Papers by Caroline Pinet-charvet
European Journal of Cancer, 2012
Background: Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor i... more Background: Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. Median survival of patients is 15 months and effective therapies are needed. Abnormal activation of the developmental Hedgehog (Hh) pathway is observed in GBM and other cancers. An inhibitor of this pathway is cyclopamine, an antagonist of the Hh pathway component Smoothened (SMO). To avoid the toxicity of cyclopamine in somatic stem cells that are also Hh-dependent, we previously synthetized a glucuronide prodrug (2b) designed to deliver cyclopamine in the presence of b-glucuronidase, an enzyme found at high levels in the necrotic area of numerous tumors. The expected reduced cytotoxicity of the prodrug and the release of active cyclopamine in the presence of b-glucuronidase were confirmed using U87 human GBM cells. Cancer stem cells (CSC) are known to be resistant to conventional therapies and are found in GBM. Here, we describe the effects of the prodrug 2b in C6 rat GBM cells which present a large population of CSC. Material and Methods: CSC were identified in the C6 cell line using tumorsphere cultures (C6-CSC) and immunodetection of stem cell markers. The effects of the prodrug 2b were examined using MTS cell viability, clonogenic survival and caspase-3/7activity assays. Targeting of the Hh pathway was assessed by measuring the expression of the Hh target gene, GLI1, by real time RT-PCR. Results: SMO expression was observed in C6 cells by immunofluorescence. Viability, clonogenicity and apoptosis tests indicated that the prodrug 2b in the presence of b-glucuronidase was toxic for C6 cells and C6-CSC. This toxicity was specific to the Hh pathway, since GLI1 gene expression was downregulated by the prodrug 2b in the presence of b-glucuronidase in C6 cells and C6-CSC. None of these effects was observed with the prodrug 2b in the absence of b-glucuronidase. Finally, the prodrug even in the presence of b-glucuronidase did not alter the viability of normal rat astrocytes. Conclusions: The cyclopamine glucuronide prodrug 2b is converted to an active drug by b-glucuronidase, an enzyme found in the necrotic area of GBM. This active cyclopamine is toxic to glioblastoma cells and glioblastoma stem cells by targeting the Hh pathway, but does not impair viability of normal astrocytes. The prodrug 2b is a good candidate for in vivo investigations using C6 cells that generate tumors presenting a necrotic area when injected into the rat brain.
Scientific Reports
The control of ovulation helps guarantee the success of reproduction and as such, contributes to ... more The control of ovulation helps guarantee the success of reproduction and as such, contributes to the fitness of a species. In mammals, two types of ovulation are observed: induced and spontaneous ovulation. Recent work on camelids, that are induced ovulators, highlighted the role of a factor present in seminal plasma, beta Nerve Growth Factor (β-NGF), as the factor that triggers ovulation in a GnRH dependent manner. In the present work, we characterized alpaca β-NGF (aβ-nGf) and its 3D structure and compared it with human recombinant β-NGF (hβ-NGF). We showed that the β-NGF enriched fraction of alpaca semen and the human recombinant protein, both stimulated spontaneous electrical activity of primary GnRH neurons derived from mouse embryonic olfactory placodes. This effect was dose-dependent and mediated by p75 receptor signaling. P75 receptors were found expressed in vitro by olfactory ensheathing cells (OEC) in close association with GnRH neurons and in vivo by tanycytes in close vicinity to GnRH fibers in adult mouse. Altogether, these results suggested that β-NGF induced ovulation through an increase in GnRH secretion provoked by a glial dependent P75 mediated mechanism. Two modes of ovulation have been described in mammals: spontaneous ovulation and induced ovulation. Spontaneous ovulation occurs in many mammalian species (mouse, rat, bovine, ovine, equine, primates). In these species, ovulation occurs spontaneously whether the female copulates or not. Induced ovulation has been described in a great variety of species belonging to various mammalian Orders. Induced ovulation has been particularly well studied in camelids 1 . In these species, ovulation occurs after mating in more than 95% of females. These observations drove to the hypothesis of a physical stimulation of the female genitalia and the existence of an ovulation reflex mediated through the spine medulla. Spontaneous ovulators have been intensively studied since most laboratory species belong to this category. In these species, it is acknowledged that ovulation is triggered by an increase in luteinizing hormone (LH) levels (preovulatory surge) as the consequence of an increase in gonadotropin releasing hormone (GnRH) release. The timing of ovulation is controlled by kisspeptin neurons
INRA Productions Animales
Le déclenchement de l’ovulation peut se faire selon deux modes chez les mammifères : spontané et ... more Le déclenchement de l’ovulation peut se faire selon deux modes chez les mammifères : spontané et provoqué. L’ovulation spontanée intervient au cours du cycle œstral sous l’effet de facteurs internes hormonaux. L’ovulation provoquée est déclenchée par l’accouplement avec un mâle. Dans les deux cas, c’est une augmentation de la sécrétion de GnRH qui conduit à l’augmentation de LH qui provoque l’ovulation. Les facteurs qui conduisent à l’augmentation de sécrétion de GnRH sont différents selon les deux modalités : principalement la kisspeptine (ovulation spontanée) et le b-NGF (ovulation provoquée). Les protocoles d’induction de l’ovulation actuels reposent sur une action directe sur l’ovaire, grâce à l’utilisation de gonadotropines hétérologues, ou bien par une action sur l’hypophyse grâce aux agonistes du GnRH. Ces protocoles présentent différents inconvénients : perte d’efficacité dans le temps, stimulation supra-physiologique qui peut être délétère, problème éthique posé par l’obten...
Journal of Molecular Neuroscience, 2014
Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Activation o... more Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Activation of the developmental hedgehog (Hh) pathway is observed in GBM, particularly in the so-called glioma stem cells (GSCs). An inhibitor of this pathway is the steroidal alkaloid cyclopamine, an antagonist of the Hh coreceptor Smoothened (SMO). To limit the toxicity of cyclopamine toward Hh-dependent non-tumor cells, our group previously reported the synthesis of a prodrug (called 1b), designed to deliver cyclopamine in the presence of β-glucuronidase, an enzyme found in the necrotic area of GBM. Here, we aimed to analyze the in vitro, ex vivo, and in vivo cytotoxic properties of this prodrug in the C6 rat GBM cells. In the presence of β-glucuronidase, the activated prodrug 1b was toxic and downregulated expression of Gli1, a Hh target gene, in C6 cells and C6-GSCs, but not in normal rat astrocytes in which the Hh pathway is weakly activated. In the absence of β-glucuronidase, prodrug 1b displayed no obvious toxicity toward rat brain tissue explants while cyclopamine clearly affected brain tissue viability. When administered to rats bearing fluorescent C6-derived GBM, the prodrug 1b reduced the tumor density more efficiently than cyclopamine. Prodrug 1b thus appears as a promising concept to optimize confinement of cyclopamine cytotoxicity within the tumors, with more limited effects in the surrounding normal brain tissue.
Endocrinology, Jan 12, 2015
Episodic release of gonadotropin hormone (GnRH) is essential for reproductive function. In vitro ... more Episodic release of gonadotropin hormone (GnRH) is essential for reproductive function. In vitro studies have established that this episodic release is an endogenous property of GnRH neurons, and that GnRH secretory pulses are associated with synchronization of GnRH neuron activity. The cellular mechanisms by which GnRH neurons synchronize remain largely unknown. There is no clear evidence of physical coupling of GnRH neurons through gap junctions to explain episodic synchronization. However, coupling of glial cells through gap junctions has been shown to regulate neuron activity in their microenvironment. The present study investigated whether glial cell communication through gap junctions plays a role in GnRH neuron activity and secretion in the mouse. Our findings show that GFAP expressing glial cells located in the median eminence in close vicinity to GnRH fibers expressed Gja1 encoding connexin-43 (Cx-43). To study the impact of glial-gap junction coupling on GnRH neuron activi...
Bulletin de l'Académie vétérinaire de France, 2014
Cardiac Cell Biology, 2003
A number of data are consistent with the hypothesis that increases in intracellular Na + concentr... more A number of data are consistent with the hypothesis that increases in intracellular Na + concentration (Na + i ) during ischemia and early reperfusion lead to calcium overload and exacerbation of myocardial injury. However, the mechanisms underlying the increased Na + i remain unclear. 23 Na nuclear magnetic resonance spectroscopy was used to monitor Na + i in isolated rat hearts perfused with a high concentration of fatty acid as can occur under some pathological conditions. Whole-cell patch-clamp experiments were also performed on isolated cardiomyocytes in order to investigate the role of voltage-gated sodium channels. Na + i increased to substantially above control levels during no-flow ischemia. The results show that a pharmacological reduction of Na + i increase by cariporide (1 µmol/L, a Na + /H + exchange blocker) is not the only protection against ischemiareperfusion damage, but that such protection may also be brought about by metabolic action aimed at reducing fatty acid utilization by myocardial cells. This action was obtained in the presence of etomoxir (0.1 µmol/L), an inhibitor of carnitine palmitoyltransferase-1 (the key enzyme involved in fatty acid uptake by the mitochondria) which also decreases long-chain acyl carnitine accumulation. The possibility of Na + channels participating in Na + i increase as a consequence of alterations in cardiac metabolism was studied in isolated cells. Sustained I Na was stimulated by the presence of lysophosphatidylcholine (LPC, 10 µmol/L) whose accumulation during ischemia is, at least partly, dependent on increased long-chain acyl carnitine. Current activation was particularly significant in the range of potentials between -60 and -20 mV. This may have particular relevance in ischemia. The quantity of charge carried by sustained I Na was reduced by 24% in the presence of 1 µmol/L cariporide. Therefore, limitation of long-chain fatty acid metabolism, and consequent limitation of ischemia-induced long-chain acyl carnitine accumulation, may contribute to reducing intracellular Na + increase during ischemia-reperfusion. (Mol Cell Biochem 242: [115][116][117][118][119][120] 2003)
Journal of Molecular Neuroscience, 2014
Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Activation o... more Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Activation of the developmental hedgehog (Hh) pathway is observed in GBM, particularly in the so-called glioma stem cells (GSCs). An inhibitor of this pathway is the steroidal alkaloid cyclopamine, an antagonist of the Hh coreceptor Smoothened (SMO). To limit the toxicity of cyclopamine toward Hh-dependent non-tumor cells, our group previously reported the synthesis of a prodrug (called 1b), designed to deliver cyclopamine in the presence of β-glucuronidase, an enzyme found in the necrotic area of GBM. Here, we aimed to analyze the in vitro, ex vivo, and in vivo cytotoxic properties of this prodrug in the C6 rat GBM cells. In the presence of β-glucuronidase, the activated prodrug 1b was toxic and downregulated expression of Gli1, a Hh target gene, in C6 cells and C6-GSCs, but not in normal rat astrocytes in which the Hh pathway is weakly activated. In the absence of β-glucuronidase, prodrug 1b displayed no obvious toxicity toward rat brain tissue explants while cyclopamine clearly affected brain tissue viability. When administered to rats bearing fluorescent C6-derived GBM, the prodrug 1b reduced the tumor density more efficiently than cyclopamine. Prodrug 1b thus appears as a promising concept to optimize confinement of cyclopamine cytotoxicity within the tumors, with more limited effects in the surrounding normal brain tissue.
Molecular Pharmacology, 2008
After the thrombus formation in cardiac cavities or coronaries, the serine protease thrombin is p... more After the thrombus formation in cardiac cavities or coronaries, the serine protease thrombin is produced and can therefore reach the myocardial tissue by the active process of extravasation and binds to the G protein-coupled protease-activated receptor-1 (PAR1) expressed in human myocardium. The role of PAR1 was investigated in the thrombin effect on sodium current (I Na ). I Na was recorded in freshly isolated human atrial myocytes by the whole-cell patch-clamp method. Action potentials (AP) were recorded in guinea pig ventricular tissue by the conventional glass microelectrode technique. Thrombinactivated PAR1 induced a tetrodotoxin-blocked persistent sodium current, I NaP , in a concentration-dependent manner with an apparent EC 50 of 28 U/ml. The PAR1 agonist peptide SFLLR-NH 2 (50 M) was able to mimic PAR1-thrombin action, whereas PAR1 antagonists N 3 -cyclopropyl-7-((4-(1-methylethyl)phenyl)methyl)-7H-pyrrolo(3,2-f)quinazoline-1,3-diamine (SCH 203099; 10 M) and 1-(3,5-di-tert-butyl-4-hydroxy-phenyl)-2-This work was partly supported by Agence Nationale de la Recherche ANR-05-PCOD-006 -01. C.P. was a recipient of grants from the Fondation Lefoulon Delalande and Association Française contre les Myopathies.
Journal of Molecular and Cellular Cardiology, 2001
Journal of Biological Chemistry, 2008
Gap junctions are clusters of transmembrane channels allowing a passive diffusion of ions and sma... more Gap junctions are clusters of transmembrane channels allowing a passive diffusion of ions and small molecules between adjacent cells. Connexin43, the main channel-forming protein expressed in ventricular myocytes, can associate with zonula occludens-1, a scaffolding protein linked to the actin cytoskeleton and to signal transduction molecules. The possible influence of Rho GTPases, major regulators of cellular junctions and of the actin cytoskeleton, in the modulation of gap junctional intercellular communication (GJIC) was examined. The activation of RhoA by cytoxic necrotizing factor 1 markedly enhanced GJIC, whereas its specific inhibition by the Clostridium botulinum C3 exoenzyme significantly reduced it. RhoA activity affects GJIC without major cellular redistribution of junctional plaques or changes in the Cx43 phosphorylation pattern. As these GTPases frequently act via the cortical cytoskeleton, the importance of F-actin in the modulation of GJIC was investigated by means of agents interfering with actin polymerization. Cytoskeleton stabilization by phalloidin slowed down the kinetics of channel rundown in the absence of ATP, whereas its disruption by cytochalasin D rapidly and markedly reduced GJIC despite ATP presence. Cytoskeleton stabilization by phalloidin markedly reduced the consequences of RhoA activation or inactivation. This mechanism appears to be the first described capable to both up-or down-regulate GJIC through RhoA activation or, conversely, inhibition. The inhibition of Rho downstream kinase effectors had no effect on GJIC. The present results provide further insight into the gating and regulation of junctional channels and identify a new downstream target for the small G-protein RhoA.
Circulation, 2002
Background-Thrombin plays a role in mediating ischemic injury and cardiac arrhythmias, but the me... more Background-Thrombin plays a role in mediating ischemic injury and cardiac arrhythmias, but the mechanisms involved are poorly understood. Because voltage-gated sodium channels (VGSCs) have not previously been considered, putative effects of thrombin on VGSC function were investigated in human isolated cardiomyocytes. Methods and Results-Sodium current (I Na ) was recorded by the whole-cell patch-clamp method. Thrombin increased peak I Na amplitude in an activity-dependent manner, from 1 to 100 U/mL, with an apparent EC 50 of 91Ϯ16 U/mL. When tested at 32 U/mL, thrombin-increased I Na was abolished by tetrodotoxin (50 mol/L). Thrombin effects on I Na were reversible and repeatable, and 100 U/mL doubled peak I Na amplitude. Thrombin (32 U/mL) shifted I Na activation to hyperpolarized potentials without affecting steady-state inactivation, producing unusually large increases in window current. Hirudin (320 U/mL) or haloenol lactone suicide substrate (10 mol/L) failed to significantly affect these effects of thrombin. In current-clamped cardiomyocytes, thrombin (32 U/mL) depolarized resting membrane potential by 10 mV. Conclusions-Facilitation of VGSC activation causing large increases in window current is a major mechanism by which thrombin may promote ischemic sodium loading and injury.
The American Journal of Pathology, 2007
We examined whether mutation of the ␦-sarcoglycan gene, which causes dilated cardiomyopathy, also... more We examined whether mutation of the ␦-sarcoglycan gene, which causes dilated cardiomyopathy, also alters the vascular smooth muscle cell (VSMC) phenotype and arterial function in the Syrian hamster CHF 147. Thoracic aorta media thickness showed marked variability in diseased hamsters with zones of atrophy and hypertrophied segments. CHF-147 VSMCs displayed a proliferating/"synthetic" phenotype characterized by the absence of the smooth muscle myosin heavy chain SM2, dystrophin, and Ca 2؉ -handling proteins, and the presence of cyclin D1. In freshly isolated VSMCs from CHF 147 hamsters, voltage-independent basal Ca 2؉ channels showed enhanced activity similar to that in proliferating wild-type (WT) cells.
European Journal of Cancer, 2012
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Papers by Caroline Pinet-charvet