Jacobi Janin, Lang Elisabeth, Bissinger Rosi, Frauenfeld Leonie, Modicano Paola, Faggio Caterina, Abed Majed, Lang Florian
Department of Physiology, University of Tuebingen, Tuebingen, Germany.
Cell Physiol Biochem. 2014;33(5):1516-26. doi: 10.1159/000358715. Epub 2014 May 14.
background: Mitotane (1,1-dichloro-2-[o-chlorophenyl]-2-[p-chlorophenyl]ethane), a cytostatic drug used for the treatment of adrenocortical carcinomas, is effective by triggering tumor cell apoptosis. In analogy to apoptosis of nucleated cells, eryptosis is the suicidal death of erythrocytes, which is typically paralleled by cell shrinkage and breakdown of cell membrane phosphatidylserine asymmetry with subsequent phosphatidylserine exposure at the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca(2+) concentration ([Ca(2+)]i). The present study tested, whether treatment of human erythrocytes with mitotane is followed by eryptosis.
[Ca(2+)]i was estimated from Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, and hemolysis from hemoglobin release.
Exposure to mitotane (≥ 5 µg/ml ≈ 16 µM) significantly increased [Ca(2+)]i, increased annexin V binding and triggered hemolysis, but did not significantly modify forward scatter. The effect on annexin V binding was significantly blunted in the absence of extracellular Ca(2+). Within 30 min Ca(2+) ionophore ionomycin (1 µM) decreased forward scatter, an effect virtually abolished in the presence of mitotane (15 µg/ml).
Mitotane increases [Ca(2+)]i with subsequent phosphatidylserine translocation. By the same token mitotane inhibits Ca(2+) induced cell shrinkage.
背景：米托坦（1,1 - 二氯 - 2 - [邻氯苯基] - 2 - [对氯苯基]乙烷），一种用于治疗肾上腺皮质癌的细胞生长抑制剂，通过触发肿瘤细胞凋亡发挥作用。与有核细胞凋亡类似，红细胞凋亡是红细胞的自杀性死亡，其特征通常是细胞收缩以及细胞膜磷脂酰丝氨酸不对称性破坏，随后磷脂酰丝氨酸暴露于红细胞表面。红细胞凋亡可能由胞质钙离子浓度（[Ca(2 +)]i）升高引发。本研究测试了用米托坦处理人红细胞后是否会引发红细胞凋亡。
通过Fluo3荧光估计[Ca(2 +)]i，通过前向散射估计细胞体积，通过膜联蛋白V结合估计磷脂酰丝氨酸暴露，通过血红蛋白释放估计溶血。
暴露于米托坦（≥5μg/ml≈16μM）显著增加[Ca(2 +)]i，增加膜联蛋白V结合并引发溶血，但未显著改变前向散射。在无细胞外钙离子的情况下，对膜联蛋白V结合的影响显著减弱。在30分钟内，钙离子载体离子霉素（1μM）降低前向散射，在存在米托坦（15μg/ml）的情况下该效应几乎完全消失。
米托坦增加[Ca(2 +)]i，随后导致磷脂酰丝氨酸易位。同样，米托坦抑制钙离子诱导的细胞收缩。
