DT-diaphorase activity in normal and neoplastic human tissues; an indicator for sensitivity to bioreductive agents?

DT-diaphorase (DTD) is an important enzyme for the bioreductive activation of the new alkylating indoloquinone E09. In preclinical studies, E09 has shown selective anti-tumour activity against solid tumours and under hypoxic conditions. The levels of three reductive enzymes have been determined in three types of human solid tumours, together with corresponding normal tissues and normal liver. DTD enzyme activities were measured in tumour extracts using 2,6-dichlorophenolindophenol (DCPIP) and NADH as substrates; cytochrome P450 reductase or cytochrome b5 reductase activities were assessed with cytochrome c and NADPH or NADH respectively. DTD activity was highest in non-small-cell lung (NSCLC) tumours (mean 123 nmol DCPIPmin^-1mg^-1), followed by colon carcinoma (mean 75 nmol min^-1 mg^-1) and squamous cell carcinoma of the head and neck (6-fold lower than NSCLC). DTD activity was very low in normal liver and normal lung (4-6 nmol min^-1 mg^-1), while the levels in normal colon mucosa or normal mucosa of the head and neck region were in the same range as the corresponding tumours. The levels of the two other reductive enzymes, cytochrome P450 reductase (CP450R) and cytochrome b5 reductase (Cb5R), were 5 to 25-fold lower than those of DTD in all the tissues, except for normal liver, in which DTD was 2 to 4-fold lower. The degree of variation found for DTD (range 4-250 nmol min^-1 mg^-1), was not observed for these enzymes (CP450R, 0.8-7.8 nmol cytochrome c min^-1 mg^-1; Cb5R, 3.5-27.6 nmol min^-1 mg^-1). It is anticipated that NSCLC patients are more likely to respond to E09 and other bioreductive agents, owing to the high levels of the activating enzyme in this tumour type. To prove that this assay has predictive value we need to study these enzyme levels in tumour samples obtained from patients on clinical studies with bioreductive agents.