Carbonyl reductase and NADPH cytochrome P450 reductase activities in human tumoral versus normal tissues

doi:10.1016/S0959-8049(98)00372-4

European Journal of Cancer

Volume 35, Issue 2, February 1999, Pages 320-324

https://doi.org/10.1016/S0959-8049(98)00372-4 Get rights and content

Abstract

The use of bioreductive agents in enzyme-directed bioreductive therapy has been proposed to take advantage not only of hypoxia in tumours, but also of the presence of reductases that metabolise such compounds. In this study, we studied the activities of NADPH cytochrome P450 reductase (P450R) and carbonyl reductase (CR) in 17 human lung tumours and 18 human breast tumours, together with the corresponding normal tissues. For lung cancer but not for breast cancer there was a significant difference in the CR activity between normal and tumour tissue. CR activity was increased with respect to the normal tissue between 2-fold and 40-fold indicating heterogeneity in tumour samples. No relationship was found between CR activity and the histological type, tumoral grade or TNM stage of the tumours. Although some variation in P450R activity in tumoral versus normal tissues was found in the majority of the samples studied, no significant differences could be demonstrated.

Introduction

Several attempts have been made to take advantage of the presence of hypoxic cells in solid tumours for therapeutic purposes. A strategy was proposed, which consisted of developing compounds that are activated by reductive mechanisms, and therefore, are favoured in an environment of low oxygen concentration; these are the so-called bioreductive agents[1]. Mitomycin C, considered to be the bioreductive agent prototype, and a series of analogues of mitomycin C, as well as the nitroimidazoles, were the first compounds of this type to be developed. At present, some of these compounds, such as the indoloquinone E09 and the N-oxide SR 4233 or tirapazamine, are in advanced phases of clinical evaluation2, 3, 4.

In the first preclinical and clinical trials, it was clear that the effectiveness of these compounds depended not only on the oxygen level but also on the presence of reductases which metabolise the compounds. Up to now, cytochrome P450 (several isoenzymes), NADPH cytochrome P450 reductase (P450R), NADH cytochrome b5 reductase, xanthine oxidase/dehydrogenase, aldehyde oxidase, carbonyl reductase (CR) and DT diaphorase have been identified, although other enzymes may also be implicated. Another variable to be considered is the enzyme activity in non-target tissues, where the metabolism of the bioreductive agent could produce undesirable effects. Therefore, some authors proposed the use of bioreductive agents, taking advantage of the differences that might exist in the levels of expression of the reductases involved in their bioactivation in different tissues and human tumours[5]. In order to develop this strategy termed ‘enzyme-directed bioreductive therapy’, it is necessary to determine the activities of the enzymes in normal as well as in tumoral tissue.

CR (EC 1.1.1.184) is a cytosolic monomeric, NADPH-dependent oxidoreductase with broad specificity for carbonyl compounds. It differs from aldehyde reductase (EC 1.1.1.2) and aldose reductase (EC 1.1.1.21) in its ability to reduce aromatic ketones and in its sensitivity to specific inhibitors[6]. Like DT diaphorase, CR catalyses the 2 electron (e⁻) reduction of quinones. It has been isolated from a number of human tissues: liver, brain, testis and others6, 7, 8, 9. The enzyme has been implicated in the metabolism of a variety of endogenous and xenobiotic carbonyl compounds: prostaglandins[10], anthracycline antibiotics, such as daunorubicin[11]and quinones derived from polycyclic aromatic hydrocarbons[12]. It seems to be the predominant reductase for the anticancer drug daunorubicin in human liver cytosol[13].

P450R (EC 1.6.2.3) is a microsomal enzyme found in a great number of human tissues, especially in lung and pancreas, which shows a coinciding distribution pattern with the principal sites of tumour formation. Studies using liver microsomes and tumour cells have shown that both cytochrome P450 and P450R contribute to the reductive activation of tirapazamine14, 15. Moreover, tirapazamine is reduced by purified rat liver cytochrome P450 reductase16, 17.

Some previous studies have shown abnormally high DT diaphorase levels (both activity or mRNA) in human tumours18, 19, 20, 21, 22. This enzyme has been implicated in the metabolism of E09 and mitomycin C amongst others. To advance the knowledge of human tumour enzyme profiling, we studied differences in expression or activity of CR and P450R in human tumoral samples versus normal adjacent tissues.

Section snippets

Tissue specimens

Biopsy specimens of lung solid tumours were obtained from 11 patients at the Hospital of Navarra and from 6 patients at the University Clinic, both in Pamplona, Spain. Tissue specimens of breast solid tumours were obtained from 18 patients at the University Clinic. Primary solid tumours were obtained together with macroscopically normal tissue from the same subject. Tissues were frozen in liquid nitrogen immediately after each operation. They were maintained at −80 °C in the pathology laboratory

Lung

Final CR activities found in paired lung tumour and normal tissues are shown in Fig. 1a. The mean activity in normal tissue was 15.2±2.8 nmol/min/mg protein, with the highest value being 47.9±0.9 nmol/min/mg protein (found in patient 1) and the lowest value being 2.1±0.4 nmol/min/mg protein (found in patient 9). There was more heterogeneity in the tumour samples. The mean was equal to 50.0±14.0 nmol/min/mg protein, the range being between 10.1±0.7 nmol/min/mg protein (patient 13) and 248.1±3.1

Discussion

There have been few studies on CR activity in human tumours. The results obtained in this study show that the activity of such an enzyme is clearly augmented in some lung and breast tumours. Nevertheless, the behaviour is totally heterogeneous: while in some patients the enzyme activity was comparable to that of normal tissue, in others there was an increase of two to 40 times the normal value. Other authors have found a mean value of CR activity in human tumours which was superior to that of

Acknowledgements

We are grateful to Celia Goñi for preparing the figures. This work was supported by Zeneca Pharmaceuticals within the National Plan of Scientific and Technological Investigation of Spain.

References (31)

V.A Miller et al.
Phase II study of the combination of the novel bioreductive agent, tirapazamine, with cisplatin in patients with advanced non-small-cell lung cancer
Ann Oncol
(1997)
N Pavlidis et al.
A randomized phase II study with two schedules of the novel indoloquinone E09 in non-small-cell lung cancer: a study of the EORTC Early Clinical Studies Group (ECSG)
Ann Oncol
(1996)
A.Y Bedikian et al.
Phase II trial of tirapazamine combined with cisplatin in chemotherapy of advanced malignant melanoma
Ann Oncol
(1997)
B Wermuth
Purification and properties of an NADPH-dependent carbonyl reductase from human brain. Relationship to prostaglandin 9-ketoreductase and xenobiotic ketone reductase
J Biol Chem
(1981)
T Nakayama et al.
Reductases from carbonyl compounds in human liver
Biochem Pharmacol
(1985)
N Inazu et al.
Carbonyl reductase from human testis: purification and comparison with carbonyl reductase from human brain and rat testis
Biochem Biophys Acta
(1992)
S.C Lee et al.
Prostaglandin metabolism I. Cytoplasmic reduced nicotinamide adenine dinucleotide phosphate-dependent and microsomal reduced nicotinamide adenine dinucleotide-dependent prostaglandin E9-ketoreductase activities in monkey and pigeon tissues
J Biol Chem
(1974)
N.K Ahmed et al.
Heterogeneity of anthracycline antibiotic carbonyl reductases in mammalian livers
Biochem Pharmacol
(1978)
B Wermuth et al.
Carbonyl reductase provides the enzymatic basis of quinone detoxification in man
Biochem Pharmacol
(1986)
H Ohara et al.
Reduction of drug ketones by dihydrodiol dehydrogenases, carbonyl reductase and aldehyde reductase of human liver
J Biochem
(1995)

M.I Walton et al.

The role of cytochrome P450 and cytochrome P450 reductase in the reductive bioactivation of the novel benzotriazine di-N-oxide hypoxic cytotoxin 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR4233, WIN59075) by mouse liver

Biochem Pharmacol

(1992)

T Cresteil et al.

High levels of expression of the NAD(P)H: quinone oxidoreductase (NQO1) gene in tumor cells compared to normal cells of the same origin

Biochem Pharmacol

(1991)

P.K Smith et al.

Measurement of protein using bicinchoninic acid

Anal Biochem

(1985)

L Ernster

DT-diaphorase

Methods Enzymol

(1967)

B Schott et al.

Comparative activity of anthracycline 13-dihydrometabolites against rat glioblastoma cells in culture

Biochem Pharmacol

(1989)

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Original paperCarbonyl reductase and NADPH cytochrome P450 reductase activities in human tumoral versus normal tissues

Abstract

Introduction

Section snippets

Tissue specimens

Lung

Discussion

Acknowledgements

Ann Oncol

Ann Oncol

Ann Oncol

J Biol Chem

Biochem Pharmacol

Biochem Biophys Acta

J Biol Chem

Biochem Pharmacol

Biochem Pharmacol

J Biochem

Biochem Pharmacol

Biochem Pharmacol

Anal Biochem

Methods Enzymol

Biochem Pharmacol

Original paper
Carbonyl reductase and NADPH cytochrome P450 reductase activities in human tumoral versus normal tissues