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

细胞毒性T淋巴细胞（CTL）通过穿孔素依赖性和Fas配体依赖性途径杀死病毒感染的细胞和转化的细胞。环氧环己烯酮衍生物，ECH和RKTS-33，抑制半胱天冬酶-8的活化，并特异性阻断Fas依赖性细胞凋亡。ECH和RKTS-33对细胞表面Fas配体的诱导表达仅有微弱的抑制作用，但对穿孔素缺陷型CD 4 ^+ CTL和Concanamycin A处理的CD 8 ^+ 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未能阻止由CD 8 ^+ CTL介导的穿孔素依赖性杀伤途径。上述结果表明，ECH和RKTS-33是CTL介导的细胞毒作用中Fas配体依赖性杀伤通路的特异性抑制剂，而蛋白质合成抑制剂乙酰氧基环己酰亚胺（E-73）可抑制TNF-α诱导的转录因子NF-κB的活化，但对IL-1无影响。当用E-73处理人肺癌A549细胞时，TNF受体1的细胞水平下降，同时培养基中切割的TNF受体1增加。金属蛋白酶抑制剂GM 6001和TACE（TNF-α转化酶）抑制剂TAPI-2可阻断E-73诱导的TNF受体1的细胞外蓄积。这些结果表明，E-73通过诱导TACE依赖性脱落减少细胞表面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.