Generation of multidrug resistant human tissues by overexpression of the ABCG2 multidrug transporter in embryonic stem cells

The ABCG2 multidrug transporter provides resistance against various endo- and xenobiotics, and protects the stem cells against toxins and stress conditions. We have shown earlier that a GFP-tagged version of ABCG2 is fully functional and may be used to follow the expression, localization and function of this transporter in living cells. In the present work we have overexpressed GFP-ABCG2, driven by a constitutive (CAG) promoter, in HUES9 human embryonic stem cells. Stem cell clones were generated to express the wild-type and a substrate-mutant (R482G) GFP-ABCG2 variant, by using the Sleeping Beauty transposon system. We found that the stable overexpression of these transgenes did not change the pluripotency and growth properties of the stem cells, nor their differentiation capacity to hepatocytes or cardiomyocytes. ABCG2 overexpression provided increased toxin resistance in the stem cells, and protected the derived cardiomyocytes against doxorubicin toxicity. These studies document the potential of a stable ABCG2 expression for engineering toxin-resistant human pluripotent stem cells and selected stem cell derived tissues

Design, synthesis and biological evaluation of thiosemicarbazones, hydrazinobenzothiazoles and arylhydrazones as anticancer agents with a potential to overcome multidrug resistance

There is a constant need for new therapies against multidrug resistant (MDR) cancer. An attractive strategy is to develop chelators that display significant antitumor activity in multidrug resistant cancer cell lines overexpressing the drug efflux pump P-glycoprotein. In this study we used a panel of sensitive and MDR cancer cell lines to evaluate the toxicity of picolinylidene and salicylidene thiosemicarbazone, arylhydrazone, as well as picolinylidene and salicylidene hydrazino-benzothiazole derivatives. Our results confirm the collateral sensitivity of MDR cells to isatin-β-thiosemicarbazones, and identify several chelator scaffolds with a potential to overcome multidrug resistance. Analysis of structure-activity-relationships within the investigated compound library indicates that NNS and NNN donor chelators show superior toxicity as compared to ONS derivatives regardless of the resistance status of the cells. © 2016 Elsevier Masson SAS

Celecoxib Prevents Doxorubicin-Induced Multidrug Resistance in Canine and Mouse Lymphoma Cell Lines

Background: Treatment of malignancies is still a major challenge in human and canine cancer, mostly due to the emergence of multidrug resistance (MDR). One of the main contributors of MDR is the overexpression P-glycoprotein (Pgp), which recognizes and extrudes various chemotherapeutics from cancer cells. Methods: To study mechanisms underlying the development of drug resistance, we established an in vitro treatment protocol to rapidly induce Pgp-mediated MDR in cancer cells. Based on a clinical observation showing that a 33-day-long, unplanned drug holiday can reverse the MDR phenotype of a canine diffuse large B-cell lymphoma patient, our aim was to use the established assay to prevent the emergence of drug resistance in the early stages of treatment. Results: We showed that an in vitro drug holiday results in the decrease of Pgp expression in MDR cell lines. Surprisingly, celecoxib, a known COX-2 inhibitor, prevented the emergence of drug-induced MDR in murine and canine lymphoma cell lines. Conclusions: Our findings suggest that celecoxib could significantly improve the efficiency of chemotherapy by preventing the development of MDR in B-cell lymphoma

Celecoxib Prevents Doxorubicin-Induced Multidrug Resistance in Canine and Mouse Lymphoma Cell Lines.

BACKGROUND: Treatment of malignancies is still a major challenge in human and canine cancer, mostly due to the emergence of multidrug resistance (MDR). One of the main contributors of MDR is the overexpression P-glycoprotein (Pgp), which recognizes and extrudes various chemotherapeutics from cancer cells. METHODS: To study mechanisms underlying the development of drug resistance, we established an in vitro treatment protocol to rapidly induce Pgp-mediated MDR in cancer cells. Based on a clinical observation showing that a 33-day-long, unplanned drug holiday can reverse the MDR phenotype of a canine diffuse large B-cell lymphoma patient, our aim was to use the established assay to prevent the emergence of drug resistance in the early stages of treatment. RESULTS: We showed that an in vitro drug holiday results in the decrease of Pgp expression in MDR cell lines. Surprisingly, celecoxib, a known COX-2 inhibitor, prevented the emergence of drug-induced MDR in murine and canine lymphoma cell lines. CONCLUSIONS: Our findings suggest that celecoxib could significantly improve the efficiency of chemotherapy by preventing the development of MDR in B-cell lymphoma

The importance of drug transporters in human pluripotent stem cells and in early tissue differentiation

Introduction: Drug transporters are large transmembrane proteins which catalyse the movement of a wide variety of chemicals, including drugs as well as xeno- and endobiotics through cellular membranes. The major groups of these proteins include the ATP-binding cassette transporters which in eukaryotes work as ATP-fuelled drug exporters and the Solute Carrier transporters, with various transport directions and mechanisms.Areas covered: In this review, we discuss the key ATP-binding cassette and Solute Carrier drug transporters which have been reported to contribute to the function and/or protection of undifferentiated human stem cells and during tissue differentiation. We review the various techniques for studying transporter expression and function in stem cells, and the role of drug transporters in foetal and placental tissues is also discussed. We especially focus on the regulation of transporter expression by factors modulating cell differentiation properties and on the function of the transporters in adjustment to environmental challenges.Expert opinion: The relatively new and as yet unexplored territory of transporters in stem cell biology may rapidly expand and bring important new information regarding the metabolic and epigenetic regulation of stemness and the early differentiation properties. Drug transporters are clearly important protective and regulatory components in stem cells and differentiation. © 2015 Taylor & Francis

Identification and Validation of Compounds Selectively Killing Resistant Cancer: Delineating Cell Line-Specific Effects from P-Glycoprotein-Induced Toxicity.

Despite significant progress, resistance to chemotherapy is still the main reason why cancer remains a deadly disease. An attractive strategy is to target the collateral sensitivity of otherwise multidrug resistant (MDR) cancer. In this study, our aim was to catalog various compounds that were reported to elicit increased toxicity in P-glycoprotein (Pgp)-overexpressing MDR cells. We show that the activity of most of the serendipitously identified compounds reported to target MDR cells is in fact cell-line specific, and is not influenced significantly by the function of Pgp. In contrast, novel 8-hydroxyquinoline derivatives that we identify in the National Cancer Institute (NCI) drug repository possess a robust Pgp-dependent toxic activity across diverse cell lines. Pgp expression associated with the resistance of the doxorubicin-resistant Brca1-/-;p53-/- spontaneous mouse mammary carcinoma cells could be eliminated by a single treatment with NSC57969, suggesting that MDR-selective compounds can effectively revert the MDR phenotype of cells expressing Pgp at clinically relevant levels. The discovery of new MDR-selective compounds shows the potential of this emerging technology and highlights the 8-hydroxyquinoline scaffold as a promising starting point for the development of compounds targeting the Achilles heel of drug-resistant cancer. Mol Cancer Ther; 16(1); 45-56. (c)2016 AACR

Expression pattern of the human ABC transporters in pluripotent embryonic stem cells and in their derivatives

Background: ATP-binding Cassette (ABC) transporters have key roles in various physiological functions as well as providing chemical defense and stress tolerance in human tissues. In this study we have examined the expression pattern of all ABC proteins in pluripotent human embryonic stem cells (hESCs) and in their differentiated progenies. We paid special attention to the cellular expression and localization of multidrug transporter ABC proteins. Methods: Stem cell differentiation was carried out without chemical induction or cell sorting, and specialized cell types were separated mechanically. Cellular features regarding pluripotency and tissue identity, as well as ABC transporter expression were studied by flow cytomtery, immuno-microscopy and qPCR-based low-density arrays. Results: Pluripotent hESCs and differentiated cell types (cardiomyocytes, neuronal cells and mesenchymal stem cells) were distinguished by morphology, immunostaining markers and selected mRNA expression patterns. We found that the mRNA expression levels of the 48 human ABC proteins also clearly distinguished the pluripotent and the respective differentiated cell types. When multidrug and lipid transporter ABC protein expression was examined by using well characterized specific antibodies by flow cytometry and confocal microscopy, the protein expression data corresponded well to the mRNA expression results. Moreover, the cellular localization of these important human ABC transporter proteins could be established in the pluripotent and differentiated hESC derived samples. Conclusions: These studies provide valuable information regarding ABC protein expression in human stem cells and their differentiated offspring. The results may also help to obtain further information concerning the specialized cellular functions of selected ABC transporter

ABC transzporterek vizsgálata emberi őssejtekben és a sejt-differenciálódás során = Expression and function of ABC transporters in human stem cells and during cell differentiation

A projektben az ABC (ATP-Binding Cassette) transzporterek kifejeződését és funkcióját vizsgáltuk emberi őssejtekben, valamint követtük a normális és daganatos sejtdifferenciálódás során bekövetkező molekuláris szintű változásokat. Elsősorban a gyógyszerek hatásában, anyagcseréjében, az ellenük kifejeződő rezisztenciában szereplő transzportereket, valamint a sejtmembrán lipid-anyagcseréjében szerepet játszó ABC transzportereket vizsgáltuk. A projekt fontos része volt az emberi embrionális őssejtek speciális tenyésztési és differenciálási módszereinek adaptálása, továbbfejlesztése. Az ABC transzporterek vizsgálata során igazoltuk, hogy a humán embrionális őssejtekben az ABCG2 a legjelentősebb xenobiotikum transzporter, a korai sejtdifferenciálódás során az ABCG2 kifejeződése először emelkedik, majd jelentősen csökken. Megállapítottuk, hogy az ABCG2 mRNS használata különbözik a tumorsejtekben, a differenciálódott szöveti sejtekben, illetve a nem-differenciálódott őssejtekben. Új, transzpozon-alapú módszereket dolgoztunk ki az őssejtek stabil genetikai módosítására, fluoreszcens riporter fehérjék bevitelére. Az ABCG2 és az ABCA1 fehérjék címkézett változatainak kifejezése lehetővé tette a transzporterek sejten belüli lokalizációjának követését. Részletesen elemeztük az ABCG2 multidrog transzporter szerkezet-funkció összefüggéseit, szubsztrátokkal jelentkező kölcsönhatásait, alkalmazásokat fejlesztettünk ki a gyógyszerhatások vizsgálatára. | In this project we have studied the expression and function of ABC (ATP-Binding Cassette) transporters in human embryonic and tumor stem cells and followed the changes during normal and tumor cell differentiation at a molecular level. We have focused on the investigation of ABC transporters involved in drug metabolism, cancer multidrug resistance and cellular lipid metabolism. An important part of the project was to establish and further develop proper laboratory conditions and methodologies for culturing and differentiating human pluripotent stem cells. When studying human ABC transporters in human embryonic stem cells we demonstrated the major role of the ABCG2 protein. We found that the expression of this transporter first increases, then greatly decreases during early stem cell differentiation. The use of mRNA was found to be different in the tumor cells, differentiated cells, and pluripotent stem cells, respectively. We have developed new, transposon-based methods for the stable genetic modification of pluripotent stem cells and for the expression of fluorescent reporter proteins. By using tagged versions of the ABCG2 and ABCA1 proteins we could follow the intracellular localization of these transporters. We have studied in detail the structure-function relationships and substrate interactions of the ABCG2 transporter and developed new assays for studying drug interactions

Establishment and Characterization of a Brca1-/-, p53-/- Mouse Mammary Tumor Cell Line.

Breast cancer is the most commonly occurring cancer in women and the second most common cancer overall. By the age of 80, the estimated risk for breast cancer for women with germline BRCA1 or BRCA2 mutations is around 80%. Genetically engineered BRCA1-deficient mouse models offer a unique opportunity to study the pathogenesis and therapy of triple negative breast cancer. Here we present a newly established Brca1-/-, p53-/- mouse mammary tumor cell line, designated as CST. CST shows prominent features of BRCA1-mutated triple-negative breast cancers including increased motility, high proliferation rate, genome instability and sensitivity to platinum chemotherapy and PARP inhibitors (olaparib, veliparib, rucaparib and talazoparib). Genomic instability of CST cells was confirmed by whole genome sequencing, which also revealed the presence of COSMIC (Catalogue of Somatic Mutations in Cancer) mutation signatures 3 and 8 associated with homologous recombination (HR) deficiency. In vitro sensitivity of CST cells was tested against 11 chemotherapy agents. Tumors derived from orthotopically injected CST-mCherry cells in FVB-GFP mice showed sensitivity to cisplatin, providing a new model to study the cooperation of BRCA1-KO, mCherry-positive tumor cells and the GFP-expressing stromal compartment in therapy resistance and metastasis formation. In summary, we have established CST cells as a new model recapitulating major characteristics of BRCA1-negative breast cancers