Cancer cells have a high iron requirement and many experimental studies, as well as clinical trials, have demonstrated that iron chelators are potential anti-cancer agents. The ligand, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT), demonstrates both potent anti-neoplastic and anti-retroviral properties. In this study, Bp4eT and its recently identified amidrazone and semicarbazone metabolites were examined and compared with respect to their anti-proliferative activity towards cancer cells (HL-60 human promyelocytic leukemia, MCF-7 human breast adenocarcinoma, HCT116 human colon carcinoma and A549 human lung adenocarcinoma), non-cancerous cells (H9c2 neonatal rat-derived cardiomyoblasts and 3T3 mouse embryo fibroblasts) and their interaction with intracellular iron pools. Bp4eT was demonstrated to be a highly potent and selective anti-neoplastic agent that induces S phase cell cycle arrest, mitochondrial depolarization and apoptosis in MCF-7 cells. Both semicarbazone and amidrazone metabolites showed at least a 300-fold decrease in cytotoxic activity than Bp4eT towards both cancer and normal cell lines. The metabolites also lost the ability to: (1) promote the redox cycling of iron; (2) bind and mobilize iron from labile intracellular pools; and (3) prevent 59Fe uptake from 59Fe-labeled transferrin by MCF-7 cells. Hence, this study demonstrates that the highly active ligand, Bp4eT, is metabolized to non-toxic and pharmacologically inactive analogs, which most likely contribute to its favorable pharmacological profile. These findings are important for the further development of this drug candidate and contribute to the understanding of the structure-activity relationships of these agents.

Department of Biochemical Sciences Charles University Prague Faculty of Pharmacy Hradec Králové Czech Republic

Department of Inorganic and Organic Chemistry Charles University Prague Faculty of Pharmacy Hradec Králové Czech Republic

Department of Pharmaceutical Chemistry and Drug Analysis Charles University Prague Faculty of Pharmacy Hradec Králové Czech Republic

Molecular Pharmacology and Pathology Program Bosch Institute and Department of Pathology University of Sydney Sydney Australia

See more in PubMed

Richardson DR, Ponka P (1997) The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells. Biochim Biophys Acta 1331: 1–40. PubMed

Le NT, Richardson DR (2002) The role of iron in cell cycle progression and the proliferation of neoplastic cells. Biochim Biophys Acta 1603: 31–46. PubMed

Merlot AM, Kalinowski DS, Richardson DR (2013) Novel chelators for cancer treatment: where are we now? Antioxid Redox Signal 18: 973–1006. 10.1089/ars.2012.4540 PubMed DOI

Lane DJ, Mills TM, Shafie NH, Merlot AM, Saleh Moussa R, Kalinowski DS, et al. (2014) Expanding horizons in iron chelation and the treatment of cancer: Role of iron in the regulation of ER stress and the epithelial-mesenchymal transition. Biochim Biophys Acta 1845: 166–181. 10.1016/j.bbcan.2014.01.005 PubMed DOI

Mortazavi A, Ling Y, Martin LK, Wei L, Phelps MA, Liu Z, et al. (2013) A phase I study of prolonged infusion of triapine in combination with fixed dose rate gemcitabine in patients with advanced solid tumors. Invest New Drugs 31: 685–695. 10.1007/s10637-012-9863-1 PubMed DOI PMC

Chao J, Synold TW, Morgan RJ Jr., Kunos C, Longmate J, Lenz HJ, et al. (2012) A phase I and pharmacokinetic study of oral 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, NSC #663249) in the treatment of advanced-stage solid cancers: a California Cancer Consortium Study. Cancer Chemother Pharmacol 69: 835–843. 10.1007/s00280-011-1779-5 PubMed DOI PMC

Ocean AJ, Christos P, Sparano JA, Matulich D, Kaubish A, Siegel A, et al. (2011) Phase II trial of the ribonucleotide reductase inhibitor 3-aminopyridine-2-carboxaldehydethiosemicarbazone plus gemcitabine in patients with advanced biliary tract cancer. Cancer Chemother Pharmacol 68: 379–388. 10.1007/s00280-010-1481-z PubMed DOI PMC

West DX, Ives JS, Krejci J, Salberg MM, Zumbahlen TL, Bain GA, et al. (1995) Copper(Ii) Complexes of 2-Benzoylpyridine N-4-Substituted Thiosemicarbazones. Polyhedron 14: 2189–2200.

Kalinowski DS, Yu Y, Sharpe PC, Islam M, Liao YT, Lovejoy DB, et al. (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: 3716–3729. PubMed

Stefani C, Punnia-Moorthy G, Lovejoy DB, Jansson PJ, Kalinowski DS, Sharpe PC, et al. (2011) Halogenated 2'-benzoylpyridine thiosemicarbazone (XBpT) chelators with potent and selective anti-neoplastic activity: relationship to intracellular redox activity. J Med Chem 54: 6936–6948. 10.1021/jm200924c PubMed DOI

Debebe Z, Ammosova T, Breuer D, Lovejoy DB, Kalinowski DS, Kumar K, et al. (2011) Iron chelators of the di-2-pyridylketone thiosemicarbazone and 2-benzoylpyridine thiosemicarbazone series inhibit HIV-1 transcription: identification of novel cellular targets—iron, cyclin-dependent kinase (CDK) 2, and CDK9. Mol Pharmacol 79: 185–196. 10.1124/mol.110.069062 PubMed DOI PMC

Debebe Z, Nekhai S, Ashenafi M, Lovejoy DB, Kalinowski DS, Gordeuk VR, et al. (2012) Development of a sensitive HPLC method to measure in vitro permeability of E- and Z-isomeric forms of thiosemicarbazones in Caco-2 monolayers. J Chromatogr B Analyt Technol Biomed Life Sci 906: 25–32. 10.1016/j.jchromb.2012.08.011 PubMed DOI PMC

Delie F, Rubas W (1997) A human colonic cell line sharing similarities with enterocytes as a model to examine oral absorption: advantages and limitations of the Caco-2 model. Crit Rev Ther Drug Carrier Syst 14: 221–286. PubMed

Merlot AM, Pantarat N, Lovejoy DB, Kalinowski DS, Richardson DR (2010) Membrane transport and intracellular sequestration of novel thiosemicarbazone chelators for the treatment of cancer. Mol Pharmacol 78: 675–684. 10.1124/mol.110.066126 PubMed DOI

Merlot AM, Pantarat N, Menezes SV, Sahni S, Richardson DR, Kalinowski DS (2013) Cellular uptake of the antitumor agent Dp44mT occurs via a carrier/receptor-mediated mechanism. Mol Pharmacol 84: 911–924. 10.1124/mol.113.088393 PubMed DOI

Stariat J, Kovarikova P, Klimes J, Kalinowski DS, Richardson DR (2010) Development of an LC-MS/MS method for analysis of interconvertible Z/E isomers of the novel anticancer agent, Bp4eT. Anal Bioanal Chem 397: 161–171. 10.1007/s00216-009-3448-7 PubMed DOI

Stariat J, Sestak V, Vavrova K, Nobilis M, Kollarova Z, Klimes J, et al. (2012) LC-MS/MS identification of the principal in vitro and in vivo phase I metabolites of the novel thiosemicarbazone anti-cancer drug, Bp4eT. Anal Bioanal Chem 403: 309–321. 10.1007/s00216-012-5766-4 PubMed DOI

Stariat J, Suprunova V, Roh J, Sestak V, Eisner T, Filipsky T, et al. (2013) Simultaneous determination of the novel thiosemicarbazone anti-cancer agent, Bp4eT, and its main phase I metabolites in plasma: Application to a pilot pharmacokinetic study in rats. Biomed Chromatogr 28: 621–629. 10.1002/bmc.3080 PubMed DOI

Chiu SH, Thompson KA, Vincent SH, Alvaro RF, Huskey SW, Stearns RA, et al. (1995) The role of drug metabolism in drug discovery: a case study in the selection of an oxytocin receptor antagonist for development. Toxicol Pathol 23: 124–130. PubMed

Lin JH, Lu AY (1997) Role of pharmacokinetics and metabolism in drug discovery and development. Pharmacol Rev 49: 403–449. PubMed

Richardson DR, Tran EH, Ponka P (1995) The potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective antiproliferative agents. Blood 86: 4295–4306. PubMed

Baker E, Richardson D, Gross S, Ponka P (1992) Evaluation of the iron chelation potential of hydrazones of pyridoxal, salicylaldehyde and 2-hydroxy-1-naphthylaldehyde using the hepatocyte in culture. Hepatology 15: 492–501. PubMed

Glickstein H, El RB, Link G, Breuer W, Konijn AM, Hershko C, et al. (2006) Action of chelators in iron-loaded cardiac cells: Accessibility to intracellular labile iron and functional consequences. Blood 108: 3195–3203. PubMed

Richardson D, Baker E (1992) Two mechanisms of iron uptake from transferrin by melanoma cells. The effect of desferrioxamine and ferric ammonium citrate. J Biol Chem 267: 13972–13979. PubMed

Richardson DR, Baker E (1990) The uptake of iron and transferrin by the human malignant melanoma cell. Biochim Biophys Acta 1053: 1–12. PubMed

Richardson DR, Sharpe PC, Lovejoy DB, Senaratne D, Kalinowski DS, Islam M, et al. (2006) Dipyridyl thiosemicarbazone chelators with potent and selective antitumor activity form iron complexes with redox activity. J Med Chem 49: 6510–6521. PubMed

Becker E, Richardson DR (1999) Development of novel aroylhydrazone ligands for iron chelation therapy: 2-pyridylcarboxaldehyde isonicotinoyl hydrazone analogs. J Lab Clin Med 134: 510–521. PubMed

Mladenka P, Kalinowski DS, Haskova P, Bobrovova Z, Hrdina R, Simunek T, et al. (2009) The novel iron chelator, 2-pyridylcarboxaldehyde 2-thiophenecarboxyl hydrazone, reduces catecholamine-mediated myocardial toxicity. Chem Res Toxicol 22: 208–217. PubMed

Potuckova E, Jansova H, Machacek M, Vavrova A, Haskova P, Tichotova L, et al. (2014) Quantitative analysis of the anti-proliferative activity of combinations of selected iron-chelating agents and clinically used anti-neoplastic drugs. PLoS One 9: e88754 10.1371/journal.pone.0088754 PubMed DOI PMC

Kralova V, Benesova S, Cervinka M, Rudolf E (2012) Selenite-induced apoptosis and autophagy in colon cancer cells. Toxicol In Vitro 26: 258–268. 10.1016/j.tiv.2011.12.010 PubMed DOI

Munafo DB, Colombo MI (2001) A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation. J Cell Sci 114: 3619–3629. PubMed

van Engeland M, Nieland LJ, Ramaekers FC, Schutte B, Reutelingsperger CP (1998) Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 31: 1–9. PubMed

Krysko DV, Vanden Berghe T, D'Herde K, Vandenabeele P (2008) Apoptosis and necrosis: detection, discrimination and phagocytosis. Methods 44: 205–221. 10.1016/j.ymeth.2007.12.001 PubMed DOI

Honda O, Kuroda M, Joja I, Asaumi J, Takeda Y, Akaki S, et al. (2000) Assessment of secondary necrosis of Jurkat cells using a new microscopic system and double staining method with annexin V and propidium iodide. Int J Oncol 16: 283–288. PubMed

Gao J, Richardson DR (2001) The potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective antiproliferative agents, IV: The mechanisms involved in inhibiting cell-cycle progression. Blood 98: 842–850. PubMed

Lui GY, Obeidy P, Ford SJ, Tselepis C, Sharp DM, Jansson PJ, et al. (2013) The iron chelator, deferasirox, as a novel strategy for cancer treatment: oral activity against human lung tumor xenografts and molecular mechanism of action. Mol Pharmacol 83: 179–190. 10.1124/mol.112.081893 PubMed DOI

Becton DL, Roberts B (1989) Antileukemic effects of deferoxamine on human myeloid leukemia cell lines. Cancer Res 49: 4809–4812. PubMed

Rodriguez-Lucena D, Gaboriau F, Rivault F, Schalk IJ, Lescoat G, Mislin GL (2010) Synthesis and biological properties of iron chelators based on a bis-2-(2-hydroxy-phenyl)-thiazole-4-carboxamide or -thiocarboxamide (BHPTC) scaffold. Bioorg Med Chem 18: 689–695. 10.1016/j.bmc.2009.11.057 PubMed DOI

Stefani C, Jansson PJ, Gutierrez E, Bernhardt PV, Richardson DR, Kalinowski DS (2013) Alkyl substituted 2'-benzoylpyridine thiosemicarbazone chelators with potent and selective anti-neoplastic activity: novel ligands that limit methemoglobin formation. J Med Chem 56: 357–370. 10.1021/jm301691s PubMed DOI

Miao Q, Xu D, Wang Z, Xu L, Wang T, Wu Y, et al. (2010) Amphiphilic hyper-branched co-polymer nanoparticles for the controlled delivery of anti-tumor agents. Biomaterials 31: 7364–7375. 10.1016/j.biomaterials.2010.06.012 PubMed DOI

Allegretti PE, Peroncini V, Castro EA, Furlong JP (2003) Study of the occurrence of tautomeric forms of ureas and thioureas by mass spectrometry. Int J Chem Sci 1: 1–12.

Yuan J, Lovejoy DB, Richardson DR (2004) Novel di-2-pyridyl-derived iron chelators with marked and selective antitumor activity: in vitro and in vivo assessment. Blood 104: 1450–1458. PubMed

Chaston TB, Watts RN, Yuan J, Richardson DR (2004) Potent antitumor activity of novel iron chelators derived from di-2-pyridylketone isonicotinoyl hydrazone involves fenton-derived free radical generation. Clin Cancer Res 10: 7365–7374. PubMed

Lederman HM, Cohen A, Lee JW, Freedman MH, Gelfand EW (1984) Deferoxamine: a reversible S-phase inhibitor of human lymphocyte proliferation. Blood 64: 748–753. PubMed

Mackova E, Hruskova K, Bendova P, Vavrova A, Jansova H, Haskova P, et al. (2012) Methyl and ethyl ketone analogs of salicylaldehyde isonicotinoyl hydrazone: novel iron chelators with selective antiproliferative action. Chem Biol Interact 197: 69–79. 10.1016/j.cbi.2012.03.010 PubMed DOI

Biederbick A, Kern HF, Elsasser HP (1995) Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur J Cell Biol 66: 3–14. PubMed

Bampton ET, Goemans CG, Niranjan D, Mizushima N, Tolkovsky AM (2005) The dynamics of autophagy visualized in live cells: from autophagosome formation to fusion with endo/lysosomes. Autophagy 1: 23–36. PubMed

Gutierrez E, Richardson DR, Jansson PJ (2014) The anticancer agent di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes prosurvival autophagy by two mechanisms: persistent induction of autophagosome synthesis and impairment of lysosomal integrity. J Biol Chem 289: 33568–33589. 10.1074/jbc.M114.599480 PubMed DOI PMC

Klionsky DJ, Cuervo AM, Seglen PO (2007) Methods for monitoring autophagy from yeast to human. Autophagy 3: 181–206. PubMed

Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140: 313–326. 10.1016/j.cell.2010.01.028 PubMed DOI PMC

Sahni S, Bae DH, Lane DJ, Kovacevic Z, Kalinowski DS, Jansson PJ, et al. (2014) The metastasis suppressor, N-myc downstream-regulated gene 1 (NDRG1), inhibits stress-induced autophagy in cancer cells. J Biol Chem 289: 9692–9709. 10.1074/jbc.M113.529511 PubMed DOI PMC

Korolchuk VI, Rubinsztein DC (2012) On signals controlling autophagy: It’s time to eat yourself healthy Biochemist 34: 8–13.

Fulda S, Debatin KM (2006) Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25: 4798–4811. PubMed

Earnshaw WC, Martins LM, Kaufmann SH (1999) Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 68: 383–424. PubMed

Liang Y, Yan C, Schor NF (2001) Apoptosis in the absence of caspase 3. Oncogene 20: 6570–6578. PubMed