BDNF/TrkB signaling protects HT-29 human colon cancer cells from EGFR inhibition

The clinical success of targeted treatment of colorectal cancer (CRC) is often limited by resistance to anti-epidermal growth factor receptor (EGFR) therapy. The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor TrkB have recently emerged as anticancer targets, and we have previously shown increased BDNF levels in CRC tumor samples. Here we report the findings from in vitro experiments suggesting that BDNF/TrkB signaling can protect CRC cells from the antitumor effects of EGFR blockade. The anti-EGFR monoclonal antibody cetuximab reduced both cell proliferation and the mRNA expression of BDNF and TrkB in human HT-29 CRC cells. The inhibitory effect of cetuximab on cell proliferation and survival was counteracted by the addition of human recombinant BDNF. Finally, the Trk inhibitor K252a synergistically enhanced the effect of cetuximab on cell proliferation, and this effect was blocked by BDNF. These results provide the first evidence that increased BDNF/TrkB signaling might play a role in resistance to EGFR blockade. Moreover, it is possible that targeting TrkB could potentiate the anticancer effects of anti-EGFR therapy.     

TrkB在胆管癌中的表达及其临床意义

目的:研究胆管癌和正常胆管组织中TrkB蛋白的表达及其与临床病理特征的关系。方法:免疫组化检测42例胆管癌和10例正常胆管石蜡切片标本中TrkB蛋白的表达并分析其与临床病理因素的关系。结果:正常胆管组织中无TrkB蛋白的表达,胆管癌组织TrkB蛋白的表达率为69.0%(29/42)。TrkB表达与分化程度、有无淋巴结转移和远处转移有关,而与性别、年龄、肿瘤大小无关。TrkB蛋白在低分化组、有淋巴结转移组和有远处转移组中的表达率均高于高中分化组,无淋巴结转移组和无远处转移组(P<0.05)。结论:TrkB在胆管癌组织中的表达可作为预测胆管癌发生淋巴结转移和远处转移的指标。     

Expression of TrkB and BDNF in human cochlea-an immunohistochemical study

Surgical human cochlear specimens were obtained during the removal of large posterior cranial fossa meningioma by a transcochlear approach in which the cochlea was removed for maximal exposure of the tumor and protection of important structures, such as the brainstem, cranial nerves, and pivotal blood vessels. The cochlear tissue was fixed and cryo-sectioned for tyrosine kinase receptor B (TrkB) and brain-derived neurotrophic factor (BDNF) immunohistochemistry. TrkB receptor protein was expressed in both neuronal somata and the processes of human spiral ganglion neurons (SGNs). In the human organ of Corti, TrkB immunoreactivity was mainly present in nerve fibers underneath outer hair cells. BDNF expression was found neither in the organ of Corti nor in the spiral ganglion of human cochlea. For antibody specificity and for control and comparative purposes, TrkB immunocytochemistry was performed in primary cultures of cochlear neuron/glia from adult guinea pig. Confocal laser scanning microscopy showed that TrkB was homogeneously distributed in the cytoplasm of both neuronal somata and axons. Knowledge of the expression of TrkB receptor in human cochlea should help to determine the target structures for neuron preservation in hearing-impaired patients. Our results indicate that the regeneration of SGNs under pathological conditions can be enhanced with BDNF/TrkB-based pharmaceutical or genetic strategies.     

"青春期"大鼠前额皮质内BDNF与trkB的表达

目的:探讨"青春期"大鼠前额皮质内BDNF与trkB的表达。方法:出生后35天大鼠作为"青春期"大鼠,出生后15天、75天大鼠分别作为幼年期与成年期对照,每组大鼠各6只,用ABC免疫组织化学方法与图像分析相结合技术检测前额皮质内BDNF与trkB免疫反应的强度和免疫阳性产物平均光密度值的变化。结果:各时间点大鼠前额皮质内均可见BDNF与trkB免疫阳性产物。35天组BDNF与trkB免疫反应最强,阳性产物平均光密度值最高,与其它两组相比差异有显著性(p<0.05)。结论:"青春期"大鼠前额皮质内BDNF与trkB高表达,提示在此时期内前额皮质对BDNF的需求最多。     

YB-1与EGFR在子宫内膜异位症中表达的研究进展

YB-1,EGFR各自作为冷激蛋白和糖蛋白家族的成员表达于各种生理和环境的损伤之后,保护细胞的生存。近年来发现YB-1和EGFR在多种癌症及内异症中高表达,并且参与肿瘤和子宫内膜异位组织的发生,发展,分化及转移的各个方面。因而YB-1和EGFR的高表达能影响子宫内膜异位症的发生和发展;也说明YB-1和EGFR可以作为子宫内膜异位症患者一个潜在的诊断和治疗靶点。     

Expression of the neurotrophin receptor TrkB in rat spleen macrophages

Increasing evidence suggests that some members of the family of the neurotrophins could be involved in immune system functioning. Both neurotrophins and their tyrosine-kinase signal-transducing receptors, the so-called Trk receptors, have been detected in various lymphoid tissues in a number of species. Nevertheless, their cellular localisation remains unclear in most cases. In this study, we used immunohistochemical techniques to localise TrkB in the rat spleen (from 0 days to 2 years). Cells expressing TrkB-like immunoreactivity were found exclusively within the white pulp of the spleen, along the marginal zone-follicle border and inside the follicles and periarteriolar lymphoid sheaths. These cells probably represented macrophage subpopulations, since they expressed the ED3 rat macrophage antigen. No evidence of TrkB-like protein expression in lymphocytes or follicular dendritic cells could be found. Furthermore, the density of TrkB-immunoreactive cells was observed to increase with age. Although the role of TrkB ligands in these cells remains to be clarified, the present findings provide further evidence for the supposed role of neurotrophins in immune system homeostasis.     

The mechanical and pharmacological regulation of glioblastoma cell migration in 3D matrices

The invasion of glioblastoma is a complex process based on the interactions of tumor cells and the extracellular matrix. Tumors that are engineered using biomaterials are more physiologically relevant than a two-dimensional (2D) cell culture system. Matrix metalloproteinases and the plasminogen activator generated by tumor cells regulate a tumor’s invasive behavior. In this study, microtumors were fabricated by encapsulating U87 glioma cells in Type I collagen and then glioma cell migration in the collagen hydrogels was investigated. Crosslinking of collagen with 8S-StarPEG increased the hydrogel viscosity and reduced the tumor cell migration speed in the hydrogels. The higher migration speed corresponded to the increased gene expression of MMP-2, MMP-9, urokinase plasminogen activator (uPA), and tissue plasminogen activator (tPA) in glioma cells grown in non-crosslinked collagen hydrogels. Inhibitors of these molecules hindered U87 and A172 cell migration in collagen hydrogels. Aprotinin and tranexamic acid did not inhibit U87 and A172 migration on the culture dish. This study demonstrated the differential effect of pharmacologic molecules on tumor cell motility in either a 2D or three-dimensional culture environment.     

Computational analysis of EGFR inhibition by Argos

Argos, a secreted inhibitor of the Drosophila epidermal growth factor receptor, and the only known secreted receptor tyrosine kinase inhibitor, acts by sequestering the EGFR ligand Spitz. We use computational modeling to show that this biochemically-determined mechanism of Argos action can explain available genetic data for EGFR/Spitz/Argos interactions in vivo. We find that efficient Spitz sequestration by Argos is key for explaining the existing data and for providing a robust feedback loop that modulates the Spitz gradient in embryonic ventral ectoderm patterning. Computational analysis of the EGFR/Spitz/Argos module in the ventral ectoderm shows that Argos need not be long-ranged to account for genetic data, and can actually have very short range. In our models, Argos with long or short length scale functions to limit the range and action of secreted Spitz. Thus, the spatial range of Argos does not have to be tightly regulated or may act at different ranges in distinct developmental contexts.     

EGFR和Ki-67在胶质瘤中表达的研究进展

胶质瘤是颅内常见的原发恶性肿瘤,而某些癌基因的激活、过表达或扩增、重排导致脑胶质瘤的形成。主要讨论脑胶质瘤的生物学特点,指出当前研究某些与肿瘤增殖活性及侵袭能力相关的基因改变、蛋白表达,推测其增殖和侵袭活动的具体过程,这是攻克脑胶质瘤的基础和关键。在众多与肿瘤相关的蛋白和基因中,选择了主要反映脑胶质瘤增殖活性和侵袭能力的相关基因——EGFR、Ki-67进行综述。从分子生物学水平评估脑胶质瘤细胞增殖和侵袭状态,在分析和判断胶质瘤的生长、分化程度,指导治疗方案的选择及预后的判断等方面有着重要的实用价值;但对于患者的预后,还需结合年龄、肿瘤位置、病理分级以及其他标记物等进行综合评价。     

Maintenance of EGFR and EGFRvIII expressions in an in vivo and in vitro model of human glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common, and most aggressive primary brain tumor among adults. A vast majority of the tumors express high levels of the epidermal growth factor receptor (EGFR) as a consequence of gene amplification. Furthermore, gene amplification is often associated with mutation of EGFR, and the constitutive activated deletion variant EGFRvIII is the most common EGFR mutation found in GBM. Activated EGFR signaling, through overexpression and/or mutation, is involved in increased tumorigenic potential. As such, EGFR is an attractive target for GBM therapy. However, clinical studies with EGFR inhibitors have shown inconsistent results, and as such, further knowledge regarding the role of EGFR and EGFRvIII in GBM is needed. For this, an appropriate in vivo/in vitro tumor model is required. Here, we report the establishment of an experimental GBM model in which the expressions of EGFR and EGFRvIII are maintained both in xenograft tumors growing subcutaneously on mice and in cell cultures established in stem cell conditions. With this model it will be possible to further study the role of EGFR and EGFRvIII, and response to targeted therapy, in GBM.     

PTEN和EGFR在胃癌中的表达及相关性研究

目的:检测第10号染色体缺失的磷酸酶和张力蛋白同源物基因PTEN及表皮生长因子受体EGFR在胃癌组织中的表达,探讨两者与胃癌肿瘤行为的关系及意义。方法:采用免疫组织化学染色法(S-P法)检测105例胃癌组织及癌旁正常胃组织中EGFR和PTEN蛋白的表达水平,并将检测结果与临床病理参数进行综合分析。结果:胃癌组织PTEN的表达56.19%明显低于正常组织中的表达的表达92.3%,而胃癌组织EGFR的表达46.67%明显高于正常组织7.62%,(P<0.01)。二者的表达与性别和年龄无相关性(P>0.05),与胃癌的分化程度、浸润深度、淋巴结转移及临床分期有相关性(P<0.05)。结论:PTEN在胃癌组织中呈低表达,EGFR在胃癌组织中存在高表达,与胃癌的发生、发展有一定关系,并与胃癌的分化程度、浸润深度、淋巴结转移及临床分期密切相关,二者呈负相关。     

Substrate viscosity impairs temozolomide-mediated inhibition of glioblastoma cells' growth

BackgroundThe mechanical state of the extracellular environment of the brain cells considerably affects their phenotype during the development of central nervous system (CNS) pathologies, and when the cells respond to drugs. The reports on the evaluation of the viscoelastic properties of different brain tumors have shown that both tissue stiffness and viscosity can be altered during cancer development. Although a compelling number of reports established the role of substrate stiffness on the proliferation, motility, and drug sensitivity of brain cancer cells, there is a lack of parallel data in terms of alterations in substrate viscosity.MethodsBased on viscoelasticity measurements of rat brain samples using strain rheometry, polyacrylamide (PAA) hydrogels mimicking elastic and viscous parameters of the tissues were prepared. Optical microscopy and flow cytometry were employed to assess the differences in glioblastoma cells morphology, proliferation, and cytotoxicity of anticancer drug temozolomide (TMZ) due to increased substrate viscosity.ResultsOur results indicate that changes in substrate viscosity affect the proliferation of untreated glioma cells to a lesser extent, but have a significant impact on the apoptosis-associated depolarization of mitochondria and level of DNA fragmentation. This suggests that viscosity sensing and stiffness sensing machinery can activate different signaling pathways in glioma cells.ConclusionCollected data indicate that viscosity should be considered an important parameter in in vitro polymer-based cell culture systems used for drug screening.     

Expression of TrkB Receptors in Developing Visual Cortex Is Not Regulated by Light

1. Neurotrophins are very good candidates which relate electrical activity to molecular changes in activity-dependent phenomena. They exert their action through binding to specific tyrosine-kinase receptors: Trk receptors. It is important to consider Trk distribution in order to understand better the role of neurotrophins in the Central Nervous System (CNS). We focused our attention on brain-derived neurotrophic factor (BDNF) Trk receptors (TrkB) during development of the rat visual cortex, since this neurotrophin has been shown to play an important role in visual system development and plasticity.2. We investigated the full length form of TrkB receptors considering both its total amount and its cellular distribution. To address this issue we used an antibody that recognizes the full length form of TrkB and we used it both in Western blot and immunohistochemistry.3. We found that the expression of TrkB receptor increases during development, but that there is no effect on visual experience, since dark-reared animals show the same protein level and pattern of TrkB expression compared to age-matched, normally reared controls.     

Chronic Administration of Cyclosporine A Changes Expression of BDNF and TrkB in Rat Hippocampus and Midbrain

Neurotrophins, including the brain-derived neurotrophic factor (BDNF), are essential for regulating neuronal differentiation in developing brains. BDNF and its receptor tyrosine kinase receptor B (TrkB) are involved in neuronal signaling, survival and plasticity. Cyclosporine A (CsA) is a potent immunosuppressive agent which prevents allograft rejection in organ transplantation and various immunological diseases. We investigated whether chronic administration of CsA decreases BDNF gene expression in rats, and the influence of CsA on mRNA levels of TrkB receptors was also examined. For 30 days of CsA (10 mg/kg/day) administration, the expression of BDNF and TrkB mRNA was significantly decreased in the hippocampus and midbrain, but there was no significant difference in the cortex. CsA (0, 1, 5 10, 15 ug/ml) down-regulated BDNF and TrkB gene expression through cultured SH-SY5Y cells, as did all-trans retinoic acid (ATRA), and there was no effect on cell viability. These experimental results indicate that suppression of the BDNF and TrkB mRNA, protein level of BDNF expression in the hippocampus and midbrain may be related to altered behavior observed following chronic administration of CsA. A common mechanism of adverse effects of CsA induced depressive symptoms may involve neurotoxicity mediated by down-regulation of brain BDNF and TrkB.     

The Relative Expression of Mig6 and EGFR Is Associated with Resistance to EGFR Kinase Inhibitors

The sensitivity of only a few tumors to anti-epidermal growth factor receptor EGFR tyrosine kinase inhibitors (TKIs) can be explained by the presence of EGFR tyrosine kinase (TK) domain mutations. In addition, such mutations were rarely found in tumor types other than lung, such as pancreatic and head and neck cancer. In this study we sought to elucidate mechanisms of resistance to EGFR-targeted therapies in tumors that do not harbor TK sensitizing mutations in order to identify markers capable of guiding the decision to incorporate these drugs into chemotherapeutic regimens. Here we show that EGFR activity was markedly decreased during the evolution of resistance to the EGFR tyrosine kinase inhibitor (TKI) erlotinib, with a concomitant increase of mitogen-inducible gene 6 (Mig6), a negative regulator of EGFR through the upregulation of the PI3K-AKT pathway. EGFR activity, which was more accurately predicted by the ratio of Mig6/EGFR, highly correlated with erlotinib sensitivity in panels of cancer cell lines of different tissue origins. Blinded testing and analysis in a prospectively followed cohort of lung cancer patients treated with gefitinib alone demonstrated higher response rates and a marked increased in progression free survival for patients with a low Mig6/EGFR ratio (approximately 100 days, P = 0.01).