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Therapeutic Concentrations of Mitoxantrone Elicit Energetic Imbalance in H9c2 Cells as an Earlier Event

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Abstract

Mitoxantrone (MTX) is a chemotherapeutic agent that emerged as an alternative to anthracycline therapy. However, MTX also causes late cardiotoxicity, being oxidative stress and mitochondrial-impaired function proposed as possible mechanisms. This work aimed to investigate the relevance of these mechanisms to the MTX toxicity in H9c2 cells, using therapeutic concentrations. The observed cytotoxicity of MTX was time and concentration dependent in both lactate dehydrogenase leakage assay and MTT reduction assay. Two therapeutic concentrations (100 nM and 1 μM) and three time points were selected (24, 48, and 96 h) for further studies. Both MTX concentrations caused a significant increase in caspase-3 activity, which was not prevented by inhibiting MTX CYP450-metabolism. Significant decreases were observed in the total and reduced glutathione levels only in MTX 100 nM at 96 h; however, neither alterations in oxidized glutathione nor increases in the malondialdehyde levels were observed at any time or concentrations tested. On the other hand, changes in the intracellular ATP levels, mitochondrial membrane potential, and intracellular calcium levels were observed in both concentrations and all time tested. Noteworthy, decreased levels of ATP-synthase expression and activity and increases in the reactive species generation were observed at 96 h in both working concentrations. However, the radical scavenger N-acetylcysteine or the mitochondrial function enhancer L-carnitine did not prevent MTX cytotoxicity. Thus, this work evidenced the early MTX-induced energetic crisis as a possible key factor in the cell injury.

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Abbreviations

DCFH-DA:

Dichlorodihydrofluorescein diacetate

DHR:

Dihydrorhodamine 123

DMEM:

Dulbecco’s modified eagle’s medium

DMSO:

Dimethyl sulfoxide

DTNB:

5,5-Dithio-bis(2-nitrobenzoic) acid

FBS:

Fetal bovine serum

FI:

Fluorescence intensity

GSH:

Reduced glutathione

GSHt:

Total glutathione

i.p:

Intraperitoneal

GSSG:

Oxidized glutathione

LDH:

Lactate dehydrogenase

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

MTX:

Mitoxantrone

NAC:

N-acetylcysteine

β-NADH:

Reduced β-nicotinamide adenine dinucleotide

Pi:

Inorganic phosphate

SD:

Standard deviation

TBA:

Thiobarbituric acid

TMRM:

Tetramethylrhodamine

References

  1. Seiter, K. (2005). Toxicity of the topoisomerase II inhibitors. Expert Opinion on Drug Safety, 4, 219–234.

    Article  PubMed  CAS  Google Scholar 

  2. Kingwell, E., Koch, M., Leung, B., Isserow, S., Geddes, J., Rieckmann, P., et al. (2010). Cardiotoxicity and other adverse events associated with mitoxantrone treatment for MS. Neurology, 74, 1822–1826.

    Article  PubMed  CAS  Google Scholar 

  3. Khan, S. N., Lai, S. K., Kumar, P., & Khan, A. U. (2010). Effect of mitoxantrone on proliferation dynamics and cell cycle progression. Bioscience Reports, 30, 375–381.

    Article  PubMed  CAS  Google Scholar 

  4. Hajihassan, Z., & Rabbani-Chadegani, A. (2011). Interaction of mitoxantrone, as an anticancer drug, with chromatin proteins, core histones and H1, in solution. International Journal of Biological Macromolecules, 48, 87–92.

    Article  PubMed  CAS  Google Scholar 

  5. Alderton, P. M., Gross, J., & Green, M. D. (1992). Comparative study of doxorubicin, mitoxantrone, and epirubicin in combination with ICRF-187 (ADR-529) in a chronic cardiotoxicity animal model. Cancer Research, 52, 194–201.

    PubMed  CAS  Google Scholar 

  6. Batra, V. K., Morrison, J. A., Woodward, D. L., Siverd, N. S., & Yacobi, A. (1986). Pharmacokinetics of mitoxantrone in man and laboratory animals. Drug Metabolism Reviews, 17, 311–329.

    Article  PubMed  CAS  Google Scholar 

  7. Sereno, M., Brunello, A., Chiappori, A., Barriuso, J., Casado, E., Belda, C., et al. (2008). Cardiac toxicity: Old and new issues in anti-cancer drugs. Clinical and Translational Oncology, 10, 35–46.

    Article  PubMed  CAS  Google Scholar 

  8. Wallace, K. B. (2003). Doxorubicin-induced cardiac mitochondrionopathy. Pharmacology and Toxicology, 93, 105–115.

    Article  PubMed  CAS  Google Scholar 

  9. Brück, T. B., & Brück, D. W. (2011). Oxidative metabolism of the anti-cancer agent mitoxantrone by horseradish, lacto-and lignin peroxidase. Biochimie, 93, 217–226.

    Article  PubMed  Google Scholar 

  10. Duthie, S. J., & Grant, M. H. (1989). The role of reductive and oxidative metabolism in the toxicity of mitoxantrone, adriamycin and menadione in human liver derived Hep G2 hepatoma cells. British Journal of Cancer, 60, 566–571.

    Article  PubMed  CAS  Google Scholar 

  11. Panousis, C., Kettle, A., & Phillips, D. R. (1995). Myeloperoxidase oxidises mitoxantrone to metabolites which bind covalently DNA and RNA. Anticancer drug designer, 10, 593–605.

    CAS  Google Scholar 

  12. Mewes, K., Blanz, J., Ehninger, G., Gebhardt, R., & Zeller, K. P. (1993). Cytochrome P-450-induced cytotoxicity of mitoxantrone by formation of electrophilic intermediates. Cancer Research, 53, 5135–5142.

    PubMed  CAS  Google Scholar 

  13. Li, S. J., Rodgers, E. H., & Grant, M. H. (1995). The activity of xenobiotic enzymes and the cytotoxicity of mitoxantrone in MCF 7 human breast cancer cells treated with inducing agents. Chemico Biological Interactions, 97, 101–118.

    Article  PubMed  CAS  Google Scholar 

  14. Rossato, L., Costa, V., De Pinho, P., Freitas, V., Viloune, L., Bastos, M., et al. (2013). The metabolic profile of mitoxantrone and its relation with mitoxantrone-induced cardiotoxicity. Archives of Toxicology. doi:10.1007/s00204-013-1040-6.

  15. Shipp, N. G., Dorr, R. T., Alberts, D. S., Dawson, B. V., & Hendrix, M. (1993). Characterization of experimental mitoxantrone cardiotoxicity and its partial inhibition by ICRF-187 in cultured neonatal rat heart cells. Cancer Research, 53, 550–556.

    PubMed  CAS  Google Scholar 

  16. Bachmann, E., Weber, E., & Zbinden, G. (1987). Effect of mitoxantrone and doxorubicin on energy metabolism of the rat heart. Cancer Treatment Reports, 71, 361–366.

    PubMed  CAS  Google Scholar 

  17. Kimes, B. W., & Brandt, B. L. (1976). Properties of a clonal muscle from rat heart. Experimental Cell Research, 98, 367–381.

    Article  PubMed  CAS  Google Scholar 

  18. Zordoky, B. N. M., & El-Kadi, A. O. S. (2007). H9c2 cell line is a valuable in vitro model to study the drug metabolizing enzymes in the heart. Journal of Pharmacological and Toxicological Methods, 56, 317–322.

    Article  PubMed  CAS  Google Scholar 

  19. Costa, V. M., Silva, R., Ferreira, R., Amado, F., Carvalho, F., Bastos, M. L., et al. (2009). Adrenaline in pro-oxidant conditions elicits intracellular survival pathways in isolated rat cardiomyocytes. Toxicology, 257, 70–79.

    Article  PubMed  CAS  Google Scholar 

  20. Rossato, L. G., Costa, V. M., De Pinho, P. G., Carvalho, F., Bastos, M. L., & Remião, F. (2011). Structural isomerization of synephrine influences its uptake and ensuing glutathione depletion in rat-isolated cardiomyocytes. Archives of Toxicology, 85, 929–939.

    Article  PubMed  CAS  Google Scholar 

  21. Sayed-Ahmed, M. M., Shaarawy, S., Shouman, S. A., & Osman, A. M. (1999). Reversal of doxorubicin-induced cardiac metabolic damage by L-carnitine. Pharmacological Research, 39, 289–295.

    Article  PubMed  CAS  Google Scholar 

  22. Angeloni, C., Spencer, J. P. E., Leoncini, E., Biagi, P. L., & Hrelia, S. (2007). Role of quercetin and its in vivo metabolites in protecting H9c2 cells against oxidative stress. Biochimie, 89, 73–82.

    Article  PubMed  CAS  Google Scholar 

  23. Grotto, D., Santa Maria, L. D., Boeira, S., Valentini, J., Charão, M. F., Moro, A. M., et al. (2007). Rapid quantification of malondialdehyde in plasma by high performance liquid chromatography-visible detection. Journal of Pharmaceutical and Biomedical Analysis, 43, 619–624.

    Article  PubMed  CAS  Google Scholar 

  24. Costa, V. M., Silva, R., Ferreira, L. M., Branco, P. S., Carvalho, F., Bastos, M. L., et al. (2007). Oxidation process of adrenaline in freshly isolated rat cardiomyocytes: Formation of adrenochrome, quinoproteins, and GSH adduct. Chemical Research in Toxicology, 20, 1183–1191.

    Article  PubMed  CAS  Google Scholar 

  25. Floryk, D., & Houstĕk, J. (1999). Tetramethyl rhodamine methyl ester (TMRM) is suitable for cytofluorometric measurements of mitochondrial membrane potential in cells treated with digitonin. Bioscience Reports, 19, 27–34.

    Article  PubMed  CAS  Google Scholar 

  26. Carvalho, M., Remião, F., Milhazes, N., Borges, F., Fernandes, E., Monteiro, M. D. C., et al. (2004). Metabolism is required for the expression of ecstasy-induced cardiotoxicity in vitro. Chemical Research in Toxicology, 17, 623–632.

    Article  PubMed  CAS  Google Scholar 

  27. Canal, P., Attal, M., Chatelut, E., Guichard, S., Huguet, F., Muller, C., et al. (1993). Plasma and cellular pharmacokinetics of mitoxantrone in high-dose chemotherapeutic regimen for refractory lymphomas. Cancer Research, 53, 4850–4854.

    PubMed  CAS  Google Scholar 

  28. Neuhaus, O., Kieseier, B. C., & Hartung, H.-P. (2006). Therapeutic role of mitoxantrone in multiple sclerosis. Pharmacology & Therapeutics, 109, 198–209.

    Article  CAS  Google Scholar 

  29. Kluza, J., Marchetti, P., Gallego, M.-A., Lancel, S., Fournier, C., Loyens, A., et al. (2004). Mitochondrial proliferation during apoptosis induced by anticancer agents: Effects of doxorubicin and mitoxantrone on cancer and cardiac cells. Oncogene, 23, 7018–7030.

    Article  PubMed  CAS  Google Scholar 

  30. Fisher, G. R., & Patterson, L. H. (1992). Lack of involvement of reactive oxygen in the cytotoxicity of mitoxantrone, CI941 and ametantrone in MCF-7 cells: comparison with doxorubicin. Cancer Chemotherapy and Pharmacology, 30, 451–458.

    Article  PubMed  CAS  Google Scholar 

  31. Chen, F., Lewis, W., Hollander, J. M., Baseler, W., & Finkel, M. S. (2012). N-acetylcysteine reverses cardiac myocyte dysfunction in HIV-Tat proteinopathy. Journal of Applied Physiology, 113, 105–113.

    Article  PubMed  CAS  Google Scholar 

  32. Chen, F., Hadfield, J., Berzingi, C., Hollander, J., Miller, D., Nichols, C., et al. (2013). N-acetylcysteine reverses cardiac myocyte dysfunction in a rodent model of behavioral stress. Journal of Applied Physiology, 115, 514–524.

    Article  PubMed  CAS  Google Scholar 

  33. Arnaiz, S. L., & Llesuy, S. (1993). Oxidative stress in mouse heart by antitumoral drugs: A comparative study of doxorubicin and mitoxantrone. Toxicology, 77, 31–38.

    Article  CAS  Google Scholar 

  34. Novak, R. F., & Kharasch, E. D. (1985). Mitoxantrone: Propensity for free radical formation and lipid peroxidation-implications for cardiotoxicity. Investigational New Drugs, 3, 95–99.

    Article  PubMed  CAS  Google Scholar 

  35. Dong, Z., Saikumar, P., Weinberg, J. M., & Venkatachalam, M. A. (2006). Calcium in cell injury and death. Annual Review of Pathology: Mechanisms of Disease, 1, 405–434.

    Article  CAS  Google Scholar 

  36. Li, R., Beebe, T., Cui, J., Rouhanizadeh, M., Ai, L., Wang, P., et al. (2009). Pulsatile shear stress increased mitochondrial membrane potential: Implication of Mn-SOD. Biochemical and Biophysical Research Communications, 388, 406–412.

    Article  PubMed  CAS  Google Scholar 

  37. Acton, B. M. (2004). Alterations in mitochondrial membrane potential during preimplantation stages of mouse and human embryo development. Molecular Human Reproduction, 10, 23–32.

    Article  PubMed  CAS  Google Scholar 

  38. Zammit, V. A., Ramsay, R. R., Bonomini, M., & Arduini, A. (2009). Carnitine, mitochondrial function and therapy. Advanced Drug Delivery Reviews, 61, 1353–1362.

    Article  PubMed  CAS  Google Scholar 

  39. Mijares, A., & López, J. R. (2001). L-carnitine prevents increase in diastolic [Ca2+] induced by doxorubicin in cardiac cells. European Journal of Pharmacology, 425, 117–120.

    Article  PubMed  CAS  Google Scholar 

  40. Newman, R., Hacker, M., & Krakoff, I. (1981). Amelioration of adriamycin and daunorubicin myocardial toxicity by adenosine. Cancer Research, 41, 3483–3488.

    PubMed  CAS  Google Scholar 

  41. Costa, V. M., Carvalho, F., Bastos, M. L., Carvalho, R. A., Carvalho, M., & Remião, F. (2011). Contribution of catecholamine reactive intermediates and oxidative stress to the pathologic features of heart diseases. Current Medical Chemistry, 18, 2272–2314.

    Article  CAS  Google Scholar 

  42. Trump, B. F., & Berezesky, I. K. (1995). Calcium-mediated cell injury and cell death. FASEB, 9, 219–228.

    CAS  Google Scholar 

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Acknowledgments

This work received financial support from “Fundação para a Ciência e Tecnologia (FCT),” Portugal (EXPL/DTP-FTO/0290/2012) and by “Fundo Comunitário Europeu” (FEDER) under the frame of “Eixo I do Programa Operacional Fatores de Competitividade (POFC) do QREN” (COMPETE: FCOMP-01-0124-FEDER-027749). The work was also supported by FCT within the framework of Strategic Projects for Scientific Research Units of R&D (project PEst-C/EQB/LA0006/2011). LGR and VVB thank FCT for their PhD Grant (SFRH/BD/63473/2009 and SFRH/BD/82556/2011, respectively) and VMC thank FCT for her Post-doc Grant (SFRH/BPD/63746/2009). Authors are grateful to Dr. Vilma Sardão, from University of Coimbra, for gently providing us with the cellular model used in this work.

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Authors and Affiliations

  1. REQUIMTE, Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal

    Luciana Grazziotin Rossato, Vera Marisa Costa, Vânia Vilas-Boas, Maria de Lourdes Bastos & Fernando Remião

  2. Departamento de Ciências da Vida, Centro de Neurociências e Biologia Celular de Coimbra, Universidade de Coimbra, Coimbra, Portugal

    Anabela Rolo & Carlos Palmeira

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  1. Luciana Grazziotin Rossato
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  2. Vera Marisa Costa
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  3. Vânia Vilas-Boas
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  4. Maria de Lourdes Bastos
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Correspondence to Luciana Grazziotin Rossato or Fernando Remião.

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Rossato, L.G., Costa, V.M., Vilas-Boas, V. et al. Therapeutic Concentrations of Mitoxantrone Elicit Energetic Imbalance in H9c2 Cells as an Earlier Event. Cardiovasc Toxicol 13, 413–425 (2013). https://doi.org/10.1007/s12012-013-9224-0

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