<p> The MT1 receptor is involved in the oncostatic action of melatonin and valproic acid (VPA) in human MCF-7 breast cancer cells and VPA can upregulate this receptor in C6 glioma cells. Therefore, the effect of VPA on the expression of the MT1 was examined in MCF-7 cells. Treatment of MCF-7 cells in low serum conditions with VPA (0.5 or 1mM) for 24 or 72 h caused a significant increase in MT1 receptor expression, as shown by reverse transcription-polymerase chain reaction analysis (RT-PCR). MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays performed in high serum conditions revealed a significant concentration-dependent inhibition of MCF-7 cell proliferation by VPA (0.5 - 5 mM), whereas melatonin (1 or 10 nM) showed modest effects alone. However, a combination of VPA and melatonin produced a marked synergistic inhibition of cell proliferation. In subsequent experiments, under high serum conditions, VPA treatment for 24h on these cells resulted in a significant decline of MT1 mRNA while the protein levels were still increasing, as seen by RT-PCR and western blotting respectively. The involvement of multiple biochemical events, such as: induction of the p53 tumor suppressor gene and repression of the estrogen receptor (ER)-alpha might be responsible for the synergistic inhibition of these cells after the simultaneous exposure to VPA and melatonin. These results indicate that clinically relevant concentrations of VPA upregulate melatonin MT1 receptor expression in human breast cancer cells. Moreover, the enhanced antiproliferative effect observed with a combination of VPA and melatonin suggests that a similar therapeutic approach may be beneficial in human breast cancer.</p> / Thesis / Master of Science (MSc)

Le récepteur humain de la mélatonine MT1 appartient à la famille des récepteurs couplés aux protéines G (RCPG). En raison du rôle majeur qu'il joue dans la régulation du rythme circadien, ce récepteur est impliqué dans les troubles du sommeil et la dépression et présente donc un intérêt thérapeutique important. Afin de progresser vers une meilleure caractérisation structurale et fonctionnelle de ce récepteur, nous avons élaboré une stratégie globale visant à générer les échantillons et méthodes nécessaires pour de telles études. Nous avons ainsi mis au point un ensemble original de conditions de production et de purification permettant d'isoler les récepteurs MT1 sous forme relativement pure, homogène et fonctionnelle. Parallèlement, à partir d'un RCPG de référence, le récepteur de l’adénosine A2A, nous avons mis au point un panel de techniques d’analyses biochimiques et biophysiques qui contribuent à une caractérisation fine des récepteurs purifiés et leur interaction avec des ligands. / The human MT1 melatonin receptor belongs to the family of G protein-coupled receptors (GPCRs). It plays a major role in the regulation of circadian rhythm and is involved in sleep disorders and depression. This receptor is thus of significant therapeutic interest. However, very few in vitro studies have been conducted on this receptor and in particular no structural characterization and interactio studies by biophysical methods. In order to progress on these aspects, we developed conditions of production and purification of MT1 receptors for obtaining samples compatible with this type of study. Furthermore, we initiated stabilization tests of the purified receptors. Meanwhile, we have developed biochemical and biophysical analysis techniques to characterize the purified receptors and study their interactions with ligands, on a reference GPCR, the A2A adenosine receptor.