Al Mamun Bhuyan Abdulla, Bissinger Rosi, Cao Hang, Lang Florian
Departments of Cardiology, Cardiovascular Medicine and Physiology, Eberhard-Karls-University of Tuebingen, Tuebingen, Germany.
Department of Molecular Medicine II, Medical Faculty, Heinrich Heine University, Duesseldorf, Germany.
Cell Physiol Biochem. 2017;42(1):1-12. doi: 10.1159/000477224. Epub 2017 May 11.
BACKGROUND/AIMS: The steroidal aromatase inactivator exemestane blocks estrogen biosynthesis and is thus employed for the prevention and treatment of breast cancer. Exemestane is in part effective by stimulation of suicidal cell death or apoptosis. Side effects of exemestane treatment include anemia. At least in theory, exemestane induced anemia could be secondary to stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, several kinases and caspases. The present study explored, whether exemestane is able to trigger eryptosis and, if so, to shed some light on the signaling involved.
Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCF fluorescence, and ceramide abundance utilizing specific antibodies.
A 48 hours exposure of human erythrocytes to exemestane (≥ 10 µg/ml) significantly increased the percentage of annexin-V-binding cells without significantly modifying forward scatter. Exemestane significantly increased Fluo3-fluorescence (10 and 20, but not 40 µg/ml), DCF fluorescence (40 µg/ml), and ceramide abundance (40 µg/ml). The effect of exemestane (40 µg/ml) on annexin-V-binding was significantly blunted by antioxidant N-acetylcysteine (1mM), but was not significantly modified by removal or increase of extracellular Ca2+, by p38 kinase inhibitor SB203580 (2 µM), casein kinase inhibitor D4476 (10 µM) and caspase inhibitor zVAD (10 µM).
Exemestane triggers phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by enhanced [Ca2+]i, oxidative stress, and increased ceramide abundance.
背景/目的：甾体类芳香化酶灭活剂依西美坦可阻断雌激素生物合成，因此被用于预防和治疗乳腺癌。依西美坦部分通过刺激自杀性细胞死亡或凋亡发挥作用。依西美坦治疗的副作用包括贫血。至少在理论上，依西美坦诱导的贫血可能继发于自杀性红细胞死亡或红细胞凋亡的刺激，其特征为细胞皱缩和细胞膜磷脂酰丝氨酸易位导致的细胞膜紊乱。刺激红细胞凋亡涉及的信号包括胞质Ca2+活性（[Ca2+]i）增加、氧化应激、神经酰胺、多种激酶和半胱天冬酶。本研究探讨了依西美坦是否能够引发红细胞凋亡，如果可以，则阐明其中涉及的信号。
采用流式细胞术通过膜联蛋白V结合来定量细胞表面磷脂酰丝氨酸暴露情况，通过前向散射来定量细胞体积，通过Fluo3荧光来定量[Ca2+]i，通过DCF荧光来定量活性氧（ROS）丰度，并使用特异性抗体来定量神经酰胺丰度。
将人红细胞暴露于依西美坦（≥10μg/ml）48小时可显著增加膜联蛋白V结合细胞的百分比，而对前向散射无显著影响。依西美坦显著增加Fluo3荧光（10和20μg/ml，但40μg/ml时无此作用）、DCF荧光（40μg/ml）和神经酰胺丰度（40μg/ml）。抗氧化剂N-乙酰半胱氨酸（1mM）可显著减弱依西美坦（40μg/ml）对膜联蛋白V结合的作用，但细胞外Ca2+的去除或增加、p38激酶抑制剂SB203580（2μM）、酪蛋白激酶抑制剂D4476（10μM）和半胱天冬酶抑制剂zVAD（10μM）对其作用无显著影响。
依西美坦引发红细胞细胞膜磷脂紊乱，此效应伴随着[Ca2+]i增强、氧化应激和神经酰胺丰度增加。
