, Volume 29, Issue 1, pp 157–170 | Cite as

Improved cytotoxicity of pyridyl-substituted thiosemicarbazones against MCF-7 when used as metal ionophores

  • Fady N. Akladios
  • Scott D. Andrew
  • Christopher J. Parkinson


Zinc is the second most abundant transition metal in the human body, between 3 and 10 % of human genes encoding for zinc binding proteins. We have investigated the interplay of reactive oxygen species and zinc homeostasis on the cytotoxicity of the thiosemicarbazone chelators against the MCF-7 cell line. The cytotoxicity of thiosemicarbazone chelators against MCF-7 can be improved through supplementation of ionic zinc provided the zinc ion is at a level exceeding the thiosemicarbazone concentration. Elimination of the entire cell population can be accomplished with this regime, unlike the plateau of cytotoxicity observed on thiosemicarbazone monotherapy. The cytotoxic effects of copper complexes of the thiosemicarbazone are not enhanced by zinc supplementation, displacement of copper from the complex being disfavoured. Treatment of MCF-7 with uncomplexed thiosemicarbazone initiates post G1 blockade alongside the induction of apoptosis, cell death being abrogated through subsequent supplementation with zinc ion after drug removal. This would implicate a metal depletion mechanism in the cytotoxic effect of the un-coordinated thiosemicarbazone. The metal complexes of the species, however, fail to initiate similar G1 blockade and apparently exert their cytotoxic effect through generation of reactive oxygen species, suggesting that multiple mechanisms of cytotoxicity can be associated with the thiosemicarbazones dependant on the level of metal ion association.


Copper Zinc Cytotoxicity Reactive oxygen species (ROS) Thiosemicarbazone 



The authors acknowledge the contribution of Dr Gregg Maynard for assistance in setting up flow cytometry studies. F Akladios acknowledges the receipt of an Australian Postgraduate Award (APA). CJP wishes to thank the CSU Pharmacy Foundation for a grant partially funding this study. CJP and SDA thank the Kolling Institute (Royal North Shore Hospital) for the donation and characterization of the MCF-7 cell line employed in this study.

Author contribution

CJP and SDA designed the research programme. FNA conducted all research, analysed data and assembled the draft manuscript. All authors read and approved the final content.


  1. Akladios FN, Andrew SD, Parkinson CJ (2015) Selective induction of oxidative stress in cancer cells via synergistic combinations of agents targeting redox homeostasis. Bioorgan Med ChemGoogle Scholar
  2. Andreini C, Banci L, Bertini I, Rosato A (2006) Counting the zinc-proteins encoded in the human genome. J Proteome Res 5(1):196–201CrossRefPubMedGoogle Scholar
  3. Anzellotti A, Farrell N (2008) Zinc metalloproteins as medicinal targets. Chem Soc Rev 37(8):1629–1651CrossRefPubMedGoogle Scholar
  4. Bernhardt PV, Sharpe PC, Islam M, Lovejoy DB, Kalinowski DS, Richardson DR (2008) Iron chelators of the dipyridylketone thiosemicarbazone class: precomplexation and transmetalation effects on anticancer activity. J Med Chem 52(2):407–415CrossRefGoogle Scholar
  5. Butler JS, Loh SN (2007) Zn2 + -dependent misfolding of the p53 DNA binding domain. Biochemistry 46(10):2630–2639CrossRefPubMedGoogle Scholar
  6. Darzynkiewicz Z, Huang X, Okafuji M, King MA (2005) Cytometric methods to detect apoptosis. Cytometry 75:307Google Scholar
  7. Gaál A, Orgován G, Polgári Z, Réti A, Mihucz VG, Bősze S, Streli C (2014) Complex forming competition and in vitro toxicity studies on the applicability of di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) as a metal chelator. J Inorg Biochem 130:52–58CrossRefPubMedGoogle Scholar
  8. Jansson PJ, Sharpe PC, Bernhardt PV, Richardson DR (2010) Novel thiosemicarbazones of the ApT and DpT series and their copper complexes: identification of pronounced redox activity and characterization of their antitumor activity. J Med Chem 53(15):5759–5769CrossRefPubMedGoogle Scholar
  9. Kalinowski DS, Yu Y, Sharpe PC, Islam M, Liao Y-T, Lovejoy DB, Richardson DR (2007) Design, synthesis, and characterization of novel iron chelators: structure-activity relationships of the 2-benzoylpyridine thiosemicarbazone series and their 3-nitrobenzoyl analogues as potent antitumor agents. J Med Chem 50(15):3716–3729CrossRefPubMedGoogle Scholar
  10. Kim DH, Kundu JK, Surh YJ (2011) Redox modulation of p53: mechanisms and functional significance. Mol Carcinog 50(4):222–234CrossRefPubMedGoogle Scholar
  11. King MA, Radicchi-Mastroianni MA (2002) Effects of caspase inhibition on camptothecin-induced apoptosis of HL-60 cells. Cytometry 49(1):28–35CrossRefPubMedGoogle Scholar
  12. Loh SN (2010) The missing zinc: p53 misfolding and cancer. Metallomics 2(7):442–449CrossRefPubMedGoogle Scholar
  13. Maret W (2008) Metallothionein redox biology in the cytoprotective and cytotoxic functions of zinc. Exp Gerontol 43(5):363–369. doi: 10.1016/j.exger.2007.11.005 CrossRefPubMedGoogle Scholar
  14. Maret W (2009) Molecular aspects of human cellular zinc homeostasis: redox control of zinc potentials and zinc signals. Biometals 22(1):149–157CrossRefPubMedGoogle Scholar
  15. Maret W, Vallee BL (1998) Thiolate ligands in metallothionein confer redox activity on zinc clusters. Proc Natl Acad Sci 95(7):3478–3482CrossRefPubMedCentralPubMedGoogle Scholar
  16. Méplan C, Richard M-J, Hainaut P (2000) Metalloregulation of the tumor suppressor protein p53: zinc mediates the renaturation of p53 after exposure to metal chelators in vitro and in intact cells. Oncogene 19(46):5227–5236CrossRefPubMedGoogle Scholar
  17. Richardson DR, Sharpe PC, Lovejoy DB, Senaratne D, Kalinowski DS, Islam M, Bernhardt PV (2006) Dipyridyl thiosemicarbazone chelators with potent and selective antitumor activity form iron complexes with redox activity. J Med Chem 49(22):6510–6521CrossRefPubMedGoogle Scholar
  18. Siriwardana G, Seligman PA (2015) Iron depletion results in Src kinase inhibition with associated cell cycle arrest in neuroblastoma cells. Physiol Rep 3(3):e12341CrossRefPubMedCentralPubMedGoogle Scholar
  19. Verhaegh GW, Parat MO, Richard MJ, Hainaut P (1998) Modulation of p53 protein conformation and DNA-binding activity by intracellular chelation of zinc. Mol Carcinog 21(3):205–214CrossRefPubMedGoogle Scholar
  20. Williams, O. (2004). Flow cytometry-based methods for apoptosis detection in lymphoid cells . In: Apoptosis methods and protocols. Springer, New York, pp. 31-42Google Scholar
  21. Yu X, Vazquez A, Levine AJ, Carpizo DR (2012) Allele-specific p53 mutant reactivation. Cancer Cell 21(5):614–625CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Fady N. Akladios
    • 1
  • Scott D. Andrew
    • 1
  • Christopher J. Parkinson
    • 1
  1. 1.School of Biomedical SciencesCharles Sturt UniversityOrangeAustralia

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