Withaferin A-stimulated Ca2+ entry, ceramide formation and suicidal death of erythrocytes - PubMed (original) (raw)

Withaferin A-stimulated Ca2+ entry, ceramide formation and suicidal death of erythrocytes

Kashif Jilani et al. Toxicol In Vitro. 2013 Feb.

Abstract

Withaferin A, a triterpenoid component from Withania somnifera, counteracts malignancy, an effect attributed to stimulation of apoptosis. Withaferin A is partially effective through induction of oxidative stress, altered gene expression and mitochondrial depolarization. Erythrocytes lack mitochondria and nuclei but may enter apoptosis-like eryptosis, a suicidal cell death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+)-activity [Ca(2+)](i) following activation of oxidant-sensitive Ca(2+)-permeable cation channels, ceramide formation and/or ATP-depletion. The present study explored, whether withaferin A triggers eryptosis. To this end, [Ca(2+)](i) was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin-V-binding, hemolysis from hemoglobin release, oxidative stress from DCFDA-fluorescence and ceramide abundance utilizing antibodies. A 48 h exposure to withaferin A significantly decreased forward scatter (at ≥ 10 μM withaferin concentration) and increased [Ca(2+)](i) (≥ 5 μM), ROS-formation (≥ 10 μM) ceramide-formation ( ≥ 10 μM) as well as annexin-V-binding ( ≥ 5 μM). Withaferin A treatment was followed by slight but significant increase of hemolysis. Extracellular Ca(2+) removal, amiloride, and the antioxidant N-acetyl-l-cysteine significantly blunted withaferin A-triggered annexin-V-binding. The present observations reveal that withaferin A triggers suicidal erythrocyte death despite the absence of gene expression and key elements of apoptosis such as mitochondria.

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