Amiodarone inhibits the Toll-like receptor 3-mediated nuclear factor κB signaling pathway by blocking organelle acidification.

Toll-like receptor (TLR) agonists or pro-inflammatory cytokines converge to activate the nuclear factor κB (NF-κB) signaling pathway, which provokes inflammatory responses. In the present study, we identified amiodarone hydrochloride as a selective inhibitor of the TLR3-mediated NF-κB signaling pathway by screening the RIKEN NPDepo Chemical Library. In human umbilical vein endothelial cells (HUVEC), amiodarone selectively inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) induced by polyinosinic-polycytidylic acid (Poly(I:C)), but not tumor necrosis factor-α, interleukin-1α, or lipopolysaccharide. In response to a Poly(I:C) stimulation, amiodarone at 20 µM reduced the up-regulation of mRNA expression encoding ICAM-1, vascular cell adhesion molecule-1, and E-selectin. The nuclear translocation of the NF-κB subunit RelA was inhibited by amiodarone at 15-20 µM in Poly(I:C)-stimulated HUVEC. Amiodarone diminished the fluorescent dots of LysoTracker® Red DND-99 scattered over the cytoplasm of HUVEC. Therefore, the present study revealed that amiodarone selectively inhibited the TLR3-mediated NF-κB signaling pathway by blocking the acidification of intracellular organelles.

Structure-Activity Relationship of Oleanane-Type Pentacyclic Triterpenoids on Nuclear Factor κB Activation and Intracellular Trafficking and N-Linked Glycosylation of Intercellular Adhesion Molecule-1.

In our previous study, two oleanane-type pentacyclic triterpenoids (oleanolic acid and maslinic acid) were reported to affect the N-glycosylation and intracellular trafficking of intercellular adhesion molecule-1 (ICAM-1). The present study was aimed at investigating the structure-activity relationship of 13 oleanane-type natural triterpenoids with respect to the nuclear factor κB (NF-κB) signaling pathway and the expression, intracellular trafficking, and N-glycosylation of the ICAM-1 protein in human lung adenocarcinoma A549 cells. Hederagenin, echinocystic acid, erythrodiol, and maslinic acid, which all possess two hydroxyl groups, decreased the viability of A549 cells. Celastrol and pristimerin, both of which possess an α,ß-unsaturated carbonyl group, decreased cell viability but more strongly inhibited the interleukin-1α-induced NF-κB signaling pathway. Oleanolic acid, moronic acid, and glycyrrhetinic acid interfered with N-glycosylation without affecting the cell surface expression of the ICAM-1 protein. In contrast, α-boswellic acid and maslinic acid interfered with the N-glycosylation of the ICAM-1 protein, which resulted in the accumulation of high-mannose-type N-glycans. Among the oleanane-type triterpenoids tested, α-boswellic acid and maslinic acid uniquely interfered with the intracellular trafficking and N-glycosylation of glycoproteins.

Sesquiterpene Lactones Containing an α-Methylene-γ-Lactone Moiety Selectively Down-Regulate the Expression of Tumor Necrosis Factor Receptor 1 by Promoting Its Ectodomain Shedding in Human Lung Adenocarcinoma A549 Cells.

Alantolactone is a eudesmane-type sesquiterpene lactone containing an α-methylene-γ-lactone moiety. Previous studies showed that alantolactone inhibits the nuclear factor κB (NF-κB) signaling pathway by targeting the inhibitor of NF-κB (IκB) kinase. However, in the present study, we demonstrated that alantolactone selectively down-regulated the expression of tumor necrosis factor (TNF) receptor 1 (TNF-R1) in human lung adenocarcinoma A549 cells. Alantolactone did not affect the expression of three adaptor proteins recruited to TNF-R1. The down-regulation of TNF-R1 expression by alantolactone was suppressed by an inhibitor of TNF-α-converting enzyme. Alantolactone increased the soluble forms of TNF-R1 that were released into the culture medium as an ectodomain. The structure-activity relationship of eight eudesmane derivatives revealed that an α-methylene-γ-lactone moiety was needed to promote TNF-R1 ectodomain shedding. In addition, parthenolide and costunolide, two sesquiterpene lactones with an α-methylene-γ-lactone moiety, increased the amount of soluble TNF-R1. Therefore, the present results demonstrate that sesquiterpene lactones with an α-methylene-γ-lactone moiety can down-regulate the expression of TNF-R1 by promoting its ectodomain shedding in A549 cells.

PGAM5 interacts with Bcl-rambo and regulates apoptosis and mitophagy.

Bcl-rambo, also known as BCL2L13, has been reported to regulate apoptosis, mitochondrial fragmentation, and mitophagy. However, the molecular mechanisms by which Bcl-rambo regulates these processes currently remain unclear. In the present study, we identified phosphoglycerate mutase member 5 (PGAM5) as an emerging partner interacting with Bcl-rambo through phenotypic Drosophila screening. The rough eye phenotype induced by human Bcl-rambo was partly rescued by the knockdown of pgam5-2, a mammalian ortholog of PGAM5. Bcl-rambo bound to PGAM5, and their interaction required the Bcl-rambo transmembrane domain. The co-expression of Bcl-rambo and PGAM5 promoted effector caspase activity in human embryonic kidney 293T cells. The transient overexpression of Bcl-rambo increased LC3B-II levels, which had been decreased by the co-expression of PGAM5. These results suggest that PGAM5 promotes Bcl-rambo-dependent apoptosis, but conversely interferes with Bcl-rambo-dependent mitophagy.

Cucurbitacin B Down-Regulates TNF Receptor 1 Expression and Inhibits the TNF-α-Dependent Nuclear Factor κB Signaling Pathway in Human Lung Adenocarcinoma A549 Cells.

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,ß-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.

Bioactive Evaluation of Ursane-Type Pentacyclic Triterpenoids: ß-Boswellic Acid Interferes with the Glycosylation and Transport of Intercellular Adhesion Molecule-1 in Human Lung Adenocarcinoma A549 Cells.

Ursane-type pentacyclic triterpenoids exert various biological effects, including anticancer and anti-inflammatory activities. We previously reported that ursolic acid, corosolic acid, and asiatic acid interfered with the intracellular trafficking and glycosylation of intercellular adhesion molecule-1 (ICAM-1) in human lung adenocarcinoma A549 cells stimulated with the pro-inflammatory cytokine interleukin-1α. However, the structure-activity relationship of ursane-type pentacyclic triterpenoids remains unclear. In the present study, the biological activities of seven ursane-type pentacyclic triterpenoids (ß-boswellic acid, uvaol, madecassic acid, 3-O-acetyl-11-keto-ß-boswellic acid, ursolic acid, corosolic acid, and asiatic acid) were investigated. We revealed that the inhibitory activities of ursane-type pentacyclic triterpenoids on the cell surface expression and glycosylation of ICAM-1 and α-glucosidase activity were influenced by the number of hydroxy groups and/or the presence and position of a carboxyl group. We also showed that ß-boswellic acid interfered with ICAM-1 glycosylation in a different manner from other ursane-type pentacyclic triterpenoids.

Small Molecule Inhibitors Targeting Nuclear Factor κB Activation Markedly Reduce Expression of Interleukin-2, but Not Interferon-γ, Induced by Phorbol Esters and Calcium Ionophores.

The T-box transcription factor Eomesodermin (Eomes) promotes the expression of interferon-γ (IFN-γ). We recently reported that the small molecule inhibitors, TPCA-1 and IKK-16, which target nuclear factor κB (NF-κB) activation, moderately reduced Eomes-dependent IFN-γ expression in mouse lymphoma BW5147 cells stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM). In the present study, we investigated the direct effects of NF-κB on IFN-γ expression in mouse lymphoma EL4 cells and primary effector T cells. Eomes strongly promoted IFN-γ expression and the binding of RelA and NFATc2 to the IFN-γ promoter when EL4 cells were stimulated with PMA and IM. Neither TPCA-1 nor IKK-16 reduced IFN-γ expression; however, they markedly decreased interleukin (IL)-2 expression in Eomes-transfected EL4 cells. Moreover, TPCA-1 markedly inhibited the binding of RelA, but not that of Eomes or NFATc2 to the IFN-γ promoter. In effector CD4+ and CD8+ T cells activated with anti-CD3 and anti-CD28 antibodies, IFN-γ expression induced by PMA and A23187 was not markedly decreased by TPCA-1 or IKK-16 under conditions where IL-2 expression was markedly reduced. Therefore, the present results revealed that NF-κB is dispensable for IFN-γ expression induced by PMA and calcium ionophores in EL4 cells expressing Eomes and primary effector T cells.

Different localization of lysosomal-associated membrane protein 1 (LAMP1) in mammalian cultured cell lines.

Lysosomal-associated membrane protein 1 (LAMP1) mainly localizes to lysosomes and late endosomes. We herein investigated the intracellular localization of lysosomal membrane proteins in five mammalian cultured cell lines. Rat LAMP1 fused to enhanced green fluorescent protein (EGFP) mostly accumulated at a particular cytoplasmic area and barely co-localized with LysoTracker® Red DND-99 in golden hamster kidney BHK-21 cells and Chinese hamster ovary CHO-K1 cells. Golden hamster, Chinese hamster, and human LAMP1-EGFP showed a similar intracellular distribution to rat LAMP1-EGFP in BHK-21 cells. Endogenous LAMP1 was also detected in a perinuclear area in BHK-21 cells and CHO-K1 cells, and co-localized with rat CD63-EGFP in BHK-21 cells. Moreover, rat LAMP1-DsRed-Monomer co-localized well with the human trans-Golgi network protein 2-EGFP in BHK-21 cells. These results reveal that LAMP1 predominantly localizes to the trans-Golgi network in BHK-21 cells.

The human Bcl-2 family member Bcl-rambo and voltage-dependent anion channels manifest a genetic interaction in Drosophila and cooperatively promote the activation of effector caspases in human cultured cells.

We previously reported that the Bcl-2 family member human Bcl-rambo, also known as BCL2L13, induces apoptosis in human embryonic kidney 293T cells. Mouse Bcl-rambo has recently been reported to mediate mitochondrial fragmentation and mitophagy. In the present study, we showed that the transfection of human Bcl-rambo and its microtubule-associated protein light chain 3-interacting region motif mutant (W276A/I279A) caused mitochondrial fragmentation and the perinuclear accumulation of fragmented mitochondria in human lung adenocarcinoma A549 cells. In comprehensive screening using the Drosophila model in which human Bcl-rambo was ectopically expressed in eye imaginal discs, voltage-dependent anion channels (VDAC), also known as mitochondrial porin, were found to manifest a genetic interaction with human Bcl-rambo. In addition to human adenine nucleotide translocase (ANT) 1 and ANT2, the human Bcl-rambo protein bound to human VDAC1, albeit to a lesser extent than ANT2. Moreover, human VDAC1 and human VDAC2 in particular promoted the activation of effector caspases only when they were co-expressed with human Bcl-rambo in 293T cells. Bcl-rambo induced the perinuclear accumulation of fragmented mitochondria by the knockdown of VDAC1, VDAC2, and VDAC3 in A549 cells. Thus, the present study revealed that human Bcl-rambo and VDAC cooperatively promote the activation of effector caspases in human cultured cells.

4-Hydroxypanduratin A and Isopanduratin A Inhibit Tumor Necrosis Factor α-Stimulated Gene Expression and the Nuclear Factor κB-Dependent Signaling Pathway in Human Lung Adenocarcinoma A549 Cells.

Tumor necrosis factor α (TNF-α), a pro-inflammatory cytokine, regulates inflammatory and immune responses by up-regulating gene expression in a manner that is dependent on the transcription factor nuclear factor κB (NF-κB). In the present study, we found that 4-hydroxypanduratin A and isopanduratin A, constituents of the rhizomes of Boesenbergia pandurata, inhibited the TNF-α-stimulated up-regulation of intercellular adhesion molecule-1 (ICAM-1) in human lung adenocarcinoma A549 cells. 4-Hydroxypanduratin A and isopanduratin A also reduced ICAM-1 mRNA expression and NF-κB-responsive luciferase activity in TNF-α-stimulated A549 cells. Moreover, 4-hydroxypanduratin A and isopanduratin A prevented the TNF-α-stimulated translocation of the NF-κB subunit p65 to the nucleus and the phosphorylation and proteasomal degradation of the inhibitor of the NF-κB α protein. The present results revealed that 4-hydroxypanduratin A and isopanduratin A inhibit TNF-α-stimulated gene expression and the NF-κB-dependent signaling pathway in A549 cells.

Asiatic Acid, Corosolic Acid, and Maslinic Acid Interfere with Intracellular Trafficking and N-Linked Glycosylation of Intercellular Adhesion Molecule-1.

The pentacyclic triterpenoid ursolic acid was previously shown to inhibit the intracellular trafficking of intercellular adhesion molecule-1 (ICAM-1) from the endoplasmic reticulum (ER) to the Golgi apparatus. In the present study, we further investigated the biological activities of three pentacyclic triterpenoids closely related to ursolic acid on the interleukin 1α-induced expression and intracellular trafficking of ICAM-1. In human lung adenocarcinoma A549 cells, asiatic acid, corosolic acid, and maslinic acid interfered with the intracellular transport of ICAM-1 to the cell surface. Endoglycosidase H-sensitive glycans were linked to ICAM-1 in asiatic acid-, corosolic acid-, and maslinic acid-treated cells. Unlike corosolic acid, asiatic acid and maslinic acid increased the amount of the ICAM-1 protein. Moreover, asiatic acid increased the co-localization of ICAM-1 with calnexin (an ER marker), but not GM130 (a cis-Golgi marker). Asiatic acid, corosolic acid, and maslinic acid inhibited yeast α-glucosidase activity, but not Jack bean α-mannosidase activity. These results indicate that asiatic acid, corosolic acid, and maslinic acid interfere with the intracellular transport of ICAM-1 to the cell surface and cause the accumulation of ICAM-1 linked to endoglycosidase H-sensitive glycans.

Eomesodermin promotes interferon-γ expression and binds to multiple conserved noncoding sequences across the Ifng locus in mouse thymoma cell lines.

The T-box transcription factors T-bet and eomesodermin (Eomes) have been shown to regulate the lineage-specific expression of interferon-γ (IFN-γ). However, in contrast to T-bet, the role of Eomes in the expression of IFN-γ remains unclear. In this study, we investigated the Eomes-dependent expression of IFN-γ in the mouse thymoma BW5147 and EL4 cells, which do not express T-bet or Eomes. The ectopic expression of Eomes induced BW5147 and EL4 cells to produce IFN-γ in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM). In BW5147 cells, Eomes augmented luciferase activity driven by the Ifng promoter encoding from -2500 to +113 bp; however, it was not increased by a stimulation with PMA and IM. A chromatin immunoprecipitation assay showed that Eomes bound to the Ifng promoter and conserved noncoding sequence (CNS) -22 kb across the Ifng locus with high efficacy in BW5147 cells. Moreover, Eomes increased permissive histone modifications in the Ifng promoter and multiple CNSs. The stimulation with PMA and IM greatly augmented Eomes binding to CNS-54, CNS-34, CNS+19 and CNS+30, which was inhibited by FK506. These results indicated that Eomes bound to the Ifng promoter and multiple CNSs in stimulation-dependent and stimulation-independent manners.

Eudesmane-Type Sesquiterpene Lactones Inhibit Nuclear Translocation of the Nuclear Factor κB Subunit RelB in Response to a Lymphotoxin ß Stimulation.

The transcription factor nuclear factor κB (NF-κB) regulates various biological processes, including inflammatory responses. We previously reported that eudesmane-type sesquiterpene lactones inhibited multiple steps in the canonical NF-κB signaling pathway induced by tumor necrosis factor-α and interleukin-1α. In contrast, the biological activities of eudesmane-type sesquiterpene lactones on the non-canonical NF-κB signaling pathway remain unclear. In the present study, we found that (11S)-2α-bromo-3-oxoeudesmano-12,6α-lactone, designated santonin-related compound 2 (SRC2), inhibited NF-κB luciferase reporter activity induced by lymphotoxin ß (LTß) in human lung carcinoma A549 cells. Although SRC2 did not prevent the processing of the NF-κB subunit p100 induced by LTß, it inhibited the nuclear translocation of RelB and p52 in response to the LTß stimulation. In contrast to (-)-dehydroxymethylepoxyquinomicin, SRC2 inhibited the LTß-induced nuclear translocation of the RelB (C144S) mutant in a manner similar to wild-type RelB. While eudesmane derivatives possessing an α-bromoketone moiety or α,ß-unsaturated carbonyl moieties inhibited LTß-induced NF-κB luciferase reporter activity, eudesmane derivatives possessing an α-bromoketone moiety exhibited stronger inhibitory activity on the LTß-induced nuclear translocation of RelB than those possessing a single α-methylene-γ-lactone moiety. The results of the present study revealed that SRC2 inhibits the nuclear translocation of RelB in the non-canonical NF-κB signaling pathway induced by LTß.

Quinacrine Inhibits ICAM-1 Transcription by Blocking DNA Binding of the NF-κB Subunit p65 and Sensitizes Human Lung Adenocarcinoma A549 Cells to TNF-α and the Fas Ligand.

Quinacrine has been used for therapeutic drugs in some clinical settings. In the present study, we demonstrated that quinacrine decreased the expression of intercellular adhesion molecule-1 (ICAM-1) induced by tumor necrosis factor (TNF)-α and interleukin-1 (IL-1) α in human lung adenocarcinoma A549 cells. Quinacrine inhibited ICAM-1 mRNA expression and nuclear factor κB (NF-κB)-responsive luciferase reporter activity following a treatment with TNF-α and IL-1α. In the NF-κB signaling pathway, quinacrine did not markedly affect the TNF-α-induced degradation of the inhibitor of NF-κB or the TNF-α-induced phosphorylation of the NF-κB subunit, p65, at Ser-536 and its subsequent translocation to the nucleus. In contrast, a chromatin immunoprecipitation assay showed that quinacrine prevented the binding of p65 to the ICAM-1 promoter following TNF-α stimulation. Moreover, TNF-α and the Fas ligand effectively reduced the viability of A549 cells in the presence of quinacrine only. Quinacrine down-regulated the constitutive and TNF-α-induced expression of c-FLIP and Mcl-1 in A549 cells. These results revealed that quinacrine inhibits ICAM-1 transcription by blocking the DNA binding of p65 and sensitizes A549 cells to TNF-α and the Fas ligand.

The C-terminal domain of the long form of cellular FLICE-inhibitory protein (c-FLIPL) inhibits the interaction of the caspase 8 prodomain with the receptor-interacting protein 1 (RIP1) death domain and regulates caspase 8-dependent nuclear factor κB (NF-κB) activation.

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.

Cardenolide aglycones inhibit tumor necrosis factor α-induced expression of intercellular adhesion molecule-1 at the translation step by blocking Na⁺/K⁺-ATPase.

Cardiac glycosides, which are inhibitors of Na(+)/K(+)-ATPase, are classified into cardenolides and bufadienolides. We have recently shown that two cardenolide glycosides, ouabain and odoroside A, inhibit Na(+)/K(+)-ATPase, thereby preventing nuclear factor κB-inducible protein expression by blocking Na(+)-dependent amino acid transport. In this study, we investigated the mechanism of action of cardenolide aglycones in tumor necrosis factor α (TNF-α)-induced gene expression. Ouabagenin, digitoxigenin, and digoxigenin were found to inhibit the TNF-α-induced cell-surface expression of intercellular adhesion molecule-1 (ICAM-1) in human lung carcinoma A549 cells. Those cardenolide aglycones did not inhibit the TNF-α-induced expression of ICAM-1 mRNA, but strongly inhibited the TNF-α-induced expression of ICAM-1 as translation product. The inhibition of the TNF-α-induced ICAM-1 expression by ouabagenin, digitoxigenin, and digoxigenin was significantly reversed by the ectopic expression of ouabain-resistant rat Na(+)/K(+)-ATPase α1 isoform. Moreover, knockdown of Na(+)/K(+)-ATPase α1 isoform augmented the inhibition of the TNF-α-induced ICAM-1 expression by ouabagenin or ouabain. These results clearly indicate that cardenolide aglycones inhibit the TNF-α-induced ICAM-1 expression at the translation step by blocking Na(+)/K(+)-ATPase.

Irciniastatin A induces potent and sustained activation of extracellular signal-regulated kinase and thereby promotes ectodomain shedding of tumor necrosis factor receptor 1 in human lung carcinoma A549 cells.

Irciniastatin A is a pederin-type marine product that potently inhibits translation. We have recently shown that irciniastatin A induces ectodomain shedding of tumor necrosis factor (TNF) receptor 1 with slower kinetics than other translation inhibitors. In human lung carcinoma A549 cells, irciniastatin A induced a marked and sustained activation of extracellular signal-regulated kinase (ERK) and induced little activation of p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK). Moreover, the TNF receptor 1 shedding induced by irciniastatin A was blocked by the MAP kinase/ERK kinase inhibitor U0126, but not by the p38 MAP kinase inhibitor SB203580 or the JNK inhibitor SP600125. Thus unlike other translation inhibitors that trigger ribotoxic stress response, our results show that irciniastatin A is a unique translation inhibitor that induces a potent and sustained activation of the ERK pathway, and thereby promotes the ectodomain shedding of TNF receptor 1 in A549 cells.

Alantolactone derivatives inhibit the tumor necrosis factor α-induced nuclear factor κB pathway by a different mechanism from alantolactone.

Alantolactone is a eudesmane-type sesquiterpene lactone that exerts various biological effects, including anti-inflammatory activity. In the present study, screening using the RIKEN Natural Products Depository chemical library identified alantolactone derivatives that inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells stimulated with proinflammatory cytokines and Toll-like receptor ligands. In human lung adenocarcinoma A549 cells stimulated with tumor necrosis factor-α (TNF-α), six alantolactone derivatives inhibited ICAM-1 expression in a dose-dependent manner and at IC50 values of 13-21 µM, whereas that of alantolactone was 5 µM. Alantolactone possesses an α-methylene-γ-lactone moiety, whereas alantolactone derivatives do not. In the nuclear factor κB (NF-κB) signaling pathway, alantolactone prevented the TNF-α-induced phosphorylation and degradation of the inhibitor of NF-κB α (IκBα) protein, and its downstream signaling pathway. In contrast, alantolactone derivatives neither reduced TNF-α-induced IκBα degradation nor the nuclear translocation of the NF-κB subunit RelA, but inhibited the binding of RelA to the ICAM-1 promoter. The inhibitory activities of alantolactone and alantolactone derivatives were attenuated by glutathione. These results indicate that alantolactone derivatives inhibit the TNF-α-induced NF-κB pathway by a different mechanism from alantolactone.