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NADH shuttles, including malate-aspartate shuttle (MAS) and glycerol-3-phosphate shuttle, can shuttle the reducing equivalents of cytosolic NADH into mitochondria. It is widely accepted that the major function of NADH shuttles is to increase mitochondrial energy production. Our study tested the hypothesis that the novel major function of NADH shuttles in cancer cells is to maintain glycolysis by decreasing cytosolic NADH/NAD(+) ratios. We found that AOAA, a widely used MAS inhibitor, led to decreased intracellular ATP levels, altered cell cycle and increased apoptosis and necrosis of C6 glioma cells, without affecting the survival of primary astrocyte cultures. AOAA also decreased the glycolytic rate and the levels of extracellular lactate and pyruvate, without affecting the mitochondrial membrane potential of C6 cells. Moreover, the toxic effects of AOAA were completely prevented by pyruvate treatment. Collectively, our study has suggested that AOAA may be used to selectively decrease glioma cell survival, and the major function of MAS in cancer cells may be profoundly different from its major function in normal cells: The major function of MAS in cancer cells is to maintain glycolysis, instead of increasing mitochondrial energy metabolism. [ABSTRACT FROM AUTHOR]

Abstract: Confocal fluorescence imaging and fluorescence resonance energy transfer (FRET) technology have been widely used to study protein–protein interactions in living cells. However, it is very difficult to quantitatively analyze FRET efficiency due to the excitation spectral crosstalk and emission spectral crosstalk between donor and acceptor. In this study, we developed a novel method to quantitatively obtain the FRET efficiency by fitting the emission spectra (FES) of donor–acceptor pair, and this method is free from both excitation and emission spectral crosstalk. We used the FES method to quantitatively monitor the FRET efficiency of SCAT3, a caspase-3 indicator based on FRET, inside living cells stably expressing SCAT3 during STS-induced apoptosis. At 0, 6 and 12h after STS treatment, the FRET efficiency of SCAT3 obtained by FES are consistent with that by two-photon excitation (TPE) fluorescence lifetime imaging microscopy (FLIM) in living cells stably expressing SCAT3. In this study, the FES was also used to analyze the caspase-3 activation in living cells during anti-cancer drug such as taxol, Artesunate (ART) or Dihydroartemisinin (DHA) treatment. Our results showed that ART or DHA induced apoptosis by a caspase-3-dependent manner, while caspase-3 was not involved in taxol-induced cell death. [Copyright &y& Elsevier]