Experimental Study On Apoptosis Induced By Simvastatin In Clioma Cells

Objective: To study the effect of cholesterol inhibitor-Simvastatin(SIM) on proliferate inhibitory effect and the apoptosis-inducing activity on glioma cells(U251 cells),and elucidate its mechanism.Methods: Selecting human glioma cell lines U251, using MTT assay to detect the growth rate among different SIM concentration groups (1,5,10,20,30,40,50,60μg/ml) and different time groups (24,48,72,96h), in order to choose proper drug concentration and action time. According to the result of MTT, establishing control and experimental group(5,10,20μg/ml ) .Apoptosis and distribution of cell cycle were examined with flow cytometry. The morphological alterations were confirmed by light microscopy and Hoechst33258 staining. Extracting total RNA of each group cell, assessing the integrality and content of RNA, the level of Survivin,Bax mRNA expression was examined by semiquantitative RT-PCR technique in the U251 cells treated before and after with SIM. The expression of Survivin,Bax protein was qualitationly studied by immunocytochemistry staining and was semi-quantitately examined by flow cytometry in cell lines treated before and after with SIM.Results: 1 MTT assay results:SIM(1,5,10,20,30,40,50,60μg/ml) could inhibit the proliferation of U251 cells.After treated with SIM for 24h to 96h,compared with control group,the OD values of SIM-treated groups decreased, and there was statistically significant difference between control group and every treatment group(P<0.05).Furthermore,with the increasing concentration of SIM and prolonging of treatment time,the OD values decreased gradually,that was, SIM inhibited the proliferation of U251 cells significantly in a dose-and time-dependent manner.Treatment with SIM for 24,48,72,96h,the IC50 was 19.25μg/ml,9.74μg/ml,5.75μg/ml,3.52μg/ml separately.2When treated with SIM, U251 cells had significant morphological changes: U251 cells were minificated, nucleonic pycnosis, cell bodys refraction were weaken, intracytoplasm could see grana. With the increasing concentration of SIM and prolonging of treatment time,the cells rounded gradually and more and more detached from the wall of culture flask,and some cells were broke.At the same time,the typical morphological changes of apoptosis were observed: U251 cells shrinked and broke into pieces,there were punctiform-lamellar cell pieces around the cells.Vacuolus could be observed in the kytoplasm of some cells.At last,so many cell pieces appeared in culture medium with increasing of the number of apoptotic cells.Observed at fluorescence microscope: U251 cells treated with 10μg/ml SIM for 48h,the nuclei of most cells were condensed and fragmented ,condensation of chromatin arranged under the nuclear membrane , with a low nuclear/cytoplasmic ratio, the characteristic nucleolus alternations , apoptotic bodies were observed.3 When cells were harvested for the analysis on distribution of cell cycle and apoptosis by flow cytometry,the results were:After treatment with 0,5,10,20μg/ml SIM for 48h,the number of cells in G0/G1 phase increased gradually,while the number of cells in S phase and G2/M phase decreased gradually.That was, SIM could induce an arrest of cell cycle in G0/G1 phase by a dose-dependent manner.In addition,after treated with 0,5,10,20μg/ml SIM for 48h,72h,the typical apoptotic peak which enhanced gradually with the increasing concentration of SIM was observed and the largest apoptotic percentage was 21.63%.Analysis on expression of Survivin and Bax by FCM showed that:Treating U251 cells with 0,5,10,20μg/ml SIM for 48h resulted in the FI values of Survivin decreasing with the increasing concentration of SIM and the FI value of Bax increasing.To the FI values of every kind of protein,there was statistically significant difference between every SIM-treated group and control group(P<0.05).4 RT-PCR detection results:Before being treated with SIM, Survivin,Bax mRNA were both expressed in U251 cells.Compared to the control group,the expression of Survivin mRNA was decreased markedly in U251 cells exposed to SIM(P<0.05); Bax mRNA was increased significantly(P<0.05).The ratio of Survivin toβ-actin was decreased from 0.944±0.018 to 0.237±0.009 at 20μg/ml concentration of 48h;whereas, ratio of Bax mRNA toβ-actin was increased from 0.219±0.015 to 0.939±0.012.Conclusions: 1 The reseach first time showed that simvastatin,an inhibitor of 3-hydroxy-3-methylglutaryl-CoA,had the effect of anti-glioma cell line U251,which provided a new theoretical basement for reasonable clinical treatment of human glioma.2 Within a certain drug concentration, SIM can inhibit proliferation of U251 cells which depending on a dose-and time-dependent manner concentration.3We confirm that SIM induced an arrest of cell cycle in G0/G1 phase as well as apoptosis in U251 cells.4 Within a certain drug concentration, SIM can inhibit the expression of Survivin,increase Bax in U251 cells. SIM's effects of inhibiting proliferation and inducing apoptosis in U251 cells seem to due to down-regulation of the expression of Survivin mRNA and protein and up-regulation of the expression of Bax mRNA and protein.5 This research revealed that the anti-cancer effect and mol-mechanism are mediated cell apoptosis by SIM,it provides basic theory and experiment foundation for clinical application.