ECH, an epoxycyclohexenone derivative that specifically inhibits Fas ligand-dependent apoptosis in CTL-mediated cytotoxicity.
CTL eliminate cells infected with intracellular pathogens and tumor cells by two distinct mechanisms mediated by Fas ligand (FasL) and lytic granules that contain perforin and granzymes. In this study we show that an epoxycyclohexenone derivative,(2R,3R,4S)-2,3-epoxy-4-hydroxy-5-hydroxymethyl-6-(1E)-propenyl-cyclohex-5-en-1-one (ECH) specifically inhibits the FasL-dependent killing pathway in CTL-mediated cytotoxicity. Recently, we have reported that ECH blocks activation of procaspase-8 in the death-inducing signaling complex and thereby prevents apoptosis induced by anti-Fas Ab or soluble FasL. Consistent with this finding, ECH profoundly inhibited Fas-mediated DNA fragmentation and cytolysis of target cells induced by perforin-negative mouse CD4+ CTL and alloantigen-specific mouse CD8+ CTL pretreated with an inhibitor of vacuolar type H+-ATPase concanamycin A that selectively induces inactivation and proteolytic degradation of perforin in lytic granules. However, ECH barely influenced perforin/granzyme-dependent DNA fragmentation and cytolysis of target cells mediated by alloantigen-specific mouse CD8+ CTL. The components of lytic granules and the granule exocytosis pathway upon CD3 stimulation were also insensitive to ECH. In conclusion, our present results demonstrate that ECH is a specific nonpeptide inhibitor of FasL-dependent apoptosis in CTL-mediated cytotoxicity. Therefore, ECH can be used as a bioprobe to evaluate the contributions of two distinct killing pathways in various CTL-target settings.
Division of Bioinformatics, Center for Biological Resources and Informatics, Tokyo Institute of Technology, Yokohama, Japan.
Tomokazu
T
Mitsui
Yasunobu
Y
Miyake
Hideaki
H
Kakeya
Hiroyuki
H
Osada
Takao
T
Kataoka
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000276:immunology*; D045744:Cell Line, Tumor; D002999:Clone Cells; D003512:Cyclohexanones / Q000494:pharmacology*; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods; D003602:Cytotoxicity, Immunologic / Q000187:drug effects*; D053938:DNA Fragmentation / Q000187:drug effects / Q000276:immunology; D018984:Epitopes, T-Lymphocyte / Q000502:physiology; D004852:Epoxy Compounds / Q000494:pharmacology*; D005089:Exocytosis / Q000187:drug effects / Q000276:immunology; D053222:Fas Ligand Protein; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D007519:Isoantigens / Q000502:physiology; D007940:Leukemia L5178; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors* / Q000172:deficiency / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008811:Mice, Inbred DBA; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D012995:Solubility; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000276:immunology*; D050378:T-Lymphocytes, Regulatory / Q000276:immunology / Q000378:metabolism
D003512:Cyclohexanones; D018984:Epitopes, T-Lymphocyte; D004852:Epoxy Compounds; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D007166:Immunosuppressive Agents; D007519:Isoantigens; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin; C036468:cyclohexanone