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Exendin-4 induces cell adhesion and differentiation and counteracts the invasive potential of human neuroblastoma cells.

Luciani P, Deledda C, Benvenuti S, Squecco R, Cellai I, Fibbi B, Marone IM, Giuliani C, Modi G, Francini F, Vannelli GB, Peri A - PLoS ONE (2013)

Bottom Line: More recently, additional biological properties have been associated to molecules that belong to the GLP-1 family.However, no data are currently available on the effects of exendin-4 on tumor cell motility.Furthermore, we demonstrated that exendin-4 reduced cell migration and counteracted anchorage-independent growth in neuroblastoma cells.

View Article: PubMed Central - PubMed

Affiliation: Endocrine Unit, "Center for Research, Transfer and High Education on Chronic, Inflammatory, Degenerative and Neoplastic Disorders for the Development of Novel Therapies" (DENOThe), Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy.

ABSTRACT
Exendin-4 is a molecule currently used, in its synthetic form exenatide, for the treatment of type 2 diabetes mellitus. Exendin-4 binds and activates the Glucagon-Like Peptide-1 Receptor (GLP-1R), thus inducing insulin release. More recently, additional biological properties have been associated to molecules that belong to the GLP-1 family. For instance, Peptide YY and Vasoactive Intestinal Peptide have been found to affect cell adhesion and migration and our previous data have shown a considerable actin cytoskeleton rearrangement after exendin-4 treatment. However, no data are currently available on the effects of exendin-4 on tumor cell motility. The aim of this study was to investigate the effects of this molecule on cell adhesion, differentiation and migration in two neuroblastoma cell lines, SH-SY5Y and SK-N-AS. We first demonstrated, by Extra Cellular Matrix cell adhesion arrays, that exendin-4 increased cell adhesion, in particular on a vitronectin substrate. Subsequently, we found that this molecule induced a more differentiated phenotype, as assessed by i) the evaluation of neurite-like protrusions in 3D cell cultures, ii) the analysis of the expression of neuronal markers and iii) electrophysiological studies. Furthermore, we demonstrated that exendin-4 reduced cell migration and counteracted anchorage-independent growth in neuroblastoma cells. Overall, these data indicate for the first time that exendin-4 may have anti-tumoral properties.

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Cell migration in response to different stimuli and CXCR4 expression.Representative experiment showing the effect of exendin-4 on the migration of NB cells and FNC cells. NB cells were induced to migration also by hMSC-conditioned culture medium (CM) (A). Expression of CXCR4 detected by real-time RT PCR and reported as fold-increase vs. related control. EXE = Exendin-4 (B). * = p<0.05 vs. related control.
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pone-0071716-g009: Cell migration in response to different stimuli and CXCR4 expression.Representative experiment showing the effect of exendin-4 on the migration of NB cells and FNC cells. NB cells were induced to migration also by hMSC-conditioned culture medium (CM) (A). Expression of CXCR4 detected by real-time RT PCR and reported as fold-increase vs. related control. EXE = Exendin-4 (B). * = p<0.05 vs. related control.

Mentions: Treatment with exendin-4 significantly reduced the migration of SH-SY5Y, SK-N-AS and FNC cells, as assessed by Boyden chambers. Figure 8A shows the counts of the migrated cells in three different experiments. Representative photographs of the filters are shown in figure 8 B-G. NB has as a preferential site of metastasization in the bone marrow. To test the influence of exendin-4 on the migration of SH-SY5Y and SK-N-AS towards the bone, we performed migration assays using a culture medium conditioned by hMSC, obtained from bone marrow of healthy donors and isolated as previously reported [19]. These cells secrete in vitro SDF-1, the major chemokine involved in the metastasization of NB to the bone marrow [26]. We determined the release of SDF-1 by hMSC in the culture medium by ELISA and we detected a concentration of 28.2 pg/ml. As expected, NB cells were strongly induced to migrate towards the conditioned medium, as well as towards the potent chemoactractant FBS. Exendin-4 significantly inhibited this process both in SK-N-AS and in SH-SY5Y (Fig. 9A). Moreover, migration assays were performed using SDF-1 (100 ng/ml) and different molecules known for their chemoactractant properties on several tumor cells including NB cells [e.g. Insulin like Growth Factor-1 (IGF-1) [27] and Platelet-Derived Growth Factor (PDGF) [28]]. Exendin-4 significantly reduced cell migration in all the conditions tested (Table 2). We also determined the influence of exendin-4 on the expression of the chemokine receptor for SDF-1 CXCR-4; the expression of CXCR4 was not affected by exendin-4 in NB cells whereas in SK-N-AS cells SDF-1 induced an increase of CXCR4 mRNA (Fig. 9B). Collectively, these results indicate that exendin-4 is able to reduce cell migration independently of the type of the chemoattractant used.


Exendin-4 induces cell adhesion and differentiation and counteracts the invasive potential of human neuroblastoma cells.

Luciani P, Deledda C, Benvenuti S, Squecco R, Cellai I, Fibbi B, Marone IM, Giuliani C, Modi G, Francini F, Vannelli GB, Peri A - PLoS ONE (2013)

Cell migration in response to different stimuli and CXCR4 expression.Representative experiment showing the effect of exendin-4 on the migration of NB cells and FNC cells. NB cells were induced to migration also by hMSC-conditioned culture medium (CM) (A). Expression of CXCR4 detected by real-time RT PCR and reported as fold-increase vs. related control. EXE = Exendin-4 (B). * = p<0.05 vs. related control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3750033&req=5

pone-0071716-g009: Cell migration in response to different stimuli and CXCR4 expression.Representative experiment showing the effect of exendin-4 on the migration of NB cells and FNC cells. NB cells were induced to migration also by hMSC-conditioned culture medium (CM) (A). Expression of CXCR4 detected by real-time RT PCR and reported as fold-increase vs. related control. EXE = Exendin-4 (B). * = p<0.05 vs. related control.
Mentions: Treatment with exendin-4 significantly reduced the migration of SH-SY5Y, SK-N-AS and FNC cells, as assessed by Boyden chambers. Figure 8A shows the counts of the migrated cells in three different experiments. Representative photographs of the filters are shown in figure 8 B-G. NB has as a preferential site of metastasization in the bone marrow. To test the influence of exendin-4 on the migration of SH-SY5Y and SK-N-AS towards the bone, we performed migration assays using a culture medium conditioned by hMSC, obtained from bone marrow of healthy donors and isolated as previously reported [19]. These cells secrete in vitro SDF-1, the major chemokine involved in the metastasization of NB to the bone marrow [26]. We determined the release of SDF-1 by hMSC in the culture medium by ELISA and we detected a concentration of 28.2 pg/ml. As expected, NB cells were strongly induced to migrate towards the conditioned medium, as well as towards the potent chemoactractant FBS. Exendin-4 significantly inhibited this process both in SK-N-AS and in SH-SY5Y (Fig. 9A). Moreover, migration assays were performed using SDF-1 (100 ng/ml) and different molecules known for their chemoactractant properties on several tumor cells including NB cells [e.g. Insulin like Growth Factor-1 (IGF-1) [27] and Platelet-Derived Growth Factor (PDGF) [28]]. Exendin-4 significantly reduced cell migration in all the conditions tested (Table 2). We also determined the influence of exendin-4 on the expression of the chemokine receptor for SDF-1 CXCR-4; the expression of CXCR4 was not affected by exendin-4 in NB cells whereas in SK-N-AS cells SDF-1 induced an increase of CXCR4 mRNA (Fig. 9B). Collectively, these results indicate that exendin-4 is able to reduce cell migration independently of the type of the chemoattractant used.

Bottom Line: More recently, additional biological properties have been associated to molecules that belong to the GLP-1 family.However, no data are currently available on the effects of exendin-4 on tumor cell motility.Furthermore, we demonstrated that exendin-4 reduced cell migration and counteracted anchorage-independent growth in neuroblastoma cells.

View Article: PubMed Central - PubMed

Affiliation: Endocrine Unit, "Center for Research, Transfer and High Education on Chronic, Inflammatory, Degenerative and Neoplastic Disorders for the Development of Novel Therapies" (DENOThe), Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy.

ABSTRACT
Exendin-4 is a molecule currently used, in its synthetic form exenatide, for the treatment of type 2 diabetes mellitus. Exendin-4 binds and activates the Glucagon-Like Peptide-1 Receptor (GLP-1R), thus inducing insulin release. More recently, additional biological properties have been associated to molecules that belong to the GLP-1 family. For instance, Peptide YY and Vasoactive Intestinal Peptide have been found to affect cell adhesion and migration and our previous data have shown a considerable actin cytoskeleton rearrangement after exendin-4 treatment. However, no data are currently available on the effects of exendin-4 on tumor cell motility. The aim of this study was to investigate the effects of this molecule on cell adhesion, differentiation and migration in two neuroblastoma cell lines, SH-SY5Y and SK-N-AS. We first demonstrated, by Extra Cellular Matrix cell adhesion arrays, that exendin-4 increased cell adhesion, in particular on a vitronectin substrate. Subsequently, we found that this molecule induced a more differentiated phenotype, as assessed by i) the evaluation of neurite-like protrusions in 3D cell cultures, ii) the analysis of the expression of neuronal markers and iii) electrophysiological studies. Furthermore, we demonstrated that exendin-4 reduced cell migration and counteracted anchorage-independent growth in neuroblastoma cells. Overall, these data indicate for the first time that exendin-4 may have anti-tumoral properties.

Show MeSH
Related in: MedlinePlus