The Interventive Effects Of Trichostatin A On Apoptosis Of The Glioma Via Regulating Expression Of Survivin

Glioma is the high incidence of the clinical brain tumor currently, due to thehigh mortality, high relapse rate, a serious threat to human health. However, due to itsanatomical site specificity, conventional surgery is extremely limited,so the treatmenteffect of the glioma is not very good. In the current conventional anticancer treatmentof glioma, although some tumor cell killing effect, but also on the normal cells andnerve tissue damage larger, and the prognosis of the course, glial tumor cells canproduce a strong resistance, thus limiting the use of a conventional anticancertreatment of glioma. Therefore, finding a new target for the treatment of gliomas andthe development of new drugs for the treatment of gliomas of great clinical value andpractical significance.Survivin has been found as one of the strongest inhibition of apoptosis proteins,can directly inhibit apoptosis by terminal effector protein Caspase-3to inhibitapoptosis and promote proliferation, so it is for tumor development has a veryimportant significance. It has been reported that, Survivin expression in glioma higher,and the same time in the normal nerve tissue and cells lower expression of it. Survivinwhich has specific expression in tumor and normal tissues is very likely to become anew therapeutic areas glioma target. But now, for the regulation mechanism ofSurvivin in glioma remains unclear.Trichostatin A (TSA) as a histone deacetylase inhibitor representatives can, byinhibiting histone deacetylase activity,which is expressing high in tumor cells, toregulating tumor deacetylation level and regulating genes transcription to the role ofan effective anti-tumor cells. TSA also has a broad-spectrum, high efficiency, lowtoxicity characteristics,while in the anti-tumor. Studies have shown that, TSAeffectively kill tumor cells, at the same time it has a protective effect for nerve tissuesand cells. The feature of TSA which is tumor and normal tissue-specific regulation ofapoptosis may be a certain correlation with Survivin in tumor and normal tissuespecificity of expression. Objective：This study was to investigate the impact of TSA for apoptosis of nerve gliomacells, and further explore for TSA by regulating Survivin mediated apoptosis inglioma-related mechanisms.Methods：Culturing PC12cells and C6cells respectively, by CCK-8detect the impact ofTSA on PC12cells and C6cell of viability at different concentrations dose, todetermine TSA is whether the killing of glioma cells while normal nerve cells withoutdamage and to determine the optimal working concentration of TSA; using flowcytometry experiments and cell morphology was observed after administration ofTSA, the changing of PC12cells and C6cell morphology and the changing of the rateof apoptosis, and further determine the effects of TSA on apoptosis of PC12cells andC6cell; the HDACs activity kit detect the activiting changes of the ClassⅠHDACsbefore and after administration of TSA; immunofluorescence staining when the ClassⅠ HDACs activity changes, the changes of Survivin and by its direct inhibitionCaspase-3expression in C6cells in PC12cells, studying the effects of TSA on theexpression of Survivin in glioma cells and normal nerve cells and the relationshipbetween the classⅠHDACs activity and Survivin expression; using Western blotanalysis the effect of different doses of TSA on Survivin and Caspase-3expressionchanges in the glioma cells and normal nerve cells, by these indicators to evaluate theeffects of TSA on regulating Survivin and the role of TSA on apoptosis of gliomacells by regulation of Survivin. And useing sodium valproate as a tool drug, byHDACs activity kits and western blot experiments for further validating theconclusions.Results:1. CCK-8experimental results showed that: compared with the Control group,the survival rate of C6cells TSA100nM group:81.33%±4.15; the survival rate of200nM group was:65.33%±2.15; the survival rate of400nM group:37.14%±2.66.Compared with the Control group, the survival rate of PC12cells TSA100nM group,200nM group was no significant difference, in the400nM, the survival rate was 84.67%±4.33.2. cell morphology experimental results showed that: compared with the Controlgroup, found no significant changes in the density of PC12cell, no significantchanges in cell morphology; whereas compared with the Control group,the C6cellswith200nM treatment group, decreased cell density, cell spacing becomes large,clear cell outline.3. Flow cytometry results showed that: compared with the apoptosis rate ofControl group (3.37%±0.36),TSA at200nM concentration,PC12cells apoptosisrate (5.42%±0.23) is no significant difference. Apoptosis in the concentration of200nM C6cells is significantly increased, the results are consistent with toxicity testresults,200nM apoptotic rate:30.11%±1.97, Control group apoptosis rate was3.12%±1.31, there is a significantly different (P <0.01).4. The ClassⅠHDAC activity assay results showed that: HDAC1, HDAC2,HDAC3, HDAC8activity in the Control group of C6cells compared with the Controlgroup of PC12cells, there were significant differences (P <0.01), significantly higherthan the activity of the Control group of PC12cell; in C6cells after treatmentadministered200nMTSA24h, compared with the Control group of C6cells, HDAC1,HDAC2, HDAC3, HDAC8activity were decreased significantly (P <0.01), and ahuge decline in the proportion. The200nM treatment group of PC12cells comparedwith the Control group of PC12cells, HDAC1, HDAC2, HDAC3, has no significantchanges in HDAC8activity.5. Immunofluorescence staining showed that: in the administration before, theControl group of C6cells was significantly higher than the fluorescence intensity ofSurvivin of the administered group of C6cells. But there was no significantdifference of the fluorescence intensity of Survivin between the Control group ofPC12cells and the treatment group of PC12cells. Meanwhile, the fluorescenceintensity of Survivin in the Control group of C6cells was also significantly higherthan the Control group of PC12cells; while the TSA administration, the Caspase-3activity of C6cells was significantly higher than the fluorescence intensity of theControl group of C6cells. But PC12cells is not. 6. The western blot results showed: the dose of TSA is100nM,200nM,400nM.the expression of Survivin in C6cells is with the increasing doses of TSA, showing agradual decline, while Survivin of the PC12cells is no significant change;and theCaspase-3expression of C6cells increased with increasing doses of TSA,100nM and200nM in the administration, the Caspase-3expression of PC12cells showed nosignificant difference with PC12cells Control group, in400nM dose wassignificantly increased (P <0.01).7. In the validation experiments, the selection of the tool drug is sodium valproate,using HDAC activity assay experiments and Western blot experiments showed thatconsistent with the experimental results of the TSA.Conclusions:1. In a certain dose range, TSA can effectively kill glioma cells but not in normalnerve cell damage;2. The ClassⅠHDACs activity of Glioma was significantly higher than normalnerve cells, TSA can effectively reduce the activity of the ClassⅠHDACs of gliomacells;3. Survivin expression in gliomas was significantly higher than normal nervecells, inhibit the expression of Survivin protein level is one of the mechanisms of TSAis killing glioma cells;4. TSA may by reducing the activity of the ClassⅠHDACs, changing the histoneacetylation levels of gliomas, regulating the expression of Survivin, thus contributingto increase the activity of Caspase-3, to promote glioma apoptosis.