Programmed erythrocyte death following in vitro Treosulfan treatment.

BACKGROUND/AIMS: The cytotoxic drug Treosulfan is clinically used for the treatment of malignancy. A common side effect of Treosulfan treatment is anemia. Treosulfan is at least partially effective by triggering apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane. Triggers of eryptosis include oxidative stress, Ca(2+)-entry and increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). The present study explored whether Treosulfan stimulates eryptosis, which may contribute to development of anemia. METHODS: Erythrocyte volume was estimated from forward scatter, phosphatidylserine abundance at the erythrocyte surface from Fluorescein isothiocyanate (FITC)-annexin-V-binding, [Ca(2+)]i from Fluo3 fluorescence and reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA)-fluorescence. RESULTS: A 48 hours exposure of human erythrocytes to Treosulfan (800 µg/ml) significantly decreased erythrocyte forward scatter, increased the percentage of annexin-V-binding cells, increased [Ca(2+)]i, and increased ROS. The effect of Treosulfan on annexin-V-binding was virtually abrogated by removal of extracellular Ca(2+). CONCLUSION: Treosulfan stimulates suicidal erythrocyte death or eryptosis at least in part by inducing oxidative stress and stimulating Ca(2+) entry.

Induction of suicidal erythrocyte death by novobiocin.

BACKGROUND: Novobiocin, an aminocoumarin antibiotic, interferes with heat shock protein 90 and hypoxia inducible factor dependent gene expression and thus compromises cell survival. Similar to survival of nucleated cells, erythrocyte survival could be disrupted by eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by phospholipd scrambling of the cell membrane with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). The Ca(2+) sensitivity of phospholipid scrambling is enhanced by ceramide. The present study explored, whether novobiocin elicits eryptosis. METHODS: [Ca(2+)]i was estimated from Fluo3-fluorescence, ceramide abundance utilizing fluorescent antibodies, cell volume from forward scatter, phosphatidylserine-exposure from annexin V binding. RESULTS: A 48 hours exposure to novobiocin (500 µM) was followed by a significant increase of [Ca(2+)]i, decrease of forward scatter, increase of annexin-V-binding and enhanced ceramide formation. Removal of extracellular Ca(2+) virtually abrogated the increase of annexin-V-binding following novobiocin exposure. CONCLUSIONS: Novobiocin stimulates eryptosis, an effect at least in part due to entry of extracellular Ca(2+) and formation of ceramide.

Breakdown of phosphatidylserine asymmetry following treatment of erythrocytes with lumefantrine.

BACKGROUND: Lumefantrine, a commonly used antimalarial drug, inhibits hemozoin formation in parasites. Several other antimalarial substances counteract parasitemia by triggering suicidal death or eryptosis of infected erythrocytes. Eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine-exposure at the erythrocyte surface. Signaling involved in eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i), formation of ceramide, oxidative stress and/or activation of p38 kinase, protein kinase C (PKC), or caspases. The present study explored, whether lumefantrine stimulates eryptosis. METHODS: Cell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence, content of reduced glutathione (GSH) from mercury orange fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. RESULTS: A 48 h exposure to lumefantrine (3 µg/mL) was followed by a significant increase of annexin-V-binding without significantly altering forward scatter, [Ca2+]i, ROS formation, reduced GSH, or ceramide abundance. The annexin-V-binding following lumefantrine treatment was not significantly modified by p38 kinase inhibitors SB203580 (2 µM) and p38 Inh III (1 µM), PKC inhibitor staurosporine (1 µM) or pancaspase inhibitor zVAD (1 or 10 µM). CONCLUSIONS: Lumefantrine triggers cell membrane scrambling, an effect independent from entry of extracellular Ca2+, ceramide formation, ROS formation, glutathione content, p38 kinase, PKC or caspases.

Stimulation of suicidal erythrocyte death by ribavirin.

Ribavirin is widely used in the treatment for viral disease such as chronic viral hepatitis. Side effects limiting the use of the drug include haemolytic anaemia. If challenged by stimulators of haemolysis, erythrocytes may enter suicidal death or eryptosis, thus preventing the release of haemoglobin into circulating blood. Eryptosis is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase in cytosolic Ca2+ activity ([Ca2+]i). This study explored whether ribavirin modifies [Ca2+]i and elicits eryptosis. Cell volume has been estimated from forward scatter, phosphatidylserine abundance at the erythrocyte surface from annexin V binding, haemolysis from haemoglobin release and [Ca2+]i from Fluo-3 fluorescence. A 48-hr exposure to ribavirin (≥8 µg/ml) was followed by a significant increase in [Ca2+]i, a significant decrease in forward scatter and a significant increase in annexin V binding. The annexin V binding after ribavirin treatment was significantly blunted but not abolished in the nominal absence of extracellular Ca2+. In conclusion, ribaverin stimulates eryptosis, an effect at least in part due to entry of extracellular Ca2+.