title: Aspirin inhibits cell viability and mTOR downstream signaling in gastroenteropancreatic and bronchopulmonary neuroendocrine tumor cells.
abstract: OBJECTIVE
  To investigate the effect of aspirin on neuroendocrine tumor (NET) cell growth and signaling in vitro.
  
  
  METHODS
  Human pancreatic BON1, bronchopulmonary NCI-H727 and midgut GOT1 neuroendocrine tumor cells were treated with different concentrations of aspirin (from 0.001 to 5 mmol/L), and the resulting effects on metabolic activity/cell proliferation were measured using cell proliferation assays and SYBR-DNA-labeling after 72, 144 and 216 h of incubation. The effects of aspirin on the expression and phosphorylation of several critical proteins that are involved in the most common intracellular growth factor signaling pathways (especially Akt protein kinase B) and mammalian target of rapamycin (mTOR) were determined by Western blot analyses. Propidium iodide staining and flow cytometry were used to evaluate changes in cell cycle distribution and apoptosis. Statistical analysis was performed using a 2-tailed Student's t-test to evaluate the proliferation assays and cell cycle analyses. The results are expressed as the mean ± SD of 3 or 4 independently performed experiments. Statistical significance was set at P < 0.05.
  
  
  RESULTS
  Treatment with aspirin suppressed the viability/proliferation of BON1, NCI-H727 and GOT1 cells in a time- and dose-dependent manner. Significant effects were observed at starting doses of 0.5-1 mmol/L and peaked at 5 mmol/L. For instance, after treatment with 1 mmol/L aspirin for 144 h, the viability of pancreatic BON1 cells decreased to 66% ± 13% (P < 0.05), the viability of bronchopulmonary NCI-H727 cells decreased to 53% ± 8% (P < 0.01) and the viability of midgut GOT1 cells decreased to 89% ± 6% (P < 0.01). These effects were associated with a decreased entry into the S phase, the induction of the cyclin-dependent kinase inhibitor p21 and reduced expression of cyclin-dependent kinase 4 and cyclin D3. Aspirin suppressed mTOR downstream signaling, evidenced by the reduced phosphorylation of the mTOR substrates 4E binding protein 1, serine/threonine kinase P70S6K and S6 ribosomal protein and inhibited glycogen synthase kinase 3 activity. We observed the (compensatory) activation of tuberous sclerosis 2, the serine/threonine specific protein kinase AKT and extracellular signal-regulated kinases.
  
  
  CONCLUSIONS
  Aspirin demonstrates promising anticancer properties for NETs in vitro. Further preclinical and clinical studies are needed.
authors.affiliation: Matilde Spampatti, George Vlotides, Gerald Spöttl, Julian Maurer, Burkhard Göke, Christoph J Auernhammer, Department of Internal Medicine II, University-Hospital Campus Grosshadern, Interdisciplinary Center of Neuroendocrine Tumours of the GastroEnteroPancreatic System (GEPNET-KUM), Ludwig-Maximilians-University of Munich, 81377 Munich, Germany.
authors.forename: Matilde
authors.initials: M
authors.lastname: Spampatti
authors.affiliation: Matilde Spampatti, George Vlotides, Gerald Spöttl, Julian Maurer, Burkhard Göke, Christoph J Auernhammer, Department of Internal Medicine II, University-Hospital Campus Grosshadern, Interdisciplinary Center of Neuroendocrine Tumours of the GastroEnteroPancreatic System (GEPNET-KUM), Ludwig-Maximilians-University of Munich, 81377 Munich, Germany.
authors.forename: George
authors.initials: G
authors.lastname: Vlotides
authors.affiliation: Matilde Spampatti, George Vlotides, Gerald Spöttl, Julian Maurer, Burkhard Göke, Christoph J Auernhammer, Department of Internal Medicine II, University-Hospital Campus Grosshadern, Interdisciplinary Center of Neuroendocrine Tumours of the GastroEnteroPancreatic System (GEPNET-KUM), Ludwig-Maximilians-University of Munich, 81377 Munich, Germany.
authors.forename: Gerald
authors.initials: G
authors.lastname: Spöttl
authors.affiliation: Matilde Spampatti, George Vlotides, Gerald Spöttl, Julian Maurer, Burkhard Göke, Christoph J Auernhammer, Department of Internal Medicine II, University-Hospital Campus Grosshadern, Interdisciplinary Center of Neuroendocrine Tumours of the GastroEnteroPancreatic System (GEPNET-KUM), Ludwig-Maximilians-University of Munich, 81377 Munich, Germany.
authors.forename: Julian
authors.initials: J
authors.lastname: Maurer
authors.affiliation: Matilde Spampatti, George Vlotides, Gerald Spöttl, Julian Maurer, Burkhard Göke, Christoph J Auernhammer, Department of Internal Medicine II, University-Hospital Campus Grosshadern, Interdisciplinary Center of Neuroendocrine Tumours of the GastroEnteroPancreatic System (GEPNET-KUM), Ludwig-Maximilians-University of Munich, 81377 Munich, Germany.
authors.forename: Burkhard
authors.initials: B
authors.lastname: Göke
authors.affiliation: Matilde Spampatti, George Vlotides, Gerald Spöttl, Julian Maurer, Burkhard Göke, Christoph J Auernhammer, Department of Internal Medicine II, University-Hospital Campus Grosshadern, Interdisciplinary Center of Neuroendocrine Tumours of the GastroEnteroPancreatic System (GEPNET-KUM), Ludwig-Maximilians-University of Munich, 81377 Munich, Germany.
authors.forename: Christoph J
authors.initials: CJ
authors.lastname: Auernhammer
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D001241:Aspirin / Q000494:pharmacology*; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology* / Q000473:pathology; D018358:Neuroendocrine Tumors / Q000201:enzymology* / Q000473:pathology; D010190:Pancreatic Neoplasms / Q000201:enzymology* / Q000473:pathology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013997:Time Factors
chemical_list: D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D047428:Protein Kinase Inhibitors; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D001241:Aspirin
keywords: Aspirin; Cell Viability; Mammalian target of rapamycin protein; Neuroendocrine tumors
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