Bilirubin-induced cell toxicity involves PTEN activation through an APE1/Ref-1-dependent pathway

J Mol Med (Berl). 2007 Oct;85(10):1099-112. doi: 10.1007/s00109-007-0204-3. Epub 2007 May 4.


Unconjugated bilirubin (UCB) is the major degradation product of the heme catabolism. A growing body of evidences suggests that UCB plays major biological effects by inhibiting cell proliferation in cancer cell lines and eliciting cell toxicity particularly in neurons and glial cells. Early molecular events responsible for bilirubin-induced cytotoxicity remain poorly understood. Using HeLa cells and mouse embryonic fibroblasts, we found that UCB at a concentration of free pigment (Bf) of 80 nM induced oxidative stress, promoting a significant increase in intracellular reactive oxygen species (ROS) and a decreased cell survival (by the MTT test). The ROS increase activated the antioxidant cell response through APE1/Ref-1, a master redox regulator in eukaryotic cells. Activation of APE1/Ref-1 was followed by a concomitant activation of Egr-1 transcription factor and by an upregulation of PTEN tumor suppressor, an Egr-1 target gene, leading to inhibition of cell growth. Blocking ROS generation with N-acetylcysteine pretreatment, restored cell survival, limited the upregulation of PTEN in response to UCB, and prevented the inhibition of cell proliferation. HeLa cells transfected with mutants of the PTEN promoter or silenced with APE1/Ref-1 small interference RNA confirmed that UCB modulates a signaling pathway involving APE1/Ref-1, Egr-1, and PTEN. These findings describe a new molecular pathway involved in the cytotoxic effects of UCB.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcysteine / metabolism
  • Animals
  • Apoptosis / drug effects
  • Bilirubin / metabolism
  • Bilirubin / toxicity*
  • Cell Communication / drug effects
  • Cell Communication / physiology
  • Cell Differentiation / drug effects
  • Cell Differentiation / physiology
  • Cell Proliferation / drug effects
  • Cell Survival / drug effects*
  • DNA-(Apurinic or Apyrimidinic Site) Lyase / genetics
  • DNA-(Apurinic or Apyrimidinic Site) Lyase / metabolism*
  • Electrophoretic Mobility Shift Assay
  • Enzyme Activation
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism
  • Gene Expression Regulation*
  • HeLa Cells / drug effects
  • HeLa Cells / metabolism
  • Humans
  • Mice
  • PTEN Phosphohydrolase / genetics
  • PTEN Phosphohydrolase / metabolism*
  • RNA, Small Interfering / genetics
  • Reactive Oxygen Species / metabolism
  • Transcription Factors / metabolism


  • RNA, Small Interfering
  • Reactive Oxygen Species
  • Transcription Factors
  • PTEN Phosphohydrolase
  • DNA-(Apurinic or Apyrimidinic Site) Lyase
  • Bilirubin
  • Acetylcysteine