Saponins, naturally occurring glycosides and triterpene glycosides in plants, are considered useful in the prophylaxis and treatment of several disorders, including malignancy. The effect of these substances is partly attributable to induction of both apoptosis and necrosis. Saponin has previously been shown to trigger hemolysis. Erythrocytes may avoid hemolysis by entering programmed cell death or eryptosis, characterized by cell shrinkage and cell membrane scrambling, leading to phosphatidylserine exposure at the erythrocyte surface. Eryptosis is triggered by increase of cytosolic Ca(2+) activity ([Ca(2+)](i)). The present study explored, whether exposure of human erythrocytes to saponin modifies [Ca(2+)](i), ceramide formation, hemolysis, and eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release, [Ca(2+)](i) from Fluo3-fluorescence, and ceramide utilizing specific antibodies. A 24 h exposure to saponin (15 µg/ml) resulted in a significant increase of annexin V binding and a significant stimulation of hemolysis. Saponin (15 µg/ml) further increased [Ca(2+)](i) and ceramide formation. Annexin V binding was significantly blunted but not abrogated in the nominal absence of extracellular Ca(2+). Saponin thus triggers cell membrane scrambling, an effect partially due to entry of extracellular Ca(2+) and ceramide formation.

Background: Mitotane (1,1-dichloro-2-[o-chlorophenyl]-2-[p-chlorophenyl]ethane), a cytostatic drug used for the treatment of adrenocortical carcinomas, is effective by triggering tumor cell apoptosis. In analogy to apoptosis of nucleated cells, eryptosis is the suicidal death of erythrocytes, which is typically paralleled by cell shrinkage and breakdown of cell membrane phosphatidylserine asymmetry with subsequent phosphatidylserine exposure at the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca²⁺ concentration ([Ca²⁺]_i). The present study tested, whether treatment of human erythrocytes with mitotane is followed by eryptosis. Methods: [Ca²⁺]_i was estimated from Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, and hemolysis from hemoglobin release. Results: Exposure to mitotane (≥ 5 µg/ml ≈ 16 µM) significantly increased [Ca²⁺]_i, increased annexin V binding and triggered hemolysis, but did not significantly modify forward scatter. The effect on annexin V binding was significantly blunted in the absence of extracellular Ca²⁺. Within 30 min Ca²⁺ ionophore ionomycin (1 µM) decreased forward scatter, an effect virtually abolished in the presence of mitotane (15 µg/ml). Conclusions: Mitotane increases [Ca²⁺]_i with subsequent phosphatidylserine translocation. By the same token mitotane inhibits Ca²⁺ induced cell shrinkage.

Nitazoxanide, a drug effective against a variety of pathogens, triggers apoptosis and is thus considered to be employed against malignancy. Similar to nucleated cells, erythrocytes may undergo an apoptosis-like suicidal cell death or eryptosis. Hallmarks of eryptosis include cell shrinkage and phospholipid scrambling of the cell membrane with translocation of phosphatidylserine to the erythrocyte surface. Stimulators of eryptosis include increase in cytosolic Ca 2+ -activity ([Ca 2+ ] i ). The Ca 2+ -sensitivity of eryptosis is increased by ceramide. This study explored whether nitazoxanide triggers eryptosis. [Ca 2+ ] i was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin-V-binding, ceramide abundance utilizing fluorescent antibodies and haemolysis from haemoglobin release. A 48-hr exposure to nitazoxanide (1-50 lg/ml) did not significantly modify [Ca 2+ ] i but significantly increased ceramide formation, decreased forward scatter (≥10 lg/ml), increased the percentage of annexin-V-binding erythrocytes (≥10 lg/ml) and, at higher concentrations (≥20 lg/ml), stimulated haemolysis. The stimulation of annexin-V-binding was significantly blunted in the absence of calcium. Nitazoxanide thus stimulates eryptosis, an effect in part due to ceramide formation.Nitazoxanide (NTZ; 2-(acetyloxy)-N-(5-nitro-2-thiazolyl)benzamide), a broad-spectrum thiazolide anti-infective agent [1,2] used for the treatment of gastrointestinal infections [1,3], is effective against a wide variety of pathogens including anaerobic bacteria, protozoa, trematodes, cestodes, nematodes and even viruses [1,[4][5][6]. The substance further counteracts inflammation and may thus be applicable in a wide variety of diseases [1]. Besides its antimicrobial and anti-inflammatory action, nitazoxanide may trigger tumour cell apoptosis with nuclear condensation, DNA fragmentation and phosphatidylserine exposure [7,8]. The pro-apoptotic effect has in part been attributed to stimulation of Bim expression [8].Despite lacking nuclei, erythrocytes could, similar to apoptosis of nucleated cells, undergo suicidal death or eryptosis [9], which is characterized by cell shrinkage [10] and breakdown of phosphatidylserine asymmetry of the erythrocyte cell membrane, leading to translocation of phosphatidylserine to the erythrocyte surface [9].Stimulators This study tested whether nitazoxanide stimulates eryptosis. To this end, the effect of nitazoxanide on erythrocyte volume and phosphatidylserine abundance at the cell surface has been determined. Additional experiments were further performed to shed light on the signalling involved. Materials and MethodsErythrocytes, solutions and chemicals. Leucocyte-depleted erythrocytes were kindly provided by the blood bank of the University of Tuebingen. The study was approved by the ethics committee of the University of Tuebingen (184/2003V). Erythrocytes were incubated in vitro at a haematocrit of 0.4% in Ringer's solution containing (in mM) 125 NaCl, 5 ...