Nuclear factor-kappaB Inhibitor Research Articles

Protective Effect of Andrographolide in 3-Nitropropionic Acid Induced Huntington Disease and Associated Neurodegenerative Changes in Rats

Aims: Oxidative stress and activation of pro-apoptotic mediators have been associated with the pathogenesis of Huntington’s disease. Andrographolide (ANDRO) is a well-known antioxidant and inhibitor of pro-apoptotic mediator, nuclear factor kappa B (NF-kB).
 Study Design: The present study was hence designed to evaluate the effect of ANDRO in Huntington’s disease.
 Place and Duration of Study: Department of Pharmacology, Khalsa College of Pharmacy, Amritsar, India between March 2018 and July 2018.
 Methodology: Five groups (n=6) of Sprague dawley rats were used. Normal group animals were kept untreated. The control group was administered 3-Nitro propionic acid (3-NP) for seven days. The three treament groups received 3-NP followed by ANDRO intraperitoneally for seven days. On 8th day, behavioral and coordination parameters were evaluated using multiple tests. Oxidative stress and anti-oxidant enzyme levels in brain tissue were also evaluated. Brain tissues were also evaluated using Haematoxylin-eosin staining.
 Results: Administration of 3-NP resulted in motor incoordination and muscle weakness as indicated by behavioral tests. Biochemically, there was an increase in the oxidative stress and depletion of free radical scavenging. Histopathologically, there was severe neuronal degeneration and neural tissue apoptotic changes in the rat striatum. ANDRO administration resulted in significant decrease in the muscle incoordination in the behavioral tests and also decreased prooxidative biochemical changes. Brain tissues of the ANDRO treated animals showed protection against neuronal damage and neurodegeneration.
 Conclusion: The results indicate that the use of ANDRO may afford protection against Huntington’s disease associated muscle incoordination and subsequent neurodegeneration. Present study provides a lead for further investigation of role of NF-kB inhibitors in Huntington’s disease and possible development of low-cost natural medication.

Silymarin Inhibits Proliferation and Induces Apoptosis in Epstein-Barr Virus-Positive Lymphoma Cells by Suppressing Nuclear Factor-Kappa B�Pathway

Epstein Barr virus-positive lymphoma results from the loss of homeostatic balance between Epstein Barr virus and immune cells. In this study, we investigated the mechanism underlying reduction in Epstein Barr virus-positive lymphoma cell proliferation and apoptosis by silymarin, an anti-inflammatory/antioxidant molecule extracted from Silybum�marianum (milk thistle). We examined the effect of silymarin on Raji cell proliferation, apoptosis, the expression of latent membrane protein�1, the proteins related to apoptosis, and the activation of nuclear factor kappa-B. Results showed that silymarin inhibited the proliferation and promoted apoptosis of Raji cells in a concentration-dependent manner. Also, silymarin reduced phosphorylated inhibitor of nuclear factor kappa-B and p65 levels. Silymarin treatment elevated latent membrane protein 1 expression and knockdown of this protein led to increased proliferation inhibition and apoptosis by silymarin. In conclusion, nuclear factor kappa-B and latent membrane protein 1 knockdown could work in concert with silymarin in suppressing Epstein Barr virus-positive lymphoma cell proliferation and inducing apoptosis.

Dietary supplement of Yunkang 10 green tea and treadmill exercise ameliorate high fat diet induced metabolic syndrome of C57BL/6\u2009J mice

BackgroundDiet and exercise play important roles in ameliorating metabolic syndrome. Yunkang 10 (Camellia sinensis var. assamica) is a most cultivated tea variety for making tea in the Southwestern China. Currently, there is no report of healthy effects of Yunkang 10 green tea (YKGT) and treadmill exercise (Ex) on high fat diet induced metabolic syndrome (MetS). We aimed to investigate the beneficial effects and molecular mechanism of YKGT and Ex using high fat diet induced MetS of C57BL/6 mice.MethodsCatechins and caffeine in water extract of YKGT were measured via high performance liquid chromatography (HPLC). 10-week old mice were fed with high fat diet (HFD) for 10 weeks to induce obese mice. Then the obese mice were fed with continuous high fat diet (HFD), HFD with YKGT, HFD with Ex, and HFD with both YKGT and Ex for 8 weeks, respectively. The another group of 10-week old mice fed with low fat diet (LFD) were used as control.ResultsHPLC data revealed that YKGT has abundantly high concentration of epigallocatechin gallate (EGCG) and caffeine compared to Longjing 43 (Camellia sinensis var. sinensis) green tea. YKGT and Ex significantly decreased the level of blood glucose, serum total cholesterol (TC), triglyceride (TG), insulin, and alanine aminotransferase activity (ALT) when compared to HFD group. The fatty liver and hepatic pro-inflammatory gene expression in the YKGT, Ex and YKGT+Ex groups was mitigated significantly compared with HFD group, respectively. The phosphorylation of inhibitor of nuclear factor kappa-B kinase α/β (IKKα/β) and inhibitor of nuclear factor kappa-B α (IkBα) protein in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) signaling pathway was also decreased in YKGT or YKGT+Ex groups. The combination of YKGT and Ex prevented gene expression for lipid synthesis in the liver tissue, and significantly upregulated mRNA level of glucose transport genes in the skeletal muscles, when compared to the HFD group.ConclusionsThis study demonstrated that YKGT supplement or exercise appeared to reverse preexisting metabolic syndrome, and effectively relieved the fatty liver and hepatic inflammatory response induced by high fat diet. YKGT supplement and treadmill exercise together had better beneficial effects than only one intervention.

Treatment of COVID-19 by Controlling the Activity of the Nuclear Factor-Kappa B

Heavy infection of the virus leads to overproduction of cytokines. The overproduction of cytokine (cytokines storms) is responsible for the critical cases and deaths of COVID-19. The nuclear factor kappa-B stimulates the expression of the genes, which is responsible for cytokines storm and RNA transcription. The COVID-19 virus can be controlled by inhibition of nuclear factor kappa-B. Nuclear factor kappa-B is controlled by inhibition of hydrogen peroxide and inhibitor kappa-B kinase enzyme.

NF-κB Inhibitor Parthenolide Promotes Renal Tubules Albumin Uptake in Type 2 Diabetic Nephropathy.

Objective Injured tubular reabsorption is highlighted as one of the causes of increased albuminuria in the early stage of diabetic nephropathy; however, the underlying mechanism has not been fully elucidated. In this study, we aimed to explore whether reducing inflammation and remodeling the insulin signaling pathway could improve albumin uptake of renal tubules. Methods 8-week-old male db/db mice (n=8), a type 2 diabetic nephropathy model, administered with nuclear factor kappa-B (NF-κB) inhibitor parthenolide (PTN, 1 mg/kg) intraperitoneally every other day for 8 weeks, were as the treatment group. Meanwhile, the age-matched male db/m mice (n=5) and db/db mice (n=8) were treated with saline as the control group and type 2 diabetic nephropathy group. When the mice were sacrificed, blood and urine were collected to examine homeostasis model assessment of insulin resistance (HOMA-IR) and urine albumin creatinine ratio, and kidney samples were used to analyze histopathologic changes with periodic acid-Schiff (PAS) staining, NF-κB p65, phosphorylation of AKT (p-AKT), amnionless and cubilin expressions with immunohistochemistry as well as western blot, and the albumin uptake of renal tubules by using immunofluorescence. In addition, HKC cells were divided into the insulin group treated with insulin alone, the TNF-α group treated with insulin and tumor necrosis factor (TNF-α), and the TNF-α+PTN group exposed to PTN, insulin and TNF-α. The levels of albumin uptake and expression levels of NF-κB p65, p-IRS-1/IRS-1, p-AKT/AKT, amnionless and cubilin in HKC cells were measured. Results Compared with the db/db group, the db/db+PTN group demonstrated decreased levels of HOMA-IR (36.83±14.09 vs. 31.07±28.05) and urine albumin creatinine ratio (190.3±7.3 vs. 143.0±97.6 mg/mmol); however, the differences were not statistically significant (P>0.05). Periodic acid-Schiff staining showed PTN could alleviate the glomerular hypertrophy and reduce the matrix in mesangial areas of db/db mice. The renal expression of NF-κB p65 was increased and p-AKT (s473) decreased in the db/db group compared with the db/m group (P<0.05). PTN significantly reduced the renal expression of NF-κB p65 and ameliorated the decline of p-AKT (s473) compared with the db/db group (P<0.05). Compared with the db/m group, the expression of amnionless and cubilin decreased and albumin uptake in tubules were reduced in the db/db group (P<0.05), and PTN could significantly increase the expression of cubilin (P<0.05), and improve albumin uptake in tubules. Insulin promoted albumin uptake and the expression of amnionless and cubilin in HKC cells (P<0.05). TNF-α stimulated the expression of NF-κB p65, increased p-IRS-1 (s307) and reduced p-AKT (s473) in HKC cells (P<0.05). In the TNF-α+PTN group, the expression of NF-κB p65 declined and p-IRS-1 (s307) and p-AKT (s473) were restored, compared with the TNF-α group (P<0.05). The expression of amnionless and cubilin decreased in the TNF-α group (P<0.05), and PTN could significantly increase the expression of cubilin (P<0.05). Conclusions Inflammation caused damage to insulin signaling, which reduced amnionless-cubilin expression and albumin uptake. PTN could reduce inflammation and remodel the impaired insulin signaling pathway, which promoted the expression of cubilin and albumin uptake. Our study can shed light on the role of inflammation in the reduction of albumin uptake of renal tubules in type 2 diabetic nephropathy.

The Effects of Orientin on Proliferation and Apoptosis of T24 Human Bladder Carcinoma Cells Occurs Through the Inhibition of Nuclear Factor-kappaB and the Hedgehog Signaling Pathway.

BackgroundOrientin is a flavone isolated from medicinal plants used in traditional Chinese medicine (TCM), which suppresses the growth of cancer cells in vitro. The effects of orientin in bladder cancer cells remains unknown. This study aimed to investigate the effect of orientin on proliferation and apoptosis of T24 human transitional cell bladder carcinoma cells in vitro in the presence of an agonist and an inhibitor of nuclear factor-kappaB (NF-κB).Material/MethodsT24 cells were cultured and divided into four study groups: an untreated control group; a group treated with 100 μM orientin; a group treated with 100 μM orientin with NF-κB agonist, phorbol 12-myristate 13-acetate (PMA); and a group treated with 100 μM orientin and the NF-κB inhibitor, IκBα. The MTT assay was performed to assess cell viability, and flow cytometry evaluated the cell cycle. The expression of proteins in the Hedgehog signaling pathway and inflammatory cytokines were determined by Western blot and enzyme-linked immunosorbent assay (ELISA).ResultsOrientin inhibited the proliferation of T24 cells, caused cell cycle arrest, reduced cell viability, and inhibited the expression of inflammatory mediators. Treatment of T24 cells with orientin inhibited the expression of NF-κB and components of the Hedgehog signaling pathway, and the NF-κB agonist, PMA, reversed these effects.ConclusionsTreatment of T24 human bladder carcinoma cells in vitro with orientin inhibited cell proliferation and promoted cell apoptosis by suppressing the Hedgehog signaling pathway and NF-κB.

Novel Functions of IFI44L as a Feedback Regulator of Host Antiviral Responses.

We describe a novel function for the interferon (IFN)-induced protein 44-like (IFI44L) gene in negatively modulating innate immune responses induced after virus infections. Furthermore, we show that decreasing IFI44L expression impairs virus production and that IFI44L expression negatively modulates the antiviral state induced by an analog of double-stranded RNA (dsRNA) or by IFN treatment. The mechanism likely involves the interaction of IFI44L with cellular FK506-binding protein 5 (FKBP5), which in turn interacts with kinases essential for type I and III IFN responses, such as inhibitor of nuclear factor kappa B (IκB) kinase alpha (IKKα), IKKβ, and IKKε. Consequently, binding of IFI44L to FKBP5 decreased interferon regulatory factor 3 (IRF-3)-mediated and nuclear factor kappa-B (NF-κB) inhibitor (IκBα)-mediated phosphorylation by IKKε and IKKβ, respectively. According to these results, IFI44L is a good target for treatment of diseases associated with excessive IFN levels and/or proinflammatory responses and for reduction of viral replication.IMPORTANCE Excessive innate immune responses can be deleterious for the host, and therefore, negative feedback is needed. Here, we describe a completely novel function for IFI44L in negatively modulating innate immune responses induced after virus infections. In addition, we show that decreasing IFI44L expression impairs virus production and that IFI44L expression negatively modulates the antiviral state induced by an analog of dsRNA or by IFN treatment. IFI44L binds to the cellular protein FKBP5, which in turn interacts with kinases essential for type I and III IFN induction and signaling, such as the kinases IKKα, IKKβ, and IKKε. IFI44L binding to FKBP5 decreased the phosphorylation of IRF-3 and IκBα mediated by IKKε and IKKβ, respectively, providing an explanation for the function of IFI44L in negatively modulating IFN responses. Therefore, IFI44L is a candidate target for reducing virus replication.

EPO promotes axonal sprouting via upregulating GDF10

Erythropoietin (EPO) has an exact neuroprotective effect on stroke. However, it remains unknown whether it participates in axonal sprouting after neuron damage. Growth and differentiation factor 10 (GDF10) has been shown to be a trigger of axonal sprouting after stroke. Hence, it was hypothesized that EPO promotes axonal sprouting mainly through GDF10. In the present in vitro experiment, it was found that EPO could promote axonal sprouting and GDF10 expression in a dose–dependent manner. The knockdown of GDF10 using siRNA abolished the effect of EPO-mediated axonal sprouting, indicating that GDF10 is the executor of EPO-mediated axonal sprouting. The treatment of neurons with nuclear factor-kappaB (NF-κB) inhibitor JSH-23 could inhibit the accumulation of NF-κB phospho-p65 (p-p65) in the nucleus, the upregualtion of GDF10 and extending of axonal length. Furthermore, the addition of Janus kinase 2 (JAK2) inhibitor CEP-33779 or phosphoinositide 3-kinase (PI3K) inhibitor LY294002 to the culture medium also blocked the nuclear translocation of p-p65, the expression of GDF10, and axonal sprouting, suggesting that EPO induces axonal sprouting via activating cellular JAK2 and PI3K signaling. Impeding JAK2 signaling with CEP-33779 can suppress the phosphorylation of PI3K, and this confirms that the upstream of PI3K signaling is JAK2. These present results provide a novel insight into the role of EPO and the molecular mechanism of axonal sprouting, which is beneficial for the development of novel approaches for neurological recovery after brain injury, including stroke.

Anti-inflammatory Action of Curcumin and Its Use in the Treatment of Lifestyle-related Diseases.

Chronic inflammation plays a significant role in lifestyle-related diseases, such as cardiovascular diseases and obesity/impaired glucose tolerance. Curcumin is a natural extract that possesses numerous physiological properties, as indicated by its anti-inflammatory action. The mechanisms underlying these effects include the inhibition of nuclear factor-kappaB and Toll-like receptor 4-dependent signalling pathways and the activation of a peroxisome proliferator-activated receptor-gamma pathway. However, the bioavailability of curcumin is very low in humans. To resolve this issue, several drug delivery systems have been developed and a number of clinical trials have reported beneficial effects of curcumin in the management of inflammation-related diseases. It is expected that evidence regarding the clinical application of curcumin in lifestyle-related diseases associated with chronic inflammation will accumulate over time.

Development of Peptide Inhibitors of Nuclear Factor KappaB as Therapeutics for Ischemic Stroke

Ischemic strokes occur when a major cerebral artery or its branches are occluded and result in activation of inflammatory processes, causing secondary tissue injury, breakdown of the blood‐brain barrier, edema, or hemorrhage. Treatments that inhibit inflammatory processes may thus be highly beneficial, but are not currently available. A key inflammatory process is the nuclear factor kappa B (NF‐κB) pathway. In its active form, NF‐κB is translocated to the nucleus of the cell where it regulates expression of proinflammatory and proapoptotic genes. The molecules that interact with NF‐κB, and the subunits that compose NF‐κB itself, allow for therapeutic targets to decrease inflammatory processes by inhibiting the nuclear localization of NF‐κB. Our goal is to develop such NF‐κB‐interacting peptides attached to the drug carrier elastin‐like polypeptide (ELP). ELP has a long plasma half‐life, can be attached to cell penetrating peptides, and has been shown to be an effective drug carrier in other models of disease. Here, we cloned five peptide inhibitors of the NF‐κB pathway into vectors to generate chimeric fusion proteins with ELP which were subsequently expressed recombinantly in E. coli and purified by inverse transition cycling. The ELP‐peptide fusions were screened using an in vitro luciferase assay in human embryonic kidney 293 cells transiently transfected with a reporter plasmid containing NF‐κB response elements driving the luciferase gene. One peptide fusion protein, targeted to the p50 subunit of NF‐κB (ELP‐p50i) resulted in attenuation of luminescence and thereby NF‐κB activity. Cellular localization of ELP‐p50i was assessed using fluorescently labeled protein after in vitro incubation with human umbilical vein endothelial cells (HUVEC) and mouse macrophages (RAW264.7) by confocal microscopy. Inhibition of NF‐κB activation by ELP‐p50i protein or a control protein lacking the NF‐κB inhibitory peptide was also confirmed in HUVECs stimulated with TNF‐a and RAW 264.7 cells stimulated with lipopolysaccharide by immunolabeling the NF‐κB complex and observing its nuclear translocation. ELP‐p50i was internalized by both HUVECs and RAW264.7 cells and localized to punctate cytoplasmic structures, likely endosomes and lysosomes, and diffusely in the cell cytoplasm. Pretreatment of cells with ELP‐p50i prevented NF‐κB nuclear localization after pro‐inflammatory stimuli. Similar analyses of the other NF‐κB inhibitory constructs are currently ongoing to determine which construct is most effective at inhibiting NF‐κB nuclear translocation. Biodistribution and pharmacokinetics comparing intravenous and intraarterial injection of these ELP‐NF‐κB peptides have been determined using healthy Wistar rats and those subjected to the middle cerebral artery occlusion (MCAO) model of stroke. In conclusion, we developed a peptide to inhibit NF‐κB nuclear translocation that we hypothesize will inhibit inflammatory processes following stroke. Future studies will test the efficacy in reducing infarct size using MCAO in rats.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NF-κB as the mediator of metformin's effect on ageing and ageing-related diseases.

Ageing can be defined as the progressive failure of repair and maintenance systems with a consequent accumulation of cellular damage in nucleic acids, proteins, and lipids. These various types of damage promote ageing by driving cellular senescence and apoptosis. The nuclear factor-kappa B (NF-kB) pathway is one of the key mediators of ageing and this pathway is activated by genotoxic, oxidative and inflammatory stress, and regulates expression of cytokines, growth factors, and genes that regulate apoptosis, cell-cycle progression, and inflammation. Therefore, NF-kB is increased in a variety of tissues with ageing, thus the inhibition of NF-kB leads to delayed onset of ageing-related symptoms and pathologies such as diabetes, atherosclerosis, and cancer. Metformin is often used as an anti-diabetic medication in type 2 diabetes throughout the world and appears to be a potential anti-ageing agent. Owing to its antioxidant, anticancer, cardio-protective and anti-inflammatory properties, metformin has become a potential candidate drug, improving in the context of ageing and ageing-related diseases. An inappropriate NF-kB activation is associated with diseases and pathologic conditions which can impair the activity of genes involved in cell senescence, apoptosis, immunity, and inflammation. Metformin, inhibiting the expression of NF-kB gene, eliminates the susceptibility to common diseases. This review underlines the pleiotropic effects of metformin in ageing and different ageing-related diseases and attributes its effects to the modulation of NF-kB.

Novel Quinolone Substituted Quinazolin-4(3H)-Ones as Anti-Inflammatory, Anticancer Agents: Synthesis and Biological Screening

Background: Quinolones and, quinazolinones are important pharmacodynamic heterocyclic nuclei which when incorporated into different heterocyclic templates,have been reported to possess potent anti-inflammatory and anticancer properties. The activity of these compounds were associated with inhibition of nuclear factorkappaB (NF-κB) which is one of the important targets studied for designing of antiinflammatory and antitumor drug. Further, the combination of two pharmacophores on the same molecule is a well-established approach for designing of potent molecules and may further enhance their activity. Objectives: Aim of the study is to synthesis a series of quinolone substituted quinazolinones and evaluation of their anti-inflammatory and anticancer activity. Methods: Quinolone substituted quinazolin-4(3H)-ones were synthesized in two steps, first step involves synthesis of 7-amino-4-methyl-quinolin- 2(1H)-one. Second step involves aminolysis of 7-amino-4-methyl-quinoline-2(1H)-one with substituted benzoxazines afforded quinolone substituted quinazolinones. Structures of synthesized compounds were characterized by spectral techniques. The synthesized compounds were evaluated for anti-inflammatory activity by carrageenan-induced rat paw oedema test and anticancer activity was assessed by evaluating the cytotoxic effect of synthesized compounds on BT-549 and HeLa, human cancer cell lines by MTT assay. Results: The synthesized compounds; 6, 7-dimethoxy-2-methyl-3-(4-methyl- 2-oxo-1, 2- ihydroquinolin-7-yl)quinazolin-4(3H)-one (6d) was the most cytotoxic compound against HeLa and BT-549 human cancer cell lines. While the compounds 6,8-dibromo-2-methyl-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)quinazolin-4(3H)-one (6f) and 2-methyl-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)-7-nitroquinazolin-4(3H)- one (6g) exhibited the highest anti- Inflammatory activity in carrageenan-induced paw oedema model. Conclusion: Molecular docking studies revealed that combination of two pharmacophores was crucial for binding of quinolone substituted quinazolin-4(3H)-ones on NF-κB and good correlation was observed between docking scores and biological activity of synthesized compounds.

Ambroxol attenuates cisplatin-induced hepatotoxicity and nephrotoxicity via inhibition of p-JNK/p-ERK.

Hepatotoxicity and nephrotoxicity are important drawbacks of cisplatin. The objective of this study is to evaluate the ability of ambroxol in 2 different doses (35 and 70 mg/kg, i.p.) to protect liver and kidney from damage induced by a single dose of cisplatin (10 mg/kg, i.p.) in comparison with N-acetylcysteine (250 mg/kg, i.p.). Inflammatory, oxidative stress, and apoptotic biomarkers were investigated to show the influence of ambroxol on hepatotoxicity and nephrotoxicity. Ambroxol decreased the elevated activity of liver enzymes (aspartate aminotransferase and alanine aminotransferase) and kidney function tests (blood urea nitrogen and creatinine). Ambroxol mitigated cisplatin inflammatory damage by inhibition of tumor necrosis factor-α, interleukin-1β, and nuclear factor kappa-B and elevation of nuclear factor erythroid 2-related factor 2. Moreover, ambroxol inhibited oxidative damage indicated by reduction of malondialdehyde and replenished the store of reduced glutathione likely by upregulating glutathione reductase and superoxide dismutase. Elevation of phosphorylated c-Jun N-terminal kinases (p-JNK) and phosphorylated extracellular signal-regulated kinase (p-ERK) were attenuated by ambroxol associated with a decrease in the expression of caspase-3; these results were consistent with histopathological results. These results recommend ambroxol to be co-administered with cisplatin in cancer patients to ameliorate liver and kidney damage, and this was confirmed by MTT assay.

Galgeun-tang Attenuates Cigarette Smoke and Lipopolysaccharide Induced Pulmonary Inflammation via IκBα/NF-κB Signaling.

Galgeun-tang water extract (GGWE) is used to treat various diseases such as the common cold, eczema and asthma in China and Korea. In this study, we investigated the anti-inflammatory effect of GGWE using a cigarette smoke (CS)- and lipopolysaccharide (LPS)-induced induced pulmonary inflammation mouse model. The mice were exposed to CS for a total of seven days (eight cigarettes per day for 1 h) and LPS was administered intranasally to mice on day 4. GGWE was administered by oral gavage at doses of 50 mg/kg or 100 mg/kg 1 h before exposure to CS. GGWE decreased inflammatory cell counts, and expression of inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α) in bronchoalveolar lavage fluid (BALF) from mice exposed to CS and LPS. GGWE reduced the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), as well as the phosphorylation of inhibitor of kappa-B subunit alpha (IκBα) and nuclear factor kappa-B (NF-κB) in CS- and LPS-exposed mice. Histological examinations revealed that GGWE suppressed inflammatory cell infiltration into lung tissue compared to untreated CS- and LPS-exposed mice. In conclusion, GGWE effectively suppressed CS- and LPS-induced pulmonary inflammation. Our results indicate that GGWE may be used as a protective drug to control pulmonary inflammation diseases such as chronic obstructive pulmonary disease.

Abstract 3513: Curcumin induces a fatal energetic impairment by inhibiting ATP-synthase activity and decreasing ATP generation and oxygen consumption in in vitro and in vivo tumor models

Abstract Curcumin has been reported to inhibit inflammation, tumor growth, angiogenesis and metastasis by decreasing cell growth and by inducing apoptosis mainly through the inhibition of nuclear factor kappa-B (NFkB), a master regulator of inflammation. Recent reports also indicate potential metabolic effects of the polyphenol and we therefore analyzed whether and how it affects the energy metabolism of tumor cells. We show that curcumin inhibits the activity of ATP-synthase in isolated mitochondrial membranes leading to a dramatic drop of ATP and a reduction of oxygen consumption in in vitro in several murine tumor cell lines (CT26 colon cancer, B16 melanoma, L1210 lymphocytic leukemia, 4T1 breast cancer) and in vivo in syngeneic tumor models. The effects of curcumin on ATP-synthase are independent of the inhibition of nuclear factor kB (NFkB) since the IkB Kinase inhibitor, SC-514, inhibits the expression of the NFkB target gene, BCL2, but does not affect the activity of the ATP-synthase. The activities of the glucose metabolism enzymes hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase are only slightly affected in a cell type specific manner. The energy impairment translates into decreased tumor cell viability. Apoptosis is induced by promoting the generation of reactive oxygen species and malondialdehyde (MDA), a marker of lipid oxidation. Tumor autophagy is induced by curcumin at least in part due to the activation of the AMP-activated protein kinase (AMPK). These activities translate into a significant delay of in vivo tumor growth likely due to a reduction of tumor angiogenesis since we observe reduced number and size of tumor vessels in vivo. These results establish the ATP-synthase, a central enzyme of the cellular energy metabolism, as a target of the anti-tumoral polyphenol leading to inhibition of cancer cell growth and a general reprogramming of tumor metabolism. Citation Format: Ulrich Pfeffer, Giovanna Bianchi, Silvia Ravera, Chiara Traverso, Adriana Amaro, Francesca Piaggio, Laura Emionite, Tiziana Bacchetti, Lizzia Raffaghello. Curcumin induces a fatal energetic impairment by inhibiting ATP-synthase activity and decreasing ATP generation and oxygen consumption in in vitro and in vivo tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3513.

Curcumin induces a fatal energetic impairment in tumor cells in vitro and in vivo by inhibiting ATP-synthase activity.

Curcumin has been reported to inhibit inflammation, tumor growth, angiogenesis and metastasis by decreasing cell growth and by inducing apoptosis mainly through the inhibition of nuclear factor kappa-B (NFκB), a master regulator of inflammation. Recent reports also indicate potential metabolic effects of the polyphenol, therefore we analyzed whether and how it affects the energy metabolism of tumor cells. We show that curcumin (10 µM) inhibits the activity of ATP synthase in isolated mitochondrial membranes leading to a dramatic drop of ATP and a reduction of oxygen consumption in in vitro and in vivo tumor models. The effects of curcumin on ATP synthase are independent of the inhibition of NFκB since the IκB Kinase inhibitor, SC-514, does not affect ATP synthase. The activities of the glycolytic enzymes hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase are only slightly affected in a cell type-specific manner. The energy impairment translates into decreased tumor cell viability. Moreover, curcumin induces apoptosis by promoting the generation of reactive oxygen species (ROS) and malondialdehyde (MDA), a marker of lipid oxidation, and autophagy, at least in part due to the activation of the AMP-activated protein kinase (AMPK). According to the in vitro anti-tumor effect, curcumin (30 mg/kg body weight) significantly delayed in vivo cancer growth likely due to an energy impairment but also through the reduction of tumor angiogenesis. These results establish the ATP synthase, a central enzyme of the cellular energy metabolism, as a target of the antitumoral polyphenol leading to inhibition of cancer cell growth and a general reprogramming of tumor metabolism.

Anti-inflammatory Effects of Cardamonin in Ovarian Cancer Cells Are Mediated via mTOR Suppression.

Cardamonin exhibits a variety of pharmacological activities including anti-inflammatory and antitumor, which are correlated with the inhibition of nuclear factor-kappaB and the mammalian target of rapamycin, respectively. However, whether the anti-inflammatory effects of cardamonin are mediated by the mammalian target of rapamycin remains unknown. In this study, ovarian cancer SKOV3 cells were cultured with lipopolysaccharide to induce inflammation, and the inhibitory effects and underlying molecular mechanisms of cardamonin were investigated using specific inhibitors of the mammalian target of rapamycin and the nuclear factor-kappaB pathway (rapamycin and pyrrolidine dithiocarbamate, respectively). Our results indicated that cardamonin inhibited the viability of normal and lipopolysaccharide-pretreated SKOV3 cells in a concentration-dependent manner. In accordance with rapamycin, the activation of the mammalian target of rapamycin and its downstream target, ribosomal protein S6 kinase 1, was inhibited by cardamonin, while pyrrolidine dithiocarbamate substantially blocked nuclear factor-kappaB activation and mildly inhibited the phosphorylation of the mammalian target of rapamycin and ribosomal protein S6 kinase 1. Pretreated with pyrrolidine dithiocarbamate, the effect of cardamonin on the mammalian target of rapamycin signalling was not affected, but the expression of inflammatory factors was further reduced. In cells pretreated with rapamycin, the inhibitory effects of cardamonin were completely suppressed with regards to the phosphorylation of the mammalian target of rapamycin, ribosomal protein S6 kinase 1, TNF-α, and interleukin-6, and nuclear factor-kappaB p65 protein expression was decreased. In conclusion, our findings indicate that the anti-inflammatory effects of cardamonin are correlated with mammalian target of rapamycin inhibition.

Lipopolysaccharide causes NFĸB-mediated induction of inducible nitric oxide synthase gene and more production of nitric oxide in air-breathing catfish, Clarias magur (Hamilton)

The magur catfish (Clarias magur) is a facultative air-breather and regularly encounters with various environmental changes along with exposure to various bacterial pathogens in its natural habitats. Occurrence of various biochemical adaptational strategies related to nitrogen metabolism in magur catfish is already known. The present investigation aimed at determining the possible induction of inducible nitric oxide synthase (inos) gene and stimulation of nitric oxide (NO) production in this catfish while challenging with lipopolysaccharide (LPS, a bacterial endotoxin) treatment, and also to determine the involvement of nuclear factor kappa B (NFkB) in induction of inos gene. Intra-peritoneal injection of LPS led to more production and accumulation of NO in different body tissues of magur catfish as a consequence of induction of iNOS activity. The induction of iNOS activity was associated with the induction of inos gene as evidenced by more expression of inos mRNA and more abundance of iNOS enzyme protein in different tissues of magur catfish with certain variations in zonal specific expression patterns. Similar observations related to more production of NO and induction of inos gene were also made when the isolated hepatocytes were treated with LPS in vitro condition. LPS treatment also led to activation of NFĸB in hepatic cells. However, in presence of a specific inhibitor of NFkB, the LPS-mediated induction of inos gene and extra production of NO were almost blocked, thereby suggesting that the induction of inos gene due to LPS treatment was mediated via the NFkB in magur catfish. It is hypothesized that the induction of iNOS activity, and more synthesis and accumulation of NO could serve as indicators to determine the pathophysiological conditions of the fish living in bacterial contaminated water bodies. Further, it can be contemplated that more synthesis of NO through iNOS enzyme probably serves as an important pharmacological tool against fish pathogen and also plays an important role in host defense mechanisms in this unique group of magur catfish.

Novel angiotensin receptor blocker, azilsartan induces oxidative stress and NFkB-mediated apoptosis in hepatocellular carcinoma cell line HepG2

Overexpression of renin angiotensin system (RAS) components and nuclear factor-kappa B (NF-kB) has a key role in various cancers. Blockade of RAS and NF-kB pathway has been suggested to reduce cancer cell proliferation. This study aimed to investigate the role of angiotensin II and NF-kB pathway in liver hepatocellular carcinoma cell line (HepG2) proliferation by using azilsartan (as a novel Ag II antagonist) and Bay 11-7082 (as NF-kB inhibitor). HepG2 cells were treated with different concentrations of azilsartan and Bay 11-7082. Cytotoxicity was determined after 24, 48, and 72 h by MTT assay. Reactive oxygen spices (ROS) generation and cytochrome c release were measured following azilsartan and Bay11- 7082 treatment. Apoptosis was analyzed qualitatively by DAPI staining and quantitatively through flow cytometry methodologies and Bax and Bcl-2 mRNA and protein levels were assessed by real time PCR and ELISA methods, respectively. The cytotoxic effects of different concentration of azilsartan and Bay11- 7082 on HepG2 cells were observed as a reduction in cell viability, increased ROS formation, cytochrome c release and apoptosis induction. These effects were found to correlate with a shift in Bax level and a downward trend in the expression of Bcl-2. These findings suggest that azilsartan and Bay11- 7082 in combination or alone have strong potential as an agent for prevention or treatment of liver cancer after further studies.

Renin-angiotensin system inhibition ameliorates CCl4-induced liver fibrosis in mice through the inactivation of nuclear transcription factor kappa B.

Therapeutic interventions for liver fibrosis are still limited due to the complicated molecular pathogenesis. Renin-angiotensin system (RAS) seems to contribute to the development of hepatic fibrosis. Therefore, we aimed to examine the effect of RAS inhibition on CCl4-induced liver fibrosis. Mice were treated with silymarin (30 mg·kg-1), perindopril (1 mg·kg-1), fosinopril (2 mg·kg-1), or losartan (10 mg·kg-1). The administration of RAS inhibitors improved liver histology and decreased protein expression of alpha smooth muscle actin (α-SMA) and hepatic content of hydroxyproline. These effects found to be mediated via inactivation of nuclear transcription factor kappa B (NFκB) pathway by the inhibition of NFκB p65 phosphorylation at the Ser536 residue and phosphorylation-induced degradation of nuclear factor kappa-B inhibitor alpha (NFκBia) subsequently inhibited NFκB-induced TNF-α and TGF-β1, leading to lower levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and vascular endothelial growth factor (VEGF). We concluded that the tissue affinity of the angiotensin converting enzyme inhibitors (ACEIs) has no impact on its antifibrotic activity and that interfering the RAS either through the inhibition of ACE or the blockade of AT1R has the same therapeutic benefit. These results suggest RAS inhibitors as promising candidates for further clinical trials in the management of hepatic fibrosis.