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

神经母细胞瘤(NB)是儿童最常见的交感神经系统实体恶性肿瘤。与许多儿童恶性肿瘤存活率的提高不同，高危肺母细胞瘤仍然是最难治愈的肿瘤之一，尽管进行了密集的多模式治疗，但长期存活率只有30%。为了提高存活率，需要新的治疗方法和对耐药机制的更好理解。神经母细胞瘤研究的一个值得注意的发现是，在340例被检测的NBS中，报告的p53肿瘤抑制基因突变不到2%(TwedleDA等人，癌症资源61：8-13,2001)。P53的细胞质隔离被认为是NB细胞失活的另一种机制，而不是突变。首先使用免疫组织化学技术(Moll UM等人，PNAS92：4407-11,1995)在冷冻的肿瘤样本中报告了隔离，后来通过免疫荧光和细胞分离实验…在NB油系中报告了隔离更多S(莫尔·乌姆等人，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，与P53结合的共识序列在体内外的结合，以及P53下游分子在Nb细胞中的上调仍未确定。因此，我们利用一组细胞系来确定Nb细胞中P53依赖的细胞死亡是否由细胞功能凋亡引起，并进一步研究了P53依赖途径诱导细胞凋亡的分子机制。我们提供了一系列证据表明，依赖于P53的压力，阿霉素(Doxo)给药，导致Nb细胞核中P53的积聚，以及在Doxo敏感和耐药的Nb细胞系中几个丝氨酸残基(丝氨酸15、丝氨酸20和丝氨酸46)的磷酸化。P53下游分子p21Cip1/Waf1的上调和促凋亡的Bcl2家族分子Noxa在线粒体中的积聚仅在对Doxo敏感的NB细胞中观察到。敏感细胞线粒体功能障碍，包括细胞色素c释放和膜电位失调，导致caspase内源性通路激活。然而，P53在细胞核内的积聚和P53的磷酸化是惰性的，可以诱导P53耐药的NB细胞NOXA积聚和线粒体功能障碍。令人惊讶的是，MDM2的过度表达使P53失活，导致线粒体中Noxa的上调被取消，并减少了细胞的凋亡。综上所述，MDM2/P53通路可能通过调控线粒体中的NoxA而在NB细胞中发挥重要作用。较少

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