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The inhibitory-kappaB kinases (IKKs) IKKα and IKKβ play central roles in regulating the non-canonical and canonical NF-κB signalling pathways. Whilst the proteins that transduce the signals of each pathway have been extensively characterised, the clear dissection of the functional roles of IKKα-mediated non-canonical NF-κB signalling versus IKKβ-driven canonical signalling remains to be fully elucidated. Progress has relied upon complementary molecular and pharmacological tools; however, the lack of highly potent and selective IKKα inhibitors has limited advances. Herein, we report the development of an aminoindazole-pyrrolo[2,3- b ]pyridine scaffold into a novel series of IKKα inhibitors. We demonstrate high potency and selectivity against IKKα over IKKβ in vitro and explain the structure-activity relationships using structure-based molecular modelling. We show selective target engagement with IKKα in the non-canonical NF-κB pathway for both U2OS osteosarcoma and PC-3M prostate cancer cells by employing isoform-related pharmacodynamic markers from both pathways. Two compounds ( SU1261 [IKKα K i = 10 nM; IKKβ K i = 680 nM] and SU1349 [IKKα K i = 16 nM; IKKβ K i = 3352 nM]) represent the first selective and potent pharmacological tools that can be used to interrogate the different signalling functions of IKKα and IKKβ in cells. Our understanding of the regulatory role of IKKα in various inflammatory-based conditions will be advanced using these pharmacological agents.

One of the most challenging issues scientists face is finding a suitable non-invasive treatment for cancer, as it is widespread around the world. The efficacy of phytochemicals that target oncogenic pathways appears to be quite promising and has gained attention over the past few years. We investigated the effect of docking phytochemicals isolated from the rhizomes of the Cimicifuga foetida plant on different domains of the IκB kinase alpha (IKK1/alpha) protein. The Cimicifugoside H-2 phytochemical registered a high docking score on the activation loop of IKK1/alpha amongst the other phytochemicals compared to the positive control. The interaction of the protein with Cimicifugoside H-2 was mostly stabilized by hydrogen bonds and hydrophobic interactions. A dynamic simulation was then performed with the Cimicifugoside H-2 phytochemical on the activation loop of IKK1/alpha, revealing that Cimicifugoside H-2 is a possible inhibitor of this protein. The pharmacokinetic properties of the drug were also examined to assess the safety of administering the drug. Therefore, in this in silico study, we discovered that the Cimicifugoside H-2 phytochemical inhibits the actively mutated conformation of IKK1/alpha, potentially suppressing the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) pathway.

Background: Oncogenic mutations in the RAS gene are associated with uncontrolled cell growth, a hallmark feature contributing to tumorigenesis. While diverse therapeutic strategies have been diligently applied to treat RAS-mutant cancers, successful targeting of the RAS gene remains a persistent challenge in the field of cancer therapy. In our study, we discover a promising avenue for addressing this challenge., Methods: In this study, we tested the viability of several cell lines carrying oncogenic NRAS, KRAS, and HRAS mutations upon treatment with IkappaBalpha (IκBα) inhibitor BAY 11-7082. We performed both cell culture-based viability assay and in vivo subcutaneous xenograft-based assay to confirm the growth inhibitory effect of BAY 11-7082. We also performed large RNA sequencing analysis to identify differentially regulated genes and pathways in the context of oncogenic NRAS, KRAS, and HRAS mutations upon treatment with BAY 11-7082., Results: We demonstrate that oncogenic NRAS, KRAS, and HRAS activate the expression of IκBα kinase. BAY 11-7082, an inhibitor of IκBα kinase, attenuates the growth of NRAS, KRAS, and HRAS mutant cancer cells in cell culture and in mouse model. Mechanistically, BAY 11-7082 inhibitor treatment leads to suppression of the PI3K-AKT signaling pathway and activation of apoptosis in all RAS mutant cell lines. Additionally, we find that BAY 11-7082 treatment results in the downregulation of different biological pathways depending upon the type of RAS protein that may also contribute to tumor growth inhibition., Conclusion: Our study identifies BAY 11-7082 to be an efficacious inhibitor for treating RAS oncogene (HRAS, KRAS, and NRAS) mutant cancer cells. This finding provides new therapeutic opportunity for effective treatment of RAS-mutant cancers., (© 2024. The Author(s).)

Background: Chronic kidney disease (CKD) is a significant worldwide health concern that leads to high mortality rates. The bioactive substance costunolide (CTD) has demonstrated several pharmacological effects and holds promise as a CKD treatment. This study aims to investigate the impact of CTD on CKD and delve into its mechanisms of action., Methods: Unilateral ureteral obstruction (UUO) methods and renal fibrosis mice models were created. Various concentrations of CTD were injected into UUO mice models to investigate the therapeutic effects of CTD on renal fibrosis of mice. Then, renal morphology, pathological changes, and the expression of genes related to fibrosis, inflammation and ferroptosis were analysed. RNA sequencing was utilized to identify the main biological processes and pathways involved in renal injury. Finally, both overexpression and inhibition of IKKβ were studied to examine their respective effects on fibrosis and inflammation in both in vitro and in vivo models., Results: CTD treatment was found to significantly alleviate fibrosis, inflammation and ferroptosis in UUO-induced renal fibrosis mice models. The results of RNA sequencing suggested that the IKKβ acted as key regulatory factor in renal injury and the expression of IKKβ was increased in vitro and in vivo renal fibrosis model. Functionally, down-regulated IKKβ expression inhibits ferroptosis, inflammatory cytokine production and collagen deposition. Conversely, IKKβ overexpression exacerbates progressive renal fibrosis. Mechanistically, CTD alleviated renal fibrosis and inflammation by inhibiting the expression of IKKβ and attenuating IKKβ/NF-κB pathway., Conclusion: This study demonstrates that CTD could mitigate renal fibrosis, ferroptosis and inflammation in CKD by modulating the IKKβ/NF-κB pathway, which indicates targeting IKKβ has an enormous potential for treating CKD., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Zhao et al.)

GSK-3β, IKK-β, and ROCK-1 kinases are implicated in the pathomechanism of Alzheimer's disease due to their involvement in the misfolding and accumulation of amyloid β (Aβ) and tau proteins, as well as inflammatory processes. Among these kinases, GSK-3β plays the most crucial role. In this study, we present compound 62 , a novel, remarkably potent, competitive GSK-3β inhibitor (IC 50 = 8 nM, K i = 2 nM) that also exhibits additional ROCK-1 inhibitory activity (IC 50 = 2.3 µM) and demonstrates anti-inflammatory and neuroprotective properties. Compound 62 effectively suppresses the production of nitric oxide (NO) and pro-inflammatory cytokines in the lipopolysaccharide-induced model of inflammation in the microglial BV-2 cell line. Furthermore, it shows neuroprotective effects in an okadaic-acid-induced tau hyperphosphorylation cell model of neurodegeneration. The compound also demonstrates the potential for further development, characterized by its chemical and metabolic stability in mouse microsomes and fair solubility.

Aberrant canonical NF-κB signaling has been implicated in diseases, such as autoimmune disorders and cancer. Direct disruption of the interaction of NEMO and IKKα/β has been developed as a novel way to inhibit the overactivation of NF-κB. Peptides are a potential solution for disrupting protein-protein interactions (PPIs); however, they typically suffer from poor stability in vivo and limited tissue penetration permeability, hampering their widespread use as new chemical biology tools and potential therapeutics. In this work, decafluorobiphenyl-cysteine S N Ar chemistry, molecular modeling, and biological validation allowed the development of peptide PPI inhibitors. The resulting cyclic peptide specifically inhibited canonical NF-κB signaling in vitro and in vivo , and presented positive metabolic stability, anti-inflammatory effects, and low cytotoxicity. Importantly, our results also revealed that cyclic peptides had huge potential in acute lung injury (ALI) treatment, and confirmed the role of the decafluorobiphenyl-based cyclization strategy in enhancing the biological activity of peptide NEMO-IKKα/β inhibitors. Moreover, it provided a promising method for the development of peptide-PPI inhibitors.

B-cell acute lymphoblastic leukemia (B-ALL) is a malignant blood disorder, particularly detrimental to children and adolescents, with recurrent or unresponsive cases contributing significantly to cancer-associated fatalities. IKBKE, associated with innate immunity, tumor promotion, and drug resistance, remains poorly understood in the context of B-ALL. Thus, this research aimed to explore the impact of the IKBKE inhibitor MCCK1 on B-ALL cells. The study encompassed diverse experiments, including clinical samples, in vitro and in vivo investigations. Quantitative real-time fluorescence PCR and protein blotting revealed heightened IKBKE mRNA and protein expression in B-ALL patients. Subsequent in vitro experiments with B-ALL cell lines demonstrated that MCCK1 treatment resulted in reduced cell viability and survival rates, with flow cytometry indicating cell cycle arrest. In vivo experiments using B-ALL mouse tumor models substantiated MCCK1's efficacy in impeding tumor proliferation. These findings collectively suggest that IKBKE, found to be elevated in B-ALL patients, may serve as a promising drug target, with MCCK1 demonstrating potential for inducing apoptosis in B-ALL cells both in vitro and in vivo.

Background: IκB kinase β (IKKβ) plays a pivotal role in the NF-κB signaling pathway and is considered a promising therapeutic target for various diseases. Materials & methods: The authors developed and validated a 3D pharmacophore model of IKKβ inhibitors via the HypoGen algorithm in Discovery Studio 2019, then performed virtual screening, molecular docking and kinase assays to identify hit compounds from the ChemDiv database. The compound with the highest inhibitory activity was further evaluated in adjuvant-induced arthritis rat models. Results: Among the four hit compounds, Hit 4 had the highest IKKβ inhibitory activity (IC 50  = 30.4 ± 3.8), and it could significantly ameliorate joint inflammation and damage in vivo . Conclusion: The identified compound, Hit 4, can be optimized as a therapeutic agent for inflammatory diseases.

Background: The inhibition of IKKβ by the inhibitor 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-(4-piperidinyl)-3-pyridine carbonitrile (ACHP) is a promising strategy for the treatment of Achilles tendinopathy. However, the poor water solubility of ACHP severely hinders its in vivo application. Moreover, the effective local delivery of ACHP to the tendon and its therapeutic effects have not been reported., Purpose: To investigate the therapeutic effects of IKKβ inhibition via injection of ACHP incorporated into a DNA supramolecular hydrogel in a collagenase-induced tendinopathy rat model., Study Design: Controlled laboratory study., Methods: Dendritic DNA, a Y-shaped monomer, and a crosslinking monomer were mixed with ACHP and self-assembled into an ACHP-DNA supramolecular hydrogel (ACHP-Gel). The effects of ACHP-Gel in tendon stem/progenitor cells were investigated via RNA sequencing and validated using quantitative reverse transcription polymerase chain reaction (qRT-PCR). A total of 120 collagenase-induced rats were randomly assigned to 5 groups: blank, phosphate-buffered saline (PBS), DNA-Gel, ACHP, and ACHP-Gel. Healing outcomes were evaluated using biomechanic and histologic evaluations at 4 and 8 weeks., Results: ACHP-Gel enhanced the solubility of ACHP and sustained its release for ≥21 days in vivo, which significantly increased the retention time of ACHP and markedly reduced the frequency of administration. RNA sequencing and qRT-PCR showed that ACHP effectively downregulated genes related to inflammation and extracellular matrix remodeling and upregulated genes related to tenogenic differentiation. The cross-sectional area ( P = .024), load to failure ( P = .002), stiffness ( P = .039), and elastic modulus ( P = .048) significantly differed between the ACHP-Gel and PBS groups at 8 weeks. The ACHP-Gel group had better histologic scores than the ACHP group at 4 ( P = .042) and 8 weeks ( P = .009). Type I collagen expression (COL-I; P = .034) and the COL-I/collagen type III ratio ( P = .015) increased while interleukin 6 expression decreased ( P < .001) in the ACHP-Gel group compared with the ACHP group at 8 weeks., Conclusion: DNA supramolecular hydrogel significantly enhanced the aqueous solubility of ACHP and increased its release-retention time. Injection frequency was markedly reduced. ACHP-Gel suppressed inflammation in Achilles tendinopathy and promoted tendon healing in a rat model., Clinical Relevance: ACHP-Gel injection is a promising strategy for the treatment of Achilles tendinopathy in clinical practice., Competing Interests: One or more of the authors has declared the following potential conflict of interest or source of funding: This study was supported by the China Postdoctoral Science Foundation (2021TQ0225, 2021M702370); the Post-Doctor Research Project, West China Hospital of Sichuan University (2021HXBH065, 2019HXBH027, 2023HXBH106); and Program of Natural Science Foundation of Sichuan (2022NSFSC1559, 2022NSFSC1569, 2023NSFSC1835). AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto.

Hepatic ischemia/reperfusion (I/R) injury plays a pivotal pathogenic role in trauma, hepatectomy, and liver transplantation. However, the whole mechanism remains undescribed. The objective of this study is to investigate the internal mechanism by which microRNA-22 (miR-22) targets family with sequence similarity 49 member B (FAM49B), thus aggravating hepatic I/R injury. Here, we found that miR-22 was upregulated while FAM49B was reduced in hepatic I/R injury. Inhibition of miR-22 in vitro was able to intensify expression of FAM49B, thus reducing phosphorylation of inhibitors of nuclear factor kappa-B kinase (IKK) and downstream pro-inflammatory proteins. A dual luciferase reporter assay indicated that miR-22 directly targeted FAM49B. Remission of hepatic pathologic alterations, apoptosis, and release of cytokines derived from constraints of miR-22 were abolished in vivo by repressing FAM49B. Further interference of Ras-related C3 botulinum toxin substrate 1 (Rac1) reversed the function of FAM49B inhibition, thus achieving anti-inflammatory consequences., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Inhibitor of kappa B (IκB) kinase β (IKKβ) has long been viewed as the dominant IKK in the canonical nuclear factor-κB (NF-κB) signalling pathway, with IKKα being more important in non-canonical NF-κB activation. Here we have investigated the role of IKKα and IKKβ in canonical NF-κB activation in colorectal cells using CRISPR-Cas9 knock-out cell lines, siRNA and selective IKKβ inhibitors. IKKα and IKKβ were redundant for IκBα phosphorylation and turnover since loss of IKKα or IKKβ alone had little (SW620 cells) or no (HCT116 cells) effect. However, in HCT116 cells IKKα was the dominant IKK required for basal phosphorylation of p65 at S536, stimulated phosphorylation of p65 at S468, nuclear translocation of p65 and the NF-κB-dependent transcriptional response to both TNFα and IL-1α. In these cells, IKKβ was far less efficient at compensating for the loss of IKKα than IKKα was able to compensate for the loss of IKKβ. This was confirmed when siRNA was used to knock-down the non-targeted kinase in single KO cells. Critically, the selective IKKβ inhibitor BIX02514 confirmed these observations in WT cells and similar results were seen in SW620 cells. Notably, whilst IKKα loss strongly inhibited TNFα-dependent p65 nuclear translocation, IKKα and IKKβ contributed equally to c-Rel nuclear translocation indicating that different NF-κB subunits exhibit different dependencies on these IKKs. These results demonstrate a major role for IKKα in canonical NF-κB signalling in colorectal cells and may be relevant to efforts to design IKK inhibitors, which have focused largely on IKKβ to date., (© 2022 The Author(s).)

Platelets play a critical role in arterial thrombosis. Rutaecarpine (RUT) was purified from Tetradium ruticarpum , a well-known Chinese medicine. This study examined the relative activity of RUT with NF-κB inhibitors in human platelets. BAY11-7082 (an inhibitor of IκB kinase [IKK]), Ro106-9920 (an inhibitor of proteasomes), and RUT concentration-dependently (1-6 μM) inhibited platelet aggregation and P-selectin expression. RUT was found to have a similar effect to that of BAY11-7082; however, it exhibits more effectiveness than Ro106-9920. RUT suppresses the NF-κB pathway as it inhibits IKK, IκBα, and p65 phosphorylation and reverses IκBα degradation in activated platelets. This study also investigated the role of p38 and NF-κB in cell signaling events and found that SB203580 (an inhibitor of p38) markedly reduced p38, IKK, and p65 phosphorylation and reversed IκBα degradation as well as p65 activation in a confocal microscope, whereas BAY11-7082 had no effects in p38 phosphorylation. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay shows that RUT and BAY11-7082 did not exhibit free radical scavenging activity. In the in vivo study, compared with BAY11-7082, RUT more effectively reduced mortality in adenosine diphosphate (ADP)-induced acute pulmonary thromboembolism without affecting the bleeding time. In conclusion, a distinctive pathway of p38-mediated NF-κB activation may involve RUT-mediated antiplatelet activation, and RUT could act as a strong prophylactic or therapeutic drug for cardiovascular diseases.

NF-κB contributes to the biosynthesis of various chemokines, cytokines, and enzymes. It plays many crucial roles in the upstream neuroinflammatory pathways. Briefly, the inhibitory IkB subunit is cleaved and phosphorylated by the IKK-α/β enzyme. It leads to the activation and translocation of the NF-κB (p50/p65) complex into the nucleus. Subsequently, the activated NF-κB interacts with the genomic DNA and contributes to expressing various proinflammatory cytokines. In the present study, we developed a novel NF-κB inhibitor encoded (D5) and investigated the efficacy of our druggable compound through several in silico, in vitro, and in situ analysis. The results demonstrated that D5 not only inhibited the mRNA expression of the IKK-α/β enzyme (around 86-96% suppression rate for both cell lines at 12 and 24 h time frames) but also by interacting to the active site of the mentioned kinase (dock score -6.14 and binding energy -23.60 kcal/mol) reduced the level of phosphorylated IkB-α in the cytosol around 96-99% and p65 subunit in the nucleus around 73-90% (among all groups in 12 and 24 h time points). Additionally, the results indicated that D5 suppressed the NF-κB target mRNA levels of TNF-α and IL-6 in a total average of around 92%. Overall, The results demonstrated that D5 in a considerably lower concentration than Dis (0.71 µM vs. 52.73 µM) showed significantly higher inhibitory efficacy on NF-κB translocation approx. 200-300%. The results suggested D5 as a potent NF-κB silencer, but further investigations are required to validate our outcomes., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Chromatographic fractionation of Sigesbeckia glabrescens led to the identification of 10 new sesquiterpene lactones, named siegesbeckialides I-O ( 1 - 7 ) and glabrescones A-C ( 8 - 10 ), along with 14 known analogues. An anti-inflammatory activity assay showed that siegesbeckialide I ( 1 ) most potently inhibited LPS-induced NO production in RAW264.7 murine macrophages. Furthermore, siegesbeckialide I suppressed the protein expression of iNOS and COX2, as well as the release of PGE 2 , IL-1β, IL-6, and TNF-α in LPS-stimulated RAW264.7 cells. Mechanistically, siegesbeckialide I directly binds to inhibitors of IKKα/β and suppresses their phosphorylation. This leads to the inhibition of IKKα/β-mediated phosphorylation and degradation of inhibitor α of NF-κB (IκBα), as well as the activation of NF-κB signaling.

Inflammatory pathway and disruption in glutamate homeostasis join at the level of the glia, resulting in various neurological disorders. In vitro studies have provided evidence that membrane proteins connexions (Cxs) are involved in glutamate release, meanwhile, excitatory amino-acid transporters (EAATs) are crucial for glutamate reuptake (clearance). Moreover, pannexin-1 (Panx-1) activation is more detrimental to neurons. Their expression patterns during inflammation and the impacts of IκB kinase (IKK) inhibition, morphine, and galantamine on the inflammatory-associated glutamate imbalance remain elusive. To investigate this, rats were injected with saline or lipopolysaccharide. Thereafter, vehicles, morphine, galantamine, and BAY-117082 were administered in different groups of animals. Subsequently, electroencephalography, enzyme-linked immunosorbent assay, western blot, and histopathological examinations were carried out and various indicators of inflammation and glutamate level were determined. Parallel analysis of Cxs, Panx-1, and EAAts in the brain was performed. Our findings strengthen the concept that unregulated expressions of Cxs, Panx-1, and EAATs contribute to glutamate accumulation and neuronal cell loss. Nuclear factor-kB (NF-κB) pathway can significantly contribute to glutamate homeostasis via modulating Cxs, Panx-1, and EAATs expressions. BAY-117082, via inhibition of IkK, promoted the anti-inflammatory effects of morphine as well as galantamine. We concluded that NF-κB is an important component of reshaping the expressions of Cxs, panx-1, and EAATs and the development of glutamate-induced neuronal degeneration., (© 2021 Wiley Periodicals LLC.)

Chloranthus oldhamii Solms (CO) is a folk medicine for treating infection and arthritis pain but its pharmacological activity and bioactive compounds remain mostly uncharacterized. In this study, the anti-inflammatory compounds of C. oldhamii were identified using an LPS-stimulated, NF-κB-responsive RAW 264.7 macrophage reporter line. Three diterpenoid compounds, 3α-hydroxy-ent-abieta-8,11,13-triene (CO-9), 3α, 7β-dihydroxy-ent-abieta-8,11,13-triene (CO-10), and decandrin B (CO-15) were found to inhibit NF-κB activity at nontoxic concentrations. Moreover, CO-9 and CO-10 suppressed the expression of IL-6 and TNF-α in LPS-stimulated RAW 264.7 cells. The inhibitory effect of CO-9 on TNF-α and IL-6 expression was further demonstrated using LPS-treated bone marrow-derived macrophages. Furthermore, CO-9, CO-10, and CO-15 suppressed LPS-triggered COX-2 expression and downstream PGE2 production in RAW 264.7 cells. CO-9 and CO-10 also reduced LPS-triggered iNOS expression and nitrogen oxide production in RAW 264.7 cells. The anti-inflammatory mechanism of the most effective compound, CO-9, was further investigated. CO-9 attenuated LPS-induced NF-κB activation by reducing the phosphorylation of IKKα/β (Ser176/180), IκBα (Ser32), and p65 (Ser534). Conversely, CO-9 did not affect the LPS-induced activation of MAPK signaling pathways. In summary, this study revealed new anti-inflammatory diterpenoid compounds from C. oldhamii and demonstrated that the IKK-mediated NK-κB pathway is the major target of these compounds.

Background: Obesity-related nephropathy (ORN) has become one of the leading causes of end-stage renal disease and has tripled over the past decade. Previous studies have demonstrated that decreased reactive oxygen species production may contribute to improving ORN by ameliorating oxidative stress injury. Here, IκB kinase (IKK) was hypothesized to inactivate the deubiquitination activity of cylindromatosis (CYLD) by activating the phosphorylation of CYLD, thus promoting the ubiquitination of NF-E2-related factor 2 (Nrf2) and further aggravating oxidative stress injury of the kidney in ORN. This study was aimed to confirm this hypothesis., Methods: Haematoxylin and eosin (HE), periodic acid-Schiff (PAS) and Oil Red O staining were performed to assess histopathology. Dihydroethidium (DHE) staining and MDA, SOD, CAT, and GSH-PX assessments were performed to measure reactive oxygen species (ROS) production. Immunohistochemical (IHC) staining, qRT-PCR and/or western blotting were performed to assess the expression of related genes. JC-1 assays were used to measure the mitochondrial membrane potential (ΔΨm) of treated HK-2 cells. Co-immunoprecipitation experiments (Co-IP) were used to analyse the interaction between CYLD and Nrf2 in ORN., Results: ORN in vivo and in vitro models were successfully constructed, and oxidative stress injury was detected in the model tissues and cells. Compared with the control groups, the phosphorylation level of CYLD increased while Nrf2 levels decreased in ORN model cells. An IKK inhibitor reduced lipid deposition, ROS production, CYLD phosphorylation levels and ΔΨm in vitro, which were reversed by knockdown of CYLD. Nrf2 directly bound to CYLD and was ubiquitinated in ORN cells. The proteasome inhibitor MG132 activated the Nrf2/ARE signalling pathway, thereby reversing the promoting effect of CYLD knockdown on oxidative stress., Conclusion: IKK inactivates the deubiquitination activity of CYLD by activating the phosphorylation of CYLD, thus promoting the ubiquitination of Nrf2 and further aggravating oxidative stress injury of the kidney in ORN. This observation provided a feasible basis for the treatment of kidney damage caused by ORN., (© 2021. The Author(s).)

Genetic models validated Inhibitor of nuclear factor (NF) kappa B kinase beta (IKKβ) as a therapeutic target for KRAS mutation associated pancreatic cancer. Phosphorylation of the activation loop serine residues (S 177 , S 181 ) in IKKβ is a key event that drives tumor necrosis factor (TNF) α induced NF-κB mediated gene expression. Here we conducted structure activity relationship (SAR) study to improve potency and oral bioavailability of a quinoxaline analog 13-197 that was previously reported as a NFκB inhibitor for pancreatic cancer therapy. The SAR led to the identification of a novel quinoxaline urea analog 84 that reduced the levels of p-IKKβ in dose- and time-dependent studies. When compared to 13-197, analog 84 was ∼2.5-fold more potent in TNFα-induced NFκB inhibition and ∼4-fold more potent in inhibiting pancreatic cancer cell growth. Analog 84 exhibited ∼4.3-fold greater exposure (AUC 0- ∞) resulting in ∼5.7-fold increase in oral bioavailability (%F) when compared to 13-197. Importantly, oral administration of 84 by itself and in combination of gemcitabine reduced p-IKKβ levels and inhibited pancreatic tumor growth in a xenograft model., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Metastasis, the main cause of breast cancer-associated fatalities, relies on many regular pathways involved in normal cell physiology and metabolism, thus, making it challenging to identify disease-specific therapeutic target(s). Chemically synthesized anti-metastatic agents are preferred for their fast and robust actions. However, these agents have adverse side effects, thus, increasingly favouring the identification of phytocompounds as suitable alternatives. Resveratrol and pterostilbene have long been established as potent anti-cancer agents. Earlier studies from our laboratory documented the anti-cancer activities associated with pterostilbene-isothiocyanate (PTER-ITC), a derivative of pterostilbene. The current study focuses on evaluating the anti-metastatic property of PTER-ITC and the underlying mechanism, by employing in silico, in vitro, and in vivo approaches. The significant anti-metastatic activity of PTER-ITC was observed in vitro against breast cancer metastatic cell line (MDA-MB-231) and in vivo in the 4T1 cell-induced metastatic mice model. Epithelial-mesenchymal transition (EMT), a hallmark of metastasis regulated by the transcription factors, Snail1 and Twist, was found to be reverted in vitro by PTER-ITC treatment. PTER-ITC blocked the activation of NF-κB/p65 and its concomitant nuclear translocation, resulting in the transcriptional repression of its target genes, Snail1 and Twist. PTER-ITC prevented the formation of IKK complex, central to NF-κB activation, by binding to the NEMO-binding domain (NBD) of IKK-β and inhibiting its interaction with NEMO (NF-κB essential modulator). According to our observations, PTER-ITC attenuated NF-κB activation selectively in cancerous cells. In conclusion, this study demonstrated that PTER-ITC is a potent anti-metastatic agent capable of targeting physiologically important pathways in a cancer-specific manner., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Inflammation has a fundamental impact on the pathophysiology of osteoarthritis (OA), a common form of degenerative arthritis. It has previously been established that curcumin, a component of turmeric ( Curcuma longa ), has anti-inflammatory properties. This research evaluates the potentials of curcumin on the pathophysiology of OA in vitro. To explore the anti-inflammatory efficacy of curcumin in an inflamed joint, an osteoarthritic environment (OA-EN) model consisting of fibroblasts, T-lymphocytes, 3D-chondrocytes is constructed and co-incubated with TNF-α, antisense oligonucleotides targeting NF-kB (ASO-NF-kB), or an IkB-kinase (IKK) inhibitor (BMS-345541). Our results show that OA-EN, similar to TNF-α, suppresses chondrocyte viability, which is accompanied by a significant decrease in cartilage-specific proteins (collagen II, CSPG, Sox9) and an increase in NF-kB-driven gene proteins participating in inflammation, apoptosis, and breakdown (NF-kB, MMP-9, Cox-2, Caspase-3). Conversely, similar to knockdown of NF-kB at the mRNA level or at the IKK level, curcumin suppresses NF-kB activation, NF-kB-promotes gene proteins derived from the OA-EN, and stimulates collagen II, CSPG, and Sox9 expression. Furthermore, co-immunoprecipitation assay shows that curcumin reduces OA-EN-mediated inflammation and chondrocyte apoptosis, with concomitant chondroprotective effects, due to modulation of Sox-9/NF-kB signaling axis. Finally, curcumin selectively hinders the interaction of p-NF-kB-p65 directly with DNA-this association is disrupted through DTT. These results suggest that curcumin suppresses inflammation in OA-EN via modulating NF-kB-Sox9 coupling and is essential for maintaining homeostasis in OA by balancing chondrocyte survival and inflammatory responses. This may contribute to the alternative treatment of OA with respect to the efficacy of curcumin.

Hepatic ischemia/reperfusion injury (IRI) is an inevitable pathological process in liver resection, shock and transplantation. However, the internal mechanism of hepatic IRI, including inflammatory transduction of multiple signaling pathways, is not fully understood. In the present study, we identified pleckstrin homology-like domain family member 1 (PHLDA1), suppressed by microRNA (miR)-194, as a critical intersection of dual inflammatory signals in hepatic IRI. PHLDA1 was upregulated in hepatic IRI with a concomitant downregulation of miR-194. Overexpression of miR-194 diminished PHLDA1 and inhibitors of the nuclear factor kappa-B kinase (IKK) pathway, thus leading to remission of hepatic pathological injury, apoptosis and release of cytokines. Further enrichment of PHLDA1 reversed the function of miR-194 both in vivo and in vitro. For an in-depth query, we verified PHLDA1 as a direct target of miR-194. Notably, inflammatory signal transduction of PHLDA1 was induced by activating TNF receptor-associated factor 6 (TRAF6), sequentially initiating IKK and mitogen-activated protein kinase (MAPK), both of which aggravate stress and inflammation in hepatic IRI. In conclusion, the miR-194/PHLDA1 axis was a key upstream regulator of IKK and MAPK in hepatic IRI. Targeting PHLDA1 might be a potential strategy for hepatic IRI therapy., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Activation of nuclear factor-kappa B (NF-κB) in microglia plays a decisive role in the progress of neuropathic pain, and the inhibitor of kappa B (IκB) is a protein that blocks the activation of NF-κB and is degraded by the inhibitor of NF-κB kinase subunit beta (IKBKB). The role of IKBKB is to break down IκB, which blocks the activity of NF-kB. Therefore, it prevents the activity of NK-kB. This study investigated whether neuropathic pain can be reduced in spinal nerve ligation (SNL) rats by reducing the activity of microglia by delivering IKBKB small interfering RNA (siRNA)-encapsulated poly (lactic- co -glycolic acid) (PLGA) nanoparticles. PLGA nanoparticles, as a carrier for the delivery of IKBKB genes silencer, were used because they have shown potential to enhance microglial targeting. SNL rats were injected with IKBKB siRNA-encapsulated PLGA nanoparticles intrathecally for behavioral tests on pain response. IKBKB siRNA was delivered for suppressing the expression of IKBKB . In rats injected with IKBKB siRNA-encapsulated PLGA nanoparticles, allodynia caused by mechanical stimulation was reduced, and the secretion of pro-inflammatory mediators due to NF-κB was reduced. Delivering IKBKB siRNA through PLGA nanoparticles can effectively control the inflammatory response and is worth studying as a treatment for neuropathic pain.

Inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ) is a key regulator of the cannonical NF-κB pathway. IKKβ has been validated as a drug target for pathological conditions, which include chronic inflammatory diseases and cancer. Pharmacological studies revealed that chronic administration of ATP-competitive IKKβ inhibitors resulted in unexpected toxicity. We previously reported the discovery of 13-197 as a non-toxic IKKβ inhibitor that reduced tumor growth. Here, we show that 13-197 inhibits IKKβ in a ATP non-competitive manner and an allosteric pocket at the interface of the kinase and ubiquitin like domains was identified as the potential binding site.

Purpose: Although enzalutamide (ENZ) has been widely used to treat de novo or castration-resistant metastatic prostate cancer, resistance develops and disease progression is ultimately inevitable. There are currently no approved targeted drugs to specifically delay or overcome ENZ resistance., Experimental Design: We selected several ENZ-resistant cell lines that replicated clinical characteristics of the majority of patients with ENZ-resistant disease. A high-throughput pharmacologic screen was utilized to identify compounds with greater cytotoxic effect for ENZ-resistant cell lines, compared with parental ENZ-sensitive cells. We validated the potential hits in vitro and in vivo , and used knockdown and overexpression assays to study the dependencies in ENZ-resistant prostate cancer., Results: ABT199 (BCL-2 inhibitor) and IMD0354 (IKKB inhibitor) were identified as potent and selective inhibitors of cell viability in ENZ-resistant cell lines in vitro and in vivo which were further validated using loss-of-function assays of BCL-2 and IKKB. Notably, we observed that overexpression of BCL-2 and IKKB in ENZ-sensitive cell lines was sufficient for the emergence of ENZ resistance. In addition, we confirmed that BCL-2 or IKKB inhibitors suppressed the development of ENZ resistance in xenografts. However, validation of both BCL-2 and IKKB in matched castration-sensitive/resistant clinical samples showed that, concurrent with the development of ENZ/abiraterone resistance in patients, only the protein levels of IKKB were increased., Conclusions: Our findings identify BCL-2 and IKKB dependencies in clinically relevant ENZ-resistant prostate cancer cells in vitro and in vivo , but indicate that IKKB upregulation appears to have greater relevance to the progression of human castrate-resistant prostate cancer., (©2021 American Association for Cancer Research.)

Low shear stress (LSS) plays a critical role in the development of atherosclerotic plaques and vascular inflammation. Previous studies have reported Akt phosphorylation induced by LSS. However, the mechanism and role of Akt activation remains unclear in LSS-induced endothelial dysfunction. In this study, our results demonstrated the increased phosphorylation of IKKε, TBK1 and Akt in HUVECs exposed to LSS. Furthermore, IKKε silencing using small interfering RNAs significantly reduced LSS-induced Akt phosphorylation. In contrast, silencing of TBK1 or inhibition of PI3K and mTORC2 had no effect on LSS-induced Akt phosphorylation. Notably, Akt inhibition markedly diminished LSS-induced expression of ICAM-1, VCAM-1 and MCP-1, as well as LSS-induced IRF3 phosphorylation and nuclear translocation, without affecting the activation of NF-κB and STAT1. Moreover, endothelial cell specific Akt overexpression mediated by adeno-associated virus markedly increased intimal ICAM-1 and IRF3 expression at LSS area of partially ligated carotid artery in mice. In brief, our findings suggest that LSS-induced Akt phosphorylation is positively regulated by IKKε and promotes IRF3 activation, leading to endothelial inflammation., (Copyright © 2020. Published by Elsevier Inc.)

Icaritin, a small molecule currently being investigated in phase III clinical trials in China (NCT03236636 and NCT03236649) for treatment of advanced hepatocellular carcinoma (HCC), is a prenylflavonoid derivative obtained from the Epimedium genus. Previously, it was found that Icaritin decreased the expression of PD-L1, but its direct molecular targets and the underlying mechanisms have not been identified. In this study, we report the identification of IKK-α as the protein target of Icaritin by biotin-based affinity binding assay. The further mutagenesis assay has provided evidence that C46 and C178 in IKK-α were essential amino acids for Icaritin binding to IKK-α, revealing the binding sites of Icaritin to IKK-α for the first time. Functionally, Icaritin inhibited the NF-κB signalling pathway by blocking IKK complex formation, which led to decreased nuclear translocation of NF-κB p65, and subsequent downregulation of PD-L1 expression in a dose-dependent manner. More importantly, PD-L1-positive patients exhibited longer overall survival upon Icaritin therapy. Finally, Icaritin in combination with checkpoints antibodies, such as α-PD-1, has demonstrated much better efficacy than any single therapy in animal models. This is the first report that anticancer effects of Icaritin are mediated, at least in part, by impairing functions of IKK-α., (© 2020 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

The NLRP3 inflammasome is a critical component of the innate immune response to sterile inflammation. Its regulation involves a priming step, required for up-regulation of inflammasome protagonists and an activation step leading to NLRP3 inflammasome complex assembly, which triggers caspase-1 activity. The IκKβ kinase regulates canonical NF-κB, a key pathway involved in transcriptional priming. We found that IκKβ also regulates the activation and function of the NLRP3 inflammasome beyond the priming step. Two unrelated IκKβ inhibitors, AFN700 and TPCA-1, when applied after priming, fully blocked IL-1β secretion triggered by nigericin in THP-1 cells. Both inhibitors prevented neither inflammasome assembly, as monitored by measuring the formation of ASC specks, nor the generation of caspase-1 p20, a hallmark of caspase-1 activity, but they impaired the initial cleavage and activation of procaspase-1. These data thus indicate that IκKβ activity is required for efficient activation of NLRP3, suggesting that IκKβ may fulfill a dual role in coupling priming and activation of the NLRP3 inflammasome., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Coronavirus disease 2019 (COVID-19) has been characterized by lymphopenia as well as a proinflammatory cytokine storm, which are responsible for the poor prognosis and multiorgan defects. The transcription factor nuclear factor-κB (NF-κB) modulates the functions of the immune cells and alters the gene expression profile of different cytokines in response to various pathogenic stimuli, while many proinflammatory factors have been known to induce NF-κB signalling cascade. Besides, NF-κB has been known to potentiate the production of reactive oxygen species (ROS) leading to apoptosis in various tissues in many diseases and viral infections. Though the reports on the involvement of the NF-κB signalling pathway in COVID-19 are limited, the therapeutic benefits of NF-κB inhibitors including dexamethasone, a synthetic form of glucocorticoid, have increasingly been realized. Considering the fact, the abnormal activation of the NF-κB resulting from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection might be associated with the pathogenic profile of immune cells, cytokine storm and multiorgan defects. Thus, the pharmacological inactivation of the NF-κB signalling pathway can strongly represent a potential therapeutic target to treat the symptomatology of COVID-19. This article signifies pharmacological blockade of the phosphorylation of inhibitor of nuclear factor kappa B kinase subunit beta (IKKβ), a key downstream effector of NF-κB signalling, for a therapeutic consideration to attenuate COVID-19.

Background: Osteoarthritis (OA), a disease with whole-joint damage and dysfunction, is the leading cause of disability worldwide. The progressive loss of hyaline cartilage extracellular matrix (ECM) is considered as its hallmark, but its exact pathogenesis needs to be further clarified. MicroRNA(miRNA) contributes to OA pathology and may help to identify novel biomarkers and therapies against OA. Here we identified miR-214-3p as an important regulator of OA., Methods: qRT-PCR and in situ hybridization were used to detect the expression level of miR-214-3p. The function of miR-214-3p in OA, as well as the interaction between miR-214-3p and its downstream mRNA target (IKBKB), was evaluated by western blotting, immunofluorescence, qRT-PCR and luciferase assay. Mice models were introduced to examine the function and mechanism of miR-214-3p in OA in vivo., Findings: In our study, we found that miR-214-3p, while being down-regulated in inflamed chondrocytes and OA cartilage, regulated ECM metabolism and cell apoptosis in the cartilage. Mechanically, the protective effect of miR-214-3p downregulated the IKK-β expression and led to the dysfunction of NF-κB signaling pathway. Furthermore, intra-articular injection of miR-214-3p antagomir in mice joints triggered spontaneous cartilage loss while miRNA-214-3p agomir alleviated OA in the experimental mouse models., Interpretation: Decreased miR-214-3p activates the NF-κB signaling pathway and aggravates OA development through targeting IKKβ, suggesting miR-214-3p may be a novel therapeutic target for OA., Funding: This study was financially supported by grants from the National Natural Science Foundation of China (81,773,532, 81,974,342)., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Aims: Diabetes is a conventional risk factor for atherosclerotic cardiovascular disease and myocardial infarction (MI) is the most common cause of death among these patients. Mesenchymal stromal cells (MSCs) in patients with type 2 diabetes mellitus (T2DM) and atherosclerosis have impaired ability to suppress activated T-cells (i.e. reduced immunopotency). This is mediated by an inflammatory shift in MSC-secreted soluble factors (i.e. pro-inflammatory secretome) and can contribute to the reduced therapeutic effects of autologous T2DM and atherosclerosis-MSC post-MI. The signalling pathways driving the altered secretome of atherosclerosis- and T2DM-MSC are unknown. Specifically, the effect of IκB kinase β (IKKβ) modulation, a key regulator of inflammatory responses, on the immunopotency of MSCs from T2DM patients with advanced atherosclerosis has not been studied., Methods and Results: MSCs were isolated from adipose tissue obtained from patients with (i) atherosclerosis and T2DM (atherosclerosis+T2DM MSCs, n = 17) and (ii) atherosclerosis without T2DM (atherosclerosis MSCs, n = 17). MSCs from atherosclerosis+T2DM individuals displayed an inflammatory senescent phenotype and constitutively expressed active forms of effectors of the canonical IKKβ nuclear factor-κB transcription factors inflammatory pathway. Importantly, this constitutive pro-inflammatory IKKβ signature resulted in an altered secretome and impaired in vitro immunopotency and in vivo healing capacity in an acute MI model. Notably, treatment with a selective IKKβ inhibitor or IKKβ knockdown (KD) (clustered regularly interspaced short palindromic repeats/Cas9-mediated IKKβ KD) in atherosclerosis+T2DM MSCs reduced the production of pro-inflammatory secretome, increased survival, and rescued their immunopotency both in vitro and in vivo., Conclusions: Constitutively active IKKβ reduces the immunopotency of atherosclerosis+T2DM MSC by changing their secretome composition. Modulation of IKKβ in atherosclerosis+T2DM MSCs enhances their myocardial repair ability., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

The contribution of many neuronal kinases to the adaptation of nerve cells to ischemic damage and their effect on functional neural network activity has not yet been studied. The aim of this work is to study the role of the four kinases belonging to different metabolic cascades (SRC, Ikkb, eEF2K, and FLT4) in the adaptive potential of the neuron-glial network for modeling the key factors of ischemic damage. We carried out a comprehensive study on the effects of kinases blockade on the viability and network functional calcium activity of nerve cells under ischemic factor modeling in vitro. Ischemic factor modelling was performed on day 14 of culturing primary hippocampal cells obtained from mouse embryos (E18). The most significant neuroprotective effect was shown in the blockade of FLT4 kinase in the simulation of hypoxia. The studies performed revealed the role of FLT4 in the development of functional dysfunction in cerebrovascular accidents and created new opportunities for the study of this enzyme and its blockers in the formation of new therapeutic strategies.

Objective: This study aimed to examine the temporal activation of NF-κB and its relationship to the development of pain-related sensitivity and behavioral changes in a non-invasive murine knee loading model of PTOA., Method: Following knee injury NF-κB activity was assessed longitudinally via in vivo imaging in FVB. Cg-Tg (HIV-EGFP,luc)8Tsb/J mice. Measures of pain-related sensitivity and behavior were also assessed longitudinally for 16 weeks. Additionally, we antagonized NF-κB signaling via intra-articular delivery of an IκB kinase two antagonist to understand how local NF-κB inhibition might alter disease progression., Results: Following joint injury NF-κB signaling within the knee joint was transiently increased and peaked on day 3 with an estimated 1.35 p/s/cm 2 /sr (95% CI 0.913.1.792 p/s/cm 2 /sr) fold increase in signaling when compared to control joints. Furthermore, injury resulted in the long-term development of hindpaw allodynia. Hyperalgesia withdrawal thresholds were reduced at injured knee joints, with the largest reduction occurring 2 days following injury (estimate of between group difference 129.1 g with 95% CI 60.9,197.4 g), static weight bearing on injured limbs was also reduced. Local delivery of an NF-κB inhibitor following joint injury reduced chondrocyte death and influenced the development of pain-related sensitivity but did not reduce long-term cartilage degeneration., Conclusion: These findings underscore the development of behavioral changes in this non-invasive loading model of PTOA and their relationships to NF-κB activation and pathology. They also highlight the potential chondroprotective effects of NF-κB inhibition shortly following joint injury despite limitations in preventing the long-term development of joint degeneration in this model of PTOA., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Human papillomavirus (HPV) is the leading cause of cervical cancer and has been implicated in several other cancer types including vaginal, vulvar, penile, and oropharyngeal cancers. Despite the recent availability of a vaccine, there are still over 310,000 deaths each year worldwide. Current treatments for HPV-mediated cancers show limited efficacy, and would benefit from improved understanding of disease mechanisms. Recently, we developed a Drosophila 'HPV 18 E6' model that displayed loss of cellular morphology and polarity, junctional disorganization, and degradation of the major E6 target Magi; we further provided evidence that mechanisms underlying HPV E6-induced cellular abnormalities are conserved between humans and flies. Here, we report a functional genetic screen of the Drosophila kinome that identified IKK[Formula: see text]-a regulator of NF-κB-as an enhancer of E6-induced cellular defects. We demonstrate that inhibition of IKK[Formula: see text] reduces Magi degradation and that this effect correlates with hyperphosphorylation of E6. Further, the reduction in IKK[Formula: see text] suppressed the cellular transformation caused by the cooperative action of HPVE6 and the oncogenic Ras. Finally, we demonstrate that the interaction between IKK[Formula: see text] and E6 is conserved in human cells: inhibition of IKK[Formula: see text] blocked the growth of cervical cancer cells, suggesting that IKK[Formula: see text] may serve as a novel therapeutic target for HPV-mediated cancers.

Aim and Objective: To evaluate a set of seventy phytochemicals for their potential ability to bind the inhibitor of nuclear factor kappaB kinase beta (IKK-β) which is a prime target for cancer and inflammatory diseases., Materials and Methods: Seventy phytochemicals were screened against IKK-β enzyme using DFT-based molecular docking technique and the top docking hits were carried forward for molecular dynamics (MD) simulation protocols. The ADME-Toxicity analysis was also carried out for the top docking hits., Results: Sesamin, matairesinol and resveratrol were found to be the top docking hits with a total score of -413 kJ/mol, -398.11 kJ/mol and 266.73 kJ/mol, respectively. Glu100 and Gly102 were found to be the most common interacting residues. The result from MD simulation observed a stable trajectory with a binding free energy of -107.62 kJ/mol for matairesinol, -120.37 kJ/mol for sesamin and -40.56 kJ/mol for resveratrol. The ADME-Toxicity prediction observed that these compounds fall within the permissible area of Boiled-Egg and it does not violate any rule for pharmacological criteria, drug-likeness etc. Conclusion: The study interprets that dietary phytochemicals are potent inhibitors of IKK-β enzyme with favorable binding affinity and less toxic effects. In fact, there is a gradual rise in the use of plant-derived molecules because of its lesser side effects compared to chemotherapy. The study has also provided an insight by which the phytochemicals inhibited the IKK-β enzyme. The investigation would also provide in understanding the inhibitory mode of certain dietary phytochemicals in treating cancer., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Triple-negative breast cancer (TNBC) remains the most challenging breast cancer subtype to treat because there are no targeted therapies. Currently, chemotherapy is the only clinical option for TNBC despite development of resistance. New therapeutic agents with unique mechanisms of action are urgently needed; therefore, this study investigated the potential anti-TNBC effects of budlein A methylacrylate (BAM), a natural sesquiterpene lactone isolated from plants of the Helianthus genus. We discovered that BAM selectively suppressed and induced apoptosis TNBC cell growth versus other breast cancer or normal mammary epithelial cells. Mechanistically, BAM co-inhibited inhibitor of nuclear factor κBα (IκBα) kinase subunit β (IKKβ) and exportin-1 (XPO-1; chromosome region maintenance 1, CRM1), which are two dysregulated onco-related proteins in TNBC cells, by covalently modifying key functional cysteine residues (Cys 179 of IKKβ, Cys 528 of XPO-1). Dual inhibition led to the stabilization and nuclear retention of IκBα, impairment of NF-κB transcriptional activity, and consequent induction of TNBC cell apoptosis. In conclusion, this study provides evidence that co-inhibition of IKKβ and XPO-1 by BAM was effective against TNBC, demonstrating it as a representative new generation inhibitor with potential for TNBC treatment., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Context: Curcumin has shown efficacy in promoting radiosensitivity combined with radiotherapy. However, the role and mechanism of curcumin on radiosensitivity in laryngeal squamous cell cancer (LSCC) is largely unknown. Objective: The aim of our study is to explore the role of IKKγ-NF-κB signaling in curcumin enhancing LSCC cell radiosensitivity in vitro . Materials and methods: Curcumin and X-ray were used to induce cell DNA damage and apoptosis, or inhibit cell clone formation. IKKγ siRNA and plasmid were used to change IKKγ expression. The CCK8 assay was used to detect cell viability. Clone formation ability was analyzed using a clonogenic assay, cell apoptosis was examined using flow cytometry, an immunofluorescence assay was used to detect DNA damage, while mRNA and protein levels were assayed using real time PCR and western blotting, respectively. Results: Curcumin significantly enhanced irradiation-induced DNA damage and apoptosis, while weakening clone-forming abilities of LSCC cell line Hep2 and Hep2-max. Compared to Hep2 cells, Hep2-max cells are more sensitive to curcumin post-irradiation. Curcumin suppressed irradiation-induced NF-κB activation by suppressing IKKγ expression, but not IKKα and IKKβ. Overexpression of IKKγ decreased irradiation-induced DNA damage and apoptosis, while promoting clone-forming abilities of Hep2 and Hep2-max cells. IKKγ overexpression further increased expression of NF-κB downstream genes, Bcl-XL, Bcl-2, and cyclin D1. Conversely, IKKγ silencing enhanced irradiation-induced DNA damage and apoptosis, but promoted clone formation in Hep2 and Hep2-max cells. Additionally, IKKγ silencing inhibited expression of Bcl-XL, Bcl-2, and cyclin D1. Conclusions: Curcumin enhances LSCC radiosensitivity via NF-ΚB inhibition by suppressing IKKγ expression.

Acute respiratory distress syndrome is an inflammatory disease with no effective pharmacological treatment. We investigated the therapeutic effect of HY1702, a new small molecule diterpene obtained from the processing and modification of Glaucocalyxin A and may exhibit anti-inflammatory activity. Specifically, we studied the anti-inflammatory effects of HY1702 on lipopolysaccharide-induced inflammatory responses in RAW264.7 and THP-1 cells in vitro and its protective efficacy on lipopolysaccharide-induced mild acute respiratory distress syndrome in mice. Our results showed that HY1702 significantly decreased lipopolysaccharide-induced inflammatory cytokine expression in RAW264.7 and THP-1 cells and attenuated the secretion of nitric oxide and prostaglandin E2 by down-regulating the expression of inducible nitric oxide synthase and cyclooxygenase 2 in RAW264.7 cells. In mice with lipopolysaccharide-induced mild acute respiratory distress syndrome, HY1702 alleviated histological alterations in the lungs and reduced the alveolar cavity protein leakage and inflammatory cytokine expression in murine bronchial alveolar lavage fluid. HY1702 decreased the myeloperoxidase activity and lung wet to dry weight ratio. In our mechanism studies in lipopolysaccharide-exposed RAW264.7 cells, HY1702 suppressed the inflammation stimulated by lipopolysaccharide through inhibiting phosphorylation of inhibitor of nuclear factor κB kinase subunit α/β (IKKα/β) and inhibitor of nuclear factor κB subunit α (IκBα), further affecting the nuclear transfer of phosphorylated p65. Meanwhile, phosphorylation of p38 mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase (ERK) was inhibited. These data suggest that HY1702 can reduce inflammation on lipopolysaccharide-stimulated macrophages and attenuate the symptoms of mild acute respiratory distress syndrome in a murine model by regulating the nuclear factor κB and MAP kinase signalling pathways., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Non-alcoholic fatty liver disease (NAFLD) was associated with increased level of lipopolysaccharides (LPS) which mechanism remained unclear on intervention between LPS and NAFLD. The aim was to explore the IKKε/NF-κB role and its intervention of LPS and high-fat diet (HFD) induced NAFLD. Male C57BL/6 mice were fed on high-fat diet (HFD) combined with or without simultaneously subcutaneous injection of LPS for 18 weeks. Body weight , blood biochemistry parameters, inflammatory mediator and liver lipid deposition were measured to evaluate LPS effect on NAFLD. Furthermore, IKKε selective inhibitor amlexanox (AM) was administrated by gavage to HFD + LPS induced mice. The indicators about metabolism and inflammation were examined and qRT-PCR, immunoblotting assay as well as immunohistochemistry were performed to assess IKKε/NF-κB activation and downstream gene expression. This study found that low-dose LPS + HFD aggravated more significant steatosis than simple HFD or high-dose LPS + HFD. Low-dose LPS exacerbated more prominent inflammation profile including increased IKKε and NF-κB expression in liver than HFD. Inhibiting IKKε/NF-κB signaling with amlexanox significantly prevented HFD + LPS induced metabolic disorders and hepatic steatosis. LPS-upregulated gene expression involved in glucolipid metabolism could be downregulated by amlexanox. Thus, the present study confirmed long-term combinational administration of subcutaneous low-dose LPS injection and HFD induced NAFLD model which had more significant phenotype in mice than simple HFD or high-dose LPS-induction. Targeting on IKKε/NF-κB signaling with its inhibitor amlexanox alleviated steatohepatitis, suggesting that IKKε/NF-κB signaling was responsible for effect of LPS and HFD on NAFLD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The common causes of Non-alcoholic fatty liver disease (NAFLD) are obesity, dyslipidemia, and insulin resistance. Metabolic disorders and lipotoxic hepatocyte damage are hallmarks of NAFLD. Even though amlexanox, a dual inhibitor of TRAF associated nuclear factor κB (NF-κB) activator-binding kinase 1 (TBK1) and IκB kinase epsilon (IKKε), has been reported to effectively improve obesity-related metabolic dysfunctions in mice models, its molecular mechanism has not been fully investigated. This study was designed to investigate the effects of amlexanox on in vitro nonalcoholic steatohepatitis (NASH) model induced by treatment of palmitic acid (PA, 0.4 mM), using a trans-well co-culture system of hepatocytes and Kupffer cells (KCs). Stimulation with PA significantly increased the phosphorylation levels of TBK1 and IKKε in both hepatocytes and KCs, suggesting a potential role of TBK1/IKKε in PA-induced NASH progression. Treatment of amlexanox (50 μM) showed significantly reduced phosphorylation of TBK1 and IKKε and hepatotoxicity as confirmed by decreased levels of lactate dehydrogenase released from hepatocytes. Furthermore, PA-induced inflammation and lipotoxic cell death in hepatocytes were significantly reversed by amlexanox treatment. Intriguingly, amlexanox inhibited the activation of KCs and induced polarization of KCs towards M2 phenotype. Mechanistically, amlexanox treatment decreased the phosphorylation of interferon regulator factor 3 (IRF3) and NF-κB in PA-treated hepatocytes. However, decreased phosphorylation of NF-κB, not IRF3, was found in PA-treated KCs upon amlexanox treatment. Taken together, our findings show that treatment of amlexanox attenuated the severity of PA-induced hepatotoxicity in vitro and lipoapoptosis by the inhibition of TBK1/IKKε-NF-κB and/or IRF3 pathway in hepatocytes and KCs., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The IKK-related kinases, IKKε and TBK1, have essential roles in innate immunity in part through modifying MYD88 signalling from the Toll-like receptors to regulate NF-κB signalling. We investigated the expression and function of IKKε and TBK1, in diffuse large B-cell lymphoma (DLBCL). DLBCL cell lines and patient-derived xenografts were used to determine their sensitivity to IKKε and TBK1 inhibitors. To understand the function of IKKε and TBK1 secreted factors were determined following administration of inhibitors. Gene expression microarrays were used to determine the transcriptional effects of inhibitors. Higher TBK1 mRNA levels associated with poorer clinical outcome but IKKε and TBK1 were expressed in both germinal centre and non-germinal centre types of DLBCL. Survival of cell lines Ly10, Ly03 and Pfeiffer, and of some primary human lymphoma cells, was suppressed by a small molecule IKKε/TBK1 inhibitor, DMX3433. DMX3433 reduced IL-10 production from Ly10 and repressed NF-κB mediated transcription. Inhibition of IKKε and TBK1 warrants further investigation as a potential therapeutic route to suppress NF-κB signalling in lymphoma., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

A new labdane-type diterpenoid, ent-19-ol-13-epi-manoyl oxide,19-undecane ester, together with ten known diterpenes, were isolated from the ethanolic crude extract of the fresh tubers of Sagittaria trifolia L. The chemical structures of these compounds were determined by extensive 2-D NMR experiments and by comparison with the data reported in the literature. These compounds showed different inhibitory effects on various human cancer cells. Among these, compound 11 exhibited potential inhibition effects against human colon cancer cells. Moreover, flow cytometry demonstrated that compound 11 arrested the cell cycle at the G1 phase and induced cellular apoptosis, accompanied by mitochondrial membrane potential reduction. Mechanistic studies revealed that treatment with compound 11 inhibited IKKα/β phosphorylation and IκBα phosphorylation, which subsequently caused the blockage of NF-κB p65 phosphorylation and nuclear translocation. Compound 11 also inhibited the expression of c-Myc, Cyclin D1, and Bcl-2, the downstream targets of NF-κB. Therefore, our findings provided insight into the anticancer components of Sagittaria trifolia L. tubers, which could facilitate their utilization as functional food ingredients.

Background: Chronic kidney disease condition requires regular dialysis; the patients have greater risk of sepsis and have high mortality rate compared to general people with sepsis. The adverse cardiac condition leads to mortality in subjects with sepsis. In the present work, we studied the consequences of chronic kidney damage by 5/6 nephrectomy on cardiac function in mice induced with sepsis and the mechanism involved., Methods: We used C57BL/6 mice and subjected them to 5/6 nephrectomy; after induction of chronic kidney damage, they were subjected to sepsis by either LPS treatment or by cecal ligation and puncture (CLP) method. The cardiac function test was done by echocardiography. Protein expression was done by western blot analysis., Results: The 5/6 nephrectomized mice showed significant increase in blood creatinine and urea levels compared to sham-operated mice; the mice also showed decreased ejection fraction and increased levels of phosphorylated IkB α and nuclear translocation of the NF- κ B and inducible nitric oxide synthase (iNOS). When subjected to CLP and LPS treatment, the 5/6 nephrectomized mice augmented cardiac abnormalities and lung inflammation and increased plasma levels of TNF- α , IL-1, IL-12, and IL-18. Also, we evidenced increased levels of p-IKK α / β and Ik βα , NF- κβ , and iNOS. Treatment of IKK inhibitor VII in 5/6 nephrectomized mice after LPS administration or CLP attenuated these effects., Conclusion: Chronic kidney disease could lead to abnormal cardiac function caused by sepsis in mice; this may be due to increased expression of NF- κβ and iNOS in cardiac tissues., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2020 Mei Ding et al.)

Purpose: Glioblastoma multiforme (GBM) is the most common and aggressive malignant type of brain tumor. Despite advances in diagnosis and therapy, the prognosis of patients with GBM has remained dismal. Multidrug resistance and high recurrence are two of the major challenges in successfully treating brain tumors. IKBKE (inhibitor of nuclear factor kappa-B kinase subunit epsilon) is a major oncogenic protein in tumors and can inhibit glioblastoma cell proliferation, migration, and tumorigenesis. Our study aimed to investigate the mechanism of IKBKE enhancing the resistance of glioma cells to temozolomide., Methods: For the in vitro experiments, LN18 and U118 glioblastoma cells were treated with a combination of sh/oe-IKBKE lentivirus and TMZ. Cell proliferation was determined by the EdU assay and colony formation assays. Apoptosis was analyzed by the TUNEL assay. In vivo, LN18 NC and LN18 sh-IKBKE cells were implanted into the cerebrums of nude mice to detect the effect of combination therapy. The protein and mRNA levels were assayed by western blot, immunohistochemistry, and qRT-PCR., Results: In this study, we demonstrated that IKBKE enhances the resistance of glioblastoma cells to temozolomide (TMZ) by activating the AKT/NF-κB signaling pathway to upregulate the expression of the DNA repair enzyme o6-methylguanine-dna methyltransferase (MGMT). In glioblastoma cells, IKBKE knockdown enhances apoptosis and suppresses cell proliferation, clone formation, and tumor development in vivo induced by TMZ. However, overexpression of IKBKE reduces the effects of TMZ., Conclusion: Our studies suggest that inhibition of IKBKE can enhance the therapeutic effect of TMZ on GBM in vitro and in vivo, providing new research directions and therapeutic targets for the treatment of GBM.

Nuclear factor kappa B (NF-κB) signaling is implicated in all major human chronic diseases, with its role in transcription of hundreds of gene well established in the literature. This has propelled research into targeting the NF-κB pathways for modulating expression of those genes and the diseases mediated by them. In-spite of the critical, but often promiscuous role played by this pathway and the inhibition causing adverse drug reaction, currently many biologics, macromolecules, and small molecules that modulate this pathway are in the market or in clinical trials. Furthermore, many marketed drugs that were later found to also have NF-κB targeting activity were repurposed for new therapeutic interventions. Despite the rising importance of biologics in drug discovery, small molecules got around 76% of US-FDA (Food and Drug Administration-US) approval in the last decade. This encouraged us to review information regarding clinically relevant small molecule inhibitors of the NF-κB pathway from cell surface receptor stimulation to nuclear signaling. We have also highlighted the underexplored targets in this pathway that have potential to succeed in clinic.

Inhibitor-kappaB kinase epsilon (IKKε) and TANK-binding kinase 1 (TBK1) are non-canonical IκB kinases, both described as contributors to tumor growth and metastasis in different cancer types. Several hints indicate that they are also involved in the pathogenesis of melanoma; however, the impact of their inhibition as a potential therapeutic measure in this "difficult-to-treat" cancer type has not been investigated so far. We assessed IKKε and TBK1 expression in human malignant melanoma cells, primary tumors and the metastasis of melanoma patients. Both kinases were expressed in the primary tumor and in metastasis and showed a significant overexpression in tumor cells in comparison to melanocytes. The pharmacological inhibition of IKKε/TBK1 by the approved drug amlexanox reduced cell proliferation, migration and invasion. Amlexanox did not affect the cell cycle progression nor apoptosis induction but significantly suppressed autophagy in melanoma cells. The analysis of potential functional downstream targets revealed that NF-кB and ERK pathways might be involved in kinase-mediated effects. In an in vivo xenograft model in nude mice, amlexanox treatment significantly reduced tumor growth. In conclusion, amlexanox was able to suppress tumor progression potentially by the inhibition of autophagy as well as NF-кB and MAP kinase pathways and might therefore constitute a promising candidate for melanoma therapy.

Animal models of inflammatory diseases support the idea that nuclear factor κB (NF-κB) activation plays a pathophysiological role and is widely implicated in multiple organ dysfunction (MOD). Indeed, the inhibition of the IκB kinase (IKK) complex, involved in the NF-κB pathway, can represent a promising approach to prevent MOD. The present work employed a rat model of systemic inflammation to investigate the preventive effects of Inhibitor of IKK complex (IKK16). In male Wistar rats, systemic inflammation was induced by a tail vein injection of lipopolysaccharides (LPS challenge; 12 mg/kg). Treatment with IKK16 (1 mg/kg body weight) was administered, by tail vein, 15 min post-LPS. Age- and sex-matched healthy rats and LPS rats without treatment were used as controls. At 24 h post-IKK16 treatment, serum enzyme levels indicative of liver, kidney, pancreas and muscle function were evaluated by biochemical analysis, and RT-PCR technique was used to analyze gene expression of pro-inflammatory cytokines. Hemodynamic parameters were also considered to assess the LPS-induced inflammation. IKK16 treatment yielded a strong therapeutic effect in preventing LPS-induced elevation of serological enzyme levels, attenuating hepatic, renal, pancreatic and muscular dysfunction after LPS challenge. Moreover, as expected, LPS promoted a significantly overexpression of TNF-α, IL-6 and IL-1β in the heart, kidney, and liver; which was diminished by IKK16 treatment. The present study provides convincing evidence that selective inhibition of the IκB kinase complex through the action of IKK16, plays a protective role against LPS-induced multiple organ dysfunction by reducing the acute inflammatory response induced by endotoxin exposure., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Lysyl oxidase (LOX) is involved in fibrosis by catalyzing collagen cross-linking. Previous work observed that Triptolide (TPL) alleviated radiation-induced pulmonary fibrosis (RIPF), but it is unknown whether the anti-RIPF effect of TPL is related to LOX. In a mouse model of RIPF, we found that LOX persistently increased in RIPF which was significantly lowered by TPL. Excessive LOX aggravated fibrotic lesions in RIPF, while LOX inhibition mitigated RIPF. Irradiation enhanced the transcription and synthesis of LOX by lung fibroblasts through IKKβ/NFκB activation, and siRNA knockdown IKKβ largely abolished LOX production. By interfering radiation induced IKKβ activation, TPL prevented NFκB nuclear translocation and DNA binding, and potently decreased LOX synthesis. Our results demonstrate that the anti-RIPF effect of TPL is associated with reduction of LOX production which mediated by inhibition of IKKβ/NFκB pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Resistance to androgen receptor (AR) targeting therapeutics in prostate cancer (PC) is a significant clinical problem. Mechanisms by which this is accomplished include AR amplification and expression of AR splice variants, demonstrating that AR remains a key therapeutic target in advanced disease. For the first time we show that IKBKE drives AR signalling in advanced PC. Significant inhibition of AR regulated gene expression was observed upon siRNA-mediated IKBKE depletion or pharmacological inhibition due to inhibited AR gene expression in multiple cell line models including a LNCaP derivative cell line resistant to the anti-androgen, enzalutamide (LNCaP-EnzR). Phenotypically, this resulted in significant inhibition of proliferation, migration and colony forming ability suggesting that targeting IKBKE could circumvent resistance to AR targeting therapies. Indeed, pharmacological inhibition in the CWR22Rv1 xenograft mouse model reduced tumour size and enhanced survival. Critically, this was validated in patient-derived explants where enzymatic inactivation of IKBKE reduced cell proliferation and AR expression. Mechanistically, we provide evidence that IKBKE regulates AR levels via Hippo pathway inhibition to reduce c-MYC levels at cis-regulatory elements within the AR gene. Thus, IKBKE is a therapeutic target in advanced PC suggesting repurposing of clinically tested IKBKE inhibitors could be beneficial to castrate resistant PC patients., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Lipopolysaccharides (LPS) are associated with various inflammatory diseases; therefore, the inhibition of LPS-induced nitric oxide (NO) production may have extensive therapeutic applications. We searched for inhibitors of NO production in the LPS-stimulated murine macrophage-like cell line RAW264.7 from MeOH extracts of marine organisms. The MeOH extract of the marine cyanobacterium Okeania sp., collected in Okinawa, Japan, showed inhibitory activity. Biseokeaniamide A was isolated from the MeOH extract by chromatographic separation. Biseokeaniamide A inhibited NO production without cytotoxicity. It reduced inducible nitric oxide synthase levels and suppressed the expression of IL-1β in LPS-stimulated RAW264.7 cells. Biseokeaniamide A did not inhibit IκBα degradation but inhibited IκBα expression. Thus, biseokeaniamide A, a naturally occurring lipopeptide, was identified as a selective inhibitor of LPS signal transduction., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)