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.     

Inhibition of IkappaB kinase activity by acetyl-boswellic acids promotes apoptosis in androgen-independent PC-3 prostate cancer cells in vitro and in vivo

Signaling through NF-kappaB has been implicated in the malignant phenotype as well as the chemoresistance of various cancers. Here we show that the natural compounds acetyl-beta-boswellic acid and acetyl-11-keto-beta-boswellic acid (AKbetaBA) inhibit proliferation and elicit cell death in chemoresistant androgen-independent PC-3 prostate cancer cells in vitro and in vivo. Induction of apoptosis was demonstrated in cultured PC-3 cells by several parameters including mitochondrial cytochrome c release and DNA fragmentation. At the molecular level these compounds inhibit constitutively activated NF-kappaB signaling by intercepting the IkappaB kinase (IKK) activity; signaling through the interferon-stimulated response element remained unaffected, suggesting specificity for IKK inhibition. The impaired phosphorylation of p65 and the reduced nuclear translocation of NF-kappaB proteins were associated with down-regulation of the constitutively overexpressed and NF-kappaB-dependent antiapoptotic proteins Bcl-2 and Bcl-x(L). In addition, expression of cyclin D1, a crucial cell cycle regulator, was reduced as well. Down-regulation of IKK by antisense oligodeoxynucleotides confirmed the essential role of IKK inhibition for the proliferation of the PC-3 cells. Both compounds tested were active in vivo, yet AKbetaBA proved to be far superior. Indeed, topical application of water-soluble AKbetaBA-gamma-cyclodextrin on PC-3 tumors xenografted onto chick chorioallantoic membranes induced concentration-dependent inhibition of proliferation as well as apoptosis. Similarly, in nude mice carrying PC-3 tumors, systemic application of AKbetaBA-gamma-cyclodextrin inhibited tumor growth and triggered apoptosis in the absence of detectable systemic toxicity. Thus, AKbetaBA and related compounds acting on IKK might provide a novel approach for the treatment of chemoresistant human tumors such as androgen-independent human prostate cancers.     

Acute alcohol exposure exerts anti-inflammatory effects by inhibiting IkappaB kinase activity and p65 phosphorylation in human monocytes

Acute alcohol use is associated with impaired immune responses and decreased proinflammatory cytokine production. Our earlier studies have shown that acute alcohol intake inhibits NF-kappaB DNA binding in an IkappaBalpha-independent manner. We report using human peripheral blood monocytes and Chinese hamster ovary cells transfected with CD14 cells that acute alcohol treatment in vitro exerts NF-kappaB inhibition by disrupting phosphorylation of p65. Immunoprecipitation of p65 and IkappaBalpha revealed that acute alcohol exposure for 1 h decreased NF-kappaB-IkappaBalpha complexes in the cytoplasm. Phosphorylation of p65 at Ser(536) is mediated by IkappaB kinase (IKK)beta and is required for NF-kappaB-dependent cellular responses. We show that acute alcohol treatment decreased LPS-induced IKKalpha and IKKbeta activity resulting in decreased phosphorylation of p65 at Ser(536). Furthermore, nuclear expression of IKKalpha increased after alcohol treatment, which may contribute to inhibition of NF-kappaB. Decreased phosphorylation of nuclear p65 at Ser(276) was likely not due to alcohol-induced inhibition of protein kinase A and mitogen- and stress-activated protein kinase-1 activity. Although decreased IkappaBalpha phosphorylation after acute alcohol treatment was attributable to reduced IKKbeta activity, degradation of IkappaBalpha during alcohol exposure was IKKbeta-independent. Alcohol-induced degradation of IkappaBalpha in the presence of a 26S proteasome inhibitor suggested proteasome-independent IkappaBalpha degradation. Collectively, our studies suggest that acute alcohol exposure modulates IkappaBalpha-independent NF-kappaB activity primarily by affecting phosphorylation of p65. These findings further implicate an important role for IKKbeta in the acute effects of alcohol in immune cells.     

Short-term modulation of interleukin-1beta signaling by hyperoxia: uncoupling of IkappaB kinase activation and NF-kappaB-dependent gene expression

We have been interested in elucidating how simultaneous stimuli modulate inflammation-related signal transduction pathways in lung parenchymal cells. We previously demonstrated that exposing respiratory epithelial cells to 95% oxygen (hyperoxia) synergistically increased tumor necrosis factor-alpha (TNF-alpha)-mediated activation of NF-kappaB and NF-kappaB-dependent gene expression by a mechanism involving increased activation of IkappaB kinase (IKK). Because the signal transduction mechanisms induced by IL-1beta are distinct to that of TNF-alpha, herein we sought to determine whether hyperoxia modulates IL-1beta-dependent signal transduction. In A549 cells, simultaneous treatment with hyperoxia and IL-1beta caused increased activation of IKK, prolonged the degradation of IkappaBalpha, and prolonged the nuclear translocation and DNA binding of NF-kappaB compared with cells treated with IL-1beta alone in room air. Hyperoxia did not affect IL-1beta-dependent degradation of the interleukin receptor-associated kinase differently from treatment with IL-beta alone. In contrast to the effects on the IKK/IkappaBalpha/NF-kappaB pathway, simultaneous treatment with hyperoxia and IL-1beta did not augment NF-kappaB-dependent gene expression compared with treatment with IL-1beta alone. Similar observations were made in a different human respiratory epithelial cell line, BEAS-2B cells. In addition, simultaneous treatment with hyperoxia and IL-1beta caused hyperphosphorlyation of the NF-kappaB p65 subunit compared with treatment with IL-1beta alone. In summary, concomitant treatment of A549 cells with hyperoxia and IL-1beta augments activation of IKK, prolongs degradation of IkappaBalpha, and prolongs nuclear translocation and DNA binding of NF-kappaB. This activation, however, is not coupled to increased expression of NF-kappaB-dependent genes, and the mechanism of this decoupling is not related to decreased phosphorylation of p65.     

Adiponectin enhances IL-6 production in human synovial fibroblast via an AdipoR1 receptor, AMPK, p38, and NF-kappa B pathway

Articular adipose tissue is a ubiquitous component of human joints, and adiponectin is a protein hormone secreted predominantly by differentiated adipocytes and involved in energy homeostasis. We investigated the signaling pathway involved in IL-6 production caused by adiponectin in both rheumatoid arthritis synovial fibroblasts and osteoarthritis synovial fibroblasts. Rheumatoid arthritis synovial fibroblasts and osteoarthritis synovial fibroblasts expressed the AdipoR1 and AdipoR2 isoforms of the adiponectin receptor. Adiponectin caused concentration- and time-dependent increases in IL-6 production. Adiponectin-mediated IL-6 production was attenuated by AdipoR1 and 5'-AMP-activated protein kinase (AMPK)alpha1 small interference RNA. Pretreatment with AMPK inhibitor (araA and compound C), p38 inhibitor (SB203580), NF-kappaB inhibitor, IkappaB protease inhibitor, and NF-kappaB inhibitor peptide also inhibited the potentiating action of adiponectin. Adiponectin increased the kinase activity and phosphorylation of AMPK and p38. Stimulation of synovial fibroblasts with adiponectin activated IkappaB kinase alpha/beta (IKK alpha/beta), IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation at Ser (276), p65 and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. Adiponectin-mediated an increase of IKK alpha/beta activity, kappaB-luciferase activity, and p65 and p50 binding to the NF-kappaB element and was inhibited by compound C, SB203580 and AdipoR1 small interference RNA. Our results suggest that adiponectin increased IL-6 production in synovial fibroblasts via the AdipoR1 receptor/AMPK/p38/IKKalphabeta and NF-kappaB signaling pathway.     

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.     

IkappaB kinases phosphorylate NF-kappaB p65 subunit on serine 536 in the transactivation domain.

Recent investigations have elucidated the cytokine-induced NF-kappaB activation pathway. IkappaB kinase (IKK) phosphorylates inhibitors of NF-kappaB (IkappaBs). The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, allowing the nuclear translocation of NF-kappaB. We have examined the possibility that IKK can phosphorylate the p65 NF-kappaB subunit as well as IkappaB in the cytokine-induced NF-kappaB activation. In the cytoplasm of HeLa cells, the p65 subunit was rapidly phosphorylated in response to TNF-alpha in a time dependent manner similar to IkappaB phosphorylation. In vitro phosphorylation with GST-fused p65 showed that a p65 phosphorylating activity was present in the cytoplasmic fraction and the target residue was Ser-536 in the carboxyl-terminal transactivation domain. The endogenous IKK complex, overexpressed IKKs, and recombinant IKKbeta efficiently phosphorylated the same Ser residue of p65 in vitro. The major phosphorylation site in vivo was also Ser-536. Furthermore, activation of IKKs by NF-kappaB-inducing kinase induced phosphorylation of p65 in vivo. Our finding, together with previous observations, suggests dual roles for IKK complex in the regulation of NF-kappaB.IkappaB complex.     

Interference effect of epigallocatechin-3-gallate on targets of nuclear factor kappaB signal transduction pathways activated by EB virus encoded latent membrane protein 1

AIM: To elucidate the interference effect of epigallocatechin-3-gallate (EGCG) on targets of nuclear factor kappaB (NF-kappaB) signal transduction pathway activated by EB virus encoded latent membrane protein 1 (LMP1) in nasopharyngeal carcinoma (NPC) cell lines. METHODS: The survival rates of CNE1 and CNE-LMP1 cell lines after the EGCG treatment were determined by MTT assay. NF-kappaB activation in CNE1 and CNE-LMP1 cells after EGCG treatment was analyzed by promoter luciferase reporter system. And then nuclear translocation of NF-kappaB (p65) after the EGCG treatment was analyzed by immunofluorescence and western blotting. Meanwhile, the changes of IkappaBalpha phosphorylation were observed after the EGCG treatment. EGFR promoter activity was analyzed by promoter luciferase reporter system and EGFR phosphorylation was observed by western blotting after the EGCG treatment. RESULTS: EGCG inhibited the survival rates of CNE1 and CNE-LMP1 cells and NF-kappaB activation caused by LMP1 in CNE-LMP1 cells. EGCG also suppressed the nuclear translocation of NF-kappaB (p65) and IkappaBalpha phosphorylation. Meanwhile, EGCG inhibited EGFR promoter activity and EGFR phosphorylation. CONCLUSIONS: EGCG inhibited not only the dose-dependent survival rate of NPC cells, but also the dose-dependent activation of NF-kappaB. This inhibition of LMP1-caused NF-kappaB activation was mediated via the phosphorylative degradation of its inhibitory protein IkappaBalpha, and then EGCG inhibited EGFR activity which was a downstream gene from NF-kappaB. This study suggests that interference effect of EGCG on targets of signal transduction pathway plays an important role in the anticancer function.     

NF-kappaB signaling regulates functional expression of the MHC class I-related neonatal Fc receptor for IgG via intronic binding sequences

The neonatal Fc receptor for IgG (FcRn) functions to transport maternal IgG to a fetus or newborn and to protect IgG from degradation. Although FcRn is expressed in a variety of tissues and cell types, the extent to which FcRn expression is regulated by immunological and inflammatory events remains unknown. Stimulation of intestinal epithelial cell lines, macrophage-like THP-1, and freshly isolated human monocytes with the cytokine TNF-alpha rapidly up-regulated FcRn gene expression. In addition, the TLR ligands LPS and CpG oligodeoxynucleotide enhanced the level of FcRn expression in THP-1 and monocytes. Treatment of TNF-stimulated THP-1 cells with the NF-kappaB-specific inhibitor or overexpression of a dominant negative mutant inhibitory NF-kappaB (IkappaBalpha; S32A/S36A) resulted in down-regulation of FcRn expression. By using chromatin immunoprecipitation we identified three NF-kappaB binding sequences within introns 2 and 4 of the human FcRn gene. An EMSA confirmed the p50/p50 and/or p65/p50 complex (s) bound to intron 2- or 4-derived oligonucleotides containing putative NF-kappaB binding sequences, respectively. The intronic NF-kappaB sequences in combination with the promoter or alone regulated the expression of a luciferase reporter gene in response to TNF-alpha stimulation or overexpression of NF-kappaB p65 and p50. DNA looping interactions potentially occurred after the stimulation between intronic NF-kappaB sequences and the FcRn promoter as shown by a chromosome conformation capture assay. Finally, TNF-alpha stimulations enhanced IgG transport across an intestinal Caco-2 epithelial monolayer. Together, these data provide the first evidence that NF-kappaB signaling via intronic sequences regulates FcRn expression and function.     

Astaxanthin inhibits nitric oxide production and inflammatory gene expression by suppressing I(kappa)B kinase-dependent NF-kappaB activation

Astaxanthin, a carotenoid without vitamin A activity, has shown anti-oxidant and anti-inflammatory activities; however, its molecular action and mechanism have not been elucidated. We examined in vitro and in vivo regulatory function of astaxanthin on production of nitric oxide (NO) and prostaglandin E2 (PGE2) as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta). Astaxanthin inhibited the expression or formation production of these proinflammatory mediators and cytokines in both lipopolysaccharide (LPS)-stimulated RAW264.7 cells and primary macrophages. Astaxanthin also suppressed the serum levels of NO, PGE2, TNF-alpha, and IL-1beta in LPS-administrated mice, and inhibited NF-kappaB activation as well as iNOS promoter activity in RAW264.7 cells stimulated with LPS. This compound directly inhibited the intracellular accumulation of reactive oxygen species in LPS-stimulated RAW264.7 cells as well as H2O2-induced NF-kappaB activation and iNOS expression. Moreover, astaxanthin blocked nuclear translocation of NF-kappaB p65 subunit and I(kappa)B(alpha) degradation, which correlated with its inhibitory effect on I(kappa)B kinase (IKK) activity. These results suggest that astaxanthin, probably due to its antioxidant activity, inhibits the production of inflammatory mediators by blocking NF-kappaB activation and as a consequent suppression of IKK activity and I(kappa)B-alpha degradation.     

Tumor necrosis factor-alpha-induced IKK phosphorylation of NF-kappaB p65 on serine 536 is mediated through the TRAF2, TRAF5, and TAK1 signaling pathway

The activation of NF-kappaB has been shown to be regulated by multiple phosphorylations of IkappaBs and the NF-kappaB p65 subunit. Here, we characterized the intracellular signaling pathway leading to phosphorylation of p65 on Ser-536 using a novel anti-phospho-p65 (Ser-536) antibody. The Ser-536 of endogenous p65 was rapidly phosphorylated in response to a wide variety of NF-kappaB stimulants including TNF-alpha in the cytoplasm and rapidly dephosphorylated in the nucleus. The TNF-alpha-but not IL-1beta-induced Ser-536 phosphorylation was severely impaired in murine embryonic fibroblasts derived from traf2-/-traf5-/- mice. Bay 11-7082, an inhibitor of IkappaB phosphorylation, inhibited the TNF-alpha-induced phosphorylation in vivo. In addition, overexpression of TGF-beta-activated kinase 1 (TAK1), IKKalpha and IKKbeta stimulated the phosphorylation, and their dominant negative mutants blocked the TNF-alpha-induced phosphorylation. Moreover, small interfering RNAs (siRNAs) against TAK1, IKKalpha and IKKbeta blocked the phosphorylation of endogenous p65. On the other hand, calyculin-A, a protein phosphatase inhibitor, blocked the dephosphorylation in the nucleus in vivo. These results indicate that similar signaling pathways were utilized for the phosphorylations of IkappaBalpha and p65, which further support the idea that both IkappaB and NF-kappaB are substrates for the IKK complex in the activation of NF-kappaB.