| Objective: This experiment aimed to evaluate the effect of thalidomide on proliferation and the expression of HIF-1 (hyp- oxia inducible factor-1 ),VEGF(vascular endothelial grow- th factor) and the concertration of calcium in glioma cell line in vitro, to investigate possible mechanism of its role as angiogenesis inhibitor and activity of anti-tumor, meanwhile, to investigate the effects of thalidomide combined with anti-cancer agents DDP on human glioma cell line U251 in vitro.Methods:1 Human glioma cell line U251 was incubated in culture medium in vitro, using MTT assay to detect the growth rate among different thalidomide concentration groups (10,25,50,100μg/ml) and different time groups (24,48,72h). Apoptosis and distribution of cell cycle were examined with flow cytometry. 2 According to the result of MTT, establishing control and experimental group, extracting total RNA of each group, assessing the integrality and content of RNA, the level of HIF-1,VEGFmRNA expression was examined by semiquanti- tative RT-PCR technique in the U251 cells treated before and after with thalidomide. The expression of HIF-1,VEGF protein was semi-quantitately examined by flow cytometry in U251cells treated before and after with thalidomide.3 MTT colorimetric method was performed to evaluate the potential cytostatic effect of combining thalidomide with DDP on human glioma cell line U251 and the OD values were computed. To determine whether the combination of thalidomide with DDP results in a synergistic cytostatic effect, Isobolanalysis formula was performed. In the analysis, there is synergy when the interaction index is less than 1; additivity when the interaction index is equal to 1; and antagonism when the interaction index is more than 1. 4 Confocal laser scanning microscope(LSM)was performed to evaluate the calcium concentration change by different thalidomide concentration groups (10,25,50,100μg/ml) 72h.Results: 1 MTT assay results:Within the concentration of (10~100)μg/ml, thalidomide can obviously inhibit proliferation of U251 in vitro. The inhibition ratio was stepping up as the concentration increasing, and the highest inhibition ratio was in the group with the concentration of 100μg/ml and 72h. There was significantly statistical significance of inhibition ratio in different concentration groups of U251 cells after being treated thalidomide (P<0.01). Treatment with thalidomide for 48h,72h, the IC50 was 51.62μg/ml,35.76μg/ml,separately. When cells were harvested for the analysis on distribution of cell cycle and apoptosis by flow cytometry, the results were: After treatment with10,25,50,100μg/ml thalidomide for 72h, the number of cells in G0/G1 phase increased gradually, while the number of cells in S phase and G2/M phase decreased grudually. That was, thalidomide could induce an arrest of cell cycle in G1 phase by a dose-dependent manner. In addition, after being treated with 10,25,50,100μg/ml thalidomide for 72h, the typical apoptotic peak which enhanced gradually with the increasing concentration of thalidomide could be observed ,and the apoptotic percentage was 9.31 %,14.53%,19.36%,28.30%.seperately,and there was statistically significant difference between control group and every treatment group(P<0.01). 2,RT-PCR detection results:There was different depression of the HIF-1,VEGF mRNA expression in different concentration groups of U251 cells after being treated thalidomide. Thalidomide obviously repressed the HIF-1,VEGF mRNA expression in U251 at 100μg/ml after treating 72h, and there was significantly statistical significance between before and after treatment (P<0.01). FCM assay results:The expression of HIF-1,VEGF protein had been examined in U251 cells being treated with 0,10,25,50,100μg/ml thalidomide for 72h, the FI-value of HIF-1 in U251 was 1.719,1.645,1.329,1.202,1.081,the FI-value of VEGF in U251 was 2.248,1.976,1.553,1.337,1.097.respectively. The difference was statistically significant (P<0.05). The expression of HIF-1,VEGF protein was significantly inhibited after treament with thalidomide of 100μg/ml,72h. There was consistent with the RT-PCR results. 3 MTT colorimetric method was performed to evaluate the potential cytostatic effect of combining thalidomide with anti-cancer agents cisplatin in U251 cells. The results showed: thalidomide excerted a synergistic cytostatic effect in U251cells when combined with cisplatin. In the range of concentration from 10μg/ml to 100μg/ml, thalidomide had the enhancing synergistic effect combined with cisplatin with the increasing concentration. The IC50 of cisplatin in U251 cells alone was22.31μg/ml,When combined with 10,25,50μg/ml thalidomide, the IC50 decreased to9.62,4.68,2.41μg/ml; Thus, there was synergistic interaction between thalidomide and cisplatin in U251 cells. 4 The analysis of isobolanalysis showed that the interaction index for thalidomide combined with cisplatin used in U251 cells was 0.86 as the inhibition ratio was 50% respectively. It proved that thalidomide indeed had a synergistic effect on inhibiting the proliferation of U251 cells combined with cisplatin. 5,The fluorescence intensity of calcium was increased with the concentration of thalidomide by confocal laser scanning microscope. The fluorescence intensity of calcium was stepping up as the concentration increasing, and the highest intensity was in the group with each concentration and 72h.Conclusions: 1,We comfirmed that thalidomide had the capability of inhibiting the proliferation of U251 cells in vitro by a dose and time-dependent manner. 2,We confirmed that thalidomide induced an arrest of cell cycle in G1 phase as well as apoptosis in U251 cells, expressing the inducement apoptotic is one of the mechanisms of its anti-tumor. 3,Within a certain drug concentration, thalidomide can inhibit the mRNA and protein expression of HIF–1 and VEGF in U 251 cells. 4,Thalidomide can hoist the calcium concertration of the U251 cell.It was proved that thalidomide has anti-angiogenesis and directly anti-tumor dual effects. 5,The thalidomide combined with cisplatin had a synergistic effect on inhibiting the proliferation of U251 cells.6,This research proved that thalidomide has the effect of anti- glioma and inriched the treatment of glioma through combining thalidomide with cisplatin.This provide a new theoretic way for glioma integrational therapy. |
