Endostatin induces autophagic cell death in EAhy926 human endothelial cells

Histol Histopathol. 2003 Jul;18(3):715-26. doi: 10.14670/HH-18.715.

Abstract

Endostatin, a proteolytic fragment of collagen XVIII, is a potent inhibitor of angiogenesis and suppresses neovascularization and tumor growth. However, the inhibitory mechanism of endostatin in human endothelial cells has not been characterized yet. Electron microscopic analysis revealed that endostatin induced formation of numerous autophagic vacuoles in endothelial in 6 to 24 h after treatment. Moreover, there was only a 2- to 3-fold increase in intracellular reactive oxygen species after endostatin treatment. Endostatin-induced cell death was not prevented by antioxidants (vitamin C, vitamin E, or propyl gallate) or caspase inhibitors, suggesting that the increase of oxidative stress or the activation of caspases may not be the crucial factors in the anti-angiogenic mechanism of endostatin. However, the cytotoxicity of endostatin was significantly reduced by 3-methyladenine (a specific inhibitor of autophagy) and serine and cysteine lysosomal protease inhibitors (leupeptin and aprotinin). Taken together, these results suggest that in human endothelial cells: (1) endostatin predominantly causes autophagic, rather than apoptotic, cell death, (2) endostatin-induced autophagic cell death occurs in the absence of caspase activation and through an oxidative-independent pathway, and (3) endostatin-induced "autophagic cell death" or "type 2 physiological cell death" is regulated by serine and cysteine lysosomal proteases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acridine Orange / pharmacology
  • Adenine / analogs & derivatives*
  • Adenine / pharmacology
  • Animals
  • Antineoplastic Agents / pharmacology
  • Apoptosis
  • Aprotinin / pharmacology
  • Blotting, Western
  • CHO Cells
  • Caspases / metabolism
  • Cell Death
  • Cells, Cultured
  • Cloning, Molecular
  • Cricetinae
  • Cysteine / chemistry
  • Cysteine Endopeptidases / metabolism
  • Dose-Response Relationship, Drug
  • Endostatins / metabolism
  • Endostatins / pharmacology*
  • Endothelial Cells / cytology
  • Endothelial Cells / pathology*
  • Endothelium, Vascular / cytology
  • Enzyme Activation
  • Flow Cytometry
  • Glutathione Transferase / metabolism
  • Humans
  • Immunohistochemistry
  • Leupeptins / pharmacology
  • Lysosomes / enzymology
  • Microscopy, Electron
  • Microscopy, Phase-Contrast
  • Oxidative Stress
  • Reactive Oxygen Species
  • Recombinant Proteins / metabolism
  • Serine / chemistry
  • Time Factors

Substances

  • Antineoplastic Agents
  • Endostatins
  • Leupeptins
  • Reactive Oxygen Species
  • Recombinant Proteins
  • Serine
  • 3-methyladenine
  • Aprotinin
  • Glutathione Transferase
  • Caspases
  • Cysteine Endopeptidases
  • Acridine Orange
  • leupeptin
  • Adenine
  • Cysteine