Limits...
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.

Show MeSH

Related in: MedlinePlus

Analysis of cyclin D1 expression in SH-SY5Y treated with SI 34. (A) Immunoblot of SH-SY5Y cells exposed to 10 μM SI 34 for 6-72 hours: a representative of three separate experiments is shown. (B) Quantification of cyclin D1 expression from blots in panels A achieved with ImageJ software. Histogram shows the results of the densitometric analysis of autoradiographic bands in which the protein levels were normalized for β-actin (black bars for the untreated cultures and white bars for the cells exposed to SI 34 10 μM for the indicated times). Levels are extrapolated as percentages of the values detected in control cells, which are arbitrarily assigned as 100%.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2992519&req=5

Figure 5: Analysis of cyclin D1 expression in SH-SY5Y treated with SI 34. (A) Immunoblot of SH-SY5Y cells exposed to 10 μM SI 34 for 6-72 hours: a representative of three separate experiments is shown. (B) Quantification of cyclin D1 expression from blots in panels A achieved with ImageJ software. Histogram shows the results of the densitometric analysis of autoradiographic bands in which the protein levels were normalized for β-actin (black bars for the untreated cultures and white bars for the cells exposed to SI 34 10 μM for the indicated times). Levels are extrapolated as percentages of the values detected in control cells, which are arbitrarily assigned as 100%.

Mentions: It is well known the crucial role of the cyclins within cell division cycle and their frequent deregulations in cancer. Cyclin D1 governs the transit through the G1 phase of the cell cycle and is amplified and/or over-expressed in a relevant proportion of human cancers, including NB [25]. In order to estimate the contribution of cyclins in the mechanisms by which SI 34 blocks the SH-SY5Y cell cycle at G0/G1 phase, we examined the expression of cyclin D1 and E in SH-SY5Y cells treated with SI 34 by western blot assays. As shown in Figure 5, when the cultures were exposed to SI 34 10 μM, a time-dependent decrease of the cyclin D1 expression was observed with the maximal reduction detected after 72 h of treatment (P < 0.05 vs untreated cultures). These data demonstrate that SI 34 is able to reduce the cyclin D1 expression in SH-SY5Y cells, suggesting a correlation between the reduction of this protein level, the cells cycle arrest and the inhibition of cellular proliferation. Cyclin E is another rate limiting regulator in G1 phase of the cell cycle and its appropriate regulation is essential to drive the cells in S phase. Cyclin E appears in late G1 and disappears in early S phase. Interestingly, no modulation of the cyclin E expression by SI 34 was observed (data not shown), strengthening the hypothesis that the block of the cell cycles induced by SI 34 occurs at the early G1 phase.


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)

Analysis of cyclin D1 expression in SH-SY5Y treated with SI 34. (A) Immunoblot of SH-SY5Y cells exposed to 10 μM SI 34 for 6-72 hours: a representative of three separate experiments is shown. (B) Quantification of cyclin D1 expression from blots in panels A achieved with ImageJ software. Histogram shows the results of the densitometric analysis of autoradiographic bands in which the protein levels were normalized for β-actin (black bars for the untreated cultures and white bars for the cells exposed to SI 34 10 μM for the indicated times). Levels are extrapolated as percentages of the values detected in control cells, which are arbitrarily assigned as 100%.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Analysis of cyclin D1 expression in SH-SY5Y treated with SI 34. (A) Immunoblot of SH-SY5Y cells exposed to 10 μM SI 34 for 6-72 hours: a representative of three separate experiments is shown. (B) Quantification of cyclin D1 expression from blots in panels A achieved with ImageJ software. Histogram shows the results of the densitometric analysis of autoradiographic bands in which the protein levels were normalized for β-actin (black bars for the untreated cultures and white bars for the cells exposed to SI 34 10 μM for the indicated times). Levels are extrapolated as percentages of the values detected in control cells, which are arbitrarily assigned as 100%.
Mentions: It is well known the crucial role of the cyclins within cell division cycle and their frequent deregulations in cancer. Cyclin D1 governs the transit through the G1 phase of the cell cycle and is amplified and/or over-expressed in a relevant proportion of human cancers, including NB [25]. In order to estimate the contribution of cyclins in the mechanisms by which SI 34 blocks the SH-SY5Y cell cycle at G0/G1 phase, we examined the expression of cyclin D1 and E in SH-SY5Y cells treated with SI 34 by western blot assays. As shown in Figure 5, when the cultures were exposed to SI 34 10 μM, a time-dependent decrease of the cyclin D1 expression was observed with the maximal reduction detected after 72 h of treatment (P < 0.05 vs untreated cultures). These data demonstrate that SI 34 is able to reduce the cyclin D1 expression in SH-SY5Y cells, suggesting a correlation between the reduction of this protein level, the cells cycle arrest and the inhibition of cellular proliferation. Cyclin E is another rate limiting regulator in G1 phase of the cell cycle and its appropriate regulation is essential to drive the cells in S phase. Cyclin E appears in late G1 and disappears in early S phase. Interestingly, no modulation of the cyclin E expression by SI 34 was observed (data not shown), strengthening the hypothesis that the block of the cell cycles induced by SI 34 occurs at the early G1 phase.

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