Inhibition of colon carcinogenesis by post-initiation induction of NQO1 in Sprague-Dawley rats.

Inducers of phase II detoxifying enzymes have been studied as chemopreventive agents for a variety of cancers. Phase II detoxifying enzymes may play a significant role in preventing carcinogen-induced colon cancer at the initiation and post-initiation stage, but the contribution of NAD(P) H:quinone oxidoreductase 1 (NQO1) to this effect remains unclear. Using the carcinogen-induced colon cancer Sprague-Dawley rat model, we previously showed that oltipraz selectively induces NQO1 in the colons of these rats without inducing other phase II detoxifying enzymes. We demonstrated that selective induction of NQO1 in the rat colon prior to treatment with a carcinogen significantly inhibited the formation of aberrant crypt foci (ACF). Using the same rat model, we found that rats fed oltipraz containing diet following treatment with the colon carcinogen, azoxymethane (AOM), had 60% fewer ACF after 12 weeks compared with rats fed a control diet. In addition, rats fed oltipraz containing diet after AOM treatment developed 40% fewer colon adenomas and fewer colon tumors than rats fed a control diet. There was also a 60% increase in the percentage of apoptotic cells in ACF from oltipraz fed rats compared with ACF from control fed rats. Together, these results suggest that NQO1 can contribute to inhibition of colon carcinogenesis at the post-initiation stage. A possible mechanism for this effect may be that induction of NQO1 increases apoptosis in carcinogen initiated colonic epithelial cells that prevents these cells from progressing to a neoplastic state. Thus, NQO1 may be an important target for chemoprevention of colon cancer.

Investigation of an NQO1 polymorphism as a possible risk and prognostic factor for chronic lymphocytic leukemia.

NAD(P)H:quinoneoxidoreductase 1 (NQO1) inhibits some cancers and increases p53 and apoptosis in cells. Due to an inactivating polymorphism, 10% of humans have no NQO1 activity. A case:control study suggested that chronic lymphocytic leukemia (CLL) patients may have an increased incidence of the NQO1 null genotype compared with controls. NQO1 genotype did not correlate with various CLL prognostic factors, but we observed a trend toward lower drug response in patients with the NQO1 null genotype. Inhibiting NQO1 activity decreased p53 levels and drug induced apoptosis in CLL cells. These results raise the possibility that the NQO1 polymorphism may be a risk factor for CLL and a predictor of response to chemotherapy.

A Model for NAD(P)H:Quinoneoxidoreductase 1 (NQO1) Targeted Individualized Cancer Chemotherapy.

NQO1 (NAD(P)H:quinoneoxidoreductase 1) is a reductive enzyme that is an important activator of bioreductive antitumor agents. NQO1 activity varies in individual tumors but is generally higher in tumor cells than in normal cells. NQO1 has been used as a target for tumor specific drug development. We investigated a series of bioreductive benzoquinone mustard analogs as a model for NQO1 targeted individualized cancer chemotherapy. We compared the tumor cell growth inhibitory activity of benzoquinone mustard analogs with sterically bulky groups of different size and placed at different positions on the benzoquinone ring, using tumor cell lines with different levels of NQO1. We demonstrated that functional groups of different steric size could be used to produce a series of bioreductive antitumor agents that were activated by different levels of NQO1 in tumor cells. This series of drugs could then be used to target cells with specific levels of NQO1 for growth inhibition and to avoid damage to normal cells, like bone marrow cells, that have low levels of NQO1. This approach could be used to develop new bioreductive antitumor agents for NQO1 targeted individualized cancer chemotherapy.

Delayed sodium thiosulphate administration reduces cisplatin efficacy on mouse EMT6 tumour cells in vitro.

OBJECTIVE: To determine whether simultaneous and/or delayed administration of sodium thiosulphate (STS) affects the oncologic effect of cisplatin or cisdiaminedichloroplatinum (CDDP) in EMT6 tumour cells in vitro. SETTING: Cell biology research laboratory. METHODS: Clonogenic assays of EMT6 tumour cells with CDDP alone, CDDP plus simultaneous STS, and CDDP plus a 4-hour delay of STS were performed. Growth fractions under these three conditions were compared. RESULTS: Tumour growth was statistically significantly increased when CDDP and STS were administered compared with CDDP alone. There was no statistically significant difference between simultaneous and 4-hour delay of STS administration. We conclude that in EMT6 cells, either simultaneous administration or a 4-hour delay of STS administration significantly decreases CDDP efficacy. CONCLUSION: STS adversely affects CDDP's oncologic efficacy in EMT6 cell cultures in vitro.

Role of NADPH cytochrome P450 reductase in activation of RH1.

PURPOSE: RH1 is a new bioreductive agent that is an excellent substrate for the two-electron reducing enzyme, NAD(P)H quinone oxidoreductase 1 (NQO1). RH1 may be an effective NQO1-directed antitumor agent for treatment of cancer cells having elevated NQO1 activity. As some studies have indicated that RH1 may also be a substrate for the one-electron reducing enzyme, NADPH cytochrome P450 reductase (P450 Red), P450 Red may contribute to the activation of RH1 where NQO1 activities are low and P450 Red activities are high. The mean P450 Red activity in the human tumor cell line panel used by NCI for evaluation of new anticancer agents is 14.8 nmol min(-1) mg prot(-1), while the mean NQO1 activity in these cell lines is 199.5 nmol min(-1) mg prot(-1). Thus, we investigated whether P450 Red could play a role in activating RH1. METHODS: Reduction of RH1 by purified human P450 Red was investigated using electron paramagnetic resonance and spectroscopic assays. The ability of RH1 to produce DNA damage following reduction by P450 Red was studied using gel assays. To determine the role of P450 Red in activation of RH1 in cells, cell growth inhibition studies with inhibitors of P450 Red and NQO1 were carried out in T47D human breast cancer cells and T47D cells transfected with the human P450 Red gene (T47D-P450) that have P450 Red activities of 11.5 and 311.8 nmol min(-1) mg prot(-1), respectively. RESULTS: Reduction studies using purified P450 Red and NQO1 confirmed that RH1 can be reduced by both enzymes, but redox cycling was slower following reduction by NQO1. RH1 produced DNA strand breaks and crosslinks in isolated DNA after reduction by either P450 Red or NQO1. DPIC, an inhibitor of P450 Red, had no effect on cell growth inhibition by RH1 in T47D cells, and had only a small effect on cell growth inhibition by RH1 in the presence of the NQO1 inhibitor, dicoumarol, in T47D-P450 cells. CONCLUSIONS: These results demonstrated that P450 Red does not contribute to the activation of RH1 in cells with normal P450 Red activity and plays only a minor role in activating this agent in cells with high levels of this enzyme. These studies confirmed that P450 Red could activate RH1 and provided the first direct evidence that RH1 could produce both DNA strand breaks and DNA crosslinks after reduction by P450 Red. However, the results strongly suggest that P450 Red does not play a significant role in activating RH1 in cells with normal P450 Red activity.

A NAD(P)H:quinone oxidoreductase 1 polymorphism is a risk factor for human colon cancer.

Colon cancer is one of the most common cancers in North America and generally develops from colonic epithelial cells following initiation by carcinogens. We have shown that the phase II detoxifying enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1) contributes to the inhibition of carcinogen-induced colon cancer in rats at both the initiation and postinitiation stages. An inactivating polymorphism at base 609 of the NQO1 gene, (609)C (NQO1 *1) --> (609)T (NQO1 *2), occurs at high frequency in the human population. Thus, we carried out a case-control study to determine if this polymorphism is associated with an increased risk of developing colon cancer. A total of 298 patients with colon cancer and 349 healthy controls matched for age, gender, and ethnic origin were enrolled in the study. There was an increased incidence of the NQO1 *2/*2 genotype in patients with colon cancer, with a gender and age-adjusted odds ratio of 2.68 (95% confidence intervals, 1.14-6.28). However, the incidence of the NQO1 *1/*2 genotype was not increased in patients with colon cancer compared with controls. When the patient and control groups were stratified by tobacco and alcohol use, the incidences of the NQO1 *2/*2 genotype were increased in patients with colon cancer for tobacco and alcohol users and nonusers, suggesting that there is no interaction between the NQO1 base 609 polymorphism and tobacco or alcohol use. These results strongly suggest that NQO1 plays a significant role in preventing the development of colon cancer, and individuals with an NQO1 *2/*2 genotype are at an increased risk of developing this disease.

The reductive activation of the antitumor drug RH1 to its semiquinone free radical by NADPH cytochrome P450 reductase and by HCT116 human colon cancer cells.

RH1 (2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), which is currently in clinical trials, is a diaziridinyl benzoquinone bioreductive anticancer drug that was designed to be activated by the obligate two-electron reductive enzyme NAD(P)H quinone oxidoreductase 1 (NQO1). In this electron paramagnetic resonance (EPR) study we showed that RH1 was reductively activated by the one-electron reductive enzyme NADPH cytochrome P450 reductase and by a suspension of HCT116 human colon cancer cells to yield a semiquinone free radical. As shown by EPR spin trapping experiments RH1 was reductively activated by cytochrome P450 reductase and underwent redox cycling to produce damaging hydroxyl radicals in reactions that were both H2O2- and iron-dependent. Thus, reductive activation by cytochrome P450 reductase or other reductases to produce a semiquinone that can redox cycle to produce damaging hydroxyl radicals and/or DNA-reactive alkylating species may contribute to the potent cell growth inhibitory effects of RH1. These results also suggest that selection of patients for treatment with RH1 based on their expression levels of NQO1 may be problematic.

Structure-activity studies with cytotoxic anthrapyrazoles.

Anthrapyrazoles have been investigated as cancer chemotherapeutic agents. The mechanism of action of these compounds is thought to involve inhibition of DNA topoisomerase II. A structure-activity study was carried out to determine the in vitro cytotoxic activity of nine novel anthrapyrazoles against human breast carcinoma, head and neck squamous cell carcinoma and leukemia cells, and against Chinese hamster ovary cells. The activity of these anthrapyrazole analogues was compared with that of two clinically tested anthrapyrazoles, losoxantrone and piroxantrone. Inhibition of topoisomerase II as a mechanism of action for the analogues was also investigated. The cytotoxic activity of the analogues was determined in vitro by MTT cell growth inhibition assay and inhibition of catalytic topoisomerase II activity by each compound was measured using a fluorometric DNA decatenation assay. All of the anthrapyrazole analogues inhibited the growth of the four cell lines with IC50 values that ranged from 0.1 to 45.2 microM. Losoxantrone was the most potent of the anthrapyrazole analogues studied. A tertiary amine in the basic side chain at N-2 increased the cytotoxic activity compared with a secondary amine in this side chain for many of the analogues, but not if there was a basic side chain at the C-5 position. A chlorine substituent on the basic side chain at N-2 did not have a consistent effect on activity. Moving the position of a chlorine substituent from C-5 to C-7 or introducing a basic side chain at C-5 did not have a consistent effect on cytotoxic activity. Anthrapyrazole analogues showed a broad range of activity for inhibiting topoisomerase II decatenation activity. Losoxantrone and piroxantrone were the most potent inhibitors of topoisomerase II activity. There was no significant correlation between the cytotoxic activity of the anthrapyrazole analogues and their ability to inhibit decatenation by topoisomerase II.

Sodium thiosulphate impairs the cytotoxic effects of cisplatin on FADU cells in culture.

OBJECTIVES: To study the effect of cisplatin (CDDP) on FADU human squamous cell carcinoma cells and to determine if sodium thiosulphate (STS), an agent protective against CDDP ototoxicity, affects the tumoricidal activity of CDDP. METHOD: FADU tumour cells were grown in culture. Cells were exposed to varying concentrations of CDDP with and without concomitant STS, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assays were then performed to determine the effect of treatment on FADU cell growth. RESULTS: Dose-response curves were generated for the FADU cells exposed to CDDP with and without concomitant STS. Concomitant STS was found to inhibit the tumoricidal activity of CDDP in vitro. CONCLUSIONS: Simultaneous administration of STS to in vitro culture of FADU tumor cells leads to inhibition of CDDP's tumoricidal activity.

Structure-activity study of the interaction of bioreductive benzoquinone alkylating agents with DNA topoisomerase II.

PURPOSE: Quantitative structure-activity studies were performed on a series of benzoquinone mustard (BM) bifunctional alkylating agents to determine whether DNA topoisomerase II (topo II) inhibition was responsible for cell growth inhibition. METHODS: Topo II inhibition was evaluated by decatenation and agarose gel electrophoresis assays. RESULTS: The BM compounds were shown to potently inhibit the decatenation activity of topo II. Though BM compounds promoted the formation of protein-DNA complexes in isolated nuclei and cells, this effect was undiminished when levels of topo II varied. The BM compounds had little activity in a topo II-mediated DNA cleavage assay, suggesting that they do not function as topo II poisons. Rather, BM-induced protein-DNA complex formation was likely due to the bifunctional alkylating reactivity of these compounds. Finally, the growth inhibitory properties of these compounds did not correlate with their ability to inhibit topo II, indicating that these compounds did not exert their cellular activity through inhibition of topo II. Some BM compounds reacted very quickly with glutathione and cysteine, likely initially through an electrophilic Michael addition. In the absence of cysteine, the growth inhibitory effects of BM were increased tenfold, indicating the modulatory effect of cysteine sulfhydryl adducts. EPR studies showed that a semiquinone-free radical was produced by some BM compounds. CONCLUSIONS: BM compounds likely exert their action through DNA cross-linking and/or by inducing oxidative stress. Although topo II is not a direct target of these agents, this enzyme may play a role in processing the consequences of direct DNA adduction and/or oxidative DNA damage.

Effect of sodium thiosulphate and cis-diamminedichloroplatinum on FADU tumour cells in nude mice.

OBJECTIVES: To study the effect of cis-diamminedichloroplatinum (CDDP) on FADU squamous cell carcinoma cells in a nude mouse model and to determine the effect of sodium thiosulphate (STS) on CDDP activity. METHODS: CD1 nude mice were inoculated with FADU tumour cells to both flanks. They were then randomized to four treatment groups: control, CDDP only, STS only, or CDDP and STS. Tumour growth was measured using calipers and charted at 3-day intervals. RESULTS: Tumour volumes were calculated as an ellipsoid and charted against time. CONCLUSIONS: CDDP inhibited FADU tumour cell growth compared with saline controls (p < .005). The addition of STS did not inhibit the CDDP activity when compared with CDDP-alone activity (p = .989). Compared with saline control solution, STS alone also inhibited tumour growth significantly (p < .005).

Role of NQO1 polymorphisms as risk factors for squamous cell carcinoma of the head and neck.

A case: control study was carried out to determine if inactivating polymorphisms of the NQO1 gene at bases 609 and 465 are associated with altered risk of developing squamous cell carcinoma of the head and neck (SCCHN). Genotyping was carried out by PCR RFLP analysis on whole blood samples. The frequency of the inactive 609T and active 609C forms, and the inactive 465T and active 465C forms, of NQO1 were compared in patient and control groups by a logistic regression analysis and odds ratios (ORs) were calculated. Participants were stratified by tobacco and alcohol use, and genotype distributions in these sub-groups were compared. There were no significant differences in genotype distribution between SCCHN patients and the control population for the base 609 or 465 polymorphisms. There were also no significant differences in genotype distributions between patient and control groups for tobacco and/or alcohol users and non-users. Genotype distributions were similar for SCCHN patients at all disease sites with the exception of the nasopharynx where there was a higher incidence of the 609C:609T and 609T:609T genotypes. These results suggest that individuals having either 609T or 465T alleles generally do not have an altered risk of developing SCCHN.

Effect of NQO1 induction on the antitumor activity of RH1 in human tumors in vitro and in vivo.

NQO1 is a reductive enzyme that is important for the activation of many bioreductive agents and is a target for an enzyme-directed approach to cancer therapy. It can be selectively induced in many tumor types by a number of compounds including dimethyl fumarate and sulforaphane. Mitomycin C is a bioreductive agent that is used clinically for treatment of solid tumors. RH1 (2,5-diaziridinyl-3-(hydroxymethyl)- 6-methyl-1,4-benzoquinone) is a new bioreductive agent currently in clinical trials. We have shown previously that induction of NQO1 can enhance the antitumor activity of mitomycin C in tumor cells in vitro and in vivo. As RH1 is activated selectively by NQO1 while mitomycin C is activated by many reductive enzymes, we investigated whether induction of NQO1 would produce a greater enhancement of the antitumor activity of RH1 compared with mitomycin C. HCT116 human colon cancer cells and T47D human breast cancer cells were incubated with or without dimethyl fumarate or sulforaphane followed by mitomycin C or RH1 treatment, and cytotoxic activity was measured by a clonogenic (HCT116) or MTT assay (T47D). Dimethyl fumarate and sulforaphane treatment increased NQO1 activity by 1.4- to 2.8-fold and resulted in a significant enhancement of the antitumor activity of mitomycin C, but not of RH1. This appeared to be due to the presence of a sufficient constitutive level of NQO1 activity in the tumor cells to fully activate the RH1. Mice were implanted with HL60 human promyelocytic leukemia cells, which have low levels of NQO1 activity. The mice were fed control or dimethyl fumarate-containing diet and were treated with RH1. NQO1 activity in the tumors increased but RH1 produced no antitumor activity in mice fed control or dimethyl fumarate diet. This is consistent with a narrow window of NQO1 activity between no RH1 activation and maximum RH1 activation. This study suggests that selective induction of NQO1 in tumor cells is not likely to be an effective strategy for enhancing the antitumor activity of RH1. In addition, we found that RH1 treatment produced significant leukopenia in mice that may be of concern in the clinic. These results suggest that the ease of reduction of RH1 by NQO1 makes it a poor candidate for an enzyme-directed approach to cancer therapy.

Structure-activity study with bioreductive benzoquinone alkylating agents: effects on DT-diaphorase-mediated DNA crosslink and strand break formation in relation to mechanisms of cytotoxicity.

PURPOSE: Structure-activity studies were carried out with the model bioreductive alkylating agent benzoquinone mustard (BM) and its structural analogs. The specific objectives were: (1) to investigate the effects of functional group substitutions to the benzoquinone ring on DNA crosslink and strand break formation subsequent to reduction of the analogs by DT-diaphorase (DTD) in vitro, (2) to correlate DNA crosslink and strand break formation by the analogs with anaerobic reduction of the BM analogs by DTD and their redox cycling in vitro, and (3) to correlate DNA crosslink and strand break formation by the BM analogs with their cytotoxic effects in cancer cells. METHODS: DNA interstrand crosslink and single-strand break formation were assessed using agarose gel assays. To determine DNA interstrand crosslinks or single-strand breaks, linearized or supercoiled plasmid DNA, respectively, were incubated with purified human DTD and increasing concentrations of each BM analog. Subsequently, DNA was electrophoresed on an agarose gel and DNA crosslink and strand break formation were quantified using densitometry. The rates of reduction of the BM analogs by purified human DTD were measured in vitro under hypoxic conditions, and the redox cycling potential was determined under aerobic conditions using HPLC analysis. The cytotoxic activities of these agents in human tumor cell lines were measured by the MTT assay, with and without the DTD inhibitor, dicoumarol. RESULTS: BM analogs with electron-donating groups (MeBM, MBM, m-MeBM), electron-withdrawing groups (CBM, FBM), sterically bulky groups (PBM, m-PBM, m-TBM) and positional isomers (MeBM, m-MeBM, PBM, m-PBM) were synthesized. After reduction by DTD, the BM analogs produced a concentration-dependent increase in DNA crosslink and DNA strand break formation. The E(10) (extent of DNA crosslink formation produced by 10 micro M BM analog) for DNA crosslink formation displayed the rank order MeBM approximately MBM>m-MeBM approximately PBM approximately BM>CBM>FBM>m-PBM approximately m-TBM. For DNA strand break formation, the E(10) values (extent of DNA strand break formation produced by 10 micro M BM analog) displayed the rank order MeBM>MBM>m-MeBM>PBM>BM approximately CBM>FBM>m-PBM approximately m-TBM. Importantly, the cytotoxic activity of the BM analogs in SK-Mel-28 human melanoma cells correlated positively with the E(10) values for DTD-mediated DNA crosslink formation ( r(s)=0.87, P<0.05) and DNA strand break formation ( r(s)=0.95, P<0.05). Similar correlations were observed in NCI-H661 human lung carcinoma cells. Furthermore, the D(10) values (concentration of BM analog that decreased the surviving cell fraction to 0.1) for cytotoxic activity of the BM analogs correlated with the maximum levels of DNA crosslinks formed with each BM analog, with r(s) values of -0.85 ( P<0.05) for the NCI-H661 cell line, and -0.81 ( P<0.05) for the SK-MEL-28 cell line. The half-time of reduction (t(1/2)) of the BM analogs by DTD did not correlate with DNA crosslink formation, DNA strand break formation, or cytotoxic potency of the analogs. CONCLUSIONS: Functional groups on the benzoquinone ring affect the ability of BM to produce DNA crosslinks and strand breaks following reduction by DTD. Electron-donating groups increased DNA damage, whereas electron-withdrawing groups and sterically bulky groups at the C6 position had no effect or decreased the ability of the compounds to produce DNA damage compared to BM. Moreover, both DNA crosslink and strand break formation appear to have an important impact on the cytotoxicity of the BM analogs. These results may have significance for optimal use of BM-based antitumor agents and for rationalization of the development of novel therapeutic compounds that require bioactivation by DTD.

Role of the TRAIL/APO2-L death receptors in chlorambucil- and fludarabine-induced apoptosis in chronic lymphocytic leukemia.

The standard treatments for chronic lymphocytic leukemia (CLL) include the alkylating agent chlorambucil (CLB) and the nucleoside analog fludarabine (F-ara-AMP, Flu). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death receptor ligand that induces apoptosis preferentially in tumors. However, CLL cells seem to be resistant to TRAIL-induced apoptosis. The TRAIL apoptotic signaling pathway has also been implicated in genotoxin-induced apoptosis through upregulation of TRAIL death receptors DR4 and DR5. In the present study, we demonstrate that the treatment of primary CLL cells with CLB or Flu increases the mRNA, protein and cell surface expression levels of DR4 and DR5 in a dose-dependent manner. In contrast to CLL cells, drug treatment fails to increase significantly the expression of DR4 or DR5 in normal lymphocytes. CLL cells are, however, resistant to TRAIL-induced apoptosis compared to B-cell lines. In contrast, combinational treatment using CLB or Flu with TRAIL (100 ng/ml) gave a synergistic apoptotic response. Furthermore, TRAIL is readily detectable on the cell surface of CLL cells, but TRAIL expression fails to increase following drug treatment. Preventing TRAIL from interacting with DR4 and DR5 decreases CLB-induced apoptosis in CLL cells. A similar, but less marked effect is observed with Flu. These findings indicate the involvement of the TRAIL apoptotic pathway in the mechanism of action of chemotherapy, and this mechanism could be utilized to sensitize CLL cells to TRAIL-induced apoptosis.

Induction of NAD(P)H quinone: oxidoreductase1 inhibits carcinogen-induced aberrant crypt foci in colons of Sprague-Dawley rats.

Phase II detoxifying enzymes like NAD(P)H (quinone acceptor)oxidoreductase1 (NQO1), glutathione S-transferases (GST), and UDP-glucuronyltransferases (UGT) may play an important role in preventing carcinogen-induced cancers. Inducers of these enzymes have been shown to inhibit carcinogen-induced colon tumors in rat and mouse models. However, it has not been clearly demonstrated that NQO1 contributes to this effect. We examined the effect of NQO1 inducers on colon carcinogenesis using an aberrant crypt foci (ACF) rat model. Sprague-Dawley rats were fed control diet or diet containing 400 ppm dimethyl fumarate or 200 ppm oltipraz for 7 days, and Phase II enzymes in rat colon and liver were measured. Dimethyl fumarate significantly increased NQO1 and GST activities in colon and liver but did not increase UGT activities in these tissues. In contrast, oltipraz significantly increased NQO1 activities in colon and liver and produced a small increase in GST activity in the liver but did not increase GST activity in the colon or UGT activities in the liver or colon. Sprague Dawley rats were fed control diet or diet containing 200 ppm oltipraz and then treated with the carcinogens azoxymethane or methyl nitrosourea. Both carcinogens produced ACF in all of the rat colons, but rats fed oltipraz diet had significantly fewer ACF than those fed control diet. This protective effect was reversed in rats treated with the NQO1 inhibitor, dicoumarol. However, treatment with oltipraz did not alter the distribution of crypt multiplicities in the ACF. These studies demonstrated that induction of NQO1 plays a significant role in inhibiting initiation of carcinogen-induced ACF in Sprague-Dawley rats. This provides the first direct evidence that NQO1 may play a role in preventing colon cancer. The study also found that oltipraz added to the diet of Sprague-Dawley rats selectively increased NQO1 activity in colon mucosa with no increase in GST and UGT activities in these tissues. Thus, this model will be useful for further investigating the role of NQO1 in prevention of colon cancer.

Lack of NQO1 induction in human tumor cells is not due to changes in the promoter region of the gene.

DT-diaphorase is a two-electron reducing enzyme that is an important detoxifying enzyme and activator of bioreductive antitumor agents. Expression of the DT-diaphorase gene, NQO1, appears to be transcriptionally regulated, and the gene is induced by a wide variety of compounds. We showed that 1,2-dithiole-3-thione can selectively increase DT-diaphorase activity in human and murine tumors, and that this enhanced the antitumor activity of bioreductive antitumor agents. However, we found that DT-diaphorase activity was not increased in some human tumor cell lines after treatment with the inducer, and this appeared to be due to a lack of increased transcription. To determine if this lack of increased transcription was due to a mutation in the promoter region of the NQO1 gene in these cells, we sequenced approximately 2000 bases of the NQO1 promoter region from non-induced cells and compared these with sequences from human HT29 colon cancer cells, which showed significant increases in NQO1 transcription, and the sequence reported for human liver cells in Genbank. Sequence analysis showed no changes in the sequences of the major transcriptional elements, XRE, CAT box, ARE, AP1 site or AP2 site, in the tumor cells compared with the Genbank sequences. The only major change was a deletion of a 20 base repeat region approximately 400 bases 5' to the XRE element in all the cells, including the HT29 cells, compared with the sequence reported in Genbank. There were also several insertions of a single base in various parts of the sequences which occurred in most, or all, of the cell lines compared with the reported sequence, and a small number of single base changes, insertions or deletions that occurred in a single cell line. However, these changes did not appear to correlate with differences in induced transcription of the NQO1 gene. These results suggest that the differences in transcription of the NQO1 gene after treatment with DT-diaphorase inducers was not due to alterations in the promoter region of the gene.

The effect of functional groups on reduction and activation of quinone bioreductive agents by DT-diaphorase.

PURPOSE: Bioreductive antitumor agents are an important class of anticancer drugs that include the clinically used drug, mitomycin C, and new agents such as EO9 and tirapazamine that have recently been tested in clinical trials. These agents require activation by reductive enzymes such as DT-diaphorase or NADPH:cytochrome P450 reductase. A major focus for improving cancer chemotherapy has been to increase the selectivity and targeting of antitumor drugs to tumor cells. Bioreductive antitumor agents are ideally suited to improving tumor selectivity by an enzyme-directed approach to tumor targeting. However, none of the bioreductive agents developed to date has been specific for activation by a single reductive enzyme. This is in part due to a lack of knowledge about structural factors that confer selectivity for activation by reductive enzymes. The purpose of this study was to investigate the ability of specific functional groups to modify reduction and activation of quinone bioreductive agents by DT-diaphorase. METHODS: We used a series of model benzoquinone mustard (BM) bioreductive agents and compared the parent compound BM to MBM, which has a strong electron-donating methoxy group, MeBM, which has a weaker electron-donating methyl group, CBM, which has an electron-withdrawing chloro group, and PBM and its structural isomer, meta-PBM (m-PBM), which both have sterically bulky benzene rings attached to the quinone moiety. We determined the rate of reduction of these agents by purified human DT-diaphorase under hypoxic and aerobic conditions. We also measured the cytotoxic activity of these agents in human tumor cell lines with and without the DT-diaphorase inhibitor, dicoumarol. RESULTS: Under hypoxic conditions in vitro, the t(1/2) values for reduction of the analogs by purified DT-diaphorase were 4, 6, 8, 9, 10 and 21 min for BM, MeBM, CBM, MBM, PBM and m-PBM, respectively. Under aerobic conditions the rank order of redox cycling after two-electron reduction by DT-diaphorase was MBM > MeBM > BM approximately CBM approximately PBM approximately m-PBM. The rate of reduction by DT-diaphorase of HBM, a non-alkylating analog of BM, was similar to that of BM under hypoxic conditions, and the rate of redox cycling under aerobic conditions was comparable to that of BM, suggesting that structural changes to the cytotoxic group of these BMs do not affect DT-diaphorase-mediated reduction and redox cycling potential. MBM, MeBM and PBM were more toxic than BM in the NCI-H661 human non-small-cell lung cancer cells and SK-MEL-28 human melanoma cells, while CBM displayed significantly increased cytotoxic activity compared to BM only in the NCI H661 cells. m-PBM had similar cytotoxic activity compared with BM in both cell lines. These cell lines have moderate to high levels of DT-diaphorase activity. When cells were pretreated with the DT-diaphorase inhibitor, dicoumarol, the cytotoxic activity of BM increased while that of MBM decreased in both cell lines, suggesting that BM was inactivated by DT-diaphorase while MBM was activated by this enzyme. Pretreatment of the SK-MEL-28 melanoma cells with dicoumarol resulted in an increased cytotoxic activity of MeBM, but pretreatment of the NCI-H661 cells did not affect the cytotoxicity of MeBM. This suggests, that similar to the results with BM, DT-diaphorase is an inactivating enzyme for MeBM in the SK-MEL-28 cell line. Dicoumarol had no significant effect on the cytotoxicity of CBM, PBM or m-PBM in both cell lines. CONCLUSIONS: These studies demonstrated that functional groups can significantly affect the reduction and activation of bioreductive agents by DT-diaphorase. All the functional groups decreased the rate of reduction of the quinone group by DT-diaphorase. Since MeBM and MBM, with electron-donating functional groups, and CBM with an electron-withdrawing functional group had similar half-lives of reduction by DT-diaphorase, steric rather than electronic effects of the functional groups appear to be more important for modifying the rate of reduction by DT-diaphorase. Steric effects on reduction by DT-diaphorase were also influenced by the position of the functional group on the quinone ring moiety, as the reduction of m-PBM was much slower than the reduction of PBM. The electron-donating methoxy and methyl functional groups increased the ability of the reduced products of MBM and MeBM to undergo redox cycling. DT-diaphorase appeared to be an activating enzyme for MBM. This may have resulted in part from increased formation of reactive oxygen species resulting from the increased redox cycling by MBM. In contrast, DT-diaphorase was an inactivating enzyme for BM, and for MeBM in the SK-MEL-28 melanoma cells, possibly because the hydroquinone product of BM and MeBM may be less cytotoxic than the semiquinone produced by one-electron reduction by NADPH:cytochrome P450 reductase.