Palytoxin induces dissociation of HSP 27 oligomers through a p38 protein kinase pathway.

Palytoxin (PlTX) induces a stress response in MCF-7 cells that involves the phosphorylation of HSP 27 at serines 15, 78, and 82 by an as yet undetermined mechanism. We have studied the involvement of major groups of the mitogen-activated protein kinase (MAPK) family in this molecular response and focused our analyses on the ERK1/2, JNK, p38 protein kinase (p38K), and ERK5 pathways. The results show that PlTX induces the activation of JNK and p38 kinase but not ERK1/2 and 5 in MCF-7 cells. Through the use of protein kinase inhibitors, we established that blocking p38K, but not JNK, prevents the phosphorylation of HSP 27 induced by PlTX and that MAPKAPK2 participates in the response induced by the toxin under our experimental conditions. The cell death response induced by PlTX was inhibited by preventing JNK phosphorylation but not by blocking p38K/MAPKAPK2 and HSP 27 phosphorylation. Sucrose density gradient centrifugation revealed that MCF-7 cell extracts contain a heterodisperse population of HSP 27, including oligomers and smaller forms. Treating MCF-7 cells with PlTX caused the dissociation of HSP 27 oligomers, and using inhibitors of the JNK and p38K pathways showed that the dissociation of HSP 27 oligomers induced by PlTX involves a p38K-dependent process. We conclude that the changes induced by PlTX in the HSP 27 stress response protein system proceed through a molecular mechanism involving the activation of the p38 kinase pathway and its substrate, MAPKAK2, leading to dissociation of HSP 27 oligomers and the stabilization of a cellular pool of monomers phosphorylated at serines 15, 78 and 82, which could play a protective role against the death response induced by PlTX.

Azaspiracid-1 inhibits the maturation of cathepsin D in mammalian cells.

Azaspiracid-1 (AZA-1) inhibits endocytosis, but the consequences of this alteration on cellular processes are unknown. We hypothesized that the inhibition of endocytosis is a key step of the mode of action of AZA-1, leading to perturbation of cellular processes dependent on proper functioning of endocytic machinery. We tested this working hypothesis by probing whether AZA-1 can alter the maturation of cathepsin D in MCF-7 epithelial cells, as a model system. We found that cell treatment with AZA-1 inhibited the conversion of 52 kDa procathepsin D into the mature 30 kDa protein. The effects induced by AZA-1 were similar to those elicited by chlorpromazine and other agents preventing proper maturation of lysosomal enzymes, indicating that the inhibition of endocytic transfer of proforms to late endosomes/lysosomess is responsible for the effect induced by the toxin. By immunofluorescence microscopy, we found no colocalization of cathepsin D and the early endosomal marker EEA-1 in control cells, where most of cathepsin D resides in late endosomes/lysosomes. Co-localization of cathepsin D and EEA-1 immunoreactivity, in turn, was found in cells exposed to AZA-1, indicating that the toxin blocks protein maturation at the early steps of endocytosis, causing accumulation of procathepsin D in early endosomes. The molecular alteration induced by AZA-1 involved both secreted and intracellular pools of procathepsin D, showing that the toxin effect does not result from a general impairment of vesicular trafficking but is the outcome of a perturbed centripetal process. Furthermore, AZA-1 was found to inhibit procathepsin D maturation also in normal fibroblasts, showing that this molecular response is induced by this toxin in different cell types. The data we obtained corroborated our hypothesis and provide a unified molecular frame for the mode of action of AZAs in animal models, involving a primary alteration of endocytic processes.

Towards tailored assays for cell-based approaches to toxicity testing.

The call for a new toxicology is mainly a call for cellular approaches and their computational integration. This article reflects on cell models, which are necessary to facilitate the transition. A mechanistic perspective has prompted the characterization of toxicity pathways and toxicity networks in order to develop robust cell-based assays for toxicity testing. Differing use scenarios for cell systems require higher degrees of sophistication, e.g., human-on-a-chip approaches are based on complex organotypic cultures to approximate the repertoire of human physiological reactions and high-throughput tests require simplicity and robustness. The new paradigm emerging under the branding of Toxicology for the 21(st) Century needs complex models for pathway of toxicity identification and simpler assays for testing the perturbation of any given pathway. With increasing knowledge about underlying mechanisms, the needs for complexity and test specificity will change. Selective cell-based assays are desirable, especially for the detection of novel toxicants and biothreats. Examples from endocrine disruption, pyrogenicity, and especially shellfish toxin testing are used to illustrate such developments.

Palytoxin induces cell lysis by priming a two-step process in mcf-7 cells.

The cytolytic action of palytoxin (PlTX) was recognized long ago, but its features have remained largely undetermined. We used biochemical, morphological, physiological, and physical tools, to study the cytolytic response in MCF-7 cells, as our model system. Cytolysis represented a stereotyped response induced by the addition of isotonic phosphate buffer (PBS) to cells that had been exposed to PlTX, after toxin removal and under optimal and suboptimal experimental conditions. Cytolysis was sensitive to osmolytes present during cell exposure to PlTX but not in the course of the lytic phase. Fluorescence microscopy showed that PlTX caused cell rounding and rearrangement of the actin cytoskeleton. Atomic force microscopy (AFM) was used to monitor PlTX effects in real time, and we found that morphological and mechanical properties of MCF-7 cells did not change during toxin exposure, but increased cell height and decreased stiffness at its surface were observed when PBS was added to PlTX-treated cells. The presence of an osmolyte during PlTX treatment prevented the detection of changes in morphological and mechanical properties caused by PBS addition to toxin-treated cells, as detected by AFM. By patch-clamp technique, we confirmed that PlTX action involved the transformation of the Na(+),K(+)-ATPase into a channel and found that cell membrane capacitance was not changed by PlTX, indicating that the membrane surface area was not greatly affected in our model system. Overall, our findings show that the cytolytic response triggered by PlTX in MCF-7 cells includes a first phase, which is toxin-dependent and osmolyte-sensitive, priming cells to lytic events taking place in a separate phase, which does not require the presence of the toxin and is osmolyte-insensitive but is accompanied by marked reorganization of actin-based cytoskeleton and altered mechanical properties at the cell's surface. A model of the two-step process of PlTX-induced cytolysis is presented.

Palytoxin action on the Na(+),K(+)-ATPase and the disruption of ion equilibria in biological systems.

Palytoxin-group toxins (PlTX) exert their potent biological activity by altering mechanisms of ion homeostasis in excitable and non-excitable tissues. This review will describe major aspects that led to the relatively early identification of the Na(+),K(+)-ATPase as the molecular target and receptor of the toxin in sensitive systems. The importance of this pump in the normal functioning of animal cells has driven extensive investigative efforts. The recognized molecular mechanism of action of PlTX involves its binding to the extracellular portion of alpha subunit of this plasma membrane protein, which converts an enzyme carrying ions against their concentration gradients at the expense of chemical energy (ATP) into a non-selective cation channel, allowing passive flow of ions following their concentration gradients. More recent findings have indicated that PlTX would interfere with the normal strict coupling between inner and outer gates of the pump controlling the ion access to the Na(+),K(+)-ATPase, allowing the gates to be simultaneously open. The ability of PlTX to make internal portions of the Na(+),K(+)-ATPase accessible to relatively large molecules has been exploited to characterize the structure-function relationship of the pump, leading to a better understanding of its ion translocation pathway. Thus, forty years from the isolation of this potent marine biotoxin, a considerable understanding of its mode of action and of its potential as a research tool have been achieved and are the basis for promising future advancement in the characterization of biological systems and their alteration by PlTX.

Azaspiracid-1 inhibits endocytosis of plasma membrane proteins in epithelial cells.

The effect of azaspiracid-1 (AZA-1) on the plasma membrane proteins E-cadherin, Na(+)/K(+)-ATPase, and prolactin receptor (R(prl)) has been investigated in MCF-7 cells. Cell treatment for 24 h with 1nM AZA-1 induced the accumulation of a proteolytic fragment of E-cadherin and significant increases in the levels of Na(+)/K(+)-ATPase and R(prl) at the level of membranous structures. The effect induced by AZA-1 was mimicked by latrunculin A, suggesting that the toxin might act by blocking the endocytosis of plasma membrane proteins. The exposure of intact cells to a biotinylation reagent that does not permeate the plasma membrane provided data showing that AZA-1 treatment of MCF-7 cells doubled the levels of total protein located on the cell surface. The exposure of intact cells to exogenous proteases (trypsin and proteinase K) showed that AZA-1 treatment of MCF-7 cells modifies the availability of the three membrane protein markers to proteolytic attacks, providing evidence that significant portions of the protein pools are located in structures that are not exposed to the cell surface after the treatment with AZA-1. Distinct subcellular locations of the membrane protein markers in MCF-7 cells exposed to AZA-1 were confirmed by immunofluorescence microscopy. Direct evidence that AZA-1 inhibits endocytosis was obtained by showing that AZA-1 blocked the intracellular transfer of E-cadherin-bound antibody in MCF-7 cells. The effects of AZA-1 on the E-cadherin system were confirmed in Caco-2 and Madin Darby canine kidney epithelial cell lines. We conclude that AZA-1 inhibits endocytosis of plasma membrane proteins in epithelial cells.

Phycotoxins: chemistry, mechanisms of action and shellfish poisoning.

Phycotoxins are natural metabolites produced by micro-algae. Through accumulation in the food chain, these toxins may concentrate in different marine organisms, including filter-feeding bivalves, burrowing and grazing organisms, herbivorous and predatory fish. Human poisoning due to ingestion of seafood contaminated by phycotoxins has occurred in the past, and harmful algal blooms (HABs) are naturally occurring events. Still, we are witnessing a global increase in HABs and seafood contaminations, whose causative factors are only partially understood. Phycotoxins are small to medium-sized natural products and belong to many different groups of chemical compounds. The molecular mass ranges from approximately 300 to over 3000 Da, and the compound classes represented include amino acids, alkaloids and polyketides. Each compound group typically has several main compounds based on the same or similar structure. However, most groups also have several analogues, which are either produced by the algae or through metabolism in fish or shellfish or other marine organisms. The different phycotoxins have distinct molecular mechanisms of action. Saxitoxins, ciguatoxins, brevetoxins, gambierol, palytoxins, domoic acid, and, perhaps, cyclic imines, alter different ion channels and/or pumps at the level of the cell membrane. The normal functioning of neuronal and other excitable tissues is primarily perturbed by these mechanisms, leading to adverse effects in humans. Okadaic acid and related compounds inhibit serine/threonine phosphoprotein phosphatases, and disrupt major mechanisms controlling cellular functions. Pectenotoxins bind to actin filaments, and alter cellular cytoskeleton. The precise mechanisms of action of yessotoxins and azaspiracids, in turn, are still undetermined. The route of human exposure to phycotoxins is usually oral, although living systems may become exposed to phycotoxins through other routes. Based on recorded symptoms, the major poisonings recognized so far include paralytic, neurotoxic, amnesic, diarrheic shellfish poisonings, ciguatera, as well as palytoxin and azaspiracid poisonings.

Yessotoxin inhibits phagocytic activity of macrophages.

Yessotoxin (YTX) is a sulphated polyether compound produced by some species of dinoflagellate algae, that can be accumulated in bivalve mollusks and ingested by humans upon eating contaminated shellfish. Experiments in mice have demonstrated the lethal effect of YTX after intraperitoneal injection, whereas its oral administration has only limited acute toxicity, coupled with an alteration of plasma membrane protein turnover in the colon of the animals. In vitro studies have shown that this effect is due to the inhibition of endocytosis induced by the toxin. In this work, we investigated the effects of YTX on phagocytosis by using the J774 macrophage cell line. We found that macrophages exposed to 10 or 1 nM YTX display a reduced phagocytic activity against Candida albicans; moreover, phagosome maturation is also inhibited in these cells. Such results were confirmed with resident peritoneal macrophages from normal mice. The inhibition of both phagocytosis and phagosome maturation likely involves cytoskeletal alterations, since a striking rearrangement of the F-actin organization occurs in YTX-treated J774 macrophages. Surprisingly, YTX also enhances cytokine production (TNF-alpha, MIP-1alpha and MIP-2) by J774 macrophages. Overall, our results show that low doses of YTX significantly affect both effector and secretory functions of macrophages.

The cytotoxic pathway triggered by palytoxin involves a change in the cellular pool of stress response proteins.

We have analyzed the proteome of MCF-7 cells exposed to palytoxin (PlTX), to characterize protein components involved in the death response induced by the toxin. The protein profiles of cell lysates were obtained by two-dimensional (2D) electrophoresis, and we found that four components were increased by PlTX treatment. By tryptic digestion of protein spots in the gels and LC-ESI-MS/MS analysis of resulting peptides, those four components were found to include three isoforms of the heat shock protein (hsp) 27 differing with regard to their phosphrylation state, as well as DJ-1/PARK7. The effects exerted by PlTX on hsp 27 and DJ-1 proteins were further quantified by immunoblotting analyses of proteins separated by monodimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and using antibodies recognizing total hsp 27, the hsp 27 forms phosphorylated in Ser(82), and DJ-1 protein. Dose-response and time-course experiments yielded results that only partially confirmed those found by protein staining after 2D electrophoresis. These findings were further checked by immunoblotting of proteins after fractionation by 2D electrophoresis, and we found that only some forms of those comigrating in a single band upon monodimensional SDS-PAGE were actually increased in extracts from PlTX-treated cells. We obtained evidence that the three hsp 27 isoforms whose relative abundance was increased in MCF-7 cells exposed to PlTX comprised two proteins phosphorylated in Ser(82), whereas the third form most likely contains a phosphorylated amino acid residue other than Ser(82). Moreover, we could show that PlTX treatment determined the accumulation of an oxidized isoform of DJ-1 in MCF-7 cells. We conclude that the toxicity pathway of PlTX in MCF-7 cells involves post-translational modifications of hsp 27 and DJ-1 stress response proteins, comprising a shift in the equilibria among hsp 27 isoforms toward those phosphorylated in Ser(82), as well as the oxidation of DJ-1.

The total activity of a mixture of okadaic acid-group compounds can be calculated by those of individual analogues in a phosphoprotein phosphatase 2A assay.

Monitoring of okadaic acid (OA)-group toxins in seafood is of paramount importance for the protection of consumer health from diarrheic shellfish poisoning. The property of OA-group compounds to inhibit type 2A serine/threonine phosphoprotein phosphatase (PP2A) has been exploited for the detection of OA in several experimental settings, but the performance of PP2A inhibition assays in the quantification of mixtures of OA-group compounds has not been reported yet. We have used a PP2A inhibition assay to analyze the total effect of mixtures including OA and one of its analogues, okadaol (OOH), by measuring the activity of individual compounds and of toxin mixtures through the inhibition they exert on the PP2A enzyme. We found that both OA and OOH inhibit PP2A under our experimental conditions, with IC50 values of 0.37 +/- 0.04 nM and 4.3 +/- 0.8 nM, respectively, confirming that OOH is about ten-fold less potent than OA. PP2A assays were also carried out with predefined mixtures of OA and OOH, covering the full dose-response of one compound in the presence of increasing concentrations of the other toxin. The experimental data we obtained were used to analyze their correlation with those that could be calculated by adding the relative effects exerted by individual analogues, and we found that a good correlation exists between the observed and the expected data, when the predicted effect was calculated on the basis of toxicity equivalence factors. Our findings show that an additive model based on the use of toxicity equivalence factors of individual toxins is appropriate for the calculation of the total activity of multi-component mixtures of OA-group compounds in unknown samples.

Proteomic analysis reveals multiple patterns of response in cells exposed to a toxin mixture.

We have used proteomic analyses to probe the responses induced by a pair of marine biotoxins, okadaic acid (OA) and gambierol (GB), added alone or in combination to a cultured cell line and carried out a preliminary investigation into the possible interactions between toxins possessing two different molecular mechanisms of action at a cellular level. When MCF-7 cells were treated with OA, we found that cellular levels of 30 proteins were significantly affected, including several isoforms of nonphosphorylated and phosphorylated hsp 27, as well as enzymes involved in the maintenance of nucleoside triphosphate pools and the control of redox states of the cell. When we repeated our analysis using GB, nine proteins were significantly affected, including some isoforms of nonphosphorylated hsp 27, as well as semenogelin-1, myosin-7, and the ATP synthase subunit delta. The combined addition of OA and GB to MCF-7 cells, in turn, affected 14 proteins, including some isoforms of nonphosphorylated and phosphorylated hsp 27, as well as myosin-7, the ATP synthase subunit delta, and enzymes involved in the control of redox states of the cell. If components affected by either OA or GB, as well as by the combined treatment, were classified according to the detected changes, two sets of data were obtained, including the components whose levels were found affected by the combined treatment, regardless of the effect observed after addition of only one agent, and those that had been found affected in cells that had been challenged with only one toxin but not when cells had been subjected to the combined treatment. Multiple patterns of responses to the toxin mixture were recorded in the two sets, consisting of both independent and interacting actions, among which we detected synergistic, similar, and antagonistic effects.

Protein markers of algal toxin contamination in shellfish.

Filter-feeding bivalve molluscs are often contaminated by algal toxins. We have probed whether proteomic analysis of extracts from the digestive gland (DG) of mussels could be employed to identify biomarkers of contamination due to okadaic acid-group toxins. The protein extracts were obtained from 18 separate mussel samples and were analyzed by two-dimensional gel electrophoresis. When samples were divided into four different classes based on the content of OA-group toxins in the starting material, we found that two proteins varied as a function of OA contamination. By BLAST analysis, the two proteins were identified as a component of photosystem II and a subunit of NADH dehydrogenase. The analysis of peptide homologies showed that the peptide of photosystem II we detected in extracts from the DG of mussels contaminated by OA-group toxins is identical to its counterpart in Dinophysis algae, which are the producers of this group of toxins. We concluded that proteomic analysis can be used for the detection and identification of biomarkers of biotoxin contamination in shellfish, including both proteins expressed by the toxin producers and components that participate to the tissue response to the exogenous bioactive contaminant.

Yessotoxin induces the accumulation of altered E-cadherin dimers that are not part of adhesive structures in intact cells.

We have studied the alteration induced by yessotoxin in the E-cadherin-catenin system of epithelial cells by stabilizing the protein-protein interactions in oligomers, through the introduction of covalent bonds between subunits in vitro and in vivo. The E-cadherin-catenin complexes that we have stabilized by crosslinking comprise multiple forms of dimeric, trimeric, tetrameric and hexameric complexes, with different subunit compositions. A 1-day treatment of MCF-7 cells with yessotoxin resulted in an increase in cellular levels of the complexes including a 100kDa fragment of E-cadherin (ECRA100), with a relative increase in cellular E-cadherin .ECRA100 heterodimers, as opposed to the E-cadherin homodimer that represents the core structure of the E-cadherin-catenin system of adhesive structures in normal cells. The high MW oligomers of cell adhesive structures, in turn, were not appreciably altered by cell treatment with yessotoxin. Most of these oligomers partitioned in a fraction that cannot be solubilized by non-ionic detergents after crosslinking of intact cells. Yessotoxin treatment did not significantly alter the levels of ECRA100 in the Triton X-100 resistant fraction of plasma membrane, but increased the relative abundance of ECRA100 in the Triton X-100 soluble pool of crosslinked cells. We have concluded that cell exposure to yessotoxin leads to increased cellular contents of E-cadherin .ECRA100 heterodimers that are not participating to cell adhesive structures but are located in other membranous fractions of intact cells.

Yessotoxin inhibits the complete degradation of E-cadherin.

Previous in vitro and in vivo toxicological studies on the effects of yessotoxin (YTX) on E-cadherin have provided conflicting results with regard to alterations of its turnover. We have then studied the effects of YTX on the degradation pathway of E-cadherin in intact cells under controlled conditions, and found that the 100kDa E-cadherin fragment (ECRA100) accumulated in cells exposed to YTX is an intermediate degradation product, detectable when the process is altered by agents interfering with endocytosis and lysosomal functioning. Cell treatment with YTX slows down the degradation of ECRA100, without affecting the half-lives of intact E-cadherin and its associated proteins beta-catenin and gamma-catenin. When cells have been treated with an inhibitor of proteasomes (lactacystin), the accumulation of ECRA100 induced by YTX was reduced. Accumulation of ECRA100, in turn, was observed after cells were exposed to inhibitors of lysosomal functioning (chloroquine) and of clathrin-mediated endocytosis (chloropromazine), but not to agents interfering with the caveolae-mediated pathway (nystatin and filipin III). The actin cytoskeleton was involved in endocytosis of ECRA100, because its accumulation was detected after MCF-7 cells had been treated with cytochalasin D. Nocodazole treatment of MCF-7 cells, in turn, did not lead to detection of ECRA100, indicating that microtubules should not be involved in its degradation. We have concluded that YTX interferes with the degradation pathway of E-cadherin by slowing down the endocytosis and complete disposal of the ECRA100 intermediate proteolytic fragment, whose cell levels are consequently increased in cells exposed to the toxin. These findings reconcile the apparent contradictions of previous studies, showing that YTX does not promote E-cadherin degradation per se, but interferes with its complete disposal.

A cytolytic assay for the measurement of palytoxin based on a cultured monolayer cell line.

A cytolytic assay that could detect palytoxin and its congeners has been developed by the use of an established cell line grown as monolayer to replace the current hemolytic method. We used MCF-7 cells and cytolysis was measured by the release of cytosolic lactate dehydrogenase (LDH) in the buffer added to treated cells (culture supernatant). A dose-dependent increase in LDH activity in culture supernatants was detected when MCF-7 cells were exposed to palytoxin and its analogue ostreocin D. The cytolytic response induced by palytoxin and ostreocin D was specific for this group of compounds, acting on Na+/K+-ATPase, as it was prevented when cells were preincubated with ouabain. The specificity of our assay for palytoxin and its congeners was confirmed by the finding that cytolysis was not detected when MCF-7 cells were exposed to unrelated toxins such as maitotoxin, tetrodotoxin, okadaic acid, and yessotoxin, even in the case of compounds that elicit cytotoxic responses under our experimental conditions. Using extracts from biological materials after spiking with the palytoxin standard, we found a good correlation between palytoxin levels measured by our cytolytic assay and the expected values. Our cytolytic assay detected palytoxin in naturally contaminated materials, but estimates were significantly higher than the palytoxin contents determined by LC-MS, indicating that naturally contaminated materials contain biologically active palytoxin congeners. We conclude that our cytolytic assay based on the use of MCF-7 cell monolayers is a viable alternative to animal-based methods for the determination of palytoxin and its congeners in contaminated materials.

A slot blot procedure for the measurement of yessotoxins by a functional assay.

We originally developed a functional assay for the detection of yessotoxins (YTX) based on its capacity to induce dose-dependent changes in cellular levels of two marker proteins, consisting of E-cadherin and an E-cadherin fragment (ECRA100) in epithelial cells. The procedure is time-consuming and we have shortened it by a slot blot format, using antibodies recognizing two different epitopes of E-cadherin (HECD-1 and C20820), thereby discriminating those markers. The best performing membrane under our conditions, in terms of binding capacity and even absorption of proteins, was a positively charged nylon membrane. Treatment of the membrane with 0.5mug of Ab/ml was appropriate for maximal detection of antigens by our slot blot procedure with both HECD-1 and C20820 antibodies. The treatment of cells with YTX, resulting in a relative increase in the cellular levels of ECRA100, led to a dose-dependent increase of the signal detected by Ab HECD-1 without a concomitant increase in the signal detected by Ab C20820 in our slot blot format, and the concentrations of YTX were correlated to both the increase of the signal detected through Ab HECD-1 and to the decrease in the ratio of the signals obtained with the two Abs (C20820 over HECD-1). Upon analyses of extracts from cells treated with shellfish samples, we could detect and quantify YTX in naturally contaminated materials. The slot blot format of our functional assay allows a substantial shortening of its analytical step (about seven hr, as compared to the two working days of the original method), providing YTX measurements that are accurate but show large standard deviations.

Azaspiracid-1 alters the E-cadherin pool in epithelial cells.

Azaspiracids cause severe damages in the epithelium of several organs. In this study we have investigated the effects of azaspiracid-1 (AZA-1) on two epithelial cell lines. Nanomolar concentrations of AZA-1 reduced MCF-7 cell proliferation and impaired cell-cell adhesion. AZA-1 altered the cellular pool of the adhesion molecule E-cadherin by inducing a dose- and time-dependent accumulation of an E-cadherin fragment (E-cadherin-related antigen [ECRA(100)]), with a concentration inducing the half-maximal effect (EC(50)) of 0.47nM. The immunological characterization of ECRA(100) revealed that it consists of an E-cadherin molecule lacking the intracellular domain, and these data showed that the effect induced by AZA-1 in MCF-7 cells is undistinguishable from that induced by yessotoxin (YTX) in the same experimental system. A comparison of toxin effects in MCF-7 and Caco 2 cells confirmed that the effects induced by AZA-1 and YTX are undistinguishable in these cells. Treatment of fibroblasts with AZA-1 did not affect the cellular pool of N-cadherin showing that the toxin effect is cadherin-specific. A comparison of the effects induced by AZA-1, YTX, and okadaic acid on F-actin and E-cadherin in MCF-7 and Caco 2 cells showed that 1nM AZA-1 did not cause significant changes in F-actin and that accumulation of ECRA(100) did not correlate with decreased levels of F-actin under our experimental conditions. Matching our results with those available in literature, we notice that, when molecular effects induced by AZA-1 and YTX have been studied in the same in vitro systems, experimental data show that they are undistinguishable in terms of sensitive cellular parameters, effective doses, and kinetics of responses in several cell lines. The possibility that azaspiracids and YTXs might share their molecular mechanism(s) of action in defined biological settings should be considered.