| Gliomas are the most common primary brain tumors. The prognosis of the patients with malignant glioma has not been improved in recent twenty years, even though combined therapeutic modalities including surgical resection, radiotherapy and chemotherapy are currently available. As the knowledge of tumor biology and molecular genetics increased, it has been shown that the development of gliomas, just the same as the tumors in the other site of the body, results from the activation of proto-oncogenes and inactivation of tumor suppressor genes, and involves multiple genetic and molecular alterations.hi a number of studies of our previous work, it had been demonstrated that EGFR gene amplification or overexpression is the early initiating molecular event in the development of malignant gliomas. EGFR overexpression will induce the activation of downstream AKT and its signaling pathways via PI3K. Phosphorylated AKT play a central role in the promotion of cell proliferation and invasion. However, as to our knowledge, the expression and AKT and its'role in glioma progression has not been fully investigated, so the present study aims at better understanding of the role of AKT and inactivation of EGFR-Pi3K-AKTpathway in the prevention of progression of gliomas.Protocol of the present studyThe first part of this study focused on the expression of AKT2 and its' relevant genes in 50 samples of gliomas as well as on the relationship among the expression level of EGFR, p-AKT, AKT2, MMP2, MMP9, Ki67 and their correlation with degree of malignancy of gliomas. Immunohistochemcal study was used for EGFR, AKT2, MMP2, 9 and Ki67 expression, p-AKT and AKT2expression were also studied by Western blotting.In the second part, anti-sense and dominant-negative (E299 K) form of AKT2 constructs in pLXSN retrovirus vectors were transfected to human glioblastoma cell line TJ905 and rat glioblastoma cell line C6, and empty pLXSN vector transfected as control. The positive clones were randomly selected and identified by Western blot and in situ hybridization. MTT and TUNEL methods were used to evaluate cell proliferation and apoptosis. Tumor invasion was examined by Transwell/ spheroid-Matrigel method, and tumor cell migration by spheroid/ wound-healing method. Flow cytometry was used for cell cycle analysis, and GFAP expression was also detected for evaluation of cell differentiation by Western blot.In the third part, in vivo study was carried out. Parental C6 cells and C6 cells trasfected with antisense AKT2 cDNA were implanted stereotactically into the right caudate nucleus of SD rats as control and transfected group. Rats with well-established cerebral gliomas were treated with antisense AKT2 cDNA and LXSN empty vector as treated and LXSN group. Each group consisted often rats. The general behavior and survival of the rats were observed, the dynamic MRI and histopathological changes of the rumors and the expression of AKT2, MMP2/9, PCNA, cyclin D1 and apoptosis in glioma cells were examined. A control rat with large intracranial tumor was treated with high dose of antisense AKT2 cDNA-lipofectamine complex before death via intratumoral and intramuscular route to further test the effect of antisense AKT2 therapy, while another rat treated with antisense AKT2 cDNA was retreated with active AKT2 to demonstrate the importance of AKT pathway in the progression of gliomas.ResultsExpression analysis of EGFR-PI3K-AKT2-MMP2/9-Ki67AKT2 expression increased correspondingly to the ascending of tumor grade, the protein expression level in grade III and IV was higher than that in low-grade gliomas, and so did p-AKT.. In immunohistochemical study, the positive stained-cell rate of AKT2 ,EGFR, MMP2/9 and Ki67 also increased with the degree of malignancy of tumors, and their differences of positive rate and expression level between the low-grade and high-grade tumors were statisticallysignificant. In addition, the expression of EGFR, p-AKT,AKT2, MMP2/9 and Ki67 correlated positively with each other. Such results imp...
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