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Antiproliferative and pro-apoptotic effects afforded by novel Src-kinase inhibitors in human neuroblastoma cells.

Navarra M, Celano M, Maiuolo J, Schenone S, Botta M, Angelucci A, Bramanti P, Russo D - BMC Cancer (2010)

Bottom Line: Furthermore, our data indicate that SI 34 reduces the SH-SY5Y cells adhesion and invasiveness.Evidence that SI 34 inhibits the Src and the ERK-phosphorylation, suggests the mechanism through which it exerts its effects in SH-SY5Y cells.Our study shows the ability of this pyrazolo-pyrimidine Src inhibitor in reducing the growth and the invasiveness of human NB cells, suggesting a promising role as novel drug in the treatment of neuroblastoma.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pharmaco-Biological Department, University of Messina, viale Annunziata, 98100 Messina, Italy. mnavarra@unime.it

ABSTRACT

Background: Neuroblastoma (NB) is the second most common solid malignancy of childhood that usually undergoes rapid progression with a poor prognosis upon metastasis. The Src-family tyrosine kinases (SFKs) are a group of proteins involved in cancer development and invasiveness that seem to play an important role in the NB carcinogenesis.

Methods: To determine cell proliferation, the growth rate was evaluated by both MTT test and cells counted. Analysis of DNA content was performed for the evaluation of the cell cycle and apoptosis. To characterize the mechanisms underlying the antiproliferative effects induced by SI 34, a novel pyrazolo-pyrimidine derivative provided with Src inhibitory activity, the involvement of some cellular pathways that are important for cell proliferation and survival was investigated by western blot assays. In particular, the contribution of cyclins, Src and ERK were examined. Finally, experiments of cell adhesion and invasiveness were performed.

Results: Treatment of SH-SY5Y human NB cells and CHP100 human neuroepithelioma (NE) cultures with three novel pyrazolo[3,4-d]pyrimidine derivatives, namely SI 34, SI 35 and SI 83, inhibits the cell proliferation in a time and concentration-dependent manner. The maximal effect was obtained after 72 hours incubation with SI 34 10 μM. Fluorescence microscopy experiments, flow cytometry analysis and determination of caspase-3 activity by fluorimetric assays showed that SI 34 induced SH-SY5Y apoptosis. Moreover, SI 34 determined cell cycle arrest at the G0/G1 phase, paralleled by a decreased expression of cyclin D1. Furthermore, our data indicate that SI 34 reduces the SH-SY5Y cells adhesion and invasiveness. Evidence that SI 34 inhibits the Src and the ERK-phosphorylation, suggests the mechanism through which it exerts its effects in SH-SY5Y cells.

Conclusions: Our study shows the ability of this pyrazolo-pyrimidine Src inhibitor in reducing the growth and the invasiveness of human NB cells, suggesting a promising role as novel drug in the treatment of neuroblastoma.

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SI 34 decreases SH-SY5Y cell adhesion. (A) Changes of cellular morphology in SH-SY5Y cultures exposed to 10 μM SI 34 for 72 hours. (B) Detached cells from cultures exposed (white bars) or not (black bars) to SI 34 were collected and counted as described in materials and methods. The results are expressed as percentage of detached cells (subtracted the percentage of dead cells from the full amount of detached cells) with respect to the total number of cells present in the well. Each value is the mean ± S.E.M. of 6 different sets of experiments made in triplicate. ***P < 0.001 vs respective controls. (C) Adhesion assay performed by plating SH-SY5Y cells on two different physiological substrates (Matrigel and collagen I) and on non-coated plastic surface for 30 min. Cells were treated with increasing concentrations of SI 34 (0, 1, 5, 10 μM) for 24 or 48 hours prior the adhesion assay. The values are expressed as mean percentage with respect to control (black bar) of at least three different measurements (± S.E.M). *, ** and ***P < 0.05, P < 0.01 and P < 0.001 vs respective control.
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Figure 7: SI 34 decreases SH-SY5Y cell adhesion. (A) Changes of cellular morphology in SH-SY5Y cultures exposed to 10 μM SI 34 for 72 hours. (B) Detached cells from cultures exposed (white bars) or not (black bars) to SI 34 were collected and counted as described in materials and methods. The results are expressed as percentage of detached cells (subtracted the percentage of dead cells from the full amount of detached cells) with respect to the total number of cells present in the well. Each value is the mean ± S.E.M. of 6 different sets of experiments made in triplicate. ***P < 0.001 vs respective controls. (C) Adhesion assay performed by plating SH-SY5Y cells on two different physiological substrates (Matrigel and collagen I) and on non-coated plastic surface for 30 min. Cells were treated with increasing concentrations of SI 34 (0, 1, 5, 10 μM) for 24 or 48 hours prior the adhesion assay. The values are expressed as mean percentage with respect to control (black bar) of at least three different measurements (± S.E.M). *, ** and ***P < 0.05, P < 0.01 and P < 0.001 vs respective control.

Mentions: In parallel with the decrease in cell proliferation, we observed that the presence of SI 34 determined a modification in cellular morphology. The cells acquired a round shape morphology, associated with a marked increase of susceptibility in their detachment (Figure 7A). After treatment or not with SI 34, the cells were detached by gentle agitation and counted. The weakly adherent cells reached up to over 20% when the SH-SY5Y cultures were treated for 72 h with 10 μM of the test compound (Figure 7B). Moreover SH-SY5Y cells were treated for 24 and 48 h with increasing concentrations of SI 34 (1, 5 and 10 μM) and then we evaluated their adhesive capacity on two different physiologic substrates, matrigel and collagen I (Figure 7C). The same experimental protocol was executed with non-coated surface. Results demonstrated an evident trend towards a decrease in adhesive capacity of treated cells in presence of all substrates and at higher concentrations of SI 34. In particular, after 48 h, the percentages of adherent cells on matrigel were significantly lower in treated cells than in non-treated cells for all concentrations of SI 34 (-30%, -32%, -47% respectively; P < 0.001).


Antiproliferative and pro-apoptotic effects afforded by novel Src-kinase inhibitors in human neuroblastoma cells.

Navarra M, Celano M, Maiuolo J, Schenone S, Botta M, Angelucci A, Bramanti P, Russo D - BMC Cancer (2010)

SI 34 decreases SH-SY5Y cell adhesion. (A) Changes of cellular morphology in SH-SY5Y cultures exposed to 10 μM SI 34 for 72 hours. (B) Detached cells from cultures exposed (white bars) or not (black bars) to SI 34 were collected and counted as described in materials and methods. The results are expressed as percentage of detached cells (subtracted the percentage of dead cells from the full amount of detached cells) with respect to the total number of cells present in the well. Each value is the mean ± S.E.M. of 6 different sets of experiments made in triplicate. ***P < 0.001 vs respective controls. (C) Adhesion assay performed by plating SH-SY5Y cells on two different physiological substrates (Matrigel and collagen I) and on non-coated plastic surface for 30 min. Cells were treated with increasing concentrations of SI 34 (0, 1, 5, 10 μM) for 24 or 48 hours prior the adhesion assay. The values are expressed as mean percentage with respect to control (black bar) of at least three different measurements (± S.E.M). *, ** and ***P < 0.05, P < 0.01 and P < 0.001 vs respective control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2992519&req=5

Figure 7: SI 34 decreases SH-SY5Y cell adhesion. (A) Changes of cellular morphology in SH-SY5Y cultures exposed to 10 μM SI 34 for 72 hours. (B) Detached cells from cultures exposed (white bars) or not (black bars) to SI 34 were collected and counted as described in materials and methods. The results are expressed as percentage of detached cells (subtracted the percentage of dead cells from the full amount of detached cells) with respect to the total number of cells present in the well. Each value is the mean ± S.E.M. of 6 different sets of experiments made in triplicate. ***P < 0.001 vs respective controls. (C) Adhesion assay performed by plating SH-SY5Y cells on two different physiological substrates (Matrigel and collagen I) and on non-coated plastic surface for 30 min. Cells were treated with increasing concentrations of SI 34 (0, 1, 5, 10 μM) for 24 or 48 hours prior the adhesion assay. The values are expressed as mean percentage with respect to control (black bar) of at least three different measurements (± S.E.M). *, ** and ***P < 0.05, P < 0.01 and P < 0.001 vs respective control.
Mentions: In parallel with the decrease in cell proliferation, we observed that the presence of SI 34 determined a modification in cellular morphology. The cells acquired a round shape morphology, associated with a marked increase of susceptibility in their detachment (Figure 7A). After treatment or not with SI 34, the cells were detached by gentle agitation and counted. The weakly adherent cells reached up to over 20% when the SH-SY5Y cultures were treated for 72 h with 10 μM of the test compound (Figure 7B). Moreover SH-SY5Y cells were treated for 24 and 48 h with increasing concentrations of SI 34 (1, 5 and 10 μM) and then we evaluated their adhesive capacity on two different physiologic substrates, matrigel and collagen I (Figure 7C). The same experimental protocol was executed with non-coated surface. Results demonstrated an evident trend towards a decrease in adhesive capacity of treated cells in presence of all substrates and at higher concentrations of SI 34. In particular, after 48 h, the percentages of adherent cells on matrigel were significantly lower in treated cells than in non-treated cells for all concentrations of SI 34 (-30%, -32%, -47% respectively; P < 0.001).

Bottom Line: Furthermore, our data indicate that SI 34 reduces the SH-SY5Y cells adhesion and invasiveness.Evidence that SI 34 inhibits the Src and the ERK-phosphorylation, suggests the mechanism through which it exerts its effects in SH-SY5Y cells.Our study shows the ability of this pyrazolo-pyrimidine Src inhibitor in reducing the growth and the invasiveness of human NB cells, suggesting a promising role as novel drug in the treatment of neuroblastoma.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pharmaco-Biological Department, University of Messina, viale Annunziata, 98100 Messina, Italy. mnavarra@unime.it

ABSTRACT

Background: Neuroblastoma (NB) is the second most common solid malignancy of childhood that usually undergoes rapid progression with a poor prognosis upon metastasis. The Src-family tyrosine kinases (SFKs) are a group of proteins involved in cancer development and invasiveness that seem to play an important role in the NB carcinogenesis.

Methods: To determine cell proliferation, the growth rate was evaluated by both MTT test and cells counted. Analysis of DNA content was performed for the evaluation of the cell cycle and apoptosis. To characterize the mechanisms underlying the antiproliferative effects induced by SI 34, a novel pyrazolo-pyrimidine derivative provided with Src inhibitory activity, the involvement of some cellular pathways that are important for cell proliferation and survival was investigated by western blot assays. In particular, the contribution of cyclins, Src and ERK were examined. Finally, experiments of cell adhesion and invasiveness were performed.

Results: Treatment of SH-SY5Y human NB cells and CHP100 human neuroepithelioma (NE) cultures with three novel pyrazolo[3,4-d]pyrimidine derivatives, namely SI 34, SI 35 and SI 83, inhibits the cell proliferation in a time and concentration-dependent manner. The maximal effect was obtained after 72 hours incubation with SI 34 10 μM. Fluorescence microscopy experiments, flow cytometry analysis and determination of caspase-3 activity by fluorimetric assays showed that SI 34 induced SH-SY5Y apoptosis. Moreover, SI 34 determined cell cycle arrest at the G0/G1 phase, paralleled by a decreased expression of cyclin D1. Furthermore, our data indicate that SI 34 reduces the SH-SY5Y cells adhesion and invasiveness. Evidence that SI 34 inhibits the Src and the ERK-phosphorylation, suggests the mechanism through which it exerts its effects in SH-SY5Y cells.

Conclusions: Our study shows the ability of this pyrazolo-pyrimidine Src inhibitor in reducing the growth and the invasiveness of human NB cells, suggesting a promising role as novel drug in the treatment of neuroblastoma.

Show MeSH
Related in: MedlinePlus