Study On The Expression Of Rac1in Glioma And Its Role In Malignant Glioma Invasion

Malignant gliomas, accounting for30%～50%of intracranial neoplasms, are the most common and lethal primary brain tumors. Although considerable progress has been made in technical proficiencies of surgical and radiation treatment for malignant gliomas patients, the impact of these advances on clinical outcome has been disappointing, with median survival time not exceeding15months. Malignant gliomas are poorly differentiated, lack of apoptosis, highly proliferated and aggressive. A fundamental source of the management challenge presented in glioma patients is the insidious propensity of tumor invasion into distant brain tissue. The potential of single cells to invade normal brain tissue, blurring tumor margins and establishing numerous’micro-tumors’at a distance from the primary tumor, which make surgical resection palliative and not curative. Previous studies have shown that these cells are tumor stem cells.The invasion of malignant gliomas is a complicate and consecutive process. The invasive process in the brain shares characteristics with that occurring in the stromal infiltration of non-neural carcinomas, namely cell detachment from the primary tumor mass, receptor-mediated adhesion to surrounding ECM, degradation of ECM to allow for cell passage, and active motility processes. Of many factors involved, cell moving plays an important role in this process. Cell migration involves multiple processes that are regulated by various signaling molecules. The actin cytoskeleton and its regulatory proteins are crucial for cell migration in most cells. During cell migration, the actin cytoskeleton is dynamically remodeled, and this reorganization produces the force necessary for cell migration. Racl is one of key regulators of actin assembly and control the formation of lamellipodia. Racl interacts with specific downstream effectors, leading to the translocation and activation of the effectors, and induction of cytoskeletal reorganization and cell migration. It is reported that Racl is over expressed in many tumors including mammary tumor, colon carcinoma and pancreatic cancer.The present study included three parts:1. The characteristics of Racl expression and distribution in gliomas. sixty-five tumor specimens were collected from patients with newly diagnosed gliomas from June,2009to June,2010, while5control brain tissues were collected from brain injury patients were served as control. In12of the21glioblastoma patients, multiple specimens were obtained under assistance of neuronavigation system during the operation including specimens from central tumor tissue and from border zone between tumor and peri-tumor edema. The expression of Racl in specimens was detected by western blotting and immnunohisological staining. Elevated expression of Racl was detected in gliomas of different WHO grades (F=41.18, P<0.0001) by western blotting, while Racl expressed negatively or very lowly in control brain tissue. The difference of Racl expression in different-grade gliomas were statistically significant in immnunohisological staining, and its expression strength was correlated positively with pathological grades of the gliomas.(rs=0.682, P=0.000). In the glioblastoma masses, Racl expressed highly and diffusely, and the Racl positive cells palisaded the necrotic areas. In border zone between tumor and peri-tumor edema, Racl positive cells aligned radially around the vessels and parallel to the white matter tracts.2. The role of Racl in migration and invasion of U251cells induced by SDF-1. Human glioma cell line U251treated with SDF-1or specific Racl inhibitor NSC23766plus SDF-1were used in this study. The morphological changes of U251cells were observed under inverted phase contrast microscope. The migration and invasion capacities of cells were assessed in2D cell migration/3D invasion assay. Western blotting was conducted to quantify the expression of Racl in cell lysates and the Rac1activity was measured by Rac1activation assays. Immunofluorescence was conducted to identify expression and intracellular location of Racl in U251cells. Results:In comparison with control group, the formation of lamellipodia was significantly increased in SDF-1treated group. SDF-1significantly increased the migration and invasion capacities of U251cells (P<0.05). The stimulation of SDF-1boosted the activity of Racl versus the unstimulated cells (P<0.05), and Racl was recruited to protruding edge in SDF-1-stimulated cells. In comparison with SDF-1treated group, the formation of lamellipodia was significantly decreased in NSC23766pre-treated group. Inhibition of Racl with specific Racl inhibitor decreased the migration and invasion capacities of SDF-1-induced U251cells (P<0.05). In comparison with SDF-1treated group, the activity of Racl were significantly decreased (P<0.05) and the recruitment of Racl to protruding edge was significantly reduced in the NSC23766pre-treated group.3. The relationship between Racl positive cells and glioma stem cells (GSCs) as well as the role of Racl in GCS migration and invasion. MACS was used to isolate CD133+cells from U251cells. Immunofluorescence was conducted to identify stem cells. Glioma stem cells were treated with specific Racl inhibitor. The migration and invasion capacities of cells were assessed in Transwell cell migration/invasion assay. The Racl activity was measured by Racl activation assays and Western blotting was conducted to quantify the expression of hMena and MMP9in cell lysates. Results: GCSs formed cell spheres, which floated in the medium. Immunofluorescence indicated that the cell sphere expressed CD133. Racl activation assays indicated that GTP-Racl was over expressed in CD133+cells comparing to CD133-cells (P<0.05). In comparison with control group, the expressions of GTP-Racl were significantly decreased in inhibitor treated group (P<0.05). Transwell cell migration/invasion assay indicated that CD133+cells are more motional than CD133-cells (P<0.05). Inhibition of Racl with specific Racl inhibitor decreased the migration and invasion capacities of GSCs (P<0.05). Western blotting indicated that the expressions of hMena and MMP9were significantly decreased in the NSC23766treated group (P<0.05) Conclusions:1. The expression rate of Racl in gliomas was positively correlated with the increasing grade of WHO classification and Racl expression was negative in control brain tissue. Racl positive cells aligned around the long axis of vascular and parallelled to the white matter tracts.2. Racl modulates the glioma cells migration and invasion induced by SDF-1.3. The glioma stem cells have an outstanding ability of migration and invasion through strongly expressing Racl. The Racl positive staining cells that aligned radially around the vessels may be the glioma stem cells. Racl modulates the glioma stem cells migration and invasion.