Evodiamine abolishes constitutive and inducible NF-kappaB activation by inhibiting IkappaBalpha kinase activation, thereby suppressing NF-kappaB-regulated antiapoptotic and metastatic gene expression, up-regulating apoptosis, and inhibiting invasion

Evodiamine, an alkaloidal component extracted from the fruit of Evodiae fructus (Evodia rutaecarpa Benth., Rutaceae), exhibits antiproliferative, antimetastatic, and apoptotic activities through a poorly defined mechanism. Because several genes that regulate cellular proliferation, carcinogenesis, metastasis, and survival are regulated by nuclear factor-kappaB (NF-kappaB), we postulated that evodiamine mediates its activity by modulating NF-kappaB activation. In the present study, we investigated the effect of evodiamine on NF-kappaB and NF-kappaB-regulated gene expression activated by various carcinogens. We demonstrate that evodiamine was a highly potent inhibitor of NF-kappaB activation, and it abrogated both inducible and constitutive NF-kappaB activation. The inhibition corresponded with the sequential suppression of IkappaBalpha kinase activity, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, p65 nuclear translocation, and p65 acetylation. Evodiamine also inhibited tumor necrosis factor (TNF)-induced Akt activation and its association with IKK. Suppression of Akt activation was specific, because it had no effect on JNK or p38 MAPK activation. Evodiamine also inhibited the NF-kappaB-dependent reporter gene expression activated by TNF, TNFR1, TRADD, TRAF2, NIK, and IKK but not that activated by the p65 subunit of NF-kappaB. NF-kappaB-regulated gene products such as Cyclin D1, c-Myc, COX-2, MMP-9, ICAM-1, MDR1, Survivin, XIAP, IAP1, IAP2, FLIP, Bcl-2, Bcl-xL, and Bfl-1/A1 were all down-regulated by evodiamine. This down-regulation potentiated the apoptosis induced by cytokines and chemotherapeutic agents and suppressed TNF-induced invasive activity. Overall, our results indicated that evodiamine inhibits both constitutive and induced NF-kappaB activation and NF-kappaB-regulated gene expression and that this inhibition may provide a molecular basis for the ability of evodiamine to suppress proliferation, induce apoptosis, and inhibit metastasis.     

Suberoylanilide hydroxamic acid potentiates apoptosis, inhibits invasion, and abolishes osteoclastogenesis by suppressing nuclear factor-kappaB activation

Because of its ability to suppress tumor cell proliferation, angiogenesis, and inflammation, the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) is currently in clinical trials. How SAHA mediates its effects is poorly understood. We found that in several human cancer cell lines, SAHA potentiated the apoptosis induced by tumor necrosis factor (TNF) and chemotherapeutic agents and inhibited TNF-induced invasion and receptor activator of NF-kappaB ligand-induced osteoclastogenesis, all of which are known to require NF-kappaB activation. These observations corresponded with the down-regulation of the expression of anti-apoptotic (IAP1, IAP2, X chromosome-linked IAP, Bcl-2, Bcl-x(L), TRAF1, FLIP, and survivin), proliferative (cyclin D1, cyclooxygenase 2, and c-Myc), and angiogenic (ICAM-1, matrix metalloproteinase-9, and vascular endothelial growth factor) gene products. Because several of these genes are regulated by NF-kappaB, we postulated that SAHA mediates its effects by modulating NF-kappaB and found that SAHA suppressed NF-kappaB activation induced by TNF, IL-1beta, okadaic acid, doxorubicin, lipopolysaccharide, H(2)O(2), phorbol myristate acetate, and cigarette smoke; the suppression was not cell type-specific because both inducible and constitutive NF-kappaB activation was inhibited. We also found that SAHA had no effect on direct binding of NF-kappaB to the DNA but inhibited sequentially the TNF-induced activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, IkappaBalpha ubiquitination, IkappaBalpha degradation, p65 phosphorylation, and p65 nuclear translocation. Furthermore, SAHA inhibited the NF-kappaB-dependent reporter gene expression activated by TNF, TNFR1, TRADD, TRAF2, NF-kappaB-inducing kinase, IkappaBalpha kinase, and the p65 subunit of NF-kappaB. Overall, our results indicated that NF-kappaB and NF-kappaB-regulated gene expression inhibited by SAHA can enhance apoptosis and inhibit invasion and osteoclastogenesis.     

Acetyl-11-keto-beta-boswellic acid potentiates apoptosis, inhibits invasion, and abolishes osteoclastogenesis by suppressing NF-kappa B and NF-kappa B-regulated gene expression

Acetyl-11-keto-beta-boswellic acid (AKBA), a component of an Ayurvedic therapeutic plant Boswellia serrata, is a pentacyclic terpenoid active against a large number of inflammatory diseases, including cancer, arthritis, chronic colitis, ulcerative colitis, Crohn's disease, and bronchial asthma, but the mechanism is poorly understood. We found that AKBA potentiated the apoptosis induced by TNF and chemotherapeutic agents, suppressed TNF-induced invasion, and inhibited receptor activator of NF-kappaB ligand-induced osteoclastogenesis, all of which are known to require NF-kappaB activation. These observations corresponded with the down-regulation of the expression of NF-kappaB-regulated antiapoptotic, proliferative, and angiogenic gene products. As examined by DNA binding, AKBA suppressed both inducible and constitutive NF-kappaB activation in tumor cells. It also abrogated NF-kappaB activation induced by TNF, IL-1beta, okadaic acid, doxorubicin, LPS, H2O2, PMA, and cigarette smoke. AKBA did not directly affect the binding of NF-kappaB to the DNA but inhibited sequentially the TNF-induced activation of IkappaBalpha kinase (IKK), IkappaBalpha phosphorylation, IkappaBalpha ubiquitination, IkappaBalpha degradation, p65 phosphorylation, and p65 nuclear translocation. AKBA also did not directly modulate IKK activity but suppressed the activation of IKK through inhibition of Akt. Furthermore, AKBA inhibited the NF-kappaB-dependent reporter gene expression activated by TNFR type 1, TNFR-associated death domain protein, TNFR-associated factor 2, NF-kappaB-inducing kinase, and IKK, but not that activated by the p65 subunit of NF-kappaB. Overall, our results indicated that AKBA enhances apoptosis induced by cytokines and chemotherapeutic agents, inhibits invasion, and suppresses osteoclastogenesis through inhibition of NF-kappaB-regulated gene expression.     

Honokiol potentiates apoptosis, suppresses osteoclastogenesis, and inhibits invasion through modulation of nuclear factor-kappaB activation pathway

Recent reports have indicated that honokiol can induce apoptosis, suppress tumor growth, and inhibit angiogenesis. In this report, we found that honokiol potentiated the apoptosis induced by tumor necrosis factor (TNF) and chemotherapeutic agents, suppressed TNF-induced tumor cell invasion, and inhibited RANKL-induced osteoclastogenesis, all of which are known to require nuclear factor-kappaB (NF-kappaB) activation. Honokiol suppressed NF-kappaB activation induced by a variety of inflammatory stimuli, and this suppression was not cell type specific. Further studies showed that honokiol blocked TNF-induced phosphorylation, ubiquitination, and degradation of IkappaBalpha through the inhibition of activation of IkappaBalpha kinase and of Akt. This led to suppression of the phosphorylation and nuclear translocation of p65 and NF-kappaB-dependent reporter gene expression. Magnolol, a honokiol isomer, was equally active. The expression of NF-kappaB-regulated gene products involved in antiapoptosis (IAP1, IAP2, Bcl-x(L), Bcl-2, cFLIP, TRAF1, and survivin), proliferation (cyclin D1, cyclooxygenase-2, and c-myc), invasion (matrix metalloproteinase-9 and intercellular adhesion molecule-1), and angiogenesis (vascular endothelial growth factor) were also down-regulated by honokiol. Honokiol also down-regulated NF-kappaB activation in in vivo mouse dorsal skin model. Thus, overall, our results indicate that NF-kappaB and NF-kappaB-regulated gene expression inhibited by honokiol enhances apoptosis and suppresses osteoclastogenesis and invasion.     

Butein, a tetrahydroxychalcone, inhibits nuclear factor (NF)-kappaB and NF-kappaB-regulated gene expression through direct inhibition of IkappaBalpha kinase beta on cysteine 179 residue

Although butein (3,4,2',4'-tetrahydroxychalcone) is known to exhibit anti-inflammatory, anti-cancer, and anti-fibrogenic activities, very little is known about its mechanism of action. Because numerous effects modulated by butein can be linked to interference with the NF-kappaB pathway, we investigated in detail the effect of this chalcone on NF-kappaB activity. As examined by DNA binding, we found that butein suppressed tumor necrosis factor (TNF)-induced NF-kappaB activation in a dose- and time-dependent manner; suppressed the NF-kappaB activation induced by various inflammatory agents and carcinogens; and inhibited the NF-kappaB reporter activity induced by TNFR1, TRADD, TRAF2, NIK, TAK1/TAB1, and IKK-beta. We also found that butein blocked the phosphorylation and degradation of IkappaBalpha by inhibiting IkappaBalpha kinase (IKK) activation. We found the inactivation of IKK by butein was direct and involved cysteine residue 179. This correlated with the suppression of phosphorylation and the nuclear translocation of p65. In this study, butein also inhibited the expression of the NF-kappaB-regulated gene products involved in anti-apoptosis (IAP2, Bcl-2, and Bcl-xL), proliferation (cyclin D1 and c-Myc), and invasion (COX-2 and MMP-9). Suppression of these gene products correlated with enhancement of the apoptosis induced by TNF and chemotherapeutic agents; and inhibition of cytokine-induced cellular invasion. Overall, our results indicated that antitumor and anti-inflammatory activities previously assigned to butein may be mediated in part through the direct inhibition of IKK, leading to the suppression of the NF-kappaB activation pathway.     

Targeted deletion of MKK4 gene potentiates TNF-induced apoptosis through the down-regulation of NF-kappa B activation and NF-kappa B-regulated antiapoptotic gene products

MAPK kinase 4 (MKK4) is a dual-specificity kinase that activates both JNK and p38 MAPK. However, the mechanism by which MKK4 regulates TNF-induced apoptosis is not fully understood. Therefore, we used fibroblasts derived from MKK4 gene-deleted (MKK4-KO) mice to determine the role of this kinase in TNF signaling. We found that when compared with the wild-type cells, deletion of MKK4 gene enhanced TNF-induced apoptosis, and this correlated with down-regulation of TNF-induced cell-proliferative (COX-2 and cyclin D1) and antiapoptotic (survivin, IAP1, XIAP, Bcl-2, Bcl-x(L), and cFLIP) gene products, all regulated by NF-kappaB. Indeed we found that TNF-induced NF-kappaB activation was abrogated in MKK4 gene-deleted cells, as determined by DNA binding. Further investigation revealed that TNF-induced I kappaB alpha kinase activation, I kappaB alpha phosphorylation, I kappaB alpha degradation, and p65 nuclear translocation were all suppressed in MKK4-KO cells. NF-kappaB reporter assay revealed that NF-kappaB activation induced by TNF, TNFR1, TRADD, TRAF2, NIK, and I kappaB alpha kinase was modulated in gene-deleted cells. Overall, our results indicate that MKK4 plays a central role in TNF-induced apoptosis through the regulation of NF-kappaB-regulated gene products.     

Interleukin-1-induced NF-kappaB activation is NEMO-dependent but does not require IKKbeta

Activation of NF-kappaB by the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the IkappaB kinase (IKK) complex, which contains two kinases named IKKalpha and IKKbeta and a critical regulatory subunit named NEMO. Although we have previously demonstrated that NEMO associates with both IKKs, genetic studies reveal that only its interaction with IKKbeta is required for TNF-induced NF-kappaB activation. To determine whether NEMO and IKKalpha can form a functional IKK complex capable of activating the classical NF-kappaB pathway in the absence of IKKbeta, we utilized a panel of mouse embryonic fibroblasts (MEFs) lacking each of the IKK complex subunits. This confirmed that TNF-induced IkappaBalpha degradation absolutely requires NEMO and IKKbeta. In contrast, we consistently observed intact IkappaBalpha degradation and NF-kappaB activation in response to IL-1 in two separate cell lines lacking IKKbeta. Furthermore, exogenously expressed, catalytically inactive IKKbeta blocked TNF- but not IL-1-induced IkappaBalpha degradation in wild-type MEFs, and reconstitution of IKKalpha/beta double knockout cells with IKKalpha rescued IL-1- but not TNF-induced NF-kappaB activation. Finally, we have shown that incubation of IKKbeta-deficient MEFs with a cell-permeable peptide that blocks the interaction of NEMO with the IKKs inhibits IL-1-induced NF-kappaB activation. Our results therefore demonstrate that NEMO and IKKalpha can form a functional IKK complex that activates the classical NF-kappaB pathway in response to IL-1 but not TNF. These findings further suggest NEMO differentially regulates the fidelity of the IKK subunits activated by distinct upstream signaling pathways.     

TNAP, a novel repressor of NF-kappaB-inducing kinase, suppresses NF-kappaB activation

NF-kappaB-inducing kinase (NIK) has been implicated as an essential component of NF-kappaB activation. However, the regulatory mechanism of NIK signaling remains elusive. We have identified a novel NIK interacting protein, TNAP (for TRAFs and NIK-associated protein). In mammalian cells, TNAP physically interacts with NIK, TRAF2, and TRAF3 but not IKK1 or IKK2. TNAP specifically inhibits NF-kappaB activation induced by tumor necrosis factor (TNF)-alpha, TNF receptor 1, TRADD, RIP, TRAF2, and NIK but does not affect IKK1- and IKK2-mediated NF-kappaB activation. Knockdown of TNAP by lentiviral-mediated small interference RNA potentiates TNF-alpha-induced NF-kappaB activation. TNAP suppresses NIK kinase activity and subsequently reduces p100 processing, p65 phosphorylation, and IkappaBalpha degradation. These data suggest that TNAP is a repressor of NIK activity and regulates both the classical and alternative NF-kappaB signaling pathways.     

Melanoma differentiation-associated gene-7/IL-24 gene enhances NF-kappa B activation and suppresses apoptosis induced by TNF

Melanoma differentiation-associated gene-7 (mda-7), also referred to as IL-24, is a novel growth regulatory cytokine that has been shown to regulate the immune system by inducing the expression of inflammatory cytokines, such as TNF, IL-1, and IL-6. Whether the induction of these cytokines by MDA-7 is mediated through activation of NF-kappaB or whether it regulates cytokine signaling is not known. In the present report we investigated the effect of MDA-7 on NF-kappaB activation and on TNF-induced NF-kappaB activation and apoptosis in human embryonic kidney 293 cells. Stable or transient transfection with mda-7 into 293 cells failed to activate NF-kappaB. However, TNF-induced NF-kappaB activation was significantly enhanced in mda-7-transfected cells, as indicated by DNA binding, p65 translocation, and NF-kappaB-dependent reporter gene expression. Mda-7 transfection also potentiated NF-kappaB reporter activation induced by TNF receptor-associated death domain and TNF receptor-associated factor-2. Cytoplasmic MDA-7 with deleted signal sequence was as effective as full-length MDA-7 in potentiating TNF-induced NF-kappaB reporter activity. Secretion of MDA-7 was not required for the potentiation of TNF-induced NF-kappaB activation. TNF-induced expression of the NF-kappaB-regulated gene products cyclin D1 and cyclooxygenase-2, were significantly up-regulated by stable expression of MDA-7. Furthermore, MDA-7 expression abolished TNF-induced apoptosis, and suppression of NF-kappaB by IkappaBalpha kinase inhibitors enhanced apoptosis. Overall, our results indicate that stable or transient MDA-7 expression alone does not substantially activate NF-kappaB, but potentiates TNF-induced NF-kappaB activation and NF-kappaB-regulated gene expression. Potentiation of NF-kappaB survival signaling by MDA-7 inhibits TNF-mediated apoptosis.