Cytochrome P450 3A4 (CYP3A4) is a key enzyme responsible for the metabolism of drugs and endogenous compounds in human liver and intestine. CYP3A4 gene expression is mainly regulated by Pregnane X receptor (PXR) which is a ligand-dependent nuclear receptor. It is a long-standing observation that inflammatory responses and infections decrease drug metabolism capacity in human and experimental animals. In this study, I reported that NF-κB activation by LPS and TNF-α plays a pivotal role in the suppression of CYP3A4 through interactions of NF-κB with PXR/RXR complex. Inhibition of NF-κB by NF-κB specific suppressor SRIκBα reversed the suppressive effects of LPS and TNF-α. Furthermore, I showed that NF-κB p65 disrupted the association of PXR/RXRα complex with DNA sequences as determined by EMSA and chromatin immunoprecipitation assays. NF-κB p65 directly interacted with DNA binding domain of RXRα and DNA binding domain, hinge domain and ligand-binding domain of PXR and may prevent its binding to the consensus DNA sequences, thus inhibiting the transactivation by PXR/RXRα complex. This mechanism of suppression by NF-κB activation may be extended to other nuclear receptor-regulated systems where RXRα is a dimerization partner. Many genes regulated by PXR and AhR are important for phase I, II and III drug metabolism. In this study I reported a crosstalk between PXR and AhR pathways. AhR physically and functionally interacted with PXR and enhanced the PXR transcriptional activity, and the interaction repressed the AhR transcriptional activity. AhR also physically interacted with RXRα. The synergistic induction of Gsta1 in the liver of mice by PCN and TCDD might assume a different mechanism. The results suggested the metabolism kinetics of mixture drugs was different from and more complicated than that of single compound. Using a HepG2 cell-based PXR-driven CYP3A4-Luciferase assay, I reported that E/F domain of PXR was responsible for ligand-dependant activation. A/B domain was necessary for co-activating the ligand-dependent activation and D domain was suppressive. High doses of Valerian Root extraction were PXR-dependent CYP3A4 inducers. Green tea polyphenols, aflatoxin B1, CuSO4 and MnCl2 enhanced the PXR transcription activity activated by rifampicin. The results suggested PXR-mediated drug metabolism kinetics altered on xenobiotic exposure.

Pregnane X receptor (PXR) is a ligand-activated transcription factor that plays a role in a broad array of biological processes, including drug metabolism and transport. Ginkgo biloba is an herb commonly used to improve cognitive function. In primary cultures of rat hepatocytes, Ginkgo biloba induces the mRNA expression of CYP3A23, a target gene for rat PXR. The present study tested the hypothesis that Ginkgo biloba activates PXR. Cultured HepG2 human hepatoma cells were transfected with the full-length human PXR (pCR3-hPXR), the full-length mouse PXR (pCR3-mPXR), or an empty vector (pCR3) in addition to a reporter plasmid (XREM-CYP3A4-LUC; firefly luciferase) and an internal control plasmid (phRL-TK; Renilla luciferase). At 24 h after transfection, cells were treated for 24 h with Ginkgo biloba extract and luciferase activity was measured. The extract at 200 µg/ml increased mouse and human PXR activity by 3.0-fold and 9.5-fold, respectively, indicating that Ginkgo biloba more effectively activates human PXR. Dose-response experiments showed that the extract produced a log-linear increase over the range of 200–800 µg/ml. To determine whether Ginkgo biloba extract induces human PXR target gene expression, cultured LS180 human colon adenocarcinoma cells were treated for 72 h with the extract. Total cellular RNA was isolated and reverse transcribed. CYP3A4, CYP3A5, and ABCB1 cDNAs were amplified by real-time PCR. Ginkgo biloba extract at 200, 400, and 800 µg/ml increased CYP3A4 mRNA expression by 1.7-, 2.4-, and 2.5-fold, respectively. The extract at the same concentrations increased the mRNA expression of CYP3A5 (1.3 to 3.6-fold) and ABCB1 (2.7 to 6.4-fold). To determine whether the increased expression involved PXR activation, cells were treated with a PXR antagonist, L-sulforaphane, and Ginkgo biloba extract. L-sulforaphane at 5, 10, and 20 µM decreased CYP3A4 mRNA expression by 54%, 78%, and 93%, respectively, in cells co-treated with the extract. A similar pattern of response was obtained with CYP3A5 and ABCB1. In cells co-treated with the extract, L-sulforaphane (5 and 10 µM) was not cytotoxic and did not decrease PXR mRNA expression. Our data from cell culture experiments indicate that Ginkgo biloba activates PXR and increases CYP3A4, CYP3A5, and ABCB1 mRNA expression.

Cytochrome P450 3A4 (CYP3A4) is a key enzyme responsible for the metabolism of drugs and endogenous compounds in human liver and intestine. CYP3A4 gene expression is mainly regulated by Pregnane X receptor (PXR) which is a ligand-dependent nuclear receptor. It is a long-standing observation that inflammatory responses and infections decrease drug metabolism capacity in human and experimental animals. In this study, I reported that NF-κB activation by LPS and TNF-α plays a pivotal role in the suppression of CYP3A4 through interactions of NF-κB with PXR/RXR complex. Inhibition of NF-κB by NF-κB specific suppressor SRIκBα reversed the suppressive effects of LPS and TNF-α. Furthermore, I showed that NF-κB p65 disrupted the association of PXR/RXRα complex with DNA sequences as determined by EMSA and chromatin immunoprecipitation assays. NF-κB p65 directly interacted with DNA binding domain of RXRα and DNA binding domain, hinge domain and ligand-binding domain of PXR and may prevent its binding to the consensus DNA sequences, thus inhibiting the transactivation by PXR/RXRα complex. This mechanism of suppression by NF-κB activation may be extended to other nuclear receptor-regulated systems where RXRα is a dimerization partner. Many genes regulated by PXR and AhR are important for phase I, II and III drug metabolism. In this study I reported a crosstalk between PXR and AhR pathways. AhR physically and functionally interacted with PXR and enhanced the PXR transcriptional activity, and the interaction repressed the AhR transcriptional activity. AhR also physically interacted with RXRα. The synergistic induction of Gsta1 in the liver of mice by PCN and TCDD might assume a different mechanism. The results suggested the metabolism kinetics of mixture drugs was different from and more complicated than that of single compound. Using a HepG2 cell-based PXR-driven CYP3A4-Luciferase assay, I reported that E/F domain of PXR was responsible for ligand-dependant activation. A/B domain was necessary for co-activating the ligand-dependent activation and D domain was suppressive. High doses of Valerian Root extraction were PXR-dependent CYP3A4 inducers. Green tea polyphenols, aflatoxin B1, CuSO4 and MnCl2 enhanced the PXR transcription activity activated by rifampicin. The results suggested PXR-mediated drug metabolism kinetics altered on xenobiotic exposure.

To understand the mechanisms of transcriptional regulation of PXR, we performed yeast two-hybrid screenings to search for PXR-interacting proteins in a human liver cDNA library using the PXR ligand binding domain as the bait. More than one million independent clones were screened. One positive clone was a partial cDNA of CNOT2 (amino acid 183-540). CNOT2 is a component of CCR4-NOT that is a multi-subunit protein complex highly conserved from yeast to humans. Using a mammalian two-hybrid system in CV-1 cells and GST-pull down assays, we confirmed the direct interaction between PXR and CNOT2 and mapped the specific domains of association. In HepG2 cells, over expression of CNOT2 suppressed the PXR-regulated luciferase reporter gene activity. siRNA knockdown of CNOT2 potentiated PXR-transcriptional activity. These results strongly suggest that the CCR4-NOT complex is significantly involved in transcriptional regulation of PXR. The immuno-precipitated CNOT2 complex contained deadenylase activity as determined by an in vitro RNA decay assay. The presence of transfected PXR inhibited the cNOT2-associated deadenylase activity, as demonstrated by poly(A) tail PCR. Cellular localization of PXR and cNOT2 by immuno-fluorescence microscopy indicates that the interaction might occur within Cajal Bodies. Taken together, these results suggest that PXR regulates the mRNA turnover through direct interaction with the NOT2 component of the CCR4-NOT complex. PXR is also involved in colon cancer progression. Our results indicate that the evolutionarily conserved PXR protects organisms from carcinogenesis by inhibiting tumor growth as well as eliminating carcinogenic substances. Our laboratory proposes that pregnane X receptor has an important role in maintaining the balance of cells progressing through the cell cycle. In vitro and in vivo experiments demonstrate expression of PXR in colon cancer cells slows the progression of tumor formation. Colony growth of the PXR-transfected HT29 cells was suppressed in soft agar assay. In the xenograft assay, the tumor size formed in nude mice was significantly suppressed in HT29 cells stably transfected with PXR (310 mg /- 6.2 vs. 120 mg±6, p<0.01). The number of Ki-67 positive cells were significantly decreased in PXR-transfected HT29 xenograft tumor tissue compared vector-transfected HT29 controls (p<0.01) as determined by immuno-histochemistry suggesting that PXR inhibits proliferation of colon cancer cells. Results of flow cytometry analysis indicated that PXR-transfection in HT29 cells caused G0/G1 arrest. The growth inhibitory effects of PXR are likely mediated through the E2F/Rb-regulated check point since E2F1 nuclear expression was significantly inhibited by PXR over expression.

Pregnane X receptor (PXR) is a ligand-activated transcription factor that plays a role in a broad array of biological processes, including drug metabolism and transport. Ginkgo biloba is an herb commonly used to improve cognitive function. In primary cultures of rat hepatocytes, Ginkgo biloba induces the mRNA expression of CYP3A23, a target gene for rat PXR. The present study tested the hypothesis that Ginkgo biloba activates PXR. Cultured HepG2 human hepatoma cells were transfected with the full-length human PXR (pCR3-hPXR), the full-length mouse PXR (pCR3-mPXR), or an empty vector (pCR3) in addition to a reporter plasmid (XREM-CYP3A4-LUC; firefly luciferase) and an internal control plasmid (phRL-TK; Renilla luciferase). At 24 h after transfection, cells were treated for 24 h with Ginkgo biloba extract and luciferase activity was measured. The extract at 200 µg/ml increased mouse and human PXR activity by 3.0-fold and 9.5-fold, respectively, indicating that Ginkgo biloba more effectively activates human PXR. Dose-response experiments showed that the extract produced a log-linear increase over the range of 200–800 µg/ml. To determine whether Ginkgo biloba extract induces human PXR target gene expression, cultured LS180 human colon adenocarcinoma cells were treated for 72 h with the extract. Total cellular RNA was isolated and reverse transcribed. CYP3A4, CYP3A5, and ABCB1 cDNAs were amplified by real-time PCR. Ginkgo biloba extract at 200, 400, and 800 µg/ml increased CYP3A4 mRNA expression by 1.7-, 2.4-, and 2.5-fold, respectively. The extract at the same concentrations increased the mRNA expression of CYP3A5 (1.3 to 3.6-fold) and ABCB1 (2.7 to 6.4-fold). To determine whether the increased expression involved PXR activation, cells were treated with a PXR antagonist, L-sulforaphane, and Ginkgo biloba extract. L-sulforaphane at 5, 10, and 20 µM decreased CYP3A4 mRNA expression by 54%, 78%, and 93%, respectively, in cells co-treated with the extract. A similar pattern of response was obtained with CYP3A5 and ABCB1. In cells co-treated with the extract, L-sulforaphane (5 and 10 µM) was not cytotoxic and did not decrease PXR mRNA expression. Our data from cell culture experiments indicate that Ginkgo biloba activates PXR and increases CYP3A4, CYP3A5, and ABCB1 mRNA expression.

Pregnane X receptor (PXR) is a ligand-activated transcription factor that plays a role in a broad array of biological processes, including drug metabolism and transport. Ginkgo biloba is an herb commonly used to improve cognitive function. In primary cultures of rat hepatocytes, Ginkgo biloba induces the mRNA expression of CYP3A23, a target gene for rat PXR. The present study tested the hypothesis that Ginkgo biloba activates PXR. Cultured HepG2 human hepatoma cells were transfected with the full-length human PXR (pCR3-hPXR), the full-length mouse PXR (pCR3-mPXR), or an empty vector (pCR3) in addition to a reporter plasmid (XREM-CYP3A4-LUC; firefly luciferase) and an internal control plasmid (phRL-TK; Renilla luciferase). At 24 h after transfection, cells were treated for 24 h with Ginkgo biloba extract and luciferase activity was measured. The extract at 200 µg/ml increased mouse and human PXR activity by 3.0-fold and 9.5-fold, respectively, indicating that Ginkgo biloba more effectively activates human PXR. Dose-response experiments showed that the extract produced a log-linear increase over the range of 200–800 µg/ml. To determine whether Ginkgo biloba extract induces human PXR target gene expression, cultured LS180 human colon adenocarcinoma cells were treated for 72 h with the extract. Total cellular RNA was isolated and reverse transcribed. CYP3A4, CYP3A5, and ABCB1 cDNAs were amplified by real-time PCR. Ginkgo biloba extract at 200, 400, and 800 µg/ml increased CYP3A4 mRNA expression by 1.7-, 2.4-, and 2.5-fold, respectively. The extract at the same concentrations increased the mRNA expression of CYP3A5 (1.3 to 3.6-fold) and ABCB1 (2.7 to 6.4-fold). To determine whether the increased expression involved PXR activation, cells were treated with a PXR antagonist, L-sulforaphane, and Ginkgo biloba extract. L-sulforaphane at 5, 10, and 20 µM decreased CYP3A4 mRNA expression by 54%, 78%, and 93%, respectively, in cells co-treated with the extract. A similar pattern of response was obtained with CYP3A5 and ABCB1. In cells co-treated with the extract, L-sulforaphane (5 and 10 µM) was not cytotoxic and did not decrease PXR mRNA expression. Our data from cell culture experiments indicate that Ginkgo biloba activates PXR and increases CYP3A4, CYP3A5, and ABCB1 mRNA expression. / Pharmaceutical Sciences, Faculty of / Graduate

CAR (Constitutive Androstane Receptor, NR1I3) et PXR (Pregnane X Receptor, NR1I2) sont deux récepteurs nucléaires dédiés à la reconna issance et à l'élimination de molécules lipophiles potentiellement toxiques pour l'organisme. Ces facteurs de transcription peuvent être activés par des ligands d'origines et de structures diverses (médicaments, polluants environnementaux, produits de l'alimentation et de phytothérapies). L'activation de ces récepteurs entraîne l'expression des gènes majeurs de la fonction de détoxication entéro-hépatique (CYP450, transférases, transporteurs) permettant l'élimination de ces toxiques. Dans ce travail, nous avons dans un premier temps 1) montré que CAR contrôle l'expression de Spot14, une protéine pro-lipogénique, et 2) nous avons identifié une nouvelle isoforme de PXR (PXR-small) codant uniquement pour le domaine de liaison des ligands de PXR. Nous avons pu déterminer les origines de transcription par 5'-RACE PCR et montrer que PXR-small représente environ 10% de l'ensemble des transcrits de PXR dans le tissu hépatique sain par une approche de PCR qua ntitative. Nous avons pu détecter sa présence par western-blot sur des extraits de protéines nucléaires issus de tissus hépatiques et de lignées cellulaires hépatiques. Par des expériences de gel retard, nous avons observé que cette nouvelle isoforme tronquée, qui ne code que pour le LBD de PXR, ne peut pas se lier à l'ADN. Des expériences de gènes rapporteurs suggèrent que cette isoforme se comporte comme un dominant négatif de PXR. Enfin, la présence d'un ilot CpG situé juste en amont de PXR-small suggère que cette nouvelle isoforme pourrait être régulée épigénétiquement par méthylation, notamment dans les cellules tumorales. / CAR (Constitutive Androstane Receptor, NR1I3) and PXR (Pregnane X Receptor, NR1I2) are two nuclear receptors devoted to the recognition and elimination of lipohilic molecules potentially toxic to the body.These transcription factors can be activated by ligands of different origins and structures (drugs, environmental pollutants, food products and herbal medicine...). The activation of these receptors leads to the expression of major genes of the detoxification process (CYP450, transferases, transporters) leading to the elimination of these toxics. In this work, we 1) showed that Spot14, a pro-lipogenic protein, is a target gene of CAR, then 2) we identified a novel isoform of PXR (PXR-small), coding only the ligand binding domain of PXR. By using 5'-RACE PXR, we established the origins of transcription of PXR-small and by quantitative PCR we observed that PXR-small represents about 10% of all PXR transcripts in human liver. By using western blo t, we detect its presence on nuclear protein extracts from liver tissues and hepatic cell lines. In Electromobility shift essays experiments, we observed that PXR-small cannot bind to DNA, while reporter essay experiments suggest that this isoform acts as a dominant negative of PXR. Finally, the presence of a CpG island just upstream of PXR-small suggests that this novel isoform might be regulated epigenetically by methylation, more particularly in tumor cells.

Le pregnane X receptor (PXR) est un récepteur nucléaire associé à la réponse au stresscellulaire. Des travaux in vitro de notre groupe ont démontré que les activateurs de cerécepteur (spironolactone (SPIR), clotrimazole (CTZ)) inhibent significativement l’apoptosespontanée ou induite, dans des primo-cultures d’hépatocytes de rat ou d’origine humaine.Les données in vitro sont en faveur d’un rôle clé du PXR dans la protection hépatique contreles xénobiotiques et endobiotiques en régulant de façon concertée leur détoxication(transport, métabolisme) et en augmentant leur résistance à l’apoptose. D’autres travauxrécents ont démontré le rôle majeur de ce récepteur dans la régulation de l’homéostasielipidique et glucidique, d’une part en favorisant la lipogenèse, d’autre part inhibant la lipolyseet la néoglucogenèse. L’induction du PXR peut être responsable de l’accumulation de lipidesdans le foie (NAFLD non alcoholic fatty liver disease). La NAFLD est fortement associée àl’obésité. La chirurgie bariatrique est à ce jour le seul traitement efficace à long terme pourl’obésité morbide. L’évolution des lésions hépatiques de la NAFLD après chirurgiebariatrique n’est pas complètement élucidée. L’objectif de ce travail de thèse, a été d’analyser si, in vivo, les agonistes du PXR tels que le CTZ et la SPIR, présentaient chez l’animal les mêmes effets que ceux décrits in vitro, et s’ilspouvaient conduire à une protection similaire du foie, lors d’atteintes pathologiques commeles lésions hépatiques induites par l’ischémie reperfusion. Dans un autre chapitre les effetsde deux procédures bariatriques, le court circuit gastrique (gastric bypass- GBP) et lagastrectomie en gouttière (sleeve gastrectomy- SG), sur le comorbiditées liées à la obésitéont été analysés. Le modèle d’ischémie reperfusion normothermique partielle et totale du foie chez le rongeur(rat et souris) a été utilisé pour étudier le rôle protecteur du PXR contre les lésionsapoptotiques. Les effets du court circuit gastrique sur les comorbiditées associées à l’obésitéont été étudiés dans deux groupes de patients obèses (index de masse corporelle > 50Kg/m2 et < 50 Kg/m2). Les résultats du GBP et de la SG ont été étudiés dans deux groupescomparables de sujets super obèses (IMC > 50 Kg/m2).Résultats 1/ Le traitement par les activateurs du PXR (CTZ et SPIR), chez le rat et chezla souris, provoque l’induction d’expression du CYP 3A1, enzyme sous le contrôle du PXR. Ilest associé à une réduction significative du nombre d’hépatocytes apoptotiques, du niveaudes transaminases, de la caspases activée et de son substrat PARP (poly-ADP-ribosepolymérase).Les mécanismes impliqués comprennent l’induction d’expression de la protéineantiapoptotique Bcl-xL, l’activation de la voie MAP kinase ERK ½, l’inhibition de l’activationde JNK et la sous-expression des heat schock proteins 27, 70, et 90, dans le cas del’ischémie complète du foie. 2/ Un an après l’intervention bariatique, le GBP a montré uneefficacité comparable sur la réversibilité du syndrome métabolique, de l’inflammationsystémique et l’insulino résistance chez les femmes super-obèses et obèses morbides,même si l’IMC moyen des patientes super obèses est resté significativement plus élevé. LeGBP et la SG conduisent à des résultats comparables, 6 mois après la chirurgie, mais lepremier est significativement plus efficace en termes d’amélioration du profil lipidique,d’inflammation à bas grade et de syndrome métabolique.L’activation du PXR est associée à un effet de protection hépatique contre les lésionsd’ischémie reperfusion. Le GBP est efficace sur les comorbiditées chez le super obèseautant que chez l’obèse morbide. ll donne également de meilleurs résultats à un an, sur laperte de poids, l’inflammation systémique et la régression du syndrome métabolique. / The pregnane X receptor (PXR) is a nuclear receptor associated with cellularresponse to xeno and endobiotics. Recently, the involvement of PXR in controlling otherfunctions has been clarified. We previously showed in our laboratory that the PXR activators(spironolactone (SPIR) and clotrimazole (CTZ) protect primoculture of human or rathépatocytes against apoptosis. In vitro data are indicate that the PXR plays a major role inthe protection of the liver against xeno and endobiotics trough the régulation of theirélimination (détoxication) and increasing their resistance against apoptosis. It has also beenshown that the PXR is implicated in the controls of lipid and carbohydrates metabolism. Theinduction of PXR increases lipogenesis, inhibits lipolysis and neoglucogenesis. PXRinduction may be responsible for the accumulations of lipids into the liver (NAFLD, nonalcoholic liver disease). This disease is strongly associated with obesity. To date bariatricsurgery is only effective treatment in the long term for morbid obesity. The evolution ofNAFLD following bariatric surgery has not been fully clarified.Aims The aims of this thesis were to ascertain whether PXR agonists such as CTZ andSPIR resulted in the same effects in the animal model than those observed in vitro andwhether the administration of these drugs was associated with any protection againstnormothermic ischemia reperfusion injury of the liver. In another chapter the effects onobesity related comorbidities of two bariatric procedures such as the gastric bypass and thesleeve gastrectomy were investigated. The animal model used to investigate the role of PXR against apoptosis wasthe partial and total normothermic ischemia reperfusion of the liver in the rodent (rat andmouse). The effects of the GBP and SG on obesity related comorbidities wereinvestigated in two groups of obese women (BMI > 50 Kg/m2 and < 50kg/m2). The results ofGBP and SG in two comparable groups of super obese patients (BMI > 50 Kg/m2) wereinvestigated. Treatment with PXR activators such as (CTZ and SPIR) in the rat and in themouse was associated with the induction of CYP 3A1, an enzyme directly controlled by thePXR, with a significant reduction of the apoptotic hepatocytes, with a significantly lowerlevels of transaminases, activated caspase 3 and its substrate PARP (poly-ADP-ribosepolymérase).The involved mécanismes include the induction of the expression of theantiapoptotic protein Bcl-xL, the activation of the extracellular regulated kinase (ERK ½), theinhibition of JNK and the down régulation of heat shock proteins (hsp 27, 70, and 90) geneexpression in the case of total liver ischemia. One year after surgery GBP showedcomparable results in terms of résolution of metabolic syndrome, systemic inflammation andinsuline resistance in morbdly obese as well as super obese women even if the mean BMI ofsuper obese women remained higher (34.7 Kg/m2 vs 28.1 Kg/m2). GBP and SG areassociated with the same results six months after surgery but GBP is more effective inimproving lipid disturbances, low-grade systemic inflammation and the metabolic syndromeat one year. PXR induction results in a protective effect against normothermic inschemiareperfusion injury in the rodent. Results of GBP in terms of resolution of obesity relatedcomorbidities are comparable in the super obese and morbidly patients. GBP is moreeffective than SG one year after surgery in terms of weight loss, and resolution of lipiddisturbances, low-grade systemic inflammation and metabolic syndrome.

Le cancer colorectal est marqué par une importante mortalité dans les stades avancés du fait du fort taux de récidives tumorales après chimiothérapie. La prédiction de l'efficacité et de la toxicité des cytotoxiques s'impose comme un des enjeux majeurs de ces prochaines années. Parce que la majorité des anticancéreux sont pris en charge par les enzymes et transporteurs dont l'expression est contrôlée par le niveau d'expression et d'activation des xénosenseurs CAR et PXR, il est fort probable que ces xénosenseurs puissent représenter des facteurs prédictifs à prendre en compte dans la prise en charge des cancers. Notre équipe a récemment montré que les récepteurs des xénobiotiques PXR (NR1I2) (Raynal et al, 2010) et CAR (NR1I3) sont exprimés dans des lignées cellulaires et des tissus coliques humains. Leur surexpression dans les lignées coliques LS174T et T84 entraine leur résistance à l'irinotécan et à son métabolite actif le SN38 alors que leur inhibition antagonise cette résistance. Des dosages intra- et extra-cellulaires du SN38 et du SN38-G, ainsi que la quantification des ARNm des l'UGT1As et du transporteur MDR1, montrent que CAR et PXR augmentent le métabolisme détoxifiant et l'efflux du SN38. L'impact de la surexpression de ces xénosenseurs sur la viabilité des cellules LS174T à différentes classes de cytotoxiques (anti-métabolites, intercalants, inhibiteurs de topoisomérases, poisons du fuseau) a ensuite été évaluée. Nous avons observé que l'expression de CAR ou PXR conduit à une forte chimiorésistance au paclitaxel, au docétaxel et au 4-hydroxy-cyclophosphamide alors que PXR entraîne une sensibilisation marquée au cisplatine et au carboplatine en augmentant la quantité d'adduits de platine sur l'ADN. Les études du transcriptome de nos modèles cellulaires nous ont permis d'identifier les gènes cibles impliqués dans ces variations de cytotoxicité. Des études de confirmation par modulation pharmacologique ou ARNs interférents de ces gènes cibles sont en cours et nous permettront de préciser les mécanismes mis en jeu dans les variations de chimiosensibilité. Ces travaux devraient permettent de mieux appréhender le rôle des xénosenseurs CAR et PXR sur le métabolisme intra-tumoral des cytotoxiques et potentiellement sur la réponse à des chimiothérapies variées. / Colorectal cancer is characterized by high mortality in advanced stages due to the high rate of tumor recurrence after chemotherapy. The prediction of the efficacy and toxicity of cytotoxic drugs represents a major challenge in the coming years. Because the majority of cancer drugs are supported by the enzymes and transporters whose expression is controlled by the level of expression and activation of the xenosensors CAR (NR1I3) and PXR (NR1I2), it is likely that they may represent predictive factors in the management of cancer. Our team has recently shown that xenobiotic receptors PXR (Raynal et al, 2010) and CAR are expressed in cell lines and human colon tissues. Their overexpression in colon cancer cell lines LS174T and T84 leads to resistance to irinotecan and to its active metabolite SN38, while their inhibition reverse this resistance. Irinotecan metabolites detection assays of SN38 and SN38G, and the quantification of UGT1As and MDR1 mRNA, show that CAR and PXR increase the detoxifying metabolism and the efflux of SN38. The impact of overexpression of these xenosensors on LS174T cell viability to different classes of cytotoxic agents (anti-metabolites, DNA intercalators, topoisomerase inhibitors, antimitotic agents) was then evaluated. We observed that the expression of CAR or PXR results in a significant drug resistance to paclitaxel, docetaxel and 4-hydroxy-cyclophosphamide whereas PXR leads to a marked sensitization to cisplatin and carboplatin by increasing the amount of platinum adducts the DNA. Microarray studies of our cell models allowed us to identify the target genes potentially involved in these changes in cytotoxicity. Further studies by pharmacological modulation or interfering RNAs of these target genes are in progress and will allow us to clarify the mechanisms involved in the changes in chemosensitivity. This work should help us to understand the impact of CAR and PXR xenosensors on the intratumoral metabolism of cytotoxic drugs and potentially on the response to various chemotherapies.

No description available.