PEGylated dendritic polyglycerol conjugate targeting NCAM-expressing neuroblastoma: Limitations and challenges

Neural cell adhesion molecule (NCAM) is found to be a stem-cell marker in several tumor types and its overexpression is known to correlate with increased metastatic capacity. To combine extravasation- and ligand-dependent targeting to NCAM overexpressing-cells in the tumor microenvironment, we developed a PEGylated NCAM-targeted dendritic polyglycerol (PG) conjugate. Here, we describe the synthesis, physico-chemical characterization and biological evaluation of a PG conjugate bearing the mitotic inhibitor paclitaxel (PTX) and an NCAM-targeting peptide (NTP). PG-NTP-PTX-PEG was evaluated for its ability to inhibit neuroblastoma progression in vitro and in vivo as compared to non-targeted derivatives and free drug. NCAM-targeted conjugate inhibited the migration of proliferating endothelial cells, suggesting it would be able to inhibit tumor angiogenesis. The targeting conjugate provided an improved binding and uptake on IMR-32 cells compared to non-targeted control. However, these results did not translate to our in vivo model on orthotopic neuroblastoma bearing mice.     

MYCN Directly Targets NeuroD1 to Promote Cellular Proliferation in Neuroblastoma

NeuroD1 is a neuronal differentiation factor that contains a basic helix–loop–helix (bHLH) motif. Recently, NeuroD1 was found to be associated with tumorigenesis in neuroblastoma (NB) and is known to promote cell proliferation and migration in these cells. Here we found that MYCN regulates the expression of NeuroD1 in NB cells and that the downregulation of MYCN using short hairpin RNAs (shRNA) results in the inhibition of cellular proliferation in NB cells. Moreover, the phenotype induced by MYCN shRNA was rescued by the exogenous expression of NeuroD1. Chromatin immunoprecipitation (ChIP) assay showed that MYCN directly binds to the E-box element in the NeuroD1 promoter region. In addition, our evaluation of two clinical databases showed that there was a positive correlation between the expression of MYCN and NeuroD1 in NB patients, which supports our in vitro data. In conclusion, this study demonstrates that MYCN-regulated NeuroD1 expression is one of the important mechanisms underlying enhanced cellular proliferation induced by the increase in MYCN expression in NB, and our results provide an important therapeutic target for NB in the future.     

Identifying altered gene expression in neuroblastoma cells preceding apoptosis

Purpose Concomitant differentiation and partial inhibition of proteasome trigger cell death in a neuroblastoma cell line (NBP2). Neither induction of differentiation nor partial inhibition of proteasome alone affects the viability of NBP2 cells. We wanted to identify genes whose expression alters under concomitant conditions and may account for cell death. Methods We used gel electrophoresis to analyze total genomic DNA for the detection of DNA fragmentation. Affymetrix Murine Genome U74A version 2 microarray was used to screen for ∼6,000 functionally characterized genes and ∼6,000 expressed sequence tags (ESTs). Real time PCR (RT-PCR) was performed to provide an accurate assessment of changes in gene expression. Results Concomitant differentiation and partial inhibition of proteasome trigger apoptosis, characterized by genomic DNA fragmentation in NBP2 cells. We found that the expression of 41 genes changed 2.5-fold or more primarily under concomitant conditions midway through apoptosis. Based on real time PCR, the expression of galectin-3, glycosylated 96, a leucine zipper protein (LRG-21), and endothelial cell activated protein C receptor (EPCR) increased between 50–500-fold, whereas the expression of Polo serine/threonine kinase, N-myc, and Histone H2A.1 decreased ranging from 8 to 37 fold. Altered expression of galectin-3, EPCR, and LRG-21 was detected as early as 2–8 h post simultaneous conditions. Conclusion We identified new genes that might be involved in apoptotic events in neuroblastoma cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

GM-CSF-tk融合基因转导人神经母细胞瘤株SH-SY5Y的抗瘤作用

目的:将单纯疱疹病毒胸苷激酶和粒细胞-单核细胞集落刺激因子融合基因(Lxpsp-hGM-CSF-Hytk)转导人恶性神经母细胞瘤株SH-SY5Y,观察外源融合基因的表达及其对肿瘤细胞的杀伤作用.方法:FuGENE6介导将含融合基因的逆转录病毒表达载体和仅含潮霉素磷酸转移酶基因的对照载体分别转染逆转录病毒包装细胞系PA317.以适量病毒上清转导SH-SY5Y,RT-PCR方法检测融合基因的存在;细胞因子依赖株TF-1的生长状况及丙氧鸟苷(GCV)杀伤实验鉴定融合基因的表达.结果:将重组病毒产生细胞PA317-GM-CSF-Hytk和PA317-Hy的病毒上清2ml分别转导SH-SY5Y,60 mg·L-1的潮霉素进行筛选,得到了稳定传代的阳性细胞集落SY5Y-GMHytk,SY5Y-Hy.RT-PCR检测阳性细胞集落中有外源的GM-CSF存在.用阳性细胞集落上清培养TF-1细胞,细胞成活.SY5Y,SY5Y-GMHytk,SY5Y-Hy 3种细胞的生长曲线无明显差别,但用不同浓度GCV分别作用于此3种细胞显示:0.03～300 mg·L-1的GCV对SY5Y-GMHytk有明显的杀伤作用(IC50=3 mg·L-1),而对另外两种细胞几乎无毒性作用(IC50＞300 mg·L-1).30 mg·L-1GCV作用96 h后可杀死80%以上的SY5Y-GMHytk细胞.结论:应用逆转录病毒介导,将融合基因GMCSF-tk成功地导入了SH-SY5Y肿瘤细胞株,并观察到外源融合基因的表达及HSV-tk/GCV体系对肿瘤细胞的杀伤作用.     

Ⅲ、Ⅳ期神经母细胞瘤外科治疗现状

目的：探讨Ⅲ、Ⅳ期神经母细胞瘤术前强化疗配合外科手术治疗效果及预后关系。方法：２４例手术病例（其中Ⅲ期１１例、Ⅳ期１３例）,完全切除肿瘤１７例,术前均经过化疗３～４疗程,动态观察肿瘤平均缩小率为１３．３％～９４．０％。结果：平均生存期２１个月,其中４例无瘤生存３～４年。结论：应用强化疗后延期手术,对Ⅲ期病例的完整切除,Ⅳ期病例切除原发灶,降低手术并发症,提高生存率有重要意义。     

The association between neuroblastoma and opsoclonus-myoclonus syndrome: a historical review

An association between neuroblastoma and opsoclonus-myoclonus syndrome (OMS) was described as early as 1927 within the first report on the transformation of malignant neuroblastoma to a benign ganglioneuroma. It was not recognized at that time nor was it appreciated in the subsequent follow-up report on the same patient in 1959. Myoclonic encephalopathy of infancy, an alternative name for OMS, was described by a pediatric neurologist in 1962; however, its connection to neuroblastoma was not known. It was only in 1968 that the association between these two conditions was first reported. The neuroblastoma tumors associated with OMS are almost all small, stage I–II with no associated MYCN amplification or metastases. OMS occurs in 2–3% of patients with neuroblastoma, but neuroblastoma is found in as many as 50% of children who present with OMS. Nearly 100% of the children with neuroblastoma associated with OMS survive, and this has led to speculation that the OMS is a result of an autoimmune process, not metastases. Affected children are treated with steroids, ACTH, or intravenous immunoglobulin, but many have persistent neurologic and developmental deficits. Using the original case reported in 1927, we summarize a century of literature in this review on OMS and its association with neuroblastoma.     

儿童神经母细胞瘤颅面骨转移的影像表现

目的 探讨儿童神经母细胞瘤颅面骨转移的影像表现.方法 回顾性分析12例经组织学证实的儿童神经母细胞瘤的影像表现.其中 10例患儿行CT平扫,6例行MRI,7例行全身SPECT骨扫描.结果 行CT检查的10例患儿中,9例表现为颅面骨溶骨性骨质破坏伴软组织肿块,其中8例可见骨膜反应,3例表现为特征性的针状骨膜反应;另l例cT未发现异常但骨扫描显示异常.行MR检查的6例患儿均表现为颅面骨骨髓腔信号异常伴周围软组织肿块,其中5例行增强扫描,骨髓腔异常信号影和软组织肿块呈明显不均匀强化.7例行全身骨扫描,均可见颅面部放射性浓聚区,其巾6例伴有全身其他部位的骨转移.结论神经母细胞瘤颅面骨转移有一定的影像特征,可提示诊断.     

Pharmacokinetics of oral fenretinide in neuroblastoma patients: indications for optimal dose and dosing schedule also with respect to the active metabolite 4-oxo-fenretinide

PURPOSE: Pharmacokinetic data on fenretinide (4-HPR) are scant, thus limiting the rational use of the drug. We investigated the pharmacokinetics of 4-HPR and its active metabolite 4-oxo-fenretinide (4-oxo-4-HPR). EXPERIMENTAL DESIGN: Pharmacokinetics were assessed in 18 children (3 for each dose) with neuroblastoma who received oral 4-HPR once daily for 28 days at the doses of 100, 300, 400, 600, 1,700 and 4,000 mg/m(2)/day. 4-HPR and 4-oxo-4-HPR were determined by HPLC in plasma collected up to 48 h after the first and 28th administration. RESULTS: After single administration, 4-HPR mean C (max) ranged from 0.9 to 6.6 muM and these concentrations roughly doubled at steady state (range 1.6-14.5 muM). 4-HPR mean t (1/2) was 22 h. 4-HPR pharmacokinetics were linear in the dose range 100-1,700 mg/m(2); less than dose-proportional increase in exposure was found at 4,000 mg/m(2). At steady state, pharmacologically relevant plasma concentrations (range 0.7-10 muM and 0.4-5 muM for 4-HPR and 4-oxo-4-HPR, respectively) were maintained during the 24 h dosing interval in the dose range 300-4,000 mg/m(2). CONCLUSIONS: 4-HPR pharmacokinetics supports once-daily dosing. Steady state concentrations of 4-HPR and 4-oxo-4-HPR in children with neuroblastoma are in line with those found to have in vitro growth inhibitory effects in neuroblastoma cells.