Ethanol Enhances Susceptibility to Apoptotic Cell Death via Down-Regulation of Autophagy-Related Proteins

Authors

  • Clarissa von Haefen,

    1. From the Department of Anaesthesiology and Intensive Care Medicine, (CVH, MS, MM, CDS) Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum and Campus-Mitte, Augustenburger Platz 1, 13353 Berlin, Germany.
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  • Marco Sifringer,

    1. From the Department of Anaesthesiology and Intensive Care Medicine, (CVH, MS, MM, CDS) Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum and Campus-Mitte, Augustenburger Platz 1, 13353 Berlin, Germany.
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  • Mario Menk,

    1. From the Department of Anaesthesiology and Intensive Care Medicine, (CVH, MS, MM, CDS) Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum and Campus-Mitte, Augustenburger Platz 1, 13353 Berlin, Germany.
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  • Claudia D. Spies

    1. From the Department of Anaesthesiology and Intensive Care Medicine, (CVH, MS, MM, CDS) Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum and Campus-Mitte, Augustenburger Platz 1, 13353 Berlin, Germany.
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Reprint requests: Claudia D. Spies, MD, PhD, Department of Anaesthesiology and Intensive Care Medicine, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, D-13353 Berlin, Germany; Tel.: +49 30 450 551 001; Fax: +49 30 450 551 900; E-mail: claudia.spies@charite.de

Abstract

Background:  Alcohol induces cellular stress and promotes cell death in immune cells. Molecular mechanisms by which ethanol impairs the function of immune cells are largely unknown. Autophagy is a degradation pathway, acting either as a pro-survival or pro-death mechanism activated during stress conditions. We examined whether ethanol influences autophagy in monocytic human U937, CD4 Jurkat, and MCF-7 cells.

Methods:  Effects of ethanol during starvation-induced autophagy were investigated, treating cells with ethanol alone and in combination with activation of autophagy by rapamycin or inhibition by wortmannin. Apoptotic and necrotic cell death features such as the breakdown of the mitochondrial membrane potential, DNA fragmentation, and cell permeability were assessed using FACS analyses. Expression level of Beclin-1, LC3-II, Bcl-2, and the activation of caspase-3, and PARP-1 were determined using Western blot analyses. Influence of ethanol on formation of LC3-II complexes was assessed using fluorescence microscopy in MCF-7 cells stable transfected with a GFP-LC3-II-expression vector.

Results:  Ethanol down regulated autophagy proteins such as Beclin-1 and LC3-II. Apoptosis was enhanced as shown by breakdown of mitochondrial potential, up-regulation of cleaved caspase-3 and PARP-1 and down-regulation of anti-apoptotic protein Bcl-2. Formation of LC3-II complexes was inhibited by ethanol in caspase-3 deficient MCF-7 cells. Stimulation of autophagy by rapamycin prevented ethanol-induced apoptotic cell death. Inhibition of autophagy by wortmannin aggravated ethanol-mediated necrotic cell death.

Conclusion:  Inhibition of autophagy via ethanol enhances susceptibility to cell death.

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