Mechanisms of Anthracycline-Enhanced Reactive Oxygen Metabolism in Tumor Cells

Abstract

In this investigation, we examined the effect of anthracycline antibiotics on oxygen radical metabolism in Ehrlich tumor cells. In tumor microsomes and nuclei, doxorubicin increased superoxide anion production in a dose-dependent fashion that appeared to follow saturation kinetics; the apparent K _m and V _max for superoxide formation by these organelles was 124.9 μM and 22.6 nmol/min/mg, and 103.4 μM and 4.8 nmol/min/mg, respectively. In both tumor microsomes and nuclei, superoxide formation required NADPH as a cofactor, was accompanied by the formation of hydrogen peroxide, and resulted from the transfer of electrons from NADPH to the doxorubicin quinone by NADPH:cytochrome P-450 reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4). Anthracycline antibiotics also significantly enhanced superoxide anion production by tumor mitochondria with an apparent K _m and V _max for doxorubicin of 123.2 μM and 14.7 nmol/min/mg. However, drug-stimulated superoxide production by mitochondria required NADH and was increased by rotenone, suggesting that the proximal portion of the electron transport chain in tumor cells was responsible for reduction of the doxorubicin quinone at this site. The net rate of drug-related oxygen radical production was also determined for intact Ehrlich tumor cells; in this system, treatment with doxorubicin produced a dose-related increase in cyanide-resistant respiration that was enhanced by changes in intracellular reducing equivalents. Finally, we found that in the presence of iron, treatment with doxorubicin significantly increased the production of formaldehyde from dimethyl sulfoxide, an indication that the hydroxyl radical could be produced by intact tumor cells following anthracycline exposure. These experiments suggest that the anthracycline antibiotics are capable of significantly enhancing oxygen radical metabolism in Ehrlich tumor cells at multiple intracellular sites by reactions that could contribute to the cytotoxicity of this class of drugs.

Figure 1

Effect of doxorubicin on oxygen consumption by Ehrlich tumor organelles. Oxygen consumption in tumor microsomes was examined as described in Supplementary . (a, i) is without drug; (ii) shows the results with doxorubicin. The addition of NADPH (1 mM), catalase (4500 units), or doxorubicin (135 μM) to the 3 ml vessel was performed through the access slot of the oxygen electrode and has been indicated (arrow). The numbers above each tracing indicate the rate of oxygen consumption (nmol/min/mg). (b) Effect of doxorubicin on oxygen consumption by Ehrlich tumor mitochondria. Oxygen consumption in these representative experiments was performed as described in Supplementary . (i) is the control reaction; (ii) represents the identical experiment in the presence of doxorubicin (135 μM). The addition of catalase (4500 units) to the 3 ml vessel was performed through the access slot of the oxygen electrode and has been indicated by the arrow. The numbers above each tracing indicate the rate of oxygen consumption (nmol/min/mg). (c) Effect of doxorubicin on oxygen consumption by Ehrlich tumor nuclei. Experimental conditions consisted of a 3 ml system containing 250 mM sucrose, 20 mM HEPES, pH 7.4, 100 μM EDTA, 1 mM NADPH, and 200 μg/ml of nuclear protein at 37°C. The addition of NADPH (1 mM), doxorubicin (135 μM), or catalase (4500 units) to the 3 ml reaction vessel was performed through the access slot of the oxygen electrode and has been indicated by an arrow. The number above the tracing is the rate of oxygen consumption (nmol/min/mg). (d) Doxorubicin-stimulated oxygen consumption by Ehrlich tumor cells. The effect of doxorubicin (400 μM) on oxygen consumption by Ehrlich cells (5 × 10⁶ cells/ml) is shown in representative examples from multiple experiments. The addition of acetylated cytochrome c (168 nmol) in (i) or catalase (9000 units) in (ii) to the 3 ml vessel has been indicated by an arrow. The numbers above each tracing indicate the rate of oxygen consumption (nmol/min/ml).

Figure 2

Effect of tumor cell number on the rate of cyanide-resistant oxygen consumption in the presence and absence of doxorubicin. The doxorubicin concentration used for these experiments was 400 μM. These studies were performed as described in Table 8; the data represent the mean ± S.E. of 3 determinations at each tumor cell concentration.

Figure 3

Effect of Ehrlich tumor cell number on doxorubicin-stimulated formaldehyde production. (a) Formaldehyde production from DMSO was assessed spectrophotometrically in the presence of a fixed concentration of doxorubicin (250 μM) over a 60 min period of incubation; the data represent the mean ± S.E. of three experiments for each concentration of tumor cells. (b) Effect of the duration of incubation on doxorubicin-induced formaldehyde formation by Ehrlich tumor cells. The extent of formaldehyde production by Ehrlich cells (10⁷/ml) in the presence of doxorubicin (250 μM) was determined as a function of the time after initiation of the reaction. Each time point represents the mean ± S.E. of three experiments. (c) Effect of doxorubicin concentration on formaldehyde production by Ehrlich tumor cells. In these studies, the production of formaldehyde from DMSO was examined at a tumor cell concentration of 10⁷/ml; the data represent the mean ± S.E. of three experiments at each doxorubicin level tested.