"Redox Imaging" to Distinguish Cells with Different Proliferative Indexes: Superoxide, Hydroperoxides, and Their Ratio as Potential Biomarkers

Abstract

The present study was directed to the development of EPR methodology for distinguishing cells with different proliferative activities, using "redox imaging." Three nitroxide radicals were used as redox sensors: (a) mito-TEMPO-cell-penetrating and localized mainly in the mitochondria; (b) methoxy-TEMPO-cell-penetrating and randomly distributed between the cytoplasm and the intracellular organelles; and (c) carboxy-PROXYL-nonpenetrating in living cells and evenly distributed in the extracellular environment. The experiments were conducted on eleven cell lines with different proliferative activities and oxidative capacities, confirmed by conventional analytical tests. The data suggest that cancer cells and noncancer cells are characterized by a completely different redox status. This can be analyzed by EPR spectroscopy using mito-TEMPO and methoxy-TEMPO, but not carboxy-PROXYL. The correlation analysis shows that the EPR signal intensity of mito-TEMPO in cell suspensions is closely related to the superoxide level. The described methodology allows the detection of overproduction of superoxide in living cells and their identification based on the intracellular redox status. The experimental data provide evidences about the role of superoxide and hydroperoxides in cell proliferation and malignancy.

Figure 1

Structural formulas of nitroxide probes used in our study and potential mechanism of their redox transformations in living cells and tissues. Mito-TEMPO and methoxy-TEMPO, which penetrate the cells, allow the analysis of the intracellular redox capacity, while carboxy-PROXYL, which does not penetrate the cells, allows the analysis of the extracellular redox capacity (according to Soule et al. [13]).

Figure 2

Representative EPR spectra of mito-TEMPO (0.1 mM) before and after incubation with cells (1 × 10⁶ cells/mL) at different time intervals. (a) Normal lymphocytes and (c) leukemic lymphocytes. Tables indicate the EPR signal intensity of mito-TEMPO, incubated with different numbers of cells for 2 hours, in a humidified atmosphere. (b) Normal lymphocytes and (d) leukemic lymphocytes. Control: mito-TEMPO in cultured (cell-free) medium. The data are the means ± SD from six independent experiments. The cell viability did not change at 2-hour incubation and was ~92-95% (for normal lymphocytes) or 96-99% (for leukemic lymphocytes).

Figure 3

Dynamics of the EPR signal of nitroxide radical (0.1 mM) in leukemic lymphocytes (Jurkat) and normal lymphocytes (1 × 10⁶ cells/mL) during 6-hour incubation: (a) mito-TEMPO, (b) methoxy-TEMPO, and (c) carboxy-PROXYL. The data are the means ± SD from three independent experiments. The cell viability did not change at 6-hour incubation and was ~92-95% (for normal lymphocytes) or 96-99% (for leukemic lymphocytes).

Figure 4

(a) Dynamics of the EPR signal of mito-TEMPO (0.1 mM) in cells with different proliferative indexes (1 × 10⁶ cells/mL) within 6-hour incubation. Control: mito-TEMPO in cultured medium. Mito-TEMPO did not affect cell viability at the selected experimental conditions. NL: normal lymphocytes. (b) Integrated EPR signal of mito-TEMPO in cell suspensions after 6-hour incubation. (c) Proliferative index of cells. (d) Basic intracellular levels of superoxide, analyzed by a DHE test. (e) Basic intracellular levels of hydroperoxides, analyzed by a DCF test. The data in (d) and (e) were normalized to 1 × 10⁶ cells/mL. In (a), (b), (c), and (d), the data are means ± SD from six independent experiments. In (e), the data are means ± SD from nine independent experiments.

Figure 5

Correlation between the proliferative index, cellular redox status (analyzed by EPR spectroscopy), intracellular superoxide (analyzed by a DHE test), intracellular hydroperoxides (analyzed by a DCF test). R: correlation coefficients.

Figure 6

(a) Biochemical strategy to enhance superoxide accumulation in cells by inhibition of mitochondrial electron transport chain and mitochondrial superoxide dismutase (SOD2) (according to Pelicano et al. [44]). (b) Level of superoxide and activities of antioxidant enzymes in cells, treated with 2-ME/Rot. Experimental conditions: cells (normal lymphocytes; 1 × 10⁶ cells/mL) were preincubated in the absence or presence of 600 nM 2-ME and 500 nM Rot within 6 hours in a humidified atmosphere (5% CO₂, 37°C). Control sample contained untreated cells. The superoxide level was measured by DHE assay, and the activities of antioxidant enzymes were measured as described in Materials and Methods. (c) EPR signal intensity of mito-TEMPO in untreated and 2-ME/Rot-treated cells. Experimental conditions: cells (1 × 10⁶ cells/mL) were preincubated in the absence or presence of 600 nM 2-ME and 500 nM Rot within 6 hours in a humidified atmosphere (5% CO₂, 37°C). Mito-TEMPO (0.1 mM) was added to the cell suspensions, and the incubation was continued for 1 hour at the same conditions. Aliquots of the cell suspensions were collected and subjected to EPR analysis. Control sample contained mito-TEMPO (0.1 mM) in cultured (cell-free) medium. In (b) and (c), the data are the means ± SD from three and four independent experiments, respectively. ^∗∗ p < 0.01 and ^∗∗∗ p < 0.001 versus control; ++p < 0.01 versus cells only. Dotted red lines show the control levels. ME: 2-methoxyestradiol; Rot: rotenone; SOD: superoxide dismutase; GSH-Px: glutathione peroxidase.

Figure 7

(a) Doubling time of normal (FHC) and cancer (HT29, Colon26) colon epithelial cells. ^∗The doubling time of FHC was obtained in DMEM-F12 medium, supplemented with growth factors. (b) Glucose consumption in cells (^∗ μmol/1 × 10⁶ cells/24 hours). (c) EPR signal intensity of mito-TEMPO (0.1 mM) in cell suspensions (3 × 10⁶ cells/mL) within 6-hour incubation at 37°C. Control: mito-TEMPO (0.1 mM) in cultured medium. Mito-TEMPO did not affect cell viability at the selected experimental conditions. (d) Basal intracellular levels of antioxidants and reducing equivalents, analyzed by OxiSelect™ Total Antioxidant Capacity Assay. (e) Basal intracellular levels of superoxide, analyzed by a DHE test. (f) Basal intracellular levels of hydroperoxides, analyzed by a DCF test. The data in (c), (d), (e), and (f) were normalized to 1 × 10⁶ cells/mL. All data are the means ± SD from three independent experiments. ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001 versus FHC; +p < 0.05 and ++p < 0.01 versus HT29; all other variations are insignificant.

Figure 8

(a) Basal intracellular levels of superoxide dismutase (SOD) activity. (b) Basal intracellular levels of catalase activity. (c) Basal intracellular levels of glutathione peroxidase activity. In (a), (b), and (c), the data were obtained in cell lysates with equal protein concentration. The data are the means ± SD from three independent experiments. ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001 versus FHC; +p < 0.05 versus HT29; all other variations are insignificant. HRP: horseradish peroxidase; X/XO: xanthine/xanthine oxidase.