RESUMO
PURPOSE: The functionality of many cellular proteins depends on cofactors; yet, they have only been implicated in a minority of Mendelian diseases. Here, we describe the first 2 inherited disorders of the cytosolic iron-sulfur protein assembly system. METHODS: Genetic testing via genome sequencing was applied to identify the underlying disease cause in 3 patients with microcephaly, congenital brain malformations, progressive developmental and neurologic impairments, recurrent infections, and a fatal outcome. Studies in patient-derived skin fibroblasts and zebrafish models were performed to investigate the biochemical and cellular consequences. RESULTS: Metabolic analysis showed elevated uracil and thymine levels in body fluids but no pathogenic variants in DPYD, encoding dihydropyrimidine dehydrogenase. Genome sequencing identified compound heterozygosity in 2 patients for missense variants in CIAO1, encoding cytosolic iron-sulfur assembly component 1, and homozygosity for an in-frame 3-nucleotide deletion in MMS19, encoding the MMS19 homolog, cytosolic iron-sulfur assembly component, in the third patient. Profound alterations in the proteome, metabolome, and lipidome were observed in patient-derived fibroblasts. We confirmed the detrimental effect of deficiencies in CIAO1 and MMS19 in zebrafish models. CONCLUSION: A general failure of cytosolic and nuclear iron-sulfur protein maturation caused pleiotropic effects. The critical function of the cytosolic iron-sulfur protein assembly machinery for antiviral host defense may well explain the recurrent severe infections occurring in our patients.
Assuntos
Proteínas Ferro-Enxofre , Peixe-Zebra , Animais , Humanos , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Masculino , Feminino , Fenótipo , Fibroblastos/metabolismo , Fibroblastos/patologia , Citosol/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Microcefalia/genética , Microcefalia/patologia , Lactente , MetalochaperonasRESUMO
BACKGROUND AND OBJECTIVE: Fabry disease (FD) is a rare lysosomal storage disorder caused by a deficiency of the enzyme α-galactosidase A (aGal A). Since 2001, two different enzyme replacement therapies have been authorized, with agalsidase beta being used in most parts of the Western world. Currently, biosimilars of several expensive enzyme therapies are under development to improve their accessibility for patients. We present the preclinical results of the development of a biosimilar to agalsidase beta. METHODS: Produced in a Chinese hamster ovary (CHO)-cell system, the biosimilar aGal A Biosidus (AGABIO), was compared with agalsidase beta with respect to amino acid sequence, glycosylation, specific α-galactosidase activity, stability in plasma, and effects on cultured human Fabry fibroblasts and Fabry mice. RESULTS: AGABIO had the same amino acid composition and similar glycosylation, enzymatic activity, and stability as compared with agalsidase beta. After uptake in fibroblasts, α-galactosidase A activity increased in a dose-dependent manner, with maximum uptake observed after 24 h, which remained stable until at least 48 h. Both enzymes were localized to lysosomes. Reduction of accumulated globotriaosylceramide (Gb3) and lysoGb3 in cultured Fabry fibroblasts by AGABIO and agalsidase beta showed comparable dose-response curves. In Fabry knockout mice, after a single injection, both enzymes were rapidly cleared from the plasma and showed equal reductions in tissue and plasma sphingolipids. Repeated dose studies in rats did not raise any safety concerns. Anti-drug antibodies from patients with FD treated with agalsidase beta showed equal neutralization activity toward AGABIO. CONCLUSION: These findings support the biosimilarity of AGABIO in comparison with agalsidase beta. The clinical study phase is currently under development.
Assuntos
Medicamentos Biossimilares , Doença de Fabry , Humanos , Camundongos , Ratos , Animais , Cricetinae , Doença de Fabry/tratamento farmacológico , alfa-Galactosidase/uso terapêutico , Células CHO , Resultado do Tratamento , Cricetulus , Proteínas Recombinantes/uso terapêuticoRESUMO
Glutaminase deficiency has recently been associated with ataxia and developmental delay due to repeat expansions in the 5'UTR of the glutaminase (GLS) gene. Patients with the described GLS repeat expansion may indeed remain undiagnosed due to the rarity of this variant, the challenge of its detection and the recency of its discovery. In this study, we combined advanced bioinformatics screening of ~3000 genomes and ~1500 exomes with optical genome mapping and long-read sequencing for confirmation studies. We identified two GLS families, previously intensely and unsuccessfully analyzed. One family carries an unusual and complex structural change involving a homozygous repeat expansion nested within a quadruplication event in the 5'UTR of GLS. Glutaminase deficiency and its metabolic consequences were validated by in-depth biochemical analysis. The identified GLS patients showed progressive early-onset ataxia, cognitive deficits, pyramidal tract damage and optic atrophy, thus demonstrating susceptibility of several specific neuron populations to glutaminase deficiency. This large-scale screening study demonstrates the ability of bioinformatics analysis-validated by latest state-of-the-art technologies (optical genome mapping and long-read sequencing)-to effectively flag complex repeat expansions using short-read datasets and thus facilitate diagnosis of ultra-rare disorders.
Assuntos
Glutaminase , Humanos , Regiões 5' não Traduzidas , Ataxia/diagnóstico , Ataxia/genética , Glutaminase/genéticaRESUMO
PURPOSE: Elevated urinary 3-methoxytyramine (3MT) level at diagnosis was recently put forward as independent risk factor for poor prognosis in neuroblastoma. Here, we investigated the biologic basis underlying the putative association between elevated 3MT levels and poor prognosis. METHODS: Urinary 3MT levels and prognosis were investigated in both retrospective Italian (N = 90) and prospective Dutch (N = 95) cohorts. From the Dutch Cancer Oncology Group cohort (N = 122), patients with available urinary 3MT and gene expression data (n = 90) were used to generate a 3MT gene signature. The 3MT gene signature score was then used to predict survival outcome in the Children's Oncology Group (N = 247) and German Pediatric Oncology Group (N = 498) cohorts and compared with other known gene signatures. Immunohistochemistry of MYCN and dopamine ß-hydroxylase proteins was performed on primary tumors. RESULTS: Elevated urinary 3MT levels were associated with poor prognosis in a retrospective cohort and a prospective cohort. Moreover, elevated urinary 3MT levels were associated with eight differentially expressed genes, providing a 3MT gene signature that successfully predicted poor clinical outcome. Even among low-risk patients, high 3MT signature score was associated with poor 5-year overall survival (72% v 99% among low-risk patients with a low 3MT signature score), and the 3MT signature score was correlated with MYC activity in the tumor (R = 82%, P < .0001). Finally, a strong MYCN and weak dopamine ß-hydroxylase staining of tumors derived from patients with elevated urinary 3MT levels was observed, linking MYC activity in the tumor to both catecholamine biosynthesis and elevated urinary 3MT levels. CONCLUSION: Elevated urinary 3MT is a promising biomarker for poor prognosis and reflects increased MYC activity in the tumor. Therefore, urinary 3MT levels should be measured at diagnosis and may assist in assessing risk.
Assuntos
Biomarcadores Tumorais/urina , Dopamina/análogos & derivados , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/urina , Dopamina/genética , Dopamina/urina , Humanos , Estudos Prospectivos , Estudos RetrospectivosRESUMO
Accumulation of DNA lesions causing transcription stress is associated with natural and accelerated aging and culminates with profound metabolic alterations. Our understanding of the mechanisms governing metabolic redesign upon genomic instability, however, is highly rudimentary. Using Ercc1-defective mice and Xpg knock-out mice, we demonstrate that combined defects in transcription-coupled DNA repair (TCR) and in nucleotide excision repair (NER) directly affect bioenergetics due to declined transcription, leading to increased ATP levels. This in turn inhibits glycolysis allosterically and favors glucose rerouting through the pentose phosphate shunt, eventually enhancing production of NADPH-reducing equivalents. In NER/TCR-defective mutants, augmented NADPH is not counterbalanced by increased production of pro-oxidants and thus pentose phosphate potentiation culminates in an over-reduced redox state. Skin fibroblasts from the TCR disease Cockayne syndrome confirm results in animal models. Overall, these findings unravel a mechanism connecting DNA damage and transcriptional stress to metabolic redesign and protective antioxidant defenses.
Assuntos
Trifosfato de Adenosina/metabolismo , Antioxidantes/metabolismo , Dano ao DNA/genética , Reparo do DNA/genética , Glicólise/fisiologia , NADP/metabolismo , Via de Pentose Fosfato/fisiologia , Transcrição Gênica/genética , Regulação Alostérica , Animais , Síndrome de Cockayne/metabolismo , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Fibroblastos/metabolismo , Instabilidade Genômica , Metabolômica , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Oxirredução , Pele/citologia , Fatores de Transcrição/genéticaRESUMO
We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5' untranslated region of the gene encoding glutaminase (GLS) that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated patients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of GLS messenger RNA transcribed from the expanded allele, which probably resulted from repeat-mediated chromatin changes upstream of the GLS repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Ataxia/genética , Deficiências do Desenvolvimento/genética , Glutaminase/deficiência , Glutaminase/genética , Glutamina/metabolismo , Repetições de Microssatélites , Mutação , Atrofia/genética , Cerebelo/patologia , Pré-Escolar , Feminino , Genótipo , Glutamina/análise , Humanos , Masculino , Fenótipo , Reação em Cadeia da Polimerase , Sequenciamento Completo do GenomaRESUMO
BACKGROUND: In situ hypothermic perfusion during liver resection performed under vascular inflow occlusion decreases hepatic ischemia-reperfusion injury, but technical limitations have restricted its widespread use. In situ hypothermic perfusion with retrograde outflow circumvents these impediments and thus could extend the applicability of in situ hypothermic perfusion. The safety and feasibility of in situ hypothermic perfusion with retrograde outflow were analyzed in selected patients undergoing right (extended) hepatectomy and compared to intermittent vascular inflow occlusion, the gold standard method, in this randomized pilot study. METHODS: Patients were first screened for parenchymal liver disease (exclusion criteria: steatosis ≥30%, cirrhosis, or cholestasis). Study participants were randomized intraoperatively to undergo in situ hypothermic perfusion with retrograde outflow (n = 9) or intermittent vascular inflow occlusion (n = 9). The target liver core temperature during in situ hypothermic perfusion with retrograde outflow was 28°C. The primary end point was ischemia-reperfusion injury (expressed by peak postoperative transaminase levels). Secondary outcomes included functional liver regeneration (assessed by hepatobiliary scintigraphy) and clinical outcomes. RESULTS: Peak transaminase levels, total bilirubin, and the international normalized ratio were similar between both groups, although a trend toward more rapid normalization of bilirubin levels was noted for the in situ hypothermic perfusion with retrograde outflow group. Functional liver regeneration as evaluated by hepatobiliary scintigraphy was improved on postoperative day 3 fafter in situ hypothermic perfusion with retrograde outflow but not after intermittent vascular inflow occlusion. Furthermore, in situ hypothermic perfusion with retrograde outflow (requiring continuous ischemia) was comparable to intermittent vascular inflow occlusion for all clinical outcomes, including postoperative complications and hospital stay. CONCLUSION: The use of in situ hypothermic perfusion with retrograde outflow appears to be safe and feasible in selected patients with healthy liver parenchyma and may benefit early functional liver regeneration. Future applications of in situ hypothermic perfusion with retrograde outflow include patients with damaged liver parenchyma who would require major hepatic resection with a prolonged vascular inflow occlusion duration.
Assuntos
Perda Sanguínea Cirúrgica/prevenção & controle , Hepatectomia/métodos , Hipotermia Induzida/métodos , Hepatopatias/cirurgia , Perfusão/métodos , Traumatismo por Reperfusão/prevenção & controle , Idoso , Estudos de Viabilidade , Feminino , Hepatectomia/efeitos adversos , Humanos , Regeneração Hepática , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Transaminases/metabolismo , Resultado do TratamentoRESUMO
Photodynamic therapy (PDT) is an established palliative treatment for perihilar cholangiocarcinoma that is clinically promising. However, tumors tend to regrow after PDT, which may result from the PDT-induced activation of survival pathways in sublethally afflicted tumor cells. In this study, tumor-comprising cells (i.e., vascular endothelial cells, macrophages, perihilar cholangiocarcinoma cells, and EGFR-overexpressing epidermoid cancer cells) were treated with the photosensitizer zinc phthalocyanine that was encapsulated in cationic liposomes (ZPCLs). The post-PDT survival pathways and metabolism were studied following sublethal (LC50) and supralethal (LC90) PDT. Sublethal PDT induced survival signaling in perihilar cholangiocarcinoma (SK-ChA-1) cells via mainly HIF-1-, NF-кB-, AP-1-, and heat shock factor (HSF)-mediated pathways. In contrast, supralethal PDT damage was associated with a dampened survival response. PDT-subjected SK-ChA-1 cells downregulated proteins associated with EGFR signaling, particularly at LC90. PDT also affected various components of glycolysis and the tricarboxylic acid cycle as well as metabolites involved in redox signaling. In conclusion, sublethal PDT activates multiple pathways in tumor-associated cell types that transcriptionally regulate cell survival, proliferation, energy metabolism, detoxification, inflammation/angiogenesis, and metastasis. Accordingly, tumor cells sublethally afflicted by PDT are a major therapeutic culprit. Our multi-omic analysis further unveiled multiple druggable targets for pharmacological co-intervention.
Assuntos
Redes e Vias Metabólicas , Metabolômica/métodos , Fotoquimioterapia , Proteômica/métodos , Transdução de Sinais , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Receptores ErbB/metabolismo , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Camundongos , Oxirredução/efeitos dos fármacos , Fármacos Fotossensibilizantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Excess catecholamine levels are suggested to be cardiotoxic and to underlie stress-induced heart failure. The cardiotoxic effects of norepinephrine and epinephrine are well recognized. However, although cardiac and circulating dopamine levels are also increased in stress cardiomyopathy patients, knowledge regarding putative toxic effects of excess dopamine levels on cardiomyocytes is scarce. We now studied the effects of elevated dopamine levels in H9c2 cardiomyoblasts. H9c2 cells were cultured and treated with dopamine (200 µM) for 6, 24, and 48 h. Subsequently, the effects on lipid accumulation, cell viability, flippase activity, reactive oxygen species (ROS) production, subcellular NADPH oxidase (NOX) protein expression, and ATP/ADP and GTP/GDP levels were analyzed. Dopamine did not result in cytotoxic effects after 6 h. However, after 24 and 48 h dopamine treatment induced a significant increase in lipid accumulation, nitrotyrosine levels, indicative of ROS production, and cell death. In addition, dopamine significantly reduced flippase activity and ATP/GTP levels, coinciding with phosphatidylserine exposure on the outer plasma membrane. Furthermore, dopamine induced a transient increase in cytoplasmic and (peri)nucleus NOX1 and NOX4 expression after 24 h that subsided after 48 h. Moreover, while dopamine induced a similar transient increase in cytoplasmic NOX2 and p47phox expression, in the (peri)nucleus this increased expression persisted for 48 h where it colocalized with ROS. Exposure of H9c2 cells to elevated dopamine levels induced lipid accumulation, oxidative stress, and a proinflammatory status of the plasma membrane. This can, in part, explain the inflammatory response in patients with stress-induced heart failure.
Assuntos
Dopaminérgicos/farmacologia , Dopamina/farmacologia , Inflamação/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Mioblastos Cardíacos/efeitos dos fármacos , NADPH Oxidases/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular , Citometria de Fluxo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Microscopia de Fluorescência , Mioblastos Cardíacos/metabolismo , Mioblastos Cardíacos/ultraestrutura , NADH NADPH Oxirredutases/efeitos dos fármacos , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1 , NADPH Oxidase 4 , NADPH Oxidases/metabolismo , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Peroxidase/efeitos dos fármacos , Peroxidase/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Tirosina/análogos & derivados , Tirosina/efeitos dos fármacos , Tirosina/metabolismoRESUMO
OBJECTIVE: Creatine kinase is reported to be a main predictor of blood pressure (BP) in the general population, with a strong correlation between resistance artery creatine kinase expression and clinical BP in humans. The enzyme rapidly regenerates ATP near cytoplasmic ATPases involved in pressor responses, including resistance artery contractility and renal sodium retention. Therefore, we assessed whether creatine kinase inhibition reduces BP. METHODS: We implemented the 'Animal Research: Reporting of In Vivo Experiments' guideline. In a 4-week randomized controlled trial, male 16-week-old spontaneously hypertensive rats (Nâ=â16) were randomly assigned to the specific competitive creatine kinase inhibitor beta-guanidinopropionic acid (3%)-supplemented chow vs. standard chow. BP measured by the tail-cuff method was the main outcome. Other outcomes included vasodilation in isolated arteries and renal renin expression. RESULTS: Creatine kinase inhibition reduced BP safely and reversibly. Mean baseline BP of, respectively, 191.5 (standard error 4.3)âmmHg SBP and 143.1 (4.1)âmmHg DBP was reduced by, respectively, 42.7 (5.5)âmmHg SBP and 35.6 (5.0)âmmHg DBP (Pâ<â0.001) compared with controls, with evidence of enhanced vasodilation and a diuretic effect. CONCLUSION: To our knowledge, this is the first report on the BP-lowering effect of creatine kinase inhibition. Our data indicate that modulation of the creatine kinase system is a potential novel treatment target for hypertension.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Creatina Quinase/antagonistas & inibidores , Guanidinas/farmacologia , Propionatos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Artérias/efeitos dos fármacos , Artérias/fisiopatologia , Diurese/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Rim/metabolismo , Masculino , Distribuição Aleatória , Ratos , Ratos Endogâmicos SHR , Renina/metabolismo , Vasodilatação/efeitos dos fármacosRESUMO
The efficacy and mechanism of action of cisplatin and gemcitabine were investigated in a panel of neuroblastoma cell lines and multicellular tumor spheroids. In neuroblastoma spheroids, the combination of cisplatin and gemcitabine induced a complete cytostasis at clinical relevant concentrations. A synergistic effect was observed when cells were coincubated with both drugs or preincubated with gemcitabine first. These administration sequences resulted in NASS cells in decreased ERCC1 and XPA expression, two key proteins of the NER DNA repair system, and increased platinum adduct formation in DNA. Most of these phenomena were not observed in SJNB8 cells which might explain the lack of synergy between cisplatin and gemcitabine in SJNB8 cells. Our results showed favorable interactions between cisplatin and gemcitabine in 4 out of 5 cell lines. Therefore, we feel that inclusion of gemcitabine into cisplatin-containing regiments might be a promising new strategy for the treatment of neuroblastoma.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Desoxicitidina/análogos & derivados , Neuroblastoma/tratamento farmacológico , Esferoides Celulares/efeitos dos fármacos , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Humanos , Células Tumorais Cultivadas , GencitabinaRESUMO
To enhance the efficacy of fenretinide (4HPR)-induced reactive oxygen species (ROS) in neuroblastoma, 4HPR was combined with buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, in neuroblastoma cell lines and spheroids, the latter being a three-dimensional tumor model. 4HPR exposure (2.5-10 µM, 24 h) resulted in ROS induction (114-633%) and increased GSH levels (68-120%). A GSH depletion of 80% of basal levels was observed in the presence of BSO (25-100 µM, 24 h). The 4HPR-BSO combination resulted in slightly increased ROS levels (1.1- to 1.3-fold) accompanied by an increase in cytotoxicity (110-150%) compared to 4HPR treatment alone. A correlation was observed between the ROS-inducing capacity of each cell line and the increase in cytotoxicity induced by 4HPR-BSO compared to 4HPR. No significant correlation between baseline antioxidant levels and sensitivity to 4HPR or BSO was observed. In spheroids, 4HPR-BSO induced a strong synergistic growth retardation and induction of apoptosis. Our data show that BSO increased the cytotoxic effects of 4HPR in neuroblastoma monolayers and spheroids in ROS-producing cell lines. This indicates that the 4HPR-BSO combination might be a promising new strategy in the treatment of neuroblastoma.
Assuntos
Butionina Sulfoximina/farmacologia , Fenretinida/farmacologia , Glutationa/metabolismo , Inibidores do Crescimento/farmacologia , Neuroblastoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose/efeitos dos fármacos , Linhagem Celular , Sinergismo Farmacológico , Glutationa/antagonistas & inibidores , Humanos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Espécies Reativas de Oxigênio/metabolismo , Esferoides CelularesRESUMO
Fenretinide induces apoptosis in neuroblastoma by induction of reactive oxygen species (ROS). In this study, we investigated the role of mitochondria in fenretinide-induced cytotoxicity and ROS production in six neuroblastoma cell lines. ROS induction by fenretinide was of mitochondrial origin, demonstrated by detection of superoxide with MitoSOX, the scavenging effect of the mitochondrial antioxidant MitoQ and reduced ROS production in cells without a functional mitochondrial respiratory chain (Rho zero cells). In digitonin-permeabilized cells, a fenretinide concentration-dependent decrease in ATP synthesis and substrate oxidation was observed, reflecting inhibition of the mitochondrial respiratory chain. However, inhibition of the mitochondrial respiratory chain was not required for ROS production. Co-incubation of fenretinide with inhibitors of different complexes of the respiratory chain suggested that fenretinide-induced ROS production occurred via complex II. The cytotoxicity of fenretinide was exerted through the generation of mitochondrial ROS and, at higher concentrations, also through inhibition of the mitochondrial respiratory chain.
Assuntos
Transporte de Elétrons/efeitos dos fármacos , Fenretinida/farmacologia , Mitocôndrias/efeitos dos fármacos , Neuroblastoma/patologia , Espécies Reativas de Oxigênio/metabolismo , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Mitocôndrias/metabolismo , Modelos Biológicos , Neuroblastoma/metabolismo , Compostos Organofosforados/farmacologia , Células Tumorais Cultivadas , Ubiquinona/análogos & derivados , Ubiquinona/farmacologiaRESUMO
Cyclopentenyl cytosine (CPEC), targetting the de novo biosynthesis of cytidine triphosphate (CTP), increases the cytotoxicity of gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) alone and in combination with irradiation in several human tumour cells in vitro. We investigated whether CPEC enhances the therapeutic ratio of gemcitabine and irradiation in human pancreatic BxPC-3 xenografts and in rat syngeneic L44 lung tumours. These models were selected because gemcitabine and radiation are used to treat both pancreatic and lung cancer patients and both models differ in growth capacity and in gemcitabine-induced radiosensitisation. A profound dose-dependent CTP-depletion was observed after a single injection of CPEC in both tumour tissue and in normal jejunum. In both models, CPEC alone induced a slight but significant tumour growth delay. The combination of CPEC with gemcitabine, at time intervals that showed CTP-depletion after CPEC, enhanced neither tumour growth delay nor toxicity as compared to gemcitabine alone. In addition, no beneficial effect of CPEC was observed in combination with gemcitabine and radiation. These results suggest that CPEC and gemcitabine alone as well as in combination with radiation target a similar cell population in both tumour models. In conclusion, future clinical development of CPEC as a modulator of gemcitabine combined with radiation is unlikely.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Citidina/análogos & derivados , Desoxicitidina/análogos & derivados , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/radioterapia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/radioterapia , Animais , Linhagem Celular Tumoral , Terapia Combinada , Citidina/farmacologia , Citidina Trifosfato/biossíntese , Desoxicitidina/farmacologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Ratos , Ratos Endogâmicos BN , GencitabinaRESUMO
The cytotoxic drug cyclopentenyl cytosine (CPEC) is currently being investigated in early clinical trials. Monitoring of plasma levels is required for pharmacokinetic analysis and management of toxicity. This paper describes the analysis of CPEC and cyclopentenyl uracil (CPEU) in plasma by HPLC-electrospray ionization tandem mass spectrometry. The calibration curves for CPEC and the metabolite CPEU were linear up to 10 microm with correlation coefficients of 0.997 (SD = 0.002, n = 10) and 0.997 (SD = 0.004, n = 10), respectively. The detection limit for CPEC was 0.03 and 0.12 microM for CPEU. The intra- and interassay coefficients of variation for CPEC and CPEU were less then 10%. The usefulness of the method was demonstrated by analyzing CPEC and CPEU in plasma of a patient treated with CPEC. HPLC with electrospray ionization tandem mass spectrometry allowed rapid and sensitive determination of CPEC and CPEU levels in plasma.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Citidina/análogos & derivados , Espectrometria de Massas em Tandem/métodos , Citidina/sangue , Citidina/química , Citidina/metabolismo , Humanos , Estrutura Molecular , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray/métodos , Uracila/análogos & derivados , Uracila/sangue , Uracila/química , Uracila/metabolismoRESUMO
The efficacy and mechanism of action of fenretinide (4-HPR), a vitamin A analogue, was investigated in a panel of six neuroblastoma cell lines and multicellular tumor spheroids. The latter are three dimensional cell aggregates and as such, a model for micrometastases. In all cell lines, the production of reactive oxygen species (ROS) increased with 163-680% after 1 h of treatment with 4-HPR. In addition, a decrease of the mitochondrial membrane potential of 30-75% was observed after 4 h of incubation with 4-HPR. A 6-12-fold difference was observed between the IC50 values for cell proliferation and viability between the most sensitive (IMR32) and most resistant (NASS) cell line towards 4-HPR. Flow cytometric analysis showed an increased amount of apoptotic bodies and no cell-cycle arrest. The antioxidant Trolox completely inhibited the accumulation of 4HPR-induced ROS and prevented the 4HPR-associated cytotoxicity. In all neuroblastoma spheroids, 4-HPR induced a complete cytostasis at clinical relevant concentrations (3-10 microM). Immunohistochemical analysis of 4-HPR-treated spheroids showed a decreased staining for proliferation marker Ki-67 and an increased staining for cleaved-PARP, a marker of apoptosis. Our results suggest that 4-HPR might be a promising agent for the treatment of micrometastases and high-risk neuroblastoma.
Assuntos
Antineoplásicos/farmacologia , Fenretinida/farmacologia , Neuroblastoma/patologia , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromanos/farmacologia , Relação Dose-Resposta a Droga , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Genes myc , Humanos , Imuno-Histoquímica , Concentração Inibidora 50 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Esferoides Celulares , Fatores de TempoRESUMO
Additive to synergistic induction of apoptosis has been reported as a result of sequential incubation of cancer cells with a histone deacetylase inhibitor (HDACi) and gemcitabine (dFdC), a deoxycytidine analogue with proven anti-tumour activity. This study shows that sequential treatment of two neuroblastoma cell lines with BL1521, an HDACi, and dFdC resulted in strong antagonism despite a minor increase of dFdCTP incorporation into the DNA of one cell line. Furthermore, no difference in the deoxycytidine kinase activity was observed in response to BL1521. In conclusion, cancer cells that respond to HDACi with a cell cycle arrest and differentiation may no longer be sensitive to dFdC.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Desoxicitidina/análogos & derivados , Inibidores Enzimáticos/administração & dosagem , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/administração & dosagem , Neuroblastoma/tratamento farmacológico , Antimetabólitos Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Desoxicitidina/administração & dosagem , Desoxicitidina/farmacologia , Combinação de Medicamentos , Inibidores Enzimáticos/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Neuroblastoma/enzimologia , Células Tumorais Cultivadas , GencitabinaRESUMO
The effect of the CTP synthetase inhibitor cyclopentenyl cytosine (CPEC) on the metabolism and cytotoxicity of 2',2'-difluorodeoxycytidine (dFdC, gemcitabine) and the expression and activity of deoxycytidine kinase (dCK) was studied in human neuroblastoma cell lines. The cytotoxicity of dFdC and CPEC was studied in a panel of MYCN-amplified and MYCN-single-copy neuroblastoma cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-assays. dFdC-metabolism was studied in SK-N-BE(2)c cells using [3H]-radiolabeled dFdC. dCK activity and expression were measured using enzyme assays, immunoblot and quantitative PCR, respectively. Both MYCN-amplified and MYCN-single-copy neuroblastoma cell lines were highly sensitive to dFdC, with concentration of the drug resulting in 50% effect when compared to untreated controls (ED50) values in the nanomolar range after a 3-h exposure to dFdC. There was no correlation of the observed ED50 with the dCK activity. Treatment with dFdC induced cell death in MYCN-amplified cells whereas MYCN-single-copy-cell lines underwent neuronal differentiation. Pre-incubation with CPEC significantly increased dFdC-cytotoxicity from 1.3 to 5.3-fold in 13 out of 15 cell lines. [3H]dFdC-anabolism increased 6-44 fold in SK-N-BE(2)c cells after incubation with CPEC and was paralleled by a significant increase in expression and activity of dCK. In conclusion, the combination of dFdC and CPEC is highly toxic to neuroblastoma in vitro.
Assuntos
Antineoplásicos/farmacologia , Citidina/análogos & derivados , Desoxicitidina Quinase/metabolismo , Desoxicitidina/análogos & derivados , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citidina/farmacologia , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Ativação Enzimática , Humanos , Immunoblotting , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/enzimologia , Neuroblastoma/genética , Neuroblastoma/patologia , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , GencitabinaRESUMO
Proteomic analysis of mouse kidney peroxisomes resulted in the identification of a novel nudix hydrolase designated RP2p, which is encoded by the D7RP2e gene. RP2p consists of 357 amino acids and contains two conserved domains: a nudix hydrolase domain and a CoA-binding domain. In addition, a PTS (peroxisomal targeting signal) type 1 (Ala-His-Leu) was found at the C-terminus. Analysis of the enzyme characteristics revealed that RP2p is a CoA diphosphatase with activity towards CoA, oxidized CoA and a wide range of CoA esters, including choloyl-CoA and branched-chain fatty-acyl-CoA esters. The enzymatic properties of RP2p indicate that at low substrate concentrations medium and long-chain fatty-acyl-CoA esters are the primary substrates. Enzyme activity was optimal at pH 9 or above, and required the presence of Mg2+ or Mn2+ ions. Subcellular fractionation studies revealed that all CoA diphosphatase activity in mouse kidney is restricted to peroxisomes.
Assuntos
Acil Coenzima A/metabolismo , Rim/citologia , Peroxissomos/enzimologia , Proteômica , Pirofosfatases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao GTP , Masculino , Proteínas de Membrana , Camundongos , Peroxissomos/metabolismo , Pirofosfatases/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Nudix HidrolasesRESUMO
The experimental anticancer drug cyclopentenyl cytosine (CPEC) was associated with cardiotoxicity in a phase I study. The aim of the present study was twofold; first we investigated whether the observed effects could be reproduced in in-vitro and in-vivo rat models. Second, we intended to investigate the underlying mechanism of the possible cardiotoxicity of CPEC. Effects on frequency and contractility were studied on the isolated atria of 18 male Wistar rats. Atria were incubated with 0.1 mmol L(-1) (n = 6) or 1 mmol L(-1) (n = 6) CPEC for 1.5 h and compared with control atria (incubation with buffer solution, n = 6). The cardiac apoptosis-inducing potential was studied in-vivo on 66 rats by 99mTc-Annexin V scintigraphy, followed by postmortem determination of radioactivity in tissues, histological confirmation with the TUNEL assay (late-phase apoptosis), and immunohistochemical staining for cleaved caspase 3 and cytochrome C (early-phase apoptosis). Serum levels of the necrotic cardiomyopathy marker troponin T were also determined. No effect on heart frequency was found in the isolated atria after CPEC treatment. A trend towards a decrease of contraction force was observed. However, the differences were not statistically significant. 99mTc-Annexin V scintigraphy showed no increase in cardiac uptake ratio upon CPEC treatment in the in-vivo rat model, which was confirmed by determination of radioactivity in heart versus blood ratios. At each section a few individual isolated late apoptotic cells (< 5) could be identified by the TUNEL assay in the highest CPEC dose group (90 mg kg(-1)) but not in controls or in rats treated with 60 mg kg(-1) CPEC. Staining for the early apoptosis markers cleaved caspase 3 and cytochrome C did not reveal any significant differences between treated and control rats. Cardiac troponin T levels were not increased after CPEC treatment. CPEC does not affect heart frequency or contraction force in our cardiotoxicity models. Moreover, we did not find an indication of CPEC-induced apoptosis in heart tissue.