Stimulation of Eryptosis by Caspofungin
Thomas Peter, Rosi Bissinger, Florian Lang
Cellular Physiology and Biochemistry
Background/Aims: The echinocandin antifungal agent caspofungin has been shown to trigger apoptosis of fungal cells. Beyond that, caspofungin is toxic for host mitochondria. Even though lacking mitochondria, erythrocytes may enter apoptosis-like suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in triggering of eryptosis include increase of cytosolic Ca 2+
... ity ([Ca 2+ ] i ), oxidative stress, ceramide, caspase activation and/ or activation of p38 kinase, protein kinase C, and casein kinase. The present study explored, whether caspofungin induces eryptosis and, if so, to shed some light on the cellular mechanisms involved. Methods: Flow cytometry was employed to determine phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca 2+ ] i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified from hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to caspofungin (≥ 30 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly enhanced hemolysis, but did not significantly increase Fluo3-fluorescence, DCFDA fluorescence or ceramide abundance. The effect of caspofungin on annexin-V-binding was not significantly blunted by removal of extracellular Ca 2+ , by inhibition of caspases with pancaspase inhibitor zVAD (10 µM), or by addition of the antioxidant N-acetyl-cysteine (1 mM), p38 kinase inhibitor SB203580 (2 µM) or protein kinase C inhibitor staurosporine (1 µM). The effect of caspofungin on annexin-Vbinding was, however, significantly blunted in the presence of casein kinase inhibitor D4476 (10 µM). Conclusions: Caspofungin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect possibly involving activation of casein kinase. of Candida albicans  . The substance may be toxic as well for host cells and for host mitochondria  . Erythrocytes lack mitochondria but could nevertheless enter apoptosis-like suicidal death or eryptosis  , which is characterized by cell shrinkage  and cell membrane scrambling with phosphatidylserine translocation to the cell surface  . A wide variety of apoptosis inducing substances similarly trigger eryptosis [30, . Triggering of eryptosis may involve increase of cytosolic Ca 2+ activity ([Ca 2+ ] i ) , ceramide , oxidative stress , energy depletion , activated caspases [30, 77, 78] , and stimulation of casein kinase 1α, Janus-activated kinase JAK3, protein kinase C, and/or p38 kinase  . Eryptosis is inhibited following activation of AMP activated kinase AMPK, cGMP-dependent protein kinase, and sorafenib/sunitinib sensitive kinases  . The present study explored, whether caspofungin is capable to trigger eryptosis. Erythrocytes from healthy volunteers were treated with caspofungin and phosphatidylserine surface abundance, cell volume, [Ca 2+ ] i , ROS formation, and ceramide abundance determined by flow cytometry.