Original Article
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World J Gastroenterol. Aug 7, 2014; 20(29): 10038-10049
Published online Aug 7, 2014. doi: 10.3748/wjg.v20.i29.10038
Aspirin inhibits cell viability and mTOR downstream signaling in gastroenteropancreatic and bronchopulmonary neuroendocrine tumor cells
Matilde Spampatti, George Vlotides, Gerald Spöttl, Julian Maurer, Burkhard Göke, Christoph J Auernhammer
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
Matilde Spampatti, U.O.C.Gastroenterologia 2 Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Università degli Studi di Milano, 20122 Milano, Italy
Author contributions: Spampatti M and Vlotides G contributed equally to this work; Spampatti M, Vlotides G and Auernhammer CJ designed the research, Spampatti M, Spöttl G and Maurer J performed the research; Göke B contributed new reagents and analytic tools; Spampatti M, Vlotides G, Göke B and Auernhammer CJ wrote the paper.
Correspondence to: Dr. 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, Marchioninistr 15, 81377 Munich, Germany. cauernha@med.uni-muenchen.de
Telephone: +49-89-440072520 Fax: +49-89-440075514
Received: June 18, 2013
Revised: January 8, 2014
Accepted: January 20, 2014
Published online: August 7, 2014
Processing time: 414 Days and 10.8 Hours
Abstract

AIM: 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.

CONCLUSION: Aspirin demonstrates promising anticancer properties for NETs in vitro. Further preclinical and clinical studies are needed.

Keywords: Neuroendocrine tumors; Aspirin; Cell Viability; Mammalian target of rapamycin protein

Core tip: We evaluated the effects of aspirin on pancreatic (BON1), bronchopulmonary (NCI-H727) and midgut (GOT1) neuroendocrine tumor cell lines and demonstrated that aspirin has potent antitumor properties in vitro. Aspirin caused a dose-dependent reduction of cell viability/cell proliferation, the inhibition of targets downstream of mammalian target of rapamycin and the (compensatory) activation of the AKT and extracellular signal-regulated kinase signaling pathways. Treatment of BON1 and NCI-H727 cells with aspirin reduced the entry of these cells into the S phase of the cell cycle, and this effect was associated with the induction of p21 and reduced cyclin-dependent kinase 4 and cyclin D3 expression.