In Vitro Induction of Erythrocyte Phosphatidylserine Translocation by the Natural Naphthoquinone Shikonin

Shikonin, the most important component of Lithospermum erythrorhizon, has previously been shown to exert antioxidant, anti-inflammatory, antithrombotic, antiviral, antimicrobial and anticancer effects. The anticancer effect has been attributed to the stimulation of suicidal cell death or apoptosis. Similar to the apoptosis of nucleated cells, erythrocytes may experience eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include the increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)) and ceramide formation. The present study explored whether Shikonin stimulates eryptosis. To this end, Fluo 3 fluorescence was measured to quantify [Ca(2+)](i), forward scatter to estimate cell volume, annexin V binding to identify phosphatidylserine-exposing erythrocytes, hemoglobin release to determine hemolysis and antibodies to quantify ceramide abundance. As a result, a 48 h exposure of human erythrocytes to Shikonin (1 µM) significantly increased [Ca(2+)](i), increased ceramide abundance, decreased forward scatter and increased annexin V binding. The effect of Shikonin (1 µM) on annexin V binding was significantly blunted, but not abolished by the removal of extracellular Ca(2+). In conclusion, Shikonin stimulates suicidal erythrocyte death or eryptosis, an effect at least partially due to the stimulation of Ca(2+) entry and ceramide formation.