Effects Of Berbamine On The Proliferation And Apoptosis Of Osteosarcoma Cells

ObjectiveThe present study was conducted to explore the inhibitory effect of berbamine on the proliferation of UMR-106cells and its possible mechanism to provide experiment evidence for the clinical application of berbamine in osteosarcoma treatment.Methods1. The rat osteosarcoma UMR-106cells were obtained from the Fourth Military Medical University. Cells were cultured in RPMI-1640medium supplemented with10%fetal bovine,100U/mL penicillin, and100mg/L streptomycin and incubated in a humidified atmosphere of5%CO2at37℃.2. The cells were cultured with different concentrations of berbamine (0,2,4,8,16and32mg/L) for24,48and72h,respectively,and the proliferation were colori-metrically assayed by MTT.3. The cells were interfered with different concentrations of berbamine (0,4,8and16mg/L) for24h,the morphological changes of UMR-106cells were observed by laser confocal scan microscope after Hoechst33258staining.4. The cells were interfered with different concentrations of berbamine (0,4,8and16mg/L) for24h,the apoptotic rates were detected by flow cytometry (FCM) using Annexin V/PI staining.5. The cells were interfered with different concentrations of berbamine (0,4,8and16mg/L) for24h, the cell cycle were detected by FCM using PI staining.6. The cells were interfered with different concentrations of berbamine (0,4,8and16mg/L) for24h, the levels of Bcl-2and Bax were measured by ELISA.7. The cells were interfered with different concentrations of berbamine (0,4,8and16mg/L) for24h, the activity of Caspase-3was detected by spectrophotometry.8. The data were expressed as mean±standard deviation and analyzed by the SPSS17.0software to evaluate the statistical difference. One-way ANOVA was used for Multiple comparisons. LSD-t test was used to analyze the differences between the groups. P<0.05was considered statistically significant.Results 1. Berbamine could obviously inhibit the proliferation of UMR-106cells in a dose-dependent manner (P<0.01).The IC50value at24,48and72h were24.69、8.03and3.54mg/L, respectively.2. There were nuclear condensation and apoptotic body in berbamine treated group.3. Early apoptotic rates of berbamine treated group (0,2,4,8,16and32mg/L) were (1.64±0.29)%,(3.58±0.31)%,(6.27±0.47)%and (11.27±1.09)%, respectively.There was a significant difference in apoptotic rate compared with the0mg/L BBM group (P<0.01)4. Berbamine significantly increased the percentages of cells in G0/G1phase and decreased in S phase and G2/M phase compared with the0mg/L BBM group (P<0.01).5. Berbamine dose-dependently reduced Bcl-2level and increased Bax, causing the reduction of the Bcl-2/Bax ratio and increasing the Caspase-3activity in UMR-106cells significantly.ConclusionBerbamine can inhibit the proliferation of UMR-106cells in vitro, induce cell apoptosis and G0/G1phase arrest. reducing Bcl-2/Bax ratio and increasing the Caspase-3activity might contribute to the apoptotic mechanisms.