Effect of cAMP on the migration, proliferation, invasion and trans-endothelial migration of Neuroblastoma cells


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Rabiu Fage Inuwa1,John Quayle2,Diana Moss2
1Institute of Translational Medicine, University of Liverpool,2Department of cellular and molecular physiology, University of Liverpool

Abstract

Background

Neuroblastoma is a pediatric cancer of the sympathoadrenal lineage derived from neural crest cells. Many patients present with metastatic disease form. During metastasis, cancer cells from the primary tumour must cross the endothelial cell barrier. Increased cAMP has been shown to increase cell junction formation between endothelial cells, potentially enhancing the barrier to metastasis. The aim of this study is to investigate the role of cAMP levels on SK-N-BE2C and SK-N-AS neuroblastoma cell migration, proliferation, invasion and trans-endothelial migration in vitro.

Method

Migration of neuroblastoma cells was measured using scratch assays. Wound was generated in a 100% confluent cell monolayer and rate of wound closure was measured. The scratch assay was repeated with forskolin and other agonists and antagonists affecting cAMP pathways, whilst change in cell number was measured using the MTT assay. Invasion was measured using matrigel spheroid assay.  Endothelial (HDMEC) cells were co-cultured with SK-N-BE2C or SK-N-AS cells for transmigration assay. Transmigration assays were repeated with Epac agonist and antagonists, and number of non-migrated cells was measured.

Results

SK-N-BE2C cells were more migratory than the SK-N-AS cells. Forskolin had no effect on the migration of either cell line. Forskolin did not change cell number over 24hr for either cell line. SK-N-AS cells were more invasive in the matrigel than SK-N-BE2C cells, and forskolin did not alter the pattern or speed of invasion in the two cell lines. Trans-endothelial migration results show a significant decrease of migrating cells across the endothelium on treatment with Epac agonist in both SK-N-BE2C and SK-N-AS cells.

Conclusion

SK-N-BE2C are more migratory than SK-N-AS in vitro. Forskolin did not have any observable effect on migration and invasion of either Neuroblastoma cell line. An Epac agonist reduced both SK-N-BE2C or SK-N-AS trans-endothelial migration.