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1.
Biochim Biophys Acta ; 1847(3): 328-342, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25482261

RESUMO

Polyethylenimines (PEIs) are among the most efficient polycationic non-viral transfectants. PEI architecture and size not only modulate transfection efficiency, but also cytotoxicity. However, the underlying mechanisms of PEI-induced multifaceted cell damage and death are largely unknown. Here, we demonstrate that the central mechanisms of PEI architecture- and size-dependent perturbations of integrated cellular metabolomics involve destabilization of plasma membrane and mitochondrial membranes with consequences on mitochondrial oxidative phosphorylation (OXPHOS), glycolytic flux and redox homeostasis that ultimately modulate cell death. In comparison to linear PEI, the branched architectures induced greater plasma membrane destabilization and were more detrimental to glycolytic activity and OXPHOS capacity as well as being a more potent inhibitor of the cytochrome c oxidase. Accordingly, the branched architectures caused a greater lactate dehydrogenase (LDH) and ATP depletion, activated AMP kinase (AMPK) and disturbed redox homeostasis through diminished availability of nicotinamide adenine dinucleotide phosphate (NADPH), reduced antioxidant capacity of glutathione (GSH) and increased burden of reactive oxygen species (ROS). The differences in metabolic and redox imprints were further reflected in the transfection performance of the polycations, but co-treatment with the GSH precursor N-acetyl-cysteine (NAC) counteracted redox dysregulation and increased the number of viable transfected cells. Integrated biomembrane integrity and metabolomic analysis provides a rapid approach for mechanistic understanding of multifactorial polycation-mediated cytotoxicity, and could form the basis for combinatorial throughput platforms for improved design and selection of safer polymeric vectors.


Assuntos
Membrana Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Polietilenoimina/toxicidade , Transfecção/métodos , Trifosfato de Adenosina/metabolismo , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Linhagem Celular , Membrana Celular/metabolismo , Respiração Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Glutationa/metabolismo , Homeostase , Humanos , Cinética , Membranas Mitocondriais/metabolismo , Estrutura Molecular , Peso Molecular , Oxirredução , Consumo de Oxigênio/efeitos dos fármacos , Polietilenoimina/química , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade
2.
Nat Commun ; 5: 5348, 2014 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-25370744

RESUMO

ARF is a small, highly basic protein that can be induced by oncogenic stimuli and exerts growth-inhibitory and tumour-suppressive activities through the activation of p53. Here we show that, in human melanocytes, ARF is cytoplasmic, constitutively expressed, and required for maintaining low steady-state levels of superoxide under conditions of mitochondrial dysfunction. This mitochondrial activity of ARF is independent of its known autophagic and p53-dependent functions, and involves the evolutionarily conserved acidic motif GHDDGQ, which exhibits weak homology to BCL-2 homology 3 (BH3) domains and mediates interaction with BCL-xL--an important regulator of mitochondrial redox homeostasis. Melanoma-predisposing CDKN2A germline mutations, which affect conserved glycine and aspartate residues within the GHDDGQ motif, impair the ability of ARF to control superoxide production and suppress growth of melanoma cells in vivo. These results reveal an important cell-protective function of ARF that links mitochondrial dysfunction and susceptibility to melanoma.


Assuntos
Melanócitos/metabolismo , Melanoma/genética , Doenças Mitocondriais/metabolismo , Superóxidos/metabolismo , Proteína Supressora de Tumor p14ARF/metabolismo , Motivos de Aminoácidos , Respiração Celular , Células Cultivadas , Predisposição Genética para Doença , Humanos , Proteína bcl-X/metabolismo
3.
Oncotarget ; 4(4): 584-99, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23603840

RESUMO

Oncogene addiction describes how cancer cells exhibit dependence on single oncogenes to escape apoptosis and senescence. While oncogene addiction constitutes the basis for new cancer treatment strategies targeting individual kinases and pathways activated by oncogenic mutations, the biochemical basis for this addiction is largely unknown. Here we provide evidence for a metabolic rationale behind the addiction to (V600E)BRAF in two malignant melanoma cell lines. Both cell lines display a striking addiction to glycolysis due to underlying dysfunction of oxidative phosphorylation (OXPHOS). Notably, even minor reductions in glycolytic activity lead to increased OXPHOS activity (reversed Warburg effect), however the mitochondria are unable to sustain ATP production. We show that (V600E)BRAF upholds the activity of glycolysis and therefore the addiction to glycolysis de facto becomes an addiction to (V600E)BRAF. Finally, the senescence response associated with inhibition of (V600E)BRAF is rescued by overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), providing direct evidence that oncogene addiction rests on a metabolic foundation.


Assuntos
Glicólise/genética , Melanoma/genética , Melanoma/metabolismo , Fosforilação Oxidativa , Proteínas Proto-Oncogênicas B-raf/genética , Apoptose/genética , Western Blotting , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Oncogenes , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Transfecção
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