Skip to main content
NCBI home page
British Journal of Cancer logoLink to British Journal of Cancer
. 1995 Nov;72(5):1144–1150. doi: 10.1038/bjc.1995.478

Importance of P450 reductase activity in determining sensitivity of breast tumour cells to the bioreductive drug, tirapazamine (SR 4233).

A V Patterson 1, H M Barham 1, E C Chinje 1, G E Adams 1, A L Harris 1, I J Stratford 1
PMCID: PMC2033939  PMID: 7577460

Abstract

P450 reductase (NADPH:cytochrome P450 reductase, EC 1.6.2.4) is known to be important in the reductive activation of the benzotriazene-di-N-oxide tirapazamine (SR 4233). Using a panel of six human breast adenocarcinoma cell lines we have examined the relationship between P450 reductase activity and sensitivity to tirapazamine. The toxicity of tirapazamine was found to correlate strongly with P450 reductase activity following an acute (3 h) exposure under hypoxic conditions, the drug being most toxic in the cell lines with the highest P450 reductase activity. A similar correlation was also observed following a chronic (96 h) exposure to the drug in air but not following acute (3 h) exposure in air. We have also determined the ability of lysates prepared from the cell lines to metabolise tirapazamine to its two-electron reduced product, SR 4317, under hypoxic conditions using NADPH as an electron donor. The rate of SR 4317 formation was found to correlate both with P450 reductase activity and with sensitivity to tirapazamine, the highest rates of SR 4317 formation being associated with the highest levels of P450 reductase activity and the greatest sensitivity to the drug. These findings indicate a major role for P450 reductase in determining the hypoxic toxicity of tirapazamine in breast tumour cell lines.

Full text

PDF
1144

Images in this article

1147Figure 4
on p.1147

Selected References

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

  1. 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]
  2. Adams G. E., Stratford I. J. Hypoxia-mediated nitro-heterocyclic drugs in the radio- and chemotherapy of cancer. An overview. Biochem Pharmacol. 1986 Jan 1;35(1):71–76. doi: 10.1016/0006-2952(86)90560-5. [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. Biedermann K. A., Wang J., Graham R. P., Brown J. M. SR 4233 cytotoxicity and metabolism in DNA repair-competent and repair-deficient cell cultures. Br J Cancer. 1991 Mar;63(3):358–362. doi: 10.1038/bjc.1991.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Brown J. M., Lemmon M. J. Potentiation by the hypoxic cytotoxin SR 4233 of cell killing produced by fractionated irradiation of mouse tumors. Cancer Res. 1990 Dec 15;50(24):7745–7749. [PubMed] [Google Scholar]
  7. 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]
  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. Carmichael J., DeGraff W. G., Gazdar A. F., Minna J. D., Mitchell J. B. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 1987 Feb 15;47(4):936–942. [PubMed] [Google Scholar]
  10. Chaplin D. J., Horsman M. R., Aoki D. S. Nicotinamide, Fluosol DA and Carbogen: a strategy to reoxygenate acutely and chronically hypoxic cells in vivo. Br J Cancer. 1991 Jan;63(1):109–113. doi: 10.1038/bjc.1991.22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cole S., Stratford I. J., Adams G. E., Fielden E. M., Jenkins T. C. Dual-function 2-nitroimidazoles as hypoxic cell radiosensitizers and bioreductive cytotoxins: in vivo evaluation in KHT murine sarcomas. Radiat Res. 1990 Oct;124(1 Suppl):S38–S43. [PubMed] [Google Scholar]
  12. Cole S., Stratford I. J., Bowler J., Nolan J., Wright E. G., Lorimore S. A., Adams G. E. Oral (po) dosing with RSU 1069 or RB 6145 maintains their potency as hypoxic cell radiosensitizers and cytotoxins but reduces systemic toxicity compared with parenteral (ip) administration in mice. Int J Radiat Oncol Biol Phys. 1991 Jul;21(2):387–395. doi: 10.1016/0360-3016(91)90787-5. [DOI] [PubMed] [Google Scholar]
  13. Cole S., Stratford I. J., Fielden E. M., Adams G. E., Leopold W., Elliott W., Suto M., Sebolt-Leopold J. Dual function nitroimidazoles less toxic than RSU 1069: selection of candidate drugs for clinical trial (RB 6145 and/or PD 130908. Int J Radiat Oncol Biol Phys. 1992;22(3):545–548. doi: 10.1016/0360-3016(92)90872-f. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Gatenby R. A., Kessler H. B., Rosenblum J. S., Coia L. R., Moldofsky P. J., Hartz W. H., Broder G. J. Oxygen distribution in squamous cell carcinoma metastases and its relationship to outcome of radiation therapy. Int J Radiat Oncol Biol Phys. 1988 May;14(5):831–838. doi: 10.1016/0360-3016(88)90002-8. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Herscher L. L., Krishna M. C., Cook J. A., Coleman C. N., Biaglow J. E., Tuttle S. W., Gonzalez F. J., Mitchell J. B. Protection against SR 4233 (Tirapazamine) aerobic cytotoxicity by the metal chelators desferrioxamine and tiron. Int J Radiat Oncol Biol Phys. 1994 Nov 15;30(4):879–885. doi: 10.1016/0360-3016(94)90364-6. [DOI] [PubMed] [Google Scholar]
  18. Hodnick W. F., Sartorelli A. C. Reductive activation of mitomycin C by NADH:cytochrome b5 reductase. Cancer Res. 1993 Oct 15;53(20):4907–4912. [PubMed] [Google Scholar]
  19. Höckel M., Knoop C., Schlenger K., Vorndran B., Baussmann E., Mitze M., Knapstein P. G., Vaupel P. Intratumoral pO2 predicts survival in advanced cancer of the uterine cervix. Radiother Oncol. 1993 Jan;26(1):45–50. doi: 10.1016/0167-8140(93)90025-4. [DOI] [PubMed] [Google Scholar]
  20. Höckel M., Schlenger K., Knoop C., Vaupel P. Oxygenation of carcinomas of the uterine cervix: evaluation by computerized O2 tension measurements. Cancer Res. 1991 Nov 15;51(22):6098–6102. [PubMed] [Google Scholar]
  21. Jenkins T. C., Naylor M. A., O'Neill P., Threadgill M. D., Cole S., Stratford I. J., Adams G. E., Fielden E. M., Suto M. J., Stier M. A. Synthesis and evaluation of alpha-[[(2-haloethyl)amino]methyl]-2- nitro-1H-imidazole-1-ethanols as prodrugs of alpha-[(1-aziridinyl)methyl]-2- nitro-1H-imidazole-1-ethanol (RSU-1069) and its analogues which are radiosensitizers and bioreductively activated cytotoxins. J Med Chem. 1990 Sep;33(9):2603–2610. doi: 10.1021/jm00171a040. [DOI] [PubMed] [Google Scholar]
  22. Kennedy K. A. Hypoxic cells as specific drug targets for chemotherapy. Anticancer Drug Des. 1987 Oct;2(2):181–194. [PubMed] [Google Scholar]
  23. 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]
  24. Koch C. J. Unusual oxygen concentration dependence of toxicity of SR-4233, a hypoxic cell toxin. Cancer Res. 1993 Sep 1;53(17):3992–3997. [PubMed] [Google Scholar]
  25. 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]
  26. Lin A. J., Cosby L. A., Shansky C. W., Sartorelli A. C. Potential bioreductive alkylating agents. 1. Benzoquinone derivatives. J Med Chem. 1972 Dec;15(12):1247–1252. doi: 10.1021/jm00282a011. [DOI] [PubMed] [Google Scholar]
  27. Lloyd R. V., Duling D. R., Rumyantseva G. V., Mason R. P., Bridson P. K. Microsomal reduction of 3-amino-1,2,4-benzotriazine 1,4-dioxide to a free radical. Mol Pharmacol. 1991 Sep;40(3):440–445. [PubMed] [Google Scholar]
  28. Moulder J. E., Rockwell S. Tumor hypoxia: its impact on cancer therapy. Cancer Metastasis Rev. 1987;5(4):313–341. doi: 10.1007/BF00055376. [DOI] [PubMed] [Google Scholar]
  29. Murray G. I., Foster C. O., Barnes T. S., Weaver R. J., Ewen S. W., Melvin W. T., Burke M. D. Expression of cytochrome P450IA in breast cancer. Br J Cancer. 1991 Jun;63(6):1021–1023. doi: 10.1038/bjc.1991.222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Okunieff P., Hoeckel M., Dunphy E. P., Schlenger K., Knoop C., Vaupel P. Oxygen tension distributions are sufficient to explain the local response of human breast tumors treated with radiation alone. Int J Radiat Oncol Biol Phys. 1993 Jul 15;26(4):631–636. doi: 10.1016/0360-3016(93)90280-9. [DOI] [PubMed] [Google Scholar]
  31. Paine A. J. The maintenance of cytochrome P-450 in rat hepatocyte culture: some applications of liver cell cultures to the study of drug metabolism, toxicity and the induction of the P-450 system. Chem Biol Interact. 1990;74(1-2):1–31. doi: 10.1016/0009-2797(90)90055-r. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Philip P. A., Kaklamanis L., Ryley N., Stratford I., Wolf R., Harris A., Carmichael J. Expression of xenobiotic-metabolizing enzymes by primary and secondary hepatic tumors in man. Int J Radiat Oncol Biol Phys. 1994 May 15;29(2):277–283. doi: 10.1016/0360-3016(94)90275-5. [DOI] [PubMed] [Google Scholar]
  34. Plumb J. A., Gerritsen M., Milroy R., Thomson P., Workman P. Relative importance of DT-diaphorase and hypoxia in the bioactivation of EO9 by human lung tumor cell lines. Int J Radiat Oncol Biol Phys. 1994 May 15;29(2):295–299. doi: 10.1016/0360-3016(94)90278-x. [DOI] [PubMed] [Google Scholar]
  35. Plumb J. A., Milroy R., Kaye S. B. Effects of the pH dependence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay. Cancer Res. 1989 Aug 15;49(16):4435–4440. [PubMed] [Google Scholar]
  36. Rampling R., Cruickshank G., Lewis A. D., Fitzsimmons S. A., Workman P. Direct measurement of pO2 distribution and bioreductive enzymes in human malignant brain tumors. Int J Radiat Oncol Biol Phys. 1994 Jun 15;29(3):427–431. doi: 10.1016/0360-3016(94)90432-4. [DOI] [PubMed] [Google Scholar]
  37. Riley R. J., Hemingway S. A., Graham M. A., Workman P. Initial characterization of the major mouse cytochrome P450 enzymes involved in the reductive metabolism of the hypoxic cytotoxin 3-amino-1,2,4-benzotriazine-1,4-di-N-oxide (tirapazamine, SR 4233, WIN 59075). Biochem Pharmacol. 1993 Mar 9;45(5):1065–1077. doi: 10.1016/0006-2952(93)90251-q. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Robertson N., Stratford I. J., Houlbrook S., Carmichael J., Adams G. E. The sensitivity of human tumour cells to quinone bioreductive drugs: what role for DT-diaphorase? Biochem Pharmacol. 1992 Aug 4;44(3):409–412. doi: 10.1016/0006-2952(92)90429-m. [DOI] [PubMed] [Google Scholar]
  40. Rockwell S., Keyes S. R., Sartorelli A. C. Preclinical studies of porfiromycin as an adjunct to radiotherapy. Radiat Res. 1988 Oct;116(1):100–113. [PubMed] [Google Scholar]
  41. Rockwell S., Moulder J. E. Hypoxic fractions of human tumors xenografted into mice: a review. Int J Radiat Oncol Biol Phys. 1990 Jul;19(1):197–202. doi: 10.1016/0360-3016(90)90154-c. [DOI] [PubMed] [Google Scholar]
  42. Smith G., Harrison D. J., East N., Rae F., Wolf H., Wolf C. R. Regulation of cytochrome P450 gene expression in human colon and breast tumour xenografts. Br J Cancer. 1993 Jul;68(1):57–63. doi: 10.1038/bjc.1993.286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Soule H. D., Vazguez J., Long A., Albert S., Brennan M. A human cell line from a pleural effusion derived from a breast carcinoma. J Natl Cancer Inst. 1973 Nov;51(5):1409–1416. doi: 10.1093/jnci/51.5.1409. [DOI] [PubMed] [Google Scholar]
  45. Stratford I. J., O'Neill P., Sheldon P. W., Silver A. R., Walling J. M., Adams G. E. RSU 1069, a nitroimidazole containing an aziridine group. Bioreduction greatly increases cytotoxicity under hypoxic conditions. Biochem Pharmacol. 1986 Jan 1;35(1):105–109. doi: 10.1016/0006-2952(86)90566-6. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Vaupel P., Schlenger K., Knoop C., Höckel M. Oxygenation of human tumors: evaluation of tissue oxygen distribution in breast cancers by computerized O2 tension measurements. Cancer Res. 1991 Jun 15;51(12):3316–3322. [PubMed] [Google Scholar]
  48. 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]
  49. 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]
  50. 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]
  51. 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]
  52. 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]
  53. Workman P. Bioreductive mechanisms. Int J Radiat Oncol Biol Phys. 1992;22(4):631–637. doi: 10.1016/0360-3016(92)90493-2. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. 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]
  56. 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]

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

RESOURCES