Pharmaco-Biological Department, University of Messina, viale Annunziata, 98100 Messina, Italy.
BMC Cancer. 2010 Nov 4;10:602. doi: 10.1186/1471-2407-10-602.
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.
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.
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.
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.
神经母细胞瘤（NB）是儿童中第二常见的实体恶性肿瘤，通常在转移后迅速进展，预后不良。Src 家族酪氨酸激酶（SFKs）是一组参与癌症发展和侵袭的蛋白质，似乎在 NB 癌变中发挥重要作用。
为了确定细胞增殖，通过 MTT 试验和细胞计数评估生长速度。进行 DNA 含量分析以评估细胞周期和细胞凋亡。为了确定具有Src 抑制活性的新型吡唑并嘧啶衍生物 SI 34 诱导的抗增殖作用的机制，通过 Western blot 分析研究了一些对细胞增殖和存活很重要的细胞途径。特别研究了细胞周期蛋白、Src 和 ERK 的作用。最后，进行了细胞黏附和侵袭实验。
用三种新型吡唑并[3,4-d]嘧啶衍生物 SI 34、SI 35 和 SI 83 处理 SH-SY5Y 人 NB 细胞和 CHP100 人神经上皮细胞培养物，可时间和浓度依赖性方式抑制细胞增殖。用 SI 34 10 μM 孵育 72 小时后达到最大效果。荧光显微镜实验、流式细胞术分析和荧光法测定 caspase-3 活性表明 SI 34 诱导 SH-SY5Y 细胞凋亡。此外，SI 34 导致细胞周期停滞在 G0/G1 期，同时 cyclin D1 的表达降低。此外，我们的数据表明 SI 34 降低了 SH-SY5Y 细胞的黏附和侵袭能力。SI 34 抑制 Src 和 ERK 磷酸化的证据表明了它在 SH-SY5Y 细胞中发挥作用的机制。
我们的研究表明，这种吡唑并嘧啶 Src 抑制剂具有降低人 NB 细胞生长和侵袭的能力，表明它作为神经母细胞瘤治疗的新型药物具有广阔的前景。
