Alzoubi Kousi, Calabrò Salvatrice, Egler Jasmin, Faggio Caterina, Lang Florian
Department of Physiology, University of Tübingen, Gmelinstr. 5, 72076 Tuebingen, Germany.
Department of Biological and Environmental Sciences, University of Messina, Viale Ferdinando Stagno d'Alcontres 31, 98166 S. Agata-Messina, Italy.
Toxins (Basel). 2014 Dec 22;6(12):3596-612. doi: 10.3390/toxins6123596.
The sesquiterpene alantolactone counteracts malignancy, an effect at least in part due to stimulation of suicidal death or apoptosis of tumor cells. Signaling of alantolactone induced apoptosis involves altered gene expression and mitochondrial depolarization. Erythrocytes lack mitochondria and nuclei but may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Cellular mechanisms involved in triggering of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and oxidative stress. The present study explored, whether alantolactone stimulates eryptosis. To this end, erythrocyte volume was estimated from forward scatter, phosphatidylserine-exposure at the erythrocyte surface from FITC-annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, ceramide abundance from binding of fluorescent antibodies, and oxidative stress from 2',7'-dichlorodihydrofluorescein-diacetate (DCFDA) fluorescence. As a result, a 48 h exposure of human erythrocytes to alantolactone (≥20 μM) significantly decreased erythrocyte forward scatter and increased the percentage of annexin-V-binding cells. Alantolactone significantly increased Fluo3 fluorescence (60 μM), ceramide abundance (60 μM) and DCFDA fluorescence (≥40 μM). The effect of alantolactone (60 μM) on annexin-V-binding was not significantly modified by removal of extracellular Ca2+. In conclusion, alantolactone stimulates suicidal erythrocyte death or eryptosis, an effect paralleled by increase of [Ca2+]i, ceramide abundance and oxidative stress.
倍半萜内酯阿兰内酯可对抗恶性肿瘤，其作用至少部分归因于刺激肿瘤细胞的自杀性死亡或凋亡。阿兰内酯诱导凋亡的信号传导涉及基因表达改变和线粒体去极化。红细胞缺乏线粒体和细胞核，但可能进入自杀性死亡或红细胞凋亡，其特征是细胞收缩和细胞膜磷脂酰丝氨酸外翻暴露于红细胞表面。触发红细胞凋亡的细胞机制包括胞质Ca2+活性（[Ca2+]i）增加和氧化应激。本研究探讨了阿兰内酯是否刺激红细胞凋亡。为此，通过前向散射估计红细胞体积，通过FITC-膜联蛋白-V结合估计红细胞表面磷脂酰丝氨酸暴露，通过Fluo3荧光估计[Ca2+]i，通过荧光抗体结合估计神经酰胺丰度，通过2',7'-二氯二氢荧光素二乙酸酯（DCFDA）荧光估计氧化应激。结果，将人红细胞暴露于阿兰内酯（≥20 μM）48小时可显著降低红细胞前向散射并增加膜联蛋白-V结合细胞的百分比。阿兰内酯显著增加Fluo3荧光（60 μM）、神经酰胺丰度（60 μM）和DCFDA荧光（≥40 μM）。去除细胞外Ca2+后，阿兰内酯（60 μM）对膜联蛋白-V结合的影响未显著改变。总之，阿兰内酯刺激红细胞自杀性死亡或红细胞凋亡，这一作用伴随着[Ca2+]i、神经酰胺丰度和氧化应激的增加。
