Merck
  • Home
  • Search Results
  • Mitochondrial apoptosis-inducing factor is involved in doxorubicin-induced toxicity on H9c2 cardiomyoblasts.

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

Biochimica et biophysica acta (2014-10-07)
Ana C Moreira, Ana F Branco, Susana F Sampaio, Teresa Cunha-Oliveira, Tatiana R Martins, Jon Holy, Paulo J Oliveira, Vilma A Sardão
ABSTRACT

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 50kbp 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Ethidium bromide solution, BioReagent, for molecular biology, 10 mg/mL in H2O
Sigma-Aldrich
Ethidium bromide solution, BioReagent, for molecular biology, 500 μg/mL in H2O
Sigma-Aldrich
Sodium chloride solution, 0.85%
Sigma-Aldrich
Sulforhodamine B, Dye content 75 %
Sigma-Aldrich
Ethidium bromide solution, for fluorescence, ~1% in H2O
Sigma-Aldrich
1,1,2,2-Tetrabromoethane, 98%
Sigma-Aldrich
Sodium chloride-35Cl, 99 atom % 35Cl
Sigma-Aldrich
Boric acid-11B, ≥99 atom % 11B
Sigma-Aldrich
Phenylmethanesulfonyl fluoride, ≥99.0% (T)
Sigma-Aldrich
Sodium chloride, BioUltra, for molecular biology, ≥99.5% (AT)
Sigma-Aldrich
Sodium chloride, tested according to Ph. Eur.
Sigma-Aldrich
1,1,2,2-Tetrabromoethane, purum p.a., for separation (of mineral compounds), ≥98.0% (GC)
Supelco
Sodium chloride, certified reference material for titrimetry, certified by BAM, ≥99.5%
Sigma-Aldrich
DL-Dithiothreitol solution, BioUltra, for molecular biology, ~1 M in H2O
Sigma-Aldrich
Sodium chloride solution, BioUltra, for molecular biology, ~5 M in H2O
Sigma-Aldrich
Bicinchoninic acid disodium salt hydrate, ≥98% (HPLC)
Sigma-Aldrich
Sodium chloride, BioXtra, ≥99.5% (AT)
Sigma-Aldrich
Sodium chloride solution, 0.9% in water, BioXtra, suitable for cell culture
Sigma-Aldrich
Sodium chloride solution, 5 M in H2O, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
Doxorubicin hydrochloride, 98.0-102.0% (HPLC)
Sigma-Aldrich
Phenylmethanesulfonyl fluoride, ≥98.5% (GC)
Sigma-Aldrich
Doxorubicin hydrochloride, suitable for fluorescence, 98.0-102.0% (HPLC)
Sigma-Aldrich
Sodium chloride, for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)
Sigma-Aldrich
Sodium chloride, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99%
Doxorubicin hydrochloride, European Pharmacopoeia (EP) Reference Standard
SAFC
Sodium chloride solution, 5 M
USP
Doxorubicin hydrochloride, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
Resorufin, 95%
Supelco
DL-Dithiothreitol solution, 1 M in H2O
Sigma-Aldrich
Ethylenediaminetetraacetic acid, anhydrous, crystalline, BioReagent, suitable for cell culture