Inhibition Of NF-κB P65 Phosphorylation Research Articles

The indole-hydantoin derivative exhibits anti-inflammatory activity by preventing the transactivation of NF-κB through the inhibition of NF-κB p65 phosphorylation at Ser276

Indole- and hydantoin-based derivatives both exhibit anti-inflammatory activity, suggesting that the structures of indole and hydantoin are functional for this activity. In the present study, we synthesized two types of indole-hydantoin derivatives, IH-1 (5-(1H-indole-3-ylmethylene) imidazolidine-2,4-dione) and IH-2 (5-(1H-indole-3-ylmethyl) imidazolidine-2,4-dione) and examined their effects on LPS-induced inflammatory responses in murine macrophage-like RAW264.7 cells. LPS-induced inflammatory responses were not affected by indole, hydantoin, or IH-2. In contrast, IH-1 significantly inhibited the LPS-induced production of nitric oxide (NO) and secretion of CCL2 and CXCL1 by suppressing the mRNA expression of inducible NO synthase (iNOS), CCL2, and CXCL1. IH-1 markedly inhibited the LPS-induced activation of NF-κB without affecting the degradation of IκBα or nuclear translocation of NF-κB. IH-1 markedly attenuated the transcriptional activity of NF-κB by suppressing the LPS-induced phosphorylation of the NF-κB p65 subunit at Ser276. Furthermore, IH-1 prevented the LPS-induced interaction of NF-κB p65 subunit with a transcriptional coactivator, cAMP response element-binding protein (CBP). Collectively, these results revealed the potential of the novel indole-hydantoin derivative, IH-1 as an anti-inflammatory drug.

Floroindole confers protection against cecal ligation and puncture-induced sepsis via inhibition of NF-kB p65 phosphorylation

Purpose: To investigate the protective effect of floroindole against cecal ligation and puncture (CLP)- induced sepsis, as well as the underlying mechanism of action.
 Methods: Thirty-five 10–week-old male Wistar rats weighing 190 - 210 g (mean: 200.00 ± 10.10 g) were used for this study. The rats were randomly assigned to seven groups of five rats each, viz, normal control group, and six CLP groups. The CLP groups were those subjected to cecal ligation and puncture (CLP). The first 5 CLP groups received 2, 4, 6, 8 or 10 mg/kg floroindole, respectively, 1 h after CLP, via intraperitoneal route (i.p.) while the 6th CLP group served as untreated control. Western blotting, enzyme-linked immunosorbent assay (ELISA) and real-time quantitative polymerase chain reaction (qRT-PCR) were used for the assessment of the expression levels of tumor necrosis factor-α (TNF- α), interleukn-6 (IL-6), nucleotide-binding oligomerization domain 2 (NOD2) and p-NF-κB p65.
 Results: Cecal ligation and puncture (CLP) significantly and time-dependently upregulated the expressions of TNF-α, IL-6 and NOD2 in intestinal tissues of rats (p < 0.05). However, treatment with floroindole significantly, and dose-dependently down-regulated CLP-induced expressions of these proteins (p < 0.05). Treatment of rats with floroindole also significantly and dose-dependently inhibited CLP-induced phosphorylation of NF-κB p65 in rat ileum (p < 0.05).
 Conclusion: The results obtained in this study demonstrate that floroindole confers some degree of protection against CLP-induced sepsis via inhibition of NF-κB p65 phosphorylation.

S100A8/A9 induces microglia activation and promotes the apoptosis of oligodendrocyte precursor cells by activating the NF-κB signaling pathway

S100A8/A9, a heterodimer complex composed of calcium-binding proteins S100A8 and S100A9, is significantly increased in the serum of multiple sclerosis (MS) patients. Relevant reports have revealed that MS pathology is commonly associated with the activation of microglial cells and the damage of oligodendrocyte precursor cells (OPCs). Moreover, microglia activation following stimulation increases the expression of pro-inflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), which further exacerbate the damage to OPCs. In this study, we were the first to confirm that S100A8/A9 treatment induced the activation, proliferation and migration of the murine microglia cell line BV-2; moreover, this treatment caused the cells to switch from an anti-inflammatory activated (M2) phenotype to a pro-inflammatory activated (M1) phenotype. Meanwhile, the level of the phosphorylated nuclear factor-κB (p-NF-κB) P65 protein was remarkably elevated, and the production of pro-inflammatory factors (IL-1β, TNF-α, MMP-9) and chemokines (CCL2, CCL3, CXCL10) was also increased in the S100A8/A9-treated BV-2 microglial cells. Inhibition of NF-κB P65 phosphorylation reversed the effects of S100A8/A9 on the production of pro-inflammatory factors and chemokines. We also explored the effects of S100A8/A9 and S100A8/A9-activated BV-2 microglial cells on the viability of OPCs. The results showed that both the S100A8/A9 complex and the conditioned medium (CM) of the S100A8/A9-activated BV-2 microglial cells resulted in OPC apoptosis, which was more pronounced in the case of the CM treatment. However, OPC apoptosis in the CM group was obviously decreased through the inhibition of NF-κB p65 phosphorylation. This study indicates that S100A8/A9 induces the activation of BV-2 microglial cells and promotes the production of pro-inflammatory factors by activating the NF-κB signaling pathway, which further exacerbates OPC damage.

Renin-angiotensin system inhibition ameliorates CCl4-induced liver fibrosis in mice through the inactivation of nuclear transcription factor kappa B.

Therapeutic interventions for liver fibrosis are still limited due to the complicated molecular pathogenesis. Renin-angiotensin system (RAS) seems to contribute to the development of hepatic fibrosis. Therefore, we aimed to examine the effect of RAS inhibition on CCl4-induced liver fibrosis. Mice were treated with silymarin (30 mg·kg-1), perindopril (1 mg·kg-1), fosinopril (2 mg·kg-1), or losartan (10 mg·kg-1). The administration of RAS inhibitors improved liver histology and decreased protein expression of alpha smooth muscle actin (α-SMA) and hepatic content of hydroxyproline. These effects found to be mediated via inactivation of nuclear transcription factor kappa B (NFκB) pathway by the inhibition of NFκB p65 phosphorylation at the Ser536 residue and phosphorylation-induced degradation of nuclear factor kappa-B inhibitor alpha (NFκBia) subsequently inhibited NFκB-induced TNF-α and TGF-β1, leading to lower levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and vascular endothelial growth factor (VEGF). We concluded that the tissue affinity of the angiotensin converting enzyme inhibitors (ACEIs) has no impact on its antifibrotic activity and that interfering the RAS either through the inhibition of ACE or the blockade of AT1R has the same therapeutic benefit. These results suggest RAS inhibitors as promising candidates for further clinical trials in the management of hepatic fibrosis.

Modulation of the transcription factor NF-κB in antigen-presenting cells by bovine respiratory syncytial virus small hydrophobic protein.

Bovine respiratory syncytial virus (BRSV) is an important cause of respiratory disease in young cattle and is closely related to human RSV (HRSV), which causes severe respiratory disease in infants and the elderly. The RSV genome encodes a small hydrophobic (SH) protein with viroporin activity. Previous studies have shown that recombinant BRSV lacking the SH gene (rBRSVΔSH) is attenuated in the lungs, but not in the upper respiratory tract, of calves and mucosal vaccination with rBRSVΔSH induced long-lasting protective immunity. Attenuation of rBRSVΔSH may be due to the ability of this virus to induce an early innate response as rBRSVΔSH induces higher levels of pro-inflammatory cytokines than wild-type (wt) rBRSV. In this study, we investigated the effects of the BRSV SH protein on NF-κB p65 phosphorylation, a master step in the regulation of pro-inflammatory cytokines. Expression of SH resulted in the inhibition of NF-κB p65 phosphorylation in response to BRSV infection and extracellular lipopolysaccharide, and a reduction in the production of pro-inflammatory cytokines. In contrast, rBRSVΔSH does not inhibit NF-κB p65 phosphorylation in bovine antigen-presenting cells, including monocytes, macrophages and dendritic cells, resulting in increased expression of pro-inflammatory cytokines and increased activation of T cells compared to cells infected with wt BRSV. These findings highlight an important role for the BRSV SH protein in immune modulation.

Plumbagin Suppresses the Invasion of HER2-Overexpressing Breast Cancer Cells through Inhibition of IKKα-Mediated NF-κB Activation.

HER2-overexpressing breast cancers account for about 30% of breast cancer occurrences and have been correlated with increased tumor aggressiveness and invasiveness. The nuclear factor-κB (NF-κB) is overexpressed in a subset of HER2-positive breast cancers and its upregulation has been associated with the metastatic potential of HER2-overexpressing tumors. The present study aimed at determining the potential of plumbagin, a naturally occurring naphthoquinone, to inhibit the invasion of HER2-overexpressing breast cancer cells and determine the involvement of NF-κB inhibition in plumbagin-mediated cell invasion suppression. In the present research we showed that plumbagin inhibited the transcriptional activity of NF-κB in HER2-positive breast cancer cells. The suppression of NF-κB activation corresponded with the inhibition of NF-κB p65 phosphorylation and downregulation of NF-κB-regulated matrix metalloproteinase 9 (MMP-9) expression. Plumbagin suppressed the invasion of HER2-overexpressing breast cancer cells and the inhibition of cell invasion was associated with the ability of plumbagin to inhibit NF-κB transcriptional activity. The silencing of NF-κB p65 increased the sensitivity of HER2-overexpressing breast cancer cells to plumbagin-induced cell invasion inhibition. NF-κB inhibition was associated with IκB kinase α (IKKα) activity suppression and inhibition of IκBα phosphorylation and degradation. The knockdown of IKKα resulted in increased sensitivity of HER2-positive cells to plumbagin-induced suppression of NF-κB transcriptional activity and expression of MMP-9. In conclusion, plumbagin inhibits the invasion of HER2-overexpressing breast cancer cells through the inhibition of IKKα-mediated NF-κB activation and downregulation of NF-κB-regulated MMP-9 expression.

L-cystathionine inhibits oxidized low density lipoprotein-induced THP-1-derived macrophage inflammatory cytokine monocyte chemoattractant protein-1 generation via the NF-κB pathway.

This study aimed to explore whether and how L-cystathionine had any regulatory effect on the inflammatory response in THP-1-derived macrophages cultured in vitro under oxidized low-density lipoprotein (ox-LDL) stimulation. The human monocyte line THP-1 cell was cultured in vitro and differentiated into macrophages after 24 hours of PMA induction. Macrophages were pretreated with L-cystathionine and then treated with ox-LDL. The results showed that compared with the controls, ox-LDL stimulation significantly upregulated the expression of THP-1-derived macrophage MCP-1 by enhancing NF-κB p65 phosphorylation, nuclear translocation and DNA binding with the MCP-1 promoter. Compared with the ox-LDL group, 0.3 mmol/L and 1.0 mmol/L L-cystathionine significantly inhibited the expression of THP-1-derived macrophage MCP-1. Mechanistically, 0.3 mmol/L and 1.0 mmol/L L-cystathionine suppressed phosphorylation and nuclear translocation of the NF-κB p65 protein, as well as the DNA binding activity and DNA binding level of NF-κB with the MCP-1 promoter, which resulted in a reduced THP-1-derived macrophage MCP-1 generation. This study suggests that L-cystathionine could inhibit the expression of MCP-1 in THP-1-derived macrophages induced by ox-LDL via inhibition of NF-κB p65 phosphorylation, nuclear translocation, and binding of the MCP-1 promoter sequence after entry into the nucleus.

Synthetic Curcumin Analog UBS109 Inhibits the Growth of Head and Neck Squamous Cell Carcinoma Xenografts

The natural compound curcumin has been investigated as an anticancer agent in many cellular systems, in animal models and in the clinic. The overriding negative characteristics of curcumin are its low solubility, weak potency and poor bioavailability. We have examined the efficacy and mechanism of action of a synthetic curcumin analog, UBS109, in head and neck squamous cell carcinoma. By nephelometry, this analog exhibits considerably greater solubility than curcumin. Pharmacokinetic studies of a single oral dose of UBS109 in mice revealed that peak plasma concentrations were reached at 0.5 hours post-dose (Tmax) with average plasma concentrations (Cmax) of 131 and 248 ng/mL for oral doses of 50 and 150 mg/kg, respectively. The terminal elimination half-lives (T½) for these doses averaged 3.7 and 4.5 hours, respectively. In both in vitro and in vivo studies, we found that UBS109 decreased the levels of phosphorylated IKKβ and phosphorylated p65 and, unexpectedly, increased the levels of phosphorylated IκBα by Western blot analysis. These observations may suggest that UBS109 suppresses tumor growth through, in part, inhibition of NF-κB p65 phosphorylation by PKAc and not through IκBα. Finally, we demonstrate that UBS109 is efficacious in retarding the growth of Tu212 (head and neck) squamous cell carcinoma (SCC) xenograft tumors in mice and may be useful for treating head and neck SCC tumors.

The Bruton’s Tyrosine Kinase (BTK) Inhibitor PCI-32765 Inhibits Growth of ABC DLBCL Tumors In Vivo and in Vitro by Preventing Activation of Pro-Survival NF-κB pathways

Abstract 4969Bruton’s tyrosine kinase (BTK), a Tec family kinase essential to signaling via the B-cell Receptor (BCR) has emerged as a very promising drug target in lymphoma. PCI-32765, a potent and selective covalently-bound small molecule inhibitor of BTK (Honigberg, Proc. Natl Acad. Sci. USA 107:13075, 2010), has demonstrated promising clinical activity in a number of Phase I and II clinical trials in patients with chronic lymphocytic leukemia (CLL) (Byrd et al., ASCO 2011 Annual Meeting Abstracts, JCO29 (15): 8012, 2010) and several subtypes of B-cell Non-Hodgkin’s Lymphoma (NHL), including diffuse large B-cell lymphoma (DLBCL). Many cell lines derived from B-cell malignancies do not show in vitro growth inhibition by BTK inhibitors, perhaps because most established B-cell lines no longer depend on BCR stimulation for survival. However, some cell lines derived from the aggressive ABC (activated B-cell) subtype of DLBCL such as OCI-Ly10 and TMD8 are very sensitive to BTK inhibition by PCI-32765 (Davis et al, Nature463: 88, 2010). We show here that both of these cell lines are potently and irreversibly inhibited by PCI-32765 (EC50 continuous exposure < 10 nM and EC50 following a 1-hr pulse < 50 nM). Interestingly, 2 other ABC-DLBCL cell lines, OCI-Ly3 and HBL-1, in which the NF-κB pathway is activated by a mutation in CARD11 which is downstream of BTK, are not inhibited by PCI-32765. Both sensitive and resistant cells express Btk at similar levels, and the active site of BTK is fully occupied in the presence of low concentrations of the inhibitor as shown using a fluorescently-labeled affinity probe. We show that the sensitive TMD8 and OCI-Ly10 cells have chronically active BCR signaling to NF-κB which is dose-dependently inhibited by PCI-32765, as measured by comparing the phosphorylation and nuclear relocation of NF-κB p65 subunit. These cells also show inhibition of other major signaling pathways downstream of the BCR, including p-ERK, p-AKT, p-PLCγ and intracellular calcium flux, ultimately leading to NF-κB activation. The non-responsive OCI-Ly3 cells do not show inhibition of NF-κB p65 phosphorylation or nuclear relocation, although Btk is present and active, as shown by the probe assay and inhibition of p-AKT by PCI-32765. Furthermore, whole genome expression analysis reveals inhibition of mRNA expression of several NF-κB target genes in OCI-Ly10, particularly c-Myc, NF-κB subunits and several chemokines and cytokines, leading to downregulation of STAT3 signaling, NFAT signaling, cell cycle and cytokine-chemokine signaling pathways, none of which was observed in OCI-Ly3 cells. In vivo, PCI-32765 dose-dependently inhibited tumor growth in mice carrying OCI-Ly10 (and TMD8, ongoing) xenografts. Analysis of these tumors exhibited dose-dependent occupancy of the BTK active site as well as inhibition of signaling pathways. These results demonstrate the mechanistic basis of PCI-32765 inhibitory activity in ABC-DLBCL and support the ongoing clinical investigation of the therapeutic use of PCI-32765 in patients with this devastating disease. Disclosures:Balasubramanian:Pharmacyclics: Employment, Equity Ownership. Crowley:Pharmacyclics: Employment, Equity Ownership. Sirisawad:Pharmacyclics: Employment, Equity Ownership. Thiemann:Pharmacyclics: Employment, Equity Ownership. Chen:Pharmacyclics: Employment, Equity Ownership. Buggy:Pharmacyclics, Inc.: Employment, Equity Ownership.

Ectosomes Released by Polymorphonuclear Neutrophils Induce a MerTK-dependent Anti-inflammatory Pathway in Macrophages

At the earliest stage of activation, human polymorphonuclear neutrophils release vesicles derived directly from the cell surface. These vesicles, called ectosomes (PMN-Ect), expose phosphatidylserine in the outer membrane leaflet. They inhibit the inflammatory response of human monocyte-derived macrophages and dendritic cells to zymosan A (ZymA) and LPS and induce TGF-β1 release, suggesting a reprogramming toward a tolerogenic phenotype. The receptors and signaling pathways involved have not yet been defined. Here, we demonstrate that PMN-Ect interfered with ZymA activation of macrophages via inhibition of NFκB p65 phosphorylation and NFκB translocation. The MerTK (Mer receptor tyrosine kinase) and PI3K/Akt pathways played a key role in this immunomodulatory effect as shown using specific MerTK-blocking antibodies and PI3K inhibitors LY294002 and wortmannin. As a result, PMN-Ect reduced the transcription of many proinflammatory genes in ZymA-activated macrophages. In sum, PMN-Ect interacted with the macrophages by activation of the MerTK pathway responsible for down-modulation of the proinflammatory signals generated by ZymA.

Licochalcone A significantly suppresses LPS signaling pathway through the inhibition of NF-κB p65 phosphorylation at serine 276

Licorice root, Glycyrrhiza inflata, has been used as a traditional medicine for the treatment of bronchial asthma and inflammation; however, the mechanism of its anti-inflammatory activity has not been clarified. Here, we investigated the effect of Licochalcone A, a major component of G. inflata, on the LPS signaling pathway. We found that Licochalcone A remarkably inhibited LPS-induced NO production, and TNFα expression and MCP-1 expression in both RAW264.7 cells and primary macrophages. Furthermore, when injected with Licochalcone A prior to injection of LPS, the serum level of TNFα and MCP-1 in C57BL/6 mice was clearly decreased, indicating that Licochalcone A has a potent anti-inflammatory effect both in vitro and in vivo. Strikingly, Licochalcone A significantly inhibited LPS-induced NF-κB transcriptional activation; however, it had no effect on not only the phosphorylation and degradation of IκBα but also nuclear translocation and DNA binding activity of NF-κB p65. Interestingly, Licochalcone A markedly inhibited the phosphorylation of p65 at serine 276. As a result, it reduced NF-κB transactivation by preventing the interaction of p65 with p300. Taken together, Licochalcone A might contribute to the potent anti-inflammatory effect of G. inflata through the unique mechanism of NF-κB inhibition.