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. 2001 Oct;85(8):1137–1146. doi: 10.1054/bjoc.2001.2056

A novel strategy for NQO1 (NAD(P)H:quinone oxidoreductase, EC 1.6.99.2) mediated therapy of bladder cancer based on the pharmacological properties of EO9

G A Choudry 1,2,3, P A Hamilton Stewart 3, J A Double 1, M R L Krul 5, B Naylor 4, G M Flannigan 3, T K Shah 3, J E Brown 2, R M Phillips 1
PMCID: PMC2375160  PMID: 11710826

Abstract

The indolequinone EO9 demonstrated good preclinical activity but failed to show clinical efficacy against a range of tumours following intravenous drug administration. A significant factor in EO9's failure in the clinic has been attributed to its rapid pharmacokinetic elimination resulting in poor drug delivery to tumours. Intravesical administration of EO9 would circumvent the problem of drug delivery to tumours and the principal objective of this study is to determine whether or not bladder tumours have elevated levels of the enzyme NQO1 (NAD(P)H:quinone oxidoreductase) which plays a key role in activating EO9 under aerobic conditions. Elevated NQO1 levels in human bladder tumour tissue exist in a subset of patients as measured by both immunohistochemical and enzymatic assays. In a panel of human tumour cell lines, EO9 is selectively toxic towards NQO1 rich cell lines under aerobic conditions and potency can be enhanced by reducing extracellular pH. These studies suggest that a subset of bladder cancer patients exist whose tumours possess the appropriate biochemical machinery required to activate EO9. Administration of EO9 in an acidic vehicle could be employed to reduce possible systemic toxicity as any drug absorbed into the blood stream would become relatively inactive due to an increase in pH. © 2001 Cancer Research Campaign   http://www.bjcancer.com

Keywords: bioreductive drugs: EO9, mitomycin C: bladder cancer, NQO1

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Selected References

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  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  2. Butler J., Spanswick V. J., Cummings J. The autoxidation of the reduced forms of EO9. Free Radic Res. 1996 Aug;25(2):141–148. doi: 10.3109/10715769609149919. [DOI] [PubMed] [Google Scholar]
  3. Connors T. A. Bioreductive agents, hypoxic cells and therapy. Eur J Cancer. 1996 Oct;32A(11):1833–1834. doi: 10.1016/0959-8049(96)00305-x. [DOI] [PubMed] [Google Scholar]
  4. Cummings J., Spanswick V. J., Gardiner J., Ritchie A., Smyth J. F. Pharmacological and biochemical determinants of the antitumour activity of the indoloquinone EO9. Biochem Pharmacol. 1998 Feb 1;55(3):253–260. doi: 10.1016/s0006-2952(97)00265-7. [DOI] [PubMed] [Google Scholar]
  5. Cummings J., Spanswick V. J., Tomasz M., Smyth J. F. Enzymology of mitomycin C metabolic activation in tumour tissue: implications for enzyme-directed bioreductive drug development. Biochem Pharmacol. 1998 Aug 15;56(4):405–414. doi: 10.1016/s0006-2952(98)00073-2. [DOI] [PubMed] [Google Scholar]
  6. Dirix L. Y., Tonnesen F., Cassidy J., Epelbaum R., ten Bokkel Huinink W. W., Pavlidis N., Sorio R., Gamucci T., Wolff I., Te Velde A. EO9 phase II study in advanced breast, gastric, pancreatic and colorectal carcinoma by the EORTC Early Clinical Studies Group. Eur J Cancer. 1996 Oct;32A(11):2019–2022. doi: 10.1016/0959-8049(96)00226-2. [DOI] [PubMed] [Google Scholar]
  7. Fitzsimmons S. A., Workman P., Grever M., Paull K., Camalier R., Lewis A. D. Reductase enzyme expression across the National Cancer Institute Tumor cell line panel: correlation with sensitivity to mitomycin C and EO9. J Natl Cancer Inst. 1996 Mar 6;88(5):259–269. doi: 10.1093/jnci/88.5.259. [DOI] [PubMed] [Google Scholar]
  8. Gutierrez P. L. Mechanism(s) of bioreductive activation. The example of diaziquone (AZQ). Free Radic Biol Med. 1989;6(4):405–445. doi: 10.1016/0891-5849(89)90087-7. [DOI] [PubMed] [Google Scholar]
  9. Hendriks H. R., Pizao P. E., Berger D. P., Kooistra K. L., Bibby M. C., Boven E., Dreef-van der Meulen H. C., Henrar R. E., Fiebig H. H., Double J. A. EO9: a novel bioreductive alkylating indoloquinone with preferential solid tumour activity and lack of bone marrow toxicity in preclinical models. Eur J Cancer. 1993;29A(6):897–906. doi: 10.1016/s0959-8049(05)80434-4. [DOI] [PubMed] [Google Scholar]
  10. Herr H. W. Intravesical therapy. A critical review. Urol Clin North Am. 1987 May;14(2):399–404. [PubMed] [Google Scholar]
  11. Hodnick W. F., Sartorelli A. C. Measurement of dicumarol-sensitive NADPH: (menadione-cytochrome c) oxidoreductase activity results in an artifactual assay of DT-diaphorase in cell sonicates. Anal Biochem. 1997 Oct 1;252(1):165–168. doi: 10.1006/abio.1997.2313. [DOI] [PubMed] [Google Scholar]
  12. Kennedy A. S., Raleigh J. A., Perez G. M., Calkins D. P., Thrall D. E., Novotny D. B., Varia M. A. Proliferation and hypoxia in human squamous cell carcinoma of the cervix: first report of combined immunohistochemical assays. Int J Radiat Oncol Biol Phys. 1997 Mar 1;37(4):897–905. doi: 10.1016/s0360-3016(96)00539-1. [DOI] [PubMed] [Google Scholar]
  13. Kuin A., Aalders M., Lamfers M., van Zuidam D. J., Essers M., Beijnen J. H., Smets L. A. Potentiation of anti-cancer drug activity at low intratumoral pH induced by the mitochondrial inhibitor m-iodobenzylguanidine (MIBG) and its analogue benzylguanidine (BG). Br J Cancer. 1999 Feb;79(5-6):793–801. doi: 10.1038/sj.bjc.6690127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Maffezzini M., Simonato A., Zanon M., Raber M., Carmignani G. Up-front intravesical chemotherapy for low stage, low grade recurrent bladder cancer. J Urol. 1996 Jan;155(1):91–93. [PubMed] [Google Scholar]
  15. Maliepaard M., Wolfs A., Groot S. E., de Mol N. J., Janssen L. H. Indoloquinone EO9: DNA interstrand cross-linking upon reduction by DT-diaphorase or xanthine oxidase. Br J Cancer. 1995 Apr;71(4):836–839. doi: 10.1038/bjc.1995.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Malkinson A. M., Siegel D., Forrest G. L., Gazdar A. F., Oie H. K., Chan D. C., Bunn P. A., Mabry M., Dykes D. J., Harrison S. D. 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 Cł. Cancer Res. 1992 Sep 1;52(17):4752–4757. [PubMed] [Google Scholar]
  17. Mossoba M. M., Alizadeh M., Gutierrez P. L. Mechanism for the reductive activation of diaziquone. J Pharm Sci. 1985 Dec;74(12):1249–1254. doi: 10.1002/jps.2600741202. [DOI] [PubMed] [Google Scholar]
  18. Newling D. Intravesical therapy in the management of superficial transitional cell carcinoma of the bladder: the experience of the EORTC GU Group. Br J Cancer. 1990 Apr;61(4):497–499. doi: 10.1038/bjc.1990.112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Oosterlinck W., Kurth K. H., Schröder F., Bultinck J., Hammond B., Sylvester R. A prospective European Organization for Research and Treatment of Cancer Genitourinary Group randomized trial comparing transurethral resection followed by a single intravesical instillation of epirubicin or water in single stage Ta, T1 papillary carcinoma of the bladder. J Urol. 1993 Apr;149(4):749–752. doi: 10.1016/s0022-5347(17)36198-0. [DOI] [PubMed] [Google Scholar]
  20. Pan S. S., Yu F., Hipsher C. Enzymatic and pH modulation of mitomycin C-induced DNA damage in mitomycin C-resistant HCT 116 human colon cancer cells. Mol Pharmacol. 1993 Jun;43(6):870–877. [PubMed] [Google Scholar]
  21. Phillips R. M. Bioreductive activation of a series of analogues of 5-aziridinyl-3-hydroxymethyl-1-methyl-2-[1H-indole-4, 7-dione] prop-beta-en-alpha-ol (EO9) by human DT-diaphorase. Biochem Pharmacol. 1996 Dec 13;52(11):1711–1718. doi: 10.1016/s0006-2952(96)00521-7. [DOI] [PubMed] [Google Scholar]
  22. Phillips R. M., Hulbert P. B., Bibby M. C., Sleigh N. R., Double J. A. In vitro activity of the novel indoloquinone EO-9 and the influence of pH on cytotoxicity. Br J Cancer. 1992 Mar;65(3):359–364. doi: 10.1038/bjc.1992.73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Phillips R. M. Inhibition of DT-diaphorase (NAD(P)H:quinone oxidoreductase, EC 1.6.99.2) by 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and flavone-8-acetic acid (FAA): implications for bioreductive drug development. Biochem Pharmacol. 1999 Jul 15;58(2):303–310. doi: 10.1016/s0006-2952(99)00092-1. [DOI] [PubMed] [Google Scholar]
  24. Phillips R. M., Loadman P. M., Cronin B. P. Evaluation of a novel in vitro assay for assessing drug penetration into avascular regions of tumours. Br J Cancer. 1998 Jun;77(12):2112–2119. doi: 10.1038/bjc.1998.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Plumb J. A., Workman P. Unusually marked hypoxic sensitization to indoloquinone EO9 and mitomycin C in a human colon-tumour cell line that lacks DT-diaphorase activity. Int J Cancer. 1994 Jan 2;56(1):134–139. doi: 10.1002/ijc.2910560124. [DOI] [PubMed] [Google Scholar]
  26. Robertson N., Haigh A., Adams G. E., Stratford I. J. Factors affecting sensitivity to EO9 in rodent and human tumour cells in vitro: DT-diaphorase activity and hypoxia. Eur J Cancer. 1994;30A(7):1013–1019. doi: 10.1016/0959-8049(94)90134-1. [DOI] [PubMed] [Google Scholar]
  27. Saunders M. P., Jaffar M., Patterson A. V., Nolan J., Naylor M. A., Phillips R. M., Harris A. L., Stratford I. J. The relative importance of NADPH: cytochrome c (P450) reductase for determining the sensitivity of human tumour cells to the indolequinone EO9 and related analogues lacking functionality at the C-2 and C-3 positions. Biochem Pharmacol. 2000 Apr 15;59(8):993–996. doi: 10.1016/s0006-2952(99)00405-0. [DOI] [PubMed] [Google Scholar]
  28. Schellens J. H., Planting A. S., van Acker B. A., Loos W. J., de Boer-Dennert M., van der Burg M. E., Koier I., Krediet R. T., Stoter G., Verweij J. Phase I and pharmacologic study of the novel indoloquinone bioreductive alkylating cytotoxic drug E09. J Natl Cancer Inst. 1994 Jun 15;86(12):906–912. doi: 10.1093/jnci/86.12.906. [DOI] [PubMed] [Google Scholar]
  29. Schlager J. J., Powis G. Mitomycin C is not metabolized by but is an inhibitor of human kidney NAD(P)H: (quinone-acceptor)oxidoreductase. Cancer Chemother Pharmacol. 1988;22(2):126–130. doi: 10.1007/BF00257309. [DOI] [PubMed] [Google Scholar]
  30. Siegel D., Beall H., Kasai M., Arai H., Gibson N. W., Ross D. pH-dependent inactivation of DT-diaphorase by mitomycin C and porfiromycin. Mol Pharmacol. 1993 Dec;44(6):1128–1134. [PubMed] [Google Scholar]
  31. Siegel D., Franklin W. A., Ross D. Immunohistochemical detection of NAD(P)H:quinone oxidoreductase in human lung and lung tumors. Clin Cancer Res. 1998 Sep;4(9):2065–2070. [PubMed] [Google Scholar]
  32. Siegel D., Gibson N. W., Preusch P. C., Ross D. Metabolism of mitomycin C by DT-diaphorase: role in mitomycin C-induced DNA damage and cytotoxicity in human colon carcinoma cells. Cancer Res. 1990 Dec 1;50(23):7483–7489. [PubMed] [Google Scholar]
  33. Smitskamp-Wilms E., Giaccone G., Pinedo H. M., van der Laan B. F., Peters G. J. DT-diaphorase activity in normal and neoplastic human tissues; an indicator for sensitivity to bioreductive agents? Br J Cancer. 1995 Oct;72(4):917–921. doi: 10.1038/bjc.1995.433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Smitskamp-Wilms E., Peters G. J., Pinedo H. M., van Ark-Otte J., Giaccone G. Chemosensitivity to the indoloquinone EO9 is correlated with DT-diaphorase activity and its gene expression. Biochem Pharmacol. 1994 Apr 20;47(8):1325–1332. doi: 10.1016/0006-2952(94)90330-1. [DOI] [PubMed] [Google Scholar]
  35. Tolley D. A., Parmar M. K., Grigor K. M., Lallemand G., Benyon L. L., Fellows J., Freedman L. S., Grigor K. M., Hall R. R., Hargreave T. B. The effect of intravesical mitomycin C on recurrence of newly diagnosed superficial bladder cancer: a further report with 7 years of follow up. J Urol. 1996 Apr;155(4):1233–1238. [PubMed] [Google Scholar]
  36. Traver R. D., Horikoshi T., Danenberg K. D., Stadlbauer T. H., Danenberg P. V., Ross D., Gibson N. W. NAD(P)H:quinone oxidoreductase gene expression in human colon carcinoma cells: characterization of a mutation which modulates DT-diaphorase activity and mitomycin sensitivity. Cancer Res. 1992 Feb 15;52(4):797–802. [PubMed] [Google Scholar]
  37. Walton M. I., Smith P. J., Workman P. The role of NAD(P)H: quinone reductase (EC 1.6.99.2, DT-diaphorase) in the reductive bioactivation of the novel indoloquinone antitumor agent EO9. Cancer Commun. 1991 Jul;3(7):199–206. doi: 10.3727/095535491820873164. [DOI] [PubMed] [Google Scholar]
  38. Workman P. Enzyme-directed bioreductive drug development revisited: a commentary on recent progress and future prospects with emphasis on quinone anticancer agents and quinone metabolizing enzymes, particularly DT-diaphorase. Oncol Res. 1994;6(10-11):461–475. [PubMed] [Google Scholar]
  39. Yen W. C., Schmittgen T., Au J. L. Different pH dependency of mitomycin C activity in monolayer and three-dimensional cultures. Pharm Res. 1996 Dec;13(12):1887–1891. doi: 10.1023/a:1016053729362. [DOI] [PubMed] [Google Scholar]

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