Epoxycyclohexenone Inhibits Fas-mediated Apoptosis by Blocking Activation of Pro-caspase-8 in the Death-inducing Signaling Complex

Abstract

Death receptors belong to the tumor necrosis factor receptor family. They can induce apoptosis following engagement with specific ligands and are known to play an important role in the regulation of the immune system. Here we report that epoxycyclohexenone (ECH) inhibits apoptosis induced by anti-Fas antibody, Fas ligand (FasL), or tumor necrosis factor-␣ but not by staurosporine, MG-132, C2-ceramide, or UV irradiation. These results suggest that ECH specifically blocks death receptor-mediated apoptosis. Neither the surface expression of Fas nor the Fas-FasL interaction was influenced by ECH. However, ECH did block the activation of pro-caspase-8 in the death-inducing signaling complex, although recruitment of Fas-associating death domain (FADD) and pro-caspase-8 was not affected. ECH inhibited the enzymatic activity of recombinant active caspase-8 at slightly lower concentrations than it did for active caspase-3 and active caspase-9 in vitro. However, in FasL-treated cells, ECH was only able to inhibit the activation of pro-caspase-8, and it had no effect on the already activated caspase-8 at a concentration that is effective at inhibiting Fas-induced apoptosis. ECH directly bound the large subunit of active caspase-8 that contains the active center cysteine and had a relatively higher affinity to pro-caspase-8. Moreover, compared with pro-caspase-3 and pro-caspase-9, pro-caspase-8 was predominantly depleted by biotinylated ECH with avidin beads in the cell lysates, suggesting that ECH preferentially affects pro-caspase-8. Thus, our results suggest that ECH blocks the self-activation of pro-caspase-8 in the death-inducing signaling complex and thus selectively inhibits death receptor-mediated apoptosis.

Chemical biology of inflammatory cytokine signaling

“…101 ECH prevents caspase-8-dependent apoptosis induced by death receptors (Fas and TNF-R1), but not apoptosis induced by staurosporine, MG-132, C2-ceramide or UV irradiation. 99 Therefore, unlike other epoxyquinoids, ECH does not seem to be an NF-kB inhibitor, but a specific inhibitor of caspase-8. The molecular mechanism by which epoxyquinoids recognize specific cysteine residues in target proteins is currently unclear and should be clarified to develop highly selective inhibitors.…”

“…11 ECH specifically inhibits the activation of caspase-8, but not its recruitment to the deathinducing signaling complex (DISC) in Fas ligand (FasL)-stimulated cells, thereby preventing caspase-8-dependent apoptosis. 99 Although ECH is the oxidized form of the epoxyquinone A monomer, it does not seem to inhibit IKKb activity, as TNF-a-induced IkBa degradation still proceeds in the presence of ECH. 99 As a selective target in the Fas signaling pathway, ECH binds covalently to caspase-8, as revealed by an immunoprecipitation study using biotinylated ECH.…”

“…99 Although ECH is the oxidized form of the epoxyquinone A monomer, it does not seem to inhibit IKKb activity, as TNF-a-induced IkBa degradation still proceeds in the presence of ECH. 99 As a selective target in the Fas signaling pathway, ECH binds covalently to caspase-8, as revealed by an immunoprecipitation study using biotinylated ECH. 99 Thus, ECH is most likely to inactivate caspase-8 through the modification of a cysteine residue critical for its catalytic activity (Figure 5b).…”

“…99 As a selective target in the Fas signaling pathway, ECH binds covalently to caspase-8, as revealed by an immunoprecipitation study using biotinylated ECH. 99 Thus, ECH is most likely to inactivate caspase-8 through the modification of a cysteine residue critical for its catalytic activity (Figure 5b). This notion is supported by our finding that the mycotoxin penicillic acid directly binds to a critical cysteine residue within the active center in the catalytic domain of caspase-8 ( Figure 6).…”

“…98 As for the bioactive epoxyquinoids, we have shown that (2R, 3R, 4S)-2,3-epoxy-4-hydroxy-5-hydroxymethyl-6-(1E)-propenyl-cyclohex-5-en-1-one (ECH, as shown in Figure 5a) and its structural derivatives specifically inhibit Fas-mediated apoptosis. 99,100 Fas is a cell-surface receptor belonging to the TNF receptor superfamily and has a cytoplasmic death domain essential for the induction of apoptosis in a FADD-and caspase-8-dependent manner. 11 ECH specifically inhibits the activation of caspase-8, but not its recruitment to the deathinducing signaling complex (DISC) in Fas ligand (FasL)-stimulated cells, thereby preventing caspase-8-dependent apoptosis.…”