NF-κB DNA Binding Research Articles

Mitochondrial β-Carotene 9′,10′ Oxygenase Modulates Prostate Cancer Growth via NF-κB Inhibition: A Lycopene-Independent Function

This study identifies BCO2 as a tumor suppressor in prostate cancer. BCO2-mediated inhibition of NF-κB signaling implies BCO2 status is important in prostate cancer progression. Mol Cancer Res; 14(10); 966-75. ©2016 AACR.

Effect of notch1,2,3 genes silicing on NF-κB signaling pathway of macrophages in patients with atherosclerosis.

Notch and NF-κB signaling pathways both play important roles in the regulation of atherosclerosis (AS). However, the mechanisms of notch and NF-κB signaling pathways on AS are still unclear. In this study, we aimed to investigate the effects of notch1,2,3 genes silicing by siRNA on notch and NF-κB signaling pathways of macrophages in patients with atherosclerosis (AS), so as to seek the treatment of AS from genetic perspective. Peripheral blood mononuclears of 31 patients with AS were isolated by density gradient centrifugation and transformed by PMA to macrophages. Then macrophages were transfected with notch1-siRNA (notch1-siRNA group), notch2-siRNA (notch2-siRNA group), notch3-siRNA (notch3-siRNA group), negative control siRNA (NC group) and none (control group). RT-PCR and Western blot analysis were applied to assess the expression level of Delta-like-4 (DLL4), Jagged-1 (JAG1), IκBα and P52. Electrophoretic mobility shift assay (EMSA) was used to observe the NF-κB DNA binding activity. Subcellular distributions of NF-κB/P52 were detected through immunofluorescence. mRNA expression levels of TNF-α, IL-6 and IL-6 in macrophages were also determined with RT- PCR. The expression of 20S proteasome was detected by Western blot. After transfected with siRNA, there was no difference in the expression of DLL4, JAG1, IκBα and P52 between NC group and control group (p>0.05). Compared with NC group and control group, the expression of DLL4, P52 and JAG1 in notch1-siRNA group, notch2-siRNA group and notch3-siRNA group was significantly downregulated (p<0.05 or p<0.01, respectively), whereas the expression of IκBα was significantly increased (P<0.05 or p<0.01, respectively), especially in notch1-siRNA group. The binding activity of NF-κB DNA was lower in notch1- siRNA group, notch2-siRNA group and notch3-siRNA group compared with NC group and control group (p<0.05), especially in notch1-siRNA group. The fluorescence intensity of p52 was decreased significantly both in the nucleus and cytoplasm in notch1-siRNA group, notch2-siRNA group and notch3-siRNA group compared with NC group and control group (p<0.05), which decreased more obviously in the nucleus, especially in notch1-siRNA group. The TNF-α, IL-1 and IL-6 expression of notch1-siRNA group, notch2-siRNA group and notch3-siRNA group was lower compared to NC group and control group (p<0.05 or p<0.01, respectively), also especially in notch1-siRNA group. 20S proteasome level was significantly lower in notch1-siRNA group, notch2-siRNA group and notch3-siRNA group than in NC group and control group (p<0.05 or p<0.01, respectively), especially in notch1-siRNA group. There was a positive regulation between Notch and NF-κB signaling pathway in patients with AS. Notch1 may play a more important role than notch2 and notch 3 in the regulation of NF-κB signaling pathway in AS.

Effect of Notch1, 2, 3 genes silencing on Notch and nuclear factor-κB signaling pathway of macrophages derived from patients with coronary artery disease

Objective: To investigate the effects of Notch1, 2, 3 genes silencing by siRNA on Notch signaling pathway (Delta-like 4(DLL4), Jagged 1(JAG1)) and nuclear factor-κB (NF-κB) signaling pathway (IκBα, P52) of macrophages derived from patients with coronary artery disease (CAD), thus to explore the potential genetic treatment perspectives for CAD. Methods: Peripheral blood mononuclear cells of CAD patients were isolated by density gradient centrifugation and transformed by phorbol-12-myristate-13-acetate (PMA) to macrophages. Macrophages were then transfected with Notch1-small interference RNA (siRNA, Notch1-siRNA group), Notch2-siRNA (Notch2-siRNA group), Notch3-siRNA (Notch3-siRNA group), negative control siRNA (NC group) and none siRNA (control group) respectively. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were applied to assess the mRNA and protein expression levels of DLL4, JAG1, IκBα and p52, respectively. Electrophoretic mobility shift assay (EMSA) was used to observe the NF-κB DNA binding activity. Subcellular distributions of NF-κB/p52 were detected through immunofluorescence. Results: (1) The mRNA and protein expressions of DLL4, JAG1 and p52 in Notch1-siRNA group, Notch2-siRNA group and Notch3-siRNA group were significantly downregulated, while the mRNA and protein expression of IκBα was significantly upregulated compared with NC group and control group(P<0.05 or 0.01). The mRNA and protein expressions of DLL4, JAG1 and p52 in Notch1-siRNA group were significantly downregulated, while the mRNA and protein expression of IκBα was significantly upregulated compared with Notch2-siRNA group and Notch3-siRNA group(P<0.05 or 0.01). The mRNA and protein expressions of DLL4, JAG1, IκBα and p52 were similar between NC group and control group (all P>0.05). (2) The binding activity of NF-κB DNA was significantly lower in Notch1-siRNA group (613±57), Notch2-siRNA group (1 169±85) and Notch3-siRNA group (1 454±90) compared with control group (2 643±115) and NC group (2 407±100) (all P<0.01), which was also significantly lower in Notch1-siRNA group compared to Notch2-siRNA group and Notch3-siRNA group (P<0.01); was significantly lower in Notch2-siRNA group compared with Notch3-siRNA group (P<0.01) and was similar between control group and NC group (P>0.05). (3) The fluorescence intensity of NF-κB/p52 was significantly lower both in the nucleus and cytoplasm in Notch1-siRNA group, Notch2-siRNA group and Notch3-siRNA group compared with NC group and control group (all P<0.01), and the decrease was more obviously in the nucleus than in cytoplasm in Notch1-siRNA group, Notch2-siRNA group and Notch3-siRNA group (P<0.05 or 0.01). The fluorescence intensity of NF-κB/p52 was similar between control group and NC group (P>0.05). Conclusion: There is a positive regulation between Notch and NF-κB pathway in macrophages derived from CAD patients, the regulation power on NF-κB signaling pathway of Notch1 is stronger than that of Notch2 and Notch 3.

Curcumin suppresses colon cancer cell invasion via AMPK-induced inhibition of NF-κB, uPA activator and MMP9.

Curcumin, an active nontoxic ingredient of turmeric, possesses potent anti-inflammatory, antioxidant and anti-cancer properties; however, the molecular mechanisms of curcumin are not fully understood. The transcription factor nuclear factor-κB (NF-κB) is key in cellular processes, and the expression/activation of urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP9) are crucial for cell invasion. The present study investigated the hypothesis that curcumin inhibits colon cancer cell invasion by modulating NF-κB-mediated expression and activation of uPA and MMP9. Human colon cancer SW480 and LoVo cells were treated with various concentrations of curcumin. Curcumin was demonstrated to dose-dependently inhibit the adhesion and proliferation ability of LoVo and SW480 cells using Transwell and MTT assays, respectively. In addition, curcumin activated 5' AMP-activated protein kinase (AMPK) and suppressed p65 NF-κB phosphorylation, as shown by western blot analysis. Compound C, a potent AMPK inhibitor, abolished curcumin-induced inhibition of NF-κB, uPA and MMP9, suggesting that AMPK activation is responsible for curcumin-mediated NF-κB, uPA and MMP9 inhibition. The binding activity of NF-κB to DNA was examined and western blotting and quantitative polymerase reaction was performed to detect the effect of curcumin on the expression of uPA and MMP9. The present results revealed that curcumin significantly decreased the expression of uPA and MMP9 and NF-κB DNA binding activity. Furthermore, curcumin decreased the level of the p65 subunit of NF-κB binding to the promoter of the gene encoding uPA and MMP9, which suppressed transcriptional activation of uPA and MMP9. Overall, the present data suggest that curcumin inhibits colon cancer cell invasion via AMPK activation and subsequent inhibition of p65 NF-κB, uPA and MMP9. The therapeutic potential of curcumin for colon cancer metastasis required additional study.

Mitogen-Activated Protein Kinase 14 Promotes AKI.

An improved understanding of pathogenic pathways in AKI may identify novel therapeutic approaches. Previously, we conducted unbiased liquid chromatography-tandem mass spectrometry-based protein expression profiling of the renal proteome in mice with acute folate nephropathy. Here, analysis of the dataset identified enrichment of pathways involving NFκB in the kidney cortex, and a targeted data mining approach identified components of the noncanonical NFκB pathway, including the upstream kinase mitogen-activated protein kinase kinase kinase 14 (MAP3K14), the NFκB DNA binding heterodimer RelB/NFκB2, and proteins involved in NFκB2 p100 ubiquitination and proteasomal processing to p52, as upregulated. Immunohistochemistry localized MAP3K14 expression to tubular cells in acute folate nephropathy and human AKI. In vivo, kidney expression levels of NFκB2 p100 and p52 increased rapidly after folic acid injection, as did DNA binding of RelB and NFκB2, detected in nuclei isolated from the kidneys. Compared with wild-type mice, MAP3K14 activity-deficient aly/aly (MAP3K14aly/aly) mice had less kidney dysfunction, inflammation, and apoptosis in acute folate nephropathy and less kidney dysfunction and a lower mortality rate in cisplatin-induced AKI. The exchange of bone marrow between wild-type and MAP3K14aly/aly mice did not affect the survival rate of either group after folic acid injection. In cultured tubular cells, MAP3K14 small interfering RNA targeting decreased inflammation and cell death. Additionally, cell culture and in vivo studies identified the chemokines MCP-1, RANTES, and CXCL10 as MAP3K14 targets in tubular cells. In conclusion, MAP3K14 promotes kidney injury through promotion of inflammation and cell death and is a promising novel therapeutic target.

Chondroitin sulphate inhibits NF-κB activity induced by interaction of pathogenic and damage associated molecules

To evaluate the anti-inflammatory mechanism of action of Chondroitin Sulphate (CS). THP-1 macrophages were cultured with a range of sizes and concentrations of HA fragments with TLR4 (LPS in a physiologically relevant concentration determined by analyses of sera of a community clinic ascertained knee osteoarthritis (OA) cohort) or TLR2 (heat killed listeria bacteria) agonists and varying concentrations of CS in a physiologically relevant range (10-200μg/ml). We measured IL-1β release, intracellular IL-1β, proIL-1β, caspase-1 and NF-κB activity and DNA binding activity of NF-κB transcription factors from nuclear and cytoplasmic extracts. Serum LPS was significantly associated with radiographic knee joint space narrowing (JSN) (P=0.02) in the OA cohort (n=40). The priming dose of LPS used for these experiments (10ng/ml) was below the lowest serum concentration of the OA cohort (median 47.09, range 14.43-81.36ng/ml). Priming doses of LPS and HA fragments alone did not elicit an inflammatory response. However, primed with LPS, HA fragments produced large dose-dependent increases in IL-1β that were inhibitable by CS. CS did not inhibit caspase-1 activity but in physiologically achievable concentrations, attenuated NF-κB activity induced by either the TLR4 (LPS 1000ng/ml) or TLR2 agonists alone or in combination with HA fragments. LPS induced and CS significantly reduced activity of canonical NF-κB transcription factors, p65, p50, c-Rel and RelB. Subinflammatory concentrations of pathogenic (LPS, listeria) and damage associated (HA) molecules interact to induce macrophage-related inflammation. CS works upstream of the inflammasome by inhibiting activation of NF-κB transcription factors.

Abstract 084: Sodium Butyrate and Retinoic Acid Attenuate Renal Inflammation and Fibrosis in Npr1 Gene-targeted Haplotype Mice: Role of NF-κB Signaling

The objective of the present study was to determine the effect of a hybrid drug of sodium butyrate (NaBu), a histone deacetylase (HDAC) inhibitor and all-trans retinoic acid (ATRA) on the attenuation of renal inflammation and fibrosis in Npr1 gene-disrupted mutant mice. Adult (18-20 week old) male Npr1 gene-disrupted heterozygous (1-copy; Npr1 +/- ) wild-type (2-copy; Npr1 +/+ ), and gene-duplicated (3-copy; Npr1 ++/+ ) mice were treated with ATRA-NaBu hybrid drug (1.0 mg/kg/day) by intraperitoneal injections for 2-weeks. A significant decrease in systolic blood pressure was observed in ATRA-NaBu-treated haplotype Npr1 +/- mice compared with untreated controls (treated, 113.3 ± 1.5 vs. control, 130.4 ± 1.9; p &lt; 0.01). After treatment with ATRA-NaBu, a marked reduction in tubulo-interstitial fibrosis (50%, p &lt; 0.001) and decreased renal collagen type I alpha 2 expression by 55% (treated, 25.9 ± 1.2 vs. control, 57.2 ± 2.9, p &lt; 0.001) was observed in Npr1 +/- mice. Treatment with ATRA-NaBu also increased creatinine clearance (ml/24 h) in Npr1 +/- mice (245.7 ± 24.3 vs. control, 83.8 ± 3.9). Similarly, higher urinary albumin to creatinine ratio was detected in Npr1 +/- mice (0.84 ± 0.03) vs. control (0.35 ± 0.03; p &lt; 0.01) and a complete reversal was observed in drug-treated Npr1 +/- mice (0.39 ± 0.05). Significant decreases were observed in renal (pg/mg protein) tumor necrosis factor-alpha (TNF-α) (4.1 ± 0.7 vs. control, 27.2 ± 2.6; p &lt; 0.01) and interleukin (IL)-6 (11.8 ± 0.8 vs. control, 59.9 ± 3.6; p &lt; 0.01) in ATRA-NaBu-treated Npr1 +/- mice. Western blot analyses showed significant reduction in renal TNF-α and IL-6 protein expression by 54%, (p &lt; 0.001) and 61%, (p &lt; 0.01), respectively, in ATRA-NaBu-treated Npr1 +/- mice. There was 49% increase in renal NF-κB (p65) DNA binding activity in Npr1 +/- mice and 51% lower activity in Npr1 +/++ mice compared with wild-type mice. Western blot analysis revealed distinctly higher levels of renal NF-κB (p65) protein expression in Npr1 +/- mice and reduced levels in Npr1 +/++ mice compared with wild-type controls. The present results provide direct evidence that ATRA-NaBu hybrid drug acts as a potent anti-inflammatory agent, which will have important implications in the pathophysiology of renal injury and hypertension.

A tryptamine-paeonol hybridization compound inhibits LPS-mediated inflammation in BV2 cells

In the present study, we synthesized and evaluated the anti-inflammatory effects of three tryptamine (Trm) hybrid compounds, HBU-375, HBU-376 and HBU-379. The Click reaction between the azido-Trm and 2- or 4-propazylated paeonol moiety resulted in HBU-376 and HBU-375, respectively. HBU-379 was generated by hybridizing Trm with propazylated acetyl-syringic acid. HBU-376 and HBU-375 dose-dependently inhibited LPS and caused nitric oxide (NO) generation in BV2 cells, whereas HBU-379 minimally inhibited NO generation, indicating that the paeonol unit plays an important role in the anti-inflammatory effect of Trm hybrid compounds. Although HBU-375 and HBU-376 demonstrated a similar inhibitory effect on LPS-induced NO generation, HBU-376 resulted in less cellular toxicity presumably due to the free phenolic hydroxyl group of paeonol. Therefore, HBU-376 may be a promising anti-inflammatory agent conferring minimal cytotoxicity. HBU-376 significantly and dose-dependently inhibited LPS-induced NO products, NO synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-6, MCP-1 and interleukin-1β mRNA expressions and iNOS and COX-2 protein expressions. However, at the same concentrations, Trm or paeonol individually did not inhibit LPS-mediated production of inflammatory molecules. HBU-376 inhibited both LPS-induced STAT-3 phosphorylation and nuclear factor-kappa B (NF-κB) activation. Furthermore, LPS-mediated DNA binding of c-Rel, p50 and p52 to the NF-κB binding site of the iNOS promoter was inhibited by HBU-376, whereas Trm and paeonol did not inhibit LPS-induced NF-κB activation and DNA binding of c-Rel, p50 and p52. Overall, our data suggest that the Trm-paeonol hybrid compound down-regulates inflammatory responses by inhibiting NF-κB and NF-κB-dependent gene expression. This suggests that it is a potential therapeutic agent for inflammatory diseases of the central nervous system.

Pseuderanthemum palatiferum leaf extract inhibits the proinflammatory cytokines, TNF-α and IL-6 expression in LPS-activated macrophages

The anti-inflammatory potential and underlying mechanisms of an ethanol extract of Pseuderanthemum palatiferum (EEP) leaves was investigated using LPS-activated macrophages. Our results show EEP produced a concentration-dependent suppression of TNF-α and IL-6 secretion by LPS-activated mouse peritoneal macrophages. EEP also suppressed LPS-induced TNF-α and IL-6 protein and mRNA levels in mouse-derived myeloid cell line RAW264.7. To further elucidate the molecular mechanisms responsible for impaired TNF-α and IL-6 regulation by EEP, the activation of transcription factors, NF-κB, C/EBP, and AP-1, was monitored using electrophoretic mobility shift assays. EEP suppressed LPS-induced NF-κB DNA binding activity within both the TNF-α and IL-6 promoters in RAW264.7 cells with impairment being more pronounced in the IL-6 promoter. In addition, EEP exhibited a concentration-dependent suppression of C/EBP and AP-1 DNA binding activity within the IL-6 promoter. Concordantly, IL-6 luciferase promoter reporter activity was also suppressed by EEP in transiently transfected RAW264.7 cells, upon LPS activation. EEP analysis by GC-MS and HPLC DAD-MSD revealed the presence of β-sitosterol and various polyphenols, respectively, which are known to possess anti-inflammatory activity. Collectively, these results suggest that the anti-inflammatory effects of EEP are mediated, at least in part, by modulating TNF-α and IL-6 expression through impairment of NF-κB, C/EBP, and AP-1 activity.

Peridinin, a carotenoid, inhibits proliferation and survival of HTLV-1-infected T-cell lines.

Human T-cell leukemia virus type 1 (HTLV-1) causes either adult T-cell leukemia (ATL) or chronic inflammatory disorders such as HTLV-1-associated myelopathy/tropical spastic paraparesis. These diseases are not curable as yet; therefore new agents for treatment and prevention are needed. Carotenoids are natural plant compounds with anti-carcinogenic activities. Peridinin is one of the most abundant carotenoids found in nature. Based on a series of past experiments, here we investigated the effects of peridinin extracted from Okinawan coral Isis hippuris on the proliferation and survival of HTLV-1-infected T-cell lines. The results of water-soluble tetrazolium-8 assay indicated that peridinin dose-dependently inhibits cell proliferation and viability of HTLV-1-infected T-cell lines. Flow cytometry showed that low concentration of peridinin induced cell cycle arrest at G1 phase, while higher concentration induced apoptosis. Peridinin caused cleavage of caspase-3, -8 and -9. Peridinin significantly reduced the expression of G1 cell cycle regulators, including cyclinD1, cyclinD2, CDK4, CDK6 and c-Myc, and anti-apoptotic proteins, including survivin, XIAP and Bcl-2, in a dose-dependent manner. Peridinin suppressed DNA binding of NF-κB. Peridinin inhibited phosphorylation of IκBα, RelA, Akt and p70S6 kinase, and reduced protein expression level of 3-phosphoinositide-dependent protein kinase 1. Thus, peridinin exerts its anti-proliferative and pro-apoptotic effects by suppressing NF-κB and Akt signaling in HTLV-1-infected T cells. Peridinin also reduced tumor growth in mice harboring ATL xenograft tumors. The results suggested that peridinin is a potentially suitable therapeutic agent against HTLV-1-associated diseases.

Prolonged Glucocorticoid Treatment in ARDS: Impact on Intensive Care Unit-Acquired Weakness.

Systemic inflammation and duration of immobilization are strong independent risk factors for the development of intensive care unit-acquired weakness (ICUAW). Activation of the pro-inflammatory transcription factor nuclear factor-κB (NF-κB) results in muscle wasting during disuse-induced skeletal muscle atrophy (ICU bed rest) and septic shock. In addition, NF-κB-mediated signaling plays a significant role in mechanical ventilation-induced diaphragmatic atrophy and contractile dysfunction. Older trials investigating high dose glucocorticoid treatment reported a lack of a sustained anti-inflammatory effects and an association with ICUAW. However, prolonged low-to-moderate dose glucocorticoid treatment of sepsis and ARDS is associated with a reduction in NF-κB DNA-binding, decreased transcription of inflammatory cytokines, enhanced resolution of systemic and pulmonary inflammation, leading to fewer days of mechanical ventilation, and lower mortality. Importantly, meta-analyses of a large number of randomized controlled trials investigating low-to-moderate glucocorticoid treatment in severe sepsis and ARDS found no increase in ICUAW. Furthermore, while the ARDS network trial investigating methylprednisolone treatment in persistent ARDS is frequently cited to support an association with ICUAW, a reanalysis of the data showed a similar incidence with the control group. Our review concludes that in patients with sepsis and ARDS, any potential direct harmful neuromuscular effect of glucocorticoids appears outweighed by the overall clinical improvement and reduced duration of organ failure, in particular ventilator dependency and associated immobilization, which are key risk factors for ICUAW.

Glioma-secreted soluble factors stimulate microglial activation: The role of interleukin-1β and tumor necrosis factor-α

We aimed to elucidate the effect of soluble factors secreted by glioma on microglial activation. Conditioned medium (CM) from glioma cells, CRT-MG and C6, significantly induced nitric oxide (NO) production and stimulated the mRNA expression of inducible NO synthase (iNOS), interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha (TNF-α) and cyclooxygenase 2 (COX-2) in BV2 cells. Glioma CM stimulated p38 mitogen-activated protein kinase (MAPK) phosphorylation, and a p38 MAPK inhibitor, SB203580, suppressed CM-induced NO production in BV2 cells. In addition, CM stimulated nuclear factor-kappaB (NF-κB) DNA binding and transcriptional activity, which was repressed by SB203580. Gliomas displayed higher mRNA expression and release of TNF-α and IL-1β than primary astrocyte cells. Neutralization of TNF-α and IL-1β in C6-CM using a neutralizing antibody inhibited NO/iNOS expression in BV-2 cells. These results indicate potential contribution of diffusible tumor-derived factors to regulate microglial activation and subsequent tumor microenvironment.

Anti-inflammatory activity of myricetin from Diospyros lotus through suppression of NF-κB and STAT1 activation and Nrf2-mediated HO-1 induction in lipopolysaccharide-stimulated RAW264.7 macrophages.

Diospyros lotus is traditionally used for the treatment of diabetes, diarrhea, tumor, and hypertension. The purpose of this study was to investigate the anti-inflammatory effect and underlying molecular mechanisms of myricetin in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Myricetin dose-dependently suppressed the production of pro-inflammatory mediators (NO, iNOS, PGE2, and COX-2) in LPS-stimulated RAW264.7 macrophages. Myricetin administration decreased the production of NO, iNOS, TNF-α, IL-6, and IL-12 in mice. Myricetin decreased NF-κB activation by suppressing the degradation of IκBα, nuclear translocation of p65 subunit of NF-κB, and NF-κB DNA binding activity in LPS-stimulated RAW264.7 macrophages. Moreover, myricetin attenuated the phosphorylation of STAT1 and the production of IFN-β in LPS-stimulated RAW264.7 macrophages. Furthermore, myricetin induced the expression of HO-1 through Nrf2 translocation. In conclusion, these results suggest that myricetin inhibits the production of pro-inflammatory mediators through the suppression of NF-κB and STAT1 activation and induction of Nrf2-mediated HO-1 expression in LPS-stimulated RAW264.7 macrophages.

Abstract 2907: Eugenol potentiates the effect of cisplatin on cancer stem-like cells through targeting the NF-κB pathway

Abstract Triple-negative subtype of breast cancer (TNBC) contains exclusively tumorigenic and often endowed with self-renewing and resistance to cytotoxic chemotherapy, such as, paclitaxel resulting in relative increases in CSCs phenotype. Signaling through NF-kB may be essential for the CSCs self-renewal and could present potential target for novel treatment in breast cancer patients. We explored the possible involvement of NF-kB pathway that contribute to the functions and maintenance of breast CSCs and targeting this pathway by cisplatin combining with a natural product eugenol both in vitro and in vivo, using estrogen dependent (MCF-7) and independent (MDA-MB-231, MDA-MB-468 and BT-20) cell lines and xenografted both subcutaneous and orthotopic (fat pad) sites in nude mice. Chromatin immunoprecipitation (ChIP) assay was utilized to assess the NF-kB associated DNA binding capacity in promoter region. Surprisingly, neither cisplatin nor eugenol was capable of complete diminishing the CSCs populations. Cisplatin alone administration partially diminished apoptosis, mammospheres formation, aldehyde dehydrogenase 1 (ALDH1) activity, invasion and growth on nude mice. We observed cisplatin-induced activation of NF-kB associated with survival and regrowth of mamospheres. For effective elimination of CSCs phenotype by cisplatin, we cotreated cells with eugenol. Combination of eugenol and cisplatin significantly eradicated cisplatin induced NF-kB DNA binding capability assed by EMSA and ChIP assays, which was associated with abrogated apoptosis, mammospheres formation, ALDH1 activity and invasion. In vivo, combination therapy reduced the tumor size in synergistic manner. This was possibly due to induction of apoptosis, inhibition of proliferation, angiogenesis and downregulation of cisplatin-induced expression of proteins of NF-kB target genes. Although treatment of mammospheres by cisplatin partially induced the CSCs population through interleukin (IL)-6 pathway, however, only coadministration of eugenol and cisplatin reduced the numbers of CSCs to virtually undeletable levels in vitro and in vivo. Our data suggest that eugenol may be well suited to increase targeting of breast CSCs by cisplatin. Citation Format: Syed S. Islam, Al-Sharif Ibtehaj, Sultan Ahlam, Abdelilah Aboussekhra. Eugenol potentiates the effect of cisplatin on cancer stem-like cells through targeting the NF-κB pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2907.

Abstract 1154: Elevated nuclear GSK3β promotes more aggressive disease in AML

Abstract Acute myeloid leukemia (AML) is a devastating disease with a poor overall 5-yr survival rate and an antiquated and poorly tolerated therapy regimen. Despite an increasing knowledge of the molecular drivers of AML, new therapeutic targets are needed. GSK3β is a key signaling molecule that plays a role in several key oncogenic pathways including the β-catenin and NF-κB pathways, key pathways in AML and other cancers. Upregulation of GSK3β is correlated with poor prognosis in several cancers. We and others have shown GSK3β inhibition promotes AML differentiation as well as death of AML cells while sparing normal cells, suggesting GSK3β plays an important role in AML. Here we show by western blotting and confocal microscopy that GSK3β is upregulated and more nuclear in AML cells. We utilize flow cytometry to show AML cells express elevated GSK3β compared to normal marrow lineages (Average upregulation = 6 fold). We transform mouse hematopoietic progenitor cells with the MLL-AF9 oncogene and observe GSK3β upregulation as a course of AML leukemogenesis. These data suggest that elevated and nuclear GSK3β is characteristic of AML. To probe biological effects of GSK3β, we knocked down endogenous GSK3β and utilize a targetted, tetracycline-inducible GSK3β rescue model to show nuclear GSK3β specifically can promote AML growth. Nuclear GSK3β increases colony formation of OCI-AML3 and HL60 cell lines more potently than cytoplasmic (cyto) GSK3β in the same lines (OCI-AML3- Nuclear: 118% Cyto: 93% of control colony growth HL60- Nuclear: 117% Cyto: 100% of control colony growth, Nuclear difference- p&amp;lt;0.01 in both lines). Utilizing a mouse xenograft model and inducing GSK3β via doxycycline water, we show inducing nuclear GSK3β shortens mouse survival compared to uninduced controls, while inducing cytoplasmic GSK3β promotes no change from controls (HL60- Nuclear: Uninduced = 35.4 days mean survival, Induced = 28.75 days mean survival Cyto: Uninduced = 38 days mean survival, Induced = 38.6 days mean survival, Induced nuclear GSK3β difference p&amp;lt;0.01 by log rank test). At a molecular level, when compared to cells expressing cytoplasmic GSK3β, nuclear GSK3β more potently promotes NF-κB DNA binding and NF-κB mediated transcription as measured by EMSA assay and luciferase assay (Nuclear- 18 fold increase Cyto- 3 fold increase). Nuclear GSK3β more potently promotes activation of the NF-κB subunit p65, as measured by pS536, than cytoplasmic GSK3β. Nuclear GSK3β also promotes nuclear localization of p65. These data suggest that nuclear GSK3β can promote NF-κB activation in AML. Finally, we utilize imaging cytometry to show GSK3β is upregulated and more nuclear in clinical AML patient samples (N = 80) compared to normal bone marrow controls (N = 5). We also show nuclear localization of GSK3β and p65 correlate linearly in AML patient samples (N = 80 R2 = 0.44721 P&amp;lt;.01). These data suggest elevated nuclear GSK3β is observed in clinical AML and may be related to NF-κB activation. Citation Format: James J. Ignatz-Hoover, Nathan Mackowski, David N. Wald. Elevated nuclear GSK3β promotes more aggressive disease in AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1154.

Abstract 3061: AS-10: a new small molecule with promising activity against pancreatic cancer

Abstract Patients with pancreatic cancer (PC) have a median survival of only 6 months and a five-year survival of less than 5%, hence making PC one of the deadliest cancer. Severity of PC is due to its identification at late stages, rapid local invasion, early metastases, and meager response to current chemotherapeutic agents. Current therapies result in minimal survival advantage and are linked with multiple adverse events and drug resistance. Hence, there is an urgent need for novel agents which are less toxic and offer greater benefits over conventional therapy. There is ample evidences in literature demonstrating that inflammation plays a critical role in PC growth and promotion. Nuclear factor κB (NF-κB) pathway, one of the major inflammatory pathway, is well known for its inflammatory response, cell proliferation, and resistance to apoptosis. It has been demonstrated that activation of NF-κB in PC is also responsible for resistance towards first line chemotherapeutic agent, gemcitabine, in PC. Through extensive structure-activity relationship studies, we have recently identified a novel small molecule, AS-10, which was lethal to PC cells. We sought to evaluate the mechanism of action of AS-10 responsible for inhibiting the growth of PC cells. In vitro, AS-10 reduced PC cell growth with an EC50, in the range of 2.5 to 5 μM. Growth arrest was confirmed by cell cycle studies, which showed that AS-10 induced G1 and G2 cell cycle arrest. The cell cycle arrest was associated with an increase of cell cycle inhibitory markers like p21 and p27. Effect of AS-10 on cell cycle was translated into activation of apoptosis, confirmed by caspase 3/7 activity, PARP cleavage and Annexin V staining. Due to its structural characteristics, we evaluated the effect of AS-10 on inflammatory NF-κB pathway. Our studies, in Panc-1 cells, showed that AS-10 inhibited NF-κB DNA binding activity as well as NF-κB translocation to the nuclei in the presence of inflammatory stimuli (tumor necrosis factor (TNFα)). Since, up regulation of NF-κB activity has been known to be responsible for gemcitabine resistance in PC, we evaluated the activity of our NF-κB inhibitor AS-10 in combination with gemcitabine, and showed that AS-10 synergistically potentiated gemcitabine activity in PC cells. Taken together, these results suggest that AS-10, may represent a potentially promising therapeutic agent for PC. Detailed investigations regarding the efficacy and mechanism of action of these compounds will be presented. Citation Format: Deepkamal Karelia, Manoj K. Pandey, Daniel Plano, Shantu Amin, Arun K. Sharma. AS-10: a new small molecule with promising activity against pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3061.

Gene sharing between Epstein-Barr virus and human immune response genes.

Epstein-Barr virus (also termed HHV-4, EBV), a component of the human virome or metagenome, is associated as a co-factor in many common human autoimmune diseases through epidemiologic evidence. Numerous EBV genes are functional as well as structural homologues of important immune response genes. For example, EBV-encoded BCRF1 is a functional homologue of IL-10, a critical cytokine regulator of immune tolerance. BZLF-1, an EBV-encoded transcription factor, contains regions with functional homology to both AP-1 and NF-κB DNA binding immune response regulatory factors. The author proposes a paradigm of "gene sharing" between viral- and host-encoded proteins as extension of molecular mimicry that has been largely overlooked in animal models that consider only host genomic factors rather than viral pathogens and the metagenome. Gene sharing may trigger chaotic behavior in human autoimmune disease through unstable feedback loops and perturbations of immune tolerance.

Interplay between 15-lipoxygenase-1 and metastasis-associated antigen 1 in the metastatic potential of colorectal cancer.

Metastasis-associated antigen 1 (MTA1) is implicated in metastasis while 15-lipoxygenase-1 (15-LOX-1) reduces cell motility, when re-expressed in colorectal cancer (CRC). We aimed to understand any potential interplay between MTA1 and 15-LOX-1 in CRC metastasis. ALOX15 and MTA1 expression in tumour and normal samples were analysed from TCGA RNA-seq data, microarray data sets and a human CRC cDNA array. Western blots, chromatin immunoprecipitation (ChIP), luciferase assays and electrophoretic mobility shift assays (EMSA) were carried out in HT-29 and LoVo cells re-expressing 15-LOX-1 to determine NF- κB activity at the MTA1 promoter. Functional assays in cells ectopically expressing either 15-LOX-1, MTA-1 or both, were carried out to determine adhesion and cell motility. Significantly higher expression of MTA1 was observed in tumours compared to normal tissues; MTA1 overexpression resulted in reduced adhesion in CRC cell lines. Re-expression of 15-LOX-1 in the CRC cell lines reduced expression of endogenous MTA1, corroborated by negative correlation between the two genes in two independent human CRC microarray data sets, with greater significance in specific subsets of patients. DNA binding and transcriptional activity of NF-κB at the MTA1 promoter was significantly lower in cells re-expressing 15-LOX-1. Functionally, the same cells had reduced motility, which was rescued when they overexpressed MTA1, and further corroborated by expressions of E-cadherin and vimentin. Expression of MTA1 and 15-LOX-1 negatively correlated in specific subsets of CRC. Mechanistically, this is at least in part through reduced recruitment of NF-κB to the MTA1 promoter.

Characterization of TNF-α– and IL-17A–Mediated Synergistic Induction of DEFB4 Gene Expression in Human Keratinocytes through IκBζ

Human β-defensin 2 (hBD2), encoded by the DEFB4 gene, is an antimicrobial peptide playing an essential role in inflammatory processes in the skin. hBD2 expression is regulated synergistically by tumor necrosis factor-α (TNF-α) and IL-17A; however, the underlying regulatory mechanisms are unknown. The purpose of this study was to characterize the molecular mechanism by which TNF-α and IL-17A synergistically induce hBD2 expression. In cultured human keratinocytes we show that a constitutive noninducible binding of the transcription factor organic cation transporter 1 (OCT1) to the DEFB4 promoter is crucial for IL-17A/TNF-α-mediated synergistic induction of hBD2 but not the synergistic induction of CCL20, IL8, IL17C and LCN2. Interestingly, stimulation with IL-17A results in a p38 mitogen-activated protein kinase-dependent accumulation of inhibitor of nuclear factor κB ζ (IκBζ), which is a necessity for synergistic induction of hBD2. Finally, co-stimulation with TNF-α induces DNA binding of NF-κB and activator protein 1 (AP-1) to two specific sites in the DEFB4 promoter region. Hence, our study shows how two inflammatory stimuli are integrated by three different signaling pathways into the regulation of one specific target gene involving the three specific transcription factors OCT1, NF-κB, and AP-1 as well as the transcriptional cofactor IκBζ. These findings may be important in psoriasis, where TNF-α and IL-17A have been identified as key pathogenic cytokines.

MicroRNA-24 inhibits high glucose-induced vascular smooth muscle cell proliferation and migration by targeting HMGB1

Dysfunction of vascular smooth muscle cells (VSMCs) performs a key role in the pathogenesis of diabetic vascular disease. Recent studies have reported that microRNA-24 (miR-24) may be implicated in diabetes and atherosclerotic vascular diseases. This study was designed to explore the role of miR-24 on VSMC proliferation and migration under high glucose conditions mimicking diabetes, and reveal the underlying mechanism. VSMCs were isolated from rat thoracic aortas, treated with normal glucose (NG, 5.5mM) or high glucose (HG, 30mM) during an incubation period. Cell viability, proliferation and migration were detected by trypan blue staining, BrdU incorporation assay and transwell chamber assay. Gene and protein expression were analyzed by qRT-PCR and Western blot respectively. We also used electrophoretic mobility shift assay (EMSA) to detect nuclear factor kappaB (NF-κB) DNA binding. TNF-α and IL-6 levels were determined by enzyme-linked immunosorbent assay. The results showed that adenovirus-mediated miR-24 overexpression significantly inhibited HG-stimulated VSMC proliferation and migration. Meanwhile, high mobility group box-1 (HMGB1) as a target of miR-24, was also markedly suppressed after miR-24 transfection. Additionally, NF-κB nuclear translocation and DNA binding, TNF-α and IL-6 production were all decreased associated with the down-regulation of HMGB1. The above data indicated that miR-24 is a crucial regulator of high glucose-induced proliferation and migration in VSMCs, and suggests that elevation of miR-24 in vascular system may be a novel therapeutic strategy to prevent the development of diabetic atherosclerosis.