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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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Analysis of voltage-dependent Na+ and Ca2+ channels in FNC and SK-N-AS cells.Typical INa traces recorded in a SK-N-AS cell. The voltage threshold of INa was at −50 mV (A). Effect of Exendin-4 (EXE) on INa amplitude (B). In A, B numbers represent the voltages eliciting the maximal INa. C) Normalized I–V plots represent the data evaluated at the current peak in all the cells investigated; the Boltzmann fits (Eq. 1) are superimposed to the experimental data. D) Normalized data related to INa activation and inactivation and superimposed Boltzmann fit in control SK-N-AS and under exendin-4 treatment; the Boltzmann curves for activation are determined from panel C by the equation:  and inactivation from eq. 2; Boltzmann parameters listed in Table 1. Data represent mean ± SE from 26–43 cells. Representative ICa,tot traces obtained in a control (E) and in exendin-4 treated FNC cell (F). The arrow in the −50 mV trace indicates the presence of a first component as a fast-activating current, ICa,T. High-voltage-activated and slowly inactivating current (ICa,L, HVA) as a second component starting from −40 mV. Ca2+ currents elicited by a voltage step at −20 mV without (Cont) and in the presence of nifedipine (Nif), Cd2+ and Ni2+(G). Representative ICa,T recorded at a holding potential of –50 mV without (H), and with exendin-4 (I). Normalized I–V plots determined at the current peaks in control and under exendin-4 treatment related to ICa,T (L) and ICa,L (M). Normalized activation and inactivation data for T- (N) and L-type Ca2+ current (O) in control and under exendin-4 treatment, with the related Boltzmann fit superimposed to the data. The related Boltzmann parameters are listed in Table 1. In each experimental condition, data are from 18 to 23 cells.
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pone-0071716-g007: Analysis of voltage-dependent Na+ and Ca2+ channels in FNC and SK-N-AS cells.Typical INa traces recorded in a SK-N-AS cell. The voltage threshold of INa was at −50 mV (A). Effect of Exendin-4 (EXE) on INa amplitude (B). In A, B numbers represent the voltages eliciting the maximal INa. C) Normalized I–V plots represent the data evaluated at the current peak in all the cells investigated; the Boltzmann fits (Eq. 1) are superimposed to the experimental data. D) Normalized data related to INa activation and inactivation and superimposed Boltzmann fit in control SK-N-AS and under exendin-4 treatment; the Boltzmann curves for activation are determined from panel C by the equation: and inactivation from eq. 2; Boltzmann parameters listed in Table 1. Data represent mean ± SE from 26–43 cells. Representative ICa,tot traces obtained in a control (E) and in exendin-4 treated FNC cell (F). The arrow in the −50 mV trace indicates the presence of a first component as a fast-activating current, ICa,T. High-voltage-activated and slowly inactivating current (ICa,L, HVA) as a second component starting from −40 mV. Ca2+ currents elicited by a voltage step at −20 mV without (Cont) and in the presence of nifedipine (Nif), Cd2+ and Ni2+(G). Representative ICa,T recorded at a holding potential of –50 mV without (H), and with exendin-4 (I). Normalized I–V plots determined at the current peaks in control and under exendin-4 treatment related to ICa,T (L) and ICa,L (M). Normalized activation and inactivation data for T- (N) and L-type Ca2+ current (O) in control and under exendin-4 treatment, with the related Boltzmann fit superimposed to the data. The related Boltzmann parameters are listed in Table 1. In each experimental condition, data are from 18 to 23 cells.

Mentions: In 20-mM TEA solution, untreated cells at 48 h exhibited INa, as shown in a typical experiment performed with FNC (Fig. 7A). The voltage threshold of INa was about −60 mV. The maximal INa was observed at −15 mV and was greater in SK-N-AS than in FNC. The treatment with exendin-4 increased INa amplitude (1.2 and 1.2 fold in SK-N-AS and FNC) (Fig. 7A, B and 6E). The normalized I–V plot is shown in Fig. 7C. The maximal current amplitude was elicited at −15±5 mV in control SK-N-AS and FNC cells, but it was shifted at −20±5 mV in exendin-4 -treated cells (Fig. 7C). Exendin-4 treatment changed the activation and inactivation data (Fig. 7D). In particular, the maximal current to peak (INa/Cm) and Gmax/Cm values increased to a similar extent than control, suggesting that the rise in current density was related to the channel conductance increase. The half voltage activation and inactivation values, Va and Vi, obtained by the Boltzmann fit, were shifted towards more negative potentials, of about 5 and 3 mV (SK-N-AS) and 7 and 4 mV (FNC), respectively. In contrast, ka values were unchanged, whereas ki diminished (Fig. 7D; Table 1). Therefore, we suggest that exendin-4 improved the INa occurrence both by significantly shifting Va and Vi and by enhancing Na+ channels conductance. The expression of functional Ca2+ channels was assessed in TEA-Ca2+ bath solution (Fig. 7E–O). In both cell types Ca2+ currents showed a low-voltage-activated and inward transient current (T-type Ca2+ current, ICa,T) with a voltage threshold at −50 mV, and a high-voltage-activated current with a slow inactivation (IHVA), which became evident from −40 mV. The fitting procedure to the activation and inactivation curves of these two currents resulted in two Boltzmann terms that were in agreement with T- and HVAC currents (Fig. 7 N–O). To verify this suggestion we added the L-type Ca2+ blockers Cd2+ or nifedipine to the bath. In our records T-type current was not affected by both molecules (Fig. 7H–I), whereas HVAC was blocked by Cd2+ and only partly by nifedipine. In fact, the current traces still showed a HVAC having amplitude of about the 10% of the control. Consequently, we can reasonably suppose that HVAC currents consisted of a large nifedipine-sensitive L- type current with a small fraction of other HVAC Ca2+ currents superimposed, most likely N, P, Q or R- types (Fig. 7G). Exendin-4 significantly increased ICa,T, and ICa,L, amplitude (Fig. 6 F–G). The normalized I–V plots and the related normalized Boltzmann curve related to T- and L-type current are shown in figure 7 L–M. Again, changes similar to those observed for INa were induced by exendin-4 in Ca2+ currents, such as an increase in Gmax,Ca,T/Cm and Gmax,Ca,L/Cm, a shift towards a more negative potential of Va and Vi and a decrease of ka and ki. Notably, both the increase of the maximal current amplitude and the conductance of T- and L-type Ca2+ currents were greater than those of INa, and the highest increases were those related to L-type Ca2+ current (Table 1). Again, the results obtained in SK-N-AS at 48 h were similar to those observed in SH-SY5Y at the same time as previously reported [17].


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)

Analysis of voltage-dependent Na+ and Ca2+ channels in FNC and SK-N-AS cells.Typical INa traces recorded in a SK-N-AS cell. The voltage threshold of INa was at −50 mV (A). Effect of Exendin-4 (EXE) on INa amplitude (B). In A, B numbers represent the voltages eliciting the maximal INa. C) Normalized I–V plots represent the data evaluated at the current peak in all the cells investigated; the Boltzmann fits (Eq. 1) are superimposed to the experimental data. D) Normalized data related to INa activation and inactivation and superimposed Boltzmann fit in control SK-N-AS and under exendin-4 treatment; the Boltzmann curves for activation are determined from panel C by the equation:  and inactivation from eq. 2; Boltzmann parameters listed in Table 1. Data represent mean ± SE from 26–43 cells. Representative ICa,tot traces obtained in a control (E) and in exendin-4 treated FNC cell (F). The arrow in the −50 mV trace indicates the presence of a first component as a fast-activating current, ICa,T. High-voltage-activated and slowly inactivating current (ICa,L, HVA) as a second component starting from −40 mV. Ca2+ currents elicited by a voltage step at −20 mV without (Cont) and in the presence of nifedipine (Nif), Cd2+ and Ni2+(G). Representative ICa,T recorded at a holding potential of –50 mV without (H), and with exendin-4 (I). Normalized I–V plots determined at the current peaks in control and under exendin-4 treatment related to ICa,T (L) and ICa,L (M). Normalized activation and inactivation data for T- (N) and L-type Ca2+ current (O) in control and under exendin-4 treatment, with the related Boltzmann fit superimposed to the data. The related Boltzmann parameters are listed in Table 1. In each experimental condition, data are from 18 to 23 cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0071716-g007: Analysis of voltage-dependent Na+ and Ca2+ channels in FNC and SK-N-AS cells.Typical INa traces recorded in a SK-N-AS cell. The voltage threshold of INa was at −50 mV (A). Effect of Exendin-4 (EXE) on INa amplitude (B). In A, B numbers represent the voltages eliciting the maximal INa. C) Normalized I–V plots represent the data evaluated at the current peak in all the cells investigated; the Boltzmann fits (Eq. 1) are superimposed to the experimental data. D) Normalized data related to INa activation and inactivation and superimposed Boltzmann fit in control SK-N-AS and under exendin-4 treatment; the Boltzmann curves for activation are determined from panel C by the equation: and inactivation from eq. 2; Boltzmann parameters listed in Table 1. Data represent mean ± SE from 26–43 cells. Representative ICa,tot traces obtained in a control (E) and in exendin-4 treated FNC cell (F). The arrow in the −50 mV trace indicates the presence of a first component as a fast-activating current, ICa,T. High-voltage-activated and slowly inactivating current (ICa,L, HVA) as a second component starting from −40 mV. Ca2+ currents elicited by a voltage step at −20 mV without (Cont) and in the presence of nifedipine (Nif), Cd2+ and Ni2+(G). Representative ICa,T recorded at a holding potential of –50 mV without (H), and with exendin-4 (I). Normalized I–V plots determined at the current peaks in control and under exendin-4 treatment related to ICa,T (L) and ICa,L (M). Normalized activation and inactivation data for T- (N) and L-type Ca2+ current (O) in control and under exendin-4 treatment, with the related Boltzmann fit superimposed to the data. The related Boltzmann parameters are listed in Table 1. In each experimental condition, data are from 18 to 23 cells.
Mentions: In 20-mM TEA solution, untreated cells at 48 h exhibited INa, as shown in a typical experiment performed with FNC (Fig. 7A). The voltage threshold of INa was about −60 mV. The maximal INa was observed at −15 mV and was greater in SK-N-AS than in FNC. The treatment with exendin-4 increased INa amplitude (1.2 and 1.2 fold in SK-N-AS and FNC) (Fig. 7A, B and 6E). The normalized I–V plot is shown in Fig. 7C. The maximal current amplitude was elicited at −15±5 mV in control SK-N-AS and FNC cells, but it was shifted at −20±5 mV in exendin-4 -treated cells (Fig. 7C). Exendin-4 treatment changed the activation and inactivation data (Fig. 7D). In particular, the maximal current to peak (INa/Cm) and Gmax/Cm values increased to a similar extent than control, suggesting that the rise in current density was related to the channel conductance increase. The half voltage activation and inactivation values, Va and Vi, obtained by the Boltzmann fit, were shifted towards more negative potentials, of about 5 and 3 mV (SK-N-AS) and 7 and 4 mV (FNC), respectively. In contrast, ka values were unchanged, whereas ki diminished (Fig. 7D; Table 1). Therefore, we suggest that exendin-4 improved the INa occurrence both by significantly shifting Va and Vi and by enhancing Na+ channels conductance. The expression of functional Ca2+ channels was assessed in TEA-Ca2+ bath solution (Fig. 7E–O). In both cell types Ca2+ currents showed a low-voltage-activated and inward transient current (T-type Ca2+ current, ICa,T) with a voltage threshold at −50 mV, and a high-voltage-activated current with a slow inactivation (IHVA), which became evident from −40 mV. The fitting procedure to the activation and inactivation curves of these two currents resulted in two Boltzmann terms that were in agreement with T- and HVAC currents (Fig. 7 N–O). To verify this suggestion we added the L-type Ca2+ blockers Cd2+ or nifedipine to the bath. In our records T-type current was not affected by both molecules (Fig. 7H–I), whereas HVAC was blocked by Cd2+ and only partly by nifedipine. In fact, the current traces still showed a HVAC having amplitude of about the 10% of the control. Consequently, we can reasonably suppose that HVAC currents consisted of a large nifedipine-sensitive L- type current with a small fraction of other HVAC Ca2+ currents superimposed, most likely N, P, Q or R- types (Fig. 7G). Exendin-4 significantly increased ICa,T, and ICa,L, amplitude (Fig. 6 F–G). The normalized I–V plots and the related normalized Boltzmann curve related to T- and L-type current are shown in figure 7 L–M. Again, changes similar to those observed for INa were induced by exendin-4 in Ca2+ currents, such as an increase in Gmax,Ca,T/Cm and Gmax,Ca,L/Cm, a shift towards a more negative potential of Va and Vi and a decrease of ka and ki. Notably, both the increase of the maximal current amplitude and the conductance of T- and L-type Ca2+ currents were greater than those of INa, and the highest increases were those related to L-type Ca2+ current (Table 1). Again, the results obtained in SK-N-AS at 48 h were similar to those observed in SH-SY5Y at the same time as previously reported [17].

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