Inhibition of IκB kinase-β and IκB kinase-α by heterocyclic adamantyl arotinoids

We recently reported on a series of retinoid-related molecules containing an adamantyl group, a.k.a. adamantyl arotinoids (AdArs), that showed significant cancer cell growth inhibitory activity and activated RXRα (NR2B1) in transient transfection assays while devoid of RAR transactivation capacity. We have now explored whether these AdArs could also bind and inhibit IKKβ, a known target that mediates the induction of apoptosis and cancer cell growth inhibition by related AdArs containing a chalcone functional group. In addition, we have prepared and evaluated novel AdArs that incorporate a central heterocyclic ring connecting the adamantyl-phenol and the carboxylic acid at the polar termini. Our results indicate that the majority of the RXRα activating compounds lacked IKKβ inhibitory activity. In contrast, the novel heterocyclic AdArs containing a thiazole or pyrazine ring linked to a benzoic acid motif were potent inhibitors of both IKKα and IKKβ, which in most cases paralleled significant growth inhibitory and apoptosis inducing activities.     

ZIP8 Regulates Host Defense through Zinc-Mediated Inhibition of NF-κB

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Highlights? NF-κB regulates expression of the zinc transporter SLC39A8 (ZIP8) ? ZIP8 negatively regulates NF-κB through zinc-mediated down-modulation of IKK activity ? Zinc inhibits IKKβ upon binding to a specific site located in the kinase domain ? Fetal fibroblasts from Slc39a8 mutant mice cannot control NF-κB activation     

Inhibition of active HIV-1 replication by NF-κB inhibitor DHMEQ

Previous reports indicate that nuclear factor (NF)-κB regulates induction of human immunodeficiency virus type 1 (HIV-1) gene expression in latently infected cells. However, the role of NF-κB in cells with active HIV-1 replication is not well understood. In this study, we examined the effect of a new NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on HIV-1 replication in a human T cell line and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PHA-PBMCs). We further explored the mechanism of DHMEQ-mediated inhibition of HIV-1 replication. DHMEQ inhibited HIV-1 replication in HIV-1-infected Molt-4 and PHA-PBMCs. DHMEQ inhibited constitutive NF-κB activity in HIV-1-infected PHA-PBMCs and HIV long terminal repeat promoter activity driven by tumor necrosis factor (TNF)-α and the trans-activator Tat. The single-round assay using vesicular stomatitis virus-pseudotyped virus in the human T cell line M8166 indicated that DHMEQ treatment resulted in decreased integration of HIV-1 provirus into the host genome and decreased HIV-1 expression. These results indicate that NF-κB regulates early events as well as the initial and accelerated expression of HIV-1 in its life cycle. Therefore, we conclude that NF-κB is a molecular target for controlling active HIV-1 replication.     

Inhibition of matrix metalloproteinase expression and cellular invasion by NF-κB inhibitors of microbial origin

Matrix metalloproteinases (MMPs) are zinc-dependent extracellular matrix remodeling endopeptidases. MMPs cleave various matrix proteins such as collagen, elastin, gelatin and casein. MMPs are often implicated in pathological processes, such as cancer progression including metastasis. Meanwhile, microorganisms produce various secondary metabolites having unique structures. We designed and synthesized dehydroxymethylepoxyquinomicin (DHMEQ) based on the structure of epoxyquinomicin C derived from Amycolatopsis as an inhibitor of NF-κB. This compound inhibited cancer cell migration and invasion. Since DHMEQ is comparatively unstable in the body, we designed and synthesized a stable DHMEQ analog, SEMBL. SEMBL also inhibited cancer cell migration and invasion. We also looked for inhibitors of cancer cell migration and invasion from microbial culture filtrates. As a result, we isolated a known compound, ketomycin, from Actinomycetes. DHMEQ, SEMBL, and ketomycin are all NF-κB inhibitors, and inhibited the expression of MMPs in the inhibition of cellular migration and invasion. These are all compounds with comparatively low toxicity, and may be useful for the development of anti-metastasis agents.     

Inhibition of NF-κB by MG132 through ER stress-mediated induction of LAP and LIP

Proteasome inhibitor MG132 blocks activation of NF-κB by preventing degradation of IκB. In this report, we propose an alternative mechanism by which MG132 inhibits cytokine-triggered NF-κB activation. We found that MG132 induced endoplasmic reticulum (ER) stress, and attenuation of ER stress blunted the suppressive effect of MG132 on NF-κB. Through ER stress, MG132 up-regulated C/EBPβ mRNA transiently and caused sustained accumulation of its translational products liver activating protein (LAP) and liver-enriched inhibitory protein (LIP), both of which were identified as suppressors of NF-κB. Our results disclosed a novel mechanism underlying inhibition of NF-κB by MG132.     

Post-Natal Inhibition of NF-κB Activation Prevents Renal Damage Caused by Prenatal LPS Exposure

Prenatal exposure to an inflammatory stimulus has been shown to cause renal damage in offspring. Our present study explored the role of intra-renal NF-κB activation in the development of progressive renal fibrosis in offspring that underwent prenatal exposure to an inflammatory stimulus. Time-dated pregnant rats were treated with saline (control group) or 0.79 mg/kg lipopolysaccharide (LPS) through intra-peritoneal injection on gestational day 8, 10 and 12. At the age of 7 weeks, offspring from control or LPS group were treated with either tap water (Con+Ve or LPS+Ve group) or pyrollidine dithiocarbamate (PDTC, 120mg/L), a NF-κB inhibitor, via drinking water starting (Con+PDTC or LPS+PDTC group), respectively, till the age of 20 or 68 weeks. The gross structure of kidney was assessed by hematoxylin-eosin, periodic acid–Schiff staining and Sirius red staining. The expression levels of TNF-α, IL-6, α-smooth muscle actin (α-SMA) and renin-angiotensin system (RAS) genes were determined by real time polymerase chain reaction and/or immunohistochemical staining. Our data showed that post-natal persistent PDTC administration efficiently repressed intra-renal NF-κB activation, TNF-α and IL-6 expression. Post-natal PDTC also prevented intra-renal glycogen deposition and collagenous fiber generation as evident by the reduced expression of collagen III and interstitial α-SMA in offspring of prenatal LPS exposure. Furthermore, post-natal PDTC administration reversed the intra-renal renin-angiotensin system (RAS) over-activity in offspring of prenatal LPS exposure. In conclusion, prenatal inflammatory exposure results in offspring’s intra-renal NF-κB activation along with inflammation which cross-talked with excessive RAS activation that caused exacerbation of renal fibrosis and dysfunction in the offspring. Thus, early life prevention of NF-κB activation may be a potential preventive strategy for chronic renal inflammation and progressive renal damage.     

AMPK Inhibition Blocks ROS-NFκB Signaling and Attenuates Endotoxemia-Induced Liver Injury

Background

AMP-activated protein kinase (AMPK) is an important enzyme in regulation of cellular energy homeostasis. We have previously shown that AMPK activation by 5-aminoimidazole-4-carboxamide (AICAR) results in suppression of immune responses, indicating the pivotal role of AMPK in immune regulation. However, the cellular mechanism underpinning AMPK inhibition on immune response remains largely to be elucidated. The study aimed to investigate the effects of AMPK inhibition on reactive oxygen species (ROS)-nuclear factor κB (NFκB) signaling and endotoxemia-induced liver injury.

Methodology/Principal Findings

RAW 264.7 cells were pretreated with AMPK activator or inhibitor, followed by LPS challenge. In addition, LPS was injected intraperitoneally into mice to induce systemic inflammation. The parameters of liver injury and immune responses were determined, and survival of mice was monitored respectively. LPS challenge in RAW 264.7 cells resulted in AMPK activation which was then inhibited by compound C treatment. Both AMPK activation by AICAR or inhibition by compound C diminished LPS-induced ROS generation, inhibited phosphorylation of IKK, IκB, and NFκB p65, and consequently, decreased TNF production of RAW 264.7 cells. AICAR or compound C treatment decreased ALT, AST, and TNF levels in serum, reduced CD68 expression and MPO activity in liver tissue of mice with endotoxemia. Moreover, AICAR or compound C treatment improved survival of endotoxemic mice.

Conclusions

AICAR or compound C treatment attenuates LPS-induced ROS-NFκB signaling, immune responses and liver injury. Strategies to activate or inhibit AMPK signaling may provide alternatives to the current clinical approaches to inhibit immune responses of endotoxemia.     

Lipid Peroxidation Inhibition Reduces NF-κB Activation and Attenuates Cerulein-induced Pancreatitis

Increased lipid peroxidation, enhanced nuclear factor kappa-B (NF- &#115 B) activation and augmented tumor necrosis factor- &#102 (TNF- &#102 ) production have been implicated in cerulein-induced pancreatitis. We investigated whether lipid peroxidation inhibition might reduce NF- &#115 B activation and the inflammatory response in cerulein-induced pancreatitis. Male Sprague-Dawley rats of 230-250 g body weight received administration of cerulein (80 &#119 g/kg s.c. for each of four injections at hourly intervals). A control group received four s.c. injections of 0.9% saline at hourly intervals. Animals were randomized to receive either raxofelast, an inhibitor of lipid peroxidation (20 mg/kg i.p. administered with the first cerulein injection) or its vehicle (1 ml/kg of a 10% DMSO/NaCl solution). All these rats were sacrificed 2 h after the last injection of either cerulein or its vehicle. Raxofelast administration (20 mg/kg i.p. with the first cerulein) significantly reduced malondialdehyde (MDA) levels, an index of lipid peroxidation (CER+DMSO=3.075 &#45 0.54 &#119 mol/g; CER+raxofelast= 0.693 &#45 0.18 &#119 mol/g; p <0.001 ), decreased myeloperoxidase (MPO) activity ( CER+DMSO=22.2 &#45 3.54 mU/g; CER+raxofelast=9.07 &#45 2.05 mU/g; p <0.01 ), increased glutathione levels (GSH) (CER+DMSO= 5.21 &#45 1.79 &#119 mol/g; CER+raxofelast=15.71 &#45 2.14 &#119 mol/g; p <0.001 ), and reduced acinar cell damage evaluated by means of histology and serum levels of both amylase ( CER+DMSO=4063 &#45 707.9 U/l; CER+raxofelast=1198 &#45 214.4 U/l; p <0.001 ), and lipase (CER+DMSO=1654 &#45 330 U/l; CER+raxofelast= 386 &#45 118.2 U/l; p <0.001 ), Furthermore, raxofelast reduced pancreatic NF- &#115 B activation and the TNF- &#102 mRNA levels and tissue content of mature protein in the pancreas. Indeed, lipid peroxidation inhibition might be considered a potential therapeutic approach to prevent the severe damage in acute pancreatitis.