Karasavvas N, Erukulla R K, Bittman R, Lockshin R, Zakeri Z
Department of Biology, Queens College, Flushing, NY, USA.
Eur J Biochem. 1996 Mar 1;236(2):729-37. doi: 10.1111/j.1432-1033.1996.00729.x.
We investigated the ability of N-octanoyl-sphingosine (C8-Cer) stereoisomers, N-octanoyl-DL-erythro-dihydrosphingosine (DL-e-DHC8-Cer), and a new ceramide derivative, N-octyl-D-erythro-sphingosine (D-e-C8-Ceramine), to induce apoptosis in U937 cells. We found the C8-Cer stereoisomers to be stereospecific with the D- and L-threo stereoisomers being severalfold more potent than the erythro in inducing nucleosomal fragmentation. The order of potency was: D-t-C8-Cer = L-t-C8-Cer > L-e-C8-Cer > D-e-C8-Cer > DL-e-DHC8-Cer. The importance of the carbonyl group in apoptosis was investigated by using a new ceramide derivative, D-e-C8-Ceramine, in which the carbonyl group was replaced by a methylene group. The carbonyl group was not necessary for triggering apoptosis. In fact, replacement of the carbonyl group decreased substantially the time required for cells to die, with maximum DNA fragmentation occurring at 6 h as opposed to the 18 h required by D-e-C8-Cer. To explore possible mechanisms by which these compounds trigger the apoptotic pathway, we tested their ability to increase the endogenous levels of cellular ceramide and to differentially activate a ceramide-activated protein kinase (CAPK). While the potent DNA fragmentation-inducing compounds D-e-C8-Ceramine and L-t-C8-Cer failed to increase the cellular ceramide levels, D-e-C8-Cer, D-t-C8-Cer and D-e-C8-Ceramine activated the CAPK equally. These studies suggest that the DNA fragmentation-inducing ability of the threo stereoisomers and D-e-C8-Ceramine cannot be attributed either to an increase in the activity of CAPK, or, as illustrated by D-e-C8-Ceramine and L-t-C8-Cer, to the differential elevation of endogenous ceramide. The phosphatase inhibitor okadaic acid failed to protect U937 cells from apoptosis induced by D-e-C8-Cer.
我们研究了N-辛酰基-鞘氨醇（C8-神经酰胺）立体异构体、N-辛酰基-DL-赤型-二氢鞘氨醇（DL-e-DHC8-神经酰胺）以及一种新的神经酰胺衍生物N-辛基-D-赤型-鞘氨醇（D-e-C8-神经酰胺）诱导U937细胞凋亡的能力。我们发现C8-神经酰胺立体异构体具有立体特异性，D-和L-苏型立体异构体在诱导核小体片段化方面比赤型强几倍。效力顺序为：D-t-C8-神经酰胺 = L-t-C8-神经酰胺 > L-e-C8-神经酰胺 > D-e-C8-神经酰胺 > DL-e-DHC8-神经酰胺。通过使用一种新的神经酰胺衍生物D-e-C8-神经酰胺来研究羰基在凋亡中的重要性，其中羰基被亚甲基取代。触发凋亡并不需要羰基。事实上，羰基的取代显著缩短了细胞死亡所需的时间，最大DNA片段化在6小时出现，而D-e-C8-神经酰胺则需要18小时。为了探究这些化合物触发凋亡途径的可能机制，我们测试了它们增加细胞内源性神经酰胺水平以及差异激活神经酰胺激活蛋白激酶（CAPK）的能力。虽然强效诱导DNA片段化的化合物D-e-C8-神经酰胺和L-t-C8-神经酰胺未能增加细胞神经酰胺水平，但D-e-C8-神经酰胺、D-t-C8-神经酰胺和D-e-C8-神经酰胺对CAPK的激活程度相同。这些研究表明，苏型立体异构体和D-e-C8-神经酰胺诱导DNA片段化的能力既不能归因于CAPK活性的增加，也不能像D-e-C8-神经酰胺和L-t-C8-神经酰胺所示的那样归因于内源性神经酰胺的差异升高。磷酸酶抑制剂冈田酸未能保护U937细胞免受D-e-C8-神经酰胺诱导的凋亡。
