P65 DNA Binding Activity Research Articles

Isoflurane reduces endotoxin-induced oxidative, inflammatory, and apoptotic responses in H9c2 cardiomyocytes.

To investigate the protective effects of ISO on cardiomyocyte injury induced by lipopolysaccharide (LPS) in H9c2 cells. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The activities of LDH and CK in the supernatant of H9c2 cells with different treatments were determined using colorimetric assays to assess the conversion of pyruvic acid to lactic acid by LDH and that of triphosphate and creatine to phosphagen by CK. ISO significantly enhanced cell viability and alleviated the release of lactate dehydrogenase and creatine phosphate kinase in a dose-dependent manner in H9c2 cells treated with LPS. However, the protective effects of higher doses of ISO (1.4% and 2.1%) had no significant difference. Thus, 1.4% ISO was selected for subsequent experiments. ISO inhibited LPS-induced inflammatory responses, as evidenced by reduced expression of tumor necrosis factor-a, interleukin (IL)-1β, and IL-6; it also attenuated the activation of nuclear factor (NF)-kB p65, and the inhibition of NF-kB p65 DNA-binding activity in H9c2 cells. ISO also suppressed oxidative stress and enhanced antioxidant defense in LPS-treated H9c2 cells, as determined by decreased levels of reactive oxygen species and malondialdehyde, increased production of glutathione reductase, and enhanced superoxide dismutase and glutathione peroxidase activities. Moreover, ISO inhibited LPS-induced H9c2 cell apoptosis, as shown by reduced caspase-3 activity; downregulated expression of the pro-apoptotic procaspase-3, cleaved caspase-3, and Bax; and upregulated expression of the anti-apoptotic Bcl-2. These findings indicate that ISO reduced LPS-induced H9c2 cell injury via anti-inflammatory, anti-oxidative, and anti-apoptotic activities; hence, ISO may be an alternative therapy for septic heart injury.

Caffeic acid phenethyl ester inhibits the progression of ovarian cancer by regulating NF-κB signaling

Ovarian cancer is one of the most common malignancies in women and characterized by a rapid progression to metastasis, which restricts effective treatment options. Caffeic acid phenethyl estre (CAPE), a natural honeybee product, exhibits a variety of biological activities, anti-tumor is included. Our study aims to explore whether CAPE could inhibit the progression of ovarian cancer and the underlying molecular mechanism. The establishment of ovarian cancer model was set up in mice through caudal vein injection of SKOV-3 cells. Results indicated that CAPE treatment remarkably decreased the viability, migration and invasion of SKOV-3 cells. Besides that, the apoptosis of SKOV-3 cells was significantly promoted by CAPE treatment. Moreover, the growth of ovarian cancer was tremendously inhibited by CAPE in vivo and its action was accompanied by the obstructed Ki67 and PCNA expression. Furthermore, nuclear factor kappa b (NF-κB) pathway was dramatically suppressed by CAPE through the inhibition of IκB phosphorylation, nuclear translocation of p65 and NF-κB p65 DNA binding activity. By contrast, cells transfected with p65 siRNA exhibited decreased cell viability, migration and invasion along with increased cell apoptosis in SKOV-3 cells. However, CAPE treatment could enhance these alters induced by p65 siRNA in KOV-3 cells. Taken together, these findings suggested that CAPE could restrain the progression of ovarian cancer via inactivating NF-κB signaling, and may provide novel therapeutic regimens for ovarian cancer.

Modulation of nuclear factor-kappa B activation by the endoplasmic reticulum stress sensor PERK to mediate estrogen-induced apoptosis in breast cancer cells

Stress responses are critical for estrogen (E2)-induced apoptosis in E2-deprived breast cancer cells. Nuclear factor-kappa B (NF-κB) is an important therapeutic target to prevent stress responses in chronic inflammatory diseases including cancer. However, whether E2 activates NF-κB to participate in stress-associated apoptosis in E2-deprived breast cancer cells is unknown. Here, we demonstrated that E2 differentially modulates NF-κB activity according to treatment time. E2 initially has significant potential to suppress NF-κB activation; it completely blocks tumor necrosis factor alpha (TNFα)-induced activation of NF-κB. We found that E2 preferentially and constantly enhances the expression of the adipogenic transcription factor CCAAT/enhancer binding protein beta (C/EBPβ), which is responsible for the suppression of NF-κB activation by E2 in MCF-7:5C cells. Interestingly, NF-κB p65 DNA-binding activity is increased when E2 is administered for 48 h, leading to the induction of TNFα and associated apoptosis. Blocking the nuclear translocation of NF-κB can completely prevent the induction of TNFα and apoptosis induced by E2. Further examination revealed that protein kinase RNA-like endoplasmic reticulum kinase (PERK), a stress sensor of unfolded protein response (UPR), plays an essential role in the late activation of NF-κB by E2. This modulation between PERK and NF-κB is mainly mediated by a stress responsive transcription factor, transducer and activator of transcription 3 (STAT3), independently of the classic canonical IκBα signaling pathway. Thus, inhibition of PERK kinase activity completely blocks the DNA binding of both STAT3 and NF-κB, thereby preventing induction of NF-κB-dependent genes and E2-induced apoptosis. All of these findings suggest that PERK is a key regulator to convey stress signals from the endoplasmic reticulum to the nucleus and illustrate a crucial role for the novel PERK/STAT3/NF-κB/TNFα axis in E2-induced apoptosis in E2-deprived breast cancer cells.

Resolvin D1 attenuates imiquimod-induced mice psoriasiform dermatitis through MAPKs and NF-κB pathways

BackgroundResolvin D1 (RvD1), a pro-resolution lipid mediator derived from docosahexaenoic acid (DHA), has been described to promote several kinds of inflammatory resolution. However, the effects and anti-inflammatory mechanisms of RvD1 on psoriasis have not been previously reported. ObjectiveThe present study aimed to determine the protective effects and the underlying mechanisms of RvD1 on imiquimod (IMQ)-induced psoriasiform dermatitis. MethodsMice were topically treated with IMQ to develop psoriasiform dermatitis on their shaved back, pretreated intraperitoneally (i.p.) with or without RvD1 or tert-butoxycarbonyl Met-Leu-Phe peptide (Boc), a lipoxin A4 (ALX) receptor antagonist. The severity was monitored and graded using a modified human scoring system, the Psoriasis Area and Severity Index (PASI), histopathology, and the signature cytokines of psoriasis (IL-23, IL-17, IL-22 and TNF-α). The mRNA and protein levels of inflammatory cytokines were quantified by quantitative real-time PCR (QRT-PCR) and ELISA. The expressions of signaling proteins MAPKs and NF-κB p65 were analyzed using western blotting. Electrophoretic mobility shift assay (EMSA) was used to check NF-κB p65 DNA binding activity. ResultsOur study showed that RvD1 alleviated IMQ-induced psoriasiform dermatitis and improved skin pathological changes. RvD1 markedly inhibited IMQ-induced activation of ERK1/2, p38, JNK (c-Jun N-terminal protein kinase, a subfamily of MAPKs), and NF-κB. Furthermore, pretreatment with Boc, would not exacerbate skin inflammation of IMQ-induced mice, but significantly reversed the beneficial effects of RvD1 on IMQ-induced psoriasiform inflammation. ConclusionRvD1 can obviously improve skin inflammation in IMQ-induced mice psoriasiform dermatitis. The protective mechanisms might be related to its selective reaction with lipoxin A4 receptor/Formyl-peptide receptor 2 (ALX/FPR2), by downregulating relevant cytokines of the IL-23/IL-17 axis expression, the inhibition of MAPKs and NF-κB signaling transduction pathways. Thus, these results show that RvD1 could be a possible candidate for psoriasis therapy.

Clinical efficacy of recombinant human latrophilin 3 antibody in the treatment of pediatric asthma.

Pediatric asthma is a chronic pulmonary inflammatory disease featuring hypersecretion of mucus and inflammation in the airway, resulting in dysfunction of the airway smooth muscle. Previous evidence demonstrated that latrophilins, a novel family of receptors, present a beneficial effect on airway smooth muscle cells. In the present study, the therapeutic effects of recombinant human latrophilin 3 (rhLPHN3) antibody (Ab) in patients with pediatric asthma were investigated, and the molecular mechanism underlying the function of LPHN3 in the treatment of asthma in clinical practice was examined. A total of 342 pediatric asthma cases were recruited and randomly divided into three groups, receiving treatment with rhLPHN3 Ab (n=134), salbutamol (n=108) or montelukast (n=100) by nasal aerosolization. Each group received the respective clinically tested dose for 16 weeks. Inflammatory factors interleukin (IL)-10, IL-17, IL-4, matrix metallopeptidase-9 (MMP-9), interferon-γ (IFN-γ) and transforming growth factor-β (TGF-β) levels in peripheral blood mononuclear cells were analyzed prior to and post treatment. The clinical outcomes revealed that pathological alterations were significantly improved following treatment with rhLPHN3 Ab for patients with pediatric asthma when compared with those receiving salbutamol and montelukast. It was also observed that rhLPHN3 Ab downregulated the plasma concentration levels of IL-10, IL-17, IL-4 and MMP-9, and upregulated IFN-γ and TGF-β levels in the three groups. In addition, clinical data demonstrated that rhLPHN3 Ab significantly promoted E-selectin and mucin 5AC expression, as well as improved the activation of nuclear factor (NF)-κB p65 DNA binding activity and the phosphorylation levels of protein kinase A. Furthermore, rhLPHN3 Ab markedly improved adhesion and proliferation of airway smooth muscle cells, which led to promotion of the contraction of these cells. In conclusion, these clinical data suggest that rhLPHN3 Ab serves an important role in the inhibition of inflammatory mediators through downregulation of NF-κB signaling pathway, which contributes to airway remodeling and bronchodilation in patients with pediatric asthma.

Berberine suppresses LPS-induced inflammation through modulating Sirt1/NF-κB signaling pathway in RAW264.7 cells

Chronic inflammation is a major contributing factor in the pathogenesis of many diseases. Natural product berberine (BBR) exhibits potent anti-inflammatory effect in vitro and in vivo, while the underlying mechanisms remain elusive. Sirt1, a NAD+-dependent protein deacetylase, was recently found to play an important role in modulating the development and progression of inflammation. Thus, we speculate that Sirt1 might mediate the inhibitory effect of BBR on inflammation. In LPS-stimulated RAW264.7 macrophages, BBR treatment significantly downregulated the expression of proinflammatory cytokines such as MCP-1, IL-6 and TNF-α. Importantly, BBR potently reversed LPS-induced down-regulation of Sirt1. Consistently, the inhibitory effects of BBR on proinflammatory cytokines expression was largely abrogated by Sirt1 inhibition either by EX527, a Sirt1 inhibitor or Sirt1 siRNA. Further mechanistic studies revealed that BBR-induced inhibition of NF-κB is Sirt1-dependent, as either pharmacologically or genetically inactivating Sirt1 enhanced the IκΒα degradation, IKK phosphorylation, NF-κB p65 acetylation and DNA-binding activity. Taken together, our results provide the first evidence that BBR potently suppressed inflammatory responses in macrophages through inhibition of NF-κB signaling via Sirt1-dependent mechanisms.

Thiamine antagonists trigger p53-dependent apoptosis in differentiated SH-SY5Y cells

Accumulating evidences suggest that p53 is a key coordinator of cellular events triggered by oxidative stress often associated with the impairment in thiamine metabolism and its functions. However, there are limited data regarding the pursuant feedback between p53 transactivation and thiamine homeostasis. Impairment in thiamine metabolism can be induced experimentally via interference with the thiamine uptake and/or inhibition of the thiamin pyrophosphate–dependent enzymes using thiamine antagonists - amprolium (AM), oxythiamine (OT) or pyrithiamine (PT). We found that exposure of neuronally differentiated SH-SY5Y cells to AM, OT and PT triggered upregulation of p53 gene expression, post-translational modification of p53 via phosphorylation and activation of p53 DNA-binding activity. Phosphorylation of p53 at Ser20 was equally efficient in upregulation of thiamine transporter 1 (THTR1) by all antagonists. However, induction of the expressions of the pyruvate dehydrogenase E1 component subunit beta (PDHB) and oxoglutarate dehydrogenase (OGDH) required dual phosphorylation of p53 at Ser9 and Ser20, seen in cells treated with PT and OT. Moreover, pretreatment of the cells with a decoy oligonucleotide carrying wild-type p53-response element markedly attenuated OT-induced THTR1, PDHB and OGDH gene expression suggesting an important role of p53 in transactivation of these genes. Finally, analysis of gene and metabolic networks showed that OT triggers cell apoptosis through the p53-dependent intrinsic pathway.

Abstract LB-016: Angiogenin contributes to bladder cancer tumorigenesis by facilitating p53 mediated activation of MMP2

Abstract Angiogenin (ANG) has been reported to be upregulated and associated with poor prognosis in some human cancers. Previous studies revealed ANG expression stimulated matrix metallopeptidase-2 (MMP2) expression through a) phosphorylation of ERK1/2 (ANG-ERK1/2-MMP2 pathway), which led to a pronounced tumorigenesis, and b) hypomethylation of the MMP2 gene by negatively regulating DNA methyltransferase 3b (DNMT3b) expression (ANG-DNMT3b-MMP2 pathway). However, since ANG-DNMT3b-MMP2 pathway is independent of ANG-ERK1/2-MMP2, the exact mechanism through which ANG-induced MMP2 expression remains unclear. Here we show that ANG-induced p53 expression is regulated by ERK1/2 and is a missing link between ERK1/2 and MMP2 in ANG-ERK1/2-MMP2 axis. The analysis of the serial deletion mutant MMP2 promoters demonstrated that the p53-binding site is critical for ANG to induce MMP2 promoter activity. We also found that ANG increased the DNA binding activity of p53, which was suppressed by knockdown of p53 leading to the reduction in MMP2 expression. Remarkably, we revealed that mutated p53 isoforms were able to induce MMP2 expression, illustrating that in this setting, the primary driver of MMP2 expression is the amount of p53 protein (wild-type and mutants) available to bind to the MMP2 promoter. ChIP-qPCR and promoter assay using MMP2 promoter constructs confirmed that both wild-type and p53 mutants (V143A, R175H, R248W, R249S and R273H) increased MMP2 promoter transcriptional activities in BCa cell lines. We also performed computational modeling to support energetic and structural feasibility of our wild-type and mutant p53 observations. We predicted that p53 mutants can bind to p53 binding sites on the MMP2 promoter with binding energy comparable to wild type. The mutants may have induced, however, changes on the expression of other p53 targets. In addition, we present data that the p53 mutants are able to induce cell growth in bladder cells, in opposition to the wild type p53. Finally, the results from human bladder tumors a) confirmed the strong correlation between ANG, p53 and MMP2 and b) reduced survival when ANG, p53 and/or MMP2 were overexpressed in human bladder tumors. We provide multiple lines of evidence indicting ANG oncogenic potential. Thus, we suggest that targeting ANG and associated factors could provide a novel strategy to inhibit tumor angiogenesis and growth. Citation Format: Rafael Peres, Hideki Furuya, Kanani Hokutan, Yoshiko Shimizu, Owen T. Chan, Ian Pagano, Lars Dyrskjøt, Jørgen B. Jensen, Per-Uno Malmström, Ulrika Segersten, Ho Leung Ng, Filip Janku, Charles J. Rosser. Angiogenin contributes to bladder cancer tumorigenesis by facilitating p53 mediated activation of MMP2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-016. doi:10.1158/1538-7445.AM2017-LB-016

Abstract 1534: The significance of a p53 gain-of-function mutation in the acquisition of metastatic traits in the SW13 cell line

Abstract The therapeutic potential of histone deacetylase inhibitors (HDACi), shown to epigenetically regulate a variety of genes, has yet to be fully realized due to their documented pleiotropic effects in vivo. Currently, four HDACi are FDA-approved for the treatment of hematological malignancies, but their effectiveness against other types of cancers have been shown to be limited at best and, as a result, most of these clinical trials have failed. A comprehensive review of the recent literature has identified that approximately 50% of all cell lines treated with an HDACi demonstrate an increase in metastatic potential as measured by increases in migration and invasion. Of these cell lines that respond with increased metastatic potential to HDACi treatment, 60% have a gain-of-function mutation within p53 (p=0.03). As HDACi have been shown to destabilize mutant p53, they have been used in studies to examine the effects of HDACi treatment on cancers with an identified p53 mutation. This is concerning as the specific p53 mutation in these cancers is not always known, and thus treatment could potentially result in an increase in cancer cell growth. Previous work in our lab demonstrates that the human SW13 adrenal adenocarcinoma cell line, which harbors a homozygous H193Y gain-of-function mutation, does have an HDACi-induced metastatic phenotype; treated cells demonstrate significant increases in MMP activity and ~50% reduction in p53 protein expression compared to untreated cells. To investigate the role that a decrease in mutant p53 expression plays in the acquisition of metastatic characteristics we utilized stable shRNA plasmids to knock-down p53 expression in the SW13 cell line. HeLa cells, which possess a wild-type p53 gene, were used as a positive control. We found that a decrease in p53 expression alone was not sufficient to induce changes in SW13 growth or invasiveness. In fact, even though p53 expression was knocked down by ~70% in the shRNA lines, HDAC inhibition still induced the acquisition of metastatic properties in these cells. Intriguingly, while the DNA binding activity of mutant p53 was correlated with protein expression and was significantly decreased following HDAC inhibition, the same response was not observed in HeLa cells. These findings suggest that p53 status may be an important determinant of therapeutic response to HDAC inhibition. Note: This abstract was not presented at the meeting. Citation Format: McKale Montgomery, Elizabeth Hull. The significance of a p53 gain-of-function mutation in the acquisition of metastatic traits in the SW13 cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1534. doi:10.1158/1538-7445.AM2017-1534

Kaempferol ameliorates H9N2 swine influenza virus-induced acute lung injury by inactivation of TLR4/MyD88-mediated NF-κB and MAPK signaling pathways.

Kaempferol, a very common type of dietary flavonoids, has been found to exert antioxidative and anti-inflammatory properties. The purpose of our investigation was designed to reveal the effect of kaempferol on H9N2 influenza virus-induced inflammation in vivo and in vitro. In vivo, BALB/C mice were infected intranasally with H9N2 influenza virus with or without kaempferol treatment to induce acute lung injury (ALI) model. In vitro, MH-S cells were infected with H9N2 influenza virus with or without kaempferol treatment. In vivo, kaempferol treatment attenuated pulmonary edema, the W/D mass ratio, pulmonary capillary permeability, myeloperoxidase (MPO) activity, and the numbers of inflammatory cells. Kaempferol reduced ROS and Malondialdehyde (MDA) production, and increased the superoxide dismutase (SOD) activity. Kaempferol also reduced overproduction of TNF-α, IL-1β and IL-6. In addition, kaempferol decreased the H9N2 viral titre. In vitro, ROS, MDA, TNF-α, IL-1β and IL-6 was also reduced by kaempferol. Moreover, our data showed that kaempferol significantly inhibited the upregulation of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), phosphorylation level of IκBα and nuclear factor-κB (NF-κB) p65, NF-κB p65 DNA binding activity, and phosphorylation level of MAPKs, both in vivo and in vitro. These results suggest that kaempferol exhibits a protective effect on H9N2 virus-induced inflammation via suppression of TLR4/MyD88-mediated NF-κB and MAPKs pathways, and kaempferol may be considered as an effective drug for the potential treatment of influenza virus-induced ALI.

Apocynin suppressed the nuclear factor-κB pathway and attenuated lung injury in a rat hemorrhagic shock model.

The aim of this study was to investigate whether a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) inhibitor, apocynin, reduces reactive oxygen species (ROS) production, suppresses the nuclear factor κB (NF-κB) pathway, attenuates lung injury, and improves survival in rat hemorrhagic shock (HS) model. Blood was drawn from male Sprague-Dawley rats (290-340 g) to maintain a mean arterial pressure of 20-25 mm Hg for 40 minutes. The rats were resuscitated with the drawn blood, and a vehicle (HS), a low dose of apocynin (20 mg/kg, LD-Apo), or a high dose of apocynin (40 mg/kg, HD-Apo) was administered intraperitoneally. The survival of the rats was observed for 72 hours. Then, a separated set of rats was euthanized at 6 hours post-HS induction. We measured gp91-phox (Nox2) expression, Nox activity, cytoplasmic phosphorylated inhibitor κB-α (p-IκB-α) expression, NF-κB p65 DNA-binding activity, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) gene expressions, malondialdehyde (MDA) level, myeloperoxidase (MPO) activity, and histological damage in the lung tissues. The survival rates of the sham, HS, HS + LD-Apo, and HS + HD-Apo groups were 100% (5/5), 30% (3/10), 40% (4/10), and 70% (7/10), respectively. A high dose of apocynin decreased gp91-phox expression, Nox activity, and MDA level in the lung tissues during HS and resuscitation. It also decreased p-IκB-α expression, NF-κB p65 DNA-binding activity, TNF-α and IL-6 gene expressions, and MPO activity in the lung tissues and attenuated histological lung injuries. However, a low dose of apocynin failed to show these benefits. The administration of a high dose of apocynin inhibited Nox2 expression and Nox activity, reduced lipid peroxidation, suppressed the NF-κB pathway and subsequent pro-inflammatory cytokines transcription in the lung tissues, and attenuated lung injury during HS and resuscitation in rats.

Ribosomal protein S3 gene silencing protects against experimental allergic asthma.

Ribosomal protein S3 (RPS3) is a 40S ribosomal protein of the S3P family essential for implementing protein translation. RPS3 has recently been found to interact with the p65 subunit of the NF-κB complex and promote p65 DNA-binding activity. Persistent activation of the NF-κB pathway is evident in allergic asthma. We hypothesized that gene silencing of lung RPS3 can ameliorate allergic airway inflammation. The gene silencing efficacy of RPS3 siRNA was screened in three different mouse cell lines by real-time PCR and immunoblotting. Protective effects of intratracheal RPS3 siRNA in a house dust mite (HDM) mouse asthma model were determined by measuring cell counts in lung lavage fluid and lung sections, lung cytokine profiles and airway hyperresponsiveness (AHR). RPS3 siRNA markedly knocked down RPS3 levels in all mouse cell lines tested, and in mouse lung tissues, blocked TNF-α- or HDM-induced release of mediators by the cultured cells and reduced eosinophil counts in lung lavage fluid from the HDM mouse asthma model. RPS3 siRNA lessened HDM-induced airway mucus hypersecretion, cytokine production and serum IgE elevation. Moreover, RPS3 knockdown significantly suppressed methacholine-induced AHR in experimental asthma. RPS3 siRNA disrupted TNF-α-induced NF-κB activation in a NF-κB reporter gene assay in vitro and prevented the nuclear accumulation of p65 subunit and p65 transcriptional activation in HDM-challenged lungs and cells. RPS3 gene silencing ameliorates experimental asthma, probably by disrupting NF-κB activity. RPS3 could be a novel therapeutic target for allergic airway inflammation.

The transient manifold structure of the p53 extreme C-terminal domain: insight into disorder, recognition, and binding promiscuity by molecular dynamics simulations.

The p53 tumour suppressor is a transcription activator that signals for cell cycle arrest and apoptosis. In its active form p53 is a tetramer, with each monomer organised in domains with different degrees of structural stability, ranging from the well folded DNA-binding domain (DBD) and tetramerization domain (TET), to the intrinsically disordered transactivation domain (TAD), and extreme C-terminal domain (CTD). Compared to all other domains, the structure/function relationship of the p53-CTD within the full-length p53 tetramer is still poorly understood due to its high degree of conformational disorder. Meanwhile, the structure of p53-CTD-like peptides has been well characterized when in complex with a variety of receptors, where, as other intrinsically disordered regions (IDR), it adopts specific, while diverse, conformations. Receptor-specific folding is likely to occur upon binding, either from a random coil, or as a result of an initial recognition of a pre-formed structural motif, known as molecular recognition feature (MoRF), selected by the receptor within the conformational ensemble of the IDP in solution. In this latter case, MoRFs act as nucleation sites, favouring the initiation of the folding process within the binding site. In this work we show the results of over 20 μs of cumulative molecular dynamics (MD) simulations of a 22 residue peptide unbound in solution with sequence corresponding to the p53-CTD 367-388 section. Such extensive sampling allowed us to identify and characterize the structure of specific sets of minimal structural MoRFs within the p53-CTD peptide conformational ensemble at equilibrium. These motifs are short, involving only 3 to 4 residues, and specifically localized within the peptide sequence. Corresponding patterns of secondary structure propensity along the p53-CTD sequence are also predicted by disorder prediction calculations. Based on these findings we discuss how the structural complementarity of specific minimal structural MoRFs to the binding site of different receptors could regulate the p53-CTD binding promiscuity.

Concurrent Treatment with Taxifolin and Cilostazol on the Lowering of β-Amyloid Accumulation and Neurotoxicity via the Suppression of P-JAK2/P-STAT3/NF-κB/BACE1 Signaling Pathways

Taxifolin is a potent flavonoid that exerts anti-oxidative effect, and cilostazol increases intracellular cAMP levels by inhibiting phosphodiesterase 3 that shows antiinflammatory actions. BACE1 (β-site APP cleaving enzyme 1) is the rate-limiting enzyme responsible for the β-cleavage of amyloid precursor proteins to Aβ peptides. In this study, endogenous Aβ and C99 accumulation was explored in N2a Swe cells exposed to 1% FBS medium. Increased Aβ and C99 levels were significantly attenuated by taxifolin alone and in combination with cilostazol. Increased phosphorylated JAK2 at Tyr1007/1008 (P-JAK), phosphorylated STAT3 at Tyr 705 (P-STAT3) expressions and increased expressions of BACE1 mRNA and protein in the activated N2a Swe cells were significantly attenuated by taxifolin (10~50 μM), cilostazol (10~50 μM) alone and in combination at minimum concentrations. In these cells, decreased cytosol IκBα expression was elevated, and increased nuclear NF-κB p65 level and nuclear NF-κB p65 DNA binding activity were significantly reduced by taxifolin and cilostazol in a similar manner. Following STAT3 gene (~70% reduction) knockdown in N2a cells, Aβ-induced nuclear NF-κB and BACE1 expressions were not observed. Taxifolin, cilostazol, or resveratrol significantly stimulated SIRT1 protein expression. In SIRT1 gene-silenced (~50%) N2a cells, taxifolin, cilostazol, and resveratrol all failed to suppress Aβ1-42-stimulated P-STAT3 and BACE1 expression. Consequently, taxifolin and cilostazol were found to significantly decrease lipopolysaccharide (1–10 μg/ml)-induced iNOS and COX-2 expressions, and nitrite production in cultured BV-2 microglia cells and to increase N2a cell viability. In conclusion, taxifolin and cilostazol strongly inhibited amyloidogenesis in a synergistic manner by suppressing P-JAK2/P-STAT3-coupled NF-κB-linked BACE1 expression via the up-regulation of SIRT1.

Gambogic acid inhibits the growth of ovarian cancer tumors by regulating p65 activity

Ovarian cancer patients often have poor prognosis, therefore, it is important to search for more effective therapeutic strategies to treat them. Gambogic acid (GA) exhibits an anti-tumor effect through various mechanisms, and has multiple targets in tumor cells. The present study aimed to elucidate the efficacy of GA in the treatment of ovarian cancer both in vivo and in vitro by analyzing its impact on cell survival and tumor growth through cell cycle and apoptosis analysis. GA inhibited the growth of ovarian cancer cells in a dose and time dependent manner, and arrested the cell cycle in ovarian cancer cells. Furthermore, GA increased caspase-3 and caspase-9 activity and inhibited RELA/NF-κB p65 (p65) DNA binding activity. Finally, GA suppressed tumor growth in vivo. Therefore, the current study suggests that GA inhibits the growth of ovarian cancer by regulating p65 activity, and may be developed as a novel therapeutic strategy to treat ovarian cancer.

Abstract 084: Sodium Butyrate and Retinoic Acid Attenuate Renal Inflammation and Fibrosis in Npr1 Gene-targeted Haplotype Mice: Role of NF-κB Signaling

The objective of the present study was to determine the effect of a hybrid drug of sodium butyrate (NaBu), a histone deacetylase (HDAC) inhibitor and all-trans retinoic acid (ATRA) on the attenuation of renal inflammation and fibrosis in Npr1 gene-disrupted mutant mice. Adult (18-20 week old) male Npr1 gene-disrupted heterozygous (1-copy; Npr1 +/- ) wild-type (2-copy; Npr1 +/+ ), and gene-duplicated (3-copy; Npr1 ++/+ ) mice were treated with ATRA-NaBu hybrid drug (1.0 mg/kg/day) by intraperitoneal injections for 2-weeks. A significant decrease in systolic blood pressure was observed in ATRA-NaBu-treated haplotype Npr1 +/- mice compared with untreated controls (treated, 113.3 ± 1.5 vs. control, 130.4 ± 1.9; p < 0.01). After treatment with ATRA-NaBu, a marked reduction in tubulo-interstitial fibrosis (50%, p < 0.001) and decreased renal collagen type I alpha 2 expression by 55% (treated, 25.9 ± 1.2 vs. control, 57.2 ± 2.9, p < 0.001) was observed in Npr1 +/- mice. Treatment with ATRA-NaBu also increased creatinine clearance (ml/24 h) in Npr1 +/- mice (245.7 ± 24.3 vs. control, 83.8 ± 3.9). Similarly, higher urinary albumin to creatinine ratio was detected in Npr1 +/- mice (0.84 ± 0.03) vs. control (0.35 ± 0.03; p < 0.01) and a complete reversal was observed in drug-treated Npr1 +/- mice (0.39 ± 0.05). Significant decreases were observed in renal (pg/mg protein) tumor necrosis factor-alpha (TNF-α) (4.1 ± 0.7 vs. control, 27.2 ± 2.6; p < 0.01) and interleukin (IL)-6 (11.8 ± 0.8 vs. control, 59.9 ± 3.6; p < 0.01) in ATRA-NaBu-treated Npr1 +/- mice. Western blot analyses showed significant reduction in renal TNF-α and IL-6 protein expression by 54%, (p < 0.001) and 61%, (p < 0.01), respectively, in ATRA-NaBu-treated Npr1 +/- mice. There was 49% increase in renal NF-κB (p65) DNA binding activity in Npr1 +/- mice and 51% lower activity in Npr1 +/++ mice compared with wild-type mice. Western blot analysis revealed distinctly higher levels of renal NF-κB (p65) protein expression in Npr1 +/- mice and reduced levels in Npr1 +/++ mice compared with wild-type controls. The present results provide direct evidence that ATRA-NaBu hybrid drug acts as a potent anti-inflammatory agent, which will have important implications in the pathophysiology of renal injury and hypertension.

Berberine improves airway inflammation and inhibits NF-κB signaling pathway in an ovalbumin-induced rat model of asthma

Objectives: Berberine has been reported for its various activities including anti-inflammatory effects and has been used in treating many diseases. However, its effects on airway inflammation in asthma have not been investigated. This study mainly aimed to detect its effects on the airway inflammation and the nuclear factor-κB (NF-κB) signaling pathway activity in a rat model of asthma. Methods: Asthma was induced by ovalbumin (OVA) sensitization and challenge. The asthmatic rats were respectively treated with vehicle PBS or berberine (100 mg/kg or 200 mg/kg) for 28 days. The control rats were treated with PBS. Inflammatory cells in bronchoalveolar lavage fluid (BALF) were counted and the lung inflammation was scored. Levels of NF-κB p65 (mRNA and protein), phosphorylated NF-κB p65 (p-NF-κB p65), inhibitory κB alpha (IκBα) (mRNA and protein) and phosphorylated IκBα (p-IκBα), as well as NF-κB p65 DNA-binding activity, were measured to assess the activity of NF-κB signaling pathway. Levels of the downstream inflammatory mediators of NF-κB signaling, IL-1β, IL-4, IL-5, IL-6, IL-13 and IL-17 in BALF, were measured. Besides, the serum levels of OVA-specific immunoglobulin (Ig)E were measured. Results: Results showed that OVA increased the number of inflammatory cells in BALF, elevated lung inflammation scores, enhanced the NF-κB signaling activity and promoted the production of IgE in rats. Berberine dose-dependently reversed the alterations induced by OVA in the asthmatic rats. Conclusions: The findings suggested a therapeutic potential of berberine on OVA- induced airway inflammation. The ameliorative effects on the OVA-induced airway inflammation might be associated with the inhibition of the NF-κB signaling pathway.

Aronia melanocarpa Concentrate Ameliorates Pro-Inflammatory Responses in HaCaT Keratinocytes and 12-O-Tetradecanoylphorbol-13-Acetate-Induced Ear Edema in Mice.

Abnormal expression of pro-inflammatory mediators such as cell adhesion molecules and cytokines has been implicated in various inflammatory skin diseases, including atopic dermatitis. In this study, we investigated the anti-inflammatory activity of Aronia melanocarpa concentrate (AC) and its action mechanisms using in vivo and in vitro skin inflammation models. Topical application of AC on mouse ears significantly suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema formation, as judged by measuring ear thickness and weight, and histological analysis. Topical administration of AC also reduced the expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 in TPA-stimulated mouse ears. Pretreatment with AC suppressed TNF-α-induced ICAM-I expression and subsequent monocyte adhesiveness in human keratinocyte cell line HaCaT. In addition, AC significantly decreased intracellular reactive oxygen species (ROS) generation as well as mitogen-activated protein kinase (MAPK) activation in TNF-α-stimulated HaCaT cells. AC and its constituent cyanidin 3-glucoside also attenuated TNF-α-induced IKK activation, IκB degradation, p65 phosphorylation/nuclear translocation, and p65 DNA binding activity in HaCaT cells. Overall, our results indicate that AC exerts anti-inflammatory activities by inhibiting expression of pro-inflammatory mediators in vitro and in vivo possibly through suppression of ROS-MAPK-NF-κB signaling pathways. Therefore, AC may be developed as a therapeutic agent to treat various inflammatory skin diseases.

Optineurin suppression activates the mediators involved in the terminal effector pathways of human labour and delivery

Spontaneous preterm birth remains the major cause of neonatal death and morbidity. Studies in non-gestational tissues report that optineurin (OPTN) is critical in the termination of NFKB1 activity and control of inflammation, central features of spontaneous preterm birth. The aims of the present study were to determine: (1) OPTN expression in fetal membranes and the myometrium during labour; (2) the effects of IL1B on OPTN expression in primary myometrial cells; and (3) the effects of OPTN short interference (si) RNA on IL1B-stimulated proinflammatory and prolabour mediators. OPTN mRNA and protein expression was significantly decreased with spontaneous term labour in fetal membranes and the myometrium. Although there was no effect of spontaneous preterm labour on OPTN expression in fetal membranes, there was decreased OPTN expression in membranes with chorioamnionitis and myometrial cells treated with 1ng mL-1 IL1B for 1 or 6h. In cells transfected with OPTN siRNA, significant increases were seen in IL1B-stimulated IL6, tumour necrosis factor, CXCL8 and monocyte chemoattractant protein-1 mRNA expression and release, cyclo-oxygenase-2 and prostanoid PTGFR receptor mRNA expression and the release of prostaglandin F2α. There was no change in IL1B-stimulated NFKBIA expression; however, there was increased NFKB1 p65 DNA-binding activity. The results of the present study suggest that OPTN is a negative regulator of inflammation-induced prolabour mediators.

Secondary interaction between MDMX and p53 core domain inhibits p53 DNA binding

The MDMX oncoprotein is an important regulator of tumor suppressor p53 activity during embryonic development. Despite sequence homology to the ubiquitin E3 ligase MDM2, MDMX depletion activates p53 without significant increase in p53 level, implicating a degradation-independent mechanism. We present evidence that MDMX inhibits the sequence-specific DNA binding activity of p53. This function requires the cooperation between MDMX and CK1α, and phosphorylation of S289 on MDMX. Depletion of MDMX or CK1α increases p53 DNA binding without stabilization of p53. A proteolytic fragment release assay revealed that in the MDMX-p53 complex, the MDMX acidic domain and RING domain interact stably with the p53 DNA binding domain. These interactions are referred to as secondary interactions because they only occur after the canonical-specific binding between the MDMX and p53 N termini, but exhibit significant binding stability in the mature complex. CK1α cooperates with MDMX to inhibit p53 DNA binding by further stabilizing the MDMX acidic domain and p53 core domain interaction. These results suggest that secondary intermolecular interaction is important in p53 regulation by MDMX, which may represent a common phenomenon in complexes containing multidomain proteins.