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1.
Eur J Pharmacol ; 883: 173377, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32687920

RESUMO

Lithium, commonly used to treat bipolar disorder, potentiates the ability of the muscarinic agonist pilocarpine to induce seizures in rodents. As this potentiation by lithium is reversed by the administration of myo-inositol, the potentiation may be mediated by inhibition of inositol monophosphatase (IMPase), a known target of lithium. Recently, we demonstrated that ebselen is a 'lithium mimetic' in regard to behaviours in both mice and man. Ebselen inhibits IMPase in vitro and lowers myo-inositol in vivo in the brains of mice and men, making ebselen the only known inhibitor of IMPase, other than lithium, that penetrates the blood-brain barrier. Our objective was to determine the effects of ebselen on sensitization to pilocarpine-induced seizures and neural activity. We administered ebselen at different doses and time intervals to mice, followed by injection of a sub-seizure dose of pilocarpine. We assessed seizure and neural activity by a subjective seizure rating scale, by monitoring tremors, and by induction of the immediate early gene c-fos. In contrast to lithium, ebselen did not potentiate the ability of pilocarpine to induce seizures. Unexpectedly, ebselen inhibited pilocarpine-induced tremor as well as pilocarpine-induced increases in c-fos mRNA levels. Both lithium and ebselen inhibit a common target, IMPase, but only lithium potentiates pilocarpine-induced seizures, consistent with their polypharmacology at diverse molecular targets. We conclude that ebselen does not potentiate pilocarpine-induced seizures and instead, reduces pilocarpine-mediated neural activation. This lack of potentiation of muscarinic sensitization may be one reason for the lack of side-effects observed with ebselen treatment clinically.


Assuntos
Anticonvulsivantes/farmacologia , Azóis/farmacologia , Encéfalo/efeitos dos fármacos , Cloreto de Lítio/toxicidade , Neurônios/efeitos dos fármacos , Compostos Organosselênicos/farmacologia , Pilocarpina , Convulsões/prevenção & controle , Animais , Anticonvulsivantes/toxicidade , Azóis/toxicidade , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Células CHO , Sinalização do Cálcio/efeitos dos fármacos , Cricetulus , Modelos Animais de Doenças , Fosfatos de Inositol/metabolismo , Masculino , Camundongos , Neurônios/metabolismo , Compostos Organosselênicos/toxicidade , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Convulsões/induzido quimicamente , Convulsões/metabolismo , Convulsões/fisiopatologia
2.
EMBO Mol Med ; 11(7): e9982, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31273933

RESUMO

Due to compromised homologous recombination (HR) repair, BRCA1- and BRCA2-mutated tumours accumulate DNA damage and genomic rearrangements conducive of tumour progression. To identify drugs that target specifically BRCA2-deficient cells, we screened a chemical library containing compounds in clinical use. The top hit was chlorambucil, a bifunctional alkylating agent used for the treatment of chronic lymphocytic leukaemia (CLL). We establish that chlorambucil is specifically toxic to BRCA1/2-deficient cells, including olaparib-resistant and cisplatin-resistant ones, suggesting the potential clinical use of chlorambucil against disease which has become resistant to these drugs. Additionally, chlorambucil eradicates BRCA2-deficient xenografts and inhibits growth of olaparib-resistant patient-derived tumour xenografts (PDTXs). We demonstrate that chlorambucil inflicts replication-associated DNA double-strand breaks (DSBs), similarly to cisplatin, and we identify ATR, FANCD2 and the SNM1A nuclease as determinants of sensitivity to both drugs. Importantly, chlorambucil is substantially less toxic to normal cells and tissues in vitro and in vivo relative to cisplatin. Because chlorambucil and cisplatin are equally effective inhibitors of BRCA2-compromised tumours, our results indicate that chlorambucil has a higher therapeutic index than cisplatin in targeting BRCA-deficient tumours.


Assuntos
Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Clorambucila/farmacologia , Sistemas de Liberação de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Receptores Ativados por Proliferador de Peroxissomo/antagonistas & inibidores , Ftalazinas/farmacologia , Piperazinas/farmacologia , Animais , Linhagem Celular Tumoral , Cricetinae , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Camundongos , Camundongos SCID , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Cell Res ; 28(7): 719-729, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29795445

RESUMO

Activation of the mitogen-activated protein kinase (MAPK) pathway is frequent in cancer. Drug development efforts have been focused on kinases in this pathway, most notably on RAF and MEK. We show here that MEK inhibition activates JNK-JUN signaling through suppression of DUSP4, leading to activation of HER Receptor Tyrosine Kinases. This stimulates the MAPK pathway in the presence of drug, thereby blunting the effect of MEK inhibition. Cancers that have lost MAP3K1 or MAP2K4 fail to activate JNK-JUN. Consequently, loss-of-function mutations in either MAP3K1 or MAP2K4 confer sensitivity to MEK inhibition by disabling JNK-JUN-mediated feedback loop upon MEK inhibition. In a panel of 168 Patient Derived Xenograft (PDX) tumors, MAP3K1 and MAP2K4 mutation status is a strong predictor of response to MEK inhibition. Our findings suggest that cancers having mutations in MAP3K1 or MAP2K4, which are frequent in tumors of breast, prostate and colon, may respond to MEK inhibitors. Our findings also suggest that MAP3K1 and MAP2K4 are potential drug targets in combination with MEK inhibitors, in spite of the fact that they are encoded by tumor suppressor genes.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , MAP Quinase Quinase 4/genética , MAP Quinase Quinase Quinase 1/genética , Neoplasias da Próstata/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Feminino , Xenoenxertos , Humanos , Mutação com Perda de Função , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase Quinase 1/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Neoplasias da Próstata/genética , Inibidores de Proteínas Quinases/farmacologia
4.
BMC Genomics ; 19(1): 19, 2018 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-29304755

RESUMO

BACKGROUND: Patient-Derived Tumour Xenografts (PDTXs) have emerged as the pre-clinical models that best represent clinical tumour diversity and intra-tumour heterogeneity. The molecular characterization of PDTXs using High-Throughput Sequencing (HTS) is essential; however, the presence of mouse stroma is challenging for HTS data analysis. Indeed, the high homology between the two genomes results in a proportion of mouse reads being mapped as human. RESULTS: In this study we generated Whole Exome Sequencing (WES), Reduced Representation Bisulfite Sequencing (RRBS) and RNA sequencing (RNA-seq) data from samples with known mixtures of mouse and human DNA or RNA and from a cohort of human breast cancers and their derived PDTXs. We show that using an In silico Combined human-mouse Reference Genome (ICRG) for alignment discriminates between human and mouse reads with up to 99.9% accuracy and decreases the number of false positive somatic mutations caused by misalignment by >99.9%. We also derived a model to estimate the human DNA content in independent PDTX samples. For RNA-seq and RRBS data analysis, the use of the ICRG allows dissecting computationally the transcriptome and methylome of human tumour cells and mouse stroma. In a direct comparison with previously reported approaches, our method showed similar or higher accuracy while requiring significantly less computing time. CONCLUSIONS: The computational pipeline we describe here is a valuable tool for the molecular analysis of PDTXs as well as any other mixture of DNA or RNA species.


Assuntos
Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Perfilação da Expressão Gênica , Humanos , Camundongos , Mutação , Alinhamento de Sequência , Análise de Sequência de DNA , Análise de Sequência de RNA
5.
EMBO Mol Med ; 9(10): 1398-1414, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28729482

RESUMO

Maintenance of genome integrity requires the functional interplay between Fanconi anemia (FA) and homologous recombination (HR) repair pathways. Endogenous acetaldehyde, a product of cellular metabolism, is a potent source of DNA damage, particularly toxic to cells and mice lacking the FA protein FANCD2. Here, we investigate whether HR-compromised cells are sensitive to acetaldehyde, similarly to FANCD2-deficient cells. We demonstrate that inactivation of HR factors BRCA1, BRCA2, or RAD51 hypersensitizes cells to acetaldehyde treatment, in spite of the FA pathway being functional. Aldehyde dehydrogenases (ALDHs) play key roles in endogenous acetaldehyde detoxification, and their chemical inhibition leads to cellular acetaldehyde accumulation. We find that disulfiram (Antabuse), an ALDH2 inhibitor in widespread clinical use for the treatment of alcoholism, selectively eliminates BRCA1/2-deficient cells. Consistently, Aldh2 gene inactivation suppresses proliferation of HR-deficient mouse embryonic fibroblasts (MEFs) and human fibroblasts. Hypersensitivity of cells lacking BRCA2 to acetaldehyde stems from accumulation of toxic replication-associated DNA damage, leading to checkpoint activation, G2/M arrest, and cell death. Acetaldehyde-arrested replication forks require BRCA2 and FANCD2 for protection against MRE11-dependent degradation. Importantly, acetaldehyde specifically inhibits in vivo the growth of BRCA1/2-deficient tumors and ex vivo in patient-derived tumor xenograft cells (PDTCs), including those that are resistant to poly (ADP-ribose) polymerase (PARP) inhibitors. The work presented here therefore identifies acetaldehyde metabolism as a potential therapeutic target for the selective elimination of BRCA1/2-deficient cells and tumors.


Assuntos
Acetaldeído/metabolismo , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Rad51 Recombinase/metabolismo , Aldeído-Desidrogenase Mitocondrial/genética , Aldeído-Desidrogenase Mitocondrial/metabolismo , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , Dano ao DNA , Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Fibroblastos , Recombinação Homóloga , Humanos , Camundongos , Camundongos Nus , Rad51 Recombinase/genética , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Cell ; 167(1): 260-274.e22, 2016 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-27641504

RESUMO

The inter- and intra-tumor heterogeneity of breast cancer needs to be adequately captured in pre-clinical models. We have created a large collection of breast cancer patient-derived tumor xenografts (PDTXs), in which the morphological and molecular characteristics of the originating tumor are preserved through passaging in the mouse. An integrated platform combining in vivo maintenance of these PDTXs along with short-term cultures of PDTX-derived tumor cells (PDTCs) was optimized. Remarkably, the intra-tumor genomic clonal architecture present in the originating breast cancers was mostly preserved upon serial passaging in xenografts and in short-term cultured PDTCs. We assessed drug responses in PDTCs on a high-throughput platform and validated several ex vivo responses in vivo. The biobank represents a powerful resource for pre-clinical breast cancer pharmacogenomic studies (http://caldaslab.cruk.cam.ac.uk/bcape), including identification of biomarkers of response or resistance.


Assuntos
Bancos de Espécimes Biológicos , Neoplasias da Mama , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Biomarcadores Farmacológicos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Testes Farmacogenômicos , Células Tumorais Cultivadas
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