Study On The Role And Mechanism Of S100B In Growth Of Glioma

Glioma is the most familiar malignant tumor in Neurosurgery. It is a neuroepithelial tumor which is characterized by the growth of invasion. Its common clinical symptoms consist of increased intracranial pressure and the corresponding focal symptoms. It is of high malignant degree and without a clear boundary to surrounding tissue. Gioma grow rapaidly and has a strong aggressive character. It cannot be get rid of completely through resection so far and easy to relapse after surgery, and effect of chemotherapy is poor due to the blood-brain barrier. The useful therapy methods nowdays are surgery, radiotherapy, chemotherapy, immunotherapy, gene therapy, microwave therapy and some other treatment methods available. The prognosis of patients with glioma is very poor; the median survival time is18months.S100B is one of the calcium-binding protein gene family members and plays a role in cells’ metabolism, proliferation and movement. Studies have shown that astrocytes in the nervous system release S100B protein with mmol concentrations to the extracellular fluid, thereby promoting axonal growth and to protect neurons. S100B protein concentration increased to micromolar concentration levels under pathological conditions including cancer, inflammation, so as to stimulate microglia and astrocytes, and can increase the expression of pro-inflammatory cytokines. S100B protein is overexpressed in the majority of gliomas. Studies generally thought that cancer occurs for the inhibition of the tumor suppressor protein p53, and through stimulates the mitogenic kinase Ndr and Akt to regulate cell’s proliferation and differentiation. S100B protein concentration is related to the growth of glioma and function of astrocytes. S100B protein may contribute to the differentiation of astrocytes and may promote the activation and motility of astrocyte in the central nervous system. However, the role of S100B in gliomagensis, as well as the mechanism of action of S100B, is not widely studied and still unclear.ObjectiveCurrently the role of S100B protein in gliomagensis and proliferation has not been extensively studied. Earlier studies have shown that even low concentrations of S100B protein can activate Stat3thereby regulating the activity of tumor-associated macrophages (TAM). Therefore, we assumed that S100B protein expression also can promote the growth and proliferation of glioma in vivo by regulating the activity of TAM.In this study, we used a variety of testing methods to observe the role of S100B protein in glioma tumor growth and angiogenesis, in order to figure out the role of S100B in gliomagensis and proliferation, clarify the mechanism of S100B promote the growth of glioma, and to explore the potential value of S100B inhibitors in the treatment of glioma targeting S100B protein.Methods1. Three stablely transfected cell lines:stablely transfected cell lines S100Bwt, S1OOBlow and S1OOBhigh.2. Effect of S100B to growth of glioma:2.1S100B effections on cells growth of GL261cell lines in vitro:Using Western blot and Protein Quantitation, ELISA to detect the expression of S100B in three cell lines. Using the technology of Cell counting and Flow cytometry to detect the growth rate of three cell lines.2.2Effections of S100B to growth of three cell lines in animal model:Using fluorescent immunohistochemistry, the actual tumor size, the survival test, angiography in living animals to detect the effects of S100B on the glioma in animal models.2.3Effect of inhibited S100B on growth of glioma in animal models:Using Western blot, mouse survival test, flow cytometry to detect the influence of inhibited S100B on tumor growth.2.4Effects of S100B on inflammation and macrophage infiltration:Use NF-KB assay, RT-PCR and flow cytometry to detect the influencea of S100B on infammation and macrophage infiltration conditions.3. RAGE affects the growth of glioma:Using Western blot, immunofluorescence, RT-PCR, flow cytometry to detect the influences of RAGE in the growth of glioma.4. CLL2affects the growth of glioma:Using ELISA, Western blot, immunofluorescence, RT-PCR to detect the influences of CLL2in growth of glioma.5. Correaltion of S100B with glioma:Using The Cancer Genome Atlas analysis to detect the relationship between S100B and tumor subtypes and correlation of S100B and CLL2in all glioma subtypes.Results1. S100B promotes growth of glioma in vivo:We found that the growth rate of glioma cells in three GL261cell lines are silimar in vitro. On the contrary, tumor growth rate and tumor angiogenesis speed is different in three cell lines in vivo.2. S100B promotes development of inflammation and enhances macrophage infiltration into gliomas.S1OOB-overexpressed (S100Bhigh) tumor cell line exhibits enhanced inflammatory response in vivo, flow cytometry also confirmed that macrophages (CD11bhighCD45high) was highly expressed in S100Bhigh and S100Bwt than in the S100Blowcell line, and found that inhibited S100B could reduce the infiltration of TAM and extend the survival of the tumor-implanted animals.3. Activation of RAGE by S100BS100B can induce activation of RAGE in tumor. S100B is the only one which can stimulate the expression of RAGE in glioma, but RAGE may not essential in S100B-mediated TAM infiltration model.4. Chemokine productionIn vitro and in vivo experiments showed that upregulation of S100B expression increased secrection of CCL2, and it is essential to the process of TAM infiltration into glioma tissue. In contrast, the secretion of CXCL12from these three cell linesshow no significant differences. Another related cell line also confirmed that expression of CCL2correlates to expression of S100B, so as to TAM infiltration.5. Correlation to human gliomasIn order to evaluate the correlation between the results we found and human gliomas, we used data from TCGA database to analyze the correlation. Expression of S100B showed positive correlations with the original proneural, neural, classic subtypes. Expression of S100B showed correlation with CCL2in all TCGA HTU133A array, and showed a stronger relation in neuronal and proneuronal subtypes of gliomas. These results confirmed the S100B may promotes TAM infiltration by upregulate the expression of CCL2in some glioma subtypes.ConclusionExperiments in vivo and in vitro demonstrated that S100B is most likely to promote tumor growth by upregulate expression of CCL2and chemotaxis of TAM. Glioma can secrete S100B, which can attract bone marrow-derived macrophages through chemoattraction of CCL2. So gliomas S100B and CCL2plays n important role in infiltration of macrophage and growth of glioma. This provides a potential application in the treatment of glioma S100B inhibitors.