Introduction: Colorectal cancer (CRC) is one of the most frequent malignancies and affects approximately 5% of worldwide population. More than 75% of CRC cases represent sporadic forms. Susceptibility to nonhereditary CRC is significantly influenced by polymorphisms and mutations in low-penetrance genes. Variations in biotransformation and DNA repair genes may result in acumulation of toxins and DNA damage in cells leading to the development of cancer. Furthermore, different gene expression profiles of membrane transporters affecting the accumulation of anticancer drugs in tumour cells, e.g. ABC drug transporters, may largely influence inter-individual variability in drug response and chemotherapy outcome. The aim of this study was to evaluate the role of genetic and lifestyle factors in the risk of onset and progression of colorectal cancer. This study followed selected genetic alterations in xenobiotic-metabolizing enzymes (CYP1B1, GSTM1, GSTT1, GSTP1, NQO1 and EPHX1) and genes involved in response to DNA damage (CHEK2 and NBN), as potential CRC susceptibility factors. Another aim of this study was to investigate expression profile of all human ABC transporter genes to follow their prognostic and predictive potential in colorectal carcinoma. Materials and methods: The polymorphisms and other...

Le glioblastome (GBM) est le cancer du cerveau le plus fréquent et grave chez l’adulte. Son pronostic sombre est en partie dû à la résistance de ces tumeurs aux chimiothérapies. L’une des principales causes de ces résistances est l’incapacité des chimiothérapies à pénétrer dans le cerveau depuis le sang à cause de la barrière hémato-encéphalique (BHE), une spécificité des vaisseaux sanguins cérébraux. Par l’injection de microbulles et l’envoi d’ultrasons dans le cerveau, il est possible d’ouvrir cette BHE pour permettre à des chimiothérapies d’entrer dans le cerveau. Nous avons montré quela chimiothérapie carboplatine gagnait en efficacité lorsqu’elle était injectée après ce procédé. Les GBM peuvent aussi montrer une résistance aux chimiothérapies par des mécanismes génétiques intrinsèques à la tumeur. Nous avons étudié l’expression et l’impact sur le pronostique des patients atteints de GBM des gènes de la famille des transporteurs ABC, dont le membre ABCA13 s’est avéré important. / Glioblastoma (GBM) is the most frequent and severe brain cancer for adults. Its dark prognosis in partly due to the resistance of these tumors to chemotherapies. One of the main causes of these resistances is the incapacity of chemotherapies to enter the brain from the blood circulation because of the bloodbrain barrier (BBB), a specificity of cerebral blood vessels. By the injection of microbubbles and the delivery of ultrasound into the brain, it is possible to open this BBB to allow chemotherapies to enter the brain. We have showed that the chemotherapeutic agent carboplatin gained efficacy when it was injectedafter this procedure. GBM can also exhibit resistance to chemotherapies by genetic mechanisms intrinsic to the tumor. We studied the expression and the impact on prognosis for GBM patients of the genes of the ABC transporters family, which member ABCA13 appeared important.

Introduction: Colorectal cancer (CRC) is one of the most frequent malignancies and affects approximately 5% of worldwide population. More than 75% of CRC cases represent sporadic forms. Susceptibility to nonhereditary CRC is significantly influenced by polymorphisms and mutations in low-penetrance genes. Variations in biotransformation and DNA repair genes may result in acumulation of toxins and DNA damage in cells leading to the development of cancer. Furthermore, different gene expression profiles of membrane transporters affecting the accumulation of anticancer drugs in tumour cells, e.g. ABC drug transporters, may largely influence inter-individual variability in drug response and chemotherapy outcome. The aim of this study was to evaluate the role of genetic and lifestyle factors in the risk of onset and progression of colorectal cancer. This study followed selected genetic alterations in xenobiotic-metabolizing enzymes (CYP1B1, GSTM1, GSTT1, GSTP1, NQO1 and EPHX1) and genes involved in response to DNA damage (CHEK2 and NBN), as potential CRC susceptibility factors. Another aim of this study was to investigate expression profile of all human ABC transporter genes to follow their prognostic and predictive potential in colorectal carcinoma. Materials and methods: The polymorphisms and other...

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Cancer continues to be a leading cause death in the United States despite improved treatments. Cancerous lesions form after acquiring oncogenic driver mutations or losing tumor suppressor function in normal cells. Traditional therapies have included use of genotoxic substances that take advantage of the increased growth rate and loss of tumor suppressor function to cause cell death. One such drug is the anthracycline antibiotic doxorubicin (DOX). DOX interchelates into DNA and disrupts transcriptional machinery while also poisoning topoisomerase II. This results in single and double stranded DNA breaks, which if severe enough leads to either necrotic or apoptotic cell death. DOX has been very effective at treating several different cancers and is still widely used today however its clinical use is limited due to cumulative dose dependent cardiotoxicity. Therefore, combination therapy targeting survival pathways is utilized to minimize the cumulative dose of DOX without ameliorating its anti-tumor effects. We investigated the potential anti-cancer effects of combining the dual PI3K/mTOR inhibitor, BEZ235 (BEZ), with DOX in pancreatic, breast and other cancer cells lines as well as its associated effects on the heart. Our results showed that co-treatment of BEZ with DOX increased apoptosis in a manner that was dependent on inhibition of the AKT survival pathway. Moreover, BEZ co-treatment with DOX had additive effects towards cell viability while it significantly enhanced necrotic cell death compared to either drug alone. Furthermore, we observed that physiological concentrations of BEZ inhibited ABCB1 efflux resulting in increased intracellular accumulation of DOX, which led to increased DNA damage. In addition, BEZ in combination with gemcitabine (Gem) reduced cell proliferation but did not enhance necrosis or apoptosis. Treatment with BEZ and DOX in mice bearing tumor xenographs reduced tumor growth as compared to BEZ, DOX or Gem. Moreover, BEZ reduced DOX toxicity in rat myoblast cells and did not potentiate the effects of DOX in tumor-bearing mice. We propose that combining BEZ with DOX could be a novel therapeutic approach for the treatment of patients with cancer in the hope of improving the prognosis of this deadly disease.

ARE subfamily proteins belonging to ABC transporters confers a different degree of resistance to macrolides, linkosamides and streptogramins antibiotics. Among the most clinically ARE subfamily proteins in staphylococci is Vga(A) protein lead to the award resistance to streptogtramins A. In 2006, discovered the new variant called the Vga(A)LC, which in addition to streptogramins A resistance also confers linkosamides. Vga(A) and Vga(A)LC differ in only 7 amino acids, yet confer different resistance phenotypes. In previous experiments it was found that the central role in determining substrate specificity play a 4 amino acid differences that accumulate in the section of 15 amino acids within the linker connecting the two ABC domains (positions 212, 219, 220 and 226). The combination of amino acids LGAG Vga(A) increases resistance to streptogramins A while present in combination SVTS Vga(A)LC increased resistance to linkosamides. Although in this subfamily includes a large number of resistance proteins, the mechanism of resistance has not yet been established with certainty. The aim was to create a new Vga(A) variants that contain specific combinations of amino acids for Vga(A) and Vga(A)LC protein at positions 212, 219, 220 and 226 and compared their ability to grant resistance to linkosamides. We also...

L’efficacité des chimiothérapies est limitée par la surexpression de pompes d’efflux adressées à la membrane plasmique des cellules cibles. En effet, celles-ci réduisent le taux intracellulaire des médicaments anticancéreux, antiviraux, antifongiques et antibactériens. La P-gp/ABCB1 est la plus impliquée dans ce phénomène, suivie de MRP1/ABCC1 et de BCRP/ABCG2. Une approche pour surmonter ce phénomène est de développer des médicaments qui ne soient pas expulsés par ces pompes. Dans ce contexte, nous avons développé une nouvelle classe d’inhibiteurs de la protéase du VIH-1 qui ne sont ni transportés par P-gp ni par BCRP. Ils sont ainsi des candidats intéressants aux trithérapies contre le SIDA. Un point clé pour comprendre comment ces transporteurs font traverser les médicaments à travers la membrane est d’identifier des nouvelles structures. Ainsi, nous avons résolu trois structures de P-gp de souris. Une d’entre-elles est complexée à un nano-anticorps lié au premier NBD («nucleotide-binding domain»), qui fige la P-gp dans une nouvelle conformation ouverte vers l'intérieur. Pour finir, nous avons localisé deux sites de liaison de P-gp en caractérisant les modes d'inhibition de deux inhibiteurs précédemment cocristallises avec la pompe. Ceci permet de mieux comprendre le mécanisme de translocation et offre la possibilité de cibler plus précisément ces sites pour développer des modulateurs de cette pompe / Resistance to chemotherapy is partly due to efflux pumps expressed in the plasma membrane which prevents the accumulation of anticancer, antiviral, antifungal and antibacterial drugs in target cells. Three human ABC transporters are particularly involved in MDR phenotype: P-gp/ABCB1, MRP1/ABCC1 and BCRP/ABCG2. Among the different approaches used to overcome the resistance linked to these transporters, the development of non-substrate drugs MDR-ABC transporters has been described. Here, new class of HIV-1 protease inhibitors not recognized by P-gp/BCRP were identified, promising to be attractive candidates to HAART therapy. Since the determination of the X-ray structures in different conformations is a key point to understand how MDR-ABC transporters translocate drugs across the plasma membrane, the crystal structures of three inward-facing conformations of mouse P-gp were resolved. One structure has a camel nanobody bound to the C-terminal side of the first nucleotide-binding domain, revealing a unique epitope on P-gp and freezing a new open-inward conformation. Finally, the enzymatic characterization of two inhibitors co-crystallized with the mouse P-gp has allowed to localize two main binding sites by which drugs efflux occurs. These results bring new findings of the drug-efflux mechanism and offer the possibility to target more precisely those sites to develop modulators of this pump

La surexpression de pompes d'efflux par les cellules cancéreuses permet l'élimination d'agents cytotoxiques, induisant alors une résistance à la chimiothérapie. Trois transporteurs ABC sont principalement impliqués dans cette résistance : P-gp/ABCB1, MRP1-ABCC1 et BCRP/ABCG2. La surexpression des ces transporteurs peut également être le "talon d'Achille" des cellules cancéreuses résistantes en les sensibilisant à certains composés. Ce phénomène, appelé sensibilité collatérale, pourrait constituer un nouvel outil thérapeutique conter les les cancers intrinséquement ou rendus résistants en éliminant sélectivement les cellules cancéreuse résistances. Ainsi, le S-vérapamil provoque la mort sélective par apoptose des cellules surexprimant suite à l'extrusion rapide et massive du glutathion 5GSH) intracellulaire par MRP1. Nous avons démontré que le vérapamil est capable de dépléter s"lectivement de leur contenu en GSH les tumeurs de cancer du poumon H69AR, MRP1 positives et résistantes, dès 3 heures d'exposition aiguë. Le vérapamil étant fortemnt carditoxique, nous avons développé de nouveaux agents de sensibilité collatérale, plus sélectif que le vérapamil, comme le xanthone 9, le flavonoïde 36 et le dimère de flavonoïde 4e. Enfin, grâce à l'étude de chimères MRP1/MRP2, nous avons démontré que la région comprenant les boucles L0 et L1-TM12 pourrait constituter les sites modualteurs et substrat du GSH sur MRP1. / Resistance to chemotherapy is partly due to efflux pumps expressed in the plasma membrane, which prevent the accumulation of anticancer drugs in tumor cells. Three human ABC transporters are particulary involved in this chemoresistance : P-gp/ABCB1, MRP1-ABCC1 and BCRP/ABCG2. The overexpression of these trnasporters can also be an "Achille heel" for resistant cancer cells by sensitizing them to various drugs. This phenomenom, called collateral sensitivity, could constitute a new chemotherapy to eradicate cancers becoming resistant or cancer which ara resistant prioir to any treatment. Thus, S-verapamil triggers selective apoptosis of MRP1 overexpressing correlated to the massive and rapide extrusion of cellular glutathione by MRP1. We showed that verapamil is able to selectivity deprive H69AR MRP1 positive and resistant lung tumors, as soon as 3 hours of acute exposure. Verapamil being highly cardiotoxic, we have developed new collateral sensitivity drugs, more selective than verapamil, such as xanthone 9, flavonoïdd 36 and flavonoïd dimer 4e. Finally, thanks to the characterization of MRP1/MRP2 chimera, we showed that the MRP1 region including the intracellular loop L0 L1-TM12 might constitute the substrate and the modulator binding sites for GSH.

ABC-Transmembrantransporter sind an der Aufnahme, Verteilung und Ausscheidung vieler Arznei- und Fremdstoffe beteiligt. Sie spielen eine Schlüsselrolle in der Pharmakokinetik und in der Ausscheidung toxischer endogener oder exogener Substanzen. Das Ziel der hier präsentierten Untersuchungen war deshalb, den Einfluss genetischer Polymorphismen des bekanntesten Vertreters dieser Proteinfamilie, MDR1 (ABCB1) zu untersuchen. Darüberhinaus sollte der ebenfalls zur ABC-Transporterfamilie gehörende hepatozelluläre Exporter für monoanionische Gallensäuren identifiziert und charakterisiert werden. MDR1 erwies sich als ein hochpolymorphes Gen mit zahlreichen Einzelbasenaustauschen (SNPs). Die meisten SNPs waren intronisch oder stumm. Für den nichtkodierenden SNP im Exon 26 3435C>T ergab sich bei homozygoten Trägern des T-Allels eine im Vergleich zum Wildtyp geringere intestinale P-Glykoprotein Expression mit einer entsprechend höheren und schnelleren Absorption von Digoxin. Die Auswertung pharmakokinetischer Profile von Digoxin in Individuen mit MDR1-Haplotypen der miteinander verbundenen SNPs in Exon 21 2677 und Exon 26 3435 untermauerte die beobachteten pharmakogenetischen Effekte. Nach oraler Einzelgabe von 1 mg Digoxin konnten wahrscheinlich aufgrund der Überschreitung der P-Glykoprotein Transportkapazität keine genotypischen Unterschiede beobachtet werden. Der biliäre Exporter für monoanionische Gallensäuren (Bsep) konnte als ein 160 kDa Glykoprotein aus einer Rattenleber cDNA-Bibliothek identifiziert werden und gehört ebenfalls zur ABC Transporter-Familie. Die transkriptionelle Regulation und Möglichkeiten der Modulation der Expression des Bsep-Gens wurden in vitro und in Tiermodellen der Cholestase untersucht. Dabei zeigte sich, dass Gallensäuren über ein proximales FXRE-Motiv die Bsep Promotoraktivität stimulierten. Arzneistoffe hatten ebenfalls einen Einfluss auf die Transkription des Bsep-Gens. Die adaptive Regulation hepatozellulärer Transporter während der Cholestase ergab eine verminderte Expression der meisten basolateralen Aufnahmetransporter und eine unveränderte oder heraufregulierte Proteinmasse kanalikulärer (apikaler) Efflux-Transporter. Dieses Regulationsmuster dient dem Schutz der Leberzelle, indem eine intrazelluläre Anreicherung toxischer Gallensäuren vermindert und der Gallefluss für eine intakte biliäre Clearance aufrechterhalten wird. / ABC transmembrane transporters are involved in absorption, distribution and excretion of diverse drugs and xenobiotics. They are key factors in pharmacokinetics and in the elimination of toxic endogenous or exogenous compounds. Therefore, the aim of the present study was to investigate the influence of genetic polymorphisms of the best known member of this protein family, MDR1 (ABCB1). In addition, the identity of another ABC transporter assumed to be the major hepatocellular export pump for monoanionic bile acids should be revealed and characterized. MDR1 turned out as a highly polymorphic gene with many single nucleotide polymorphisms (SNPs). Most of the SNPs were intronic or silent. Homozygous carriers of the non-coding SNP in exon 26 3435C>T had lower intestinal P-glycoprotein expression rates and thus enhanced absorption of the model compound digoxin as compared to wildtype controls. The analysis of pharmacokinetic profiles in different MDR1-haplotypes of the linked SNPs in exon 21 2677 and exon 26 3435 supported the above data. An oral single dose of 1 mg digoxin did not result in genotypic differences of tested genotypes, probably because this dose was above the maximal transport capacity of P-glycoprotein. The biliary export pump for monoanionic bile acids (Bsep) was identified as an 160 kDa glycoprotein of the ABC transporter family by screening a rat liver cDNA library. The transcriptional regulation and modulatory factors of Bsep (Abcb11) gene expression were analyzed in vitro and in animal models of cholestasis. The promoter activity of Bsep was stimulated by bile acids via a proximal FXRE motif. Drugs were also able to modify Bsep gene transcription. Adaptive regulation of hepatocellular transporters during cholestasis followed a pattern of diminished expression of most basolateral uptake carrier systems and maintained or even upregulated protein mass of canalicular (apical) exporters. This pattern serves as a protective mechanism of the liver cells preventing intracellular accumulation of toxic bile acids and providing unimpaired biliary flow and clearance.