Role of dt-diaphorase as a determinant of sensitivity to mitomycin analogs in nonsmall cell lung-cancer cell-lines

DT-diaphorase (DT-D) is regarded as a two-electron reductase that plays an important role in the biotransformation of mitomycin C (MMC) to antitumor metabolites, which is enhanced under hypoxic conditions. To evaluate the role of DT-D as a bioactivator of MMC and its analogue, KW-2149, in non-small cell lung cancer (NSCLC) cell lines under an aerobic or hypoxic condition, we examined the inhibitory effect of dicumarol which was regarded as an inhibitor of DT-D on the sensitivity to these anticancer agents in vitro. In this study, we used an MMC-resistant NSCLC cell line (PC-9/MC4) which was established in our laboratory from a PC-9 cell line as a parent cell line by continuous exposure to MMC. The subline PC-9/MC4 was 6.7-fold more resistant to MMC than PC-9, the parent cell line, under aerobic conditions, and 5.2-fold more resistant even under hypoxic conditions. PC-9/MC4 cell lines did not show collateral resistance to KW-2149, a newly developed MMC analogue. The IC50 value of PC-9 against MMC significantly decreased by co-incubation with dicumarol under aerobic, but not under hypoxic conditions. KW-2149 was cytotoxic to PC-9/MC4 as well as PC-9 cells, and the sensitivity to KW-2149 was not altered by coincubation with dicumarol or exposure to hypoxia. There were no significant differences in intracellular uptake of MMC and the activities of cytosolic detoxification enzymes, GST and GSH, between PC-9 and PC-9/MC4 cell lines under aerobic conditions. These findings suggest a partial role of DT-D in bioactivation of MMC, but not of KW-2149, under aerobic conditions. However, the detailed mechanisms of drug resistance to MMC under hypoxic conditions are still not clear.     

Establishment and Characterization of Non-small Cell Lung Cancer Cell Lines Resistant to Mitomycin C under Aerobic Conditions

To elucidate the mechanisms of acquired resistance to mitomycin C (MMC) in non-small cell lung cancer (NSCLC), we established two MMC-resistant NSCLC sublines by continuous exposure to MMC, using PC-9 as a parent cell line. The sublines, PC-9/MC2 and PC-9/MC4, were 6.4- and 10-fold more resistant to MMC than their parent cell line, respectively, at the IC₅₀ value as determined by MTT assay. They exhibited cross-resistance to EO9, but were not resistant to cisplatin, vindesine, etoposide, carboquone, or KW-2149, a novel MMC derivative. They were collaterally sensitive to adriamycin and menadione. Accumulation of the drug was decreased in the resistant sublines to about 60% of that in the parent cells. Cytosolic DT-diaphorase (DTD) activities were decreased to 13.5±3.2 in PC9/MC2 and 1.3±0.6 in PC-9/MC4 from 261.5±92.7 nmol/min/mg protein in the parent PC-9. NADH:cytochrome b ₅ reductase activities in both of the resistant cell lines were significantly decreased as compared to that in the parent cell line. Addition of dicumarol resulted in a two-fold increase in IC₅₀ value in PC-9, whereas the IC₅₀ value showed no change in PC-9/MC4. Moreover, dicumarol did not affect the sensitivities to KW-2149 but decreased the sensitivities to EO9 in both the parent and the resistant cell lines. Formation of an alkylating metabolite was significantly decreased in the resistant cells, in parallel to the degree of resistance. We concluded that deficient drug activation due to decreased DTD activity was important as a mechanism of resistance to MMC in PC-9, a relatively DTD-rich NSCLC cell line.     

Cytotoxicity of the bioreductive agent RH1 and its lack of interaction with radiation

BACKGROUND AND PURPOSE: RH1 is a new bioreductive agent that was developed as a cytotoxic agent with selectivity for tumour cells expressing high levels of the enzyme DT-diaphorase (DTD). The aim of the present study was to investigate the cytotoxicity of RH1 in relation to cellular levels of reducing enzymes and any interaction of RH1 with ionizing radiation under oxic and hypoxic conditions. PATIENTS AND METHODS: The MB-MDA231 human breast cancer cell line (WT) and WT cells transfected with the NQO1 gene encoding DTD (the D7 cell line) were used to examine the dependency of RH1's cytotoxicity on cellular DTD activity. The role of the 1-electron reducing enzyme P450 reductase was also studied using a P450 reductase-transfected isogenic cell line (R4). A clonogenic assay was used to investigate the cytotoxicity of RH1 with and without irradiation in air and in nitrogen. In all cases drug exposure was for 3 h. RESULTS: DTD levels were around 300-fold higher in D7 compared to WT and R4 cells. RH1 was cytotoxic at nanomolar concentrations to all the cell lines, and was 2-3 times more toxic in the D7 cells with high DTD than in the other two cell lines. Doses of RH1 was around 2-fold more effective in hypoxic than in oxic WT cells, but not by as much in D7 cells. RH1 did not radiosensitise the cells but showed an additive effect when combined with irradiation under oxic and hypoxic conditions. CONCLUSIONS: RH1 shows high clonogenic cytotoxicity to MDA231 cells with high DTD activity but its selectivity based on the presence of DTD is much less than as shown in previous reports. RH1 showed an additive cell killing effect when combined with irradiation under both oxic and hypoxic conditions.     

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 EO9. In preclinical studies, EO9 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 DCPIP min-1 mg-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).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of three bioreductive drugs (mitomycin C, RSU-1069 and SR4233) on cell lines selected for their sensitivity to mitomycin C or ionising radiation

We have investigated the cross-sensitivity of a number of cell lines to three different classes of bioreductive drugs under both aerobic and hypoxic conditions. The cell lines used were selected for their sensitivity to DNA-damaging agents and fall into two groups. One group, MMC cells derived from CHO-K1 cells (Robson et al., 1985), show a range of sensitivities to mitomycin C in air. The second group, irs cells were cloned from V79 Chinese hamster fibroblasts (Jones et al., 1987) and exhibit sensitivity to ionising radiation. The sensitivity of both groups of cells to mitomycin C (MMC), RSU-1069 and SR4233 was assessed under aerobic and hypoxic conditions. No difference in aerobic or hypoxic toxicity of MMC was observed for CHO-K1 or MMC sensitive cell lines (MMC-2 and MMC-3). However, the MMC-resistant cell line (MMCr) was 10 times more sensitive under hypoxic than aerobic conditions. This suggests that MMCr cells lack or are deficient in the enzymes responsible for activating MMC under aerobic conditions compared to other MMC cells. In contrast, differential toxicities of between 3 and 30 have been observed for all CHO cells treated with RSU-1069 and SR4233. Treatment of V79 and irs cells with RSU-1069 and SR4233 also resulted in selective toxicity towards hypoxic cells. Differential toxicities between 50 and 100 were observed for V79 cells. For both RSU-1069 and SR4233, the hypoxic toxicities were similar in V79 and irs cells but in air, the radiation sensitive cells were up to 10 times more sensitive than wild type cells.     

Cytotoxicity of mitomycin C on clonogenic human carcinoma cells is not enhanced by hypoxia

Summary The bioreductive alkylating agent mitomycin C (mitomycin) has been shown to have greater activity under hypoxic than oxic conditions on murine cell lines such as the EMT-6 fibrosarcoma cell line. Solid tumors are known to contain hypoxic cells and are relatively resistant to ionizing radiation and some chemotherapeutic agents. We tested the cytotoxicity of mitomycin against fresh biopsies of human carcinomas under both hypoxic and oxic conditions in the human tumor clonogenic assay (HTCA). Additionally, we examined the metabolism of mitomycin by sonicates of the murine EMT-6 cells and the human WiDR colon carcinoma cells. We confirmed that under our clonogenic assay conditions the EMT-6 cell line was more sensitive to mitomycin under hypoxic than oxic conditions. Additionally, we established that EMT-6 cells also metabolize mitomycin at a more rapid rate under hypoxic than oxic conditions. However, these effects of hypoxia on mitomycin activity were not demonstrable for the human WiDR colon cancer cell line. In addition to these findings, the cytoxicity of mitomycin was either unchanged or reduced under hypoxic conditions for ten fresh human tumors tested for mitomycin sensitivity in HTCA. Based on these observations, we conclude that the potentiating effect of hypoxia on mitomycin metabolism and biological activity may be peculiar to the murine EMT-6 and S-180 cell lines and that mitomycin C is not likely to have differential efficacy against hypoxic human carcinoma cells.     

BIM induction of apoptosis triggered by EGFR-sensitive and resistance cell lines of non-small-cell lung cancer

We sought to improve the understanding of oncogene-dependent and independent non-small-cell lung cancer (NSCLC), which could provide insight into mechanism of sensitivity and/or resistance to tyrosine kinase inhibitors or chemotherapeutics. NSCLC cell lines with different EGFR genotypes were used in this study; MTT assay and flow cytometry were applied to study the sensitivities of these cell lines to gefitinib and cisplatin. Western blot was performed to determine the expression levels of BIM and other Bcl-2 family proteins pre- and pro-treatment. Gefitinib provoked apoptosis of caspase activation via the intrinsic pathways and significantly up-regulated expression of BIM protein in drug-sensitive PC-9 cell line, but not resistant PC-9/BB4 cell line. The knockdown of BIM expression by RNA interference virtually eliminated gefitinib-induced cell killing in PC-9 cells in vitro. Cisplatin could induce apoptosis of the cell lines, including H1299, A549, PC-9, and PC-9/BB4 cells, but which was not associated with overexpression of BIM. BIM is involved in TKI-induced apoptosis in sensitive EGFR-mutant cell line. Down-regulation of BIM and resistance to gefitinib were both seen in the acquired resistant PC-9/BB4 cell line. The induction of BIM may have a role in the treatment of TKI-resistant tumors.     

Biochemical characterization of a mitomycin C-resistant human bladder cancer cell line

This study describes characteristics of a mitomycin C (MMC)-resistant human bladder cancer cell line, J82/MMC-2, which was established by repeated in vitro exposures of a 6-fold MMC-resistant variant (J82/MMC) to 18 nM MMC. A 9.6-fold higher concentration of MMC was required to kill 50% of the J82/MMC-2 sub-line compared with parental cells (J82/WT). NADPH cytochrome P450 reductase and DT-diaphorase activities were significantly lower in J82/MMC-2 cells compared with J82/WT, suggesting that reduced sensitivity of J82/MMC-2 cells to MMC resulted from impaired drug activation. Consistent with this hypothesis, the formation of MMC-alkylating metabolites was significantly lower in J82/MMC-2 cells compared with J82/WT. Furthermore, DT-diaphorase activity in J82/MMC-2 cells was significantly lower compared with the 6-fold MMC-resistant variant. Glutathione (GSH) levels were comparable in all 3 cell lines. Although GSH transferase (GST) activity was significantly higher in the J82/MMC-2 cells compared with J82/WT, this enzyme activity did not differ between 6- and 9.6-fold MMC-resistant variants. Whereas DNA polymerase α mRNA expression was comparable in these cell lines, levels of DNA ligase I mRNA were slightly lower in both MMC-resistant variants relative to J82/WT. However, the DNA polymerase β mRNA level was markedly higher in the J82/MMC-2 cell line compared with either J82/WT or J82/MMC. Thus, emergence of a higher level of resistance to MMC in J82/MMC-2 cells compared with J82/MMC may be attributed to (i) impaired drug activation through further reduction in DT-diaphorase activity and (ii) enhanced DNA repair through over-expression of DNA polymerase β. © 1996 Wiley-Liss, Inc.     

The Induction of Cytotoxic T Lymphocytes against HLA-A Locus-matched Lung Adenocarcinoma in Patients with Non-small Cell Lung Cancer

To induce cytotoxic T lymphocytes (CTL) against non-small cell lung cancer (NSCLC) efficiently, the induction of CTL was attempted using HLA-A locus-shared allogeneic NSCLC cells. T cells derived from either tumor tissue specimens or the regional lymph nodes of patients with NSCLC were stimulated twice or three times with an HLA-A2/A24-positive NSCLC cell line (PC-9), and thereafter the cytotoxic activity was examined by ⁵¹Cr-release assay. In patients with HLA-A24/ adenocarcinoma, anti-PC-9 cytotoxicity was induced in all 6 patients tested. Anti-PC-9 cytotoxicity was induced in 2 out of 5 patients with HLA-A2 (A24⁻)/adenocarcinoma, in 2 out of 4 patients with HLA-A24/squamous cell carcinoma, and 1 of 2 patients with HLA-A2/squamous cell carcinoma. The cytotoxic activity was observed to kill PC-9 selectively, not other NSCLC lines, and the activity was substantially blocked by anti-MHC class I antibody, but not by anti-MHC class II antibody. The PC-9-specific CTL produced γ-interferon in response to autologous tumor cells. These results indicated that the anti-PC-9 cytotoxicity was mediated by cytotoxic T lymphocytes that may recognize the T cell epitope(s) shared and presented by HLA-A2 and/or HLA-A24-positive NSCLC.     

Decreased NADPH:cytochrome P-450 reductase activity and impaired drug activation in a mammalian cell line resistant to mitomycin C under aerobic but not hypoxic conditions

Mitomycin C (MMC) is regarded as the prototype bioreductive alkylating agent in clinical use. To elucidate the biochemical basis of MMC resistance, we isolated a drug resistant derivative (designated CHO-MMC) of a Chinese hamster ovary cell line (CHO-K1) by exposure to progressively higher concentrations of MMC. CHO-MMC cells exhibited a 17-fold increase in resistance to MMC and were 33-fold cross-resistant to the monofunctional derivative, decarbamoyl mitomycin C. In contrast, CHO-MMC cells showed only a 2-fold level of resistance to BMY 25282, a more easily activated analogue of MMC, and exhibited parental sensitivity to MMC under radiobiologically hypoxic conditions. CHO-MMC cells showed no increased resistance to a range of DNA damaging agents including several other alkylating agents (e.g., melphalan and methyl methanesulfonate). Cross-resistance to drugs associated with the multidrug resistant phenotype (e.g., Adriamycin and vincristine) was present only at very low levels. Using a specific high performance liquid chromatography technique, we examined the rates of reduction of MMC and BMY 25282 in cell extracts from CHO-K1 and CHO-MMC cells under both aerobic (air) and hypoxic (N2) conditions. Reduction rates for both drugs were at least 30-fold faster under nitrogen than in air. Metabolism of MMC was undetectable in air but was readily detectable under nitrogen and was 2- 3-fold slower in CHO-MMC cell extracts than in CHO-K1 cell extracts. Although BMY 25282 was more readily reduced under nitrogen, no difference was detected between extracts from CHO-K1 or CHO-MMC cells in the rate of reduction of BMY 25282 under either air or nitrogen. The activity of NADPH:cytochrome P-450 (cytochrome c) reductase, an enzyme implicated in the bioreductive activation of MMC, was 3-4-fold lower in CHO-MMC cells than in the parental line. These findings suggest that the resistance of CHO-MMC cells to MMC under aerobic conditions may be due to impaired metabolic activation of the drug as a result of a decrease in NADPH:cytochrome P-450 reductase activity. This supports the view that decreased bioreductive enzyme activity may be a significant mechanism for acquired resistance to MMC in tumor cells in vivo and that more readily activated analogues may be potentially useful in overcoming this specific form of resistance.     

Immunological Quantitation of DT-Diaphorase in Carcinoma Cell Lines and Clinical Colon Cancers: Advanced Tumors Express Greater Levels of DT-Diaphorase

NAD(P)H:quinone oxidoreductase (DT-diaphorase; DTD) plays a major role in activating mitomycin C (MMC) in human colon and gastric carcinoma cell lines. Thus, measurement of DTD in clinical tumor samples could be beneficial in designing adjuvant chemotherapy. We explored immunological quantitation of DTD protein using a monoclonal antibody against DTD, demonstrating a close correlation between protein expression and enzyme activity of DTD in colon and gastric carcinoma cell lines and in colorectal tumor samples. This indicates that such immunoblot analysis is a simple alternative method for quantitating DTD in clinically excised samples. In most colorectal tumor samples, the tumors expressed larger amounts of DTD than did the peripheral normal tissues, suggesting a selective toxicity of MMC toward tumor cells. Also tumors with nodal metastases showed significantly higher DTD levels than did tumors without metastasis. These results raise the possibility that DTD expression is related to tumorigenesis and malignant progression of colorectal tumors. Measurement of DTD by the immunological method described here could be beneficial in designing a rational adjuvant chemotherapy with MMC.     

Effect of hypoxia on 1,3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity in 9L cells

The cytotoxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on 9L rat brain tumor cells were studied under oxic and hypoxic conditions. Acute hypoxia was produced by gassing exponentially growing monolayer cells with 95% nitrogen/5% CO2 for 2 h, after which cells were treated with graded concentrations of BCNU for 1 h. Cell survival was assayed with a colony forming-efficiency assay and the extent of DNA cross-linking was measured with the alkaline elution assay. BCNU was more cytotoxic to hypoxic than to oxic cells. There were far more interstrand cross-links formed in hypoxic than in oxic cells, and the number of cross-links could be measured readily in hypoxic cells at very low concentrations of BCNU. This allowed cell survival and cross-linking to be compared at the same dose levels, a correlation not possible for oxic cells in which cross-links are not measurable at low doses. The total intracellular levels of glutathione were lower in hypoxic cells, but the reduced glutathione levels may not be related to the enhanced cell kill because survival of oxic cells treated with BCNU was not affected when cells were depleted of glutathione with buthionine sulfoximine. These results indicate that the additional cell kill produced in 9L cells under hypoxic conditions is related to increased cross-link formation.     

Relevance of DT-diaphorase activity to mitomycin C (MMC) efficacy on human cancer cells: Differences in in vitro and in vivo systems

Using 4 human cancer cell lines, 4 tumors xenografted into nude mice, and 11 fresh tumor specimens removed at surgery, we investigated the relevance of NAD(P)H:quinone oxidoreductase (DT-diaphorase, DTD) activity (nmoles/min/mg protein) to mitomycin C (MMC)-induced cytotoxicity. In culture cell lines, KB cells had significantly higher levels of DTD activity (8260) than PH101 (1934), SH101 (1805) or K562 (1796), and the highest sensitivity to MMC. In contrast, the higher the DTD activity of xenografts, the greater their resistance to MMC, while the inhibition rate of relative tumor growth for MMC, as evaluated by the NCI protocol, was highest in SH-6, high in CH-5, lower in CH-4 and lowest in EH-6. The investigation using 11 fresh tumor specimens also showed an inverse relationship between IC⁵⁰ values after a 30-min MMC treatment, as evaluated by ATP assay and DTD activities. Moreover, a non-toxic DTD inhibitor, dicoumarol (DIC), or flavin adenine dinucleotide (FAD), suppressed the efficacy of MMC in culture cells, but enhanced it in xenografts. Thus, we suggest that DTD may play an important role in MMC-induced cytotoxicity but MMC metabolism by DTD in solid tumors may differ from that in culture cells.     

Modification of the cytotoxic activity of mitomycin C by oxygen and ascorbic acid in Chinese hamster ovary cells and a repair-deficient mutant

The cellular and molecular damage produced by mitomycin C (MMC) in Chinese hamster ovary cells, AA8-4, and a repair deficient mutant of this line, UV-20, was studied by utilizing a system in which oxygen levels could be altered and monitored in solution during acute drug exposures. The cytotoxic activity of MMC decreased from hypoxic conditions to 1% oxygen in solution, while from 1 to 20% there was little change. The relative level of DNA cross-linking in cells was examined under these conditions by alkaline elution and found to increase as cell survival decreased. Utilizing a cell-free assay which detects formation of alkylating species it was confirmed that, while alkylation was observed under aerobic conditions, overall levels increased in the absence of oxygen. The presence of ascorbic acid in the exposure medium (0.284 mM) increased the aerobic but not the hypoxic cytotoxicity of MMC. This resulted in a diminished differential toxicity for cells exposed under aerobic versus hypoxic conditions in the presence of ascorbic acid. When ascorbic acid was present, net alkylation increased under aerobic conditions but was unchanged under hypoxic conditions. One interpretation of these results is that at least two mechanisms of activation of MMC to toxic intermediates may be present in these cells.     

Overexpression of Cathepsin L is associated with gefitinib resistance in non-small cell lung cancer

Lung cancer, the most common malignancy, is still the leading cause of cancer-related death worldwide. Non-small-cell lung cancer (NSCLC) accounts for 80 % of all lung cancers. Recent studies showed Cathepsin L (CTSL) is overexpressed in various cancerous tissues; however, the association between CTSL expression and EGFR-TKI resistance remains unknown. In this study, we investigated the expression of CTSL in lung cancer specimens and matched normal tissues by quantitative real-time PCR and IHC. The functional role of CTSL in resistant PC-9/GR cell line was investigated by proliferation and apoptosis analysis compared with control PC-9 cells. Our results found that the level of CTSL expression was higher in NSCLC tissues compared with matched normal adjacent tissue samples, and CTSL was more highly expressed in PC-9/GR cells compared to PC-9 cells. Knocking-down of CTSL in PC-9/GR cells could decrease cell proliferation and potentiate apoptosis induced by gefitinib, suggesting CTSL may contribute to gefitinib resistance in NSCLC. CTSL might be explored as a candidate of therapeutic target for modulating EGFR-TKI sensitivity in NSCLC.     

The role of human and rodent DT-diaphorase in the reductive metabolism of hypoxic cell cytotoxins.

DT-diaphorase is a unique two electron (2e) donating reductase catalyzing either bioactivation or bioprotection reactions. Using human and rodent DT-diaphorase preparations (cell extracts and purified enzyme) we have characterized the reductive metabolism of the hypoxic cell cytotoxins EO9, mitomycin C (MMC), CB 1954, and SR 4233 in vitro. Drug metabolism was assayed spectrophotometrically or by HPLC, with dicoumarol as a selective inhibitor. DNA damage was measured using an agarose gel mobility technique with plasmid pBR322 DNA. The developmental indoloquinone, EO9, was metabolized by both rat Walker and human HT29 tumor DT-diaphorases. Reduction proceeded 5-fold more efficiently with the rat than the human tumor enzyme and resulted in single-strand breaks in plasmid DNA. The structurally related MMC was metabolized much more slowly than EO9 by the rat Walker tumor enzyme and there was no detectable reaction with the human HT29 tumor DT-diaphorase. No DNA damage was seen with MMC for either enzyme. The dinitrophenylaziridine CB 1954 was reduced by both human and rat enzymes forming, preferentially, the highly toxic 4-hydroxylamine as a 4e reduction product. Rates were 3-fold lower than for the human tumor enzyme. SR 4233 was also reduced by the rat tumor enzyme predominantly via 4e reduction to the benzotriazine SR 4330, in a novel reaction mechanism. This appears to be a bioprotection pathway that bypasses the toxic 1e radical formed by other reductases. Such information may be valuable in the selection of hypoxic cell cytoxins to treat human tumors high or low in DT-diaphorase and should facilitate 'enzyme-directed' analogue development.     

Elevated DT-diaphorase activity and messenger RNA content in human non-small cell lung carcinoma: relationship to the response of lung tumor xenografts to mitomycin C?.

The enzyme DT-diaphorase (DTD; NAD(P)H:quinone oxidoreductase, EC 1.6.99.2), is an obligate two electron reductase which catalyzes reduction of a broad range of substrates, including quinones. We report here variations in DTD concentrations among different classes of lung tumors known also to vary in their responsiveness to cytotoxic agents. Small cell lung carcinomas (SCLCs) and cell lines derived from them have the low DTD activities and mRNA content characteristic of normal human lung, whereas non-small cell lung carcinomas (NSCLCs) have greatly elevated levels. DTD activity was increased up to 80-fold in NSCLC tumors relative to normal lung and 20-35-fold in NSCLC relative to SCLC cell lines. Increased DTD activity appeared to be a function of the NSCLC phenotype rather than a result of derivation from a cell type rich in DTD, since all histological classes of NSCLC showed this phenotype. In addition, where transfection of SCLC cell lines with the v-Ha-ras protooncogene caused a transition to a NSCLC phenotype, DTD activity was also elevated. Neuroendocrine-positive cells (SCLC, carcinoids, and a few NSCLC lines) typically had far lower DTD activities than did cell lines which lacked neuroendocrine markers (most NSCLC cells and mesotheliomas). High DTD activity may be exploited in the design of drugs which undergo bioreductive activation by this enzyme. Consistent with this, xenografts derived from NSCLC cell lines with high DTD that were grown in athymic nude mice were more susceptible to the antitumor quinone, mitomycin C, than were xenografts derived from SCLC cells containing low DTD. These data provide a mechanistic basis for the rational design of more effective bioreductive antitumor agents for use against NSCLC.     

DT-diaphorase protects cells from the hypoxic cytotoxicity of indoloquinone EO9.

Aerobic sensitivity to indoloquinone EO9 has been shown to correlate with cellular levels of the two-electron reducing enzyme DT-diaphorase. However, little is known about the relative roles of one- and two-electron reducing enzymes in the hypoxic cytotoxicity of EO9. We have characterised a panel of 23 human tumour cell lines for both bioreductive enzyme activities and aerobic sensitivity to EO9. Eight cell lines were then selected for a comparison of aerobic and hypoxic sensitivities. Activities of DT-diaphorase showed a wide range (> 10,000-fold), while activities of the one-electron reducing cytochrome b5 and cytochrome P450 reductases were generally lower and showed only a 15- and 25-fold range respectively. The aerobic cytotoxicity of EO9 was clearly related to the cellular levels of DT-diaphorase (r = 0.87), with higher levels giving increased sensitivity, but not to the levels of one-electron reducing enzymes. In contrast, there was no relationship between sensitivity to BCNU, cisplatin or the bioreductive agent SR 4233 (tirapazamine) and activities of any of these reducing enzymes. Under hypoxic conditions sensitivity to EO9 was markedly increased in cell lines with low levels of DT-diaphorase activity, while cell lines with high levels show only a small increase in sensitivity. This is reflected by a clear correlation (r = 0.98) between cellular DT-diaphorase activity and the ratio of aerobic to hypoxic sensitivity to EO9. However, we have now for the first time demonstrated an inverse correlation (r = 0.93) between the cellular activity of DT-diaphorase and hypoxic sensitivity to EO9, that is sensitivity decreases with increasing DT-diaphorase activity. Moreover, this correlation was lost when cells were exposed to drug in the presence of dicoumarol, supporting an involvement of DT-diaphorase in this relationship. These observations question the previously straightforward role for DT-diaphorase in the metabolic activation of EO9. Whereas DT-diaphorase is associated with increased toxicity in air, it appears to reduce the cytotoxicity of EO9 in hypoxic conditions. This suggests either that the one-electron reduction product of EO9 metabolism, the semiquinone, is more toxic than the two-electron reduction product, the hydroquinone, or that the hydroquinone is not cytotoxic and aerobic toxicity is due to the transient appearance of the semiquinone upon back oxidation of the hydroquinone.     

Gold nanoparticle cellular uptake,toxicity and radiosensitisation in hypoxic conditions

Background and purpose

Gold nanoparticles (GNPs) are novel agents that have been shown to cause radiosensitisation in vitro and in vivo. Tumour hypoxia is associated with radiation resistance and reduced survival in cancer patients. The interaction of GNPs with cells in hypoxia is explored.

Materials and methods

GNP uptake, localization, toxicity and radiosensitisation were assessed in vitro under oxic and hypoxic conditions.

Results

GNP cellular uptake was significantly lower under hypoxic than oxic conditions. A significant reduction in cell proliferation in hypoxic MDA-MB-231 breast cancer cells exposed to GNPs was observed. In these cells significant radiosensitisation occurred in normoxia and moderate hypoxia. However, in near anoxia no significant sensitisation occurred.

Conclusions

GNP uptake occurred in hypoxic conditions, causing radiosensitisation in moderate, but not extreme hypoxia in a breast cancer cell line. These findings may be important for the development of GNPs for cancer therapy.