Question:

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 Ca²⁺-activity ([Ca²⁺]_i) following activation of oxidant-sensitive Ca²⁺-permeable cation channels, ceramide formation and/or ATP-depletion. The present study explored, whether withaferin A triggers eryptosis.

Methods:

[Ca²⁺]_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.

Results:

A 48h exposure to withaferin A significantly decreased forward scatter (at =10 µM withaferin concentration) and increased [Ca²⁺]_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 Ca²⁺ removal, amiloride, and the antioxidant N-acetyl-L-cysteine significantly blunted withaferin A-triggered annexin-V-binding.

Conclusions:

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.