Nuclear NF-κB P65 Expression Research Articles

Abstract 5428: Consumption of S-allylcysteine inhibits tumor growth and progression in a mouse xenograft model of oral cancer

Abstract Oral cancer is a prevalent type of cancer in many countries. Although studies indicated that garlic extracts such as diallyl disulfide and diallyl trisulfide have anti-cancer effects, the chemopreventive effects of aqueous garlic extracts, S-allylcysteine (SAC), on oral cancer have not been studied yet. To this specific aim, we investigated the inhibitory effects of SAC on the proliferation and progression of oral cancer cells in both in vitro and in vivo models. In the present study, our results demonstrated that SAC dose-dependently inhibited the proliferation of human oral squamous cancer CAL-27 cells in vitro. The results indicated that SAC stabilized the adherent junction complex of E-cadherin/β-catenin and suppressed epithelial-mesenchymal transition (EMT) in oral cancer cells through suppression of MAPK/ERK signaling pathways. By study end (4th week), consumption of SAC (at doses of 5 and 40 mg / kg of body weight per day) significantly prevented tumor growth of oral cancer in a mouse xenograft model. Histopathological and immunohistochemical (IHC) staining results indicated that SAC could effectively suppress tumor progression including EMT of oral cancer in vivo. The chemopreventive effects of SAC were associated with decreases in cyclin D1 expression and suppression of Akt, mTOR, IκBα, and ERK 1/2 signaling cascades in tumor tissues. SAC consumption could inhibit the expression of cyclooxygenase-2 (COX-2), Vimentin, and nuclear NF-κB p65 (RelA) proteins. These results suggested that SAC could act as an effective agent to prevent tumor growth, progression, and EMT of oral cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5428. doi:1538-7445.AM2012-5428

High basal NF-κB activity in nonpigmented melanoma cells is associated with an enhanced sensitivity to vitamin D3 derivatives

Background:Melanoma is highly resistant to current modalities of therapy, with the extent of pigmentation playing an important role in therapeutic resistance. Nuclear factor-κB (NF-κB) is constitutively activated in melanoma and can serve as a molecular target for cancer therapy and steroid/secosteroid action.Methods:Cultured melanoma cells were used for mechanistic studies on NF-κB activity, utilising immunofluorescence, western blotting, EMSA, ELISA, gene reporter, and estimated DNA synthesis assays. Formalin-fixed, paraffin-embedded specimens from melanoma patients were used for immunocytochemical analysis of NF-κB activity in situ.Results:Novel 20-hydroxyvitamin (20(OH)D3) and classical 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) secosteroids inhibited melanoma cell proliferation. Active forms of vitamin D were found to inhibit NF-κB activity in nonpigmented cells, while having no effect on pigmented cells. Treatment of nonpigmented cells with vitamin D3 derivatives inhibited NF-κB DNA binding and NF-κB-dependent reporter assays, as well as inhibited the nuclear translocation of the p65 NF-κB subunit and its accumulation in the cytoplasm. Moreover, analysis of biopsies of melanoma patients showed that nonpigmented and slightly pigmented melanomas displayed higher nuclear NF-κB p65 expression than highly pigmented melanomas.Conclusion:Classical 1,25(OH)2D3 and novel 20(OH)D3 hydroxyderivatives of vitamin D3 can target NF-κB and regulate melanoma progression in nonpigmented melanoma cells. Melanin pigmentation is associated with the resistance of melanomas to 20(OH)D3 and 1,25(OH)2D3 treatment.

The regulatory effects of polyporus polysaccharide on the nuclear factor kappa B signal pathway of bladder cancer cells stimulated by Bacillus Calmette-Guerin

To detect the effects of Polyporus polysaccharide (PPS), Bacillus Calmette-Guerin (BCG), and their combination on the nuclear factor kappa B (NF-κB) signaling pathway associated-gene expression and investigate the molecular mechanisms of the toxic-reducing effect of PPS in coordination with BCG against bladder cancer. After T739 cells were treated with PPS, BCG and their combination, the changes in mRNA and protein expression of inhibitor of kappa B kinase beta (IKKβ), NF-κB subunit p65 (NF-κB p65), intracellular adhesion molecule 1 (ICAM1) and chemokine (C-c motif) ligand 2 (CCL2) in bladder cancer cell line T739 were determined by relative quantitative real-time PCR, Western blot, and flow cytometry (FCM). NF-κB p65 DNA-binding activity in T739 cell was detected by biotinylated probe-ELISA, and NF-κB p65 nuclear expression in T739 cell was observed by immunohistochemistry. Compared with the T739 control group, the mRNA expression of IKBKB (IKKβ), Rel A (NF-κB p65), ICAM1 and CCL2 in T739 cells treated with BCG were increased obviously (Ratio>2.0), as well as the expression of IKKβ, CCL2 and ICAM1 proteins. Meanwhile, NF-κB p65 DNA-binding activity and NF-κB p65 nuclear expression in T739 cells treated with BCG were up-regulated significantly (P<0.05). Compared with the control, the increased expression in T739 cells were simultaneously down-regulated after PPS treatment, except for ICAM1 protein expression. With cells treated with a combination of BCG and PPS, the expression of genes associated with the NF-κB signaling pathway, such as IKBKB, ICAM1 and CCL2, were all down-regulated compared to the BCG group, as well as Rel A mRNA expression, NF-κB p65 DNA-binding activity and NF-κB p65 nuclear expression. PPS could inhibit the over-activation of the NF-κB signaling pathway induced by BCG in bladder cancer cells and accordingly attenuate the adverse reactions to BCG therapy.

The Biphasic Role of NF-κB in Progression and Chemoresistance of Ovarian Cancer

NF-κB is a transcription factor known to promote tumorigenesis. However, NF-κB is also known to be proapoptotic and may potentially function as a tumor suppressor, although such a functional role has not been extensively investigated in human cancer. A dominant-negative mutant of IκBα with mutations at S32A and S36A was used to inhibit the function of NF-κB in ovarian cancer cell lines. The transcription ability, tumorigenesis, apoptosis, and drug sensitivity were examined in derivative cell lines in comparison with parental cells. We also analyzed the association of nuclear expression of NF-κB p65 with patient survival in an ovarian cancer tissue array. We show that NF-κB functions as a tumor suppressor in four ovarian cancer cell lines, but it functions as an oncogene in their aggressive chemoresistant isogenic variants. NF-κB can exert its proapoptotic or antiapoptotic effect by activating or repressing mitogen-activated protein kinase (MAPK) phosphorylation in parental or aggressive chemoresistant variant cell lines. We also show that the nuclear accumulation of p65 in epithelial cancer tissue is associated with a good response to chemotherapy and can predict longer overall survival for patients with ovarian cancer. Our data provide strong evidence that NF-κB can function as a biphasic regulator, either suppressing or enhancing ovarian cancer growth through the regulation of MAPK and cellular apoptosis.

Niacin attenuates lung inflammation and improves survival during sepsis by downregulating the nuclear factor-κB pathway*

To examine whether niacin attenuates lung inflammation and improves survival during sepsis and to determine whether the beneficial effects of niacin are associated with downregulation of the nuclear factor (NF)-κB pathway. Prospective laboratory study. University laboratory. Male Sprague-Dawley rats (n = 119). To induce endotoxemia in rats, lipopolysaccharide (Escherichia coli, O26:B6) at a dosage of 10 mg/kg was injected into a tail vein and 10 mins later, vehicle, a low dose of niacin (360 mg/kg), or a high dose of niacin (1180 mg/kg) was administered once through an orogastric tube, respectively. We observed the survival of the subjects for 72 hrs. At 6 hrs postlipopolysaccharide, we euthanized animals and measured cytoplasmic phosphorylated inhibitor κB-α and inhibitor κB-α expressions, nuclear NF-κB p65 expression, NF-κB p65 DNA-binding activity, tumor necrosis factor-α, and interleukin-6 gene expressions and histologic damages in lung tissues. We also measured nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide phosphate, reduced glutathione, and malondialdehyde levels in lung tissues. High dose of niacin suppressed NF-κB activation and proinflammatory cytokine gene expressions in lung tissues, reduced histologic lung damages, and improved survival in endotoxemic rats. Furthermore, it increased nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and glutathione levels and decreased malondialdehyde level in lung tissues. High dose of niacin attenuated lung inflammation, reduced histologic lung damages, and improved survival during sepsis in rats. These therapeutic benefits were associated with downregulation of the NF-κB pathway.

MicroRNA-10a regulation of proinflammatory phenotype in athero-susceptible endothelium in vivo and in vitro

A chronic proinflammatory state precedes pathological change in arterial endothelial cells located within regions of susceptibility to atherosclerosis. The potential contributions of regulatory microRNAs to this disequilibrium were investigated by artery site-specific profiling in normal adult swine. Expression of endothelial microRNA10a (miR-10a) was lower in the athero-susceptible regions of the inner aortic arch and aorto-renal branches than elsewhere. Expression of Homeobox A1 (HOXA1), a known miR-10a target, was up-regulated in the same locations. Endothelial transcriptome microarray analysis of miR-10a knockdown in cultured human aortic endothelial cells (HAEC) identified IkappaB/NF-kappaB-mediated inflammation as the top category of up-regulated biological processes. Phosphorylation of IkappaBalpha, a prerequisite for IkappaBalpha proteolysis and NF-kappaB activation, was significantly up-regulated in miR-10a knockdown HAEC and was accompanied by increased nuclear expression of NF-kappaB p65. The inflammatory biomarkers monocyte chemotactic protein 1 (MCP-1), IL-6, IL-8, vascular cell adhesion molecule 1 (VCAM-1), and E-selectin were elevated following miR-10a knockdown. Conversely, knockin of miR-10a (a conservative 25-fold increase) inhibited the basal expression of VCAM-1 and E-selectin in HAEC. Two key regulators of IkappaBalpha degradation--mitogen-activated kinase kinase kinase 7 (MAP3K7; TAK1) and beta-transducin repeat-containing gene (betaTRC)--contain a highly conserved miR-10a binding site in the 3' UTR. Both molecules were up-regulated by miR-10a knockdown and suppressed by miR-10a knockin, and evidence of direct miR-10a binding to the 3' UTR was demonstrated by luciferase assay. Comparative expression studies of endothelium located in athero-susceptible aortic arch and athero-protected descending thoracic aorta identified significantly up-regulated MAP3K7, betaTRC, phopho-IkappaBalpha, and nuclear p65 expression suggesting that the differential expression of miR-10a contributes to the regulation of proinflammatory endothelial phenotypes in athero-susceptible regions in vivo.

Anti-inflammatory effect of SD-01 in RAW 264.7 cells

ABSTRACT Objective : The aim of this study was to investigate anti-inflammatory act ivity of SD-01 methanol extract in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.Methods : Cytotoxic activity of SD-01 methanol extract on RAW 264.7 cell s was measured using 5-(3- caroboxymeth-oxyphenyl)-2H-tetra-zolium inner salt (MTS) assay. The nitric oxide (NO) production was measured by Griess reagent system. And proinflammatory cytokine s and PGE 2 were measured by ELISA method. The levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), Iκ-B-alpha and nuclear NF-κB p65 expression were detected by western blot.Results : Our results indicated that methanol extract of SD-01 significantly inhibited the LPS-induced NO, PGE 2 production and iNOS, COX-2 expression accompanied by an attenuation of TNF-α, IL-1β, IL-6 and MCP-1 production in RAW 264.7 cells. Moreover, methanol extract of SD-01 treatment also blocked LPS-induced NF-κB activation. Conclusion : These findings indicate that methanol extract of SD-01 inhibits the production of pro-inflammatory mediators and cytokines via suppression of NF-κB activation. Take together, these results indicate that methanol extract of SD-01 has the potential for use as an agent of anti-chronic inflammatory diseases. Key Words : SD-01, oxidative stress, inflammation, macrophage

Nuclear factor κB‐associated inflammation mediates impaired vascular endothelial function in non‐diabetic middle‐aged and older overweight/obese men

Healthy middle‐aged/older (MA/O) sedentary men demonstrate vascular endothelial dysfunction with increased endothelial expression of nuclear factor‐kappa B (NFκB), a key proinflammatory nuclear transcription factor. However, there is no direct evidence that NFκB‐associated vascular inflammation mediates endothelial dysfunction in MA/O adults. High doses of Salsalate (4500 mg/d; therapeutic [plasma] 22.2 ± 1.7 mg/100ml, mean ± SE), an aspirin‐like agent that inhibits NFκB, was ingested for 4 days (randomized, placebo‐controlled, double‐blind crossover design) by 10 non‐diabetic overweight or obese MA/O men (62 ± 2 y; BMI 31.4 ± 2 kg/m2) with low‐grade inflammation (plasma C‐reactive protein 2.0 ± 0.7 mg/L). Salsalate reduced total (−30%: 0.42 ± 0.07 vs. 0.60 ± 0.09 intensity/HUVEC intensity, P=0.03) and nuclear (−23%: 128 ± 25 vs. 167 ± 24 total intensity, P=0.06) expression of NFκB p65 in vascular endothelial cells (quantitative immunofluorescence), and increased endothelium‐dependent dilation (EDD, brachial artery flow‐mediated dilation) by 100% (6.2 ± 0.6 vs. 3.1 ± 0.6%, P&lt;0.001) without affecting endothelium‐independent dilation (brachial artery dilation to sublingual nitroglycerin) (19.5 ± 2.2 vs. 19.3 ± 1.6%, P=0.80). These results support the idea that NFκB‐associated vascular inflammation mediates endothelial dysfunction in MA/O overweight/obese men. NIH AG006537, AG013038, AG022241

Pitavastatin inactivates NF-κB and decreases IL-6 production through Rho kinase pathway in MCF-7 cells

The aim of the present study was to provide new mechanistic insight into the effect of pitavastatin at low dose on NF-kappaB activated by TNF-alpha in the human breast cancer cell line (MCF-7). We found that treatment of MCF-7 with 1 microM pitavastatin inhibited the proliferation and suppressed the nuclear expression of NF-kappaB p65 induced by TNF-alpha with Western blotting. Furthermore, EMSA showed that pitavastatin significantly reduced the DNA binding activity of NF-kappaB induced by TNF-alpha. Subsequently, luciferase assay revealed that pitavastatin (1 microM) inhibited the transcriptional activity of the NF-kappaB promoter, which was clearly related to the HMG-CoA reductase activity because addition of mevalonic acid (MEV) could elevate the NF-kappaB activity. Moreover, the Rho kinase inhibitor Y27632 abolished the effect of pitavastatin on NF-kappaB activity. Finally, the addition of TNF-alpha significantly increased IL-6 protein production, which was suppressed by the addition of pitavastatin. These results suggest that pitavastatin at low dose (1 microM) inhibits NF-kappaB activation and decreases IL-6 production induced by TNF-alpha. It is dependent on Rho kinase pathway in human breast cancer cells.

Nuclear factor‐κB (nf‐κB) is frequently expressed in lung cancer and preneoplastic lesions

Nuclear factor-kappaB (NF-kappaB), a key transcription factor thought to play a major role in carcinogenesis, regulates many important signaling pathways involved in tumor promotion. Although NF-kappaB can be activated in lung cancer cell lines by tobacco exposure, there have been no studies of the expression of NF-kappaB in lung cancer pathogenesis. The immunohistochemical expression of NF-kappaB p65 was investigated in 394 lung cancers (370 nonsmall cell lung carcinomas [NSCLC]; and 24 small cell lung carcinomas [SCLC]) and 269 lung normal epithelium and preneoplastic lesions, including hyperplasias, squamous metaplasias, dysplasias, and atypical adenomatous hyperplasias. High levels of nuclear immunohistochemical expression of NF-kappaB p65 were detected in the lung cancers, with significantly higher levels in SCLCs compared with NSCLCs (P<.0001). In adenocarcinomas the NF-kappaB p65 expression level was significantly higher in advanced TNM stages (III-IV) than in earlier stages (I-II) (P<.0001), and when NF-kappaB p65 is dichotomized using 50% as the cutoff point (high vs low), a higher NF-kappaB p65 expression level was detected in tumors having either K-RAS (P = .02) or EGFR (P = .009) mutations compared with wildtype tumors. A relatively high level of nuclear NF-kappaB p65 expression was detected in normal and mildly abnormal epithelium, and a progression with increasing histology severity was detected in preneoplastic lesions. NF-kappaB p65 nuclear expression is an early and frequent phenomenon in the pathogenesis of lung cancer. The findings indicate that NF-kappaB activation plays an important role in lung cancer pathogenesis and is a suitable target for the development of new lung cancer therapies and chemoprevention strategies.

Rosiglitazone, an agonist of peroxisome proliferator-activated receptor gamma, reduces chronic colonic inflammation in rats

Recent studies have shown the implication of the peroxisome proliferator-activated receptor gamma (PPARγ) in control of inflammation, immune and apoptotic responses during early experimental colitis. However, there is little information about the effects of these agents on colonic mucosa under chronic inflammatory conditions. In this study, we have evaluated the effects of rosiglitazone, a PPAR-γ agonist, on the chronic injury caused by intra-colonic administration of trinitrobenzensulfonic acid (TNBS) in rats. Rosiglitazone (1 and 5 mg/kg p.o.) was administered by oral gavage, 24 h after TNBS instillation and daily during 2 weeks before killing the rats. Colons were removed for histological and biochemical analysis. Administration of rosiglitazone corrected the disorders in morphology associated to lesions, significantly reduced the ulceration index, the rise of myeloperoxidase (MPO) and the levels of tumour necrosis factor alpha (TNF-α). In addition, rosiglitazone treatment increased prostaglandin (PG)E 2 production and returned PGD 2 to basal levels. Also, reduced cyclooxygenase (COX)-2 and nuclear transcription factor NF-kappa B (NF-κB) p65 proteins expression. Furthermore, treatment of rats with rosiglitazone caused a significant increase of TNBS-induced apoptosis. In summary, rosiglitazone exerts protective effects in chronic experimental colitis. The anti-inflammatory effects seem to be related to impairment of neutrophil function, absence of up-regulation of TNF-α and decrease of nuclear NF-κB p65 expression. Our results also suggest that the activation of the PPARγ pathway reduces COX-2 overexpression, returns the increased PGD 2 values to basal levels and induces a significant increase of TNBS-induced apoptosis. We conclude that rosiglitazone represents a novel approach to the treatment of ulcerative colitis.