OBJECTIVETo assess the effects of both TrkA and p75(NTR) on nerve growth factor (NGF)-induced differentiation of neuroblastoma cells.

METHODSRetroviral vectors were constructed to express the high affinity NGF receptor (TrkA) and low affinity NGF receptor (p75(NTR)). Neuroblastoma cell line IMR-32 was transfected by the vectors expressing either TrkA or p75(NTR) or both by using lipofectmine trade mark reagent separately or cotransfected at the same time. Southern blot, Northern blot, RT-PCR and flow cytometry were used to determine the success of the transfection. MTT technique was to monitor the cell proliferation. Colony formation in soft agar and tumor forming assay in nude mice were used to test the biological characteristics of the tumor cells. Terminal-deoxynucleotidytransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was used to test the apoptosis of the tumor cells.

RESULTSStable transformant cell lines expressing TrkA, p75(NTR) or both genes were established. Studies on these transformant cell lines have shown different NGF responses. The p75(NTR) transfection only resulted in the mild differentiation response, and transfection of TrkA gene caused remarkable neurite extension, up-regulation of neurofilament and decreased expression of N-myc oncogene after NGF treatment. The cotransfection of the two genes into this cell line resulted in the more rapid and more apparent morphological changes than single TrkA transfected cells after NGF treatment. The cotransfected cells underwent apoptosis after withdrawal of NGF.

CONCLUSIONSThe results indicate that coexpression of both low- and high-affinity NGF receptors are not only more efficient in restoration of NGF-induced differentiation pathway, but also be able to activate the pro-apoptotic activity of low-affinity NGF receptor and make the tumor cells become NGF-dependent and irreversibly differentiated.

Animals ; Apoptosis ; Cell Differentiation ; Genes, myc ; Humans ; Mice ; Mice, Nude ; Nerve Growth Factor ; pharmacology ; Neuroblastoma ; pathology ; Receptor, Nerve Growth Factor ; Receptor, trkA ; physiology ; Receptors, Nerve Growth Factor ; physiology ; Transfection ; Tumor Cells, Cultured

The neurotrophin receptor kinase (NTRK) gene encodes neurotrophic factor receptor tyrosine kinase (NTRK), which plays an important role in the development and function of the nervous system. NTRK gene fusion mutation results in the production of chimeric NTRK proteins, which have carcinogenic potential through constitutive activation or overexpression. NTRK gene fusion mutation can lead to a special type of wild type gastrointestinal stromal tumor (GIST), whose clinical manifestations and treatment are completely different from other types of GIST. This fusion mutation can be detected clinically by a variety of methods, including tumor DNA and RNA sequencing and immunohistochemical staining. In patients with NTRK fusion positive tumors, NTRK inhibitors such as larotrectinib and entrectinib have shown good antitumor efficacy, with clinical response rates as high as 75%. Therefore, there is a need to improve the recognition and detection of fuch patients and to improve their prognosis by individualized and precise treatment with TRK inhibitors.
Gastrointestinal Stromal Tumors/genetics* ; Gene Fusion ; Humans ; Neoplasms ; Nerve Growth Factors ; Protein Kinase Inhibitors ; Receptor, trkA/genetics* ; Receptors, Nerve Growth Factor/genetics*

OBJECTIVETo investigate the role of p75 neurotrophin receptor (p75NTR) in the regeneration of facial nerve crush injury.

METHODSIn p75NTR knockout mice and wild type mice, the regenerating fibres in the facial nerve were also labelled by an anterograde tracer cholera toxin B (CTB). The next day after injury of facial nerve, CTB was injected into the trunk of the nerve in the proximal side of the crush, and then anterograde tracing and immunohistochemistry were used to examine the regeneration of axons after facial nerve crush injury. In p75NTR knockout mice and wild type mice, the facial nerves on one side were crushed and regenerating neurons in the facial nerve nucleus were labelled by Fast Blue. The facial nerve trunk was cut in the bifurcated region in the 4th day after injury and the stump was inserted into a small polymer tube containing Fast Blue. Retrograde tracing and labling motoneuron counting were used to examine the survival of motoneurons in the facial nerve nucleus after facial nerve crush injury.

RESULTSThe results showed that the axonal growth of injured axons in the facial nerve of p75NTR knockout mice was significantly retarded. The number of regenerated neurons in the facial nerve nucleus in p75NTR knockout mice was significantly reduced (P < 0.05). Immunohistochemical staining of regenerating axons also showed the reduction in nerve regeneration in p75NTR knockout mice (P < 0.01).

CONCLUSIONp75NTR plays an important role in the regeneration of injured peripheral nerves after injury.

Animals ; Axons ; Facial Nerve ; Mice ; Motor Neurons ; Nerve Regeneration ; Neurons ; Receptor, Nerve Growth Factor ; Receptors, Nerve Growth Factor

OBJECTIVETo investigate the expressions and action mechanisms of nerve growth factor (NGF) receptors TrkA and p75NTR in the oncogenesis and progression of prostate cancer (PCa).

METHODSUsing immunohistochemistry, we detected the expressions of TrkA and p75NTR in 62 PCa and 35 benign prostatic hyperplasia (BPH) samples, and conducted statistical analysis on the basis of clinical data.

RESULTSIndependent-samples t-test showed that, along with poorer tissue differentiation or higher clinical stage of PCa, the expression of TrkA was significantly up-regulated, that of p75NTR remarkably down-regulated, and the expression ratio of TrkA to p75NTR markedly increased. The TrkA/p75NTR ratio was 0.32 in the BPH, 0.52 in the PCa tissue with Gleason score of 6, 1.65 in the PCa tissue with Gleason score of 7, 5.75 in the PCa tissue with Gleason score ≥ 8, 0.89 in the clinical stage of pT2, 1.5 in pT3 a, 3.75 in pT3b, and 7.00 in pTxN1.

CONCLUSIONThe abnormally increased expression ratio of TrkA to p75NTR might be one of the essential features of malignant transformation of prostate cells. A higher TrkA/p75NTR expression ratio may be associated with a lower tissue differentiation, a higher clinical stage or Gleason score, and therefore a poorer prognosis.

Humans ; Immunohistochemistry ; Male ; Neoplasm Grading ; Neoplasm Staging ; Nerve Tissue Proteins ; metabolism ; Prognosis ; Prostatic Hyperplasia ; pathology ; Prostatic Neoplasms ; pathology ; Receptor, trkA ; metabolism ; Receptors, Nerve Growth Factor ; metabolism ; Up-Regulation

OBJECTIVE: To determine the expression of nerve growth factor (NGF), tyrosine kinase receptor A (trkA), and pan-neurotrophin receptor (p75) in the lung tissues in asthmatic rats, and to explore their effects on the airway inflammation. METHODS: Thirty-two SD rats were randomly divided into 4 groups: the control, asthma, NGF and anti-NGF groups. The asthmatic model was established by the inhalation and injection of ovalbumin. The total cell count and differential cell count in the bronchoalveolar lavage fluid (BALF) were performed. The pathologic changes in the lung tissues of the 4 groups was detected by HE staining. The NGF mRNA expression in the lung tissues of the asthma and control groups was determined by reverse transcription-polymerase chain reaction (RT-PCR). The changes of trkA and p75 mRNA expressions in the lung tissues in the 4 groups were also investigated by RT-PCR. RESULTS: Compared with the control group, the BALF total cell, the BALF eosinophils (Eos), and the BALF lymphocytes (Lyms) significantly increased (All P <0. 001) in the asthma group; and the lung tissues of the asthma group had more infiltrating inflammatory cells. Not only the expression of NGF mRNA, but also its receptors trkA and p75 mRNA in the lung tissues were significantly higher in the asthma group than those in the control group (All P < 0.01). Positive correlation was found between the expression of NGF mRNA and the BALF total cell, the BALF Lyms in the asthma group. Compared with the asthma group, the total cell, the Eos, and the lyms in BALF in the NGF group significantly increased (All P < 0.01), and the lungs of the NGF group had apparent inflammatory changes. The expre-ssions of p75 and trkA mRNA were enhanced significantly (All P < 0.05). Compared with the asthma group, the total cell, the Eos, and the lyms in BALF in the anti-NGF group significantly decreased (All P < 0.001), and the lungs of the anti-NGF group showed alleviative inflammatory changes. The expre-ssions of p75 and trkA mRNA significantly decreased (All P < 0.01). CONCLUSION In lungs of asthmatic rats, the elevated expression of NGF mRNA is closely related to the airway inflammation. NGF can upregulate the expressions of p75 and trkA mRNA in asthmatic rats, and then may promote their role in the airway neuronal inflammation in asthma.
Animals ; Asthma ; chemically induced ; metabolism ; Bronchitis ; metabolism ; Lung ; metabolism ; Male ; Nerve Growth Factor ; metabolism ; Ovalbumin ; RNA, Messenger ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptor, trkA ; metabolism ; Receptors, Nerve Growth Factor ; metabolism

OBJECTIVE: Nerve growth factor (NGF) is a member of the neurotrophic factor family and plays a vital role in the physiological processes of organisms, especially in the nervous system. Many recent studies have reported that NGF is also involved in the regulation of tumourigenesis by either promoting or suppressing tumor growth, which depends on the location and type of tumor. However, little is known regarding the effect of NGF on interspinal schwannoma (IS). In the present study, we aimed to explored whether mouse nerve growth factor (mNGF), which is widely used in the clinic, can influence the growth of interspinal schwannoma cells (ISCs) isolated from IS in vitro.METHODS: ISCs were isolated, cultured and identified by S-100 with immunofluorescence analysis. S-100-positive cells were divided into five groups, and separately cultured with various concentrations of mNGF (0 [phosphate buffered saline, PBS], 40, 80, 160, and 320 ng/mL) for 24 hours. Western blot and quantantive real time polymerase chain reaction (PCR) were applied to detect tyrosine kinase A (TrkA) receptor and p75 neurotrophin receptor (p75(NTR)) in each group. Crystal violet staining was selected to assess the effect of mNGF (160 ng/mL) on ISCs growth.RESULTS: ISCs growth was enhanced by mNGF in a dose-dependent manner. The result of crystal violet staining revealed that it was significantly strengthened the cells growth kinetics when cultured with 160 ng/mL mNGF compared to PBS group. Western blot and quantantive real time PCR discovered that TrkA receptor and mRNA expression were both up-regualated under the condition of mNGF, expecially in 160 ng/mL, while the exoression of p75(NTR) demonstrated no difference among groups.CONCLUSION: From these data, we conclude that exogenous mNGF can facilitate ISC growth by activating both TrkA receptor and p75(NTR). In addition, patients who are suffering from IS should not be administered mNGF in the clinic.
Animals ; Blotting, Western ; Fluorescent Antibody Technique ; Gentian Violet ; Humans ; In Vitro Techniques ; Kinetics ; Mice ; Nerve Growth Factor ; Nervous System ; Neurilemmoma ; Physiological Processes ; Protein-Tyrosine Kinases ; Real-Time Polymerase Chain Reaction ; Receptor, Nerve Growth Factor ; Receptor, trkA ; Receptors, Nerve Growth Factor ; RNA, Messenger

BACKGROUND: Nerve growth factor (NGF) promotes the survival and differentiation of vertebrate neurons, and their actions are mediated by two classes of cell surface receptors: tyrosine kinase A receptor (TrkA) and p75 neurotrophic receptor (p75NTR). We evaluated the role of NGF receptors in neuronal survival and the physical interactions between them. METHODS: Organotypic hippocampal slices were obtained from 5 to 7-day-old rat pups and were grown for 14 days in vitro. The expression of the TrkA and p75NTR was evaluated by the western blot and immunohistochemical methods. The neuroprotective effect of NGF on the blocking of antibody-induced neuronal cell death was tested by the application of NGF (0, 50 and 150 ng/ml) to the culture media in the presence of 200 ng/ml of blocking antibodies against TrkA and p75NTR. Functional interactions between the two receptors were examined using the immunoprecipitation method. RESULTS: TrkA and p75NTR were co-expressed in the principal neurons of the hippocampal slice culture, and the expression level was increased time dependently until 14 days of culture. The blocking antibody against each receptor induced neuronal damage in time and dose-dependent manners. NFG delayed or prevented the blocking antibody from inducing neuronal damage. Results from the immunoprecipitation experiment showed physical interactions between the two NGF receptors. CONCLUSIONS: Our results indicate that the co-expressed NGF receptors, TrkA and p75NTR, might have protective roles in the survival of neuronal cells through the cooperative interactions between them.
Animals ; Antibodies, Blocking ; Blotting, Western ; Cell Death ; Culture Media ; Immunoprecipitation ; Nerve Growth Factor ; Neurons* ; Neuroprotective Agents ; Protein-Tyrosine Kinases ; Rats ; Receptor, Nerve Growth Factor ; Receptors, Cell Surface ; Receptors, Nerve Growth Factor ; Vertebrates

OBJECTIVETo investigate the effects of high-affinity tyrosine kinase receptors TrkB, TrkC and the low-affinity neurotrophin receptor p75 in spiral ganglion cell (SGC) of cisplatin-induced ototoxicity.

METHODSThe 50 adult Wistar rats were divided randomly into 5 groups received intraperitoneal injection of cisplatin with vary dose. Control group was received equivalent volumes of saline. The group received 1 day intraperitoneal injection was cisplatin treated at a dose of 5 mg/kg and killed at next day. The group received 3 days was cisplatin treated for 3 days at same dose daily and then killed at next day. The group A received 5 days was cisplatin treated for 5 days and killed at next day. The group B received 5 days was cisplatin treated for 5 days and then were sacrificed after 7 days. The change of mRNA level of neurotrophin receptors in cochlear tissue were examined by RT-PCR. The expressing pattern of TrkB, TrkC, P75 in damaged cochlea were study by immunochemistry using antibodies against TrkB, TrkC, P75 protein.

RESULTSThe research data showed the expression of Trk B, Trk C, p75 exhibited in SGC was dynamic along with the administration lasting. The mRNA and protein level of Trk B (x(-) +/- s) at day 1 and 3 after cisplatin treatment were 0.76 +/- 0.06, 88.78 +/- 4.28, 0.82 +/- 0.09 and 91.64 +/- 4.06, with significant difference among those and other groups (P < 0.05). The mRNA and protein level of TrkC at day 1 after cisplatin treatment were 0.80 +/- 0.06 and 89.66 +/- 2.76, with significant difference among that and other groups (P < 0.05). The mRNA and protein level of p75 at the control group and cisplatin treated groups were 0.64 +/- 0.04, 55.16 +/- 3.10, 0.77 +/- 0.04, 78.46 +/- 3.86, 1.01 +/- 0.09, 105.02 +/- 6.61, 1.18 +/- 0.09, 111.10 +/- 6.08, 0.51 +/- 0.04 and 42.74 +/- 5.20, with significant difference among the control group and cisplatin treated groups (P < 0.05).

CONCLUSIONSThe expression of Trk B increased to peak at day 1 - 3 after cisplatin treatment and decreased at day 5 early and following weeks. The expression of Trk C went up to peak at day 1 after cisplatin treatment and went down during subsequently time. P75 kept a trend of continuance increased during the drug treatment and decrease at drug stopped. The expression of Trk B, Trk C and P75 may be involved in cochlear insult with cisplatin-induced. Trk B and Trk C may play an important role in the reparative process of cochlear, especially at early stage of the damage. P75 could promote SGC apoptosis in cisplatin-induced neurotoxicity.

Animals ; Cisplatin ; toxicity ; Male ; Rats ; Rats, Wistar ; Receptor, Nerve Growth Factor ; metabolism ; Receptor, trkB ; metabolism ; Receptor, trkC ; metabolism ; Spiral Ganglion ; drug effects ; metabolism

Adeonoid cystic carcinoma (ACC) is one of the most common malignant tumors of salivary glands. It is characterized by a relentless regrowth especially around nerve tissues and a high rate of hematogenous distant metastasis. Clinically most deaths from salivary ACC are caused by delayed lung metastases that are resistant to conventional chemotherapy. So, knowledge of cellular and molecular properties that influence the dissemination of metastatic tumor cells, is important for new treatment strategies of metastatic lesions. We determined expressions of angiogenic signaling molecules microvessel density (MVD) using surgical specimens of human salivary ACC. Protein expressions of vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)-2, activated VEGFR-2, and human CD31 were assessed in 20 cases of salivary ACC by immunohistochemical staining. Most of the tumors, especially ACC with a tubulocribriform pattern, were positive for antibodies of VEGF, VEGFR-2, and activated VEGFR-2. The overall percentages of the 20 specimens expressing VEGF, VEGFR-2, activated VEGFR-2 were 90, 95, and 95%, respectively. Immunoreactivities of the biomarkers in salivary ACC were higher than those in normal salivary gland. Furthermore, immune-related cells as well as tumor cells expressed VEGF/VEGFR-2. Microvessel density of salivary ACC was higher than that of normal salivary gland (P<0.05). Taken together, angiogenic signaling molecules are actively expressed in salivary ACC. And we suggest that these molecules may have critical role in the hematogenous spread of salivay ACC, which has a propensity for delayed lung metastasis. Therefore, these biomarkers can be molecular targets for therapy of metastasis of salivary ACC.
Adenoids* ; Antibodies ; Biomarkers ; Carcinoma, Adenoid Cystic* ; Drug Therapy ; Humans* ; Lung ; Microvessels* ; Neoplasm Metastasis ; Nerve Tissue ; Receptors, Vascular Endothelial Growth Factor ; Salivary Glands* ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factor Receptor-2

Enhancing adult nerve regeneration is a potential therapeutic strategy for treating spinal cord injury. Vascular endothelial growth factor (VEGF) is a major contributor to angiogenesis, which can reduce the spinal cord injury by inhibiting the inflammation and improve recovery after spinal cord injury. We have previously demonstrated that exogenous VEGF has neurotrophic effects on injured spinal nerves in organotypic spinal cord slice cultures. However, the mechanisms underlying the neurite growth by exogenous VEGF remain to be explored in spinal cord. In this study, we found out that exogenous VEGF mediated axonal outgrowth through VEGF receptor 1 (VEGFR1) and VEGFR2, both of which were expressed on organotypic spinal cord slices. Although VEGFR1 and VEGFR2 were constitutively expressed in some cells of control spinal cord slices, VEGF treatment upregulated expression of VEGFR1 and VEGFR2. Both VEGFR1 and VEGFR2 were expressed in neuronal cells as well as glial cells of organotypic spinal cord slices. We also observed that VEGF-induced axonal outgrowth was attenuated by a specific mitogen-activated protein kinase (MAPK) inhibitor PD98059 and a specific phosphoinositide 3-kinase (PI3K) inhibitor wortmannin. Thus, these findings suggest that these MAPK and PI3K pathways have important roles in regulating VEGF-induced axonal outgrowth in the postnatal spinal cord.
Adult ; Axons* ; Humans ; Inflammation ; Nerve Regeneration ; Neurites ; Neuroglia ; Neurons ; Protein Kinases ; Receptors, Vascular Endothelial Growth Factor* ; Spinal Cord Injuries ; Spinal Cord* ; Spinal Nerves ; Vascular Endothelial Growth Factor A* ; Vascular Endothelial Growth Factor Receptor-1*