The Role Of Tumor Marker Hsp90 In The Proliferation And Invasion Of Glioma Cells And Its Molecular Mechanism

Part 1 The role of tumor marker Hsp90 in the growth and metastasis of glioma in vitro and in vivo[Objective]Glioma is one of the most common malignant tumors. Hsp90 plays an important role in the growth and metastasis of glioma. The purpose of this study is to detect the effect of Hsp90 on the occurrence and development of glioma.[Method]The expresssion of Hsp90α was detected in three glioma cell lines SHG44, C6 and U251. Rabbit anti-Hsp90α polyclonal antibody and mouse anti-Hsp90α monclonal antibody were prified by Ni-NTA agrose gel assay. In vitro cell invasion assay was used to investigated the inhibitory effect of Hsp90a antibody on glioma cell invasion. Through gene transfection to overexpress MMP-2 and Hsp90α, the synergistic effects between the both factors was detected by in vitro cell invasion. Finally, human glioma xenograft model was established in nude mice. The role of Hsp90α in metastasis of glioma was tested by immunofluorescence staining.[Results]1 The expression level of secretory Hsp90α is postively correlated with the malignant degree of glioma cells.Western blot shown that there were no significant difference in the expression level of intracellular Hsp90α. In contrast, the level of secretory Hsp90α was postively correlated with the malignant degree of glioma cells.2. Hsp90α promotes the invasion of glioma cells, which partly depends on the MMP-2In vitro cell invasion assay shown that Rabbit anti-Hsp90α polyclonal antibody and mouse anti-Hsp90α monclonal antibody significantly inhibited invasiveness of glioma cells, compared with the control group.Via gene transfection, the invasive ability of Hsp90α overexpression glima cells (Hsp90α WT) was sigificantly higher than that of Hsp90α mutant glima cells (Hsp90α T90A). And what is more, co-transfection of MMP-2 further enhanced the invasive ability of Hsp90α WT cell.[Conclusion]The expression level of secretory Hsp90α was postively correlated with the malignant degree of glioma cells. Hsp90α promoted the invasion of glioma cells, which partly depends on the MMP-2.Part Ⅱ Correlationship between tumor marker Hsp90a and the malignant degree of glioma in vivo[Objective]According the data of Part I, the expression level of secretory Hsp90a is demonstraed to be postively correlated with the malignant degree of glioma cells and Hsp90a promoted the invasion of glioma cells in vitro. This part is mainly to investigate the Hsp90a as a tumor specific bio-marker, and its correlationship with the malignant degree of tumor in vivo.[Methods]Human glioma xenograft model was established in nude mice by subcutaneous implantation. Immunoblot assay was used to deteced by rabbit anti-Hsp90a polyclonal antibody and mouse anti-Hsp90a monclonal antibody in mice serum. Enzyme linked immunosorbent assay (ELISA) was used to deteced the Hsp90a level in human serum, its correlationship with the metastasis of glioma and inflammation. Through establishment of nude mice glima model, the source of Hsp90a was also clarified by immune reaction assay.[Results]1. Expression level of secretory Hsp90a is postively correlated with the malignant degree of glioma.Immunoblot assay in nude mice shown the higher level of plasma Hsp90a in tumor mice than the control mice. Immunoblot assay in human shown the higher level of plasma Hsp90a in tumor-beared patients than the nomal people. ELISA method showed the higher level of plasma Hsp90a in tumor-beared patients, compared with the nomal people benign brain tumor patients (P<0.05).2. The level of Hsp90α is correlated with the metastasis of glioma.ELISA method showed that the plasma levels of Hsp90α in patients with metastasis were significantly higher than those in patients without metastasis (=0.003 P).3. No correlation between the level of Hsp90a and inflammationPlasma of patients with pneumonia or hepatitis were collected and ELISA method was used to detect the concentration of the Hsp90. Results showed that there was no significant difference among the three groups:pneumonia and hepatitis patients and normal people (P value were 0.2988,0.5177,0.138).4. Plasma Hsp90α is secreted by glioma cells.Human glioma xenograft model was established in nude mice by subcutaneous implantation. Immunoblot staining showed that cultivated from human glioma in mouse plasma Hsp90 can be identified by the human source Hsp90 antibody, suggested that plasma Hsp90 is secreted by glioma cells.5. No correlation between plasma level of Hsp90α and other clinical variables of patients with gliomaThe relevance of Hsp90a with clinical variables, such as age, tumor size, the plasma concentration levels of ER and PR, and so on. The results showed that there was no correlation between plasma level of Hsp90α and other clinical variables of patients with glioma (P>0.05).[Conclusion]Hsp90α may be a potential tumor specific marker, and its expression level is positively correlated with the malignant degree of the tumor.Part III The mechanism of Hsp90a in metastasis of glioma[Objective]The study above showed the positve role of Hsp90a in the metastasis of glioma. This study aims to explore the underlying mechanism of Hsp90a in metastasis of glioma.[Methods]Immunofluorescence staining was used to detect the concentration of Hsp90a in tumor cell surface. The localization in cell surface of Hsp90a was used to detect by laser confocal. The double-staining of Hsp90α and extracellular matrix was used to detect by laser confocal. The double-staining of Hsp90a and invadopodia was used to detect by laser confocal. The detection of invadopodia-related regulatory factors on the secretion of Hsp90 effect was used to detect by immunofluorescence staining and Western blot.[Results]1. Hsp90a is mainly distributed in the boundary and synapse of glioma cells.Immunofluorescence staining shown that after cultived in the extracellular matrix-uncoated coverslips, Hsp90a was diffusely distributed in the cell surface, and lightly stained, suggesting that the distribution of Hsp90a in cell surface was nospecific and low level.After cultived in the fibronectin-coated coverslips, Hsp90a was mainly distributed in the cell boundary, surface and synapse.On coverslips coated with Gelatin staining, the tumor cell membrane surface was enhanced, and mostly near the cell boundary and synapse. Using a laser confocal microscope, immunofluorescence staining showed that Hsp90α was localaized in the junction between the cell and extracellular matrix.2. Co-localization of Hsp90α and glioma cell surface invadopodiaCommonly used in fluorescent labeling of invadopodia main component of microfilament and invadopodia protein. The results showed the fine co-localization of Hsp90α and microfilament formation of synaptic structure, and the fine co-localization of Hsp90α and specific markers of invasive invadopodia.3 Invadopodia-related regulatory factors affects the secretion of Hsp90αImmunofluorescence staining and Western blot showed that knockdown of c-Src, N-WASP and Cortactin decreased the level of Hsp90a in cell surface.4. Hsp90α enhances invadopodia to degradate extracellular matrixIn vitro invadopodia formation assay was used to measured the effect of Hsp90α on tumor cell invasion pseudopod formation. Results are shown that the inhibitory effect of Hsp90α antibody on EGF induced invadopodia formation.5. Co-localization of Hsp90α and invadopodia marked-protein in human glioma tissues.Immunofluorescence staining showed the co-localization of Hsp90α and Cortactin, invadopodia marked-protein in the invasive frontier of human glioma tissues.[Conclusion]Tumor marker Hsp90α may enhance glioma metastasis mainly through promoting the formation of invadopodia.Summary1. The expression level of secretory Hsp90α is postively correlated with the malignant degree of glioma cells in vitro.2. Hsp90α promotes the invasion of glioma cells, which partly depends on the MMP-23 Hsp90α may be used as a tumor specific marker, and expression level of secretory Hsp90α is postively correlated with the malignant degree of tumor.4. Tumor markers of Hsp90α is colocalized with invadopodia.