Nuclear Accumulation Of NF-κB Research Articles

The Hepatic Antisteatosis Effect of Xanthohumol in High-Fat Diet-Fed Rats Entails Activation of AMPK as a Possible Protective Mechanism.

Obesity is the leading cause of non-alcoholic fatty liver disease by provoking hyperglycemia, hyperlipidemia, insulin resistance, oxidative stress, and inflammation. Low activity of AMP-activated protein kinase (AMPK) is linked to obesity, liver injury, and NAFLD. This study involves examining if the anti-steatosis effect of Xanthohumol (XH) in high-fat diet (HFD)-fed rats involves the regulation of AMPK. Adult male rats were divided into five groups (n = 8 each) as control (3.85 kcal/g); XH (control diet + 20 mg/kg), HFD (4.73 kcl/g), HFD + XH (20 mg/kg), and HFD + XH (30 mg/kg) + compound c (cc) (0.2 mg/kg). All treatments were conducted for 12 weeks. Treatment with XH attenuated the gain in body weight, fat pads, fasting glucose, and insulin in HFD rats. It also lowered serum leptin and free fatty acids (FFAs) and improved glucose and insulin tolerances in these rats. It also attenuated the increase in serum livers of liver marker enzymes and reduced serum and hepatic levels of triglycerides (TGs), cholesterol (CHOL), FFAs, as well as serum levels of low-density lipoproteins cholesterol (LDL-c) oxidized LDL-c. XH also reduced hepatic levels of malondialdehyde (MDA), nuclear accumulation of NF-κB, and the levels of tumor necrosis-factor-α (TNF-α) and interleukin-6 (IL-6) while stimulating the nuclear levels of Nrf2 and total levels of glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) in these HFD-fed rats. At the molecular levels, XH increased hepatic mRNA expression and phosphorylation of AMPK (Thr72) and reduced the expression of lipogenic genes SREBP1c and ACC-1. In concomitance, XH reduced hepatic liver droplet accumulation, reduced the number of apoptotic nuclei, and improved the structures of nuclei, mitochondria, and rough endoplasmic reticulum. Co-treatment with CC, an AMPK inhibitor, completely abolished all these effects of XH. In conclusion, XH attenuates obesity and HFD-mediated hepatic steatosis by activating hepatic AMPK.

Astaxanthin protects tilapia head kidney cells against polystyrene microplastics-induced inflammation through MAPK and NF-κB signaling pathways

Microplastic particles (MPs) are emerging as global pollutants, causing oxidative stress and inflammation in aquaculture. Astaxanthin is classified as a xanthophyll carotenoid compound with many functions, including anti-inflammatory properties. However, astaxanthin has yet to be proven to counteract the proinflammatory effects of MPs. In this study, we used the polystyrene (PS)-MPs-treated Nile tilapia (Oreochromis niloticus) head kidney cells as a model to explore the anti-inflammatory effect of astaxanthin. In primary cell cultures from the head kidney, the administration of PS-MPs dose-dependently inhibited cell viability, whereas the application of astaxanthin negated the viability-suppressive effect of PS-MPs by inhibiting the cellular reactive oxygen species (ROS) production. Furthermore, astaxanthin treatment repressed PS-MPs-induced interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) secretion. In parallel studies, the inhibitory effects of PS-MPs on these cytokines release were prevented by the p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinases (ERK1/2), and nuclear factor-kappa B (NF-κB) inhibitors. However, the c-Jun N-terminal kinase (JNK1/2) inhibition did not influence cytokines secretion in PS-MPs-treated head kidney cells. Using Western blot analysis, we demonstrated that astaxanthin dose-dependently inhibited the PS-MPs-induced stimulation of phosphorylation of the p38 MAPK, ERK1/2, and Iκκα/β. Additionally, astaxanthin noticeably prevented PS-MPs-induced NF-κB nuclear accumulation. These findings suggest that astaxanthin can repair PS-MPs-triggered inflammatory responses in tilapia head kidney cells via downregulating the MAPK (p38 MAPK and ERK1/2) and NF-κB signaling pathways.

Exercise and nitrite prevent and Nω-nitrol-L-arginine methyl ester reproduces imbalance in the nuclear factor-κB/NADPH oxidase 2 and nuclear factor erythroid 2-related factor 2/NADPH oxidase 4/endothelial nitric oxide synthase systems in diabetes.

Diabetes-induced vasculopathies are linked to inflammation mediated by mutually inhibitory nuclear factor-kappaB (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2). NF-κB is activated by superoxide (O2 ˙-)- producing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase homologues, including NADPH oxidase 2 (Nox2), and vice versa, with NF-κB inducing Nox2. Nrf2 is activated by H2O2-producing Nox4 and nitric oxide (NO), but also induces NADPH oxidase 4 (Nox4) and endothelial nitric oxide synthase (eNOS). The NF-κB/Nox2 system is upregulated and Nrf2/Nox4/eNOS is downregulated in diabetes. We hypothesized that this vascular-deleterious imbalance results from the reduced vascular NO signaling, and so may be prevented by exercise training and sodium nitrite (interventions known to replenish vascular NO), and be reproduced by nitric oxide synthase (NOS) inhibition. Streptozotocin diabetic rats were examined on days 4, 10, 49 and 84 of diabetes. From day 4 onwards, plasma nitrite was reduced while NF-κB nuclear accumulation in the heart and kidneys gradually increased, while Nrf2 decreased. In parallel, the cardiac expression of signatures of the NF-κB (inducible nitric oxide synthase (iNOS), vascular cell adhesion molecule-1 (VCAM-1), NADPH oxidase 2 Nox2) increased and of the Nrf2 (Nox4, eNOS, heme-oxygenase-1 (HO-1)) decreased. Exercise training and dietary nitrite prevented this phenotype in the 49-day diabetes model. 7-day treatment of non-diabetic rats with NOS inhibitor of Nω-nitro-L-arginine methyl ester (L-NAME) recapitulated the NF-κB/Nox2 and Nrf2/Nox4/eNOS imbalance, as seen in diabetic rats. Nitrite failed to prevent the changes induced by L-NAME. The coherence of changes in NF-κB, Nox2, Nrf2, Nox4 and eNOS under the various settings of this study aimed at modifying the vascular NO leads us to propose that NF-κB/Nox2 and Nrf2/Nox4/eNOS are two crosstalking functional subsystems of one larger regulatory network, with NOS-derived NO ensuring the balance between these subsystems, and thus preventing vascular oxidative stress, endothelial dysfunction and inflammation.

Abstract 1403: HOTAIR functions through NF-κB pathway in regulating ovarian cancer stem cells

Abstract Ovarian cancer (OC) is the fifth leading cause of cancer-related death among American women. Persistence of OC stem cells (OCSCs) is believed to contribute to resistance to platinum-based chemotherapy and disease relapse. Long non-coding RNA HOXC transcript antisense RNA (HOTAIR) has been shown to be associated with chemoresistance and overexpressed in many types of cancers, including high-grade serous OC (HGSOC). Previously published work has demonstrated that NF-κB was activated by HOTAIR through I-κB inhibition via trimethylation of histone H3 lysine K27, which contributes to chemoresistance in HGSOC. NF-κB-medicated signaling pathways involved in maintaining characteristics of CSCs, such as targeting stem cell markers, CD44, CD133, and ALDH1 has been demonstrated but not well defined in OC. The goals of this study are to understand the mechanism of HOTAIR-mediated NF-κB signaling pathway in regulating OCSCs and develop novel strategies to target OCSCs and overcome OC recurrence and drug resistance. Quantitative RT-PCR analysis revealed that HOTAIR was overexpressed in OCSCs compared to non-OCSCs. In order to produce loss-of-function phenotypes of HOTAIR and investigate the function of this gene, we utilized the paired CRISPR guide RNA design to delete the functional sites of HOTAIR without affecting nearby protein-coding gene. Knockout of HOTAIR re-sensitized OC cells to platinum treatment and significantly decreased (P<0.001) OCSC population and stemness-related phenotypes, including spheroid formation and colony formation ability. ALDH1A1 and expression of other stemness-related genes, including Notch3, PROM1 were significantly decreased by HOTAIR knockout. Overexpression of HOTAIR in OC cells significantly increased (P<0.05) these stem-like characteristics. Furthermore, we showed NF-κB nuclear accumulation and activation in OCSCs compared to non-OCSCs. Knockout of HOTAIR decreased (P<0.01) nuclear localization of NF-κB in OCSCs. Inhibiting NF-κB in using a commercially available pharmacological approach as well as NF-κB knockout both significantly decreased OCSC population and stemness-related phenotypes, as well as ALDH1 protein expression (P< 0.01). However, inhibition of NF-κB has no effect on OCSC characteristics in HOTAIR knockout cells, indicating that HOTAIR may function through constitutively activated NF-κB in regulating OCSCs. We suggest a better understanding of HOTAIR- NF-κB axis in regulating OCSCs will facilitate identifying the key therapeutic target to eliminate residual tumor cells after conventional chemotherapy and prevent OC recurrence and drug resistance. Citation Format: Weini Wang, Fang Fang, Ali R. Özeş, Kenneth P. Nephew. HOTAIR functions through NF-κB pathway in regulating ovarian cancer stem cells [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 1403.

Emodin Protects Against Acute Pancreatitis-Associated Lung Injury by Inhibiting NLPR3 Inflammasome Activation via Nrf2/HO-1 Signaling.

AimLung injury is a common complication of acute pancreatitis (AP), which leads to the development of acute respiratory distress syndrome and causes high mortality. In the present study, we investigated the therapeutic effect of emodin on AP-induced lung injury and explored the molecular mechanisms involved.Materials and MethodsThirty male Sprague-Dawley rats were randomly divided into AP (n=24) and normal (n=6) groups. Rats in the AP group received a retrograde injection of 5% sodium taurocholate into the biliary-pancreatic duct and then randomly assigned to untreated, emodin, combined emodin and ML385, and dexamethasone (DEX) groups. Pancreatic and pulmonary injury was assessed using H&E staining. In in vitro study, rat alveolar epithelial cell line L2 cells were exposed to lipopolysaccharide and treated with emodin. Nrf2 siRNA pool was applied for the knockdown of Nrf2. The contents of the pro-inflammatory cytokines in the bronchoalveolar lavage fluid and lung were determined using enzyme-linked immunosorbent assay. The expressions of related mRNAs and proteins in the lung or L2 cells were detected using real-time polymerase chain reaction, Western blot, immunohistochemistry and immunofluorescence.Key FindingsEmodin administration alleviated pancreatic and pulmonary injury of rats with AP. Emodin administration suppressed the production of proinflammatory cytokines, downregulated NLRP3, ASC and caspase-1 expressions and inhibited NF-κB nuclear accumulation in the lung. In addition, Emodin increased Nrf2 nuclear translocation and upregulated HO-1 expression. Moreover, the anti-inflammatory effect of emodin was blocked by Nrf2 inhibitor ML385.ConclusionEmodin effectively protects rats against AP-associated lung injury by inhibiting NLRP3 inflammasome activation via Nrf2/HO-1 signaling.

Clonal Mutations Activate the NF-κB Pathway to Promote Recurrence of Nasopharyngeal Carcinoma.

The genetic events occurring in recurrent nasopharyngeal carcinoma (rNPC) are poorly understood. Here, we performed whole-genome and whole-exome sequencing in 55 patients with rNPC and 44 primarily diagnosed NPC (pNPC), with 7 patients having paired rNPC and pNPC samples. Previously published pNPC exome data were integrated for analysis. rNPC and pNPC tissues had similar mutational burdens, however, the number of clonal mutations was increased in rNPC samples. TP53 and three NF-κB pathway components (TRAF3, CYLD, and NFKBIA) were significantly mutated in both pNPC and rNPC. Notably, mutations in TRAF3, CYLD, and NFKBIA were all clonal in rNPC, however, 55.6% to 57.9% of them were clonal in pNPC. In general, the number of clonal mutations in NF-κB pathway-associated genes was significantly higher in rNPC than in pNPC. The NF-κB mutational clonality was selected and/or enriched during NPC recurrence. The amount of NF-κB translocated to the nucleus in samples with clonal NF-κB mutants was significantly higher than that in samples with subclonal NF-κB mutants. Moreover, the nuclear abundance of NF-κB protein was significantly greater in pNPC samples with locoregional relapse than in those without relapse. Furthermore, high nuclear NF-κB levels were an independent negative prognostic marker for locoregional relapse-free survival in pNPC. Finally, inhibition of NF-κB enhanced both radiosensitivity and chemosensitivity in vitro and in vivo. In conclusion, NF-κB pathway activation by clonal mutations plays an important role in promoting the recurrence of NPC. Moreover, nuclear accumulation of NF-κB is a prominent biomarker for predicting locoregional relapse-free survival. SIGNIFICANCE: This study uncovers genetic events that promote the progression and recurrence of nasopharyngeal carcinoma and has potential prognostic and therapeutic implications.See related commentary by Sehgal and Barbie, p. 5915.

Liquiritigenin and liquiritin alleviated monocrotaline-induced hepatic sinusoidal obstruction syndrome via inhibiting HSP60-induced inflammatory injury

Hepatic sinusoidal obstruction syndrome (HSOS) is a life-threatening liver disease caused by the damage to liver sinusoidal endothelial cells (LSECs). Liquiritigenin and liquiritin are two main compounds in Glycyrrhizae Radix et Rhizoma (Gan-cao). Our previous study has shown that both liquiritigenin and liquiritin alleviated monocrotaline (MCT)-induced HSOS in rats via inducing the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant signaling pathway. This study aims to further investigate whether inhibiting liver inflammatory injury also contributed to the liquiritigenin and liquiritin-provided alleviation on MCT-induced HSOS. The results of serum alanine/aspartate aminotransferases (ALT/AST) activities and total bilirubin (TBil) amount, liver histological evaluation, scanning electron microscope observation and hepatic metalloproteinase-9 (MMP9) expression showed that liquiritigenin and liquiritin both alleviated MCT-induced HSOS in rats. Liquiritigenin and liquiritin reduced the increased liver myeloperoxidase (MPO) activity, mRNA expression of pro-inflammatory factors, hepatic infiltration of immune cells, hepatic toll-like receptor 4 (TLR4) expression and nuclear factor κB (NFκB) nuclear accumulation induced by MCT in rats. Furthermore, liquiritigenin and liquiritin attenuated MCT-induced liver mitochondrial injury, increased the decreased Lon protein expression and reduced the release of heat shock protein 60 (HSP60). Moreover, liquiritigenin and liquiritin also reduced NFκB nuclear accumulation and decreased the elevated cellular mRNA expression of NFκB-downstream pro-inflammatory cytokines induced by HSP60 in macrophage RAW264.7 cells. In conclusion, our study revealed that both liquiritigenin and liquiritin alleviated MCT-induced HSOS by inhibiting hepatic inflammatory responses triggered by HSP60.

Vitamin D attenuates lipopolysaccharide-induced inflammatory response in endothelial cells through inhibition of PI3K/Akt/NF-κB signaling pathway.

Inflammation and endothelial cells contribute to the pathogenesis of various cardiovascular diseases (CVDs). Vitamin D is associated with a decreased risk of inflammation related diseases including CVDs. However, the links between vitamin D, endothelial cells and vascular inflammation remain to be elucidated. In this study, we investigated the anti-inflammatory effect of vitamin D in lipopolysaccharide (LPS)-activated endothelial cells and the related signaling pathways. 1,25(OH)₂D₃ treatment significantly suppressed LPS-induced upregulation of COX-2, TNF-α, IL-6 and PGE2 in Human umbilical vein endothelial cells (HUVECs). 1,25(OH)₂D₃ inhibited LPS-stimulated phosphorylation of Akt at S473 and T308, IκBα phosphorylation, and nuclear accumulation of NF-κB. Blocking PI3K signaling abolished the inhibitory effect of 1,25(OH)₂D₃ on LPS-induced increase of Akt S473 phosphorylation and COX-2 expression. Blocking NF-κB signaling blunted the suppression effect of 1,25(OH)₂D₃ on IκBα phosphorylation, NF-κB accumulation in the nucleus and COX-2 expression. These results indicate that 1,25(OH)₂D₃ suppresses inflammatory response in LPS-induced HUVECs through PI3K/Akt/NF-κB signaling pathway.

Abstract 737: NF-κB pathway activation by clonal mutations contributes to the recurrence of nasopharyngeal carcinoma

Abstract Objectives: Identification and characterization of the genomic features that potentially drive NPC recurrence. Methods: We performed whole-genome/whole-exome sequencing of fresh tumors and corresponding blood samples from 55 rNPC and 44 pNPC patients. Additionally, previously published pNPC exome data were integrated for analysis. Immunohistochemistry and histologic analyses were performed to identify novel biomarkers among the differential mutational events between pNPC and rNPC in independent cohorts of 148 rNPC and 237 pNPC samples with complete clinical follow-up. Results: rNPC and pNPC tissues had similar mutational burdens; however, the number of clonal mutations was increased in rNPC samples. TP53 and three NF-κB pathway components (TRAF3, CYLD and NFKBIA) were significantly mutated in both pNPC and rNPC. Notably, mutations in TRAF3, CYLD and NFKBIA were all clonal in rNPC; however, 55.6-57.9% of them were clonal in pNPC (P < 0.05). In general, the number of clonal mutations in NF-κB pathway-associated genes was significantly higher in rNPC than in pNPC (P < 0.05). Moreover, the nuclear abundance of NF-κB protein was significantly greater in pNPC samples with local regional relapse than in those without relapse (P = 0.006). Further, in both the pNPC training and validation cohorts, high nuclear NF-κB levels were an independent negative prognostic marker for locoregional relapse-free survival (LRRFS) in pNPC. Accordingly, concurrent chemotherapy for patients with high nuclear NF-κB reduced the rate of locoregional relapse. Lastly, we functionally validated that inhibition of NF-κB enhanced both radiosensitivity and chemosensitivity in NPC cells, strongly supporting our in vivo results. Conclusion: NF-κB pathway activation by clonal mutations plays an important role in promoting the recurrence of NPC. Moreover, nuclear accumulation of NF-κB is a prominent biomarker for predicting LRRFS and suggesting treatment for pNPC patients. Finally, blocking NF-κB activity may be a novel and effective therapeutic modality for treating NPC. Citation Format: Rui You, You-Ping Liu, Ming-Yuan Chen. NF-κB pathway activation by clonal mutations contributes to the recurrence of nasopharyngeal carcinoma [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 737.

Protective effects of hydrogen inhalation during the warm ischemia phase against lung ischemia-reperfusion injury in rat donors after cardiac death

BackgroundSuccessful amelioration of long-term warm ischemia lung injury in donors after cardiac death (DCDs) can remarkably improve outcomes. Hydrogen gas provides potent anti-inflammatory and antioxidant effects against ischemia-reperfusion injury (IRI). This study observed the effects of hydrogen inhalation on lung grafts during the warm ischemia phase in cardiac death donors. MethodsAfter cardiac death, rat donor lungs (n = 8) underwent mechanical ventilation with 40% oxygen plus 60% nitrogen (control group) or 3% hydrogen and 40% oxygen plus 57% nitrogen (hydrogen group) for 2 h during the warm ischemia phase in situ. Then, lung transplantation was performed after 2 h of cold storage and 3 h of recipient reperfusion prior to lung graft assessment. Rats that underwent left thoracotomy without transplantation served as the sham group (n = 8). The results of static compliance and arterial blood gas analysis were assessed in the recipients. The wet-to-dry weight ratio (W/D), inflammation, oxidative stress, cell apoptosis and histologic changes were evaluated after 3 h of reperfusion. Nuclear factor kappa B (NF-κB) protein expression in the graft was analyzed by Western blotting. ResultsCompared with the sham group, lung function, W/D, inflammatory reaction, oxidative stress and histological changes were decreased in both transplant groups (control and hydrogen groups). However, compared with the control group, exposure to 3% hydrogen significantly improved lung graft static compliance and oxygenation and remarkably decreased the wet-to-dry weight ratio, inflammatory reactions, and lipid peroxidation. Furthermore, hydrogen improved the lung graft histological changes, decreased the lung injury score and apoptotic index and reduced NF-κB nuclear accumulation in the lung grafts. ConclusionLung inhalation with 3% hydrogen during the warm ischemia phase attenuated lung graft IRI via NF-κB-dependent anti-inflammatory and antioxidative effects in rat donors after cardiac death.

Abstract P2-06-12: Oncogenic potential of Trefoil factor 3 in initiation of mammary carcinoma through suppression of p53 pathway

Abstract Background Oncogenic transformation is a complex multistep process where normal cells acquire the hallmarks of cancer, leading to unrestrained outgrowth of malignant clones. Trefoil Factor 3 (TFF3) is a clinically validated and functionally potent oncogene in mammary carcinoma. Elevated TFF3 expression has been consistently observed in mammary carcinoma, being involved in cancer progression. The present study investigates the potential functional role and the underlying mechanisms of TFF3 in promoting oncogenic transformation early in the onset of mammary carcinoma. Material and method Immortalized human mammary epithelial cells (HMECs): HMEC-hTERT, MCF10A and MCF12A, with forced expression of TFF3, were used as in vitro models and in an orthotopic xenograft model to study the oncogenic roles of TFF3. Furthermore, microarray analysis, immunofluorescence, and ubiquitination and CHX chase assays were used to examine the involvement of p53 pathway in TFF3 mediated-oncogenic transformation. Results Immortalized HMECs with forced expression of TFF3 exhibited the capacity of anchorage independent growth in the soft agar colony formation assay, which is a hallmark of oncogenic transformation. The forced expression of TFF3 also enhanced 3D growth of the immortalized HMECs in matrigel. Furthermore, immortalized HMECs with forced expression of TFF3 gaverise to orthotopic xenograft tumors in nude mice, which are not observed in mice injected with immortalized HMECs. These observations suggest that TFF3 stimulates the oncogenic transformation of non-malignant immortalized HMECs. In addition, the forced expression of TFF3 promoted aberrant cell proliferation, resistance to apoptosis, and increased cell migration and invasion of the HMECs, all these being important hallmarks of cancer. Here, we showed that TFF3-mediated oncogenic transformation of the immortalized HMEC-hTERT cells is dependent on p53 signaling pathway suppression. Mechanistically, TFF3 downregulated NF-κB (p65)-mediated transcription of p53 through decreasing NF-κB (p65) expression and nuclear accumulation. TFF3 also decreased p53 protein levels through post-transcriptional regulation. The forced expression of TFF3 increased MDM2 expression, resulting in an increased ubiquitin-mediated proteasomal degradation of p53. Moreover, forced expression of TFF3 decreased the cleaved form of MDM2, which is responsible for stabilizing p53 protein. Concordantly, HMECs with forced expression of TFF3 exhibited shorter p53 protein half-life as compared to vector control HMECs . Conclusion In summary, our study highlights the oncogenic potential of TFF3 in the initiation of mammary carcinoma through the suppression of the p53 pathway. Citation Format: Chen RM, Pandey V, Chong QY, Poh HM, Zhang MY, Kumar AP, Lobie PE. Oncogenic potential of Trefoil factor 3 in initiation of mammary carcinoma through suppression of p53 pathway [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-12.

Signal Distortion: How Intracellular Pathogens Alter Host Cell Fate by Modulating NF-κB Dynamics.

By uncovering complex dynamics in the expression or localization of transcriptional regulators in single cells that were otherwise hidden at the population level, live cell imaging has transformed our understanding of how cells sense and orchestrate appropriate responses to changes in their internal state or extracellular environment. This has proved particularly true for the nuclear factor-kappaB (NF-κB) family of transcription factors, key regulators of the inflammatory response and innate immune function, which are capable of encoding information about the mode and intensity of stimuli in the dynamics of NF-κB nuclear accumulation and loss. While live cell imaging continues to serve as a useful tool in ongoing efforts to characterize the feedbacks that shape these dynamics and to connect dynamics to downstream gene expression, it is also proving invaluable for recent studies that seek to determine how intracellular pathogens subvert NF-κB signaling to survive and replicate within host cells by providing quantitative information about the pathogen and changes in NF-κB activity during different stages of an infection. Here, we provide a brief overview of NF-κB signaling in innate immune cells and review recent literature that uses live imaging to investigate the mechanisms by which bacterial and yeast pathogens modulate NF-κB in a variety of different host cell types to evade destruction or maintain the viability of an intracellular growth niche.

Betulinic acid inhibits the migration and invasion of fibroblast-like synoviocytes from patients with rheumatoid arthritis

The aggressive phenotype displayed by fibroblast-like synoviocytes (FLSs) contributes to cartilage and bone destruction in rheumatoid arthritis (RA). Betulinic acid has been demonstrated to have a positive therapeutic effect on tumor, inflammation and immune disorder, however, the effects of betulinic acid on RA FLSs have not been verified. Therefore, in the present study, we observed the effect of betulinic acid on the migration and invasion of RA FLSs and explored its underlying signal mechanisms. Our results showed that betulinic acid treatment suppressed the migration, invasion and reorganization of the actin cytoskeleton of RA FLSs. In addition, we found that the mRNA expression of IL-1β, IL-6, IL-8 and IL-17A were markedly down-regulated by treatment with betulinic acid in TNF-α-induced RA FLSs. To gain insight into the molecular mechanisms, we evaluated the effect of betulinic acid on NF-κB activation in RA FLSs. The results indicated that betulinic acid treatment reduced the TNF-α-induced activation of NF-κB signal pathway and the NF-κB nuclear accumulation. We also observed that treatment with betulinic acid attenuated synovial inflammation and joint destruction in mice with CIA. Taken together, these results suggest that betulinic acid inhibits the migration and invasion of RA FLSs by blocking NF-κB signal pathway activation.

Scutellarin alleviates blood-retina-barrier oxidative stress injury initiated by activated microglia cells during the development of diabetic retinopathy

The breakdown of blood-retinal barrier (BRB) is an early and typical event during the development of diabetic retinopathy (DR). Scutellarin (SC) is a natural flavonoid. This study aims to investigate the protection of SC from BRB damage via focusing on inhibiting microglia-initiated inflammation and subsequent oxidative stress injury. SC attenuated BRB breakdown and the reduced expression of claudin-1 and claudin-19 in STZ-induced diabetic mice. SC reduced microglia cells activation both in vivo and in vitro. The results of transendothelial/transepithelial electrical resistance (TEER/TER) and fluorescein isothiocyanate (FITC)-conjugated dextran cell permeability assay showed that SC attenuated BRB damage induced by d-glucose (25 mM)-stimulated microglia BV2 cells. SC suppressed nuclear factor κB (NFκB) activation and tumor necrosis factor (TNF)-α expression induced by d-glucose (25 mM) in BV2 cells. SC decreased the phosphorylation of extracellular regulated protein kinase (ERK)1/2 both in vivo and in vitro. MEK1/2 inhibitor U0126 reduced the d-glucose-induced NFκB nuclear accumulation and TNFα expression in BV2 cells. Next, SC improved the decreased expression of claudin-1 and claudin-19, the increased BRB damage and cellular reactive oxygen species (ROS) formation, and enhanced nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) in TNFα-treated human retinal endothelial cells (HRECs) and APRE19 cells. Moreover, the SC-provided alleviation on BRB breakdown in STZ-induced diabetic mice was diminished in Nrf2 knock-out mice. In conclusion, SC alleviates BRB breakdown via abrogating retinal inflammatory responses and subsequent oxidative stress injury initiated by microglia cells that is activated by hyperglycemia during DR development.

The role of miRNAs 34a, 146a, 320a and 542 in the synergistic anticancer effects of methyl 2-(5-fluoro-2-hydroxyphenyl)-1H- benzo[d]imidazole-5-carboxylate (MBIC) with doxorubicin in breast cancer cells.

Combination Index (CI) analysis suggested that MBIC and doxorubicin synergistically inhibited up to 97% of cell proliferation in ER+/PR+MCF-7 and triple negative MDA-MB-231 breast cancer cell lines. Moreover, treatment of the breast cancer cells with the combined drugs resulted in lower IC50 values in contrast to the individual drug treatment. Small noncoding microRNAs (miRNA) may function as non-mutational gene regulators at post-transcriptional level of protein synthesis. In the present study, the effect of the combined treatment of MBIC and doxorubicin on the expression level of several miRNAs including miR-34a, miR-146a, miR-320a and miR-542 were evaluated in MCF-7 and MDA-MB-231 breast cancer cell lines. These miRNAs have the potential to alter the protein level of survivin, the anti-apoptotic protein and reduce the metastatic activity in human breast cancer cell lines by interfering with the nuclear accumulation of NF-κB. Our results demonstrated the several fold changes in expression of miRNAs, which is drug and cell line dependent. This finding demonstrated a functional synergistic network between miR-34a, miR-320a and miR-542 that are negatively involved in post-transcriptional regulation of survivin in MCF-7 cells. While in MDA-MB-231 cells, changes in expression level of miR-146a was correlated with inhibition of the nuclear translocation of NF-κB. The overall result suggested that alteration in protein level and location of survivin and NF-κB by miR-34a, miR-320a, miR-146a and miR-542, remarkably influenced the synergistic enhancement of combined MBIC and doxorubicin in treatment of aggressive and less aggressive human breast cancer cell lines.

A Rationally Designed Multifunctional Antibiotic for the Treatment of Drug-Resistant Acne

Acne is a multifactorial skin disease, underpinned by colonization of Propionibacterium acnes and inflammation. The emergence of resistant P.acnes strains has affected the current acne treatment algorithm. This setback served as an impetus for rationally designing a library of next-generation antibiotics that exhibit a bactericidal effect on resistant P.acnes and exert an immunomodulatory function to reduce inflammation. In silico screening showed that one of the molecules, VCD-004, exhibits improved mode of binding to bacterial DNA gyrase. VCD-004 shows high potency against clinical isolates of resistant P.acnes and excellent efficacy invivo. Furthermore, VCD-004 exhibits a superior mutant prevention index, suggesting that it impedes the development of resistance better than clindamycin. Additionally, it shows optimal skin penetration and has a potent anti-inflammatory effect via reduction of proinflammatory cytokines (IL-6) independent of its antibacterial action. VCD-004 affects P.acnes-induced nuclear accumulation of NF-κB in THP-1 cells. The invitro viability of human keratinocytes in the presence of VCD-004 indicates a desirable therapeutic window for topical use. Such rationally designed bactericidal and immunomodulatory dual pharmacophore-based lipophilic molecule(s) can emerge as the next-generation topical therapy for acne with underlying resistant P.acnes etiology.

Curcumin ameliorates the in vitro efficacy of carfilzomib in human multiple myeloma U266 cells targeting p53 and NF-κB pathways

Multiple myeloma (MM) is a malignant B-cell neoplasm with accumulation of malignant plasma cells in bone marrow. Pharmacological therapy improves response frequency even if with various associated toxicities. Herein, we investigated if combination of curcumin with carfilzomib (CFZ) can induce a better cytotoxic effect on in vitro cultured U266 cells. Cell viability data showed that curcumin significantly ameliorates CFZ cytotoxic effect. Furthermore, curcumin alone did not affect proteasome at the tested dose, confirming the involvement of different mechanisms in the observed effects. U266 cells exposure to curcumin or CFZ increased reactive species (RS) levels, although their production did not appear further potentiated following drugs combination. Interestingly, NF-κB nuclear accumulation was reduced by treatment with CFZ or curcumin, and was more deeply decreased in cells treated with CFZ-curcumin combinations, very likely due to the different mechanisms through which they target NF-κB. Our results confirmed the induction of p53/p21 axis and G0/G1 cell cycle arrest in anticancer activities of both drugs, an effect more pronounced for the CFZ-curcumin tested combinations. Furthermore, curcumin addition enhanced CFZ proapoptotic effect. These findings evidence that curcumin can ameliorate CFZ efficacy, and lead us to hypothesize that this effect might be useful to optimize CFZ therapy in MM patients.

Sphingosine kinase 1 mediates diabetic renal fibrosis via NF-κB signaling pathway: involvement of CK2α.

Sphingosine kinase 1 (SphK1) plays a pivotal role in regulating diabetic renal fibrotic factors such as fibronectin (FN) and intercellular adhesion molecule-1 (ICAM-1). Especially, activation of SphK1 is closely linked to the body inflammatory reaction. Casein kinase 2α subunit (CK2α), a protein kinase related to inflammatory reaction, influences diabetic renal fibrosis and expressions of FN and ICAM-1 via NF-κB pathway. However, the mechanism by which SphK1 mediates diabetic renal fibrosis has not yet fully elucidated. The current study is aimed to investigate if SphK1 mediates diabetic renal fibrotic pathological process via inflammatory pathway and activation of CK2α. The following findings were observed: (1) Expressions of SphK1 were upregulated in kidneys of diabetic mice and rats; (2) Knockdown of SphK1 expression suppressed high glucose (HG)-induced NF-κB nuclear translocation and expressions of FN and ICAM-1; (3) Compared with C57 diabetic mice, SphK1-/- diabetic mice exhibited less renal fibrotic lesions, FN accumulation and NF-κB nuclear accumulation in glomeruli of kidneys; (4) SphK1 mediated phosphorylation of CK2α, while CK2α knockdown depressed SphK1-induced activation of NF-κB pathway. This study indicates the essential role of SphK1 in regulating activation of CK2α and diabetic renal fibrotic pathological process via NF-κB.

Tert-buthylhydroquinone pre-conditioning exerts dual effects in old female rats exposed to 3-nitropropionic acid

The brain is a very susceptible organ to structural and functional alterations caused by oxidative stress and its vulnerability increases with age. Understanding the antioxidant response activated by the transcription factor Nrf2 has become very important in the aging field in order to activate cellular protection. However, the role of Nrf2 inducers during old age has not been completely understood. Our aim was to activate the Nrf2 pathway by pre-treating old rats with a widely used Nrf2-inducer, tert-buthylhydroquinone (tBHQ), prior to 3-nitropropionic acid (3-NP) insult, in order to evaluate its effects at a behavioral, morphological and biochemical levels. 3-NP has been used to reproduce the biochemical and pathophysiological characteristics of Huntington's disease due to an oxidative effect. Our results suggest that tBHQ confers an important protective effect against 3-NP toxicity; nevertheless, Nrf2 seems not to be the main protective pathway associated to neuroprotection. Hormetic responses include the activation of more than one transcription factor. Nrf2 and NFκB are known to simultaneously initiate different cellular responses against stress by triggering parallel mechanisms, therefore NFκB nuclear accumulation was also evaluated.

Therapeutic implication of ‘Iturin A’ for targeting MD-2/TLR4 complex to overcome angiogenesis and invasion

Tumor angiogenesis and invasion are deregulated biological processes that drive multistage transformation of tumors from a benign to a life-threatening malignant state activating multiple signaling pathways including MD-2/TLR4/NF-κB. Development of potential inhibitors of this signaling is emerging area for discovery of novel cancer therapeutics. In the current investigation, we identified Iturin A (A lipopeptide molecule from Bacillus megaterium) as a potent inhibitor of angiogenesis and cancer invasion by various in vitro and in vivo methods. Iturin A was found to suppress VEGF, a powerful inducer of angiogenesis and key player in tumor invasion, as confirmed by ELISA, western blot and real time PCR. Iturin A inhibited endothelial tube arrangement, blood capillary formation, endothelial sprouting and vascular growth inside the matrigel. In addition, Iturin A inhibited MMP-2/9 expression in MDA-MB-231 and HUVEC cells. Cancer invasion, migration and colony forming ability were significantly hampered by Iturin A. Expressions of MD-2/TLR4 and its downstream MyD88, IKK-α and NF-κB were also reduced in treated MDA-MB-231 and HUVEC cells. Western blot and immunofluorescence study showed that nuclear accumulation of NF-κB was hampered by Iturin A. MD-2 siRNA or plasmid further confirmed the efficacy of Iturin A by suppressing MD-2/TLR4 signaling pathway. The in silico docking study showed that the Iturin A interacted well with the MD-2 in MD-2/TLR4 receptor complex. Conclusively, inhibition of MD-2/TLR4 complex with Iturin A offered strategic advancement in cancer therapy.