RESUMO
Microphthalmia-associated transcription factor (MITF) is a master regulator of melanocyte function, development and plays a significant role in melanoma pathogenesis. MITF genomic amplification promotes melanoma development, and it can facilitate resistance to multiple therapies. Here, we show that MITF regulates a global antioxidant program that increases survival of melanoma cell lines by protecting the cells from reactive oxygen species (ROS)-induced damage. In addition, this redox program is correlated with MITF expression in human melanoma cell lines and patient-derived melanoma samples. Using a zebrafish melanoma model, we show that MITF decreases ROS-mediated DNA damage in vivo. Some of the MITF target genes involved, such as IDH1 and NNT, are regulated through direct MITF binding to canonical enhancer box (E-BOX) sequences proximal to their promoters. Utilizing functional experiments, we demonstrate the role of MITF and its target genes in reducing cytosolic and mitochondrial ROS. Collectively, our data identify MITF as a significant driver of the cellular antioxidant state.
Assuntos
Regulação Neoplásica da Expressão Gênica , Isocitrato Desidrogenase , Melanoma , Fator de Transcrição Associado à Microftalmia , Espécies Reativas de Oxigênio , Peixe-Zebra , Fator de Transcrição Associado à Microftalmia/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Espécies Reativas de Oxigênio/metabolismo , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Animais , Humanos , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Linhagem Celular Tumoral , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Dano ao DNA , Transcrição GênicaRESUMO
Microphthalmia-associated transcription factor (MITF) plays pivotal roles in melanocyte development, function, and melanoma pathogenesis. MITF amplification occurs in melanoma and has been associated with resistance to targeted therapies. Here, we show that MITF regulates a global antioxidant program that increases survival of melanoma cell lines by protecting the cells from reactive oxygen species (ROS)-induced damage. In addition, this redox program is correlated with MITF expression in human melanoma cell lines and patient-derived melanoma samples. Using a zebrafish melanoma model, we show that MITF decreases ROS-mediated DNA damage in vivo . Some of the MITF target genes involved, such as IDH1 and NNT , are regulated through direct MITF binding to canonical enhancer box (E-BOX) sequences proximal to their promoters. Utilizing functional experiments, we demonstrate the role of MITF and its target genes in reducing cytosolic and mitochondrial ROS. Collectively, our data identify MITF as a significant driver of the cellular antioxidant state. One Sentence Summary: MITF promote melanoma survival via increasing ROS tolerance.
RESUMO
Many cancer-associated mutations that deregulate cellular metabolic responses to hypoxia also reprogram carbon metabolism to promote utilization of glutamine. In renal cell carcinoma (RCC), cells deficient in the von Hippel-Lindau (VHL) tumor suppressor gene use glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate (αKG). Glutamine can also generate aspartate, the carbon source for pyrimidine biosynthesis, and glutathione for redox balance. Here we have shown that VHL-/- RCC cells rely on RC-derived aspartate to maintain de novo pyrimidine biosynthesis. Glutaminase 1 (GLS1) inhibitors depleted pyrimidines and increased ROS in VHL-/- cells but not in VHL+/+ cells, which utilized glucose oxidation for glutamate and aspartate production. GLS1 inhibitor-induced nucleoside depletion and ROS enhancement led to DNA replication stress and activation of an intra-S phase checkpoint, and suppressed the growth of VHL-/- RCC cells. These effects were rescued by administration of glutamate, αKG, or nucleobases with N-acetylcysteine. Further, we observed that the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib synergizes with GLS1 inhibitors to suppress the growth of VHL-/- cells in vitro and in vivo. This work describes a mechanism that explains the sensitivity of RCC tumor growth to GLS1 inhibitors and supports the development of therapeutic strategies for targeting VHL-deficient RCC.
Assuntos
Glutaminase/antagonistas & inibidores , Neoplasias Renais/tratamento farmacológico , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Animais , Carcinoma de Células Renais , Glutamatos/genética , Glutamatos/metabolismo , Glutaminase/genética , Glutaminase/metabolismo , Glutamina/genética , Glutamina/metabolismo , Humanos , Neoplasias Renais/enzimologia , Neoplasias Renais/genética , Neoplasias Renais/patologia , Camundongos , Camundongos Nus , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase S do Ciclo Celular/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The objective of this study is to prepare a non-viral carrier of gene transfection from various polysaccharides and evaluate the feasibility in gene expression for mesenchymal stem cells (MSCs). Various amounts of spermine were chemically introduced into pullulan, dextran and mannan with a molecular weight of around 40 000 or pullulan with different molecular weights to prepare cationized polysaccharides with different extents of spermine introduced (spermine-polysaccharide). Each cationized polysaccharide was complexed with a plasmid DNA at various ratios and in vitro gene transfection was investigated for rat bone marrow-derived MSCs. The level of gene expression depended on the type of cationized polysaccharide. The highest level was observed for the complex of spermine-pullulan and plasmid DNA. Additionally, the level also depended on the molecular weight of pullulan and the extent of spermine introduced to pullulan. Suppression of gene expression with chlorpromazine and methyl-beta-cyclodextrin of endocytosis inhibitors demonstrated that the cellular uptake of spermine-pullulan-plasmid DNA complexes was mediated by clathrin- and raft/caveolae-dependent endocytic pathways. The cationized pullulan is a promising non-viral carrier of plasmid DNA for MSCs.
Assuntos
Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Transfecção/métodos , Animais , Sobrevivência Celular/efeitos dos fármacos , DNA/genética , DNA/metabolismo , Endocitose/efeitos dos fármacos , Glucanos/química , Glucanos/metabolismo , Glucanos/toxicidade , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Peso Molecular , Plasmídeos/genética , Polissacarídeos/toxicidade , Ratos , Ratos Wistar , Espermina/químicaRESUMO
The objective of this study was to prepare a novel gene carrier from pullulan, a polysaccharide with an inherent affinity for the liver, and evaluate the feasibility in gene transfection. Pullulan with different molecular weights was cationized by chemical introduction of spermine. The cationized pullulan derivative was complexed with a plasmid DNA and applied to HepG2 cells for in vitro gene transfection. The level of gene expression depended on the molecular weight of cationized pullulan derivatives and the highest level was observed for the cationized pullulan derivative with a molecular weight of 47.3 x 10(3). Pre-treatment of cells with asialofetuin decreased the level of gene expression by the complexes. These findings indicate that the cationized pullulan derivative is a promising non-viral carrier of plasmid DNA which is internalized in a receptor-mediated fashion.
Assuntos
Expressão Gênica , Vetores Genéticos/química , Glucanos/química , Plasmídeos/química , Transfecção/métodos , Assialoglicoproteínas/farmacologia , Cátions/química , Linhagem Celular Tumoral , DNA/química , DNA/genética , Fetuínas , Vetores Genéticos/efeitos dos fármacos , Vetores Genéticos/genética , Humanos , Fígado/metabolismo , Luciferases/análise , Luciferases/genética , Peso Molecular , Plasmídeos/efeitos dos fármacos , Plasmídeos/genética , Espermina/química , alfa-Fetoproteínas/farmacologiaRESUMO
The feasibility and mechanism of gene delivery by pullulan-spermine, a recently developed cationic polysaccharide, were investigated. Pullulan-spermine-mediated transfection of plasmid DNA resulted in greatly reduced cytotoxicity and a 10-fold increase in the level of gene expression when compared to Lipofectamine 2000, a commercially available cationic lipid. Additionally, after transfection of p53-expressing plasmid DNA by pullulan-spermine but not Lipofectamine 2000, the in vitro proliferation of T24 cells was significantly reduced. Pullulan-spermine-mediated gene expression was inhibited by both chlorpromazine of clathrin-mediated endocytosis inhibitor and methyl-beta-cyclodextrin and filipin of raft/caveolae inhibitors. We conclude that pullulan-spermine is a promising carrier for gene transfection, and that cellular uptake of pullulan-spermine-plasmid DNA complexes is mediated by clathrin- and raft/caveolae-dependent endocytotic pathways.