Posadas I, Vellecco V, Santos P, Prieto-Lloret J, Ceña V
Unidad Asociada Neurodeath, UCLM-CSIC, Universidad de Castilla-La Mancha, Avda. de Almansa, Albacete, Spain.
Br J Pharmacol. 2007 Mar;150(5):577-85. doi: 10.1038/sj.bjp.0706993. Epub 2007 Jan 22.
Neuroblastoma is the most common solid tumour in infants characterized by a high resistance to apoptosis. Recently, the cyclo-oxygenase pathway has been considered a potential target in the treatment of different kinds of tumours. The aim of the present work was to investigate a possible relationship between cyclo-oxygenase pathway and stauroporine-induced apoptosis in the neuroblastoma cell line SH-SY5Y.
Cellular viability was measured by release of LDH. DNA fragmentation was visualized by electrophoresis on agarose gel containing ethidium bromide. Cyclo-oxygenase activity was measured in microsomal fractions obtained from cells by quantification of its final product PGE2 by RIA. Caspase-3 activity was measured fluorimetrically and Western blot analysis was performed to assess cytochrome c expression.
We have found that staurosporine (500 nM) induced cellular death in a time-dependent manner in SH-SY5Y human neuroblastoma cells. Cyclo-oxygenase enzymatic activity was present in SH-SY5Y human neuroblastoma cells under basal conditions and pharmacological experiments using COX inhibitors indicate that cyclo-oxygenase-1 and cyclo-oxygenase-3 are the active isoforms in these cells. Co-incubation of SH-SY5Y cells with staurosporine (500 nM) and acetaminophen for 24 h potentiated staurosporine-mediated cellular death in a concentration-dependent manner. This process is mediated by an increase in cytochrome c release and caspase 3 activation and is prevented by N-acetylcysteine or the superoxide dismutase mimetic, MnTBAP.
Acetaminophen potentiates staurosporine-mediated neuroblastoma cell death. The mechanism of action of acetaminophen seems to be related to production of reactive oxygen species and decreased intracellular glutathione levels.
神经母细胞瘤是婴儿最常见的实体瘤，其特点是对细胞凋亡具有高度抗性。最近，环氧化酶途径被认为是治疗不同类型肿瘤的潜在靶点。本研究的目的是探讨环氧化酶途径与星形孢菌素诱导神经母细胞瘤细胞系SH-SY5Y凋亡之间的可能关系。
通过乳酸脱氢酶释放量测定细胞活力。在含有溴化乙锭的琼脂糖凝胶上进行电泳，观察DNA片段化情况。通过放射免疫分析法对从细胞中获得的微粒体部分的最终产物前列腺素E2进行定量，测定环氧化酶活性。采用荧光法测定半胱天冬酶-3活性，并进行蛋白质印迹分析以评估细胞色素c的表达。
我们发现，星形孢菌素（500 nM）在SH-SY5Y人神经母细胞瘤细胞中以时间依赖性方式诱导细胞死亡。在基础条件下，SH-SY5Y人神经母细胞瘤细胞中存在环氧化酶活性，使用环氧化酶抑制剂的药理实验表明，环氧化酶-1和环氧化酶-3是这些细胞中的活性同工型。将SH-SY5Y细胞与星形孢菌素（500 nM）和对乙酰氨基酚共同孵育24小时，以浓度依赖性方式增强了星形孢菌素介导的细胞死亡。这个过程由细胞色素c释放增加和半胱天冬酶3激活介导，并被N-乙酰半胱氨酸或超氧化物歧化酶模拟物MnTBAP所抑制。
对乙酰氨基酚增强星形孢菌素介导的神经母细胞瘤细胞死亡。对乙酰氨基酚的作用机制似乎与活性氧的产生和细胞内谷胱甘肽水平降低有关。
