No description available.

Les anthracyclines tels que la doxorubicin et la daunorubicin sont une famille de médicaments anticancéreux hydrophiles qui doivent être transportés dans les cellules afin d’exercer leur action par intercalation à l’ADN dans le noyau cellulaire. Ceci mène à la perturbation du métabolisme de l’ADN et entraine la mort cellulaire. Les anthracyclines sont utilisés pour le traitement d’une variété de cancers incluant la leucémie, les lymphomes, le cancer du sein, le cancer des poumons et le cancer des ovaires. Étant donné que le transport actif des anthracyclines dans les cellules a partiellement été démontré, le transporteur spécifique impliqué dans ce processus n’est pas encore connu. En utilisant un modèle de cancer des ovaires, la lignée cellulaire TOV2223G, nous avons démontré que des substrats spécifiques au transporteur de cations organiques 1 (OCT1), notamment la ergothionéine, la thiamine et la phenformin, ont partiellement inhibé l’absorption de la daunorubicin en différence de la carnitine qui est un substrat de haute affinité des transporteurs CT2 et OCTN2. Ces résultats suggèrent que les transporteurs organiques spécifiques au transport de la carnitine ne sont pas impliqués dans le transport des anthracyclines. Ainsi, nos résultats ont démontré que l’absorption de la daunorubicin est orchestrée par le transporteur OCT1 dans les cellules TOV2223G (Km ~ 5 μM) et des concentrations micromolaires de choline ont complètement abolies l’absorption de la drogue. De plus, un ARN sh dirigé contre OCT1 a réprimé son expression protéique, ce qui a été confirmé par la technique d’immuno-buvardage en utilisant un anti-OCT1 anticorps. Les cellules déficientes en OCT1 n’ont pas été capables d’absorber la daunorubicin et ont été plus résistantes à l’action de la drogue par rapport aux cellules contrôle. La transfection des cellules HEK293T avec un plasmide construit de façon à faire exprimer OCT1 comme protéine de fusion avec la protéine fluorescente EYFP a montré que celle-ci est localisée dans la membrane plasmique. Les cellules transfectées ont été capables d’absorber cinq fois plus de daunorubicin comparé aux cellules contrôles. Cette étude est, selon nous, la première à démontrer que OCT1 est un transporteur de haute affinité des anthracyclines. Ainsi, nous avons émis l’hypothèse que des défauts de OCT1 peuvent contribuer à l’efficacité de la réponse des cellules cancéreuses à la chimiothérapie avec les anthracyclines. / Anthracyclines such as doxorubicin and daunorubicin are hydrophilic anticancer agents that must be transported into cells. These drugs accumulate in the nucleus where they intercalate with DNA, thereby interfering with DNA replication in turn leading to cell death. Anthracyclines are used for treating a variety of cancers including leukemia, lymphomas, breast, lung, and ovarian. Despite evidence for active uptake of anthracyclines, the specific transporter has not been identified. Using the ovarian cancer cell line TOV2223G, we show that substrates reported for the organic cation transporter OCT1, such as ergothioneine, thiamine and phenformin, partially compete with uptake of daunorubicin, but not of L-carnitine, i.e., a high affinity substrate transported by hCT2 and OCTN2. These findings exclude the involvement of the L-carnitine organic cation family of transporters in anthracycline uptake. Moreover, we show that OCT1 actively mediates high affinity (Km ~ 5 μM) transport of daunorubicin into TOV2223G cells, whereas micromolar amounts of choline completely abolish drug uptake. shRNA-mediated downregulation of OCT1 causes defective uptake of daunorubicin, as well as significant resistance to the drug, as compared to the vector control. Transfection of HEK293T cells with a plasmid expressing OCT1 as a GFP fusion protein revealed that OCT1-EYFP was predominantly localized to the plasma membrane. These transfected cells manifested nearly 5-fold increased uptake of daunorubicin compared to the empty vector control. In summary, we show for the first time that human OCT1 is a high affinity transporter for anthracyclines. As such, we postulate that OCT1 status represents a critical determinant in the response of cancer cells to chemotherapy with anthracyclines

No description available.

Les interactions entre facteurs génétiques et environnementaux, comme le stress, jouent un rôle important dans la physiopathologie de maladies psychiatriques telles que la dépression. Les transporteurs de cation organiques (OCTs) sont des transporteurs polyspécifiques considérés comme sensibles à la corticostérone. Au cours de ma thèse, je me suis intéressé au rôle du transporteur de cation organique 2 (OCT2) dans la réponse au stress et la vulnérabilité à la dépression. OCT2 est exprimé dans de nombreuses régions impliquées dans la réponse au stress et le long de l’axe hypothalamo-hypophyso-surrénalien (HPA). L’absence d’OCT2 entraîne une augmentation de la sécrétion de corticostérone (156%) en réponse à un stress aigu, sans altération de la sensibilité des surrénales à l’hormone adrénocorticotrope (ACTH). En conséquence, les souris OCT2-/- sont plus sensibles aux effets du stress chronique imprédictible léger et développent des anomalies comportementales transitoires de la mémoire spatiale et de l’interaction sociale dans la phase précoce du stress chronique. De plus, nous avons montré que l’état fonctionnel de la GSK3β, une voie de signalisation profondément modulée par le stress et la dépression, est altéré dans l’hippocampe des souris OCT2-/-. Des expériences pharmacologiques in vivo et des Western blot suggèrent qu’une augmentation du tonus sérotoninergique chez les souris OCT2-/- pourrait expliquer la dérégulation de la GSK3β lors du stress. Ce travail a permis d’identifier OCT2 comme un déterminant important de la réponse au stress, suggérant que chez l’homme des polymorphismes ou son inhibition pharmacologique lors de traitements thérapeutiques de longue durée pourraient altérer l’activité de l’axe HPA et rendre les individus vulnérables aux effets délétères du stress. / Interactions between genetic and environmental factors like exposure to stress play an important role in the pathogenesis of mood-related psychiatric disorders such as major depressive disorder. The polyspecific organic cation transporters (OCTs) were shown previously to be sensitive to the stress hormone corticosterone in vitro, suggesting these transporters might play a physiological role in the response to stress. During my PhD thesis I investigated the role of organic cation transporter 2 (OCT2) during stress and vulnerability to depression. OCT2 is expressed in several stress-related circuits in the brain and along the hypothalamic-pituitary-adrenocortical (HPA) axis. Genetic deletion of OCT2 in mice enhanced hormonal response to acute stress (156%) without altering adrenal sensitivity to adrenocorticotropic hormone (ACTH). As a consequence, OCT2-/- mice were potently more sensitive to the action of unpredictable chronic mild stress and developed a transient aggravation of depression-related behaviors involving spatial memory and social interaction. We showed that the functional state of the glycogen synthase kinase-3β (GSK3β) signaling pathway, highly responsive to acute stress, was altered in the hippocampus of OCT2-/- mice. In vivo pharmacology and Western blot experiments argue for increased serotonin tonus as a main mechanism for impaired GSK3β signaling in OCT2-/- mice brain during acute response to stress. Our findings identify OCT2 as an important determinant of the response to stress in the brain, suggesting that in man OCT2 mutations or blockade by certain therapeutic drugs could interfere with HPA axis function and enhance vulnerability to repeated adverse events leading to stress-related disorders.

No description available.

No description available.

The organic cation transporters (OCTs) play a critical role in the absorption, distribution and elimination of many drugs, hormones, herbal medicines, and environmental toxins. Given the broad substrate specificity of OCTs, they fall victim to the high susceptibility for contributing to harmful drug-drug interactions. Further defining how human (h)OCTs mechanistically bind to its broad array of substrates will provide significant insight to the understanding and prediction of drug-drug interactions in polypharmacy patients and the advancement of future rational drug design for therapeutics targeting OCTs. The goal of the current study was to elucidate the critical amino acid residues for transporter-substrate binding interactions on human (h)OCT1 and 2 utilizing in silico molecular modeling techniques (homology modeling and automated docking), as well as in vitro mutagenesis and kinetic transport experiments. Three-dimensional homology models were generated for hOCT1 and 2 using Piriformospora indica phosphate transporter (PiPT) serving as template. A putative binding pocket was identified and used to dock the prototypical substrate MPP+. Docking studies revealed five residues for each transporter (hOCT1 and hOCT2) that may be critical for substrate-transporter interactions. The in silico data was used to guide subsequent in vitro site-directed mutagenesis and kinetic analysis. Four hOCT1 mutants (Gln241Lys, Thr245Lys, Tyr361Ala, and Glu447Lys) and three hOCT2 mutants (Gln242Lys, Tyr362Phe, and Tyr362Ala) showed complete loss of MPP+ transporter activity. Decreased affinity for MPP+ was observed for Phe244Ser and Thr245Ser in hOCT1, and Tyr245Ala in hOCT2. All amino acid residues highlighted in the in vitro experiments may be potentially critical for substrate-transporter interactions particularly Tyr361, Phe244 and Thr245 in hOCT1; and Tyr362 and Tyr245 in hOCT2. Docking of known structurally divergent hOCT1 and hOCT2 substrates revealed similar binding interactions as that identified for MPP+, albeit with some unique residues, suggesting the presence of a large central cavity within both transporters. Through the combination of in silico and in vitro experiments, a putative binding pocket was defined and several residues important for substrate-transporter interaction were identified and verified for hOCT1 and hOCT2. Further defining how OCTs biochemically interact with their broad array of substrates will provide significant insight to the understanding and prediction of drug-drug interactions in polypharmacy patients and the advancement of future rational drug design for therapeutics targeting OCT1 and OCT2.

<p>The aim of this study was to investigate the genetic diversity of the SLC22A1 gene and to deduce its possible pharmacogenetic implications within the Cape Coloured population of South&nbsp / Africa / a uniquely admixed population of immigrant Europeans, Asians and the indigenous populations. Recent studies have reported an abundance of polymorphic variants within this solute&nbsp / carrier transporter gene encoding for the organic cation transporter 1, as well as evidence linking these variants to an effect on metformin uptake. This study included establishing baseline&nbsp / frequency distribution of previously reported alleles for 20 SNP variants within the SLC22A1 gene, as well as the development of SNaPshot&reg / and Multiplex AS-PCR genotyping assays, and&nbsp / also exploring the possibility of using High-resolution melt (HRM) analysis as a costeffective alternative for SNP genotyping. Ethics clearance was obtained from the Ethics Committee of the&nbsp / University of the Western Cape. Biological samples in the form of buccal (oral) swabs were collected from 132 unrelated voluntary donors from the Cape Coloured population residing in the&nbsp / Cape Metropolitan area. Two SNaPshot&reg / Multiplex Systems were specifically designed for the study,successfully optimized and used for genotyping. Hundred genetic profiles were then generated for a total of 20 SNP variants on SLC22A1 gene, using this primer extension-based genotyping method that enables multiplexing up 10 SNPs. Population genetics data obtained for&nbsp / the investigated SNPs were analysed using various statistical analysis software. Important population genetic parameters were calculated, and possible pharmacogenetics implications were then discussed. Among others, allelic and genotypic frequencies, as well as linkage disequilibrium were determined and compared with world populations. Minor deviation from Hardy- Weinberg equilibrium was observed in the Cape Coloured population. No significantLinkage Disequilibrium between the investigated SNPs was observed in this population. A Multiplex allele specific &ndash / PCR (MAS-PCR) genotyping&nbsp / system was successfully designed and optimized for the genotyping of 10 SNPs from the SLC22A1. This system, also developed specifically for this study, was made of 2 multiplexes each covering 5 SNPs. It is an inexpensive genotyping assay that allows for efficient discrimination of SNP polymorphisms in one reaction tube with standard PCR conditions. A pilot study was&nbsp / conducted to explore the possibility of using High-resolution melt (HRM) analysis as a cost-effective alternative for SNP genotyping. In addition to genotyping, HRM analysis can be used to scan&nbsp / large numbers of samples for novel genetic variations.&nbsp / </p>

<p>The aim of this study was to investigate the genetic diversity of the SLC22A1 gene and to deduce its possible pharmacogenetic implications within the Cape Coloured population of South&nbsp / Africa / a uniquely admixed population of immigrant Europeans, Asians and the indigenous populations. Recent studies have reported an abundance of polymorphic variants within this solute&nbsp / carrier transporter gene encoding for the organic cation transporter 1, as well as evidence linking these variants to an effect on metformin uptake. This study included establishing baseline&nbsp / frequency distribution of previously reported alleles for 20 SNP variants within the SLC22A1 gene, as well as the development of SNaPshot&reg / and Multiplex AS-PCR genotyping assays, and&nbsp / also exploring the possibility of using High-resolution melt (HRM) analysis as a costeffective alternative for SNP genotyping. Ethics clearance was obtained from the Ethics Committee of the&nbsp / University of the Western Cape. Biological samples in the form of buccal (oral) swabs were collected from 132 unrelated voluntary donors from the Cape Coloured population residing in the&nbsp / Cape Metropolitan area. Two SNaPshot&reg / Multiplex Systems were specifically designed for the study,successfully optimized and used for genotyping. Hundred genetic profiles were then generated for a total of 20 SNP variants on SLC22A1 gene, using this primer extension-based genotyping method that enables multiplexing up 10 SNPs. Population genetics data obtained for&nbsp / the investigated SNPs were analysed using various statistical analysis software. Important population genetic parameters were calculated, and possible pharmacogenetics implications were then discussed. Among others, allelic and genotypic frequencies, as well as linkage disequilibrium were determined and compared with world populations. Minor deviation from Hardy- Weinberg equilibrium was observed in the Cape Coloured population. No significantLinkage Disequilibrium between the investigated SNPs was observed in this population. A Multiplex allele specific &ndash / PCR (MAS-PCR) genotyping&nbsp / system was successfully designed and optimized for the genotyping of 10 SNPs from the SLC22A1. This system, also developed specifically for this study, was made of 2 multiplexes each covering 5 SNPs. It is an inexpensive genotyping assay that allows for efficient discrimination of SNP polymorphisms in one reaction tube with standard PCR conditions. A pilot study was&nbsp / conducted to explore the possibility of using High-resolution melt (HRM) analysis as a cost-effective alternative for SNP genotyping. In addition to genotyping, HRM analysis can be used to scan&nbsp / large numbers of samples for novel genetic variations.&nbsp / </p>

No description available.