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Inhibition des phosphatases CDC 25 dans le cadre d'une thérapie anticancéreuse: étude mécanistique de nouveaux inhibiteurs

Abstract : Within the context of research for new targets for cancer therapy, Cdc25 phosphatases are interesting candidates, the inhibition of which being able to slow down tumor growth and eventually improve the cancer treatments currently in use. The objectives of this PhD project are to design and synthesize new compounds able to inhibit CDC25 and to determine efficiency of identified compounds in breast cancer cell lines. In vitro evaluation of inhibitory potential of compound is realized through a high sensitivity fluorometric method (3-OMFP substrate). Cellular effects were evaluated in MCF-7 and MDA-MB-231 cell lines. Effects on cell viability are assessed through MTT assays, and cytotoxicity is evaluated through trypan blue assays and microscopic observations with Incucyte videomicroscopy system. Cell death was characterized by detection of apoptotic markers (caspases) and DNA damages markers (PARP Histone H.2AX) by Western Blotting. The analyses of mechanisms underlying cell death were explored through cytometric detection of reactive oxygen species (ROS) with H2DCFDA and Redox Sensor Red probes. Inhibition of CDC25 in cells was indirectly evaluated through detection of phosphorylated forms of CDK by Western Blotting. In vitro evaluation of 93 synthesized compounds allowed us to find new active compound in various chemical families including steroid, thiophene, coumarinic, imidazole and quinone derivatives. The coumarinic derivatives showed potent CDC25 inhibition. A new coumarin-sulfurquinone combined structure, named SV37, was designed to optimize efficiency of inhibition. In vitro tests on this compound, showed a strong CDC25 inhibitory potential (IC50 under 5µM for CDC25 A and C). Effect of SV37 on cell growth was evaluated on various cell lines (MCF-7, MDA-MB-231, hTERT-HME1 and HepG2). Results indicate inhibition of cell growth (IC50 values from 9 to 18 µM). Analysis of cell viability indicates no remarkable cell death in MCF7 at IC50 value whereas in MDA-MB-231 the cell growth decrease was characterized by an increase of cell death. For deeper investigations on the cell death and on the underlying mechanisms, we focused the study on the triple negative model MDA-MB-231. The morphological changes of MDA-MB-231 cells during the treatment were characterized by the appearance of cellular alterations compatible with a cellular demise and culminating with a disruption of cells after 20h. Caspase-3 and 7 cleavages were observed 16h after beginning of the treatment, suggesting an apoptotic cell death. A ROS induction was observed 15 min after the beginning of the treatment and was totally prevented by Nacetylcysteine (NAC) pretreatment. DNA damage markers were detected between 16 and 28 hours after beginning of treatment, a timing falling with caspase activation and with the appearance of cell demise observed by video microscopy. Accumulation of pCDK in cells was observed after 4 and 8 hr of treatment by SV37 at IC50 suggesting an inhibition of CDC25 activity, and cells pretreated with NAC showed no accumulation of pCDK after SV37 treatment. This strongly suggests a direct link between ROS generation by the compound SV37 and the accumulation of pCDK. This project increased knowledge on inhibitors of CDC25 phosphatases and allowed the identification of coumarine compound as new CDC25 inhibitors. This work will enable the development of ever more efficient inhibitors, leading to efficient inhibition of CDC25 and inhibition of tumor development
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Émilie Bana. Inhibition des phosphatases CDC 25 dans le cadre d'une thérapie anticancéreuse: étude mécanistique de nouveaux inhibiteurs. Médecine humaine et pathologie. Université de Lorraine, 2013. Français. ⟨NNT : 2013LORR0105⟩. ⟨tel-01750092⟩

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