Publications by authors named "Christophe Orvain"

13 Publications

  • Page 1 of 1

Bifunctional ligand design for modulating mutant p53 aggregation in cancer.

Chem Sci 2019 Dec 7;10(46):10802-10814. Epub 2019 Oct 7.

Department of Chemistry , Simon Fraser University , Burnaby , British Columbia V5A 1S6 , Canada . Email:

Protein misfolding and aggregation contributes to the development of a wide range of diseases. In cancer, over 50% of diagnoses are attributed to p53 malfunction due to missense mutations, many of which result in protein misfolding and accelerated aggregation. p53 mutations also frequently result in alteration or loss of zinc at the DNA-binding site, which increases aggregation nucleation with zinc-bound p53. Herein, we designed two novel bifunctional ligands, and , to modulate mutant p53 aggregation and restore zinc binding using a metallochaperone approach. Interestingly, only the incorporation of iodine function in resulted in modulation of mutant p53 aggregation, both in recombinant and cellular environments. Native mass spectrometry shows a protein-ligand interaction for , as opposed to , which is hypothesized to lead to the distinct difference in the p53 aggregation profile for the two ligands. Incorporation of a di-2-picolylamine binding unit into the ligand design provided efficient intracellular zinc uptake, resulting in metallochaperone capability for both and . The ability of to reduce mutant p53 aggregation results in increased restoration of p53 transcriptional function and mediates both caspase-dependent and -independent cell death pathways. We further demonstrate that exhibits minimal toxicity in non-cancerous organoids, and that it is well tolerated in mice. These results demonstrate that iodination of our ligand framework restores p53 function by interacting with and inhibiting mutant p53 aggregation and highlights as a suitable candidate for comprehensive anticancer preclinical evaluations.
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http://dx.doi.org/10.1039/c9sc04151fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7006507PMC
December 2019

HDAC4 Levels Control Sensibility toward Cisplatin in Gastric Cancer via the p53-p73/BIK Pathway.

Cancers (Basel) 2019 Nov 7;11(11). Epub 2019 Nov 7.

Laboratory STREINTH (Stress Response and Innovative Therapies), Inserm IRFAC UMR_S1113, Université de Strasbourg, 3 av. Molière, 67200 Strasbourg, France.

Gastric cancer (GC) remains a health issue due to the low efficiency of therapies, such as cisplatin. This unsatisfactory situation highlights the necessity of finding factors impacting GC sensibility to therapies. We analyzed the cisplatin pangenomic response in cancer cells and found HDAC4 as a major epigenetic regulator being inhibited. HDAC4 mRNA repression was partly mediated by the cisplatin-induced expression of miR-140. At a functional level, HDAC4 inhibition favored cisplatin cytotoxicity and reduced tumor growth. Inversely, overexpression of HDAC4 inhibits cisplatin cytotoxicity. Importantly, HDAC4 expression was found to be elevated in gastric tumors compared to healthy tissues, and in particular in specific molecular subgroups. Furthermore, mutations in HDAC4 correlate with good prognosis. Pathway analysis of genes whose expression in patients correlated strongly with HDAC4 highlighted DNA damage, p53 stabilization, and apoptosis as processes downregulated by HDAC4. This was further confirmed by silencing of HDAC4, which favored cisplatin-induced apoptosis characterized by cleavage of caspase 3 and induction of proapoptotic genes, such as , in part via a p53-dependent mechanism. Altogether, these results reveal HDAC4 as a resistance factor for cisplatin in GC cells that impacts on patients' survival.
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http://dx.doi.org/10.3390/cancers11111747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6896094PMC
November 2019

Multifunctional Compounds for Activation of the p53-Y220C Mutant in Cancer.

Chemistry 2018 Dec 9;24(67):17734-17742. Epub 2018 Nov 9.

Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, Canada.

The p53 protein plays a major role in cancer prevention, and over 50 % of cancer diagnoses can be attributed to p53 malfunction. The common p53 mutation Y220C causes local protein unfolding, aggregation, and can result in a loss of Zn in the DNA-binding domain. Structural analysis has shown that this mutant creates a surface site that can be stabilized using small molecules, and herein a multifunctional approach to restore function to p53-Y220C is reported. A series of compounds has been designed that contain iodinated phenols aimed for interaction and stabilization of the p53-Y220C surface cavity, and Zn-binding fragments for metallochaperone activity. Their Zn-binding affinity was characterized using spectroscopic methods and demonstrate the ability of compounds L4 and L5 to increase intracellular levels of Zn in a p53-Y220C-mutant cell line. The in vitro cytotoxicity of our compounds was initially screened by the National Cancer Institute (NCI-60), followed by testing in three stomach cancer cell lines with varying p53 status', including AGS (WTp53), MKN1 (V143A), and NUGC3 (Y220C). Our most promising ligand, L5, is nearly 3-fold more cytotoxic than cisplatin in a large number of cell lines. The impressive cytotoxicity of L5 is further maintained in a NUGC3 3D spheroid model. L5 also induces Y220C-specific apoptosis in a cleaved caspase-3 assay, reduces levels of unfolded mutant p53, and recovers p53 transcriptional function in the NUGC3 cell line. These results show that these multifunctional scaffolds have the potential to restore wild-type function in mutant p53-Y220C.
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http://dx.doi.org/10.1002/chem.201802677DOI Listing
December 2018

Rollover Cyclometalated Bipyridine Platinum Complexes as Potent Anticancer Agents: Impact of the Ancillary Ligands on the Mode of Action.

Inorg Chem 2018 Mar 14;57(5):2851-2864. Epub 2018 Feb 14.

School of Chemical Sciences , University of Auckland , Private Bag 92019, Auckland 1142 , New Zealand.

Platinum-based anticancer coordination compounds are widely used in the treatment of many tumor types, where they are very effective but also cause severe side effects. Organoplatinum compounds are significantly less investigated than the analogous coordination compounds. We report here rollover cyclometalated Pt compounds based on 2,2'-bipyridine which are demonstrated to be potent antitumor agents both in vitro and in vivo. Variation of the co-ligands on the Pt(2,2'-bipyridine) backbone resulted in the establishment of structure-activity relationships. They showed that the biological activity was in general inversely correlated with the reaction kinetics to biomolecules as shown for amino acids, proteins, and DNA. The less stable compounds caused higher reactivity with biomolecules and were shown to induce p53-dependent DNA damage. In contrast, the presence of bulky PTA and PPh ligands was demonstrated to cause lower reactivity and increased antineoplastic activity. Such compounds were devoid of DNA-damaging activity and induced ATF4, a component of the endoplasmic reticulum (ER) stress pathway. The lead complex inhibited tumor growth similar to oxaliplatin while showing no signs of toxicity in test mice. Therefore, we demonstrated that it is possible to fine-tune rollover-cyclometalated Pt(II) compounds to target different cancer pathways and be a means to overcome the side effects associated with cisplatin and analogous compounds in cancer chemotherapy.
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http://dx.doi.org/10.1021/acs.inorgchem.7b03210DOI Listing
March 2018

Monomeric Ti(iv)-based complexes incorporating luminescent nitrogen ligands: synthesis, structural characterization, emission spectroscopy and cytotoxic activities.

Dalton Trans 2016 Nov;45(47):19072-19085

Laboratoire de Chimie Moléculaire de l'Etat Solide, UMR 7140 UDS-CNRS, Université de Strasbourg, 4 rue Blaise Pascal, F-67000 Strasbourg, France.

This manuscript describes the synthesis of a series of neutral titanium(iv) monomeric complexes constructed around a TiON core. The two nitrogen atoms that compose the coordination sphere of the metallic center belong to 2,2'-bipyrimidine ligands homo-disubstituted in the 4 and 4' positions by methyl (2a), phenylvinyl (2b), naphthylvinyl (2c) or anthrylvinyl (2d) groups. The crystal structures of these complexes named [Ti(1)(2a)], [Ti(1)(2b)], [Ti(1)(2c)] and [Ti(1)(2d)] (where 1 is a 2,2'-biphenolato ligand substituted in the 6 and 6' positions by phenyl groups) are reported. The hydrolytic stability of the four complexes is evaluated by monitoring the evolution of the free 2a-d signals by H NMR spectroscopy. For the conditions tested (6 mM, DMSO-d/DO: 8/1), a rather good stability with t ranging from 180 to 300 min is determined for the complexes. In the presence of an acid (DCl), the hydrolysis of [Ti(1)(2a)] is faster than without an acid. The cytotoxic activity against gastric cancer cells of the titanium-based compounds and the free disubstituted 2,2'-bipyrimidine ligands is tested, showing IC ranging from 6.2 ± 1.2 μM to 274 ± 56 μM. The fluorescence studies of the ligands 2a-d, and the complexes [Ti(1)(2a-d)] reveal an important fluorescence loss of the ligands 2c and 2d upon coordination with the Ti(1) fragment. Frontier orbitals obtained by DFT calculations permit us to explain this fluorescence quenching.
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http://dx.doi.org/10.1039/c6dt03477bDOI Listing
November 2016

Impact of cyclometalated ruthenium(II) complexes on lactate dehydrogenase activity and cytotoxicity in gastric and colon cancer cells.

J Inorg Biochem 2016 10 26;163:28-38. Epub 2016 Jul 26.

Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA.

Lactate dehydrogenase (LDH) is a redox enzyme often overexpressed in cancer cells allowing their survival in stressful metabolic tumor environment. Ruthenium(II) complexes have been shown to impact on the activity of purified horseradish peroxidase and glucose oxidase but the physiological relevance remains unclear. In this study we investigated how ruthenium complexes impact on the activity of LDH in vitro and in cancer cells and performed a comparative study using polypyridine ruthenium(II) complex [Ru(bpy)] (1) and its structurally related cyclometalated 2-phenylpyridinato counterpart [Ru(phpy)(bpy)] (2) (bpy=2,2'-bipyridine, phpyH=2-phenylpyridine). We show that the cytotoxicity in gastric and colon cancer cells induced by 2 is significantly higher compared to 1. The kinetic inhibition mechanisms on purified LDH and the corresponding inhibition constants K or i values were calculated. Though complexes 1 and 2 are structurally very similar (one Ru-C bond in 2 replaces one Ru-N bond in 1), their inhibition modes are different. Cyclometalated complex 2 behaves exclusively as a non-competitive inhibitor of LDH from rabbit muscle (LDH, strongly suggesting that 2 does not interact with LDH in the vicinities of either lactate/pyruvate or NAD/NADH binding sites. Sites of interaction of 1 and 2 with LDH were revealed theoretically through computational molecular docking. Inhibition of LDH activity by 2 was confirmed in cancer cells. Altogether, these results revealed an inhibition of LDH activity by ruthenium complex through a direct interaction structurally tuned by a Ru-C bond.
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http://dx.doi.org/10.1016/j.jinorgbio.2016.07.014DOI Listing
October 2016

Expression and functional studies of the GDNF family receptor alpha 3 in the pancreas.

J Mol Endocrinol 2016 Feb 17;56(2):77-90. Epub 2015 Nov 17.

Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC)Institut National de la Santé et de la Recherche Médicale (INSERM) U964, Centre National de Recherche Scientifique (CNRS) UMR 7104, Université de Strasbourg (UdS), 1 Rue Laurent Fries, 67404 Illkirch, France

The generation of therapeutic β-cells from human pluripotent stem cells relies on the identification of growth factors that faithfully mimic pancreatic β-cell development in vitro. In this context, the aim of the study was to determine the expression and function of the glial cell line derived neurotrophic factor receptor alpha 3 (GFRα3) and its ligand artemin (Artn) in islet cell development and function. GFRα3 and Artn expression were characterized by in situ hybridization, immunochemistry, and qRT-PCR. We used GFRα3-deficient mice to study GFRα3 function and generated transgenic mice overexpressing Artn in the embryonic pancreas to study Artn function. We found that GFRα3 is expressed at the surface of a subset of Ngn3-positive endocrine progenitors as well as of embryonic α- and β-cells, while Artn is found in the pancreatic mesenchyme. Adult β-cells lack GFRα3 but α-cells express the receptor. GFRα3 was also found in parasympathetic and sympathetic intra-islet neurons as well as in glial cells in the embryonic and adult pancreas. The loss of GFRα3 or overexpression of Artn has no impact on Ngn3 and islet cell formation and maintenance in the embryo. Islet organization and innervation as well as glucose homeostasis is normal in GFRα3-deficient mice suggesting functional redundancy.
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http://dx.doi.org/10.1530/JME-15-0213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5911917PMC
February 2016

Rfx6 is an Ngn3-dependent winged helix transcription factor required for pancreatic islet cell development.

Development 2010 Jan;137(2):203-12

Institute of Genetics and Molecular and Cell Biology (IGBMC), Inserm U-964, CNRS UMR7104, University of Strasbourg, Illkirch, France.

The transcription factor neurogenin 3 (Neurog3 or Ngn3) controls islet cell fate specification in multipotent pancreatic progenitor cells in the mouse embryo. However, our knowledge of the genetic programs implemented by Ngn3, which control generic and islet subtype-specific properties, is still fragmentary. Gene expression profiling in isolated Ngn3-positive progenitor cells resulted in the identification of the uncharacterized winged helix transcription factor Rfx6. Rfx6 is initially expressed broadly in the gut endoderm, notably in Pdx1-positive cells in the developing pancreatic buds, and then becomes progressively restricted to the endocrine lineage, suggesting a dual function in both endoderm development and islet cell differentiation. Rfx6 is found in postmitotic islet progenitor cells in the embryo and is maintained in all developing and adult islet cell types. Rfx6 is dependent on Ngn3 and acts upstream of or in parallel with NeuroD, Pax4 and Arx transcription factors during islet cell differentiation. In zebrafish, the Rfx6 ortholog is similarly found in progenitors and hormone expressing cells of the islet lineage. Loss-of-function studies in zebrafish revealed that rfx6 is required for the differentiation of glucagon-, ghrelin- and somatostatin-expressing cells, which, in the absence of rfx6, are blocked at the progenitor stage. By contrast, beta cells, whose number is only slightly reduced, were no longer clustered in a compact islet. These data unveil Rfx6 as a novel regulator of islet cell development.
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http://dx.doi.org/10.1242/dev.041673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2799156PMC
January 2010

The transcription factor c-Myb affects pre-mRNA splicing.

Biochem Biophys Res Commun 2008 Jul 20;372(2):309-13. Epub 2008 May 20.

University of Oslo, Department of Molecular Biosciences, P.O. Box 1041 Blindern, N-0316 Oslo, Norway.

c-Myb is a transcription factor which plays a key role in haematopoietic proliferation and lineage commitment. We raised the question of whether c-Myb may have abilities beyond the extensively studied transcriptional activation function. In this report we show that c-Myb influences alternative pre-mRNA splicing. This was seen by its marked effect on the 5'-splice site selection during E1A alternative splicing, while no effect of c-Myb was observed when reporters for the 3'-splice site selection or for the constitutive splicing process were tested. Moreover, co-immunoprecipitation experiments provided evidence for interactions between c-Myb and distinct components of the splicing apparatus, such as the general splicing factor U2AF(65) and hnRNPA1 involved in the 5'-splice site selection. The effect on 5'-splice site selection was abolished in the oncogenic variant v-Myb. Altogether, these data provide evidence that c-Myb may serve a previously unappreciated role in the coupling between transcription and splicing.
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http://dx.doi.org/10.1016/j.bbrc.2008.05.054DOI Listing
July 2008

Transcription factors in pancreatic development. Animal models.

Endocr Dev 2007 ;12:24-32

Inserm U682, Development and Physiopathology of the Intestine and Pancreas, Université Louis Pasteur, Strasbourg , France.

Through the analysis of genetically modified mice a hierarchy of transcription factors regulating pancreas specification, endocrine destiny as well as endocrine subtype specification and differentiation has been established. In addition to conventional approaches such as transgenic technologies and gene targeting, recombinase fate mapping in mice has been key in establishing the lineage relationship between progenitor cells and their progeny in understanding pancreas formation. Moreover, the design of specific mouse models to conditionally express transcription factors in different populations of progenitor cells has revealed to what extent transcription factors required for islet cell development are also sufficient to induce endocrine differentiation and the importance of the competence of progenitor cells to respond to the genetic program implemented by these factors. Taking advantage of this basic science knowledge acquired in rodents, immature insulin-producing cells have recently been differentiated in vitro from human embryonic stem cells. Taken together these major advances emphasize the need to gain further in-depth knowledge of the molecular and cellular mechanisms controlling beta-cell differentiation in mice to generate functional beta-cells in the future that could be used for cell therapy in diabetes.
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http://dx.doi.org/10.1159/000109602DOI Listing
December 2007

Pancreatic islet progenitor cells in neurogenin 3-yellow fluorescent protein knock-add-on mice.

Mol Endocrinol 2004 Nov 5;18(11):2765-76. Epub 2004 Aug 5.

Institut National de la Santé et de la Recherche Médicale Unité 381, 3 avenue Molière, 67200 Strasbourg, France.

The basic helix-loop-helix transcription factor Neurogenin 3 (NGN3) controls endocrine cell fate specification in uncommitted pancreatic progenitor cells. Ngn3-deficient mice do not develop any islet cells and are diabetic. All the major islet cell types, including insulin-producing beta-cells, derive from Ngn3-positive endocrine progenitor cells. Therefore, the characterization of this population of immature cells is of particular interest for the development of novel strategies for cell replacement therapies in type 1 diabetes. To explore further the biology of islet progenitor cells we have generated a mouse in which Ngn3-expressing cells are labeled with the enhanced yellow fluorescent protein (EYFP) using a knock-add-on strategy. In this approach, the EYFP cDNA is introduced into the 3'-untranslated region of the proendocrine transcription factor, Neurogenin 3, without deleting any endogenous coding or regulatory sequences. In Ngn3(EYFP/+) and Ngn3(EYFP/EYFP) mice, the EYFP protein is targeted to Ngn3-expressing progenitors in the developing pancreas, and islets develop normally. Islet progenitors can be purified from whole embryonic pancreas by fluorescence-activated cell sorting from Ngn3(EYFP/+) mice and their development can be monitored in real time in pancreas explant cultures. These experiments showed that endocrine progenitors can form de novo and expand, in vitro, in the absence of signals from the surrounding mesenchyme, suggesting that endocrine commitment is a default pathway. The Ngn3(EYFP) mice represent a valuable tool to study islet cell development and neogenesis in normal and diabetic animals as well as for the determination of the conditions to generate beta-cells in vitro.
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http://dx.doi.org/10.1210/me.2004-0243DOI Listing
November 2004

Multiple functional domains of the oncoproteins Spi-1/PU.1 and TLS are involved in their opposite splicing effects in erythroleukemic cells.

Oncogene 2004 May;23(25):4389-99

Inserm U528, Section de recherche, Institut Curie, Paris, France.

The hematopoietic transcription factor Spi-1/PU.1 is an oncoprotein participating to the malignant transformation of proerythroblasts in the Friend erythroleukemia or in the erythroleukemic process developed in spi-1 transgenic mice. Overexpression of Spi-1 in proerythroblasts blocks their differentiation. We have shown that Spi-1 promotes the use of the proximal 5'-splice site during the E1A pre-mRNA splicing and interferes with the effect of TLS (Translocated in LipoSarcoma) in this splicing assay. TLS was identified from chromosomal translocations in human liposarcoma and acute myeloid leukemia. Here, we determine the function of Spi-1 domains in splicing and in the interference with TLS. In transient transfection assays in erythroid cells, we show that the DNA binding domain cooperates with the transactivation domain or the PEST region of Spi-1 to modify the function of TLS in splicing. Interestingly, the 27 C-terminal amino acids, which determine the DNA binding activity of Spi-1, are necessary for the splicing function of Spi-1 as well as for its ability to interfere with TLS. Finally, we demonstrate that in leukemic proerythroblasts overexpressing Spi-1, TLS has lost its splicing effect. Thus, we hypothesize that oncogenic pathways in proerythroblasts may involve the ability of Spi-1 to alter splicing.
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http://dx.doi.org/10.1038/sj.onc.1207578DOI Listing
May 2004

The human neuroendocrine thyrotropin-releasing hormone receptor promoter is activated by the haematopoietic transcription factor c-Myb.

Biochem J 2003 Jun;372(Pt 3):851-9

Department of Biochemistry, University of Oslo, P.O. Box 1041 Blindern, Norway.

Thyrotropin-releasing hormone (TRH) receptor (TRHR) is a G-protein-coupled receptor playing a crucial role in the anterior pituitary where it controls the synthesis and secretion of thyroid-stimulating hormone and prolactin. Its widespread presence not only in the central nervous system, but also in peripheral tissues, including thymus, indicates other important, but unknown, functions. One hypothesis is that the neuropeptide TRH could play a role in the immune system. We report here that the human TRHR promoter contains 11 putative response elements for the haematopoietic transcription factor c-Myb and is highly Myb-responsive in transfection assays. Analysis of Myb binding to putative response elements revealed one preferred binding site in intron 1 of the receptor gene. Transfection studies of promoter deletions confirmed that this high-affinity element is necessary for efficient Myb-dependent transactivation of reporter plasmids in CV-1 cells. The Myb-dependent activation of the TRHR promoter was strongly suppressed by expression of a dominant negative Myb-Engrailed fusion. In line with these observations, reverse transcriptase PCR analysis of rat tissues showed that the TRHR gene is expressed both in thymocytes and bone marrow. Furthermore, specific, high-affinity TRH agonist binding to cell-surface receptors was demonstrated in thymocytes and a haematopoietic cell line. Our findings imply a novel functional link between the neuroendocrine and the immune systems at the level of promoter regulation.
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http://dx.doi.org/10.1042/BJ20030057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1223435PMC
June 2003