title: Docosahexanoic acid antagonizes TNF-α-induced necroptosis by attenuating oxidative stress, ceramide production, lysosomal dysfunction, and autophagic features.
abstract: OBJECTIVE
  It was previously reported that docosahexanoic acid (DHA) reduces TNF-α-induced necrosis in L929 cells. However, the mechanisms underlying this reduction have not been investigated. The present study was designed to investigate cellular and biochemical mechanisms underlying the attenuation of TNF-α-induced necroptosis by DHA in L929 cells.
  
  
  METHODS
  L929 cells were pre-treated with DHA prior to exposure to TNF-α, zVAD, or Necrostatin-1 (Nec-1). Cell death and survival were assessed by MTT and caspase activity assays, and microscopic visualization. Reactive oxygen species (ROS) were measured by flow cytometry. C16- and C18-ceramides were measured by mass spectrometry. Lysosomal membrane permeabilization (LMP) was evaluated by fluorescence microscopy and flow cytometry using Acridine Orange. Cathepsin L activation was evaluated by immunoblotting and fluorescence microscopy. Autophagy was assessed by immunoblotting of LC3-II and Beclin.
  
  
  RESULTS
  Exposure of L929 cells to TNF-α alone for 24 h induced necroptosis, as evidenced by the inhibition of cell death by Nec-1, absence of caspase-3 activity and Lamin B cleavage, and morphological analysis. DHA attenuated multiple biochemical events associated with TNF-α-induced necroptosis, including ROS generation, ceramide production, lysosomal dysfunction, cathepsin L activation, and autophagic features. DHA also attenuated zVAD-induced necroptosis but did not attenuate the enhanced apoptosis and necrosis induced by the combination of TNF-α with Actinomycin D or zVAD, respectively, suggesting that its protective effects might be limited by the strength of the cell death insult induced by TNF-α.
  
  
  CONCLUSIONS
  DHA effectively attenuates TNF-α-induced necroptosis and autophagy, most likely via its ability to inhibit TNF-α-induced sphingolipid metabolism and oxidative stress. These results highlight the role of this Omega-3 fatty acid in antagonizing inflammatory cell death.
authors.affiliation: Center for Health Disparities and Molecular Medicine and Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA, 92350, USA.
authors.forename: Fabio J
authors.initials: FJ
authors.lastname: Pacheco
authors.forename: Frankis G
authors.initials: FG
authors.lastname: Almaguel
authors.forename: Whitney
authors.initials: W
authors.lastname: Evans
authors.forename: Leslimar
authors.initials: L
authors.lastname: Rios-Colon
authors.forename: Valery
authors.initials: V
authors.lastname: Filippov
authors.forename: Lai S
authors.initials: LS
authors.lastname: Leoh
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Rook-Arena
authors.forename: Melanie
authors.initials: M
authors.lastname: Mediavilla-Varela
authors.forename: Marino
authors.initials: M
authors.lastname: De Leon
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Casiano
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy; D002460:Cell Line; D002518:Ceramides / Q000378:metabolism*; D004281:Docosahexaenoic Acids / Q000494:pharmacology*; D008247:Lysosomes / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D009336:Necrosis / Q000139:chemically induced / Q000188:drug therapy*; D018384:Oxidative Stress / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha
chemical_list: D002518:Ceramides; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D004281:Docosahexaenoic Acids
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