BACKGROUND: Abnormal upregulation of prostaglandin E₂ (PGE₂) is considered to be a key oncogenic event in the development and progression of inflammation-associated human colon cancer. It has been reported that 15-hydroxyprostaglandin dehydrogenase (15-PGDH), an enzyme catabolizing PGE₂, is ubiquitously downregulated in human colon cancer. 15-Deoxy-Δ(12,14)-prostaglandin J₂ (15d-PGJ₂), a peroxisome proliferator-activated receptor γ ligand, has been shown to have anticarcinogenic activities. In this study, we investigate the effect of 15d-PGJ₂ on expression of 15-PGDH in human colon cancer HCT116 cells. METHODS: HCT116 cells were treated with 15d-PGJ₂ analysis. The expression of 15-PGDH in the treated cells was measured by Western blot analysis and RT-PCR. In addition, the cells were subjected to a 15-PGDH activity assay. To determine which transcription factor(s) and signaling pathway(s) are involved in 15d-PGJ₂-induced 15-PGDH expression, we performed a cDNA microarray analysis of 15d-PGJ₂-treated cells. The DNA binding activity of AP-1 was measured by an electrophoretic mobility shift assay. To determine whether the AP-1 plays an important role in the 15d-PGJ₂-induced 15-PGDH expression, the cells were transfected with siRNA of c-Jun, a major subunit of AP-1. To elucidate the upstream signaling pathways involved in AP-1 activation by 15d-PGJ₂, we examined its effect on phosphorylation of Akt by Western blot analysis in the presence or absence of kinase inhibitor. RESULTS: 15d-PGJ₂ (10 μM) significantly upregulated 15-PGDH expression at the mRNA and protein levels in HCT-116 cells. 15-PGDH activity was also elevated by 15d-PGJ₂. We observed that genes encoding C/EBP delta, FOS-like antigen 1, c-Jun, and heme oxygenase-1 (HO-1) were most highly induced in the HCT116 cells following 15d-PGJ₂ treatment. 15d-PGJ₂ increased the DNA binding activity of AP-1. Moreover, transfection with specific siRNA against c-Jun significantly reduced 15-PGDH expression induced by 15d-PGJ₂. 15d-PGJ₂ activates Akt and a pharmacological inhibitor of Akt, LY294002, abrogated 15d-PGJ₂-induced 15-PGDH expression. We also observed that an inhibitor of HO-1, zinc protoporphyrin IX, also abrogated upregulation of 15-PGDH and down-regulation of cyclooxygenase-2 expression induced by 15d-PGJ₂. CONCLUSIONS: These finding suggest that 15d-PGJ₂ upregulates the expression of 15-PGDH through AP-1 activation in colon cancer HCT116 cells.
Blotting, Western ; Colon ; Colonic Neoplasms ; Cyclooxygenase 2 ; DNA ; Down-Regulation ; Electrophoretic Mobility Shift Assay ; HCT116 Cells ; Heme Oxygenase-1 ; Heme ; Humans ; Oligonucleotide Array Sequence Analysis ; Oxidoreductases ; Peroxisomes ; Phosphorylation ; Phosphotransferases ; RNA, Messenger ; RNA, Small Interfering ; Transcription Factor AP-1 ; Transfection ; Up-Regulation ; Zinc

PURPOSE: To explore the molecular mechanism of the upregulation of multidrug resistance-associated protein 4 (MRP4) in cholestasis. MATERIALS AND METHODS: The mRNA and protein levels of MRP4 in liver samples from cholestatic patients were determined by quantitative real-time PCR and Western blot. In human hepatoma HepG2 cells, electrophoretic mobility shift assay (EMSA) was used to determine the affinity of nuclear factor-E2-related factor (Nrf2) binding to MRP4 promoter. Dual-luciferase reporter assay was used to detect the binding of tumor necrosis factor α (TNFα) to the promotor of E2F1. The bile duct ligation mouse models were established using male C57BL/6 mice. RESULTS: The mRNA and protein levels of MRP4 were significantly increased in cholestatic patients. TNFα treatment induced the expression of MRP4 and Nrf2 and enhanced cell nuclear extract binding activity to MRP4 promoter, as demonstrated by EMSA. Nrf2 knockdown reduced MRP4 mRNA levels in both HepG2 and Hep-3B cells. In addition, TNFα increased Rb phosphorylation and expression of MRP4 and Nrf2 and activated E2F1 and phosphorylated p38 in HepG2 and Hep-3B cells. These effects were markedly inhibited by pretreatment with E2F1 siRNA. Dual-luciferase reporter assay validated that TNFα induces the transcription of E2F1. Furthermore, the expression of MRP4, Nrf2, E2F1, and p-p38 proteins was improved with treatment of TNFα in a mouse model of cholestasis. E2F1 siRNA lentivirus or SB 203580 (p38 inhibitor) inhibited these positive effects. CONCLUSION: Our findings indicated that TNFα induces hepatic MRP4 expression through activation of the p38-E2F1-Nrf2 signaling pathway in human obstructive cholestasis.
Animals ; Bile Ducts ; Blotting, Western ; Carcinoma, Hepatocellular ; Cholestasis ; Electrophoretic Mobility Shift Assay ; Hep G2 Cells ; Humans ; Lentivirus ; Ligation ; Liver ; Male ; Mice ; Multidrug Resistance-Associated Proteins ; Phosphorylation ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; RNA, Small Interfering ; Tumor Necrosis Factor-alpha ; Up-Regulation

PURPOSE: Peroxisome proliferator-activated receptor γ (PPARγ) is involved in the pathology of numerous diseases including atherosclerosis, diabetes, obesity, and cancer. Matrix metalloproteinases (MMPs) play a significant role in tissue remodeling related to various processes such as morphogenesis, angiogenesis, tissue repair, invasion, and metastasis. We investigated the effects of PPARγ on MMP expression and invasion in breast cancer cells. METHODS: MCF-7 cells were cultured and then cell viability was monitored in an MTT assay. Western blotting, gelatin zymography, real-time polymerase chain reaction, and luciferase assays were performed to investigate the effect of the synthetic PPARγ ligand troglitazone on MMP expression. Transcription factor DNA binding was analyzed by electrophoretic mobility shift assay. A Matrigel invasion assay was used to assess the effects of troglitazone on MCF-7 cells. RESULTS: Troglitazone did not affect MCF-7 cell viability. 12-O-tetradecanoylphorbol-13-acetate (TPA) induced MMP-9 expression and invasion in MCF-7 cell. However, these effects were decreased by troglitazone. TPA increased nuclear factor κB and activator protein-1 DNA binding, while troglitazone inhibited these effects. The selective PPARγ antagonist GW9662 reversed MMP-9 inhibition by troglitazone in TPA-treated MCF-7 cells. CONCLUSION: Troglitazone inhibited nuclear factor κB and activator protein-1-mediated MMP-9 expression and invasion of MCF-7 cells through a PPARγ-dependent mechanism.
Atherosclerosis ; Blotting, Western ; Breast Neoplasms* ; Breast* ; Cell Survival ; DNA ; Electrophoretic Mobility Shift Assay ; Gelatin ; Luciferases ; Matrix Metalloproteinase 9* ; Matrix Metalloproteinases ; MCF-7 Cells ; Morphogenesis ; Neoplasm Metastasis ; NF-kappa B ; Obesity ; Pathology ; Peroxisomes* ; PPAR gamma ; Real-Time Polymerase Chain Reaction ; Transcription Factor AP-1 ; Transcription Factors

Four isoforms of calcium binding proteins containing 2 EF hand motifs and a dynein light chain-like domain in the human liver fluke Opisthorchis viverrini, namely OvCaBP1, 2, 3, and 4, were characterized. They had molecular weights of 22.7, 21.6, 23.7, and 22.5 kDa, respectively and showed 37.2–42.1% sequence identity to CaBP22.8 of O. viverrini. All were detected in 2- and 4-week-old immature and mature parasites. Additionally, OvCaBP4 was found in newly excysted juveniles. Polyclonal antibodies against each isoform were generated to detect the native proteins in parasite extracts by Western blot analysis. All OvCaBPs were detected in soluble and insoluble crude worm extracts and in the excretory-secretory product, at approximate sizes of 21–23 kDa. The ion-binding properties of the proteins were analyzed by mobility shift assays with the divalent cations Ca²⁺, Mg²⁺, Zn²⁺, and Cu²+. All OvCaBPs showed mobility shifts with Ca²⁺ and Zn²⁺. OvCaBP1 showed also positive results with Mg²⁺ and Cu²⁺. As tegumental proteins, OvCaBP1, 2, and 3 are interesting drug targets for the treatment of opisthorchiasis.
Antibodies ; Blotting, Western ; Calcium-Binding Proteins ; Cations, Divalent ; Dyneins ; EF Hand Motifs ; Electrophoretic Mobility Shift Assay ; Fasciola hepatica ; Humans ; Molecular Weight ; Opisthorchiasis ; Opisthorchis ; Parasites ; Protein Isoforms

Loss of response to anti-tumor necrosis factor (anti-TNF) agents in the treatment of inflammatory bowel disease (IBD) is a major consideration to maintain sustained response. Reversal of immunogenicity can re-establish response and increase the durability of these agents. Strategies to reverse immunogenicity include dose-intensification and/or the addition of an immunomodulator. However, there is a relative paucity of data on the efficacy of such interventions in pediatric IBD patients. Available reports have not strictly utilized homogenous mobility shift assay, which reports on anti-drug antibodies even in the presence of detectable drug, whereas prior studies have been confounded by the use of drug sensitive assays. We report four pediatric inflammatory bowel disease patients with successful reversal of immunogenicity on an anti-TNF agent using dose intensification and/or addition of an immunomodulator.
Antibodies ; Biological Products ; Child ; Electrophoretic Mobility Shift Assay ; Humans ; Inflammatory Bowel Diseases* ; Necrosis*

BACKGROUND: Oxidative stress occurs in white adipose tissue and dysregulates the expression of adipokines secreted from adipocytes. Since adipokines influence inflammation, supplementation with antioxidants might be beneficial for preventing oxidative stress-mediated inflammation in adipocytes and inflammation-associated complications. β-Carotene is the most prominent antioxidant carotenoid and scavenges reactive oxygen species in various tissues. The purpose of this study was to determine whether β-carotene regulates the expression of adipokines, such as adiponectin, monocyte chemoattractant protein-1 (MCP-1), and regulated on activation, normal T cell expressed and secreted (RANTES) in 3T3-L1 adipocytes treated with glucose/glucose oxidase (G/GO). METHODS: 3T3-L1 adipocytes were cultured with or without β-carotene and treated with G/GO, which produces H2O2. mRNA and protein levels in the medium were determined by a real-time PCR and an ELISA. DNA binding activities of transcription factors were assessed using an electrophoretic mobility shift assay. RESULTS: G/GO treatment increased DNA binding affinities of redox-sensitive transcription factors, such as NF-κB, activator protein-1 (AP-1), and STAT3. G/GO treatment reduced the expression of adiponectin and increased the expression of MCP-1 and RANTES. G/GO-induced activations of NF-κB, AP-1, and STAT3 were inhibited by β-carotene. G/GO-induced dysregulation of adiponectin, MCP-1, and RANTES were significantly recovered by treatment with β-carotene. CONCLUSIONS: β-Carotene inhibits oxidative stress-induced inflammation by suppressing pro-inflammatory adipokines MCP-1 and RANTES, and by enhancing adiponectin in adipocytes. β-Carotene may be beneficial for preventing oxidative stress-mediated inflammation, which is related to adipokine dysfunction.
Adipocytes ; Adipokines ; Adiponectin ; Adipose Tissue, White ; Antioxidants ; beta Carotene ; Chemokine CCL2 ; Chemokine CCL5 ; DNA ; Electrophoretic Mobility Shift Assay ; Enzyme-Linked Immunosorbent Assay ; Inflammation ; Oxidative Stress ; Oxidoreductases ; Reactive Oxygen Species ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; Transcription Factor AP-1 ; Transcription Factors

PURPOSE: Cockroach exposure is a pivotal cause of asthma. Tight junctions are intercellular structures required for maintenance of the barrier function of the airway epithelium, which is impaired in this disease. Matrix metalloproteinases (MMPs) digest extracellular matrix components and are involved in asthma pathogenesis: MMP1 is a collagenase with a direct influence on airway obstruction in asthmatics. This study aimed to investigate the mechanism by which German cockroach extract (GCE) induces MMP1 expression and whether MMP1 release alters cellular tight junctions in human airway epithelial cells (NCI-H292). MATERIALS AND METHODS: mRNA and protein levels were determined using real-time PCR and ELISA. Tight junction proteins were detected using immunofluorescence staining. Epithelial barrier function was measured by transepithelial electrical resistance (TEER). The binding of a transcription factor to DNA molecules was determined by electrophoretic mobility shift assay, while the levels of tight junction proteins and phosphorylation were determined using Western blotting. RESULTS: GCE was shown to increase MMP1 expression, TEER, and tight junction degradation. Both an inhibitor and small interfering RNA (siRNA) of MMP1 significantly decreased GCE-induced tight junction disruption. Furthermore, transient transfection with ETS1 and SP1 siRNA, and anti-TLR2 antibody pretreatment prevented MMP1 expression and tight junction degradation. An extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) inhibitor also blocked MMP1 release, ETS1/SP1 DNA binding, and tight junction alteration. CONCLUSION: GCE treatment increases MMP1 expression, leading to tight junction disruption, which is transcriptionally regulated and influenced by the ERK/MAPK pathway in airway epithelial cells. These findings may contribute to developing novel therapeutic strategies for airway diseases.
Airway Obstruction ; Asthma ; Blattellidae* ; Blotting, Western ; Cockroaches ; Collagenases ; DNA ; Electric Impedance ; Electrophoretic Mobility Shift Assay ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells* ; Epithelium ; Extracellular Matrix ; Fluorescent Antibody Technique ; Humans* ; Matrix Metalloproteinase 1* ; Matrix Metalloproteinases ; Phosphorylation ; Phosphotransferases ; Protein Kinases ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; RNA, Small Interfering ; Tight Junction Proteins ; Tight Junctions* ; Transcription Factors ; Transfection

Aberrant regulation of miRNA genes contributes to pathogenesis of a wide range of human diseases, including cancer. The TAR DNA binding protein 43 (TDP-43), a RNA/DNA binding protein associated with neurodegeneration, is involved in miRNA biogenesis. Here, we systematically examined miRNAs regulated by TDP-43 using RNA-Seq coupled with an siRNA-mediated knockdown approach. TDP-43 knockdown affected the expression of a number of miRNAs. In addition, TDP-43 down-regulation led to alterations in the patterns of different isoforms of miRNAs (isomiRs) and miRNA arm selection, suggesting a previously unknown role of TDP-43 in miRNA processing. A number of TDP-43 associated miRNAs, and their candidate target genes, are associated with human cancers. Our data reveal highly complex roles of TDP-43 in regulating different miRNAs and their target genes. Our results suggest that TDP-43 may promote migration of lung cancer cells by regulating miR-423-3p. In contrast, TDP-43 increases miR-500a-3p expression and binds to the mature miR-500a-3p sequence. Reduced expression of miR-500a-3p is associated with poor survival of lung cancer patients, suggesting that TDP-43 may have a suppressive role in cancer by regulating miR-500a-3p. Cancer-associated genes LIF and PAPPA are possible targets of miR-500a-3p. Our work suggests that TDP-43-regulated miRNAs may play multifaceted roles in the pathogenesis of cancer.
Animals ; Cells, Cultured ; DNA-Binding Proteins ; metabolism ; Electrophoretic Mobility Shift Assay ; Humans ; Immunoprecipitation ; Mice ; MicroRNAs ; genetics ; metabolism ; Neoplasms ; genetics ; metabolism

Among a myriad of pathogen-associated molecular pattern-sensing receptors, toll-like receptors (TLRs) are the principal core sensors of the host. Despite intensive studies for the expression of TLRs and their roles in the central nervous system, controversies remain regarding the expression and the function of TLR4 in human astrocytes. In order to clarify this issue, we attempted to verify functional expression of TLR4 in human astrocytes. Using Reverse transcription-polymerase chain reaction (RT-PCR), we confirmed that the human astrocytes express the TLR4 constitutively. To determine the function of TLR4, astrocytes were treated with TLR4 ligand or lipopolysaccharide (LPS), and then inflammatory cytokines expressions were checked using RT-PCR and enzyme-linked immunosorbent assay. Nuclear factor (NF)-κB activation was checked using electrophoretic mobility shift assay. Treatment of astrocytes with LPS increased tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8 expression and induced NF-κB activation. Neutralizing anti-TLR4 antibody blocked the effect of LPS on cytokine production and NF-κB activation in astrocytes. The effect of LPS on cytokine production and NF-κB activation was shown in the presence of serum but not in the absence of serum. Therefore, we investigated the sensing mechanism of LPS in human astrocytes. Human astrocytes were treated with LPS following neutralizing anti-CD14 antibody treatment in the presence of serum. Neutralizing anti-CD14 antibody treatment abolished the effect of LPS on cytokine expression and NF-κB activation. Additionally, supplement of recombinant CD14 in serum-free media induced LPS effect on cytokine production and NF-κB activation. In these results, we showed that human astrocytes constitutively express functional TLR4 and require soluble CD14 to recognize LPS.
Astrocytes* ; Central Nervous System ; Culture Media, Serum-Free ; Cytokines ; Electrophoretic Mobility Shift Assay ; Enzyme-Linked Immunosorbent Assay ; Humans* ; Interleukin-8 ; Interleukins ; Toll-Like Receptors ; Tumor Necrosis Factor-alpha

BACKGROUND: Isoflavones are biologically active compounds that occur naturally in a variety of plants, with relatively high levels in soybean. Tectorigenin, an isoflavone, protects against hydrogen peroxide (H2O2)-induced cell damage. However, the underlying mechanism is unknown. METHODS: The MTT assay was performed to determine cell viability. Catalase activity was assessed by determining the amount of enzyme required to degrade 1 μM H2O2. Protein expression of catalase, phospho-extracellular signal-regulated kinase (ERK), IκB-α, and NF-κB were evaluated by Western blot analysis. A mobility shift assay was performed to assess the DNA-binding ability of NF-κB. Transient transfection and a NF-κB luciferase assay were performed to assess transcriptional activity. RESULTS: Tectorigenin reduced H2O2-induced death of Chinese hamster lung fibroblasts (V79-4). In addition, tectorigenin increased the activity and protein expression of catalase. Blockade of catalase activity attenuated the protective effect of tectorigenin against oxidative stress. Furthermore, tectorigenin enhanced phosphorylation of ERK and nuclear expression of NF-κB, while inhibition of ERK and NF-κB attenuated the protective effect of tectorigenin against oxidative stress. CONCLUSIONS: Tectorigenin protects cells against oxidative damage by activating catalase and modulating the ERK and NF-κB signaling pathway.
Animals ; Blotting, Western ; Catalase* ; Cell Death* ; Cell Survival ; Cricetinae ; Cricetulus ; Electrophoretic Mobility Shift Assay ; Extracellular Signal-Regulated MAP Kinases ; Fibroblasts ; Hydrogen Peroxide ; Isoflavones ; Luciferases ; Lung ; NF-kappa B ; Oxidative Stress ; Phosphorylation ; Phosphotransferases ; Soybeans ; Transfection