| 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[1]. Surgery and chemotherapy play significant and crucial roles in clinical anti-osteosarcoma cancer treatment to achieve prolonged patient survival[2]. However, a high failure rate and low median survival rate are observed in patients undergoing chemotherapy with recurrent, intractable osteosarcoma[3]. To improve the patient survival rate, investigation of the mechanism of chemotherapy for osteosarcoma treatment is very important. Pirarubicin has been clinically used for the treatment of osteosarcoma while the paclitaxel was used as the secondary chemical drug for osteosarcoma and the application was less than pirarubicin[4]. The antitumor potency of pirarubicin and paclitaxel have been well documented in clinical application[5~7]. However, very few studies have carried out on the analysis of the mechanisms of inducing bone tumor cell death. Therefore, in order to find more reasonable and effective combination treatment, here we investigated the possible apoptosis-inducing activity of treatment with pirarubicin and paclitaxel.Our experiment was about the molecules mechanism of the apoptosis of osteosarcoma which might had been used by pirarubicin and paclitaxelfor clinical treatment.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 growth phase .(1) Light microscopy observation of cell morphological changes: different concentrations of paclitaxel and THP were roled in the MG-63 cells and to observat the growth of cell regularly in inverted phase contrast microscope and to photo .(2) MTT essay : The cultures were initiated in 96-well plates at a density of 5×104. Cells were treated for 72h with various concentrations (10 nmol/L,102 nmol/L,103 nmol/L,104 nmol/L) of paclitaxel or (102nmol/L, 103 nmol/L,104 nmol/L,105 nmol/L) of pirarubicin with three replicate wells for each concentration. The relative cell number of adherent cells was then determined by the MTT method.(3)Flow cytometry analysis: The cells were treated with various concentrations of paclitaxel and pirarubicin for 24h. Then we gathered cells from control group (without medication), identical concentrations (102nmol/L) paclitaxel group, and identical concentrations(103nmol/L) pirarubicin group on 12h, 48h and 72h respectively. We got 5×106cells and centrifugation at the rate of 1000r/min , then washed once with PBS and harvested by trypsinization, washed in 1% bovine serum albumin (BSA) and fixed with 75% ethanol containing 0.5% between 20 for at least 1 hat 4℃. The cells were washed in 1% BSA and resuspended in a cold Pirarubicin staining solution (100μg/ml Rnase A and 10μg/ml Pirarubicin in PBS) 1 ml for 40 min at 4℃. Data acquisition and analysis were carried out by using a flow cytometry system (Beckman Coulter, Inc.,Fullerton, CA,USA).(4) Western blot analysis: We observed the expression of PCNA,cyclinD1,cyclinE,Bcl-2 and bax in paclitaxel and Pirarubicin treated MG-63 by western blot..Results: (1) After the MG-63 cells treated by Paclitaxel about 12h, we observed that under the inverted phase contrast microscope, the cells refraction became strong, the volume of some cells became round and small, and after 24h, cells began to shrink, the adhere ability became weak, volume of them smaller, a few cells floated, after 48h, the volume of cells became smaller universally, cytoplast concentrated, a lot of cells became round and floated, and when it came to 72h, most of the cells floated and the change became significant. After treated by 103nmol/L Pirarubicin 12h, the increasing number of cells decreased and volume enlarged, cell membrane broke, and cytoplast overflew, necrotic cells floated. After 24h, cells at fission stage could rarely be seen, cell membrane broke, more necrotic cells floated, and after 48h, the type of most cell became irregular, volume of them became larger, necrotic cells floated, even the living cells were tarnish, stopped growing. After 72h, necrotic cells floated increasingly.(2) The MTT results showed that after we treated MG-63 cells in different time 12h,24h,48h and 72h and with drugs in different concentration,(10 nmol/L,102 nmol/L,103 nmol/L,104 nmol/L)Paclitaxel,(102 nmol/L, 103 nmol/L,104 nmol/L,105 nmol/L)Pirarubicin, both of the drugs could significantly retrain the multiplication of MG-63, and the effectiveness depended on time of experiment and dose of drugs(p<0.01).(3) After FCM analysis, we found that when the cells were treated in different concentrat(10nmol/L,102nmol/L,103 nmol/L,104nmol/L)24h and then by identical concentrations(102nmol/L) Paclitaxel at 12h,24h,48h and 72h, the peak of apoptosis came out before G0/G1 stage, the percentage of G2/M stage increased associated with increasing of time and concentration, and the percentage of G1 stage had the tendency of decrease. And when the cells were treated byPirarubicin in different concentration (10nmol/L,102 nmol/L,103 nmol/L,104 nmol/L,105 nmol/L)24h and then by identical concentrations(103nmol/L) Pirarubicin at 12h, 24h,48h and 72h, there was not obvious apoptosis peak, and the percentage of S stage had the tendency of increase with increasing of time and concentration. (4) Result of Western-blotting: Compared with the control group, the protein expression of PCNA, cyclinD, cyclinE, bax and Bcl-2 were down regulated in pacitaxel and Pirarubicin groups, and bax protein expression up regulated.Conclusion: (1) The MG-63 osteoscarcoma cell could be inhibited by Paclitaxel and Pirarubicin depended on time and dose.The final inhibit rate was the same in different concentration by increasing the time. The concentration at apoptosis peak might be the suitable concentration. The inhibit rate of Paclitaxel in low concentration and long treating time was same as that in high concentration and short treating. So, concentration and treating time were not the point, the best effect of the drug was just the concentration and treating time at the apoptosis peak.(2) Paclitaxel could blockade the MG-63 cell at G2/M stage, and had time-dose dependency. The drug could blockade the cycle of cells and further induce the apoptosis. Pirarubicin could stop cells in the G2 stage, stay the cells down in S stage, and inhibit the synthesis of DNA to interfere the cells'fission. The drug also had time-dose dependency in a certain range, and was more dependent on dose. After treated by Paclitaxel in different concentration, the percentage of G0/G1 stage decreased entirely. It showed that Paclitaxel could blockade the cells'multiplication on G0/G1 stage. The mechanism of the blockage needed to be researched. When cells were treated by Pirarubicin in different concentration, the percentage of G0/G1 stage decreased initially and then increased. It showed that Pirarubicin could prolong the cells'multiplication on G0/G1 stage. (3) If we stopped using the drug on oversize S stage, the GM cells might fission unceasingly and fast, and if on the G2/M stage, the cells came to S stage fast after stopped using the drug. It was most efficient if cells were blockaded on G0 stage. There was not apoptosis peak when concentration of the drug was very low. It revealed that the drug could kill normal cells but not tumor cells. The situation was the same as before when concentration of the drug was very high. It displayed that apoptosis of cells decreased when the concentration was high and the side effect of the drug was so obvious. Only if the concentration of the drug was the same as it on apoptosis peak, all tumor cells could be killed by destroying the MG-63's cell membrane and its function.(4) Paclitaxel and Pirarubicin could be used in the treatment of osteoscarcoma together. |
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