PMID- 28793293 OWN - NLM STAT- MEDLINE DCOM- 20171026 LR - 20211204 IS - 1421-9778 (Electronic) IS - 1015-8987 (Linking) VI - 42 IP - 4 DP - 2017 TI - Triggering of Eryptosis, the Suicidal Erythrocyte Death by Mammalian Target of Rapamycin (mTOR) inhibitor Temsirolimus. PG - 1575-1591 LID - 10.1159/000479398 [doi] AB - BACKGROUND/AIMS: The mammalian target of rapamycin (mTOR) inhibitor temsirolimus is utilized for the treatment of malignancy. Temsirolimus is at least in part effective by triggering suicidal tumor cell death. The most common side effect of temsirolimus treatment is anemia. At least in theory, the anemia following temsirolimus treatment could result from stimulation of eryptosis, the suicidal erythrocyte death. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the orchestration of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, as well as activation of staurosporine and chelerythrine sensitive protein kinase C, SB203580 sensitive p38 kinase, D4476 sensitive casein kinase 1, and zVAD sensitive caspases. The purpose of the present study was to test whether temsirolimus influences eryptosis and, if so, to shed light on the signaling involved. METHODS: Flow cytometry was employed to estimate cell volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was determined from hemoglobin concentration in the supernatant. RESULTS: A 48 hours exposure of human erythrocytes to temsirolimus (5 - 20 microg/ml) significantly decreased forward scatter and significantly increased the percentage of annexin-V-binding cells. Temsirolimus significantly increased Fluo3-fluorescence, DCFDA fluorescence and ceramide abundance at the erythrocyte surface. The effect of temsirolimus on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+ and by addition of staurosporine (1 microM) or chelerythrine (10 microM) but not significantly modified by addition of SB203580 (2 microM), D4476 (10 microM), or zVAD (10 microM). Chelerythrine (10 microM) further significantly blunted the effect of temsirolimus on DCFDA fluorescence but not ceramide formation. Removal of extracellular Ca2+ had no effect on temsirolimus induced ROS formation or ceramide abundance. CONCLUSIONS: Temsirolimus triggers eryptosis with cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress, ceramide and activation of staurosporine/Chelerythrine sensitive kinase(s). CI - (c) 2017 The Author(s). Published by S. Karger AG, Basel. FAU - Al Mamun Bhuyan, A AU - Al Mamun Bhuyan A AD - Department of Internal Medicine III, Tuebingen, Germany. FAU - Cao, Hang AU - Cao H AD - Department of Internal Medicine III, Tuebingen, Germany. FAU - Lang, Florian AU - Lang F AD - Department of Physiology I, Eberhard-Karls-University of Tuebingen, Tuebingen, Germany. AD - Department of Molecular Medicine II, Medical Faculty, Heinrich Heine University, Duesseldorf, Germany. LA - eng PT - Journal Article DEP - 20170724 PL - Germany TA - Cell Physiol Biochem JT - Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology JID - 9113221 RN - 0 (4-(4-(2,3-dihydrobenzo(1,4)dioxin-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl)benzamide) RN - 0 (Aniline Compounds) RN - 0 (Annexin A5) RN - 0 (Antineoplastic Agents) RN - 0 (Benzamides) RN - 0 (Benzophenanthridines) RN - 0 (Ceramides) RN - 0 (Fluoresceins) RN - 0 (Imidazoles) RN - 0 (Oligopeptides) RN - 0 (Phosphatidylserines) RN - 0 (Protein Kinase Inhibitors) RN - 0 (Pyridines) RN - 0 (Reactive Oxygen Species) RN - 0 (Xanthenes) RN - 0 (benzyloxycarbonyl-valyl-alanyl-aspartic acid) RN - 2044-85-1 (diacetyldichlorofluorescein) RN - 23D4W0B50Y (Fluo-3) RN - 624KN6GM2T (temsirolimus) RN - E3B045W6X0 (chelerythrine) RN - EC 2.7.1.1 (MTOR protein, human) RN - EC 2.7.11.1 (Casein Kinase I) RN - EC 2.7.11.1 (TOR Serine-Threonine Kinases) RN - EC 2.7.11.24 (p38 Mitogen-Activated Protein Kinases) RN - EC 3.4.22.- (Caspases) RN - H88EPA0A3N (Staurosporine) RN - OU13V1EYWQ (SB 203580) RN - SY7Q814VUP (Calcium) RN - W36ZG6FT64 (Sirolimus) SB - IM MH - Aniline Compounds/chemistry MH - Annexin A5/metabolism MH - Antineoplastic Agents/*pharmacology MH - Benzamides/pharmacology MH - Benzophenanthridines/pharmacology MH - Calcium/metabolism MH - Casein Kinase I/antagonists & inhibitors/genetics/metabolism MH - Caspases/genetics/metabolism MH - Ceramides/metabolism MH - Eryptosis/*drug effects/genetics MH - Erythrocytes/cytology/*drug effects/metabolism MH - Fluoresceins/chemistry MH - Gene Expression Regulation MH - Hemolysis/*drug effects MH - Humans MH - Imidazoles/pharmacology MH - Oligopeptides/pharmacology MH - Phosphatidylserines/metabolism MH - Primary Cell Culture MH - Protein Kinase Inhibitors/*pharmacology MH - Pyridines/pharmacology MH - Reactive Oxygen Species/metabolism MH - Signal Transduction MH - Sirolimus/*analogs & derivatives/pharmacology MH - Staurosporine/pharmacology MH - TOR Serine-Threonine Kinases/*antagonists & inhibitors/genetics/metabolism MH - Xanthenes/chemistry MH - p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/genetics/metabolism OTO - NOTNLM OT - Calcium OT - Ceramide OT - Eryptosis OT - Oxidative stress OT - Phosphatidylserine OT - Staurosporine EDAT- 2017/08/10 06:00 MHDA- 2017/10/27 06:00 CRDT- 2017/08/10 06:00 PHST- 2017/02/15 00:00 [received] PHST- 2017/06/12 00:00 [accepted] PHST- 2017/08/10 06:00 [pubmed] PHST- 2017/10/27 06:00 [medline] PHST- 2017/08/10 06:00 [entrez] AID - 000479398 [pii] AID - 10.1159/000479398 [doi] PST - ppublish SO - Cell Physiol Biochem. 2017;42(4):1575-1591. doi: 10.1159/000479398. Epub 2017 Jul 24.