Stimulation of Eryptosis, the Suicidal Erythrocyte Death, by Costunolide

Abstract

Background/Aims: The sesquiterpene lactone Costunolide is effective against various disorders including inflammation and malignancy. The substance is effective in part by triggering suicidal death or apoptosis of tumor cells. Mechanisms involved include altered function of transcription factors and mitochondria. Erythrocytes lack nuclei and mitochondria but are – in analogy to apoptosis of nucleated cells – able to enter suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), oxidative stress and ceramide. The present study explored, whether Costunolide induces eryptosis and, if so, to shed light on the 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)-dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to Costunolide (15 µg/ml) significantly enhanced the percentage of annexin-V-binding cells, significantly decreased forward scatter and significantly increased Fluo3-fluorescence, DCF-fluorescence, and ceramide abundance. The effect of Costunolide on annexin-V-binding was significantly blunted by removal of extracellular Ca²⁺. Conclusion: Costunolide triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca²⁺ entry and paralleled by oxidative stress and ceramide formation.

The specific PKC-α inhibitor chelerythrine blunts costunolide-induced eryptosis

“…Eryptosis-inducing substances may dose-dependently cause both cell swelling and cell shrinkage [ 36 ]. Costunolide induces a sustained influx of Ca 2+ ions [ 40 ], which activates the Gardos channel, a Ca 2+ -sensitive and K + -selective channel, by which cellular K + ions leave the cell followed by chloride ions and water, thus leading to erythrocyte shrinkage [ 48 ]. Volume-sensitive transport pathways are regulated by kinase(s) and phosphatase(s) activities [ 49 , 50 ].…”

“…Due to a wide range of biological activities, costunolide has been extensively studied over the past 20 years. Our laboratory previously published that costunolide induces eryptosis [ 40 ]. The present study explored the effects of costunolide on pro-survival factors and enzymes in human erythrocytes, e.g.…”

Stimulation of Erythrocyte Membrane Blebbing by Bifenthrin Induced Oxidative Stress

“…Leukocyte free erythrocytes were obtained by using a protocol described by Fink et al 16 Blood samples were centrifuged at 12 298 g for 20 minutes to collect erythrocytes. A 48 h in-vitro incubation (at 37°C) of .4% hematocrit were done in ringer solution containing (in mM) MgSO 4 1, NaCl 125, KCl 5, Glucose 5, CaCl 2 1, N-2-hydroxy-piperacine-N-2-ethanesulphonic acid (HEPES) 32.…”

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“…Leukocyte-depleted cells were prepared by following protocol explained by Fink et al 11 Isolated erythrocytes were stored in separate micro-centrifuge tubes. In vitro incubations of erythrocytes were performed at a hematocrit of 0.4% in Ringer solution (pH 7.4) that contain (in mM) MgSO 4 1, NaCl 125, KCl 5, glucose 5, CaCl 2 1, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid 32 at 37°C for 48 hours.…”