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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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Src- and ERK-phosphorylation in SH-SY5Y treated with SI 34. (A) SH-SY5Y cells were stimulated with insulin (100 nM; 30 min) in the presence or absence of SI 34 (10 μM; 30 min), and then western blotting analysis of Src and phospho-Src was performed. A Western blot, representative of three independent experiments showing similar results, is presented. (C) A representative gel (out of three) showed the ERK and phospho-ERK expression in presence or not of SI 34 is illustrated. (B and D) Densitometric analysis of immunoreactive bands corresponding to the Src-phosphorylated and ERK phosphorylated forms from blots A and C are reported. Autoradiographic bands were quantified by ImageJ software and normalized for β-actin levels. Data are reported as percentages of the values detected in untreated cultures.
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Figure 6: Src- and ERK-phosphorylation in SH-SY5Y treated with SI 34. (A) SH-SY5Y cells were stimulated with insulin (100 nM; 30 min) in the presence or absence of SI 34 (10 μM; 30 min), and then western blotting analysis of Src and phospho-Src was performed. A Western blot, representative of three independent experiments showing similar results, is presented. (C) A representative gel (out of three) showed the ERK and phospho-ERK expression in presence or not of SI 34 is illustrated. (B and D) Densitometric analysis of immunoreactive bands corresponding to the Src-phosphorylated and ERK phosphorylated forms from blots A and C are reported. Autoradiographic bands were quantified by ImageJ software and normalized for β-actin levels. Data are reported as percentages of the values detected in untreated cultures.

Mentions: To better understand the mechanisms underlying the antiproliferative effects induced by SI 34, the involvement of some cellular pathways that are important for cell proliferation and survival like Src and extracellular receptor kinases (ERK) phosphorylation was investigated by western blot assays. In normal conditions, the level of Src-phosphorylation in SH-SY5Y cells is low. Thus, to enhance Src phosphorylation the cells were stimulated with 100 nM insulin for 30 minutes, obtaining an increase of phosphorylated Src level compared to the untreated cultures (Figure 6A and 6B). SH-SY5Y cells stimulated with insulin were treated with SI 34 (10 μM; 30 min) and the levels of phosphorylated and non-phosphorylated Src were examined. As a result, Src-phosphorylation promoted in SH-SY5Y cells by insulin was inhibited using 10 μM SI 34 (P < 0.05) while the basal levels of Src were not affected (Figure 6A and 6B).


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)

Src- and ERK-phosphorylation in SH-SY5Y treated with SI 34. (A) SH-SY5Y cells were stimulated with insulin (100 nM; 30 min) in the presence or absence of SI 34 (10 μM; 30 min), and then western blotting analysis of Src and phospho-Src was performed. A Western blot, representative of three independent experiments showing similar results, is presented. (C) A representative gel (out of three) showed the ERK and phospho-ERK expression in presence or not of SI 34 is illustrated. (B and D) Densitometric analysis of immunoreactive bands corresponding to the Src-phosphorylated and ERK phosphorylated forms from blots A and C are reported. Autoradiographic bands were quantified by ImageJ software and normalized for β-actin levels. Data are reported as percentages of the values detected in untreated cultures.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Src- and ERK-phosphorylation in SH-SY5Y treated with SI 34. (A) SH-SY5Y cells were stimulated with insulin (100 nM; 30 min) in the presence or absence of SI 34 (10 μM; 30 min), and then western blotting analysis of Src and phospho-Src was performed. A Western blot, representative of three independent experiments showing similar results, is presented. (C) A representative gel (out of three) showed the ERK and phospho-ERK expression in presence or not of SI 34 is illustrated. (B and D) Densitometric analysis of immunoreactive bands corresponding to the Src-phosphorylated and ERK phosphorylated forms from blots A and C are reported. Autoradiographic bands were quantified by ImageJ software and normalized for β-actin levels. Data are reported as percentages of the values detected in untreated cultures.
Mentions: To better understand the mechanisms underlying the antiproliferative effects induced by SI 34, the involvement of some cellular pathways that are important for cell proliferation and survival like Src and extracellular receptor kinases (ERK) phosphorylation was investigated by western blot assays. In normal conditions, the level of Src-phosphorylation in SH-SY5Y cells is low. Thus, to enhance Src phosphorylation the cells were stimulated with 100 nM insulin for 30 minutes, obtaining an increase of phosphorylated Src level compared to the untreated cultures (Figure 6A and 6B). SH-SY5Y cells stimulated with insulin were treated with SI 34 (10 μM; 30 min) and the levels of phosphorylated and non-phosphorylated Src were examined. As a result, Src-phosphorylation promoted in SH-SY5Y cells by insulin was inhibited using 10 μM SI 34 (P < 0.05) while the basal levels of Src were not affected (Figure 6A and 6B).

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