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

细胞毒性T淋巴细胞(CTL)通过穿孔素依赖和Fas配体依赖的途径杀伤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抑制caspase-8的激活，并特异性地阻断Fas依赖的细胞凋亡。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，但对穿孔素缺陷的CD4^+CTL和刀豆素A诱导的CD8^+CTL介导的Fas配体依赖的杀伤途径有较强的抑制作用。但ECH和RKTS-33不能阻断CD8~+CTL介导的穿孔素依赖的杀伤途径。这些结果表明，ECH和RKTS-33是CTL介导的细胞毒作用中Fas配体依赖的杀伤途径的特异性抑制剂。蛋白质合成抑制剂乙酰氧环己亚胺(E-73)可以抑制由肿瘤坏死因子-κ诱导的转录因子NF-α的激活，但不能抑制IL-1的激活。E-73作用于人肺癌A549细胞后，细胞内肿瘤坏死因子受体1的表达水平降低，裂解后的肿瘤坏死因子受体1表达增加。金属蛋白酶抑制剂GM6001和肿瘤坏死因子-α转换酶抑制剂TAPI2可阻断E-73诱导的肿瘤坏死因子受体1的胞外聚集。这些结果表明，E-73通过诱导血管紧张素转换酶依赖性脱落减少细胞表面肿瘤坏死因子受体1，从而降低A549细胞对肿瘤坏死因子-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.