Protection by ascorbic acid from denaturation and release of cytochrome c, alteration of mitochondrial membrane potential and activation of multiple caspases induced by H(2)O(2), in human leukemia cells

Biochem Pharmacol. 2002 Apr 1;63(7):1325-35. doi: 10.1016/s0006-2952(02)00863-8.


We investigated peroxide and superoxide accumulation, cytochrome c nature and release from mitochondria, as well as caspase activation during exposure of HL-60 cells to H(2)O(2) and the protective effect of ascorbic acid. Exposure of the cells to 100 microM H(2)O(2) resulted in intracellular accumulation of peroxides, denaturation of cytochrome c that was identified in the mitochondria and cytosol, release of native cytochrome c to the cytosol and fall in mitochondrial membrane potential (DeltaPsi(m)). Loading of cells with ascorbic acid before exposure to H(2)O(2) resulted in a dose-dependent protective effect against: intracellular accumulation of peroxides, DeltaPsi(m) alteration, cytochrome c denaturation and release. The accumulation of peroxides induced processings and activations of procaspase-8, -9 and -3. Double staining with antiserum which recognizes the p18 subunit of cleaved caspase-3 and with Hoechst had shown that a high percentage of cells exposed to 100 microM H(2)O(2) stained positively with the antibody and showed features of apoptosis. Ascorbic acid loading prevented caspase activation induced by H(2)O(2). We conclude that ascorbic acid protects against activation of apoptotic cascades in HL-60 cells exposed to H(2)O(2) and against apoptosis.

MeSH terms

  • Apoptosis
  • Ascorbic Acid / pharmacology*
  • Caspase 3
  • Caspases / metabolism*
  • Cytochrome c Group / drug effects
  • Cytochrome c Group / metabolism*
  • Drug Interactions
  • Enzyme Activation
  • HL-60 Cells
  • Humans
  • Hydrogen Peroxide / pharmacology*
  • Immunoblotting
  • Leukemia / pathology
  • Membrane Potentials / drug effects
  • Mitochondria / drug effects*
  • Mitochondria / physiology
  • Protective Agents / pharmacology*
  • Protein Denaturation / drug effects
  • Superoxides / metabolism
  • Tumor Cells, Cultured


  • Cytochrome c Group
  • Protective Agents
  • Superoxides
  • Hydrogen Peroxide
  • CASP3 protein, human
  • Caspase 3
  • Caspases
  • Ascorbic Acid