NF-kappaB Pathway Research Articles

Tumor extracelluar hyroxyapatite: a potential biomarker for imaging ovarian cancer

<h3>Objectives:</h3> Identifying new strategies to improve response to poly ADP-ribose polymerase inhibitors (PARPi) in women with homologous recombination (HR)-proficient ovarian cancer is a key clinical challenge. The nuclear orphan receptor NR4A1 has pro-tumor effects in ovarian cancer cells through transcriptional regulation of key genes promoting growth, survival and HR DNA repair. NR4A1 is also expressed in immune cells in the tumor microenvironment and may play a role in inhibiting re-polarization of pro-tumor M2-like tumor-associated macrophages (TAMs) to a more effective anti-tumor M1-like TAM phenotype. Our group has shown that the NF-kappaB (NF-ĸB) pathway is a key regulator of TAM phenotype, with upregulated NF-ĸB signaling promoting M1-like functions. Our goal therefore was to determine whether inhibition of NR4A1 can improve responses to PARPi both by increased tumor DNA damage and apoptosis, and by promoting an anti-tumor M1 phenotype in TAMs by modulating NF-ĸB activity. <h3>Methods:</h3> Cultured HR-proficient ID8 mouse ovarian cancer cells, mouse ovarian TAMs and mouse bone marrow-derived macrophages were treated with the established NR4A1 antagonist C-DIM-pPHOH (C-DIM) and/or the PARPi olaparib (OLA) for 24-72 hours. C57BL/6 mice with established intra-peritoneal ID8 tumors were treated with vehicle, C-DIM, OLA or combined C-DIM/OLA for 3 weeks. Standard techniques were used to determine cell growth (Sulforhodamine B assay), HR efficiency (<i>BRCA1</i> and RAD51 foci formation), NF-ĸB activity (nuclear p65, luciferase reporter activity), and expression of markers of DNA damage (pH2AX), apoptosis (cleaved PARP), proliferation (PCNA), and macrophage M1 (CCL3, iNOS) and M2 (CD206, arginase-1) polarization. Statistical significance (p<0.05) between group means was determined by Student's t test for <i>in vitro</i> studies and Mann-Whitney test for mouse studies. <h3>Results:</h3> In ID8 cells, co-treatment with C-DIM and OLA significantly reduced cell growth and HR efficiency, and increased DNA-damage-induced apoptosis, compared to OLA treatment alone. NR4A1 was highly expressed in macrophages, and C-DIM increased NF-ĸB activity and induced M1 polarization alone and when combined with olaparib. Similar effects of combined treatment were observed <i>in vivo.</i> Compared to mice treated with OLA alone, there was significantly reduced ascites volume and omental tumor mass in C-DIM/OLA mice at the time of sacrifice. Combined treatment also reduced proliferation and increased DNA damage and apoptosis in harvested tumors, and increased M1 polarization of TAMs in ascites fluid, compared to OLA mice. <h3>Conclusions:</h3> NR4A1 inhibition sensitizes ovarian tumors to PARPi though direct effects on tumors by targeting the HR pathway, and by modulating TAM function towards an anti-tumor phenotype. These findings have important implications for expanding the use of PARPi in women with HR-proficient ovarian tumors, which would benefit a substantial number of women with this devastating disease.

A Pilot Study of Lower Doses of Ibrutinib in Patients (pts) with Chronic Lymphocytic Leukemia (CLL)

A Pilot Study of Lower Doses of Ibrutinib in Patients (pts) with Chronic Lymphocytic Leukemia (CLL)

SARS-CoV-2 Switches 'on' MAPK and NFκB Signaling via the Reduction of Nuclear DUSP1 and DUSP5 Expression.

Mitogen-activated protein kinases (MAPK) and NF-kappaB (NF-κB) pathway regulate many cellular processes and are essential for immune cells function. Their activity is controlled by dual-specificity phosphatases (DUSPs). A comprehensive analysis of publicly available gene expression data sets of human airway epithelial cells (AECs) infected with SARS-CoV-2 identified DUSP1 and DUSP5 among the lowest induced transcripts within these pathways. These proteins are known to downregulate MAPK and NF-κB pathways; and their lower expression was associated with increased activity of MAPK and NF-κB signaling and enhanced expression of proinflammatory cytokines such as TNF-α. Infection with other coronaviruses did not have a similar effect on these genes. Interestingly, treatment with chloroquine and/or non-steroidal anti-inflammatory drugs counteracted the SARS-CoV-2 induced reduction of DUSP1 and DUSP5 genes expression. Therapeutically, impeding this evasion mechanism of SARS-CoV-2 may help control the exaggerated activation of these immune regulatory pathways during a COVID-19 infection.

Molecular mechanisms of An-Chuan Granule for the treatment of asthma based on a network pharmacology approach and experimental validation.

An-Chuan Granule (ACG), a traditional Chinese medicine (TCM) formula, is an effective treatment for asthma but its pharmacological mechanism remains poorly understood. In the present study, network pharmacology was applied to explore the potential mechanism of ACG in the treatment of asthma. The tumor necrosis factor (TNF), Toll-like receptor (TLR), and Th17 cell differentiation-related, nucleotide-binding oligomerization domain (NOD)-like receptor, and NF-kappaB pathways were identified as the most significant signaling pathways involved in the therapeutic effect of ACG on asthma. A mouse asthma model was established using ovalbumin (OVA) to verify the effect of ACG and the underlying mechanism. The results showed that ACG treatment not only attenuated the clinical symptoms, but also reduced inflammatory cell infiltration, mucus secretion and MUC5AC production in lung tissue of asthmatic mice. In addition, ACG treatment notably decreased the inflammatory cell numbers in bronchoalveolar lavage fluid (BALF) and the levels of pro-inflammatory cytokines (including IL-6, IL-17, IL-23, TNF-alpha, IL-1beta and TGF-beta) in lung tissue of asthmatic mice. In addition, ACG treatment remarkably down-regulated the expression of TLR4, p-P65, NLRP3, Caspase-1 and adenosquamous carcinoma (ASC) in lung tissue. Further, ACG treatment decreased the expression of receptor-related orphan receptor (RORγt) in lung tissue but increased that of Forkhead box (Foxp3). In conclusion, the above results demonstrate that ACG alleviates the severity of asthma in a ´multi-compound and multi-target’ manner, which provides a basis for better understanding of the application of ACG in the treatment of asthma.

Differential Signals From TNFα-Treated and Untreated Embryos in Uterine Tissues and Splenic CD4+ T Lymphocytes During Preimplantation Pregnancy in Mice.

The main aim of this study was to examine if a female mouse body in preimplantation pregnancy can distinguish between embryos of normal and impaired biological quality in the local and peripheral compartments. Normal (control group) and TNFα (tumor necrosis factor-α)-treated embryos (experimental group) at the morula stage were non-surgically transferred into the uteri of CD-1 strain [Crl:CD1(Icr)] female murine recipients. Twenty-four hours after the embryo transfer, females were euthanised, and uteri and spleens were dissected. In uterine tissues (local compartment), we assessed the expression of 84 genes comprising nine signal transduction pathways, using a modified RT2 Profiler PCR Array. In the spleen (peripheral compartment), we determined the proteome of splenic CD4+ lymphocytes using 2D protein electrophoresis with subsequent protein identification by mass spectrometry. Sample clustering and differential gene expression analyses within individual signal transduction pathways revealed differential expression of genes in the uteri of females after transplantation of normal vs. TNFα-treated embryos. The most affected signal transduction cascade was the NFKB (Nuclear factor NF-kappa-B) pathway, where 87.5% of the examined genes were significantly differentially expressed. Proteomic analysis of splenic CD4+ T lymphocytes revealed significant differential expression of 8 out of 132 protein spots. Identified proteins were classified as proteins influenced by cell stress, proteins engaged in the regulation of cytoskeleton stabilization and cell motility, and proteins having immunomodulatory function. These results support the hypothesis that even before embryo implantation, the body of pregnant female mice can sense the biological quality of an embryo both at the local and peripheral level.

HELQ and EGR3 expression correlate with IGHV mutation status and prognosis in chronic lymphocytic leukemia

BackgroundIGHV mutation status is a crucial prognostic biomarker for CLL. In the present study, we investigated the transcriptomic signatures associating with IGHV mutation status and CLL prognosis.MethodsThe co-expression modules and hub genes correlating with IGHV status, were identified using the GSE28654, by ‘WGCNA’ package and R software (version 4.0.2). The over-representation analysis was performed to reveal enriched cell pathways for genes of correlating modules. Then 9 external cohorts were used to validate the correlation of hub genes expression with IGHV status or clinical features (treatment response, transformation to Richter syndrome, etc.). Moreover, to elucidate the significance of hub genes on disease course and prognosis of CLL patients, the Kaplan–Meier analysis for the OS and TTFT of were performed between subgroups dichotomized by the median expression value of individual hub genes.Results2 co-expression modules and 9 hub genes ((FCRL1/FCRL2/HELQ/EGR3LPL/LDOC1/ZNF667/SOWAHC/SEPTIN10) correlating with IGHV status were identified by WGCNA, and validated by external datasets. The modules were found to be enriched in NF-kappaB, HIF-1 and other important pathways, involving cell proliferation and apoptosis. The expression of hub genes was revealed to be significantly different, not only between CLL and normal B cell, but also between various types of lymphoid neoplasms. HELQ expression was found to be related with response of immunochemotherapy treatment significantly (p = 0.0413), while HELQ and ZNF667 were expressed differently between stable CLL and Richter syndrome patients (p < 0.0001 and p = 0.0278, respectively). By survival analysis of subgroups, EGR3 expression was indicated to be significantly associated with TTFT by 2 independent cohorts (GSE39671, p = 0.0311; GSE22762, p = 0.0135). While the expression of HELQ and EGR3 was found to be associated with OS (p = 0.0291 and 0.0114 respectively).The Kras, Hedgehog and IL6-JAK-STAT3 pathways were found to be associating with the expression of hub genes, resulting from GSEA.ConclusionsThe expression of HELQ and EGR3 were correlated with IGHV mutation status in CLL patients. Additionally, the expression of HELQ/EGR3 were prognostic markers for CLL associating with targetable cell signaling pathways.

Methods for Modulating the Pathway of NF-κB Using Short Hairpin RNA (ShRNA).

Target gene silencing is a strategy that can be used to turn off pathways or genes which are difficult to turn off pharmacologically, both because of lack of targeting drugs, or because of the risk of wider off-target effects. Here we describe the design and use of short hairpin RNA (ShRNA) and lentiviral vectors as an efficient technique for silencing NF-kappaB (NF-κB) pathway in cultured cells. This method can be used also in hard to transfect primary cell cultures.

SNP-adjacent super enhancer network mediates enhanced osteogenic differentiation of MSCs in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a rheumatic disease with pathological osteogenesis that causes bony ankylosis and even deformity over time. Mesenchymal stem cells (MSCs) are multipotent stem cells that are the main source of osteoblasts. We previously demonstrated that enhanced osteogenic differentiation of MSCs from AS patients (ASMSCs) is related to pathological osteogenesis in AS. However, the more concrete mechanism needs further exploration. Super enhancers (SEs) are dense clusters of stitched enhancers that control cell identity determination and disease development. Single-nucleotide polymorphisms (SNPs) regulate the formation and interaction of SEs and denote genes accounting for AS susceptibility. Via integrative analysis of multiomic data, including histone 3 lysine 27 acetylation (H3K27ac), chromatin immunoprecipitation sequencing (ChIP-seq), SNPs and RNA sequencing (RNA-seq) data, we discovered a transcription network mediated by AS SNP-adjacent SEs (SASEs) in ASMSCs and identified key genes, such as Toll-like receptor 4 (TLR4), interleukin 18 receptor 1 (IL18R1), insulin-like growth factor binding protein 4 (IGFBP4), transportin 1 (TNPO1) and proprotein convertase subtilisin/kexin type 5 (PCSK5), which are pivotal in osteogenesis and AS pathogenesis. The SASE-regulated network modulates the enhanced osteogenic differentiation of ASMSCs by synergistically activating the PI3K-Akt, NF-kappaB and Hippo signaling pathways. Our results emphasize the crucial role of the SASE-regulated network in pathological osteogenesis in AS, and the preferential inhibition of ASMSC osteogenic differentiation by JQ1 indicates that SEs may be attractive targets in future treatment for new bone formation in AS.

Abstract PO-055: Signaling regulation of epithelial-mesenchymal transition in the hypoxic tumor microenvironment of pancreas cancer

Abstract Epithelial-mesenchymal transition (EMT) is a normal developmental process by which epithelial cells lose their polarity and cell-cell adhesions to become more motile and invasive mesenchymal cells. EMT is associated with the earliest stages in metastasis and promotes chemoresistance in a variety of carcinomas, including pancreatic ductal adenocarcinoma (PDAC). While specific growth factors (e.g., transforming growth factor beta) are perhaps the most well studied inducers of EMT, the low oxygen tension (hypoxia) characteristic of the PDAC tumor microenvironment has also been reported to drive EMT. The signaling pathways that promote EMT in the hypoxic PDAC tumor microenvironment may therefore represent useful drug targets for combination therapy approaches as adjuvants to augment the efficacy of chemotherapy. However, hypoxia-driven EMT has not been well characterized in PDAC, nor have the relevant hypoxia-driven signaling pathways been identified. Our lab recently developed a statistical model of the multivariate signaling regulation of growth factor-induced EMT to identify that the ERK, JNK, and NF-kappaB pathways coordinate to drive EMT in PDAC cells. In the present study, we sought to determine the roles of those pathways in promoting EMT in PDAC cells in response to hypoxia. We created an in vitro model of hypoxia-driven EMT by culturing PDAC cells in 1% oxygen and characterizing dynamic changes in the expression of hypoxia inducible factors (HIF) 1 and 2 alpha, as well as the concomitant loss of E-cadherin protein and transcripts, increased abundance of vimentin-positive cells, and increased expression of the mesenchymal transcripts VIM, SNAI1, and SNAI2. Hypoxia-driven EMT was observed in human PDAC cell lines, patient-derived xenograft cell lines, and KPC murine cell lines. To probe for the activation of specific signaling pathways, we used a combination of immunoblotting and immunofluorescence microscopy. Automated image analysis of immunofluorescence images was used to understand the relevance of specific signaling pathways for explaining the heterogeneity of EMT within populations of cells. We further identified potential kinase targets for antagonizing EMT in the hypoxic microenvironment and confirmed their roles by pharmacological inhibition and/or siRNA-mediated knockdown. We also found that specific kinase pathways are involved in the stabilization of HIF proteins in hypoxia. The relevance of these pathways in driving EMT is being further validated through analyses of a patient-derived xenograft mouse model of PDAC and a HIF1A deletion/Kras mutant autochthonous mouse model of PDAC. Citation Format: Brooke A. McGirr, Nicholas M. Seyler, Matthew J. Lazzara. Signaling regulation of epithelial-mesenchymal transition in the hypoxic tumor microenvironment of pancreas cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-055.

CXXC5 variant in an immunodeficient patient with a progressive loss of hematopoietic cells

Partial loss of function variant in CXXC5 identified in an immunodeficient patient with progressive bone marrow failure, corrected by bone marrow transplant.

Abstract 6679: Immunophenotypic and transcriptome analyses of CT26 and 4T1 murine tumor models following anti-CTLA-4 treatment

Abstract Model selection for evaluation of immuno-oncology agents is often aided by both response to therapy and changes in immune cell subsets following treatment. To identify underlying factors that influence differences in therapeutic response, CT26 colorectal and 4T1-Luc (4T1) breast tumor models, which are immunologically warm and cold, respectively, were treated with anti-cytotoxic T-lymphocyte associated protein 4 (CTLA-4). Flow cytometry and transcriptome analysis were used to describe treatment-related changes in immune cell infiltrate, TIL activation status, cytokine/granzyme B production, and overall changes in gene expression. Response to anti-CTLA-4 therapy was distinct between these models. Tumor growth inhibition was observed in CT26 (74%), but not 4T1. In CT26, CD8+ and NK T cell infiltration increased, while M2 macrophages decreased. In 4T1 tumors, only increased NKT cell infiltration was noted after treatment, and no demonstrable changes were observed in any other cell type or activation marker. Several pharmacodynamic changes in immune cell activation were observed in CT26. Notably, CT26-derived CD4+, CD8+, and NK T cell subsets had increased IFNγ production (70%, 25%, and 17%, respectively, versus control). An increase in polyfunctional T cells producing both IFNγ and TNFα was also noted for CT26 and was correlated with slower tumor growth. Granzyme B was reduced and CD107a was increased in CT26-derived NK T cells, suggesting functionally active T cells. This was also supported by upregulation of genes in the cytotoxicity, antigen presentation, and NFkappaB pathways. Following CT26 treatment with anti-CTLA-4, transcriptome analysis with the nCounter® Mouse PanCancer IO 360 Panel (NanoString) showed an 8-10 fold induction of several T cell-related genes as well as an 8-14 fold induction of several chemokines and their receptors. These data complement the flow cytometry data and support stimulation of T cell activation and recruitment to CT26 tumors following anti-CTLA-4 treatment. In contrast, analysis of 4T1 tumors indicated only a 2 to 3-fold induction of CCL4, CCL11, and Granzyme A after treatment. Interestingly, genes in the matrix remodeling pathway were upregulated in 4T1 tumors upon anti-CTLA-4 treatment. In summary, CT26 tumors were responsive to anti-CTLA-4 treatment, showing increased polyfunctional T cell recruitment, NK T cell activity, and reduced M2 macrophage infiltration, as well as marked upregulation of several genes involved in T cell recruitment and activation in tumor. In contrast, 4T1 tumors did not display any remarkable response to therapy, changes to TIL infiltrates, and only limited changes to gene expression with treatment. Collectively, these results suggest tumor profiles following treatment can provide insight into potential targets of interest and are important for preclinical model selection. Citation Format: Sheri R. Barnes, David Draper, Sarah E. Church, Maryland Rosenfeld Franklin. Immunophenotypic and transcriptome analyses of CT26 and 4T1 murine tumor models following anti-CTLA-4 treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6679.

ARMCX Family Gene Expression Analysis and Potential Prognostic Biomarkers for Prediction of Clinical Outcome in Patients with Gastric Carcinoma.

Armadillo gene subfamily members (ARMCX1-6) are well-known to regulate protein-protein interaction involved in nuclear transport, cellular connection, and transcription activation. Moreover, ARMCX signals on cell pathways also implicated in carcinogenesis and tumor progression. However, little is known about the associations of the ARMCX subfamily members with gastric carcinoma. This study investigated the prognostic value of ARMCX subfamily mRNA expression levels with the prognosis of gastric carcinoma (GC). We retrieved the data of a total of 351 GC patients from TCGA database. Survival and gene set enrichment analyses were employed to explore the predictive value and underlying mechanism of ARMCX genes in GC. The multivariate survival analysis revealed that individually low expressions of ARMCX1 (adjusted P = 0.006, HR = 0.620, CI = 0.440 − 0.874) and ARMCX2 (adjusted P = 0.005, HR = 0.610, 95%CI = 0.432–0.861) were related to preferable overall survival (OS). The joint-effects analysis shown that combinations of low level expression of ARMCX1 and ARMCX2 were correlated with favorable OS (adjusted P = 0.003, HR = 0.563, 95%CI = 0.384–0.825). ARMCX1 and ARMCX2 were implicated in WNT and NF-kappaB pathways, and biological processes including cell cycle, apoptosis, RNA modification, DNA replication, and damage response. Our results suggest that mRNA expression levels of ARMCX subfamily are potential prognostic markers of GC.

P0512SKLB023 ATTENUATED LIPOPOLYSACCHARIDE-INDUCED ACUTE KIDNEY INJURY VIA INHIBITION OF NF-KAPPPAB AND MAPKS PATHWAYS

Abstract Background and Aims Excessive nitric oxide (NO) production by the activation of inducible nitric oxide synthase (iNOS) during sepsis was considered to contribute to acute kidney injury (AKI) and selective inhibition of iNOS activity may be a promising strategy for the treatment of sepsis-induced AKI. The novel small molecule compound (Z)-N-(3-Chlorophenyl)-2-(4-((2,4-dioxothiazolidin-5-ylidene) methyl) phenoxy) acet-amide (SKLB023) as a selective iNOS inhibitor was designed, synthesized by our lab and exhibited therapeutic potential in arthritis and non-alcoholic steatohepatitis models with potent anti-inflammatory effects. This study aimed to evaluate whether SKLB023 as a drug candidate could offer renal protective effect against sepsis-induced AKI. Method 21 C57BL6 mice (20-22g, 6-8 weeks old) were randomly divided into three groups: control, model, SKLB023 pretreatment. AKI was induced by a single injection of 10mg/kg LPS, and SKLB023 was orally administrated at the dose of 50 mg/kg for three consecutive days and one hour before LPS injection. All mice were sacrificed 16h after LPS injection and blood and kidney tissues were collected for further evaluation. LPS-induced renal dysfunction was assessed by measurement of blood creatinine and urea nitrogen (BUN) as well as histopathological changes. Moreover, the kidney mRNA and protein expression of neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule 1(KIM-1), newly identified kidney injury markers, were tested by polymerase chain reaction (PCR) and western blot. To explore underlying mechanisms, the renal expression of inflammatory cytokine genes IL-1β, IL-6, mcp-1 and proteins iNOS, COX-2, IL-1β, TNF-α, IL-6, HMGB1 in were measured. The total expression of nuclear factor-kappa B (NF-κB), IκBα and MAPKs proteins (ERK1/2, p38 MAPK) and their corresponding phosphoproteins (p-NF-κB, p-IκBα, p-ERK1/2, p-P38 MAPK) in mice kidneys were subsequently analyzed by western blot. Results 16hours following LPS challenge, mice developed AKI as evidenced by elevated creatinine and BUN levels and corresponding pathological changes. Additionally, kidney expression of NGAL and KIM-1 were remarkably increased after LPS injection. SKLB023 pretreatment, however, alleviated LPS-induced renal dysfunction. Moreover, the excessive production of inflammatory cytokines during sepsis was significantly suppressed by SKLB023. Further observation from PCR and immunoblot results indicated that SKLB023 attenuated the activity of NF-kB and the phosphorylation of ERK1/2 and p38 MAPK in septic AKI mice. Conclusion These data demonstrated that SKLB023 exhibited renoprotective effect in LPS-induced AKI, which was associated with its anti-inflammatory activities by inhibiting the NF-kappaB and MAPK signaling pathways

Nicardipine Inhibits Priming of the NLRP3 Inflammasome via Suppressing LPS-Induced TLR4 Expression.

The Nod-like receptor protein 3 (NLRP3) inflammasome is a multi-protein complex composed of NLRP3, pro-caspase-1, and apoptosis-associated speck-like protein that contains a caspase recruitment domain (ASC). After NLRP3 priming by lipopolysaccharide (LPS), the ligand of toll-like receptor 4 (TLR4), activation of the NLRP3 inflammasome triggers caspase-1 maturation, leading to pyroptosis and release of interleukin-1beta (IL-1beta). Expression of TLR4 modulates LPS-triggered inflammatory cascades as well as the NLRP3 signaling. L-type calcium channel antagonists are widely used as anti-hypertensive drugs and also exert anti-inflammatory effects through inhibiting release of cytokines including IL-1beta. However, few studies reveal effects of L-type calcium channel antagonists on the NLRP3 inflammasome. In this study, we investigated the effects of nicardipine and verapamil, both L-type calcium channel antagonists, on the NLRP3 inflammasome using differentiated THP-1 cells. Pyroptosis or levels of IL-1beta and caspase-1 were assayed by flow cytometry or enzyme-linked immunosorbent assay, respectively. ASC oligomerization was assayed by immunofluorescence microscopy. Expression of NLRP3 or TLR4 was assayed by polymerase chain reaction and immunoblotting. Nuclear factor-kappaB (NF-kappaB) pathway was also studied. Our results showed that pyroptosis and IL-1beta release were attenuated by nicardipine, but not verapamil. Nicardipine also mitigated caspase-1 activation, inhibited ASC oligomerization, and reduced NLRP3 expression. Furthermore, nicardipine downregulated phosphorylation or nuclear translocation of NF-kappaB p65, consistent with the inhibitory effect of nicardipine on LPS-induced TLR4 expression. In conclusion, nicardipine exerted anti-inflammatory effects through inhibiting NLRP3 inflammasome pathway. Nicardipine may mitigate NLRP3 priming via inhibiting NF-kappaB activation, mediated by suppressing LPS-induced TLR4 expression.

Jak-Stat Signaling Pathway Related Gene Expressions and Blood Biochemical indicators in Acute Promyelocytic Leukemia

Jak-Stat Signaling Pathway Related Gene Expressions and Blood Biochemical indicators in Acute Promyelocytic Leukemia

Investigation of miRNA and mRNA Co-expression Network in Ependymoma.

Ependymoma (EPN) is a rare primary tumor of the central nervous system (CNS) that affects both children and adults. Despite the definition and classification of distinct molecular subgroups, there remains a group of EPNs with a balanced genome, which makes it difficult to predict a prognosis of patients with EPN. The role of miRNA-mRNA network on EPN is still poorly understood. We assessed the involvement of miRNA-mRNA pairs in EPN by applying a weighted co-expression network analysis (WGCNA) approach. Using whole genome expression profile analysis followed by functional enrichment, we detected hub genes involved in active proliferation and DNA replication of nerve cells. Key genes including CYP11B1, KRT33B, RUNX1T1, SIK1, MAP3K4, MLANA, and SFRP5 identified in co-expression networks were regulated by miR-15a and miR-24-1. These seven miRNA-mRNA pairs were considered to influence not only pathways in cancer and tumor suppression process, but also MAPK, NF-kappaB, and WNT signaling pathways which were associated with tumorigenesis and development. This study provides a novel insight into potential diagnostic biomarkers of EPN and may have value in choosing therapeutic targets with clinical utility.

Bifenthrin insecticide promotes oxidative stress and increases inflammatory mediators in human neuroblastoma cells through NF-kappaB pathway

The extensive application of bifenthrin (BF) insecticide in agriculture has raised serious concerns with regard to increased risks of developing neurodegenerative diseases. Recently, our group showed that BF exposure in rodent models induced oxidative stress and inflammation markers in various regions of the brain (frontal cortex, striatum and hippocampus) and this was associated with behavioral changes. This study aimed to confirm such inflammatory and oxidative stress in an in vitro cell culture model of SK-N-SH human neuroblastoma cells. Markers of oxidative stress (ROS, NO, MDA, H2O2), antioxidant enzyme activities (CAT, GPx, SOD) and inflammatory response (TNF-α, IL-6, PGE2) were analyzed in SK-N-SH cells after 24 h of exposure to different concentrations of BF (1–20 μM). Protein synthesis and mRNA expression of the enzymes implicated in the synthesis of PGE2 were also measured (COX-2, mPGES-1) as well as nuclear factor κappaB (NF-κBp65) and antioxidant nuclear erythroid-2 like factor-2 (Nrf-2). Cell viability was analyzed by MTT-tetrazolio (MTT) and lactate dehydrogenase (LDH) assays. Exposure of SK-N-SH cells to BF resulted in a concentration-dependent reduction in the number of viable cells (reduction of MTT and increase in LDH activity). There was also a BF concentration-dependent increase in oxidative stress markers (ROS release, NO, MDA and H2O2) and decrease in the activity of antioxidant enzymes (CAT and GPx activities). There was further a concentration-dependent increase in pro-inflammatory cytokines (TNF-α and IL-6) and inflammatory mediator PGE2, increase in protein synthesis and mRNA expression of inflammatory markers (COX-2, mPGES-1 and NF-κBp65) and decrease in protein synthesis and mRNA expression of antioxidant Nrf-2. Our data shows that BF induces various oxidative stress and inflammatory markers in SK-N-SH human neuroblastoma cells as well as the activation of NF-κBp65 signaling pathway. This is in line with prior results in brain regions of rodents exposed in vivo to BF showing increased oxidative stress in response to BF exposure, occurring in pro-inflammatory conditions and likely activating programmed cell death.

Rosanortriterpenes A-B, Two Promising Agents from Rosa laevigata var. leiocapus, Alleviate Inflammatory Responses and Liver Fibrosis in In Vitro Cell Models.

Rosanortriterpenes A–B (RTA and RTB), two nortriterpenoids, are characteristic constituents in the fruits of Rosa laevigata var. leiocapus. However, pharmacological studies on these compounds are still scarce. In the present study, we aim to investigate the anti-inflammatory mechanisms associated with the effects of RTA–B in RAW264.7 macrophages and LO2 cells by detecting cell viabilities, nitric oxide (NO) production, tumour necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) production. Simultaneously, the anti-inflammatory action mechanisms of these two compounds were illustrated through western blot assay. Besides, the antihepatic fibrosis activities of these compounds have also been explored. The results demonstrated that RTA and RTB inhibited the production of NO, TNF-α, and IL-6 and suppressed liver fibrosis. RTA and RTB treatment also greatly inhibited the activation of NF-kappaB (NF-κB) pathway. Our study confirmed the promising anti-inflammatory and anti-liver fibrosis actions of RTA–B, suggesting that they might be developed as alternative and promising drugs for the treatment of hepatic inflammatory and fibrotic diseases.

Comparative Analysis of the Biosimilar and Innovative G-CSF Modulated Pathways on Umbilical Cord Blood-Derived Mononuclear Cells.

Biosimilars of granulocyte colony-stimulating factor (G-CSF) have been routinely introduced into clinical practice. However, not functional genomics characterization has been performed yet in comparison with the innovator G-CSF. This study aimed to evaluate the transcriptomic changes in an in vitro model of umbilical cord blood cells (UBC) exposed to G-CSF for the identification of their modulated pathways. Umbilical cord blood cells–derived mononuclear cells (MNCs) were treated with biosimilar and innovator G-CSF for further gene expression profiling analysis using a microarray-based platform. Comparative analysis of biosimilar and innovator G-CSF gene expression signatures allowed us to identify the most commonly modulated pathways by both drugs. In brief, we observed predominantly upmodulation of transcripts related to PI3K-Akt, NF-kappaB, and tumor necrosis factor (TNF) signaling pathways as well as transcripts related to negative regulation of apoptotic process among others. In addition, hematopoietic colony-forming cell assays corroborate the G-CSF phenotypic effects over UBC-derived MNCs. In conclusion, our study suggests that G-CSF impacts UBC-derived cells through the modulation of several signaling pathways associated with cell survival, migration, and proliferation. The concordance observed between biosimilar and innovator G-CSF emphasizes their similarity in regards to their specificity and biological responses.

Abstract C34: Signaling regulation of epithelial-mesenchymal transition in pancreas cancer cells cultured under hypoxic conditions

Abstract Pancreatic ductal adenocarcinoma (PDAC) tumors exhibit hypoxia due to high oxygen demand and oxygen diffusional limitations (1). Hypoxia has been reported as a driver of epithelial-mesenchymal transition (EMT) (2), which occurs early in pancreas cancer dissemination and promotes resistance to chemotherapy (3,4). Druggable signaling pathways that promote EMT in the hypoxic PDAC microenvironment may therefore represent useful targets for combination therapy. Our lab previously studied the roles of ERK and SHP2 in driving EMT in response to growth factors (5,6). More recently, our lab employed an unbiased data-driven modeling approach and found that the ERK, JNK, and NF-kappaB pathways coordinate to drive robust EMT in PDAC cells treated with combinations of growth factors (unpublished). In the present study, we sought to determine the roles of those pathways in promoting EMT in PDAC cells in response to a shift from normoxic to hypoxic conditions. The immortalized human PDAC cell line HPAF-II, which exhibits baseline epithelial characteristics, was used for these studies. In simple two-dimensional cell culture, a shift from 20% to 1% oxygen for ≤120 hr led to several indications of EMT, including decreased epithelial cell cluster circularity, reduced junctional E-cadherin expression and an increased fraction of vimentin-positive cells (determined by an automated image analysis pipeline), and increased expression of the mesenchymal transcripts VIM, SNAI1, and SNAI2 (determined by RT-qPCR). A hypoxic cellular response was confirmed by immunoblotting, which showed an early (4 hr) peak in HIF-1alpha expression and a relatively late (48 hr) peak in HIF-2alpha expression, as well as increased expression of the HIF target genes PGK1 and SLC2A1. In three-dimensional cultures of HPAF-II cell spheroids in a collagen I matrix, a shift to hypoxia promoted cell invasion into the matrix, a behavior also observed when HPAF-II cells were treated with exogenous growth factors that drive EMT. To probe for the activation of the ERK, JNK, and NF-kappaB pathways, we used a combination of immunoblotting and immunofluorescence microscopy. A shift from 20% to 1% oxygen increased the phosphorylation of ERK and promoted the nuclear localization of c-Jun, a substrate of JNK. Inhibition of MEK or JNK at the same time as the shift to 1% oxygen prevented the loss of junctional E-cadherin that otherwise occurred, with JNK inhibition having the greater effect. JNK inhibition also abrogated loss of CDH1 transcripts and the increase of VIM transcripts otherwise observed in response to hypoxia. Thus, activation of the ERK and JNK pathways in PDAC cells in response to a shift to low oxygen conditions promoted EMT. These pathways may therefore represent useful targets for antagonizing EMT in the hypoxic microenvironment characteristic of PDAC tumors.