Decreased NF-κB Activation Research Articles

Abstract LB568: Protein phosphatase 4 deficiency protects mice against cerulein-induced acute pancreatitis

Abstract Background: Pancreatitis is an inflammatory disease of the pancreas that causes significant morbidity and mortality and is one of the major risk factors for pancreatic cancer. Protein phosphatase 4 (PP4) has been reported to be overexpressed in breast, lung and pancreatic cancers. PP4 plays an important role in the regulation of centrosome maturation, DNA repair, NF-κB, and c-jun-NH2-kinase (JNK) signaling pathways. However, the expression and functions of PP4 in pancreatitis have not been studied. Methods: To examine the functions of PP4 in cerulein-induced acute pancreatitis (AP), we crossed PP4flox/+ mice with pancreatic acinar cell specific cre, CreERT, which was driven by a full length pancreatic acinar specific elastase I promoter. PP4flox/flox, CreERT mice and control mice were treated with 8 hourly intraperitoneal injections of cerulein (50 µg/kg/h). The pancreata were harvested at different time points for histopathological examination and molecular analysis. Results: PP4 expression was increased at both protein and mRNA levels in mouse pancreas with AP. Compared to CreERT mice, PP4flox/flox, CreERT mice demonstrated significantly less edema, less inflammation, less necrosis or loss of pancreatic acinar cells in pancreas after cerulein treatment. The serum amylase and lipase levels were lower in cerulein-treated PP4flox/flox, CreERT mice compared with controls. The pancreata of PP4flox/flox, CreERT mice exhibited decreased STAT3 Tyr705 phosphorylation and decreased NF-κB activation cerulein-treated PP4flox/flox, CreERT mice than control mice. Conclusion: Our findings demonstrated that PP4 plays an important role in cerulein-induced AP through STAT3 and NF-κB signaling pathways. Inhibition of PP4 may help to reduce the pancreatic injuries and to prevent the development of chronic pancreatitis, which increases the risk of pancreatic cancer. Citation Format: Hua Wang, Reza Abbasgholizadeh, Rohan Moniruzzaman, Baoan Ji, Anirban Maitra, Huamin Wang. Protein phosphatase 4 deficiency protects mice against cerulein-induced acute pancreatitis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB568.

The link between vitamin D status and NF-κB-associated renal dysfunction in experimental diabetes mellitus

BackgroundType 1 diabetes (T1D) is accompanied by numerous side effects, including renal dysfunction. Mounting evidence suggests that overactivation of nuclear factor ĸB (NF-κB) is one of the key triggers of diabetes-associated chronic kidney disease. Vitamin D3 is considered as a strong modulator of a number of transcription factors, including NF-κB. The purpose of our study was to assess the contribution of NF-κB to type 1 diabetes (T1D)-induced kidney dysfunction and to determine if vitamin D3 supplementation can correct the changes associated with T1D. MethodsThe following animal groups were used: control, diabetic (induced by single i.p. injection of streptozocin at dose 55 mg/kg b.w.), T1D group treated with vitamin D3 (600 IU/kg b.w.), T1D group treated with NF-κB inhibitor – BAY 11‐7082. ResultsDiabetes led to a decrease in serum 25(OH)D that was accompanied by down-regulation of vitamin D receptor (VDR) expression and up-regulation of hydroxylases CYP24A1 and CYP27B1 synthesis in the kidneys. Diabetes activated the transcription factor NF-κB and increased cleaved (p17) caspase-3 level in renal tissue. Restoration of vitamin D status normalized vitamin D-endocrine system, decreased NF-κB activation and caspase-3 protein level in the kidneys of diabetic animals. BAY 11–7082 partially mimicked the effects of vitamin D3. General significanceVitamin D3 supplementation counteracts diabetes-induced kidney damage, most likely through VDR-mediated inhibition of NF-κB activation.

Inclisiran inhibits oxidized low-density lipoprotein-induced foam cell formation in Raw264.7 macrophages via activating the PPARγ pathway

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a well-known proprotein convertase that influences foam cell formation and modulates atherosclerosis. Inclisiran is a novel chemosynthetic small interfering RNA that inhibits PCSK9 synthesis. This study aimed to explore the effect of inclisiran on oxidized low-density lipoprotein (ox-LDL)-induced foam cell formation in Raw264.7 macrophages and to investigate the underlying mechanisms. Raw264.7 cells were treated with ox-LDL to induce the formation of macrophage-derived foam cells. Oil Red O staining and high-performance liquid chromatography were performed to detect lipid accumulation and cholesterol levels. Dil-ox-LDL uptake assay, CCK-8, RT-qPCR, and Western blotting analysis were performed to examine ox-LDL uptake, cell viability, and expression of scavenger receptor-related factors. Inclisiran reduced lipid accumulation in ox-LDL-treated macrophages in a dose-dependent manner. Inclisiran significantly inhibited the levels of total cholesterol, free cholesterol, and cholesterol ester in the supernatant of Raw264.7 cells. Inclisiran reduced ox-LDL uptake and increased Raw264.7 cell viability. Meanwhile, inclisiran downregulated the expression of SR-A, LOX-1, and CD36 and upregulated SR-BI, ApoE, and ABCA1. Furthermore, inclisiran increased PPARγ activity and decreased NF-κB activity. An inhibitor of PPARγ (T0070907) reversed the beneficial effects of inclisiran on ox-LDL uptake, NF-κB inactivation, and cytokine expression. In conclusion, these data suggested that inclisiran inhibited the formation of macrophage-derived foam cells by activating the PPARγ pathway. Highlights Inclisiran reduces lipid accumulation in Raw264.7 cells; Inclisiran reduces ox-LDL uptake and increases Raw264.7 cell viability; Inclisiran inhibits foam cell formation by activating the PPARγ pathway.

The phytoactive constituents of Eugenia selloi B.D. Jacks (pitangatuba): Toxicity and elucidation of their anti-inflammatory mechanism(s) of action

We determined the phytochemical composition, anti-inflammatory mechanism of action, ROS/RNS scavenging capacity and systemic toxicity of a purified subfraction (S8) of Eugenia selloi. The composition of S8 was assessed by LC-ESI-QTOF-MS; the anti-inflammatory activity in RAW264.7 macrophages through NF-κB activation and biomarkers by multiplex in THP-1 cells; neutrophil migration, intravital microscopy and ICAM-1 expression in mice; NETs formation and CD11b expression; S8 scavenging capacity of ROS/RNS; toxicity in Galleria mellonella larvae model. Coumaric acid, quercetrin and vanillic acid were identified. S8 decreased NF-κB activation, IL-1β, IL-6, IL-10, MDC and MCP-1 levels, reduced neutrophil migration and ICAM-1 expression in mice; S8 did not interfere NET formation and CD11b expression, exhibited high antioxidant and showed negligible toxicity. E. selloi proved to be a promising, yet underexplored source of bioactive compounds, which can be useful employed in agribusiness and in the pharmaceutical and food industry to develop new products or human health supplies.

MiR-27b inhibition contributes to cytotoxicity in patulin-exposed HEK293 cells

Patulin (PAT) is a mycotoxin produced by Penicillium and other fungi that contaminate fruit. PAT targets the kidney and is associated with nephrotoxicity. Micro-RNAs (miRNA) may offer new insights into PAT-induced nephrotoxicity. Cytochrome P450 family 1, subfamily B, polypeptide 1 (CYP1B1), involved in metabolism of dietary toxins is negatively regulated by miR-27b and linked with the nuclear factor kappa B (NF-κB) pathway and peroxisome proliferator activated receptor gamma (PPARɣ) in renal fibrosis. This study investigated the effects of PAT on miR-27b, CYP1B1, PPARɣ and cytotoxicity in human kidney (HEK293) cells.HEK293 cells were exposed to PAT (2.5 μM, 24h). Protein expression of CYP1B1, PPARɣ, NF-κB (p65), pNF-κB (p65) (phospho-Ser563) and cleaved PARP-1 was quantified using western blotting. QPCR evaluated mRNA levels of CYP1B1, IL-6, miR-27b, OGG1, mtDNA, TFAM and UCP2. Mitochondrial membrane potential and phosphatidylserine (PS) externalization was evaluated by flow cytometry while levels of ATP and caspase −9, −8, −3/7 activity was measured using luminometry.PAT significantly decreased miR-27b levels (p = 0.0014) and increased CYP1B1 mRNA (p = 0.0015) and protein (p = 0.0013) levels. PPARɣ protein expression was significantly increased (p = 0.0002) and associated with decreased NF-κB activation (p = 0.0273) and IL-6 mRNA levels (p = 0.0265). Finally, PAT significantly compromised mitochondrial repair mechanisms and increased apoptotic biomarkers.PAT altered miR-27b levels and PPARɣ, with associated changes to NF-κB activation, downstream IL-6 and CYP1B1 expression. These results show that PAT impairs detoxification mechanisms leading to mitochondrial damage and apoptosis. In conclusion, PAT altered the epigenetic environment and impaired detoxification processes, supporting a mechanism for nephrotoxic outcomes.

Bone Marrow-Derived RIPK3 Mediates Kidney Inflammation in Acute Kidney Injury.

Receptor-interacting protein kinase 3 (RIPK3), a component of necroptosis pathways, may have an independent role in inflammation. It has been unclear which RIPK3-expressing cells are responsible for the anti-inflammatory effect of overall Ripk3 deficiency and whether Ripk3 deficiency protects against kidney inflammation occurring in the absence of tubular cell death. We used chimeric mice with bone marrow from wild-type and Ripk3-knockout mice to explore RIPK3's contribution to kidney inflammation in the presence of folic acid-induced acute kidney injury AKI (FA-AKI) or absence of AKI and kidney cell death (as seen in systemic administration of the cytokine TNF-like weak inducer of apoptosis [TWEAK]). Tubular and interstitial cell RIPK3 expressions were increased in murine AKI. Ripk3 deficiency decreased NF-κB activation and kidney inflammation in FA-AKI but did not prevent kidney failure. In the chimeric mice, RIPK3-expressing bone marrow-derived cells were required for early inflammation in FA-AKI. The NLRP3 inflammasome was not involved in RIPK3's proinflammatory effect. Systemic TWEAK administration induced kidney inflammation in wild-type but not Ripk3-deficient mice. In cell cultures, TWEAK increased RIPK3 expression in bone marrow-derived macrophages and tubular cells. RIPK3 mediated TWEAK-induced NF-κB activation and inflammatory responses in bone marrow-derived macrophages and dendritic cells and in Jurkat T cells; however, in tubular cells, RIPK3 mediated only TWEAK-induced Il-6 expression. Furthermore, conditioned media from TWEAK-exposed wild-type macrophages, but not from Ripk3-deficient macrophages, promoted proinflammatory responses in cultured tubular cells. RIPK3 mediates kidney inflammation independently from tubular cell death. Specific targeting of bone marrow-derived RIPK3 may limit kidney inflammation without the potential adverse effects of systemic RIPK3 targeting.

Activation of α7nAChR Protects Against Gastric Inflammation and Dysmotility in Parkinson's Disease Rats.

The cholinergic anti-inflammatory pathway (CAIP) has been proposed to regulate gastrointestinal inflammation via acetylcholine released from the vagus nerve activating α7 nicotinic receptor (α7nAChR) on macrophages. Parkinson’s disease (PD) patients and PD rats with substantia nigra (SN) lesions exhibit gastroparesis and a decayed vagal pathway. To investigate whether activating α7nAChR could ameliorate inflammation and gastric dysmotility in PD rats, ELISA, western blot analysis, and real-time PCR were used to detect gastric inflammation. In vitro and in vivo gastric motility was investigated. Proinflammatory mediator levels and macrophage numbers were increased in the gastric muscularis of PD rats. α7nAChR was located on the gastric muscular macrophages of PD rats. The α7nAChR agonists PNU-282987 and GTS-21 decreased nuclear factor κB (NF-κB) activation and monocyte chemotactic protein-1 mRNA expression in the ex vivo gastric muscularis of PD rats, and these effects were abolished by an α7nAChR antagonist. After treatment with PNU-282987 in vivo, the PD rats showed decreased NF-κB activation, inflammatory mediator production, and contractile protein expression and improved gastric motility. The present study reveals that α7nAChR is involved in the development of gastroparesis in PD rats and provides novel insight for the treatment of gastric dysmotility in PD patients.

Incorporation of a Nitric Oxide Donating Motif into Novel PC-PLC Inhibitors Provides Enhanced Anti-Proliferative Activity.

Inhibition of phosphatidylcholine-specific phospholipase C (PC-PLC) has previously been shown to be a potential target for novel cancer therapeutics. One downstream consequence of PC-PLC activity is the activation of NF-κB, a nuclear transcription factor responsible for transcribing genes related to oncogenic traits, such as proliferation, angiogenesis, metastasis, and cancer cell survival. Another biological pathway linked to NF-κB is the exogenous delivery of nitric oxide (NO), which decreases NF-κB activity through an apparent negative-feedback loop. In this study, we designed and synthesised 13 novel NO-releasing derivatives of our previously reported class of PC-PLC inhibitors, 2-morpholinobenzoic acids. These molecules contained a secondary benzylamine group, which was readily nitrosylated and subsequently confirmed to release NO in vitro using a DAF-FM fluorescence-based assay. It was then discovered that these NO-releasing derivatives possessed significantly improved anti-proliferative activity in both MDA-MB-231 and HCT116 cancer cell lines compared to their non-nitrosylated parent compounds. These results confirmed that the inclusion of an exogenous NO-releasing functional group onto a known PC-PLC inhibitor enhances anti-proliferative activity and that this relationship can be exploited in order to further improve the anti-proliferative activity of current/future PC-PLC inhibitors.

Heat Shock Protein 27, a Novel Downstream Target of Collagen Type XI alpha 1, Synergizes with Fatty Acid Oxidation to Confer Cisplatin Resistance in Ovarian Cancer Cells.

Simple SummaryCollagen type XI alpha 1 (COL11A1) is a novel biomarker associated with poor survival in ovarian cancer and a promoter of ovarian cancer cell resistance to cisplatin. However, it is poorly understood how COL11A1 promotes ovarian cancer cisplatin resistance. We performed assays to discover the biological molecules that are activated by COL11A1 in ovarian cancer cells. We found that heat shock protein 27 (HSP27), a cellular stress response protein, is activated by COL11A1. Furthermore, we observed that depletion and drug inhibition of HSP27 makes ovarian cancer cells grown on COL11A1 to be more susceptible to cisplatin treatment. We also discovered that ovarian cancer cells upregulate fatty acid oxidation (FAO), a metabolic process that breaks down fats to generate energy and biomolecules, to compensate for the loss of HSP27. Our findings have therapeutic implications for clinicians who wish to treat ovarian tumors that maintain high levels of COL11A1 and HSP27.Collagen type XI alpha 1 (COL11A1) is a novel biomarker associated with cisplatin resistance in ovarian cancer. We have previously reported that COL11A1 activates Src-Akt signaling through the collagen receptors discoidin domain receptor 2 (DDR2) and integrin α1β1 to confer cisplatin resistance to ovarian cancer cells. To identify the potential signaling molecules downstream of COL11A1 signaling, we performed protein kinase arrays and identified heat shock protein 27 (HSP27) as a potential mediator of COL11A1-induced cisplatin resistance. Through receptor knockdown and inhibitor experiments, we demonstrated that COL11A1 significantly upregulates HSP27 phosphorylation and expression via DDR2/integrin α1β1 and Src/Akt signaling in ovarian cancer cells. Furthermore, genetic knockdown and pharmacological inhibition of HSP27, via ivermectin treatment, significantly sensitizes ovarian cancer cells cultured on COL11A1 to cisplatin treatment. HSP27 knockdown or inhibition also decreases NFκB activity as well as the expression of inhibitors of apoptosis proteins (IAPs), which are known downstream effector molecules of COL11A1 that promote cisplatin resistance. Interestingly, HSP27 knockdown or inhibition stimulates ovarian cancer cells to upregulate fatty acid oxidation (FAO) for survival and cisplatin resistance, and dual inhibition of HSP27 and FAO synergistically kills ovarian cancer cells that are cultured on COL11A1. Collectively, this study identifies HSP27 as a novel and druggable COL11A1 downstream effector molecule that may be targeted to overcome cisplatin resistance in recurrent ovarian cancer, which often overexpress COL11A1.

Disruption of endothelial Pfkfb3 ameliorates diet-induced murine insulin resistance.

Overnutrition-induced endothelial inflammation plays a crucial role in high-fat diet (HFD)-induced insulin resistance in animals. Endothelial glycolysis plays a critical role in endothelial inflammation and proliferation, but its role in diet-induced endothelial inflammation and subsequent insulin resistance has not been elucidated. PFKFB3 is a critical glycolytic regulator, and its increased expression has been observed in adipose vascular endothelium of C57BL/6J mice fed with HFD in vivo, and in palmitate (PA)-treated primary human adipose microvascular endothelial cells (HAMECs) in vitro. We generated mice with Pfkfb3 deficiency selective for endothelial cells to examine the effect of endothelial Pfkfb3 in endothelial inflammation in metabolic organs and in the development of HFD-induced insulin resistance. EC Pfkfb3-deficientmice exhibited mitigated HFD-induced insulin resistance, including decreased body weight and fat mass, improved glucose clearance and insulin sensitivity, and alleviated adiposity and hepatic steatosis. Mechanistically, cultured PFKFB3 knockdown HAMECs showed decreased NF-κB activation induced by PA, and consequent suppressed adhesion molecule expression and monocyte adhesion. Taken together, these results demonstrate that increased endothelial PFKFB3 expression promotes diet-induced inflammatory responses and subsequent insulin resistance, suggesting that endothelial metabolic alteration plays an important role in the development of insulin resistance.

IRE1 and PERK signaling regulates inflammatory responses in a murine model of contact hypersensitivity.

Contact sensitizers may interfere with correct protein folding. Generation of un-/misfolded proteins can activate the IRE-1 or PERK signaling pathways initiating the unfolded protein response (UPR) and thereby determine inflammatory immune responses. We have analyzed the effect of sensitizers with different potencies on the induction of UPR activation/inhibition and the subsequent generation of a pro-inflammatory micromilieu in vitro as well as the effect of UPR modulation on the inflammatory response in the murine contact hypersensitivity (CHS) in vivo. Semi-quantitative and quantitative PCR, fluorescence microscopy, ELISA, NF-κB activation and translocation assays, DC/keratinocyte co-culture assay, FACS, and in vivo CHS experiments were performed. Sensitizers and irritants activate IRE-1 and PERK in murine and human keratinocytes. Synergistic effects occur after combination of different weak sensitizers / addition of irritants. Moreover, tolerogenic dinitrothiocyanobenzene can be converted into a strong sensitizer by pre-activation of the UPR. Blocking UPR signaling results in decreased NF-κB activation and cytokine production in keratinocytes and in activation marker downregulation in a HaCaT/THP-1 co-culture. Interestingly, not only systemic but also topical application of UPR inhibitors abrogates CHS responses in vivo. These observations highlight an important role of the UPR in determination of the inflammatory response in vitro and in vivo further underlining the importance of tissue stress and damage responses in the development of ACD and provide mechanistically based concepts as a basis for the development of new therapeutic approaches to treat allergic contact dermatitis.

Mechanism of preservation of the intestinal mucosa architecture and NF-κB/PGE2 reduction by hydrogen sulfide on cholera toxin-induced diarrhea in mice

AimsInvestigate the involvement of Hydrogen sulfide (H2S) in inflammatory parameters and intestinal morphology caused by cholera toxin (CT) in mice. Main methodsMice were subjected to the procedure of inducing diarrhea by CT in the isolated intestinal loop model. The intestinal loops were inoculated with H2S donor molecules (NaHS and GYY 4137) or saline and CT. To study the role of EP2 and EP4 prostaglandin E2 (PGE2) receptors in the H2S antisecretory effect, PAG (DL-propargylglycine - inhibitor of cystathionine-γ-lyase (CSE)), PF-04418948 (EP2 antagonist) and ONO-AE3-208 (EP4 antagonist) were used. The intestinal loops were evaluated for intestinal secretion, relation of the depth of villi and intestinal crypts, and real-time PCR for the mRNA of the CXCL2, IL-6, NOS-2, IL-17, NF-κB1, NF-κBIA, SLC6A4 and IFN-γ genes. Key findingsH2S restored the villus/crypt depth ratio caused by CT. NaHS and GYY 4137 increased the expression of NF-κB1 and for the NF-κBIA gene, only GYY 4137 increased the expression of this gene. The increased expression of NF-κB inhibitors, NF-κB1 and NF-κBIA by H2S indicates a possible decrease in NF-κB activity. The pretreatment with PAG reversed the protective effect of PF-04418948 and ONO-AE3-208, indicating that H2S probably decreases PGE2 because in the presence of antagonists of this pathway, PAG promotes intestinal secretion. SignificanceOur results point to a protective activity of H2S against CT for promoting a protection of villus and crypt intestine morphology and also that its mechanism occurs at least in part due to decreasing the activity of NF-κB and PGE2.

MCPIP1 Enhances TNF-α-Mediated Apoptosis through Downregulation of the NF-κB/cFLIP Axis

Simple SummaryTNF-α is considered to be a potential therapeutic drug for cancer, but the systemic toxicity problem still exists in clinical use. MCPIP1 plays a crucial role in anti-inflammatory and anti-viral contexts, as well as in the inhibition of cell growth. This study aims to investigate the roles of MCPIP1 in TNF-α-treated cells and their underlying molecular mechanisms. MCPIP1 was found to enhance TNF-α-induced apoptosis by activating the caspase cascade, and pan-caspase inhibitor and the caspase-1/-4 inhibitor could reverse TNF-α/MCPIP1-mediated apoptosis. MCPIP1 was first identified with cleavage site of caspase-1/-4 and could be cleaved during apoptosis. Downregulation of NF-κB activation and a caspase-8 inhibitor, cFLIP, were associated with TNF-α/MCPIP1-mediated apoptosis.Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) is rapidly produced under proinflammatory stimuli, thereby feeding back to downregulate excessive inflammation. In this study, we used the stable, inducible expressions of wild-type (WT) MCPIP1 and an MCPIP1-D141N mutant in T-REx-293 cells by means of a tetracycline on (Tet-on) system. We found that WT MCPIP1 but not MCPIP1-D141N mutant expression dramatically increased apoptosis, caspase-3, -7, -8, and -9 activation, and c-Jun N-terminal kinase (JNK) phosphorylation in TNF-α-treated cells. The pan-caspase inhibitor, z-VAD-fmk, and the caspase-1 inhibitor, z-YVAD-fmk, but not the JNK inhibitor, SP600125, significantly reversed apoptosis and caspase activation in TNF-α/MCPIP1-treated cells. Surprisingly, MCPIP1 itself was also cleaved, and the cleavage was suppressed by treatment with the pan-caspase inhibitor and caspase-1 inhibitor. Moreover, MCPIP1 was found to contain a caspase-1/-4 consensus recognition sequence located in residues 234~238. As expected, the WT MCPIP1 but not the MCPIP1-D141N mutant suppressed NF-κB activation, as evidenced by inhibition of IκB kinase (IKK) phosphorylation and IκB degradation using Western blotting, IKK activity using in vitro kinase activity, and NF-κB translocation to nuclei using an immunofluorescence assay. Interestingly, MCPIP1 also significantly inhibited importin α3 and importin α4 expressions, which are major nuclear transporter receptors for NF-κB. Inhibition of NF-κB activation further downregulated expression of the caspase-8 inhibitor, cFLIP. In summary, the results suggest that MCPIP1 could enhance the TNF-α-induced apoptotic pathway through decreasing NF-κB activation and cFLIP expression.

NF-κB and neutrophil extracellular traps cooperate to promote breast cancer progression and metastasis

Aberrant NF-κB activation and neutrophil extracellular traps (NETs) are associated with breast cancer progression. How NF-κB and NETs modulate each other in breast cancer development remains unclear. Here, we found that NETs induced by phorbol 12-myristate 13-acetate promote breast cancer cell progression. In turn, cancer cells–derived factors, such as IL-8 and granulocyte colony-stimulating factor, stimulate neutrophils to form NETs. Mechanistically, NETs increased the interaction of NF-κB essential modifier (NEMO) with IκB kinase (IKK)α/β and enhanced NF-κB activation. We then employed a cell-permeable peptide corresponding to the NEMO-binding domain (NBD) of IKKα/β, termed NBD peptide, which disrupts NETs-mediated NEMO interaction with IKKα/β and abolished NF-κB activation in vitro. NBD peptide also reduced IL-8 level and NETs formation, and suppressed primary tumor growth and/or lung metastasis in human breast cancer mouse xenograft models and mouse spontaneous breast cancer model. Blockade of NET formation using a peptidylarginine deiminase 4 (PAD4) pharmacologic inhibitor decreased NF-κB activation and tumor metastasis. Collectively, these data suggest that NF-κB associates with NETs to form a positive loop facilitating breast tumor progression and metastasis, and that selective inhibition of NF-κB and PAD4-dependent NETs provides an effective therapeutic approach for treating breast cancer.

Quorum Sensing and NF-κB Inhibition of Synthetic Coumaperine Derivatives from Piper nigrum.

Bacterial communication, termed Quorum Sensing (QS), is a promising target for virulence attenuation and the treatment of bacterial infections. Infections cause inflammation, a process regulated by a number of cellular factors, including the transcription Nuclear Factor kappa B (NF-κB); this factor is found to be upregulated in many inflammatory diseases, including those induced by bacterial infection. In this study, we tested 32 synthetic derivatives of coumaperine (CP), a known natural compound found in pepper (Piper nigrum), for Quorum Sensing Inhibition (QSI) and NF-κB inhibitory activities. Of the compounds tested, seven were found to have high QSI activity, three inhibited bacterial growth and five inhibited NF-κB. In addition, some of the CP compounds were active in more than one test. For example, compounds CP-286, CP-215 and CP-158 were not cytotoxic, inhibited NF-κB activation and QS but did not show antibacterial activity. CP-154 inhibited QS, decreased NF-κB activation and inhibited bacterial growth. Our results indicate that these synthetic molecules may provide a basis for further development of novel therapeutic agents against bacterial infections.

The nuclear retinoid-related orphan receptor RORα controls adipose tissue inflammation in patients with morbid obesity and diabetes.

Inflammation governs adipose tissue (AT) dysfunction in obesity. Retinoic acid receptor-related orphan receptor alpha (RORα) is associated with inflammation and insulin resistance in animal studies, but its role in human obesity remains elusive. We investigated the expression and function of RORα on AT inflammation in patients with morbid obesity with/without diabetes. We assessed RORα expression in paired biopsies of subcutaneous and omental AT from 41 patients (body mass index (BMI) 43.3 ± 0.8 kg/m2) during Roux-en-Y-gastric surgery and explored the functional consequences of pharmacological RORα blockade in AT ex vivo. RORα expression was significantly higher in omental AT than in subcutaneous AT (p = 0.03) and was positively associated with BMI (r = 0.344, p = 0.027) and homeostasis model assessment of insulin resistance (r = 0.319, p = 0.041). In ex vivo assays, IL-8/CXCL8 and MCP-1/CCL2 chemokine release was significantly higher in omental fat explants from diabetic patients than from non-diabetics and was significantly diminished by RORα blockade (p < 0.05). Inhibition of RORα improved protein kinase B signaling and decreased NF-κB activity in omental AT from patients with diabetes (p < 0.05). Under dynamic flow conditions, RORα blockade prevented mononuclear cell attachment to human dysfunctional endothelial cells. RORα blockade represents a potential therapy to prevent AT dysfunction and inflammation associated with insulin resistance in human obesity.

Retraction: KSHV vFLIP Is Essential for the Survival of Infected Lymphoma Cells.

Primary effusion lymphomas (PELs) associated with infection by the Kaposi's sarcoma–associated herpesvirus (KSHV/HHV-8) have constitutive nuclear factor (NF)–κB activity that is essential for their survival, but the source of this activity is unknown. We report that viral FADD-like interleukin-1-β–converting enzyme [FLICE/caspase 8]-inhibitory protein (FLIP) activates NF-κB more potently than cellular FLIP in B cells and that it is largely responsible for NF-κB activation in latently infected PEL cells. Elimination of vFLIP production in PEL cells by RNA interference results in significantly decreased NF-κB activity, down-regulation of essential NF-κB–regulated cellular prosurvival factors, induction of apoptosis, and enhanced sensitivity to external apoptotic stimuli. vFLIP is the first virally encoded gene shown to be essential for the survival of naturally infected tumor cells.

Antinociceptive and anti-inflammatory activities of Copaifera pubiflora Benth oleoresin and its major metabolite ent-hardwickiic acid

Ethnopharmacological relevanceCopaifera species folkloric names are “copaíbas, copaibeiras, copaívas or oil stick”, which are widely used in Brazilian folk medicine. Among all ethnopharmacological applications described for Copaifera spp oleoresins, their anti-inflammatory effect stands out. However, the knowledge of anti-inflammatory and antinociceptive properties of Copaifera pubiflora Benth is scarce. Aim of the studyTo investigate the cytotoxic, anti-inflammatory, and antinociceptive activities of C. pubiflora oleoresin (CPO), and its major compound ent-hardwickiic acid (HA). Material and methodsThe phosphatase assay was used to evaluate the cytotoxicity of CPO and HA in three different cell lines. CPO and HA doses of 1, 3, and 10 mg/kg were employed in the biological assays. The assessment of motor activity was performed using open-field and rotarod tests. Anti-inflammatory activity of CPO and HA was assessed through luciferase assay, measurement of INF-γ, IL-1β, IL-6, IL-10, and TNF-α in a multi-spot system with the immortalized cell line THP-1, zymosan-induced arthritis, and carrageenan-induced paw edema. Acetic acid-induced abdominal writhing and formalin tests were undertaken to evaluate the antinociceptive potential of CPO and HA. In addition, the evaluation using carrageenan was performed to investigate the effect of CPO in pain intensity to a mechanical stimulus (mechanical hyperalgesia), using the von Frey filaments. A tail-flick test was used to evaluate possible central CPO and HA actions. ResultsIn the cytotoxicity evaluation, CPO and HA were not cytotoxic to the cell lines tested. CPO and HA (10 mg/kg) did not affect animals' locomotor capacity in both open-field and rotarod tests. In the luciferase assay, CPO and HA significantly reduced luciferase activity (p < 0.05). This reduction indicates a decrease in NF-κB activity. HA and CPO decreased INF-γ, IL-1β, IL-6, IL-10, and TNF-α at 24 and 72 h in the multi-spot system. In zymosan-induced arthritis, CPO and HA decreased the number of neutrophils in the joint of arthritic mice and the number of total leukocytes (p < 0.05). In experimental arthritis HA significantly decreased joint swelling (p < 0.05). CPO and HA also increased the mechanical threshold during experimental arthritis. HA and CPO significantly inhibited the carrageenan-induced paw edema, being the doses of 10 mg/kg the most effective, registering maximum inhibitions of 58 ± 8% and 76 ± 6% respectively, p < 0.05. CPO and HA reduced the nociceptive behavior in both phases of formalin at all tested doses. The highest doses tested displayed inhibitions of 87 ± 1% and 72 ± 4%, respectively, p < 0.001, in the first phase, and 87 ± 1% and 81 ± 2%, respectively, p < 0.001, in the second phase. Oral treatment of CPO and HA (1, 3, 10 mg/kg) significantly reduced the nociceptive response in acetic acid-induced abdominal writhings, and the 10 mg/kg dose was the most effective with maximum inhibitions of 86 ± 2% and 82 ± 1%, respectively, p < 0.001. Both HA and CPO significantly decreased the intensity of mechanical inflammatory hyper-nociception on carrageenan-induced hyperalgesia at all tested doses, and 10 mg/kg was the most effective dose with maximum inhibitions of 73 ± 5% and 74 ± 7%, respectively, p < 0.05.CPO increased the tail-flick latencies in mice, and concomitant administration of naloxone partially reduced its effect. ConclusionsCPO and HA may inhibit the production of inflammatory cytokines by suppressing the NF-κB signaling pathway, resulting in anti-inflammatory and antinociceptive activities.

6-Gingerol attenuates ventilator-induced lung injury via anti-inflammation and antioxidative stress by modulating the PPARγ/NF- κB signalling pathway in rats

Although mechanical ventilation (MV) is indispensable to life-support therapy in critically ill patients, it may promote or aggravatelunginjury known asventilator-inducedlunginjury(VILI). 6-Gingerol is the principal ingredient of ginger with potential anti-inflammatory and antioxidant properties in various diseases. Nevertheless, the role and mechanism of 6-gingerol in the process of VILI has not been explicitly investigated. In the study, we found that pre-treatment with 6-gingerol significantly improved the histological changes and pulmonary oedema, inhibited neutrophil accumulation and the release of early pro-inflammatory cytokines and MPO, and reduced oxidative stress reactions after high MV. Moreover, 6-gingerol treatment also increased PPARγ expression and decreased NF-κB activation in rats subjected to high MV. Furthermore, GW9662, a specific PPARγ inhibitor, was demonstrated to activatethe NF-κB pathway and cancele the protective role of 6-gingerol in VILI. This indicates that 6-gingerol exerted anti-inflammatory and antioxidative stress effects in VILI by activating PPARγ and inhibiting the NF-κBsignalling pathway.

Curcumin suppresses the proliferation of oral squamous cell carcinoma through a specificity protein 1/nuclear factor-κB-dependent pathway.

Oral squamous cell carcinoma (OSCC) is the most common cancer of the oral cavity. Curcumin (Cur), a naturally derived compound, is reported to have broad-spectrum anticancer activity and is considered as an effective nuclear factor-κB (NF-κB) inhibitor. The present study aimed to clarify the detailed molecular mechanism though which Cur regulates NF-κB pathway activity in OSCC. The viability of HSC3 and CAL33 cells following treatment with Cur was determined using a Cell Counting Kit-8 assay. The protein and mRNA expression of specificity protein 1 (Sp1), p65 and heat shock factor 1 (HSF1) was determined by western blotting and reverse transcription-quantitative PCR analysis, respectively. The NF-κB activity was measured by Dual-Luciferase reporter assay. Short hairpin RNA targeting Sp1 or control RNA was transfected into HSC3 cells using X-treme GENE HP DNA Transfection System. Colony formation assays were performed using crystal violet staining. The results demonstrated that Cur significantly inhibited the viability and colony formation ability of HSC3 and CAL33 cells. In addition, Cur decreased the expression of Sp1, p65 and HSF1 by suppressing their transcription levels. Cur decreased NF-κB activity in OSCC cells, and Sp1 downregulation enhanced the effect of Cur. The findings from the present study suggested that Cur may inhibit the proliferation of OSCC cells via a Sp1/NF-κB-dependent mechanism.