Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Effects of hypoxia and limited diffusion in tumor cell microenvironment on bystander effect of P450 prodrug therapy

Cancer Gene Therapy volume 13, pages 771–779 (2006)Cite this article

Abstract

Cytochrome P450 (CYP) enzyme 2B1 metabolizes the anticancer prodrug cyclophosphamide (CPA) to 4-hydroxy-CPA, which decomposes to the cytotoxic metabolites acrolein and phosphoramide mustard. We have evaluated the bystander cytotoxicity of CPA in combination with CYP2B1 gene-directed enzyme prodrug therapy using a cell culture-based agarose overlay technique. This method mimics the tumor microenvironment by limiting the diffusion of metabolites and by reducing the oxygen concentration to levels similar to those found in solid tumors. Under these conditions, the CYP activity of CYP2B1-expressing tumor cells was decreased by 80% compared to standard aerobic conditions. Despite this decrease in metabolic activity, a potent bystander effect was observed, resulting in up to 90% killing by CPA of a tumor cell population comprised of only ∼20% CYP-expressing tumor cells. Similarly, transient transfection of a small fraction (∼14%) of a human hepatoma Huh7 cell population with a CYP2B1 expression plasmid followed by short-term treatment with CPA (5 h) led to an eradication of 95% of the cells. No such bystander effect was observed without the agarose overlay. These findings suggest that the agarose overlay technique is very useful as an in vitro test system for investigation of the bystander effect of CYP/CPA and other enzyme/prodrug combinations under conditions that mimic the hypoxic conditions present in solid tumors in vivo.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 3
Figure 1
Figure 2
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

Abbreviations

CPA:

cyclophosphamide

CYP:

cytochrome P450

GDEPT:

gene-directed enzyme prodrug therapy

FBS:

fetal bovine serum

HBS:

150 mM NaCl,20 mM HEPES pH 7.4

IFA:

ifosfamide

MTT:

(3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide)

PEI:

polyethylenimine

References

  1. Dachs GU, Tupper J, Tozer GM . From bench to bedside for gene-directed enzyme prodrug therapy of cancer. Anticancer Drugs 2005; 16: 349–359.

    Article  CAS  PubMed  Google Scholar 

  2. Gunther M, Wagner E, Ogris M . Specific targets in tumor tissue for the delivery of therapeutic genes. Curr Med Chem Anti-cancer Agents 2005; 5: 157–171.

    Article  PubMed  Google Scholar 

  3. Roy P, Waxman DJ . Activation of oxazaphosphorines by cytochrome P450: application to gene-directed enzyme prodrug therapy for cancer. Toxicol in Vivo 2005; 20: 176–186.

    Article  Google Scholar 

  4. Wei MX, Tamiya T, Rhee RJ, Breakefield XO, Chiocca EA . Diffusible cytotoxic metabolites contribute to the in vitro bystander effect associated with the cyclophosphamide/cytochrome P450 2B1 cancer gene therapy paradigm. Clin Cancer Res 1995; 1: 1171–1177.

    CAS  PubMed  Google Scholar 

  5. Chen L, Waxman DJ . Intratumoral activation and enhanced chemotherapeutic effect of oxazaphosphorines following cytochrome P-450 gene transfer: development of a combined chemotherapy/cancer gene therapy strategy. Cancer Res 1995; 55: 581–589.

    CAS  PubMed  Google Scholar 

  6. Jounaidi Y, Waxman DJ . Use of replication-conditional adenovirus as a helper system to enhance delivery of P450 prodrug-activation genes for cancer therapy. Cancer Res 2004; 64: 292–303.

    Article  CAS  PubMed  Google Scholar 

  7. Wright JE, Tretyakov O, Ayash LJ, Elias A, Rosowsky A, Frei III E . Analysis of 4-hydroxycyclophosphamide in human blood. Anal Biochem 1995; 224: 154–158.

    Article  CAS  PubMed  Google Scholar 

  8. Mahoney BP, Raghunand N, Baggett B, Gillies RJ . Tumor acidity, ion trapping and chemotherapeutics. I. Acid pH affects the distribution of chemotherapeutic agents in vitro. Biochem Pharmacol 2003; 66: 1207–1218.

    Article  CAS  PubMed  Google Scholar 

  9. Jahde E, Roszinski S, Volk T, Glusenkamp KH, Wiedemann G, Rajewsky MF . Metabolic response of AH13r rat tumours to cyclophosphamide as monitored by pO2 and pH semi-microelectrodes. Eur J Cancer 1992; 29A: 116–122.

    CAS  PubMed  Google Scholar 

  10. Gillies RJ, Schornack PA, Secomb TW, Raghunand N . Causes and effects of heterogeneous perfusion in tumors. Neoplasia 1999; 1: 197–207.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 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.

    CAS  PubMed  Google Scholar 

  12. Braybrooke JP, Slade A, Deplanque G, Harrop R, Madhusudan S, Forster MD et al. Phase I study of MetXia-P450 gene therapy and oral cyclophosphamide for patients with advanced breast cancer or melanoma. Clin Cancer Res 2005; 11: 1512–1520.

    Article  CAS  PubMed  Google Scholar 

  13. Boeckle S, von Gersdorff K, van der PS, Culmsee C, Wagner E, Ogris M . Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer. J Gene Med 2004; 6: 1102–1111.

    Article  CAS  PubMed  Google Scholar 

  14. Donato MT, Gomez-Lechon MJ, Castell JV . A microassay for measuring cytochrome P450IA1 and P450IIB1 activities in intact human and rat hepatocytes cultured on 96-well plates. Anal Biochem 1993; 213: 29–33.

    Article  CAS  PubMed  Google Scholar 

  15. Kim MH, Jung YS, Moon CH, Lee SH, Baik EJ, Moon CK . High-glucose induced protective effect against hypoxic injury is associated with maintenance of mitochondrial membrane potential. Jpn J Physiol 2003; 53: 451–459.

    Article  CAS  PubMed  Google Scholar 

  16. von Gersdorff K, Ogris M, Wagner E . Cryoconserved shielded and EGF receptor targeted DNA polyplexes: cellular mechanisms. Eur J Pharm Biopharm 2005; 60: 279–285.

    Article  CAS  PubMed  Google Scholar 

  17. Rao J, Otto WR . Fluorimetric DNA assay for cell growth estimation. Anal Biochem 1992; 207: 186–192.

    Article  CAS  PubMed  Google Scholar 

  18. Yuan F, Krol A, Tong S . Available space and extracellular transport of macromolecules: effects of pore size and connectedness. Ann Biomed Eng 2001; 29: 1150–1158.

    Article  CAS  PubMed  Google Scholar 

  19. Ding Q, Kestell P, Baguley BC, Palmer BD, Paxton JW, Muller G et al. Potentiation of the antitumour effect of cyclophosphamide in mice by thalidomide. Cancer Chemother Pharmacol 2002; 50: 186–192.

    Article  CAS  PubMed  Google Scholar 

  20. Sladek NE . Metabolism and pharmacokinetic behavior of cyclophosphamide and related oxazophosphorines. In: Powis G (ed). Anticancer Drugs: Reactive Metabolism and Drug Interactions. Pergamon Press Ltd: Oxford, 1994, pp 79–156.

    Google Scholar 

  21. Chen L, Waxman DJ . Cytochrome P450 gene-directed enzyme prodrug therapy (GDEPT) for cancer. Curr Pharm Des 2002; 8: 1405–1416.

    Article  CAS  PubMed  Google Scholar 

  22. Denny WA . Prodrugs for gene-directed enzyme-prodrug therapy (suicide gene therapy). J Biomed Biotechnol 2003; 2003: 48–70.

    Article  PubMed  PubMed Central  Google Scholar 

  23. 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.

    CAS  PubMed  Google Scholar 

  24. Yuan F, Krol A, Tong S . Available space and extracellular transport of macromolecules: effects of pore size and connectedness. Ann Biomed Eng 2001; 29: 1150–1158.

    Article  CAS  PubMed  Google Scholar 

  25. Ding Q, Kestell P, Baguley BC, Palmer BD, Paxton JW, Muller G et al. Potentiation of the antitumour effect of cyclophosphamide in mice by thalidomide. Cancer Chemother Pharmacol 2002; 50: 186–192.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported in part by Grant CA49248 from the NIH (DJW) and by a Grant from the Wilhelm Sander Stiftung (MG and MO).

Author information

Authors and Affiliations

  1. Department of Pharmacy, Pharmaceutical Biology-Biotechnology, Ludwig-Maximilians-Universität, Munich, Germany

    M Günther, E Wagner & M Ogris

  2. Department of Biology, Division of Cell and Molecular Biology, Boston University, Boston, MA, USA

    D J Waxman

Authors
  1. M Günther
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. D J Waxman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. E Wagner
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M Ogris
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M Ogris.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Günther, M., Waxman, D., Wagner, E. et al. Effects of hypoxia and limited diffusion in tumor cell microenvironment on bystander effect of P450 prodrug therapy. Cancer Gene Ther 13, 771–779 (2006). https://doi.org/10.1038/sj.cgt.7700955

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.cgt.7700955

Keywords

This article is cited by

Search

Advanced search

Quick links