37 research outputs found
RNAi-Based Strategies for Cyclooxygenase-2 Inhibition in Cancer
No full textCyclooxygenase-2 (COX-2) enzyme has been involved in the tumorigenesis and in the progression of colorectal cancer (CRC). The use of traditional nonsteroidal anti-inflammatory drugs (NSAIDs) or selective COX-2 inhibitors has been proposed for the prevention and the treatment of this relevant neoplastic disease. In the light of an innovative alternative to these pharmacological approaches, we review here the possible strategies to achieve a strong and selective inhibition of COX-2 enzyme by using the mechanism of RNA Interference (RNAi) targeted against its mRNA. Anti-COX-2 siRNA molecules (siCOX-2) can be generated in CRC cells from short hairpin RNA (shRNA) precursors, delivered in vitro by a retroviral expression system, and induce a significant and stable silencing of overexpressed COX-2 in human colon cancer cells. As a safer alternative to viral approach, nonpathogenic bacteria (E. coli) can be engineered to invade eukaryotic cells and to generate siCOX-2 molecules in cancer cells. Moreover, the involvement of miRNAs in COX-2 posttranscriptional regulation opens up the possibility to exploit an endogenous silencing mechanism to knockdown overexpressed COX-2. Thus, these recent strategies disclose new challenging perspectives for the development of clinically compatible siRNA or miRNA capable of selectively inhibiting COX-2 enzyme.</jats:p
Selective inhibition of prostacyclin synthase activity by rofecoxib
No full textThe development of cyclooxygenase-2 (COX-2) selective inhibitors prompted studies aimed at treating chronic inflammatory diseases and cancer by using this new generation of drugs.Yet, several recent reports pointed out that long-term treatment of patients with COX-2 selective inhibitors (especially rofecoxib) caused severe cardiovascular complicances. The aim of this study was to ascertain whether, in addition to inhibiting COX-2, rofecoxib may also affect prostacyclin (PGI2) level by inhibiting PGI2 forming enzyme (prostacyclin synthase, PGIS). In order to evaluate if selective (celecoxib, rofecoxib) and non-selective (aspirin, naproxen) anti-inflammatory compounds could decrease PGI2 production in endothelial cells by inhibiting PGIS, we analyzed the effect of anti-inflammatory compounds on the enzyme activity by ELISA assay after addition of exogenous substrate, on PGIS protein levels by Western blotting and on its subcellular distribution by confocal microscopy. We also analyzed the effect of rofecoxib on PGIS activity in bovine aortic microsomal fractions enriched in PGIS. This study demonstrates an inhibitory effect of rofecoxib on PGIS activity in human umbilical vein endothelial (HUVE) cells and in PGIS-enriched bovine aortic microsomal fractions, which is not observed by using other anti-inflammatory compounds. The inhibitory effect of rofecoxib is associated neither to a decrease of PGIS protein levels nor to an impairment of the enzyme intracellular localization. The results of this study may explain the absence of a clear relationship between COX-2 selectivity and cardiovascular side effects. Moreover, in the light of these results we propose that novel selective COX-2 inhibitors should be tested on PGI2 synthase activity inhibition
RNA interference as a key to knockdown overexpressed cycloxygenase 2 gene in tumour cells
No full textSilencing those genes that are overexpressed in cancer and contribute to the survival and progression of tumour cells is the aim of several researches. Cyclooxygenase-2 (COX-2) is one of the most intensively studied genes since it is overexpressed in most tumours, mainly in colon cancer. The use of specific COX-2 inhibitors to treat colon cancer has generated great enthusiasm. Yet, the side effects of some inhibitors emerging during long-term treatment have caused much concern. Genes silencing by RNA interference (RNAi) has led to new directions in the field of experimental oncology. In this study, we detected sequences directed against COX-2 mRNA, that potently downregulate COX-2 gene expression and inhibit phorbol 12-myristate 13-acetate-induced angiogenesis in vitro in a specific, nontoxic manner. Moreover, we found that the insertion of a specific cassette carrying anti-COX-2 short hairpin RNA sequence into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT29) without activating any interferon response. Phenotypically, COX-2 deficient HT29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, the retroviral approach enhancing COX-2 knockdown, mediated by RNAi, proved to be an useful tool to better understand the role of COX-2 in colon cancer. Furthermore, the higher infection efficiency we observed in tumour cells, if compared to normal endothelial cells, may disclose the possibility to specifically treat tumour cells without impairing endothelial COX-2 activit
Caveolae and caveolae constituents in mechanosensing: effect of modelled microgravity on cultured human endothelial cells.
No full textStudies in modeled microgravity or during orbital space flights have clearly demonstrated that endothelial cell physiology is strongly affected by the reduction of gravity. Nevertheless, the molecular mechanisms by which endothelial cells may sense gravity force remain unclear. We previously hypothesized that endothelial cell caveolae could be a mechanosensing system involved in hypergravity adaptation of human endothelial cells. In this study, we analyzed the effect on the physiology of human umbilical vein endothelial cell monolayers of short exposure to modeled microgravity (24-48 h) obtained by clinorotation. For this purpose, we evaluated the levels of compounds, such as nitric oxide and prostacyclin, involved in vascular tone regulation and synthesized starting from caveolae-related enzymes. Furthermore, we examined posttranslational modifications of Caveolin (Cav)-1 induced by simulated microgravity. The results we collected clearly indicated that short microgravity exposure strongly affected endothelial nitric oxide synthase activity associated with Cav-1 (Tyr 14) phosphorylation, without modifying the angiogenic response of human umbilical vein endothelial cells. We propose here that one of the early molecular mechanisms responsible for gravity sensing of endothelium involves endothelial cell caveolae and Cav-1 phosphorylation
Effect of copper on extracellular levels of key pro-inflammatory molecules in hypothalamic GN11 and primary neurons
No full textCopper dyshomeostasis is responsible for the neurological symptoms observed in the genetically inherited copper-dependent disorders (e.g., Menkes' and Wilson's diseases), but it has been also shown to have an important role in neurodegenerative diseases such as Alzheimer disease, prion diseases, Parkinson's disease and amyotrophic lateral sclerosis. It is widely accepted that increased extracellular copper levels contribute to neuronal pathogenic process by increasing the production of dangerous radical oxygen species, but the existence of other molecular mechanisms explaining copper neurotoxicity has not been investigated yet. By using a cellular model based on hypothalamic GN11 cultured neurons exposed to copper supplementation and by analysing the cell conditioned media, we try here to identify new molecular events explaining the association between extracellular copper accumulation and neuronal damages. We show here that increased extracellular copper levels produce a wide complex of alterations in the neuronal extracellular environment. In particular, copper affects the secretion of molecules involved in the protection of neurons against oxidative stress, such as cyclophilin A (CypA), or of molecules capable of shifting neuronal cells towards a pro-inflammatory state, such as IL-1 alpha, IL-12, Rantes, neutrophil gelatinase-associated lipocalin (NGAL) and secreted protein acidic and rich in cysteine (SPARC). Copper pro-inflammatory properties have been confirmed by using primary neurons. (C) 2009 Elsevier Inc. All rights reserved
Drosophila Awd/Nm23 expression during larval development in normal and pathological conditions.
No full textSelective inhibition of prostacyclin synthase activity by rofecoxib
No full textThe development of cyclooxygenase-2 (COX-2) selective inhibitors prompted studies aimed at treating chronic
inflammatory diseases and cancer by using this new generation of drugs.Yet, several recent reports pointed
out that long-term treatment of patients with COX-2 selective inhibitors (especially rofecoxib) caused
severe cardiovascular complicances. The aim of this study was to ascertain whether, in addition to inhibiting
COX-2, rofecoxib may also affect prostacyclin (PGI2) level by inhibiting PGI2 forming enzyme (prostacyclin
synthase, PGIS). In order to evaluate if selective (celecoxib, rofecoxib) and non-selective (aspirin, naproxen)
anti-inflammatory compounds could decrease PGI2 production in endothelial cells by inhibiting PGIS, we
analyzed the effect of anti-inflammatory compounds on the enzyme activity by ELISA assay after addition of
exogenous substrate, on PGIS protein levels by Western blotting and on its subcellular distribution by confocal
microscopy. We also analyzed the effect of rofecoxib on PGIS activity in bovine aortic microsomal fractions
enriched in PGIS. This study demonstrates an inhibitory effect of rofecoxib on PGIS activity in human
umbilical vein endothelial (HUVE) cells and in PGIS-enriched bovine aortic microsomal fractions, which is not
observed by using other anti-inflammatory compounds. The inhibitory effect of rofecoxib is associated neither
to a decrease of PGIS protein levels nor to an impairment of the enzyme intracellular localization. The results
of this study may explain the absence of a clear relationship between COX-2 selectivity and cardiovascular
side effects. Moreover, in the light of these results we propose that novel selective COX-2 inhibitors should
be tested on PGI2 synthase activity inhibition
Assays for membrane and intracellular signalling events. in Angiogenesis Assays
No full textThe growth of new blood vessels from existing vessels, familiar to most as angiogenesis or neovascularization, has acquired an importance that would have been difficult to imagine a few years ago. The proven efficacy of recently approved drugs that block angiogenesis in tumours and age-related macular degeneration has heightened the visibility and relevance of research on blood vessel growth and regression. The promise of factors that stimulate functional revascularization of organs, starved of their blood supply by ischaemic vascular disease or other conditions, is also increasing. Success in the clinic has shifted into high gear the search for even more efficacious drugs. How can these agents be identified, screened and tested? How can their mechanism of action be determined? The seemingly ideal approach for evaluating agents would be through pre-clinical models of the targeted diseases. However, few pre-clinical models faithfully mimic human disease. There-fore, the search continues for faster, easier, more relevant ways of assessing agents that stimulate or inhibit angiogenesis
The German Shorthair Pointer Dog Breed (Canis lupus familiaris) : Genomic Inbreeding and Variability
No full textThe German Shorthaired Pointer (GSHP) is a breed worldwide known for its hunting versatility. Dogs of this breed are appreciated as valuable companions, effective trackers, field trailers and obedience athletes. The aim of the present work is to describe the genomic architecture of the GSHP breed and to analyze inbreeding levels under a genomic and a genealogic perspective. A total of 34 samples were collected (24 Italian, 10 USA), and the genomic and pedigree coefficients of inbreeding have been calculated. A total of 3183 runs of homozygosity (ROH) across all 34 dogs have been identified. The minimum and maximum number of Single Nucleotide Polymorphisms (SNPs) defining all ROH are 40 and 3060. The mean number of ROH for the sample was 93.6. ROH were found on all chromosomes. A total of 854 SNPs (TOP_SNPs) defined 11 ROH island regions (TOP_ROH), in which some gene already associated with behavioral and morphological canine traits was annotated. The proportion of averaged observed homozygotes estimated on total number of SNPs was 0.70. The genomic inbreeding coefficient based on ROH was 0.17. The mean inbreeding based on genealogical information resulted 0.023. The results describe a low inbred population with quite a good level of genetic variability
