Gian Paolo Rossini

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.

The toxic responses induced by okadaic acid involve processing of multiple caspase isoforms

The recognized role of caspases as executioners of apoptosis, led us to investigate their involvement in death responses induced by okadaic acid (OA) in HeLa S(3) and MCF-7 cells. A one-day treatment with OA induced accumulation of the 85kDa poly(ADP-ribose) polymerase (PARP) fragment in cell lysates but the response was prevented if cells were treated with OA in the presence of the caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK. The HeLa S(3) and MCF-7 cells were found to contain measurable levels of the intact caspase-2, -7, -8 and -9 zymogens, whereas caspase-3 was found only in HeLa cells. After one day of OA treatment, pro-caspase-2, -3, -7 and -9 isoforms were found processed in HeLa cells, whereas only pro-caspase-2 was processed in MCF-7 cells. Pro-caspase-8, in turn, was mostly unprocessed in both cell lines. The possible interference of caspase inhibitors on cell death was also evaluated, and we found that both Z-VAD-FMK and Z-DEVD-FMK could contribute different extents of protection of MCF-7 and HeLa cells from toxic effects caused by OA. We concluded that OA triggers multiple pathways of caspase processing, contributing to death responses triggered by OA in HeLa S(3) and MCF-7 cells.

Functional assay to measure yessotoxins in contaminated mussel samples.

Yessotoxin (YTX) treatment of MCF-7 cells results in the accumulation of a 100-kDa fragment of E-cadherin (ECRA(100)) without a parallel loss of the intact protein in cytosoluble extracts. As a consequence, concentration-dependent increases in the total immunoreactivity detectable by anti-E-cadherin antibodies relative to controls (RTI) and in the relative immunoreactivity of ECRA(100) (RI) are observed. These responses have been exploited to develop a functional assay to measure YTX in samples from contaminated mussels by a three-step procedure, consisting of (i) treatment of MCF-7 cells with YTX standard in the concentration range 0-1nM and of unknown samples; (ii) preparation of cellular extracts, fractionation of proteins by polyacrylamide gel electrophoresis under denaturing conditions, and immunoblotting with anti-E-cadherin antibodies, followed by densitometric analyses of autoradiographies and calculation of RI of ECRA(100) and of RTI of the samples; and (iii) interpolation of the YTX concentrations in unknown samples on standard curves, by the RI of ECRA(100) and the RTI of the samples. The procedure has been used to measure yessotoxins in contaminated mussel samples, and the results obtained show that this functional assay is very sensitive (limit of detection of about 100ng equivalent YTX/g of digestive gland), and robust, as (i) it is insensitive to matrix effects in the range of toxin concentrations relevant for risk assessment to protect humans from exposure to YTX, (ii) calculations are based on a molecular parameter (the RI of ECRA(100)) which is not affected by errors in sample preparation, (iii) it can be performed by the use of antibodies commercially available from different companies, and (iv) it does not show an absolute need of calibration by a pure standard within each assay.

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.

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.

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.

Recovery of cellular E-cadherin precedes replenishment of estrogen receptor and estrogen-dependent proliferation of breast cancer cells rescued from a death stimulus

Loss of estrogen‐responsiveness and impaired E‐cadherin expression/function has been linked to increased metastatic potential of breast cancer cells. In this study, we report that proliferation of breast cancer cells can resume following removal of a toxic stimulus causing severe impairment of cell adhesion and estrogen responsiveness. This type of response was induced by okadaic acid (OA) in MCF‐7 cells, and was accompanied by an almost complete block of DNA synthesis, loss of cell–cell contact and cell detachment from culture dishes, loss of estrogen receptor (ER), progesterone receptor (PR) and E‐cadherin, whereas only a weak, if any, inhibition of protein synthesis could be observed. These responses were detected in MCF‐7 cells after a 1‐day treatment with 50 nM OA, and could be reversed if OA‐treated cells were recovered in a culture medium devoid of the toxin, so that rescued cells resumed growth 8–12 days after replating. By pulse‐chase experiments, we found that protein synthesis was not significantly affected in rescued cells, whose DNA synthesis, instead, was almost completely blocked during the first days of MCF‐7 cell rescue from OA treatment. We also analyzed E‐cadherin, mitogen activated protein kinase isoforms ERK1 and ERK2, Bcl‐2 and BAX proteins during the rescue of MCF‐7 cells from OA‐induced cell death, and found that their expression followed temporally defined patterns. Cellular levels of E‐cadherin returned to control levels within the first days of the rescue, followed by ER, ERK1, and ERK2, and finally by Bcl‐2 and BAX proteins. Under our experimental conditions, restoration of cell adhesion did not require a functional ER system, but recovery of a normal ER pool accompanied resumption of estrogen‐dependent proliferation of OA‐treated MCF‐7 cells.

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.

Inhibitors of phosphoprotein phosphatases 1 and 2A cause activation of a 53 kDa protein kinase accompanying the apoptotic response of breast cancer cells

Treatment of MCF‐7 breast cancer cells with 50 nM okadaic acid triggers an apoptotic response which is accompanied by a 7‐fold increase in the activity of a protein kinase with a relative molecular mass of 53 kDa. The activity of the kinase was stimulated by cell treatment with inhibitors of phosphoprotein phosphatase 1 and 2A, but not by stressing conditions. Okadaic acid‐induced stimulation of the 53 kDa protein kinase was not abolished by coincubation of cells with cycloheximide. We conclude that stimulation of the 53 kDa protein kinase by inhibitors of phosphoprotein phosphatases involves pre‐existing molecular components whose activity depends on the phosphorylation state of serine/threonine residues.