At 2000 M concentration of 16-DS, all cells were necrotic, but also polyploidisation and formation of two nuclei in some of the cells were noted (Physique 3A and Physique 4). 2.2.2. were reduced slower than alone. The investigated compounds, administered with DOX, enhanced DOX-induced cell death and exhibited concentration-dependent biphasic influence on membrane fluidity. A-Tocopherol showed weaker effects than DSs, regardless the mode of its applicationalone or with DOX. High concentrations of -Tocopherol and DSs decreased DOX-induced LPO. Substantial cytotoxicity of the DSs suggests that they should be used more carefully in the investigations performed on sensitive cells. Enhancement of DOX toxicity by DSs showed their potential to act as chemosensitizers of cancer cells to anthracycline chemotherapy. < 0.05 vs. control; ** < 0.005 vs. control; *** < 0.001 vs. control; # < 0.05 vs. DOX; ## < 0.005 vs. DOX; ### < 0.001 vs. DOX. 2.1.2. The Effects of the Investigated Compounds Applied in Combination With 0.5 M DOX Pretreatment of cells with the investigated compounds before their incubation with DOX, differently affected DOX cytotoxicity. Only -Tocopherol and 16-DS at PP1 Analog II, 1NM-PP1 100 M concentration acted protectively and caused a moderate (about 13%) increase in the fraction of live cells. Neither 5-DS nor Met-12-DS at this concentration displayed any effect. Higher concentrations of nitroxides and -Tocopherol enhanced DOX cytotoxicity to a different degree. The PP1 Analog II, 1NM-PP1 greatest effects were observed for 200 and 500 M 5-DS (45 and 70% reduction in fraction of live cells, respectively, compared to 20% reduction Nid1 caused by 0.5 M DOX). Met-12-DS was also considerably toxic, and at 2000 M concentration, it eliminated more than 95% of viable cells. Significant enhancement of DOX toxicity was also evident in cells preincubated with 1000 and 2000 M 16-DS and 2000 M -Tocopherol, which caused a loss of about 45% of the live cell population (Physique 2). 2.2. Changes in Cell Morphology and Induction of Cell Death 2.2.1. The Effects of Doxyl Stearate Nitroxides and -Tocopherol Our study showed that 5-DS, Met-12-DS, and 16-DS, as well as a reference compound -Tocopherol, can trigger apoptosis, which intensity increased with an increase in compound concentrations. The level of spontaneous cell death observed in control cells was negligible and did not exceed 2% (Physique 3A and Physique 4). A concentration of 10 M of 5-DS, 16-DS, and -Tocopherol induced minor changes (the fraction of apoptotic and necrotic cells PP1 Analog II, 1NM-PP1 did not exceed 10%). A significant, progressive increase in the percentage of apoptotic cells was observed after treatment with high concentrations of nitroxides. The highest intensity of apoptosis was found in cells incubated with 5-DS. Low and intermediate concentrations (10 and 100 M) of -Tocopherol had a small influence on cell viability. Some features common for early apoptosis, such as cell shrinkage, however, were visible in cells incubated with 10 M concentration of -Tocopherol and to a greater extent in cells treated with its 100 M concentration (Physique 3B and Physique 4). A high concentration of -Tocopherol (1000 M) caused cells to lose their normal morphological features and enter the apoptosis pathway. Besides shrunken apoptotic cells, enlarged necrotic cells were also present, however, most of PP1 Analog II, 1NM-PP1 the cells were in late apoptosis (Physique 3B and Physique 4). Incubation with 2000 M -Tocopherol considerably intensified cell death processes (Physique 3B and Physique 4). Open in a separate window Open in a separate window Physique 3 Morphology and cell death of B14 cells at 24 h after cell treatment with the investigated compounds administered alone (0.5 M doxorubicin (DOX), -Tocopherol, and doxyl stearate nitroxides: 5-DS, Met-12-DS, and 16-DS) or in combination: 0.5 M DOX with -Tocopherol or with the nitroxides. Cell death was visualized using fluorescent double staining with propidium iodide (PI) and Hoechst 33258. Cell monolayers were analysed using an optical microscopy (morphology) or with the use of an inverted fluorescence microscopy (cell death) (Olympus IX70, Tokyo, Japan) under magnification 150. Cells were classified as viable (blue fluorescent), early apoptotic (bright blue fluorescent), late apoptotic (blue-violet fluorescent), and necrotic (red fluorescent cells). (A) Control cells, DOX alone, Met-12-DS or 16-DS-treated cells; (B) -Tocopherol, 5-DS. Open in a separate window Physique 4 Percentages of live, early apoptotic, late apoptotic, and necrotic cells at 24 h after B14 cell treatment with -Tocopherol; doxyl stearate nitroxides: 5-DS, Met-12-DS, and 16-DS, administered alone or in combination with 0.5 M doxorubicin (DOX). Changes in cell morphology associated with apoptosis/necrosis were determined based on fluorescent double staining with propidium iodide (PI) and Hoechst 33258 (Section 2.2 and Section 4.2.3, Determine.