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Naphthoquinoxaline metabolite of mitoxantrone is less cardiotoxic than the parent compound and it can be a more cardiosafe drug in anticancer therapy

  • Organ Toxicity and Mechanisms
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Abstract

Mitoxantrone (MTX) is an antineoplastic agent used to treat several types of cancers and on multiple sclerosis, which shows a high incidence of cardiotoxicity. Still, the underlying mechanisms of MTX cardiotoxicity are poorly understood and the potential toxicity of its metabolites scarcely investigated. Therefore, this work aimed to synthesize the MTX-naphthoquinoxaline metabolite (NAPHT) and to compare its cytotoxicity to the parent compound in 7-day differentiated H9c2 cells using pharmacological relevant concentrations (0.01–5 µM). MTX was more toxic in equivalent concentrations in all cytotoxicity tests performed [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction, neutral red uptake, and lactate dehydrogenase release assays] and times tested (24 and 48 h). Both MTX and NAPHT significantly decreased mitochondrial membrane potential in 7-day differentiated H9c2 cells after a 12-h incubation. However, energetic pathways were affected in a different manner after MTX or NAPHT incubation. ATP increased and lactate levels decreased after a 24-h incubation with MTX, whereas for the same incubation time and concentrations, NAPHT did not cause any significant effect. The increased activity of ATP synthase seems responsible for MTX-induced increases in ATP levels, as oligomycin (an inhibitor of ATP synthase) abrogated this effect on 5 µM MTX-incubated cells. 3-Methyladenine (an autophagy inhibitor) was the only molecule to give a partial protection against the cytotoxicity produced by MTX or NAPHT. To the best of our knowledge, this was the first broad study on NAPHT cardiotoxicity, and it revealed that the parent drug, MTX, caused a higher disruption in the energetic pathways in a cardiac model in vitro, whereas autophagy is involved in the toxicity of both compounds. In conclusion, NAPHT is claimed to largely contribute to MTX-anticancer properties; therefore, this metabolite should be regarded as a good option for a safer anticancer therapy since it is less cardiotoxic than MTX.

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Abbreviations

ATP:

Adenosine triphosphate

DiO6 :

3,3′-Dihexyloxacarbocyanine iodide

DMEM:

Dulbecco’s modified eagle medium

DMSO:

Dimethyl sulphoxide

FBS:

Foetal bovine serum

FU:

Fluorescence units

h:

Hours

HPLC:

High-performance liquid chromatography

HRP:

Horseradish peroxidase

k :

Retention factor

LDH:

Lactate dehydrogenase

min:

Minutes

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

MTX:

Mitoxantrone

m :

Multiplet

NADH:

Nicotinamide adenine dinucleotide reduced form

NAPHT:

Naphthoquinoxaline

NR:

Neutral red

NMR:

Nuclear magnetic resonance

PBS:

Phosphate-buffered saline

RA:

Retinoic acid

s :

Singlet

SD:

Standard deviation

SDS:

Sodium dodecyl sulphate

t :

Triplet

TLC:

Thin-layer chromatography

UV:

Ultraviolet

References

  • Albini A, Pennesi G, Donatelli F, Cammarota R, De Flora S, Noonan DM (2010) Cardiotoxicity of anticancer drugs: the need for cardio-oncology and cardio-oncological prevention. J Natl Cancer Inst 102(1):14–25

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Allemani C, Weir HK, Carreira H et al (2015) Global surveillance of cancer survival 1995–2009: analysis of individual data for 25,676,887 patients from 279 population-based registries in 67 countries (CONCORD-2). The Lancet 385(9972):977–1010

    Article  Google Scholar 

  • Avasarala JR, Cross AH, Clifford DB, Singer BA, Siegel BA, Abbey EE (2003) Rapid onset mitoxantrone-induced cardiotoxicity in secondary progressive multiple sclerosis. Mult Scler 9(1):59–62

    Article  CAS  PubMed  Google Scholar 

  • Blanz J, Mewes K, Ehninger G et al (1991) Evidence for oxidative activation of mitoxantrone in human, pig, and rat. Drug Metab Dispos 19(5):871–880

    CAS  PubMed  Google Scholar 

  • Bruck TB, Bruck DW (2011) Oxidative metabolism of the anti-cancer agent mitoxantrone by horseradish, lacto-and lignin peroxidase. Biochimie 93:217–226

    Article  PubMed  Google Scholar 

  • Bruck TB, Harvey PJ (2003) Oxidation of mitoxantrone by lactoperoxidase. Biochim Biophys Acta 1649:154–163

    Article  CAS  PubMed  Google Scholar 

  • Capela JP, Meisel A, Abreu AR et al (2006) Neurotoxicity of Ecstasy metabolites in rat cortical neurons, and influence of hyperthermia. J Pharmacol Exp Ther 316(1):53–61

    Article  CAS  PubMed  Google Scholar 

  • Capela JP, da Costa Araujo S, Costa VM et al (2013) The neurotoxicity of hallucinogenic amphetamines in primary cultures of hippocampal neurons. Neurotoxicology 34:254–263

    Article  CAS  PubMed  Google Scholar 

  • Carver JR, Desai CJ (2010) Cardiovascular toxicity of antitumor drugs: dimension of the problem in adult settings. In: Minotti G (ed) Cardiotoxicity of non-cardiovascular drugs. Wiley, Hoboken, pp 127–199

    Chapter  Google Scholar 

  • Chiccarelli FS, Morrison JA, Cosulich DB et al (1986) Identification of human urinary mitoxantrone metabolites. Cancer Res 46(9):4858–4861

    CAS  PubMed  Google Scholar 

  • Coleman RE, Maisey MN, Knight RK, Rubens RD (1984) Mitoxantrone in advanced breast cancer—a phase II study with special attention to cardiotoxicity. Eur J Cancer Clin Oncol 20(6):771–776

    Article  CAS  PubMed  Google Scholar 

  • Costa VM, Silva R, Ferreira LM et al (2007) Oxidation process of adrenaline in freshly isolated rat cardiomyocytes: formation of adrenochrome, quinoproteins, and GSH adduct. Chem Res Toxicol 20(8):1183–1191

    Article  CAS  PubMed  Google Scholar 

  • Costa VM, Silva R, Ferreira R et al (2009a) Adrenaline in pro-oxidant conditions elicits intracellular survival pathways in isolated rat cardiomyocytes. Toxicology 257(1–2):70–79

    Article  CAS  PubMed  Google Scholar 

  • Costa VM, Silva R, Tavares LC et al (2009b) Adrenaline and reactive oxygen species elicit proteome and energetic metabolism modifications in freshly isolated rat cardiomyocytes. Toxicology 260(1–3):84–96

    Article  CAS  PubMed  Google Scholar 

  • Dietel M, Arps H, Lage H, Niendorf A (1990) Membrane vesicle formation due to acquired mitoxantrone resistance in human gastric carcinoma cell line EPG85-257. Cancer Res 50(18):6100–6106

    CAS  PubMed  Google Scholar 

  • Dores-Sousa JL, Duarte JA, Seabra V, Bastos ML, Carvalho F, Costa VM (2015) The age factor for mitoxantrone’s cardiotoxicity: multiple doses render the adult mouse heart more susceptible to injury. Toxicology 329:106–119

    Article  CAS  PubMed  Google Scholar 

  • Ehninger G, Schuler U, Proksch B, Zeller KP, Blanz J (1990) Pharmacokinetics and metabolism of mitoxantrone. A review. Clin Pharmacokinet 18(5):365–380

    Article  CAS  PubMed  Google Scholar 

  • Feofanov A, Sharonov S, Fleury F, Kudelina I, Nabiev I (1997a) Quantitative confocal spectral imaging analysis of mitoxantrone within living K562 cells: intracellular accumulation and distribution of monomers, aggregates, naphtoquinoxaline metabolite, and drug-target complexes. Biophys J 73(6):3328–3336

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Feofanov A, Sharonov S, Kudelina I, Fleury F, Nabiev I (1997b) Localization and molecular interactions of mitoxantrone within living K562 cells as probed by confocal spectral imaging analysis. Biophys J 73(6):3317–3327

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ferlay J, Steliarova-Foucher E, Lortet-Tieulent J et al (2013) Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012. Eur J Cancer 49(6):1374–1403

    Article  CAS  PubMed  Google Scholar 

  • Ferreira PS, Nogueira TB, Costa VM et al (2013) Neurotoxicity of “ecstasy” and its metabolites in human dopaminergic differentiated SH-SY5Y cells. Toxicol Lett 216(2–3):159–170

    Article  CAS  PubMed  Google Scholar 

  • Fotakis G, Timbrell JA (2006) In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicol Lett 160(2):171–177

    Article  CAS  PubMed  Google Scholar 

  • Freitas M, Costa VM, Ribeiro D et al (2013) Acetaminophen prevents oxidative burst and delays apoptosis in human neutrophils. Toxicol Lett 219(2):170–177

    Article  CAS  PubMed  Google Scholar 

  • Goldman SDB, Funk RS, Rajewski RA, Krise JP (2009) Mechanisms of amine accumulation in, and egress from, lysosomes. Bioanalysis 1(8):1445–1459

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gupta MK, Neelakantan TV, Sanghamitra M et al (2006) An assessment of the role of reactive oxygen species and redox signaling in norepinephrine-induced apoptosis and hypertrophy of H9c2 cardiac myoblasts. Antioxid Redox Signal 8(5–6):1081–1093

    Article  CAS  PubMed  Google Scholar 

  • Kimes BW, Brandt BL (1976) Properties of a clonal muscle cell line from rat heart. Exp Cell Res 98(2):367–381

    Article  CAS  PubMed  Google Scholar 

  • Kluza J, Marchetti P, Gallego M-A et al (2004) Mitochondrial proliferation during apoptosis induced by anticancer agents: effects of doxorubicin and mitoxantrone on cancer and cardiac cells. Oncogene 23(42):7018–7030

    Article  CAS  PubMed  Google Scholar 

  • Kolodziejczyk P, Reszka K, Lown JW (1988) Enzymatic oxidative activation and transformation of the antitumor agent mitoxantrone. Free Radic Biol Med 5:13–25

    Article  CAS  PubMed  Google Scholar 

  • Martins JB, Bastos Mde L, Carvalho F, Capela JP (2013) Differential effects of methyl-4-phenylpyridinium ion, rotenone, and paraquat on differentiated SH-SY5Y cells. J Toxicol 2013:347312

    PubMed  PubMed Central  Google Scholar 

  • Menna P, Salvatorelli E, Minotti G (2008) Cardiotoxicity of antitumor drugs. Chem Res Toxicol 21(5):978–989

    Article  CAS  PubMed  Google Scholar 

  • Mewes K, Blanz J, Ehninger G, Gebhardt R, Zeller KP (1993) Cytochrome P-450-induced cytotoxicity of mitoxantrone by formation of electrophilic intermediates. Cancer Res 53(21):5135–5142

    CAS  PubMed  Google Scholar 

  • Ndolo RA, Luan Y, Duan S, Forrest ML, Krise JP (2012) Lysosomotropic properties of weakly basic anticancer agents promote cancer cell selectivity in vitro. PLoS ONE 7(11):e49366

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Panousis C, Kettle AJ, Phillips DR (1994) Oxidative metabolism of mitoxantrone by the human neutrophil enzyme myeloperoxidase. Biochem Pharmacol 48(12):2223–2230

    Article  CAS  PubMed  Google Scholar 

  • Panousis C, Kettle AJ, Phillips DR (1997) Neutrophil-mediated activation of mitoxantrone to metabolites which form adducts with DNA. Cancer Lett 113(1–2):173–178

    Article  CAS  PubMed  Google Scholar 

  • Pereira SL, Ramalho-Santos J, Branco AF, Sardao VA, Oliveira PJ, Carvalho RA (2011) Metabolic remodeling during H9c2 myoblast differentiation: relevance for in vitro toxicity studies. Cardiovasc Toxicol 11(2):180–190

    Article  CAS  PubMed  Google Scholar 

  • Pratt CB, Vietti TJ, Etcubanas E et al (1986) Novantrone for childhood malignant solid tumors. A pediatric oncology group phase II study. Invest New Drugs 4(1):43–48

    Article  CAS  PubMed  Google Scholar 

  • Reis-Mendes A, Sousa E, Bastos ML, Costa VM (2015) Metabolism of anticancer drugs and cardiotoxicity: a missing link? Curr Drug Metab 17(1):75–90

    Article  PubMed  Google Scholar 

  • Reszka KJ, Chignell CF (1996) Acid-catalyzed oxidation of the anticancer agent mitoxantrone by nitrite ions. Mol Pharmacol 50:1612–1618

    CAS  PubMed  Google Scholar 

  • Reszka K, Kolodziejczyk P, Lown JW (1986) Horseradish peroxidase-catalyzed oxidation of mitoxantrone: spectrophotometric and electron paramagnetic resonance studies. J Free Radic Biol Med 2:25–32

    CAS  PubMed  Google Scholar 

  • Rossato LG, Costa VM, de Pinho PG et al (2013a) The metabolic profile of mitoxantrone and its relation with mitoxantrone-induced cardiotoxicity. Arch Toxicol 87:1809–1820

    Article  CAS  PubMed  Google Scholar 

  • Rossato LG, Costa VM, Vilas-Boas V et al (2013b) Therapeutic concentrations of mitoxantrone elicit energetic imbalance in H9c2 cells as an earlier event. Cardiovasc Toxicol 13(4):413–425

    Article  CAS  PubMed  Google Scholar 

  • Rossato LG, Costa V, Dallegrave E et al (2014) Mitochondrial cumulative damage induced by mitoxantrone: late onset cardiac energetic impairment. Cardiovasc Toxicol 14(1):30–40

    Article  CAS  PubMed  Google Scholar 

  • Ruiz M, Courilleau D, Jullian JC et al (2012) A cardiac-specific robotized cellular assay identified families of human ligands as inducers of PGC-1alpha expression and mitochondrial biogenesis. PLoS ONE 7(10):e46753

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Seiter K (2005) Toxicity of the topoisomerase II inhibitors. Expert Opin Drug Saf 4(2):219–234

    Article  CAS  PubMed  Google Scholar 

  • Shi J, Shen HM (2008) Critical role of Bid and Bax in indirubin-3′-monoxime-induced apoptosis in human cancer cells. Biochem Pharmacol 75(9):1729–1742

    Article  CAS  PubMed  Google Scholar 

  • Shipp NG, Dorr RT, Alberts DS, Dawson BV, Hendrix M (1993) Characterization of experimental mitoxantrone cardiotoxicity and its partial inhibition by ICRF-187 in cultured neonatal rat heart cells. Cancer Res 53(3):550–556

    CAS  PubMed  Google Scholar 

  • Smith PJ, Sykes HR, Fox ME, Furlong IJ (1992) Subcellular distribution of the anticancer drug mitoxantrone in human and drug-resistant murine cells analyzed by flow cytometry and confocal microscopy and its relationship to the induction of DNA damage. Cancer Res 52(14):4000–4008

    CAS  PubMed  Google Scholar 

  • Soares AS, Costa VM, Diniz C, Fresco P (2013) Potentiation of cytotoxicity of paclitaxel in combination with Cl-IB-MECA in human C32 metastatic melanoma cells: a new possible therapeutic strategy for melanoma. Biomed Pharmacother 67(8):777–789

    Article  CAS  PubMed  Google Scholar 

  • Soares AS, Costa VM, Diniz C, Fresco P (2014) Combination of Cl-IB-MECA with paclitaxel is a highly effective cytotoxic therapy causing mTOR-dependent autophagy and mitotic catastrophe on human melanoma cells. J Cancer Res Clin Oncol 140(6):921–935

    Article  CAS  PubMed  Google Scholar 

  • Zhitomirsky B, Assaraf YG (2015) Lysosomal sequestration of hydrophobic weak base chemotherapeutics triggers lysosomal biogenesis and lysosome-dependent cancer multidrug resistance. Oncotarget 6(2):1143–1156

    Article  PubMed  Google Scholar 

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Acknowledgments

We would like to thank Dr. Sara Cravo for her technical assistance in obtaining the HPLC—diode array detector data and Centro de Apoio Científico e Tecnolóxico á Investigation (CACTI, University of Vigo, Spain) for enabling measurements at the API III mass spectrometer. NMR data was collected at the UC-NMR facility which is supported in part by FEDER—European Regional Development Fund through the COMPETE Programme (Operational Programme for Competitiveness) and by National Funds through FCT—Fundação para a Ciência e a Tecnologia (Portuguese Foundation for Science and Technology) through grants RECI/QEQ-QFI/0168/2012, CENTRO-07-CT62-FEDER-002012, and Rede Nacional de Ressonância Magnética Nuclear (RNRMN). This work was supported by the Fundação para a Ciência e Tecnologia (FCT)—projects EXPL/DTP-FTO/0290/2012 and PTDC/DTP-FTO/1489/2014—QREN initiative with EU/FEDER financing through COMPETE—Operational Programme for Competitiveness Factors and partially supported by the Strategic Funding UID/Multi/04423/2013 through national funds provided by FCT—Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of the programme PT2020. The authors are also grateful to “Fundação para a Ciência e a Tecnologia” for Grant Nos. UID/MULTI/04378/2013 and UID/Multi/04423/2013, ERDF, and PT2020. V.M.C. (Post doc) acknowledges “Fundação para a Ciência e Tecnologia (FCT)” for her post doc Grants (SFRH/BPD/63746/2009 and SFRH/BPD/110001/2015).

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Authors and Affiliations

  1. UCIBIO-REQUIMTE (Rede de Química e Tecnologia), Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal

    A. Reis-Mendes, F. Carvalho, F. Remião, M. L. Bastos & V. M. Costa

  2. UCIBIO-REQUIMTE, Laboratory of Microbiology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313, Porto, Portugal

    A. S. Gomes

  3. Centre for Functional Ecology, Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra, Coimbra, Portugal

    R. A. Carvalho

  4. Lab. Química Orgânica e Farmacêutica, Dep. Química, Faculdade de Farmácia, U. Porto, Porto, Portugal

    A. S. Gomes, M. Pinto & E. Sousa

  5. CIIMAR – Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal

    M. Pinto & E. Sousa

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  1. A. Reis-Mendes
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  2. A. S. Gomes
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  3. R. A. Carvalho
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  4. F. Carvalho
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  5. F. Remião
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  6. M. Pinto
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  7. M. L. Bastos
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  8. E. Sousa
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  9. V. M. Costa
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Corresponding authors

Correspondence to A. Reis-Mendes or V. M. Costa.

Additional information

Part of this article has been taken from the first author’s thesis entitled “The synthesis and in vitro toxicological evaluation of the mitoxantrone naphthoquinoxaline metabolite: a comparative study with the parent drug” printed last September 2014. The thesis document is available online at the university repository (available at http://repositorio-aberto.up.pt/bitstream/10216/77095/2/103849.pdf).

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Reis-Mendes, A., Gomes, A.S., Carvalho, R.A. et al. Naphthoquinoxaline metabolite of mitoxantrone is less cardiotoxic than the parent compound and it can be a more cardiosafe drug in anticancer therapy. Arch Toxicol 91, 1871–1890 (2017). https://doi.org/10.1007/s00204-016-1839-z

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  • DOI: https://doi.org/10.1007/s00204-016-1839-z

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