Reduced NF-κB Activation Research Articles

Abstract P5-04-14: The covalent JNK inhibitor, JNK-IN-8, synergizes with lapatinib to cause cell death in basal-like breast cancer cell lines

Abstract Basal-like and claudin-low breast cancers have the worst prognosis and represent 15-20% of breast cancers diagnosed each year. Endocrine and molecularly targeted therapies, such as trastuzumab, are ineffective due to lack of ER expression or HER2 amplification in the tumors. They also have a high frequency of p53 mutations, low BRCA1 expression and high EGFR expression. Our lab has shown that high expression of JNK2 in human basal-like breast cancers leads to significantly decreased disease-free survival. JNK2 also promotes basal-like tumor progression in mice by increasing EGFR-mediated migration through facilitating internalization of EGFR, upregulating EMT gene expression and promoting metastasis. The ATP-competitive JNK inhibitor SP600125 has been commonly used by researchers to elucidate JNK-specific mechanisms, but this inhibitor was found to have high affinity to many other intracellular kinases. A new JNK inhibitor, JNK-IN-8, has been developed that binds covalently to all three JNK gene products and is more selective than the SP600125 compound (Zhang, et al. 2012). Using this inhibitor, we have found that basal-like breast cancer cell lines can become sensitized to lapatinib. This combination is synergistic and causes apoptotic cell death, while as single agents at these concentrations, these drugs have little effect on cell viability. Treatment with either lapatinib or JNK-IN-8 decreases transcriptional activity of NF-κB significantly, but combination of the two drugs reduces NF-κB activity to an almost negligible amount compared to vehicle treatment. Combination treatment also led to a 6-fold increase in ROS production that may be activating apoptosis. We hypothesize that inhibition by both JNK-IN-8 and lapatinib cause independent decreases in NF-κB activation that, when combined, cause a synergistic decrease in NF-κB-mediated survival mechanisms. Use of the JNK-IN-8 inhibitor with lapatinib in humans may increase survival in patients with basal-like or claudin-low breast cancers. Zhang, T. et al. "Discovery of potent and selective covalent inhibitors of JNK". Chem Biol. 2012 Jan 27;19(1):140-54. Citation Format: Ebelt ND, Van Den Berg CL. The covalent JNK inhibitor, JNK-IN-8, synergizes with lapatinib to cause cell death in basal-like breast cancer cell lines. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-14.

Receptor-Interacting Protein Kinase-2 Inhibition by CYLD Impairs Antibacterial Immune Responses in Macrophages.

Upon infection with intracellular bacteria, nucleotide oligomerization domain protein 2 recognizes bacterial muramyl dipeptide and binds, subsequently, to receptor-interacting serine/threonine kinase 2 (RIPK2), which activates immune responses via the nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) and extracellular signal-regulated kinase (ERK) pathways. Activation of RIPK2 depends on its K63 ubiquitination by E3 ligases, whereas the deubiquitinating enzyme A20 counter regulates RIPK2 activity by cleaving K63-polyubiquitin chains from RIPK2. Here, we newly identify the deubiquitinating enzyme CYLD as a new inhibitor of RIPK2. We show that CYLD binds to and removes K63-polyubiquitin chains from RIPK2 in Listeria monocytogenes (Lm) infected murine bone marrow-derived macrophages. CYLD-mediated K63 deubiquitination of RIPK2 resulted in an impaired activation of both NF-κB and ERK1/2 pathways, reduced production of proinflammatory cytokines interleukin-6 (IL-6), IL-12, anti-listerial reactive oxygen species (ROS) and nitric oxide (NO), and, finally, impaired pathogen control. In turn, RIPK2 inhibition by siRNA prevented activation of NF-κB and ERK1/2 and completely abolished the protective effect of CYLD deficiency with respect to the production of IL-6, NO, ROS, and pathogen control. Noteworthy, CYLD also inhibited autophagy of Listeria in a RIPK2-ERK1/2-dependent manner. The protective function of CYLD deficiency was dependent on interferon gamma (IFN-γ) prestimulation of infected macrophages. Interestingly, the reduced NF-κB activation in CYLD-expressing macrophages limited the protective effect of IFN-γ by reducing NF-κB-dependent signal transducers and activators of transcription-1 (STAT1) activation. Taken together, our study identifies CYLD as an important inhibitor of RIPK2-dependent antibacterial immune responses in macrophages.

Two COX-2 inhibitors induce apoptosis in human erythroleukemia K562cells by modulating NF-κB and FHC pathways.

BackgroundLeukemia is distinguished by abnormal proliferation of leukocytes. Although there has been some progress in developing novel cancer therapies, no significant improvement was observed in the overall survival rate over the last decade. Selective cyclooxygenase-2 (COX-2) inhibitors are known to inhibit tumor growth by exerting antimetastatic and antiangiogenic effects through inhibition of COX –dependent and independent pathways. The ability of two new triaryl-oxadiazole derivatives, compounds A (3-(4-chlorophenyl) -5-(4-flurophenyl)-4-Phenyl-4,5-dihydro-1,2,4-oxadiazole) and B (3,5-bis(4-chlorophenyl)-4-Phenyl-4,5-dihydro-1,2,4-oxadiazole), to induce apoptosis in human erythroleukemia K562 cells was evaluated and the upstream mechanism was investigated.MethodsK562 cells were treated with compounds A and B at their IC50 concentrations and analyzed by DAPI staining and Annexin-V-FLUOS labelling solution. Nuclear factor kappa-B (NF-κB) activation was evaluated by TransAM kit. Cyclooxygenase-2 (COX-2), Caspase-3, Bax, Bcl-2, ferritin heavy chain (FHC), extra cellular signal-regulated kinase (ERK), p-ERK and early growth response protein-1 (Egr1) levels were determined using Western blotting, while c-Myc mRNA level was investigated by RT-PCR.ResultsChanges in nuclear morphology and the increased annexin-V/PI staining revealed the apoptotic cell death in compounds A- and B-treated K562 cells. A significant reduction in NF-κB activity as well as FHC and p-ERK levels were detected in these cells. No change was observed in the levels of Bax, Bcl-2, Caspase-3, COX-2, c-Myc and Egr1, following treatment with the two compounds. Collectively, compounds A and B potentiate apoptosis as shown by DAPI staining, flowcytometry, FHC and p-ERK downregulation and NF-κB inactivation.ConclusionTwo compounds induce apoptosis in a COX-2-independent manner which also appears to be independent from mitochondria, caspase and c-Myc/Egr1 pathways.

The Sphingosine-1-Phosphate Lyase (LegS2) Contributes to the Restriction of Legionella pneumophila in Murine Macrophages.

L. pneumophila is the causative agent of Legionnaires’ disease, a human illness characterized by severe pneumonia. In contrast to those derived from humans, macrophages derived from most mouse strains restrict L. pneumophila replication. The restriction of L. pneumophila replication has been shown to require bacterial flagellin, a component of the type IV secretion system as well as the cytosolic NOD-like receptor (NLR) Nlrc4/ Ipaf. These events lead to caspase-1 activation which, in turn, activates caspase-7. Following caspase-7 activation, the phagosome-containing L. pneumophila fuses with the lysosome, resulting in the restriction of L. pneumophila growth. The LegS2 effector is injected by the type IV secretion system and functions as a sphingosine 1-phosphate lyase. It is homologous to the eukaryotic sphingosine lyase (SPL), an enzyme required in the terminal steps of sphingolipid metabolism. Herein, we show that mice Bone Marrow-Derived Macrophages (BMDMs) and human Monocyte-Derived Macrophages (hMDMs) are more permissive to L. pneumophila legS2 mutants than wild-type (WT) strains. This permissiveness to L. pneumophila legS2 is neither attributed to abolished caspase-1, caspase-7 or caspase-3 activation, nor due to the impairment of phagosome-lysosome fusion. Instead, an infection with the legS2 mutant resulted in the reduction of some inflammatory cytokines and their corresponding mRNA; this effect is mediated by the inhibition of the nuclear transcription factor kappa-B (NF-κB). Moreover, BMDMs infected with L. pneumophila legS2 mutant showed elongated mitochondria that resembles mitochondrial fusion. Therefore, the absence of LegS2 effector is associated with reduced NF-κB activation and atypical morphology of mitochondria.

CFTR Controls the Activity of NF-κB by Enhancing the Degradation of TRADD

Background/Aims: Chronic lung infection in cystic fibrosis leads to an inflammatory response that persists because of the chronic presence of bacteria and ultimately leads to a catastrophic failure of lung function. Methods: We use a combination of biochemistry, cell and molecular biology to study the interaction of TRADD, a key adaptor molecule in TNFα signaling, with CFTR in the regulation of NFκB. Results: We show that Wt CFTR binds to and colocalizes with TRADD. TRADD is a key signaling intermediate connecting TNFα with activation of NFκB. By contrast, ΔF508 CFTR does not bind to TRADD. NF-κB activation is higher in CFBE expressing ΔF508 CFTR than in cells expressing Wt CFTR. However, this differential effect is abolished when TRADD levels are knocked down. Transfecting Wt CFTR into CFBE cells reduces NF-κB activity. However the reduction is abolished by the CFTR chloride transport inhibitor-172. Consistently, transfecting in the correctly trafficked CFTR conduction mutants G551D or S341A also fail to reduce NFκB activity. Thus CFTR must be functional if it is to regulate NF-κB activity. We also found that TNFα produced a greater increase in NF-κB activity in CFBE cells than in the same cell when Wt CFTR-corrected. Consistently, the effect is also abolished when TRADD is knocked down by shRNA. Thus, Wt CFTR control of TRADD modulates the physiological activation of NF-κB by TNFα. Based on studies with proteosomal and lysosomal inhibitors, the mechanism by which Wt CFTR, but not ΔF508 CFTR, suppresses TRADD is by lysosomal degradation. Conclusion: We have uncovered a novel mechanism whereby Wt CFTR regulates TNFα signaling by enhancing TRADD degradation. Thus by reducing the levels of TRADD, Wt CFTR suppresses downstream proinflammatory NFκB signaling. By contrast, suppression of NF-κB activation fails in CF cells expressing ΔF508 CFTR.

�Dip and dry� micropattern capable bioactive coatings for modulating biomaterial associated inflammation

Event Abstract Back to Event ‘Dip and dry’ micropattern capable bioactive coatings for modulating biomaterial associated inflammation Paul J. Taylor1, Nicholas H. Williams1 and John W. Haycock2 1 University of Sheffield, Chemistry, United Kingdom 2 University of Sheffield, Materials Science & Engineering, United Kingdom Introduction: As quality of life improves there is an increase in the use of implantable biomaterials as medical devices. Although biomaterials are designed to be inert they can trigger host inflammation at the interface, which left untreated can lead to device failure. α-Melanocyte stimulating hormone (α-MSH) is a pleiotropic peptide hormone recognised for its anti-inflammatory properties. Our initial work successfully immobilised a synthetic version of α-MSH [GKP(D)V] onto glass using calixarene surface chemistry which attenuated an inflammatory response in vitro[1]. Our latest work incorporates α-MSH onto aryl azide functionalised silanes offering micropatterning capabilities via photolithography based on an established method within the group[2]. The present aim of this work is to optimise the photochemistry of the aryl azide silanes by immobilising a variety of bioactive molecules to provide a direct method for the surface modification of biomaterials. As well as observing the effects of surface patterning we aim to create highly ordered coatings with multifunctional bioactive properties. Materials and Methods: Glass coverslips were surface functionalised with calixarene-PEG-3-GKP(D)V and subjected to biological inflammatory testing (Fig.1). Thereafter, glass coverslips were coated with a photoactive aryl azide organosilane and the surface irradiated to immobilise a modified azido-PEG-6-GKP(D)V tether for micropatterning and subjected to inflammatory testing. Figure 1: A) Synthetic α-MSH (GKP(D)V) attached to a calixarene via PEG-3 tether for surface modification. B) Summary of surface patterning on aryl azide silane surfaces via photolithography[2]. Surfaces were characterised by XPS, SIMS, ellipsometry, and contact angle goniometry. Human fibroblasts, Schwann cells, and NIH-3T3 fibroblasts were seeded onto surface treated coverslips before stimulation with TNF-α or LPS. To monitor inflammatory cell-signalling activity, the p65 subunit of NF-κB was immunolabeled and quantified by confocal microscopy. Results and Discussion: XPS and SIMS confirmed that GKPV(D) was immobilised onto calixarene and aryl azide coated surfaces. Calixarene-PEG-GKP(D)V surface coatings inhibited TNF-α stimulated NF-κB activity by 23% ± 4% (n=3, p=0.001). Inhibition was comparable to control untreated surfaces stimulated with TNF-α and 10-9M GKP(D)V (39% ± 3%; n=3, p=0.003) indicating that coatings require relatively a low peptide load[1]. Similarly, the aryl azide silanated surfaces with N3-HEG-GKP(D)V also reduced NF-κB activity by 28% ± 4% (n=3, p=0.01). Conclusions: Data demonstrates that both calixarene and silane immobilisation methods can inhibit NF-κB cell signalling activity involved in the inflammatory response. Current work is focussed on how surface patterning controls anti-inflammatory properties and extends through to carbon monoxide releasing molecules using inorganic carbonyl compounds as a therapeutic approach. Engineering and Physical Sciences Research Council (EPSRC), UK

Reduced Hsp70 and Glutamine in Pediatric Severe Malaria Anemia: Role of Hemozoin in Suppressing Hsp70 and NF-κB activation.

Severe malarial anemia [SMA, hemoglobin (Hb) <5.0 g/dL] is a leading cause of global morbidity and mortality among children residing in Plasmodium falciparum transmission regions. Exploration of molecular pathways through global gene expression profiling revealed that SMA was characterized by decreased HSPA1A, a heat shock protein (Hsp) 70 coding gene. Hsp70 is a ubiquitous chaperone that regulates Nuclear Factor-kappa B (NF-κB) signaling and production of pro-inflammatory cytokines known to be important in malaria pathogenesis (e.g., IL-1β, IL-6 and TNF-α). Since the role of host Hsp70 in malaria pathogenesis is unexplored, we investigated Hsp70 and molecular pathways in children with SMA. Validation experiments revealed that leukocytic HSP70 transcripts were reduced in SMA relative to non-severe malaria, and that intraleukocytic hemozoin (PfHz) was associated with lower HSP70. HSP70 was correlated with reticulocyte production and Hb. Since glutamine (Gln) up-regulates Hsp70, modulates NF-κB activation, and attenuates over-expression of pro-inflammatory cytokines, circulating Gln was measured in children with malaria. Reduced Gln was associated with increased risk of developing SMA. Treatment of cultured peripheral blood mononuclear cells (PBMCs) with PfHz caused a time-dependent decrease in Hsp70 transcripts/protein, and NF-κB activation. Gln treatment of PBMCs overcame PfHz-induced suppression of HSP70 transcripts/protein, reduced NF-κB activation, and suppressed over-expression of IL-1β, IL-6 and TNF-α. Findings here demonstrate that SMA is characterized by reduced intraleukocytic HSP70 and circulating Gln, and that PfHz-induced suppression of HSP70 can be reversed by Gln. Thus, Gln supplementation may offer important immunotherapeutic options for futures studies in children with SMA.

A low toxicity synthetic cinnamaldehyde derivative ameliorates renal inflammation in mice by inhibiting NLRP3 inflammasome and its related signaling pathways

Uncontrolled inflammation is a leading cause of various chronic diseases. Cinnamaldehyde (CA) is a major bioactive compound isolated from the essential oil of the leaves of Cinnamomum osmophloeum kaneh that exhibits anti-inflammatory activity; however, the use of CA is limited by its cytotoxicity. Here, we synthesized three CA derivatives and identified 4-hydroxycinnamaldehyde-galactosamine (HCAG) as a low toxicity anti-inflammatory compound in vitro (HCAG IC50≫1600µM; CA IC50=40µM) and in vivo. HCAG reduced pro-inflammatory mediator expression in LPS-activated macrophages by inhibiting MAPK and PKC-α/δ phosphorylation, decreasing ROS generation and reducing NF-κB activation. HCAG also reduced NLRP3 inflammasome-derived IL-1β secretion by inhibiting the ATP-mediated phosphorylation of AKT and PKC-α/δ. In a mouse model of LPS-induced renal inflammation, we observed reduced albuminuria and a mild degree of glomerular proliferation, glomerular sclerosis and periglomerular inflammation in the HCAG-treated mice compared with the vehicle-treated mice. The underlying mechanisms for these renoprotective effects involved: (1) inhibited NLRP3 inflammasome activation; (2) decreased superoxide anion levels and apoptosis; and (3) suppressed activation of NF-κB and related downstream inflammatory mediators.

Haemophilus influenzae induces steroid-resistant inflammatory responses in COPD.

BackgroundChronic obstructive pulmonary disease (COPD) is an inflammatory disorder partially resistant to glucocorticoids. A reduced histone deacetylase (HDAC) activity has been proposed to explain this resistance. Haemophilus influenzae frequently colonizes the airways of COPD patients, where it enhances inflammation. The effects of Haemophilus influenzae on HDAC activity have not been investigated before.MethodsThe effects of the presence or absence of Haemophilus influenzae ex-vivo and in vitro were studied. To this end, we determined: (1) cytokine release in alveolar macrophages (AM) from 7 patients with COPD, 5 healthy smokers, 6 healthy non-smokers and (2) HDAC activity, nuclear factor kappa B (NF-κB) activation in a macrophage-like cell line (PMA-transformed U937 cells) co-cultured with epithelial cells. Experiments were repeated with dexamethasone (1 μM) and/or the HDAC enhancer theophylline (10 μM).ResultsHaemophilus influenzae induced a steroid-resistant inflammatory response in AM from COPD and controls and decreased HDAC activity, activated NF-κB and induced the secretion of several cytokines (IL-6, IL-8, IL-1β, IL-10 and TNF-α) (p < 0.001 for all comparisons) in the macrophage-like cell line. Dexamethasone reduced NF-κB activation but it did not modify HDAC activity. The addition of theophylline to dexamethasone increased HDAC activity and suppressed cytokine release completely, without modifying NF-κB activation.ConclusionsThese results indicate that Haemophilus influenzae reduces HDAC activity and induces a NF-κB mediated inflammatory response that is only partially suppressed by glucocorticoids irrespective of having COPD. Yet, the latter can be fully restored by targeting HDAC activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12890-015-0155-3) contains supplementary material, which is available to authorized users.

Pin1 facilitates NF-κB activation and promotes tumour progression in human hepatocellular carcinoma.

Background:NF-κB promotes HCC progression; however, therapies targeting NF-κB are not used due to severe adverse reactions. Pin1 is reported to induce tumour progression in vitro. However, the role of Pin1 in HCC is unclear. Moreover, little is known about the mechanism of Pin1-mediated NF-κB activation.Methods:Fresh surgical specimens were collected from 144 HCC patients. Pin1 and NF-κB-p65 expression was evaluated by immunohistochemistry and western blotting. NF-κB activation was assessed by EMSA.Results:Pin1 was increased in HCC compared to adjacent liver tissue. The multivariate analysis revealed that high Pin1 expression was an independent factor for poor prognosis. In HCC with high Pin1 expression, tumour size was larger and portal vein invasion was increased. Pin1 expression was correlated with phosphorylated (p−) NF-κB-p65(Thr254) and p-NF-κB-p65(Ser276), and thereby NF-κB activation. Pin1-induced NF-κB activation accelerated cell cycle progression, induced angiogenesis, and inhibited apoptosis. Pin1 knockdown in HCC cells inhibited the phosphorylation of NF-κB-p65(Ser276), and reduced NF-κB activation, which resulted in inhibiting tumour cell progression. When HCC cells were treated with the Pin1 inhibitors, p-NF-κB-p65(Ser276) expression and NF-κB activation was reduced, and cell proliferation was inhibited.Conclusions:Pin1 is associated with aggressive tumour progression and poor prognosis in HCC by mediating NF-κB activation.

Curcumin inhibits superoxide anion-induced pain-like behavior and leukocyte recruitment by increasing Nrf2 expression and reducing NF-κB activation.

This study aimed at evaluating the activity of curcumin in superoxide anion-induced pain-like behavior and leukocyte recruitment in mice. Administration of curcumin 10 mg/kg subcutaneously 1 h before stimulus. KO2 was used as superoxide anion donor. Overt pain-like behaviors were determined by the number of abdominal writhings, paw flinches and time spent licking the paw. Mechanical and thermal hyperalgesia were determined using an electronic anesthesiometer and hot plate, respectively. Cytokine concentration and NF-κB activity were determined by ELISA, antioxidant effect by nitrobluetretrazolium assay and ABTS radical scavenging ability. Myeloperoxidase activity was measured by colorimetric assay. The Nrf2, heme oxygenase-1 (HO-1) and gp91phox mRNA expression was determined by quantitative PCR. Data were analyzed by ANOVA followed by Tukey's post hoc and considered significant when p<0.05. Curcumin inhibited superoxide anion-induced overt pain-like behaviors as well as mechanical and thermal hyperalgesia. Curcumin also inhibited superoxide anion-induced leukocyte recruitment in the peritoneal cavity and in the paw skin inhibited myeloperoxidase activity, oxidative stress, IL-1β and TNF-α production and NF-κB activation as well as enhanced IL-10 production, and HO-1 and Nrf2 mRNA expression. Curcumin inhibits superoxide anion-induced inflammatory pain-like behaviors and leukocyte recruitment by targeting inflammatory molecules and oxidative stress; and inducing antioxidant and anti-inflammatory pathways.

Dexamethasone induces apoptosis in pulmonary arterial smooth muscle cells.

BackgroundDexamethasone suppressed inflammation and haemodynamic changes in an animal model of pulmonary arterial hypertension (PAH). A major target for dexamethasone actions is NF-κB, which is activated in pulmonary vascular cells and perivascular inflammatory cells in PAH. Reverse remodelling is an important concept in PAH disease therapy, and further to its anti-proliferative effects, we sought to explore whether dexamethasone augments pulmonary arterial smooth muscle cell (PASMC) apoptosis.MethodsAnalysis of apoptosis markers (caspase 3, in-situ DNA fragmentation) and NF-κB (p65 and phospho-IKK-α/β) activation was performed on lung tissue from rats with monocrotaline (MCT)-induced pulmonary hypertension (PH), before and after day 14–28 treatment with dexamethasone (5 mg/kg/day). PASMC were cultured from this rat PH model and from normal human lung following lung cancer surgery. Following stimulation with TNF-α (10 ng/ml), the effects of dexamethasone (10−8–10−6 M) and IKK2 (NF-κB) inhibition (AS602868, 0–3 μM (0-3×10−6 M) on IL-6 and CXCL8 release and apoptosis was determined by ELISA and by Hoechst staining. NF-κB activation was measured by TransAm assay.ResultsDexamethasone treatment of rats with MCT-induced PH in vivo led to PASMC apoptosis as displayed by increased caspase 3 expression and DNA fragmentation. A similar effect was seen in vitro using TNF-α-simulated human and rat PASMC following both dexamethasone and IKK2 inhibition. Increased apoptosis was associated with a reduction in NF-κB activation and in IL-6 and CXCL8 release from PASMC.ConclusionsDexamethasone exerted reverse-remodelling effects by augmenting apoptosis and reversing inflammation in PASMC possibly via inhibition of NF-κB. Future PAH therapies may involve targeting these important inflammatory pathways.

PP42 - Fisetin regulates TPA-induced breast cancer cell invasion by suppressing matrix metalloproteinase-9 activation via the PKC/ROS/MAPK pathways

Invasion and metastasis are among the main causes of death in patients with malignant tumors. Fisetin (3,3′,4′,7-tetrahydroxyflavone), a natural flavonoid found in the smoke tree (Cotinus coggygria), is known to have antimetastatic effects on prostate and lung cancers; however, the effect of fisetin on breast cancer metastasis is unknown. The aim of this study was to determine the anti-invasive activity of fisetin in human breast cancer cells. Matrix metalloproteinase (MMP)-9 is a major component facilitating the invasion of many cancer tumor cell types, and thus the inhibitory effect of fisetin on MMP-9 expression in 12- O -tetradecanoylphorbol-13-acetate (TPA)-stimulated human breast cancer cells was investigated in this study. Fisetin significantly attenuated TPA-induced cell invasion in MCF-7 human breast cancer cells, and was found to inhibit the activation of the PKCa/ROS/ERK1/2 and p38 MAPK signaling pathways. This effect was furthermore associated with reduced NF-κB activation, suggesting that the anti-invasive effect of fisetin on MCF-7 cells may result from inhibited TPA activation of NF-κB and reduced TPA activation of PKCα/ROS/ERK1/2 and p38 MAPK signals, ultimately leading to the downregulation of MMP-9 expression. Our findings indicate the role of fisetin in MCF-7 cell invasion, and clarify the underlying molecular mechanisms of this role, suggesting fisetin as a potential chemopreventive agent for breast cancer metastasis.

Ameliorative effect of trimetazidine on cisplatin-induced hepatotoxicity in rats.

This study was designed to evaluate the protective effects of trimetazidine (TMZ) against cisplatin (CP) induced liver damage in rats. Animals were distributed among 4 groups as follows: control group; TMZ group (20 mg/kg body mass, per oral), which was treated for 10 days; CP group (6 mg/kg, by intraperitoneal injection), which received a single injection; and the CP + TMZ group (20 mg/kg, per oral), which received TMZ 4 days before and 6 days after CP injection. The extent of hepatic damage was studied by assessing biochemical parameters and histopathological evaluation of the extracted liver tissue. The results revealed that liver enzymes were markedly elevated after injection of CP, as evident from significant increases in the serum levels of alanine transaminase (AST), alanine aminotransferase (ALT), gamma glutamyl transferase (γ-GT), and lactate dehydrogenase (LDH), as well as marked changes to the liver architecture, with a significant decrease in serum levels of albumin. There were also marked changes to the antioxidant defense system, as indicated by significant decreases in total antioxidants and hepatic levels of reduced glutathione (GSH) and superoxide dismutase (SOD), together with a significant increase in lipid peroxidation. However, there was a significant increase in the activity of hepatic nuclear factor kappa B (NF-κB) as well as hepatic Bax protein expression. We conclude that TMZ protects against CP-induced liver damage through scavenging free radicals and anti-inflammatory and antiapoptotic effects, as well as through reducing NF-κB activation.

The human papillomavirus (HPV) E7 protein antagonises an Imiquimod-induced inflammatory pathway in primary human keratinocytes.

High-risk human papillomaviruses (HPV) are the etiological pathogen of cervical and a number of ano-genital cancers. How HPVs overcome the significant barriers of the skin immune system has been the topic of intensive research. The E6 and E7 oncoproteins have emerged as key players in the deregulation of host innate immune pathways that are required for the recruitment of effector cells of the immune response. Here we demonstrate that E7, and to a lesser extend E6, strongly reduce NFκB activation in response to the inflammatory mediator imiquimod. Moreover, we establish that undifferentiated keratinocytes do not express the putative receptor for imiquimod, TLR7, and as such are stimulated by imiquimod through a novel pathway. Inhibition of imiquimod induced cytokine production required residues in the CR1 and CR3 regions of E7 and resulted in reduced nuclear translocation and acetylation of the p65 sub-unit of NFκB. The results provide further evidence for a TLR7-independent role of imiquimod in the epithelial immune response and reinforce the ability of the HPV oncoproteins to disrupt the innate immune response, which may have important consequences for establishment of a chronic infection.

Pathological concentrations of homocysteine increases IL-1β production in macrophages in a P2X7, NF-ĸB, and erk-dependent manner.

Elevated plasma levels of homocysteine (Hcy) are associated with the development of coronary artery disease (CAD), peripheral vascular disease, and atherosclerosis. Hyperhomocysteinemia is likely related to the enhanced production of pro-inflammatory cytokines including IL-1β. However, the mechanisms underlying the effects of Hcy in immune cells are not completely understood. Recent studies have established a link between macrophage accumulation, cytokine IL-1β, and the advance of vascular diseases. The purpose of the present study is to investigate the effects of Hcy on IL-1β secretion by murine macrophages. Hcy (100 μM) increases IL-1β synthesis via enhancement of P2X7 expression and NF-ĸB and ERK activation in murine macrophages. In addition, the antioxidant agent N-acetylcysteine (NAC) reduces NF-κB activation, ERK phosphorylation, and IL-1β production in Hcy-exposed macrophages, indicating the importance of ROS in this pro-inflammatory process. In summary, our results show that Hcy may be involved in the synthesis and secretion of IL-1β via NF-ĸB, ERK, and P2X7 stimulation in murine macrophages.

Abstract 1271: MDM2 small molecule inhibitors synergize with chemotherapeutics to attenuate senescence-driven inflammatory secretion

Abstract An attractive therapeutic approach for treatment of cancers with wild-type (wt) p53 is the emergent class of small molecule MDM2 inhibitors, including the imidazoline nutlin-3 and more potent spiro-oxindole MI-63. Both compounds rapidly increase intracellular wt p53 levels and induce apoptosis or a senescence growth arrest, depending on the target cell. A critical question in the clinical development of MDM2 inhibitors is their potential for normal tissue toxicity owing to their ability to cause p53-dependent apoptosis or cellular senescence. Recent work suggest senescent cells exhibit a senescence-associated secretory phenotype (SASP) that can stimulate cancer progression. We critically examined the effect of MDM2 small molecule inhibitors Nutlin-3a and MI-63 on non-senescent and senescent cells. Both compounds caused normal cells to undergo a transient growth arrest. Continuous incubation with MDM2 small molecule inhibitors caused normal cells to exhibit some hallmarks of senescence (arrested growth, SA-β-Gal expression and secretion of High Mobility Group Box 1 protein), but the cells resumed growth upon removal of the MDM2 inhibitors. Senescent cells cultured with MDM2 inhibitors failed to resume growth upon removal of the compounds, as expected. However, their SASP was markedly dampened, as determined by multiplex ELISA assays. Since NF-κB and IL-1α are major drivers of the SASP, we examined their status in senescent cells cultured with MDMD2 inhibitors. The small molecule MDM2 inhibitors significantly reduced NF-κB activity and IL-1α mRNA expression in senescent cells. Our data suggest using combinatorial therapy of chemotherapeutics or irradiation and MDMD2 small molecule inhibitors may provide a potent therapeutic strategy to promote apoptosis of p53 positive tumor cells after radio- or chemo-therapy and attenuate the potentially deleterious effects of the SASP. Citation Format: Nicholas Schaum, Fatouma Almirah, Gary Scott, Christopher Benz, Judith Campisi, Albert R. Davalos. MDM2 small molecule inhibitors synergize with chemotherapeutics to attenuate senescence-driven inflammatory secretion. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1271. doi:10.1158/1538-7445.AM2015-1271

Fisetin regulates TPA-induced breast cell invasion by suppressing matrix metalloproteinase-9 activation via the PKC/ROS/MAPK pathways

Invasion and metastasis are among the main causes of death in patients with malignant tumors. Fisetin (3,3′,4′,7-tetrahydroxyflavone), a natural flavonoid found in the smoke tree (Cotinus coggygria), is known to have antimetastatic effects on prostate and lung cancers; however, the effect of fisetin on breast cancer metastasis is unknown. The aim of this study was to determine the anti-invasive activity of fisetin in human breast cancer cells. Matrix metalloproteinase (MMP)-9 is a major component facilitating the invasion of many cancer tumor cell types, and thus the inhibitory effect of fisetin on MMP-9 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated human breast cancer cells was investigated in this study. Fisetin significantly attenuated TPA-induced cell invasion in MCF-7 human breast cancer cells, and was found to inhibit the activation of the PKCα/ROS/ERK1/2 and p38 MAPK signaling pathways. This effect was furthermore associated with reduced NF-κB activation, suggesting that the anti-invasive effect of fisetin on MCF-7 cells may result from inhibited TPA activation of NF-κB and reduced TPA activation of PKCα/ROS/ERK1/2 and p38 MAPK signals, ultimately leading to the downregulation of MMP-9 expression. Our findings indicate the role of fisetin in MCF-7 cell invasion, and clarify the underlying molecular mechanisms of this role, suggesting fisetin as a potential chemopreventive agent for breast cancer metastasis.

Hydrogen water alleviates lung injury induced by one-lung ventilation

BackgroundWith the development of thoracic surgeries, one-lung ventilation (OLV) has been routinely used to facilitate surgical exposure. However, OLV can cause lung injury during the surgical process and becomes an important factor affecting the outcomes. To date, effective treatments for the prevention of lung injury caused by OLV are lacking. Hydrogen has been demonstrated to have effective protection against tissue injuries caused by oxidative stress, inflammation, and apoptosis. This study investigated the efficacy of hydrogen water consumption on the prevention of lung injury induced by OLV in rats. Materials and MethodsMale Sprague–Dawley rats (n = 32, 240–260 g) were divided randomly into the following four groups: sham group, sham + H2 group, OLV group, OLV + H2 group. The rats drank hydrogen water or degassed hydrogen water for 4 wk before the operation and received OLV for 60 min and two-lung ventilation for 60 min. Lung tissues were assayed for wet-to-dry ratio, oxidative stress variables, proinflammatory cytokines, and hematoxylin–eosin staining. ResultsHydrogen water consumption reduced wet-to-dry weight ratio, malondialdehyde and myeloperoxidase activity and decreased the concentration of TNF-α, IL-1β, and IL-6 in the lung tissues compared with sham group and sham + H2 group. Hydrogen water consumption further attenuated NF-κB activation and caused histopathologic alterations. ConclusionsOur data demonstrated that hydrogen water consumption ameliorated OLV-induced lung injury, and it may exert its protective role by its anti-inflammation, antioxidation and reducing NF-κB activity in the lung tissues.

Cross-talk between α7 nAChR-mediated cholinergic pathway and acylation stimulating protein signaling in 3T3-L1 adipocytes: role of NFκB and STAT3.

Inflammation is a key feature in adipose tissue, especially in association with obesity comorbidies. The novel adipokine acylation stimulating protein (ASP) is one factor implicated in the inflammatory response. The disruption of the α7 nicotine acetylcholine receptor (α7nAChR), an important component of the endogenous non-neural cholinergic defense system, may exacerbate sustained inflammatory phenotype. We examined cholinergic regulation of ASP-initiated inflammatory response in 3T3-L1 adipocytes. Our results show that preincubation of 3T3-L1 cells with α7nAChR agonist GTS-21 significantly reduces ASP-mediated chemokine MCP-1 secretion, which is regulated though nuclear factor κB (NFκB) and signal transducer and activator of transcription 3 (STAT3). Treatment of 3T3-L1 cells with GTS-21 significantly reduced NFκB activation by DNA binding and STAT3 activation by disturbing post-translational modification.