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.

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.

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 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.

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 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.

Oral administration of yessotoxin stabilizes E-cadherin in mouse colon.

YTX has been shown to disrupt the E-cadherin-catenin system in cultured epithelial cells, raising some concern that ingestion of seafood contaminated by YTX might favour tumour spreading and metastasis formation in vivo. In order to probe whether YTX might affect cadherin systems in vivo, we have set up a study involving repeated oral dosing of the toxin in mice (1mg/kg/day, for 7 days) and analysis of E-cadherin and N-cadherin in tissue extracts obtained at the end of the dosing scheme, as well as 1 and 3 months after YTX administration. We found that the E-cadherin pools obtained from lung and kidney were not altered by YTX in any of our experimental conditions. Extracts from mouse colon contained intact E-cadherin and an E-cadherin fragment of about 90 kDa (ECRA(90)), displaying a molecular alteration resembling that caused by YTX in cultured cells. We found that the relative proportion of ECRA(90), as compared to intact E-cadherin, was higher in colon extracts from control mice than from YTX-treated animals, indicating that oral administration of YTX to mice stabilizes E-cadherin of mouse colon. No significant difference could be detected in samples prepared from colons obtained 30 or 90 days after termination of YTX treatment. Oral administration of YTX to mice did not lead to a significant increase in the fragments of E-cadherin detectable in serum, neither it altered the N-cadherin pool of mouse heart. Electron microscopy analysis showed no substantial ultrastructural differences between controls and YTX-treated mice. Our findings show that ingestion of food contaminated by YTX poses a low risk of disruption of the E-cadherin system in vivo.