Identification of protein targets underlying dietary nitrate-induced protection against doxorubicin cardiotoxicity

J Cell Mol Med. 2011 Nov;15(11):2512-24. doi: 10.1111/j.1582-4934.2011.01257.x.


We recently demonstrated protective effect of chronic oral nitrate supplementation against cardiomyopathy caused by doxorubicin (DOX), a highly effective anticancer drug. The present study was designed to identify novel protein targets related to nitrate-induced cardioprotection. Adult male CF-1 mice received cardioprotective regimen of nitrate (1 g NaNO(3) per litre of drinking water) for 7 days before DOX injection (15 mg/kg, i.p.) and continued for 5 days after DOX treatment. Subsequently the heart samples were collected for proteomic analysis with two-dimensional differential in-gel electrophoresis with 3 CyDye labelling. Using 1.5 cut-off ratio, we identified 36 proteins that were up-regulated by DOX in which 32 were completely reversed by nitrate supplementation (89%). Among 19 proteins down-regulated by DOX, 9 were fully normalized by nitrate (47%). The protein spots were further identified with Matrix Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-TOF)/TOF tandem mass spectrometry. Three mitochondrial antioxidant enzymes were altered by DOX, i.e. up-regulation of manganese superoxide dismutase and peroxiredoxin 3 (Prx3), and down-regulation of Prx5, which were reversed by nitrate. These results were further confirmed by Western blots. Nitrate supplementation also significantly improved animal survival rate from 80% in DOX alone group to 93% in Nitrate + DOX group 5 days after the DOX treatment. In conclusion, the proteomic analysis has identified novel protein targets underlying nitrate-induced cardioprotection. Up-regulation of Prx5 by nitrate may explain the observed enhancement of cardiac antioxidant defence by nitrate supplementation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibiotics, Antineoplastic / adverse effects
  • Antibiotics, Antineoplastic / toxicity
  • Antioxidants
  • Cardiomyopathies / chemically induced
  • Cardiomyopathies / mortality
  • Cardiomyopathies / prevention & control*
  • Cardiotonic Agents
  • Cardiotoxins / toxicity*
  • Doxorubicin / toxicity*
  • Heart / drug effects*
  • Male
  • Mice
  • Myocytes, Cardiac / drug effects
  • Myocytes, Cardiac / metabolism
  • Nitrates / administration & dosage*
  • Nitric Oxide / metabolism*
  • Oxidative Stress / drug effects
  • Peroxiredoxin III / biosynthesis
  • Peroxiredoxins / biosynthesis
  • Proteomics
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Superoxide Dismutase / biosynthesis
  • Survival


  • Antibiotics, Antineoplastic
  • Antioxidants
  • Cardiotonic Agents
  • Cardiotoxins
  • Nitrates
  • Nitric Oxide
  • Doxorubicin
  • Peroxiredoxin III
  • Peroxiredoxins
  • Superoxide Dismutase