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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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Expression of GLP1R and cell proliferation assays.RT-PCR analysis of the expression of the GLP1R in all the neuronal cell lines and in pancreas taken as the positive control. Amplicon length = 240 basepairs M 100 bp = Molecular weight marker 100 basepairs (A). Cell proliferation assay performed in SK-N-AS and SH-SY5Ycells after treatment with 0.3 µM exendin-4 (in 2% FBS) for 24 h. Cpm = counts per minute; C 2% = control cells in 2% FBS, EXE = exendin-4. +EXE vs. C 2%, p>0.05). C 10% = control cells in 10% FBS (standard medium, used as the positive control) (B).
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pone-0071716-g001: Expression of GLP1R and cell proliferation assays.RT-PCR analysis of the expression of the GLP1R in all the neuronal cell lines and in pancreas taken as the positive control. Amplicon length = 240 basepairs M 100 bp = Molecular weight marker 100 basepairs (A). Cell proliferation assay performed in SK-N-AS and SH-SY5Ycells after treatment with 0.3 µM exendin-4 (in 2% FBS) for 24 h. Cpm = counts per minute; C 2% = control cells in 2% FBS, EXE = exendin-4. +EXE vs. C 2%, p>0.05). C 10% = control cells in 10% FBS (standard medium, used as the positive control) (B).

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)

Expression of GLP1R and cell proliferation assays.RT-PCR analysis of the expression of the GLP1R in all the neuronal cell lines and in pancreas taken as the positive control. Amplicon length = 240 basepairs M 100 bp = Molecular weight marker 100 basepairs (A). Cell proliferation assay performed in SK-N-AS and SH-SY5Ycells after treatment with 0.3 µM exendin-4 (in 2% FBS) for 24 h. Cpm = counts per minute; C 2% = control cells in 2% FBS, EXE = exendin-4. +EXE vs. C 2%, p>0.05). C 10% = control cells in 10% FBS (standard medium, used as the positive control) (B).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750033&req=5

pone-0071716-g001: Expression of GLP1R and cell proliferation assays.RT-PCR analysis of the expression of the GLP1R in all the neuronal cell lines and in pancreas taken as the positive control. Amplicon length = 240 basepairs M 100 bp = Molecular weight marker 100 basepairs (A). Cell proliferation assay performed in SK-N-AS and SH-SY5Ycells after treatment with 0.3 µM exendin-4 (in 2% FBS) for 24 h. Cpm = counts per minute; C 2% = control cells in 2% FBS, EXE = exendin-4. +EXE vs. C 2%, p>0.05). C 10% = control cells in 10% FBS (standard medium, used as the positive control) (B).
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