Skip to main content
NCBI home page
British Journal of Cancer logoLink to British Journal of Cancer
. 1997;76(10):1338–1347. doi: 10.1038/bjc.1997.558

Overexpression of human NADPH:cytochrome c (P450) reductase confers enhanced sensitivity to both tirapazamine (SR 4233) and RSU 1069.

A V Patterson 1, M P Saunders 1, E C Chinje 1, D C Talbot 1, A L Harris 1, I J Strafford 1
PMCID: PMC2228151  PMID: 9374381

Abstract

P450 reductase (NADPH: cytochrome c (P450) reductase, EC 1.6.2.4) plays an important role in the reductive activation of the bioreductive drug tirapazamine (SR4233). Thus, in a panel of human breast cancer cell lines, expression of P450 reductase correlated with both the hypoxic toxicity and the metabolism of tirapazamine [Patterson et al (1995) Br J Cancer 72: 1144-1150]. To examine this dependence in more detail, the MDA231 cell line, which has the lowest activity of P450 reductase in our breast cell line panel, was transfected with the human P450 reductase cDNA. Isolated clones expressed a 78-kDa protein, which was detected with anti-P450 reductase antibody, and were shown to have up to a 53-fold increase in activity of the enzyme. Using six stable transfected clones covering the 53-fold range of activity of P450 reductase, it was shown that the enzyme activity correlated directly with both hypoxic and aerobic toxicity of tirapazamine, and metabolism of the drug under hypoxic conditions. No metabolism was detected under aerobic conditions. For RSU1069, toxicity was also correlated with P450 reductase activity, but only under hypoxic conditions. Measurable activity of P450 reductase was found in a selection of 14 primary human breast tumours. Activity covered an 18-fold range, which was generally higher than that seen in cell lines but within the range of activity measured in the transfected clones. These results suggest that if breast tumours have significant areas of low oxygen tension, then they are likely to be highly sensitive to the cytotoxic action of tirapazamine and RSU 1069.

Full text

PDF
1338

Images in this article

1340Figure 1
on p.1340

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams G. E., Ahmed I., Sheldon P. W., Stratford I. J. Radiation sensitization and chemopotentiation: RSU 1069, a compound more efficient than misonidazole in vitro and in vivo. Br J Cancer. 1984 May;49(5):571–577. doi: 10.1038/bjc.1984.91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adams G. E., Stratford I. J. Bioreductive drugs for cancer therapy: the search for tumor specificity. Int J Radiat Oncol Biol Phys. 1994 May 15;29(2):231–238. doi: 10.1016/0360-3016(94)90267-4. [DOI] [PubMed] [Google Scholar]
  3. Baker M. A., Zeman E. M., Hirst V. K., Brown J. M. Metabolism of SR 4233 by Chinese hamster ovary cells: basis of selective hypoxic cytotoxicity. Cancer Res. 1988 Nov 1;48(21):5947–5952. [PubMed] [Google Scholar]
  4. Barham H. M., Inglis R., Chinje E. C., Stratford I. J. Development and validation of a spectrophotometric assay for measuring the activity of NADH: cytochrome b5 reductase in human tumour cells. Br J Cancer. 1996 Oct;74(8):1188–1193. doi: 10.1038/bjc.1996.515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Belcourt M. F., Hodnick W. F., Rockwell S., Sartorelli A. C. Differential toxicity of mitomycin C and porfiromycin to aerobic and hypoxic Chinese hamster ovary cells overexpressing human NADPH:cytochrome c (P-450) reductase. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):456–460. doi: 10.1073/pnas.93.1.456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brown J. M. SR 4233 (tirapazamine): a new anticancer drug exploiting hypoxia in solid tumours. Br J Cancer. 1993 Jun;67(6):1163–1170. doi: 10.1038/bjc.1993.220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brown JM, Siim BG. Hypoxia-Specific Cytotoxins in Cancer Therapy. Semin Radiat Oncol. 1996 Jan;6(1):22–36. doi: 10.1053/SRAO0060022. [DOI] [PubMed] [Google Scholar]
  8. Cahill A., White I. N. Reductive metabolism of 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) and the induction of unscheduled DNA synthesis in rat and human derived cell lines. Carcinogenesis. 1990 Aug;11(8):1407–1411. doi: 10.1093/carcin/11.8.1407. [DOI] [PubMed] [Google Scholar]
  9. Denny W. A., Wilson W. R., Hay M. P. Recent developments in the design of bioreductive drugs. Br J Cancer Suppl. 1996 Jul;27:S32–S38. [PMC free article] [PubMed] [Google Scholar]
  10. Fitzsimmons S. A., Lewis A. D., Riley R. J., Workman P. Reduction of 3-amino-1,2,4-benzotriazine-1,4-di-N-oxide (tirapazamine, WIN 59075, SR 4233) to a DNA-damaging species: a direct role for NADPH:cytochrome P450 oxidoreductase. Carcinogenesis. 1994 Aug;15(8):1503–1510. doi: 10.1093/carcin/15.8.1503. [DOI] [PubMed] [Google Scholar]
  11. Griffith O. W. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem. 1980 Jul 15;106(1):207–212. doi: 10.1016/0003-2697(80)90139-6. [DOI] [PubMed] [Google Scholar]
  12. Kennedy K. A. Hypoxic cells as specific drug targets for chemotherapy. Anticancer Drug Des. 1987 Oct;2(2):181–194. [PubMed] [Google Scholar]
  13. Keohane A., Godden J., Stratford I. J., Adams G. E. 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. Br J Cancer. 1990 May;61(5):722–726. doi: 10.1038/bjc.1990.162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Laderoute K., Wardman P., Rauth A. M. Molecular mechanisms for the hypoxia-dependent activation of 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233). Biochem Pharmacol. 1988 Apr 15;37(8):1487–1495. doi: 10.1016/0006-2952(88)90010-x. [DOI] [PubMed] [Google Scholar]
  15. Mason R. P., Holtzman J. L. The mechanism of microsomal and mitochondrial nitroreductase. Electron spin resonance evidence for nitroaromatic free radical intermediates. Biochemistry. 1975 Apr 22;14(8):1626–1632. doi: 10.1021/bi00679a013. [DOI] [PubMed] [Google Scholar]
  16. Morgenstern J. P., Land H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 1990 Jun 25;18(12):3587–3596. doi: 10.1093/nar/18.12.3587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983 Dec 16;65(1-2):55–63. doi: 10.1016/0022-1759(83)90303-4. [DOI] [PubMed] [Google Scholar]
  18. Noss M. B., Panicucci R., McClelland R. A., Rauth A. M. Preparation, toxicity and mutagenicity of 1-methyl-2-nitrosoimidazole. A toxic 2-nitroimidazole reduction product. Biochem Pharmacol. 1988 Jul 1;37(13):2585–2593. doi: 10.1016/0006-2952(88)90250-x. [DOI] [PubMed] [Google Scholar]
  19. O'Neill P., McNeil S. S., Jenkins T. C. Induction of DNA crosslinks in vitro upon reduction of the nitroimidazole-aziridines RSU-1069 and RSU-1131. Biochem Pharmacol. 1987 Jun 1;36(11):1787–1792. doi: 10.1016/0006-2952(87)90239-5. [DOI] [PubMed] [Google Scholar]
  20. Osborne W. R., Miller A. D. Design of vectors for efficient expression of human purine nucleoside phosphorylase in skin fibroblasts from enzyme-deficient humans. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6851–6855. doi: 10.1073/pnas.85.18.6851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Patterson A. V., Barham H. M., Chinje E. C., Adams G. E., Harris A. L., Stratford I. J. Importance of P450 reductase activity in determining sensitivity of breast tumour cells to the bioreductive drug, tirapazamine (SR 4233). Br J Cancer. 1995 Nov;72(5):1144–1150. doi: 10.1038/bjc.1995.478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Patterson A. V., Robertson N., Houlbrook S., Stephens M. A., Adams G. E., Harris A. L., Stratford I. J., Carmichael J. The role of DT-diaphorase in determining the sensitivity of human tumor cells to tirapazamine (SR 4233). Int J Radiat Oncol Biol Phys. 1994 May 15;29(2):369–372. doi: 10.1016/0360-3016(94)90291-7. [DOI] [PubMed] [Google Scholar]
  23. Plumb J. A., Gerritsen M., Workman P. DT-diaphorase protects cells from the hypoxic cytotoxicity of indoloquinone EO9. Br J Cancer. 1994 Dec;70(6):1136–1143. doi: 10.1038/bjc.1994.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Riley R. J., Workman P. Enzymology of the reduction of the potent benzotriazine-di-N-oxide hypoxic cell cytotoxin SR 4233 (WIN 59075) by NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2) purified from Walker 256 rat tumour cells. Biochem Pharmacol. 1992 Jan 22;43(2):167–174. doi: 10.1016/0006-2952(92)90274-m. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Shephard E. A., Palmer C. N., Segall H. J., Phillips I. R. Quantification of cytochrome P450 reductase gene expression in human tissues. Arch Biochem Biophys. 1992 Apr;294(1):168–172. doi: 10.1016/0003-9861(92)90152-m. [DOI] [PubMed] [Google Scholar]
  27. Siim B. G., van Zijl P. L., Brown J. M. Tirapazamine-induced DNA damage measured using the comet assay correlates with cytotoxicity towards hypoxic tumour cells in vitro. Br J Cancer. 1996 Apr;73(8):952–960. doi: 10.1038/bjc.1996.187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Silver A. R., O'Neill P. Interaction of the aziridine moiety of RSU-1069 with nucleotides and inorganic phosphate. Implications for alkylation of DNA. Biochem Pharmacol. 1986 Apr 1;35(7):1107–1112. doi: 10.1016/0006-2952(86)90146-2. [DOI] [PubMed] [Google Scholar]
  29. Stratford I. J., Stephens M. A. The differential hypoxic cytotoxicity of bioreductive agents determined in vitro by the MTT assay. Int J Radiat Oncol Biol Phys. 1989 Apr;16(4):973–976. doi: 10.1016/0360-3016(89)90898-5. [DOI] [PubMed] [Google Scholar]
  30. Stratford I. J., Walling J. M., Silver A. R. The differential cytotoxicity of RSU 1069: cell survival studies indicating interaction with DNA as a possible mode of action. Br J Cancer. 1986 Mar;53(3):339–344. doi: 10.1038/bjc.1986.57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Walton M. I., Wolf C. R., Workman P. Molecular enzymology of the reductive bioactivation of hypoxic cell cytotoxins. Int J Radiat Oncol Biol Phys. 1989 Apr;16(4):983–986. doi: 10.1016/0360-3016(89)90900-0. [DOI] [PubMed] [Google Scholar]
  32. Walton M. I., Wolf C. R., Workman P. The role of cytochrome P450 and cytochrome P450 reductase in the reductive bioactivation of the novel benzotriazine di-N-oxide hypoxic cytotoxin 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233, WIN 59075) by mouse liver. Biochem Pharmacol. 1992 Jul 22;44(2):251–259. doi: 10.1016/0006-2952(92)90007-6. [DOI] [PubMed] [Google Scholar]
  33. Walton M. I., Workman P. Enzymology of the reductive bioactivation of SR 4233. A novel benzotriazine di-N-oxide hypoxic cell cytotoxin. Biochem Pharmacol. 1990 Jun 1;39(11):1735–1742. doi: 10.1016/0006-2952(90)90119-6. [DOI] [PubMed] [Google Scholar]
  34. Walton M. I., Workman P. Nitroimidazole bioreductive metabolism. Quantitation and characterisation of mouse tissue benznidazole nitroreductases in vivo and in vitro. Biochem Pharmacol. 1987 Mar 15;36(6):887–896. doi: 10.1016/0006-2952(87)90181-x. [DOI] [PubMed] [Google Scholar]
  35. Wang J., Biedermann K. A., Wolf C. R., Brown J. M. Metabolism of the bioreductive cytotoxin SR 4233 by tumour cells: enzymatic studies. Br J Cancer. 1993 Feb;67(2):321–325. doi: 10.1038/bjc.1993.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Whitmore G. F., Gulyas S. Studies on the toxicity of RSU-1069. Int J Radiat Oncol Biol Phys. 1986 Jul;12(7):1219–1222. doi: 10.1016/0360-3016(86)90262-2. [DOI] [PubMed] [Google Scholar]
  37. Wilson J. M., Johnston D. E., Jefferson D. M., Mulligan R. C. Correction of the genetic defect in hepatocytes from the Watanabe heritable hyperlipidemic rabbit. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4421–4425. doi: 10.1073/pnas.85.12.4421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Workman P., Stratford I. J. The experimental development of bioreductive drugs and their role in cancer therapy. Cancer Metastasis Rev. 1993 Jun;12(2):73–82. doi: 10.1007/BF00689802. [DOI] [PubMed] [Google Scholar]
  39. Zeman E. M., Brown J. M., Lemmon M. J., Hirst V. K., Lee W. W. SR-4233: a new bioreductive agent with high selective toxicity for hypoxic mammalian cells. Int J Radiat Oncol Biol Phys. 1986 Jul;12(7):1239–1242. doi: 10.1016/0360-3016(86)90267-1. [DOI] [PubMed] [Google Scholar]
  40. van den Hoff M. J., Moorman A. F., Lamers W. H. Electroporation in 'intracellular' buffer increases cell survival. Nucleic Acids Res. 1992 Jun 11;20(11):2902–2902. doi: 10.1093/nar/20.11.2902. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from British Journal of Cancer are provided here courtesy of Cancer Research UK

RESOURCES