Piperlongumine-Induced Phosphatidylserine Translocation in the Erythrocyte Membrane

Background: Piperlongumine, a component of Piper longum fruit, is considered as a treatment for malignancy. It is effective by inducing apoptosis. Mechanisms involved in the apoptotic action of piperlongumine include oxidative stress and activation of p38 kinase. In analogy to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine-exposure at the erythrocyte surface. Signaling involved in eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)), formation of ceramide, oxidative stress and activation of p38 kinase. Methods: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, [Ca(2+)](i) from Fluo3 fluorescence, reactive oxygen species from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. Results: A 48 h exposure to piperlongumine (30 µM) was followed by significant decrease of forward scatter and increase of annexin-V-binding. Piperlongumine did not significantly modify [Ca(2+)](i) and the effect was not dependent on presence of extracellular Ca(2+). Piperlongumine significantly increased ROS formation and ceramide abundance. Conclusions: Piperlongumine triggers cell membrane scrambling, an effect independent from entry of extracellular Ca(2+) but at least partially due to ROS and ceramide formation.