Investigation of a new molecular target-based treatment of caneer-drug resistant pediatric solid tumors

神经母细胞瘤（NB）是最常见的儿童来自交感神经系统的实体恶性肿瘤。与许多儿童恶性肿瘤的生存率提高不同，高风险成空细胞瘤仍然是最难治愈的肿瘤之一，尽管进行了强化多模式治疗，但长期生存率仅为30%。为了提高生存率，需要新的治疗方法和更好地了解耐药机制。神经母细胞瘤研究的一个值得注意的发现是，在340个测试的NBs中，据报道p53肿瘤抑制基因突变不到2% （Tweddle DA et al., Cancer Res 61: 8-13, 2001）。p53的细胞质隔离已被认为是NB细胞失活的另一种机制，而不是突变。这种分离首先是用免疫组织化学技术在冷冻肿瘤样本中报道的（Moll UM et al., PNAS 92: 4407- 11,1995），后来用免疫荧光和细胞分离实验在NB油系中报道的……More s （Moll UM et al., MCB 16: 1126- 37,1996）。然而，DNA损伤刺激前后p53的定位、p53的磷酸化稳定和激活、体外和体内与p53结合共识序列的结合、p53下游分子在NB细胞中的上调等仍有待研究。因此，我们通过一组细胞系来确定NB细胞中p53依赖性细胞死亡是否由细胞凋亡功能引起，并进一步研究p53依赖性途径诱导细胞凋亡的分子机制。我们提供了一系列证据，证明p53依赖的应激，多柔比星（Doxo）给药，导致p53在NB细胞细胞核中积累，并在多柔比星敏感和耐药的NB细胞系中磷酸化几个丝氨酸残基（丝氨酸15、丝氨酸20和丝氨酸46）。仅在doxo敏感的NB细胞中观察到p53下游分子p2lCip 1/Waf1的上调和线粒体中促凋亡的Bcl-2家族分子Noxa的积累。在敏感细胞中引起线粒体功能障碍，包括细胞色素c释放和膜电位失调，导致内在caspase途径激活。然而，在耐p53的NB细胞中，细胞核内的积累和p53的磷酸化并不能诱导Noxa的积累和线粒体功能障碍。令人惊讶的是，过度表达MDM2导致p53失活，从而取消线粒体中Noxa的上调，减少凋亡细胞的死亡。综上所述，MDM2/p53通路似乎通过线粒体中的Noxa调控在NB细胞中发挥重要作用。少

Neuroblastoma (NB) is the most common pediatric solid malignant tumor derived from the sympathetic nervous system. Unlike the improvements in survival in many childhood malignancies, high-risk neumblastoma is still one of the most difficult tumors to cure, with only 30% long-term survival despite intensive multi-modal therapy. New treatments and a better understanding of drug resistance mechanisms are required for improvement of the survival rates. A noteworthy finding of neuroblastoma research is that mutations of p53 tumor suppressor have been reported less than 2% of NBs out of 340 tested (Tweddle DA et al., Cancer Res 61 : 8-13, 2001). Instead of mutation, cytoplasmic sequestration of p53 has been proposed as an alternative mechanism of inactivation in NB cells. The sequestration was first reported in frozen tumor samples using immuno-histodremical techniques (Moll UM et al., PNAS 92 : 4407-11, 1995) and later in NB oil lines by immuno-fluorescence and cell fractionation experiment … More s (Moll UM et al., MCB 16 : 1126-37, 1996). However, the p53 localization before and after DNA damage stimulation, p53 stabilization and activation by phosphorylation, binding to the p53-binding consensus sequences in vitro and in vivo, and up-regulation of p53-dowmstream molecules in NB cells have remained to be determined.Therefore, we employed a panel of cell lines to determine whether the p53-dependent cell death in NB cells is caused by apoptotic cellular function, and we further studied the molecular mechanism of apoptosis induced via p53-dependent pathway. We provide lines of evidence that a p53-dependent stress, doxorubicin (Doxo) administration, causes accumulation of p53 in nucleus of NB cells and phosphorylation of several serine residues (serine 15, serine 20, and serine 46) in both Doxo-sensitive and -resistant NB cell lines. Up-regulation of a p53-downstream molecule, p2lCip 1/Waf1 and accumulation of Noxa, a pro-apoptotic Bcl-2 family molecule, in mitochondria are observed in only Doxo-sensitive NB cells. Mitochondrial dysfunction including cytochrome c release and membrane potential dysregulation was caused in the sensitive cells, resulting in the activation of the intrinsic caspase pathway. However, the accumulation in nucleus and phosphorylation of p53 were inert to induce the Noxa accumulation and the mitochondrial dysfunction in p53-resistant NB cells. Surprisingly, inactivation of the p53 by over-expression of MDM2 resulted in the canceling of up-regulation of Noxa in mitochondria and reduced the apoptotic cell death. Taken together, MDM2/p53 pathway seems to play an important role in NB cells by Noxa regulation in mitochondria. Less