title: 6-Mercaptopurine reduces cytokine and Muc5ac expression involving inhibition of NFκB activation in airway epithelial cells.
abstract: BACKGROUND
  Mucus hypersecretion and excessive cytokine synthesis is associated with many of the pathologic features of chronic airway diseases such as asthma. 6-Mercaptopurine (6-MP) is an immunosuppressive drug that is widely used in several inflammatory disorders. Although 6-MP has been used to treat asthma, its function and mechanism of action in airway epithelial cells is unknown.
  
  
  METHODS
  Confluent NCI-H292 and MLE-12 epithelial cells were pretreated with 6-MP followed by stimulation with TNFα or PMA. mRNA levels of cytokines and mucins were measured by RT-PCR. Western blot analysis was performed to assess the phosphorylation of IκBα and luciferase assays were performed using an NFκB reporter plasmid to determine NFκB activity. Periodic Acid Schiff staining was used to assess the production of mucus.
  
  
  RESULTS
  6-MP displayed no effect on cell viability up to a concentration of 15 μM. RT-PCR analysis showed that 6-MP significantly reduces TNFα- and PMA-induced expression of several proinflammatory cytokines in NCI-H292 and MLE-12 cells. Consistent with this, we demonstrated that 6-MP strongly inhibits TNFα-induced phosphorylation of IκBα and thus attenuates NFκB luciferase reporter activity. In addition, 6-MP decreases Rac1 activity in MLE-12 cells. 6-MP down-regulates gene expression of the mucin Muc5ac, but not Muc2, through inhibition of activation of the NFκB pathway. Furthermore, PMA- and TNFα-induced mucus production, as visualized by Periodic Acid Schiff (PAS) staining, is decreased by 6-MP.
  
  
  CONCLUSIONS
  Our data demonstrate that 6-MP inhibits Muc5ac gene expression and mucus production in airway epithelial cells through inhibition of the NFκB pathway, and 6-MP may represent a novel therapeutic target for mucus hypersecretion in airway diseases.
authors.affiliation: Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands. k.b.kurakula@lumc.nl.|Present address: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands. k.b.kurakula@lumc.nl.
authors.forename: Kondababu
authors.initials: K
authors.lastname: Kurakula
authors.affiliation: Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands. a.a.hamers@amc.uva.nl.
authors.forename: Anouk A
authors.initials: AA
authors.lastname: Hamers
authors.affiliation: Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands. p.b.vanloenen@amc.uva.nl.
authors.forename: Pieter
authors.initials: P
authors.lastname: van Loenen
authors.affiliation: Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands. c.j.devries@amc.uva.nl.
authors.forename: Carlie J M
authors.initials: CJ
authors.lastname: de Vries
mesh_terms: D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D016207:Cytokines / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D015122:Mercaptopurine / Q000494:pharmacology*; D055271:Mucin 5AC / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000187:drug effects / Q000378:metabolism*
chemical_list: D016207:Cytokines; C524678:MUC5AC protein, human; D055271:Mucin 5AC; D016328:NF-kappa B; D015122:Mercaptopurine