Effects Of Transient Axonal Glycoprotein-1 On Activity Of U251 Cells And It's Genetic Regulatory Mechanism

BackgroundGlioblastomas are the most highly frequent and malignant brain tumors, accounting for about 51% of all central nerve system (CNS) tumors. The incidence of primary brain tumours proximately seven per one hundred thousands indibidulas per year, accouting 7% of the years of life lost from cancer before the age of 70. Glioblastomas possess some of characteristics such as progressive growth, extensive angiogenesis, recurrence, diffuse and restless invasion and so on, which cause severe neurological dysfunction. General therapeutic ways including neurosurgical techniques, radiation and chemotherapy have progressed a great development in the past two decades, but the treatment of malignant glioma and therapy remains mostly palliative and the prognosis remains dismal. And a report indicated that the survival of patients with glioblastoma was extremely poor, with survival rates of 42.4% at six months,17.7% at one year, and 3.3% at two years. Therefore, it is necessary to explore new effective ways for therapying the glioma,by studying some molecular mechanisms of glioma genesis.p53 was considered as the "guardian of the genome", which would transcribe and prevent the cells from malignant transformation by regulating the cellular cycle, controlling the senescence, and promoting the cellular apoptosis, as soon as it was actived by some stress stimulus such as DNA damage, inflammation, hypoxia, etc. During the procedure, it is the main function for gene P53 to promote the cellular apoptosis.Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor and belongs to the ErbB family of tyrosine kinase receptors, Activated EGFR propagates proliferative signaling via two major pathways, the Ras pathway and the PI3K pathway. It was well known that the EGFR pathway plays a crucial role in pathogenesis of glioblastoma. Several new therapeutic strategies including blocking the celluar surface of EGFR by monoclones such as Trastuzumab, or locking the intracellular signal pathways by some micromolecule drugs such as ZD-or OS-774. this research focused on the tumor suppressor p53, and the EGFR in order to explore the mechanisms of glioma development and to search for a new way to enhance the gene therapeutic effects.New treatment options bring new hope for glioma people such as molecular therapies, gene therapy and so on. This research focused on the tumor suppressor p53 and epidermal growth factor receptor (EGFR) to explore the mechanisms of glioma development and search for a new method to strength the gene therapeutic effects.It was well known that the brain tumor in slow growth and long course of disease might induce the Alzheimer's disease (AD), which related to the types and genesis nositions of the brain tumors closelv (http://baike.aiii.cn/Detailed/5102.html). Hippocampus was generally considered as the important location of AD genesis, in which the brain tumor with large size and quick growth velocity would cause AD easily. On the other hand, it was noted in clinic that few patients with AD suffered in brain tumor again, which implied that a signal pathway relating to AD genesis maybe inhibited the formation and development of glioma. As all known, the one of the main pathological markers of AD just was the deposit ofβ-amyloid peptide (Aβ) that came from the enzymolysis from amyloid precursor protein (APP). As soon as the Aβproduced, the APP would release the APP intracellular domain (AICD) which might enhance the effects of both appoptosis and tumor depression by the signal pathways such as APP/AICD/p53 or APP/AICD/EGFR. Especially, it was found by our research team that the transient axonal glycoprotein-1 (TAG1) could attend the development of AD by enhancing the release of AICD as the ligand of APP (Nature Cell Biology,2008, 10 (3):283-94). Then, whether or not there was a relationship between the Signal (TAG-1/APP/AICD/p53 or TAG-1/APP/AICD/EGFR) primed by TAG1 and the genesis of glioma? What are the effects of these signals primed by TAG1, during the glioma development? All of the questions are not well known yet. This research took the tumor suppressor genes (p53 and EGFF) as the focus, the signal pathways (TAG-1/APP/AICD/p53 and TAG-1/APP/AICD/EGFR) as the study tracks to explore the effects of TAG-1 on activity of U251 cells and gene expression of AICD, p53 or EGFF, in order to get the possible new way for giolma therapy.Objective To explore the effects of TAG-1 on activity of U251 cells and gene expression of AICD, p53 and EGFR in them, by which the new signal pathway in glioma proliferating will be confirmed as the new therapeutic way for clinical.Methods The survival activity of U251 cells was tested by MTT assay at 48h following adding various concentrations of TAG-1. APP positive expression was detected by immunocytofluorescence. The apoptotic cells were examined by TUNEL. The gene expression of AICD,p53 or EGFR on U251 cells following TAG-1 stimualtion was assayed by real-time PCR.Results APP expressed aboundently in U251 cells memerane and dendron (but not in cytoplasm) of U251 cell. TAG-1 didn't inhibit the growth of U251 cells, oppositely to promote it, following stimulation with different concentration of TAG-1. The results showed no significance (P=0.429) among the different groups. U251 cells did not show the apoptosis when treated with TAG-1 at 10μg/ml of final concentration comparing with the control (0μg/ml of TAG-1), while the gene expresses of AICD, p53 and EGFR increased at different dgrees.Discussion The experimental results implied that the TAG-1 did not inhibit the growth of U251 cells, and did not reduce the cellular apoptosis either but promoted their growth and apoptosis upon the MTT assay and TUNEL test respectively. At the same time, the gene expresses of AICD, p53 and EGFR showed the increase comparing with the control (0μg/ml of TAG-1). All of results above were so different with our primary expect, which suggested us some research clues as below. The first, TAG-1 might induce the over-express of EGFR to promote the proliferation of U251 cells by other different signal pathways. The second, the upregulated gene express of p53 might result from the emergency reaction of tumor cells proliferation. In other words, the over-proliferating tumor cells induced the up-regulation of p53 gene express. Therefore, the one of the effective therapeutic ways might be considered as blocking the TAG-1 signal pathway.This study reported the effects of TAG-1 on AICD, p53 and EGFR of U251 cells at the first, which implied that the signal pathways including TAG-1/APP/AICD/p53 and AG-1/APP/AICD/EGFR primed by TAG-1 played the important positive role in glioma proliferation (but not inhibition). It might be understand that the molecular signal pathway itself was not as the straight line-style but the different signal pathways could be actived by the same primer, then the different levels of effects from them would be showed. On the other hand, the effects of any kind of molecules just depended on the types and status of the efficacy cells. Therefore, the different stimulators for the same cell or the different cells for the same signal pathway would result in the different phenomenia of researches. Lots of studies about EGFR mechanisms enhanced by TAG-1 and effective mechanisms of TAG1-APP signal pathway in cellular proliferation for glioma therapy will be performed futher.Conclusions1.TAG-1 didn't induce glioma cells apoptosis through TAG-1/APP/AICD/p53 or TAG-1/APP/AICD/EGFR signal pathway to inhibit glioma growth, but it might promote U251 cells proliferate by activiting unknowen signal pathway.2.TAG-1 might induce the over-expression of EGFR to promote the proliferation of U251 cells by other different signal pathways. The upregulated gene express of p53 might result from the emergency reaction of tumor cells proliferation. The over-proliferating tumor cells induced the up-regulation of p53 gene express. P53 undergone mutate and inactivation so that couldn't induce apoptosis.Therefore, the one of the effective therapeutic ways might be considered as blocking the TAG-1 signal pathway.