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Cellular redox status and the level of reactive oxygen species (ROS) are important regulators of apoptotic potential, playing a crucial role in the growth of cancer cell and their resistance to apoptosis. However, the relationships between the redox status and ROS production during apoptosis remain poorly explored. In this study, we present an investigation on the correlations between the production of ROS, the redox ratio FAD/NAD(P)H, the proportions of the reduced nicotinamide cofactors NADH and NADPH, and caspase-3 activity in cancer cells at the level of individual cells. Two-photon excitation fluorescence lifetime imaging microscopy (FLIM) was applied to monitor simultaneously apoptosis using the genetically encoded sensor of caspase-3, mKate2-DEVD-iRFP, and the autofluorescence of redox cofactors in colorectal cancer cells upon stimulation of apoptosis with staurosporine, cisplatin or hydrogen peroxide. We found that, irrespective of the apoptotic stimulus used, ROS accumulation correlated well with both the elevated pool of mitochondrial, enzyme-bound NADH and caspase-3 activation. Meanwhile, a shift in the contribution of bound NADH could develop independently of the apoptosis, and this was observed in the case of cisplatin. An increase in the proportion of bound NADPH was detected only in staurosporine-treated cells, this likely being associated with a high level of ROS production and their resulting detoxification. The results of the study favor the discovery of new therapeutic strategies based on manipulation of the cellular redox balance, which could help improve the anti-tumor activity of drugs and overcome apoptotic resistance. [ABSTRACT FROM AUTHOR]

Previously, we have demonstrated that increased NADH-a SB 2 sb and FAD/NADH ratios are common responses of cancer cells to chemotherapeutic drugs, including cisplatin SP 34 sp ." "On the other hand, results by Brentnall et al. suggested that caspase-3 inhibits ROS production, while caspase-7 may be required for ROS accumulation during intrinsic apoptosis SP 27 sp ." now reads: "On the other hand, results by Brentnall et al. suggested that caspase-3 inhibits ROS production, while caspase-7 may be required for ROS accumulation during intrinsic apoptosis SP 28 sp ." "It has previously been verified that the intensity-based redox ratios FAD/(NADH + FAD) or FAD/NADH are highly correlated with biochemical assessments of the NAD SP + sp /(NADH + NAD SP + sp ) ratio using mass spectrometry SP 28 sp or indicated by assessing cellular oxygen consumption SP 29 sp ." now reads: "It has previously been verified that the intensity-based redox ratios FAD/(NADH + FAD) or FAD/NADH are highly correlated with biochemical assessments of the NAD SP + sp /(NADH + NAD SP + sp ) ratio using mass spectrometry SP 29 sp or indicated by assessing cellular oxygen consumption SP 30 sp .". [Extracted from the article]

Abstract: Background: Genetically encoded photosensitizers are a promising optogenetic instrument for light-induced production of reactive oxygen species in desired locations within cells in vitro or whole body in vivo. Only two such photosensitizers are currently known, GFP-like protein KillerRed and FMN-binding protein miniSOG. In this work we studied phototoxic effects of miniSOG in cancer cells. Methods: HeLa Kyoto cell lines stably expressing miniSOG in different localizations, namely, plasma membrane, mitochondria or chromatin (fused with histone H2B) were created. Phototoxicity of miniSOG was tested on the cells in vitro and tumor xenografts in vivo. Results: Blue light induced pronounced cell death in all three cell lines in a dose-dependent manner. Caspase 3 activation was characteristic of illuminated cells with mitochondria- and chromatin-localized miniSOG, but not with miniSOG in the plasma membrane. In addition, H2B-miniSOG-expressing cells demonstrated light-induced activation of DNA repair machinery, which indicates massive damage of genomic DNA. In contrast to these in vitro data, no detectable phototoxicity was observed on tumor xenografts with HeLa Kyoto cell lines expressing mitochondria- or chromatin-localized miniSOG. Conclusions: miniSOG is an excellent genetically encoded photosensitizer for mammalian cells in vitro, but it is inferior to KillerRed in the HeLa tumor. General significance: This is the first study to assess phototoxicity of miniSOG in cancer cells. The results suggest an effective ontogenetic tool and may be of interest for molecular and cell biology and biomedical applications. [Copyright &y& Elsevier]