Abstract
Intracellular singlet oxygen generation in photofrin-loaded cells caused cell death without discrimination between nonmalignant and malignant cells. In contrast, extracellular singlet oxygen generation caused apoptosis induction selectively in tumor cells through singlet oxygen-mediated inactivation of tumor cell protective catalase and subsequent reactivation of intercellular ROS-mediated apoptosis signaling through the HOCl and the NO/peroxynitrite signaling pathway. Singlet oxygen generation by extracellular photofrin alone was, however, not sufficient for optimal direct inactivation of catalase, but needed to trigger the generation of cell-derived extracellular singlet oxygen through the interaction between H2O2 and peroxynitrite. Thereby, formation of peroxynitrous acid, generation of hydroxyl radicals and formation of perhydroxyl radicals (HO2.) through hydroxyl radical/H2O2 interaction seemed to be required as intermediate steps. This amplificatory mechanism led to the formation of singlet oxygen at a sufficiently high concentration for optimal inactivation of membrane-associated catalase. At low initial concentrations of singlet oxygen, an additional amplification step needed to be activated. It depended on singlet oxygen-dependent activation of the FAS receptor and caspase-8, followed by caspase-8-mediated enhancement of NOX activity. The biochemical mechanisms described here might be considered as promising principle for the development of novel approaches in tumor therapy that specifically direct membrane-associated catalase of tumor cells and thus utilize tumor cell-specific apoptosis-inducing ROS signaling.
Highlights
Reactive oxygen and nitrogen species (ROS) can cause destructive effects through their mutagenic potential and through their ability to react with proteins and lipids, but they can establish specific and fine-tuned signaling pathways [1,2]
Addition of photofrin without illumination had no apoptosis-inducing effect, confirming the role of singlet oxygen for the biological effect observed. These findings indicate that photofrindependent singlet oxygen generation within the cells caused apoptosis induction directly, whereas singlet oxygen generated outside the cells seemed to trigger an apoptosis-inducing intercellular signaling mechanism, as indicated by its dependence on the density of the cells
Treatment of human gastric carcinoma cells MKN-45 and human normal diploid fibroblasts Alpha-1 with increasing concentrations of photofrin, followed by illumination two hours after addition of photofrin, caused apoptosis induction dependent of the concentration of photofrin and with similar efficiency in malignant and nonmalignant cells (Fig. 2B). Apoptosis induction under these conditions was not inhibited by the NOX1 inhibitor AEBSF, indicating that NOX1-derived extracellular superoxide anions played no role for apoptosis induction under these conditions
Summary
Reactive oxygen and nitrogen species (ROS) can cause destructive effects through their mutagenic potential and through their ability to react with proteins and lipids, but they can establish specific and fine-tuned signaling pathways [1,2]. Tumor progression in vivo requires the acquisition of the “H2O2-catabolizing phenotype”, i.e. resistance against ROS-mediated apoptosis induction [49,50,51,52,53] This resistance is established through expression of membrane-associated catalase, which interferes with HOCl signaling through decomposition of H2O2 [54,55,56], and with NO/peroxynitrite signaling through oxidation of NO [57] and decomposition of peroxynitrite [56,58] (Fig. 1B).
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