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.

No description available.

Drug transport across cells and cell membranes in the human body is crucial for the pharmacological effect of drugs. Active transport governed by transport proteins plays an important role in this process. A vast number of transport proteins with a wide tissue distribution have been identified during the last 15 years. Several important examples of their role in drug disposition and drug-drug interactions have been described to date. Investigation of drug-drug interactions at the transport protein level are therefore of increasing interest to the academic, industrial and regulatory research communities. The gene expression of transport proteins involved in drug transport was investigated in the jejunum, liver, kidney and colon to better understand their influence on the ADMET properties of drugs. In addition, the gene and protein expression of transport proteins in cell lines, widely used for predictions of drug transport and metabolism, was examined. The substrate and inhibitor heterogeneity of many transport proteins makes it difficult to foresee whether the transport proteins will cause drug-drug interactions. Therefore, in vitro assays for OCT1 and OATP1B1, among the highest expressed transport proteins in human liver, were developed to allow investigation of the inhibitory patterns of these proteins. These assays were used to investigate two data sets, consisting of 191 and 135 registered drugs and drug-like molecules for the inhibition of OCT1 and OATP1B1, respectively. Numerous new inhibitors of the transport proteins were identified in the data sets and the properties governing inhibition were determined. Further, antidepressant drugs and statins displayed strong inhibition of OCT1 and OATP1B1, respectively. The inhibition data was used to develop predictive in silico models for each of the two transport proteins. The highly polymorphic nature of some transport proteins has been shown to affect drug response and may lead to an increased risk of drug-drug interactions, and therefore, the OCT1 in vitro assay was used to study the effect of common genetic variants of OCT1 on drug inhibition and drug-drug interactions. The results indicated that OCT1 variants with reduced function were more susceptible to inhibition. Further, a drug-drug interaction of potential clinical significance in the genetic OCT1 variant M420del was proposed. In summary, gene expression of transport proteins was investigated in human tissues and cell lines. In vitro assays for two of the highest expressed liver transport proteins were used to identify previously unknown SLC transport protein inhibitors and to develop predictive in silico models, which may detect previously known drug-drug interactions and enable new ones to be identified at the transport protein level. In addition, the effect of genetic variation on inhibition of the OCT1 was investigated.

No description available.