Azaspiracid-1 Inhibits Endocytosis of Plasma Membrane Proteins in Epithelial Cells

Abstract

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.

Collaborative study for the detection of toxic compounds in shellfish extracts using cell-based assays. Part I: screening strategy and pre-validation study with lipophilic marine toxins

“…This effect is consistent with the in vivo effects inducing complete degradation of gastrointestinal lining and confirmed by TEER experiments on monolayer Caco2 model [30] or by disturbance of filament organization in Caco2 cells [31]. Even if some data indicated that AZA1 induced the fragmentation of E-cadherin [21] and inhibited endocytosis [32], its molecular target is still unknown.…”

Towards tailored assays for cell-based approaches to toxicity testing

“…If veratridine/ouabain-treated cells are exposed to Stx, the toxin blocks NaV and sodium entrance, preventing sodium accumulation into neuroblastoma cells and allowing their survival. the knowledge of the mechanism of action of Stx and of cellular physiology, as well as the use of pharmacological tools targeting specific cellular components and processes, has The implications of those findings have supported further studies on the molecular mechanisms of action of Ytx and AZA-groups of compounds, leading to the conclusion that the accumulation of the 100 kDa e-cadherin fragment is caused by inhibition of endocytosis (Callegari, and Rossini, 2008;Bellocci et al, 2010). These specific issues will not be discussed further here; instead we will confine our attention to some heuristic indications provided by those findings that are relevant for the framework of cell-based methods for the detection of toxic agents/stressors.…”

Derivation of toxicity equivalency factors for marine biotoxins associated with Bivalve Molluscs

“…In humans, they cause vomiting, nausea, diarrhoea and stomach cramps within a few hours after ingestion (Klontz, Abraham, Plakas, & Dickey, 2009) AZAs target several apoptotic modulators (Botana, et al, 2014;Roman, et al, 2002;Twiner, et al, 2005), such as caspase, cytoskeleton (Vilarino, Nicolaou, Frederick, Vieytes, & Botana, 2007), cytochrome release (Twiner, Hanagriff, Butler, Madhkoor, & Doucette, 2012), c-jun-N-terminal protein kinase (JNK), calcium levels (Cao, LePage, Frederick, Nicolaou, & Murray, 2010;Vale, Wandscheer, et al, 2008), fatty acid biosynthesis (Twiner, et al, 2008). AZAs decrease cell volume mediated by potassium and chloride efflux (Vale, Nicolaou, Frederick, Vieytes, & Botana, 2010), deplete ATP (Kellmann, et al, 2009), inhibit endocytosis (Bellocci, Sala, Callegari, & Rossini, 2010) and decrease procathepsin pools in endocytosis (Sala, Bellocci, Callegari, & Rossini, 2013) (Chevallier, et al, 2015), which could explain the neurotoxicity linked to AZA (Twiner, et al, 2014).…”