Biomedical subjects

Sylvie G Bernier

Publications and source records attributed to Sylvie G Bernier.

10 recordsLinked to original sources

PubMed2006

Inhibition of melanoma tumor growth by a pharmacological inhibitor of MetAP-2, PPI-2458.

Over the past few decades, melanoma has shown the fastest growing incidence rate of all cancers. This malignancy is clinically defined by its potential to rapidly metastasize, and advanced metastatic melanomas are highly resistant to existing therapeutic regimens. Here, we report that PPI-2458, a novel, orally active agent of the fumagillin class of irreversible methionine aminopeptidase-2 (MetAP-2) inhibitors, potently inhibited the proliferation of B16F10 melanoma cells in vitro, with a growth inhibitory concentration 50% (GI50) of 0.2 nM. B16F10 growth inhibition was correlated with the inhibition of MetAP-2 enzyme, in a dose-dependent fashion, as determined by a pharmacodynamic assay, which measures the amount of uninhibited MetAP-2 following PPI-2458 treatment. Prolonged exposure of B16F10 cells to PPI-2458 at concentrations of up to 1 microM, 5,000-fold above the GI50, did not alter their sensitivity to PPI-2458 growth inhibition and no drug resistance was observed. Moreover, prolonged exposure to this agent induced melanogenesis, concomitant with the elevated expression of the melanocyte-specific enzymes tyrosinase and tyrosinase-related proteins (TRP) 1 and 2, a morphological feature associated with differentiated melanocytes. PPI-2458, when administered orally (p.o.), significantly inhibited B16F10 tumor growth in mice in a dose-dependent fashion, with a maximum inhibition of 62% at 100 mg/kg. This growth inhibition was directly correlated to the amount of irreversibly inhibited MetAP-2 (80% at 100 mg/kg PPI-2458) in tumor tissue. These data demonstrate that PPI-2458 has potent antiproliferative activity against B16F10 cells in vitro and in vivo, and that both activities are directly correlated with levels of MetAP-2 enzyme inhibition. This antiproliferative activity, coupled with additional observations from studies in vitro (absence of detectable resistance to PPI-2458 and induction of morphological features consistent with differentiated melanocytes), provides a rationale for assessing the therapeutic potential of PPI-2458 in the treatment of melanoma.

Administration, Oral↗

PubMed2005

Methionine aminopeptidases type I and type II are essential to control cell proliferation.

The dependence of cell growth on methionine aminopeptidase (MetAP) function in bacteria and yeast is firmly established. Here we report experimental evidence that the control of cell proliferation in mammalian cells is directly linked and strictly dependent on the activity of both MetAP-1 and MetAP-2. The targeted downregulation of either methionine aminopeptidase MetAP-1 or MetAP-2 protein expression by small interfering RNA (siRNA) significantly inhibited the proliferation of human umbilical vein endothelial cells (HUVEC) (70%-80%), while A549 human lung carcinoma cell proliferation was less inhibited (20%-30%). The cellular levels of MetAP-2 enzyme were measured after MetAP-2 siRNA treatment and found to decrease over time from 4 to 96 h, while rapid and complete depletion of MetAP-2 enzyme activity was observed after 4 h treatment with two pharmacological inhibitors of MetAP-2, PPI-2458 and fumagillin. When HUVEC and A549 cells were treated simultaneously with MetAP-2 siRNA and PPI-2458, or fumagillin, which irreversibly inhibit MetAP-2 enzyme activity, no additive effect on maximum growth inhibition was observed. This strongly suggests that MetAP-2 is the single critical cellular enzyme affected by either MetAP-2 targeting approach. Most strikingly, despite their significantly different sensitivity to growth inhibition after targeting of either MetAP-1 or MetAP-2, HUVEC, and A549 cells, which were made functionally deficient in both MetAP-1 and MetAP-2 were completely or almost completely inhibited in their growth, respectively. This closely resembled the observed growth inhibition in genetically double-deficient map1map2 yeast strains. These results suggest that MetAP-1 and MetAP-2 have essential functions in the control of mammalian cell proliferation and that MetAP-dependent growth control is evolutionarily highly conserved.

Aminopeptidases↗

PubMed2004

A methionine aminopeptidase-2 inhibitor, PPI-2458, for the treatment of rheumatoid arthritis.

The hallmark of rheumatoid arthritis (RA) is the progressive destruction of articular joints, characterized by invasive synovial hyperplasia and pathological neovascularization. Here we report that PPI-2458, a member of the fumagillin class of irreversible methionine aminopeptidase-2 (MetAP-2) inhibitors, potently inhibits the proliferation of human fibroblast-like synoviocytes (HFLS-RA), derived from RA patients, with a growth inhibitory concentration 50 (GI(50)) of 0.04 nM and a maximum inhibition of >95% at 1 nM. Human umbilical vein endothelial cells (HUVEC) are similarly inhibited in proliferation by PPI-2458 (GI(50), 0.2 nM). We developed a method to measure the level of MetAP-2 enzyme inhibition after exposure to PPI-2458 and demonstrate that growth inhibition of PPI-2458-sensitive HFLS-RA and HUVEC is linked to MetAP-2 enzyme inhibition, in a dose-dependent fashion. The secretion of several inflammatory mediators such as IL-6 and vascular endothelial growth factor from activated HFLS-RA was not inhibited by PPI-2458. The CNS toxicity profile of PPI-2458, determined by the incidence of seizures, is significantly improved over that of the parental compound TNP-470. In the rat model of peptidoglycan-polysaccharide-induced arthritis, PPI-2458 significantly attenuated paw swelling when therapeutically administered after the onset of chronic disease. We suggest that the mechanism of PPI-2458 action, highly selective and potent anti-proliferative activity on HFLS-RA and HUVEC in vitro, a significantly improved CNS toxicity profile, and marked attenuation of chronic disease in the rat peptidoglycan-polysaccharide arthritis model in vivo, positions this compound as a drug for the treatment of RA.

Aminopeptidases↗

PubMed2003

Modulation of pro-inflammatory gene expression by nuclear lysophosphatidic acid receptor type-1.

Lysophosphatidic acid (LPA) is a bioactive molecule involved in inflammation, immunity, wound healing, and neoplasia. Its pleiotropic actions arise presumably by interaction with their cell surface G protein-coupled receptors. Herein, the presence of the specific nuclear lysophosphatidic acid receptor-1 (LPA1R) was revealed in unstimulated porcine cerebral microvascular endothelial cells (pCMVECs), LPA1R stably transfected HTC4 rat hepatoma cells, and rat liver tissue using complementary approaches, including radioligand binding experiments, electron- and cryomicroscopy, cell fractionation, and immunoblotting with three distinct antibodies. Coimmunoprecipitation studies in enriched plasmalemmal fractions of unstimulated pCMVEC showed that LPA1Rs are dually sequestrated in caveolin-1 and clathrin subcompartments, whereas in nuclear fractions LPA1R appeared primarily in caveolae. Immunofluorescent assays using a cell-free isolated nuclear system confirmed LPA1R and caveolin-1 co-localization. In pCMVEC, LPA-stimulated increases in cyclooxygenase-2 and inducible nitric-oxide synthase RNA and protein expression were insensitive to caveolea-disrupting agents but sensitive to LPA-generating phospholipase A2 enzyme and tyrosine kinase inhibitors. Moreover, LPA-induced increases in Ca2+ transients and/or iNOS expression in highly purified rat liver nuclei were prevented by pertussis toxin, phosphoinositide 3-kinase/Akt inhibitor wortmannin and Ca2+ chelator and channel blockers EGTA and SK&F96365, respectively. This study describes for the first time the nucleus as a potential organelle for LPA intracrine signaling in the regulation of pro-inflammatory gene expression.

Androstadienes↗

PubMed2003

Platelet activating factor receptors.

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Animals↗

PubMed2003

Increased platelet-activating factor-induced periventricular brain microvascular constriction associated with immaturity.

Oxidant stress contributes to the pathogenesis of hypoxic-ischemic encephalopathies. Platelet-activating factor (PAF) is generated during oxidant stress. We studied the vasomotor mode of actions of PAF on periventricular (PV) microvessels of fetal ( approximately 75% of term), newborn (1-3 days), and adult pigs. PAF constricted PV microvessels from fetal (29.27 +/- 2.6%) and newborn (22.14 +/- 3.2%) pigs but was ineffective in adults (<2.5%). Specific [(3)H]PAF binding was greater in fetus and newborn than in adults; a concordant developmental PAF-induced inositol phosphate formation was observed. PAF-induced vasoconstriction was abrogated by thromboxane A(2) (TXA(2)) synthase and receptor inhibitors, calcium channel blockers, and by removal of endothelium; vasoconstriction to TXA(2) mimetic U-46619 did not differ with age. Immunoreactive TXA(2) synthase expression and PAF-evoked TXA(2) formation revealed a fetus> newborn>adult profile. Thus the greater PAF-induced PV microvascular constriction in younger subjects seems attributable to greater PAF receptor density and mostly secondary to TXA(2) formation from endothelium. The resulting decrease in blood flow may contribute to the increased vulnerability of the PV brain regions to oxidant stress-induced injury in immature subjects.

Aging↗

PubMed2002

Proinflammatory gene induction by platelet-activating factor mediated via its cognate nuclear receptor.

It has been postulated that intracellular binding sites for platelet-activating factor (PAF) contribute to proinflammatory responses to PAF. Isolated nuclei from porcine cerebral microvascular endothelial cells (PCECs) produced PAF-molecular species in response to H(2)O(2). Using FACS analysis, we demonstrated the expression of PAF receptors on cell and nuclear surfaces of PCECs. Confocal microscopy studies performed on PCECs, Chinese hamster ovary cells stably overexpressing PAF receptors, and isolated nuclei from PCECs also showed a robust nuclear distribution of PAF receptors. Presence of PAF receptors at the cell nucleus was further revealed in brain endothelial cells by radioligand binding experiments, immunoblotting, and in situ in brain by immunoelectron microscopy. Stimulation of nuclei with methylcarbamate-PAF evoked a decrease in cAMP production and a pertussis toxin-sensitive rise in nuclear calcium, unlike observations in plasma membrane, which exhibited a pertussis toxin-insensitive elevation in inositol phosphates. Moreover, on isolated nuclei methylcarbamate-PAF evoked the expression of proinflammatory genes inducible nitric oxide synthase and cyclooxygenase-2 (COX-2) and was associated with augmented extracellular signal-regulated kinase 1/2 phosphorylation and NF-kappaB binding to the DNA consensus sequence. COX-2 expression was prevented by mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 and NF-kappaB inhibitors. This study describes for the first time the nucleus as a putative organelle capable of generating PAF and expresses its receptor, which upon stimulation induces the expression of the proinflammatory gene COX-2.

Animals↗

PubMed2002

Regulation of eNOS expression in brain endothelial cells by perinuclear EP(3) receptors.

We reported upregulation of endothelial nitric oxide synthase (eNOS) by PGE(2) in tissues and presence of perinuclear PGE(2) receptors (EP). We presently studied mechanisms by which PGE(2) induces eNOS expression in cerebral microvessel endothelial cells (ECs). 16,16-Dimethyl PGE(2) and selective EP(3) receptor agonist M&B28767 increased eNOS expression in ECs and the NO-dependent vasorelaxant responses induced by substance P on cerebral microvessels. These effects could be prevented by prostaglandin transporter blocker bromcresol green and actinomycin D. EP(3) immunoreactivity was confirmed on plasma and perinuclear membrane of ECs. M&B28767 increased eNOS RNA expression in EC nuclei, and this effect was augmented by overexpression of EP(3) receptors. M&B28767 also induced increased phosphorylation of Erk-1/2 and Akt, as well as changes in membrane potential revealed by the potentiometric fluorescent dye RH421, which were prevented by iberiotoxin; perinuclear K(Ca) channels were detected, and their functionality corroborated by NS1619-induced Ca(2+) signals and nuclear membrane potential changes. Moreover, pertussis toxin, Ca(2+) chelator, and channel blockers EGTA, BAPTA, and SK&F96365, as well as K(Ca) channel blocker iberiotoxin, protein-kinase inhibitors wortmannin and PD 98059, and NF-kappaB inhibitor pyrrolidine dithiocarbamate prevented M&B28767-induced increase in Ca(2+) transients and/or eNOS expression in EC nuclei. We describe for the first time that PGE(2) through its access into cell by prostaglandin transporters induces eNOS expression by activating perinuclear EP(3) receptors coupled to pertussis toxin-sensitive G proteins, a process that depends on nuclear envelope K(Ca) channels, protein kinases, and NF-kappaB; the roles for nuclear EP(3) receptors seem different from those on plasma membrane.

Animals↗

PubMed2002

PGE(2)-mediated eNOS induction in prolonged hypercapnia.

PURPOSE: Because prostaglandins (PGs) are implicated in acute hypercapnia-induced hyperemia, this study was conducted to test the hypothesis that prolonged hypercapnia may cause a sustained increase in retinal blood flow (RBF) through a PG-dependent induction of endothelial nitric oxide synthase (eNOS). METHODS: Time-dependent RBF (microsphere technique), PGE(2), nitrite (NO(2)(-)), and NOS protein (reduced nicotinamide adenine dinucleotide phosphate [NADPH]-diaphorase staining) production were measured in hypercapnia (6% CO(2))-treated piglets. From the same species, PGE(2), eNOS mRNA, NOS protein, and vasomotor responses were measured in eyecup preparations, as were Ca(2+) transients in neuroretinovascular endothelial cells. RESULTS: Hypercapnia caused biphasic (at 0.5 hours and 6-8 hours) increases in RBF that were abolished with normalization of the pH. The early phase (0.5 hour) was associated with an increase in PGE(2) levels and the latter phase (6-8 hours) with an increase in NO(2)(-) and NOS protein. Inhibition of cyclooxygenase by diclofenac prevented the early and late increase in RBF. NOS inhibitor L-nitro-arginine prevented only the latter. Hypercapnic acidosis increased retinal PGE(2) levels and eNOS-dependent vasorelaxation ex vivo. The ex vivo time course of eNOS mRNA expression corresponded with the late-phase increase in RBF and was blocked by the transcription inhibitor actinomycin D and the receptor-operated Ca(2+) channel blocker SK&F96365. In neuroretinovascular cells, acidosis increased Ca(2+) transients, which were inhibited by SK&F96365, but not diclofenac. CONCLUSIONS: This study discloses a previously unexplored mechanism for late retinal hyperemia during sustained hypercapnia that appears secondary to the induced expression of eNOS mediated by PGE(2).

Animals↗

PubMed2002

Platelet-activating factor in vasoobliteration of oxygen-induced retinopathy.

PURPOSE: To test whether platelet-activating factor (PAF) directly causes retinovascular endothelial cell (EC) death. METHODS: Retinovascular density was calculated in rat pups exposed to 80% O(2) from postnatal days (P)6 to P14 (to produce oxygen-induced retinopathy [OIR]), using the adenosine diphosphatase (ADPase) technique, in animals treated with distinct PAF receptor blockers (PCA-4248, BN52021, or THG315). PAF levels were then measured in the retinas. Viability of ECs from piglets and humans in response to C-PAF (a stable PAF analogue) was determined by the reduction of the tetrazolium salt 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) by viable cells, incorporation of propidium iodide (PI), TUNEL assay, and release of lactate dehydrogenase. Release of thromboxane (TX) was measured in the cell media. RESULTS: PAF levels in retina were markedly increased by exposure of isolated rat retinas to H(2)O(2) (1 micro M) and of rat pups placed in 80% O(2). Exposure to 80% O(2) induced retinal vasoobliteration, which was equally significantly inhibited ( approximately 60%) by all PAF receptor blockers tested. C-PAF increased incorporation of PI by isolated rat retinal microvasculature. Also, C-PAF caused time- and concentration-dependent death of cultured retinal ECs, which was prevented by the PAF receptor antagonist CV-3988. This effect of C-PAF was selective on retinal and neurovascular ECs, but not on other ECs. DNA fragmentation (TUNEL) was hardly detected, and inhibition of apoptosis-related processes by nicotinamide, cyclosporin A, and Z-DEVD-FMK and Z-VAD-FMK (caspase inhibitors) barely protected against death in EC, whereas C-PAF increased release of lactate dehydrogenase, implying that necrosis is the nature of EC death. Finally, C-PAF-induced cell death was preceded by an increase in TXB(2) levels and was prevented by TXA(2) synthase inhibition (with CGS12970). CONCLUSIONS: The data suggest PAF plays a major role in vasoobliteration in OIR by triggering death of neuroretinal microvascular ECs. The cell death seems to be mediated at least in part by TXA(2). These effects of PAF may participate in ischemic retinopathies such as diabetes and retinopathy of prematurity.

Animals↗