Pan-caspase inhibitors induce necroptosis via ROS-mediated activation of mixed lineage kinase domain-like protein and p38 in classically activated macrophages

Exp Cell Res. 2019 Jul 15;380(2):171-179. doi: 10.1016/j.yexcr.2019.04.027. Epub 2019 Apr 27.


Classically activated macrophages (CAMs) play a crucial protective role in the host by killing the invading pathogens. However, excessive activation of CAMs causes chronic inflammation leading to host tissue damage. Thus, control of macrophage activity is necessary to prevent chronic inflammation. To date, regulation of CAMs in the development of chronic inflammatory diseases has not been elucidated. In this study, we investigated the effect of a pan-caspase inhibitor, zVAD-fmk, in cell death in lipopolysaccharide (LPS)-activated macrophages, CAMs. Necrostatin-1, an inhibitor of necroptosis, inhibited zVAD-fmk-induced cell death in CAMs. The expression of mixed lineage kinase domain-like protein (MLKL) involved in the necroptosis pathway was up-regulated by LPS in CAMs. zVAD-fmk enhanced the phosphorylation of MLKL in CAMs. Moreover, inhibition of activation of mitogen activated protein kinase p38 and generation of reactive oxygen species (ROS) reduced zVAD-fmk-induced cell death in CAMs. Inhibition of ROS generation decreased the activation of MLKL and p38 in zVAD-fmk-treated CAMs. These results, taken together, indicate that zVAD-fmk-induced cell death occurred by necroptosis through ROS-mediated activation of MLKL and p38 in CAMs. Elucidation of the molecular mechanism underlying zVAD-fmk-induced necroptosis in CAMs might help in better understanding its significance in chronic inflammatory diseases.

Keywords: Classically activated macrophages; MLKL; Necroptosis; Reactive oxygen species; p38.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Chloromethyl Ketones / pharmacology*
  • Animals
  • Caspase Inhibitors / pharmacology*
  • Cell Death / drug effects
  • Cells, Cultured
  • Dose-Response Relationship, Drug
  • Lipopolysaccharides / pharmacology
  • Macrophage Activation / drug effects
  • Macrophages / cytology
  • Macrophages / drug effects*
  • Macrophages / immunology
  • Macrophages / metabolism
  • Mice
  • Necroptosis / drug effects*
  • Phosphorylation / drug effects
  • Protein Kinases / metabolism*
  • Reactive Oxygen Species / metabolism*
  • p38 Mitogen-Activated Protein Kinases / metabolism*


  • Amino Acid Chloromethyl Ketones
  • Caspase Inhibitors
  • Lipopolysaccharides
  • Reactive Oxygen Species
  • benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone
  • MLKL protein, mouse
  • Protein Kinases
  • p38 Mitogen-Activated Protein Kinases