NF-κB Luciferase Reporter Assay Research Articles

Cerium Oxide Nanoparticles Biosynthesized Using Fresh Green Walnut Shell in Microwave Environment and their Anticancer Effect on Breast Cancer Cells.

In this study, cerium oxide nanoparticles (CONPs) were synthesized using fresh green walnut shell extract in microwave environment. The morphology and structure of the CONPs were determined using ultraviolet-visible (UV/VIS), attenuated total reflection-Fourier transform infrared (ATR-FT-IR), X-ray diffraction (XRD), energy-dispersive X-ray (EDX) spectroscopy, and scanning electron microscopy (SEM). Crystal purple staining, Annexin V-FITC detection, RT-PCR, P53, and NF-κB luciferase reporter assays were performed to evaluate the mechanism of action of CONPs in breast cancer cell lines (MCF7). The biosynthesized CONPs showed cytotoxic effects and induced apoptosis in MCF7 cells. Furthermore, CONPs induced P53 expression and suppressed NF-κB gene expression, both of which were confirmed using reporter assays. Based on the present results, it was concluded that CONPs can induce apoptosis by acting on P53 at the transcriptional level and may cause cell death by suppressing NF-κB-mediated transcription.

The lncARSR/PTEN/Akt/nuclear factor-kappa B feedback regulatory loop contributes to doxorubicin resistance in hepatocellular carcinoma.

Chemoresistance is a major obstacle to hepatocellular carcinoma (HCC) chemotherapy. Our previous study found that long noncoding RNA lncARSR (lncRNA Activated in RCC with Sunitinib Resistance) activated Akt signaling via repressing phosphatase and tensin homolog (PTEN) during doxorubicin resistance in HCC. The purpose of this study is to further explore lncARSR-mediated mechanisms and roles during doxorubicin resistance in HCC. The expression of lncARSR was detected by real-time quantitative polymerase chain reaction (qPCR). Nuclear factor-kappa B (NF-κB) activity was detected by NF-κB luciferase reporter assays, western blot, and NF-κB transcription factor assays. The effects of NF-κB on lncARSR were detected by chromatin immunoprecipitation assay, promoter luciferase reporter assay, and real-time qPCR. The effects of lncARSR/Akt/NF-κB on doxorubicin resistance were detected by Cell Counting Kit-8 assay, capsase-3 activity assay, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelingassay. lncARSR activated NF-κB signaling through activation of Akt. NF-κB transactivated lncARSR through directly binding lncARSR promoter and increasing lncARSR promoter activity. Akt transactivated lncARSR via activating NF-κB signaling. Thus, lncARSR, Akt, and NF-κB formed a positive feedback regulatory loop in HCC. Through this feedback loop, lncARSR auto-regulated its transcription. Drug sensitivity assays showed that the lncARSR/Akt/NF-κB feedback regulatory loop promoted doxorubicin resistance in HCC. These findings identified the lncARSR/Akt/NF-κB feedback regulatory loop in HCC, which represent potential therapeutic targets for improving doxorubicin sensitivity in HCC.

YB-1 Interferes with TNFα-TNFR Binding and Modulates Progranulin-Mediated Inhibition of TNFα Signaling.

Inflammation and an influx of macrophages are common elements in many diseases. Among pro-inflammatory cytokines, tumor necrosis factor α (TNFα) plays a central role by amplifying the cytokine network. Progranulin (PGRN) is a growth factor that binds to TNF receptors and interferes with TNFα-mediated signaling. Extracellular PGRN is processed into granulins by proteases released from immune cells. PGRN exerts anti-inflammatory effects, whereas granulins are pro-inflammatory. The factors coordinating these ambivalent functions remain unclear. In our study, we identify Y-box binding protein-1 (YB-1) as a candidate for this immune-modulating activity. Using a yeast-2-hybrid assay with YB-1 protein as bait, clones encoding for progranulin were selected using stringent criteria for strong interaction. We demonstrate that at physiological concentrations, YB-1 interferes with the binding of TNFα to its receptors in a dose-dependent manner using a flow cytometry-based binding assay. We show that YB-1 in combination with progranulin interferes with TNFα-mediated signaling, supporting the functionality with an NF-κB luciferase reporter assay. Together, we show that YB-1 displays immunomodulating functions by affecting the binding of TNFα to its receptors and influencing TNFα-mediated signaling via its interaction with progranulin.

LncRNA ARFRP1 knockdown inhibits LPS-induced the injury of chondrocytes by regulation of NF-κB pathway through modulating miR-15a-5p/TLR4 axis.

AimsLong non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been reported as the important regulators in osteoarthritis (OA). However, the detailed mechanism is implicated. The aim of this study is to reveal the functional mechanism of lncRNA ARFRP1 and miR-15a-5p in osteoarthritis. Materials and methodsThe expression level of genes was detected by quantitative real time polymerase chain reaction (qRT-PCR) or western blot assay. Cell Counting Kit-8 (CCK-8) was used to assess cell viability. Cell apoptosis rate was analyzed by flow cytometry analysis. Furthermore, Enzyme-linked immunosorbent assay (ELISA) was performed to measure tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β contents. The interaction between miR-15a-5p and ARFRP1 or Toll-like receptor 4 (TLR4) was predicted by miRcode or PITA, and then confirmed by the dual luciferase reporter assay or pull down assay. Besides, NF-κB-driven luciferase activity was determined using NF-κB luciferase reporter assay. Key findingsARFRP1 and TLR4 levels were increased and miR-15a-5p level was decreased in OA cartilage tissues and lipopolysaccharides (LPS)-induced chondrocytes. ARFRP1 knockdown inhibited LPS-induced the injury of chondrocytes. Interestingly, miR-15a-5p downregulated by ARFRP1 negatively modulated TLR4 expression through interaction. ARFRP1 mediated LPS-induced the injury of chondrocytes via regulating miR-15a-5p/TLR4 axis. Furthermore, ARFRP1 exerted function by modulation of NF-κB pathway. SignificanceOur findings confirmed that ARFRP1 mediated LPS-induced the injury of chondrocytes through regulating NF-κB pathway by modulation of miR-15a-5p/TLR4 axis, providing theoretical basis for the treatment of OA patients.

Development and characterization of a novel anti-OX40 antibody for potent immune activation.

With the great success of anti-CTLA-4 and anti-PD-1 therapeutics in cancer immunotherapy, tumor necrosis factor receptor superfamily members have been recognized as ideal targets to provide co-stimulatory signals in combination with immune checkpoint blocking antibodies. Among these is OX40 (CD134), a co-stimulatory molecule expressed by activated immune cells. Recently, several anti-OX40 agonistic monoclonal antibodies, pogalizumab as the most advanced, have entered early phase clinical trials. Using ayeast platform and multiple screening methods, we identified a fully human anti-OX40 antibody (IBI101) with distinct modes of action. Unlike pogalizumab, IBI101 partially blocks the binding of OX40 to its ligand OX40L and exhibits both FcγR-dependent and independent agonistic activities in NF-κB luciferase reporter assays. IBI101 also promotes T cell activation and proliferation in vitro. These unique properties partially explain the more potent anti-tumor activity of IBI101 than that of pogalizumab in humanized NOG mice bearing LoVo tumors. In addition, IBI101 shows efficacious anti-tumor activity in mice when administrated alone or in combination with anti-PD-1 antibodies. In human OX40 knock-in mice bearing MC38 colon carcinoma, IBI101 treatment induces tumor antigen-specific CD8+ T-cell responses, decreases immunosuppressive regulatory T cells in tumor, and enhances the immune response to PD-1 inhibition. Preclinical studies of IBI101 in non-human primates demonstrate typical pharmacokinetic characteristics of an IgG antibody and no drug-related toxicity. Collectively, IBI101 has desirable preclinical attributes which support its clinical development for cancer treatment.

293 Pityriasis rubra pilaris type V with a heterozygous mutation in CARD14

Pityriasis rubra pilaris (PRP) is a rare chronic inflammatory skin disorder clinically characterized by keratotic follicular papules, well-demarcated scaly erythematous plaques interspersed with distinct islands of uninvolved skin, and palmoplantar keratoderma. The autosomal dominant subtype, which is classified as PRP type V (PRPV), is associated with gain-of-function mutations in CARD14. Although around 30 cases with PRPV have been reported in the literature, diagnosis and treatment remain difficult. In this study, we analysed a 60-year-old Japanese male with erythroderma, palmoplantar keratoderma, ectropion and joint contractures of the fingers, all of which had appeared since birth. Notably, he has experienced multiple skin tumours including malignant melanoma and squamous cell carcinoma. Although the patient was initially diagnosed with congenital ichthyosiform erythroderma (CIE), mutation analysis unexpectedly revealed a heterozygous mutation c.356T>C (p.Met119Thr) in CARD14, whereas no pathogenic mutations were detected in any of the 12 genes responsible for CIE. This de novo mutation was absent from available databases. Overexpression of this mutant CARD14 formed aggregations in the cytoplasm, which exhibited a completely different distribution from that of the wild type. Furthermore, the NF-κB luciferase reporter assay revealed that mutant CARD14 showed a 2-fold increase in NF-κB activity compared with the wild type, suggesting the pathogenicity of the mutation. These findings led to the final diagnosis of PRPV. We have treated the present case with ustekinumab, which we have found partially effective. This study further expands our understanding of clinical and genetic features of PRPV. The multiple occurrences of skin tumours with this patient may suggest potential tumorigenesis associated with the disease.

Abstract 885: Induction of PRDX2 by H. pylori reduces ROS and promotes cancer cell survival and resistance to cisplatin

Abstract Background & Aims: The antioxidant enzyme peroxiredoxin 2 (PRDX2) plays a critical role in regulating reactive oxygen species (ROS) levels in several diseases. Helicobacter pylori (H. pylori) infection is a well-known risk factor of gastric cancer. The role of PRDX2 in gastric tumorigenesis remains largely unknown. We investigated the molecular function and regulation of PRDX2 in response to infection with H. pylori and cisplatin treatment in gastric cancer cells. Methods:Western blots (WB) and quantitative real-time PCR (qPCR) analysis were performed on AGS, SNU-1 and MKN28 gastric cancer cell lines to detect PRDX2 expression levels with or without H. pyloriinfection (7.13 or J166 strain). We evaluated the levels of ROS by H2DCFDA staining. WB analysis was used to determine oxidative DNA damage and double stranded DNA breaks by using antibodies against 8-Oxo-guanine and p-H2AX. WB and NF-κb luciferase reporter assay were performed to investigate the link between PRDX2 and NF-κb signaling. ATP-GLO cell titer analysis was utilized to determine cell viability. Results:Our data indicated that PRDX2 mRNA and protein expression levels were induced byH. pyloriin AGS and SNU-1 cells. PRDX2 knockdown significantly increased ROS levels and 8-Oxoguanine staining followingH. pyloriinfection in both cells. These results were further confirmed by Western blot data showing that p-H2AX protein level was strongly induced in PRDX2 knockdown and H. pyloriinfected cells. Interestingly, Western blot data showed that TNF-αtreatment induced PRDX2 protein levels while Bay 11-7082 treatment decreased PRDX2 protein level in AGS and SNU-1 cells. At the same time, PRDX2 transient knockdown in both AGS and SNU-1 cells decreased p-P65 (S536) protein expression levels, nuclear localization of NF-κB-p65, and luciferase reporter activity (P<0.05). We also found that inhibition of PRDX2 significantly sensitized AGS and SNU-1 cells to cisplatin treatment. Conclusion:Our data indicates that knockdown of PRDX2 enhances ROS and DNA damage with H. pylori infection on gastric cancer cells after H. pylori infection and sensitizes gastric cancer cells to cisplatin treatment. Our results suggest a positive feedforward loop between PRDX2 and NF-kB to reduce ROS levels. KEY WORDS: PRDX2,H. pylori, gastric cancer, reactive oxygen species, DNA damage, NF-κb Citation Format: Sen Wang, Zheng Chen, Heng Lu, Shoumin Zhu, Dunfa Peng, Mohammed Soutto, Ahmed Gomma, Nadeem Bhat, Huma Naz, Zekuan Xu, Wael El-Rifai. Induction of PRDX2 by H. pylori reduces ROS and promotes cancer cell survival and resistance to cisplatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 885.

Pantoprazole Induces Mitochondrial Apoptosis and Attenuates NF-κB Signaling in Glioma Cells.

Gastric H+/K+-ATPase or vacuolar-ATPases (V-ATPases) are critical for the cancer cells survival and growth in the ischemic microenvironment by extruding protons from the cell. The drugs which inhibit V-ATPases are known as proton pump inhibitors (PPIs). In the present study, we aimed to evaluate the anticancer efficacy of pantoprazole (PPZ) and its consequences on NF-κB signaling in glioma cells. We have used MTT and clonogenic assay to show PPZ effect on glioma cell growth. Propidium iodide and rhodamine 123 staining were performed to demonstrate cell cycle arrest and mitochondrial depolarization. TUNEL staining was used to evidence apoptosis after PPZ treatment. Immunoblotting and immunofluorescence microscopy were performed to depict protein levels and localization, respectively. Luciferase assay was performed to confirm NF-κB suppression by PPZ. Our results revealed PPZ treatment inhibits cell viability or growth and induced cell death in a dose- and time-dependent manner. PPZ exposure arrested G0/G1 cyclic phase and increased TUNEL positivity, caspase-3 and PARP cleavage with altered pro and anti-apoptotic proteins. PPZ also induced ROS levels and depolarized mitochondria (Δψm) with increased cytosolic cytochrome c level. Further, PPZ suppressed TNF-α stimulated NF-κB signaling by repressing p65 nuclear translocation. NF-κB luciferase reporter assays revealed significant inhibition of NF-κB gene upon PPZ treatment. PPZ exposure also reduced the expression of NF-κB-associated genes, such as cyclin-D1, iNOS, and COX-2, which indicate NF-κB inhibition. Altogether, the present study disclosed that PPZ exerts mitochondrial apoptosis and attenuates NF-κB signaling suggesting PPZ can be an effective and safe anticancer drug for glioma.

Nuclear Factor κB Activation in a Type V Pityriasis Rubra Pilaris Patient Harboring Multiple CARD14 Variants.

Pityriasis rubra pilaris (PRP) is a rare papulosquamous skin disorder, which is phenotypically related to psoriasis. Some familial PRP cases show autosomal dominant inheritance due to CARD14 mutations leading to increased nuclear factor κB (NFκB) activation. Moreover, CARD14 polymorphisms have also been implicated in sporadic PRP. A Hungarian PRP patient with childhood onset disease showing worsening of the symptoms in adulthood with poor therapeutic response underwent genetic screening for the CARD14 gene, revealing four genetic variants (rs117918077, rs2066964, rs28674001, and rs11652075). To confirm that the identified genetic variants would result in altered NFκB activity in the patient, functional studies were carried out. Immunofluorescent staining of the NFκB p65 subunit and NFκB-luciferase reporter assay demonstrated significantly increased NFκB activity in skin samples and keratinocytes from the PRP patient compared to healthy samples. Characterization of the cytokine profile of the keratinocytes and peripheral blood mononuclear cells demonstrated that the higher NFκB activation in PRP cells induces enhanced responses to inflammatory stimuli. These higher inflammatory reactions could not be explained solely by the observed CARD14 or other inflammation-related gene variants (determined by whole exome sequencing). Thus our study indicates the importance of investigations on other genetic factors related to PRP and their further functional characterization to bring us closer to the understanding of cellular and molecular background of disease pathogenesis.

Inflammatory Response and PM2.5 Exposure of Urban Traffic Conductors

Human exposure to airborne PM2.5 has been linked to an increased risk of respiratory and cardiovascular diseases, possibly via the activation of systemic inflammation. However, the associations between airborne PM2.5 and systemic inflammation in humans remain inconclusive. Traffic-related air pollutants (TRAPs) are the major source of PM2.5 in urban areas; the adverse health effect of PM2.5 from TRAPs is currently a critical issue of public concern. The present cross-sectional study examines the relationship between PM2.5 exposure and systemic inflammation in order to consider the health impacts of TRAP PM2.5 on urban traffic conductors. All study participants, viz., office-based police officers (the reference) and traffic conductors (the exposure), were requested to carry a personal sampler to determine individual PM2.5 exposure. An adenovirus-based NF-κB luciferase reporter assay was used to determine the proinflammatory activity in serum samples collected from the study participants. The blood proinflammatory activity was presented as tumor necrosis factor-α (TNFα) equivalence (TNFα-EQ), which was extrapolated from the sigmoidal semi-logarithmic dose-response curve of the NF-κB reporter assay by TNFα. The levels of both personal PM2.5 exposure and blood proinflammatory activity (TNFα-EQ) in the exposure group (traffic conductors) were significantly higher than in the reference group (office-based police officers) (p < 0.05). The present study reveals a positive and significant association between personal PM2.5 exposure levels and blood TNFα-EQ levels in a linear regression model of y = 0.511x – 3.062 (y = log TNFα-EQ and x = log PM2.5; R = 0.231 and p = 0.047); the results suggest that exposure to TRAP PM2.5 significantly contributes to increased systemic inflammation in humans. This research provides clear evidence that long-term occupational exposure to TRAPs causes adverse health impacts, i.e., inflammation, on traffic conductors.

Anti-Inflammatory Activity of Chemical Constituents Isolated from the Willow Bracket Medicinal Mushroom Phellinus igniarius (Agaricomycetes).

Nuclear factor-κB (NF-κB) has an important role in immune response and inflammation. Phellinus igniarius (known as Sang huang in traditional Chinese medicine) has various pharmacologic effects. In this study, 19 chemical constituents (1-19) were isolated from the fruiting bodies of Ph. igniarius. Their structures were elucidated based on data from nuclear magnetic resonance spectroscopy and comparison with the literature. Overall, 3 compounds (2, 7, and 11) from this species were reported, to our knowledge for the first time. Two compounds (4 and 15) from the genus Phellinus and 9 compounds (1, 3, 5, 8-10, and 12-14) from the Hymenochaetaceae family were also reported for the first time. An NF-κB luciferase reporter assay of these compounds was carried out in tumor necrosis factor-α-induced HeLa cells-again, to our knowledge for the first time. Among them, compounds 5 and 7 showed moderate inhibition of NF-κB, with fold values of 0.48 ± 0.02 and 0.55 ± 0.09, respectively, in HeLa cells at 100 μmol/L. These results suggest that some of the ingredients from Ph. igniarius could be developed into antiinflammatory agents for use in clinical applications.

Abstract 463: The Orphan G Protein-Coupled Receptor C5B is Regulated by Hyperglycemia and Activates Pro-Inflammatory Signaling in Endothelial Cells Via the Tyrosine Kinase Fyn

Background: Atherosclerosis is driven by an inflammatory process of the vascular wall in which endothelial cells (EC) are decisively involved. The orphan G protein-coupled receptor GPRC5B activates inflammatory pathways in adipocytes and regulates the glucose and lipid metabolism in Drosophila melanogaster . Hypothesis: We hypothesize that GPRC5B is involved in inflammatory signaling of EC, particularly in hyperglycemia. Methods &amp; Results: Stimulation of mouse brain endothelial cells (bEnd.3) and primary human umbilical vein endothelial cells (HUVEC) with high glucose concentration (25 mM), Tumor Necrosis Factor α (TNFα 0,1 μg/ml) or Lipopolysaccharides (LPS 1 μg/ml), respectively, leads to a significant upregulation of GPRC5B mRNA and protein level compared to unstimulated cells (mRNA / protein: gluc +254% / +86%, p&lt;0.001; TNFα +203% / +124%, p&lt;0.001; LPS +195%, p&lt;0.05, n=6). Similar results are obtained in HUVECs after stimulation with high glucose and cytokines. Overexpression of GPRC5B in EC induced a significant increase in Interleukin 6 (IL6), Intercellular Adhesion Molecule 1 (ICAM1) and Vascular Cell Adhesion Molecule 1 (VCAM1) mRNA and protein expression. Furthermore, activation of NFκB (luciferase reporter assay) and phosphorylation of extracellular signal–regulated kinases 1/2 (Erk 1/2) were significantly increased compared to controls (mRNA: IL6 +42%, p&lt;0.01; ICAM1 +37%, p&lt;0.01; VCAM1 +40%, p&lt;0.001, n=8. Protein: ICAM1 +48%, p&lt;0.05; VCAM1 +52%, p&lt;0.05; pErk 1/2 +67%, p&lt;0.01, n=5. NFκB: +165%, p&lt;0.001, n=18). Conversely, the siRNA-mediated GPRC5B knockdown resulted in diminished expression of IL6 and VCAM1 as well as decreased NFκB activity compared to controls (IL6 -55%, p&lt;0.05, n=3, NFκB -36%, p&lt;0.05, n=6) . The knockdown results were confirmed in HUVECs. Co-immunoprecipitations in EC revealed the tyrosine kinase Fyn as interaction partner of GPRC5B. Conclusion: Our data suggest that GPRC5B is not only regulated by glucose and pro-inflammatory cytokines but also activates pro-inflammatory and pro-atherogenic pathways in mouse and human endothelial cells. Therefore, GPRC5B might play an important role in hyperglycemia-accelerated vascular inflammation and pathogenesis of atherosclerosis.

Porcine parvovirus induces activation of NF-κB signaling pathways in PK-15 cells mediated by toll-like receptors.

Porcine parvovirus (PPV) is a pathogenic factor that primarily induces severe reproductive failure of pregnant swine, which results in extensive losses to the swine industry worldwide. In this study, a potential mechanism of PPV-induced activation of the nuclear transcription factor-kappaB (NF-κB) by infection in porcine kidney cells (PK-15) was elucidated for the first time. The subcellular localization of p65 analyzed by immunofluorescence assay (IFA) showed that PPV infection induced p65 translocation from the cytoplasm to the nucleus. p65 phosphorylation was detected in PK-15 cells with progression of PPV infection. NF-κB-regulated gene expression was enhanced in a viral dose-dependent manner using the NF-κB luciferase reporter assay system. Furthermore, PPV-induced NF-κB activation was closely related to the inhibitory kappa B alpha (IκBα) degradation. Treatment with a NF-κB-specific inhibitor demonstrated that the production of PPV progeny viruses was enhanced to some extent. In addition, these results demonstrated that the adapter molecule TIR domain-containing adapter inducing IFN-β (TRIF) and myeloid differentiation primary-response protein 88 (MyD88)-dependent signaling pathways were involved in PPV-induced NF-κB activation. Together, these results provide evidence that the toll-like receptor (TLR) pathway participates in recognition of PPV and induction of NF-κB activation, and add to understanding of the molecular mechanisms underlying PPV infection.

Bioactivity-based UPLC/Q-TOF/MS strategy for screening of anti-inflammatory components from Cimicifugae Rhizoma

In this study, the protective effects of Cimicifugae Rhizoma (CR) was demonstrated in Pseudomonas aeruginosa-induced pneumonia mouse model. To identify the anti-inflammatory ingredients, an ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) integrated nuclear factor κB (NF-κB) luciferase reporter assay screening system was carried out. As a result, some caffeic acid derivatives, including caffeic acid, ferulic acid/isoferulic acid, fukinolic acid, and cimicifugic acid ingredients were identified as the potential effective compounds from CR. For testing the anti-inflammatory capacity, caffeic acid was demonstrated to inhibit NF-κB and reduce the levels of IL-6 and IL-8 in TNF-α-treated BEAS-2B cells in a dose-dependent manner. Hence, CR preparations and its cinnamic acid derivatives have the possibility to be developed as a complementary therapy in the treatment of respiratory system infection in clinics.

Baicalein inhibits TNF-α-induced NF-κB activation and expression of NF-κB-regulated target gene products.

The nuclear factor-κB (NF-κB) transcription factors control many physiological processes including inflammation, immunity, apoptosis and angiogenesis. In our search for NF-κB inhibitors from natural resources, we identified baicalein from Scutellaria baicalensis as an inhibitor of NF-κB activation. As examined by the NF-κB luciferase reporter assay, we found that baicalein suppressed TNF-α-induced NF-κB activation in a dose-dependent manner. It also inhibited TNF-α-induced nuclear translocation of p65 through inhibition of phosphorylation and degradation of IκBα. Furthermore, baicalein blocked the TNF-α-induced expression of NF-κB target genes involved in anti-apoptosis (cIAP-1, cIAP-2, FLIP and BCL-2), proliferation (COX-2, cyclinD1 and c-Myc), invasion (MMP‑9), angiogenesis (VEGF) and major inflammatory cytokines (IL-8 and MCP1). The flow cytometric analysis indicated that baicalein potentiated TNF-α-induced apoptosis and induced G1 phase arrest in HeLa cells. Moreover, baicalein significantly blocked activation of p38, extracellular signal-regulated kinase 1/2 (ERK1/2). Our results imply that baicalein could be a lead compound for the modulation of inflammatory diseases as well as certain cancers in which inhibition of NF-κB activity may be desirable.

TIPE2, a negative regulator of TLR signaling, regulates p27 through IRF4-induced signaling.

Targeted inhibition of specific toll-like receptor (TLR) pathways may provide an effective strategy to prevent the development of selected gastric malignancies. Tumor necrosis factor (TNF)-α-induced protein 8-like-2 (TIPE2) was identified as a novel negative regulator of TLR signaling. Our previous study identified TIPE2 as an inhibitor of gastric cancer cell growth; it promotes p27 expression, which leads to restored control of the cell cycle and cell division. However, the molecular mechanism by which TIPE2 regulates p27 remains unclear. In the present study, we examined the expression patterns of TIPE2 in serial clinical gastritis tissues as well as gastric cancer, and found a negative correlation between TIPE2 expression and progression of gastritis to gastric cancer. This negative correlation verified the role of TIPE2 in preventing the occurrence and development of gastric cancer, suggesting that TIPE2 may be a potential biomarker for gastric cancer progression. To determine the mechanism employed by TIPE2 in gastric cell carcinogenesis, a TIPE2-expressing plasmid was introduced into gastric cell lines, and microarray and western blot analysis revealed that TIPE2 selectively upregulates the expression of interferon regulatory factor 4 (IRF4). Variations in IRF4 expression were additionally verified in knockout mice. Next, the effect of IRF4 on p27 expression was tested by an IRF4 siRNA interference assay. Finally, we explored the signaling pathways used by TIPE2 to regulate IRF4. An experiment using pathway inhibitors and a nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) luciferase reporter assay showed that NF-κB plays a crucial role in regulating IRF4 expression. Our data provide evidence that TIPE2, a potential biomarker for gastric cancer progression, stimulates an IRF4-associated signaling cascade that promotes p27 expression and controls cell growth. To the best of our knowledge, this is the first study to demonstrate that IRF4 acts as an inhibitor of epithelial cell proliferation and mediates the expression of TIPE2, a negative regulator of TLR signaling, to control cell growth.

Epstein-Barr Virus (EBV) Tegument Protein BGLF2 Promotes EBV Reactivation through Activation of the p38 Mitogen-Activated Protein Kinase.

Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus associated with both B cell and epithelial cell malignancies. EBV infection of B cells triggers activation of several signaling pathways that are critical for cell survival, virus latency, and growth transformation. To identify EBV proteins important for regulating cell signaling, we used a proteomic approach to screen viral proteins for AP-1 and NF-κB promoter activity in AP-1- and NF-κB-luciferase reporter assays. We found that EBV BGLF2 activated AP-1 but not NF-κB reporter activity. Expression of EBV BGLF2 in cells activated p38 and c-Jun N-terminal kinase (JNK), both of which are important for mitogen-activated protein kinase (MAPK) signaling. Deletion of the carboxyl-terminal 66 amino acids of BGLF2 reduced the ability of BGLF2 to activate JNK and p38. Expression of BGLF2 enhanced BZLF1 expression in latently EBV-infected lymphoblastoid cell lines, and knockdown of BGLF2 reduced EBV reactivation induced by IgG cross-linking. Expression of BGLF2 induced BZLF1 expression and virus production in EBV-infected gastric carcinoma cells. BGLF2 enhanced BZLF1 expression and EBV production by activating p38; chemical inhibition of p38 and MAPK/ERK kinases 1 and 2 (MEK1/2) reduced expression of BZLF1 and virus production induced by BGLF2. In summary, the EBV tegument protein BGLF2, which is delivered to the cell at the onset of virus infection, activates the AP-1 pathway and enhances EBV reactivation and virus production. Epstein-Barr virus (EBV) is associated with both B cell and epithelial cell malignancies, and the virus activates multiple signaling pathways important for its persistence in latently infected cells. We identified a viral tegument protein, BGLF2, which activates members of the mitogen-activated protein kinase signaling pathway. Expression of BGLF2 increased expression of EBV BZLF1, which activates a switch from latent to lytic virus infection, and increased production of EBV. Inhibition of BGFL2 expression or inhibition of p38/MAPK, which is activated by BGLF2, reduced virus reactivation from latency. These results indicate that a viral tegument protein which is delivered to cells upon infection activates signaling pathways to enhance virus production and facilitate virus reactivation from latency.

Inflammatory MAPK and NF-κB signaling pathways differentiated hepatitis potential of two agglomerated titanium dioxide particles

TiO2 nanoparticles (TiO2NPs) consumption and deposit in liver have possible implications for hepatitis risks. Specific signal dysregulation at early inflammatory responses needs to be characterized in TiO2NP-induced hepatopathy. MAPK and NF-κB signaling pathways are known to participate in inflammation and respond sensitively to chemical agents, making them preferable biomarkers for hepatitis. In the present study, dynamic activation of MAPK and NF-κB pathways were explored by immunoblotting and NF-κB luciferase reporter assay depending on characterization of TiO2NP agglomeration in human HepG2 cells. Inflammatory and cytotoxic potential of TiO2NPs were determined by qRT-PCR and WST-1 assay. AFM and TEM analyses uncovered ultrastructure damages underlying hepatotoxicity of TiO2NPs. Rod-like rutile agglomerated smaller and induced more pronounced cytotoxicity and immunogenicity than anatase. Correspondingly, though both rutile and anatase significantly activated p38, ERK1/2 and NF-κB pathways, rutile accelerated the maximum phosphorylation of ERK1/2 and elevated the potency of IκBα phosphorylation to its bell curve shape in comparison with a lower and sigmoid type of IκBα phosphorylation for anatase. Furthermore, cell elasticity indicated by Young’s modulus and adhesion force increased accompanied with mitochondria damage, contributing to signal dysregulation and hepatotoxicity. The results suggested that differential activation of MAPK and NF-κB pathways could be early predictors for distinct hepatitis risks of two agglomerated TiO2NPs.

Magnolin inhibits cell migration and invasion by targeting the ERKs/RSK2 signaling pathway.

BackgroundMagnolin is a natural compound abundantly found in Magnolia flos, which has been traditionally used in oriental medicine to treat headaches, nasal congestion and anti-inflammatory reactions. Our recent results have demonstrated that magnolin targets the active pockets of ERK1 and ERK2, which are important signaling molecules in cancer cell metastasis. The aim of this study is to evaluate the effects of magnolin on cell migration and to further explore the molecular mechanisms involved.MethodsMagnolin-mediated signaling inhibition was confirmed by Western blotting using RSK2+/+ and RSK2−/− MEFs, A549 and NCI-H1975 lung cancer cells, and by NF-κB and Cox-2 promoter luciferase reporter assays. Inhibition of cell migration by magnolin was examined by wound healing and/or Boyden Chamber assays using JB6 Cl41 and A549 human lung cancer cells. The molecular mechanisms involved in cell migration and epithelial-to-mesenchymal transition were determined by zymography, Western blotting, real-time PCR and immunocytofluorescence.ResultsMagnolin inhibited NF-κB transactivation activity by suppressing the ERKs/RSK2 signaling pathway. Moreover, magnolin abrogated the increase in EGF-induced COX-2 protein levels and wound healing. In human lung cancer cells such as A549 and NCI-H1975, which harbor constitutive active Ras and EGFR mutants, respectively, magnolin suppressed wound healing and cell invasion as seen by a Boyden chamber assay. In addition, it was observed that magnolin inhibited MMP-2 and −9 gene expression and activity. The knockdown or knockout of RSK2 in A549 lung cancer cells or MEFs revealed that magnolin targeting ERKs/RSK2 signaling suppressed epithelial-to-mesenchymal transition by modulating EMT marker proteins such as N-cadherin, E-cadherin, Snail, Vimentin and MMPs.ConclusionsThese results demonstrate that magnolin inhibits cell migration and invasion by targeting the ERKs/RSK2 signaling pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1580-7) contains supplementary material, which is available to authorized users.

PWE-177 The functional relevance of toll-like receptor 4 mutations in oesophageal adenocarcinoma

Introduction Altered innate immune signalling may disrupt host-microbe homeostasis and promote an inflammatory microenvironment that favours tumourigenesis. The aim of this project was to assess whether somatic mutations in oesophageal adenocarcinoma (OAC) could interfere with Toll-like receptor (TLR) signalling, with a specific focus on TLR4. Method We interrogated the mutational profiles of TLR genes in 170 OAC samples, which had undergone whole genome sequencing as part of the oesophageal ICGC study. We performed site-directed mutagenesis and created 13 plasmids expressing mutant TLR4 with a CMV promoter (12 single nucleotide variants (SNVs), and one plasmid combined two SNVs). Luciferase reporter gene assays were performed in HEK293 cells stimulated with synthetic monophosphoryl lipid A (MPLA) to assess how the mutants altered TLR4 signalling and downstream activation of nuclear factor kappa B (NF-κB). Two of the TLR4 mutants were taken forward for transfection into the OAC cell line OE33, and ELISA was used to measure cytokine secretion after stimulation with MPLA. Results The TLR signalling pathway appeared to be recurrently mutated with missense mutations in 23/170 (13.5%) of tumour samples, including mutations in TLR1 (1.2%), TLR2 (0.6%), TLR4 (4.7%), TLR5 (0.6%), TLR7 (1.8%), TLR9 (1.2%), and MYD88 (1.5%). Based on our data and another recent study by Dulak et al . 1 , TLR4 mutations were the most frequent (5.4% combined data). One tumour sample contained two TLR4 mutations, E439G and F703C. Interestingly, the mutation E439G was identified both in our dataset and in the study by Dulak et al . Furthermore, in silico modelling suggested that the E439G mutation disrupts hydrogen bonds in the binding site of LPS and MD2. The use of NF-κB luciferase reporter assays demonstrated a significant decrease in ligand-dependent signalling for 8/12 of the TLR4 mutations tested in HEK293 cells. A double mutation of E439G with F703C revealed a further decrease in TLR4 signalling. Similarly, the concentration of IL-8 was lower for TLR4 mutants E439G and E439G+F703C transfected into OE33 cells stimulated with MPLA, in comparison to wild-type TLR4. Conclusion TLR4 is recurrently mutated in OAC, and the majority of these mutations showed decreased TLR4 signalling in response to ligand stimulation, both in HEK293 and OE33 cells. The biological relevance of this in relation to host-microbe homeostasis and carcinogenesis is being explored. Disclosure of interest None Declared. Reference Dulak AM, et al . Exome and whole-genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity. Nat Genet. 2013;45(5):478–486.