ABSTRACT
Scabrosin esters (SEs), which have been recently isolated from the lichen Xanthoparmelia scabrosa, belong to the epipolythiodioxopiperazine (ETP) class of secondary metabolites characterized by possession of a reactive disulfide bond. Colony forming assays show that these toxins are active against human tumor cell lines at nanomolar concentrations. Other members of the ETP class of toxins such as gliotoxin have been shown to induce apoptosis in cells, although the cellular target(s) of the ETP toxins is currently unknown. ETP toxins have been shown to inhibit a variety of enzymes via interaction with sensitive cysteine residues. Here we show that the typical scabrosin ester acetate butyrate induces early mitochondrial membrane hyperpolarization assessed by JC-1 staining accompanied by apoptotic cell death. The toxin lowers ATP in intact cells and inhibits the rate of ATP synthesis in permeabilzed cells. Comparison with the effects of the known ATP synthase inhibitor oligomycin B is consistent with ATP synthase as an early target in scabrosin ester-induced cell death.
Subject(s)
Heterocyclic Compounds, 4 or More Rings/toxicity , Lichens/physiology , Macrophages/drug effects , Mitochondria/drug effects , Mitochondrial Proton-Translocating ATPases/biosynthesis , Plant Extracts/toxicity , Animals , Apoptosis , Benzimidazoles , Carbocyanines , Caspase 3 , Caspases/metabolism , Cells, Cultured , DNA Fragmentation , Esters/toxicity , Fluorescent Dyes , Intracellular Membranes/drug effects , Intracellular Membranes/physiology , Macrophages/enzymology , Macrophages/pathology , Membrane Potentials/drug effects , Mitochondria/enzymology , Mitochondrial Proton-Translocating ATPases/antagonists & inhibitors , Oligomycins/pharmacologyABSTRACT
Gliotoxin, a member of the epipolythiodioxopiperazine (ETP) class of toxins, induces both apoptotic and necrotic cell death in a concentration-dependent manner. Whereas the specific trigger for apoptotic death caused by these toxins is unclear, the reactive disulfide bond in the ETP toxins is required for biological activity. Thus it is likely that it is the interaction of this disulfide moiety with macromolecules in cells that was responsible for activity of ETP toxins. Here we present evidence that necrosis induced by gliotoxin and a simple synthetic ETP toxin is largely because of an influx of extracellular calcium through a redox-sensitive calcium channel in the plasma membrane of murine thymocytes. The calcium rises are strongly dependent on the pH of the external medium and the presence of external calcium and are abrogated and/or reversed by the presence of dithiothreitol, cell impermeant glutathione, and the calcium channel blocker Ni(2+). Comparisons with thapsigargin, which indirectly causes release of calcium from internal stores, indicates that ETP toxins do not provoke calcium rises by store depletion. A mechanism of oxidation by ETP toxins of cell surface thiol groups resulting in direct entry of calcium through a redox active channel in the plasma membrane is proposed. Necrotic but not apoptotic cell death was abrogated by inhibition of calcium entry.
