title: Protoporphyrin IX induces a necrotic cell death in human THP-1 macrophages through activation of reactive oxygen species/c-Jun N-terminal protein kinase pathway and opening of mitochondrial permeability transition pore.
abstract: BACKGROUND
  Protoporphyrin IX (PpIX) and its derivatives are widely used in photodynamic therapy (PDT) to kill cancer cells. Studies showed that the application of these drugs could cause systemic toxic effects in human. However, the molecular pathways involved in PpIX-induced cytotoxicity are not well-defined. Macrophages represent the primary system for protecting tissues from toxicants and initiating the resolution of inflammation. Thus, this study aims to investigate the toxicity of PpIX on macrophages and provide strategies to prevent the toxic effects.
  
  
  METHODS
  THP-1 macrophages were incubated with PpIX and cell death was measured by MTT assay and Annexin V-PI staining. Intracellular reactive oxygen species (ROS) were evaluated by 2', 7'-Dichlorodihydrofluorescin diacetate (DCFH-DA) and MitoSOX® Red staining and mitochondrial membrane potential (ΔΨm) was detected by tetramethylrhodamine methyl ester (TMRM) staining. Mitogen-activated protein (MAP) kinase activation was assayed by western blotting. Mitochondrial permeability transition pore (mPTP) opening was measured by calcein loading/Co(2+) quenching technique and evaluating the release of mitochondrial content.
  
  
  RESULTS
  PpIX reduced cell viability in a dose- and time-dependent manner. The cell death was characterized by increasing PI-positive cells, ATP depletion, LDH releasing and rapid ΔΨm loss favoring necrotic features. In addition, PpIX successively induced ROS production, c-Jun N-terminal protein kinase (JNK) activation and mPTP opening. ROS scavengers, N-acetylcysteine (NAC) and deferoxamine (DFX), JNK inhibitor, SP600125, and mPTP inhibitor, cyclosporin A (CsA), all significantly rescued this cell death. Furthermore, mPTP opening was directly regulated by ROS/JNK pathway.
  
  
  CONCLUSIONS
  PpIX induces a necrotic cell death in THP-1 macrophages through ROS production, JNK activation, and mPTP opening. It is tempting to speculate that blocking the pathways involved in the cytotoxic effects of PpIX will alleviate its side effects.
authors.affiliation: Division of Cardiology, the First Affiliated Hospital, Cardiovascular Institute, Harbin Medical University, Harbin, China.
authors.forename: Haobo
authors.initials: H
authors.lastname: Xu
authors.forename: Yan
authors.initials: Y
authors.lastname: Sun
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Juhua
authors.initials: J
authors.lastname: Dan
authors.forename: Jianting
authors.initials: J
authors.lastname: Yao
authors.forename: Haibo
authors.initials: H
authors.lastname: Chen
authors.forename: Fang
authors.initials: F
authors.lastname: Tian
authors.forename: Xin
authors.initials: X
authors.lastname: Sun
authors.forename: Shuyuan
authors.initials: S
authors.lastname: Guo
authors.forename: Zhen
authors.initials: Z
authors.lastname: Tian
authors.forename: Ye
authors.initials: Y
authors.lastname: Tian
mesh_terms: D000873:Anthracenes / Q000008:administration & dosage; D016923:Cell Death / Q000235:genetics; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism / Q000473:pathology; D008928:Mitochondria / Q000378:metabolism / Q000473:pathology; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D000083162:Mitochondrial Permeability Transition Pore; D009336:Necrosis / Q000235:genetics / Q000378:metabolism*; D011524:Protoporphyrins / Q000008:administration & dosage* / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D000873:Anthracenes; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D011524:Protoporphyrins; D017382:Reactive Oxygen Species; C432165:pyrazolanthrone; C028025:protoporphyrin IX; D048031:JNK Mitogen-Activated Protein Kinases