Background/Aims: The antihelminthic, contraceptive, anti-inflammatory and anticancer phytochemical embelin is at least in part effective against malignancy by inducing suicidal death or apoptosis of tumor cells. Erythrocytes are similarly able to enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling of eryptosis includes increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i), ceramide formation, oxidative stress as well as activation of p38 kinase and protein kinase C (PKC). The present study tested, whether and how embelin induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, ceramide abundance utilizing specific antibodies and reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. Results: A 48 hours exposure of human erythrocytes to embelin (≥25 µM) significantly increased the percentage of annexin-V-binding cells and hemolysis. Embelin did not significantly modify [Ca²⁺]_i. The effect of embelin on annexin-V-binding was not blunted by removal of extracellular Ca²⁺, by p38 kinase inhibitor SB203580 (2 µM) or by PKC inhibitor staurosporine (1 µM). Embelin did, however, significantly increase the ceramide abundance. Conclusions: Embelin stimulates phospholipid scrambling of the erythrocyte cell membrane, an effect involving ceramide formation.

The 1,4-naphthoquinone derivative naphthazarin may trigger apoptosis and is thus considered for the treatment of malignancy. On the other hand, naphthazarin decreases neurotoxicity. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by cell membrane scrambling with translocation of phosphatidylserine to the erythrocyte surface. Signalling leading to triggering of eryptosis include increase in cytosolic Ca 2+ -activity ([Ca 2+ ] i ), ceramide and oxidative stress. The present study explored whether naphthazarin impacts on eryptosis and, if so, to unravel underlying mechanisms. To this end, erythrocyte volume was estimated from forward scatter, phosphatidylserine abundance at the erythrocyte surface from FITC-annexin-V-binding, [Ca 2+ ] i from Fluo3 fluorescence, reactive oxidant species (ROS) from 2 0 ,7 0 -dichlorodihydrofluorescein diacetate (DCFDA) fluorescence and ceramide abundance at the erythrocyte surface from binding of fluorescent antibodies in flow cytometry. As a result, a 24-hr exposure of human erythrocytes to naphthazarin (10 lM) significantly decreased erythrocyte forward scatter, significantly increased the percentage of annexin-V-binding cells, significantly increased ceramide abundance at the erythrocyte surface and significantly increased ROS. The effect of naphthazarin on annexin-V-binding was not significantly blunted by removal of extracellular Ca 2+ . In conclusion, naphthazarin stimulates eryptosis, an effect at least in part due to oxidative stress and enhanced ceramide abundance at the erythrocyte surface.Naphthazarin (5,8-dihydroxy-l,4-naphthoquinone), a naturally available [1,2] 1,4-naphthoquinone derivative [3], has the capacity to induce apoptosis of tumour cells and is thus considered for the treatment of malignancy [3][4][5]. Naphthazarin sensitizes breast cancer cells to the pro-apoptotic effect of radiation [6]. Naphthazarin is effective by various cellular mechanisms including oxidative stress [7,8] [13,[15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33] and accelerated eryptosis contributes to the pathophysiology of several clinical conditions, such as iron deficiency, phosphate depletion, malignancy, metabolic syndrome, diabetes, hepatic and renal insufficiency, dehydration, hyperphosphataemia, haemolytic uraemic syndrome, sepsis, malaria, sickle cell disease, thalassaemia and Wilson's disease [13,[34][35][36].The present study explored whether naphthazarin is able to trigger suicidal death of erythrocytes, that is cells devoid of mitochondria and nuclei, key organelles in the execution of apoptosis. To this end, erythrocytes drawn from healthy volunteers were treated with naphthazarin, and phosphatidylserine surface abundance, cell volume, [Ca 2+ ] i , oxidative stress and ceramide abundance were determined. Materials and MethodsErythrocytes, solutions and chemicals. Fresh Li-Heparinanticoagulated blood samples were kindly provided by the blood...