Expression Of Nuclear factor-kappaB P65 Research Articles

Pankreas duktal adenokarsinomunda NF-Kappa B ekspresyonu

Purpose: In this study we investigated the expression of the p65 subunit of the nuclear factor-kappaB (NF-kB) complex and the activation status of NF-kB by phospho-IkB-alpha antibody.Materials and Methods: A tissue microarray based on material obtained from 107 patients was utilized. The antibody staining was scored by combining staining intensity with percentage of tumor staining. The antibodies used were NF-kB p65 and phospho-IkB-alpha(ser32/36), both from Cell Signaling Technology. The staining scores were correlated with the archival data available on some patients on margin and lymph node status, stage, tumor size, as well as clinical data including survival.Results: The staining was nuclear (p65) and cytoplasmic (p-IkBalpha) respectively. In general there was an increased expression and activation of NF-kB in the carcinomas, compared to non-tumoral regions. None of the markers had a significant correlation with the overall survival. NF-kB(p65) expression had a correlation with positive lymph node status.Conclusion: The correlation with the positive lymph node status suggests a role in invasive properties of the tumor. Activation of NF-kB is most likely an early event in pancreatic carcinogenesis. Despite the lack of an effect on overall survival, due to its increased activation in pancreatic cancer, NF-kB is still a good target for therapeutic interventions.

MicroRNA hsa-let-7g targets lectin-like oxidized low-density lipoprotein receptor-1 expression and inhibits apoptosis in human smooth muscle cells

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) has been identified as a major receptor for oxidatively modified low density lipoprotein (ox-LDL) in endothelial cells and smooth muscle cells (SMCs). MicroRNAs are small non-coding RNAs that regulate gene expression. Very few studies have reported regulation of LOX-1 expression by microRNAs in SMCs. The present study demonstrates that the microRNA hsa-let-7g can inhibit LOX-1 expression in a time- and dose-dependent fashion, and subsequently inhibit ox-LDL uptake in and proliferation of human aortic SMCs. We also show that hsa-let-7g can reduce SMC apoptosis by down-regulation of cytochrome c and Smac/Diablo and upregulation of Bcl-xL and Bcl-2 expression. Furthermore, we show that hsa-let-7g reduces ox-LDL-induced increase in the expression of NADPH oxidase (p22(phox) and p47(phox) subunits) and subsequent intracellular reactive oxygen species generation, phosphorylation of p44/42 mitogen-activated protein kinase and nuclear factor-kappaB p65 expression. These observations suggest that hsa-let-7g is a critical regulator of SMC apoptosis, and may be suitable for therapeutic intervention in disease states characterized by LOX-1 over-expression.

Role of nuclear factor‐kappa B in mitochondria‐derived superoxide‐lowered protein expression of voltage‐gated sodium channels in nodose neurons from heart failure rats

Our previous study has shown that chronic heart failure (CHF) reduces protein expression of voltage‐gated sodium (Nav) channels in rat nodose neurons. In the present study, we investigated the involvements of mitochondrial superoxide and nuclear factor‐kappa B (NF‐kB) in CHF‐decreased Nav channel expression in rat nodose neurons. CHF was induced by left coronary ligation. CHF reduced the protein expression of manganese superoxide dismutase (MnSOD) and elevated mitochondrial superoxide level in the nodose neurons. Adenoviral MnSOD (Ad.MnSOD) gene transfection (50 multiplicity of infection) into the nodose neurons normalized the MnSOD expression, reduced the elevation of mitochondrial superoxide, and partially reversed the reduced protein expression of Nav channels in the CHF nodose neurons. Additionally, protein expression of NF‐kB p65 and phosphorylated NF‐kB p65 in the nodose neurons was higher in CHF rats than that in sham rats. Treatment with an NF‐kB inhibitor (caffeic acid phenethyl ester, 10 ìM, 24 h) significantly increased Nav channel density in CHF nodose neurons. Furthermore, Ad.MnSOD inhibited the NF‐kB p65 binding to Nav channel promoter in CHF nodose neurons. These results indicate that mitochondrial superoxide lowered protein expression of Nav channels in CHF nodose neurons via activating NF‐kB.

Abstract P276: Cardiac-Specific Knockout NF-kB Mice Resist Dysfunction Induced by Global Ischemia-Reperfusion

Subjective: Nuclear factor Kappa B (NF-kB) is a pluripotent transcriptional factor that has been implicated as mediator of cardiac injury after ischemia-reperfusion (IR). Multiple approaches have been used to target inhibition of NF-kB, yet these strategies are often non-specific. P65-NF-kB exists as a heterodimer with its p50 subunit and is the dominant transcriptional subunit of NF-kB. In order to better understand its role in the heart, we have recently generated mice that have cardiomyocyte (CMC) specific deletion of p65 NF-kB and hypothesized that these animals would be protected from ischemic stress. Methods: Transgenic mice were generated with (WT) or without CMC p65 (KO). NF-kB p65 expression was assessed with Western Blots. We subjected the mice to global IR using Langendorf system with baseline 20 min, ischemia 45 min and reperfusion 60 min. Cardiac contractile functional recovery was analyzed using rate pressure product (RPP), contractility (+dp/dt), and relaxation (-dp/dt). Infarct size was assessed with TTC staining. LDH and CK were measured in reperfused solutions. Results: 1. No differences were found between groups of WT (n=7) and KO (n=8) in body weight and heart weight. 2. KO mice express significant less cardiac-specific NF-kB p65. 3. During reperfusion, RPP, +dp/dt, -dp/dt recovery to base line were all better in p65 KO mice (RPP: 0.841±0.074 in KO vs 0.635±0.080 in WT; +dp/dt: 0.997±0.096 in KO vs 0.731±0.108 in WT; -dp/dt: 0.869±0.096 in KO vs 0.630±0.090 in WT; p<0.05 respectively). 4. After ischemia, both LDH and CK release were markedly less in solutions collected from p65 KO heart (LDH: 45.2±28.0 u/L; CK: 28.1±23.6 u/L) than those in p65 WT heart (LDH: 143.2±25.9 u/L; CK: 107.2±26.2 u/L; p,0.01 respectively). 5. TTC staining shown significant less necrotic tissue in KO heart vs WT heart (0.314±0.052 vs 0.653±0.061, p<0.05). Conclusion: CMC-specific knockout NF-kB p65 mice have improved cardiac recovery function, less heart necrosis, and decreased CMC injury compared to WT after global heart ischemia. These studies confirm many of the previous studies suggesting a protective role for anti-NF-kB strategies for ischemia. It furthermore provides a template to further define the mechanisms of p65-NF-kB afforded cardioprotection.

Abstract LB-44: Effect of modulation of I kappa B kinases (IKKs) in prostate cancer cell growth and apoptosis

Abstract Prostate cancer is the most common cancer in men. Although a wide variety of treatments such as surgery, radiation and hormone therapy exist, patients diagnosed with locally advanced or metastatic disease eventually progress to a castrate resistant state [1]. Understanding the mechanism by which castrate resistant prostate cancer (CRPC) develops remains a major issue. Elevated nuclear factor kappa B (NFkB) activity has been observed in many tumors including prostate cancer [2]. In this study, we employed patient samples in combination with in vitro methods to examine the role of NFkB and IKKs in prostate cancer growth and apoptosis. Tissue microarray technology was employed to assess NFkB p65 protein expression in 255 prostate cancer specimens obtained at diagnosis. Immunohistochemistry was performed using an antibody to p65 and expression assessed using the weighted histoscore method. IKKα and/or IKKβ were down regulated in PC3 cells by electroporation with specific siRNA. The effects of IKKα and/or IKKβ silencing on cell growth, cell cycle markers and apoptosis were examined. Full clinical follow-up was available for the patient cohort, mean follow-up was 8.05 years, 129 patients died of their disease and 56 patients had metastases at diagnosis. Gleason sum (p=0.001), presence of metastasis (p=0.001), stage (p=0.016) and androgen receptor status (p=0.002) significantly correlated with decreased disease specific survival. NFkB p65 expression was observed in both the tumor cell cytoplasm and nucleus (location when activated). Cytoplasmic p65 expression was not associated with any known clinical parameters, however high (supra medium) nuclear p65 expression was associated with decrease in disease specific survival (p=0.005) and this was independent when combined with Gleason sum, presence of metastasis stage and androgen receptor status on multivariate analysis (p=0.004, hazard ratio 4.5 (1.6-12.8)). We further designed a series of experiments to assess the effect of inhibiting NFkB activation on prostate cancer cells via knocking down IKKα and/or IKKβ expression. When IKKα and IKKβ alone or combined were silenced, p65 expression in PC3 cells was observed only in the cell cytoplasm and not in the nucleus, suggesting NFkB activation had been inhibited. However, no significant effect on cell proliferation (as measured by WST-1) or apoptosis (Annexin V/ ViaProbe) was observed. Furthermore, no significant change was observed in protein levels of cyclin D1, A, B1 or E as measured by western blotting, however an inhibition of Wee 1, a known G2 checkpoint regulator, was observed following IKKβ silencing revealing a novel cell cycle target for IKKs. In conclusion, NFkB p65 nuclear expression is a negative prognostic marker for prostate cancer at diagnosis and modification of IKKα and IKKβ alone or combined may offer a potential therapeutic target by modifying the functions of cell cycle regulators.

Effects of penehyclidine pretreatment on nuclear factor kappa B activity during lipopolysaccharide-induced acute lung injury in neonate rats

Objective To investigate the effects of penehyclidine (PHCD) pretreatment on nuclear factor kappa B ( NF-kB ) activity during lipopolysaccharide ( LPS )-induced acute lung injury ( ALl ) in neonate rats.Methods Thirty 7-day old Wistar rats of both sexes weighing 18-21 g were randomly divided into 3 groups ( n =10 each): group Ⅰ control (group C); group Ⅱ LPS; group Ⅲ PHCD. Group Ⅱ and Ⅲ received intraperitoneal ( group IP) LPS 3 mg/kg. In group Ⅲ PHCD 5 mg/kg was administered IP at 30 min before LPS respectively. The animals were killed at 4 h after LPS administration. The lungs were immediately removed. The W/D lung weight ratio was measured. The TNF-α, IL-1 βand IL-10 content in the lung were detected by ELISA and expression of NF-kB p65 was detected by immuno-histochemical staining.Results LPS significantly increased W/D lung weight ratio, TNF-α, IL-1 β, IL-10 content and NF-kB p65 expression in the lung as compared with control group. PHCD administered before LPS significantly attenuated the LPS-induced changes. Electron microscopy showed that PHCD before LPS significandy ameliorated the LPS-induced histological damages. Conclusion Pretreatment with PHCD can attenuate LPS-induced acute lung injury though inhibition of NF-kB activation and inflammatory response of lung tissue in neonate rats. Key words: Cholinergic antagonism; Respiratory distress syndrome,adult; NF-kappa B; Infant,nwwborn

Protective effects of mildronate in indinavir- and efavirenz-induced toxicity in mice

Protective effects of mildronate in indinavir- and efavirenz-induced toxicity in micePreviously we showed that mildronate effectively protected mice heart tissue against the toxic influence of anti-HIV drugs azidothymidine, stavudine and lamivudine, which belong to nucleosideanalogue reverse transcriptase inhibitor (NRTI) class. Recently we also demonstrated that mildronate protected isolated rat liver mitochondria against mitochondrial damage caused by azidothymidine. The present study was devoted to examine the possible protective effectiveness of mildronate in cardio-, hepato- and neurotoxicity models caused by anti-HIV drugs of other classes: indinavir, a representative of protease inhibitor (PI) class, and efavirenz, a non-nucleoside-analogue reverse transcriptase inhibitor (NNRTI). Drugs were administered intraperitoneally for two weeks, at the dose of 50 mg/kg of anti-HIV drugs and 100 mg/kg for mildronate. Afterwards, mice heart, liver and brain tissue were examined morphologically and immunohistochemically. The results showed that indinavir in heart tissue caused inflammatory and degenerative changes, manifested as increased expression of nuclear factor kappaBp65 (NF-kBp65), as well as cardiomyocyte necrosis and cellular infiltration. Efavirenz did not cause pathological changes in mice heart tissue, whereas it induced marked expression of caspase-3 and glial fibrillary acidic protein (GFAP) in mice brain tissue and small degenerative alterations in mice liver tissue. The data obtained show mildronate's protective properties in indinavir-induced cardiotoxicity and efavirenz-induced neurotoxicity.

Ghrelin Inhibits the Development of Acute Pancreatitis and Nuclear Factor κB Activation in Pancreas and Liver

To investigate the influence of ghrelin on the development of severe acute pancreatitis (SAP) and the expression of nuclear factor kappaB (NF-kappaB) p65 in the pancreas and liver. Severe acute pancreatitis was induced in rat by sodium taurocholate injection in the pancreaticobiliary duct. Ghrelin was administrated twice at the dose 10 or 20 nmol/kg per injection, respectively. Then, serum amylase activity; serum tumor necrosis factor alpha, interleukin 1beta, and interleukin 6 concentrations; and morphological signs of pancreatitis and hepatic damage were measured. Meanwhile, determination of pancreatic and hepatic NF-kappaB p65 expression was performed by Western blotting and immunohistochemistry. The serumal parameters increased, and morphological damages were observed in the pancreas and liver in SAP rats. Nuclear factor kappaB p65 expression was significantly higher in the pancreas and liver than sham-operated rats (P < 0.05). Treatment with ghrelin attenuated the morphological damages, and reduced the serumal parameters. Nuclear factor kappaB p65 expression was also significantly reduced by ghrelin (P < 0.05), both in the pancreas and liver. Ghrelin inhibits the development of acute pancreatitis induced by sodium taurocholate. It exerts the therapeutic effects through inhibiting NF-kappaB expression, thereby blocks the inflammatory signal transduction pathway and reduces the release of inflammatory media and cytokines.

Cleaved caspase-3 and nuclear factor- κB p65 are prognostic factors in metastatic serous ovarian carcinoma

Tumor progression and treatment failure in ovarian carcinoma are frequently associated with metastasis to effusions. The present study analyzed the expression and clinical role of nuclear factor-kappaB p65, nuclear factor-kappaB inhibitor alpha, and parameters of apoptosis in serous carcinoma. Cleaved caspase-3 and caspase-8 levels and deoxyuridine triphosphate incorporation were measured in 65 effusions using flow cytometry. Effusions (n = 209) and corresponding primary carcinomas and solid metastases (n = 114) were immunohistochemically analyzed for nuclear factor-kappaB p65 and nuclear factor-kappaB inhibitor alpha expression. Effusions (n = 75) were further analyzed for nuclear factor-kappaB phospho-p65 (Ser536) levels using immunoblotting. Results were analyzed for association with anatomic site, clinicopathologic parameters, and survival. Caspase cleavage and deoxyuridine triphosphate incorporation were limited to less than 10% of cells in most effusions. Nuclear factor-kappaB p65 expression was frequently detected at all anatomic sites, with less frequent cytoplasmic nuclear factor-kappaB p65 and nuclear factor-kappaB inhibitor alpha expressions. Immunoblotting showed nuclear factor-kappaB p65 phosphorylation in 72 (96%) of 75 effusions. Higher than median cleaved caspase-3 levels correlated with improved overall and progression-free survival in univariate analysis of all patients (P = .024 and P = .046, respectively) and of those with postchemotherapy effusions (P = .042 and P = .036, respectively). Cleaved caspase-3 expression was an independent predictor of longer progression-free survival for patients with postchemotherapy effusions (P = .029). Nuclear factor-kappaB p65 expression correlated with poor progression-free survival for all patients (P = .048) and for those with postchemotherapy effusions (P = .025). Ovarian carcinoma cells in effusions undergo little apoptosis, but high levels of cleaved caspase-3 are associated with improved survival. Nuclear factor-kappaB p65 is frequently expressed in advanced-stage serous ovarian carcinoma, and its nuclear localization is associated with poor progression-free survival.

Protection of Azidothymidine‐Induced Cardiopathology in Mice by Mildronate, a Mitochondria‐Targeted Drug

Azidothymidine, a nucleoside-analogue reverse transcriptase inhibitor (NRTI), is a commonly used antiretroviral drug in AIDS treatment, however its use is limited by severe toxic side effects due to its influence on mitochondria that result in myopathy, particularly affecting the cardiac muscle. We suggest that effective protection of azidothymidine-induced cardiopathology can be expected from drugs that are capable of targeting mitochondria. Therefore the present study in mice was carried out with mildronate, a cardioprotective drug of the aza-butyrobetaine class, which previously has been shown to act as a highly potent protector of mitochondrial processes. In our study, saline (control), azidothymidine (50 mg/kg), mildronate (50, 100 and 200 mg/kg), and azidothymidine + mildronate (at the doses mentioned) were injected intraperitoneally daily in separate groups of mice for two weeks. At the termination of the experiment, mice were sacrificed, the hearts were removed and cardiac tissue was examined morphologically and immunohistochemically. It was found that azidothymidine, compared to control and mildronate groups, induced major morphologic changes in cardiac tissue, which were manifestated as degeneration and inflammation. These changes were prevented when mildronate was co-administered with azidothymidine. Mildronate also reduced the azidothymidine-induced expression of nuclear factor kappaBp65 (NF-kappaBp65). The obtained data demonstrate a high ability of mildronate of preventing azidothymidine-induced cardiopathologic changes, and suggest mildronate's indirect action on azidothymidine-caused oxidative stress reactions leading to mitochondrial dysfunction. This offers a rational combination of mildronate with azidothymidine or other anti-HIV drugs for beneficial application in AIDS therapy.