Triggering of Suicidal Erythrocyte Death Following Boswellic Acid Exposure

Abstract

Background/Aims: The antinflammatory natural product boswellic acid is effective against cancer at least in part by inducing tumor cell apoptosis. Similar to apoptosis of nucleated cells erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include oxidative stress, increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i), energy depletion, ceramide formation and p38 kinase activation. The present study tested, whether and how boswellic acid induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca²⁺]_i from Fluo3-fluorescence, ceramide abundance utilizing specific antibodies, reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofuorescein diacetate (DCFDA) fluorescence, and cytosolic ATP concentration utilizing a luciferin-luciferase assay kit. Results: A 24 hours exposure of human erythrocytes to boswellic acid (5 µg/ml) significantly increased the percentage of annexin-V-binding cells (to 9.3 ±0.9 %) and significantly decreased forward scatter. Boswellic acid did not significantly modify [Ca²⁺]_i, cytosolic ATP, ROS, or ceramide abundance. The effect of boswellic acid on annexin-V-binding was significantly blunted, but not abolished by p38 kinase inhibitors skepinone (2 µM) and SB203580 (2 µM). Conclusions: Boswellic acid stimulates cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part dependent on p38 protein kinase activity.