Effects Of As₂O₃ And Vincristine On The Apoptosis In Human Osteosarcoma MG63 Cells And The Mechanisms Involved

Osteosarcoma , a malignant bone tumor which has high malignancy level, fast progress is common in clinic. It more often attacks Adolescents and usually with a bad prognosis. Although the chemotherapy achieved large improvement, a number of patients have medicine resistant. The drugs them-selves have so many side effects . As a result the patiants may get low survival rate for long. In order to reduce the adverse factors, we must find some new drugs from which can we get better effects. Both of As2O3 and Vincristine have ability to define human tumor and are using in clinical theropy.as reported. But no report systematically indicated that they were used on osteosarcoma. To elucidate the molecular mechanisms through which the As2O3 and Vincristine can promot apoptosis in human osteosarcoma MG63 cells, we designed a series experiments to detect the effect of As2O3 and Vincristine of different doses on the apoptosis, the oxidative stress and apoptosis respectively and the possibly involved mechanisms, using MG63 as the model system. We screen the expression of early responsive genes by the effect of As2O3 and Vincristine, and approach the mechanisms of effects of As2O3 and Vincristine on apoptosis in human osteosarcoma MG63 cells .Methods:After treated with different concentrations of As2O3 and Vincristine, changes by apoptosis of MG63 is assessed by the retention of Hoechst33342 a specific fluorescent cationic dye that is readily sequestered by live cells. Also the ROS content is detected by the DCFH-DA staining and the cell apoptosis is measured using Annexin V-PI kit. Real time-PCR analysis is performed to screen the possible target genes which are responsive when MG63 are treated with As2O3 and Vincristine of different doses.Results:One: Laser microscope images show that cells modulate from polygonal shape to the round shape as the concentration of As2O3 and Vincristine increases while the cell adhesiveness decreases, and the cell debris without clear outlines is observed .Two: Flow cytometry analysis shows that the apoptosis pattern is dose-dependent in which the apoptosis increases as the concentration of As2O3 and Vincristine increases.There: the ROS expression pattern is also dose-dependent in which the ROS increases as the concentration of As2O3 and Vincristine increases. Four: Some responsive genes are identified by real time-PCR analysis that the expressions of caspase-9, GADD45 and HO1 go up obviously and CAT goes down when M63 treated As2O3 and Vincristine of different concentrations.Conclusions:One: As2O3 and Vincristine is able to induce apoptosis in human osteosarcoma MG63 cells, and the apoptosis pattern is dose-dependent in which the apoptosis increases in the concentration of As2O3 and Vincristine increases.Two: The ROS level is also dose-dependent in which the ROS increases in the different concentration of As2O3 and Vincristine, suggesting that antioxidant defense system is active when MG63 treated with As2O3 and Vincristine.Three: The expression of CAT goes down and dose-dependent when MG63 treated with different concentration of As2O3 and Vincristine, which can increase the ROS in the cells.Four: The expressions of caspase-9 increases when MG63 treated with different concentration of Vincristine. This fact might indicate the increasing ROS regulates caspase-9 up to induce apoptosis by effect of Vincristine in human osteosarcoma MG63 cells.Five: GADD45 goes up when MG63 treated with different concentration of As2O3. This fact might indicate a pathway which is p53 independent that regulated DADD45 up can induce apoptosis by effect of As2O3 in human osteosarcoma MG63 cells.Six: HO1 increase strikingly when MG63 treated with different concentration of As2O3 sensitively and exceptionally.Collectively, the manifest apoptosis and the ROS level alteration are caused by the different concentration of As2O3 and Vincristine in human osteosarcoma MG63 cells. The increasing ROS and a pathway which is p53 independent can induce apoptosis by effect of As2O3 and Vincristine in human osteosarcoma MG63 cells.