RESUMEN
The reduction in androgen synthesis and the blockade of the androgen receptor (AR) function by chemical castration and AR signaling inhibitors represent the main treatment lines for the initial stages of prostate cancer. Unfortunately, resistance mechanisms ultimately develop due to alterations in the AR pathway, such as gene amplification or mutations, and also the emergence of alternative pathways that render the tumor less or, more rarely, completely independent of androgen activation. An essential oncogenic axis activated in prostate cancer is the phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, as evidenced by the frequent alterations of the negative regulator phosphatase and tensin homolog (PTEN) and by the activating mutations in PI3K subunits. Additionally, crosstalk and reciprocal feedback loops between androgen signaling and the PI3K/AKT/mTOR signaling cascade that activate pro-survival signals and play an essential role in disease recurrence and progression have been evidenced. Inhibitors addressing different players of the PI3K/AKT/mTOR pathway have been evaluated in the clinic. Only a limited benefit has been reported in prostate cancer up to now due to the associated side effects, so novel combination approaches and biomarkers predictive of patient response are urgently needed. Here, we reviewed recent data on the crosstalk between AR signaling and the PI3K/AKT/mTOR pathway, the selective inhibitors identified, and the most advanced clinical studies, with a focus on combination treatments. A deeper understanding of the complex molecular mechanisms involved in disease progression and treatment resistance is essential to further guide therapeutic approaches with improved outcomes.
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Neoplasias de la Próstata , Receptores Androgénicos , Masculino , Humanos , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Andrógenos/farmacología , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismoRESUMEN
The branched-chain amino acid transaminases (BCATs) are enzymes that catalyze the first reaction of catabolism of the essential branched-chain amino acids to branched-chain keto acids to form glutamate. They are known to play a key role in different cancer types. Here, we report a new structural class of BCAT1/2 inhibitors, (trifluoromethyl)pyrimidinediones, identified by a high-throughput screening campaign and subsequent optimization guided by a series of X-ray crystal structures. Our potent dual BCAT1/2 inhibitor BAY-069 displays high cellular activity and very good selectivity. Along with a negative control (BAY-771), BAY-069 was donated as a chemical probe to the Structural Genomics Consortium.
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Aminoácidos de Cadena Ramificada , Transaminasas , Transaminasas/metabolismo , Aminoácidos de Cadena Ramificada/metabolismo , Cetoácidos/metabolismoRESUMEN
PIP4K2A is an insufficiently studied type II lipid kinase that catalyzes the conversion of phosphatidylinositol-5-phosphate (PI5P) into phosphatidylinositol 4,5-bisphosphate (PI4,5P2). The involvement of PIP4K2A/B in cancer has been suggested, particularly in the context of p53 mutant/null tumors. PIP4K2A/B depletion has been shown to induce tumor growth inhibition, possibly due to hyperactivation of AKT and reactive oxygen species-mediated apoptosis. Herein, we report the identification of the novel potent and highly selective inhibitors BAY-091 and BAY-297 of the kinase PIP4K2A by high-throughput screening and subsequent structure-based optimization. Cellular target engagement of BAY-091 and BAY-297 was demonstrated using cellular thermal shift assay technology. However, inhibition of PIP4K2A with BAY-091 or BAY-297 did not translate into the hypothesized mode of action and antiproliferative activity in p53-deficient tumor cells. Therefore, BAY-091 and BAY-297 serve as valuable chemical probes to study PIP4K2A signaling and its involvement in pathophysiological conditions such as cancer.
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Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Naftiridinas/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
PURPOSE: 5' adenosine monophosphate-activated kinase (AMPK) is an essential regulator of cellular energy homeostasis and has been associated with different pathologies, including cancer. Precisely defining the biological role of AMPK necessitates the availability of a potent and selective inhibitor. METHODS: High-throughput screening and chemical optimization were performed to identify a novel AMPK inhibitor. Cell proliferation and mechanistic assays, as well as gene expression analysis and chromatin immunoprecipitation were used to investigate the cellular impact as well as the crosstalk between lipid metabolism and androgen signaling in prostate cancer models. Also, fatty acid turnover was determined by examining lipid droplet formation. RESULTS: We identified BAY-3827 as a novel and potent AMPK inhibitor with additional activity against ribosomal 6 kinase (RSK) family members. It displays strong anti-proliferative effects in androgen-dependent prostate cancer cell lines. Analysis of genes involved in AMPK signaling revealed that the expression of those encoding 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), fatty acid synthase (FASN) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), all of which are involved in lipid metabolism, was strongly upregulated by androgen in responsive models. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) analysis identified several androgen receptor (AR) binding peaks in the HMGCR and PFKFB2 genes. BAY-3827 strongly down-regulated the expression of lipase E (LIPE), cAMP-dependent protein kinase type II-beta regulatory subunit (PRKAR2B) and serine-threonine kinase AKT3 in responsive prostate cancer cell lines. Also, the expression of members of the carnitine palmitoyl-transferase 1 (CPT1) family was inhibited by BAY-3827, and this was paralleled by impaired lipid flux. CONCLUSIONS: The availability of the potent inhibitor BAY-3827 will contribute to a better understanding of the role of AMPK signaling in cancer, especially in prostate cancer.
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Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Neoplasias de la Próstata , Línea Celular Tumoral , Humanos , Masculino , Transducción de Señal/efectos de los fármacosRESUMEN
Structural mutants of p53 induce global p53 protein destabilization and misfolding, followed by p53 protein aggregation. First evidence indicates that p53 can be part of protein condensates and that p53 aggregation potentially transitions through a condensate-like state. We show condensate-like states of fluorescently labeled structural mutant p53 in the nucleus of living cancer cells. We furthermore identified small molecule compounds that interact with the p53 protein and lead to dissolution of p53 structural mutant condensates. The same compounds lead to condensation of a fluorescently tagged p53 DNA-binding mutant, indicating that the identified compounds differentially alter p53 condensation behavior depending on the type of p53 mutation. In contrast to p53 aggregation inhibitors, these compounds are active on p53 condensates and do not lead to mutant p53 reactivation. Taken together our study provides evidence for structural mutant p53 condensation in living cells and tools to modulate this process.
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Chemoresistance is an important factor in the treatment failure of childhood acute lymphoblastic leukemia (ALL). One underlying mechanism of chemoresistance involves (over)expression of ATP-dependent drug efflux transporters such as multidrug resistance protein 1-5 (MRP1-5) and breast cancer resistance protein (BCRP), which can extrude the important antileukemia drug methotrexate (MTX). Survival of childhood ALL critically depends on the leukemic blasts' capacity for intracellular retention of MTX and MTX-polyglutamates. This pilot study assessed whether expression of MRP1, MRP4, MRP5 and BCRP (real-time PCR) in primary childhood ALL blasts (n = 23) correlated with ex vivo resistance to MTX (assayed by in situ thymidylate synthase inhibition assay (TSIA)), ex vivo accumulation of (radioactive) MTX polyglutamates, and patient survival. Results show that high MRP4 expression is correlated with ex vivo MTX resistance assayed by TSIA (P = 0.01). Moreover, elevated MRP4 and BCRP expression correlated with lower accumulation of MTX-PGs (P = 0.004 and P = 0.03, respectively). Combined high expression of BCRP and MRP4 even further impacted reduced MTX-PG accumulation (P = 0.02). Overall survival was lower (P logrank = 0.04) in children with ALL cells which featured a relatively high expression of both BCRP and MRP4 transporters. These results underscore the impact of high drug efflux transporter expression, notably MRP4 and BCRP, in diminished MTX response in childhood ALL.
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Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Resistencia a Antineoplásicos/genética , Metotrexato/análogos & derivados , Metotrexato/uso terapéutico , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas de Neoplasias/genética , Ácido Poliglutámico/análogos & derivados , Leucemia-Linfoma Linfoblástico de Células Precursoras , Transportadoras de Casetes de Unión a ATP/genética , Adolescente , Niño , Preescolar , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Lactante , Recién Nacido , Masculino , Metotrexato/metabolismo , Proyectos Piloto , Ácido Poliglutámico/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Cultivo Primario de Células , Células Tumorales CultivadasRESUMEN
PURPOSE: We have previously identified the gene MACC1 as a strong prognostic biomarker for colorectal cancer metastasis and patient survival. Here, we report for the first time the generation of transgenic mouse models for MACC1. EXPERIMENTAL DESIGN: We generated mice with transgenic overexpression of MACC1 in the intestine driven by the villin promoter (vil-MACC1) and crossed them with Apc(Min) mice (vil-MACC1/Apc(Min)). RESULTS: vil-MACC1/Apc(Min) mice significantly increased the total number of tumors (P = 0.0056). This was particularly apparent in large tumors (≥3-mm diameter; P = 0.0024). A detailed histopathologic analysis of these lesions demonstrated that the tumors from the vil-MACC1/Apc(Min) mice had a more invasive phenotype and, consequently, showed a significantly reduced survival time than Apc(Min) mice (P = 0.03). Molecular analysis revealed an increased Wnt and pluripotency signaling in the tumors of vil-MACC1/Apc(Min) mice. Specifically, we observed a prominent upregulation of the pluripotency markers Oct4 and Nanog in these tumors compared with Apc(Min) controls. Finally, we could also validate that Oct4 and Nanog are regulated by MACC1 in vitro and strongly correlate with MACC1 levels in a cohort of 60 tumors of colorectal cancer patients (r = 0.7005 and r = 0.6808, respectively; P > 0.0001 and P > 0.0002, respectively). CONCLUSIONS: We provide proof of principle that MACC1-induced tumor progression in colorectal cancer acts, at least in part, via the newly discovered MACC1/Nanog/Oct4 axis. These findings might have important implications for the design of novel therapeutic intervention strategies to restrict tumor progression. Clin Cancer Res; 22(11); 2812-24. ©2016 AACR.
Asunto(s)
Adenoma/metabolismo , Neoplasias Colorrectales/metabolismo , Proteína Homeótica Nanog/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción/fisiología , Adenoma/patología , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Femenino , Expresión Génica , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína Homeótica Nanog/genética , Células Madre Neoplásicas/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Células Madre Pluripotentes/metabolismo , Transactivadores , Vía de Señalización WntRESUMEN
Colorectal cancer is one of the most common cancers worldwide and one of the leading causes of cancer-related death in the Western world. Tumor progression towards metastasis affects a large number of patients with colorectal cancer and seriously affects their clinical outcome. Therefore, considerable effort has been made towards the development of therapeutic strategies that can decrease or prevent colorectal cancer metastasis. Standard treatment of metastatic colorectal cancer with chemotherapy has been improved in the last 10 years by the addition of new targeted agents. The currently used antibodies bevacizumab, cetuximab and panitumumab target the VEGF and EGFR signaling pathways, which are crucial for tumor progression and metastasis. These antibodies have shown relevant efficacy in both first- and second-line treatment of metastatic colorectal cancer. Additionally, other signaling pathways, including the Wnt and HGF/Met pathways, have a well-established role in colorectal cancer progression and metastasis and constitute, therefore, promising targets for new therapeutic approaches. Several new drugs targeting these pathways, including different antibodies and small-molecule tyrosine kinase inhibitors, are currently being developed and tested in clinical trials. In this review, we summarize the new developments in this field, focusing on the inhibitors that show more promising results for use in colorectal cancer patients.
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Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Terapia Molecular Dirigida , Animales , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Diseño de Fármacos , Humanos , Metástasis de la Neoplasia/prevención & control , Transducción de Señal/efectos de los fármacosRESUMEN
The calcium-binding protein S100A4 is a central mediator of metastasis formation in colon cancer. S100A4 is a target gene of the Wnt/ß-catenin pathway, which is constitutively active in the majority of colon cancers. In this study a high-throughput screen was performed to identify small-molecule compounds targeting the S100A4-promoter activity. In this screen calcimycin was identified as a transcriptional inhibitor of S100A4. In colon cancer cells calcimycin treatment reduced S100A4 mRNA and protein expression in a dose- and time-dependent manner. S100A4-induced cellular processes associated with metastasis formation, such as cell migration and invasion, were inhibited by calcimycin in an S100A4-specific manner. Calcimycin reduced ß-catenin mRNA and protein levels despite the expression of Δ45-mutated ß-catenin. Consequently, calcimycin inhibited Wnt/ß-catenin pathway activity and the expression of prominent ß-catenin target genes such as S100A4, cyclin D1, c-myc, and dickkopf-1. Finally, calcimycin treatment of human colon cancer cells inhibited metastasis formation in xenografted immunodeficient mice. Our results demonstrate that targeting the expression of S100A4 with calcimycin provides a functional strategy to restrict cell motility in colon cancer cells. Therefore calcimycin may be useful for studying S100A4 biology, and these studies may serve as a lead for the development of treatments for colon cancer metastasis.
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Antineoplásicos/farmacología , Calcimicina/farmacología , Movimiento Celular/efectos de los fármacos , Proteínas S100/metabolismo , Vía de Señalización Wnt/efectos de los fármacos , Animales , Antineoplásicos/uso terapéutico , Calcimicina/uso terapéutico , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Expresión Génica/efectos de los fármacos , Genes Reporteros , Células HCT116 , Humanos , Neoplasias Hepáticas/prevención & control , Neoplasias Hepáticas/secundario , Luciferasas/biosíntesis , Luciferasas/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica , Regiones Promotoras Genéticas , Proteína de Unión al Calcio S100A4 , Proteínas S100/antagonistas & inhibidores , Proteínas S100/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A series of novel N(10)-substituted acridone derivatives bearing alkyl side-chain with tertiary amine groups at the terminal position were evaluated for their in vitro cytotoxic effects against drug sensitive and resistant cancer cell lines. All the molecules were designed on the basis of hydrogen bond acceptors, carbonyl, fluoro groups with precise spatial separation and structural features of lipophilicity, positive charge at neutral pH and presence of aromatic rings. The in vitro cytotoxic effects in comparison with reference drugs doxorubicin (DX) and C(1311) against cancer cell lines SW 1573, SW 1573 2R 160 (Pg-P expressing) which are non-small cell lung cancer cells, human embryo kidney cells HEK 293, HEK 293 MRP4, HEK 293 MRP5i, human promyelocytic leukemia sensitive cell line HL-60, including its multidrug cross-resistant of two main (P-gp and MRP) phenotype sublines vincristine resistant HL-60/VINC and doxorubicin resistant HL-60/DX cancer cell lines are presented. Compounds 14, 15 and 16 exhibited highest cytotoxicity among the derivatives. On the other hand, the in vitro cytotoxic activity of compound 14 (with butyl side-chain and tertiary amino group ß-hydroxy ethyl piperizine) against resistant cancer cell lines indicate that it might be a promising new hit for further development as an anti-MDR agent. The non-covalent interaction of these molecules with DNA duplexes have been investigated by ESI-MS technique. The results indicate, these acridone derivatives interact with duplex DNA by intercalation, possesses higher affinity to GC than AT base pairs of the DNA and they could not interact non-covalently with the minor grooves of the DNA. The ability of acridones to inhibit calmodulin dependent cAMP phosphodiesterase has been determined. The results suggest that acridones inhibit the Ca(2+)/calmodulin stimulated cAMP-phosphodiesterase activity and have no direct effects on the enzyme itself and a strong correlation between calmodulin inhibition and cytotoxicity against HL-60/VINC and HL-60/DX MDR cancer cell lines.
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Acridonas/química , Acridonas/farmacología , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Aminoacridinas/farmacología , Animales , Calcio/metabolismo , Calmodulina/metabolismo , Línea Celular Tumoral , ADN/química , ADN/metabolismo , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Células HEK293 , Células HL-60 , Humanos , Masculino , Relación Estructura-ActividadRESUMEN
A Avaliação Ambiental Estratégica (AAE) surge como uma ferramenta que fornece oportunidades para a formulação de políticas, planos e programas mais sensíveis às questões ambientais; facilita a integração e coordenação entre vários atores institucionais; e aumenta e fortalece a participação pública. As agências multilaterais de desenvolvimento, como o Banco Interamericano de Desenvolvimento e o Banco Mundial, vêm estimulando a adoção da AAE nos países em desenvolvimento como recomendação ou requisito para aprovação de investimentos e, ainda, por meio de eventos e atividades de capacitação. O presente trabalho discutiu a situação atual da AAE no Brasil, e o papel que essas agências estão desempenhando na sua implementação, a fim de identificar possíveis melhoras práticas e limitações. Defende-se que a prática da AAE no Brasil seja resultado de um modelo autônomo, baseado nas características e demandas internas.
Strategic Environmental Assessment (SEA) has emerged as a tool that provides opportunities for making policies, plans and programs more sensitive to environmental issues. It can also assist integration and coordination between various institutional actors, and the increasing and strengthening of public participation. Multilateral development agencies such as the Interamerican Development Bank and the World Bank have been encouraging the adoption of SEA in developing countries as a recommendation or requirement for investments approval. This paper presents and discusses the role Multilateral Agencies are actually playing in SEA application in Brazil in order to identify possible best practices and constraints. It is argued that SEA in Brazil should be developed as an autonomous instrument, based on Brazilian institutional context, specific characteristics and internal demands.
RESUMEN
AIMS: The current study investigates whether or not functional polymorphisms in the ATP-binding cassette transporter gene ABCG2 might affect gefitinib activity and/or toxicity in non-small-cell lung cancer (NSCLC) patients. MATERIALS & METHODS: Towards this end, ABCG2 polymorphisms and expression were assessed in DNA and tumors from 94 NSCLC patients treated with gefitinib, whereas their associations with toxicity/response and time-to-progression/overall survival were evaluated using Pearson-χ(2) and log-rank-test, respectively. RESULTS: Patients carrying an ABCG2 -15622T/T genotype or harboring at least one TT copy in the ABCG2 (1143C/T, -15622C/T) haplotype developed significantly more grade 2/3 diarrhea (p < 0.01). No associations were found between polymorphisms and outcome. Consistently, ABCG2 protein levels in tumors were not significantly different between patients harboring different ABCG2 variants. CONCLUSION: The ABCG2 -15622C/T polymorphism and ABCG2 (1143C/T, -15622C/T) haplotype resulted in a gefitinib-dependent, moderate-to-severe diarrhea suggesting that these pharmacogenetic markers should be considered to optimize NSCLC treatment.
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Transportadoras de Casetes de Unión a ATP/genética , Antineoplásicos/efectos adversos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Diarrea/inducido químicamente , Diarrea/genética , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas de Neoplasias/genética , Quinazolinas/efectos adversos , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Antineoplásicos/uso terapéutico , Femenino , Gefitinib , Estudio de Asociación del Genoma Completo , Genotipo , Haplotipos/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo Genético/genética , Medicina de Precisión , Quinazolinas/uso terapéutico , Resultado del TratamientoRESUMEN
A series of 2-fluoro N(10)-substituted acridone derivatives with varying alkyl side chain length with propyl, butyl substitution, and a tertiary amine group at the terminal end of the alkyl side chain were synthesized and screened against cancer cell lines SW 1573, SW 1573 2R 160 (P-gp substrate) which are non-small lung cancer cell lines, MCF-7, MCF-7/MR (BCRP substrate) are breast cancer cell lines, 2008 WT, 2008MRP1, 2008MRP2, 2008MRP3 are ovarian cancer cell lines, and human embryo kidney cell lines like HEK293, HEK293 MRP4, and HEK293 MRP5i. The propyl-series compounds showed lipophilicity in the range of 1.93 to 4.40 and the butyl series in the range of 2.37 to 4.78. The compounds 4, 7, and 8 showed good cytotoxicity against the 60 human cancer cell line panel of the National Cancer Institute, USA. The compounds 14 and 15 showed a better cytotoxicity in most of the cancer cell lines compared to other compounds tested. The DNA-binding properties of the compounds were evaluated based on their affinity or intercalation with CT-DNA measured with absorption titration. The compound 11 bearing planar tricyclic ring linked with a butyl methylpiperazino side chain showed the highest binding affinity with a binding constant (K(i)) of 10.38 x 10 M(-1). Evaluation of the compounds in cell lines with an overexpression of various multidrug resistance-related protein (MRP), P-glycoprotein (P-gp), or Breast Cancer Resistance Protein (BCRP) showed that all compounds are not substrates for any of these transporters.
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Acridonas/síntesis química , Sustancias Intercalantes/síntesis química , Acridonas/farmacología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Sustancias Intercalantes/farmacología , Relación Estructura-ActividadRESUMEN
Multi-drug resistance (MDR) frequently contributes to the failure of chemotherapeutic treatments in cancer patients. Mechanisms underlying the development of MDR have been extensively studied and are considered multifactorial. Among them, the ATP-Binding Cassette (ABC) family of proteins plays a pivotal role. Processes of cellular distribution and subcellular localization of MDR-ABC proteins are not yet well explored and to enlighten these topics could be crucial to understand cellular drug uptake and retention. In this review, we analysed literature data concerning i) intracellular trafficking of MDR-ABC proteins (BCRP, P-gp and MRP1) and ii) mechanisms altering their cellular localization and trafficking. Moreover, we describe single nucleotide polymorphisms (SNP) that have been reported for some multidrug resistance (MDR) transporters, such as BCRP and P-gp, emphasizing their ability to affect the expression, function and localization of the transporters, with implications on drug resistance phenotypes.
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Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Polimorfismo de Nucleótido Simple , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Resistencia a Múltiples Medicamentos/genética , Humanos , Modelos Biológicos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/análisis , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismoRESUMEN
As cellular folate levels seem to have a different effect on cancer cells from different origins, we extended our initial study to a broader panel of cancer cells. BCRP and MRP1-5 expression was determined in KB, OVCAR-3, IGROV-1, ZR75-1/R/MTX, SCC-11B, SCC-22B, and WiDr either grown in standard RPMI 1640 containing 2.3 micromol/L supraphysiologic concentration of folic acid [high folate (HF)] or adapted to more physiologic concentrations [1-5 nmol/L folic acid or leucovorin; low folate (LF)]. Compared with the HF counterparts, KB LF cells displayed 16.1-fold increased MRP3 and OVCAR-3 LF cells showed 4.8-fold increased MRP4 mRNA levels along with increased MRP3 and MRP4 protein expression, respectively. A marked increase on BCRP protein and mRNA expression was observed in WiDr LF cells. These cells acquired approximately 2-fold resistance to mitoxantrone compared with the HF cell line, a phenotype that could be reverted by the BCRP inhibitor Ko143. Of note, WiDr cells expressed BCRP in the intracellular compartment, similarly to what we have described for Caco-2 cells. Our results provide further evidence for an important role of cellular folate status in the modulation of the expression of multidrug resistance transporters in cancer cells. We show that up-regulation of intracellularly localized BCRP in response to adaptation to LF conditions may be a common feature within a panel of colon cancer cell lines. Under these circumstances, folate supplementation might improve the efficacy of chemotherapeutic drugs by decreasing BCRP expression.
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Transportadoras de Casetes de Unión a ATP/genética , Ácido Fólico/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes MDR , Proteínas de Neoplasias/genética , Neoplasias/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/metabolismo , Adaptación Fisiológica/efectos de los fármacos , Adaptación Fisiológica/genética , Antineoplásicos/farmacología , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Ácido Fólico/metabolismo , Ácido Fólico/fisiología , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mitoxantrona/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Distribución TisularRESUMEN
Folates can induce the expression and activity of the breast-cancer-resistance-protein (BCRP) and the multidrug-resistance-protein-1 (MRP1). Our aim was to study the time-dependent effect of folate deprivation/supplementation on (i) BCRP and MRP expression and (ii) on drug resistance mediated by these transporters. Therefore Caco-2 colon cancer cells usually grown in standard RPMI-medium containing supraphysiological folic acid (FA) concentrations (2.3 muM; high-folate, HF) were gradually adapted to more physiological folate concentrations (1 nM leucovorin (LV) or 1 nM FA; low-folate, LF), resulting in the sublines Caco-2-LF/LV and Caco-2-LF/FA. Caco-2-LF/LV and LF/FA cells exhibited a maximal increase of 5.2- and 9.6-fold for BCRP-mRNA and 3.9- and 5.7-fold for BCRP protein expression, respectively, but no major changes on MRP expression. Overexpression of BCRP in the LF-cells resulted in 3.6- to 6.3-fold resistance to mitoxantrone (MR), which was completely reverted by the BCRP inhibitor Ko143. On the other hand, LF-adapted cells were markedly more sensitive to methotrexate than the HF-counterpart, both after 4-hr (9,870- and 23,923-fold for Caco-2-LF/LV and LF/FA, respectively) and 72-hr (11- and 22-fold for Caco-2-LF/LV and LF/FA, respectively) exposure. Immunofluorescent staining observed with a confocal-laser-scan-microscope revealed that in Caco-2 cells (both HF and LF), BCRP is mainly located in the cytoplasm. In conclusion, folate deprivation induces BCRP expression associated with MR resistance in Caco-2 cells. The intracellular localization of BCRP in these cells suggests that this transporter is not primarily extruding its substrates out of the cell, but rather to an intracellular compartment where folates can be kept as storage.
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Transportadoras de Casetes de Unión a ATP/genética , Antineoplásicos/farmacología , Ácido Fólico/administración & dosificación , Mitoxantrona/farmacología , Proteínas de Neoplasias/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Células CACO-2 , Resistencia a Antineoplásicos , Humanos , Proteínas de Neoplasias/antagonistas & inhibidores , ARN Mensajero/genéticaRESUMEN
The proteasome inhibitor bortezomib is a novel anticancer drug that has shown promise in the treatment of refractory multiple myeloma. However, its clinical efficacy has been hampered by the emergence of drug-resistance phenomena, the molecular basis of which remains elusive. Toward this end, we here developed high levels (45- to 129-fold) of acquired resistance to bortezomib in human myelomonocytic THP1 cells by exposure to stepwise increasing (2.5-200 nM) concentrations of bortezomib. Study of the molecular mechanism of bortezomib resistance in these cells revealed (1) an Ala49Thr mutation residing in a highly conserved bortezomib-binding pocket in the proteasome beta5-subunit (PSMB5) protein, (2) a dramatic overexpression (up to 60-fold) of PSMB5 protein but not of other proteasome subunits including PSMB6, PSMB7, and PSMA7, (3) high levels of cross-resistance to beta5 subunit-targeted cytotoxic peptides 4A6, MG132, MG262, and ALLN, but not to a broad spectrum of chemotherapeutic drugs, (4) no marked changes in chymotrypsin-like proteasome activity, and (5) restoration of bortezomib sensitivity in bortezomib-resistant cells by siRNA-mediated silencing of PSMB5 gene expression. Collectively, these findings establish a novel mechanism of bortezomib resistance associated with the selective overexpression of a mutant PSMB5 protein.
Asunto(s)
Ácidos Borónicos/farmacología , Resistencia a Antineoplásicos/genética , Mutación , Complejo de la Endopetidasa Proteasomal/genética , Pirazinas/farmacología , Sitios de Unión , Ácidos Borónicos/uso terapéutico , Bortezomib , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Pirazinas/uso terapéutico , ARN Interferente Pequeño/farmacologíaRESUMEN
Because the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib and the multitargeted antifolate pemetrexed are registered in the treatment of second-line non-small-cell lung cancer (NSCLC), empirical combinations of these drugs are being tested. This study investigated molecular mechanisms underlying their combination in six NSCLC cell lines. Cells were characterized by heterogeneous expression of pemetrexed determinants, including thymidylate synthase (TS) and dihydrofolate reductase (DHFR), and mutations potentially affecting chemosensitivity. Pharmacological interaction was studied using the combination index (CI) method, whereas cell cycle, apoptosis induction, and EGFR, extracellular signal-regulated kinases 1 and 2, and Akt phosphorylation were studied by flow cytometry, fluorescence microscopy, and enzyme-linked immunosorbent assays. Reverse-transcriptase polymerase chain reaction (RT-PCR), Western blot, and activity assays were performed to assess whether erlotinib influenced TS. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assays demonstrated that EGFR and k-Ras mutations were related to erlotinib sensitivity, whereas TS and DHFR expression were related to pemetrexed sensitivity. Synergistic cytotoxicity was found in all cells, most pronounced with pemetrexed + erlotinib (24 h) --> erlotinib (48 h) sequence (CI, 0.09-0.40), which was associated with a significant induction of apoptosis. Pemetrexed increased EGFR phosphorylation and reduced Akt phosphorylation, which was additionally reduced by drug combination (-70.6% in H1650). Erlotinib significantly reduced TS expression and activity, possibly via E2F-1 reduction, as detected by RT-PCR and Western blot, and the combination decreased TS in situ activity in all cells. Erlotinib and pemetrexed showed a strong synergism in NSCLC cells, regardless of their genetic characteristics. Induction of apoptosis, modulation of EGFR and Akt phosphorylation, and changes in the expression of critical genes involved in pemetrexed activity contribute to this synergistic interaction and support the clinical investigation of these markers.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/patología , Receptores ErbB/antagonistas & inhibidores , Antagonistas del Ácido Fólico/farmacología , Glutamatos/farmacología , Guanina/análogos & derivados , Neoplasias Pulmonares/patología , Quinazolinas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/genética , Catálisis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Extractos Celulares , Línea Celular Tumoral , Cromonas/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Clorhidrato de Erlotinib , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Guanina/farmacología , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Morfolinas/farmacología , Pemetrexed , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Timidilato Sintasa/genética , Timidilato Sintasa/metabolismoRESUMEN
Folate is a water-soluble B vitamin with a crucial role in the synthesis and methylation of DNA and in the metabolism of several amino acids. In the present study we investigated whether beverages like wine, beer and tea, or some of their specific constituents, affect the intestinal uptake of (3)H-folic acid or (3)H-methotrexate (an antifolate). All tested beverages significantly inhibited the uptake of (3)H-folic acid by Caco-2 cells. Most of these beverages, with the exception of wines (not tested), also inhibited (3)H-methotrexate uptake in these cells. Additionally, ethanol, when tested separately, inhibited the uptake of both compounds. Some of the tested phenolic compounds, namely myricetin, epigallocatechin gallate (EGCG) and isoxanthohumol, markedly inhibited (3)H-folic acid uptake. Myricetin and EGCG also had a concentration-dependent inhibitory effect upon the uptake of (3)H-methotrexate by Caco-2 cells. Resveratrol, quercetin and kaempferol were able to inhibit the transport of both compounds, but only in the concentration of 100 microM. In conclusion, dietary constituents may impact on intestinal folate uptake, as here shown for phenolic compounds.
Asunto(s)
Antioxidantes/farmacología , Bebidas , Células CACO-2/metabolismo , Flavonoides/farmacología , Antagonistas del Ácido Fólico/farmacología , Ácido Fólico/farmacocinética , Fenoles/farmacología , Cerveza/análisis , Células CACO-2/efectos de los fármacos , Humanos , Absorción Intestinal/efectos de los fármacos , Polifenoles , Té/química , Tritio , Vino/análisisRESUMEN
The aim of this work was to test the putative involvement of members of the ABC superfamily of transporters on folic acid (FA) cellular homeostasis in the human placenta. [(3)H]FA uptake and efflux in BeWo cells were unaffected or hardly affected by multidrug resistance 1 (MDR1) inhibition (with verapamil), multidrug resistance protein (MRP) inhibition (with probenecid) or breast cancer resistance protein (BCRP) inhibition (with fumitremorgin C). However, [(3)H]FA uptake and efflux were inhibited by progesterone (200 microM). An inhibitory effect of progesterone upon [(3)H]FA uptake and efflux was also observed in human cytotrophoblasts. Moreover, verapamil and ss-estradiol also reduced [(3)H]FA efflux in these cells. Inhibition of [(3)H]FA uptake in BeWo cells by progesterone seemed to be very specific since other tested steroids (beta-estradiol, corticosterone, testosterone, aldosterone, estrone and pregnanediol) were devoid of effect. However, efflux was also inhibited by beta-estradiol and corticosterone and stimulated by estrone. Moreover, the effect of progesterone upon the uptake of [(3)H]FA by BeWo cells was concentration-dependent (IC(50 )= 65 [range 9-448] microM) and seems to involve competitive inhibition. Also, progesterone (1-400 microM) did not affect either [(3)H]FA uptake or efflux at an external acidic pH. Finally, inhibition of [(3)H]FA uptake by progesterone was unaffected by either 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid (SITS), a known inhibitor of the reduced folate carrier (RFC), or an anti-RFC antibody. These results suggest that progesterone inhibits RFC. In conclusion, our results show that progesterone, a sterol produced by the placenta, inhibits both FA uptake and efflux in BeWo cells and primary cultured human trophoblasts.