Al Mamun Bhuyan Abdulla, Signoretto Elena, Bissinger Rosi, Lang Florian
Departments of Cardiology, Vascular Medicine and Physiology, Eberhard-Karls-University of Tuebingen, Tuebingen, Germany.
Cell Physiol Biochem. 2016;39(2):639-50. doi: 10.1159/000445655. Epub 2016 Jul 21.
BACKGROUND/AIMS: The viral integrase enzyme inhibitor dolutegravir is utilized for the treatment of immunodeficiency virus (HIV) infection. Knowledge on cytotoxicity of dolutegravir is limited. The present study thus explored, whether dolutegravir is able to trigger suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms involved in the triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, and activation of protein kinase C, p38 kinase, casein kinase, and caspases. The present study explored, whether Dolutegravir induces eryptosis and, if so, to gain insight into cellular mechanisms involved.
Utilizing flow cytometry, phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified from haemoglobin concentration in the supernatant.
A 48 hours exposure of human erythrocytes to dolutegravir significantly increased the percentage of annexin-V-binding cells (≥ 4.8 µM), significantly increased hemolysis (19.1 µM), but did not significantly modify forward scatter. Dolutegravir significantly increased Fluo3-fluorescence (≥ 4.8 µM), DCFDA fluorescence (19.1 µM) and ceramide abundance (19.1 µM). The effect of dolutegravir on annexin-V-binding was significantly blunted by removal of extracellular Ca2+, but was not significantly modified by protein kinase C inhibitor staurosporine (1 µM), p38 kinase inhibitor SB203580 (2 µM), casein kinase inhibitor D4476 (10 µM) or pancaspase inhibitor zVAD (10 µM).
Dolutegravir triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, ceramide formation and oxidative stress.
背景/目的：病毒整合酶抑制剂多替拉韦用于治疗免疫缺陷病毒（HIV）感染。关于多替拉韦细胞毒性的知识有限。因此，本研究探讨了多替拉韦是否能够引发自杀性红细胞死亡或红细胞凋亡，其特征为细胞皱缩和细胞膜磷脂酰丝氨酸易位至红细胞表面。引发红细胞凋亡的细胞机制包括胞质Ca2+活性（[Ca2+]i）增加、氧化应激、神经酰胺以及蛋白激酶C、p38激酶、酪蛋白激酶和半胱天冬酶的激活。本研究探讨了多替拉韦是否诱导红细胞凋亡，若如此，则深入了解其中涉及的细胞机制。
利用流式细胞术，通过膜联蛋白-V结合估计细胞表面磷脂酰丝氨酸暴露情况，通过前向散射估计细胞体积，通过Fluo3荧光估计[Ca2+]i，通过DCFDA依赖性荧光估计活性氧形成，并利用特异性抗体估计神经酰胺丰度。通过上清液中的血红蛋白浓度定量溶血情况。
人红细胞暴露于多替拉韦48小时后，膜联蛋白-V结合细胞百分比显著增加（≥4.8 μM），溶血显著增加（19.1 μM），但前向散射未显著改变。多替拉韦显著增加Fluo3荧光（≥4.8 μM）、DCFDA荧光（19.1 μM）和神经酰胺丰度（19.1 μM）。去除细胞外Ca2+可显著减弱多替拉韦对膜联蛋白-V结合的影响，但蛋白激酶C抑制剂星形孢菌素（1 μM）、p38激酶抑制剂SB203580（2 μM）、酪蛋白激酶抑制剂D4476（10 μM）或泛半胱天冬酶抑制剂zVAD（10 μM）对其影响未产生显著改变。
多替拉韦引发红细胞细胞膜皱缩和磷脂易位，这一效应至少部分归因于Ca2+内流、神经酰胺形成和氧化应激。
