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.     

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.     

Methotrexate suppresses NF-kappaB activation through inhibition of IkappaBalpha phosphorylation and degradation

Methotrexate (MTX), a folate antagonist, is a commonly used anti-inflammatory, antiproliferative, and immunosuppressive drug whose mode of action is not fully established. Due to the central role of NF-kappaB in these responses, we postulated that MTX must mediate its effects through suppression of NF-kappaB activation. We investigated the effects of MTX on NF-kappaB activation induced by TNF in Jurkat cells. The treatment of these cells with MTX suppressed TNF-induced NF-kappaB activation with optimum effects occurring at 10 microM MTX for 60 min. These effects were not restricted to Jurkat cells because other cell types were also inhibited. Besides TNF, MTX also suppressed the NF-kappaB activation induced by various other inflammatory stimuli. The suppression of TNF-induced NF-kappaB activation by MTX correlated with inhibition of IkappaBalpha degradation, suppression of IkappaBalpha phosphorylation, abrogation of IkappaBalpha kinase activation, and inhibition of NF-kappaB-dependent reporter gene expression. Because ecto 5' nucleotidase inhibitor (alpha,beta-methylene adenosine-5'-diphosphate) blocked the effect of MTX, adenosine mimicked the effect of MTX, and adenosine A2b receptor antagonist (3,7-dimethyl-1-propargylxanthine) reversed the inhibitory effect of MTX, we suggest that MTX suppresses NF-kappaB activation by releasing adenosine. A partial reversal of MTX-induced NF-kappaB suppression by thymidine and folinic acid indicates the role of the thymidylate synthase pathway also. Overall, our results clearly demonstrate that MTX suppresses NF-kappaB activation through the release of adenosine, which may contribute to the role of MTX in anti-inflammatory, immunomodulatory, and antiproliferative effects.     

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.     

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.     

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.     

Leflunomide suppresses TNF-induced cellular responses: effects on NF-kappa B, activator protein-1, c-Jun N-terminal protein kinase, and apoptosis

Leflunomide is a pyrimidine biosynthesis inhibitor that has recently been approved for treatment of rheumatoid arthritis. However, the mechanism of leflunomide's antiarthritis activity and is not fully understood. The critical role that TNF plays in rheumatoid arthritis led us to postulate that leflunomide blocks TNF signaling. Previously, we have demonstrated that leflunomide inhibits TNF-induced NF-kappaB activation by suppressing I-kappaBalpha (inhibitory subunit of NF-kappaB) degradation. We in this study show that leflunomide also blocks NF-kappaB reporter gene expression induced by TNFR1, TNFR-associated factor 2, and NF-kappaB-inducing kinase (NIK), but not that activated by the p65 subunit of NF-kappaB, suggesting that leflunomide acts downstream of NIK. Leflunomide suppressed TNF-induced phosphorylation of I-kappaBalpha, as well as activation of I-kappaBalpha kinase-beta located downstream to NIK. Leflunomide also inhibited TNF-induced activation of AP-1 and the c-Jun N-terminal protein kinase activation. TNF-mediated cytotoxicity and caspase-induced poly(ADP-ribose) polymerase cleavage were also completely abrogated by treatment of Jurkat T cells with leflunomide. Leflunomide suppressed TNF-induced reactive oxygen intermediate generation and lipid peroxidation, which may explain most of its effects on TNF signaling. The suppressive effects of leflunomide on TNF signaling were completely reversible by uridine, indicating a critical role for pyrimidine biosynthesis in TNF-mediated cellular responses. Overall, our results suggest that suppression of TNF signaling is one of the possible mechanisms for inhibitory activity of leflunomide against rheumatoid arthritis.     

TNF activates Syk protein tyrosine kinase leading to TNF-induced MAPK activation, NF-kappaB activation, and apoptosis

Spleen tyrosine kinase (Syk), a nonreceptor protein kinase initially found to be expressed only in hemopoietic cells, has now been shown to be expressed in nonhemopoietic cells and to mediate signaling of various cytokines. Whether Syk plays any role in TNF signaling was investigated. Treatment of Jurkat T cells with TNF activated Syk kinase but not ZAP70, another member of Syk kinase family, and the optimum activation occurred at 10 s and with 1 nM TNF. TNF also activated Syk in myeloid and epithelial cells. TNF-induced Syk activation was abolished by piceatannol (Syk-selective inhibitor), which led to the suppression of TNF-induced activation of c- JNK, p38 MAPK, and p44/p42 MAPK. Jurkat cells that did not express Syk (JCaM1, JCaM1/lck) showed lack of TNF-induced Syk, JNK, p38 MAPK, and p44/p42 MAPK activation, as well as TNF-induced IkappaBalpha phosphorylation, IkappaBalpha degradation, and NF-kappaB activation. TNF-induced NF-kappaB activation was enhanced by overexpression of Syk by Syk-cDNA and suppressed when Syk expression was down-regulated by expression of Syk-small interfering RNA (siRNA-Syk). The apoptotic effects of TNF were reduced by up-regulation of NF-kappaB by Syk-cDNA, and enhanced by down-regulation of NF-kappaB by siRNA-Syk. Immunoprecipitation of cells with Syk Abs showed TNF-dependent association of Syk with both TNFR1 and TNFR2; this association was enhanced by up-regulation of Syk expression with Syk-cDNA and suppressed by down-regulation of Syk using siRNA-Syk. Overall, our results demonstrate that Syk activation plays an essential role in TNF-induced activation of JNK, p38 MAPK, p44/p42 MAPK, NF-kappaB, and apoptosis.