title: Glycogen synthase kinase-3 inhibitor AR-A014418 suppresses pancreatic cancer cell growth via inhibition of GSK-3-mediated Notch1 expression.
abstract: BACKGROUND
  Glycogen synthase kinase-3 (GSK-3) can act as either a tumour promoter or suppressor by its inactivation depending on the cell type. There are conflicting reports on the roles of GSK-3 isoforms and their interaction with Notch1 in pancreatic cancer. It was hypothesized that GSK-3α stabilized Notch1 in pancreatic cancer cells thereby promoting cellular proliferation.
  
  
  METHODS
  The pancreatic cancer cell lines MiaPaCa2, PANC-1 and BxPC-3, were treated with 0-20 μM of AR-A014418 (AR), a known GSK-3 inhibitor. Cell viability was determined by the MTT assay and Live-Cell Imaging. The levels of Notch pathway members (Notch1, HES-1, survivin and cyclinD1), phosphorylated GSK-3 isoforms, and apoptotic markers were determined by Western blot. Immunoprecipitation was performed to identify the binding of GSK-3 specific isoform to Notch1.
  
  
  RESULTS
  AR-A014418 treatment had a significant dose-dependent growth reduction (P < 0.001) in pancreatic cancer cells compared with the control and the cytotoxic effect is as a result of apoptosis. Importantly, a reduction in GSK-3 phosphorylation lead to a reduction in Notch pathway members. Overexpression of active Notch1 in AR-A014418-treated cells resulted in the negation of growth suppression. Immunoprecipitation analysis revealed that GSK-3α binds to Notch1.
  
  
  CONCLUSIONS
  This study demonstrates for the first time that the growth suppressive effect of AR-A014418 on pancreatic cancer cells is mainly mediated by a reduction in phosphorylation of GSK-3α with concomitant Notch1 reduction. GSK-3α appears to stabilize Notch1 by binding and may represent a target for therapeutic development. Furthermore, downregulation of GSK-3 and Notch1 may be a viable strategy for possible chemosensitization of pancreatic cancer cells to standard therapeutics.
authors.affiliation: Division of Surgical Oncology and Medical College of Wisconsin Cancer Center, Department of Surgery, Medical College of Wisconsin, Milwaukee, WI, USA.
authors.forename: Selvi
authors.initials: S
authors.lastname: Kunnimalaiyaan
authors.affiliation: Division of Surgical Oncology and Medical College of Wisconsin Cancer Center, Department of Surgery, Medical College of Wisconsin, Milwaukee, WI, USA.
authors.forename: T Clark
authors.initials: TC
authors.lastname: Gamblin
authors.affiliation: Division of Surgical Oncology and Medical College of Wisconsin Cancer Center, Department of Surgery, Medical College of Wisconsin, Milwaukee, WI, USA.
authors.forename: Muthusamy
authors.initials: M
authors.lastname: Kunnimalaiyaan
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D010179:Pancreas / Q000378:metabolism / Q000473:pathology*; D010190:Pancreatic Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology; D051881:Receptor, Notch1 / Q000096:biosynthesis*; D013844:Thiazoles / Q000494:pharmacology*; D014508:Urea / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: C497761:NOTCH1 protein, human; D051881:Receptor, Notch1; D013844:Thiazoles; C479831:N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea; D014508:Urea; D038362:Glycogen Synthase Kinase 3; C448021:glycogen synthase kinase 3 alpha