Selective Hypoxia-Sensitive Oxomer Formation by FIH Prevents Binding of the NF-κB Inhibitor IκBß to NF-κB Subunits.

Pharmacologic inhibitors of cellular hydroxylase oxygen sensors are protective in multiple preclinical in vivo models of inflammation. However, the molecular mechanisms underlying this regulation are only partly understood, preventing clinical translation. We previously proposed a new mechanism for cellular oxygen sensing: oxygen-dependent, (likely) covalent protein oligomer (oxomer) formation. Here, we report that the oxygen sensor factor inhibiting HIF (FIH) forms an oxomer with the NF-κB inhibitor ß (IκBß). The formation of this protein complex required FIH enzymatic activity and was prevented by pharmacologic inhibitors. Oxomer formation was highly hypoxia-sensitive and very stable. No other member of the IκB protein family formed an oxomer with FIH, demonstrating that FIH-IκBß oxomer formation was highly selective. In contrast to the known FIH-dependent oxomer formation with the deubiquitinase OTUB1, FIH-IκBß oxomer formation did not occur via an IκBß asparagine residue, but depended on the amino acid sequence VAERR contained within a loop between IκBß ankyrin repeat domains 2 and 3. Oxomer formation prevented IκBß from binding to its primary interaction partners p65 and c-Rel, subunits of NF-κB, the master regulator of the cellular transcriptional response to pro-inflammatory stimuli. We therefore propose that FIH-mediated oxomer formation with IκBß contributes to the hypoxia-dependent regulation of inflammation.

HOXA1 silencing inhibits cisplatin resistance of oral squamous cell carcinoma cells via IκB/NF-κB signaling pathway.

The resistance of oral squamous cell carcinoma (OSCC) cells to cisplatin remains a tough nut to crack in OSCC therapy. Homeobox A1 (HOXA1) overexpression has been detected in head and neck squamous carcinoma (HNSC). Accordingly, this study aims to explore the potential role and mechanism of HOXA1 on cisplatin resistance in OSCC. The expression of HOXA1 in HNSC and its role in overall survival (OS) rate of OSCC patients were analyzed by bioinformatic analysis. Following transfection as needed, OSCC cells were induced by different concentrations of cisplatin, and the cell viability and apoptosis were evaluated by cell counting kit-8 and flow cytometry assays. The mRNA and protein expression levels of HOXA1 and the phosphorylation of IκBα and p65 were determined by real-time quantitative PCR and western blot. HOXA1 expression level was upregulated in HNSC tissues and OSCC cells. Overexpressed HOXA1 was correlated with a low OS rate of OSCC patients. Cisplatin exerted an anti-cancer effect on OSCC cells. HOXA1 silencing or cisplatin suppressed OSCC cell viability, boosted the apoptosis, and repressed the phosphorylation of IκBα and p65. Intriguingly, the combination of HOXA1 silencing and cisplatin generated a stronger anti-cancer effect on OSCC cells than their single use. HOXA1 silencing attenuates cisplatin resistance of OSCC cells via IκB/NF-κB signaling pathway, hinting that HOXA1 is a biomarker associated with OSCC and HOXA1 silencing can enhance the sensitivity of OSCC cells to cisplatin.

Botulinum toxin type A inhibits M1 macrophage polarization by deactivation of JAK2/STAT1 and IκB/NFκB pathway and contributes to scar alleviation in aseptic skin wound healing.

The non-neuronal and non-muscular effects of botulinum toxin type A (BTXA) on scar reduction has been discovered. This study was designed to investigate the effects of BTXA on macrophages polarization during the early stage of skin repair. A skin defect model was established on the dorsal skin of SD rats. BTXA was intracutaneous injected into the edge of wound immediately as the model was established. Histological examinations were performed on scar samples. Raw 264.7 was selected as the cell model of recruited circulating macrophages, and was induced for M1 polarization by LPS. Identify the signaling pathways that primarily regulated M1 polarization and respond to BTXA treatment. Application of BTXA at early stage of injury significantly reduced the scar diameter without delaying wound closure. BTXA treatment improved fiber proliferation and arrangement, and inhibited angiogenesis in scar granular tissue. The number of M1 macrophages and the levels of pro-inflammation were decreased after treated with BTXA in scar tissues. LPS activated JAK2/STAT1 and IκB/NFκB pathways were downregulated by BTXA, as well as LPS induced M1 polarization. At early stage of skin wound healing, injection of BTXA effectively reduced the number of M1 macrophages and the levels of pro-inflammatory mediators which contributes to scar alleviation. BTXA resisted the M1 polarization of macrophages induced by LPS via deactivating the JAK2/STAT1 and IκB/NFκB pathways.

Polyphenols Targeting NF-κB Pathway in Neurological Disorders: What We Know So Far?

Polyphenolic compounds have shown promising neuroprotective properties, making them a valuable resource for identifying prospective drug candidates to treat several neurological disorders (NDs). Numerous studies have reported that polyphenols can disrupt the nuclear factor kappa B(NF-κB) pathway by inhibiting the phosphorylation or ubiquitination of signaling molecules, which further prevents the degradation of IκB. Additionally, they prevent NF-κB translocation to the nucleus and pro-inflammatory cytokine production. Polyphenols such as curcumin, resveratrol, and pterostilbene had significant inhibitory effects on NF-κB, making them promising candidates for treating NDs. Recent experimental findings suggest that polyphenols possess a wide range of pharmacological properties. Notably, much attention has been directed towards their potential therapeutic effects in NDs such as Alzheimer's disease (AD), Parkinson's disease (PD), cerebral ischemia, anxiety, depression, autism, and spinal cord injury (SCI). Much preclinical data supporting the neurotherapeutic benefits of polyphenols has been developed. Nevertheless, this study has described the significance of polyphenols as potential neurotherapeutic agents, specifically emphasizing their impact on the NF-κB pathway. This article offers a comprehensive analysis of the involvement of polyphenols in NDs, including both preclinical and clinical perspectives.

Rabdosichuanin C inhibits productions of pro-inflammatory mediators regulated by NF-κB signaling in LPS-stimulated RAW264.7 cells.

Chronic pharyngitis (CP) is an inflammatory disease of the pharyngeal mucosa and its lymphatic tissues that is difficult to treat clinically. However, research on the exact therapeutic agents and molecular mechanisms of CP is still unclear. In this study, we investigated Rabdosichuanin C (RC) to attenuate lipopolysaccharide (LPS)-induced inflammatory damage in RAW264.7 cells by a combination of targeted virtual screening and in vitro activity assay and further clarified its molecular mechanism of action centering on the IκB/nuclear factor kappa B (NF-κB) pathway. Molecular docking and pharmacophore simulation methods were used to screen compounds with IκB inhibitory effects. Expression of genes and proteins related to the IκB/NF-κB signaling pathway by RC in LPS-induced inflammatory injury model of RAW264.7 cells was detected by PCR, enzyme-linked immunosorbent assay, and Western blot. The docking of RC with IκB protein showed good binding energy, and pharmacophore simulations further confirmed the active effect of RC in inhibiting IκB protein. RC intervention in LPS-induced RAW264.7 cells significantly reduced the expression levels of inflammatory factors tumor necrosis factor-α, interleukins-6, iNOS, and CD-86 at the messenger RNA and protein levels, downregulated IκB, p65 protein phosphorylation levels, and significantly inhibited IκB/NF-κB signaling pathway activation. Virtual screening provided us with an effective method to rapidly identify compounds RC that target inhibit the action of IκB, and the activity results showed that RC inhibits NF-κB signaling pathway activation. It is suggested that RC may play a role in the treatment of CP by inhibiting the IκB/NF-κB signaling pathway.

NF-κB: a mediator that promotes or inhibits angiogenesis in human diseases?

The nuclear factor of κ-light chain of enhancer-activated B cells (NF-κB) signaling pathway, which is conserved in invertebrates, plays a significant role in human diseases such as inflammation-related diseases and carcinogenesis. Angiogenesis refers to the growth of new capillary vessels derived from already existing capillaries and postcapillary venules. Maintaining normal angiogenesis and effective vascular function is a prerequisite for the stability of organ tissue function, and abnormal angiogenesis often leads to a variety of diseases. It has been suggested that NK-κB signalling molecules under pathological conditions play an important role in vascular differentiation, proliferation, apoptosis and tumourigenesis by regulating the transcription of multiple target genes. Many NF-κB inhibitors are being tested in clinical trials for cancer treatment and their effect on angiogenesis is summarised. In this review, we will summarise the role of NF-κB signalling in various neovascular diseases, especially in tumours, and explore whether NF-κB can be used as an attack target or activation medium to inhibit tumour angiogenesis.

The central inflammatory regulator IκBζ: induction, regulation and physiological functions.

IκBζ (encoded by NFKBIZ) is the most recently identified IkappaB family protein. As an atypical member of the IkappaB protein family, NFKBIZ has been the focus of recent studies because of its role in inflammation. Specifically, it is a key gene in the regulation of a variety of inflammatory factors in the NF-KB pathway, thereby affecting the progression of related diseases. In recent years, investigations into NFKBIZ have led to greater understanding of this gene. In this review, we summarize the induction of NFKBIZ and then elucidate its transcription, translation, molecular mechanism and physiological function. Finally, the roles played by NFKBIZ in psoriasis, cancer, kidney injury, autoimmune diseases and other diseases are described. NFKBIZ functions are universal and bidirectional, and therefore, this gene may exert a great influence on the regulation of inflammation and inflammation-related diseases.

KLF7 promotes adipocyte inflammation and glucose metabolism disorder by activating the PKCζ/NF-κB pathway.

In the obesity context, inflammatory cytokines secreted by adipocytes lead to insulin resistance and are key to metabolic syndrome development. In our previous study, we found that the transcription factor KLF7 promoted the expression of p-p65 and IL-6 in adipocytes. However, the specific molecular mechanism remained unclear. In the present study, we found that the expression of KLF7, PKCζ, p-IκB, p-p65, and IL-6 in epididymal white adipose tissue (Epi WAT) in mice fed a high-fat diet (HFD) was significantly increased. In contrast, the expression of PKCζ, p-IκB, p-p65, and IL-6 was significantly decreased in Epi WAT of KLF7 fat conditional knockout mice. In 3T3-L1 adipocytes, KLF7 promoted the expression of IL-6 via the PKCζ/NF-κB pathway. In addition, we performed luciferase reporter and chromatin immunoprecipitation assays, which confirmed that KLF7 upregulated the expression of PKCζ transcripts in HEK-293T cells. Collectively, our results show that KLF7 promotes the expression of IL-6 by upregulating PKCζ expression and activating the NF-κB signaling pathway in adipocytes.

Inhibition of MLCK­mediated migration and invasion in human endometriosis stromal cells by NF­κB inhibitor DHMEQ.

Endometriosis is initiated by the movement of endometrial cells in the uterus to the fallopian tubes, the ovaries and the peritoneal cavity after the shedding of the uterus lining. To cause endometriosis, it is often necessary for these endometrial cells to migrate, invade and grow at the secondary site. In the present study, immortalized human endometriosis stromal cells (HESC) were employed to look for the inhibitors of migration and invasion. Using a chemical library of bioactive metabolites, it was found that an NF­κB inhibitor, DHMEQ, inhibited the migration and invasion of HESC. Both whole­genome array and metastasis PCR array analyses suggested the involvement of myosin light chain kinase (MLCK) in the mechanism of inhibition. DHMEQ was confirmed to inhibit the expression of MLCK and small inhibitory RNA knockdown of MLCK reduced cellular migration and invasion. The addition of DHMEQ to the knockdown cells did not further inhibit migration and invasion. DHMEQ is particularly effective in suppressing disease models by intraperitoneal (IP) administration and this therapy is being developed for the treatment of inflammation and cancer. DHMEQ IP therapy may also be useful for the treatment of endometriosis.

ATP Consumption Is Coupled with Endocytosis in Exudated Neutrophils.

Neutrophil energy metabolism during phagocytosis has been previously reported, and adenosine triphosphate (ATP) plays a crucial role in endocytosis. Neutrophils are prepared by intraperitoneal injection of thioglycolate for 4 h. We previously reported a system established for measuring particulate matter endocytosis by neutrophils using flow cytometry. In this study, we utilized this system to investigate the relationship between endocytosis and energy consumption in neutrophils. A dynamin inhibitor suppressed ATP consumption triggered by neutrophil endocytosis. In the presence of exogenous ATP, neutrophils behave differently during endocytosis depending on ATP concentration. The inhibition of ATP synthase and nicotinamide adenine dinucleotide phosphate oxidase but not phosphatidylinositol-3 kinase suppresses neutrophil endocytosis. The nuclear factor kappa B was activated during endocytosis and inhibited by I kappa B kinase (IKK) inhibitors. Notably, IKK inhibitors restored endocytosis-triggered ATP consumption. Furthermore, data from the NLR family pyrin domain containing three knockout mice suggest that inflammasome activation is not involved in neutrophil endocytosis or concomitant ATP consumption. To summarize, these molecular events occur via endocytosis, which is closely related to ATP-centered energy metabolism.

Anti-inflammatory Activity of Gegen Decoction and Its Modulatory Mechanism on the NF-κB and MAPK Signaling Pathways.

Gegen Decoction as anti-inflammatory medicine is used in clinic widespread, however the specific anti-inflammatory molecular mechanism of Gegen Decoction is still unclear. The purpose was to study the anti-inflammatory activity of Gegen Decoction in vivo and to research its anti-inflammatory molecular mechanism. The content of main essential components in Gegen Decoction were determined by HPLC method. The anti-inflammatory activity of Gegen Decoction was confirmed through in vivo animal experiments. Furthermore, RAW 264.7 cells were stimulated by lipopolysaccharides to induce inflammatory reaction, the modulatory effect of Gegen Decoction on the activation process of mitogen-activated protein kinases and nuclear factor-κB signaling pathways was investigated. The content of puerarin was the highest among all the index components. Gegen Decoction inhibited carrageenan-induced paw edema in rats and xylene-induced ear swelling in mice. Gegen Decoction had no obvious toxicity against RAW 264.7 cells at the concentrations of 10-40 mg/mL; significantly inhibited the release of nitric oxide, prostaglandin E2, tumor necrosis factor-α and interleukin-6; down-regulated the high expression of inflammatory proteins inducible nitric oxide synthase and cyclooxygenase-2. It inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs)/extracellular regulated protein kinases (ERK)/c-Jun N-terminal kinase (JNK), the degradation of nuclear factor-κB (NF-κB)/inhibitor of NF-κB-α (IκB-α) and the nuclear translocation of NF-κB/p65 into nucleus. Gegen Decoction exerts significant anti-inflammatory activity, mainly by blocking the activation of both MAPKs and NF-κB pathway.

Oxymatrine attenuated isoproterenol-induced heart failure via the TLR4/NF-κB and MAPK pathways in vivo and in vitro.

Oxymatrine (OMT) is a quinoline alkaloid isolated from the root of the Sophora flavescens that has a variety of biological activities. However, the effect and potential mechanism of OMT on isoproterenol (ISO)-induced heart failure (HF) are not clear. In this study, we found that OMT improved the survival of HL-1 cells induced by ISO. We also demonstrated that OMT significantly inhibited the levels of the inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). OMT decreased the levels of the TLR4 and reduced the phosphorylation levels of nuclear factor-κB (NF-κB) inhibitor (IκB), p65, c-Jun N-terminal kinases (JNK) and p38. The inhibitory effect of the TLR4 inhibitor TAK242 on HL-1 cells was evaluated. The results showed that the effect of OMT on the phosphorylation levels of IκBα and p65 was enhanced in HL-1 cells treated with TAK242. Using animal models, OMT significantly reduced ISO-induced cardiac injury, myocardial necrosis, interstitial edema, and fibrosis. In addition, OMT attenuated TNF-α and IL-6 and inhibited the expression of TLR4/NF-κB and MAPK pathway-related proteins. This finding suggests that OMT may alleviate HF by interfering with the TLR4/NF-κB and MAPK pathways.

Corylin inhibits the progression of Non-small cell lung cancer cells by regulating NF-κB signaling pathway via targeting p65.

BACKGROUND: Lung cancer is characterized by high-risk and high mortality, among which non-small cell lung cancer (NSCLC) conquers a dominant position. Previous studies have reported that corylin has anti-inflammatory, anti-oxidant, and anti-tumor effects; however, its role in NSCLC cells remains unclear. HYPOTHESIS: Corylin inhibits the progression of NSCLC cells. METHODS: A lentivector NF-κB luciferase reporter was constructed by molecular cloning. Corylin was screened and identified as an NF-κB pathway inhibitor by luciferase reporter assay. Corylin inhibited the expression of NF-κB downstream genes, which was detected by qRT-PCR. The effect of corylin on NSCLC cells was detected by colony formation assay, cell apoptosis, cell proliferation, in vitro invasion, and cell scratch assay. Corylin inhibited p65 nuclear translocation and was detected by molecular docking, immunofluorescence assay, and Western blot analysis. RESULTS: We constructed a lentiviral expression vector, containing an NF-κB luciferase reporter and established a stable A549 cell line for its expression. Using this cell line, corylin was screened and identified as an NF-κB pathway inhibitor. It was found that corylin inhibited the expression of NF-κB downstream genes and inhibited the proliferation and migration of NSCLC cells. Meanwhile, it was also found that corylin significantly reversed the increased proliferation of NSCLC cell lines induced by p65 overexpression. Molecular docking analysis showed that corylin could bind to p65 by hydrogen bonding. Further study showed that corylin inhibited the NF-κB signaling pathway by blocking p65 nuclear translocation. CONCLUSIONS: Our study screened and identified corylin as an NF-κB inhibitor and elucidated the molecular mechanism by which corylin inhibits the growth of NSCLC cells. The present study provides a novel strategy for improving the prognosis and treatment of NSCLC patients.

23-O-acetylshengmanol-3-O-α-L-arabinoside alleviates lipopolysaccharide-induced acute lung injury through inhibiting IκB/NF-κB and MAPK/AP-1 signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Cimicifuga foetida L. is a well-established traditional Chinese medicine with heat-clearing and detoxifying effects and has good therapeutic effect on oral mucosal ulcer and pharyngitis. The rhizome of this herb is rich in triterpenoid glycosides, including 23-O-acetylshengmanol-3-o-α-L-arabinoside (DA). AIM OF THE STUDY: Whether and how DA attenuates acute lung injury (ALI) are unclear. Accordingly, we focused on its anti-inflammatory effects and underlying molecular mechanisms in lipopolysaccharide (LPS)-stimulated ALI mice and RAW264.7 cells. MATERIALS AND METHODS: The model of ALI mice was established by exposed intratracheal instillation of LPS. Lung pathological changes were evaluated by hematoxylin and eosin staining. Pulmonary function was assessed by whole-body plethysmography. Total protein content in bronchoalveolar lavage fluid (BALF) was detected by bicinchoninic acid method. Wet/dry lung ratio was used to evaluate the degree of pulmonary edema in mice. The levels of pro-inflammatory mediators were measured using enzyme-linked immunosorbent assay. The relative expression of pro-inflammatory gene mRNA was examined by RT-qPCR. The expression of inflammatory-related proteins was detected by Western blot. RAW264.7 cells were used to test the anti-inflammatory effects of DA in vitro. Cytotoxicity was assessed using a MTT assay. Nitric oxide production was measured by Griess assay. The production and expression of inflammatory mediators and the protein levels of inflammatory signaling molecules in the NF-κB and MAPK pathways were measured. Furthermore, immunofluorescence staining was used to analyze the expression of p-IκBα, p-ERK, and p-p38 in lung macrophages and the nuclear translocation of NF-κB p65 and AP-1 in cells. RESULTS: DA evidently alleviated histopathological changes and ameliorated pulmonary edema. Moreover, DA could reduce excessive inflammatory reaction in lung tissue as manifested by the reduction of proinflammatory mediators (IL-1ß, IL-6, TNF-α, MCP-1, iNOS, and COX-2) in BALF, serum, and lung tissues. Further, DA inhibited the activation of the NLRP3/caspase-1 pathway in the lung. DA reduced the production and expression of the proinflammatory mediators above in RAW264.7 cells. Mechanistically, DA remarkably blocked the nuclear translocation of NF-κB p65, suppressed IκBα phosphorylation, and markedly reduced the nuclear translocation of AP-1 and the phosphorylation of ERK and p38. CONCLUSIONS: The findings demonstrated that DA exerts anti-inflammatory effects in LPS-stimulated ALI mice and macrophages by downregulating the NLRP3/caspase-1 signaling pathway in lung tissue and the IκB/NF-κB and MAPKs/AP-1 pathways in macrophages, suggesting that DA may be promising in ALI treatment.

Engineered nanoparticles as emerging gene/drug delivery systems targeting the nuclear factor-κB protein and related signaling pathways in cancer.

The transcription factor nuclear factor-κB (NF-κB) is a critical regulator of the immune response, inflammation, cell growth, and survival. Canonical and non-canonical pathways, two NF-κB pathways, are activated through diverse stimulators and receptors. NF-κB activity is dysregulated in various inflammation-related diseases and cancers. It was found that the persistent NF-κB activity has a major role in proliferation, apoptosis inhibition, metastasis, and cell cycle disruption in cancer cells and also the survival of cancer stem cells (CSCs) within the tumors. Therefore, suppression of the NF-κB pathway could be a promising therapeutic target for cancer therapy. Different biological inhibitors (e.g., peptides, small molecules, antisense oligonucleotides (ASOs), and antibodies (Abs)) have been demonstrated to inhibit the NF-κB pathway. Low stability in the circulation system, weak availability, and poor cellular uptake of some inhibitors limit their therapeutic applications. To address these drawbacks nanocarrier systems are often formulated and applied in drug delivery as an effective therapeutic approach. Targeted nanosystems (i.e., small molecules, peptides, Abs and Aptamers (Aps) conjugated nanocarriers), as well as smart responsive nanocarriers, can improve the efficiency of therapeutics while reducing the off-target toxicity. This review describes the NF-κB signaling pathways and mechanisms of their over-activation in tumor initiation and progression. The NF-κB inhibitors and their clinical applications are also discussed. It also overviews different nanocarriers used as robust vehicles for the delivery of NF-κB inhibitors and anti-tumor agents to improve the bioavailability of drugs and selective targeting of cancer cells to repress NF-κB activity in tumor cells.

ZC3H11A loss of function enhances NF-κB signaling through defective IκBα protein expression.

ZC3H11A is a cellular protein associated with the transcription export (TREX) complex that is induced during heat-shock. Several nuclear-replicating viruses exploit the mRNA export mechanism of ZC3H11A protein for their efficient replication. Here we show that ZC3H11A protein plays a role in regulation of NF-κB signal transduction. Depletion of ZC3H11A resulted in enhanced NF-κB mediated signaling, with upregulation of numerous innate immune related mRNAs, including IL-6 and a large group of interferon-stimulated genes. IL-6 upregulation in the absence of the ZC3H11A protein correlated with an increased NF-κB transcription factor binding to the IL-6 promoter and decreased IL-6 mRNA decay. The enhanced NF-κB signaling pathway in ZC3H11A deficient cells correlated with a defect in IκBα inhibitory mRNA and protein accumulation. Upon ZC3H11A depletion The IκBα mRNA was retained in the cell nucleus resulting in failure to maintain normal levels of the cytoplasmic IκBα mRNA and protein that is essential for its inhibitory feedback loop on NF-κB activity. These findings indicate towards a previously unknown mechanism of ZC3H11A in regulating the NF-κB pathway at the level of IkBα mRNA export.

CYP24A1 Involvement in Inflammatory Factor Regulation Occurs via the Wnt Signaling Pathway.

OBJECTIVE: While the upregulation of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) gene expression has been reported in colon cancer, its role in tumorigenesis remains largely unknown. In this study, we aimed to investigate the involvement of CYP24A1 in Wnt pathway regulation via the nuclear factor kappa B (NF-κB) pathway. METHODS: The human colon cancer cell lines HCT-116 and Caco-2 were subjected to stimulation with interleukin-6 (IL-6) as well as tumor necrosis factor alpha (TNF-α), with subsequent treatment using the NF-κB pathway-specific inhibitor ammonium pyrrolidinedithiocarbamate (PDTC). Furthermore, CYP24A1 expression was subjected to knockdown via the use of small interfering RNA (siRNA). Subsequently, NF-κB pathway activation was determined by an electrophoretic mobility shift assay, and the transcriptional activity of ß-catenin was determined by a dual-luciferase reporter assay. A mouse ulcerative colitis (UC)-associated carcinogenesis model was established, wherein TNF-α and the NF-κB pathway were blocked by anti-TNF-α monoclonal antibody and NF-κB antisense oligonucleotides, respectively. Then the tumor size and protein level of CYP24A1 were determined. RESULTS: IL-6 and TNF-α upregulated CYP24A1 expression and activated the NF-κB pathway in colon cancer cells. PDTC significantly inhibited this increase in CYP24A1 expression. Additionally, knockdown of CYP24A1 expression by siRNA could partially antagonize Wnt pathway activation. Upregulated CYP24A1 expression was observed in the colonic epithelial cells of UC-associated carcinoma mouse models. Anti-TNF-α monoclonal antibody and NF-κB antisense oligonucleotides decreased the tumor size and suppressed CYP24A1 expression. CONCLUSION: Taken together, this study suggests that inflammatory factors may increase CYP24A1 expression via NF-κB pathway activation, which in turn stimulates Wnt signaling.

Inhibition of NF-κB DNA Binding Suppresses Myeloma Growth via Intracellular Redox and Tumor Microenvironment Modulation.

Multiple myeloma is a plasma cell malignancy that is still largely incurable, despite considerable progress in recent years. NF-κB is a well-established therapeutic target in multiple myeloma, but none of the currently available treatment options offer direct, specific pharmacologic targeting of NF-κB transcriptional activity. Thus, we designed a novel direct NF-κB inhibitor (IT848) as a drug candidate with strong potential for clinical translation and conducted comprehensive in vitro and in vivo mechanistic studies in multiple myeloma cell lines, primary multiple myeloma cells, xenograft models, and immunocompetent mouse models of multiple myeloma. Here, we show that IT848 inhibits NF-κB activity through inhibition of DNA binding of all five NF-κB subunits. IT848 treatment of multiple myeloma cell lines and patient samples inhibited proliferation and induced caspase-dependent and independent apoptosis. In addition to direct NF-κB inhibitory effects, IT848 treatment altered the redox homeostasis of multiple myeloma cells through depletion of the reduced glutathione pool, selectively inducing oxidative stress in multiple myeloma but not in healthy cells. Multiple myeloma xenograft studies confirmed the efficacy of IT848 as single agent and in combination with bortezomib. Furthermore, IT848 significantly improved survival when combined with programmed death protein 1 inhibition, and correlative immune studies revealed that this clinical benefit was associated with suppression of regulatory T-cell infiltration of the bone marrow microenvironment. In conclusion, IT848 is a potent direct NF-κB inhibitor and inducer of oxidative stress specifically in tumor cells, displaying significant activity against multiple myeloma cells in vitro and in vivo, both as monotherapy as well as in combination with bortezomib or immune checkpoint blockade.

A Novel De Novo NFKBIA Missense Mutation Associated to Ectodermal Dysplasia with Dysgammaglobulinemia.

Background: Inborn errors of immunity (IEIs) are comprised of heterogeneous groups of genetic disorders affecting immune function. In this report, a 17-month-old Malay patient suspected of having Hyper IgM syndrome, a type of IEIs, was described. However, the diagnosis of Hyper IgM syndrome was excluded by the normal functional studies and the mild features of ectodermal dysplasia observed from a further clinical phenotype inspection. Methods: Whole-exome sequencing (WES) was performed to unravel the causative mutation in this patient. Results: The variant analysis demonstrated a novel missense mutation in NFKBIA (NM_020529:c.94A > T,NP_065390:p.Ser32Cys) and was predicted as damaging by in silico prediction tools. The NFKBIA gene encodes for IκBα, a member of nuclear factor kappa B (NF-κB) inhibitors, playing an important role in regulating NF-κB activity. The mutation occurred at the six degrons (Asp31-Ser36) in IκBα which were evolutionarily conserved across several species. Prediction analysis suggested that the substitution of Ser32Cys may cause a loss of the phosphorylation site at residue 32 and a gain of the sumoylation site at residue 38, resulting in the alteration of post-translational modifications of IκBα required for NF-κB activation. Conclusion: Our analysis hints that the post-translational modification in the NFKBIA Ser32Cys mutant would alter the signaling pathway of NF-κB. Our findings support the usefulness of WES in diagnosing IEIs and suggest the role of post-translational modification of IκBα.

18ß-Glycyrrhetinic acid ameliorates endoplasmic reticulum stress-induced inflammation in pulmonary arterial hypertension through PERK/eIF2α/NF-κB signaling.

Endoplasmic reticulum stress (ERS)-induced inflammation participates in the occurrence of pulmonary arterial hypertension (PAH) by promoting pulmonary vascular remodeling, which involved in the activation of PERK/eIF2α/NF-κB signaling pathway. 18ß-Glycyrrhetinic acid (18ß-GA) has been found efficacious for attenuating PAH through its anti-remodeling effects in our previous research and it remains unclear whether 18ß-GA has an effect on the remodeling caused by ERS-induced inflammation. In this study, we made observations in monocrotaline-induced PAH rats and found improvement of hemodynamic and histopathological parameters, decreases in the right ventricular hypertrophy index, and alleviation of pulmonary vascular remodeling after 18ß-GA administration in vivo. Moreover, 18ß-GA could significantly inhibit the proliferation and DNA synthesis of human pulmonary arterial smooth muscle cells (HPASMCs) induced by platelet-derived growth factor BB. At the cellular and molecular levels, we found that 18ß-GA could significantly reduce the accumulation of misfolded protein in rat lung tissue, inhibit ERS activation, reduce the expression of GRP78, p-PERK, p-eIF2α, and p-NF-κB p65, and increase IκB protein expression. 18ß-GA could inhibit the migration of NF-κB into the nucleus, reduce the contents of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and monocyte chemoattractant protein-1 (MCP-1) in the culture supernatant of HPASMCs, and reduce GRP78, p-PERK, p-eIF2α, p-NF-κB p65, TNF-α, IL-6, and MCP-1 protein expression, increase IκB protein expression in HPASMCs. According to what we observed, this study indicated that 18ß-GA could treat PAH, which is related to the inhibition of PERK/eIF2α/NF-κB signaling pathway.