Molecular Medicine

, Volume 20, Issue 1, pp 417–426 | Cite as

Regulation of Autophagy by α1-Antitrypsin: “A Foe of a Foe Is a Friend”

  • Michal G. Shapira
  • Boris Khalfin
  • Eli C. Lewis
  • Abraham H. Parola
  • Ilana Nathan
Research Article


Autophagy is involved in both the cell protective and the cell death process but its mechanism is largely unknown. The present work unravels a novel intracellular mechanism by which the serpin α1-antitrypsin (AAT) acts as a novel negative regulator of autophagic cell death. For the first time, the role of intracellularly synthesized AAT, other than in liver cells, is demonstrated. Autophagic cell death was induced by N-α-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and tamoxifen. By utilizing a fluorescently tagged TPCK analog, AAT was “fished out” (pulled out) as a TPCK intracellular protein target. The interaction was further verified by competition binding experiments. Both inducers caused downregulation of AAT expression associated with activation of trypsin-like proteases. Furthermore, silencing AAT by siRNA induced autophagic cell death. Moreover, AAT administration to cultured cells prevented autophagic cell death. This new mechanism could have implications in the treatment of diseases by the regulation of AAT levels in which autophagy has a detrimental function. Furthermore, the results imply that the high synthesis of endogenous AAT by cancer cells could provide a novel resistance mechanism of cancer against autophagic cell death.



We are grateful to Zbigniew Darzynkiewicz from the Brander Cancer Research Institute at the New York Medical College for providing us with FSFCK and TRFCK and for his giving advice. The financial support of the James-Frank Center for Laser-Matter Interaction, the Edmund Safra Foundation for Functional Biopolymers and the NYUSH research grant to AH Parola are gratefully acknowledged.


  1. 1.
    Codogno P, Meijer AJ. (2005) Autophagy and signaling: their role in cell survival and cell death. Cell Death Differ. 12 Suppl 2:1509–18.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Klionsky DJ, Emr SD. (2000) Autophagy as a regulated pathway of cellular degradation. Science. 290:1717–21.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Shintani T, Klionsky DJ. (2004) Autophagy in health and disease: a double-edged sword. Science. 306:990–5.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Levine B, Kroemer G. (2008) Autophagy in the pathogenesis of disease. Cell. 132:27–42.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Chen N, Karantza V. (2011) Autophagy as a therapeutic target in cancer. Cancer Biol. Ther. 11:157–68.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Marino G, et al. (2003) Human autophagins, a family of cysteine proteinases potentially implicated in cell degradation by autophagy. J. Biol. Chem. 278:3671–8.CrossRefPubMedGoogle Scholar
  7. 7.
    Yousefi S, et al. (2006) Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nat. Cell. Biol. 8:1124–32.CrossRefPubMedGoogle Scholar
  8. 8.
    Nakashima A, et al. (2006) A starvation-specific serine protease gene, isp6+, is involved in both autophagy and sexual development in Schizosac-charomyces pombe. Curr. Genet. 49:403–13.CrossRefPubMedGoogle Scholar
  9. 9.
    Ohmuraya M, et al. (2005) Autophagic cell death of pancreatic acinar cells in serine protease inhibitor Kazal type 3-deficient mice. Gastroenterology. 129:696–705.CrossRefPubMedGoogle Scholar
  10. 10.
    Li B, et al. (2010) Omi/HtrA2 is a positive regulator of autophagy that facilitates the degradation of mutant proteins involved in neurodegenerative diseases. Cell Death Differ. 17:1773–84.CrossRefPubMedGoogle Scholar
  11. 11.
    Hallak M, et al. (2008) A molecular mechanism for mimosine-induced apoptosis involving oxidative stress and mitochondrial activation. Apoptosis. 13:147–55.CrossRefPubMedGoogle Scholar
  12. 12.
    Zhang B, et al. (2007) Alpha1-antitrypsin protects beta-cells from apoptosis. Diabetes. 56:1316–23.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Ballif BA, Shimamura A, Pae E, Blenis J. (2001) Disruption of 3-phosphoinositide-dependent kinase 1 (PDK1) signaling by the anti-tumorigenic and anti-proliferative agent n-alpha-tosyl-lphenylalanyl chloromethyl ketone. J. Biol. Chem. 276:12466–75.CrossRefPubMedGoogle Scholar
  14. 14.
    Karahashi H, Nagata K, Ishii K, Amano F. (2000) A selective inhibitor of p38 MAP kinase, SB202190, induced apoptotic cell death of a lipopolysaccharide-treated macrophage-like cell line, J774.1. Biochim. Biophys. Acta. 18:207–23.CrossRefGoogle Scholar
  15. 15.
    Lotem J, Sachs L. (1996) Differential suppression by protease inhibitors and cytokines of apoptosis induced by wild-type p53 and cytotoxic agents. Proc. Natl. Acad. Sci. U. S. A. 93:12507–12.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Eitel K, Wagenknecht B, Weller M. (1999) Inhibition of drug-induced DNA fragmentation, but not cell death, of glioma cells by non-caspase protease inhibitors. Cancer Lett. 142:11–6.CrossRefPubMedGoogle Scholar
  17. 17.
    Huang Y, Sheikh MS, Fornace AJ Jr., Holbrook NJ. (1999) Serine protease inhibitor TPCK prevents Taxol-induced cell death and blocks c-Raf-1 and Bcl-2 phosphorylation in human breast carcinoma cells. Oncogene. 18:3431–9.CrossRefPubMedGoogle Scholar
  18. 18.
    Heussler VT, Fernandez PC, Machado J Jr., Botteron C, Dobbelaere DA. (1999) N-acetylcysteine blocks apoptosis induced by N-alpha-tosyl-L-phenylalanine chloromethyl ketone in transformed T-cells. Cell Death Differ. 6:342–50.CrossRefPubMedGoogle Scholar
  19. 19.
    Zhu H, Dinsdale D, Alnemri ES, Cohen GM. (1997) Apoptosis in human monocytic THP.1 cells involves several distinct targets of N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK). Cell Death Differ. 4:590–9.CrossRefPubMedGoogle Scholar
  20. 20.
    Biederbick A, Kern HF, Elsasser HP. (1995) Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur. J. Cell Biol. 66:3–14.PubMedGoogle Scholar
  21. 21.
    McGahon AJ, et al. (1995) The end of the (cell) line: methods for the study of apoptosis in vitro. Methods Cell Biol. 46:153–85.CrossRefPubMedGoogle Scholar
  22. 22.
    Weber K, Osborn M. (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406–12.PubMedGoogle Scholar
  23. 23.
    Bergmeyer HU (ed.). (1974) Methods of Enzymatic Analysis. 2nd English edition. New York: Academic Press. 4 vols.Google Scholar
  24. 24.
    Lavens SE, Proud D, Warner JA. (1993) A sensitive colorimetric assay for the release of tryptase from human lung mast cells in vitro. J. Immunol. Methods. 166:93–102.CrossRefPubMedGoogle Scholar
  25. 25.
    Yasothornsrikul S, Hook VY. (2000) Detection of proteolytic activity by fluorescent zymogram ingel assays. Biotechniques. 28:1166–8, 70, 72–3.CrossRefPubMedGoogle Scholar
  26. 26.
    Tanida I, Minematsu-Ikeguchi N, Ueno T, Kominami E. (2005) Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy. Autophagy. 1:84–91.CrossRefGoogle Scholar
  27. 27.
    Grabarek J, Darzynkiewicz Z. (2002) In situ activation of caspases and serine proteases during apoptosis detected by affinity labeling their enzyme active centers with fluorochrome-tagged inhibitors. Exp. Hematol. 30:982–9.CrossRefPubMedGoogle Scholar
  28. 28.
    Wu M, et al. (1996) Inhibition of NF-kappaB/Rel induces apoptosis of murine B cells. EMBO J. 15:4682–90.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Maiuri MC, Zalckvar E, Kimchi A, Kroemer G. (2007) Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat. Rev. Mol. Cell. Biol. 8:741–52.CrossRefPubMedGoogle Scholar
  30. 30.
    Higashiyama M, Doi O, Kodama K, Yokouchi H, Tateishi R. (1992) An evaluation of the prognostic significance of alpha-1-antitrypsin expression in adenocarcinomas of the lung: an immunohistochemical analysis. Br. J. Cancer. 65:300–2.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Vercaigne-Marko D, Carrere J, Guy-Crotte O, Figarella C, Hayem A. (1989) Human cationic and anionic trypsins: differences of interaction with alpha 1-proteinase inhibitor. Biol. Chem. Hoppe Seyler. 370:1163–71.CrossRefPubMedGoogle Scholar
  32. 32.
    Yousef GM, et al. (2003) The human kallikrein protein 5 (hK5) is enzymatically active, glycosylated and forms complexes with two protease inhibitors in ovarian cancer fluids. Biochim. Biophys. Acta. 1628:88–96.CrossRefPubMedGoogle Scholar
  33. 33.
    Tseng IC, et al. (2008) Purification from human milk of matriptase complexes with secreted serpins: mechanism for inhibition of matriptase other than HAI-1. Am. J. Physiol. Cell Physiol. 295:C423–31.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Petrache I, et al. (2006) Alpha-1 antitrypsin inhibits caspase-3 activity, preventing lung endothelial cell apoptosis. Am. J. Pathol. 169:1155–66.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Lewis EC. (2012) Expanding the clinical indications for a(1)-antitrypsin therapy. Mol. Med. 18:957–70.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Louie SG, Sclar DA, Gill MA. (2005) Aralast: a new alpha1-protease inhibitor for treatment of alpha-antitrypsin deficiency. Ann. Pharmacother. 39:1861–9.CrossRefPubMedGoogle Scholar
  37. 37.
    Yang S, et al. (2011) Pancreatic cancers require autophagy for tumor growth. Genes. Dev. 25:717–29.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Rubinsztein DC, Codogno P, Levine B. (2012) Autophagy modulation as a potential therapeutic target for diverse diseases. Nat. Rev. Drug. Discov. 11:709–30.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Kagawa S, et al. (2001) Deficiency of caspase-3 in MCF7 cells blocks Bax-mediated nuclear fragmentation but not cell death. Clin. Cancer Res. 7:1474–80.PubMedGoogle Scholar
  40. 40.
    Huang H, et al. (2004) Alpha1-antitrypsin inhibits angiogenesis and tumor growth. Int. J. Cancer. 112:1042–8.CrossRefPubMedGoogle Scholar

Copyright information

© The Author(s) 2014

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, and provide a link to the Creative Commons license. You do not have permission under this license to share adapted material derived from this article or parts of it.

The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this license, visit (

Authors and Affiliations

  1. 1.Department of Chemistry, Faculty of Natural SciencesBen-Gurion University of the NegevBeer-ShevaIsrael
  2. 2.Department of Clinical Biochemistry and Pharmacology, Faculty of Health SciencesBen-Gurion University of the NegevBeer-ShevaIsrael
  3. 3.New York University ShanghaiShanghaiPeople’s Republic of China
  4. 4.Soroka University Medical CenterBeer-ShevaIsrael

Personalised recommendations