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

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Abstract

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.

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Abbreviations

AP-1:

activator protein-1

APE1/Ref-1:

apurinic apyrimidinic endonuclease/redox effector factor-1

Egr-1:

early growth response protein-1

H2DCFDA:

2,7-dichlorofluorescein diacetate

HSA:

human serum albumin

MEF:

mouse embryonic fibroblasts

Mrp1:

multidrug resistance-associated protein 1

MTT:

3(4,5-dimethylthiazolyl-2)-2,5 diphenyl tetrazolium

NAC:

N-acetyl-cysteine

NF-κB:

nuclear factor-κB

PTEN:

protein phosphatase and tensin homolog

ROS:

reactive oxygen species

siRNA:

small interference RNA

UCB:

unconjugated bilirubin

Bf:

free unconjugated bilirubin

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Acknowledgement

The financial support of Telethon–Italy (grant GGP 05062) of MiUR (PRIN 2005 to GT, CT and FQ) and of Fondo Studi Fegato (Grant to CT) is gratefully acknowledged. SC was supported by a PhD fellowship by Ministero Affari Esteri, Rome, Italy. The authors wish to thank E. Adamson for the PTEN promoter plasmids.

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Authors and Affiliations

  1. Department of Biomedical Sciences and Technologies, University of Udine, 33100, Udine, Italy

    Laura Cesaratto, Carlo Vascotto, Marta Deganuto, Franco Quadrifoglio & Gianluca Tell

  2. Department BBCM, University of Trieste, Centro Studi Fegato (CSF), AREA Science Park, 34012, Trieste, Italy

    Sebastian D. Calligaris, Cristina Bellarosa & Claudio Tiribelli

  3. Gastroenterology/Hepatology Division, University of Washington School of Medicine and VA Puget Sound Health Care System/Seattle Division, Seattle, WA, 98195-6424, USA

    J. Donald Ostrow

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  1. Laura Cesaratto
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  2. Sebastian D. Calligaris
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  3. Carlo Vascotto
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  9. Gianluca Tell
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Correspondence to Gianluca Tell.

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L. Cesaratto and S. D. Calligaris contributed equally to the present work.

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Cesaratto, L., Calligaris, S.D., Vascotto, C. et al. Bilirubin-induced cell toxicity involves PTEN activation through an APE1/Ref-1-dependent pathway. J Mol Med 85, 1099–1112 (2007). https://doi.org/10.1007/s00109-007-0204-3

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  • DOI: https://doi.org/10.1007/s00109-007-0204-3

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