During ischemia-reperfusion injury, brief pre-exposure to oxidative stress renders organs resistant to subsequent severe damage. NF-kappaB is a transcription factor that is involved in reperfusion-induced inflammatory and immune responses. The activity of NF-kappaB has been shown to be modulated by oxidative stress in various cell types through different pathways. We studied the effect of pre-exposure to oxidative stress on subsequent NF-kappaB activation in TNFalpha-stimulated HEK293 cells. The cells were transiently exposed to 0.5 mM H2O2 for 20 min, prior to stimulation with TNFalpha, and the subsequent expression of NF-kappaB-dependent genes and the levels of NF-kappaB signaling molecules were measured. Pre-exposure to H2O2 significantly delayed the TNFalpha-induced expression of an NF-kappaB reporter gene and inflammatory proteins (intercellular adhesion molecule-1 and IL-1beta). The degradation of inhibitor of NF-kappaB alpha (IkappaBalpha) and the nuclear translocation of NF-kappaB were also delayed by H2O2 treatment, whereas IkappaBalpha phosphorylation and IkappaB kinase activity were not changed. When we examined the ubiquitin/proteosome pathway in H2O2-treated cells, we could not detect significant changes in proteosomal peptidase activities, but we were able to detect a delay of IkappaBalpha poly-ubiquitination. Our results suggest that transient exposure to oxidative stress temporally inhibits NF-kappaB-dependent gene expression by suppressing the poly-ubiquitination of phosphorylated IkappaBalpha in HEK293 cells.
Active Transport, Cell Nucleus ; Cell Nucleus/metabolism ; Enzyme Activation/drug effects ; HEK293 Cells ; Humans ; Hydrogen Peroxide/*pharmacology ; I-kappa B Kinase/antagonists & inhibitors/*metabolism ; Phosphorylation/drug effects ; Protein Transport ; Tumor Necrosis Factor-alpha/*pharmacology ; Ubiquitination/*drug effects

Carbon monoxide (CO), as a vital small molecule in signaling pathways, is found to be involved in ischemia-reperfusion injury (IRI) in renal transplantation. CO-releasing molecule-2 (CORM-2), a CO-releasing molecule, is a type of metal carbonyl complexes which can quickly release CO in vivo. In this study, an in vitro oxidative stress injury model was established to examine the effect of CORM-2 pretreatment on the nuclear-cytoplasmic translocation of high mobility group box 1 protein (HMGB1) in mouse primary renal proximal tubular epithelial cells (RPTECs). Immunofluorescence staining showed that HMGB1 in the medium- and CORM-2-treated groups was predominantly localized in the nucleus of the cells, whereas higher amounts of HMGB1 translocated to the cytoplasm in the HO- and inactive CORM-2 (iCORM-2)-treated groups. Western blotting of HMGB1 showed that the total amounts of cytoplasmic HMGB1 in the HO-treated (0.59±0.27) and iCORM-2-treated (0.57±0.22) groups were markedly higher than those in the medium-treated (0.19±0.05) and CORM-2-treated (0.21±0.10) groups (P<0.05). Co-immunoprecipitation showed that the levels of acetylated HMGB1 in the HO-treated (642.98±57.25) and iCORM-2-treated (342.11±131.25) groups were markedly increased as compared with the medium-treated (78.72±74.17) and CORM-2-treated (71.42±53.35) groups (P<0.05), and no significant difference was observed between the medium-treated and CORM-2-treated groups (P>0.05). In conclusion, our study demonstrated that in the in vitro oxidative stress injury model of primary RPTECs, CORM-2 can significantly inhibit the nuclear-cytoplasmic translocation of HMGB1, which is probably associated with the prevention of HMGB1 acetylation.
Active Transport, Cell Nucleus ; drug effects ; Animals ; Carbon Monoxide ; pharmacology ; Cell Nucleus ; metabolism ; Cells, Cultured ; Epithelial Cells ; drug effects ; metabolism ; HMGB1 Protein ; metabolism ; Kidney Tubules ; cytology ; Mice ; Organometallic Compounds ; pharmacology ; Oxidative Stress

Wound healing is initiated and progressed by complex integrated process of cellular, physiologic, and biochemical events, such as inflammation, cell migration and proliferation. Interleukin 6 (IL-6) is a multifunctional cytokine, and it could regulate the inflammatory response of wound healing process in a timely manner. Hyaluronic acid (HA) is an essential component of the extracellular matrix, and contributes significantly to cell proliferation and migration. The purpose of this study was to investigate the effects of IL-6 or/and HA on the cell migration process in human keratinocytes. Combining IL-6 and HA significantly increased the cell migration in scratch based wound healing assay. The phosphorylation of extracellular-signal-regulated kinase (ERK) was significantly increased after 1 hr of IL-6 and HA treatment, but the phosphorylation of p38 mitogen-activated protein kinase (MAPK) was not. We also found that significant increase of the NF-kappaB translocation from cytoplasm into nucleus after 1 hr of IL-6 or/and HA treatments. This study firstly showed that synergistic effects of combining IL-6 and HA on the cell migration of wound healing by activation of ERK and NF-kappaB signaling. Further studies might be required to confirm the synergistic effects of HA and IL-6 in the animal model for the development of a novel therapeutic mixture for stimulation of wound healing process.
Active Transport, Cell Nucleus/drug effects ; Cell Line ; Cell Movement/*drug effects ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Enzyme Activation/drug effects ; Extracellular Signal-Regulated MAP Kinases/*metabolism ; Humans ; Hyaluronic Acid/*pharmacology ; Interleukin-6/*pharmacology ; Keratinocytes/*metabolism ; MAP Kinase Signaling System/drug effects ; NF-kappa B/metabolism ; Phosphorylation/drug effects ; Protein Transport/drug effects ; Wound Healing ; p38 Mitogen-Activated Protein Kinases/metabolism

Here, we report that B-cell lymphoma 2 (Bcl-2) is a novel target molecule of aspirin in breast cancer cells. Aspirin influenced the formation of a complex by Bcl-2 and FKBP38 and induced the nuclear translocation of Bcl-2 and its phosphorylation. These events inhibited cancer cell proliferation and subsequently enhanced MCF-7 breast cancer cell apoptosis. Bcl-2 knockdown using small interfering RNA (siRNA) delayed apoptotic cell death, which correlated with increased proliferation following aspirin exposure. In contrast, Bcl-2 overexpression enhanced the onset of aspirin-induced apoptosis, which was also associated with a significant increase in Bcl-2 phosphorylation in the nucleus. Therefore, this study may provide novel insight into the molecular mechanism of aspirin, particularly its anticancer effects in Bcl-2- and estrogen receptor-positive breast cancer cells.
Active Transport, Cell Nucleus/drug effects ; *Apoptosis ; Aspirin/*pharmacology ; Cell Nucleus/*metabolism ; Humans ; MCF-7 Cells ; Phosphorylation ; Protein Binding ; Proto-Oncogene Proteins c-bcl-2/genetics/*metabolism ; Tacrolimus Binding Proteins/metabolism

OBJECTIVETo study the effect of eucalyptus globulus oil on the activity of nuclear factor-kappaB(NF-kappaB) in THP-1 cell line.

METHODSTHP-1 cells were cultured with or without eucalyptus globulus oil at different concentrations (1, 10, 100 mg x L(-1), 30 min) before being stimulated with lipopolysaccharide (LPS, 1 mg x L(-1), 30 min). The location of NF-kappaB p65 subunit (NF-kappaB/p65) in THP-1 cells was detected by indirect immunofluorescence and laser scanning confocal microscope. The expression of NF-kappaB/p65 in nuclei was measured by Western-blot analysis.

RESULTThe FITC-label NF-kappaB/p65 was mainly located in the nuclei after THP-1 cells were stimulated with LPS. Whereas, no fluorescence were seen in the nuclei of cells pretreated with eucalyptus globulus oil. This effect on NF-kappaB/p65 nuclear translocation was in a concentration dependent manner.

CONCLUSIONEucalyptus globulus oil inhibits the nuclear translocation of NF-kappaB induced by LPS in THP-1 cells.

Active Transport, Cell Nucleus ; drug effects ; Blotting, Western ; Cell Line ; Dose-Response Relationship, Drug ; Eucalyptus ; chemistry ; Humans ; Lipopolysaccharides ; pharmacology ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Plant Oils ; pharmacology ; Tosyllysine Chloromethyl Ketone ; pharmacology

Annexin A2, a multifunctional tumor associated protein, promotes nuclear factor-kappa B (NF-κB) activation by interacting with NF-κB p50 subunit and facilitating its nuclear translocation. Here we demonstrated that two ginsenosides Rg5 (G-Rg5) and Rk1 (G-Rk1), with similar structure, directly bound to Annexin A2 by molecular docking and cellular thermal shift assay. Both Rg5 and Rk1 inhibited the interaction between Annexin A2 and NF-κB p50 subunit, their translocation to nuclear and NF-κB activation. Inhibition of NF-κB by these two ginsenosides decreased the expression of inhibitor of apoptosis proteins (IAPs), leading to caspase activation and apoptosis. Over expression of K302A Annexin A2, a mutant version of Annexin A2, which fails to interact with G-Rg5 and G-Rk1, effectively reduced the NF-κB inhibitory effect and apoptosis induced by G-Rg5 and G-Rk1. In addition, the knockdown of Annexin A2 largely enhanced NF-κB activation and apoptosis induced by the two molecules, indicating that the effects of G-Rg5 and G-Rk1 on NF-κB were mainly mediated by Annexin A2. Taken together, this study for the first time demonstrated that G-Rg5 and G-Rk1 inhibit tumor cell growth by targeting Annexin A2 and NF-κB pathway, and G-Rg5 and G-Rk1 might be promising natural compounds for targeted cancer therapy.
Active Transport, Cell Nucleus ; drug effects ; Annexin A2 ; chemistry ; deficiency ; genetics ; metabolism ; Antineoplastic Agents ; chemistry ; metabolism ; pharmacology ; Apoptosis ; drug effects ; Biological Products ; chemistry ; metabolism ; pharmacology ; Cell Nucleus ; drug effects ; metabolism ; Down-Regulation ; drug effects ; Drug Discovery ; Gene Knockdown Techniques ; Ginsenosides ; chemistry ; Hep G2 Cells ; Humans ; Molecular Docking Simulation ; Molecular Targeted Therapy ; NF-kappa B p50 Subunit ; metabolism ; Protein Conformation

OBJECTIVETo study the effect of curcumin on the activity of matrix metalloproteinases (MMPs) and nuclear expression of RelA in rat hepatic stellate cells (HSCs) by activating peroxisome proliferator-activated receptor gamma (PPARgamma) signal in vitro.

METHODSHSCs were isolated from SD rats through in situ perfusion of liver, digestion with pronase E and density-gradient centrifugation with Nycodenz. The lytic HSCs were collected after treatment to extract the total protein and nucleoprotein for detecting the expression of PPARgamma, alphaSMA, collagen type I and RelA by Western blot, and the supernatant was collected to measure the activity of MMP2 and MMP 9 by gelatin zymograph method.

RESULTSThe PPARgamma expression decreased gradually with increasing of HSC activation, which was up-regulated by curcumin (P < 0.01); curcumin inhibited the expression of aSMA, the production of collagen type I, and the nuclear expression of activated RelA (P < 0.01), and elevated the activity of MMP2 and MMP9 significantly (P < 0.01). However, these effects were weakened by the PPARgamma antagonist, GW9662, significantly (P < 0.01).

CONCLUSIONBy activating PPARgamma signal transduction pathway curcumin treatment can inhibit HSC activation, increase the activity of MMP2 and MMP9 and inhibit/ interfere nuclear translocation of NFkappaB.

Active Transport, Cell Nucleus ; drug effects ; Animals ; Blotting, Western ; Cell Nucleus ; drug effects ; metabolism ; Curcumin ; pharmacology ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; PPAR gamma ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Transcription Factor RelA ; metabolism

Monocyte chemoattractant protein-1 (MCP1) plays a key role in monocyte/macrophage infiltration to the sub-endothelial space of the blood vessel wall, which is a critical initial step in atherosclerosis. In this study, we examined the intracellular signaling pathway of IL-1beta-induced MCP1 expression using various chemical inhibitors. The pretreatment of a phosphatidylcholine (PC)-specific PLC (PC-PLC) inhibitor (D609), PKC inhibitors, or an NF-kappaB inhibitor completely suppressed the IL-1beta-induced MCP1 expression through blocking NF-kappaB translocation to the nucleus. Pretreatment with inhibitors of tyrosine kinase or PLD partially suppressed MCP1 expression and failed to block nuclear NF-kappaB translocation. These results suggest that IL-1beta induces MCP1 expression through activation of NF-kappaB via the PC-PLC/PKC signaling pathway.
Active Transport, Cell Nucleus/drug effects ; Aorta/pathology ; Atherosclerosis/immunology/metabolism ; Bridged Compounds/pharmacology ; Cell Nucleus/*metabolism ; Cells, Cultured ; Chemokine CCL2/*biosynthesis ; Estrenes/pharmacology ; Genistein/pharmacology ; Humans ; Interleukin-1beta/metabolism ; Myocytes, Smooth Muscle/drug effects/immunology/*metabolism/pathology ; NF-kappa B/*metabolism ; Phospholipases/antagonists & inhibitors ; Protein-Tyrosine Kinases/antagonists & inhibitors ; Pyrrolidinones/pharmacology ; Recombinant Proteins/metabolism ; Signal Transduction/*drug effects ; Thiones/pharmacology

The HIV-1 Rev protein facilitates nuclear export of unspliced and singly spliced viral transcripts containing RRE RNA through the CRM1 export pathway. Inhibition of Rev-mediated RNA nuclear export can arrest HIV-1 transcriptional process, which clearly, reveals a target for anti-HIV drug development. In this work, a cell-based assay has been established for screening anti-HIV compounds targeting the Rev-mediated RNA nuclear export. This assay utilized a codon-optimized green fluorescent protein (GFP) as reporter gene, which expression is in a Rev-dependent manner. Any compound that inhibits the Rev-mediated RNA nuclear export is identified by reducing emission of GFP. The Z' score of this model is 0.8220. Three thousands compounds were screened and the positive rate was 9.3% with a cutoff at 50% inhibition. IMB7C7, one of the positive compounds, efficiently inhibits viral production from HIV-1 infected cells.
Active Transport, Cell Nucleus ; drug effects ; Anti-HIV Agents ; pharmacology ; Cell Nucleus ; metabolism ; Codon ; Fatty Acids, Unsaturated ; pharmacology ; Genes, Reporter ; Green Fluorescent Proteins ; genetics ; metabolism ; HEK293 Cells ; HIV-1 ; drug effects ; genetics ; High-Throughput Screening Assays ; Humans ; Karyopherins ; genetics ; metabolism ; RNA, Viral ; Receptors, Cytoplasmic and Nuclear ; genetics ; metabolism ; Transfection ; Virus Replication ; drug effects ; rev Gene Products, Human Immunodeficiency Virus ; genetics ; metabolism

Scoparone, which is a major constituent of Artemisia capillaries, has been identified as an anticoagulant, hypolipidemic, vasorelaxant, anti-oxidant and anti-inflammatory drug, and it is used for the traditional treatment of neonatal jaundice. Therefore, we hypothesized that scoparone could suppress the proliferation of VSMCs by interfering with STAT3 signaling. We found that the proliferation of these cells was significantly attenuated by scoparone in a dose-dependent manner. Scoparone markedly reduced the serum-stimulated accumulation of cells in the S phase and concomitantly increased the proportion of cells in the G0/G1 phase, which was consistent with the reduced expression of cyclin D1, phosphorylated Rb and survivin in the VSMCs. Cell adhesion markers, such as MCP-1 and ICAM-1, were significantly reduced by scoparone. Interestingly, this compound attenuated the increase in cyclin D promoter activity by inhibiting the activities of both the WT and active forms of STAT3. Similarly, the expression of a cell proliferation marker induced by PDGF was decreased by scoparone with no change in the phosphorylation of JAK2 or Src. On the basis of the immunofluorescence staining results, STAT3 proteins phosphorylated by PDGF were predominantly localized to the nucleus and were markedly reduced in the scoparone-treated cells. In summary, scoparone blocks the accumulation of STAT3 transported from the cytosol to the nucleus, leading to the suppression of VSMC proliferation through G1 phase arrest and the inhibition of Rb phosphorylation. This activity occurs independent of the form of STAT3 and upstream of kinases, such as Jak and Src, which are correlated with abnormal vascular remodeling due to the presence of an excess of growth factors following vascular injury. These data provide convincing evidence that scoparone may be a new preventative agent for the treatment of cardiovascular diseases.
Active Transport, Cell Nucleus ; Animals ; Biomarkers ; Cell Cycle Proteins/genetics/metabolism ; Cell Movement/drug effects ; Cell Proliferation/drug effects ; Cells, Cultured ; Coumarins/*pharmacology ; Gene Expression Regulation/drug effects ; Hep G2 Cells ; Humans ; Muscle, Smooth, Vascular/*cytology ; Myocytes, Smooth Muscle/*metabolism ; Proto-Oncogene Proteins c-sis/metabolism ; Rats ; STAT3 Transcription Factor/genetics/*metabolism ; Signal Transduction/drug effects ; Transcription, Genetic