title: Sorafenib inhibits intracellular signaling pathways and induces cell cycle arrest and cell death in thyroid carcinoma cells irrespective of histological origin or BRAF mutational status.
abstract: BACKGROUND
  Patients with dedifferentiated or anaplastic thyroid carcinomas currently lack appropriate treatment options. Kinase inhibitors are among the most promising new agents as alternative strategies. The BRAF- and multi-kinase inhibitor, sorafenib, has already shown antitumor effects in thyroid carcinoma patients in a phase III clinical trial. In this study we aim to better characterize molecular effects and efficacy of sorafenib against thyroid carcinoma cells with various histological origins and different BRAF mutational status. Analysis of different signaling pathways affected by sorafenib may contribute to assist a more specific therapy choice with fewer side effects. Twelve thyroid carcinoma cell lines derived from anaplastic, follicular and papillary thyroid carcinomas with wildtype or mutationally activated BRAF were treated with sorafenib. Growth inhibition, cell cycle arrest, cell death induction and inhibition of intracellular signaling pathways were then comprehensively analyzed.
  
  
  METHODS
  Cell viability was analyzed by MTT assay, and the cell cycle was assessed by flow cytometry after propidium iodide staining. Cell death was assessed by lactate dehydrogenase liberation assays, caspase activity assays and subG1 peak determinations. Inhibition of intracellular pathways was analyzed in dot blot and western blot analyses.
  
  
  RESULTS
  Sorafenib inhibited proliferation of all thyroid carcinoma cell lines tested with IC50 values ranging between 1.85 and 4.2 μM. Cells derived from papillary carcinoma harboring the mutant BRAF (V600E) allele were slightly more sensitive to sorafenib than those harboring wildtype BRAF. Cell cycle analyses and caspase assays showed a sorafenib-dependent induction of apoptosis in all cell lines, whereas increased lactate dehydrogenase release suggested cell membrane disruption. Sorafenib treatment caused a rapid inhibition of various MAP kinases in addition to inhibiting AKT and receptor tyrosine kinases.
  
  
  CONCLUSIONS
  Sorafenib inhibited multiple intracellular signaling pathways in thyroid carcinoma cells, which resulted in cell cycle arrest and the initiation of apoptosis. Sorafenib was effective against all thyroid carcinoma cell lines regardless of their tumor subtype origin or BRAF status, confirming that sorafenib is therapeutically beneficial for patients with any subtype of dedifferentiated thyroid cancer. Inhibition of single intracellular targets of sorafenib in thyroid carcinoma cells may allow the development of more specific therapeutic intervention with less side effects.
authors: Department of Endocrinology and Metabolism, and Division of Laboratory Research, University Hospital Essen, Hufelandstr. 55, Essen, Germany. martina.broecker@uni-due.de.|Present address: Department of Clinical Chemistry, University Hospital Essen, Hufelandstr. 55, 45122, Essen, Germany. martina.broecker@uni-due.de.
authors: Martina
authors: M
authors: Broecker-Preuss
authors: Department of Nuclear Medicine, University Hospital Essen, Hufelandstr. 55, Essen, Germany. stefan.mueller@uni-due.de.
authors: Stefan
authors: S
authors: Müller
authors: Department of Endocrinology and Metabolism, and Division of Laboratory Research, University Hospital Essen, Hufelandstr. 55, Essen, Germany. martin.britten@uk-essen.de.|Present address: University Hospital Essen, Klinik für Anästhesiologie & Intensivmedizin, Hufelandstr. 55, 45122, Essen, Germany. martin.britten@uk-essen.de.
authors: Martin
authors: M
authors: Britten
authors: Institute of Pathology at the University Hospital Essen, Hufelandstr. 55, Essen, Germany. karl.worm@uk-essen.de.
authors: Karl
authors: K
authors: Worm
authors: Institute of Pathology at the University Hospital Essen, Hufelandstr. 55, Essen, Germany. kw.schmid@uk-essen.de.
authors: Kurt Werner
authors: KW
authors: Schmid
authors: Department of Endocrinology and Metabolism, and Division of Laboratory Research, University Hospital Essen, Hufelandstr. 55, Essen, Germany. klaus.mann@ezah.de.|Present address: Center of Endocrinology Alter Hof München, Dienerstr. 12, 80331, München, Germany. klaus.mann@ezah.de.
authors: Klaus
authors: K
authors: Mann
authors: Department of Endocrinology and Metabolism, and Division of Laboratory Research, University Hospital Essen, Hufelandstr. 55, Essen, Germany. dagmar.fuehrer-sakel@uk-essen.de.
authors: Dagmar
authors: D
authors: Fuhrer
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D059447:Cell Cycle Checkpoints / Q000187:drug effects* / Q000235:genetics*; D016923:Cell Death / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009154:Mutation; D009536:Niacinamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D010671:Phenylurea Compounds / Q000494:pharmacology*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D000077157:Sorafenib; D013964:Thyroid Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology
chemical_list: D000970:Antineoplastic Agents; D010671:Phenylurea Compounds; D047428:Protein Kinase Inhibitors; D009536:Niacinamide; D000077157:Sorafenib; D020794:Receptor Protein-Tyrosine Kinases; D048493:Proto-Oncogene Proteins B-raf; D020928:Mitogen-Activated Protein Kinases