High resolution respirometry analysis of polyethylenimine-mediated mitochondrial energy crisis and cellular stress: Mitochondrial proton leak and inhibition of the electron transport system.
Biochim Biophys Acta
; 1827(10): 1213-25, 2013 Oct.
Article
em En
| MEDLINE
| ID: mdl-23850549
ABSTRACT
Polyethylenimines (PEIs) are highly efficient non-viral transfectants, but can induce cell death through poorly understood necrotic and apoptotic processes as well as autophagy. Through high resolution respirometry studies in H1299 cells we demonstrate that the 25kDa branched polyethylenimine (25k-PEI-B), in a concentration and time-dependent manner, facilitates mitochondrial proton leak and inhibits the electron transport system. These events were associated with gradual reduction of the mitochondrial membrane potential and mitochondrial ATP synthesis. The intracellular ATP levels further declined as a consequence of PEI-mediated plasma membrane damage and subsequent ATP leakage to the extracellular medium. Studies with freshly isolated mouse liver mitochondria corroborated with bioenergetic findings and demonstrated parallel polycation concentration- and time-dependent changes in state 2 and state 4o oxygen flux as well as lowered ADP phosphorylation (state 3) and mitochondrial ATP synthesis. Polycation-mediated reduction of electron transport system activity was further demonstrated in 'broken mitochondria' (freeze-thawed mitochondrial preparations). Moreover, by using both high-resolution respirometry and spectrophotometry analysis of cytochrome c oxidase activity we were able to identify complex IV (cytochrome c oxidase) as a likely specific site of PEI mediated inhibition within the electron transport system. Unraveling the mechanisms of PEI-mediated mitochondrial energy crisis is central for combinatorial design of safer polymeric non-viral gene delivery systems.
Palavras-chave
((R-L)/E); (L/E); (R/E); Anti-A; Asc; CCCP; CI; CII; CIII; CIV; Cell death; Cytochrome c oxidase; DIC; ETS; Electron transport system; FBS; L; LEAK control ratio; LEAK respiration; MTG; MitoTracker Green; Mitochondrial membrane potential; Mitochondrial uncoupling; Mna; N,N,N',N'-tetramethyl-p-phenylenediamine; NADH; NaN(3); OXPHOS; PEI; PI; Polyethylenimine; R; RCR; RFRs; ROUTINE control ratio; ROUTINE respiration; ROX; Rote; TMPD; TMRM; antimycin A; ascorbate; carbonyl cyanide m-chlorophenylhydrazone; complex I; complex II; complex III; complex IV; cyt c; cytochrome c; differential interference contrast; electron transport system; fetal bovine serum; malonic acid; netROUTINE control ratio; nicotinamide adenine dinucleotide; oxidative phosphorylation; polyethylenimine; propidium iodide; residual oxygen consumption; respiratory control ratio; respiratory flux ratios; rotenone; sodium azide; tetramethyl rhodamine methyl ester
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Polietilenoimina
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Prótons
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Mitocôndrias Hepáticas
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Carcinoma Pulmonar de Células não Pequenas
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Respiração Celular
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Complexo de Proteínas da Cadeia de Transporte de Elétrons
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Neoplasias Pulmonares
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article