Stimulation of Eryptosis, the Suicidal Erythrocyte Death by Piceatannol

Background/aims: Piceatannol, an analog and metabolite of resveratrol, is effective against various disorders including malignancy. It is in part effective by triggering suicidal death or Apoptosis of tumor cells. Cellular mechanisms mediating the proapoptotic effect of Piceatannol include mitochondrial depolarization and cytochrome c release. Erythrocytes lack mitochondria but may nevertheless enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms involved in the triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide formation. The present study explored, whether Piceatannol induces eryptosis and, if so, to shed some light on the cellular mechanisms involved.

Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, Reactive Oxygen Species (ROS) formation from 2',7'-dichlorodihydrofluorescein (DCF) diacetate-dependent fluorescence, and ceramide abundance utilizing specific Antibodies. Hemoglobin concentration in the supernatant was taken as measure of hemolysis.

Results: A 48 hours exposure of human erythrocytes to Piceatannol (10 - 20 µM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly increased DCFDA-fluorescence, significantly increased ceramide abundance, but did not significantly increase Fluo3-fluorescence. Removal of extracellular Ca2+ slightly blunted but did not abolish the effect of Piceatannol on annexin-V-binding and forward scatter. Piceatannol (20 µM) significantly augmented the increase of annexin-V-binding, but significantly blunted the decrease of forward scatter following treatment with the Ca2+ ionophore ionomycin.

Conclusions: Piceatannol triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part downstream of Ca2+ and involving oxidative stress and ceramide formation.