Antiproliferative and pro-apoptotic effects afforded by novel Src-kinase inhibitors in human neuroblastoma cells
BMC Cancer
Antiproliferative and pro-apoptotic effects afforded by novel Src-kinase inhibitors in human neuroblastoma cells
Michele Navarra 0 3
Marilena Celano 2
Jessica Maiuolo 2
Silvia Schenone 1
Maurizio Botta 5
Adriano Angelucci 4
Placido Bramanti 3
Diego Russo 2
0 Pharmaco-Biological Department, University of Messina , viale Annunziata, 98100, Messina , Italy
1 Department of Pharmaceutical Sciences, University of Genova , viale Benedetto XV, 16132 Genova , Italy
2 Department of Pharmacobiological Sciences, University “Magna Graecia” of Catanzaro , viale Europa, 88100, Catanzaro , Italy
3 IRCCS centro neurolesi “Bonino-Pulejo” , via Palermo C/da Casazza, 98124, Messina , Italy
4 Department of Experimental Medicine, University of L'Aquila , via Vetoio, 67100 Coppito, L'Aquila , Italy
5 Pharmaco-Chemical-Technological Department, University of Siena , via Aldo Moro 2, 53100, Siena , Italy
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 ERKphosphorylation, 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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Background
Neuroblastoma (NB) is the most common extracranial
pediatric solid tumour. It accounts for more than 7% of
malignancies in patients younger than 15 years and
around 15% of all paediatric oncologic deaths. NB
originates from neural crest precursor cells as the results of
genetic alterations occurring in neural crest cells that
affect the normal developmental program [1,2]. NB may
present with a broad spectrum of clinical behaviour and
may have various prognosis depending on the
assignment to a risk group. However, about half of patients
present with evidence of metastasis and the majority of
tumors usually undergo rapid progression with a fatal
outcome. Although an aggressive and intensive
multimodality approach (surgery, cytotoxic chemotherapy,
radio-metabolic treatment) has produced some
improvements in the overall cure rate of these patients, the
treatment strategies are still far from satisfaction [1,2].
Thus, innovative drugs are needed to develop novel
therapeutic strategies acting to ameliorate the prognosis
of NB patients.
Several studies have identified the protein tyrosine
kinases (TKs) as targets for cancer therapy, since
enhancement of TK activity has been correlated with
cancer and other proliferative diseases [3]. For this
reason, many TK inhibitors (TKIs) have been tested for
their in vitro and in vivo anticancer activity [4], and
some of them have been approved in clinical trials or
are in clinical use [5,6]. A subclass of TKIs with strong
antiproliferative activity is represented by the inhibitors
of Src-family tyrosine kinases (SFK), a group of
nonreceptor TKs involved in cancer development and
invasivity [7,8]. Src can stimulate cell proliferation, migration
and invasion as well as angiogenesis [9]. Moreover,
recent studies have suggested that Src may be
implicated in the development of drug resistance [10].
Overexpression or aberrant activation of Src has been
detected in a variety of human cancers [11], including
NB [12,13], thus representing an attractive (...truncated)