Increased sensitivity to platinum drugs of cancer cells with acquired resistance to trabectedin.

BACKGROUND: In order to investigate the mechanisms of acquired resistance to trabectedin, trabectedin-resistant human myxoid liposarcoma (402-91/T) and ovarian carcinoma (A2780/T) cell lines were derived and characterised in vitro and in vivo. METHODS: Resistant cell lines were obtained by repeated exposures to trabectedin. Characterisation was performed by evaluating drug sensitivity, cell cycle perturbations, DNA damage and DNA repair protein expression. In vivo experiments were performed on A2780 and A2780/T xenografts. RESULTS: 402-91/T and A2780/T cells were six-fold resistant to trabectedin compared with parental cells. Resistant cells were found to be hypersensitive to UV light and did not express specific proteins involved in the nucleotide excision repair (NER) pathway: XPF and ERCC1 in 402-91/T and XPG in A2780/T. NER deficiency in trabectedin-resistant cells was associated with the absence of a G2/M arrest induced by trabectedin and with enhanced sensitivity (two-fold) to platinum drugs. In A2780/T, this collateral sensitivity, confirmed in vivo, was associated with an increased formation of DNA interstrand crosslinks. CONCLUSIONS: Our finding that resistance to trabectedin is associated with the loss of NER function, with a consequent increased sensitivity to platinum drugs, provides the rational for sequential use of these drugs in patients who have acquired resistance to trabectedin.

Phase I trial combining gemcitabine and treosulfan in advanced cutaneous and uveal melanoma patients.

Gemcitabine and treosulfan are DNA-damaging agents. Preclinical studies suggest that synergism exists when melanoma cells are exposed to both drugs concurrently. We conducted a phase I trial in advanced melanoma patients to determine the optimal dose of gemcitabine to be combined with treosulfan. Cohorts of three patients received increasing doses of gemcitabine, commencing at 0.5 g m(-2), followed by a fixed dose of 5.0 g m(-2) treosulfan on day one of a 21-day cycle. Patients alternately received a first cycle of single-agent gemcitabine or treosulfan before subsequent cycles of both drugs. Peripheral blood lymphocytes were collected in cycles 1 and 2 at various time points until 48 h post-treatment. The single-cell gel electrophoresis (Comet) assay was used to measure chemotherapy-induced DNA damage. A total of 27 patients were enrolled, no objective responses were observed, but two uveal melanoma patients had minor responses. Dose-limiting myelosuppression was reached at 3.0 g m(-2) gemcitabine. DNA single-strand breaks were detected 4 h post-gemcitabine, repaired by 24 h. DNA interstrand crosslinks were detected 4 h post-treosulfan, fully removed by 48 h. Following combination chemotherapy, treosulfan-induced DNA crosslinks persisted, still being detectable 48 h post-treatment, supporting the hypothesis that gemcitabine potentiates treosulfan-induced cytotoxicity. The recommended regimen for further study is 2.5 g m(-2) gemcitabine combined with 5.0 g m(-2) treosulfan.

Autologous plasma activates Akt/protein kinase B and enhances basal survival and resistance to DNA damage-induced apoptosis in B-chronic lymphocytic leukaemia cells.

We have studied the actions of autologous plasma on both basal and DNA damage-induced apoptosis in B-chronic lymphocytic leukaemia (B-CLL) cells. Apoptosis was quantified using morphological criteria and Western blot analysis for the apoptosis-specific p85 fragment of poly(ADP ribose) polymerase. Cell viability was estimated using the methyl thiazol tetrazolium bromide dye reduction assay. Plasma cultures showed lower rates of basal apoptosis as well as a decreased cytotoxic response to chlorambucil and gamma-radiation compared with cultures in fetal calf serum. Experiments using neutralizing antibodies suggested that the protective actions of plasma could not be accounted for by interleukin 4, the interferons alpha or gamma or stromal cell-derived factor 1, each of which have been shown to protect B-CLL cells from apoptosis in vitro. Plasma addition to B-CLL cells resulted in rapid activation of the Akt protein kinase, a key signalling enzyme that has been implicated in anti-apoptotic signalling. LY294002, an inhibitor of phosphatidylinositol 3'-kinase, blocked Akt activation by plasma. To the best of our knowledge, this is the first report to show that factors present in plasma promote basal survival of B-CLL cells and resistance to cytotoxic drugs via stimulation of the Akt cytoprotective-signalling pathway. Pharmacological blockade of this pathway may have potential in the development of novel therapeutic strategies for B-CLL treatment.

Repair of DNA interstrand crosslinks: molecular mechanisms and clinical relevance.

Drugs that produce DNA interstrand crosslinks (ICLs), between the two complementary strands of the double helix, have an important role in chemotherapy regimens for cancer. Novel crosslinking agents, and targeting strategies involving DNA crosslinking agents, continue to be developed. The ability of cells to repair DNA ICLs is a critical determinant of sensitivity, and recent dinical studies indicate that DNA repair capacity is strongly implicated in both inherent tumour sensitivity and acquired drug resistance. A detailed understanding of the cellular mechanisms that act to eliminate these critical DNA lesions is clearly important. DNA ICLs present a complex challenge to DNA repair mechanisms because of the involvement of both DNA strands. It is now clear that cells from bacteria and yeast to mammals eliminate interstrand ICLs through the coordinated action of several DNA repair pathways. Recently, a model of ICL repair has been proposed, in which mammalian cells use novel excision repair reactions (requiring the XPF and ERCC1 proteins) to uncouple the crosslink. This is followed by a homologous recombination step to provide the genetic information needed to complete repair. This new knowledge may permit the development of screens for tumour response to crosslinking agents, and should also aid the design of more effective crosslinking agents that evade DNA repair. In addition, the proteins mediating the repair reactions represent potential targets for therapeutic intervention.

Measurement of DNA cross-linking in patients on ifosfamide therapy using the single cell gel electrophoresis (comet) assay.

The single cell gel electrophoresis comet assay has become established as a sensitive technique for measuring DNA strand breaks. The technique has been modified to allow the sensitive detection and quantitation of DNA interstrand cross-linking at the single cell level. Cells are irradiated immediately before analysis to deliver a fixed level of random strand breakage. After embedding of cells in agarose and lysis, the presence of cross-links retards the electrophoretic mobility of the alkaline denatured cellular DNA. Cross-links are, therefore, quantitated as the decrease in the comet tail moment compared with irradiated controls. Using this method, a linear response of cross-linking versus dose of chlorambucil over a wide dose range was demonstrated in human lymphocytes after drug treatment ex vivo. The method was also sensitive enough to determine cross-linking in clinical samples after chemotherapy. For example, crosslinking was observed in the lymphocytes of patients receiving ifosfamide (3 g/m2/day) as a continuous infusion for 3-5 days or as a 3-h infusion daily for 3 days. Cross-links were detected in all patients within 3 h, with no evidence of DNA single strand break formation. In patients receiving continuous infusion, a plateau of cross-linking was reached by 24 h. In the patients receiving ifosfamide over 3 h, a clear decrease in the peak level of cross-linking was observed before subsequent infusions.

Measurement of drug-induced DNA interstrand crosslinking using the single-cell gel electrophoresis (comet) assay.

DNA damaging agents have been widely used in cancer chemotherapy for many years and have proved successful in the treatment of both solid tissue and haematological malignancies. Many commonly used clinical agents, such as members of the nitrogen mustard, chloroethylnitrosourea, dimethane-sulphonate and platinum classes, are bifunctional. DNA interstrand crosslinks (ISC) formed in cells are clearly critical cytotoxic lesions and the formation of DNA ISC has been shown to correlate with cytotoxicity in vitro (1-5). Acquired resistance in vitro to such agents can occur by a number of mechanisms, for example altered drug transport (6), intracellular detoxification via enhanced glutathione and glutathione-S-transferase activity (7), but enhanced DNA repair capacity can also play an important role (3). Clinically the mechanisms of acquired resistance to DNA damaging agents are less clear but enhanced repair of ISC has been suggested to play a role in the acquired resistance of some cancers, e.g., chronic lymphocytic leukaemia to nitrogen mustards (8). In addition, the inherent sensitivity (and curability) of some tumors, e.g., testicular cancer, to DNA damaging agents may result in part from their inability to repair critical DNA lesions (9).

Pharmacological determinants of the antitumour activity of mitomycin C.

Recent investigations into bioreductive anticancer drugs have focused on profiling reductase enzymes and relating their expression to therapeutic activity in an approach referred to as enzyme directed drug development. However, few studies have attempted to validate this approach in vivo and even less is known about how the expression of reductases relates quantitatively and qualitatively to metabolic activation. In the present study, the antitumour activity, pharmacokinetics and metabolism of mitomycin C (MMC) has been determined in vivo in two murine adenocarcinomas of the colon, MAC 16 (high DT-diaphorase activity) and MAC 26 (low DT-diaphorase activity) after intra-tumoural injection of drug. Over a broad range of drug concentrations (50-250 microg), MAC 16 proved to be consistently the more sensitive tumour (e.g. 75 microg of MMC, T/C 11% for MAC 16 and 31% for MAC 26). Higher levels of parent drug (peak concentration 103 microg/tumour compared to 58 microg/tumour) were maintained over 45 min in MAC 16 after which time clearance was rapid from both tumours. Four metabolites were detected in both tumours characteristic of different pathways of metabolism. However, by far the major metabolite was 2,7-diaminomitosene (2,7-DM), an accurate indicator of metabolic activation of MMC. Despite higher reductase levels and greater sensitivity to the drug, there was 4-fold less production of 2,7-DM in MAC 16. These results indicate a lack of a simple relationship in vivo between reductase expression and metabolic activation and suggest factors other than pharmacological determinants being responsible for the chemosensitivity of the MAC tumours to MMC.

Current issues in the enzymology of mitomycin C metabolic activation.

1. Mitomycin C (MMC) is considered to be the prototype bioreductive drug undergoing activation to toxic species preferentially under hypoxic conditions. Therefore a proper understanding of the enzymology of bioreduction in tumor tissue is of great importance. 2. DT-diaphorase and NADPH:cytochrome P-450 reductase (quinone reductases) are believed to have established roles in this activation pathway, but these roles are now undergoing revision. 3. It is emerging, however, that different reductases prevail under different physiological conditions. Indeed, DT-diaphorase has been found to protect cells from the hypoxic cytotoxicity of MMC in cell lines expressing high levels of the enzyme. 4. A novel mitochondrial reductase(s) has been identified in solid tumor tissue and is active only under hypoxic conditions and is more efficient at metabolizing MMC than are the other reductases identified. 5. Thus, this newly identified mitochondrial reductase(s) is a potential new target for enzyme-directed bioreductive drug therapy if tumor hypoxia can be achieved. However, because most tumors overexpress DT-diaphorase, this enzyme may prove optimal for MMC drug therapy.

Enzymology of mitomycin C metabolic activation in tumour tissue: implications for enzyme-directed bioreductive drug development.

Mitomycin C (MMC) is the prototype bioreductive DNA alkylating agent. To exploit its unique properties and maximize patient responses, different therapeutic approaches have been investigated. Recently, the focus has concentrated on monitoring the levels of the proteins metabolizing the drug and relating these to activity in a regimen referred to as enzyme-directed bioreductive drug development. To be successful, it is important to understand the enzymology of metabolic activation not only in cell lines but also in solid tumour models. A general mechanism of action for MMC has now emerged that is activated regardless of the source of reducing equivalents, comprising three competing pathways that give rise to unique reactive intermediates and different DNA adducts. Partitioning into the pathways is dictated by chemical considerations such as pH and drug concentration. DT-diaphorase stands out in this mechanism, since it is much less effective at metabolizing MMC at neutral pH. At least five different enzymes can catalyse MMC bioreduction in vitro, and as many activities may be present in solid tumours, including a series of novel mitochondrial reductases such as a cytochrome P450 reductase. Competition between reductases for MMC appears to be based solely on protein levels rather than enzyme kinetics. Consequentially, DT-diaphorase can occupy a central role in MMC metabolic activation since it is often highly overexpressed in cancer cells. Although a good correlation has been observed in cell lines between DT-diaphorase expression and aerobic cytotoxicity, this does not hold consistently in vivo for any single bioreductive enzyme, suggesting revision of the enzyme-directed hypothesis as originally formulated.

Pharmacological and biochemical determinants of the antitumour activity of the indoloquinone EO9.

EO9 is a novel bioreductive drug which has recently undergone extensive clinical evaluation. Its mechanism of action remains to be clearly defined. Antitumour activity of EO9 has been determined in 2 human colon cancer xenografts (HT-29 and BE) and 2 murine colon adenocarcinomas (MAC 16 and 26) after intratumoural injection of 250 microg of drug. Levels of the major bioreductive enzymes (DT-diaphorase, cytochrome P-450 reductase and cytochrome b5 reductase) were measured in tumours using cytochrome c reduction and menadione as the intermediate electron acceptor. There was no correlation between chemosensitivity (T/C: HT-29, 15%; BE, 27%; MAC 16, 33% and MAC 26, 60%) and enzyme activity (r2 = 0.47 for DT-diaphorase, r2 = 0.1 for cytochrome P-450 reductase and r2 = 0.52 for cytochrome b5 reductase). Drug metabolism was followed in vitro using tumour homogenates incubated under aerobic and anaerobic conditions. Four metabolites were identified by HPLC and characterised bv UV-visible spectroscopy. With the exception of the hydrolysis product EO5A, all other metabolites appeared to be drug adducts. No correlation was observed between the kinetics of metabolite formation and antitumour activity. A good correlation (r2 = 0.86) was found with the rate of disappearance of parent drug and antitumour activity. These data show that the overall capacity of a tumour to metabolise EO9 is the most important determinant of antitumour activity rather than the expression of the major bioreductive enzymes and that the parent drug rather than a metabolite leads to the active form of the drug.

The autoxidation of the reduced forms of EO9.

The properties of the semiquinone radical from [3-hydroxy-5-aziridinyl-1-methyl-2-(1H-indole-4,7-indi one)-prop-beta-en-alpha-ol], EO9, have been studied using pulse-radiolysis techniques. The reduction potential of the semiquinone of EO9 at pH7.4, E(EO9/EO9-), is -253 +/- 6 mV and hence this quinone can be readily reduced by one-electron reducing enzymes such as cytochrome P450 reductase and xanthine oxidase. However, the radical is unstable in the presence of oxygen (k = 1.3 +/- 0.15 x 10(8) M-1 s-1). The semiquinone radicals and the hydroquinone are in equilibrium although the formation of the hydroquinone is favoured t physiologically relevant pH. The hydroquinone of EO9 is also unstable in the presence of oxygen and it is predicted that in fully aerated solutions, its half life is 1.5 +/- 0.3 seconds. These results are discussed in view of the selective cytotoxicity of EO9 and its ability to undergo bioreductive activation by one-electron reducing enzymes and DT-diaphorase.

Enzymology of mitomycin C metabolic activation in tumour tissue. Characterization of a novel mitochondrial reductase.

In this study, the enzymology of mitomycin C (MMC) bioactivation in two murine colon adenocarcinomas, MAC 16 and MAC 26, was examined. Subcellular quinone reductase assessment via cytochrome c reduction confirmed a number of active enzymes. MAC 16 exhibited 22-fold greater levels of cytosolic DT-diaphorase than MAC 26, while microsomal NADPH:cytochrome P-450 reductase levels were similar in both tumour types. Metabolism of MMC by subcellular fractions isolated from both MAC 16 and MAC 26 was quantitated by monitoring the formation of the principle metabolite 2,7-diaminomitosene (2,7-DM) via high-performance liquid chromatography (HPLC). In MAC 16 only, activity displaying the properties of cytosolic DT-diaphorase and microsomal NADPH:cytochrome P-450 reductase was detected and confirmed, using the enzyme inhibitors dicoumarol and cytochrome P-450 reductase antiserum, respectively. The highest level of MMC metabolism was associated with the mitochondrial fraction from both tumours and was the sole enzyme activity detected in MAC 26. The greatest mitochondrial drug metabolism was achieved in the presence of NADPH as cofactor and hypoxia (MAC 16-specific activity, 3.67 +/- 0.58 nmol/30 min/mg; MAC 26 specific-activity, 3.87 +/- 0.71 nmol/30 min/mg) and was unaffected by the addition of the inhibitors dicoumarol and cytochrome P-450 reductase antiserum. NADH-dependent mitochondrial activity was only observed in MAC 16 at approximately 4-fold less than that seen with NADPH. MAC 26 homogenate incubations displayed enhanced metabolism under hypoxia, presumably due to the presence of the identified mitochondrial enzyme. MAC 16 homogenates showed no increase in metabolism under hypoxia, suggesting that other enzyme(s) may be predominant. These data indicate the presence of a novel mitochondrial one-electron reductase capable of metabolising MMC in MAC 16 and MAC 26.