Gamma Secretase Inhibtor-Ⅰ Exerted Cytotoxic Effects Onmalignant Glioma Cell Lines By Inducing Cell Cycle Arrest And Apoptosis

BACKGROUND & OBJECTIVEGlioma is a type of malignant brain tumors, due to the highly invasion, drug resistance, resistance to radiotherapy, early metastasis, local recurrence and other features, most of the patients with very short survival,Only about 1 year. The study confirmed, notch signaling pathway may play a very important role in glioma genesis and development, Activation of Notch signaling pathway may be an important factor that result in malignant glioma,γ-secretase plays an important role in the notch signaling pathway activation process. Gamma secretase is a multi-subunit protease complex its exact composition is still unclear but minimally consists of four individual proteins:presenilin, nicastrin, APH-1, and PEN-2. Presenilin, an aspartyl protease, is the catalytic subunit; Nicastrin's primary role is in maintaining the stability of the assembled complex and regulating intracellular protein trafficking; PEN-2 associates with the complex via binding of a transmembrane domain of presenilin and, among other possible roles, helps to stabilize the complex after presenilin proteolysis has generated the activated N-terminal and C-terminal fragments。APH-1, which is required for proteolytic activity, binds to the complex via a conserved alpha helix interaction motif and aids in initiating assembly of premature components。Gamma-secretase is close with a variety of diseases, such as Alzheimer's disease (Alzheimer's disease, AD), brain gliomas and so on.Alzheimer's disease is an autosomal dominant genetic disease, is one of old age frequently-occurring disease, AD incidence may be associated with y-secretase dysfunction when encoding Presenilin 1 and Presenilin 2.In view of y-secretase is the notch signaling pathway important regulatory protein, therefore, useγ-secretase inhibitors(Gamma secretase inhibtor-Ⅰ, GSI-I)to inhibit the activity ofγ-secretase, thus inhibiti notch signaling pathway activation has some of the value on the treatment of glioma. Bleomycin is a commonly used antineoplastic agents, clinical primarily for the treatment of lung cancer, cervical cancer, vaginal cancer, esophageal cancer, head-neck and skin squamous carcinoma, BLM mainly inhibit thymidine incorporate into DNA, and binding to DNA so that the decomposition, effect the proliferation of the cell cycle at S phase, thus inhibiting the proliferation of tumor cells, So can BLM also inhibit the proliferation of glioma cells? In this study, we use U87, U251 glioma cell line as a model, investigate the effect of GSI-I and BLM on proliferation and apoptosis in malignant glioma cell line.In our study, firstly, RT-PCR was performed to analysis the expression of Notch receptor and its target gene Hes-1 in U87, U251 glioma cells. as Hes-1 is one of the main target gene that regulate Notch pathway, by detecting the expression levels of Hes-1 that can determine Notch pathway activation degree. Therefore, fluorescent quantitative RT-PCR was perfoumed to quantitative analysis the inhibition effect of GSI-Ⅰon the Notch pathway; meanwhile, MTT technology was applied to investigate the inhibition effect of GSI-Ⅰand BLM on the two cells proliferation. Next, flow cytometry was done to study the effect of cell cycle and induced apoptosis of the GSI-Ⅰand BLM on the U251, U87 two cell lines. Finally, Western blot technique for further analysis of the GSI-Ⅰon the U251, U87 cell cycle protein expression.METHODS1. U87 and U251 glioma cells in cultureU87, U251 glioma cells were cultured in DMEM containing 10% fetal bovine serum medium, incubated in37℃,5% co2,use logarithmic growth phase cells for experiment.2. Detection of Notch receptor and its downstream signaling pathway gene Hes-1Logarithmic growth phase of U87, U251 glioma cells, extract total RNA according to Trizol instructions, RT-PCR detect the expression of notch receptor and its downstream gene Hes-1.3. Detection of the expression levels of Hes-1after treated by GSIU87 glioma cells were treated by 1μmol/L GSI and 2.5μmol/L GSI for 4h, 24,48 h, respectively. then isolate total RNA, line quantitative RT-PCR detect Hes-1 expression.4. Detect the effect of GSI on proliferation ability in U87 and U251 glioma cellsTake logarithmic growth phase U87 and U251 cells, with different concentrations of GSI treatment, results also set the negative control group,48h after the home microplate reader measured OD values at 570nm wavelength of the hole, analysis cells proliferation ability.5. Detection the cooperativity of GSI+Bleocin on the U87, U251 glioma cells the experment Were divided into negative control group, different GSI concentration group, different Bleocin concentration group and GSI+Bleocin each concentration group, treatment MTT method were used to investigate the effect of GSI, Bleocin and GSI+Bleocin on U87 and U251 cell proliferation after 48 hour.6. Detect the effect of GSI on U87 and U251 cell cycleafter treated byGSI for 48 hour,U87 and U251 cells were collected and fixed with 70% ethanol for at least 6 hours, flow cytometry analysis the effect of GSI on U87 and U251 in each cell cycle phases respectively,while Western blot was used to detect the effect of GSI on U87 and U251 cell cycle proteins.7. Detect the effect of GSI, Bleocin, GSI+Bleocin on U87 and U251 cell apoptosisDifferent concentrations of GSI, Bleocin, GSI+Bleocin treatment, cells were collected, Annexin V/PI double staining flow cytometry was adopt to study the effect of GSI on the induction of apoptosis in U87, U251 glioma cell lines.8. Statistical Methodsstatistical analysis was done using SPSS 10.0 software, quantitative RT-PCR assay were analyzed by repeated measure square analyze, cell cycle assay were analyzed by independent samples t-test (Independent t Test), cell proliferation inhibition and apoptosis assay were analyze by One-way ANOVA. GSI+Bleocin synergy assay were analyzed by factorial designed square analyzed.the main results and conclusions are as follows:1. Successfully cultured human origin of U87, U251 glioma cells, by RT-PCR method, we detected the notch receptor and notch signaling pathway downstream of the target gene Hes-1 in U87, U251 glioma cell line were expressed, indicating notch signaling pathway in U87, U251 glioma cells exist2. GSI-Ⅰcan inhibit y-secretase activity, thereby inhibiting notch signaling pathway, after notch signaling pathway is blocked, the downstream target gene Hes-1 expression would be reduced, therefore, we use GSI-ⅠTreat U87, U251 glioma cells 4 to 48 hours, quantitative RT-PCR showed that different concentrations of GSI treated for 4h,24h,48h, Hes-1 expression was significantly decreased, These results suggest that after GSI treatment, notch signaling pathway downstream target gene Hes-1 expression compared with the control group was significantly lower, and showed a dose-dependent manner, indicating GSI-Ⅰcan inhibit The notch signaling pathway in U87, U251 glioma cells.3. Since the GSI-Ⅰcan inhibit U87, U251 glioma cells in the notch signaling pathway, then whether GSI-Ⅰcan inhibit the proliferation of U87, U251 glioma cells? We used different concentrations of GSI-I treat U87, U251 glioma cell for 48h. MTT assay showed that 2.5μmol/L and above concentrations of GSI-Ⅰcould significantly inhibit the proliferation of U87 and U251 glioma cells( F=11.174, P=0.000) and (F=92.755, P=0.000), and this effect showed dose-dependent increase. Low concentrations of GSI-I (0.5μmol/L or 1μmol/L) on the cell line was not obvious, indicating GSI-I have toxic effects on glioma cells, but must reach a certain concentration.4. Bleomycin can inhibit the proliferation of some of the common tumor cells, whether it can be used for the treatment of glioma is still no such reports. To this end, we investigated whether BLM+GSI treatments have cooperative effect in glioma cells. We found BLM+GSI inhibit the proliferation of U87 and U251 cells more significantly than single treatment with GSI(F=4.217,P=0.000) and(F=3.863,P=0.001). Indicated that BLM combined to GSI have synergistic effects on proliferation of glioma cells, we supposed that the mechanism of BLM inhibited the proliferation of glioma cells and other tumor may be similar, but also by acting on the cell cycle of glioma cells and inhibited their proliferation.5.To further investigate the mechanism of GSI-Ⅰinhibit the proliferation of glioma cell.firstly, we detected the effect of cell cycle on the U87 and U251 cell after treated with GSI by flow cytometry. By Adding DMSO or 2.5μmol/L and above the concentration of GSI-Ⅰ,treated 48h, cells were collected to analysis the cell cycle by flow cytometry, the results showed that the U87 cells in S phase was significantly reduced after treated with GSI, while the G1 phase cells increased,indicated that U87 cells were arrested in G1 phase. In U251 cells was also detected in S phase decreased, but the G1 phase cells also decreased, and G2 phase cells were significantly increased.implyed GSI-Ⅰon the U251 cells is mainly G2 arrest. G1 phase arrest demonstratied that the cell proliferation was inhibited, but G2 phase arrest means apoptosis.Next, Western blot was done to analysis the effect of GSI on the expression of U87 and U251 cell cycle protein Akt, S6K, CDK2, CyclinB1 and CyclinD1. U87, U251 glioma cells treated with different concentrations of GSI (1μmol,2.5μmol, 5μmol, 10μmol) 48h after treatment, total protein extracted from different treatment groups, testing the expression level of Akt, S6K, CDK2, CyclinB1 and CyclinD1. Western blot analysis showed that the phosphorylation level of Akt and S6K decreased slightly, yclin D1 expression is down in U87 cells lines, the expression of Cyclin B1 reduced in U251 cells, CDK2. significantly reduced these two cell lines, Suggest that GSI have effects in both anti-apoptosis protein and the activity of cell cycle protein,this may be one of the molecular mechanisms that GSI anti-toumor cell. The results from the above proved that the Toxic effects of GSI on glioma cells not only in inhibition of proliferation, and may directly induce apoptosis. Therefore,we further examined the apoptosis effect of GSI-Ⅰon these two glioma cell line.6. in the experiment, U87 and U251 glioma cells were treated with different concentrations of GSI for 24h and 48h.the results showed:after 24h treated by GSI,the apoptosis rate increased as the concentration of GSI increased in U87 and U251 cell lines, These results suggest that GSI-Ⅰcan significantly induce apoptosis in glioma cell,and the effect are different in different glioma cell lines. U251 cells were more easily induced apoptosis than the U87 cells by GSI—Ⅰ7.We further analyzed the GSI,Bleocin,Bleocin+GSI on the U87,U251 glioma cell apoptosis,with 1μmol／L GSI—Ⅰ,1μmol／L Bleocin,1μmol／L GSI—Ⅰ+1μmol／L Bleocin handling U87 glioma cells,with 2.5μmol／L GSI—Ⅰ,1μmol／L Bleocin, 2.5μmol／L GSI—Ⅰ+1μmol／L Bleocin processing U251 cells for 48h,then detect the death rate of the cells by flow cytometry,the results showed that 1μmol／L of GSI-Ⅰon the U87 cell apoptosis rate was 9.217±0.440％(P=0.000),1μmol／L of Bleocin on U87 cell apoptosis rate was 8.343±0.778％(P=0.001),1μmol／L GSI—Ⅰ+1μmol／L Bleocin on the U87 cell apoptosis rate was 21.160±1.423％(P=0.000).2.5μmol／L GSI—I on apoptosis of U251 cells was 21.883±2.054％(P =0.000),1μmol／L of Bleocin apoptosis on U251 cells was 8.343±0.776％(P= 0.015),2.5μmol／L GSI—I+1μmol／L Bleocin apoptosis on U251 cells was 60.980±2.015％(P=0.000).These results further suggested that GSI with BLM have cooperative effect on the proliferation inhibit in glioma cells,the underlying molecular mechanism remains to be further studied.