Brekke O L, Sagen E, Bjerve K S
Department of Clinical Chemistry, University Hospital, Norwegian University of Science and Technology, N-7006 Trondheim, Norway.
J Lipid Res. 1999 Dec;40(12):2223-33.
Recombinant tumor necrosis factor alpha (rTNF-alpha)-induced release of endogenous fatty acids was examined in WEHI 164 clone 13 fibrosarcoma cells using a highly sensitive HPLC method. The initial rTNF-alpha-induced extracellular release of endogenous fatty acids was dominated by 20:4n;-6, 22:4n;-6, 24:4n;-6, and 18:1n;-9 showing relative rates of 2.9, 0.9, 1.1, and 1.0, respectively. Release of endogenous AA and DNA fragmentation occurred simultaneously and preceded cell death by approx. 2 h. Methyl arachidonoyl fluorophosphonate and LY311727, specific inhibitors of Ca(2+)-dependent cytosolic PLA(2) (cPLA(2)) and secretory PLA(2) (sPLA(2)), respectively, neither blocked rTNF-alpha-induced cytotoxicity or endogenous AA release. However, both inhibitors reduced rTNF-alpha-induced release of other endogenous fatty acids. In comparison, the antioxidant butylated hydroxyanisole (BHA) completely inhibited the rTNF-alpha-induced cytotoxicity as well as AA release mediated through the TNF receptor p55, while the very similar antioxidant butylated hydroxytoluene had no effect. BHA did not inhibit recombinant cPLA(2) or sPLA(2) enzyme activity in vitro. Furthermore, stimulation of cells with rTNF-alpha for 4 h did not increase cPLA(2) enzyme activity. The data indicate that neither cPLA(2) or sPLA(2) mediate rTNF-alpha-induced apoptosis and extracellular AA release in WEHI cells. The results suggest that a BHA-sensitive signaling pathway coupled to AA release is a key event in TNF-induced cytotoxicity in these cells.
利用一种高灵敏度的高效液相色谱法，在WEHI 164克隆13纤维肉瘤细胞中检测了重组肿瘤坏死因子α（rTNF-α）诱导的内源性脂肪酸释放。rTNF-α诱导的内源性脂肪酸的初始细胞外释放以20:4n;-6、22:4n;-6、24:4n;-6和18:1n;-9为主，其相对释放率分别为2.9、0.9、1.1和1.0。内源性花生四烯酸（AA）的释放和DNA片段化同时发生，且在细胞死亡前约2小时出现。甲基花生四烯酰基氟磷酸酯和LY311727分别是钙依赖性胞质磷脂酶A2（cPLA2）和分泌型磷脂酶A2（sPLA2）的特异性抑制剂，二者均未阻断rTNF-α诱导的细胞毒性或内源性AA释放。然而，两种抑制剂都降低了rTNF-α诱导的其他内源性脂肪酸的释放。相比之下，抗氧化剂丁基羟基茴香醚（BHA）完全抑制了rTNF-α诱导的细胞毒性以及通过肿瘤坏死因子受体p55介导的AA释放，而非常相似的抗氧化剂丁基羟基甲苯则没有效果。BHA在体外不抑制重组cPLA2或sPLA2的酶活性。此外，用rTNF-α刺激细胞4小时并未增加cPLA2的酶活性。数据表明，cPLA2或sPLA2均不介导rTNF-α诱导的WEHI细胞凋亡和细胞外AA释放。结果表明，与AA释放相关的BHA敏感信号通路是这些细胞中肿瘤坏死因子诱导细胞毒性的关键事件。
