Condense logarithms radical free#

The catalase inhibitior 3-aminotriazole (3-AT) ( 32) as stock solution (2 M in steril PBS) was stored at −20☌. 4-(2-Aminoethyl-benzenesulfonyl fluoride (AEBSF) is a specific inhibitor of NADPH oxidases ( 31) and was stored as a stock solution of 10 mM in phosphate-buffered saline (PBS) at −20☌. The following compounds were obtained from Sigma Aldrich (Schnelldorf, Germany). It, thus, significantly extends our experimentally-derived concept of tumor cell-specific resistance mechanisms directed towards ROS-mediated induction of apoptosis. This approach allowed the unraveling of the additional strong potential of HOCl to modulate the apoptotic response through fostering singlet oxygen generation and inactivation of antioxidant enzymes of tumor cells. The experimental design underlying this study conceptionally dissects the HOCl signaling pathway into the initial step ‘HOCl synthesis' and the subsequent step ‘HOCl/superoxide anion interaction’, focusing specifically on the latter. Non-transformed cells are also not sensitive to exogenously added HOCl in the micromolar concentration range, as they lack abundant extracellular superoxide anion generation requried for HOCl/superoxide anion interaction. In contrast, despite the presence of DUOX, non-transformed cells cannot achieve HOCl synthesis as they lack active NOX1 to support generation of hydrogen peroxide. However, tumor cells are sensitive to apoptosis induction by exogenously added HOCl or HOCl generated by attacking neutrophils. They thus escape the HOCl signaling pathway. Whereas transformed cells are readily induced to die by apoptosis through this mechanism, tumor cells prevent HOCl synthesis through decomposition of hydrogen peroxide by membrane-associated catalase ( 21, 22). HOCl then reacts with superoxide anions generated by the membrane-associated NADPH oxidase (NOX1), yielding apoptosis-inducing hydroxyl radicals. The peroxidase domain of DUOX can be supplied by neighboring non-transformed cells or the population of transformed cells themselves. In an initial step, superoxide anions dismutate to hydrogen peroxide, which is subsequently used by the peroxidase domain of dual oxidase (DUOX) to generate HOCl. Extracellular superoxide anions generated by malignant cells drive both the efficiency and selectivity of intercellular induction of apoptosis and autocrine apoptotic self-destruction (reviewed in 21, 23, 30). HOCl signaling depends on the generation of extracellular superoxide anions through membrane-associated NADPH oxidase (NOX1) that is characteristic and essential for cells transformed in vitro and for bona fide tumor cells ( 19, 22- 29). The HOCl signaling pathway is established during intercellular induction of apoptosis and autocrine apoptotic self-destruction, two recently described potential controling steps during oncogenesis ( 19- 23). Site-specific generation of hydroxyl radicals through HOCl/superoxide anion interaction also represents an essential and critical step for selective reactive oxygen species (ROS)-driven apoptosis induction specifically in malignant cells, through the HOCl signaling pathway. Due to the relatively short free-diffusion path length of superoxide anions and the extremely narrow free-diffusion path length of hydroxyl radicals, HOCl allows for site-specific generation of hydroxyl radicals defined by the locus of superoxide anion generation ( 16- 18). The reaction of HOCl with superoxide anions leads to the generation of hydroxyl radicals ( 13- 15) that efficiently destroy microbial structures or cause induction of apoptosis of malignant cells through lipid peroxidation. HOCl is used by neutrophils for their attack against microbes and tumor cells ( 1- 12).

The data presented here show a complex, but coherent picture of interactions between defined reactive oxygen species and protective enzymes on the surface of tumor cells. Higher concentrations of singlet oxygen inactivate membrane-associated catalase and thus lead to partial inhibition of apoptosis induction by exogenous HOCl, due to consumption of HOCl by H 2O 2.

Condense logarithms radical free#

The interaction between HOCl and H 2O 2 causes singlet oxygen generation that inactivates superoxide dismutase (SOD) on the surface of the tumor cells and thus enhances HOCl-mediated apoptosis through an increase in free superoxide anions. Tumor cells prevent HOCl synthesis through expression of membrane-associated catalase, but their extracellular superoxide anions readily react with exogenous HOCl. In contrast, transformed cells generate extracellular superoxide anions and HOCl, and die by apoptosis after HOCl/superoxide-dependent hydroxyl radical generation at their membrane. The lack of extracellular superoxide anion production by non-transformed cells prevents H 2O 2/peroxidase-mediated HOCl synthesis by these cells, as well as apoptosis induction by exogenous HOCl.