Enhanced Ca2+ Entry, Ceramide Formation, and Apoptotic Death of Erythrocytes Triggered by Plumbagin

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone, 1), a natural product from plants with potential anticancer potency, induces apoptosis. Mechanisms involved in 1-induced apoptosis' include. mitochondrial depolarization, inactivation of NF-kappa B, and altered expression of anti- and proapolitotic Bcl proteins. Similar to nucleated cells, erythrocytes may undergo suicidal death or eryptosis, which, like apoptosis, results in cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+](i)) and ceramide formation. The present study explored whether 1 stimulates eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin-V-binding, hemolysis from hemoglobin release; [Ca2+](i) from Fluo-3 fluorescence, and ceramide abundance utilizing antibodies. A 48 h exposure to 1 (2 mu M) decreased forward scatter and increased annexin-V-binding significantly, events paralleled by increased [Ca2+](i) and ceramide formation. Exposure to 1 was followed by a slight but significant increase of hemolysis. Removal of extracellular Ca2+ slightly, but significantly blunted the effect of 1 (2 mu M) on annexin-V-binding. The present observations demonstrate that 1 may trigger suicidal death of erythrocytes, cells devoid of mitochondria and nuclei.