salicylates and 3-methoxyphthalic-acid

We have previously shown that the anticancer agent doxorubicin undergoes oxidation and inactivation when exposed to myeloperoxidase-containing human leukemia HL-60 cells, or to isolated myeloperoxidase, in the presence of hydrogen peroxide and nitrite. In the current study we report that commercial fetal bovine serum (FBS) alone oxidizes doxorubicin in the presence of hydrogen peroxide and that nitrite accelerates this oxidation. The efficacy of inactivation was dependent on the concentration of serum present; no reaction was observed when hydrogen peroxide or serum was omitted. Peroxidase activity assays, based on oxidation of 3,3',5,5'-tetramethylbenzidine, confirmed the presence of a peroxidase in the sera from several suppliers. The peroxidative activity was contained in the >10000 MW fraction. We also found that hemoglobin, a heme protein likely to be present in commercial FBS, is capable of oxidizing doxorubicin in the presence of hydrogen peroxide and that nitrite further stimulates the reaction. In contrast to intact doxorubicin, the serum + hydrogen peroxide + nitrite treated drug appeared to be nontoxic for PC3 human prostate cancer cells. Together, this study shows that (pseudo)peroxidases present in sera catalyze oxidation of doxorubicin by hydrogen peroxide and that this diminishes the tumoricidal activity of the anthracycline, at least in in vitro settings. Finally, this study also points out that addition of H2O2 to media containing FBS will stimulate peroxidase-type of reactions, which may affect cytotoxic properties of studied compounds.

Topics: Aniline Compounds; Anthracyclines; Antibiotics, Antineoplastic; Benzidines; Blood Proteins; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Doxorubicin; Doxylamine; Hemeproteins; Humans; Hydrogen Peroxide; Mass Spectrometry; Methemoglobin; Methimazole; Molecular Structure; Oxidation-Reduction; Peroxidases; Phthalic Acids; Salicylates; Sodium Nitrite

The effect of doxorubicin on oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) by lactoperoxidase and hydrogen peroxide has been investigated. It was found that: (1) oxidation of ABTS to its radical cation (ABTS*(+)) is inhibited by doxorubicin as evidenced by its induction of a lag period, duration of which depends on doxorubicin concentration; (2) the inhibition is due to doxorubicin hydroquinone reducing the ABTS*(+) radical (stoichiometry 1: 1.8); (3) concomitant with the ABTS*(+) reduction is oxidation of doxorubicin; only when the doxorubicin concentration decreases to a near zero level, net oxidation of ABTS could be detected; (4) oxidation of doxorubicin leads to its degradation to 3-methoxysalicylic acid and 3-methoxyphthalic acid; (5) the efficacy of doxorubicin to quench ABTS*(+) is similar to the efficacy of p-hydroquinone, glutathione and Trolox C. These observations support the assertion that under certain conditions doxorubicin can function as an antioxidant. They also suggest that interaction of doxorubicin with oxidants may lead to its oxidative degradation.

Topics: Antibiotics, Antineoplastic; Benzothiazoles; Chromans; Doxorubicin; Free Radicals; Glutathione; Hydrogen Peroxide; Hydroquinones; Lactoperoxidase; Oxidants; Oxidation-Reduction; Phthalic Acids; Salicylates; Sulfonic Acids; Thiazoles

The anticancer anthracyclines, doxorubicin and daunorubicin, are highly cytotoxic to both cancer and normal cells. In this work, we have investigated the capacity of cellular myeloperoxidase to inactivate these agents. We show that incubation of human leukemia HL-60 cells with the anthracyclines in the presence of hydrogen peroxide and nitrite causes irreversible oxidation of the drugs, suggesting an extensive modification of their chromophores. Methimazole, 4-aminobenzoic acid hydrazide, or azide inhibits the reaction, suggesting that it is mediated by the cellular myeloperoxidase, an enzyme naturally present in large amounts in HL-60 cells. In contrast to the intact drugs, the oxidatively transformed anthracyclines were substantially less cytotoxic for HL-60 (assayed by apoptosis) and PC3 prostate cancer cells and H9c2 rat cardiac myoblasts in vitro (assayed by clonogenic survival), indicating that the oxidative metabolism of these agents leads to their inactivation. Using tandem mass spectrometry, we identified two specific metabolic products of the anthracycline degradation, 3-methoxyphthalic acid and 3-methoxysalicylic acid. These two metabolic products were obtained as authentic compounds and were nontoxic to HL-60 leukemic cells and cardiac myocytes. These findings may have important implications for the cellular pharmacology of anthracyclines and for clinical oncology.

Topics: Animals; Antibiotics, Antineoplastic; Biotransformation; Cell Line, Tumor; Daunorubicin; Doxorubicin; Drug Screening Assays, Antitumor; HL-60 Cells; Humans; Male; Myocytes, Cardiac; Oxidation-Reduction; Peroxidase; Phthalic Acids; Prostatic Neoplasms; Rats; Salicylates