Resistance to TRAIL-induced apoptosis in neuroblastoma cells correlates with a loss of caspase-8 expression

BACKGROUND: Disruption of apoptotic pathways may be involved in tumor formation, regression, and treatment resistance of neuroblastoma (NB). TNF-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in cancer cell lines. PROCEDURE: In this study we analyzed the expression and function of TRAIL, its agonistic and antagonistic receptors, and important intracellular signaling elements in 18 NB cell lines. RESULTS: Semiquantitative RT-PCR revealed that TRAIL-R2 and TRAIL-R3 are the main TRAIL-receptors used by NB cells. Sensitivity to TRAIL-induced apoptosis did not correlate with mRNA expression of TRAIL receptors or cFLIP. Surprisingly, caspase-8 and caspase-10 mRNA was detected in only 5 of 18 NB cell lines. Interestingly, only these five NB cell lines were susceptible to TRAIL-induced apoptosis in a time- and dose-dependent manner. CONCLUSIONS: Treatment with 5-aza-2'-deoxycytidine restored mRNA expression of caspase-8 and -10 and TRAIL sensitivity of resistant cell lines, suggesting that gene methylation is involved in caspase inactivation. Since many cytotoxic drugs induce caspase-dependent apoptosis, failure to express caspase-8 and/or caspase-10 might be an important mechanism of resistance to chemotherapy in NB.     

Caspase-8在TRAIL诱导神经母细胞瘤细胞凋亡中的作用

目的：应用干扰素(IFNγ)诱导神经母细胞瘤（neuroblastoma, NB）细胞caspase 8的表达并观察其是否可以恢复NB细胞对肿瘤坏死因子相关凋亡诱导配体（TRAIL）的敏感性。方法：应用RT PCR方法检测IFNγ作用前后NB细胞caspase-8 mRNA的表达;应用Alamar Blue法及流式细胞术检测IFNγ、TRAIL、IFNγ+TRAIL、IFNγ+caspase-8抑制剂zIETD-FMK+TRAIL对NB细胞生长及凋亡的影响;应用比色法测定NB细胞caspase-8相对活性。结果：对TRAIL敏感的CHP212细胞表达caspase-8,且经IFNγ处理后caspase-8 表达水平逐步增加;对TRAIL不敏感的SY5Y细胞不表达caspase-8,IFNγ作用后其caspase-8 mRNA表达明显增加。IFNγ与TRAIL联用对SY5Y细胞有明显诱导凋亡作用。CHP212细胞caspase-8相对活性随TRAIL作用时间的延长逐步升高;IFNγ与TRAIL联合作用的SY5Y细胞caspase-8相对活性明显高于未加药物处理的对照组、IFNγ组、TRAIL组及抑制剂组。结论：表达caspase-8的NB细胞对TRAIL的诱导凋亡作用敏感,TRAIL诱导NB细胞凋亡过程中伴随caspase-8活性的增加。［中国当代儿科杂志,2010,12(11)：902-907］     

Caspase-8和DR5在TRAIL诱导神经母细胞瘤细胞凋亡中的作用

目的：肿瘤坏死因子相关凋亡诱导配体(TRAIL)能诱导多种肿瘤细胞凋亡而对正常细胞无诱导凋亡作用。大多数神经母细胞瘤细胞株对TRAIL的诱导凋亡作用耐受与其Caspase8表达缺失有关,也与细胞表面TRAIL受体的表达和分布有关。该文主要探讨Caspase8及TRAIL受体DR5的表达在TRAIL诱导神经母细胞瘤细胞株SKNDZ凋亡中的作用及其发生机制。方法：应用RTPCR方法检测IFNγ作用前后SKNDZ细胞Caspase8的表达;应用WesternBlot方法检测化疗药作用前后SKNDZ细胞DR5的表达;应用四甲基偶氮唑蓝(MTT)比色法及流式细胞仪(FCM)检测TRAIL、IFNγ+TRAIL、化疗药+TRAIL及化疗药+IFNγ+TRAIL对SKNDZ细胞生长及凋亡的影响。结果：SKNDZ细胞不表达Caspase8,IFNγ作用后的SKNDZ细胞Caspase8表达明显增加。对照组未检测到DR5蛋白表达,而阿霉素和依托泊苷处理后检测到DR5蛋白表达。表达Caspase8的SKNDZ细胞对TRAIL的诱导凋亡作用仍不敏感,而同时表达Caspase8和DR5的SKNDZ细胞对TRAIL的诱导凋亡作用敏感。阿霉素/依托泊苷+IFNγ+TRAIL组早期凋亡率为:(17.9±3.6)%、(14.8±3.3)%,与IFNγ+TRAIL组(3.9±1.2)%比较,差异有显著性(F=26.233,P<0.01)。结论：同时表达Caspase8和DR5的SKNDZ细胞恢复了对TRAIL的敏感性,Caspase8和DR5在TRAIL诱导SKNDZ细胞凋亡中起着十分关键的作用。     

Caspase-8沉默--神经母细胞瘤耐药的新机制

神经母细胞瘤(neuroblastorma,NB)是小儿常见的恶性肿瘤之一,Ⅰ、Ⅱ期NB预后较好,而进展期NB对目前的治疗方案仍耐药.NB耐药涉及多种机制,该文介绍一种NB耐药的新机制,即Caspase-8沉默.Caspase-8沉默是由于Caspase-8基因超甲基化所致,如何恢复Caspase-8基因的表达,进而逆转NB的耐药性将成为NB治疗的一个新的研究方向.     

p75 mediated apoptosis in neuroblastoma cells is inhibited by expression of TrkA

BACKGROUND: Neurotrophins mediate their effects by binding to members of the Trk family of receptor tyrosine kinases and to the low-affinity nerve growth factor receptor p75. Nerve growth factor (NGF) has been demonstrated to support survival and differentiation of neuroblastoma (NB) cells by activation of the TrkA receptor. The p75 receptor belongs to the tumor necrosis factor (TNF) family of death receptors and has been suggested as a receptor that mediates apoptosis in neuronal and NB cells. PROCEDURE: To investigate the effect of p75 expression in NB, we transfected the p75 cDNA into SH-SY5Y cells, an NB cell line lacking expression of both p75 and TrkA. RESULTS: Cell clones expressing elevated levels of p75 showed a high degree of apoptosis even in 10% serum-supplemented medium. Apoptotic signaling by p75 was ligand-independent and only partly caspase-dependent. The level of apoptosis correlated directly with the expression level of the receptor, indicating that p75 may activate the cell death program directly. However, additional transfection of TrkA into SY5Y-p75 cells resulted in a significantly reduced rate of apoptosis even in the absence of NGF. CONCLUSIONS: Thus, expression of the TrkA receptor itself inhibits p75 mediated apoptosis in NB cells.     

Interferon-gamma sensitizes osteosarcoma cells to Fas-induced apoptosis by up-regulating Fas receptors and caspase-8

BACKGROUND: Osteosarcoma is the third most frequent neoplasm in adolescents. Although chemotherapy, frequently used in pre- and post-operative settings, has resulted in significant improvement in disease-free survival, some patients show little sensitivity to chemotherapy and alternative therapeutic strategies are needed. Because the Fas ligand/Fas receptor (CD95, APO-1) apoptosis pathway is a potential therapeutic target in osteosarcomas, we examined the effect of IFN-gamma on Fas-induced apoptosis in four osteosarcoma cell lines. PROCEDURE AND RESULTS: As measured by flow cytometry, all cell lines expressed cell surface IFN-gamma receptors, and when cultured for 2 days in the presence of IFN-gamma, all cell lines exhibited a significant increase in expression of Fas receptors. By flow cytometric detection of intracellular fragmented DNA as a marker of apoptosis, all cell lines cultured with either IFN-gamma or anti-Fas antibody (clone CH-11) alone showed only moderate apoptosis, whereas significantly high levels of apoptosis occurred in cells cultured with both IFN-gamma and CH-11. Western blotting analysis also revealed that IFN-gamma caused up-regulation of caspase-8 in all cell lines, but no change in Fas-associated death domain protein (FADD/MORT1) or caspase-3. Both caspase-8 and caspase-3 were activated when apoptosis was induced with both IFN-gamma and CH-11. Addition to cultures of z-IETD-fmk, an inhibitor of caspase-8, significantly blocked this apoptosis. CONCLUSIONS: IFN-gamma sensitizes osteosarcoma cells to Fas-induced apoptosis through up-regulation of Fas receptor and caspase-8. Combined immunotherapy with IFN-gamma and either anti-Fas monoclonal antibody or cytotoxic T cells that bear Fas ligand might be a useful adjunctive therapy for patients with osteosarcoma.     

短链RNA抑制神经母细胞瘤MYCN基因表达诱发肿瘤细胞凋亡的研究

目的 探讨MYCN高扩增神经母细胞瘤肿瘤细胞中MYCN基因的表达改变对肿瘤细胞凋亡、增殖的影响.方法 采用RNA干扰抑制MYCN基因表达,荧光定量PCR法及Westernblot法检验基因表达受抑制情况;ELISA法检验肿瘤细胞凋亡情况,Western-blot法检验神经元特异性烯醇酶表达变化.结果 siRNA转染12 h、24 h MYCN mRNA表达,相对灰度值分别为0.53±0.10(对照组为1)、0.28±0.09(对照组为1.12±0.31),表达显著降低(P＜0.05);Western-blot结果显示转染12 h、24 h MYCN蛋白表达,相对灰度值分别为0.76±0.13,对照组为1.25±0.21、0.44±0.07,对照组为1.39±0.29,表达显著降低(P＜0.05);MYCN基因表达抑制后肿瘤细胞凋亡增加,抑制组DNA碎片值1.90±0.12,对照组1.13±0.09,P＜0.05;神经元特异性烯醇酶表达增高,抑制组相对灰度值为1.04±0.14,对照组相对灰度值为0.47±0.10,P＜0.05.结论 抑制MYCN基因的表达可以促进神经母细胞瘤细胞凋亡及分化.     

二烯丙基二硫诱导人白血病K562细胞凋亡及其对Fas、FasL及caspase-8表达的影响

目的：探讨二烯丙基二硫（DADS）诱导白血病 K562 细胞凋亡的作用及其机制。方法：采用 Hoechst 33258 染色法观察细胞凋亡形态学变化;流式细胞术分析不同浓度及不同作用时间 DADS 对 K562 细胞凋亡的诱导作用;RT-PCR法检测DADS作用48 h后 Fas、FasL、caspase-8 mRNA的表达。结果：DADS可诱导K562 细胞凋亡。DADS(作用细胞24 h)浓度从10 mg/L增至40 mg/L时,其对K562细胞的凋亡率由（11.60±0.83）%增至（37.94±0.87）%;DADS(40 mg/L浓度组)作用时间从24 h增至72 h,其对K562细胞的凋亡率由（37.94±0.87）% 增至（47.02±0.66）%,各实验组随时间、浓度增加凋亡率明显增加 (P<0.05)。DADS作用48 h后,Fas、caspase-8 mRNA 表达水平较对照组上调;FasL mRNA 较对照组下调（P＜0.05）。结论：DADS 呈时间和浓度依赖性诱导白血病K562 细胞凋亡,其凋亡机制可能与上调 Fas、caspase-8,下调 FasL有关。     

糖皮质激素抵抗及其受体表达的相关性研究进展

糖皮质激素(GC)被广泛应用于许多炎症性疾病和自身免疫性疾病的治疗并取得很好疗效,然而也有部分患儿用药过程中表现出GC效应不明显甚至出现激素抵抗,使得这些患儿的治疗复杂化.阐明GC的抵抗机制对于减轻长期大剂量使用GC带来的不良反应及优化治疗策略尤为重要.就小儿肾病综合征而言,GC抵抗可以发生于激素信号途径的多个环节,包括GC受体(GR)基因的表达、数量、GRα/GRB的相对水平、GR翻译后的修饰对其核转位及与配体亲和力的影响、炎症介质增高对GR诱导的基因转录的影响等,也有部分GC抵抗的患儿,其机制并非GC信号途径本身,而与编码裂孔隔膜蛋白的基因突变有关.     

半胱氨酸蛋白酶3和白细胞介素8在高氧暴露下早产大鼠肺组织中的动态表达及其意义

目的 探讨半胱氨酸蛋白酶-3(Caspase-3)及白细胞介素-8(IL-8)在早产大鼠高氧肺损伤中的动态表达及其意义.方法 孕21 d的SD早产大鼠生后第2天,随机分为空气组和高氧组(均n=40).高氧组大鼠予以85%高氧持续暴露,空气组大鼠置于空气中.于暴露1、4、7、14和21 d每组各处死8只大鼠,收集肺组织标本,苏木精-伊红染色观察肺组织病理形态学变化,双抗夹心ELISA法检测IL-8含量,免疫组化和Western blot检测Caspase-3表达.结果 高氧暴露后肺泡腔内可见有坏死脱落细胞、炎症细胞渗出增多、间质水肿,肺组织结构紊乱,肺泡形成明显滞后,肺泡结构简单化和囊泡化;与空气对照组比较,高氧暴露4、7和14 d肺组织Caspase-3和IL-8含量均明显增高(P＜0.01).结论 在高氧所致早产大鼠肺损伤中细胞凋亡和坏死共存,两者共同参与了早产大鼠高氧肺损伤的病理过程.     

IFNγ联合TRAIL诱导神经母细胞瘤细胞凋亡的研究

目的探讨IENγ（γ干扰素）对TRAIL（肿瘤坏死因子相关凋亡诱导配体）诱导神经母细胞瘤细胞株SMS-KCNR（KCNR）细胞凋亡的影响及其发生机制。方法应用RT-PCR方法检测IFNγ作用前后KCNR细胞Caspase8的表达；应用四甲基偶氮唑蓝（MTT）比色法及流式细胞仪（FCM）检测IFNγ、TRAIL、IFNγ＋TRAIL及IFNγ＋Caspase8抑制剂（zIETD-FMK）＋TRAIL对KCNR细胞生长及凋亡的影响；应用比色法测定Caspase8相对活性。结果KCNR细胞不表达Caspase8，IFNγ作用48h后的KCNR细胞Caspase8表达明显增加；KCNR细胞对TRAIL不敏感，IFNγ诱导表达Caspase8的KCNR细胞对TRAIL敏感；IFNγ＋TRAIL组Caspase8相对活性明显高于TRAIL组及抑制剂组；zIETD-FMK能阻断Caspase8的活化而抑制TRAIL对KCNR细胞的诱导凋亡作用。结论IFNγ通过诱导Caspase8表达而逆转KCNR细胞对TRAIL诱导凋亡的耐受。     

熊果酸对神经母细胞瘤细胞增殖凋亡及MYCN表达的影响

目的实验研究熊果酸作用于体外培养的神经母细胞瘤细胞株（SH—SY-5Y）,对肿瘤细胞增殖、凋亡及MYCN基因mRNA表达的影响,探讨熊果酸对儿童神经母细胞瘤的治疗的作用。方法以终浓度为4uM／L、8uM／L、12uM／L的熊果酸作用于神经母细胞瘤细胞SH—SY-5Y,WST-1法检测SH—SY-5Y细胞增殖情况,流式细胞术检测SH—SY-5Y细胞凋亡情况,采用RealtimePCR方法检测MYCNmRNA的表达情况。结果熊果酸对SH—SY-5Y细胞增殖的抑制呈明显的剂量、时间依赖性,随剂量的增加,作用时间的延长,抑制效果逐渐加强;熊果酸对SHSY-5Y细胞凋亡的促进呈明显的剂量依赖性,随剂量的增加,促进凋亡效果逐渐加强;熊果酸对SH—SY-5Y细胞MYCN表达有抑制作用并有明显的剂量依赖性,随剂量的增加,抑制效果逐渐加强。12uM／L作用72h后神经母细胞瘤细胞增殖抑制效率达98．3％,MYCN基因mRNA表达抑制率达52．37％。结论应用熊果酸在体外环境作用SH—SY-5Y细胞,可以有效抑制该细胞的增殖、促进细胞凋亡、抑制原癌基因MYCNmRNA的表达。     

Caspase-3,-8,-9在铜诱导大鼠原代皮层神经元凋亡中的作用研究

目的通过建立体外铜诱导神经元模型,观察醋酸铜对大鼠原代皮层神经元凋亡及活化caspase-3,caspase-8和caspase-9蛋白表达的影响,探索高浓度铜导致大鼠神经元损伤的作用机制。方法采用不同浓度(20,40,80μM)醋酸铜诱导体外培养72h的大鼠原代皮层神经元;MTT法检测神经元活力,Hoechst33258染色和流式Annexin-V/PI法检测神经元凋亡,Western blot法检测不同浓度和时间点活化的caspase-3、caspase-8和caspase-9蛋白表达。结果体外培养的原代皮层神经元经醋酸铜诱导48h后,凋亡率上升,细胞活力呈浓度梯度下降,活化的caspase-8和caspase-9在铜诱导4h20μM组开始升高,48h各浓度组表达最高,呈时间与浓度梯度依赖;活化caspase-3在铜诱导24h20μM组才开始升高,激活的时间晚于caspase-8和caspase-9,48h各浓度组表达最高。结论过量铜可导致体外培养大鼠原代皮层神经元发生凋亡,caspase-3、caspase-8和caspase-9级联反应在铜诱导皮层神经元凋亡过程中发挥重要调控作用。     

Caspase-3,-8,-9在铜诱导大鼠原代皮层神经元凋亡中的作用研究

目的:通过建立体外铜诱导神经元模型,观察醋酸铜对大鼠原代皮层神经元凋亡及活化caspase-3,caspase-8和caspase-9蛋白表达的影响,探索高浓度铜导致大鼠神经元损伤的作用机制。方法:采用不同浓度（20,40,80 μM）醋酸铜诱导体外培养72 h的大鼠原代皮层神经元;MTT法检测神经元活力,Hoechst33258染色和流式Annexin-V/PI法检测神经元凋亡,Western blot法检测不同浓度和时间点活化的caspase-3、caspase-8和caspase-9蛋白表达。结果:体外培养的原代皮层神经元经醋酸铜诱导48 h后,凋亡率上升,细胞活力呈浓度梯度下降,活化的caspase-8和caspase-9在铜诱导4 h 20 μM组开始升高,48 h各浓度组表达最高,呈时间与浓度梯度依赖;活化caspase-3在铜诱导24 h 20 μM组才开始升高,激活的时间晚于caspase-8和caspase-9,48 h各浓度组表达最高。结论:过量铜可导致体外培养大鼠原代皮层神经元发生凋亡,caspase-3、caspase-8和caspase-9级联反应在铜诱导皮层神经元凋亡过程中发挥重要调控作用。［中国当代儿科杂志,2009,11（11）：917-922］     

Somatostatin receptor type 2 gene expression in neuroblastoma, measured by competitive RT-PCR, is related to patient survival and to somatostatin receptor imaging by indium -111-pentetreotide

BACKGROUND: We previously reported that human neuroblastoma cell lines and primary neuroblastoma tumors expressed a variable amount of mRNA for type 2 somatostatin (sst2) receptor gene. We also found that high level of sst2 expression were positively related to patient survival. PROCEDURE: We studied retrospectively 49 primary neuroblastomas. To detect and measure sst2 mRNA expression we developed a quantitative RT-PCR based on competitive PCR. When possible the number of MYCN copies was also measured with competitive PCR. RESULTS;. We found that the lowest level of sst2 mRNA was detected in advanced stages of neuroblastomas (stage IV) when compared with the other stages (P< 0.005). Patients with high levels of sst2 expression (>7 x 10(7) molecules/microg RNA) had a cumulative survival better than those with low sst2 expression (P < 0.0005). This predictive independent value of sst2 (P= 0.005) is retained after stratification for N-myc amplification. Finally we verified that the ex vivo sst2 gene expression in tumor samples was positively related (P < 0.01) to the in vivo semiquantitative determination of sst2 protein, assessed by 111In-pentetreotide imaging. CONCLUSIONS: Our data indicate that the measurement of sst2 mRNA measurement could represent a relevant tool in the prediction of neuroblastoma outcome, independently from MYCN amplification.     

Clinicopathologic factors related to apoptosis in retinoblastoma

PURPOSE: To investigate the presence of apoptosis in retinoblastoma and its correlation with other pathologic and prognostic factors. METHODS: The pathologic and admission records of 25 patients with a pathologically confirmed diagnosis of retinoblastoma were reviewed. TUNEL (TdT dUTP nick end labeling) staining was used to examine apoptosis in the pathologic slides of these 25 patients. The association between apoptosis and clinicopathologic factors was examined with chi-square, Fisher's exact, and Student's t tests. RESULTS: Of the 25 specimens tested, 11 were TUNEL stain-positive for the presence of apoptotic cells. Apoptosis was found more frequently in younger patients and within rosettes, although these associations were not statistically significant. Apoptosis was not associated with tumor invasion or metastasis. CONCLUSION: Apoptotic cells were found in 11 of 25 retinoblastoma specimens. Apoptosis tends to occur in young patients and be distributed within rosettes.     

Loss of heterozygosity for chromosome 14q in neuroblastoma

BACKGROUND: Neuroblastoma is a genetically heterogeneous disease, with subsets of tumors demonstrating rearrangements of several genomic regions. Preliminary studies from several groups have identified loss of heterozygosity (LOH) for the long arm of chromosome 14 (14q) in 20-25% of primary neuroblastomas. PROCEDURE: To determine precisely the frequency and extent of 14q deletions, we performed LOH analysis for a large series of primary neuroblastomas using a panel of 11 highly polymorphic markers. RESULTS: LOH was detected in 83 of 372 tumors (22%). Although the majority of tumors with allelic loss demonstrated allelic loss for all informative markers, 13 cases showed LOH for only a portion of 14q. A single consensus region of deletion, which was shared by all tumors with 14q LOH, was defined within 14q23-q32 between D14S588 and the 14q telomere. Allelic loss for 14q was strongly correlated with the presence of 11q LOH (P < 0.001 ) and inversely correlated with MYCN amplification (P= 0.04). CONCLUSIONS: LOH for 14q was evident in all clinical risk groups, indicating that this abnormality may be a universal feature of neuroblastoma tumor development. These findings suggest that a tumor suppressor gene involved in the initiation or progression of neuroblastoma is located within distal 14q.     

Leptin is closely related to body fat in prepubertal children aged 8-11 years

BACKGROUND: In adults and obese children, serum leptin concentrations are closely related to body fat. AIM: To investigate whether such a relationship between leptin concentrations and body fat is also evident in children with a relatively normal body composition. METHODS: The study was a cross-sectional population study in 170 Caucasian children (91 boys and 79 girls), with a mean age of 9.9+/-0.6 y (range 8.5-10.9 y) and a mean BMI of 17.4+/-2.6 (range 12.8-28.1). Serum leptin was measured and compared to total body fat as determined by dual-energy X-ray absorptiometry. RESULTS: In the whole population, serum leptin concentrations were highly correlated with total body fat (r=0.83, p<0.001). A stepwise forward multi-regression analysis revealed that the inclusion of other anthropometrical data did not add any significance to the model. Leptin concentrations were significantly higher in girls (5.2 ng/ml) than in boys (3.2 mg/ml; p=0.003). Gender differences still prevailed (p=0.007) after adjusting for number of kilograms of fat tissue. CONCLUSION: This study shows that, already at the young age of 9-11 y, an adult-like pattern of regulation of leptin exists. This indicates similar risk factor dependency of leptin across all age groups.