The MTOR Pathway Inhibitor Everolimus In Vivo Studies Of The Different Glioma Cell Autophagy And Cell Cycle

BACKGROUNDGlioma is a central nervous system is the most good, highly malignant tumor, the present study to give the surgery plus radiotherapy, and chemotherapy treatment programs, senior other glioma patients with a median survival of only16.7its due in large part because of the recurrence and the formation of metastases. Traditional chemotherapy drugs to inhibit tumor growth by promoting cell apoptosis, but the rapid emergence of resistance. In order to reverse this phenomenon, we need to find a new cellular protein targets to improve the current chemotherapy regimens prolong survival of patients. Cell death is considered to include three types:necrosis, apoptosis and autophagic cell death. Apoptotic proteins in cancer cells, have varying degrees of impairment (restricted drugs on the activation of the apoptotic pathway), autophagy (now considered to be type Ⅱ cell death, unlike apoptosis) refers to the cells by single or bilayers wrapped to be degradation products to the formation of autophagosomes, and the formation of autophagic lysosomes and the digestion and degradation, as a cellular protection mechanism, and its protein are preferentially retained. mTOR signaling pathway of cell growth, proliferation and cell cycle regulation, the PI3K/AKT signaling pathway (regulation of autophagy) is the central link. Everolimus mTOR signaling pathway-specific blocker, in recent years significant role in cancer of the liver, pancreas and other tumors. On the mTOR pathway and its inhibitors on glioma less, the following shortcomings:①of mTOR-related pathway protein expression in human gliomas and its mechanism is still not entirely clear;②mTOR inhibitor on different genetic background glioma cell lines and the relationship between apoptosis and autophagy remains unclear;(3) mTOR inhibitor-induced gliomas subcutaneously into nude mice transplanted tumor efficacy remains unclear. In this study, on the basis of previous research, trying in vitro from different genetic backgrounds glioma cell line U-87MG, of U251, of SHG44treated with different concentrations of everolimus to detect the administration of glioma cell lines before and after cell growth, cell cycle and cell autophagy; and through the establishment of the three kinds of glioma cell lines subcutaneously into nude mice xenograft model, given by intraperitoneal injection of everolimus, immunohistochemistry, cell cycle, tumor vascular density and other methods of measurement, to detect everolimus inhibition of different cell lines. Trying to prove that the mTOR signaling pathway can be used as a new target for glioma therapy, a new experimental basis for the treatment of glioma.OBJECTIVEExplore the role of everolimus people in U87-MG, of U251-the MG, the SHG-44glioma cells in vivo and in vitro and its possible mechanism.1. cultured glioma cell lines in U87, of U251, of SHG44with different concentrations of drug intervention in cell growth, clear apoptosis and autophagy.2.the establishment of strains in U87glioma cells of U251, of SHG44nude mouse xenograft tumor model, tumor growth with different concentrations of drug intervention, clear and its mechanism of inhibition of different cell lines according to Weimo Secretary.METHODS1. The mTOR signaling pathway related protein expression and mTOR protein expression by Western Blot Detection of human glioma specimens, an immunohistochemical staining detected32cases of human glioma specimens.2. Fostering stable growth in glioma cell lines in U87, of U251, of SHG44, MTT assay was used to observe different doses of the mTOR inhibitor everolimus inhibition of human glioma cells; apoptosis fluorescent Hoechst33342/PI hair Determination of cell apoptosis; single dansyl cadaverine (MDC), the fluorescence staining of autophagic vesicles; flow cytometry for quantitative cell cycle; Western blot for further analysis to verify the pathways of everolimus on the role of glioma cells and its mechanism.3. Through the establishment of the glioma cell lines in U87, of U251, of SHG44nude mouse subcutaneous xenograft model, and given intraperitoneal injection of everolimus, immunohistochemistry, cell cycle and apoptosis, tumor vascular density were measured, understanding of everolimus inhibition of transplanted tumors of different glioma cell lines and to explore its possible mechanism.RESULTS1. There were significant differences in the expression of mTOR protein between the control and grade Ⅱ, grade Ⅲ or grade Ⅳ (all P<0.01). There was no significant difference in the expression of those protein between the control and grade Ⅰ (P>0.05)..2. Glioma cell lines in vitro in different cell lines by the degree of inhibition increased with the in accordance with the increase in the concentration of Weimo Secretary. In U87-MG cell line is most sensitive; U251cell lines with moderate sensitivity; the SHG-44cell line is the least sensitive.3. Apoptosis fluorescent Hoechst33342/PI double staining results showed that the glioma cell a different apoptotic forms of death, may be autophagy. OK MDC live cell staining showed that the glioma cells to increase the role of everolimus in the acidic vesicle-like organelles (from macrophage foam) formation that everolimus can promote the formation of glioma cell autophagy. The three cell lines are given to the IC50dose of everolimus, flow cytometry cycle analysis after48hours. We found that the three cell lines are specific cells in G0/G1phase arrest. Using the AnnexinV detection did not find significant apoptosis.4. Western blot analysis of the role of everolimus in glioma cell lines after the non-phosphorylation of mTOR, of p70S6K,4E-BP-1and p70S6K and phosphorylation levels. In all three cell lines, with the increase in everolimus dose of mTOR, of p70S6K,4E-BP-1phosphorylation expression levels were significantly reduced phosphorylation of p70S6K and did not find significant changes, only The phosphorylation of4E-BP-1expression levels tandem according Weimo Secretary concentration increased and decreased.5. Established in U87glioma cell lines of U251, of SHG44nude mice subcutaneous xenograft model, to draw the tumor growth curve. Give nude everolimus found at the end of12days, the transplanted tumor volume of each cell line the experimental group compared with the control group was significantly reduced, and by a formula measuring the inhibition rate of the different cell lines, U-87MG the SHG-44and U251cells lines, tumor inhibition rates were30.6%,42.1%,29.7%, everolimus significantly inhibited glioma.6. Determination of different glioma cell lines line apoptosis, each cell line the experimental group and control group, apoptosis, and there is no statistically significant difference (P>0.05). OK cell cycle measurement, each cell line in the experimental group than in the control group were significantly G0/G1phase arrest (P<0.05). U-87MG cell lines of the G0/G1phase of stagnation than the control group, the biggest difference, the SHG-44, followed by the smallest of U251.7. Determination of tumor vascular density, cell line U-87MG, U-251in tumor vascular density values of the experimental group than the control group did not differ significantly (P>0.05), tumor blood vessels of the cell line SHG-44experimental group MVD value of31.1(25.0-37.2) compared with the control group20.9(14.8-27.0) were significantly different (P <0.05). Further measurement of the different cell lines the expression of VEGF in the SHG-44cell line the experimental group, VEGF expression level compared with the control group was significantly lowered, and found no significant difference in the remaining two cell lines.8. Separation of tumor tissue, the immunohistochemical analysis of various specimens, which the U251cell line the experimental group than in the control group pmTOR expression increase in p70S6K1, pAKT expression was decreased, VEGF expression did not differ significantly; U-87MG cell line the experimental group than the control group expression of p70S6K1expression to reduce pAKT pmTOR, VEGF expression did not differ significantly; SHG-44cell line the experimental group than the control group pmTOR, p70S6K1expression did not differ significantly, pAKT expression was decreased, VEGF expression significantly reduced.CONCLUSION1. mTOR inhibitor everolimus by blocking the cell cycle, thereby inducing autophagy of glioma cells, and inhibit tumor angiogenesis, which play the role of anti-glioma.2. mTOR signaling pathway can be used as a new target in the treatment of gliomas, and to provide new ideas for the treatment of glioma.