Selective inhibition of cyclooxygenase-2 by C-phycocyanin, a biliprotein from Spirulina platensis

We report data from two related assay systems (isolated enzyme assays and whole blood assays) that C-phycocyanin a biliprotein from Spirulina platensis is a selective inhibitor of cyclooxygenase-a (COX-2) with a very low IC50 COX-2/IC50 COX-1 ratio (0.04). The extent of inhibition depends on the period of preincubation of phycocyanin with COX-2, but without any effect on the period of preincubation with COX-1. The IC50 value obtained for the inhibition of COX-2 by phycocyanin is much lower (180 nM) as compared to those of celecoxib (255 nM) and rofecoxib (401 nM), the well-known selective COX-2 inhibitors. In the human whole blood assay, phycocyanin very efficiently inhibited COX-2 with an IC50 value of 80 nM. Reduced phycocyanin and phycocyanobilin, the chromophore of phycocyanin are poor inhibitors of COX-2 without COX-2 selectivity. This suggests that apoprotein in phycocyanin plays a key role in the selective inhibition of COX-2. The present study points out that the hepatoprotective, anti-inflammatory, and anti-arthritic properties of phycocyanin reported in the literature may be due, in part, to its selective COX-2 inhibitory property, although its ability to efficiently scavenge free radicals and effectively inhibit lipid peroxidation may also be involved. (C) 2000 Academic Press.

PIM2 Induced COX-2 and MMP-9 Expression in Macrophages Requires PI3K and Notch1 Signaling

Activation of inflammatory immune responses during granuloma formation by the host upon infection of mycobacteria is one of the crucial steps that is often associated with tissue remodeling and breakdown of the extracellular matrix. In these complex processes, cyclooxygenase-2 (COX-2) plays a major role in chronic inflammation and matrix metalloproteinase-9 (MMP-9) significantly in tissue remodeling. In this study, we investigated the molecular mechanisms underlying Phosphatidyl-myo-inositol dimannosides (PIM2), an integral component of the mycobacterial envelope, triggered COX-2 and MMP-9 expression in macrophages. PIM2 triggers the activation of Phosphoinositide-3 Kinase (PI3K) and Notch1 signaling leading to COX-2 and MMP-9 expression in a Toll-like receptor 2 (TLR2)-MyD88 dependent manner. Notch1 signaling perturbations data demonstrate the involvement of the cross-talk with members of PI3K and Mitogen activated protein kinase pathway. Enforced expression of the cleaved Notch1 in macrophages induces the expression of COX-2 and MMP-9. PIM2 triggered significant p65 nuclear factor-kappa B (NF-kappa B) nuclear translocation that was dependent on activation of PI3K or Notch1 signaling. Furthermore, COX-2 and MMP-9 expression requires Notch1 mediated recruitment of uppressor of Hairless (CSL) and NF-kappa B to respective promoters. Inhibition of PIM2 induced COX-2 resulted in marked reduction in MMP-9 expression clearly implicating the role of COX-2 dependent signaling events in driving the MMP-9 expression. Taken together, these data implicate PI3K and Notch1 signaling as obligatory early proximal signaling events during PIM2 induced COX-2 and MMP-9 expression in macrophages.

ESAT-6 induced COX-2 expression involves coordinated interplay between PI3K and MAPK signaling

Macrophages, as sentinels of robust host immunity, are key regulators of innate immune responses against invading mycobacteria; however, pathogenic mycobacteria survive in the infected host by subverting host innate immunity. Infection dependent expression of early secreted antigenic target protein 6 (ESAT-6) by Mycobacterium tuberculosis is strongly correlated with subversion of innate immune responses against invading mycobacteria. As a part of multifaceted immunity to mycobacterial infection, induced expression of cyclooxygenase-2 (COX-2) may act as an important influencing factor towards effective host immunity. In the current investigation, we demonstrate that ESAT-6 triggers COX-2 expression both in vitro and in vivo in a TLR2 dependent manner. Signaling perturbation data suggest that signaling dynamics of PI3K and p38 and JNK1/2 MAPK assume critical importance in ESAT-6 triggered expression of COX-2 in macrophages. Interestingly, ESAT-6 triggered PI3K-MAPK signaling axis holds the capacity to regulate coordinated activation of NF-kappa B and AP-1. Overall, current investigation provides mechanistic insights into ESAT-6 induced COX-2 expression and unravels TLR2 mediated interplay of PI3K and MAPK signaling axis as a rate-determining step during intricate host immune responses. These findings would serve as a paradigm to understand pathogenesis of mycobacterial infection and clearly pave a way towards development of novel therapeutics. (C) 2011 Elsevier Ltd. All rights reserved.

Polymorphs and hydrates of Etoricoxib, a selective COX-2 inhibitor

The crystal structures of two polymorphs and two polymorphic hemihydrates of Etoricoxib are reported. Etoricoxib is a non-steroidal anti-inflammatory drug (NSAID) that is a selective inhibitor of COX-2. It is used in the treatment of various types of inflammation, pain and fever. Clas et al. have reported four polymorphs (labeled I through IV) and two solvates (hemi-and sesquihydrate) of the API in US patent 6,441,002 (Clas et al, US patent 6,441,002, 2002). However, no crystal structures have been reported for any of these forms. A comparison was made between the PXRD patterns reported in patent `002 and the powder spectra simulated from single crystal data. The two polymorphs characterized here correspond to form I and form IV of the patent. Form II of the patent could not be obtained by us with a variety of experimental conditions. Form III of the patent corresponds to hemihydrate II of this study. Form III is therefore not a polymorph of form I and form IV. What we have termed hemihydrate I in this study is obtained under a wide variety of conditions and it is also the only hemihydrate reported as such in the patent. Because the Etoricoxib molecule contains no conventional hydrogen bond donors, there cannot be any strong hydrogen bonds in the crystal structures of forms I and IV. The packing is accordingly characterized by weak hydrogen bonds of the C-H center dot center dot center dot O=S and C-H center dot center dot center dot N type. Thermal data were collected for form I, form IV and hemihydrate I to shed some light on relative stabilities. PXRD diffractograms show the transformation of form IV to form I at elevated temperature, indicating that form I is more stable than form IV. However, this transformation occurs only in samples of form IV that contain some form I; it does not occur in pure form IV. The formation of the two hemihydrates could follow from the known tendency of an acceptor-rich molecule to crystallize as a hydrate.

STAT-1 expression is regulated by IGFBP-3 in malignant glioma cells and is a strong predictor of poor survival in patients with glioblastoma Laboratory investigation

Object. Insulin-like growth factor binding proteins (IGEBPs) have been implicated in the pathogenesis of glioma. In a previous study the authors demonstrated that IGFBP-3 is a novel glioblastoma biomarker associated with poor survival. Since signal transducer and activator of transcription 1 (STAT-1) has been shown to be regulated by IGFBP-3 during chondrogenesis and is a prosurvival and radioresistant molecule in different tumors, the aim in the present study was to explore the functional significance of IGFBP-3 in malignant glioma cells, to determine if STAT-1 is indeed regulated by IGFBP-3, and to study the potential of STAT-1 as a biomarker in glioblastoma. Methods. The functional significance of IGFBP-3 was investigated using the short hairpin (sh)RNA gene knockdown approach on U251MG cells. STAT-1 regulation by IGFBP-3 was tested on U251MG and U87MG cells by shRNA gene knockdown and exogenous treatment with recombinant IGFBP-3 protein. Subsequently, the expression of STAT-1 was analyzed with real-time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) in glioblastoma and control brain tissues. Survival analyses were done on a uniformly treated prospective cohort of adults with newly diagnosed glioblastoma (136 patients) using Kaplan-Meier and Cox regression models. Results. IGFBP-3 knockdown significantly impaired proliferation, motility, migration, and invasive capacity of U251MG cells in vitro (p < 0.005). Exogenous overexpression of IGFBP-3 in U251MG and U87MG cells demonstrated STAT-1 regulation. The mean transcript levels (by real-time RT-PCR) and the mean labeling index of STAT-1 (by IHC) were significantly higher in glioblastoma than in control brain tissues (p = 0.0239 and p < 0.001, respectively). Multivariate survival analysis revealed that STAT-1 protein expression (HR 1.015, p = 0.033, 95% CI 1.001-1.029) along with patient age (HR 1.025, p = 0.005, 95% CI 1.008-1.042) were significant predictors of shorter survival in patients with glioblastoma. Conclusions. IGFBP-3 influences tumor cell proliferation, migration, and invasion and regulates STAT-1 expression in malignant glioma cells. STAT-1 is overexpressed in human glioblastoma tissues and emerges as a novel prognostic biomarker.

SIRT6 Promotes COX-2 Expression and Acts as an Oncogene in Skin Cancer

SIRT6 is a SIR2 family member that regulates multiple molecular pathways involved in metabolism, genomic stability, and aging. It has been proposed previously that SIRT6 is a tumor suppressor in cancer. Here, we challenge this concept by presenting evidence that skin-specific deletion of SIRT6 in the mouse inhibits skin tumorigenesis. SIRT6 promoted expression of COX-2 by repressing AMPK signaling, thereby increasing cell proliferation and survival in the skin epidermis. SIRT6 expression in skin keratinocytes was increased by exposure to UVB light through activation of the AKT pathway. Clinically, we found that SIRT6 was upregulated in human skin squamous cell carcinoma. Taken together, our results provide evidence that SIRT6 functions as an oncogene in the epidermis and suggest greater complexity to its role in epithelial carcinogenesis. (C) 2014 AACR.

Ac2PIM-responsive miR-150 and miR-143 Target Receptor-interacting Protein Kinase 2 and Transforming Growth Factor Beta-activated Kinase 1 to Suppress NOD2-induced Immunomodulators

Specific and coordinated regulation of innate immune receptor-driven signaling networks often determines the net outcome of the immune responses. Here, we investigated the cross-regulation of toll-like receptor (TLR)2 and nucleotide-binding oligomerization domain (NOD)2 pathways mediated by Ac2PIM, a tetra-acylated form of mycobacterial cell wall component and muramyl dipeptide (MDP), a peptidoglycan derivative respectively. While Ac2PIM treatment of macrophages compromised their ability to induce NOD2-dependent immunomodulators like cyclooxygenase (COX)-2, suppressor of cytokine signaling (SOCS)-3, and matrix metalloproteinase (MMP)-9, no change in the NOD2-responsive NO, TNF-alpha, VEGF-A, and IL-12 levels was observed. Further, genome-wide microRNA expression profiling identified Ac2PIM-responsive miR-150 and miR-143 to target NOD2 signaling adaptors, RIP2 and TAK1, respectively. Interestingly, Ac2PIM was found to activate the SRC-FAK-PYK2-CREB cascade via TLR2 to recruit CBP/P300 at the promoters of miR-150 and miR-143 and epigenetically induce their expression. Loss-of-function studies utilizing specific miRNA inhibitors establish that Ac2PIM, via the miRNAs, abrogate NOD2-induced PI3K-PKC delta-MAPK pathway to suppress beta-catenin-mediated expression of COX-2, SOCS-3, and MMP-9. Our investigation has thus underscored the negative regulatory role of Ac2PIM-TLR2 signaling on NOD2 pathway which could broaden our understanding on vaccine potential or adjuvant utilities of Ac2PIM and/or MDP.

Sonic hedgehog-responsive lipoxygenases and cyclooxygenase-2 modulate Dectin-1-induced inflammatory cytokines

Immune responses during fungal infections are predominately mediated by 5/15-lipoxygenases (LO)-or cyclooxygenase (COX)-2-catalysed bioactive eicosanoid metabolites like leukotrienes, lipoxins and prostaglandins. Although few host mediators of fungi-triggered eicosanoid production have been established, the molecular mechanism of expression and regulation of 5-LO, 15-LO and COX-2 are not well-defined. Here, we demonstrate that, macrophages infected with representative fungi Candida albicans, Aspergillus flavus or Aspergillus fumigatus or those treated with Curdlan, a selective agonist of pattern recognition receptor for fungi Dectin-1, displays increased expression of 5-LO, 15-LO and COX-2. Interestingly, Dectin-1-responsive Syk pathway activates mTOR-sonic hedgehog (SHH) signaling cascade to stimulate the expression of these lipid metabolizing enzymes. Loss-of-function analysis of the identified intermediaries indicates that while Syk-mTOR-SHH pathway-induced 5-LO and 15-LO suppressed the Dectin-l-responsive pro-inflammatory signature cytokines like TNE-alpha, IL-1 beta and IL-12, Syk-mTOR-SHH-induced COX-2 positively regulated these cytokines. Dectin-1-stimulated IL-6, however, is dependent on 5-LO, 15-LO and COX-2 activity. Together, the current study establishes Dectin-1-arbitrated host mediators that direct the differential regulation of immune responses during fungal infections and thus are potential candidates of therapeutic intervention. (C) 2015 Elsevier Ltd. All rights reserved.

Mycobacteria-responsive sonic hedgehog signaling mediates programmed death-ligand 1-and prostaglandin E-2-induced regulatory T cell expansion

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) are exploited by mycobacteria to subvert the protective host immune responses. The Treg expansion in the periphery requires signaling by professional antigen presenting cells and in particularly dendritic cells (DC). However, precise molecular mechanisms by which mycobacteria instruct Treg expansion via DCs are not established. Here we demonstrate that mycobacteria-responsive sonic hedgehog (SHH) signaling in human DCs leads to programmed death ligand-1 (PD-L1) expression and cyclooxygenase (COX)-2-catalyzed prostaglandin E-2 (PGE(2)) that orchestrate mycobacterial infection-induced expansion of Tregs. While SHH-responsive transcription factor GLI1 directly arbitrated COX-2 transcription, specific microRNAs, miR-324-5p and miR-338-5p, which target PD-L1 were downregulated by SHH signaling. Further, counter-regulatory roles of SHH and NOTCH1 signaling during mycobacterial-infection of human DCs was also evident. Together, our results establish that Mycobacterium directs a fine-balance of host signaling pathways and molecular regulators in human DCs to expand Tregs that favour immune evasion of the pathogen.

X-ray Structure of Gelonin at 1.8 Å Resolution

Gelonin is a single chain ribosome inactivating protein (RIP) with potential application in the treatment of cancer and AIDS. Diffraction quality crystals grown using PEG3350, belong to the space group P2(1), with it a = 49.4 Angstrom b = 44.9 Angstrom, c = 137.4 Angstrom and beta = 98.4 degrees, and contain two molecules in the asymmetric unit. Diffraction data collected to 1.8 Angstrom resolution has a R(m) value of 7.3%. Structure of gelonin has been solved by the molecular replacement method, using ricin A chain as the search model. Crystallographic refinement using X-PLOR resulted in a model for which the r.m.s deviations from ideal bond lengths and bond angles are 0.012 Angstrom and 2.7 degrees, respectively The final R-factor is 18.4% for 39,806 reflections for which I > 1.0 sigma(I).The C-alpha atoms of the two molecules in the asymmetric unit superpose to within 0.38 Angstrom for 247 atom pairs. The overall fold of gelonin is similar to that of other RIPs such as ricin A chain and alpha-momorcharin, the r.m.s.d. for C-alpha superpositions being 1.3 and 1.4 Angstrom, respectively The-catalytic residues (Glu166, Arg169 and Tyr113) in the active site form a hydrogen bond scheme similar to that observed in other RIPs. The conformation of Tyr74 in the active site, however, is significantly different from that in alpha-momorcharin. Three well defined water molecules are located in the active site cavity and one of them, X319, superposes to within 0.2 Angstrom of a corresponding water molecule in the structure of alpha-momorcharin. Any of the three could be the substrate water molecule in the hydrolysis reaction catalysed by gelonin.Difference electron density for a N-linked sugar moiety has been observed near only one of the two potential glycosylation sites in the sequence. The amino acid at position 239 has been established as Lys by calculation of omit electron density maps.The two cysteine residues in the sequence, Cys44 and Cys50, form a disulphide bond, and are therefore not available for disulphide conjugation with antibodies. Based on the structure, the region of the molecule that is involved in intradimer interactions is suggested to be suitable for introducing a Cys residue for purposes of conjugation with an antibody to produce useful immunotoxins.

Ethyl 1-(2-cyanoethyl)-3,5-dimethyl-1H-indole-2-carboxylate

The title compound, C16H18N2O2, is an important precursor in the synthesis of 1,2,3,4-tetrahydropyrazinoindoles, which show excellent antihistamine, antihypertensive and central nervous system depressant properties. The carbethoxy group attached to C2 and the planar cyanoethyl group attached to N1 make dihedral angles of 11.0(4) and 75.0(3)degrees, respectively, with the mean plane of the indole ring, The C-C=N chain is linear with a bond angle of 179.3 (4)degrees.

3-Phenylisoquinolin-1(2H)-one

The title compound, C15H11NO, consists of a planar isoquinolinone group to which a phenyl ring is attached in a twisted fashion [dihedral angle = 39.44 (4)degrees]. The crystal packing is dominated by intermolecular N-H center dot center dot center dot O and C-H center dot center dot center dot O hydrogen bonds which define centrosymmetric dimeric entitities.

Methyl 1H-1,2,3-triazole-4-carboxylate

The title compound, C4H5N3O2, features an essentially planar molecule (r.m.s. deviation for all non-H atoms = 0.013 angstrom). The crystal structure is stabilized by intermolecular N-H center dot center dot center dot O hydrogen bonds and pi-pi stacking interactions (centroid centroid distance 3.882 angstrom).

1-(4-Chloro-3-fluorophenyl)-2-[(3-phenylisoquinolin-1-yl)sulfanyl]ethanone

In the title compound, C23H15ClFNOS, the isoquinoline system and the 4-chloro-3-fluorophenyl ring are aligned at 80.4 (1)degrees. The dihedral angle between the isoquinoline system and the pendant (unsubstituted) phenyl ring is 19.91 (1)degrees.