title: Identification and validation of Notch pathway activating compounds through a novel high-throughput screening method.
abstract: BACKGROUND
  Carcinoids are neuroendocrine (NE) tumors with limited treatment options. Notch activation has been shown to suppress growth and hormone production in carcinoid cells.
  
  
  METHODS
  The purpose of this study was to provide a process for identifying Notch activating compounds via high-throughput screening (HTS) and to validate the effects of the strongest hit from the 7264 compounds analyzed: resveratrol (RESV).
  
  
  RESULTS
  Treatment of carcinoid cells with RESV resulted in up-regulation of the Notch signaling pathway as measured by suppression of its downstream target achaete-scute complex-like 1. Luciferase reporter assays incorporating the centromere-binding factor 1 binding site also confirmed the functional activity of RESV-induced Notch. Because activation of the Notch pathway has been shown to suppress carcinoid proliferation, RESV treatment of carcinoid cells led to a dose-dependent inhibition of cellular growth. Immunoblotting revealed phosphorylation of cdc2 (Tyr15) and up-regulation of p21Cip1/Waf, markers of cell cycle arrest, with RESV treatment. Flow cytometry confirmed the mechanism of RESV-induced growth inhibition is S phase cell cycle arrest. Furthermore, because Notch has been shown to inhibit bioactive hormone production from NE tumors, RESV also suppressed expression of the NE peptides/hormones chromogranin A and serotonin. RNA interference assays demonstrated that the hormone suppressing capacity of RESV was due to up-regulation of the Notch2 isoform.
  
  
  CONCLUSIONS
  HTS can be used to identify novel Notch activating compounds, which may have the potential to suppress carcinoid tumor growth and the associated endocrinopathies. Cancer 2011. © 2010 American Cancer Society.
authors.affiliation: Endocrine Surgery Research Laboratory, Department of Surgery and the University of Wisconsin Carbone Cancer Center, Madison, Wisconsin 53792, USA.
authors.forename: Scott N
authors.initials: SN
authors.lastname: Pinchot
authors.forename: Renata
authors.initials: R
authors.lastname: Jaskula-Sztul
authors.forename: Li
authors.initials: L
authors.lastname: Ning
authors.forename: Noel R
authors.initials: NR
authors.lastname: Peters
authors.forename: Mackenzie R
authors.initials: MR
authors.lastname: Cook
authors.forename: Muthusamy
authors.initials: M
authors.lastname: Kunnimalaiyaan
authors.forename: Herbert
authors.initials: H
authors.lastname: Chen
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D002276:Carcinoid Tumor / Q000188:drug therapy / Q000378:metabolism*; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D057166:High-Throughput Screening Assays; D006801:Humans; D051880:Receptors, Notch / Q000378:metabolism*; D000077185:Resveratrol; D015398:Signal Transduction; D013267:Stilbenes / Q000494:pharmacology; D015854:Up-Regulation
chemical_list: D000970:Antineoplastic Agents; D051880:Receptors, Notch; D013267:Stilbenes; D000077185:Resveratrol