title: PMX464, a thiol-reactive quinol and putative thioredoxin inhibitor, inhibits NF-kappaB-dependent proinflammatory activation of alveolar epithelial cells.
abstract: OBJECTIVE
  Subtle changes in the intracellular reduction-oxidation (redox) state can modulate nuclear factor-kappaB (NF-kappaB) activity. Thioredoxin-1 (Trx) is a small, ubiquitous, redox-active thiol (-SH) protein that, with thioredoxin reductase-1 (TrxR), modifies the redox status of NF-kappaB pathway components. PMX464 is a novel thiol-reactive quinol thought to inhibit the Trx/TrxR system. The aim of this work was to investigate whether PMX464 inhibited NF-kappaB-mediated proinflammatory activation of human type II alveolar epithelial cells (A549).
  
  
  METHODS
  Intercellular adhesion molecule-1 (ICAM-1), granulocyte-macrophage colony-stimulating factor (GM-CSF) and CXCL8, NF-kappaB DNA binding, nuclear translocation of NF-kappaB p65 subunit, IkappaBalpha degradation, IkappaB phosphorylation and IkappaB kinase (IKK) activity were assessed in A549 cells stimulated with IL-1beta with or without PMX464 pretreatment. Effects of PMX464 on ICAM-1 expression in human lung microvascular endothelial cells (HLMVEC) were also investigated. For comparison, selected measurements (ICAM-1 and IkappaB-alpha phospho-IkappaB-alpha) were made on A549 cells after RNA interference-mediated silencing (siRNA) of Trx.
  
  
  RESULTS
  PMX464 reduced ICAM-1, GM-CSF and CXCL8 expression in IL-1beta-stimulated A549 cells and ICAM-1 in HLMVEC. PMX464 inhibited IL-1beta-induced NF-kappaB DNA binding, nuclear translocation of NF-kappaB p65 subunit and factors involved in NF-kappaB activation; specifically, IkappaBalpha degradation, IkappaB phosphorylation and IkappaB kinase (IKK) activity in A549. By contrast, Trx siRNA did not alter ICAM-1 expression or IkappaBalpha degradation/phosphorylation in IL-1beta-stimulated A549 cells.
  
  
  CONCLUSIONS
  PMX464 inhibits a proinflammatory response in A549 cells targeting the NFkappaB pathway above IKK. The lack of effect with Trx siRNA suggests that PMX464 acts on thiol proteins, in addition to Trx, to elicit anti-inflammatory responses in lung epithelial cells.
authors.affiliation: Critical Care, Pulmonary Vascular and Sleep Science, Respiratory Science, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, London, UK.
authors.forename: M E
authors.initials: ME
authors.lastname: Callister
authors.forename: L
authors.initials: L
authors.lastname: Pinhu
authors.forename: M C
authors.initials: MC
authors.lastname: Catley
authors.forename: A D
authors.initials: AD
authors.lastname: Westwell
authors.forename: R
authors.initials: R
authors.lastname: Newton
authors.forename: S K
authors.initials: SK
authors.lastname: Leaver
authors.forename: G J
authors.initials: GJ
authors.lastname: Quinlan
authors.forename: T W
authors.initials: TW
authors.lastname: Evans
authors.forename: M J
authors.initials: MJ
authors.lastname: Griffiths
authors.forename: A
authors.initials: A
authors.lastname: Burke-Gaffney
mesh_terms: D000818:Animals; D052160:Benzothiazoles / Q000737:chemistry / Q000494:pharmacology*; D002448:Cell Adhesion / Q000187:drug effects; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003512:Cyclohexanones / Q000737:chemistry / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D006873:Hydroquinones / Q000737:chemistry / Q000494:pharmacology*; D015151:Immunoblotting; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000378:metabolism*; D009504:Neutrophils / Q000166:cytology; D011650:Pulmonary Alveoli / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D013879:Thioredoxins / Q000037:antagonists & inhibitors* / Q000235:genetics
chemical_list: C515226:AW 464; D052160:Benzothiazoles; D003512:Cyclohexanones; D006873:Hydroquinones; D016328:NF-kappa B; D034741:RNA, Small Interfering; D013879:Thioredoxins