In this study, we investigated the properties of ascorbic acid (vitamin C), which is a naturally occurring water-soluble vitamin. Our goal is to evaluate its pro-oxidative and/or antioxidant capabilities. To do this, we initially used a confocal laser scanning microscope (CLSM) to visualize the differentiation pattern in U-937 cells under the treatment of variable concentrations of ascorbic acid. Prior to induction, U-937 cells showed a spherical morphology. After treatment, significant morphological changes were observed in the form of prominent pseudopodia and amoeboid structures. Interestingly, pseudopodia incidences increased with an increase in ascorbic acid concentrations. In addition, our analysis of protein modification using anti-malondialdehyde antibodies showed changes in more than one protein. The findings reveal the link between the differentiation of U-937 cells into macrophages and the protein modifications triggered by the production of reactive oxygen species when U-937 cells are exposed to ascorbic acid. Furthermore, the transformation of ascorbic acid from a pro-oxidative to an antioxidant property is also demonstrated.

• Keywords
• Antioxidants, Human cells, Pro-oxidant, Reactive oxygen species, Vitamin C,
• Publication type
• Journal Article MeSH

N-acetylcysteine (NAC), often used as an antioxidant-scavenging reactive oxygen species (ROS) in vitro, was recently shown to increase the cytotoxicity of other compounds through ROS-dependent and ROS-independent mechanisms. In this study, NAC itself was found to induce extensive ROS production in human leukemia HL-60 and U937 cells. The cytotoxicity depends on ROS-modulating enzyme expression. In HL-60 cells, NAC activated NOX2 to produce superoxide (O2•-). Its subsequent conversion into H2O2 by superoxide dismutase 1 and 3 (SOD1, SOD3) and production of ClO- from H2O2 by myeloperoxidase (MPO) was necessary for cell death induction. While the addition of extracellular SOD potentiated NAC-induced cell death, extracellular catalase (CAT) prevented cell death in HL-60 cells. The MPO inhibitor partially reduced the number of dying HL-60 cells. In U937 cells, the weak cytotoxicity of NAC is probably caused by lower expression of NOX2, SOD1, SOD3, and by the absence of MOP expression. However, even here, the addition of extracellular SOD induced cell death in U937 cells, and this effect could be reversed by extracellular CAT. NAC-induced cell death exhibited predominantly apoptotic features in both cell lines. Conclusions: NAC itself can induce extensive production of O2•- in HL-60 and U937 cell lines. The fate of the cells then depends on the expression of enzymes that control the formation and conversion of ROS: NOX, SOD, and MPO. The mode of cell death in response to NAC treatment bears apoptotic and apoptotic-like features in both cell lines.

• Keywords
• HL-60 cells, MPO, N-acetylcysteine, NOX, SOD, U937 cells, oxidative stress,
• MeSH
• Acetylcysteine pharmacology   MeSH
• HL-60 Cells MeSH
• Catalase genetics   MeSH
• Leukemia drug therapy   genetics   metabolism   MeSH
• Humans MeSH
• NADPH Oxidase 2 genetics   MeSH
• Oxidative Stress drug effects   MeSH
• Peroxidase genetics   MeSH
• Cell Proliferation drug effects   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Gene Expression Regulation, Neoplastic drug effects   MeSH
• Gene Expression Profiling MeSH
• Superoxide Dismutase genetics   MeSH
• U937 Cells MeSH
• Cell Survival drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Acetylcysteine MeSH
• CYBB protein, human MeSH Browser
• Catalase MeSH
• MPO protein, human MeSH Browser
• NADPH Oxidase 2 MeSH
• Peroxidase MeSH
• Reactive Oxygen Species MeSH
• Superoxide Dismutase MeSH

Free radical-mediated activation of inflammatory macrophages remains ambiguous with its limitation to study within biological systems. U-937 and HL-60 cell lines serve as a well-defined model system known to differentiate into either macrophages or dendritic cells in response to various chemical stimuli linked with reactive oxygen species (ROS) production. Our present work utilizes phorbol 12-myristate-13-acetate (PMA) as a stimulant, and factors such as concentration and incubation time were considered to achieve optimized differentiation conditions. ROS formation likely hydroxyl radical (HO●) was confirmed by electron paramagnetic resonance (EPR) spectroscopy combined with confocal laser scanning microscopy (CLSM). In particular, U-937 cells were utilized further to identify proteins undergoing oxidation by ROS using anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibodies. Additionally, the expression pattern of NADPH Oxidase 4 (NOX4) in relation to induction with PMA was monitored to correlate the pattern of ROS generated. Utilizing macrophages as a model system, findings from the present study provide a valuable source for expanding the knowledge of differentiation and protein expression dynamics.

• Keywords
• HL-60 cells, NADPH oxidase, NOX4, U-937 cells, macrophages, phorbol 12-myristate 13-acetate, protein-centered radicals,
• MeSH
• Acetophenones pharmacology   MeSH
• Staining and Labeling MeSH
• Cell Differentiation * drug effects   MeSH
• Electron Spin Resonance Spectroscopy MeSH
• HL-60 Cells MeSH
• Hydroxyl Radical MeSH
• Humans MeSH
• Monocytes cytology   drug effects   metabolism   MeSH
• NADP metabolism   MeSH
• Cell Proliferation drug effects   MeSH
• Proteins metabolism   MeSH
• Tetradecanoylphorbol Acetate pharmacology   MeSH
• Cell Shape drug effects   MeSH
• U937 Cells MeSH
• Cell Survival drug effects   MeSH
• Free Radicals metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Acetophenones MeSH
• acetovanillone MeSH Browser
• Hydroxyl Radical MeSH
• NADP MeSH
• Proteins MeSH
• Tetradecanoylphorbol Acetate MeSH
• Free Radicals MeSH