title: Neuronal differentiation by indomethacin and IBMX inhibits proliferation of small cell lung cancer cells in vitro.
abstract: BACKGROUND
  Small cell lung cancer (SCLC) is one of the most aggressive malignancies implying a very poor prognosis for patients even under therapy. Since it is known that SCLC cells exhibit neurone-like characteristics, we investigated whether a neuronal induction medium (NID) consisting of indomethacin (200 μM), 3-isobutyl-1-methylxanthine (IBMX, 500 μM) and insulin (5 μg/ml) induces neuronal differentiation and by this reduces malignancy of SCLC in vitro.
  
  
  METHODS
  Anti-proliferative effects were tested by incubating five SCLC cell lines (OH1, OH3, SW2, H69 and H82) with NID for 72 h (XTT-assay). Afterwards, anti-proliferative as well as cytotoxic effects (lactate dehydrogenase [LDH] assay, electron microscopy) of a range of drug concentrations (indomethacin 6.25-800 μM, IBMX 15.625-2000 μM and combinations of both) regarding H82 and SW2 were analysed. We further investigated the presence of cyclooxygenase- (COX-) 1 and 2 (IHC, Western blot) as well as levels of COX-2 before and after treatment. Neuronal differentiation was evaluated by morphological analyses (electron microscopy), detection of CD 56 and CD 171 (FACS) and recording Na(+) and K(+) currents (patch clamp).
  
  
  RESULTS
  Proliferation of all cell lines was inhibited significantly in a dose dependent manner (linear regression), whereas SW2 and H82 were most sensitive. Treatment with insulin alone had no effect at all. Cytotoxic effects were only observed after incubation with high concentrations of indomethacin (H82) and combined treatment (SW2). COX-1 and 2 were detectable in H82 and SW2, whereas the level of COX-2 remained unaffected under treatment. By electron microscopy, we could not observe distinct neurone-like morphological changes after 72 h of treatment. However, the majority of H82 and SW2 cells expressed both CD 56 (NCAM) and CD 171 (L1), showing an increase of NCAM and L1 intensity at the cell surface after 7 and 14 days of treatment. We further demonstrated an up-regulation of neurone-specific Na(+) currents as well as a significant down-regulation of herg K(+) currents after NID treatment.
  
  
  CONCLUSIONS
  Our findings demonstrate significant anti-proliferative, non-toxic effects of indomethacin and IBMX on SCLC cells in vitro. Treated SCLC cells further possess increased neuronal characteristics in vitro, possibly leading to a reduced malignant potential.
authors.affiliation: Institute of Anatomy II: Experimental Morphology and University Cancer Center Hamburg (UCCH), University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. annikalange@o2online.de
authors.forename: Annika
authors.initials: A
authors.lastname: Lange
authors.forename: Heike
authors.initials: H
authors.lastname: Gustke
authors.forename: Günter
authors.initials: G
authors.lastname: Glassmeier
authors.forename: Markus
authors.initials: M
authors.lastname: Heine
authors.forename: Uwe
authors.initials: U
authors.lastname: Zangemeister-Wittke
authors.forename: Jürgen R
authors.initials: JR
authors.lastname: Schwarz
authors.forename: Udo
authors.initials: U
authors.lastname: Schumacher
authors.forename: Tobias
authors.initials: T
authors.lastname: Lange
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology*; D019002:CD56 Antigen / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D048708:Cell Growth Processes / Q000187:drug effects; D045744:Cell Line, Tumor; D051545:Cyclooxygenase 1 / Q000235:genetics / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology*; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000473:pathology / Q000503:physiopathology; D039842:Neural Cell Adhesion Molecule L1 / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D055752:Small Cell Lung Carcinoma / Q000188:drug therapy* / Q000473:pathology / Q000503:physiopathology; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism
chemical_list: D019002:CD56 Antigen; D039842:Neural Cell Adhesion Molecule L1; D015222:Sodium Channels; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D015056:1-Methyl-3-isobutylxanthine; D007213:Indomethacin