Stimulation of Erythrocyte Death by Phloretin

Abstract

Background: Phloretin, a natural component of apples, pears and strawberries, has previously been shown to stimulate apoptosis of nucleated cells. Erythrocytes may similarly enter suicidal death or eryptosis, which is characterized by cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i), ceramide, ATP depletion, and activation of protein kinase C (PKC) as well as p38 mitogen activated protein kinase (p38 kinase). Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, and ceramide abundance from binding of specific antibodies. Results: A 48 h exposure of human erythrocytes to phloretin significantly increased the percentage of annexin-V-binding cells (≥100 µM) without significantly influencing forward scatter. Phloretin did not significantly modify [Ca²⁺]_i and the stimulation of annexin-V-binding by phloretin (300 µM) did not require presence of extracellular Ca²⁺. Phloretin did not significantly modify erythrocyte ATP levels, and the effect of phloretin on annexin-V-binding was not significantly altered by PKC inhibitor staurosporine (1 µM) or p38 kinase inhibitor SB2203580 (2 µM). However, phloretin significantly increased the ceramide abundance at the cell surface. Conclusions: Phloretin stimulates phospholipid scrambling of the erythrocyte cell membrane, an effect at least partially due to up-regulation of ceramide abundance.