Skip to main content

Advertisement

Log in

An evaluation of the interaction of pixantrone with formaldehyde-releasing drugs in cancer cells

  • Original Article
  • Published:
Cancer Chemotherapy and Pharmacology Aims and scope Submit manuscript

Abstract

Purpose

Pixantrone is a synthetic aza-anthracenedione currently used in the treatment of non-Hodgkin’s lymphoma. The drug is firmly established as a poison of the nuclear enzyme topoisomerase II, however, pixantrone can also generate covalent drug-DNA adducts following activation by formaldehyde. While pixantrone-DNA adducts form proficiently in vitro, little evidence is presently at hand to indicate their existence within cells. The molecular nature of these lesions within cancer cells exposed to pixantrone and formaldehyde-releasing prodrugs was characterized along with the cellular responses to their formation.

Methods

In vitro crosslinking assays, [14C] scintillation counting analyses and alkaline comet assays were applied to characterize pixantrone-DNA adducts. Flow cytometry, cell growth inhibition and clonogenic assays were used to measure cancer cell kill and survival.

Results

Pixantrone-DNA adducts were not detectable in MCF-7 breast cancer cells exposed to [14C] pixantrone (10–40 µM) alone, however the addition of the formaldehyde-releasing prodrug AN9 yielded readily measurable levels of the lesion at ~ 1 adduct per 10 kb of genomic DNA. Co-administration with AN9 completely reversed topoisomerase II-associated DNA damage induction by pixantrone yet potentiated cell kill by the drug, suggesting that pixantrone-DNA adducts may promote a topoisomerase II-independent mechanism of cell death. Pixantrone-DNA adduct-forming treatments generally conferred mild synergism in multiple cell lines in various cell death and clonogenic assays, while pixantrone analogues either incapable or relatively defective in forming DNA adducts demonstrated antagonism when combined with AN9.

Conclusions

The features unique to pixantrone-DNA adducts may be leveraged to enhance cancer cell kill and may be used to guide the design of pixantrone analogues that generate adducts with more favorable anticancer properties.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Availability of data and materials

Not applicable.

Code availability

Not applicable.

References

  1. Evison BJ, Sleebs BE, Watson KG, Phillips DR, Cutts SM (2016) Mitoxantrone, more than just another topoisomerase II poison. Med Res Rev 36(2):248–299. https://doi.org/10.1002/med.21364

    Article  CAS  PubMed  Google Scholar 

  2. Smith PJ, Morgan SA, Fox ME, Watson JV (1990) Mitoxantrone-DNA binding and the induction of topoisomerase II associated DNA damage in multi-drug resistant small cell lung cancer cells. Biochem Pharmacol 40(9):2069–2078

    Article  CAS  Google Scholar 

  3. Parker BS, Cullinane C, Phillips DR (1999) Formation of DNA adducts by formaldehyde-activated mitoxantrone. Nucleic Acids Res 27(14):2918–2923

    Article  CAS  Google Scholar 

  4. Parker BS, Buley T, Evison BJ, Cutts SM, Neumann GM, Iskander MN, Phillips DR (2004) A molecular understanding of mitoxantrone-DNA adduct formation: effect of cytosine methylation and flanking sequences. J Biol Chem 279(18):18814–18823. https://doi.org/10.1074/jbc.M400931200

    Article  CAS  PubMed  Google Scholar 

  5. Corbett TH, Griswold DP Jr, Roberts BJ, Schabel FM Jr (1981) Absence of delayed lethality in mice treated with aclacinomycin A. Cancer Chemother Pharmacol 6(2):161–168

    Article  CAS  Google Scholar 

  6. Krapcho AP, Petry ME, Getahun Z, Landi JJ Jr, Stallman J, Polsenberg JF, Gallagher CE, Maresch MJ, Hacker MP, Giuliani FC et al (1994) 6,9-Bis[(aminoalkyl)amino]benzo[g]isoquinoline-5,10-diones. A novel class of chromophore-modified antitumor anthracene-9,10-diones: synthesis and antitumor evaluations. J Med Chem 37(6):828–837

    Article  CAS  Google Scholar 

  7. Pean E, Flores B, Hudson I, Sjoberg J, Dunder K, Salmonson T, Gisselbrecht C, Laane E, Pignatti F (2013) The European Medicines Agency review of pixantrone for the treatment of adult patients with multiply relapsed or refractory aggressive non-Hodgkin’s B cell lymphomas: summary of the scientific assessment of the committee for medicinal products for human use. Oncologist 18(5):625–633. https://doi.org/10.1634/theoncologist.2013-0020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Cavalletti E, Crippa L, Mainardi P, Oggioni N, Cavagnoli R, Bellini O, Sala F (2007) Pixantrone (BBR 2778) has reduced cardiotoxic potential in mice pretreated with doxorubicin: comparative studies against doxorubicin and mitoxantrone. Invest New Drugs 25(3):187–195. https://doi.org/10.1007/s10637-007-9037-8

    Article  CAS  PubMed  Google Scholar 

  9. Longo M, Della Torre P, Allievi C, Morisetti A, Al-Fayoumi S, Singer JW (2014) Tolerability and toxicological profile of pixantrone (Pixuvri(R)) in juvenile mice. Comparative study with doxorubicin. Reprod Toxicol 46:20–30. https://doi.org/10.1016/j.reprotox.2014.02.006

    Article  CAS  PubMed  Google Scholar 

  10. De Isabella P, Palumbo M, Sissi C, Capranico G, Carenini N, Menta E, Oliva A, Spinelli S, Krapcho AP, Giuliani FC et al (1995) Topoisomerase II DNA cleavage stimulation, DNA binding activity, cytotoxicity, and physico-chemical properties of 2-aza- and 2-aza-oxide-anthracenedione derivatives. Mol Pharmacol 48(1):30–38

    PubMed  Google Scholar 

  11. Zwelling LA, Mayes J, Altschuler E, Satitpunwaycha P, Tritton TR, Hacker MP (1993) Activity of two novel anthracene-9,10-diones against human leukemia cells containing intercalator-sensitive or -resistant forms of topoisomerase II. Biochem Pharmacol 46(2):265–271

    Article  CAS  Google Scholar 

  12. Evison BJ, Mansour OC, Menta E, Phillips DR, Cutts SM (2007) Pixantrone can be activated by formaldehyde to generate a potent DNA adduct forming agent. Nucleic Acids Res 35(11):3581–3589. https://doi.org/10.1093/nar/gkm285

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Rephaeli A, Rabizadeh E, Aviram A, Shaklai M, Ruse M, Nudelman A (1991) Derivatives of butyric acid as potential anti-neoplastic agents. Int J Cancer J Int Cancer 49(1):66–72

    Article  CAS  Google Scholar 

  14. Rephaeli A, Entin-Meer M, Angel D, Tarasenko N, Gruss-Fischer T, Bruachman I, Phillips DR, Cutts SM, Haas-Kogan D, Nudelman A (2006) The selectivty and anti-metastatic activity of oral bioavailable butyric acid prodrugs. Invest New Drugs 24(5):383–392. https://doi.org/10.1007/s10637-006-6213-1

    Article  CAS  PubMed  Google Scholar 

  15. Entin-Meer M, Rephaeli A, Yang X, Nudelman A, VandenBerg SR, Haas-Kogan DA (2005) Butyric acid prodrugs are histone deacetylase inhibitors that show antineoplastic activity and radiosensitizing capacity in the treatment of malignant gliomas. Mol Cancer Ther 4(12):1952–1961. https://doi.org/10.1158/1535-7163.Mct-05-0087

    Article  CAS  PubMed  Google Scholar 

  16. Mansour OC, Evison BJ, Sleebs BE, Watson KG, Nudelman A, Rephaeli A, Buck DP, Collins JG, Bilardi RA, Phillips DR, Cutts SM (2010) New anthracenedione derivatives with improved biological activity by virtue of stable drug-DNA adduct formation. J Med Chem 53(19):6851–6866. https://doi.org/10.1021/jm901894c

    Article  CAS  PubMed  Google Scholar 

  17. Salti GI, Das Gupta TK, Constantinou AI (2000) A novel use for the comet assay: detection of topoisomerase II inhibitors. Anticancer Res 20(5A):3189–3193

    CAS  PubMed  Google Scholar 

  18. Olive PL (1989) Cell proliferation as a requirement for development of the contact effect in Chinese hamster V79 spheroids. Radiat Res 117(1):79–92

    Article  CAS  Google Scholar 

  19. Olive PL, Banath JP, Durand RE (1990) Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the “comet” assay. Radiat Res 122(1):86–94

    Article  CAS  Google Scholar 

  20. Chou TC, Talalay P (1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22:27–55. https://doi.org/10.1016/0065-2571(84)90007-4

    Article  CAS  PubMed  Google Scholar 

  21. Harker WG, Slade DL, Drake FH, Parr RL (1991) Mitoxantrone resistance in HL-60 leukemia cells: reduced nuclear topoisomerase II catalytic activity and drug-induced DNA cleavage in association with reduced expression of the topoisomerase II beta isoform. Biochemistry 30(41):9953–9961

    Article  CAS  Google Scholar 

  22. Evison BJ, Pastuovic M, Bilardi RA, Forrest RA, Pumuye PP, Sleebs BE, Watson KG, Phillips DR, Cutts SM (2011) M2, a novel anthracenedione, elicits a potent DNA damage response that can be subverted through checkpoint kinase inhibition to generate mitotic catastrophe. Biochem Pharmacol 82(11):1604–1618. https://doi.org/10.1016/j.bcp.2011.08.013

    Article  CAS  PubMed  Google Scholar 

  23. Valiathan C, McFaline JL, Samson LD (2012) A rapid survival assay to measure drug-induced cytotoxicity and cell cycle effects. DNA Repair 11(1):92–98. https://doi.org/10.1016/j.dnarep.2011.11.002

    Article  CAS  PubMed  Google Scholar 

  24. Evison BJ, Chiu F, Pezzoni G, Phillips DR, Cutts SM (2008) Formaldehyde-activated Pixantrone is a monofunctional DNA alkylator that binds selectively to CpG and CpA doublets. Mol Pharmacol 74(1):184–194. https://doi.org/10.1124/mol.108.045625

    Article  CAS  PubMed  Google Scholar 

  25. Thorndike J, Beck WS (1977) Production of formaldehyde from N5-methyltetrahydrofolate by normal and leukemic leukocytes. Can Res 37(4):1125–1132

    CAS  Google Scholar 

  26. Liu J, Liu FY, Tong ZQ, Li ZH, Chen W, Luo WH, Li H, Luo HJ, Tang Y, Tang JM, Cai J, Liao FF, Wan Y (2013) Lysine-specific demethylase 1 in breast cancer cells contributes to the production of endogenous formaldehyde in the metastatic bone cancer pain model of rats. PLoS ONE 8(3):e58957. https://doi.org/10.1371/journal.pone.0058957

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. DeGregorio MW, Dingley KH, Wurz GT, Ubick E, Turteltaub KW (2006) Accelerator mass spectrometry allows for cellular quantification of doxorubicin at femtomolar concentrations. Cancer Chemother Pharmacol 57(3):335–342. https://doi.org/10.1007/s00280-005-0060-1

    Article  CAS  PubMed  Google Scholar 

  28. Dhareshwar SS, Stella VJ (2008) Your prodrug releases formaldehyde: should you be concerned? No! J Pharm Sci 97(10):4184–4193. https://doi.org/10.1002/jps.21319

    Article  CAS  PubMed  Google Scholar 

  29. Nitiss JL (2009) Targeting DNA topoisomerase II in cancer chemotherapy. Nat Rev Cancer 9(5):338–350. https://doi.org/10.1038/nrc2607

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Swift LP, Rephaeli A, Nudelman A, Phillips DR, Cutts SM (2006) Doxorubicin-DNA adducts induce a non-topoisomerase II-mediated form of cell death. Can Res 66(9):4863–4871. https://doi.org/10.1158/0008-5472.CAN-05-3410

    Article  CAS  Google Scholar 

  31. Coldwell KE, Cutts SM, Ognibene TJ, Henderson PT, Phillips DR (2008) Detection of Adriamycin-DNA adducts by accelerator mass spectrometry at clinically relevant Adriamycin concentrations. Nucleic Acids Res 36(16):e100. https://doi.org/10.1093/nar/gkn439

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Reid T, Valone F, Lipera W, Irwin D, Paroly W, Natale R, Sreedharan S, Keer H, Lum B, Scappaticci F, Bhatnagar A (2004) Phase II trial of the histone deacetylase inhibitor pivaloyloxymethyl butyrate (Pivanex, AN-9) in advanced non-small cell lung cancer. Lung Cancer 45(3):381–386. https://doi.org/10.1016/j.lungcan.2004.03.002

    Article  PubMed  Google Scholar 

  33. Li M, Wang Y, Li M, Wu X, Setrerrahmane S, Xu H (2021) Integrins as attractive targets for cancer therapeutics. Acta Pharm Sin B 11(9):2726–2737. https://doi.org/10.1016/j.apsb.2021.01.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Konda SK, Kelso C, Medan J, Sleebs BE, Phillips DR, Cutts SM, Collins JG (2016) Isolation and structural analysis of the covalent adduct formed between a bis-amino mitoxantrone analogue and DNA: a pathway to major-minor groove cross-linked adducts. Org Biomol Chem 14(43):10217–10221. https://doi.org/10.1039/c6ob02100j

    Article  CAS  PubMed  Google Scholar 

  35. Konda SK, Kelso C, Pumuye PP, Medan J, Sleebs BE, Cutts SM, Phillips DR, Collins JG (2016) Reversible and formaldehyde-mediated covalent binding of a bis-amino mitoxantrone analogue to DNA. Org Biomol Chem 14(20):4728–4738. https://doi.org/10.1039/c6ob00561f

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Health and Medical Research Council (S.C and D.P) and the CASS Foundation (S.C and B.E).

Funding

This work was supported by the National Health and Medical Research Council (S.C and D.P) and the CASS Foundation (S.C and B.E).

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Suzanne M. Cutts or Benny J. Evison.

Ethics declarations

Conflict of interest

B.E. is an employee of Nyrada Inc. and declares no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mansour, O.C., Nudelman, A., Rephaeli, A. et al. An evaluation of the interaction of pixantrone with formaldehyde-releasing drugs in cancer cells. Cancer Chemother Pharmacol 89, 773–784 (2022). https://doi.org/10.1007/s00280-022-04435-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00280-022-04435-1

Keywords

Navigation