Mechanisms of Anthracycline-Enhanced Reactive Oxygen Metabolism in Tumor Cells

Table 7

Requirements for anthracycline-stimulated superoxide formation by the nuclear fraction. Superoxide formation by tumor nuclei was examined using paired 1 ml reaction mixtures containing 250 mM sucrose, 20 mM HEPES, pH 7.4, 100 μM EDTA, 56 μM acetylated cytochrome c, 200 μg of nuclear protein, and either 0 or 10 μg of SOD. The reaction was carried out at 37°C and was initiated by the addition of 1 mM NADPH after the chemotherapeutic agent was added.

Reaction mixture	Superoxide formation (nmol/min/mg)
Control	(7)^a
Using NADH (1 mM) rather than NADPH	(3)
Doxorubicin (135 μM)	(12)^b
-NADPH	N.D. (3)^c,d
-Acetylated cytochrome c	N.D. (3)^d
Using heat-denatured nuclei	N.D. (3)^d
Using NADH (1 mM) rather than NADPH	(3)^d
+Heat-denatured SOD	(3)
+DMSO (13 mM)	(3)
+Catalase (1500 units/ml)	(3)
Using FAD (1 mM) rather than NADPH	N.D. (3)^d
Using FMN (1 mM) rather than NADPH	N.D. (3)^d
Daunorubicin (135 μM)	4 (3)^b
Rubidazone (135 μM)	(3)^b
Aclacinomycin A (135 μM)	(3)^b

^a.; numbers in parentheses are numbers of experiments; ^bsignificantly different from control (); ^cN.D. is not detectable; ^dsignificantly different from complete system containing doxorubicin alone ().