BACKGROUND & AIMS: :Circumvention of the p53 checkpoint in neuroblastoma (NB) might arise from increased expression of its main negative regulator MDM2. The SNP309, a T-to-G substitution in the MDM2 promoter, was associated with higher levels of MDM2 mRNA and protein, with consequent attenuation of the p53 pathway. The association between MDM2 SNP309 and disease progression and survival was evaluated in a cohort of 142 children with stage 4 NB. The SNP309 GG patients had a worse overall survival and a worse survival after relapse than the TT ones, whereas the heterozygotes showed an intermediate behaviour (p=0.043 and p=0.049, respectively, log-rank test for trend). No evident association between SNP309 and event free survival was found. The lack of association between SNP309 and MYCN status indicates that MDM2 SNP309 may be a new independent prognostic factor for stage 4 NB.

背景与目标： ：在神经母细胞瘤（NB）中p53检查点的规避可能是由于其主要的负调节因子MDM2的表达增加所致。 SNP309是MDM2启动子中的T-to-G取代，与更高水平的MDM2 mRNA和蛋白质有关，因此减弱了p53途径。在142名4级NB儿童中评估了MDM2 SNP309与疾病进展和生存之间的关联。 SNP309 GG患者的总体生存率和复发后生存率均比TT差，而杂合子表现出中等的行为（趋势的对数检验分别为p = 0.043和p = 0.049）。没有发现SNP309与无事件生存之间的明显关联。 SNP309和MYCN状态之间缺乏关联性表明MDM2 SNP309可能是4期NB的新的独立预后因素。

BACKGROUND & AIMS: :Secondary expansion and/or evolution of aggressive subclones are associated with the disease progression and resistance to chemotherapy in neuroblastoma, and it is important to track the clonal changes during the treatment period. Cell-free (cf) DNA analysis, namely liquid biopsy, can detect the genomic change of tumor cells without surgical procedures. In this report, we showed that serial polymerase chain reaction-based cf DNA neuroblastoma proto-oncogene quantification is sensitive enough to evaluate the aggressive cellular characteristics of ALK/MYCN-coamplified neuroblastoma and stressed the promise of cf DNA analyses as a reliable molecular marker in advanced neuroblastoma.

背景与目标： ：侵袭性亚克隆的继发扩增和/或进化与神经母细胞瘤的疾病进展和对化疗的耐药性有关，因此追踪治疗期间的克隆变化非常重要。无细胞（cf）DNA分析（即液体活检）无需手术即可检测肿瘤细胞的基因组变化。在本报告中，我们显示了基于连续聚合酶链反应的cf DNA神经母细胞瘤原癌基因定量方法足够灵敏，可以评估ALK / MYCN扩增的神经母细胞瘤的侵袭性细胞特征，并强调了cf DNA分析作为一种可靠的分子标记的前景。晚期神经母细胞瘤。

BACKGROUND & AIMS: OBJECTIVE:The objective of this study was to evaluate the overall diagnostic value of PET(CT) in patients with neuroblastoma (NB) based on qualified studies. METHODS:PubMed, Cochrane, and Embase database were searched by the index words to identify the qualified studies, and relevant literature sources were also searched. The latest research was performed in April 2019. Heterogeneity of the included studies was tested, which was used to select proper effect model to calculate pooled weighted sensitivity, specificity, and diagnostic odds ratio (DOR). Summary receiver operating characteristic (SROC) analyses were also performed. RESULTS:Eleven studies with 580 patients were involved in the meta-analysis to explore the diagnostic accuracy of PET(CT) for NB. PET(CT) has high diagnostic accuracy of NB: the global sensitivity was 91% (95% confidence interval [CI], 86%-94%), the global specificity was 78% (95% CI, 66%-86%), the global positive likelihood ratio was 4.07 (95% CI, 2.54-6.50), the global negative likelihood ratio was 0.12 (95% CI, 0.08-0.18), the global DOR was 27.43 (95% CI, 14.45-52.07), and the area under the SROC was high (area under the curve, 0.93; 95% CI, 0.90-0.95). Besides this, PET(CT) has high diagnostic accuracy of primary NB: the global sensitivity was 86% (95% CI, 73%-93%), the global specificity was 82% (95% CI, 57%-94%), the global positive likelihood ratio was 4.90 (95% CI, 1.63-14.72), the global negative likelihood ratio was 0.17 (95% CI, 0.07-0.40), the global DOR was 25.427 (95% CI, 3.988-162.098), and the area under the SROC was high (area under the curve, 0.91; 95% CI, 0.88-0.93). However, there has no significant accuracy of PET(CT) in NB with bone marrow. CONCLUSIONS:This study provides a systematic review and meta-analysis of diagnostic accuracy studies of PET(CT) for NB. The results indicated that PET(CT) is a highly accurate diagnostic tool for NB.

背景与目标： 目的：本研究的目的是根据合格研究评估PET（CT）对神经母细胞瘤（NB）患者的总体诊断价值。
方法：通过关键词检索PubMed，Cochrane和Embase数据库，以鉴定合格的研究，并检索相关文献来源。最新研究于2019年4月进行。对纳入研究的异质性进行了测试，用于选择适当的效应模型以计算合并的加权敏感性，特异性和诊断比值比（DOR）。还进行了摘要接收器工作特性（SROC）分析。
结果：11项研究对580例患者进行了荟萃分析，以探讨PET（CT）对NB的诊断准确性。 PET（CT）对NB的诊断准确性很高：总体敏感性为91％（95％置信区间[CI]，86％-94％），总体特异性为78％（95％CI，66％-86％） ，全局正似然比为4.07（95％CI，2.54-6.50），全局负似然比为0.12（95％CI，0.08-0.18），全局DOR为27.43（95％CI，14.45-52.07）， SROC下的面积较高（曲线下的面积为0.93； 95％CI为0.90-0.95）。除此之外，PET（CT）对原发性NB的诊断准确性很高：整体敏感性为86％（95％CI，73％-93％），整体特异性为82％（95％CI，57％-94％） ，全局正似然比为4.90（95％CI，1.63-14.72），全局负似然比为0.17（95％CI，0.07-0.40），全局DOR为25.427（95％CI，3.988-162.098）， SROC下的面积较高（曲线下的面积为0.91； 95％CI为0.88-0.93）。但是，在患有骨髓的NB患者中，PET（CT）的准确性不高。
结论：本研究提供了对PET（CT）NB诊断准确性研究的系统评价和荟萃分析。结果表明，PET（CT）是用于NB的高度准确的诊断工具。

BACKGROUND & AIMS: :Neuroblastoma (NB) is a common intracranial solid tumor with high mortality. Small nucleolar RNA host gene 16 (SNHG16), one of the long noncoding RNAs (lncRNAs), has been reported to be linked to the poor prognosis of NB. However, the mechanisms of SNHG16 in regulating NB progression remain poorly understood. The expression level of SNHG16 was measured by quantitative real time polymerase chain reaction (qRT-PCR). The starBase was employed to predict the interaction of miR-128-3p and SNHG16 or HOXA7, which was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Cell proliferation and apoptosis were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry, respectively. Transwell assay was used to detect cell invasion or migration. The mRNA and protein levels of homeobox protein A7 (HOXA7) were determined by qRT-PCR and western blot, respectively. The levels of SNHG16 and HOXA7 were conspicuously increased in NB tissues and cells, while the expression of miR-128-3p was obviously declined, compared with corresponding normal tissues and cells. SNHG16 silencing inhibited proliferation, migration and invasion and induced apoptosis of NB cells. We identified that SNHG16 directly interacted with miR-128-3p, and miR-128-3p could target the 3'UTR of HOXA7 in NB cells. Simultaneously, miR-128-3p expression was negatively associated with SNHG16 or HOXA7. Further studies indicated that SNHG16 overexpression rescued the effects of miR-128-3p-mediated on inhibiting proliferation, migration, invasion and promoting apoptosis of NB cells. Moreover, SNHG16 could modulate HOXA7 by sponging miR-128-3p in NB cells. Besides, SNHG16 silencing suppressed tumor growth in vivo. Knockdown of SNHG16 impeded proliferation, migration, invasion and induced apoptosis through the SNHG16/miR-128-3p/HOXA7 axis in NB cells.

背景与目标： 神经母细胞瘤（NB）是常见的颅内实体瘤，死亡率高。小核仁RNA宿主基因16（SNHG16），长的非编码RNA（lncRNAs）之一，据报道与NB的不良预后有关。但是，尚不清楚SNHG16调节NB进程的机制。通过定量实时聚合酶链反应（qRT-PCR）测量SNHG16的表达水平。使用starBase预测miR-128-3p与SNHG16或HOXA7的相互作用，这已通过双荧光素酶报告基因检测和RNA免疫沉淀（RIP）检测得到验证。细胞增殖和凋亡分别通过3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物（MTT）和流式细胞仪进行评估。 Transwell测定法用于检测细胞侵袭或迁移。同源盒蛋白A7（HOXA7）的mRNA和蛋白水平分别通过qRT-PCR和western blot测定。与相应的正常组织和细胞相比，NB组织和细胞中SNHG16和HOXA7的含量明显增加，而miR-128-3p的表达则明显下降。 SNHG16沉默抑制NB细胞的增殖，迁移和入侵，并诱导其凋亡。我们确定SNHG16直接与miR-128-3p相互作用，而miR-128-3p可以靶向NB细胞中HOXA7的3'UTR。同时，miR-128-3p表达与SNHG16或HOXA7负相关。进一步的研究表明，SNHG16的过表达挽救了miR-128-3p介导的抑制NB细胞增殖，迁移，侵袭和促进细胞凋亡的作用。此外，SNHG16可以通过在NB细胞中海绵化miR-128-3p来调节HOXA7。此外，SNHG16沉默抑制体内肿瘤的生长。敲低SNHG16阻碍了NB细胞中SNHG16 / miR-128-3p / HOXA7轴的增殖，迁移，侵袭并诱导了细胞凋亡。

BACKGROUND & AIMS: BACKGROUND:Gangliosicle GD2 is abundant on human neuroblastoma (NB). Monoclonal antibody 3F8 targeted to GD2 may have imaging and therapeutic potential. Antigen-negative clones can escape immune-mediated attack leading to clinical resistance or recurrence. PROCEDURE:Among 95 evaluable patients treated intravenously with 3F8 (94 Stage 4, 1 Stage 3), 66 received nonradiolabeled 3F8, 11 received 131-iodine-labeled-3F8 (8-28 mCi/kg) with autologous bone marrow rescue, and 18 received both forms of treatment. Prior to treatment, 90 patients tested positive for GD2 reactivity by bone marrow immunofluorescence (n = 68), tumor immunohistochemistry (n = 20), or diagnostic radioimmunoscintigraphy (n = 2). RESULTS:Of 62 patients who had refractory or recurrent neuroblastoma following 3F8 treatment, 61 (98%) tested positive for GD2 reactivity by bone marrow immunofluorescence (n = 51) or tumor immunohistochemistry (n = 10). The sole tumor that lost GD2 expression underwent phenotypic transformation into a pheochromocytoma-like tumor. CONCLUSIONS:The persistence of GD2 expression in refractory or recurrent NB suggests that complete antigen loss is an uncommon event and cannot account for treatment failure.

背景与目标： 背景：神经节GD2在人神经母细胞瘤（NB）中含量丰富。靶向GD2的单克隆抗体3F8可能具有成像和治疗潜力。抗原阴性克隆可以逃脱免疫介导的攻击，从而导致临床耐药或复发。
程序：在95例可评估的患者中，静脉注射3F8（94阶段4，1阶段3），66例接受了非放射性标记的3F8，11例接受了131碘标记的3F8（8-28 mCi / kg）的自体骨髓拯救术，18例接受了两种形式的治疗。在治疗之前，有90名患者通过骨髓免疫荧光检查（n = 68），肿瘤免疫组织化学检查（n = 20）或诊断性放射免疫闪烁照相术（n = 2）检测出GD2反应阳性。
结果：62例3F8治疗后患有难治性或复发性神经母细胞瘤的患者中，有61例（98％）通过骨髓免疫荧光（n = 51）或肿瘤免疫组织化学（n = 10）检测为GD2反应阳性。唯一丢失GD2表达的肿瘤发生了表型转化为嗜铬细胞瘤样肿瘤。
结论：难治性或复发性NB中GD2表达的持续性提示完全抗原丢失是罕见的事件，不能解释治疗失败的原因。

BACKGROUND & AIMS: BACKGROUND:Intracellular signaling responsible for gastrin-releasing peptide (GRP) receptor-mediated neovascularization is not clearly understood. We sought to determine the cellular mechanisms involved in the GRP receptor regulation of vascular endothelial growth factor (VEGF) release in neuroblastoma cells. MATERIALS AND METHODS:BE(2)-C cells were treated with bombesin (BBS), the amphibian equivalent of GRP, Phorbol myristate acetate (PMA) a PKC agonist, or GF109293X (GFX), and analyses were performed for VEGF secretion, phosphorylated protein kinase B (AKT), extracellular signal-regulated kinases (ERK) and protein kinase D (PKD) expression. RESULTS:BBS rapidly increased VEGF secretion at 30 min. Pre-treatment with PMA alone produced similar results; this effect was synergistic with the addition of GRP. Conversely, GFX blocked PMA-stimulated increase in VEGF secretion. Immunofluorescent staining for VEGF correlated to BBS, PMA and GFX. CONCLUSION:PKC is critically responsible for rapid VEGF secretion by GRP receptor signaling in neuroblastoma cells. Inhibition of VEGF significantly reduced GRP-mediated cell proliferation, suggesting its crucial role in neuroblastoma tumorigenesis.

背景与目标： 背景：负责胃泌素释放肽（GRP）受体介导的新血管形成的细胞内信号传递尚不清楚。我们试图确定参与神经母细胞瘤细胞中血管内皮生长因子（VEGF）释放的GRP受体调节的细胞机制。
材料与方法：将BE（2）-C细胞用Bombesin（BBS），GRP的两栖类等效物，PKC激动剂或肉豆蔻酸肉豆蔻酸酯（PMA）或GF109293X（GFX）处理，并进行VEGF分泌，磷酸化的分析蛋白激酶B（AKT），细胞外信号调节激酶（ERK）和蛋白激酶D（PKD）的表达。
结果：BBS在30分钟时迅速增加了VEGF的分泌。仅用PMA进行预处理可获得相似的结果。该效果与添加GRP协同作用。相反，GFX阻止了PMA刺激的VEGF分泌增加。 VEGF的免疫荧光染色与BBS，PMA和GFX相关。
结论：PKC是神经母细胞瘤细胞中GRP受体信号传导快速分泌VEGF的关键原因。 VEGF的抑制显着降低了GRP介导的细胞增殖，表明其在神经母细胞瘤肿瘤发生中的关键作用。

BACKGROUND & AIMS: :Neuroblastoma is a pediatric malignancy with a poor prognosis at least partly attributable to an early pattern of dissemination. New approaches to treatment of micrometastases include targeted radiotherapy using radiolabeled antibodies or molecules which are taken up preferentially by tumor cells. Multicellular tumor spheroids (MTS) resemble micrometastases during the avascular phase of their development. A human neuroblastoma cell line (NBl-G) was grown as MTS and incubated briefly with a radiolabeled monoclonal antibody (131I-UJ13A) directed against neuroectodermal antigens. Spheroid response was evaluated in terms of regrowth delay or proportion sterilized. A dose-response relationship was demonstrated in terms of 131I activity or duration of incubation. Control experiments using unlabeled UJ13A, radiolabeled nonspecific antibody (T2.10), radiolabeled human serum albumin, and radiolabeled sodium iodide showed these to be relatively ineffective compared to 131I-UJ13A. The cell line NBl-G grown as MTS has also been found to preferentially accumulate the radiolabeled catecholamine precursor molecule m-[131I]iodobenzylguanidine compared to cell lines derived from other tumor types. NBl-G cells grown as MTS provide a promising laboratory model for targeted radiotherapy of neuroblastoma micrometastases using radiolabeled antibodies or m-iodobenzylguanidine.

背景与目标： ：神经母细胞瘤是一种儿童恶性肿瘤，预后较差，至少部分归因于早期传播。治疗微转移的新方法包括使用放射标记抗体或分子优先被肿瘤细胞吸收的分子进行靶向放射治疗。多细胞肿瘤球（MTS）在其发展的无血管阶段类似于微转移。将人神经母细胞瘤细胞系（NB1-G）生长为MTS，并与针对神经外胚层抗原的放射性标记单克隆抗体（131I-UJ13A）短暂孵育。根据再生延迟或灭菌比例评估球体反应。通过131I活性或孵育持续时间证明了剂量-反应关系。使用未标记的UJ13A，放射性标记的非特异性抗体（T2.10），放射性标记的人血清白蛋白和放射性标记的碘化钠进行的对照实验显示，与131I-UJ13A相比，它们相对无效。与衍生自其他肿瘤类型的细胞系相比，还发现以MTS形式生长的细胞系NB1-G优先积累放射性标记的儿茶酚胺前体分子m- [131I]碘代苄基胍。生长为MTS的NB1-G细胞为使用放射标记抗体或间碘苄基胍的神经母细胞瘤微转移靶向放疗提供了有希望的实验室模型。

BACKGROUND & AIMS: :Neuroblastoma (NB) is a type of tumor usually found in children under 5 years of age, which originates from lesions in the nervous system and has fast growth and early transformation characteristics. Similar to other cancer types, some typical tumor suppressor genes (TSGs), such as P53 and CHD5 are silenced in NB because of high methylation at promoter zones. In the present study, our results showed that genistein, an element found in soy, is an epigenetic modifier able to decrease hypermethylation levels of CHD5, and enhances the expression of CHD5 as well as p53, possibly contributing to inhibition of NB growth in vivo and tumor microvessel formation. Furthermore, genistein acts as a DNA methyltransferase (DNMT) inhibitor to significantly decrease the expression of DNMT3b. Our study indicates that genistein plays an important role in inhibiting NB growth in vivo, probably preventing tumorigenesis risk as a kind of therapeutic agent for NB treatment in the future.

背景与目标： 神经母细胞瘤（NB）是一种常见于5岁以下儿童的肿瘤，起源于神经系统病变，具有快速生长和早期转化的特征。与其他类型的癌症类似，由于启动子区域的甲基化程度较高，一些典型的肿瘤抑制基因（TSG）（例如P53和CHD5）在NB中被沉默。在本研究中，我们的结果表明，染料木黄酮（一种在大豆中发现的元素）是一种表观遗传修饰剂，能够降低CHD5的超甲基化水平，并增强CHD5和p53的表达，可能有助于抑制体内NB的生长。肿瘤微血管形成。此外，金雀异黄素起着DNA甲基转移酶（DNMT）抑制剂的作用，可显着降低DNMT3b的表达。我们的研究表明，染料木黄酮在体内抑制NB的生长中起着重要的作用，有可能作为未来NB治疗的一种治疗剂来预防肿瘤发生的风险。

BACKGROUND & AIMS: :Neuroblastoma (NB) is the most common pediatric neuroendocrine tumor, and it is characterized by a quite variable clinical course. We previously found a great variability in the expression of somatostatin receptor type 2 (sst2) in several human NB cell lines and primary tumors. In this report we investigated whether expression of sst2 is somehow related to clinical outcome. We performed a retrospective study on 54 patients with a maximum follow-up of 100 months. The concentration of specific messenger ribonucleic acid (mRNA) for sst2 was measured by competitive RT-PCR and validated, in a small subset of samples, by quantitative imaging of gene (in situ hybridization) and protein (immunohistochemistry) expression. We found that sst2 mRNA was variably expressed in all NB tumors (range, 2.5 x 10(5) to 8 x 10(9) molecules/microg RNA) with a relevant reduction in the more advanced stage (P < 0.01). Analysis of Kaplan-Meier curves indicated that sst2 expression is positively related to the overall (P < 0.0001) and event-free (P < 0.0001) survival. Expression of sst2 was negatively related to tumor stage (P < 0.02) and MYCN amplification (P < 0.001), a poor prognostic factor. However, the prognostic information derived from sst2 is apparently independent from MYCN amplification, as assessed by stratifying sst2 values according to MYCN. In addition, the expression of sst2 was the only significant prognostic factor (P < 0.02) when it was included in a multivariate model containing other well known prognostic factors such as age, stage, and MYCN amplification. Hence, we propose that sst2 expression represents a new prognostic marker for NB. The main clinical value of a quantitative measure of sst2 lies in its ability to detect patients at low risk, independently from other prognostic factor, including MYCN amplification.

背景与目标： ：神经母细胞瘤（NB）是最常见的小儿神经内分泌肿瘤，其特点是临床过程变化很大。我们先前发现生长抑素受体2型（sst2）在几种人NB细胞系和原发性肿瘤中的表达存在很大差异。在本报告中，我们调查了sst2的表达是否与临床结果相关。我们对54例患者进行了回顾性研究，最大随访时间为100个月。通过竞争性RT-PCR测量sst2的特定信使核糖核酸（mRNA）的浓度，并在一小部分样品中通过定量成像基因（原位杂交）和蛋白质（免疫组织化学）进行验证。我们发现sst2 mRNA在所有NB肿瘤中均可变表达（范围为2.5 x 10（5）至8 x 10（9）分子/ microg RNA），并且在更晚期阶段有相关的减少（P <0.01）。 Kaplan-Meier曲线的分析表明sst2表达与总体生存期（P <0.0001）和无事件生存期（P <0.0001）正相关。 sst2的表达与肿瘤分期（P <0.02）和MYCN扩增（P <0.001）呈负相关，这是不良的预后因素。但是，从sst2得出的预后信息显然与MYCN扩增无关，如通过根据MYCN分层sst2值所评估的。此外，当sst2的表达包含在包含其他众所周知的预后因素（例如年龄，阶段和MYCN扩增）的多变量模型中时，它是唯一的重要预后因素（P <0.02）。因此，我们建议sst2表达代表NB的新的预后标志物。定量检测sst2的主要临床价值在于其能够独立于其他预后因素（包括MYCN扩增）检测低危患者。

BACKGROUND & AIMS: :Carbachol-mediated activation of type M(3) muscarinic acetylcholine receptors induces the biosynthesis of the transcription factor Egr-1 in human SH-SY5Y neuroblastoma cells involving an activation of extracellular signal-regulated protein kinase. Carbachol triggered the phosphorylation of the ternary complex factor Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, and strikingly enhanced the transcriptional activation potential of Elk-1. Chromatin immunoprecipitation experiments revealed that Elk-1 binds in vivo to the 5'-upstream region of the Egr-1 gene in carbachol-stimulated neuroblastoma cells. Together, these data indicate that Elk-1 connects the intracellular signaling cascade elicited by activation of M(3) muscarinic acetylcholine receptors with the transcription of the Egr-1 gene. Lentiviral-mediated expression of either MAP kinase phosphatase-1 (MKP-1) or a constitutively active mutant of calcineurin A inhibited Egr-1 biosynthesis following carbachol stimulation, indicating that these phosphatases function as shut-off devices of muscarinic acetylcholine receptor signaling. Additionally, carbachol stimulation increased transcription of a chromatin-embedded collagenase promoter/reporter gene, showing that AP-1 activity is enhanced in carbachol-stimulated neuroblastoma. Expression experiments revealed that both MKP-1 and a constitutively active mutant of calcineurin A impaired carbachol-induced upregulation of AP-1 activity. The fact that carbachol stimulation of neuroblastoma cells activates the transcription factors Egr-1 and AP-1 suggests that changes in the gene expression pattern are an integral part of muscarinic acetylcholine receptor signaling.

背景与目标： ：Carbachol介导的M（3）型毒蕈碱乙酰胆碱受体的激活诱导人SH-SY5Y神经母细胞瘤细胞中转录因子Egr-1的生物合成，涉及细胞外信号调节蛋白激酶的激活。卡巴胆碱触发三元复合因子Elk-1的磷酸化，这是血清反应元件驱动的基因转录的关键转录调节因子，并显着增强了Elk-1的转录激活潜能。染色质免疫沉淀实验表明，Elk-1在体内与卡巴胆碱刺激的神经母细胞瘤细胞中Egr-1基因的5'上游区域结合。在一起，这些数据表明Elk-1连接通过Egr-1基因的转录激活M（3）毒蕈碱乙酰胆碱受体引起的细胞内信号传导级联。 MAP激酶磷酸酶-1（MKP-1）或钙调神经磷酸酶A的组成型活性突变体的慢病毒介导的表达在卡巴胆碱刺激后抑制了Egr-1的生物合成，表明这些磷酸酶起着毒蕈碱性乙酰胆碱受体信号转导的关闭装置的作用。此外，卡巴胆碱刺激增加了染色质嵌入的胶原酶启动子/报告基因的转录，表明AP-1活性在卡巴胆碱刺激的神经母细胞瘤中得到增强。表达实验表明，MKP-1和钙调神经磷酸酶A的组成型活性突变体均会破坏卡巴胆碱诱导的AP-1活性上调。卡巴胆碱刺激神经母细胞瘤细胞激活转录因子Egr-1和AP-1的事实表明，基因表达模式的改变是毒蕈碱型乙酰胆碱受体信号传导的组成部分。

BACKGROUND & AIMS: :According to their sugar recognition specificity, plant lectins are proposed as bioactive proteins with potential in cancer treatment and diagnosis. Helja is a mannose-specific jacalin-like lectin from sunflower which was shown to inhibit the growth of certain fungi. Here, we report its recombinant expression in a prokaryotic system and its activity in neurobalstoma cells. Helja coding sequence was fused to the pET-32 EK/LIC, the enterokinase/Ligation-independent cloning vector and a 35 kDa protein was obtained in Escherichia coli representing Helja coupled to thioredoxin (Trx). The identity of this protein was verified using anti-Helja antibodies. This chimera, named Trx-rHelja, was enriched in the soluble bacterial extracts and was purified using Ni+2-Sepharose and d-mannose-agarose chromatography. Trx-rHelja and the enterokinase-released recombinant Helja (rHelja) both displayed toxicity on human SH-SY5Y neuroblastomas. rHelja decreased the viability of these tumor cells by 75% according to the tetrazolium reduction assay, and microscopic analyses revealed that the cell morphology was disturbed. Thus, the stellate cells of the monolayer became spheroids and were isolated. Our results indicate that rHelja is a promising tool for the development of diagnostic or therapeutic methods for neuroblastoma cells, the most common solid tumors in childhood.

背景与目标： ：根据其糖识别特异性，提出了植物凝集素作为具有癌症治疗和诊断潜力的生物活性蛋白。 Helja是一种来自向日葵的甘露糖特异性贾卡林样凝集素，被证明可以抑制某些真菌的生长。在这里，我们报告其在原核系统中的重组表达及其在神经原发瘤细胞中的活性。将Helja编码序列与pET-32 EK / LIC融合，肠激酶/连接独立的克隆载体，并在大肠杆菌中获得35 kDa蛋白，代表Helja与硫氧还蛋白（Trx）偶联。使用抗Helja抗体验证了该蛋白质的身份。该嵌合体名为Trx-rHelja，富含可溶细菌提取物，并使用Ni 2-Sepharose和d-甘露糖-琼脂糖层析纯化。 Trx-rHelja和肠激酶释放的重组Helja（rHelja）均对人SH-SY5Y神经母细胞瘤具有毒性。根据四唑鎓还原法，rHelja将这些肿瘤细胞的活力降低了75％，显微镜分析表明细胞形态受到干扰。因此，单层的星状细胞变成球状并被分离。我们的结果表明，rHelja是开发成神经细胞瘤细胞（儿童时期最常见的实体瘤）的诊断或治疗方法的有前途的工具。

BACKGROUND & AIMS: PURPOSE:Neuroblastoma, a common childhood tumor, remains one of the most elusive diseases to treat. To date, high-risk neuroblastoma is associated with low survival rates. To address this, novel and more effective therapeutic strategies must continue to be explored. METHODS:We employed a bioinformatics approach corroborated with in vitro and in vivo data. Samples from neuroblastoma patients were retrieved and immuno-stained for Bruton's tyrosine kinase (BTK). To evaluate its effect on cellular functions, BTK expression in SK-N-BE(2) and SH-SY5Y neuroblastoma cells was downregulated using gene silencing or inhibition with ibrutinib or acalabrutinib. Xenograft mouse models were used to investigate the in vivo role of BTK in neuroblastoma tumorigenesis. RESULTS:We found that BTK was highly expressed in primary neuroblastoma samples, preferentially in MYCN-amplified neuroblastoma cases, and was associated with a poor prognosis. Immunohistochemical staining of tissues from our neuroblastoma cohort revealed a strong BTK immunoreactivity. We also found that neuroblastoma SK-N-BE(2) and SH-SY5Y cells were sensitive to treatment with ibrutinib and acalabrutinib. Pharmacologic or molecular inhibition of BTK elicited a reduction in the migratory and invasive abilities of neuroblastoma cells, and ibrutinib considerably attenuated the neurosphere-forming ability of neuroblastoma cells. Both inhibitors showed synergism with cisplatin. In vivo assays showed that acalabrutinib effectively inhibited neuroblastoma tumorigenesis. CONCLUSIONS:From our data we conclude that BTK is a therapeutically targetable driver of neuroblastoma.

背景与目标： 目的：神经母细胞瘤，一种常见的儿童期肿瘤，仍然是最难以治疗的疾病之一。迄今为止，高危神经母细胞瘤与低存活率有关。为了解决这个问题，必须继续探索新颖和更有效的治疗策略。
方法：我们采用了一种生物信息学方法，该方法得到了体内和体外数据的证实。取自神经母细胞瘤患者的样品并对其进行布鲁顿酪氨酸激酶（BTK）免疫染色。为了评估其对细胞功能的影响，SK-N-BE（2）和SH-SY5Y神经母细胞瘤细胞中的BTK表达可通过使用基因沉默或依鲁替尼或acalabrutinib抑制而下调。使用异种移植小鼠模型研究BTK在神经母细胞瘤肿瘤发生中的体内作用。
结果：我们发现BTK在原发性神经母细胞瘤样品中高表达，优先在MYCN扩增的神经母细胞瘤病例中表达，并且与不良预后相关。来自我们神经母细胞瘤队列的组织的免疫组织化学染色显示强大的BTK免疫反应性。我们还发现神经母细胞瘤SK-N-BE（2）和SH-SY5Y细胞对依鲁替尼和acalabrutinib的治疗敏感。 BTK的药理或分子抑制作用导致神经母细胞瘤细胞的迁移和侵袭能力降低，而依鲁替尼则大大削弱了神经母细胞瘤细胞的神经球形成能力。两种抑制剂均显示出与顺铂的协同作用。体内试验表明，acalabrutinib有效抑制神经母细胞瘤的肿瘤发生。
结论：从我们的数据，我们得出结论，BTK是神经母细胞瘤的可治疗靶向的驱动程序。

BACKGROUND & AIMS: :Neuroblastoma stem cells (NSCs) can cause drug resistance and tumor recurrence. This study aimed to enhance the lytic effect of dendritic cells (DCs) co-cultured with cytokine-induced killer (CIK) cells. NSCs were obtained by suspension culture, and DC-CIK cells were loaded with extracted NSC membrane-based microparticles (MMPs) before evaluating the lytic effect of DC-CIK cells on NSCs. After inhibiting the function or expression of human leukocyte antigen-E (HLA-E) in NSCs by anti-HLA-E monoclonal antibody or siRNA, the DC-CIK cell lytic effect on NSCs was re-assessed. NSC nestin expression was high, but glial fibrillary acid protein expression and class IIIβ-tubulin-1 expression were low. Moreover, NSCs exhibited strong tumorigenic ability in nude mice. Loading DCs with NSC-derived MMPs induced the differentiation of DCs and CIK cells and enhanced the killing of NSCs by DC-CIK cells. Inhibiting the function or expression of HLA-E in NSCs further enhanced the cytolytic capability of DC-CIK cells loaded with NSC-derived MMPs. HLA-E inhibitor can enhance the killing of NSC by DC-CIK cells loaded with NSC-derived MMPs.

背景与目标： 神经母细胞瘤干细胞（NSCs）可能导致耐药性和肿瘤复发。这项研究旨在增强与细胞因子诱导的杀伤（CIK）细胞共培养的树突状细胞（DC）的裂解作用。通过悬浮培养获得NSC，并且在评估DC-CIK细胞对NSC的裂解作用之前，将提取的基于NSC膜的微粒（MMP）加载到DC-CIK细胞中。通过抗HLA-E单克隆抗体或siRNA抑制NSC中人白细胞抗原E（HLA-E）的功能或表达后，重新评估DC-CIK细胞对NSC的裂解作用。 NSC nestin表达高，但神经胶质纤维酸蛋白表达和IIIβ-tubulin-1表达低。此外，NSCs在裸鼠中显示出强大的致瘤能力。用NSC衍生的MMP加载DC诱导DC和CIK细胞分化，并增强DC-CIK细胞对NSC的杀伤力。在NSC中抑制HLA-E的功能或表达进一步增强了装载有NSC衍生MMP的DC-CIK细胞的溶细胞能力。 HLA-E抑制剂可增强装载有NSC衍生MMP的DC-CIK细胞对NSC的杀伤作用。

BACKGROUND & AIMS: :Activation of the NPFF(2) receptor reduces the inhibitory effect of opioids on the N-type Ca(2+) channel. Although this anti-opioid effect is specific for opioid receptors in neurons and tissues, it also affects NPY Y2 and alpha(2)-adrenoreceptors in undifferentiated SH-SY5Y cells stably expressing the NPFF(2) receptor. To test whether this difference could be due to the immaturity of these cells, they were differentiated to a noradrenergic neuronal phenotype with staurosporine. The differentiated cells ceased to divide and grew long, thin neurites. The inhibition of the depolarization-triggered Ca(2+) transient by activation of G(i)-coupled receptors was either unaffected (micro-opioid), increased (NPY), reduced (NPFF(2)) or lost (alpha(2)-adrenoreceptors). Following a 20 min incubation with 1DMe, the effect of DAMGO was reduced, as in undifferentiated cells, but the effect of NPY was no longer affected. Staurosporine differentiation did not modify the coupling of the micro-opioid and NPFF(2) receptors to the G(i/o) proteins. We suggest that the specificity of the effect of NPFF may not reside in the molecular mechanism of its anti-opioid activity itself but in the organization of receptors within the membrane.

背景与目标： ：激活NPFF（2）受体减少阿片类药物对N型Ca（2）通道的抑制作用。尽管这种抗阿片类药物作用对神经元和组织中的阿片类药物受体而言是特异性的，但它也影响稳定表达NPFF（2）受体的未分化SH-SY5Y细胞中的NPY Y2和α（2）-肾上腺素受体。为了测试这种差异是否可能是由于这些细胞的不成熟，将它们分化为带有星形孢菌素的去甲肾上腺素能神经元表型。分化的细胞停止分裂并长出细长的神经突。通过激活G（i）耦合受体抑制去极化触发的Ca（2）瞬变不受影响（微阿片类药物），增加（NPY），减少（NPFF（2））或丢失（alpha（2） -肾上腺素受体）。与1DMe孵育20分钟后，与未分化细胞一样，DAMGO的作用降低了，但NPY的作用不再受到影响。 Staurosporine分化不会修改微阿片类药物和NPFF（2）受体到G（i / o）蛋白质的耦合。我们建议，NPFF作用的特异性可能不在于其自身抗阿片类药物活性的分子机制，而在于膜内受体的组织。

BACKGROUND & AIMS: :Anti-apoptotic properties of physiological and elevated levels of the cellular prion protein (PrPc) under stress conditions are well documented. Yet, detrimental effects of elevated PrPc levels under stress conditions, such as exposure to staurosporine (STS) have also been described. In the present study, we focused on discerning early apoptotic STS-induced proteome and phospho-proteome changes in SH-SY5Y human neuroblastoma cells stably transfected either with an empty or PRNP-containing vector, expressing physiological or supraphysiological levels of PrPc, respectively. PrPc-overexpression per se appears to stress the cells under STS-free conditions as indicated by diminished cell viability of PrPc-overexpressing versus control cells. However, PrPc-overexpression becomes advantageous following exposure to STS. Thus, only a short exposure (2 h) to 1 μM STS results in lower survival rates and significantly higher caspase-3 activity in control versus PrPc-overexpressing cells. Hence, by exposing both experimental groups to the same apoptotic conditions we were able to induce apoptosis in control, but not in PrPc-overexpressing cells (as assessed by caspase-3 activity), which allowed for filtering out proteins possibly contributing to protection against STS-induced apoptosis in PrPc-overexpressing cells. Among other proteins regulated by different PrPc levels following exposure to STS, those involved in maintenance of cytoskeleton integrity caught our attention. In particular, the finding that elevated PrPc levels significantly reduce profilin-1 (PFN-1) expression. PFN-1 is known to facilitate STS-induced apoptosis. Silencing of PFN-1 expression by siRNA significantly increased viability of PrPc-overexpressing versus control cells, under STS treatment. In addition, PrPc-overexpressing cells depleted of PFN-1 exhibited increased viability versus PrPc-overexpressing cells with preserved PFN-1 expression, both subjected to STS. Concomitant increase in caspase-3 activity was observed in control versus PrPc-overexpressing cells after treatment with siRNA- PFN-1 and STS. We suggest that reduction of PFN-1 expression by elevated levels of PrPc may contribute to protective effects PrPc-overexpressing SH-SY5Y cells confer against STS-induced apoptosis.

背景与目标： ：在应激条件下，生理抗性和细胞病毒蛋白（PrPc）水平升高的抗凋亡特性已得到充分证明。然而，还已经描述了在应激条件下升高的PrPc水平的有害作用，例如暴露于星形孢菌素（STS）。在本研究中，我们专注于识别早期凋亡STS诱导的蛋白质组和磷酸化蛋白质组的变化，分别用空载体或含PRNP的载体稳定转染的SH-SY5Y人成神经细胞瘤细胞表达生理或超生理学水平的PrPc。 PrPc过度表达本身似乎在无STS的条件下对细胞造成压力，这由PrPc过度表达与对照细胞的细胞活力降低所表明。但是，PrPc过表达在暴露于STS后变得有利。因此，与过表达PrPc的细胞相比，仅短暂暴露（2 h）1μMSTS会导致较低的存活率和显着较高的caspase-3活性。因此，通过将两个实验组置于相同的凋亡条件下，我们能够在对照中诱导凋亡，但不能在过表达PrPc的细胞中诱导凋亡（通过caspase-3活性评估），从而可以滤除可能有助于抵抗STS的蛋白质诱导的PrPc过表达细胞凋亡。在暴露于STS后受不同PrPc水平调节的其他蛋白质中，涉及维持细胞骨架完整性的蛋白质引起了我们的注意。特别是，发现PrPc水平升高会大大降低profilin-1（PFN-1）的表达。已知PFN-1促进STS诱导的细胞凋亡。在STS处理下，通过siRNA沉默PFN-1表达可显着增加过表达PrPc的细胞与对照细胞的生存力。另外，与均保留了PFN-1表达的PrPc过表达细胞相比，均被STS处理的细胞中，耗尽了PFN-1的PrPc过表达细胞表现出增加的生存力。在用siRNA-PFN-1和STS处理后，在对照组中与过表达PrPc的细胞中同时观察到caspase-3活性的增加。我们建议，通过升高水平的PrPc降低PFN-1表达可能有助于过表达PrPc的SH-SY5Y细胞赋予抗STS诱导的细胞凋亡的保护作用。