1 2003 Vol: 10(1):40-48. DOI: 10.1038/sj.cgt.7700522

Bioreductive GDEPT using cytochrome P450 3A4 in combination with AQ4N

The bioreductive drug, AQ4N, is metabolized under hypoxic conditions and has been shown to enhance the antitumor effects of radiation and chemotherapy drugs. We have investigated the role of cytochrome P450 3A4 (CYP3A4) in increasing the metabolism of AQ4N using a gene-directed enzyme prodrug therapy (GDEPT) strategy. RIF-1 murine tumor cells were transfected with a mammalian expression vector containing CYP3A4 cDNA. In vitro AQ4N metabolism, DNA damage, and clonogenic cell kill were assessed following exposure of transfected and parental control cells to AQ4N. The presence of exogenous CYP3A4 increased the metabolism of AQ4N and significantly enhanced the ability of the drug to cause DNA strand breaks and clonogenic cell death. Cotransfection of CYP reductase with CYP3A4 showed a small enhancement of the effect in the DNA damage assay only. A single injection of CYP3A4 into established RIF-1 murine tumors increased the metabolism of AQ4N, and when used in combination with radiation, three of nine tumors were locally controlled for >60 days. This is the first demonstration that CYPs alone can be used in a GDEPT strategy for bioreduction of the cytotoxic prodrug, AQ4N. AQ4N is the only CYP-activated bioreductive agent in clinical trials. Combination with a GDEPT strategy may offer a further opportunity for targeting radiation-resistant and chemo-resistant hypoxic tumor cells.

Mentions
Figures
Figure 1: In vitro metabolism of AQ4N in S9 protein (10 mg/mL) fractions prepared from stable CYP3A4-transfected and parental RIF-1 cells. AQ4N loss over time was determined by HPLC. Figure 2: DNA damage (mean tail moment) assessed using the alkaline comet assay in parental nontransfected RIF-1 cells, a range of experimental controls, and cells transiently transfected with CYP3A4, CYP3A4/CYPRed. Cells were assessed for DNA damage after exposure of all groups to 20 M AQ4N under anoxia/hypoxia (except one of the parental control groups) (a) immediately and (b) 24 hours later. The results are the meanSE of three independent experiments. Figure 3: Surviving fraction in cells transiently transfected with CYP3A4, CYP3A4/CYPRed, and various controls following treatment with AQ4N (0–12 M) (a) under oxic (95% air/5% CO2) conditions and (b) anoxic (95% N2/5% CO2) conditions. Surviving fraction was assessed using a clonogenic assay. The results are the meanSE of three independent experiments. Figure 4: Tumor growth in RIF-1 murine tumors in vivo following injection of CYP3A4 in combination with AQ4N and radiation. A: Kaplan-Meier survival curve showing the percent of mice with tumors reaching their VQT at various times following injection with CYP3A4/liposomal mix and exposed to AQ4N (100 mg/kg) and radiation (20 Gy) 24 hours later. Controls included mice with no tumor treatment, mice with tumors exposed to X-rays alone, mice with tumors exposed to X-rays in combination with AQ4N and injected with liposomal agent only, and mice with tumors exposed to X-rays in combination with AQ4N and injected with liposomal agent and empty vector. Each symbol represents a different animal. There were five to nine animals in each group (see Table 1). B: Photograph of two mice on day 20 of the experiment. Both mice 1 and 2 were treated with AQ4N and radiation; however, mouse 1 was also injected with the CYP3A4 construct. Figure 5: Levels of CYP3A4 protein in control and CYP3A4-injected RIF-1 tumors. Western blot showing CYP3A4 levels (top panel) in established control and treated RIF-1 tumors, 24 hours following a single injection of CYP3A4. Protein loading was assessed using a -actin antibody (lower panel).
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References
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    • . . . Research indicates that prodrug systems, together with gene therapy, may provide the desired specificity for cancer treatment.1 Gene-directed enzyme prodrug therapy (GDEPT) delivers a gene that codes for an enzyme that is capable of converting a prodrug to its cytotoxic product.2 Several GDEPT strategies have been tested to date: herpes simplex virus thymidine kinase (HSV-TK) converts the nontoxic prodrug ganciclovir (GCV) into a metabolite that kills replicating cells; cytosine deaminase (CD), which converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU); nitroreductase (NTR), which reduces the mustard prodrug, CB1954, to a toxic bifunctional alkylating agent; carboxypeptidase G2, which converts CMDA to a cytotoxic DNA cross-linking mustard and horseradish peroxidase that converts indole-3-acetic acid to a number of toxic products (reviewed in Ref. [1]) . . .
    • . . . Hypoxic cells are resistant to many of the current GDEPT strategies mentioned above; activation of CPA is an oxygen-dependent reaction and there is a loss of cytotoxicity of 5-FU and CD/5-FC in hypoxic tumor cells.1 Furthermore, in other systems, the activated prodrug requires further metabolism by endogenous enzymes (e.g., HSV-TK, CD/5-FC, and NTR/CB1954) and, in many cases, the active drugs are not membrane-permeable and depend on cell-to-cell contact for transfer into neighbouring cells to induce bystander effects (HSV/TK). . . .
    • . . . Ideally, the activated drug should be at least 100-fold more toxic than the prodrug and have the ability to diffuse to surrounding cells.1 This was apparent in this study . . .
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    • . . . Research indicates that prodrug systems, together with gene therapy, may provide the desired specificity for cancer treatment.1 Gene-directed enzyme prodrug therapy (GDEPT) delivers a gene that codes for an enzyme that is capable of converting a prodrug to its cytotoxic product.2 Several GDEPT strategies have been tested to date: herpes simplex virus thymidine kinase (HSV-TK) converts the nontoxic prodrug ganciclovir (GCV) into a metabolite that kills replicating cells; cytosine deaminase (CD), which converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU); nitroreductase (NTR), which reduces the mustard prodrug, CB1954, to a toxic bifunctional alkylating agent; carboxypeptidase G2, which converts CMDA to a cytotoxic DNA cross-linking mustard and horseradish peroxidase that converts indole-3-acetic acid to a number of toxic products (reviewed in Ref. [1]) . . .
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    • . . . For example, the CYP2B6 isoform converts the prodrug cyclophosphamide (CPA) to an alkylating toxic phosphoramide mustard that results in apoptosis.3,4,5 More recently, macrophages have been used to deliver a CYP2B6/CPA gene combination to hypoxic regions of tumor.6 Also, a CYP2B6/P450 reductase combination has been used to enhance the antitumor effects of CPA in combination with the bioreductive drug tirapazamine (TPZ).7 In the present study, we used a CYP isoform in a GDEPT strategy to metabolize the bioreductive prodrug AQ4N [1,4-bis[2-(dimethylamino-N-oxide)ethyl]amino-5,8-di-hydroxyanthracene-9,10-dione] to its cytotoxic product, AQ4. . . .
    • . . . The CYPs 2B1, 2C6, and 2C11 have been shown to be involved in the activation of CPA,27 and CYP3A has been shown to activate ifosphamide.28 In addition, tumors overexpressing CYP2B6 in vivo have been shown to increase the metabolism of CPA.3,4,21 When CYP reductase was also overexpressed, increased antitumor efficacy was observed following administration of both CPA (presumed to be metabolized by CYP2B6) and TPZ (presumed to be metabolized by CYP reductase).7 . . .
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    • . . . For example, the CYP2B6 isoform converts the prodrug cyclophosphamide (CPA) to an alkylating toxic phosphoramide mustard that results in apoptosis.3,4,5 More recently, macrophages have been used to deliver a CYP2B6/CPA gene combination to hypoxic regions of tumor.6 Also, a CYP2B6/P450 reductase combination has been used to enhance the antitumor effects of CPA in combination with the bioreductive drug tirapazamine (TPZ).7 In the present study, we used a CYP isoform in a GDEPT strategy to metabolize the bioreductive prodrug AQ4N [1 . . .
    • . . . The CYPs 2B1, 2C6, and 2C11 have been shown to be involved in the activation of CPA,27 and CYP3A has been shown to activate ifosphamide.28 In addition, tumors overexpressing CYP2B6 in vivo have been shown to increase the metabolism of CPA.3,4,21 When CYP reductase was also overexpressed, increased antitumor efficacy was observed following administration of both CPA (presumed to be metabolized by CYP2B6) and TPZ (presumed to be metabolized by CYP reductase).7 . . .
  5. Kan O, Griffiths L & Baban D, et al. Direct retroviral delivery of human cytochrome P450 2B6 for gene-directed enzyme prodrug therapy of cancer. Cancer Gene Ther. 2001; 8: 473-482 , .
    • . . . For example, the CYP2B6 isoform converts the prodrug cyclophosphamide (CPA) to an alkylating toxic phosphoramide mustard that results in apoptosis.3,4,5 More recently, macrophages have been used to deliver a CYP2B6/CPA gene combination to hypoxic regions of tumor.6 Also, a CYP2B6/P450 reductase combination has been used to enhance the antitumor effects of CPA in combination with the bioreductive drug tirapazamine (TPZ).7 In the present study, we used a CYP isoform in a GDEPT strategy to metabolize the bioreductive prodrug AQ4N [1,4-bis[2-(dimethylamino-N-oxide)ethyl]amino-5,8-di-hydroxyanthracene-9,10-dione] to its cytotoxic product, AQ4. . . .
  6. Griffiths L, Binley K & Iqball S, et al. The macrophage - a novel system to deliver gene therapy to pathological hypoxia. Gene Ther. 2000; 7: 255-262 , .
    • . . . For example, the CYP2B6 isoform converts the prodrug cyclophosphamide (CPA) to an alkylating toxic phosphoramide mustard that results in apoptosis.3,4,5 More recently, macrophages have been used to deliver a CYP2B6/CPA gene combination to hypoxic regions of tumor.6 Also, a CYP2B6/P450 reductase combination has been used to enhance the antitumor effects of CPA in combination with the bioreductive drug tirapazamine (TPZ).7 In the present study, we used a CYP isoform in a GDEPT strategy to metabolize the bioreductive prodrug AQ4N [1,4-bis[2-(dimethylamino-N-oxide)ethyl]amino-5,8-di-hydroxyanthracene-9,10-dione] to its cytotoxic product, AQ4. . . .
  7. Jounaidi Y & Waxman DJ. Combination of the bioreductive drug tirapazamine with the chemotherapeutic prodrug cyclophosphamide for P450/P450-reductase-based cancer gene therapy. Cancer Res. 2000; 60: 3761-3769 , .
  8. Hejmadi MV, McKeown SR, Friery OP, McIntyre IA, Patterson LH & Hirst DG. DNA damage following combination of radiation with the bioreductive drug AQ4N: possible selective toxicity to oxic and hypoxic tumour cells. Br J Cancer. 1996; 73: 499-505 , .
    • . . . AQ4N prodrug activation displays a high selectivity towards hypoxic cells,8,9 which are a specific feature of tumors and are known to limit the success of conventional treatments.10 We have already demonstrated in vivo that AQ4N can significantly improve antitumor efficacy in combination with oxic cell cytotoxins such as radiation, CPA, and cisplatin.9,11,12,13,14,15 Clinical trials of AQ4N have now been initiated. . . .
  9. Patterson LH, McKeown SR & Ruparelia K, et al. Enhancement of chemotherapy and radiotherapy of murine tumours by AQ4N, a bioreductively activated anti-tumour agent. Br J Cancer. 2000; 82: 1984-1990 , .
    • . . . AQ4N prodrug activation displays a high selectivity towards hypoxic cells,8,9 which are a specific feature of tumors and are known to limit the success of conventional treatments.10 We have already demonstrated in vivo that AQ4N can significantly improve antitumor efficacy in combination with oxic cell cytotoxins such as radiation, CPA, and cisplatin.9,11,12,13,14,15 Clinical trials of AQ4N have now been initiated. . . .
  10. Hockel M & Vaupel P. Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. J Natl Cancer Inst. 2001; 93: 266-276 , .
    • . . . AQ4N prodrug activation displays a high selectivity towards hypoxic cells,8,9 which are a specific feature of tumors and are known to limit the success of conventional treatments.10 We have already demonstrated in vivo that AQ4N can significantly improve antitumor efficacy in combination with oxic cell cytotoxins such as radiation, CPA, and cisplatin.9,11,12,13,14,15 Clinical trials of AQ4N have now been initiated. . . .
  11. McKeown SR, Hejmadi MV, McIntyre IA, McAleer JJ & Patterson LH. AQ4N: an alkylaminoanthraquinone N-oxide showing bioreductive potential and positive interaction with radiation in vivo. Br J Cancer. 1995; 72: 76-81 , .
    • . . . AQ4N prodrug activation displays a high selectivity towards hypoxic cells,8,9 which are a specific feature of tumors and are known to limit the success of conventional treatments.10 We have already demonstrated in vivo that AQ4N can significantly improve antitumor efficacy in combination with oxic cell cytotoxins such as radiation, CPA, and cisplatin.9,11,12,13,14,15 Clinical trials of AQ4N have now been initiated. . . .
  12. McKeown SR, Friery OP, McIntyre IA, Hejmadi MV, Patterson LH & Hirst DG. Evidence for a therapeutic gain when AQ4N or tirapazamine is combined with radiation. Br J Cancer Suppl. 1996; 27: S39-S42 , .
    • . . . AQ4N prodrug activation displays a high selectivity towards hypoxic cells,8,9 which are a specific feature of tumors and are known to limit the success of conventional treatments.10 We have already demonstrated in vivo that AQ4N can significantly improve antitumor efficacy in combination with oxic cell cytotoxins such as radiation, CPA, and cisplatin.9,11,12,13,14,15 Clinical trials of AQ4N have now been initiated. . . .
  13. Friery OP, Gallagher R & Murray MM, et al. Enhancement of the anti-tumour effect of cyclophosphamide by the bioreductive drugs AQ4N and tirapazamine. Br J Cancer. 2000; 82: 1469-1473 , .
    • . . . AQ4N prodrug activation displays a high selectivity towards hypoxic cells,8,9 which are a specific feature of tumors and are known to limit the success of conventional treatments.10 We have already demonstrated in vivo that AQ4N can significantly improve antitumor efficacy in combination with oxic cell cytotoxins such as radiation, CPA, and cisplatin.9,11,12,13,14,15 Clinical trials of AQ4N have now been initiated. . . .
  14. Patterson LH & McKeown SR. AQ4N: a new approach to hypoxia-activated cancer chemotherapy. Br J Cancer. 2000; 83: 1589-1593 , .
    • . . . AQ4N prodrug activation displays a high selectivity towards hypoxic cells,8,9 which are a specific feature of tumors and are known to limit the success of conventional treatments.10 We have already demonstrated in vivo that AQ4N can significantly improve antitumor efficacy in combination with oxic cell cytotoxins such as radiation, CPA, and cisplatin.9,11,12,13,14,15 Clinical trials of AQ4N have now been initiated. . . .
  15. Gallagher R, Hughes CM & Murray MM, et al. The chemopotentiation of cisplatin by the novel bioreductive drug AQ4N. Br J Cancer. 2001; 85: 625-629 , .
    • . . . AQ4N prodrug activation displays a high selectivity towards hypoxic cells,8,9 which are a specific feature of tumors and are known to limit the success of conventional treatments.10 We have already demonstrated in vivo that AQ4N can significantly improve antitumor efficacy in combination with oxic cell cytotoxins such as radiation, CPA, and cisplatin.9,11,12,13,14,15 Clinical trials of AQ4N have now been initiated. . . .
  16. Smith PJ, Desnoyers R, Blunt N, Giles Y, Patterson LH & Watson JV. Flow cytometric analysis and confocal imaging of anticancer alkylaminoanthraquinones and their N-oxides in intact human cells using 647-nm krypton laser excitation. Cytometry. 1997; 27: 43-53 , .
  17. Wilson WR, Denny WA & Pullen SM, et al. Tertiary amine N-oxides as bioreductive drugs: DACA N-oxide, nitracrine N-oxide and AQ4N. Br J Cancer Suppl. 1996; 27: S43-S47 , .
    • . . . AQ4N is reduced under hypoxic conditions to its toxic product, AQ4, by a process that is inhibited by molecular oxygen.17 AQ4N does not bind to DNA, but AQ4 has a high DNA affinity and is a potent topoisomerase II inhibitor.16,18 Studies using ketoconazole and carbon monoxide, global inhibitors of the CYP superfamily, implicate CYP enzymes in the metabolism of AQ4N to AQ4 in rat liver microsomes.19 Furthermore, AQ4N metabolism is correlated with CYP3A activity as determined using phenotyped human liver microsomes.20 It follows that for AQ4N to be successful in the clinic, both hypoxia and the correct CYP will need to be present in the tumor . . .
    • . . . There was a clear difference in clonogenic cell kill between oxic (Fig 3A) and anoxic (Fig 3B) conditions, confirming previous reports that AQ4N is metabolized only under hypoxic/anoxic conditions7,17 (three-way ANOVA; df=1, F ratio=58.8834, P<.0001) . . .
  18. Patterson LH. Rationale for the use of aliphatic N-oxides of cytotoxic anthraquinones as prodrug DNA binding agents: a new class of bioreductive agent. Cancer Metastasis Rev. 1993; 12: 119-134 , .
  19. Raleigh SM, Wanogho E, Burke MD & Patterson LH. Rat cytochromes P450 (CYP) specifically contribute to the reductive bioactivation of AQ4N, an alkylaminoanthraquinone-di-N-oxide anticancer prodrug. Xenobiotica. 1999; 29: 1115-1122 , .
    • . . . AQ4N is reduced under hypoxic conditions to its toxic product, AQ4, by a process that is inhibited by molecular oxygen.17 AQ4N does not bind to DNA, but AQ4 has a high DNA affinity and is a potent topoisomerase II inhibitor.16,18 Studies using ketoconazole and carbon monoxide, global inhibitors of the CYP superfamily, implicate CYP enzymes in the metabolism of AQ4N to AQ4 in rat liver microsomes.19 Furthermore, AQ4N metabolism is correlated with CYP3A activity as determined using phenotyped human liver microsomes.20 It follows that for AQ4N to be successful in the clinic, both hypoxia and the correct CYP will need to be present in the tumor . . .
    • . . . Generally, CYP activity is low or even absent in cells cultured in vitro.30 Previously, this has led to difficulties in unequivocally establishing the potential of AQ4N as a metabolically activated antitumor agent.18 Several studies have demonstrated indirectly the importance of CYPs in the bioreductive activation and cytotoxicity of AQ4N.18,19,20 However, this study has, for the first time, allowed effective assessment of CYP-mediated AQ4N cytotoxicity in vitro and in vivo using delivery of specific CYPs by molecular approaches . . .
    • . . . This supports previous inhibitor studies, which show that AQ4N metabolism correlates with CYP3A activity.19,20 Interestingly, the in vitro metabolism study demonstrated that the parental cell line was also able to metabolize AQ4N, although to a much lesser extent than the stable transfectant . . .
  20. Raleigh SM, Wanogho E, Burke MD, McKeown SR & Patterson LH. Involvement of human cytochromes P450 (CYP) in the reductive metabolism of AQ4N, a hypoxia activated anthraquinone di-N-oxide prodrug. Int J Radiat Oncol Biol Phys. 1998; 42: 763-767 , .
    • . . . AQ4N is reduced under hypoxic conditions to its toxic product, AQ4, by a process that is inhibited by molecular oxygen.17 AQ4N does not bind to DNA, but AQ4 has a high DNA affinity and is a potent topoisomerase II inhibitor.16,18 Studies using ketoconazole and carbon monoxide, global inhibitors of the CYP superfamily, implicate CYP enzymes in the metabolism of AQ4N to AQ4 in rat liver microsomes.19 Furthermore, AQ4N metabolism is correlated with CYP3A activity as determined using phenotyped human liver microsomes.20 It follows that for AQ4N to be successful in the clinic, both hypoxia and the correct CYP will need to be present in the tumor . . .
    • . . . Generally, CYP activity is low or even absent in cells cultured in vitro.30 Previously, this has led to difficulties in unequivocally establishing the potential of AQ4N as a metabolically activated antitumor agent.18 Several studies have demonstrated indirectly the importance of CYPs in the bioreductive activation and cytotoxicity of AQ4N.18,19,20 However, this study has, for the first time, allowed effective assessment of CYP-mediated AQ4N cytotoxicity in vitro and in vivo using delivery of specific CYPs by molecular approaches . . .
  21. Chen L, Yu LJ & Waxman DJ. Potentiation of cytochrome P450/cyclophosphamide-based cancer gene therapy by coexpression of the P450 reductase gene. Cancer Res. 1997; 57: 4830-4837 , .
  22. Ding S, Yao D, Burchell B, Wolf CR & Friedberg T. High levels of recombinant CYP3A4 expression in Chinese hamster ovary cells are modulated by coexpressed human P450 reductase and hemin supplementation. Arch Biochem Biophys. 1997; 348: 403-410 , .
  23. Twentyman PR. Comparative chemosensitivity of exponential- versus plateau-phase cells in both in vitro model systems. Cancer Treat Rep. 1976; 60: 1719-1722 , .
    • . . . RIF-1 murine fibrosarcomas were routinely maintained by the protocol of Twentyman et al.23 In culture, cells were maintained in RPMI 1640 with L-glutamine (10 mM) (Gibco BRL, Paisley, UK) supplemented with fetal bovine serum (15%) (Globe Pharm, Surrey, UK) and penicillin/streptomycin (1%) (Sigma, Poole, UK) . . .
  24. Singh NP, McCoy MT, Tice RR & Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 1988; 175: 184-191 , .
    • . . . The alkaline single cell gel (comet) electrophoresis assay24 was used to assess DNA damage in transfected RIF-1 cells at 0 and 24 hours after drug treatment . . .
  25. McKelvey-Martin VJ, Green MH, Schmezer P, Pool-Zobel BL, DeMeo MP & Collins A. The single cell gel electrophoresis assay (comet assay): a European review. Mutat Res. 1993; 288: 47-63 , .
    • . . . Tail moment was found to be the most reliable indicator for measuring DNA damage.25 The mean tail moment was calculated by evaluating 50 cells from each of the three slides for each treatment group . . .
  26. Swaine DJ, Loadman PM, Bibby MC, Graham MA & Patterson LH. High-performance liquid chromatographic analysis of AQ4N, an alkylaminoanthraquinone N-oxide. J Chromatogr B Biomed Sci Appl. 2000; 742: 239-245 , .
  27. Clarke L & Waxman DJ. Oxidative metabolism of cyclophosphamide: identification of the hepatic monooxygenase catalysts of drug activation. Cancer Res. 1989; 49: 2344-2350 , .
    • . . . The CYPs 2B1, 2C6, and 2C11 have been shown to be involved in the activation of CPA,27 and CYP3A has been shown to activate ifosphamide.28 In addition, tumors overexpressing CYP2B6 in vivo have been shown to increase the metabolism of CPA.3,4,21 When CYP reductase was also overexpressed, increased antitumor efficacy was observed following administration of both CPA (presumed to be metabolized by CYP2B6) and TPZ (presumed to be metabolized by CYP reductase).7 . . .
  28. Weber GF & Waxman DJ. Activation of the anti-cancer drug ifosphamide by rat liver microsomal P450 enzymes. Biochem Pharmacol. 1993; 45: 1685-1694 , .
    • . . . The CYPs 2B1, 2C6, and 2C11 have been shown to be involved in the activation of CPA,27 and CYP3A has been shown to activate ifosphamide.28 In addition, tumors overexpressing CYP2B6 in vivo have been shown to increase the metabolism of CPA.3,4,21 When CYP reductase was also overexpressed, increased antitumor efficacy was observed following administration of both CPA (presumed to be metabolized by CYP2B6) and TPZ (presumed to be metabolized by CYP reductase).7 . . .
  29. Brown JM. Exploiting the hypoxic cancer cell: mechanisms and therapeutic strategies. Mol Med Today. 2000; 6: 157-162 , .
    • . . . The evidence for hypoxic regions in tumors is now generally well accepted.29 These hypoxic cells are a significant problem in the control of solid tumors because they are two to three times more resistant to radiotherapy and are less sensitive to most chemotherapeutic agents.29 The concept of metabolic activation of a drug to a cytotoxic agent within the hypoxic environment makes direct use of this property to target treatment-resistant hypoxic cells . . .
  30. Patterson LH, McKeown SR, Robson T, Gallagher R, Raleigh SM & Orr S. Antitumour prodrug development using cytochrome P450 (CYP) mediated activation. Anticancer Drug Des. 1999; 14: 473-486 , .
    • . . . Generally, CYP activity is low or even absent in cells cultured in vitro.30 Previously, this has led to difficulties in unequivocally establishing the potential of AQ4N as a metabolically activated antitumor agent.18 Several studies have demonstrated indirectly the importance of CYPs in the bioreductive activation and cytotoxicity of AQ4N.18,19,20 However, this study has, for the first time, allowed effective assessment of CYP-mediated AQ4N cytotoxicity in vitro and in vivo using delivery of specific CYPs by molecular approaches . . .
  31. Hejmadi MV, McKeown SR, Friery OP, McIntyre IA, Patterson LH & Hirst DG. DNA damage following combination of radiation with the bioreductive drug AQ4N: possible selective toxicity to oxic and hypoxic tumour cells. Br J Cancer. 1996; 73: 499-505 , .
    • . . . Single strand breaks, as measured using the alkaline comet assay, have previously been shown to be a mechanism of AQ4N-induced cell death.31 It is likely that the DNA strand breaks observed are indicative of topoisomerase inhibition by AQ4, produced following reduction of AQ4N through a CYP-mediated process.32 Therefore, the increase in DNA damage found in the cells transfected with CYP3A4 implicates this isoform in AQ4N activation leading to tumor cell kill . . .
    • . . . This supports previous work, which showed that AQ4 does not cause strand breaks per se, but compromises the ability of cells to proceed through the cell cycle.31 This is evidenced in the comet assay by increasing numbers of damaged cells with time because the binding of AQ4 to DNA/topoisomerase II results in DNA damage/cell death . . .
  32. Smith PJ, Blunt NJ, Desnoyers R, Giles Y & Patterson LH. DNA topoisomerase II-dependent cytotoxicity of alkylaminoanthraquinones and their N-oxides. Cancer Chemother Pharmacol. 1997; 39: 455-461 , .
    • . . . Single strand breaks, as measured using the alkaline comet assay, have previously been shown to be a mechanism of AQ4N-induced cell death.31 It is likely that the DNA strand breaks observed are indicative of topoisomerase inhibition by AQ4, produced following reduction of AQ4N through a CYP-mediated process.32 Therefore, the increase in DNA damage found in the cells transfected with CYP3A4 implicates this isoform in AQ4N activation leading to tumor cell kill . . .
  33. Huber BE, Austin EA, Richards CA, Davis ST & Good SS. Metabolism of 5-fluorocytosine to 5-fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene: significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase. Proc Natl Acad Sci USA. 1994; 91: 8302-8306 , .
    • . . . In other GDEPT strategies, transfection efficiencies as low as 4–10% in solid tumors resulted in eradication.33 A number of studies suggest that tumor tissues down-regulate intercellular GAP junctions, adversely affecting the bystander effect of some agents, e.g., GCV.34,35 However, there is evidence that an AQ4-mediated bystander effect may occur independently of gap junctions by passive transportation in cellular systems.36,37,38 . . .
  34. Mesnil M, Piccoli C, Tiraby G, Willecke K & Yamasaki H. Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins. Proc Natl Acad Sci USA. 1996; 93: 1831-1835 , .
    • . . . In other GDEPT strategies, transfection efficiencies as low as 4–10% in solid tumors resulted in eradication.33 A number of studies suggest that tumor tissues down-regulate intercellular GAP junctions, adversely affecting the bystander effect of some agents, e.g., GCV.34,35 However, there is evidence that an AQ4-mediated bystander effect may occur independently of gap junctions by passive transportation in cellular systems.36,37,38 . . .
  35. Touraine RL, Ishii-Morita H, Ramsey WJ & Blaese RM. The bystander effect in the HSVtk/ganciclovir system and its relationship to gap junctional communication. Gene Ther. 1998; 5: 1705-1711 , .
    • . . . In other GDEPT strategies, transfection efficiencies as low as 4–10% in solid tumors resulted in eradication.33 A number of studies suggest that tumor tissues down-regulate intercellular GAP junctions, adversely affecting the bystander effect of some agents, e.g., GCV.34,35 However, there is evidence that an AQ4-mediated bystander effect may occur independently of gap junctions by passive transportation in cellular systems.36,37,38 . . .
  36. Freeman SM, Abboud CN & Whartenby KA, et al. The "bystander effect": tumor regression when a fraction of the tumor mass is genetically modified. Cancer Res. 1993; 53: 5274-5283 , .
  37. Frank DK, Frederick MJ, Liu TJ & Clayman GL. Bystander effect in the adenovirus-mediated wild-type p53 gene therapy model of human squamous cell carcinoma of the head and neck. Clin Cancer Res. 1998; 4: 2521-2528 , .
    • . . . In other GDEPT strategies, transfection efficiencies as low as 4–10% in solid tumors resulted in eradication.33 A number of studies suggest that tumor tissues down-regulate intercellular GAP junctions, adversely affecting the bystander effect of some agents, e.g., GCV.34,35 However, there is evidence that an AQ4-mediated bystander effect may occur independently of gap junctions by passive transportation in cellular systems.36,37,38 . . .
  38. Massaad L, deWaziers I & Ribrag V, et al. Comparison of mouse and human colon tumors with regard to phase I and phase II drug-metabolizing enzyme systems. Cancer Res. 1992; 52: 6567-6575 , .
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