We studied the effect of doxorubicin on the production of hydrogen peroxide by PC3 human prostate cancer cells, using a sensitive assay based on aminotriazole-mediated inhibition of catalase. PC3 cells exposed to increasing concentrations of doxorubicin had an increase in intracellular hydrogen peroxide that was concentration-dependent up to 1 μM doxorubicin. The apparent hydrogen peroxide concentration in the PC3 cells was 13 ± 4 pM under basal steady-state conditions and increased to 51 ± 13 pM after exposure to 1 μM doxorubicin for 30 min. The level of hydrogen peroxide in the medium as measured by Amplex Red did not increase as a result of doxorubicin treatment. PC3 cells overexpressing catalase were no more resistant to doxorubicin cytotoxicity as compared to non-transduced wild-type cells; therefore, the exact role of hydrogen peroxide in anthracycline cytotoxicity remains unproven. This study demonstrates that a specific oxidative event associated with the exposure of PC3 human prostate cancer cells to anthracyclines results in an increase in intracellular hydrogen peroxide. KeywordsHydrogen peroxide; Doxorubicin; PC3 cells; Catalase; Transduction; Amplex Red; Oxidative stress; Aminotriazole; Fluorescent probes; Reactive oxygen species There are reports that suggest that anthracycline anti-cancer drugs may increase the intracellular production of hydrogen peroxide. The metabolic reductive activation of doxorubicin to a semiquinone (Eq. 1) stimulates production of superoxide by the oneelectron reduction of oxygen (Eq. 2) [1]. The dismutation of the resulting superoxide,

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We have previously reported that H2O2-induced apoptosis in HL-60 human leukemia cells takes place in the presence of chloride, requires myeloperoxidase (MPO), and occurs through oxidative reactions involving hypochlorous acid and chloramines. We now report that when chloride is replaced by the pseudohalide thiocyanate, there is little or no H2O2-induced apoptosis. Furthermore, thiocyanate inhibits H2O2-induced apoptosis when chloride is present at physiological concentrations, and this occurs at thiocyanate concentrations that are present in human serum and saliva. In contrast, bromide can substitute for chloride in H2O2-induced apoptosis, but results in a lower percent of the cells induced into apoptosis. Hypobromous acid is likely a short-lived intermediate in this H2O2/MPO/bromide apoptosis, and reagent hypobromous acid and bromamines induce apoptosis in HL-60 cells. We conclude that the physiologic concentrations of thiocyanate found in human plasma could modulate the cytototoxicity of H2O2 and its resulting highly toxic MPO-generated hypochlorous acid by competing with chloride for MPO. Furthermore, the oxidative products of the reaction of thiocyanate with MPO are relatively innocuous for human leukemic cells in culture. In contrast, bromide can support H2O2/MPO/halide apoptosis, but is less potent than chloride and it has no effect in the presence of physiological levels of chloride.

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