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DT-diaphorase is a two-electron-reducing enzyme that is an important activator of bioreductive anti-tumour agents, such as mitomycin C (MMC) and EO9, and is inducible by many compounds, including 1,2-dithiole-3-thiones (D3Ts). We showed previously that D3T selectively increased DT-diaphorase activity in mouse lymphoma cells compared with normal mouse marrow cells, and also increased MMC or EO9 cytotoxic activity in the lymphoma cells with only minor effects in the marrow cells. In this study, we found that D3T significantly increased DT-diaphorase activity in 28 of 38 human tumour cell lines representing ten tissue types with no obvious relationships between the tumour type, or the base level of DT-diaphorase activity, and the ability of D3T to increase the enzyme activity. Induction of DT-diaphorase activity in human tumour cell lines by 12 D3T analogues varied markedly with the D3T structure. D3T also increased DT-diaphorase activity in normal human bone marrow and kidney cells but the increases were small in these cells. In addition, D3T increased the level of enzyme activity in normal human lung cells. Pretreatment of human tumour cells with D3T analogues significantly increased the cytotoxic activity of MMC or EO9 in these cells, and the level of enhancement of anti-tumour activity paralleled the level of DT-diaphorase induction. In contrast, D3T did not effect the toxicity of EO9 in normal kidney cells. These results demonstrate that D3T analogues can increase DT-diaphorase activity in a wide variety of human tumour cells and that this effect can enhance the anti-tumour activity of the bioreductive agents MMC and EO9.

NAD(P)H:(quinone acceptor)oxidoreductase (DT-diaphorase) is a two-electron reducing enzyme that activates bioreductive antitumor agents and is induced by a wide variety of compounds including 1,2-dithiole-3-thione (D3T). We investigated factors influencing DT-diaphorase induction in fourteen human tumor cell lines. Four cell lines had basal DT-diaphorase activity that was increased by D3T treatment (group A), six cell lines had basal DT-diaphorase activity but the activity was not increased by D3T (group B), and four cell lines had low enzyme activity without, or with, D3T (group C). Two cell lines in group A and two cell lines in group B had a C to T polymorphism at base 609 in the NQO(1), DT-diaphorase gene, in one allele, while all four cell lines in group C were homozygous mutants. The base 609 mutant NQO(1) gene produces a protein with little enzyme activity. In group A, D3T increased NQO(1) mRNA and wild-type protein, and also increased mutant protein in the two heterozygous cell lines. In group B, the inducer slightly increased NQO(1) mRNA, did not increase the wild-type protein, but did increase the mutant protein in the two heterozygous cell lines. In group C, D3T increased NQO(1) mRNA as well as its mutant enzyme product. Transfection of the mutant NQO(1) gene into cells with two wild-type alleles did not alter DT-diaphorase activity. The results suggest that the lack of induction of DT-diaphorase activity is transcriptional in nature, that basal and induced expression of DT-diaphorase are regulated independently, and that mutant NQO(1) does not act as a dominant-negative to suppress DT-diaphorase activity.

DT-diaphorase is a two-electron reducing enzyme that activates the bioreductive anti-tumour agent, mitomycin C (MMC). Cell lines having elevated levels of DT-diaphorase are generally more sensitive to MMC. We have shown that DT-diaphorase can be induced in human tumour cells by a number of compounds, including 1,2-dithiole-3-thione. In this study, we investigated whether induction of DT-diaphorase could enhance the cytotoxic activity of MMC in six human tumour cell lines representing four tumour types. DT-diaphorase was induced by many dietary inducers, including propyl gallate, dimethyl maleate, dimethyl fumarate and sulforaphane. The cytotoxicity of MMC was significantly increased in four tumour lines with the increase ranging from 1.4- to threefold. In contrast, MMC activity was not increased in SK-MEL-28 human melanoma cells and AGS human gastric cancer cells, cell lines that have high base levels of DT-diaphorase activity. Toxicity to normal human marrow cells was increased by 50% when MMC was combined with 1,2-dithiole-3-thione, but this increase was small in comparison with the threefold increase in cytotoxicity to tumour cells. This study demonstrates that induction of DT-diaphorase can increase the cytotoxic activity of MMC in human tumour cell lines, and suggests that it may be possible to use non-toxic inducers of DT-diaphorase to enhance the efficacy of bioreductive anti-tumour agents. © 1999 Cancer Research Campaign

DT-diaphorase (EC 1.6.99.2) is a flavoprotein that catalyses two-electron reduction of quinones, quinone imines, and nitrogen oxides. It is a Phase II detoxifying enzyme that can detoxify chemically reactive metabolites, and may be important in an early cellular defense against tumorigenesis. DT-diaphorase is also an activating enzyme for bioreductive antitumor agents like mitomycin C (MMC) and EO9. DT-diaphorase is induced in many tissues by a wide variety of compounds including dithiolethiones and isothiocyanates. Dithiolethiones are chemoprotective agents against a variety of chemical carcinogens in animal models, and the dithiolethione analogue, oltipraz, is currently in Phase I and Phase II clinical chemoprevention trials. Similarly, the isothiocyanate derivative, sulforaphane, blocks the formation of carcinogen-induced mammary tumors in rats. The low toxicity of these inducers of DT-diaphorase makes them suitable for use as chemopreventive agents in high-risk individuals. Cells with elevated DT-diaphorase levels are generally more sensitive to bioreductive antitumor agents. Thus, we suggested that the antitumor efficacy of bioreductive agents can be enhanced by selective induction of DT-diaphorase in tumor cells compared with normal cells. We showed that 1,2-dithiole-3-thione (D3T) can increase the level of DT-diaphorase activity and the cytotoxic activity of bioreductive agents in mouse lymphoma cells without increasing these activities in normal mouse marrow cells. D3T also increased DT-diaphorase activity in 24 of 33 human tumor cell lines representing nine tissue types with no obvious relationships between the tumor type, or the base level of DT-diaphorase activity, and the ability to increase enzyme activity. A series of dithiolethione analogues and dietary components were also shown to be good inducers of DT-diaphorase in human tumor cells. D3T increased DT-diaphorase activity in normal human bone marrow and kidney cells but the increases were small in these cells. Combination treatment with D3T and EO9 increased cell kill in HL-60 human leukemia cells compared with EO9 alone, but had no effect on EO9 toxicity in normal human kidney cells. Similarly, D3T increased tumor cell kill by EO9 in H661 human lung cancer cells and by MMC in T47D human breast cancer cells. Thus, inducers of DT-diaphorase may play an important role in cancer chemoprevention programs and may also be useful in enhancing the antitumor efficacy of bioreductive agents.