P65 DNA Binding Activity Research Articles

IL-17A Promotes the Migration and Invasiveness of Colorectal Cancer Cells Through NF-κB-Mediated MMP Expression.

Interleukin-17A (IL-17A) plays a significant role in many inflammatory diseases and cancers. The aim of this study is to investigate the effect of IL-17A on the invasiveness of colorectal cancer. In the study, we found that IL-17A could promote the migration and invasion of colorectal cancer cells. Furthermore, after being treated with IL-17A, the expression and activity of matrix metalloproteinase 2 (MMP-2) and MMP-9 were upregulated. Moreover, the nuclear/overall fractions and DNA-binding activity of p65 and p50 were dramatically elevated by IL-17A. Pretreatment with a nuclear factor-κB (NF-κB) inhibitor (PDTC) or PI3K/AKT inhibitor (LY294002) was proven to abolish the promoting effect of IL-17A on the invasion ability of colorectal cancer cells and upregulation of MMP-2/9. In conclusion, our findings demonstrated that IL-17A could promote the invasion of colorectal cancer cells by activating the PI3K/AKT/NF-κB signaling pathway and subsequently upregulating the expression of MMP-2/9. Our results suggest that IL-17A could serve as a promising therapeutic target for colorectal cancer.

6-Mercaptopurine attenuates tumor necrosis factor-α production in microglia through Nur77-mediated transrepression and PI3K/Akt/mTOR signaling-mediated translational regulation.

BackgroundThe pathogenesis of several neurodegenerative diseases often involves the microglial activation and associated inflammatory processes. Activated microglia release pro-inflammatory factors that may be neurotoxic. 6-Mercaptopurine (6-MP) is a well-established immunosuppressive drug. Common understanding of their immunosuppressive properties is largely limited to peripheral immune cells. However, the effect of 6-MP in the central nervous system, especially in microglia in the context of neuroinflammation is, as yet, unclear. Tumor necrosis factor-α (TNF-α) is a key cytokine of the immune system that initiates and promotes neuroinflammation. The present study aimed to investigate the effect of 6-MP on TNF-α production by microglia to discern the molecular mechanisms of this modulation.MethodsLipopolysaccharide (LPS) was used to induce an inflammatory response in cultured primary microglia or murine BV-2 microglial cells. Released TNF-α was measured by enzyme-linked immunosorbent assay (ELISA). Gene expression was determined by real-time reverse transcription polymerase chain reaction (RT-PCR). Signaling molecules were analyzed by western blotting, and activation of NF-κB was measured by ELISA-based DNA binding analysis and luciferase reporter assay. Chromatin immunoprecipitation (ChIP) analysis was performed to examine NF-κB p65 and coactivator p300 enrichments and histone modifications at the endogenous TNF-α promoter.ResultsTreatment of LPS-activated microglia with 6-MP significantly attenuated TNF-α production. In 6-MP pretreated microglia, LPS-induced MAPK signaling, IκB-α degradation, NF-κB p65 nuclear translocation, and in vitro p65 DNA binding activity were not impaired. However, 6-MP suppressed transactivation activity of NF-κB and TNF-α promoter by inhibiting phosphorylation and acetylation of p65 on Ser276 and Lys310, respectively. ChIP analyses revealed that 6-MP dampened LPS-induced histone H3 acetylation of chromatin surrounding the TNF-α promoter, ultimately leading to a decrease in p65/coactivator-mediated transcription of TNF-α gene. Furthermore, 6-MP enhanced orphan nuclear receptor Nur77 expression. Using RNA interference approach, we further demonstrated that Nur77 upregulation contribute to 6-MP-mediated inhibitory effect on TNF-α production. Additionally, 6-MP also impeded TNF-α mRNA translation through prevention of LPS-activated PI3K/Akt/mTOR signaling cascades.ConclusionsThese results suggest that 6-MP might have a therapeutic potential in neuroinflammation-related neurodegenerative disorders through downregulation of microglia-mediated inflammatory processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0543-5) contains supplementary material, which is available to authorized users.

Paeonol protects endotoxin-induced acute kidney injury: potential mechanism of inhibiting TLR4-NF-κB signal pathway.

Study design and methodsIn order to determine the therapeutic effect and mechanism of paeonol on acute kidney injury induced by endotoxin, an acute kidney injury model was established by intraperitoneal administration of lipopolysaccharide in mice in vivo and on LPS-induced dendritic cells in vitro. Renal tissues were used for histologic examination. Concentrations of blood urea nitrogen and serum creatinine were detected, inflammatory cytokines were determined by ELISA. The relative proteins' expression of TLR4-NF-κB signal pathway was assessed by Western blot, the localization and expression of phospho-NF-κB p65 in kidney was monitored by immunohistochemistry.ResultsTreatment of paeonol successfully cuts histopathological scores and dilutes the concentrations of blood urea nitrogen and serum creatinine as index of renal injury severity. In addition, paeonol reduces pro-inflammatory cytokines and increases anti-inflammatory cytokines stimulated by LPS in a dose-dependent manner. Paeonol also inhibits the expression of phosphorylated NF-κB p65, IκBα and IKKβ, and restrains NF-κB p65 DNA-binding activity. Paeonol treatment also attenuates the effects of LPS on dendritic cells, with significant inhibition of pro-inflammatory cytokines release, then TLR4 expression and NF-κB signal pathway have been suppressed.ConclusionsThese results indicated that paeonol has protective effects on endotoxin-induced kidney injury. The mechanisms underlying such effects are associated with its successfully attenuate inflammatory and suppresses TLR4 and NF-κB signal pathway. Therefore, paeonol has great potential to be a novel and natural product agent for treating AKI or septic-AKI.

Involvement of the NF-κB signaling pathway in the renoprotective effects of isorhamnetin in a type 2 diabetic rat model.

The aim of the present study was to investigate the renoprotective effects of isorhamnetin (ISO) in type 2 diabetic rats and its effects on the nuclear factor-κB (NF-κB) signaling pathway, which is associated with diabetic nephropathy. The type 2 diabetic rat model was established by a high-fat diet plus streptozocin injection and the rats were subsequently treated with two dosages of ISO, respectively. The levels of blood glucose were determined. Urinary osteopontin, kidney injury molecule-1 (KIM-1) and albumin were measured to evaluate the renal function of the rats. Renal NF-κB signaling activity was assessed by measuring the levels of NF-κB p65, phospho-NF-κB p65, inhibitor of NF-κB (IκBα) and phospho-IκBα, and the NF-κB p65 DNA-binding activity. Downstream inflammatory mediators [tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, intercellular adhesion molecule-1 (ICAM-1) and transforming growth factor-β1 (TGF-β1)] of the NF-κB signaling pathway were investigated to evaluate the renal inflammatory response. Renal levels of malondialdehyde and total superoxide dismutase were detected to access the oxidative stress. Furthermore, glomerular mesangial cells (GMCs) were treated with lipopolysaccharide and ISO. In the cellular experiment, the NF-κB signaling activity, levels of TNF-α, IL-1β, IL-6, ICAM-1 and TGF-β1, and oxidative stress were also investigated. The results showed that ISO decreased the levels of urinary osteopontin, KIM-1 and albumin. ISO also inhibited the NF-κB signaling activity, decreased the production of inflammatory mediators and attenuated oxidative stress in diabetic rats and GMCs. The present investigations revealed that ISO had ameliorative effects on diabetes-induced renal damage and the activity may be associated with the negative regulation of NF-κB signaling pathway.

Emodin suppresses LPS-induced inflammation in RAW264.7 cells through a PPARγ-dependent pathway

Inflammation is a defense and protective response to multiple harmful stimuli. Over and uncontrolled inflammation can lead to local tissues or even systemic damages and injuries. Actually, uncontrolled and self-amplified inflammation is the fundament of the pathogenesis of a variety of inflammatory diseases, including sepsis shock, acute lung injury and acute respiratory distress syndrome (ALI/ARDS). Our recent study showed that emodin, the main active component of Radix rhizoma Rhei, could significantly ameliorate LPS-induced ALI/ARDS in mice. However, its underlying signal pathway was not still very clear. Then, the aim of current study was to explore whether emodin could attenuate LPS-induced inflammation in RAW264.7 cells, and its involved potential mechanism. The mRNA and protein expression of ICAM-1, MCP-1 and PPARγ were measured by qRCR and western blotting, the production of TNF-α was evaluated by ELISA. Then, the phosphorylation of NF-κB p65 was also detected by western blotting. And NF-κB p65 DNA binding activity was analyzed by ELISA as well. Meanwhile, siRNA-PPARγ transfection was performed to knockdown PPARγ expression in cells. Our data revealed that LPS-induced the up-regulation of ICAM-1, MCP-1 and TNF-α, LPS-induced the down-regulation of PPARγ, and LPS-enhanced NF-κB p65 activation and DNA binding activity were substantially suppressed by emdoin in RAW264.7 cells. Furthermore, our data also figured out that these effects of emdoin were largely abrogated by siRNA-PPARγ transfection. Taken together, our results indicated that LPS-induced inflammation were potently compromised by emodin very likely through the PPARγ-dependent inactivation of NF-κB in RAW264.7 cells.

Effect of Cichorium intybus L. on the expression of hepatic NF-κB and IKKβ and serum TNF-α in STZ- and STZ+ niacinamide-induced diabetes in rats.

BackgroundInflammation is an early event in the development of diabetes type 2 (T2D). Cichorium intybus L. (chicory) possesses anti-inflammatory action. We compared the anti-inflammatory aspect of aqueous chicory seed extract (CSE) in early and late stage T2D in rats.MethodsWistar albino rats were divided into nine final groups (n = 6). Three main groups consisted of non-diabetic (Control), early stage diabetes (ET2D; niacinamide/streptozotocin, i.e., NIA/STZ), and late stage diabetes (LT2D; STZ). Within each main group, a subgroup was treated with CSE (125 mg/kg; i.p.); within each diabetic group (STZ and NIA/STZ) a subgroup received metformin (100 mg/kg; i.p.); another subgroup in STZ group received aspirin (120 mg/kg; oral). After 21 days, fasting blood glucose (FBS), insulin, and TNF-α level were measured in serum; IKKβ and NF-κB (p65) mRNA and protein expression were evaluated by real time PCR and Western blotting; p65 DNA binding activity was determined by ELISA, in liver tissue.ResultsThe mRNA and protein expression levels of IKKβ, and P65 genes increased in both stages of T2D (p < 0.01); CSE decreased their expression (p < 0.001, mRNAs; p < 0.05, proteins). The increased DNA-binding capacity of NF-κB (p < 0.0001) in diabetes was lowered by CSE (p < 0.001). The effect of CSE was limited to ET2D requiring insulin.ConclusionsThe anti-inflammatory action of CSE is due to a direct modulation of cytokine expression. The dependency of chicory action on the presence of insulin indicates its usefulness in the early stages of diabetes and for the purpose of preventing and delaying diabetes onset.Electronic supplementary materialThe online version of this article (doi:10.1186/s13098-016-0128-6) contains supplementary material, which is available to authorized users.

Mechanical Stretch Inhibits MicroRNA499 via p53 to Regulate Calcineurin-A Expression in Rat Cardiomyocytes.

BackgroundMicroRNAs play an important role in cardiac remodeling. MicroRNA 499 (miR499) is highly enriched in cardiomyocytes and targets the gene for Calcineurin A (CnA), which is associated with mitochondrial fission and apoptosis. The mechanism regulating miR499 in stretched cardiomyocytes and in volume overloaded heart is unclear. We sought to investigate the mechanism regulating miR499 and CnA in stretched cardiomyocytes and in volume overload-induced heart failure.Methods & ResultsRat cardiomyocytes grown on a flexible membrane base were stretched via vacuum to 20% of maximum elongation at 60 cycles/min. An in vivo model of volume overload with aorta-caval shunt in adult rats was used to study miR499 expression. Mechanical stretch downregulated miR499 expression, and enhanced the expression of CnA protein and mRNA after 12 hours of stretch. Expression of CnA and calcineurin activity was suppressed with miR499 overexpression; whereas, expression of dephosphorylated dynamin-related protein 1 (Drp1) was suppressed with miR499 overexpression and CnA siRNA. Adding p53 siRNA reversed the downregulation of miR499 when stretched. A gel shift assay and promoter-activity assay demonstrated that stretch increased p53 DNA binding activity but decreased miR499 promoter activity. When the miR499 promoter p53-binding site was mutated, the inhibition of miR499 promoter activity with stretch was reversed. The in vivo aorta-caval shunt also showed downregulated myocardial miR499 and overexpression of miR499 suppressed CnA and cellular apoptosis.ConclusionThe miR499-controlled apoptotic pathway involving CnA and Drp1 in stretched cardiomyocytes may be regulated by p53 through the transcriptional regulation of miR499.

Lipopolysaccharide represses PP2Ac expression through activation of NF-κB pathway in pancreatic cancer cells

Nuclear factor-κB (NF-κB) and the signaling pathways that regulate its activity have become a focal point for intense drug research and development efforts. NF-κB regulates the transcription of a large number of genes, particularly those involved in immune, inflammatory, and anti-apoptotic responses. In our search for NF-κB inhibitors from natural resources, we identified malloapelta B as an inhibitor of NF-κB activation from Mallotus apelta Muell-Arg. In the present study, we demonstrated the effect of malloapelta B on NF-κB activation in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. This compound suppressed NF-κB activation through the inhibition of IκB kinase (IKK) activation, thereby blocking the phosphorylation and degradation of the inhibitor of NF-κB alpha (IκBα), and the nuclear translocation and DNA-binding activity of p65. The suppression of NF-κB by malloapelta B led to the down-regulation of target genes involved in inflammation and proliferation. Taken together, this study extends our understanding on the mechanisms underlying the possible anti-inflammatory and anti-cancer activities of malloapelta B. Our findings provide new insight into its mechanisms of action and propose a potential application of malloapelta B for inflammatory diseases as well as certain cancers associated with abnormal NF-κB activation.

EGb761 attenuates depressive-like behaviours induced by long-term light deprivation in C57BL/6J mice through inhibition of NF-κB-IL-6 signalling pathway.

Our previous investigation found that Ginkgo extract EGb761 could attenuate the depressive-like behaviours induced by a single injection of lipopolysaccharide in mice. However, it has not been investigated whether EGb761 is effective on depressive-like behaviours induced by long-term light deprivation and whether its effects are associated with the inhibition of NF-κB-IL-6 signalling pathway. In this study, three groups (vehicle group, EGb761 low-dose group, and EGb761 high-dose group) of C57BL/6J male mice were exposed to constant darkness for four weeks. The control mice remained on a 12 : 12 light-dark cycle. Depressive-like behaviours were evaluated by tail suspension test (TST), forced swim test (FST), and sucrose preference test (SPT). Spontaneous locomotor activity was evaluated by open field test (OFT). Levels of IL-6, IL-6 mRNA, NF-κB p65, phospho-NF-κB p65, IκBα, and phospho-IκBα were measured using Elisa, western blotting, or PCR assays. NF-κB p65 DNA binding activity was evaluated using Chemi Transcription Factor Assay Kit. Results showed long-term light deprivation prolonged the immobile time in TST and FST, shortened the latency to immobility in FST, reduced spontaneous locomotor activity in OFT, decreased sucrose preference in SPT, and increased levels of IL-6, IL-6 mRNA, NF-κB p65, phospho-NF-κB p65, and phospho-IκBα in hippocampal tissue. EGb761 dose-dependently reversed the changes of the above parameters induced by long-term light deprivation, without affecting spontaneous locomotor activity. We conclude that EGb761 could attenuate the depressive-like behaviours and inhibit the NF-κB-IL-6 signalling pathway in a light-deprivation-induced mouse model of depression.

Phenethyl isothiocyanate potentiates anti-tumour effect of doxorubicin through Akt-dependent pathway.

The present study aims to investigate the in vivo and in vitro anti-tumour properties of phenethyl isothiocyanate (PEITC) alone and in combination with doxorubicin (Dox). The anti-tumour activity was evaluated in vitro by MTT assay using cultured human breast cancer cell line (MCF-7) and human hepatoma cell line (HepG-2) cell lines. In vivo, Ehrlich solid tumour model was used. Tumour volume, weight and antioxidant parameters were determined. Immunohistochemistry analysis for active (cleaved) caspase-3 was also performed. We tested the effect of PEITC treatment on pAkt/Akt ratio, NF-κB p65 DNA binding activity and caspase-9 enzyme activity in both MCF-7 and HepG-2 cell lines. Effect of PEITC treatment on cell migration was assessed by wound healing assay. PEITC and/or Dox treatment significantly inhibited solid tumour volume and tumour weight when compared with control mice. PEITC treatment significantly reduced oxidative stress caused by Dox treatment as indicated by significant increase in total antioxidant capacity and decrease in malondialdehyde level. Microscopic examination of tumour tissues showed a significant increase in active (cleaved) caspase-3 expression in PEITC and/or Dox treated groups. PEITC showed a dose-dependent inhibition of MCF-7 and HepG-2 cellular viability. PEITC inhibited Akt and NF-κB activation and increased caspase-9 activity in a dose-dependent manner. PEITC treatment effectively inhibited both MCF-7 and HepG-2 cell migration. We can conclude that PEITC acts via multiple molecular targets to elicit anti-carcinogenic activity. PEITC/Dox combination therapy might be a potential novel strategy, which may benefit patients with breast and liver cancers.

A novel aspirin prodrug inhibits NFκB activity and breast cancer stem cell properties.

IntroductionActivation of cyclooxygenase (COX)/prostaglandin and nuclear factor κB (NFκB) pathways can promote breast tumor initiation, growth, and progression to drug resistance and metastasis. Thus, anti-inflammatory drugs have been widely explored as chemopreventive and antineoplastic agents. Aspirin (ASA), in particular, is associated with reduced breast cancer incidence but gastrointestinal toxicity has limited its usefulness. To improve potency and minimize toxicity, ASA ester prodrugs have been developed, in which the carboxylic acid of ASA is masked and ancillary pharmacophores can be incorporated. To date, the effects of ASA and ASA prodrugs have been largely attributed to COX inhibition and reduced prostaglandin production. However, ASA has also been reported to inhibit the NFκB pathway at very high doses. Whether ASA prodrugs can inhibit NFκB signaling remains relatively unexplored.MethodsA library of ASA prodrugs was synthesized and screened for inhibition of NFκB activity and cancer stem-like cell (CSC) properties, an important PGE2-and NFκB-dependent phenotype of aggressive breast cancers. Inhibition of NFκB activity was determined by dual luciferase assay, RT-QPCR, p65 DNA binding activity and Western blots. Inhibition of CSC properties was determined by mammosphere growth, CD44+CD24−immunophenotype and tumorigenicity at limiting dilution.ResultsWhile we identified multiple ASA prodrugs that are capable of inhibiting the NFκB pathway, several were associated with cytotoxicity. Of particular interest was GTCpFE, an ASA prodrug with fumarate as the ancillary pharmacophore. This prodrug potently inhibits NFκB activity without innate cytotoxicity. In addition, GTCpFE exhibited selective anti-CSC activity by reducing mammosphere growth and the CD44+CD24−immunophenotype. Moreover, GTCpFE pre-treated cells were less tumorigenic and, when tumors did form, latency was increased and growth rate was reduced. Structure-activity relationships for GTCpFE indicate that fumarate, within the context of an ASA prodrug, is essential for anti-NFκB activity, whereas both the ASA and fumarate moieties contributed to attenuated mammosphere growth.ConclusionsThese results establish GTCpFE as a prototype for novel ASA-and fumarate-based anti-inflammatory drugs that: (i) are capable of targeting CSCs, and (ii) may be developed as chemopreventive or therapeutic agents in breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1868-7) contains supplementary material, which is available to authorized users.

Urolithins, gut microbiota-derived metabolites of ellagitannins, inhibit LPS-induced inflammation in RAW 264.7 murine macrophages.

Ellagitannin-rich food products and medicinal plant materials were shown to have beneficial effects toward intestinal inflammation. Due to the questionable bioavailability of ellagitannins their gut microbiota metabolites-urolithins have come to be regarded as potential factors responsible for biological activities observed in vivo. The aim of the study was to determine the influence of the three most abundant bioavailable ellagitannin gut microbiota metabolites-urolithins A, B, and C on inflammatory responses in RAW 264.7 murine macrophages, which are involved in the pathogenesis of intestine inflammation. Urolithins A, B, and C decreased NO production via inhibition of the iNOS protein and mRNA expression. They decreased the expression of IL-1β, TNF-α, and IL-6 mRNA in LPS challenged RAW 264.7 murine macrophages. A clear inhibition of NF-κB p65 nuclear translocation and p50 DNA-binding activity was associated with the observed anti-inflammatory activities of urolithins. Among the tested compounds urolithin A had the strongest anti-inflammatory activity. The anti-inflammatory effects of urolithins at concentrations that are physiologically relevant for gut tissues (≥40 μM), as revealed in this study, support the data from in vivo studies showing the beneficial effects of ellagitannin-rich products toward intestinal inflammation.

Glucagon Like Peptide-1 (GLP-1) Modulates OVA-Induced Airway Inflammation and Mucus Secretion Involving a Protein Kinase A (PKA)-Dependent Nuclear Factor-κB (NF-κB) Signaling Pathway in Mice.

Asthma is a common chronic pulmonary inflammatory disease, featured with mucus hyper-secretion in the airway. Recent studies found that glucagon like peptide-1 (GLP-1) analogs, including liraglutide and exenatide, possessed a potent anti-inflammatory property through a protein kinase A (PKA)-dependent signaling pathway. Therefore, the aim of current study was to investigate the value of GLP-1 analog therapy liraglutide in airway inflammation and mucus secretion in a murine model of ovalbumin (OVA)-induced asthma, and its underlying molecular mechanism. In our study, BALB/c mice were sensitized and challenged by OVA to induce chronic asthma. Pathological alterations, the number of cells and the content of inflammatory mediators in bronchoalveolar lavage fluid (BALF), and mucus secretion were observed and measured. In addition, the mRNA and protein expression of E-selectin and MUC5AC were analyzed by qPCR and Western blotting. Then, the phosphorylation of PKA and nuclear factor-κB (NF-κB) p65 were also measured by Western blotting. Further, NF-κB p65 DNA binding activity was detected by ELISA. OVA-induced airway inflammation, airway mucus hyper-secretion, the up-regulation of E-selectin and MUC5AC were remarkably inhibited by GLP-1 in mice (all p < 0.01). Then, we also found that OVA-reduced phosphorylation of PKA, and OVA-enhanced NF-κB p65 activation and NF-κB p65 DNA binding activity were markedly improved by GLP-1 (all p < 0.01). Furthermore, our data also figured out that these effects of GLP-1 were largely abrogated by the PKA inhibitor H-89 (all p < 0.01). Taken together, our results suggest that OVA-induced asthma were potently ameliorated by GLP-1 possibly through a PKA-dependent inactivation of NF-κB in mice, indicating that GLP-1 analogs may be considered an effective and safe drug for the potential treatment of asthma in the future.

Abstract 1219: ID4 and p53 cross-talk promotes restoration of mutant-p53 transcriptional activity

Abstract The physiological mechanisms that can restore biological activity of mutant p53 is an area of high interest given that mutant p53 expression is observed in one third of prostate cancer and more than 50% of all cancers. Here we demonstrate that ID4 (inhibitor of differentiation-4) a dominant negative regulator of bHLH transcription factors can restore mutant p53 transcriptional activity in prostate cancer cells. ID4 is highly expressed in the normal prostate and decreased in prostate cancer due to promoter hypermethylation. Prostate cancer cell lines: DU145 harbors mutant p53 and also lacks ID4 expression; LNCaP cells express wild-type p53 and ID4; whereas PC3 cells are null for p53 and express low levels of ID4. p53 mutants (P223L and V274F) in DU145 cells are within the DNA binding domain and abrogate p53 transcriptional activity due to structural de-stabilization and/or DNA interactions. Ectopic expression of ID4 in DU145 cells resulted in increased apoptosis and expression of BAX, PUMA and p21, transcriptional targets of p53. DNA binding, p53 luciferase reporter studies and ChIP analysis demonstrated that mutant p53 gains ID4 dependent DNA binding and transcriptional activity in part due to CBP/p300 dependent acetylation of p53 at lysine 373. Loss of ID4 in LNCaP cells also abrogated wild-type p53 DNA binding and transcriptional activity with concomitant loss of CBP/p300 requirement and decreased acetylation of p53. To further elucidate ID4 dependent restoration of biological activity of p53 we stably transfected p53-null cell line PC3, which has endogenous ID4 with mutant p53 mimicking DU145 cells. mRNA, protein levels and apoptosis assays were used to determine the effects on cell death and to determine if mutant p53 had the ability to transactivate upstream/downstream targets (MDM2, PUMA, and p21). To further validate our gene expression profile of p53 responsive targets, we performed DNA binding assays as well as p53 luciferase reporter assays to demonstrate wild-type and mutant p53 transcriptional activity in PC3 cells. Also, to elucidate the mechanism by which ID4 promotes apoptosis, we performed acetylation studies in LNCaP and DU145 xenografts (+/-) ID4 as well as p53 co-immunoprecipation studies to demonstrate interaction of both lysine residues 320 and 373. Our results indicate that transfected mutant p53 in PC3 cells were able to induce expression of downstream targets as well as effect biological processes such as apoptosis and cell cycle arrest. Also, post-translational modifications on residues lys320 and lys373 indicated activation of mutant p53. Collectively, based on our studies we demonstrate that ID4 promotes post-translational modifications of p53, which in turn regulates p53 transcriptional activity. NIH grant #R01CA128914 Citation Format: Derrick Jerone Morton, Divya Patel, Jugal Joshi, Pankaj Sharma, Ashley Knowell, Aisha Hunt, Jaideep Chaudhary. ID4 and p53 cross-talk promotes restoration of mutant-p53 transcriptional activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1219. doi:10.1158/1538-7445.AM2015-1219

The therapeutic effect and mechanism of niacin on acute lung injury in a rat model of hemorrhagic shock: Down-regulation of the reactive oxygen species-dependent nuclear factor κB pathway.

The purpose of the current study was to investigate the protective effect of niacin on acute lung injury by the down-regulation of the nuclear factor κB (NF-κB) pathway in hemorrhagic shock (HS) rats. HS was induced in male Sprague-Dawley rats by withdrawing blood to maintain a mean arterial pressure of 20 mm Hg to 25 mm Hg for 40 minutes. The rats were resuscitated by the reinfusion of the drawn blood, and a vehicle (HS), a low-dose of niacin (360 mg/kg, HS + LD-NA), or a high dose of niacin (1,080 mg/kg, HS + HD-NA) were administered orally. The survival of the subjects was observed for 72 hours, and a separate set of animals was killed at 6 hours after HS induction. We measured cytoplasmic phosphorylated inhibitor κB-α and inhibitor κB-α expressions, nuclear NF-κB p65 expression, NF-κB p65 DNA-binding activity, MEK partner 1 activity, tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), IL-8, nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide phosphate, reduced glutathione, glutathione disulfide, malondialdehyde levels, and histologic damage in the lung tissue. We also measured TNF-α, IL-6, and IL-8 levels in the serum. The survival rates of the sham, HS, HS + LD-NA, and HS + HD-NA groups were 6 of 6 (100%), 0 of 9 (0%), 1 of 9 (11.1%), and 3 of 9 (33.3%), respectively. A high dose of niacin increased lung NAD+, nicotinamide adenine dinucleotide phosphate levels, and glutathione-glutathione disulfide ratios; decreased lung malondialdehyde levels; down-regulated the NF-κB pathway; suppressed TNF-α, IL-6, and IL-8 levels in the lung tissue and serum; and attenuated histologic lung damage. A high dose of niacin attenuated lung inflammation, suppressed proinflammatory cytokine release, reduced histologic lung damage, and improved survival after HS in rats. Its therapeutic benefits were associated with the down-regulation of the reactive oxygen species-dependent NF-κB pathway.

Effect of trans-10, cis-12 Conjugated Linoleic Acid on Calcium-Dependent Reactive Oxygen Species and Nitric Oxide Production and Nuclear Factor-${kappa}B$ Activation in Lipopolysaccharide-Stimulated RAW 264.7 Cells

Trans-10, cis-12-conjugated linoleic acid (t10c12-CLA) has been shown to participate in the regulation of anti-inflammatory effects. The objectives of this study were to examine the effects of t10c12-CLA on reactive oxygen species (ROS) and nitric oxide (NO) production and nuclear factor-kappaB (NF-κB) activation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and to determine whether these effects were associated with change of intracellular calcium ion (Ca 2+ ). ROS production was increased in LPS-stimulated RAW 264.7 cells, and this effect was suppressed by 1,2-bis-(o-aminophenoxy) ethane-N,N,N,N-tetraacetic acid-acetoxymethyl ester (BAPTA/AM), a calcium chelator. t10c12-CLA suppressed ROS production in LPS-stimulated RAW 264.7 cells, which was further more decreased by treatment with BAPTA/AM. These indicated that t10c12-CLA decreases Ca 2+ -dependent ROS production in LPSstimulated RAW 264.7 cells. Similarly, NF-κB p65 DNA binding activity and NO production were decreased by treatment with either t10c12-CLA, BAPTA/AM, or t10c12-CLA and BAPTA/AM combination. However, there were no differences between t10c12-CLA and BAPTA/AM treatment in NO production of LPS-stimulated RAW 264.7 cells. These data indicate that t10c12-CLA inhibits the increases in ROS and NO production and the NF-κB activation in LPS-stimulated condition. These results suggested that CLA exerts potent anti-inflammatory effects by suppression of LPS-induced ROS and NO production, and NF-κB activationn via Ca 2+ -dependent pathway.

Down-regulation of NF kappa B activation is an effective therapeutic modality in acquired platinum-resistant bladder cancer.

BackgroundNo previous study has addressed the efficacy of NF-κB blockade when bladder tumors develop acquired resistance toward CDDP-treatments. We investigated the changes in NF-κB activation and therapeutic impact of NF-κB blockade by the novel NF-κB inhibitor dehydroxymethyl derivative of epoxyquinomicin (DHMEQ) in CDDP-resistant bladder cancer cells.MethodsTwo human invasive bladder cancer cell lines, T24 and T24PR, were used. The T24PR cell line was newly established as an acquired platinum-resistant subline by culturing in CDDP-containing medium for 6 months. Expression of intranuclear p65 protein in the fractionated two cell lines was determined by Western blotting analysis. DNA-binding activity of NF-κB was detected by electrophoretic mobility shift assay. The cytotoxic effects and induction of apoptosis were analyzed in vivo and in vitro.ResultsIntranuclear expression and DNA-binding activity of p65 were strongly enhanced in T24PR cells compared with those of T24 cells, and both were significantly suppressed by DHMEQ. Lowered cell viability and strong induction of apoptosis were observed by treatment with DHMEQ alone in these chemo-resistant cells compared with parent cells. As T24PR cells did not show dramatic cross-resistance to paclitaxel in the in vitro study, we next examined whether the combination of DHMEQ with paclitaxel could enhance the therapeutic effect of the paclitaxel treatment in T24PR tumors. Using mouse xenograft models, the mean volume of tumors treated with the combination of DHMEQ (2 mg/kg) and paclitaxel (10 mg/kg) was significantly smaller than those treated with paclitaxel alone (p < 0.05), and the reduction of tumor volume in mice treated with DHMEQ in combination with paclitaxel and paclitaxel alone as compared to vehicle control was 66.9% and 17.0%, respectively.ConclusionThere was a distinct change in the activation level of NF-κB between T24 and T24PR cells, suggesting strong nuclear localization of NF-κB could be a promising target after developing acquired platinum-resistance in bladder cancer.

Celastrol ameliorates cytokine toxicity and pro-inflammatory immune responses by suppressing NF-κB activation in RINm5F beta cells.

Upregulation of pro-inflammatory mediators contributes to β-cell destruction and enhanced infiltration of immune cells into pancreatic islets during development of type 1 diabetes mellitus. In this study, we examined the regulatory effects and the mechanisms of action of celastrol against cytotoxicity and pro-inflammatory immune responses in the RINm5F rat pancreatic β-cell line stimulated with a combination of interleukin-1 beta, tumor necrosis factor-alpha, and interferon-γ. Celastrol significantly restored cytokine-induced cell death and significantly inhibited cytokine-induced nitric oxide production. In addition, the protective effect of celastrol was correlated with a reduction in pro-inflammatory mediators, such as inducible nitric oxide synthase, cyclooxygenase-2, and CC chemokine ligand 2. Furthermore, celastrol significantly suppressed cytokine-induced signaling cascades leading to nuclear factor kappa B (NF-κB) activation, including IκB-kinase (IKK) activation, IκB degradation, p65 phosphorylation, and p65 DNA binding activity. These results suggest that celastrol may exert its cytoprotective activity by suppressing cytokine-induced expression of pro-inflammatory mediators by inhibiting activation of NF-κB in RINm5F cells. [BMB Reports 2015; 48(3): 172-177]

Total saponin from Anemone flaccida Fr. Schmidt abrogates osteoclast differentiation and bone resorption via the inhibition of RANKL-induced NF-κB, JNK and p38 MAPKs activation.

Osteoclasts, bone-specialized multinucleated cells, are responsible for bone destructive diseases such as rheumatoid arthritis and osteoporosis. Natural plant-derived products have received substantial attention given their potential therapeutic and preventive activities against bone destructive diseases. In the present study, we investigated the effects of total saponin (TS) from Anemone flaccida Fr. Schmidt, on receptor activator of nuclear factor-κB ligand (RANKL)-induced in vitro osteoclast differentiation. We observed that TS concentration-dependently inhibited RANKL-induced osteoclast formation from RAW 264.7 cell and bone marrow-derived macrophages (BMMs), as well as decreased extent of actin ring formation and lacunar resorption. The RANKL-stimulated expression of osteoclast-related transcription factors were also diminished by TS. Moreover, TS blocked the RANKL-triggered TRAF6 expression, phosphorylation of mitogen-activated protein kinases (MAPKs) and IκB-α, and inhibited NF-κB p65 DNA binding activity. Furthermore, TS almost abrogated the nuclear factor of activated T cells (NFATc1) and c-Fos expression. Taken together, our results demonstrated that TS suppresses RANKL-induced osteoclast differentiation and inflammatory bone loss via the down-regulation of TRAF6 level, suppression of JNK and p38 MAPKs and NF-κB activation, and subsequent decreased expression of c-Fos and NFATc1. Therefore, TS may be a potential agent and needs to be more evaluated in vivo or in clinical trials to become a therapeutic for lytic bone diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0440-1) contains supplementary material, which is available to authorized users.

A fresh frozen plasma to red blood cell transfusion ratio of 1:1 mitigates lung injury in a rat model of damage control resuscitation for hemorrhagic shock

BackgroundWe aimed to evaluate the effects of resuscitation with different ratios of fresh frozen plasma (FFP) to red blood cells (RBCs) on pulmonary inflammatory injury and to illuminate the beneficial effects of FFP on lung protection compared with lactated ringers (LR) using a rat model of hemorrhagic shock. MethodsRats underwent pressure-controlled hemorrhage for 60 minutes and were then transfused with LR for initial resuscitation. Thereafter, the rats were transfused with varying ratios of FFP:RBC (1:4, 1:2, 1:1, and 2:1) or LR:RBC (1:1) to hold their mean arterial pressure (MAP) at 100 ± 3 mm Hg for 30 minutes. After 4 hours of observation, lung tissue was harvested to determine the wet/dry weight, myeloperoxidase levels, tumor necrosis factor α levels, macrophage inflammatory protein 2 (MIP-2) levels, inducible nitric oxide synthase activity, and the nuclear factor κB p65 DNA-binding activity. ResultsWith an increase in the FFP:RBC ratio, the volume of required RBC to maintain the target MAP decreased. The MAP value in each group was not significantly different during the whole experiment period. The values of the wet/dry weights and MIP-2 were significantly lower in the FFP:RBC = 1:1 group than the other groups (P < .05). All parameters detected above were predominantly lower in the FFP:RBC = 1:1 group than the FFP:RBC = 1:2 group and the LR:RBC = 1:1 group (P < .05). In addition, all parameter values were lower in the FFP:RBC = 1:1 group than in the FFP:RBC = 2:1 group, but only the wet/dry weight, myeloperoxidase, and MIP-2 values were significantly different (P < .05). ConclusionsResuscitation with a 1:1 ratio of FFP to RBC results in decreased lung inflammation. Compared with LR, FFP could further mitigate lung inflammatory injury.