IKKβ Kinase Activity Research Articles

Targeting IKKβ Activity to Limit Sterile Inflammation in Acetaminophen-Induced Hepatotoxicity in Mice.

The kinase activity of inhibitory κB kinase β (IKKβ) acts as a signal transducer in the activating pathway of nuclear factor-κB (NF-κB), a master regulator of inflammation and cell death in the development of numerous hepatocellular injuries. However, the importance of IKKβ activity on acetaminophen (APAP)-induced hepatotoxicity remains to be defined. Here, a derivative of caffeic acid benzylamide (CABA) inhibited the kinase activity of IKKβ, as did IMD-0354 and sulfasalazine which show therapeutic efficacy against inflammatory diseases through a common mechanism: inhibiting IKKβ activity. To understand the importance of IKKβ activity in sterile inflammation during hepatotoxicity, C57BL/6 mice were treated with CABA, IMD-0354, or sulfasalazine after APAP overdose. These small-molecule inhibitors of IKKβ activity protected the APAP-challenged mice from necrotic injury around the centrilobular zone in the liver, and rescued the mice from hepatic damage-associated lethality. From a molecular perspective, IKKβ inhibitors directly interrupted sterile inflammation in the Kupffer cells of APAP-challenged mice, such as damage-associated molecular pattern (DAMP)-induced activation of NF-κB activity via IKKβ, and NF-κB-regulated expression of cytokines and chemokines. However, CABA did not affect the upstream pathogenic events, including oxidative stress with glutathione depletion in hepatocytes after APAP overdose. N-acetyl cysteine (NAC), the only FDA-approved antidote against APAP overdose, replenishes cellular levels of glutathione, but its limited efficacy is concerning in late-presenting patients who have already undergone oxidative stress in the liver. Taken together, we propose a novel hypothesis that chemical inhibition of IKKβ activity in sterile inflammation could mitigate APAP-induced hepatotoxicity in mice, and have the potential to complement NAC treatment in APAP overdoses.

Triptolidenol, isolated from Tripterygium wilfordii, disrupted NF-κB/COX-2 pathway by targeting ATP-binding sites of IKKβ in clear cell renal cell carcinoma

Triptolidenol (TPD) is an epoxy diterpene lactone from Tripterygium wilfordii, which has been used for chronic nephritis in China，and possessed various pharmacological properties, such as anti-inflammatory and anti-cancer activities. However, the precise molecular antitumor mechanism of TPD remains to be elucidated. In this study, we investigated the effects of TPD on human clear cell renal cell carcinoma (ccRCC) and investigated its precise anti-tumor mechanisms. It was showed that TPD significantly suppressed ccRCC cell proliferation, cell migration, and induced cell cycle arrest at S phase. Furthermore, TPD also induced apoptosis by activating the cytochrome c (cyt c)/caspase cascade signaling pathway. Moreover, using confocal immunofluorescence, a dual-luciferase reporter assay and molecular docking study, the results showed that TPD obviously reduced the expression of COX-2 by inhibiting the kinase activity of IKKβ via targeting its ATP-binding domain, and then attenuating the transactivation of NF-κB. Collectively, our study demonstrated that TPD suppressed renal cell carcinoma growth through disrupting NF-κB/COX-2 pathway by targeting ATP-binding sites of IKKβ, and provided pharmacological evidence that TPD exhibits potential use in the treatment of COX-2-mediated diseases such as ccRCC.

Venezuelan Equine Encephalitis Virus nsP3 Phosphorylation Can Be Mediated by IKKβ Kinase Activity and Abrogation of Phosphorylation Inhibits Negative-Strand Synthesis.

Venezuelan equine encephalitis virus (VEEV), a mosquito transmitted alphavirus of the Togaviridae family, can cause a highly inflammatory and encephalitic disease upon infection. Although a category B select agent, no FDA-approved vaccines or therapeutics against VEEV currently exist. We previously demonstrated NF-κB activation and macromolecular reorganization of the IKK complex upon VEEV infection in vitro, with IKKβ inhibition reducing viral replication. Mass spectrometry and confocal microscopy revealed an interaction between IKKβ and VEEV non-structural protein 3 (nsP3). Here, using western blotting, a cell-free kinase activity assay, and mass spectrometry, we demonstrate that IKKβ kinase activity can directly phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5. Alanine substitution mutations at sites 204/5, 142, or 134/5 reduced VEEV replication by >30-100,000-fold corresponding to a severe decrease in negative-strand synthesis. Serial passaging rescued viral replication and negative-strand synthesis, and sequencing of revertant viruses revealed reversion to the wild-type TC-83 phosphorylation capable amino acid sequences at nsP3 sites 204/5, 142, and 135. Generation of phosphomimetic mutants using aspartic acid substitutions at site 204/5 resulted in rescue of both viral replication and negative-strand RNA production, whereas phosphomimetic mutant 134/5 rescued viral replication but failed to restore negative-strand RNA levels, and phosphomimetic mutant 142 did not rescue VEEV replication. Together, these data demonstrate that IKKβ can phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5, and suggest that phosphorylation is essential for negative-strand RNA synthesis at site 204/5, but may be important for infectious particle production at site 134/5.

IKKβ Kinase Promotes Stemness, Migration, and Invasion in KRAS-Driven Lung Adenocarcinoma Cells.

KRAS oncogenic mutations are widespread in lung cancer and, because direct targeting of KRAS has proven to be challenging, KRAS-driven cancers lack effective therapies. One alternative strategy for developing KRAS targeted therapies is to identify downstream targets involved in promoting important malignant features, such as the acquisition of a cancer stem-like and metastatic phenotype. Based on previous studies showing that KRAS activates nuclear factor kappa-B (NF-κB) through inhibitor of nuclear factor kappa-B kinase β (IKKβ) to promote lung tumourigenesis, we hypothesized that inhibition of IKKβ would reduce stemness, migration and invasion of KRAS-mutant human lung cancer cells. We show that KRAS-driven lung tumoursphere-derived cells exhibit stemness features and increased IKKβ kinase activity. IKKβ targeting by different approaches reduces the expression of stemness-associated genes, tumoursphere formation, and self-renewal, and preferentially impairs the proliferation of KRAS-driven lung tumoursphere-derived cells. Moreover, we show that IKKβ targeting reduces tumour cell migration and invasion, potentially by regulating both expression and activity of matrix metalloproteinase 2 (MMP2). In conclusion, our results indicate that IKKβ is an important mediator of KRAS-induced stemness and invasive features in lung cancer, and, therefore, might constitute a promising strategy to lower recurrence rates, reduce metastatic dissemination, and improve survival of lung cancer patients with KRAS-driven disease.

Polyubiquitin-dependent recruitment of NEMO/IKKγ into T cell receptor signaling microclusters is controlled by IKKβ kinase activity

Abstract The IkB kinase (IKK) complex coordinates inflammatory responses by activating canonical NFkB downstream of immune receptors. This complex consists of the kinases IKKa/β and the dimeric adaptor subunit NEMO (NFkB essential modulator protein). NEMO mutations cause immunodeficiencies and impair antigen receptor function. Canonical NFkB activation by immunoreceptors, such as the T cell receptor (TCR), requires the assembly of the Carma1/Bcl10/Malt1 (CBM) signalosome. While, functional studies and in vitro interactions, place the IKK complex downstream of the CBM signalosome, the spatial relationship between these complexes has never been observed in intact immune cells. We observed that NEMO is recruited into TCR microclusters and membrane compartments within ~70 seconds of TCR engagement. Point mutations impacting the K63-linked and linear polyubiquitin ubiquitin-binding domains of NEMO selectively impair NEMO recruitment into TCR microclusters. We verified the involvement of these polyubiquitin chains by showing that inhibitors of either the K63-specific Uev1A/Ubc13 ubiquitin E2 ligase or the linear ubiquitin activating complex (LUBAC) prevent NEMO from entering TCR microclusters. To examine whether the entire IKK complex is recruited to the TCR, we co-expressed IKKb chimeras with NEMO. We observed that wild-type (WT), but not kinase-dead, IKKb caused NEMO microclusters to disappear. Similarly, the treatment of cells expressing WT IKKb with inhibitors of IKKβ or its upstream activator, TAK1, restored the recruitment of IKKβ and NEMO into microclusters. These results suggest that the IKK complex is recruited to the TCR via linear- and K63-linked polyubiquitin chains and then dissociates from the TCR via IKKβ activity.

Modulation of LPS-induced inflammation in RAW264.7 murine cells by novel isoflavonoids from Millettia pulchra.

Millettia pulchra is a renowned anti-inflammatory herbal medicine in southeast provinces of China. However, the underlying anti-inflammation mechanism remained incompletely understood. Herein, four new isoflavones, pulvones A-D and eleven reported constituents were isolated from the stems of Millettia pulchra with their structures being elucidated by HRMS and NMR analysis. The anti-inflammatory activities of pulvones A and C were further evaluated due to the better inhibitory activity on nitric oxide production in LPS-stimulated RAW264.7 cells and no obvious cytotoxicity to RAW264.7 cells. Western blot showed that pulvones A significantly decreased the levels of iNOS and COX-2 proteins and pulvones C only decreased the level of iNOS protein. ELISA analysis demonstrated that pulvones A inhibited the production of both interleukin-6 (IL-6) and IL-1β while pulvones C showed better suppression effect on IL-1β production in LPS-stimulated RAW264.7 cells. Then, their potential inhibitory effects on NF-κB pathway were tested in LPS-stimulated RAW264.7 cells. Immunofluorescence and western blot assay showed that pulvones A and C reduced the nuclear translocation of NF-κB(p65) and interrupted IκB phosphorylation. The ADP-Glo™ kinase assay showed pulvones A and C could directedly inhibit the IKKβ kinase activity with the inhibitory rate of 40%, which were also verified by docking study. Collectively, these results suggested that pulvones A and C's anti-inflammatory effects were relevant to the interruption of NF-κB activation by inhibiting IKKβ kinase.

A mutation of cysteine 46 in IKK-β promotes mPGES-1 and caveolin-1 expression to exacerbate osteoclast differentiation and osteolysis

IKK-β is indispensable for inflammatory osteolysis, the functional residues of IKK-β are therapeutic drug targets for developing inhibitors to treat multiple diseases now. Thus it remains appealing to find the new residues of IKK-β to influence osteoclasts for alleviating bone loss diseases such as rheumatoid arthritis (RA). By employing IKK-β cysteine 46-A transgenic (IKK-βC46A) mice, we found that mutation of cysteine 46 to alanine in IKK-β exacerbated inflammatory bone destruction in vivo, and increased osteoclast differentiation and bone resorption ex vivo and in vitro. Consistent with these, IKK-β kinase activity as well as c-Fos, NFATc1 were up-regulated in bone marrow macrophages (BMMs) from IKK-βC46A mice during RANKL-induced osteoclastogenesis. Of interesting, we further identified and demonstrated that the expressions of mPGES-1 and caveolin-1 were heightened in BMMs of IKK-βC46A mice compared to those in WT mice in RANKL-induced osteoclastogenesis. Together, it revealed that mutating cysteine 46 in IKK-β could increase caveolin-1 and mPGES-1 expression to facilitate osteoclast differentiation and osteolysis. Cysteine 46 can serve as a novel target in IKK-β for designing inhibitors to treat osteolysis.

Phytochemicals as potential IKK-β inhibitor for the treatment of cardiovascular diseases in plant preservation: terpenoids, alkaloids, and quinones.

Modulation of inhibitor kappa B kinase-beta (IKK-β) kinase activity could be useful for preventing inflammation that serves an efficient role in protection against cardiovascular diseases (CVDs). IKK-β induces inflammation by activating transcription factor NF-kappa B (NF-κB) through phosphorylation of IκB. Therefore, IKK-β is considered an interesting target for protecting and treating CVDs. The cardioprotective potential of terpenoids, alkaloids and quinines may be related to modulating inflammatory responses. In this study, the interactions between different classes of inhibitors and IKK-β were investigated, through the application of SystemsDock. Molecular docking results showed that Diosgenin and Ginsenoside Re were the top docking score compounds. Diosgenin and Ginsenoside Re are the most promising IKK-β inhibitors in terpenoids, alkaloids, and quinones. Diosgenin and Ginsenoside Re could be helpful to find the lead compounds on designing and developing novel cardioprotective agents.

Azithromycin Polarizes Macrophages to an M2 Phenotype via Inhibition of the STAT1 and NF-κB Signaling Pathways

Azithromycin is effective at controlling exaggerated inflammation and slowing the long-term decline of lung function in patients with cystic fibrosis. We previously demonstrated that the drug shifts macrophage polarization toward an alternative, anti-inflammatory phenotype. In this study we investigated the immunomodulatory mechanism of azithromycin through its alteration of signaling via the NF-κB and STAT1 pathways. J774 murine macrophages were plated, polarized (with IFN-γ, IL-4/-13, or with azithromycin plus IFN-γ) and stimulated with LPS. The effect of azithromycin on NF-κB and STAT1 signaling mediators was assessed by Western blot, homogeneous time-resolved fluorescence assay, nuclear translocation assay, and immunofluorescence. The drug's effect on gene and protein expression of arginase was evaluated as a marker of alternative macrophage activation. Azithromycin blocked NF-κB activation by decreasing p65 nuclear translocation, although blunting the degradation of IκBα was due, at least in part, to a decrease in IKKβ kinase activity. A direct correlation was observed between increasing azithromycin concentrations and increased IKKβ protein expression. Moreover, incubation with the IKKβ inhibitor IKK16 decreased arginase expression and activity in azithromycin-treated cells but not in cells treated with IL-4 and IL-13. Importantly, azithromycin treatment also decreased STAT1 phosphorylation in a concentration-dependent manner, an effect that was reversed with IKK16 treatment. We conclude that azithromycin anti-inflammatory mechanisms involve inhibition of the STAT1 and NF-κB signaling pathways through the drug's effect on p65 nuclear translocation and IKKβ.

The natural flavonoid glycoside vitexin displays preclinical antitumor activity by suppressing NF-κB signaling in nasopharyngeal carcinoma.

Background and objectives: Vitexin is a natural flavonoid glycoside mainly extracted from the leaves of vitex, which has a variety of physiological activities. For example, vitexin has antitumor and anti-inflammation activities, and it can also promote blood circulation in the body. However, the function and mechanism of vitexin in nasopharyngeal carcinoma (NPC) are still unclear.Materials and methods: Cell Counting Kit-8 assay and cell cycle analysis were performed to examine cell survival in response to vitexin. Immunoblotting was used to analyze relative proteins’ expression. NPC xenograft models were established to assess the effect of vitexin in vivo. The luciferase activity of pNFκB-Luc was analyzed by using Dual-Luciferase Reporter Assay System. Quantitative real-time polymerase chain reaction was performed to detect relative genes’ expression. Kinase activity of IKKβ was analyzed in a cell-free system.Results: In this study, vitexin was found to display significant antitumor activity in NPC in vitro and in vivo. In NPC cells, vitexin inhibited cell cycle progression in NPC cells and induced the cleavages of PARP and inhibited antiapoptotic proteins’ expression, including Bcl-2 and Mcl1. Further studies indicated that vitexin significantly suppressed the luciferase activity of pNF-κB-Luc and inhibited the activation of NF-κB key regulators, including p65, IκBα and IKKs in NPC cells. Moreover, the kinase activity of IKKβ could be suppressed by vitexin in a cell-free system, and overexpression of CA-IKKβ could attenuate the inhibitory effect of vitexin on p65 phosphorylation.Conclusion: These results indicated that vitexin displayed antitumor activity by suppressing NF-κB signaling in NPC, which suggested that vitexin could be as a potential drug for the treatment of NPC in the future.

Isoalantolactone inhibits IKKβ kinase activity to interrupt the NF-κB/COX-2-mediated signaling cascade and induces apoptosis regulated by the mitochondrial translocation of cofilin in glioblastoma.

Isoalantolactone (IATL), a sesquiterpene lactone compound, possesses many pharmacological and biological activities, but its role in glioblastoma (GBM) treatment is still unknown. The aim of the current study was to investigate the antiglioma effects of IATL and to explore the underlying molecular mechanisms. In the current study, the biological functions of IATL were examined by MTT, cell migration, colony formation, and cell apoptosis assays. Confocal immunofluorescence techniques, chromatin immunoprecipitation, and pull‐down assays were used to explore the precise underlying molecular mechanisms. To examine IATL activity and the molecular mechanisms by which it inhibits glioma growth in vivo, we used a xenograft tumor mouse model. Furthermore, Western blotting was used to confirm the changes in protein expression after IATL treatment. According to the results, IATL inhibited IKKβ phosphorylation, thus inhibiting both the binding of NF‐κB to the cyclooxygenase 2 (COX‐2) promoter and the recruitment of p300 and eventually inhibiting COX‐2 expression. In addition, IATL induced glioma cell apoptosis by promoting the conversion of F‐actin to G‐actin, which in turn activates the cytochrome c (Cyt c) and caspase‐dependent apoptotic pathways. In the animal experiments, IATL reduced the size and weight of glioma tumors in xenograft mice and inhibited the expression of COX‐2 and phosphorylated NF‐κB p65 in the transplanted tumors. In conclusion, the current study indicated that IATL inhibited the expression of COX‐2 through the NF‐κB signaling pathway and induced the apoptosis of glioma cells by increasing actin transformation. These results suggested that IATL could be greatly effective in GBM treatment.

Design, synthesis and biological evaluation of novel thiazolidinedione derivatives as irreversible allosteric IKK-β modulators

The kinase known as IKK-β activates NF-κB signaling pathway leading to expression of several genes contributing to inflammation, immune response, and cell proliferation. Modulation of IKK-β kinase activity could be useful for treatment and management of such diseases. Starting from a discovered weakly active hit compound, twenty four thiazolidinedione-scaffold based chemical entities belonging to five series have been designed, synthesized and evaluated as potential IKK-β modulators. Among them, compounds 6q, 6r and 6u showed low micromolar IC50 values while compounds 6v, 6w, and 6x elicited submicromolar IC50 values equal to 0.4, 0.7 and 0.9 μM respectively. These submicromolar IC50 values are 243, 139 and 105 folds the value of the reported IC50 of the starting hit compound. Kinetic study of compounds 6v and 6w confirmed this class of modulators as irreversible inhibitors. LPS-treated RAW 264.7 macrophages proved the anti-inflammatory activity of compounds 6q and 6v. Assay of hERG inhibition demonstrated a safe profile of compound 6q suggesting it as a lead for further development of IKK-β modulators.

Anti-inflammatory effect of avenanthramides via NF-κB pathways in C2C12 skeletal muscle cells.

Avenanthramides (Avns), the polyphenol compounds found only in oats, have been shown to exhibit anti-inflammatory effects mainly by inhibiting nuclear factor (NF)-κB activation in select cell lines. However, the molecular mechanism by which Avns regulate the NF-κB pathway is still unclear. The purpose of this study was to investigate (1) the molecular mechanism by which three main fractions of Avns (AvnA, AvnB and AvnC) interact with IκB Kinase β (IKKβ); and (2) whether this interaction results in reduced inflammatory responses in skeletal muscle cells. The protein-ligand docking and molecular dynamics simulation studies suggest that Avns acted as an allosteric inhibitor for modulating IKKβ's affinity for the NF-κB complex. Thus, Avns reduced IKKβ kinase activity in response to tert-butyl hydroperoxide (tBHP) stimulation and attenuated tBHP-induced TNFα and IL-1β mRNA expression. Furthermore, the three-fold increases in cyclooxygenase-2 (COX-2) protein and luciferase activity with tBHP treatment were reduced by 50% with Avns (P < .01), along with decreased prostaglandin E2 levels (P < .01). These data indicate that Avns are potent inhibitors of NFκB-mediated inflammatory response due to the downregulation of IKKβ activity in C2C12 cells.

Xanthohumol prevents dextran sulfate sodium-induced colitis via inhibition of IKKβ/NF-κB signaling in mice.

Xanthohumol (XN), a prenylated chalcone isolated from the hop plant, has been reported to exhibit multiple biological functions including anti-inflammation. However, the pharmacological function of XN on colitis remains unknown. In this study, we investigated the anti-inflammatory effect of synthesized XN and molecular mechanism on dextran sulfate sodium (DSS)-induced experimental colitis. XN attenuated the colitis symptoms along with the prevention of colonic lesions after DSS challenge. XN inhibited the production of pro-inflammatory cytokines, oxidative stress and cyclooxygenase-2 expression in DSS-treated mice. Moreover, XN inhibited the phosphorylation of IκBα, the nuclear translocation of NF-κB subunits and the transcriptional activity of NF-κB in vivo and in vitro. In contrast to XN, isoXN showed much less effects on the kinase activity of IKKβ and IκBα phosphorylation induced by XN in this study, suggesting that an electrophilic carbon center present in XN is critical for the anti-inflammation in colitis, especially inhibition of IKKβ/NF-κB signaling pathway. Consistently, our docking analysis revealed that XN could bind to the active site, presumably at the Cys99 of IKKβ. Taken together, these findings demonstrate a new function of XN to inhibit IKKβ/NF-κB signaling, suggesting XN could be the potential therapeutic agent for the prevention of colitis.

Alantolactone, a natural sesquiterpene lactone, has potent antitumor activity against glioblastoma by targeting IKK\u03b2 kinase activity and interrupting NF-\u03baB/COX-2-mediated signaling cascades

BackgroundGlioblastoma multiforme (GBM) is one of the most refractory and palindromic central nervous system (CNS) neoplasms, and current treatments have poor effects in GBM patients. Hence, the identification of novel therapeutic targets and the development of effective treatment strategies are essential. Alantolactone (ATL) has a wide range of pharmacological activities, and its anti-tumor effect is receiving increasing attention. However, the molecular mechanism underlying the anti-GBM activity of ATL remains poorly understood.MethodsThe biological functions of ATL in GBM cells were investigated using migration/invasion, colony formation and cell cycle/apoptosis assays. The localization of nuclear factor kappa B (NF-κB) p50/p65 and its binding to the cyclooxygenase 2 (COX-2) promoter were determined using confocal immunofluorescence, a streptavidin-agarose pulldown assay and a chromatin immunoprecipitation (ChIP) assay. IKKβ kinase activity was determined using a cell IKKβ kinase activity spectrophotometry quantitative detection kit and a molecular docking study. LC-MS/MS analysis was performed to determine the ability of ATL to traverse the blood-brain barrier (BBB). The in vivo anti-tumor efficacy of ATL was also analyzed in xenografted nude mice. Western blot analysis was performed to detect the protein expression levels.ResultsATL significantly suppressed the growth of GBM in vivo and in vitro. ATL significantly reduced the expression of COX-2 by inhibiting the kinase activity of IKKβ by targeting the ATP-binding site and then attenuating the binding of NF-κB to the COX-2 promoter region. Furthermore, ATL induced apoptosis by activating the cytochrome c (cyt c)/caspase cascade signaling pathway. Moreover, ATL could penetrate the BBB.ConclusionsATL exerts its anti-tumor effects in human GBM cells at least in part via NF-κB/COX-2-mediated signaling cascades by inhibiting IKKβ kinase activity. ATL, which is a natural small molecule inhibitor, is a promising candidate for clinical applications in the treatment of CNS tumors.

Aspirin Inhibits IKK-β-mediated Prostate Cancer Cell Invasion by Targeting Matrix Metalloproteinase-9 and Urokinase-Type Plasminogen Activator

Background/Aims: Aspirin has been demonstrated to possess potent chemopreventive and anticancer effects on prostate cancer. However, the more detailed molecular mechanisms of aspirin to suppress prostate cancer cell invasion have not been clearly elucidated. Methods: Transwell assays were performed to evaluate the effects of aspirin on cell invasion. Matrix metalloproteinases (MMPs) and serine proteinases activities in cell media were examined by gelatin zymography and ELISA. In addition, inhibitor of κB (IκB) kinase-β (IKK-β) phosphorylation and IKK-β kinase activity were measured to assess the effects of aspirin on IKK-β activation. Results: We found that aspirin suppressed the invasion and attachment in human prostate cancer cells. Aspirin treatment significantly resulted in reduction of matrix metalloproteinase-9 (MMP-9) and upregulation of tissue inhibitors of metalloproteinase-1 (TIMP-1) activity, which are the proteolytic enzymes contributing to the degradation of extracellular matrix and basement membrane in cell invasion and metastasis. Our data further showed that aspirin was able to inhibit both urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) expression in the cells. In addition, aspirin treatment caused a strong decrease in nuclear factor-kappa B (NF-κB) activation, inhibitor of κB (IκB)-α phosphorylation together with translocation of NF-κB p65 to nucleus and IκB kinase (IKK)- β activation. Moreover, the inhibitory effects of aspirin on cell invasion were reversed by IKK-β overexpression, while the IKK inhibitor sensitizes the anti-invasive effect of aspirin in prostate cancer cells. Conclusion: The present research concluded that aspirin suppressed prostate cancer cell invasion by reducing MMP-9 activity and uPA expression through decreasing of IKK-β-mediated NF-κB activation, indicating that the ability of aspirin to inhibit cell invasion might be useful in the chemoprevention of metastatic prostate cancer.

Mutation of cysteine 46 in IKK-beta increases inflammatory responses.

Activation of IκB kinase β (IKK-β) and nuclear factor (NF)-κB signaling contributes to cancer pathogenesis and inflammatory disease; therefore, the IKK-β-NF-κB signaling pathway is a potential therapeutic target. Current drug design strategies focus on blocking NF-κB signaling by binding to specific cysteine residues on IKK-β. However, mutations in IKK-β have been found in patients who may eventually develop drug resistance. For these patients, a new generation of IKK-β inhibitors are required to provide novel treatment options. We demonstrate in vitro that cysteine-46 (Cys-46) is an essential residue for IKK-β kinase activity. We then validate the role of Cys-46 in the pathogenesis of inflammation using delayed-type hypersensitivity (DTH) and an IKK-β C46A transgenic mouse model. We show that a novel IKK-β inhibitor, dihydromyricetin (DMY), has anti-inflammatory effects on WT DTH mice but not IKK-β C46A transgenic mice. These findings reveal the role of Cys-46 in the promotion of inflammatory responses, and suggest that Cys-46 is a novel drug-binding site for the inhibition of IKK-β.

Dimer of arfaptin 2 regulates NF-κB signaling by interacting with IKKβ/NEMO and inhibiting IKKβ kinase activity

IκB kinases (IKKs) are a therapeutic target due to their crucial roles in various biological processes, including the immune response, the stress response, and tumor development. IKKs integrate various upstream signals that activate NF-κB by phosphorylating IκB and also regulate many proteins related to cell growth and metabolism. Although they function as a heteromeric complex comprised of kinase subunits and an adaptor, these kinases produce distinct cellular responses by phosphorylating different target molecules, suggesting that they may also be regulated in a subtype-specific manner. In this study, arfaptin 2 was identified as an IKKβ-specific binding partner. Interestingly, arfaptin 2 also interacted with NEMO. Domain mapping studies revealed that the C-terminal region, including the IKKβ HLH domain and the first coiled-coil NEMO region were respectively required for interactions with the arfaptin 2 N-terminal flexible region. Overexpression of arfaptin 2 inhibited tumor necrosis factor (TNF)-α-stimulated nuclear factor-κB (NF-κB) signaling, whereas downregulation of arfaptin 2 by small interfering RNA enhanced NF-κB activity. Dimerization of arfaptin 2 through the Bin–Amphiphysin–Rvs domain may be essential to inhibit activation of NF-κB through multimodal interactions with IKKβs or IKKβ/NEMO, as ectopic expression of the arfaptin 2 fragment responsible for IKK interactions did not change TNFα-stimulated NF-κB activation. These data indicate that arfaptin 2 is the first molecule to regulate NF-κB signaling by interacting with the functional IKK complex but not by direct inhibiting IKKβ phosphorylation.

Pentacyclic Triterpenoids Inhibit IKKβ Mediated Activation of NF-κB Pathway: In Silico and In Vitro Evidences.

Pentacyclic Triterpenoids (PTs) and their analogues as well as derivatives are emerging as important drug leads for various diseases. They act through a variety of mechanisms and a majority of them inhibit the nuclear factor kappa-beta (NF-κB) signaling pathway. In this study, we examined the effects of the naturally occurring PTs on IκB kinase-β (IKKβ), which has great scientific relevance in the NF-κB signaling pathway. On virtual screening, 109 PTs were screened through the PASS (prediction of activity spectra of substances) software for prediction of NF-κB inhibitory activity followed by docking on the NEMO/IKKβ association complex (PDB: 3BRV) and testing for compliance with the softened Lipinski’s Rule of Five using Schrodinger (LLC, New York, USA). Out of the projected 45 druggable PTs, Corosolic Acid (CA), Asiatic Acid (AA) and Ursolic Acid (UA) were assayed for IKKβ kinase activity in the cell free medium. The UA exhibited a potent IKKβ inhibitory effect on the hotspot kinase assay with IC50 of 69 μM. Whereas, CA at 50 μM concentration markedly reduced the NF-κB luciferase activity and phospho-IKKβ protein expressions. The PTs tested, attenuated the expression of the NF-κB cascade proteins in the LPS-stimulated RAW 264.7 cells, prevented the phosphorylation of the IKKα/β and blocked the activation of the Interferon-gamma (IFN-γ). The results suggest that the IKKβ inhibition is the major mechanism of the PTs-induced NF-κB inhibition. PASS predictions along with in-silico docking against the NEMO/IKKβ can be successfully applied in the selection of the prospective NF-κB inhibitory downregulators of IKKβ phosphorylation.

Altered neutrophil functions in elderly patients during a 6-month follow-up period after a hip fracture

BackgroundFracture of the hip (HF) is a significant cause of morbidity and mortality in elderly individuals. HF is an acute stress that triggers a state of inflammation which may affect immune responses and physical recovery. MethodsLongitudinal study of the impact of HF on the functions of polymorphonuclear neutrophils (PMNs) in elderly subjects. Data were recorded prior to surgery, 6weeks and 6months later. ResultsPMN functions were severely impaired shortly after HF (chemotaxis, phagocytosis, superoxide production) but there was a time-related recovery of some PMN functions (chemotaxis, phagocytosis) over time, except in the case of superoxide production. Whereas FcγRII (CD32) expression remained unchanged, FcγRIII (CD16) increased from low values before surgery to levels of controls 6months post-surgery. This was also the case for the C5a complement receptor and CD11b. TLR2 and TLR4 expressions were unchanged. Cytokine and chemokine secretions by stimulated PMN were altered. TNFα and IL-10 secretions were increased following HF but IL-8 secretion was decreased. Impaired PMN functions prior to surgery were related to alterations in PI3K and NF-κB signaling pathways. Recovery of these functions paralleled increased PI3K activity, although superoxide production remained low. Sustained activation of the NF-κB pathway by TNFα has been reported to involve upregulation of IKKβ kinase activity. Activated IKKβ kinase inhibits ERK1/2 and results in concomitant downstream inhibition of NADPH oxidase complex which can account for sustained impaired production of ROS in HF patients. ConclusionOur data showed that the stress caused by HF negatively affects initial PMN responses shortly after the event and that may negatively influence clinical outcomes such as resolving long-term inflammation and recovery, as well as explaining susceptibility to opportunistic infections.