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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 adhesion assay on different ECM proteins and uPAR expression.Representative experiment on the effect of 0.3 µM exendin-4 on the adhesion of SH-SY5Y and SK-N-AS cells on different ECM proteins. * = p<0.05 vs. related control (A); Bengal rose adhesion assay performed on SK-N-AS (B) and SH-SY5Y (C) cells plated on vitronectin and treated with 0.3 µM exendin-4 for 24 h. A representative experiment of three independent experiments is shown in each case. * = p<0.05 vs. control; expression of uPAR as assessed by real-time RT-PCR in SK-N-AS and SH-SY5Y cells treated with 0.3 µM exendin-4 for 24 h. Mean percentage ± SE of four independent experiments. EXE = exendin-4. * = p<0.05 vs. control cells (D).
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pone-0071716-g003: Cell adhesion assay on different ECM proteins and uPAR expression.Representative experiment on the effect of 0.3 µM exendin-4 on the adhesion of SH-SY5Y and SK-N-AS cells on different ECM proteins. * = p<0.05 vs. related control (A); Bengal rose adhesion assay performed on SK-N-AS (B) and SH-SY5Y (C) cells plated on vitronectin and treated with 0.3 µM exendin-4 for 24 h. A representative experiment of three independent experiments is shown in each case. * = p<0.05 vs. control; expression of uPAR as assessed by real-time RT-PCR in SK-N-AS and SH-SY5Y cells treated with 0.3 µM exendin-4 for 24 h. Mean percentage ± SE of four independent experiments. EXE = exendin-4. * = p<0.05 vs. control cells (D).

Mentions: The presence of the GLP-1R in cells was evaluated by RT-PCR analysis (Fig. 1A). To test the effect of exendin-4 on cell proliferation and survival, we treated the cells with the GLP-1 receptor agonist exendin-4 at the dose of 0.3 µM. Exendin-4 did not significantly alter DNA synthesis (Fig. 1B), cell viability or cell counts (not shown). To demonstrate a possible effect of exendin-4 on cell adhesion, we performed a Bengal rose assay and we observed an increased number of adherent cells at 48 h in all the cells models (Fig. 2). In order to characterize which components of the Cell Adhesion Molecules (CAM) were involved in the increased adhesion properties, we screened the ability of the cells to adhere to different ECM proteins: collagen I, collagen II, collagen IV, fibronectin, laminin, tenascin, vitronectin. We found that both SH-SY5Y and SK-N-AS cells treated with exendin-4 augmented their interaction with vitronectin (Fig. 3A). Conversely, exendin-4 did not affect the adhesion of FNC to the proteins tested (Fig. S1A). To confirm data obtained with arrays, we performed cell adhesion tests in plates coated with vitronectin, and we found a significantly increased number of adherent NB cells (Fig. 3B and C), but not FNC cells (Fig. S1B). Moreover, since vitronectin is a major urokinase plasminogen activator surface receptor (uPAR) ligand with a role in cell adhesion, we measured the receptor expression by real-time RT-PCR, in order to evaluate whether exendin-4 increases the adhesion to vitronectin by up-regulating uPAR. As shown in figure 3D, exendin-4 did not increase uPAR expression in the two NB cell lines and actually mRNA levels appeared decreased (significantly in SK-N-AS), thus confirming that uPAR does not play a role in the stimulatory effect of exendin-4 on cell adhesion.


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 adhesion assay on different ECM proteins and uPAR expression.Representative experiment on the effect of 0.3 µM exendin-4 on the adhesion of SH-SY5Y and SK-N-AS cells on different ECM proteins. * = p<0.05 vs. related control (A); Bengal rose adhesion assay performed on SK-N-AS (B) and SH-SY5Y (C) cells plated on vitronectin and treated with 0.3 µM exendin-4 for 24 h. A representative experiment of three independent experiments is shown in each case. * = p<0.05 vs. control; expression of uPAR as assessed by real-time RT-PCR in SK-N-AS and SH-SY5Y cells treated with 0.3 µM exendin-4 for 24 h. Mean percentage ± SE of four independent experiments. EXE = exendin-4. * = p<0.05 vs. control cells (D).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750033&req=5

pone-0071716-g003: Cell adhesion assay on different ECM proteins and uPAR expression.Representative experiment on the effect of 0.3 µM exendin-4 on the adhesion of SH-SY5Y and SK-N-AS cells on different ECM proteins. * = p<0.05 vs. related control (A); Bengal rose adhesion assay performed on SK-N-AS (B) and SH-SY5Y (C) cells plated on vitronectin and treated with 0.3 µM exendin-4 for 24 h. A representative experiment of three independent experiments is shown in each case. * = p<0.05 vs. control; expression of uPAR as assessed by real-time RT-PCR in SK-N-AS and SH-SY5Y cells treated with 0.3 µM exendin-4 for 24 h. Mean percentage ± SE of four independent experiments. EXE = exendin-4. * = p<0.05 vs. control cells (D).
Mentions: The presence of the GLP-1R in cells was evaluated by RT-PCR analysis (Fig. 1A). To test the effect of exendin-4 on cell proliferation and survival, we treated the cells with the GLP-1 receptor agonist exendin-4 at the dose of 0.3 µM. Exendin-4 did not significantly alter DNA synthesis (Fig. 1B), cell viability or cell counts (not shown). To demonstrate a possible effect of exendin-4 on cell adhesion, we performed a Bengal rose assay and we observed an increased number of adherent cells at 48 h in all the cells models (Fig. 2). In order to characterize which components of the Cell Adhesion Molecules (CAM) were involved in the increased adhesion properties, we screened the ability of the cells to adhere to different ECM proteins: collagen I, collagen II, collagen IV, fibronectin, laminin, tenascin, vitronectin. We found that both SH-SY5Y and SK-N-AS cells treated with exendin-4 augmented their interaction with vitronectin (Fig. 3A). Conversely, exendin-4 did not affect the adhesion of FNC to the proteins tested (Fig. S1A). To confirm data obtained with arrays, we performed cell adhesion tests in plates coated with vitronectin, and we found a significantly increased number of adherent NB cells (Fig. 3B and C), but not FNC cells (Fig. S1B). Moreover, since vitronectin is a major urokinase plasminogen activator surface receptor (uPAR) ligand with a role in cell adhesion, we measured the receptor expression by real-time RT-PCR, in order to evaluate whether exendin-4 increases the adhesion to vitronectin by up-regulating uPAR. As shown in figure 3D, exendin-4 did not increase uPAR expression in the two NB cell lines and actually mRNA levels appeared decreased (significantly in SK-N-AS), thus confirming that uPAR does not play a role in the stimulatory effect of exendin-4 on cell adhesion.

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