Proteomic characterization of isolated Arabidopsis clathrin-coated vesicles reveals evolutionarily conserved and plant-specific components.

In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein assembly polypeptide-1 (AP-1) complex operates as part of the secretory pathway at the trans-Golgi network (TGN), while the AP-2 complex and the TPLATE complex jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched TGN/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis (Arabidopsis thaliana) cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.

Anti-inflammatory and antioxidant properties of chemical constituents of Broussonetia papyrifera.

Extensive phytochemical analysis of the CHCl3-soluble part of an ethanolic extract of branches and twigs of Broussonetia papyrifera led to the isolation of fourteen compounds, including a novel 5,11-dioxabenzo[b]fluoren-10-one derivative named broussofluorenone C (12). The isolated compounds 1-14 were characterized based on their NMR and HRMS data, and examined for their anti-inflammatory activities in LPS-stimulated THP-1 cells as well as for their cellular antioxidant effects. Compounds 7-10 and 12 showed inhibitory effects on NF-κB/AP-1 activation and compounds 7-9 were subsequently confirmed to suppress the secretion of both IL-1ß and TNF-α in LPS-stimulated THP-1 cells more significantly than the prednisone used as a positive control. In the CAA assay, compound 10 exhibited the greatest antioxidant effect, greater than that of the quercetin used as a positive control. The results show possible beneficial effects and utilization of B. papyrifera wood in the treatment of inflammatory diseases as well as oxidative stress.

Expression of activator protein-1 in papillary thyroid carcinoma and its clinical significance.

BACKGROUND: The abnormal expression of activator protein-1(AP-1) has recently been investigated in a variety of tumors. While the relationship between AP-1 and thyroid cancer is poorly studied, our study was to evaluate the protein expression and clinical value of AP-1 in papillary thyroid carcinoma (PTC). METHODS: The expression of AP-1 was examined by immunohistochemistry on paraffin-embedded tissues obtained from PTC and correspondent paracancerous tissues of 82 patients. RESULTS: Compared with paracancerous tissues, AP-1 expression was significantly elevated in PTC tissues and the positive rate was 79.3% (65/82). Our study found a linear trend relationship between the expression of AP-1 and tumor size. However, the differences in AP-1 expression among gender, age, lymph node metastasis, number of lesions, location of the lesion, and extrathyroid invasion are not statistically significant. CONCLUSIONS: The expression of AP-1 plays an important role in the proliferation process of PTC.

Role of the TGFß/PDCD4/AP-1 Signaling Pathway in Nasopharyngeal Carcinoma and Its Relationship to Prognosis.

BACKGROUND: The objective of the present study was to evaluate the role of the TGFß/PDCD4/AP-1 pathway in nasopharyngeal carcinoma (NPC) and its relationship to NPC prognosis. METHODS: NPC tissues collected from 66 NPC patients were compared to 17 nasopharyngeal mucosa biopsy specimens collected as normal tissues. Immunohistochemical staining was performed to assess expression of transforming growth factor-ß receptor I (TGFßRI), programmed cell death 4 (PDCD4) and activator protein-1 (AP-1). The Kaplan-Meier method was applied to evaluate NPC patient overall survival (OS) and progression-free-survival (PFS). Cox regression analysis was used to estimate independent prognostic factors for NPC. The human NPC cell line CNE2 was selected and treated with SB431542, an inhibitor of TGFßRI; expression of TGFßRI and PDCD4 in CNE2 cells was determined by western blotting. NPC tissues showed higher expression of TGFßRI and AP-1 but lower expression of PDCD4 than normal tissues (all P < 0.05). RESULTS: The results of Kaplan-Meier analysis showed that TGFßRI-positive patients and AP-1-positive patients had shorter OS and PFS than TGFßRI-negative patients and AP-1-negative patients; additionally, PDCD4-positive patients had higher OS and PFS than PDCD4-negative patients. Cox regression analysis revealed that advanced tumor stage, overexpression of TGFßRI and AP-1, and low expression of PDCD4 were unfavorable factors influencing OS and PFS in NPC patients. Compared with the control group, expression of TGFßRI decreased and that of PDCD4 increased significantly in CNE2 cells treated with the inhibitor (all P < 0.05). These findings indicate that the TGFß/PDCD4/AP-1 pathway may be associated with NPC development and progression. CONCLUSION: High expression of TGFßRI and AP-1 and low expression of PDCD4 may be unfavorable prognostic factors for NPC.

Evidence for Homodimerization of the c-Fos Transcription Factor in Live Cells Revealed by Fluorescence Microscopy and Computer Modeling.

The c-Fos and c-Jun transcription factors, members of the activator protein 1 (AP-1) complex, form heterodimers and bind to DNA via a basic leucine zipper and regulate the cell cycle, apoptosis, differentiation, etc. Purified c-Jun leucine zipper fragments could also form stable homodimers, whereas c-Fos leucine zipper homodimers were found to be much less stable in earlier in vitro studies. The importance of c-Fos overexpression in tumors and the controversy in the literature concerning c-Fos homodimerization prompted us to investigate Fos homodimerization. Förster resonance energy transfer (FRET) and molecular brightness analysis of fluorescence correlation spectroscopy data from live HeLa cells transfected with fluorescent-protein-tagged c-Fos indicated that c-Fos formed homodimers. We developed a method to determine the absolute concentrations of transfected and endogenous c-Fos and c-Jun, which allowed us to determine dissociation constants of c-Fos homodimers (Kd = 6.7 ± 1.7 µM) and c-Fos-c-Jun heterodimers (on the order of 10 to 100 nM) from FRET titrations. Imaging fluorescence cross-correlation spectroscopy (SPIM-FCCS) and molecular dynamics modeling confirmed that c-Fos homodimers were stably associated and could bind to the chromatin. Our results establish c-Fos homodimers as a novel form of the AP-1 complex that may be an autonomous transcription factor in c-Fos-overexpressing tissues and could contribute to tumor development.

Differential expression of AP-1 proteins in human myometrium after spontaneous term labour onset.

OBJECTIVES: The aims of this study were (i) to determine the localisation of activator protein (AP)-1 family members (cFos, FosB, cJun, JunB and JunD) in human myometrium; and (ii) to determine the effect of human term labour on the expression of AP-1 family of transcription factors in myometrium. STUDY DESIGN: This localised the AP-1 family members cFos, FosB, cJun, JunB and JunD in human myometrium was performed by immunohistochemistry. The effect of term labour on the expression of these family members at the mRNA and protein level was assessed by qRT-PCR and Western blotting, respectively. The effect of pro-inflammatory stimuli on AP-1 transcriptional activity was assessed using a luciferase assay in primary human myometrial cells. RESULTS: Immunohistochemical expression of cFos, FosB, cJun, JunB and JunD were all present in human myometrial tissue and displayed cytoplasmic staining. FosB and JunD also displayed nuclear staining. Term labour was associated with an increase in cFos and JunB mRNA and protein expression. On the other hand, JunD mRNA and protein expression was decreased with labour. FosB mRNA was increased with labour, but there was no change at the protein level. There was no change in cJun mRNA or protein expression. AP-1 transcriptional activity was increased in human myometrial cells by the pro-inflammatory cytokine TNF-α. There was, however, no effect of the bacterial products lipopolysaccharide (LPS; TLR4 ligand), iE-DAP (NOD1 ligand), MDP (NOD2 ligand), FSL-1 (TLR2 ligand) or flagellin (TLR5 ligand) on AP-1 transcriptional activity. CONCLUSION: This study shows that human labour is associated with changes in AP-1 family members. Further studies are required to determine the exact role of the AP-1 family members in myometrium.

The effects of proresolution of ellagic acid in an experimental model of allergic airway inflammation.

Asthma is a disease of airway inflammation characterized by airway hyperresponsiveness, eosinophilic inflammation, and hypersecretion of mucus. Ellagic acid, a compound derived from medicinal plants and fruits, has shown anti-inflammatory activity in several experimental disease models. We used the classical experimental model, in BALB/c mice, of sensibilization with ovalbumin to determine the effect of ellagic acid (10 mg/kg; oral route) in the resolution of allergic airways response. Dexamethasone (1 mg/kg; subcutaneous route) was used as a positive control. The control group consisted of nonimmunized mice that received challenge with ovalbumin. Ellagic acid and dexamethasone or vehicle (water) were administered before or after intranasal allergen challenge. Ellagic acid accelerated the resolution of airways inflammation by decreasing total leukocytes and eosinophils numbers in the bronchoalveolar lavage fluid (BALF), the mucus production and lung inflammation in part by reducing IL-5 concentration, eosinophil peroxidase (EPO) activity, and P-selectin expression, but not activator protein 1 (AP-1) and nuclear factor kappa B (NF-κB) pathways. In addition, ellagic acid enhanced alveolar macrophage phagocytosis of IgG-OVA-coated beads ex vivo, a new proresolving mechanism for the clearance of allergen from the airways. Together, these findings identify ellagic acid as a potential therapeutic agent for accelerating the resolution of allergic airways inflammation.

Visfatin induces stromal cell-derived factor-1 expression by ß1 integrin signaling in colorectal cancer cells.

Obesity has been shown to be associated with the risk of colorectal cancer (CRC). Adipokines produced by the adipose tissue are linked to some malignancies, including CRC. Visfatin is an adipokine shown to be a biomarker of CRC malignant potential. In addition, the stromal cell-derived factor-1 (SDF-1) has been reported to play a role in CRC progression. Although the relationship between visfatin and CRC has been established, the underlying mechanism has not been clarified. We investigated the molecular mechanism governing the interaction between visfatin stimulation and SDF-1 expression in human CRC cell lines. We found that visfatin stimulation led to an increase in the expression and secretion of SDF-1 in CRC DLD-1 and SW48 cells. Experiments involving specific inhibitors and small interfering RNA demonstrated that the activation of ERK and p38 mitogen-activated protein kinase (MAPK) pathways are critical for visfatin-induced SDF-1 expression. Analysis of transcription factor binding using ELISA and luciferase reporter assays revealed that visfatin increased NF-κB- and AP-1-DNA-binding activities in DLD-1 cells. Inhibition of NF-κB and AP-1 activation blocked the visfatin-induced expression and activity of the SDF-1 promoter. The effect of visfatin on DLD-1 signaling and SDF-1 expression was mediated by ß1 integrin. In summary, these findings provide novel insights pertaining to the pathophysiological role of visfatin in CRC.

Pimaric acid from Aralia cordata has an inhibitory effect on TNF-α-induced MMP-9 production and HASMC migration via down-regulated NF-κB and AP-1.

Many studies have indicated that activation of matrix metalloproteinase (MMP)-9 and smooth muscle cell (SMC) migration are involved in neointimal formation and atherosclerosis. In this study, we revealed that pimaric acid (PiMA) purified from Aralia cordata had an inhibitory effect on MMP-9 production and migration of human aortic smooth muscle cells (HASMCs) induced by tumor necrosis factor (TNF)-α. Down-regulated MMP-9 mRNA transcription was detected in PiMA-treated cells using RT-PCR and the luciferase-tagged MMP-9 promoter assay. Results of an electrophoretic mobility shift assay indicated that PiMA-treated HASMCs showed decreased binding activity of nuclear factor (NF)-κB and activator protein-1 transcription factors. A Western-blot analysis using nuclear extract demonstrated that PiMA reduced the levels of NF-κB p65, c-Fos, p-c-Jun, Jun-D, and p-ATF2 proteins in the nucleus. In addition, TNF-α stimulated mitogen activated protein kinase (MAPK) containing extracellular signal regulated kinase 1 and 2, p38, and c-Jun N-terminal kinase was inhibited by PiMA. Using the Transwell system, we found that PiMA inhibited TNF-α stimulated HASMC migration/invasion in a dose-dependent manner. To confirm whether MAPK mediated MMP-9 expression, we used MAPK inhibitors including U0126, SB253580, and SP600125 and found that those inhibitors reduced MMP-9 expression and HASMC migration/invasion. These results suggest that PiMA has potent anti-atherosclerotic activity with inhibitory action on MMP-9 production and cell migration in TNF-α-induced HASMCs.

Azadirachta indica exerts chemopreventive action against murine skin cancer: studies on histopathological, ultrastructural changes and modulation of NF-kappaB, AP-1, and STAT1.

The present study reports the histopathological, ultrastructural changes and modulation of NF-kappaB, AP-1, and STAT 1 during skin carcinogenesis in LACA mice and its intervention with Azadirachta indica. Skin tumors were induced by topical application of 7,12-dimethylbenz(a)anthracene (DMBA) (500 nmol/100 microl for 2 weeks) followed by 12-O-tetradecanoylphorbol-13-acetate (TPA) (1.7 nmol/100 microl of acetone, twice weekly) as a promoter. Male LACA mice were divided into four groups: Control, DMBA/TPA, aqueous Azadirachta indica leaf extract (AAILE), and AAILE + DMBA/TPA. AAILE was administered orally at a dose level of 300 mg/kg body weight three times a week for 20 weeks. Topical application of DMBA/ TPA to the skin resulted in well-developed squamous cell carcinomas characterized by hyperproliferation, hyperkeratosis, and corrugation of the epidermis. Degenerative changes were observed in the tumors of AAILE + DMBA/TPA-treated animals. Scanning electron microscopy revealed surface disruptions and certain rounded structures on the skin tumors of DMBA/TPA-treated animals. Topographical changes were also observed in the tumors of AAILE + DMBA/TPA-treated animals, which resembled regions of degeneration. Tumors obtained in DMBA/TPA group were associated with enhanced expression of NF-kappaB and AP-1 when compared to the control counterparts. Inhibition in tumorigenesis in response to A. indica treatment was accompanied by an overexpression of STAT 1 and AP-1 and decrease in NF-kappaB expression. The results of the present study provide a basis for the chemopreventive potential of A. indica against murine skin carcinogenesis.

Diosgenin inhibits macrophage-derived inflammatory mediators through downregulation of CK2, JNK, NF-kappaB and AP-1 activation.

Diosgenin is a precursor of steroid hormones, which can be found in several plant species. Diosgenin has been shown to have a variety of biological activities including anti-inflammatory activity, but through a mechanism that is unclear. Especially, the effect of this agent on macrophage function has not been characterized in detail. In the present study, we examined the effects of diosgenin on the production of inflammatory mediators in lipopolysaccharide (LPS)/interferon gamma (IFN-gamma)-activated murine macrophage. Macrophages pre-exposed to diosgenin (0.1-10 microM) were stimulated with LPS/IFN-gamma. Pretreatment with diosgenin resulted in the inhibition of NO production and inducible nitric oxide synthase (iNOS) protein and mRNA expression in a concentration-dependent manner. In addition, diosgenin inhibits production of reactive oxygen species (ROS), interleukin-1 (IL-1), and IL-6, but not that of tumor necrosis factor-alpha (TNF-alpha). Inhibition of these inflammatory mediators appears to be at the transcriptional level, since diosgenin decreased LPS/IFN-gamma-induced NF-kappaB and AP-1 activity. Diosgenin blocked CK2 activation and phosphorylation of c-Jun NH(2)-terminal kinase (JNK), but not that of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 (ERK 1/2). These results indicate that the inhibition of these signaling molecules expression was correlated with the reduced production of inflammatory mediators in macrophages. Taken together the present data suggest that diosgenin reduces the production of inflammatory meditators by inhibiting LPS/IFN-gamma-triggered CK2, JNK, NF-kappaB and AP-1 activation, thereby implicating a mechanism by which diosgenin may exert its immunosuppressive effects.

Protective effect of carbon monoxide inhalation on lung injury after hemorrhagic shock/resuscitation in rats.

BACKGROUND: Hemorrhagic shock and resuscitation (HSR) induces pulmonary inflammation that leads to acute lung injury. Carbon monoxide (CO), a by-product of heme catalysis, was shown to have potent cytoprotective and anti-inflammatory effects. The aim of this study was to examine the effects of CO inhalation at low concentration on lung injury induced by HSR in rats. METHODS: Rats were subjected to HSR by bleeding to achieve mean arterial pressure of 30 mm Hg for 60 minutes followed by resuscitation with shed blood and saline as needed to restore blood pressure. HSR animals were either maintained in room air or were exposed to CO at 250 ppm for 1 hour before and 3 hours after HSR. RESULTS: HSR caused an increase in the DNA binding activity of nuclear factor-kappaB and activator protein-1 in the lung followed by the up-regulation of pulmonary gene expression of tumor necrosis factor-alpha, inducible nitric oxide synthase, and interleukin (IL)-10. HSR also resulted in an increase in myeloperoxidase activity and wet weight to dry weight ratio in the lung, and more prominent histopathologic changes including congestion, edema, cellular infiltration, and hemorrhage. In contrast, CO inhalation significantly ameliorated these inflammatory events as judged by fewer histologic changes, less up-regulation of inflammatory mediators, and less activation of nuclear factor-kappaB and activator protein-1. Interestingly, the protective effects against lung injury afforded by CO were associated with further increases in mRNA expression of IL-10 in the lung. CONCLUSIONS: These findings suggest that inhaled CO at a low concentration ameliorated HSR-induced lung injury and attenuated inflammatory cascades by up-regulation of anti-inflammatory IL-10.

The pathophysiology of abdominal aortic aneurysm growth: corresponding and discordant inflammatory and proteolytic processes in abdominal aortic and popliteal artery aneurysms.

OBJECTIVE: There is remarkable controversy over the processes driving abdominal aneurysm growth. The inherent limitations of animal and human studies hamper elucidation of the key inflammatory and proteolytic processes. Human data are largely derived from surgical specimens that typically reflect the final stages of the disease process and thus do not allow distinction between primary and secondary processes. Clear epidemiologic and genetic associations between abdominal aortic aneurysm (AAA) and popliteal artery aneurysms (PAA) suggest that that these two pathologies share common grounds. On this basis, we reasoned that information of corresponding and discordant processes in these aneurysms might provide critical clues on the processes that are crucial for aneurysm progression. METHODS: Messenger RNA (semi-quantitative real-time polymerase chain reaction) and protein analysis (enzyme-linked immunosorbent assay, multiplex, Western blotting), and histology were performed on aneurysm wall samples obtained during elective PAA and AAA repair. Nonaneurysmal aorta tissue from organ donors was included as reference. RESULTS: Messenger RNA and protein analysis showed that PAA and AAA are both characterized by a marked activation of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) proinflammatory transcription factors, and hyperexpression of interleukin (IL)-6 and IL-8. Discordant findings were found for other inflammatory markers such as interferon-gamma, interferon-inducible protein 10, tumor necrosis factor-alpha, monocyte chemotactic protein-1, and macrophage inflammatory protein 1alpha and beta, which were all lower in PAA. On the cellular level, both pathologies exhibited profuse infiltration of macrophages, neutrophils, and T-helper cells. Results for B cells, plasma cells, and cytotoxic T cells were discordant, with minimal infiltration of these cell types in PAA. Evaluation of protease expression and activation showed that both conditions are dominated by increased matrix metalloproteinase 8 and 9, and cathepsin K, L and S expression and activation. CONCLUSION: This explorative study characterizes degenerative aneurysmal disease general inflammatory conditions that are dominated by profound activation of the NF-kappaB and AP-1 pathways, hyperexpression of IL-6 and IL-8, and neutrophil involvement. Discordant findings for interferon gamma, cytotoxic T cells, B cells, and plasma cells challenge a critical role for these factors in the process of aneurysm growth. Pharmaceutic strategies targeting the common components in AAA and PAA may prove effective for the stabilization of AAA.

KBTBD7, a novel human BTB-kelch protein, activates transcriptional activities of SRE and AP-1.

In this study, a novel member of BTB-kelch proteins, named KBTBD7, was cloned from a human embryonic heart cDNA library. The cDNA of KBTBD7 is 3,008 bp long and encodes a protein product of 684 amino acids (77.2 kD). This protein is highly conserved in evolution across different species. Western blot analysis indicates that a 77 kD protein specific for KBTBD7 is wildly expressed in all embryonic tissues examined. In COS-7 cells, KBTBD7 proteins are localized to the cytoplasm. KBTBD7 is a transcription activator when fused to GAL4 DNA-binding domain. Deletion analysis indicates that the BTB domain and kelch repeat motif are main regions for transcriptional activation. Overexpression of KBTBD7 in MCF-7 cells activates the transcriptional activities of activator protein-1 (AP-1) and serum response element (SRE), which can be relieved by siRNA. These results suggest that KBTBD7 proteins may act as a new transcriptional activator in mitogen-activated protein kinase (MAPK) signaling. [BMB reports 2010; 43(1): 17-22].

Utility of fascin and JunB in distinguishing nodular lymphocyte predominant from classical lymphocyte-rich Hodgkin lymphoma.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) and lymphocyte-rich classical Hodgkin lymphoma (LRCHL), although clinically and morphologically similar, differ biologically and in prognosis. Immunolabeling of Reed-Sternberg (RS) cells in LRCHL and lymphocytic and/or histiocytic variants (L&H cells) in NLPHL is often required to help distinguish between the 2 variants. Our aim was to evaluate fascin (a distinct 55-kd actin-bundling protein) and JunB (an activator protein-1 family transcription factor) to differentiate NLPHL from LRCHL. A total of 35 archival cases of NLPHL (n = 24) and LRCHL (n = 11) from adults and children were studied. Slides were reviewed for all cases and clinical, morphologic, and immunohistochemical features were evaluated. Each case was immunostained for fascin and JunB, and immunoreactivity of RS cells, L&H cells, and background lymphocytes were recorded. Whereas occasional L&H cells were weakly positive for fascin in 3 out of 24 (12.5%) cases of NLPHL, RS cells in LRCHL were positive for fascin in 11 out of 11 (100%) cases with a strong cytoplasmic staining pattern. JunB was positive in 10 out of 24 (41.7%) of NLPHL cases, and 11 out of 11 (100%) of LRCHL cases, showing a stippled and/or diffuse nuclear staining pattern. In addition to L & H Cells, JunB also stained small background lymphocytes, particularly in areas of progressively transformed germinal centers of NLPHL. Either stains when tested alone, if negative, or with rare L&H cell weak positivity for fascin, is indicative of NLPHL. The L&H cells of NLPHL cases were negative for concomitant staining in 24 out of 24 (100%) cases. Concomitant positive staining of classic RS cells for fascin and JunB was found in 11 out of 11 (100%) of LRCHL cases. Although fascin positivity alone supports the diagnosis of LRCHL, concomitant positivity offers stronger support and is less likely to lead to a false conclusion if aberrant fascin staining were to be encountered in a case of NLPHL. Staining for fascin and JunB provides a basis for distinguishing NLPHL from LRCHL and offers an alternative to other antibody profiles.

Antibody-based proteomics for esophageal cancer: Identification of proteins in the nuclear factor-kappaB pathway and mitotic checkpoint.

To identify the molecular background of esophageal cancer, we conducted a proteomics study using an antibody microarray consisting of 725 antibodies and surgical specimens from three cases. The microarray analysis identified 24 proteins with aberrant expression in esophageal cancer compared with the corresponding normal mucosa. The overexpression of 14 of the 24 proteins was validated by western blotting analysis of the same samples. These 14 proteins were examined by immunohistochemistry, in which nine proteins showed consistent results with those obtained by western blotting. Among the nine proteins, seven were localized in tumor cells, and two in infiltrating cells. The former included proteins associated with mitotic checkpoint control and the nuclear factor (NF)-kappaB pathway. Although mitotic checkpoint gene products (budding uninhibited by benzidazoles 1 homolog beta (BubR1) and mitotic arrest deficient-like 1 (Mad2)) have previously been reported to be involved in esophageal cancer, the association of NF-kappaB-activating kinase, caspase 10, and activator protein-1 with esophageal cancer has not been previously reported. These proteins play a key role in the NF-kappaB pathway, and NF-kappaB is a signal transduction factor that has emerged as an important modulator of altered gene programs and malignant phenotype in the development of cancer. The association of these proteins with esophageal cancer may indicate that mitotic checkpoint gene products and NF-kappaB play an important part in the carcinogenesis of esophageal cancer.

In vitro analysis of inflammatory responses following environmental exposure to pharmaceuticals and inland waters.

Pharmaceuticals are regularly released into the environment; in particular non-steroidal anti-inflammatory drugs (NSAIDs) and antibiotics. Erythromycin, naproxen, furosemide and atenolol are reported to be stable for up to 1 year in the environment, which increases the risk for accumulation. In the present study we have measured the occurrence and concentration of pharmaceuticals in river Viskan (Jössabron) downstream of a sewage treatment plant in Borås, Sweden. Pharmaceuticals and water samples were tested for potential human risk by evaluating inflammatory responses (NF-kappaB and AP-1) using human T24 bladder epithelial cells and Jurkat T-cells. NF-kappaB activity in T24 cells was significantly reduced by all NSAIDs analysed (diclofenac, ketoprofen, naproxen, ibuprophen and dextropropoxyphene), but also by trimethoprim, using environmentally relevant concentrations. NF-kappaB and AP-1 activation was further analysed in response to water samples collected from different locations in Sweden. Dose-dependent down-regulation of AP-1 activity in Jurkat cells was observed at all locations. At two locations (Jössabron and Almenäs) down-regulation of NF-kappaB was observed. In contrast, the NF-kappaB response was potentiated by exposure to water from both locations following activation of NF-kappaB by treatment with heat-killed Escherichia coli. To determine the involvement of pharmaceuticals in the responses, T24 cells were exposed to the pharmaceutical mixture, based on the determined levels at Jössabron. This resulted in reduction of the NF-kappaB response following exposure to the pharmaceutical mixture alone while no potentiation was observed when cells were co-exposed to heat killed E. coli and pharmaceuticals. The obtained results demonstrate that the identified pharmaceuticals affect the inflammatory responses and furthermore indicate the presence of unknown substance(s) with the ability to potentiate inflammatory responses.

Resveratrol analog-3,5,4'-trimethoxy-trans-stilbene inhibits invasion of human lung adenocarcinoma cells by suppressing the MAPK pathway and decreasing matrix metalloproteinase-2 expression.

Resveratrol (3,5,4'-trihydroxystilbene) is a natural polyphenol that presents various physiological activities. It has been reported that the methylated derivatives of resveratrol show better potential antifungal and antiproliferative activities than resveratrol. In the present study, we investigated the inhibitory effect of 3,5,4'-trimethoxy-trans-stilbene (MR-3), a methylated derivative of resveratrol, on the invasion of A549 cells (a human lung adenocarcinoma cell line). We found that treatment with MR-3 at the concentration of 5 muM resulted in antiadhesive, antimigratory, and antiinvasive activities on A549 cells through the suppression of matrix metalloproteinase (MMP)-2 protein expression and transcriptional levels in a time-dependent manner. The suppression of MMP-2 expression by MR-3 led to an inhibition of A549 cell invasion by inactivating phosphorylation of SAPK/c-Jun N-terminal kinase (JNK) and p38 MAPK signaling pathways. A time-dependent inhibition of protein levels for p65, c-Jun, and c-Fos in the nucleus by MR-3 treatment was also observed. In conclusion, our data demonstrate that the antiinvasive effects of MR-3 on A549 cells are likely mediated through the inhibition of phosphorylation of JNK and p38, as well as a reduction in the protein levels of nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1) in the nucleus, ultimately leading to downregulation of MMP-2 expression.

Reduced pulmonary inflammatory response during cardiopulmonary bypass: effects of combined pulmonary perfusion and carbon monoxide inhalation.

OBJECTIVE: Pulmonary inflammation induced by cardiopulmonary bypass (CPB) is one of the main causes for lung injury after cardiac surgery. Pulmonary perfusions as well as carbon monoxide (CO) inhalation are known to reduce the inflammatory reaction of the lung. We hypothesized that a combination of pulmonary perfusion and carbon monoxide inhalation leads to an even stronger reduction of the lung inflammation. METHODS: Pigs (n=7 per experimental group) were randomized to sham operation (SHAM), conventional CPB (CPB), inhalation of CO (CPB+CO, 250 ppm), pulmonary perfusion (CPB+PP) or pulmonary perfusion plus inhalation of CO (CPB+PP+CO). Various cytokine levels (TNF-alpha, IL-1, IL-6, and IL-10) and caspase-3 activity were measured using enzyme-linked immunosorbent assay (ELISA). Transcription factor activity was analyzed via electrophoretic mobility shift assay (EMSA). Blood gases and hemodynamics were measured continuously. A p value <0.05 assessed by Holm-Sidak method was considered statistically significant. RESULTS: Hemodynamic parameters and blood gas analysis showed no significant differences between the groups. While IL-1 protein expression was comparable between the groups, TNF-alpha (478+/-58 vs 869+/-95 pg/ml; p<0.001) and IL-6 protein levels in the lung (256+/-82 vs 936+/-76 pg/ml; p<0.001) showed a significant inhibition in the CPB+PP+CO group at 120 min post-bypass time compared to the CPB group. The cytokine levels were comparable to the CPB+PP and CPB+CO group. IL-10 protein expression (325+/-47 vs 65+/-27 pg/ml; p<0.05) was significantly higher in the CO-treated compared to CPB+PP and CPB-treated animals at 120 min post-bypass. Activation of the transcription factors NF-kappaB and AP-1 showed a CO-mediated induction compared to the CPB or CPB+PP group. Caspase-3 activity revealed a CO-dependent, significant inhibition in CO and CPB+PP+CO-treated animals compared to CPB animals (p<0.05). CONCLUSION: The combination of pulmonary perfusion and inhalative carbon monoxide inhibits CPB-mediated pulmonary inflammation as well as pulmonary apoptosis stronger than pulmonary perfusion or carbon monoxide alone.

A novel high-throughput screening system identifies a small molecule repressive for matrix metalloproteinase-9 expression.

Aberrant gene expression is one of the driving forces for cancer progression and is considered an ideal target for chemical intervention. Although emerging bioluminescence reporter systems allow high-throughput searches for small molecules regulatory for gene expression, frequent silencing of reporter genes by epigenetic mechanisms hinders wide application of this drug discovery strategy. Here we report a novel system that directs the integration of a promoter-reporter construct to an open chromosomal location by Flp-mediated homologous recombination, thereby overcoming reporter-gene silencing. Using this system, we have screened more than 8000 compounds in the DIVERSet chemical library for repressors of a matrix metalloproteinase-9 (MMP-9) promoter and identified 5-methyl-2-(4-methylphenyl)-1H-benzimidazole (MPBD) inhibitory for MMP-9 gene expression. Consistent with this effect, MPBD inhibits MMP-9-dependent invasion of UMSCC-1 oral cancer cells, preosteoclast migration, and receptor activator of nuclear factor-kappaB ligand-induced osteoclast activity over concentration ranges that repressed MMP-9 expression. Mechanistic studies indicated that MPBD antagonizes AP-1 function by inhibiting its transactivation activity. We conclude that the Flp-mediated homologous recombination system to direct reporter integration into open chromatin regions represents a novel strategy allowing for the development of high-throughput systems screening for lead compounds targeting aberrant gene expression in cancer.