using a GraphPad Prism version 2.01 software (GraphPad Software, Inc.), where y is the observed effect at concentration x, Emax is maximum effect, IC50 is the concentration that induces 50% of maximum effect, and n is the slope of the curve. A positive control of cytotoxicity induced by cisplatin was included in every experiment. Quantification of DNA Synthesis. DNA synthesis was measured by the incorporation of 5-bromo-2′-deoxyuridine (BrdU, a thymidine analog) to newly sythesized DNA, labeling live-cells that progressed through S-phase of the cell cycle. Cells were grown on coverslips and incubated with toxin or carrier for 48 h. During the last hour, 100 µM BrdU was added. Then, BrdU was washed, and cells were fixed and permeabilized with 4% paraformaldehyde and 0.1% Triton X-100. After blocking with 3% BSA/PBS, cells were incubated with 20 µM Texas Red DNAse I, which was used as the counterstain. The preparation was then washed, fixed, blocked again, and treated with HCl/Triton X-100 denaturing solution for 15 min at room temperature. Incubation with Alexa Fluor 488 anti-BrdU (1:100) followed several washes. Finally, the preparation was washed and mounted. Incorporation of BrdU was measured in a CompuCyte laser scanning cytometer (Cambridge, MA) using argon laser light. Detection parameters were set to identify red fluorescent cells as events. Data were collected as a histogram of event number plotted versus green fluorescence maximum pixel. BrdU labeled and unlabeled cells were equally identified as events because Alexa Fluor 488 anti-BrdU did not emit in the red channel, and the red fluorescence intensity and profile were the same in proliferating and nonproliferating cells. Texas Red DNAse I does not emit green fluorescence in this experimental setting. Experiments were performed in duplicate.

Results AZA-1 Effect on Cell Morphology and Actin Cytoskeleton. The effects of a 48 h exposure to 50 nM AZA-1 on morphology and actin cytoskeleton were tested in neuroblastoma BE(2)-M17 and Caco-2 cells. AZA-1 induced a loss of neurites in neuroblastoma cells that rounded up, losing the characteristic flattened morphology of control cells (Figure 1A and B). Stress fibers (actin microfilament bundles), which spread all over the cell cytosol in control cells, became concentrated in thick cell prolongations in AZA-1-treated cells. Morphological changes were generalized in every preparation and were observable both in fixed and live cells. However, the content of F-actin per cell was not affected by treatment with 50 nM AZA-1 after 48 h of


Figure 1. Effect of AZA-1 on actin cytoskeleton in neuroblastoma BE(2)-M17 and Caco-2 cell lines. BE(2)-M17 and Caco-2 cells were incubated for 48 h in the presence of 50 nM AZA-1 or an equivalent concentration of DMSO. Then cells were fixed and stained for F-actin labeling. Pictures show green fluorescence images. (A) DMSO-treated and (B) AZA-1-treated neuroblastoma cells (representative of 10 experiments; bar size 10 µm). (C) DMSO-treated and (D) AZA-1treated Caco-2 cells, basal image (representative of three experiments; bar size 30 µm).

incubation (Figure 6A), suggesting that AZA-1 does not induce microfilament depolymerization in these conditions. G-actin labeling with Texas Red DNAse I by LSC did not show any significant change of monomeric actin levels after toxin exposure (data not shown). Because diarrhea is one of the main sympthoms during ASP and the intestine is one of the most affected organs in mouse toxicological studies, cytoskeletal effects were also tested in an intestinal cell line. Caco-2 cell morphology did not show a clear change in shape after exposure to AZA-1 for 48 h, probably due to the fact that they grow in a tight monolayer (data not shown); however, they showed a disorganization of polymerized actin. In control cells, basal F-actin looked uniformly distributed (Figure 1C); however, in AZA-1 preincubated cells, F-actin looked disorganized with thickened stress fibers (Figures 1D). A middle section of Caco-2 cells always gave a reticulated image with most of the F-actin localized in the cortical microfilament network, after treatment with AZA-1 F-actin moved from the cortex to the cell centre (data not shown). The decrease of neurite number and the thickening of basal stress fibers suggest a decrease in focal adhesion contacts with the surface. TIRF imaging was used to study F-actin distribution in close proximity to the surface to which cells are attached. In neuroblastoma cells, 50 nM AZA-1 induced an important reduction in the amount of F-actin involved in attachment to the surface (Figure 2A and B). TIRF imaging also revealed a marked disorganization of basal F-actin in Caco-2 cells (data not shown). Interestingly, after a 48 h exposure to AZA-1, the Caco-2 monolayer appeared detached from the surface in extensive areas. Dose dependency and kinetics of AZA-1 effects on morphology and actin cytoskeleton were analyzed next in neuroblastoma cells. The loss of neurites and round shape appeared at doses of 10, 20, 50, and 100 nM AZA-1 after 48 h of incubation. No effect was observed at 1 nM (Figure 3). A 50 nM concentration was used for the kinetics experiments. Incubation times of 3 and 6 h did not show any differences from the control, but at 12 h, the cells started to round up. The typical changes induced by AZA-1 were clearly present at 24 h, looking more intense at 48 h (Figure 4).

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Figure 2. TIRF image of neuroblastoma BE(2)-M17 cells treated with AZA-1 or DMSO. Cells were incubated for 48 h in the presence of 50 nM AZA-1 or an equivalent concentration of DMSO, fixed, and stained for F-actin labeling. Pictures show green fluorescence images. TIRF image of (A) DMSO-treated and (B) AZA-treated neuroblastoma cells; insets show epifluorescence images (representative of three experiments in duplicate; bar size 10 µm).

AZA-1 Effect on Microtubules. Labeling of the microtubules in live neuroblastoma cells with a flurorescent taxol derivative demonstrated that microtubules spread along the cell cytosol (Figure 5A). In AZA-1 treated neuroblastoma cells, microtubule labeling showed no apparent change in the intracellular distribution of microtubules other than the change in cell shape (Figure 5B). Interestingly, in three experiments done in duplicate, a clearly formed mitotic spindle was observed in three cells of the AZA-1 treated preparations (Figure 5B, arrowhead), indicating the suitability of this microtubule labeling technique. AZA-1 treatment did not affect the content of polymerized tubulin per cell (Figure 6B). AZA-1 Induced Cytotoxicity and Effect on Cell Cycle Progression. We analyzed AZA-induced cytotoxicity in lung carcinoma cells and its effect on cell cycle progression into S-phase in neuroblastoma cells. Sulforhodamine B cytotoxicity experiments were performed in a microplate and showed an inhibition of lung carcinoma cell growth by AZA-1 (Figure 7A) with an EC50 of 0.22 ( 0.05 µM and an Emax of 71 ( 3% inhibition. In the same experiments, the anti-tumor drug cisplatin had an EC50 and an Emax of 1.65 ( 0.08 µM and 87 ( 2% inhibition, respectively. During cytoskeleton labeling experiments, we observed that neuroblastoma cells were loosely attached to the surface after treatment with AZA-1, causing the loss of cells during staining protocols that require multiple washes. Therefore, the sulforhodamine B protocol, which measures cell mass, was not suitable to measure cell growth inhibition by AZA-1 in neuroblastoma cells because the effect would be overstimated due to cell loss after fixation. We used the incorporation of BrdU to DNA of proliferating cells to quantify the effect of AZA-1 on cell growth in neuroblastoma cells by LSC, which allows a measurement of population proliferation on a per cell basis. The results showed that after 48 h of incubation with 50 nM AZA-1, the percentage of neuroblastoma cells undergoing DNA synthesis was considerably reduced compared to the control (Figure 7B). Cell growth was inhibited 50.9 ( 9.8% after AZA-1 treatment. Structure-Activity Relationship. AZA-1, ABCD-epiAZA-1 (enantiomeric ABCD domain), two analogs based on the originally proposed structures of AZA-1 with an open E-ring, and 12 fragments of AZA-1 with varying stereochemistry (Table 1) were compared for morphological/actin cytoskeleton effects and cytotoxicity in order to find a relationship between chemical structure and toxin activity. F-actin experiments were performed in neuroblastoma cells using 50 and 200 nM concentrations for each compound. Because of the limited amount of AZA-1 and


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Figure 3. AZA-1 dose-response of the effect on actin cytoskeleton in neuroblastoma BE(2)-M17. The BE(2)-M17 cells were incubated with (A) DMSO or (B) 1 nM, (C) 10 nM, (D) 30 nM, (E) 50 nM, and (F) 100 nM AZA-1 for 48 h, then fixed, and stained for F-actin labeling (representative of three experiments; bar size 10 µm).

Figure 4. Kinetics of AZA-1 effect on actin cytoskeleton in neuroblastoma BE(2)-M17. The BE(2)-M17 cells were incubated with (A) DMSO for 48 h or 50 nM AZA-1 for (B) 3 h, (C) 6 h, (D) 12 h, (E) 24 h, and (F) 48 h, and then fixed and stained for F-actin labeling (representative of three experiments; bar size 10 µm).

Figure 5. AZA-1 effect on neuroblastoma BE(2)-M17 microtubules. The BE(2)-M17 cells were incubated for 48 h in the presence of 50 nM AZA-1 or an equivalent concentration of DMSO. Then cells were stained for microtubule labeling. (A) DMSO; (B) AZA-1 (representative of three experiments done in duplicate; bar size 30 µm).

other compounds, cell growth inhibition experiments had to be performed in a microplate using lung tumor cells instead of neuroblastoma proliferation assays performed on coverslips. In this case, concentrations were raised up to 10 µM. Results are summarized in Table 1 for all molecules tested. No fragment of the AZA-1 molecule had any effect on either the actin

cytoskeleton or cell growth regardless of the stereochemistry of the ABCDE and FGHI rings, suggesting that both parts of the molecule are necessary for actin cytoskeleton rearrangement and cell growth inhibition (Table 1). Only AZA-1 and ABCDepi-AZA-1 inhibited cell growth and affected actin cytoskeleton/ morphology. The EC50 of ABCD-epi-AZA-1 for cell growth inhibition was 0.86 ( 0.05 µM and the Emax was 53 ( 3% inhibition (the values for cisplatin control were EC50 of 3.5 ( 0.4 µM and Emax of 85 ( 2% inhibition). In cytoskeleton experiments, a 50 nM concentration of both AZA-1 and ABCDepi-AZA-1 induced similar changes. The integrity of the E ring and/or position of the double bond in the A ring and stereochemistry of the molecule were also necessary for AZA-1 activity on neuroblastoma cytoskeleton (Table 1). Additionally, 50 nM AZA-1 was combined with 150 nM natural ABCDE or 150 nM natural FGHI to explore if either of these fragments would compete with AZA-1 for binding to its cellular target. Neither the ABCDE nor FGHI fragments inhibited the effects of AZA-1 on neuroblastoma cell morphology and actin cytoskeleton (data not shown).


Figure 6. AZA-1 effect on the content of polymerized actin and tubulin per cell in the neuroblastoma BE(2)-M17 cell line. BE(2)-M17 cells were incubated for 48 h with 50 nM AZA-1 or DMSO, and actin or tubulin cytoskeletons were labeled and images recorded in a confocal microscope. Individual cell fluorescence was quantified and averaged. (A) F-actin (n ) 5, average ( sem). (B) Microtubules (n ) 3, average ( sem).

Discussion AZA-1 is one of the most recently described marine toxins. Several cellular types are sensitive to AZA-1 toxicity both in ViVo and in Vitro; however, its mechanism of action is not known yet. An effect of AZA-1 on the actin cytoskeleton was suggested in a previous study based on changes of lymphocyte cell morphology, although at the concentrations used in that work, AZA-1 did not affect the amount of polymerized actin (17). Our results confirm that actin cytoskeleton organization is modified by AZA-1 in two adherent cell models but also demonstrate that the attachment of adherent cells to a surface is very sensitive to the presence of this toxin. Disruption of adherences can be so strong so that exposure of Caco-2 cells to AZA-1 induces detachment of the cell monolayer from the surface in extensive areas without the disruption of cell-cell attachment. Interestingly, this effect is reminiscent of the separation/spaces observed between the intestinal epithelium and lamina propria in mice toxicology studies (4). The different components of the cytoskeleton often interact at several levels of cell functionality, and although actin fibers are structurally necessary for focal adhesion assembly, microtubules are also involved in focal adhesion regulation (25, 26). In this line of thought, microtubule disregulation might be responsible for the AZA-1-induced effects on morphology and the actin cytoskeleton. Our data indicate that AZA-1 does not apparently affect tubulin polymerization or microtubule distribution. Therefore, it does not seem likely that AZA-1 exerts its toxic effects through microtubule modulation. Cytotoxicity studies demonstrated that AZA-1 inhibited lung carcinoma cell growth; however, these cells did not seem to be as sensitive to AZA-1-induced cytotoxicity as other cell lines, where cytotoxic effects were observed in the low nanomolar range (17). Cell growth inhibition was confirmed in neuroblas-

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Figure 7. AZA-1 induced cytotoxicity in lung carcinoma cells and effect on proliferation of neuroblastoma BE(2)-M17 cells. (A) Dose dependency of AZA-1-induced cytotoxicity in lung carcinoma cells incubated in the presence of the toxin or carrier for 48 h. Lung carcinoma cells were seeded in 96 well plates and exposed to several AZA-1 concentrations for 48 h. The cell mass was detected with the sulforrodamine technique, and inhibition of cell growth was calculated as described in Materials and Methods and plotted vs toxin concentration (n ) 3, mean ( sem). (B) Incorporation of BrdU to DNA during S-phase in neuroblastoma cells treated with DMSO or 50 nM AZA-1 for 48 h. Cells were incubated with BrdU during the last hour of toxin treatment and stained with an Alexa Fluor 488 anti-BrdU antibody. The percentage of neuroblastoma cells in S-phase for the control and AZA-1 treated cells is plotted (n ) 4, mean ( sem).

toma cells by measuring the amount of cells undergoing DNA synthesis after a 48 h exposure to AZA-1. Induction of lung tumors in mice has raised public health concerns regarding AZA’s tumorigenic activity, a situation agravated by the fact that AZAs usually occur with DSP toxins with tumor promoter actitity. So far, there are no in Vitro data that explain the tumorigenic activity of AZA-1. However, AZA-1-related cytotoxicity and growth inhibition are probably responsible for at least some of the organ damage observed in toxicologic studies. Focal adhesion down regulation, cell death induction, and cell growth arrest are often related. It is well known that the loss of focal adhesions and subsequent cell detachment is responsible for cell cycle arrest and a form of apoptosis called anoikis (27-31). Conversely, a round shape and the loss of adhesion, together with other morphological changes, are typically associated with cells undergoing apoptosis, where protease activation is responsible for cytoskeletal protein lysis and subsequent cell detachment (29, 32-37). Therefore, there are two possible scenarios, either neuroblastoma cells enter apoptosis and that causes morphological and cytoskeletal changes or cytoskeleton changes precede apoptosis. Previous studies showed that concentrations as high as 10 µM AZA-1 did not induce any change of mitochondrial membrane voltage, a sign of apoptotic death, in this neuroblastoma cell line after incubation times of 24 h (18), suggesting that apoptosis is not the trigger for AZA-1-related morphological changes. However, we cannot rule out completely the possibility of AZA-1-induced


apoptosis because apoptosis can be activated by mitochondrialindependent pathways (38, 39). Although the absence of AZA1-induced apoptosis seems to be against the generalized observation of a loss of adhesion triggering apoptosis, there are occasions when dettachment is not associated with anoikis/ apoptosis induction or cell cycle arrest (40-42). Further exploration is necessary to clarify whether AZA-1 activates apoptosis mechanisms and whether morphological/cytoskeletal changes are responsible for cell cycle arrest and/or apoptosis. Multiple signaling pathways are involved in the regulation of actin cytoskeleton, and therefore, besides apoptosis, many different mechanisms could be responsible for AZA-1 effects. Among them, kinases/phosphatases are key modulators of cytoskeletal processes and constitute good candidates for AZA-1 targeting. Additionally, direct binding of AZA-1 to F- or Gactin cannot be completely ruled out, although the fact that For G-actin levels are not affected by the toxin at nanomolar concentrations suggests that actin is an improbable target. The effective dose and kinetics of AZA-1 in morphology/ cytoskeleton experiments are in agreement with previously published data (17). However, AZA-1 EC50 in our cytotoxicity studies was 100-fold higher than the EC50 described for other cell lines. These differences could be due to the source of toxin, synthetic in our case and purified from mussels in the Twiner et al. study. However, it is likely that the lung carcinoma NCIH460 cell line is less sensitive to AZA-1-induced cytotoxicity, given the similarity in cytoskeletal/morphological activities in both works and the high variability of cytotoxicity among cell lines. Interestingly, Twiner et al. reported that the lung epithelial A549 cells were the most resilient to AZA-1-induced cytotoxicity among the seven cell lines tested. Considering that the toxin used in those previous studies on actin cytoskeleton and cytotoxicity was purified from mussels after a natural algae bloom and that the AZA-1 used in the present work was synthetic, the fact that the effects, doses, and kinetics are similar in both studies strongly supports the newly proposed structure of AZA-1 (6, 7, 11). Structure-activity results using several modifications of the AZA-1 molecule indicate that no fragment of AZA-1 (natural ABCD, ABCDE, FGHI, or their stereoisomers) has any effect on cell growth or morphology/cytoskeleton regardless of its stereochemistry. Moreover, the integrity of the E ring and stereochemistry of the whole molecule is relevant for AZA-1 activity on the cytoskeleton. The lack of the E ring may eliminate key moieties for interaction with its target or change the spatial arrangement so that the 3D orientation of moieties is not adequate for interaction with a target site. Finally, the correct stereochemistry of the ABCD rings is not necessary for the presence of cytoskeleton and growth effects, but it could affect the molecule’s toxic potency. Although 50 nM AZA-1 or ABCD-epi-AZA-1 had very similar effects on the cytoskeleton of neuroblastoma cells, cytoskeletal/morphological changes are difficult to quantify, and small differences in potency would probably be undetected by this technique. The EC50 values obtained in cell growth inhibition experiments point to a higher potency of AZA-1 (approximately 4 fold), although the cisplatin control also had a lower EC50 in that set of experiments (approximately 2-fold). Overall, the natural ABCD stereochemistry does not seem to determine an impressive increase of toxic activity. In summary, AZA-1-induced rearrangement of actin cytoskeleton reduces cell adhesion to the substrate in two cellular models, neuroblastoma and Caco-2 cell lines. These effects are not associated to actin depolymerization or microtubule reor-

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ganization. Additionally, we report an inhibition of cell growth caused by AZA-1, as demonstrated by a lower increase of cell mass over time and a diminished percentage of cell population progressing to the S-phase of the cell cycle. Finally, the first approach to a structure-activity relationship obtained with several modifications of AZA-1 suggests that interaction with the AZA-1 cellular target responsible for mediating cytoskeletal/ morphological and cell growth effects requires moieties located in both the ABCDE and FGHI rings. Acknowledgment. Professor Nicolau acknowledges financial support for this work from The Skaggs Institute for Chemical Biology, the National Institutes of Health (U.S.A.), a predoctoral fellowship from the National Science Foundation (to M.O.F.), and grants from Amgen and Merck. The spanish work was funded with grants from the following agencies: Ministerio de Ciencia y Tecnologıá, Grant Numbers SAF200308765-C03-02, REN2001-2959-C04-03, REN2003-06598-C0201, AGL2004-08268-02-O2/ALI, and INIA CAL01-068. Xunta de Galicia, Spain, Grant Numbers PGIDT99INN26101, PGIDIT03AL26101PR, and PGIDIT04TAL261005PR. Fondo de Investigaciones Sanitarias, Grant Number FISS REMA-G03-007. EU VIth Frame Program, Grant Number IP FOOD-CT-200406988 (BIOCOP), STREP FOOD-CT-2004-514055 (DETECTOX), and CRP 030270-2 (SPIES-DETOX).

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