Abstract
Gastrin-releasing peptide (GRP) acts as an autocrine growth factor for neuroblastoma cells. Overexpression of the GRP receptor (GRP-R), which increases binding capacity for its ligand GRP, has been shown to stimulate cell growth; however, the exact role of GRP on tumor invasion/metastatic properties is unknown. Integrin is a heterodimer composed of α- and β-subunits; 24 integrin isoforms exist, from different pairings among 18 α- and 8 β-subunits. Integrins bind to components of extracellular matrix (ECM) such as collagen, fibronectin, and laminin to mediate cell adhesion, spread, or migration. The purpose of our study was to investigate the effect of GRP on integrin expression in neuroblastoma cells. Methods: To determine the effects of autocrine growth factor, GRP, on integrin expression, human neuroblastoma cell line, SK-N-SH, was stably transfected with pEGFP (control vector) and pEGFP-GRP-R plasmids. We then performed a metastasis specific gene array (SuperArray) assay. The expression of ECM receptor integrins was analyzed by Western blot analysis. The expression of cell membrane integrin protein levels was determined by fluorescence-activated cell sorter analysis (FACS). Results: Human neuroblastoma cells overexpressing GRP-R, which we had previously shown to increase GRP binding capacity, demonstrated marked changes in their cell morphology; cells appeared flat and more adhesive to culture plates. Gene array showed increased expression of integrins β1, α2, and α3 when compared to control cells. Consistent with these data, Western blot analysis showed increased protein expression of integrins β1, α2, and α3 in GRP-R overexpressing cells. These findings were further confirmed by FACS, where GRP-R overexpression resulted in increased expression of functional cell surface membrane integrins β1, α2, and α3. Conclusions: GRP-R overexpression resulted in enhanced mRNA and protein expression of integrins, suggesting that GRP-induced neuroblastoma cell proliferation may also enhance factors responsible for tumor invasion/metastatic potential. A better understanding of this process may allow us to develop novel therapy targeted at inhibiting GRP-induced neuroblastoma cell proliferation and metastasis.
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