Mitochondrial apoptosis-inducing factor is involved in doxorubicin-induced toxicity on H9c2 cardiomyoblasts

Ana C. Moreira, Ana F. Branco, Susana F. Sampaio, Teresa Cunha-Oliveira, Tatiana R. Martins, Jon Holy, Paulo J. Oliveira, Vilma A. Sardão

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48 Scopus citations


The cardiotoxicity induced by the anti-cancer doxorubicin involves increased oxidative stress, disruption of calcium homeostasis and activation of cardiomyocyte death. Nevertheless, antioxidants and caspase inhibitors often show little efficacy in preventing cell death. We hypothesize that a caspase-independent cell death mechanism with the release of the apoptosis-inducing factor from mitochondria is involved in doxorubicin toxicity. To test the hypothesis, H9c2 cardiomyoblasts were used as model for cardiac cells. Our results demonstrate that z-VAD-fmk, a pan-caspase inhibitor, does not prevent doxorubicin toxicity in this cell line. Doxorubicin treatment results in AIF translocation to the nuclei, as confirmed by Western Blotting of cell fractions and confocal microscopy. Also, doxorubicin treatment of H9c2 cardiomyoblasts resulted in the appearance of 50. kbp DNA fragments, a hallmark of apoptosis-inducing factor nuclear effects. Apoptosis-inducing factor knockdown using a small-interfering RNA approach in H9c2 cells resulted in a reduction of doxorubicin toxicity, including decreased p53 activation and poly-ADP-ribose-polymerase cleavage. Among the proteases that could be responsible for apoptosis-inducing factor cleavage, doxorubicin decreased calpain activity but increased cathepsin B activation, with inhibition of the latter partly decreasing doxorubicin toxicity. Altogether, the results support that apoptosis-inducing factor release is involved in doxorubicin-induced H9c2 cell death, which explains the limited ability of caspase inhibitors to prevent toxicity.

Original languageEnglish (US)
Pages (from-to)2468-2478
Number of pages11
JournalBiochimica et Biophysica Acta - Molecular Basis of Disease
Issue number12
StatePublished - Dec 1 2014

Bibliographical note

Funding Information:
This work was supported by the Portuguese Foundation for Science and Technology (FCT) research grants PTDC/SAU-TOX/117912/2010 and PTDC/DTP-FTO/1180/2012 to P.J.O. and PEst-C/SAU/LA0001/2013-2014 (from FEDER/Compete/National Budget programs) as well as by FEDER/QREN program #4832, CENTRO-07-ST24-FEDER-002008 , to the CNC. The FCT also sponsored Ph.D. fellowhips SFRH/BD/33892/2009 to A.C.M. and SFRH/BD/41384/2007 to A.F.B.; V.A.S. was supported by FCT Post-doctoral fellowship SFRH/BPD/31549/2006 and afterwards by a FEDER/QREN #4832, CENTRO-07-ST24-FEDER-002008 Post-doctoral fellowship; T. C-O. was also supported by a FEDER/QREN #4832, CENTRO-07-ST24-FEDER-002008 Post-doctoral fellowship. The funding agencies had no role in the decision to publish or in the content of the manuscript. The authors are grateful to Dr Inês Araújo (University of Algarve, Portugal) for kindly providing the MDL28170 inhibitor.

Publisher Copyright:
© 2014 Elsevier B.V.


  • Apoptosis
  • Apoptosis-inducing factor
  • Cardiomyoblast
  • Doxorubicin


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