Analysis of molecular mechanism of signal transduction pathway via cytokine receptors by bioprobes

细胞毒性T淋巴细胞（ctl）通过穿孔蛋白依赖和Fas配体依赖的途径杀死病毒感染的细胞和转化的细胞。环氧环己酮衍生物ECH和RKTS-33可抑制caspase-8的激活，特异性阻断fas依赖性细胞凋亡。ECH和RKTS-33对诱导表达的细胞表面Fas配体只有微弱的抑制作用，但对穿孔缺陷的CD4^+ ctl和康那霉素a处理的CD8^+ ctl介导的Fas配体依赖性杀伤途径有强烈的抑制作用。然而，ECH和RKTS-33未能阻止CD8^+ ctl介导的穿孔依赖性杀伤途径。这些结果表明，在ctl介导的细胞毒性中，ECH和RKTS-33是Fas配体依赖性杀伤途径的特异性抑制剂。蛋白合成抑制剂acetoxycycloheximide （E-73）可抑制TNF-α诱导的转录因子NF-κB的活化，但对IL-1无抑制作用。E-73作用于人肺癌A549细胞时，细胞中TNF受体1水平下降，同时培养基中裂解的TNF受体1增加。金属蛋白酶抑制剂GM6001和TACE （TNF-α转换酶）抑制剂TAPI-2可阻断E-73诱导的TNF受体1的细胞外积累。这些结果表明，E-73通过诱导tace依赖性脱落降低细胞表面TNF受体1，从而降低A549细胞对TNF-α的反应性。

Cytotoxic T lymphocytes (CTLs) kill virus-infected cells and transformed cells via the perforin-dependent and Fas ligand-dependent pathways. Epoxycyclohexenone derivatives, ECH and RKTS-33, inhibit activation of caspase-8 and specifically block Fas-dependent apoptosis. ECH and RKTS-33 had only weak inhibitory effects on inducible expression of cell-surface Fas ligand, but strongly inhibited the Fas ligand-dependent killing pathway mediated by perforin-defective CD4^+ CTLs and concanamycin A-treated CD8^+ CTLs. However, ECH and RKTS-33 failed to prevent the perforin-dependent killing pathway mediated by CD8^+ CTLs. These results indicate that ECH and RKTS-33 are specific inhibitors for the Fas ligand-dependent killing pathway in CTL-mediated cytotoxicity.The protein synthesis inhibitor acetoxycycloheximide (E-73) inhibits activation of the transcription factor NF-κB induced by TNF-α, but not IL-1. When human lung carcinoma A549 cells were treated with E-73, the cellular level of TNF receptor 1 decreased accompanied by the increase of cleaved TNF receptor 1 in the medium. The metalloproteinase inhibitor GM6001 and the TACE (TNF-α converting enzyme) inhibitor TAPI-2 blocked the extracellular accumulation of TNF receptor 1 induced by E-73. These results indicate that E-73 decreases cell surface TNF receptor 1 by inducing TACE-dependent shedding and thereby reduces the responsiveness of A549 cells to TNF-α.