Kimura K, Markowski M, Edsall L C, Spiegel S, Gelmann E P
Department of Oncology, Georgetown University, Washington, DC 20007-2197, USA.
Cell Death Differ. 2003 Feb;10(2):240-8. doi: 10.1038/sj.cdd.4401145.
The sphingomyelin metabolites ceramide and sphingosine are mediators of cell death induced by gamma-irradiation. We studied the production of ceramide and the effects of exogenous ceramide on apoptosis in LNCaP prostate cancer cells that are highly resistant to gamma-irradiation-induced cell death. LNCaP cells can be sensitized to gamma-irradiation by tumor necrosis factor alpha (TNF-alpha) and, to a lesser degree, by the agonistic FAS antibody CH-11. TNF-alpha activated intrinsic and extrinsic apoptosis pathways and increased ceramide and sphingosine levels in irradiated LNCaP cells. CH-11 activated only the extrinsic apoptosis pathways and had a negligible effect on ceramide and sphingosine levels in irradiated LNCaP cells. Exogenous ceramide and bacterial sphingomyelinase sensitized LNCaP cells to radiation-induced apoptosis and had a synergistic effect on cell death after irradiation with TNF-alpha, but not with CH-11. Cell death effects after exposure to ceramide and irradiation were blocked by the serine protease inhibitor TLCK (Na-p-tosyl-L-lysine-chloromethylketone), but not by the caspase inhibitor z-VAD (2-val-Ala-Asp(oMe)-CH(2)F). During LNCaP cell apoptosis induced by exogenous ceramide, we observed activation of caspase-9, but not caspases-8, -3, or -7. The effect of ceramide occurred largely via the intrinsic mitochondrial apoptosis pathway and enhanced TNF-alpha, but not CH-11 effects on irradiated cells. The data show that ceramide enhanced activation of the intrinsic apoptotic pathway and enhanced cell death induced by TNF-alpha with or without gamma-irradiation. TNF-alpha and gamma-irradiation elevated levels of endogenous ceramide and activated the intrinsic cell death pathway.
鞘磷脂代谢产物神经酰胺和鞘氨醇是γ射线诱导细胞死亡的介质。我们研究了神经酰胺的产生以及外源性神经酰胺对LNCaP前列腺癌细胞凋亡的影响，该细胞对γ射线诱导的细胞死亡具有高度抗性。LNCaP细胞可被肿瘤坏死因子α（TNF-α）致敏而对γ射线敏感，且在较小程度上可被激动性FAS抗体CH-11致敏。TNF-α激活内源性和外源性凋亡途径，并增加受辐照LNCaP细胞中神经酰胺和鞘氨醇的水平。CH-11仅激活外源性凋亡途径，且对受辐照LNCaP细胞中神经酰胺和鞘氨醇水平的影响可忽略不计。外源性神经酰胺和细菌鞘磷脂酶使LNCaP细胞对辐射诱导的凋亡敏感，并在用TNF-α而非CH-11辐照后对细胞死亡具有协同作用。暴露于神经酰胺和辐射后的细胞死亡效应被丝氨酸蛋白酶抑制剂TLCK（对甲苯磺酰-L-赖氨酸氯甲基酮）阻断，但未被半胱天冬酶抑制剂z-VAD（Z-Val-Ala-Asp(OMe)-CH2F）阻断。在外源性神经酰胺诱导LNCaP细胞凋亡过程中，我们观察到半胱天冬酶-9的激活，但未观察到半胱天冬酶-8、-3或-7的激活。神经酰胺的作用主要通过内源性线粒体凋亡途径发生，并增强TNF-α对辐照细胞的作用，但不增强CH-11的作用。数据表明，神经酰胺增强内源性凋亡途径的激活，并增强TNF-α诱导的细胞死亡，无论有无γ射线照射。TNF-α和γ射线照射提高内源性神经酰胺水平并激活内源性细胞死亡途径。
