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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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Effects of exendin-4 on SK-N-AS and SH-SY5Y in 2D or 3D matrigel cultures.Representative 400X phase-contrast inverted microscope fields of: SH-SY5Y control (A) and exendin-4-treated (B) cells after 1 h plating on top of matrigel; SK-N-AS control (C) and exendin-4-treated (D) cells after 3 h plating on top of matrigel: the arrows show the adherent cells in contrast to detached, rounded and refractile cells. Representative 100X phase-contrast inverted microscope fields of SH-SY5Y control (E) and exendin-4-treated (F) cells, SK-N-AS control (G) and exendin-4-treated (H) cells, FNC control (I) and exendin-4-treated (L) cells after plating inside matrigel for 48 h; long thin neuritic protrusions are indicated by the arrows and are suggestive of a more differentiated phenotype. EXE = exendin-4.
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pone-0071716-g004: Effects of exendin-4 on SK-N-AS and SH-SY5Y in 2D or 3D matrigel cultures.Representative 400X phase-contrast inverted microscope fields of: SH-SY5Y control (A) and exendin-4-treated (B) cells after 1 h plating on top of matrigel; SK-N-AS control (C) and exendin-4-treated (D) cells after 3 h plating on top of matrigel: the arrows show the adherent cells in contrast to detached, rounded and refractile cells. Representative 100X phase-contrast inverted microscope fields of SH-SY5Y control (E) and exendin-4-treated (F) cells, SK-N-AS control (G) and exendin-4-treated (H) cells, FNC control (I) and exendin-4-treated (L) cells after plating inside matrigel for 48 h; long thin neuritic protrusions are indicated by the arrows and are suggestive of a more differentiated phenotype. EXE = exendin-4.

Mentions: In vitro 3D cultures are relevant models of the cell-cell and cell-stroma interactions in both normal and cancer tissues [24] and allow the study of cellular differentiation, organization and migration. Using matrigel, we set up 2D cultures, plating cells on top, and 3D cultures, suspending cells inside the matrigel. We observed that soon after plating cells on top of matrigel, the number of cells spreading on the matrix was higher in samples exposed to exendin-4 than in controls (62±8% vs. 36±7% for SH-SY5Y after 1 hour p<0.05; 55±6% vs. 28±5% for SK-N-AS after 3 hours, p<0.05) (Fig. 4 A–D). No modification in adhesive properties was detected in FNC cells cultured on top of matrigel (Fig. S2). Furthermore, exendin-4-treated SH-SY5Y, SK-N-AS and FNC cells suspended inside the matrigel showed a rapid adaptation to the 3D environment, resulting in a more evident presence of neurite-like protrusions vs. control cells (Fig. 4 E–L). A similar effect had been already reported on plastic support for exendin-4-treated SH-SY5Y [17], whereas no effect was observed in SK-N-AS or FNC cells (not shown). We measured the expression of the neuronal markers MAP2, Tau and Synaptophysin; a significant increase of the transcripts in the cell lines tested was found, with the only exception of Tau in FNC (Fig. 5).


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)

Effects of exendin-4 on SK-N-AS and SH-SY5Y in 2D or 3D matrigel cultures.Representative 400X phase-contrast inverted microscope fields of: SH-SY5Y control (A) and exendin-4-treated (B) cells after 1 h plating on top of matrigel; SK-N-AS control (C) and exendin-4-treated (D) cells after 3 h plating on top of matrigel: the arrows show the adherent cells in contrast to detached, rounded and refractile cells. Representative 100X phase-contrast inverted microscope fields of SH-SY5Y control (E) and exendin-4-treated (F) cells, SK-N-AS control (G) and exendin-4-treated (H) cells, FNC control (I) and exendin-4-treated (L) cells after plating inside matrigel for 48 h; long thin neuritic protrusions are indicated by the arrows and are suggestive of a more differentiated phenotype. EXE = exendin-4.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750033&req=5

pone-0071716-g004: Effects of exendin-4 on SK-N-AS and SH-SY5Y in 2D or 3D matrigel cultures.Representative 400X phase-contrast inverted microscope fields of: SH-SY5Y control (A) and exendin-4-treated (B) cells after 1 h plating on top of matrigel; SK-N-AS control (C) and exendin-4-treated (D) cells after 3 h plating on top of matrigel: the arrows show the adherent cells in contrast to detached, rounded and refractile cells. Representative 100X phase-contrast inverted microscope fields of SH-SY5Y control (E) and exendin-4-treated (F) cells, SK-N-AS control (G) and exendin-4-treated (H) cells, FNC control (I) and exendin-4-treated (L) cells after plating inside matrigel for 48 h; long thin neuritic protrusions are indicated by the arrows and are suggestive of a more differentiated phenotype. EXE = exendin-4.
Mentions: In vitro 3D cultures are relevant models of the cell-cell and cell-stroma interactions in both normal and cancer tissues [24] and allow the study of cellular differentiation, organization and migration. Using matrigel, we set up 2D cultures, plating cells on top, and 3D cultures, suspending cells inside the matrigel. We observed that soon after plating cells on top of matrigel, the number of cells spreading on the matrix was higher in samples exposed to exendin-4 than in controls (62±8% vs. 36±7% for SH-SY5Y after 1 hour p<0.05; 55±6% vs. 28±5% for SK-N-AS after 3 hours, p<0.05) (Fig. 4 A–D). No modification in adhesive properties was detected in FNC cells cultured on top of matrigel (Fig. S2). Furthermore, exendin-4-treated SH-SY5Y, SK-N-AS and FNC cells suspended inside the matrigel showed a rapid adaptation to the 3D environment, resulting in a more evident presence of neurite-like protrusions vs. control cells (Fig. 4 E–L). A similar effect had been already reported on plastic support for exendin-4-treated SH-SY5Y [17], whereas no effect was observed in SK-N-AS or FNC cells (not shown). We measured the expression of the neuronal markers MAP2, Tau and Synaptophysin; a significant increase of the transcripts in the cell lines tested was found, with the only exception of Tau in FNC (Fig. 5).

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