Caspase 8 plays an essential role in the regulation of apoptotic and non-apoptotic signaling pathways. The long form of cellular FLICE-inhibitory protein (c-FLIPL) has been shown previously to regulate caspase 8-dependent nuclear factor κB (NF-κB) activation by receptor-interacting protein 1 (RIP1) and TNF receptor-associated factor 2 (TRAF2). In this study, the molecular mechanism by which c-FLIPL regulates caspase 8-dependent NF-κB activation was further explored in the human embryonic kidney cell line HEK 293 and variant cells barely expressing caspase 8. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone greatly diminished caspase 8-dependent NF-κB activation induced by Fas ligand (FasL) when c-FLIPL, but not its N-terminal fragment c-FLIP(p43), was expressed. The prodomain of caspase 8 was found to interact with the RIP1 death domain and to be sufficient to mediate NF-κB activation induced by FasL or c-FLIP(p43). The interaction of the RIP1 death domain with caspase 8 was inhibited by c-FLIPL but not c-FLIP(p43). Thus, these results reveal that the C-terminal domain of c-FLIPL specifically inhibits the interaction of the caspase 8 prodomain with the RIP1 death domain and, thereby, regulates caspase 8-dependent NF-κB activation.

Bcl-2 family proteins play a central role in regulating apoptosis. We previously reported that human Bcl-rambo, also termed BCL2L13, localized to mitochondria and induced apoptosis when overexpressed in human embryonic kidney 293T cells. However, the physiological function of Bcl-rambo currently remains unclear. In the present study, human Bcl-rambo was ectopically expressed in Drosophila melanogaster. Bcl-rambo mainly localized to the mitochondria of Drosophila Schneider 2 (S2) cells. The overexpression of Bcl-rambo, but not Bcl-rambo lacking a C-terminal transmembrane domain, induced apoptosis in S2 cells. Moreover, the ectopic expression of Bcl-rambo by a GAL4-UAS system induced aberrant morphological changes characterized by atrophied wing, split thorax, and rough eye phenotypes. Bcl-rambo induced the activation of effector caspases in eye imaginal discs. The rough eye phenotype induced by Bcl-rambo was partly rescued by the co-expression of p35, Diap1, and Diap2. By using this Drosophila model, we showed that human Bcl-rambo interacted genetically with Drosophila homologues of adenine nucleotide translocators and the autophagy-related 8 protein. The results of the present study demonstrated that human Bcl-rambo localized to mitochondria and at least regulated an apoptosis signaling pathway in Drosophila.

Pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), induce the expression of intracellular adhesion molecule-1 (ICAM-1) by activating the nuclear factor κB (NF-κB) signaling pathway. In the present study, we found that cucurbitacin B decreased the expression of ICAM-1 in human lung adenocarcinoma A549 cells stimulated with TNF-α or interleukin-1α. We further investigated the mechanisms by which cucurbitacin B down-regulates TNF-α-induced ICAM-1 expression. Cucurbitacin B inhibited the nuclear translocation of the NF-κB subunit RelA and the phosphorylation of IκBα in A549 cells stimulated with TNF-α. Cucurbitacin B selectively down-regulated the expression of TNF receptor 1 (TNF-R1) without affecting three adaptor proteins (i.e., TRADD, RIPK1, and TRAF2). The TNF-α-converting enzyme inhibitor suppressed the down-regulation of TNF-R1 expression by cucurbitacin B. Glutathione, N-acetyl-L-cysteine, and, to a lesser extent, L-cysteine attenuated the inhibitory effects of cucurbitacin B on the TNF-α-induced expression of ICAM-1, suggesting that an α,β-unsaturated carbonyl moiety is essential for anti-inflammatory activity. The present results revealed that cucurbitacin B down-regulated the expression of TNF-R1 at the initial step in the TNF-α-dependent NF-κB signaling pathway.

Ursolic acid (3β‐hydroxy‐urs‐12‐en‐28‐oic acid) is a natural pentacyclic triterpenoid that is present in many plants, including medicinal herbs, and foods. Ursolic acid was initially identified as an inhibitor of the expression of intercellular adhesion molecule‐1 (ICAM‐1) in response to interleukin‐1α (IL‐1α). We report here a novel biological activity: ursolic acid inhibits intracellular trafficking of proteins. Ursolic acid markedly inhibited the IL‐1α‐induced cell‐surface ICAM‐1 expression in human cancer cell lines and human umbilical vein endothelial cells. By contrast, ursolic acid exerted weak inhibitory effects on the IL‐1α‐induced ICAM‐1 expression at the protein level. Surprisingly, we found that ursolic acid decreased the apparent molecular weight of ICAM‐1 and altered the structures of N‐linked oligosaccharides bound to ICAM‐1. Ursolic acid induced the accumulation of ICAM‐1 in the endoplasmic reticulum, which was linked mainly to high‐mannose‐type glycans. Moreover, in ursolic‐acid‐treated cells, the Golgi apparatus was fragmented into pieces and distributed over the cells. Thus, our results reveal that ursolic acid inhibits intracellular trafficking of proteins and induces the accumulation of ICAM‐1 linked to high‐mannose‐type glycans in the endoplasmic reticulum.