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Andrographolide Inhibits COX-2 Expression in Human Fibroblast Cells Due to its Interaction with Arginine and Histidine in Cyclooxygenase Site  [PDF]
Jutti Levita,As `ari Nawawi,Abdul Mutholib,Slamet Ibrahim
Journal of Applied Sciences , 2010,
Abstract: The aim of this study was to identify and analyze the interaction of andrographolide with COX-2, followed by in vitro study of the effect of this compound on COX-2 expression in human fibroblast cells. The molecular modeling study was performed by docking andrographolide to COX-2 enzyme at the site where SC-558, a selective inhibitor of this enzyme, was co-crystallized and compared its interaction to the enzyme with SC-558’s. The inhibition of COX-2 expression was determined by measuring PGE2 production in human fibroblast cells stimulated with LPS with and without andrographolide preincubations. Andrographolide interacted with Arg513 and His90 in the cyclooxygenase site of COX-2 and inhibited PGE2 production in human fibroblast cells (IC50 = 4 μM). These data confirm that andrographolide’s anti-inflammatory activity occurs via inhibition of COX-2 expression.
Hypoxia regulates human lung fibroblast proliferation via p53-dependent and -independent pathways
Shiro Mizuno, Herman J Bogaard, Norbert F Voelkel, Yukihiro Umeda, Maiko Kadowaki, Shingo Ameshima, Isamu Miyamori, Takeshi Ishizaki
Respiratory Research , 2009, DOI: 10.1186/1465-9921-10-17
Abstract: Normal human lung fibroblasts (NHLF) were cultured in a hypoxic chamber or exposed to desferroxamine (DFX). DNA synthesis was measured using bromodeoxyuridine incorporation, and expression of p53, p21 and p27 was measured using real-time RT-PCR and Western blot analysis.DNA synthesis was increased by moderate hypoxia (2% oxygen) but was decreased by severe hypoxia (0.1% oxygen) and DFX. Moderate hypoxia decreased p21 synthesis without affecting p53 synthesis, whereas severe hypoxia and DFX increased synthesis of both p21 and p53. p27 protein expression was decreased by severe hypoxia and DFX. Gene silencing of p21 and p27 promoted DNA synthesis at ambient oxygen concentrations. p21 and p53 gene silencing lessened the decrease in DNA synthesis due to severe hypoxia or DFX exposure. p21 gene silencing prevented increased DNA synthesis in moderate hypoxia. p27 protein expression was significantly increased by p53 gene silencing, and was decreased by wild-type p53 gene transfection.These results indicate that in NHLF, severe hypoxia leads to cell cycle arrest via the p53-p21 pathway, but that moderate hypoxia enhances cell proliferation via the p21 pathway in a p53-independent manner. In addition, our results suggest that p27 may be involved in compensating for p53 in cultured NHLF proliferation.Hypoxia is observed in many physiological and pathological conditions, including interstitial lung diseases, acute respiratory distress syndrome, chronic obstructive pulmonary diseases, asthma, wounded tissues, neoplasmas, and atherosclerosis [1-5]. Under such hypoxic conditions, fibroblast proliferation with enhanced production of extracellular matrix (ECM) and marked fibrosis are key components to understanding tissue remodeling [6,7]. Fibroblast proliferation with enhanced production of ECM is an important feature of hypoxia-associated lung diseases, and several in vitro studies have also shown that exposure to moderate hypoxia stimulates the proliferation of lung fibroblasts
Hypoxia-regulated target genes implicated in tumor metastasis  [cached]
Tsai Ya-Ping,Wu Kou-Juey
Journal of Biomedical Science , 2012, DOI: 10.1186/1423-0127-19-102
Abstract: Hypoxia is an important microenvironmental factor that induces cancer metastasis. Hypoxia/hypoxia-inducible factor-1α (HIF-1α) regulates many important steps of the metastatic processes, especially epithelial-mesenchymal transition (EMT) that is one of the crucial mechanisms to cause early stage of tumor metastasis. To have a better understanding of the mechanism of hypoxia-regulated metastasis, various hypoxia/HIF-1α-regulated target genes are categorized into different classes including transcription factors, histone modifiers, enzymes, receptors, kinases, small GTPases, transporters, adhesion molecules, surface molecules, membrane proteins, and microRNAs. Different roles of these target genes are described with regards to their relationship to hypoxia-induced metastasis. We hope that this review will provide a framework for further exploration of hypoxia/HIF-1α-regulated target genes and a comprehensive view of the metastatic picture induced by hypoxia.
Lung Myofibroblasts Are Characterized by Down-Regulated Cyclooxygenase-2 and Its Main Metabolite, Prostaglandin E2  [PDF]
Marta Gabasa, Dolores Royo, Maria Molina-Molina, Jordi Roca-Ferrer, Laura Pujols, Cesar Picado, Antoni Xaubet, Javier Pereda
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0065445
Abstract: Background Prostaglandin E2 (PGE2), the main metabolite of cyclooxygenase (COX), is a well-known anti-fibrotic agent. Moreover, myofibroblasts expressing α-smooth muscle actin (α-SMA), fibroblast expansion and epithelial-mesenchymal transition (EMT) are critical to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Our aim was to investigate the expression of COX-2 and PGE2 in human lung myofibroblasts and establish whether fibroblast-myofibroblast transition (FMT) and EMT are associated with COX-2 and PGE2 down-regulation. Methods Fibroblasts obtained from IPF patients (n = 6) and patients undergoing spontaneous pneumothorax (control, n = 6) and alveolar epithelial cell line A549 were incubated with TGF-β1 and FMT and EMT markers were evaluated. COX-2 and α-SMA expression, PGE2 secretion and cell proliferation were measured after IL-1β and PGE2 incubation. Results Myofibroblasts from both control and IPF fibroblast cultures stimulated with IL-1β showed no COX-2 expression. IPF fibroblasts showed increased myofibroblast population and reduced COX-2 expression in response to IL-1β. TGF-β1 increased the number of myofibroblasts in a time-dependent manner. In contrast, TGF-β1 induced slight COX-2 expression at 4 h (without increase in myofibroblasts) and 24 h, but not at 72 h. Both IPF and control cultures incubated with TGF-β1 for 72 h showed diminished COX-2 induction, PGE2 secretion and α-SMA expression after IL-1β addition. The latter decreased proliferation in fibroblasts but not in myofibroblasts. A549 cells incubated with TGF-β1 for 72 h showed down-regulated COX-2 expression and low basal PGE2 secretion in response to IL-1β. Immuno-histochemical analysis of IPF lung tissue showed no COX-2 immuno-reactivity in myofibroblast foci. Conclusions Myofibroblasts are associated with COX-2 down-regulation and reduced PGE2 production, which could be crucial in IPF development and progression.
F11R Expression upon Hypoxia Is Regulated by RNA Editing  [PDF]
Michal Ben-Zvi, Ninette Amariglio, Gideon Paret, Yael Nevo-Caspi
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0077702
Abstract: F11R is a cell adhesion molecule found on the surface of human platelets. It plays a role in platelet aggregation, cell migration and cell proliferation. F11R is subjected to RNA editing, a post-transcriptional modification which affects RNA structure, stability, localization, translation and splicing. RNA editing in the 3'UTR of F11R and RNA levels are increased upon hypoxia. We therefore set to examine if RNA editing plays a role in the increase of F11R RNA seen upon hypoxic conditions. We show that ADAR1, but not ADAR2, takes part in the editing of F11R however editing alone is not sufficient for obtaining an elevation in RNA levels. In addition we show that hyper-edited mature mRNAs are retained in the nucleus and are associated with p54nrb. We therefore conclude that hypoxia-induced edited RNAs of F11R are preferentially stabilized and accumulate in the nucleus preventing their export to the cytoplasm for translation. This mechanism may be used by additional proteins in the cell as part of the cell's effort to reduce metabolism upon hypoxic stress.
Identification of 9 uterine genes that are regulated during mouse pregnancy and exhibit abnormal levels in the cyclooxygenase-1 knockout mouse
Baohui Zhao, Deanna Koon, Allyson L Curtis, Jessica Soper, Kathleen E Bethin
Reproductive Biology and Endocrinology , 2007, DOI: 10.1186/1477-7827-5-28
Abstract: Gestational d18.0 uteri (n = 4) were collected from pregnant wild-type and cyclooxygenase-1 knockout mice. Part of the uterus was used for frozen sections and RNA was isolated from the remainder. Microarray analysis was performed at the Indiana University School of Medicine Genomic Core and analyzed using the Microarray Data Portal. Northern analysis was performed to confirm microarray data and the genes localized in the gravid uterus by in situ hybridization.We identified 277 genes that are abnormally expressed in the gravid d18.0 cyclooxygenase-1 knockout mouse. Nine of these genes are also regulated in the normal murine uterus during the last half of gestation. Many of these genes are involved in the immune response, consistent with an important role of the immune system in parturition. Expression of 4 of these genes; arginase I, IgJ, Tnfrsf9 and troponin; was confirmed by Northern analysis to be mis-regulated during pregnancy in the knockout mouse. In situ hybridization of these genes demonstrated a similar location in the gravid wild-type and Cox-1 knockout mouse uteri.To our knowledge, this is the first work to demonstrate the uterine location of these 4 genes in the mouse during late pregnancy. There are several putative transcription factor binding sites that are shared by many of the 9 genes identified here including; estrogen and progesterone response elements and Ets binding sites. In summary, this work identifies 9 uterine murine genes that may play a role in parturition. The function of these genes is consistent with an important role of the immune system in parturition.In 2004 12.5% of all births in the USA were preterm [1]. Preterm birth is the leading cause of all infant mortality and a major cause of morbidity [2-4]. The reason that idiopathic preterm labor remains an enigma is that the mechanisms that initiate normal labor are largely unknown. Parturition has been studied in many species, but there is no perfect animal model of human labor [5]. Mou
Multimodal hypoxia imaging and intensity modulated radiation therapy for unresectable non-small-cell lung cancer: the HIL trial  [cached]
Askoxylakis Vasileios,Dinkel Julien,Eichinger Monika,Stieltjes Bram
Radiation Oncology , 2012, DOI: 10.1186/1748-717x-7-157
Abstract: Background Radiotherapy, preferably combined with chemotherapy, is the treatment standard for locally advanced, unresectable non-small cell lung cancer (NSCLC). The tumor response to different therapy protocols is variable, with hypoxia known to be a major factor that negatively influences treatment effectiveness. Visualisation of tumor hypoxia prior to the use of modern radiation therapy strategies, such as intensity modulated radiation therapy (IMRT), might allow optimized dose applications to the target volume, leading to improvement of therapy outcome. 18 F-fluoromisonidazole dynamic positron emission tomography and computed tomography (18 F-FMISO dPET-CT) and functional magnetic resonance imaging (functional MRI) are attractive options for imaging tumor hypoxia. Methods/design The HIL trial is a single centre study combining multimodal hypoxia imaging with 18 F-FMISO dPET-CT and functional MRI, with intensity modulated radiation therapy (IMRT) in patients with inoperable stage III NSCLC. 15 patients will be recruited in the study. All patients undergo initial FDG PET-CT and serial 18 F-FMISO dPET-CT and functional MRI before treatment, at week 5 of radiotherapy and 6 weeks post treatment. Radiation therapy is performed as inversely planned IMRT based on 4D-CT. Discussion Primary objectives of the trial are to characterize the correlation of 18 F-FMISO dPET-CT and functional MRI for tumor hypoxia imaging in NSCLC and evaluate possible effects of radiation therapy on tumor re-oxygenation. Further objectives include the generation of data regarding the prognostic value of 18 F-FMISO dPET-CT and functional MRI for locoregional control, progression free survival and overall survival of NSCLC treated with IMRT, which will form the basis for larger clinical trials focusing on possible interactions between tumor oxygenation and radiotherapy outcome. Trial registration The ClinicalTrials.gov protocol ID is NCT01617980
Interleukin-1beta and fibroblast growth factor receptor 1 cooperate to induce cyclooxygenase-2 during early mammary tumourigenesis
Johanna R Reed, Ronald P Leon, Majken K Hall, Kathryn L Schwertfeger
Breast Cancer Research , 2009, DOI: 10.1186/bcr2246
Abstract: To determine the functional consequences of IL-1β induction during FGFR1-induced mammary tumourigenesis, the effects of IL-1β inhibition on the formation of epithelial hyperplasias were examined using the MMTV-iFGFR1 transgenic mouse model. Further studies used a combination of the HC-11 mammary epithelial cell line that stably expresses iFGFR1 and the MMTV-iFGFR1 transgenic mice to further define the mechanisms of IL-1β function.Inhibition of IL-1β activity in vivo resulted in reduced iFGFR1-induced epithelial proliferation and formation of hyperplastic structures. Further studies demonstrated that treatment of mammary epithelial cells with IL-1β-induced expression of cyclooxygenase (Cox)-2 both in vitro and in vivo. Finally, inhibition of Cox-2 prior to activation of iFGFR1 in the transgenic mice also resulted in decreased iFGFR1-induced formation of hyperplastic structures.The results from these studies indicate that targeting the inflammatory cytokine IL-1β partially inhibits iFGFR1-induced formation of early-stage mammary lesions, in part through induction of Cox-2. These findings demonstrate that activation of a growth factor receptor in mammary epithelial cells results in increased expression of inflammatory mediators, which cooperate to promote the initiation of hyperplastic lesions in the mammary gland.Inflammation is a well-known risk factor for tumour development and correlates with increased invasiveness and poor prognosis in a variety of cancers [1]. It is well-established that chronic inflammation that is driven by extrinsic factors promotes several types of cancer, including gastric, hepatic and gastrointestinal cancers [1]. However, inflammation has also been correlated with the development of cancers that are not typically associated with chronic inflammatory states, such as breast cancer. There has been ongoing interest in the concept that intrinsic factors, such as activation of an oncogene within epithelial cells, induce a state of localised infl
Expression of DDX3 Is Directly Modulated by Hypoxia Inducible Factor-1 Alpha in Breast Epithelial Cells  [PDF]
Mahendran Botlagunta,Balaji Krishnamachary,Farhad Vesuna,Paul T. Winnard Jr.,Guus M. Bol,Arvind H. Patel,Venu Raman
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0017563
Abstract: DEAD box protein, DDX3, is aberrantly expressed in breast cancer cells ranging from weakly invasive to aggressive phenotypes and functions as an important regulator of cancer cell growth and survival. Here, we demonstrate that hypoxia inducible factor-1α is a transcriptional activator of DDX3 in breast cancer cells. Within the promoter region of the human DDX3 gene, we identified three putative hypoxia inducible factor-1 responsive elements. By luciferase reporter assays in combination with mutated hypoxia inducible factor-1 responsive elements, we determined that the hypoxia inducible factor-1 responsive element at position -153 relative to the translation start site is essential for transcriptional activation of DDX3 under hypoxic conditions. We also demonstrated that hypoxia inducible factor-1 binds to the DDX3 promoter and that the binding is specific, as revealed by siRNA against hypoxia inducible factor-1 and chromatin immunoprecipitation assays. Thus, the activation of DDX3 expression during hypoxia is due to the direct binding of hypoxia inducible factor-1 to hypoxia responsive elements in the DDX3 promoter. In addition, we observed a significant overlap in the protein expression pattern of hypoxia inducible factor-1α and DDX3 in MDA-MB-231 xenograft tumors. Taken together, our results demonstrate, for the first time, the role of DDX3 as a hypoxia-inducible gene that exhibits enhanced expression through the interaction of hypoxia inducible factor-1 with hypoxia inducible factor-1 responsive elements in its promoter region.
Fibroblast Migration Is Regulated by Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Protein  [PDF]
Laura E. Ott, Eui Jae Sung, Adam T. Melvin, Mary K. Sheats, Jason M. Haugh, Kenneth B. Adler, Samuel L. Jones
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0066512
Abstract: Myristoylated alanine-rich C-kinase substrate (MARCKS) is a ubiquitously expressed substrate of protein kinase C (PKC) that is involved in reorganization of the actin cytoskeleton. We hypothesized that MARCKS is involved in regulation of fibroblast migration and addressed this hypothesis by utilizing a unique reagent developed in this laboratory, the MANS peptide. The MANS peptide is a myristoylated cell permeable peptide corresponding to the first 24-amino acids of MARCKS that inhibits MARCKS function. Treatment of NIH-3T3 fibroblasts with the MANS peptide attenuated cell migration in scratch wounding assays, while a myristoylated, missense control peptide (RNS) had no effect. Neither MANS nor RNS peptide treatment altered NIH-3T3 cell proliferation within the parameters of the scratch assay. MANS peptide treatment also resulted in inhibited NIH-3T3 chemotaxis towards the chemoattractant platelet-derived growth factor-BB (PDGF-BB), with no effect observed with RNS treatment. Live cell imaging of PDGF-BB induced chemotaxis demonstrated that MANS peptide treatment resulted in weak chemotactic fidelity compared to RNS treated cells. MANS and RNS peptides did not affect PDGF-BB induced phosphorylation of MARCKS or phosphoinositide 3-kinase (PI3K) signaling, as measured by Akt phosphorylation. Further, no difference in cell migration was observed in NIH-3T3 fibroblasts that were transfected with MARCKS siRNAs with or without MANS peptide treatment. Genetic structure-function analysis revealed that MANS peptide-mediated attenuation of NIH-3T3 cell migration does not require the presence of the myristic acid moiety on the amino-terminus. Expression of either MANS or unmyristoylated MANS (UMANS) C-terminal EGFP fusion proteins resulted in similar levels of attenuated cell migration as observed with MANS peptide treatment. These data demonstrate that MARCKS regulates cell migration and suggests that MARCKS-mediated regulation of fibroblast migration involves the MARCKS amino-terminus. Further, this data demonstrates that MANS peptide treatment inhibits MARCKS function during fibroblast migration and that MANS mediated inhibition occurs independent of myristoylation.

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