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Investigation of new molecular target of neuroblastoma therapy: The role of p53 pathway in neuroblastoma cell death

神经母细胞瘤治疗新分子靶点研究：p53通路在神经母细胞瘤细胞死亡中的作用

基本信息

批准号：
17591077
负责人：
KAMIJO Takehiko
金额：
$ 2.11万
依托单位：
Chiba Cancer Center Research Institute (2006)Shinshu University (2005)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591077/
关键词：
Pediatric malignancies Neuroblastoma p53 cell death apoptosis Noxa 神経芽種 Mitochondria HDM2

项目摘要

Neuroblastoma (NB) is the most common pediatric solid malignant tumor derived from the sympathetic nervous system. We investigated the inhibitor of p53-dependent cell death in NB cells. The candidate for the inhibitor, which was highly expressed in Doxorubicin-resistant cells and bound to p53 in nucleus, seemed to be p53-E3 ubiquitine ligase HDM2.Of note, both of p53 and HDM2 were accumulated in the Doxorubicin-resistant NB cells although HDM2 accelerates p53 degradation in a proteasome dependent manner. Recently, it has reported that overproduction of Mdm2, resulting from a naturally occurring SNP309, inhibits chromatin-bound p53 from activating the transcription of its target genes (JBC, Arva NC et al., 2005). These results prompted us to study the existence of SNP309 HDM2 in NB cells. However, we could detect the heterozygous SNP only in SK-N-SH (sensitive) but not in IMR32 (resistant), NB-19 (resistant) and NB-9 (sensitive) cells. Next, we sequenced the p53-binding domain of HDM2 t … More o verify the p53-HDM2 interaction in NB cells and could not found mutations in the p53 binding domain of HDM2We employed a panel of cell lines to determine whether the p53-dependent cell death in neuroblastoma (NB) cells is caused by apoptotic cellular function, and we further studied the molecular mechanism of apoptosis induced via the p53-dependent pathway. We obtained evidence that a type of p53-dependent stress, doxorubicin (Doxo) administration, causes accumulation of p53 in the nucleus of NB cells and phosphorylation of several serine residues in both Doxo-sensitive and-resistant cell lines. Up-regulation of p53-downstream molecules in cells and accumulation of Noxa in the mitochondrial fraction were observed only in Doxo-sensitive NB cells. Significance of Noxa in the Doxo-induced NB cell death was confirmed by Noxa-knockdown experiments. Mitochondrial dysfunction, including cytochrome c release and membrane potential dis-regulation, occurred and resulted in the activation of the intrinsic caspase pathway (Kurata K et al., Oncogene, revise). Intriguingly, we found that HDM2 not only relates to control of p53 protein amounts but also may regulate the kinetics of Noxa in NB cells. In HDM-2 over-expressed SK-N-SH cells, p53 downstream pathway was inactivated although p53 was accumulated. Although Noxa was accumulated in mitochondria before stimulation, Doxorubicin could not up-regulate Noxa in mitochondria and induce apoptosis in the HDM2-over-expressed SK-N-SH cells. These results indicate that HDM2 might be key molecule to control p53 stability and activities and to regulate Noxa kinetics in mitochondria in NB cells. We would like to do further analysis of the mitochondria apoptosis related molecules to develop new therapies for unfavorable neuroblastoma. Less

神经母细胞瘤（NB）是最常见的小儿交感神经系统实体恶性肿瘤。我们研究了NB细胞中p53依赖性细胞死亡的抑制剂。p53-E3泛素连接酶HDM 2在多柔比星耐药NB细胞中高表达，并与p53在细胞核中结合。值得注意的是，尽管HDM 2以蛋白酶体依赖的方式促进p53降解，但p53和HDM 2在多柔比星耐药NB细胞中均聚集。最近，已经报道了由天然存在的SNP 309引起的Mdm 2的过度产生抑制染色质结合的p53激活其靶基因的转录（JBC，Arva NC et al.，2005年）。这些结果促使我们研究SNP 309 HDM 2在NB细胞中的存在。然而，我们只能在SK-N-SH（敏感）细胞中检测到杂合SNP，而在IMR 32（耐药）、NB-19（耐药）和NB-9（敏感）细胞中检测不到杂合SNP。接下来，我们对HDM 2的p53结合结构域进行了测序， ...更多信息为了验证p53-HDM 2在NB细胞中的相互作用，未发现HDM 2的p53结合结构域的突变，我们使用一组细胞系来确定神经母细胞瘤（NB）细胞中p53依赖的细胞死亡是否由凋亡细胞功能引起，并进一步研究通过p53依赖的途径诱导凋亡的分子机制。我们获得的证据表明，一种类型的p53依赖性应激，阿霉素（Doxo）管理，导致积累的p53在NB细胞的细胞核和磷酸化的几个丝氨酸残基在Doxo敏感和耐药细胞系。仅在Doxo敏感的NB细胞中观察到细胞中p53下游分子的上调和线粒体组分中Noxa的积累。通过Noxa敲低实验证实了Noxa在Doxo诱导的NB细胞死亡中的意义。发生线粒体功能障碍，包括细胞色素c释放和膜电位失调，并导致内源性半胱天冬酶途径的激活（Kurata K等人，Oncogene，revision）.有趣的是，我们发现HDM 2不仅与p53蛋白量的控制有关，而且还可以调节NB细胞中Noxa的动力学。在HDM-2过表达的SK-N-SH细胞中，尽管p53积累，但p53下游通路被失活。虽然在刺激前Noxa在线粒体中积累，但阿霉素不能上调线粒体中的Noxa并诱导HDM 2过表达的SK-N-SH细胞凋亡。这些结果表明，HDM 2可能是控制NB细胞中p53稳定性和活性以及调节线粒体中Noxa动力学的关键分子。我们希望通过对线粒体凋亡相关分子的进一步研究，为神经母细胞瘤的治疗提供新的思路。少