Major vault protein promotes locomotor recovery and regeneration after spinal cord injury in adult zebrafish.

In contrast to mammals, adult zebrafish recover locomotor functions after spinal cord injury (SCI), in part due to axonal regrowth and regeneration permissivity of the central nervous system. Upregulation of major vault protein (MVP) expression after spinal cord injury in the brainstem of the adult zebrafish prompted us to probe for its contribution to recovery after SCI. MVP is a multifunctional protein expressed not only in many types of tumours but also in the nervous system, where its importance for regeneration is, however, unclear. Using an established zebrafish SCI model, we found that MVP mRNA and protein expression levels were increased in ependymal cells in the spinal cord caudal to the lesion site at 6 and 11 days after SCI. Double immunolabelling showed that MVP was co-localised with Islet-1 or tyrosine hydroxylase around the central canal of the spinal cord in sham-injured control fish and injured fish 11 days after surgery. MVP co-localised with the neural stem cell marker nestin in ependymal cells after injury. By using an in vivo morpholino-based knock-down approach, we found that the distance moved by MVP morpholino-treated fish was reduced at 4, 5 and 6 weeks after SCI when compared to fish treated with standard control morpholino. Knock-down of MVP resulted in reduced regrowth of axons from brainstem neurons into the spinal cord caudal to the lesion site. These results indicate that MVP supports locomotor recovery and axonal regrowth after SCI in adult zebrafish.

[Expression of gdnf and nos in adult zebrafish brain during the regeneration after spinal cord injury].

Recently, it is unclear about the mechanism of notable regenerated ability of adult zebrafish after spinal cord injury. To investigate the effects of brain on restoration from spinal cord injury, adult zebrafish spinal cord injury model was built and brain samples were dissected at different time points after the injury. Real-time quantitative PCR and in situ hybridization were applied to reveal the dynamics of glial cell line-derived neurotrophic factor (gdnf) and nitric oxide synthases (nos) mRNA expression in various regions of zebrafish brain. The results showed that, compared to sham group at each time points separately, the expression of gdnf mRNA in adult zebrafish brain during both acute phase (4 h and 12 h) and chronic phase of neuroregeneration (6 d and 11 d) increased significantly (P<0.05). The expression of nos mRNA in zebrafish brain enhanced during acute phase, and then reduced to the level lower than the sham group during the chronic phase of neuroregeneration (11 d) (P<0.05). This suggests that brain may promote neural axons regeneration in spinal cord via a more beneficial microenvironment which retains higher level of gdnf and lower level of nos.

Neuregulin 1 enhances cell adhesion molecule L1 like expression levels and promotes malignancy in human glioma.

Neural cell adhesion molecular L1-like protein (CHL1) is a member of the cell adhesion molecule L1 family and serves an important role in the development and progression of tumors. The cytokine neuregulin 1 (NRG1) has been indicated in the tumorigenesis and promotion of metastasis through the modulation of L1. However, the roles of NRG1 in regulating CHL1 in glioma have not been elucidated. The present study investigated the protein expression levels and roles of CHL1 and the possible correlation between NRG1 and CHL1 protein expression levels in human gliomas, both in vivo and in vitro. Using immunohistochemistry coupled with a human glioma tissue microarray, it was demonstrated that the percentage of CHL1-positive areas was the highest in grade II glioma tissues. Using immunofluorescence staining, a positive correlation was identified between the expression levels of CHL1 and proliferating cell nuclear antigen. In addition, CHL1 downregulation also resulted in increased senescence of U-87 MG human glioblastoma cells. In vitro, administration of NRG1α induced a significant increase in CHL1 protein expression levels in human glioma SHG-44 and U251 cells and in human glioblastoma U-87 MG cells, whereas NRG1ß failed to increase CHL1 expression levels in U251 cells. These findings were further confirmed by the downregulation of NRG1 expression levels using small interfering RNA treatment, which resulted in the reduction of CHL1 protein expression levels in U-87 MG cells. These data indicate that NRG1 can regulate CHL1 protein expression levels in gliomas, that it is correlated with malignancy, and that NRG1 may contribute to malignancy by upregulating CHL1 protein expression levels in glioma/glioblastoma cells.

Trimebutine Promotes Glioma Cell Apoptosis as a Potential Anti-tumor Agent.

Gliomas are the most common primary brain tumors with a usually fatal malignancy. They are associated with a poor prognosis although multiple therapeutic options have been available. Trimebutine is one of the prokinetic agents and it has been mainly used for treatment of disorders of the gastrointestinal (GI) tract such as irritable bowel syndrome. However, its effects on glioma cells remain unknown. Here, we used various concentrations of trimebutine to treat SHG44, U251, and U-87 MG human glioma/glioblastoma cells. And combined experiments of MTT, colony formation assay, and wound healing assay, as well as western blot and immunofluorescence staining were used to evaluate the effects of trimebutine on glioma cells. The results demonstrated that trimebutine significantly inhibited cell viability and colony formation. A significant inhibition of glioma cell migration was also indicated by wound healing assay. In addition, trimebutine promoted cell apoptosis and induced Bcl-2 downregulation, accompanied with Bax upregulation. Both immunofluorescence staining and western blot results showed that trimebutine increased the level of active Caspase-3. Moreover, trimebutine reduced the activation of both AKT and ERK signaling pathways. In subcutaneous U-87 MG cell xenograft tumors in nude mice, trimebutine significantly inhibited tumor growth. More TUNEL-positive apoptotic cells in tumor sections were observed in trimebutine-treated mice when compared to the vehicle control. Reduced Bcl-2 and upregulated Bax, as well as perturbed p-AKT and p-ERK signaling pathways were also observed in trimebutine-treated xenograft tissues. Our combined data indicated that trimebutine may be potentially applied for the clinical management of glioma/glioblastoma.

Quercetin promotes cell apoptosis and inhibits the expression of MMP-9 and fibronectin via the AKT and ERK signalling pathways in human glioma cells.

Gliomas are the most common and malignant primary brain tumours and are associated with a poor prognosis despite the availability of multiple therapeutic options. Quercetin, a traditional Chinese medicinal herb, is an important flavonoid and has anti-cancer activity. Here, we evaluated whether quercetin could inhibit glioma cell viability and migration and promote apoptosis. The treatment of U87-MG glioblastoma and U251 and SHG44 glioma cell lines with different concentrations of quercetin inhibited cell viability in a dose-dependent manner. Wound healing assays indicated that quercetin significantly decreased glioma cell migration. ß-galactosidase staining, DNA staining and Annexin V-EGF/PI double staining assays demonstrated that quercetin promoted cell senescence and apoptosis. In addition, the protein levels of p-AKT, p-ERK, Bcl-2, matrix metallopeptidase 9 (MMP-9) and fibronectin (FN) were significantly reduced following quercetin treatment. Therefore, we conclude that quercetin might inhibit the viability and migration and promote the senescence and apoptosis of glioma cells by suppressing the Ras/MAPK/ERK and PI3K/AKT signalling pathways. Quercetin might be a potential candidate for the clinical treatment of glioma.

HMGB1 contributes to regeneration after spinal cord injury in adult zebrafish.

High mobility group box 1 (HMGB1, also called amphoterin) facilitates neurite outgrowth in early development, yet can exacerbate pathology and inhibit regeneration by inducing adverse neuroinflammation when released from dying cells, suggesting that HMGB1 plays a critical, yet undefined role in neuroregeneration. We explored whether HMGB1 contributes to recovery after complete spinal cord transection in adult zebrafish. Quantitative PCR and in situ hybridization revealed that HMGB1 mRNA levels decreased between 12 h to 11 days after spinal cord injury (SCI), then returned to basal levels by 21 days. Western blot and immunohistological analyses indicated that the time course of HMGB1 protein expression after SCI parallels that of mRNA. Immunofluorescence staining revealed that HMGB1 translocates from nuclei into the cytoplasm of spinal motoneurons at 4 and 12 h (acute stage) following SCI, then accumulates in the nuclei of motoneurons during the ensuing chronic stage (after 6 days following SCI). Immunohistology of transgenic zebrafish, expressing green fluorescent protein in blood vessels, showed enhanced HMGB1 expression in blood vessels in the vicinity of motoneurons. Application of anti-sense HMGB1 morpholinos inhibited locomotor recovery by 34 % and decreased axonal regeneration by 34 % compared to fish treated with a control morpholino. The present study shows that HMGB1 expression increases in both endothelial cells and motoneurons, suggesting that HMGB1 promotes recovery from SCI not only through enhancing neuroregeneration, but also by increasing angiogenesis. The inflammatory effects of HMGB1 are minimized through the decrease in HMGB1 expression during the acute stage.

A surgery protocol for adult zebrafish spinal cord injury.

Adult zebrafish has a remarkable capability to recover from spinal cord injury, providing an excellent model for studying neuroregeneration. Here we list equipment and reagents, and give a detailed protocol for complete transection of the adult zebrafish spinal cord. In this protocol, potential problems and their solutions are described so that the zebrafish spinal cord injury model can be more easily and reproducibly performed. In addition, two assessments are introduced to monitor the success of the surgery and functional recovery: one test to assess free swimming capability and the other test to assess extent of neuroregeneration by in vivo anterograde axonal tracing. In the swimming behavior test, successful complete spinal cord transection is monitored by the inability of zebrafish to swim freely for 1 week after spinal cord injury, followed by the gradual reacquisition of full locomotor ability within 6 weeks after injury. As a morphometric correlate, anterograde axonal tracing allows the investigator to monitor the ability of regenerated axons to cross the lesion site and increasingly extend into the gray and white matter with time after injury, confirming functional recovery. This zebrafish model provides a paradigm for recovery from spinal cord injury, enabling the identification of pathways and components of neuroregeneration.

The cell neural adhesion molecule contactin-2 (TAG-1) is beneficial for functional recovery after spinal cord injury in adult zebrafish.

The cell neural adhesion molecule contactin-2 plays a key role in axon extension and guidance, fasciculation, and myelination during development. We thus asked, whether contactin-2 is also important in nervous system regeneration after trauma. In this study, we used an adult zebrafish spinal cord transection model to test the functions of contactin-2 in spinal cord regeneration. The expression patterns of contactin-2 at different time points after spinal cord injury were studied at the mRNA level by qPCR and in situ hybridization, and contactin-2 protein levels and immunohistological localization were detected by Western blot and immunofluorescence analyses, respectively. Contactin-2 mRNA and protein levels were increased along the central canal at 6 days and 11 days after spinal cord injury, suggesting a requirement for contactin-2 in spinal cord regeneration. Co-localization of contactin-2 and islet-1 (a motoneuron marker) was observed in spinal cords before and after injury. To further explore the functions of contactin-2 in regeneration, an anti-sense morpholino was used to knock down the expression of contactin-2 protein by application at the time of injury. Motion analysis showed that inhibition of contactin-2 retarded the recovery of swimming functions when compared to standard control morpholino. Anterograde and retrograde tracing at 6 weeks after injury showed that knock down of contactin-2 inhibited axonal regrowth from NMLF neurons beyond lesion site. The combined observations indicate that contactin-2 contributes to locomotor recovery and successful regrowth of axons after spinal cord injury in adult zebrafish.

Antibody fragments directed against different portions of the human neural cell adhesion molecule L1 act as inhibitors or activators of L1 function.

The neural cell adhesion molecule L1 plays important roles in neuronal migration and survival, neuritogenesis and synaptogenesis. L1 has also been found in tumors of different origins, with levels of L1 expression correlating positively with the metastatic potential of tumors. To select antibodies targeting the varied functions of L1, we screened the Tomlinson library of recombinant human antibody fragments to identify antibodies binding to recombinant human L1 protein comprising the entire extracellular domain of human L1. We obtained four L1 binding single-chain variable fragment antibodies (scFvs), named I4, I6, I13, and I27 and showed by enzyme-linked immunosorbent assay (ELISA) that scFvs I4 and I6 have high affinity to the immunoglobulin-like (Ig) domains 1-4 of L1, while scFvs I13 and I27 bind strongly to the fibronectin type III homologous (Fn) domains 1-3 of L1. Application of scFvs I4 and I6 to human SK-N-SH neuroblastoma cells reduced proliferation and transmigration of these cells. Treatment of SK-N-SH cells with scFvs I13 and I27 enhanced cell proliferation and migration, neurite outgrowth, and protected against the toxic effects of H(2)O(2) by increasing the ratio of Bcl-2/Bax. In addition, scFvs I4 and I6 inhibited and scFvs I13 and I27 promoted phosphorylation of src and Erk. Our findings indicate that scFvs reacting with the immunoglobulin-like domains 1-4 inhibit L1 functions, whereas scFvs interacting with the fibronectin type III domains 1-3 trigger L1 functions of cultured neuroblastoma cells.

Mismatch repair, minichromosome maintenance complex component 2, cyclin A, and transforming growth factor ß receptor type II as prognostic factors for colorectal cancer: results of a 10-year prospective study using tissue microarray analysis.

BACKGROUND: The expression of genes encoding a number of pathogenetic pathways involved in colorectal cancer could potentially act as prognostic markers. Large prospective studies are required to establish their relevance to disease prognosis. METHODS: We investigated the relevance of 19 markers in 790 patients enrolled in a large randomised trial of 5-fluorouracil using immunohistochemistry and chromogenic in situ hybridisation. The relationship between overall 10-year survival and marker status was assessed. RESULTS: Minichromosome maintenance complex component 2 (MCM2) and cyclin A were significantly associated with overall survival. Elevated MCM2 expression was associated with a better prognosis (HR = 0.63, 95%CI: 0.46 - 0.86). Cyclin A expression above the median predicted an improved patient prognosis (HR = 0.71, 95%CI: 0.53 - 0.95). For mismatch repair deficiency and transforming growth factor ß receptor type II (TGFBRII) overexpression there was a borderline association with a poorer prognosis (HR = 0.69, 95%CI: 0.46 - 1.04 and HR = 2.11, 95%CI: 1.02 - 4.40, respectively). No apparent associations were found for other markers. CONCLUSION: This study identified cell proliferation and cyclin A expression as prognostic indicators of patient outcome in colorectal cancer.