| Glioma is the most common primary malignant tumor in the central nervous system, which is invasive growth, hard to be removed completely by surgical resection and the clinical curative effective is poor. The five-year mortality rate of malignant glioma is the third in the whole body tumors, only surrendering pancreatic cancer and lung cancer.Five-year survival rate of malignant glioma is less than 5%.Although we have tried multiple modalities of treatment,but the curative effective is still not satisfactory.This is owning to tumor is a kind of desease that exposure to multi factor impact,multi genes regulating and multi-stage developing.because of above, to study the pathogenesis and searching an efficient therapeutic method always be one of the important projects of the neurosurgery.We should consider the initiation and development of glioma is as the same as other tumors,it is involved in various abnormal pathological process,such as cell cycle regulation,develepment, differentiation, signal transduction,apotosis,cell death,cell metabolize and so on.PI3K-Akt signal pathway is an important intracellular signal transduction pathway. It plays important roles in cell apoptosis and survival by affecting the activity of down stream effector molecules, and it is closely associated with the development and progression of human tumors.As a kind of Serine/threonine kinase,AKT/PKB phosphorylate substrate concerns with many major signal pathway.PI3K is a key second messenger. The phosphorylation of PI3K 3'end can make PIP2 convert to PIP3.PI3K is a lipid kinase, which can be activated by diverse signal,including groth factor and the interaction signal of intercellular.PI3K effect lots of cell progression through actvating a highly conserved singal tansduction pathway and their downsteam protein kinase,such as P70 S6 kinase,PDKs.Wnt is an extremely conserved signal tansduct pathway.The members of this pathway have highly homology from lowlycreature (Drosophila melanogaster) to highermammal.Wnt gene belong to proto-oncogene, coding excretion glycoproteins. This protein contains a signal peptide and 23 or 24 amino acid residues which are located in conserved site. It is generally accepted that mainly members of classical Wnt signal transduct pathway are ligand(Wnt family members),transmembrane receptor(Frizzled family members and LRP-5/6),cytoplasm regulatory protein(Dsh,APC,Axin,GSK3β,β-catenin)and intranuclear trancript factor(TCF/LEF1 family).After Wnt molecule combine with transmembrane receptor, the interaction of the transmembrane receptor and Dsh as well as a series of cytoplasm protein make P-catenin accumulate in cytoplasm and translocates to nucleus.The cooperation betweenβ-catenin and TCF/LEF 1 can activate the transcription of downstream target genes then cause biological effect. More and more reaseches showed that Wnt not only play an important role in the process of embryonic development but also have realationships with numerous different tissue stem cells'self-renewal and differentiation. It is reported that AKT/PI3K and Wnt signal pathway are concerned with cell proliferation, development and tumorigenesis in different animals or distinct organ. So far there are few studies on the change of Wnt after blockading AKT/PI3K pathway by special drug.This study will be divided to 3 sections:Part 1:Treat U251 and LN229 glioma cell lines with AKT special inhibitor LY294002.Test the expression of AKT and p-Akt protein by Western blot.To investigae the optimum inhibition concentration of this inhibitor, glioma cell lines were treated with gradient concentration of LY294002. MTT method was used to evaluate cell proliferation Flow cytometry was used for cell cycle analysis. Invasion ability was detected by Transwell analysis.Part 2:We used RNA interfrence to down-regulateβ-catenin then dectect the expression of members of Wnt pathway,includingβ-catenin,CyclinDl,Fra-1,c-Myc by Western blot.The results were compared with the changes after blockading AKT pathway.TOP/FOP flash system was used to detect the trancription activity of TCF. Immunofluorescence was used to define intracellular location ofβ-catenin.Part3:In vivo study, nude mice bearing xenografted subcutaneous LN229 gliomas were carried out to investigate the synergy effect of LY294002. The dynamic growth rate and tumor volume were observed. Proteins in neoplastic tissues were observed by immunohistochemical SABC methods to further proving the relation of protein and cell proliferation,cell cycle and cell invasion. Conclusion:1 LY294002 could restrain PI3K/Akt signal pathway and suppress cell proliferation, invasion of U251 and LN229 glioma cell lines2 The repressed proliferation activity of tumor cells is concerned with down-regulation of Wnt/β-catenin pathway, including Fra-1,c-Myc and Cyclin D1.3Wnt/β-catenin is regulated by PI3K/Akt in Malignant glioma cell.4 The speed of growth of enografted subcutaneous LN229 gliomas could be slow down by treatment with LY294002 in vivo study. |
PDF Full Text Request