| Objective: Osteosarcoma, which is mainly suffered in adolescent of 10~20 years old, is the most common primarily malignancy bone tumor with a higher malignancy and the lower prognosis. Lung metastasis and bone metastasis easily happended early in these patients. Osteosarcoma, which the 5-year survival rate is very low, severely affects physical and mental health of the young people. Although surgery and adjuvant chemotherapy made significant improvement in early prognosis of patients, it is still a malignant disease which the mortality and disability rate is very high at present. Chemotherapy is the only effective way in patients of distant metastasis. However, currently, clinic drugs that can be chosen are very limited. Thus, filtering anti-cancer drugs which are lower toxicity and higher efficacy has become a very important area. Arsenic trioxide (As2O3) has been applied to the treatment of relapsed acute promyelocytic leukemia (APL, M3), and owns a significant effect. But the use of arsenic trioxide in the treatment of osteosarcoma was rarely. Vincristine(VCR) has already been used in the treatment of osteosarcoma, but researches of the specific molecular mechanism of treatment was still very few. Our experiment is mainly about the apoptosis and molecular mechanism of apoptosis signal transduction pathway of osteosarcoma which might have been induced by arsenic trioxide and vincristine, and the experiment may help provide a theoretical basis in clinical medication in the future.Methods: MG-63 cells were cultured at 37℃in DMEM supplemented with 10% heat-inactivated FBS in the tissue culture flask under a humidified 5%CO2 atmosphere , apply to the experiment when they entered the logarithmic phase .1 Microscope observation of cell morphological changes: Different concentrations of As2O3 and VCR were roled in the MG-63 cells. We observed the growth of cell regularly in inverted phase contrast microscope.2 MTT assay: Adjusting the cell concentration to 1×105/ml, and each hole add 100μl cultured in 96-well plates. Then the cells were exposed in different concentrations of As2O3 (10, 8, 6, 4μmol/L) and VCR (4, 2, 1, 0.5μg/ml)in corresponding well when the cells entered the logarithmic phase, at last using MTT detects the inhibition of cell growth in different times.3 Flow cytometry analyses: The cells were treated with various concentrations of As2O3 and VCR for 36h. Then we gathered cells from control group (without medication), 2μg/ml VCR group on 12h, 24h ,36h and 48h, and 8μmol/L As2O3 group (24h, 36h ,48h and 60h) respectively. I harvested the cells by trypsinization at least the number of the cells(1～5)×106 and centrifugated at the rate of 500～1000r/min; Then washed once with PBS, and fixed with 70% ethanol containing 0.5% Tween 20 for at least 1h at 4℃. After that centrifugated at the rate of 500～1000r/min ,throwed away the ethanol and resuspended in a cold Propidium Iodide for 30 min at 4℃. Using Epics-XLⅡof flow cytometry measured apoptosis and cell cycle distribution of the cells. At last appling software to analysis the results.4 Western blot analyses: We observed the expression of PCNA, Caspase-3, CyclinD1, CyclinE, Bcl-2 and Bax of the MG-63 cells exposed to As2O3 and VCR by western blot.Results:1 The appearance of the MG-63 cells exposed to As2O3 or VCR were observed under inverted phase contrast microscope. As the concentration increased and the time extended, the shape of the cells began to change gradually. At first, the long shape turned round, but the cell membrane integrity. And then the cells started to shrink, the number of round cells gradually increased, the adhesion of the cells increasingly weakened, and floated. Then the volume of floated cells generally turned smaller than before, and the cytoplasm condensed, morphology became irregular, membrane got incomplete, and the number of adherent cells was fewer and fewer. In the end the majority of cells floated, more obvious changes can be seen, and adherent cells were rarely.2 The MTT results showed that after we treated MG-63 cells in different time 24h,36h,48h and 60h and with drugs in different concentration, (10,8,6,4μmol/L)As2O3,(4,2,1,0.5μg/ml)VCR, both of the drugs could significantly retrain the multiplication of MG-63, and the effectiveness depended on the time of experiment and dose of drugs(p<0.05).3 After FCM analysis, we found that when the cells were treated by As2O3 in different concentration (10,8,6,4μmol/L)at 24h ,36h, 48h and 60h, the peak of apoptosis came out before G0/G1 stage, the percentage of S stage increased associated with increasing of time and concentration, the largest proportion of S stage got up to 81.4%.But the percentage of G1 stage had decreased obviously .When the cells were treated by VCR in different concentration (4,2,1,0.5μg/ml)at 12h,24h ,36h and 48h, the peak of apoptosis came out before G0/G1 stage, the percentage of G2/M stage increased associated with extend of time and concentration relative to the control group, the largest proportion of G2/M stage got up to 92.5%.But the percentage of G1 stage had decreased progressively.4 Result of western-blotting: Compared with the control group, the protein expression of PCNA, CyclinD1, CyclinE, Bax and Bcl-2 were significantly restrained in As2O3 and VCR groups, and Bax, Caspase-3 protein expression strengthened.Conclusion:1 As2O3 and VCR had the effect of inducing apoptosis and restraining the multiplication of the MG-63 cells, and the effect of drugs depended on time and dose. With the increase of time, the final restrain effect of As2O3 and VCR were the same in different concentrations. The cells treated a long time with Low drug concentration had the same inhibition as the cells exposed short-term in a high concentration had the same. So, the concentration of the highest apoptotic peak may be the optimal concentration.2 VCR showed a significant blockade at G2/M phase on MG-63 cells and could induce apoptosis, and there was a time-dose dependency. As2O3 could block the cell cycle at S phase, the cells intercepted at the S phase could inhibit synthesis of DNA or protein.3 As2O3 and VCR might induce apoptosis through inhibiting the expression of the Bcl-2 protein and enhancing the expression of Bax, Caspase-3 protein after exposing on MG-63 cells. And the two drugs might inhibit proliferation by decreasing the expression of CyclinD1 and CyclinE.4 As2O3 combinating VCR could be used in the treatment of osteoscarcoma.
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