| Background and Objective:Glioma is a common highly-malignant intracranial neoplasm with a low survival (often less than1year) even after the therapy combining with the surgery, radiotherapy and chemotherapy. It creates huge economic and psychological burden to family and individual.Currently, various agents against the glioma can be used for glioma but are associated with many side effects and less efficacy due to the increasing resistance by tumor cells.Therefore, a novel better agent is in an urgent need.Allitricin, an anticancer component in the garlic bulb, is diallyl trisulfide (DADS) and hoped to effectively treatment glioma.Cell and animal experiments have proven that allitricin can inhibit and kill various cancer cells such as the gastric cancer, hepatic cancer, bladder cancer and leukemia.However, no studies of allitricin on glioma are reported.Objectives of this study was to (1)observe effects of allitricin on morphology and cell cycle of human brain glioma cells U251in vitro and explore its inhibition on U251cells;(2)observe effects of allitricin on the cell U251apoptosis;(3)observe changes in bcl-2/bax expressions in U251cells treated with allitricin and investigate the mechanism of allitricin inducing U251cell apoptosis.Methods:1. The CCK-8method was used to assay effects of different concentrations of allitricin on proliferation of U251cell in hours12,24and48.2. Fluorescence microscopy was used to observe the morphology of Hoechst33342-stained apoptotic cells in hours24post allitricin treatment.3. Flow cytometer was used to assay the apoptotic rate and cycle of cells stained both AnnexinV-FITC and PI.4. The immunohistochemical method was used to assay bcl-2and bax expressions in U251cells treated with different doses of allitricin.Results:1. The CCK-8method was used to assay the survival of U251cells after treated with different doses of allitricin in different time points. Statistical analysis showed that inhibition of allitricin on U251cells was time-and dose-dependent (F=25.307,P<0.001and F=80.713, P<0.001) and was positively associated with the time and concentration. The inhibition concentration50%(IC50) of allitricin on U251cells was55.1μg/ml,39.5μg/ml and21.6μg/ml in hours12,24and48, respectively.2. Flow cytometer was used to calculate percentages of Gl, S and S2phases in the cycle of U251cells post treated with0and40μg/ml allitricin for24h. One-factor analysis of variance showed that percentage of Gl, S and S2phases in the cell cycle were (54.00±2.01)%,(23.56±1.62)%and (21.26±1.08)%in the Oμg/ml group and (67.41±2.14)%,(16.35±2.98)%, significantly higher than (13.90±1.18)%in the40μ,g/ml groups, respectively (P<0.001).Results that the percentage of the Gl phase in the cycle of U251cells increased but percentages of S and G2phases in the cycle of U251cells decreased.3. Flow cytometer was used to assay apoptotic rates of U251cells after treated with0,20and40μg/ml allitricin. One-factor analysis of variance showed that the apoptotic rate was significantly different among the3groups (P<0.001),(2.54±0.57)%,(28.34±3.53)%and (46.57±2.97)%in the0,20and40μg/ml groups, respectively. These results showed that the apoptosis of allitricin on U251cells were dose-dependent and a high dose was associated with a high apoptotic rate. Therefore, it could be considered that allitricin could induce the cell apoptosis.4. After treated with40(μg/ml allitricin for24h, U251cells were stained with Hoechst33342fluorescence. Under the microscope, apoptotic cells shrank with pycnosis and condensation of nucleus and edge-set of chromatins and strongly-blue particulate fluorescent signals were visible.5. The immunohistochemical method was used to assay grey values of bcl-2and bax expressions in the cytoplasm of U251cells treated with0,20and40μg/mlallitricin. One-factor analysis of variance showed that grey values of bcl-2were84.6±3.3,124.6±3.7and147.5±3.3and grey values of bax were154.8±2.8,134.5±4.3and104.5±2.7in the3dose groups, respectively. A significant difference was noted among the3doses (P<0.001).With the concentration increasing, the grey value of bax decreased and the grey value of bcl-2increase, illustrating a gradually increasing bax expressions and a decreasing bcl-2expressions. These results suggested that allitricin could induce cell apoptosis by down-regulating bcl-2expressions and up-regulating bax expressions in U251cells.Conclusion:1. Allitricin can inhibit the proliferation of U251cells in a concentration and time-dependent manner.2. Allitricin can induce the apoptosis of U251cells in a concentration-dependent manner.3.Allitricin can change the cell cycle of U251cells, primarily manifested with the increasing Gi phase and decrease S and G2phases.4.The mechanism of allitricin to inhibit proliferation of U251cells is related to up-regulation of bax and down-regulation of bcl-2. |
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