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。     

小剂量维甲酸诱导对神经母细胞瘤耐药的影响

目的探讨小剂量维甲酸诱导对神经母细胞瘤(NB)化疗耐药的影响。方法选择人NB细胞株SH SY5Y,用RT PCR方法检测小剂量全反式维甲酸(ATRA)作用下TrkB mRNA水平;通过四甲基偶氮蓝比色(MTT)法及流式细胞仪(FCM)检测ATRA诱导前后顺铂对人NB细胞株SH SY5Y的生长及凋亡的影响。结果SH SY5Y中未检测到TrkB mRNA表达,用1、10、100nM/LATRA诱导5d后可检测到TrkB mRNA表达,且随ATRA浓度增加TrkB mRNA水平逐渐升高。100nM/LATRA诱导7d,TrkB mRNA的相对比值与诱导5d比差异无显著性意义(P>0.05);MTT分析显示:BDNF+顺铂组(无TrkB表达)、ATRA+顺铂组(无BDNF刺激)细胞存活率同单用顺铂组比较无显著性差异(P>0.05);10nM/LATRA+BDNF+顺铂组NB细胞存活率明显高于单用顺铂组(P<0.01);FCM分析显示:ATRA+BDNF+顺铂组NB细胞凋亡率明显低于单用顺铂组(P<0.01)。结论存在BDNF的条件下,小剂量ATRA能阻断顺铂对SH SY5Y的细胞毒性作用,从而使SY5Y细胞对化疗耐药。     

γ—IFN诱导神经母细胞瘤细胞分化及TrKA表达的实验研究

本实验观察不同浓度的γ－IFN对NB细胞产生增殖抑制及诱导分化作用时,TrkA MRNA表达的变化,及其与增殖抑制,分化程度的关系。首先应用,常规培养SMS－KCNR细胞;然后用三种不同浓度的γ－IFN处理这些细胞,在不同的作用时间,通过台盼蓝拒染实验判定γ－INF对NB细胞的增殖抑制作用及相差显微镜观察NB细胞形态学变化;用RNA－PCR及Southern Blot杂交法检测γ－IFN对NB细胞的Tr-kA mRNA表达的影响。结果表明：（1）1000IU／ml,2000IU/ml,γ-INF对人NB细胞的体外增殖有抑制作用。（2）γ－IFN可诱导NB细胞分化成熟及TrkA mRNA表达水平增高,其作用随时间延长,浓度增加而增强。结论：（1）γ－IFN能够抑制NB细胞增殖并诱导其分化,同时TrkA mRNA表达增加,其作用效应,呈量效依赖关系。（2）TrkA mRNA表达水平的增高可能γ－IFN体外诱导NB细胞分化逆转的分子生物学机制之一。是     

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。     

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目的探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)对人神经母细胞瘤的作用及干扰素-γ(IFN-γ)对新型凋亡分子TRAIL抗瘤活性的影响,并探讨其影响机制。方法应用MTT分析和流式细胞仪研究TRAIL对人神经母细胞瘤细胞的抑制作用及IFN-γ对TRAIL抗瘤活性的影响。用RT-PCR方法检测IFN-γ作用48h后,Caspase-8-mRNA的表达。结果TRAIL(10,50,100μg/L)单独作用、IFN-γ(1000U/mL)单独作用后,对SY5Y细胞的增殖抑制率分别为(4·38±0·89)%、(3·54±1·66)%、(5·03±1·26)%、(5·33±2·06)%;凋亡率分別为(5·18±3·33)%、(5·26±3·64)%、(5·00±2·88)%、(8·14±7·35)%。IFN-γ(1000U/mL)与TRAIL(100μg/L)联合作用后,对SY5Y细胞的增殖抑制率为(41·22±7·09)%;凋亡率为(21·29±6·20)%。RT-PCR方法发现IFN-γ作用48h后Caspase-8-mRNA的表达明显上调。结论神经母细胞瘤细胞SH-SY5Y对TRAIL不敏感,IFN-γ可以明显提高TRAIL对神经母细胞瘤细胞的敏感性,其发生机制可能是通过IFN-γ上调Caspase-8-mRNA的表达而实现的。     

γ干扰素与阿霉素联用对成神经细胞瘤细胞生长及凋亡的影响

目的探讨γ干扰素(IFN-γ)对阿霉素抗人成神经细胞瘤细胞作用的影响,并探讨其影响机制。方法应用四甲基偶氮唑蓝(MTT)分析和流式细胞仪,研究IFN-γ与化疗药阿霉素联合应用前后人成神经细胞瘤细胞SH-SY5Y的存活率及凋亡率;免疫组化方法检测IFN-γ作用前后半胱氨酸蛋白酶-8(caspase-8)蛋白的表达。结果阿霉素(0.03,0.10,0.30μg/ml)单独作用24h后,SY5Y细胞的存活率分别为(95.62±13.03)%,(82.62±7.94)%和(64.84±9.19)%,各组间差异显著。IFN-γ(10,100,1000U/ml)单独作用48h后,SY5Y细胞的存活率分别为(98.37±11.25)%,(97.15±5.36)%和(98.84±7.41)%,各组间差异无显著性。IFN-γ(1000U/ml)与不同浓度的阿霉素(0.03,0.10,0.30μg/ml)联合作用后,SY5Y细胞的存活率同单独应用阿霉素组比较明显下降,差异有显著性;阿霉素(0.30μg/ml)单独作用24h,SH-SY5Y的凋亡率为(22.00±6.55)%,IFN-γ(1000U/ml)单独作用48h,凋亡率为(8.22.±4.00)%。二者联合作用后,凋亡率较单用阿霉素组比较明显升高,差异有显著性;免疫组化方法发现SH-SY5Y细胞株不表达caspase-8,IFN-γ作用48h后caspase-8表达阳性。结论阿霉素对成神经细胞瘤细胞SH-SY5Y具有抑制增殖和诱导凋亡的作用,IFN-γ可以使其作用明显增强。其发生机制可能是通过IFN-γ上调caspase-8蛋白的表达而实现的。     

TrkB-BDNF信号通路对神经母细胞瘤细胞分泌血管内皮生长因子的影响

目的探讨TrkB-BDNF信号通路对神经母细胞瘤(NB)细胞SH-SY5Y分泌血管内皮生长因子(VEGF)的影响。方法 Western blot方法检测全反式维甲酸(ATRA)诱导前后SY5Y细胞TrkB蛋白表达及脑源性神经营养因子(BDNF)刺激后SY5Y细胞磷酸化-TrkB(p-TrkB)蛋白表达;ELISA技术检测ATRA、BDNF、特异性酪氨酸激酶抑制剂K252a及PI3K抑制剂LY294002处理后SY5Y细胞培养上清中VEGF含量。结果 ATRA诱导前,SY5Y细胞中未检测到TrkB蛋白表达;1,10,100 nM/L ATRA处理后,SY5Y细胞中可检测到TrkB蛋白表达,且TrkB蛋白表达水平随ATRA浓度增加逐渐升高,差异有统计学意义。10 nM/L ATRA单独处理组未检测到p-TrkB表达,ATRA+BDNF组可检测p-TrkB蛋白表达。ATRA+BDNF组的VEGF含量明显高于对照组及ATRA组(P<0.01);ATRA+K252a+BDNF组VEGF含量明显低于ATRA+BDNF组(P<0.05);ATRA+LY294002+BD-NF组VEGF含量亦明显低于ATRA+BDNF组(P<0.01)。结论激活TrkB-BDNF信号通路可促进NB细胞合成、分泌VEGF;用K252a阻断TrkB-BDNF信号通路或用LY294002阻断TrkB-BDNF信号下游通路PI3K/Akt均可有效抑制NB细胞合成、分泌VEGF。     

5-氮杂胞苷增强阿霉素对神经母细胞瘤细胞的抗瘤活性

目的：许多神经母细胞瘤细胞系及肿瘤组织标本中caspase-8表达缺失, caspase-8基因沉寂的主要原因是其启动子区域高度甲基化。该文探讨去甲基化药物5-氮杂胞苷对caspase-8表达及对化疗药阿霉素抗人神经母细胞瘤细胞作用的影响及其影响机制。方法：应用RT-PCR方法检测5-氮杂胞苷作用前后SH-SY5Y细胞中caspase-8 mRNＡ水平变化。MTT分析研究5-氮杂胞苷与化疗药阿霉素联合应用前后人神经母细胞瘤细胞SH-SY5Y的存活率,以及加入caspase-8活性抑制剂后存活率的变化。结果：RT-PCR方法发现SH-SY5Y细胞株不表达caspase-8 mRNA, 5-氮杂胞苷作用3 d后可检测到 caspase-8 mRNA表达,5 d后表达较第3天增加; 5-氮杂胞苷与不同浓度阿霉素(0.05, 0.1, 0.25, 0.5 μg/mL)联合应用后SH-SY5Y细胞存活率为(77.61±7.30)%,（57.35±6.64）%,（46.25±4.46）%,（35.59±5.12）%,同一浓度单独应用阿霉素组细胞存活率为（94.89±4.15）%,（80.60±8.50）%,（64.48±4.92）%,（52.32±6.71）%,5-氮杂胞苷与阿霉素联用组细胞存活率明显低于单用阿霉素组,差异均有显著性。Caspase-8抑制剂组与同一浓度的5-氮杂胞苷+阿霉素组比较,细胞存活率明显增加,依次为（92.95±3.48）%,（78.39±4.28）%,（62.31±6.50）%,（49.92±5.77）%,差异均有显著性。结论：阿霉素对神经母细胞瘤细胞SH-SY5Y具有增殖抑制作用,5-氮杂胞苷可以增强阿霉素的抗瘤活性。其发生机制可能是通过上调caspase-8 mRNA的表达而实现的。［中国当代儿科杂志,2007,9（6）：577-579］     

Smac基因过表达增强肿瘤坏死因子相关的凋亡诱导配体诱导神经母细胞瘤细胞凋亡

目的研究Smac基因在肿瘤坏死因子相关的凋亡诱导配体（TRAIL）诱导的神经母细胞瘤（NB）SK-N-SH细胞凋亡中的增强作用。方法1.利用反转录（RT）-PCR从人胚肾293细胞中扩增人Smac基因全长cDNA,通过TA克隆构建重组T载体pGEM-T/Smac。2.将目的片段从重组T载体上酶切下来,构建真核细胞表达载体pcDNA3.1/Smac,并转染人NB SK-N-SH细胞。用新霉素G418筛选稳定细胞株SK-N-SH/Smac,并用Western blot鉴定Smac基因的过表达。3.用1 000μg/L的TRAIL作用于过表达Smac基因的SK-N-SH/Smac和普通的SK-N-SH细胞24 h。采用流式细胞分析检测细胞凋亡百分率。另设无TRAIL干预的SK-N-SH/Smac及正常SK-N-SH对照组。4.比色法测定各组细胞中caspase-8酶活性。结果1.成功建立稳定过表达Smac基因的NB克隆株SK-N-SH/Smac。2.流式细胞分析显示1 000μg/L的TRAIL作用24 h后,过表达Smac基因的SK-N-SH细胞凋亡百分率显著高于正常对照的SK-N-SH细胞[（44.8&#177;4.71）%vs（23.6&#177;3.05）%P〈0.05]。3.比色法测定显示,同样在1000μg/L的TRAIL作用24 h后,过表达Smac基因的SK-N-SH/Smac细胞中,caspase-8的酶活性显著高于未转染的普通SK-N-SH细胞[（0.743&#177;0.029）vs（0.476&#177;0.031）P〈0.05]。结论转染并过表达Smac基因可增强TRAIL对神经母细胞瘤SK-N-SH细胞的促凋亡作用。     

MYCN基因表达抑制促TRAIL诱导神经母细胞瘤细胞凋亡的研究

目的神经母细胞瘤(NB)为小儿常见恶性肿瘤。MYCN基因高扩增NB细胞对TRAIL诱导凋亡多有抵抗。我们以MYCN基因高扩增NB细胞株为研究对象,抑制其MYCN基因的表达,探讨其对TRAIL诱导凋亡有无增强作用。方法构建MYCN siRNA,转染NB细胞株SK-N-BE(2),抑制MYCN基因表达后,Western-blot法检测SK-N-BE(2)细胞Bcl-2、Bcl-xL、Bid、DR4、DR5表达的变化,ELISA法检测细胞凋亡。结果转染MYCN siRNA的SK-N-BE(2)细胞中MY-CN基因表达显著降低(P<0.05)。MYCN基因表达抑制后,SK-N-BE(2)细胞DR5、Bid表达增高(P<0.05),Bcl-2、Bcl-xL表达降低(P<0.05),DR4表达无变化(P>0.05),TRAIL诱导细胞凋亡显著增强(P<0.05)。结论 MYCN基因表达抑制后,可调控Bcl家族中相关蛋白、DR5的表达,促进TRAIL诱导NB细胞凋亡。     

ROLE OF CASPASE 8 AS A DETERMINANT IN TRAIL SENSITIVITY OF NEUROBLASTOMA CELL LINES

Objective: Resistance of neuroblastoma (NB) cells to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis is thought to be caused by loss of caspase 8 expression. The aim of this study is to investigate if induction of caspase 8 by γ-interferon (IFNγ) renders NB cells sensitive to TRAIL. Methods: Caspase 8 expression was monitored by RT-PCR and Western blot analysis. The effects of IFNγ, TRAIL, IFNγ + TRAIL, and caspase 8 inhibitor + TRAIL on the growth and apoptosis of NB cells were detected with the methods of reduction rate of Alamar blue assay and flow cytometry. The relative caspase 8 activity was measured with colorimetric assay. Results: An increased expression of caspase 8 mRNA and protein was found after treatment with IFNγ. IFNγ in combination with TRAIL decreased proliferation of NB cell line SH-SY5Y cells. The killing effect of TRAIL on NB cells expressing caspase 8 was depressed by caspase 8 inhibitor. The relative caspase 8 activity of NB cells expressing caspase 8 increased with the prolongation of TRAIL action time. Conclusions: TRAIL induced apoptosis in NB cells expressing caspase 8.     

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.     

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.     

Effect of CEP-751 (KT-6587) on neuroblastoma xenografts expressing TrkB

BACKGROUND: The compound CEP-751 (KT-6587), a potent and selective inhibitor of the Trk family of tyrosine kinases, has been shown to inhibit the growth of human neuroblastoma (NB) xenografts in nude mice [1]. PROCEDURE: To address its mechanism of action, we studied SY5Y, a human NB cell line with no detectable Trk expression, and two subclones transfected with TrkB. The transfected clones, SY5Y (G8) and SY5Y (G12), expressed moderate and high levels, respectively, of TrkB mRNA and protein. These TrkB-expressing subclones and the parental line were then grown as xenografts in nude mice, and CEP-751 was used to inhibit TrkB tyrosine kinase activity in these xenografts. Animals were treated twice a day with CEP-751 (21 mg/kg), or with the carrier vehicle as a control. TrkB expression in the resultant tumors was examined by quantitative RT-PCR. The effect of CEP-751 on TrkB activation by BDNF was examined in G12 cells in culture by immunoprecipitation with antipan Trk antiserum, followed by Western blot analysis using antiphosphotyrosine antibodies. To determine if CEP-751 was causing apoptosis, the TUNEL assay was used. RESULTS: CEP-751 had little effect on the growth of SY5Y tumors, but did slow the growth rate of the C8 and G12 tumors. The daily growth rate of the treated tumors was 0.16, 0.13, and 0.10 cm3, respectively, for the SY5Y, G8, and G12 tumors. RT PCR analysis confirmed the expression of TrkB in G8 and G12, but not in SY5Y tumors. Activation of TrkB by BDNF in G12 cells was inhibited by CEP-751 in a dose dependent fashion. The treated tumors showed marked evidence of apoptosis. CONCLUSIONS: These data suggest that the effect of CEP-751 is due, at least in part, to its inhibition of TrkB kinase, and that CEP-751 may become a useful therapeutic tool for the treatment of aggressive neuroblastomas, which often express TrkB.     

阻断TrkB-BDNF信号传导通路对神经母细胞瘤细胞生长及凋亡的影响

目的：脑源性神经生长因子(BDNF)和其酪氨酸激酶受体B（TrkB）与神经母细胞瘤(NB) 细胞的化疗耐药及恶性预后密切相关。该研究探讨阻断TrkB-BDNF信号传导通路后，NB细胞对化疗药物敏感性的变化。方法：常规培养SH-SY5Y NB细胞，用nM浓度全反式维甲酸（ATRA）诱导TrkB高表达，加入BDNF、化疗药顺铂（CDDP）和特异性酪氨酸酶抑制剂K252a处理。MTT实验方法检测应用K252a前后细胞的存活率的变化；同时应用Western-blot方法检测应用K252a前后TrkB的磷酸化水平的变化；流式细胞仪（FCM）检测细胞凋亡率；透射电镜（TEM）观察凋亡细胞的形态与结构。结果：ATRA+BDNF+CDDP组细胞的存活率及凋亡率与对照组相比，差异无显著性（P>0.05）。而经K252a处理后，细胞对顺铂的敏感性增加，细胞的存活率明显降低，凋亡率明显升高，差异有显著意义。Western-blot分析显示K252a能够阻断TrkB的磷酸化。结论：阻断TrkB-BDNF信号传导通路可提高NB的化疗敏感性，逆转耐药。     

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.     

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.