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.     

Neuronal differentiation in human neuroblastoma cells by nerve growth factor following TrkA up-regulation by interferon-gamma

BACKGROUND: TrkA mRNA expression has been reported to be related to favorable outcome of neuroblastoma (NB). Previously, we found that interferon-gamma (IFN-gamma) can enhance TrkA mRNA expression in NB cell lines. In the present study, we examined the effect of nerve growth factor (NGF) on IFN-gamma-induced TrkA protein to clarify the relationship between TrkA and cell differentiation of NB. PROCEDURE: The effect of IFN-gamma on the TrkA mRNA expression was screened in six human NB cell lines and a freshly prepared sample, SK-rib, from a stage IV patient. Using two of them, we examined their morphological change during simultaneous loading of NGF and IFN-gamma. Tyrosine phosphorylation pattern after 5 min of NGF stimulation was also examined in immunoblot analysis with anti-gp140(trkA) antibody and antiphospho tyrosine antibody. RESULTS: After a 4-day treatment with 500 IU/ml IFN-gamma, TrkA mRNA increased in five cell lines and SK-rib cells in association with growth inhibition. Although the degree of morphological differentiation did not increase in proportion to the TrkA expression induced by IFN-gamma, continuous loading of both IFN-gamma and NGF caused marked morphological differentiation in a cultured KP-N-RT cell line and SK-rib cells during 10 days. Moreover, 5 min of NGF stimulation after IFN-gamma treatment caused the phosphorylation of TrkA protein and downstream proteins. CONCLUSIONS: IFN-gamma could induce the functional NGF receptor even in the aggressive phenotype of NB.     

TrkA signal transduction pathways in neuroblastoma

BACKGROUND: Favorable neuroblastomas frequently express high levels of the TrkA receptor, and these tumors have a propensity to either differentiate or regress, but the mechanisms responsible for these two fates are unclear. PROCEDURE: To study TrkA signal transduction in neuroblastoma (nb), we stably expressed wild-type TrkA and five TrkA mutants in the human nb cell line SH-SY5Y. Resulting single cell clones were characterized by TrkA mRNA and protein expression and by autophosphorylation of the receptor. RESULTS: Introduction of TrkA restored nerve growth factor (NGF) responsiveness of SH-SY5Y cells, demonstrated by morphological differentiation and induction of immediate-early genes. TrkA overexpression leads to growth inhibition in the absence of NGF, whereas NGF treatment results in increased proliferation. CONCLUSIONS: Analysis of downstream signaling elements in mutated TrkA receptors indicates that NGF-induced differentiation is dependent on TrkA kinase activity, but several redundant pathways seem to be used farther downstream. This suggests differences from TrkA pathways identified in PC12 cells.     

γ干扰素和神经生长因子诱导神经母细胞瘤细胞分化及TrkA的表达

目的　在人神经母细胞瘤 (neuroblastoma ,NB)SMS KCNR细胞系中分别应用γ干扰素(IFN γ)、神经生长因子 (NGF)及联合应用IFN γ和NGF作为诱导剂诱导NB细胞分化 ,观察细胞形态学的变化、细胞增殖情况及TrkAmRNA的表达。方法　常规方法培养人SMS KCNR细胞系细胞 ,收取第 10天培养的细胞 ,提取总RNA ,应用逆转录 聚合酶链反应 (RT PCR)方法检测TrkAmRNA的表达水平 ,应用锥虫兰染色方法计数活细胞数并用倒置相差显微镜观察细胞形态学的改变 ,计数细胞分化率。结果　在KCNR细胞中同时负荷IFN γ和NGF的细胞表现出比单独负荷IFN γ或NGF更加显著的形态学分化 (P <0 0 1)。前者细胞增殖抑制更加明显 (P <0 0 1)。在IFN γ组 ,TrkAmRNA表达增加 (P <0 0 1) ,NGF组TrkAmRNA的表达未见明显改变 (P >0 0 5 ) ,联合用药组TrkAmRNA的表达有明显降低 (P <0 0 1)。结论　在人SMS KCNR细胞中 ,联合应用IFN γ和NGF比单独应用二者有更加显著诱导分化的作用。与IFN γ诱导TrkAmRNA表达增加不同 ,联合用药导致TrkAmRNA表达下调 ,可能存在某种负调节机制。研究结果表明 ,联合应用IFN γ和NGF可能是治疗NB甚至是晚期NB的合理方案     

Different effects of TrkA expression in neuroblastoma cell lines with or without MYCN amplification

BACKGROUND: Expression of the neurotrophin receptor TrkA is associated with a favorable prognosis in neuroblastoma (NB) and promotes growth inhibition and neuronal differentiation. Aggressive, MYCN-amplified NB tumors express little or no TrkA mRNA, suggesting that MYCN overexpression may inhibit TrkA expression. PROCEDURE: To study the interactions of TrkA expression and MYCN amplification in NB, we stably expressed the TrkA receptor in the MYCN single copy cell lines SH-SY5Y and NB69 as well as in the MYCN amplified cell lines CHP134 and IMR5. RESULTS: All four transfected cell lines demonstrated high TrkA expression and similar activation of the TrkA receptor and of mitogen-activated protein kinases as well as induction of immediate-early genes in response to nerve growth factor (NGF). Introduction of TrkA restored NGF responsiveness of SH-SY5Y and NB69 cells, as demonstrated by morphologic differentiation, growth inhibition, and enhanced survival in serum-free medium. However, no morphologic, growth, or survival responses to NGF were detected in MYCN-amplified CHP134 and IMR5 TrkA transfectants. CONCLUSIONS: Thus, transfection of TrkA into MYCN amplified NB cell lines only partly restored the TrkA/NGF signaling pathway, suggesting additional inhibitory effects of MYCN overexpression on TrkA signaling.     

Expression of neurotrophin receptor TrkA inhibits angiogenesis in neuroblastoma

BACKGROUND: Mechanisms regulating the expression of angiogenic factors in tumor cells are largely unknown. High expression of the neurotrophin receptor TrkA in neuroblastomas (NB) is associated with favorable prognosis, whereas TrkB is expressed on aggressive, MYCN-amplified NB. PROCEDURE: To investigate the biological effects of TrkA and TrkB expression on angiogenesis in NB, we examined the expression of angiogenic factors in the human NB cell line SY5Y and its TrkA and TrkB transfectants. RESULTS: In comparison to parental SY5Y cells, mRNA and protein levels of angiogenic factors were significantly reduced in SY5Y-TrkA cells, whereas SY5Y-TrkB cells did not demonstrate a significant change. Conditioned medium (CM) of parental SY5Y and SY5Y-TrkB cells induced endothelial cell proliferation, but this effect was completely absent in SY5Y-TrkA cells. TrkA expression also resulted in severely impaired tumorigenicity in a mouse xenograft model, and was associated with reduced angiogenic factor expression and less vascularization of tumors, as determined by immunohistochemistry and an in vivo Matrigel assay.     

TrkA、p75NTR mRNA在肛门直肠畸形胎鼠直肠末端的表达

目的通过检测肛门直肠畸形(ARM)胎鼠直肠末端神经生长因子受体TrkA、p75NTR mRNA的表达水平,探讨ARM直肠末端肠神经系统发育情况。方法成年SD孕鼠16只。其中实验组10只,对照组6只。实验组孕鼠在孕11 d、13 d 2次给予10 g.L-1乙烯硫脲(125 mg.kg-1)灌胃,制作先天性ARM动物模型;对照组灌胃注入等量9 g.L-1盐水。2组孕鼠在孕20 d时行剖宫手术取出胎鼠,取其直肠末端并保存于-80℃冰箱;采用反转录/实时定量PCR(RT-PCR)方法检测正常胎鼠和ARM胎鼠直肠末端TrkA、p75NTR mRNA表达情况。结果 TrkA、p75NTR mRNA在正常胎鼠直肠末端的相对表达量分别是162.221±104.675,129.778±51.976,在ARM胎鼠直肠末端中表达分别是78.937±33.425,87.145±25.812,两组比较差异均有统计学意义(Pa<0.05);琼脂糖凝胶电泳显示TrkA、p75NTR基因扩增后产物cDNA片段与引物设计的扩增片段完全吻合,TrkA、p75NTR基因条带亮度对照组均高于实验组。结论 TrkA、p75NTR mRNA在ARM胎鼠直肠末端的表达异常,提示神经生长因子及受体TrkA、p75NTR可能参与ARM肠神经系统的发育。     

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.     

MycN sensitizes neuroblastoma cells for drug-triggered apoptosis

BACKGROUND: Amplification of the MYCN gene is found in a large proportion of neuroblastomas and is associated with a poor prognosis. PROCEDURE: To investigate the effect of ectopic MycN expression on the susceptibility of neuroblastoma cells to cytotoxic drugs, we used a human neuroblastoma cell line with tetracycline-controlled expression of MycN. RESULTS: Neither conditional expression of MycN alone nor low drug concentrations induced apoptosis. However, MycN and cytotoxic drugs cooperated to induce cell death. Apoptosis triggered by MycN and doxorubicin was mediated by cleavage of caspases and involved activation of the CD95 system. MycN overexpression and cytotoxic drugs also synergized to induce p53 and Bax protein expression and to trigger mitochondrial permeability transition and cytochrome c release. CONCLUSION: In that amplification of MYCN is considered an adverse prognostic factor, these findings suggest that dysfunctions in apoptosis pathways may be a mechanism by which MycN-induced apoptosis of neuroblastoma cells is inhibited.     

Loss of caspase-8 expression in neuroblastoma is related to malignancy and resistance to TRAIL-induced apoptosis

Background and Procedures NB-derived cell lines were tested for their sensitivity to apoptosis induced by the tumor-selective apoptotic ligand TRAIL. Noninvasive S-type cell lines are highly sensitive to TRAIL, whereas invasive N-type cell lines are resistant. RESULTS: Although both S- and N-type cell lines express TRAIL-R2, FADD, and caspases-3 and -10, only S-type cells express caspase-8. Reduced levels of caspase-8 protein were also observed in a stage IV NB tumor when compared to a ganglioneuroma. The caspase-8 gene is not deleted in either N-type NB cell lines or high-stage tumors, and expression can be induced by demethylation. CONCLUSIONS: Therefore, caspase-8 expression is silenced in malignant NB, which correlates to tumor severity and resistance to TRAIL-induced apoptosis.     

Prognostic and biological role of neurotrophin-receptor TrkA and TrkB in neuroblastoma

Expression of different neurotrophin receptors of the tyrosine kinase (Trk) family plays an important role in the biology and clinical behavior of neuroblastomas (NB). Observations from several independent studies suggest that high expression of TrkA is present in NB with favorable biological features and highly correlated with patient survival, whereas TrkB is mainly expressed on unfavorable, aggressive NB with MYCN-amplification. To determine expression of Trk receptors and ligands in primary NB, we developed a reliable semiquantitative duplex RT-PCR protocol, that requires only 1 microgram RNA per tumor sample. Activation of TrkA by its ligand nerve growth factor (NGF) initiates a cascade of signaling events and promotes neuronal differentiation in vitro. Activation of TrkB by its ligand brain derived neurotrophic factor (BDNF) has been associated with proliferation and survival of NB cells. To study Trk signal transduction pathways and their biological effects in NB, we stably expressed TrkA and TrkB cDNA in the human NB cell line SH-SY5Y. Introduction of TrkA and TrkB restored responsiveness of SH-SY5Y cells to the ligands NGF and BDNF, respectively, and resulted in morphological differentiation. Expression of TrkA resulted in growth inhibition of the transfectants compared to parental cells, whereas TrkB transfectants demonstrated an increased proliferation rate. Further insight into the differences of TrkA and TrkB signaling may suggest new options for the treatment of NB. As expression of TrkA is a strong prognostic factor especially in MYCN non-amplified NB, a prospective study of Trk receptor expression using RT-PCR should be performed for German neuroblastoma patients.     

三氧化二砷对人神经母细胞瘤细胞凋亡作用的影响

目的：三氧化二砷(As2O3 )通过诱导细胞凋亡和诱导细胞分化效应治疗急性早幼粒细胞白血病(APL)患者已取得很好疗效。与APL细胞相似,神经母细胞瘤(NB)细胞也是分化成熟受阻,该文对As2O3 在体外对人神经母细胞瘤细胞凋亡作用的影响及可能的机制进行探讨。方法：比色法观察As2O3 对神经母细胞瘤SK N SH细胞增殖的影响;Giemsa染色观察细胞形态学改变;Annexinⅴ/PI双染色流式细胞术检测细胞凋亡;免疫细胞化学染色方法检测bcl 2蛋白表达变化。结果：As2O3 明显抑制SK N SH细胞增殖,抑制作用呈剂量时间依赖效应,并出现细胞凋亡的形态学改变;流式细胞仪检测4. 0μmol/LAs2O3 作用SK N SH细胞72h后,早期凋亡细胞比例( 9. 9±0. 8 )%,与对照组( 3. 7±0. 2 )%相比明显增多,差异有显著性(P< 0. 05 );分别以1. 0, 2. 0,4. 0μmol/L的As2O3 处理SK N SH细胞72h后,bcl 2表达随As2O3 浓度增加呈降低趋势,bcl 2表达阳性率分别为(26. 3±8. 0)%, (15. 0±4. 5)%和(4. 8±3. 2)%,与对照组(73. 6±6. 1)%相比,差异有显著性(P<0. 01)。结论：As2O3 可在体外抑制神经母细胞瘤细胞增殖,诱导凋亡,bcl 2参与了其调节作用。     

TrkA基因在神经母细胞瘤增殖及形态分化中的作用

目的 观察酪氨酸激酶受体A(TrkA)基因抑制神经母细胞瘤生长、诱导其分化的可行性。方法 利用脂质体转染法将TrkA cDNA重组质粒转染入人神经母细胞瘤(NB)SH-SY5Y细胞系中,应用RT-PCR技术鉴定转染后基因的表达,比较转染前后SH-SY5Y细胞的增殖率及细胞的分化程度。结果 成功转染TrkA基因并在SH-SY5Y细胞中稳定表达,转染后SH-SY5Y细胞的增殖率较亲代细胞明显下降(P<0.01),细胞的分化程度增加(P<0.01)。结论 TrkA基因的高表达能有效抑制神经母细胞瘤的生长,并能促其向良性分化,为神经母细胞瘤的基因治疗提供理论依据。     

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。     

Retinoic acid-induced apoptosis of the CHP134 neuroblastoma cell line is associated with nuclear accumulation of p53 and is rescued by the GDNF/Ret signal

BACKGROUND: Neuroblastoma (NBL) is one of the most common solid malignancies in childhood and is derived from the sympathetic precursor cells. Although p53, a tumor suppressor, has been reported to be rarely mutated in NBLs, it is sequestered abnormally in the cytoplasm of the NBL cell. The mechanism and functional role of the abnormal intracellular localization of p53 remain unclear. PROCEDURE: Here, we established an in vitro system of apoptosis model using a NBL cell line CHP134 which also showed a cytoplasmic sequestration of p53. The treatment of the cells with 1 or 5 microM all-trans retinoic acid (RA) induced moderate neurite outgrowth followed by massive death of CHP134 cells by days 5 to 6. RESULTS: TUNEL staining showed that the cell death was due to apoptosis. Immunofluorescent stain demonstrated that p53 was strongly positive in the nucleus on day 5, which was accompanied with induction of p21WAF1. In addition, expression of caspase-3 was also increased during the cell death. Intriguingly, the RA treatment induced expression of Ret tyrosine kinase receptor in CHP134 cells. CONCLUSIONS: The addition of ligands, glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN), inhibited apoptosis as well as nuclear accumulation of p53 in the cell. The present results suggest that the RA-induced apoptosis of NBL cells is associated with activation of both the caspase cascade and the p53-mediated pathway with its nuclear translocation. The neurotrophic signal through the GDNF-Ret system may prevent the neuronal cell death.     

Enhanced effect of IFNgamma on the induced apoptosis of neuroblastoma cells by cytotoxic drugs

The objective of this study was to explore the antitumor effects of cytotoxic drugs combined with IFNgamma on neuroblastoma cell line SH-SY5Y cells. The expression of caspase 8 mRNA and protein was detected with RT-PCR and Western blot analysis. The effects of cytotoxic drugs and IFNgamma combined with cytotoxic drugs on the growth and apoptosis of SH-SY5Y cells were detected with the methods of MTT and flow cytometry. Caspase 8 activity was measured by colorimetric assay. Caspase 8 was undetectable in SH-SY5Y cells with an increased expression of caspase 8 after the treatment of IFNgamma. SH-SY5Y cells were sensitive to Adriamycin relatively but resistant to TNFalpha and TRAIL, while IFNgamma-pretreated SH-SY5Y cells were more sensitive to the cytotoxic drugs with an increase of caspase 8 activity. The authors conclude that IFNgamma can sensitize SH-SY5Y cells to Adriamycin-, TNFalpha-, and TRAIL-induced apoptosis and this may be realized by the upregulation of caspase 8.     

雷帕霉素对人神经母细胞瘤细胞株生长抑制及诱导凋亡的研究

目的 研究雷帕霉素对人神经母细胞瘤细胞株SH-SY5Y生长抑制及诱导凋亡的作用.方法 雷帕霉素处理SH-SY5Y细胞,并设立对照组,以CCK-8检测细胞生长活性,流式细胞法(FCM)检测细胞周期,DNA梯度条带(DNA Ladder)和罗丹明123-碘化丙啶(Rhodamine 123/PI)染色法分析细胞凋亡.结果 雷帕霉素处理后,神经母细胞瘤细胞呈浓度依赖性生长抑制.20μM雷帕霉素处理12 h后,细胞周期出现G2/M期、S期阻滞,G2/M期(24.22±2.89)%、S期(22.05±0.96)%的细胞百分比明显高于对照组.罗丹明123-碘化丙啶检测细胞凋亡率(54.35±5.39)%也明显高于对照组,并有明显的凋亡条带出现.结论 雷帕霉素具有抑制神经母细胞瘤细胞株SH-SY5Y生长、诱导其凋亡的作用.     

Etoposide toxicity on human neuroblastoma cells in vitro is enhanced by preceeding hyperthermia

BACKGROUND: Thermal enhancement has been proven in vitro for the cytotoxic effect of alkylants and platinum compounds, not, however, for etoposide, which acts synergistically to these drugs. PROCEDURE: Our in vitro study on a neuroblastoma cell line confirmed previous results in other tumor models that the cytotoxicity of etoposide (12.8% as compared to untreated controls) is not enhanced by simultaneous heating to 40 or 42 degrees C for 1 hr (11.9%), as jugded by colony forming assay. RESULTS: The same temperature applied 24 hr before the drug resulted in a significant decrease of colonies (6.1%). Double treatment with etoposide within a 24-hr-interval yielded a similar result (5.6%). The colony number could be further decreased by adding hyperthermia 24 hr before the second treatment (1.3%). CONCLUSIONS: We demonstrate in vitro that the enhancing effect of increased temperature on the cytotoxicity of etoposide on neuroblastoma cells is not absent, but depends on scheduling. The temperature range used is achievable in total body hyperthermia. Thus, our results are relevant for possible treatment of disseminated neuroblastoma.