Doxyl Nitroxide Spin Probes Can Modify Toxicity of Doxorubicin towards Fibroblast Cells

Figures

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  Figure 1. The structure of three derivatives of stearic acid applied in this study spin-labelled using an oxazolidine ring bearing a nitroxyl group: (A) 5-doxyl-stearic acid (5-DS), (B) 12-doxyl-stearic acid methyl ester (Met-12-DS), and (C) 16-doxyl-stearic acid (16-DS).

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  Figure 2. Cell viability of B14 fibroblasts determined by the trypan blue exclusion test. The cells were incubated for 3 h with α-Tocopherol and doxyl stearate nitroxides: 5-DS, Met-12-DS, and 16-DS or for 2 h with doxorubicin (DOX). In combined treatment cells before incubation with DOX were pretreated for 1 h with α-Tocopherol or with any of the doxyl stearate nitroxides. At the end of the incubation (0 h time point) with the investigated compounds, cells were stained with trypan blue (0.2% final concentration of the dye). All cells and blue stained dead cells were counted with an Olympus IX70 microscope (Tokyo, Japan), and the fraction of live cells was calculated as a percentage of all cells (100%). The data are shown as a mean ± SD of three independent experiments, each in at least three repeats.* p < 0.05 vs. control; ** p < 0.005 vs. control; *** p < 0.001 vs. control; # p < 0.05 vs. DOX; ## p < 0.005 vs. DOX; ### p < 0.001 vs. DOX.

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  Figure 3. Morphology and cell death of B14 cells at 24 h after cell treatment with the investigated compounds administered alone (0.5 μM doxorubicin (DOX), α-Tocopherol, and doxyl stearate nitroxides: 5-DS, Met-12-DS, and 16-DS) or in combination: 0.5 μM DOX with α-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) α-Tocopherol, 5-DS.

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  Figure 4. Percentages of live, early apoptotic, late apoptotic, and necrotic cells at 24 h after B14 cell treatment with α-Tocopherol; doxyl stearate nitroxides: 5-DS, Met-12-DS, and 16-DS, administered alone or in combination with 0.5 μM 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, Figure 3). The percentages of particular cell fractions were calculated in relation to all cells (100%).

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  Figure 5. The influence of doxyl stearate nitroxides: 5-DS, Met-12-DS, and 16-DS and α-Tocopherol administered alone (A,B) or in combination with 0.5 μM doxorubicin (DOX) (C,D) on fluidity of the plasma membrane of B14 cells measured fluorimetrically using 1-(4-Trimethylammoniumphenyl)-6-Phenyl-1,3,5-Hexatriene p-Toluenesulfonate (TMA-DPH) (A,C) and 12-AS (B,D) fluorescent probes (Section 4.1.1 and Section 4.2.4). The data are shown as a mean ± SD. ** p < 0.01 vs. control; *** p < 0.001 vs. control; **** p < 0.0001 vs. control; ### p < 0.001 vs. DOX; #### p < 0.0001 vs. DOX.

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  Figure 6. (A) The level of lipid peroxidation estimated on the basis of measurements of the amount of thiobarbituric acid-reactive substances (TBARS) in the plasma membrane of B14 cells treated with doxyl stearate nitroxides (5-DS, Met-12-DS, and 16-DS) and α-Tocopherol. The data are shown as a mean ± SD of three independent experiments, at least in three repeats each. ** p < 0.02 vs. control; **** p < 0.0001 vs. control. (B) The effect of α-Tocopherol and doxyl stearate nitroxides 5-DS, Met-12-DS, and 16-DS on lipid peroxidation induced in the plasma membrane of B14 cells by 10 μM doxorubicin (DOX). The data are shown as a mean ± SD of three independent experiments, at least in three repeats each. * p < 0.05 vs. control; ** p < 0.01 vs. control; *** p < 0.001 vs. control; **** p < 0.0001 vs. control; ### p < 0.001 vs. DOX; #### p < 0.0001 vs. DOX.

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  Figure 7. Kinetics of the reduction by B14 fibroblast cells of the doxyl stearate nitroxides (DSs): 5-DS, Met-12-DS, and 16-DS. The data are shown as a percentage of the height of the middle-field line (h0), calculated based on the spectra of the DSs obtained from EPR measurements (Section 4.2.6, Equation (2)). All the data are shown as a mean ± SD of at least three independent experiments. (A) Reduction by B14 cells of 5-DS, Met-12-DS, and 16-DS applied alone at 80 μM final concentration. (B) Reduction by B14 cells of 5-DS, Met-12-DS, and 16-DS applied at 80 μM final concentration together with 1 μM doxorubicin (DOX).