The Study of Effects of Monophenolic Antioxidants, Sodium Anphen and Potassium Phenosan, on Cell Apoptosis by Fluorescence and Confocal Microscopy

Currently, poly- and monophenol antioxidants should be considered not only as inhibitors that interact with free radicals, but also take into account that they are biologically active substances that affect specific targets in cells and can induce the activity of certain genes or stimulate various signaling pathways. The phenols can directly influence different points of the apoptotic process, and/or the expression of regulatory proteins. In our present study the effect of two antioxidants, sterically hindered monophenols sodium anphene (ANa) and potassium phenosan (PhK), on cell apoptosis of splenocytes was studied by fluorescence and confocal microscopy. PhK has already been introduced into medical practice in the Russian Federation because it proved effective as an anticonvulsant and was useful in treating neonatal hypoxia. The study of ANa continues; it may be a promising anticancer drug for some types of tumors. The fluorescent and confocal microscopy methods demonstrate that ANa in combination with H2O2 enhances apoptosis in suspension of Lewis carcinoma cells and to a lesser extent in splenocyte culture. We also discovered that autofluorescence of FAD and immunofluorescence of NADPH enzymatic complexes (with the AV-FITC fluorophore) in splenocytes of normal cells increases symbatically. The autofluorescence of FAD in splenocytes of Lewis carcinoma cells significantly exceeded that of splenocytes of healthy animals. The exact distinctive result was obtained when using potassium phenozan. It turned out that PhK prevents the development of apoptosis in mouse splenocyte cell culture (F1(CBA×C57B)). The combined use of ANa and PhK had no effect on splenocyte apoptosis. We show that fluorescence and confocal microscopy allow observing and quantifying the apoptotic effect of ANa and hydrogen peroxide, and make it possible to visualize metabolic changes in the cell, increased FAD fluorescence in tumor cells and NADPH -oxidase complexes in splenocytes. The data obtained indicate the possibility of using ANa in combination with hydrogen peroxide as an antitumor drug acting on certain types of cells. The different effects of sterically hindered monophenols ANa and PhK on the level of the anti-apoptotic protein Bcl-2 in the cell were established. ANa acts to lower Bcl-2 levels, signaling apoptosis, while PhK prevents the development of apoptosis and induces repair processes.