Sulindac Sulfide – Induced Stimulation of Eryptosis

Abstract

Background: Sulindac sulfide, a non-steroidal anti-inflammatory drug (NSAID), stimulates apoptosis of tumor cells and is thus effective against malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, an apoptosis-like suicidal erythrocyte death, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine-exposure at the cell surface. Stimulators of eryptosis include increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i) and ceramide formation. The present study explored, whether sulindac sulfide stimulates eryptosis. Methods: [Ca²⁺]_i was estimated from Fluo-3 fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from binding of fluorescent annexin-V, hemolysis from hemoglobin release, and ceramide abundance utilizing fluorescent antibodies. Results: A 48 h exposure to sulindac sulfide (≤ 20 µM) was followed by significant increase of [Ca²⁺]_i, enhanced ceramide abundance, decreased forward scatter and increased percentage of annexin-V-binding erythrocytes. Sulindac sulfide triggered slight but significant hemolysis. Removal of extracellular Ca²⁺ significantly blunted, but did not abrogate the effect of sulindac sulfide (20 µM) on annexin-V-binding. Conclusion: Sulindac sulfide stimulates the suicidal death of erythrocytes or eryptosis, an effect paralleled by Ca²⁺-entry, ceramide formation, cell shrinkage and phosphatidylserine-exposure.