NF-kappaB Activation Research Articles

Temsirolimus targets multiple hallmarks of cancer to impede mesothelioma growth in vivo.

The mechanistic target of rapamycin (mTOR) promotes cancer cell proliferation and survival, transduces pro-angiogenic signals and regulates immune cell differentiation and function. We hypothesized that temsirolimus, an mTOR inhibitor, would curtail experimental mesothelioma progression in vivo by limiting tumour cell growth, abrogating tumour angiogenesis and modulating immune/inflammatory tumour milieu. We produced flank and pleural syngeneic murine mesotheliomas by delivering AE17 and AB1 murine mesothelioma cells into the right flank or the pleural space of C57BL/6 and BALB/c mice, respectively. Animals were given five times/week intraperitoneal injections of 20 mg/kg temsirolimus or vehicle and were sacrificed on day 26 (flank) or on day 15 (pleural) post-tumour cell propagation. Temsirolimus limited mesothelioma growth in vivo by stimulating tumour cell apoptosis, inhibiting tumour angiogenesis, enhancing tumour lymphocyte abundance and blocking pro-tumour myeloid cell recruitment. Pleural fluid accumulation was significantly mitigated in AE17 but not in AB1 mesotheliomas. In vitro, temsirolimus hindered mesothelioma cell growth, NF-kappaB activation and macrophage migration. In conclusion, temsirolimus apart from inducing tumour cell apoptosis, targets tumour angiogenesis and influences inflammatory tumour microenvironment to halt experimental mesothelioma growth in vivo.

Differential inflammatory response of dental pulp explants and fibroblasts to saliva.

To investigate the inflammatory response of dental pulp fibroblasts and the respective explants to whole saliva. Explants from human and porcine dental pulp tissue and isolated dental pulp fibroblasts were used to investigate the inflammatory response to sterile saliva. Cytokine and chemokine expression was assessed by RT-PCR. Western blot analysis and pharmacologic inhibitors were used to determine the involvement of signalling pathways. Dental pulp explants of human and porcine origin exposed to human saliva exhibited no major changes of IL-6 and IL-8 mRNA expression (P>0.05). In contrast, isolated porcine and human dental pulp fibroblasts, when stimulated with human saliva, exhibited a vastly increased expression of IL-6 and IL-8 mRNA (P<0.05). In pulp fibroblasts, saliva also increased the expression of other cytokines and chemokines via activation of NFkappaB, ERK and p38 signalling. Notably, a significantly reduced inflammatory response was elicited when pulp fibroblasts were transiently exposed to saliva. Saliva has a potential impact on inflammation of dental pulp fibroblasts invitro but not when cells are embedded in the intrinsic extracellular matrix of the explant tissue.

Hydroxylated fullerene: a potential antiinflammatory and antioxidant agent for preventing mouse preterm birth

Intrauterine infection such as by Escherichia coli and Ureaplasma spp induce placental inflammation and are one of the leading causes of preterm birth. Here we evaluated hydroxylated fullerene (C60[OH]44) for its in vitro antiinflammatory and antioxidant effects against host cellular responses to the ureaplasma toll-like receptor 2 (TLR2) ligand, UPM-1. In addition, we investigated the preventative effects of C60(OH)44 in vivo in a mouse preterm birth model that used UPM-1. TLR2-overexpressing cell lines and the primary cultures of mouse peritoneal macrophages were pretreated with C60(OH)44. After UPM-1 addition to the cell lines, the activation of the nuclear factor kappa-light chain-enhancer of activated B cells (NF-kappaB) signaling cascade and the production of reactive oxygen species were monitored. The levels of expression of inflammatory cytokines of interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α, and the production of reactive oxygen species were quantified after stimulation with UPM-1. The in vivo preventative effects of C60(OH)44 on mice preterm birth were evaluated by analyzing the preterm birth rates and fetal survival rates in the preterm birth mouse model with placental histological analyses. Pretreatment with C60(OH)44 significantly suppressed UPM-1-induced NF-kappaB activation and reactive oxygen species production in TLR2-overexpressing cell lines. In the primary culture of mouse peritoneal macrophages, UPM-1-induced production of reactive oxygen species and the expression of inflammatory cytokines such as IL-6, IL-1β, and TNF-α were significantly reduced by pretreatment with C60(OH)44. In the UPM-1-induced preterm birth mouse model, the preterm birth rate decreased from 72.7% to 18.2% after an injection of C60(OH)44. Placental examinations of the group injected with C60(OH)44 reduced the damage of the spongiotrophoblast layer and reduced infiltration of neutrophils. C60(OH)44 was effective as a preventative agent of preterm birth in mice.

Abstract A105: Mechanisms of overcoming intrinsic resistance to gemcitabine in pancreatic ductal adenocarcinoma through redox modulation

Abstract Pancreatic ductal adenocarcinoma (PDAC) usually develops intrinsic and extrinsic resistances to gemcitabine, and the mechanisms are not well understood. Here we found gemcitabine treatment induced NOX-derived ROS generation through the increase of p22-phox expression via NF-kappaB activation. As ROS levels increased, nuclear translocation of Nrf2 stimulated transcription of cytoprotective antioxidant genes, specially, those encoding enzymes that catalyze glutathione production to reduce elevated ROS as one of intrinsic resistance counter measures. RNAi depletion of Nrf2 enhanced the sensitivity to gemcitabine in multiple PDAC cells. The addition of beta–phenylethyl isothiocyanate (PEITC) inhibited the ROS detoxification process by reducing glutathione levels; this, in turn, increased the efficacy of gemcitabine in vitro and in vivo. Thus, our results provide novel pre-clinical knowledge in pancreatic cancer chemoresistance, and combination with a redoxmodulating compound to deplete antioxidants can overcome intrinsic gemcitabine resistance. Our study suggests that a redox-mediated pathway that contributes to the intrinsic resistance of PDAC to gemcitabine and provides a basis for developing strategies to preferentially kill PDAC cells through ROS-mediated mechanism. The combination of gemcitabine and PEITC demonstrates a coordinated and selective cytotoxic effect against pancreatic cancer cells and a new avenue for further clinical studies of this deadly disease. Thus, this combination strategy could prove valuable as a cancer treatment. Note: These results were published in Molecular Cancer Therapeutics as a full-length article before the online publication of this abstract. Citation information: Mechanisms of Overcoming Intrinsic Resistance to Gemcitabine in Pancreatic Ductal Adenocarcinoma through the Redox Modulation. Huai-Qiang Ju, Takeshi Gocho, Mitzi Aguilar, Min Wu, Zhuo-Nan Zhuang, Jie Fu, Katsuhiko Yanaga, Peng Huang, and Paul J. Chiao. Mol Cancer Ther March 2015 14:788-798. doi:10.1158/1535-7163.MCT-14-0420. Citation Format: Huaiqiang Ju, Takshi Gocho, Mitzi Aguilar, Zhuo-Nan Zhuang, Jie Fu, Min Wu, Katsuhiko Yanaga, Peng Huang, Paul Chiao. Mechanisms of overcoming intrinsic resistance to gemcitabine in pancreatic ductal adenocarcinoma through redox modulation. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A105.

Anticancer effect of tectochrysin in colon cancer cell via suppression of NF-kappaB activity and enhancement of death receptor expression.

BackgroundFlavonoids are a diverse family of natural phenolic compounds commonly found in fruits and vegetables. Epidemiologic studies showed that flavonoids also reduce the risk of colon cancer. Tectochrysin is one of the major flavonoids of Alpinia oxyphylla Miquel. However, the anti-cancer effects and the molecular mechanisms of tectochrysin in colon cancer cells have not yet been reported. We investigated whether tectochrysin could inhibit colon cancer cell growth at 1, 5, 10 μg/ml. In in vivo study, we injected a tectochrysin treatment dose of 5 mg/kg to each mouse.ResultsTectochrysin suppressed the growth of SW480 and HCT116 human colon cancer cells. The expression of DR3, DR4 and Fas were significantly increased, and pro-apoptotic proteins were also increased. Tectochrysin treatment also inhibited activity of NF-κB. A docking model indicated that tectochrysin binds directly to the p50 unit. In in vivo, tumor weights and volumes in mice were reduced when treated with tectochrysin. Tectochrysin leads to apoptotic cell death in colon cancer cells through activation of death receptors expression via the inhibition of NF-κB.ConclusionsTectochrysin can be a useful agent for the treatment of colon cancer cell growth as well as an adjuvant agent for chemo-resistant cancer cells growth.

Constitutive NF-kappaB activity regulates the expression of VEGF and IL-8 and tumor angiogenesis of human glioblastoma.

Angiogenesis is a key pathologic feature of glioblastoma, which is the most common and most lethal primary brain tumor in adults. The degree of angiogenesis has been shown to be inversely related to patient survival. However, the molecular changes leading to angiogenesis in glioblastoma remain poorly understood. In the present study, we found a direct correlation between nuclear factor (NF)-kappaB activation and angiogenesis in glioblastomas. Blockade of NF-kappaB signaling significantly inhibited glioblastoma growth and angiogenesis in nude mice. These effects were consistent with significant inhibition of the expression of multiple angiogenic molecules, including vascular endothelial growth factor, and interleukin-8, in vitro and in vivo. Furthermore, blockade of NF-kappaB signaling also significantly inhibited the angiogenic potential of glioblastoma cells in vitro and angiogenesis of brain tumors in mouse xenograft models. Collectively, these results suggest that NF-kappaB activation plays a critical role in the growth and progression of glioblastoma and is a potential target for therapy for human glioblastoma.

A comparative study of mechanical strain, icariin and combination stimulations on improving osteoinductive potential via NF-kappaB activation in osteoblast-like cells

BackgroundThe combination of drugs and exercise was the effective treatment in bone injure and rebuilding in clinic. As mechanical strain has potential in inducing the differentiation of osteoblasts in our previous study, the further research to investigate the combination of mechanical strain and icariin stimulation on inducing osteoblast proliferation, differentiation and the possible mechanism in MC3T3-E1 cell line.MethodsA whole cell enzyme-linked immunosorbent assay that detects the bromodeoxyuridine incorporation during DNA synthesis was applied to evaluate the proliferation. The mRNA expression of alkaline phosphatase (ALP), osteocalcin (OCN), type I collagen (Col I), bone morphogenetic protein-2 (BMP-2) and BMP-4 was detected by real-time reverse-transcription polymerase chain reaction. The activity of ALP was analyzed by ELISA and the protein expression of OCN, Col I and BMP-2 was assessed by western blot. Moreover, the activity of nuclear transcription factor kappa-B (NF-κB) signaling pathway was investigated with the expression of inhibitor of κB (IκB) α, phosphorylation of IκB-α (P-IκB-α), p65, P-p65 by western blot.ResultsWe observed that compared to single mechanical strain or icariin stimulation, the mRNA and protein expressions of ALP (P < 0.05 or P < 0.01), OCN (P < 0.01) and Col I (P < 0.05 or P < 0.01) were increased significantly by the combination of mechanical strain and icariin stimulation. Moreover, the combination of mechanical strain and icariin stimulation could up-regulate the expression of BMP-2 (P < 0.01) and BMP-4 compared to single mechanical strain or icariin stimulation. The combination of mechanical strain and icariin stimulation could activate NF-κB signaling pathway by increasing the expression of IκB α, P-IκB-α, p65, P-p65 (P < 0.01).ConclusionThe combination of mechanical strain and icariin stimulation could activate the NF-κB pathway to improve the proliferation, differentiation of osteoblast-like cells.

Abstract 356: IKK-Beta Deletion Alleviates the Atherogenetic Effect of Intermittent Hypoxia Induced Macrophage Foam Cell Formation

Background: Obstructive sleep apnea syndrome (OSAS) is a common sleeping disorder characterized by intermittent hypoxia (IH). Clinical studies have previously shown an independent association between obstructive sleep apnea and atherosclerosis. Furthermore, it has been previously shown that such a predisposition to atherosclerosis in OSAS patient can be caused by various inflammatory mediators, particularly the NF-kappa B (NF-kB) pathway. Foam cells or lipid-laden macrophages in the atherosclerotic lesion have been well documented as a hallmark of atherosclerosis; however, the contribution of IH, such as in OSAS, to foam cell formation is not yet fully understood. Previous observations have led us to hypothesized that IH induces macrophage foam cell formation due to the activation of NF-kappa B pathway. Methods: Myeloid restricted IKK-beta deleted mice were generated by a Cre/lox recombination system to inactivate the NF-kB pathway in macrophages. Thioglycollate-elicited peritoneal macrophages were incubated with 200 μg/ml of low-density lipoprotein and simultaneously exposed to either IH (Normoxia: 8min, 0.5% O2: 10min) or normoxia for 24 hours. After exposure, the extent of foam cell formation was assessed by quantification of intracellular cholesterol. Finally, we compared the differences in gene expression using RNA-seq between wild type and IKK-beta deleted macrophages exposed to either IH or normoxia for 24 hours. Results: IH significantly increased total cholesterol in wild type macrophages (63.4±3.3 μg/mg of cellular protein, n=9) in comparison to normoxia (51.2±1.6). Interestingly, such increase in intracellular cholesterol in response to IH-exposure was abolished by IKK-beta deletion (IH 52.4±1.1; normoxia 50.0±1.6 n=8), suggesting that NF-kB pathway regulated gene expression is critical for IH-induced foam cell formation. Indeed, we have found that NF-kB knockout abolished IH-induced expressional alterations in 364 genes, which are potential candidates for regulating intracellular cholesterol. Conclusion: NF-kB activation plays a critical role in IH-induced macrophage foam cell formation.

Abstract 378: Contribution of Unfolded Protein Response in Arsenic-Induced Endothelial Activation and Atherosclerois

Epidemiological and animal studies suggest that exposure to arsenic contaminated water exacerbates atherosclerosis. However, the mechanisms by which arsenic exerts its atherogenic effects are not known. We observed that chronic (1 μM; 4 days) or acute (20 μM; 2-6 hours) exposure of sodium arsenite to human umbilical vein endothelial cells (HUVEC) increased the surface expression of adhesion molecules ICAM-1, VCAM-1 and E-Selectin by 1.2-1.5-fold; leukocyte adhesion by 1.5-3.5 fold; leukocyte trans-endothelial migration by 1.7-2.5-fold; and mRNA expression of IL-8 by 5-25-fold. Similarly, exposure of human aortic endothelial cells and mouse aortic endothelial cells to arsenic also caused endothelial activation. Arsenic also enhanced the unfolded protein response (UPR) in endothelial cells as evident by the phosphorylation of IRE-1 and activation of its downstream proteins JNK and NF-kappaB; nuclear translocation of ATF6, and increased expression of molecular chaperones GRP78 and HERP. SiRNA-mediated knockdown of IRE-1 attenuated arsenic-induced NF-kappaB activation and IL-8 expression, but did not affect JNK phosphorylation. Adenoviral transfection with ATF-6 upregulated GRP78 and decreased the expression of IL-8. Similarly, pre-incubation of endothelial cells with phenyl butyric acid (PBA; 5 mM, 16h), a chemical chaperone of protein folding, significantly (P&lt;0.05) prevented arsenic-induced leukocyte adhesion, trans-endothelial migration and IL-8 expression. Feeding of apoE-null mice with PBA for 16 weeks inhibited the arsenic-induced UPR in atherosclerotic lesions; expression of adhesion molecules on endothelial cells lining the atherosclerotic lesions; lesional and systemic inflammation; and prevented the arsenic-induced exacerbation of lesion formation by 90% (P&lt;0.05). Together, these data suggest that arsenic causes endothelial activation and exacerbates atherosclerosis by triggering UPR and chemical chaperones of protein folding prevent arsenic-induced exacerbation of atherogenesis.

Protective Effects of a Novel Probiotic Strain of Lactobacillus plantarum JSA22 from Traditional Fermented Soybean Food Against Infection by Salmonella enterica Serovar Typhimurium.

Lactobacillus species have been shown to enhance intestinal epithelial barrier function, modulate host immune responses, and suppress the growth of pathogenic bacteria, yeasts, molds, and viruses. Thus, lactobacilli have been used as probiotics for treating various diseases, including intestinal disorders, and as biological preservatives in the food and agricultural industries. However, the molecular mechanisms used by lactobacilli to suppress pathogenic bacterial infections have been poorly characterized. We previously isolated Lactobacillus plantarum JSA22 from buckwheat sokseongjang, a traditional Korean fermented soybean food, which possessed high enzymatic, fibrinolytic, and broad-spectrum antimicrobial activity against foodborne pathogens. In this study, we investigated the effects of L. plantarum JSA22 on the growth of S. Typhimurium and S. Typhimurium-induced cytotoxicity by stimulating the host immune response in intestinal epithelial cells. The results showed that coincubation of S. Typhimurium and L. plantarum JSA22 with intestinal epithelial cells suppressed S. Typhimurium infection, S. Typhimurium-induced NF-kappaB activation, and IL-8 production, and lowered the phosphorylation of both Akt and p38. These data indicated that L. plantarum JSA22 has probiotic properties, and can inhibit S. Typhimurium infection of intestinal epithelial cells. Our findings can be used to develop therapeutic and prophylactic agents against pathogenic bacteria.

A Novel Synthetic Compound, YH-1118, Inhibited LPS-Induced Inflammatory Response by Suppressing I��B Kinase/NF-��B Pathway in Raw 264.7 Cells

For the search of a potent first-in-class compound to inactivate macrophages responsible for inflammatory responses, in the present study, we investigated the anti-inflammatory effects of YH-1118, a novel synthetic compound, in a lipopolysaccharide (LPS)-stimulated mouse macrophage cell line, Raw 264.7. YH-1118 inhibited LPS-induced nitric oxide (NO) production and inducible NO synthase (iNOS) expression at both the protein and mRNA levels. The suppression of LPS-induced iNOS expression by YH-1118 was mediated via nuclear factor kappa B (NF-κB), but not activator protein-1 (AP-1) transcription factor. This was supported by the finding that YH-1118 attenuated the phosphorylation of inhibitor of κBα (IκBα) and nuclear translocation and DNA binding activity of NF-kappaB. Through the mechanisms that YH- 1118 inhibited the activation of IkappaB kinases (IKKs), upstream activators of NF-κB, or p38 MAPK, YH-1118 significantly suppressed LPS-induced production of pro-inflammatory cytokines, tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6 (p < 0.05). In conclusion, our results suggest that YH-1118 inhibits LPS-induced inflammatory responses by blocking IKK and NF-κB activation in macrophages, and may be a therapeutic candidate for the treatment of various inflammatory diseases.

Macrophages modulate migration and invasion of human tongue squamous cell carcinoma.

Oral tongue squamous cell carcinoma (OTSCC) has a high mortality rate and the incidence is rising worldwide. Despite advances in treatment, the disease lacks specific prognostic markers and treatment modality. The spreading of OTSCC is dependent on the tumor microenvironment and involves tumor-associated macrophages (TAMs). Although the presence of TAMs is associated with poor prognosis in OTSCC, the specific mechanisms underlying this are still unknown. The aim here was to investigate the effect of macrophages (Mfs) on HSC-3 tongue carcinoma cells and NF-kappaB activity. We polarized THP-1 cells to M1 (inflammatory), M2 (TAM-like) and R848 (imidazoquinoline-treated) type Mfs. We then investigated the effect of Mfs on HSC-3 cell migration and NF-kappaB activity, cytokine production and invasion using several different in vitro migration models, a human 3D tissue invasion model, antibody arrays, confocal microscopy, immunohistochemistry and a mouse invasion model. We found that in co-culture studies all types of Mfs fused with HSC-3 cells, a process which was partially due to efferocytosis. HSC-3 cells induced expression of epidermal growth factor and transforming growth factor-beta in co-cultures with M2 Mfs. Direct cell-cell contact between M2 Mfs and HSC-3 cells induced migration and invasion of HSC-3 cells while M1 Mfs reduced HSC-3 cell invasion. M2 Mfs had an excess of NF-kappaB p50 subunit and a lack of p65 subunits both in the presence and absence of HSC-3 cells, indicating dysregulation and pro-tumorigenic NF-kappaB activation. TAM-like cells were abundantly present in close vicinity to carcinoma cells in OTSCC patient samples. We conclude that M2 Mfs/TAMs have an important role in OTSCC regulating adhesion, migration, invasion and cytokine production of carcinoma cells favouring tumor growth. These results demonstrate that OTSCC patients could benefit from therapies targeting TAMs, polarizing TAM-like M2 Mfs to inflammatory macrophages and modulating NF-kappaB activity.

Resveratrol loaded gelatin nanoparticles synergistically inhibits cell cycle progression and constitutive NF-kappaB activation, and induces apoptosis in non-small cell lung cancer cells

PurposePreviously, we reported that the prepared resveratrol (RSV) loaded gelatin nanoparticles (GNPs) possessed enhanced anticancer effect than free RSV in non-small cell lung carcinoma cells and Swiss albino mice. The present study aims to explore the relevant mechanism of cell death induced by the combination of RSV-GNPs in NCI-H460 cells. Methods and resultsTo increase its bioavailability and anticancer efficacy, we have encapsulated RSV-GNPs by Coacervation method. The detailed methods of preparation and characterization of RSV-GNPs were reported in our earlier publication. RSV-GNPs treated cells showed a further increased level of lipid peroxidative markers, i.e. TBARS and LHP in NCI-H460 cells. Activities of antioxidant enzymes SOD, CAT, GPx and GSH levels were decreased upon the treatment with RSV-GNPs in NCI-H460 cells. The nuclear fragmentation was evaluated by DAPI staining and data showed condensed apoptotic bodies upon treatment with the combination of RSV-GNPs compared to RSV alone treatment group. In addition, cell death induced by RSV-GNPs was mainly due to apoptosis which was characterized by a nuclear DNA fragmentation in a ladder-pattern was obtained from the genomic DNA analysis. Moreover, Western blotting analysis showed that apoptosis induced by RSV-GNPs is associated with the increased Bax, p53, p21, caspase-3 protein levels, and decreased Bcl-2 and NF-κB proteins expression, which indicates the involvement of mitochondria-dependent apoptosis in the anticancer efficacy of RSV-GNPs in NCI-H460 cells. It was also found that this enhanced anticancer efficacy of RSV-GNPs induced cell arrest in the G0/G1 phase of cell cycle. ConclusionsTaken together, the results of our study clearly suggested that the cell death induced by the combination of RSV-GNPs would involve alteration in expression of p53, p21, caspase-3, Bax, Bcl-2 and NF-κB, indicating oxidative mechanism in NCI-H460 cells. Based on these results, it is concluded that GNPs is an ideal way to deliver RSV because of its high loading efficiency and superior efficacy in NCI-H460 cells.

HMGB1 induces angiogenesis in rheumatoid arthritis via HIF-1α activation.

High-mobility group box protein 1 (HMGB1), a nonhistone nuclear protein and a cytokine mediator, is implicated in the pathogenesis of rheumatoid arthritis (RA). Extracellular HMGB1 binds to its receptors and triggers downstream signal cascade leading to the perpetuation of synovitis and local tissue invasion. Here, we investigated a novel role of HMGB1 in regulating hypoxia-inducible factor (HIF)-1α to mediate angiogenesis in RA synovium. HIF-1α mRNA levels and activities in synovial fibroblasts from RA patients were enhanced by HMGB1. Pharmacological inhibition of TLR4 and NF-kappaB activation blocked the HMGB1-dependent upregulation of HIF-1α mRNA expression and its activity, suggesting the involvement of transcriptional regulation. HMGB1 stimulated expression of vascular endothelial growth factor (VEGF), and inhibition of HIF-1α attenuated HMGB1-induced VEGF. Conditioned media derived from HMGB1-stimulated synovial fibroblasts enhanced tube formation in human microvascular endothelial cells by upregulating HIF-1α. In the joint tissues of mice with collagen-induced arthritis, treatment with anti-HMGB1 neutralizing antibody prevented blood vessel formation in association with decreased expression of HIF-1α. These observations support the idea that increased HMGB1 induces an extension of inflamed synovium by accelerating angiogenesis in RA through enhancement of HIF-1α activation. Therefore, inhibition of HMGB1 could prove beneficial for the treatment of angiogenesis in RA.

Molecular mechanisms of constitutive and inducible NF-kappaB activation in oesophageal adenocarcinoma

BackgroundNuclear factor-kappaB (NF-κB) regulates the expression of a large number of genes involved in the immune and inflammatory response. NF-κB is constitutively activated in oesophageal tumour tissues and induced in oesophageal cells by bile and acid. The aim of the present study was to define the mechanisms underlying NF-κB activation in oesophageal adenocarcinoma. Patients and methodsFresh biopsy specimens were obtained from 20 patients with oesophageal adenocarcinoma. The activation of NF-κB in oesophageal tumour specimens and oesophageal SKGT-4 cells was assessed by gel mobility shift and Western blotting. Phosphorylation of protein kinase B (AKT/PKB), Ikappa kinase-alpha/beta (IKK-α/β) and extracellular signal-regulated kinase 1/2 (ERK1/2) was examined by Western blotting. High content analysis was used to quantify NF-κB translocation in oesophageal cells. ResultsOesophageal tumour tissues had higher levels of NF-κB. Increased levels of phosphorylated AKT and IKK-α/β and ERK1/2 were detected in tumour tissues compared with normal oesophageal mucosa. Exposure of SKGT-4 cells to deoxycholic acid (DCA) or acid resulted in NF-κB activation and phosphorylation of AKT, IKK-α/β and ERK1/2. Specific inhibitors for phosphoinositide 3-kinase; PI3K (LY294002 and worhmannin) and ERK1/2 inhibitors (PD98059 and U0126) suppressed DCA- and acid-induced NF-κB activation. The proteasome inhibitor MG-132 and the antioxidants vitamin C and pyrrolidine dithiocarbamate (PDTC) also inhibited NF-κB activation. ConclusionsOur data demonstrate a major role for PI3K/AKT–IKK-α/β–ERK1/2 signalling pathway in NF-κB activation in oesophageal adenocarcinoma. These results suggest that NF-κB may be a prognostic marker for oesophageal adenocarcinoma, and modulating of NF-κB may uncover new therapeutic strategies.

Novel Types of Small RNA Exhibit Sequence- and Target-dependent Angiogenesis Suppression Without Activation of Toll-like Receptor 3 in an Age-related Macular Degeneration (AMD) Mouse Model.

RNA interference (RNAi) has become a powerful tool for suppressing gene expression in vitro and in vivo. A great deal of evidence has demonstrated the potential for the use of synthetic small interfering RNAs (siRNAs) as therapeutic agents. However, the application of siRNA to clinical medicine is still limited, mainly due to sequence-independent suppression of angiogenesis mediated by Toll-like receptor 3 (TLR3). Here, we describe novel types of synthetic RNA, named nkRNA and PnkRNA, that exhibit sequence-specific gene silencing through RNAi without activating TLRs or RIG-I–like receptor signaling. In addition, we confirmed the therapeutic effect for the novel types of RNA in an animal model of age-related macular degeneration (AMD) without retinal degeneration. These data indicate that nkRNA and PnkRNA are of great potential utility as therapies against blinding choroidal neovascularization due to AMD.

Up-Regulation of Glioma-Associated Oncogene Homolog 1 Expression by Serum Starvation Promotes Cell Survival in ER-Positive Breast Cancer Cells

Background/Aims: Cancer cells are resistant to ischemia and starvation. Glioma-associated oncogene homolog 1 (Gli1) is a positive transcriptional activator of Hedgehog (Hh) pathway and plays an essential role in the development of cancers, including breast cancer. However, how Gli1 promotes cell survival remains elusive. The main purpose of this study is to investigate the pro-survival effect of Gli1 under serum starvation and its molecular mechanism in ER-positive breast cancer cells. Methods: Gene expression was determined by quantitative real-time PCR (QRT-PCR) and Western blot. The survival of Gli1 stably transfected ER-positive breast cancer cell lines (Gli1-MCF-7 and Gli1-T47D cells) and their untransfected control cells was estimated by WST-8 assay. Microarray analysis was performed to screen downstream Hh/Gli1 target genes in Gli1-overexpressed MCF-7 cells. Transcriptional activities of NF-kappaB were measured by luciferase assays. ChIP analysis was performed to explore whether cIAP2 was a direct target gene of Gli1. Results: Serum starvation significantly up-regulated the expression of Gli1 gene through activating PI3K/AKT pathway. Over-expression of Gli1 markedly promoted cell survival under serum starvation. Microarray analysis revealed that 338 genes were differentially expressed in Gli1-MCF-7 cells compared with those in the control cells. Among these genes, cellular inhibitor of apoptosis 2 (cIAP2), coding an anti-apoptosis and pro-survival protein, was significantly up-regulated not only by Hh/Gli1 pathway, but also by serum starvation. However, ChIP assay revealed no binding of Gli1 to cIAP2 promoter at the region of -1792 to -1568bp. Moreover, over-expression of Gli1 resulted in enhanced trans-activation of transcriptional factor NF-κB. Suppression of NF-κB signaling with NF-κB inhibitor Bay11-7082, significantly reduced the expression of cIAP2 and the cell survival under serum starvation. Conclusion: Serum starvation significantly up-regulated the expression of Gli1, which in turn increased its key target cIAP2 expression and enhanced NF-κB/cIAP2 pathway, resulting in promoting cell survival under serum starvation. These findings may provide new insights into the pro-survival mechanisms of Gli1 in breast cancer.

Inhibition of andrographolide in RAW 264.7 murine macrophage osteoclastogenesis by downregulating the nuclear factor-kappaB signaling pathway.

This study aims to investigate the effects of andrographolide (AGP) on osteoclast formation in RAW 264.7 murine macrophage cells. The effects of AGP on cell viability were determined in RAW 264.7 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of AGP on osteoclast formation were tested by osteoclast staining with tartrate-resistant acid phosphatase (TRAP). The effects of AGP on receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL)-induced, NF-kappaB-dependent transcription in RAW 264.7 cells were assessed using luciferase reporter assays. The results demonstrated that the viability of osteoclast precursor RAW 264.7 cells was not affected by AGP treatment at a concentration of 0.4 to 10 μM. Additionally, the number of TRAP-positive osteoclasts was significantly reduced by the same concentrations of AGP treatment. AGP also inhibited RANKL-induced NF-kappaB activation in a dose-dependent fashion as evidenced by luciferase reporter assays. In summary, this study demonstrates that AGP inhibits osteoclastogenesis in RAW 264.7 murine macrophage cells through downregulation of the NF-kappaB signaling pathway.

Invited Commentary

Miyahara and colleagues [1Miyahara S. Nakai H. Izawa N. et al.Influences of chronic obstructive pulmonary disease on outcomes of total arch replacement.Ann Thorac Surg. 2015; 99: 72-79Scopus (11) Google Scholar] have provided an interesting analysis on the influence of chronic obstructive pulmonary disease (COPD) on the outcome of total arch replacement. The association between aortic wall degeneration and destruction of lung tissue is under current debate. COPD as well as aortic disease are important morbidities of an aging population. The prevalence of lung disease in patients with aortic disease is high, reaching up to 47% of patients with aortic arch pathologies and to even higher rates of 55% in patients with abdominal aortic aneurysms [2LeMaire S.A. Price M.D. Parenti J.L. et al.Early outcome after aortic arch replacement by using the Y-graft technique.Ann Thorac Surg. 2011; 91: 700-707Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 3Fowkes F.G. Anandan C.L. Lee A.J. et al.Reduced lung function in patients with abdominal aortic aneurysm is associated with activation of inflammation and hemostasis, not smoking or cardiovascular disease.J Vasc Surg. 2006; 43: 474-480Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar]. For this reason, careful preoperative assessment, especially spirometry, is usually recommended but carries a potential risk for patients with thoracic aortic disease due to changes of intrathoracic pressure. To avoid potential harm, patients are not assessed by spirometry preoperatively, and the presence or severity of COPD is overlooked or remains underdiagnosed. The data of Miyahara and colleagues [1Miyahara S. Nakai H. Izawa N. et al.Influences of chronic obstructive pulmonary disease on outcomes of total arch replacement.Ann Thorac Surg. 2015; 99: 72-79Scopus (11) Google Scholar] once again underline the importance of COPD as a risk factor for in-hospital death as well for postoperative pulmonary complications. Especially patients undergoing aortic arch replacement necessitating deep hypothermic circulatory arrest should be evaluated very carefully for preoperative lung disease. Ischemia-reperfusion can be the initiating factor for pulmonary dysfunction during and after extracorporeal circulation and can possibly induce devastating damage to already diseased lung tissue. Experimental data reveal an increase of inflammation factors interleukin-8 and interleukin-6, as well as activation of nuclear factor-κB, and might explain the mechanism of deep hypothermic circulatory arrest–induced pulmonary injury [4Zheng J.H. Gao B.T. Jiang Z.M. Yu X.Q. Xu Z.W. Evaluation of early macrophage activation and NF-kappaB activity in pulmonary injury caused by deep hypothermia circulatory arrest: an experimental study.Pediatr Cardiol. 2010; 31: 215-221Crossref PubMed Scopus (12) Google Scholar]. In summary, their report reflects daily clinical routine: the vast majority of patients with mild to moderate COPD do well, even after extended aneurysm repair. Those patients presenting with severe COPD and the definitive need deep hypothermic circulatory arrest have to be assessed very carefully. Influences of Chronic Obstructive Pulmonary Disease on Outcomes of Total Arch ReplacementThe Annals of Thoracic SurgeryVol. 99Issue 1PreviewAlthough an association between chronic obstructive pulmonary disease (COPD) and adverse surgical outcomes has been proposed, the impact of COPD severity on postoperative outcomes remains unclear. Our objective was to analyze the prognostic implication of COPD severity on outcomes after total aortic arch replacement. Full-Text PDF

Mechanisms of Immune Signaling in Colitis-Associated Cancer

The inflammatory bowel diseases ulcerative colitis and Crohn’s disease are associated with an increased risk for the development of colorectal cancer. During recent years, several immune signaling pathways have been linked to colitis-associated cancer (CAC), largely owing to the availability of suitable preclinical models. Among these, chronic intestinal inflammation has been shown to support tumor initiation through oxidative stress–induced mutations. A proinflammatory microenvironment that develops, possibly as a result of defective intestinal barrier function and host–microbial interactions, enables tumor promotion. Several molecular pathways such as tumor necrosis factor/nuclear factor-κB or interleukin 6/signal transducer and activator of transcription 3 signaling have been identified as important contributors to CAC development and could be promising therapeutic targets for the prevention and treatment of CAC.