Archives of Toxicology

, Volume 91, Issue 4, pp 1871–1890 | Cite as

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

  • A. Reis-Mendes
  • A. S. Gomes
  • R. A. Carvalho
  • F. Carvalho
  • F. Remião
  • M. Pinto
  • M. L. Bastos
  • E. Sousa
  • V. M. Costa
Organ Toxicity and Mechanisms


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.


Mitoxantrone Autophagy Naphthoquinoxaline Differentiated H9c2 cells Cardiotoxicity 



Adenosine triphosphate


3,3′-Dihexyloxacarbocyanine iodide


Dulbecco’s modified eagle medium


Dimethyl sulphoxide


Foetal bovine serum


Fluorescence units




High-performance liquid chromatography


Horseradish peroxidase


Retention factor


Lactate dehydrogenase




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






Nicotinamide adenine dinucleotide reduced form




Neutral red


Nuclear magnetic resonance


Phosphate-buffered saline


Retinoic acid




Standard deviation


Sodium dodecyl sulphate




Thin-layer chromatography





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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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.UCIBIO-REQUIMTE (Rede de Química e Tecnologia), Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de FarmáciaUniversidade do PortoPortoPortugal
  2. 2.UCIBIO-REQUIMTE, Laboratory of Microbiology, Department of Biological Sciences, Faculty of PharmacyUniversity of PortoPortoPortugal
  3. 3.Centre for Functional Ecology, Department of Life Sciences, Faculty of Sciences and TechnologyUniversity of CoimbraCoimbraPortugal
  4. 4.Lab. Química Orgânica e Farmacêutica, Dep. Química, Faculdade de FarmáciaU. PortoPortoPortugal
  5. 5.CIIMAR – Interdisciplinary Centre of Marine and Environmental ResearchPortoPortugal

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