The Molecular Mechanism Of Cisplatin And Carboplatin On Osteosarcoma Cell Line

Purpose: the adoption of cisplatin and carboplatin for osteosarcoma cell (MG-63) cytotoxicity and the comparative study of the molecular mechanisms, to provide experimental basis for cisplatin and carboplatin in the selective treatment of osteosarcoma.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. Light microscopy observation of cell morphological changes: different concentrations of cisplatin and carplatin 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 assay: The cultures were initiated in 96-well plates at a density of Cells were treated for 48h with various concentrations of ciaplatin and various concentrations of carplatin 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: We gathered cells from control group, IC50 cisplatin group, andIC50 carplatin group on 24h,48h and 72h respectively. We got centrifugation at the rate of 500～1000r/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.4. Western blot analysis: We observed the expression of PCNA, Bcl-2 and Bax-2 in paclitaxel and Pirarubicin treated MG-63 by western blot..Results: 1. After the MG-63 cells treated by cisplatin about 24h, we observed that under the inverted phase contrast microscope, the cells refraction became strong, the volume of some cells became round and small, after 48h, the volume of cells became smaller universally, 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 carboplatin 24h, the increasing number of cells lessened, and volume enlarged, cell membrane broke, and cytoplast overflew, necrosis cells floated. After 48h, the type of most cell became irregular, volume of them became larger, necrosis cells floated, even the living cells were tarnish, stopped growing. After 72h, necrosis cells floated increasingly.2. The MTT results showed that after we treated MG-63 cells in different time 24h,48h and 72h and with drugs in different concentration, 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<0.05)3. After FCM analysis, we couldn't find the peak of apoptosis when the cells were treated by cisplatin in different concentration at 24h, 48h , 72h and by carplatin in different concentration at 24h, 48h , 72h,and phases of the cell cycle without significant change in the proportion.4. Western blot's results: Compared with the control group,by cisplatin and carboplatin-treated osteosarcoma cells showed PCNA, Bcl-2 expression was down regulated, Bax expression was up regulatedConclusions: 1. In this study, as was observed under inverted microscope osteosarcoma cells showed morphological changes in the situation within a certain range of the increase over time of drugs on cells gradually.2. According to the results of the analysis of cell cycle, cisplatin and carboplatin on osteosarcoma cell lines with cytotoxicity and increase their concentration increased, and affirmed the cisplatin, the anti-tumor activity of carboplatin dose-dependent manner.3. Compared with control group, there was no obvios apoptotic peak in the cell cycle, the cell cycle phases without significant change in the proportion of that of cisplatin and carboplatin on tumor cell growth inhibition both periods, the osteosarcoma cells for carboplatin and cisplatin has a similar effect.4. Under the growth curve showed that osteosarcoma cell lines to carboplatin in about 25 times the concentration of cisplatin in order to produce similar growth inhibitory effect. This is consistent with literature reports,which The concentration of carboplatin with a wide range of tumor cell line growth inhibition effects of cisplatin are 1 / 40 ~ 1 / 10, while the same tumor cells inhibited the effect of the amount generated by carboplatin vice response to cisplatin than for small.5. Proliferating cell nuclear antigen (PCNA), is a reflection of cell proliferation status of a good indicator .PCNA in this experiment cisplatin and carboplatin group expression in osteosarcoma compared with the control group decreased significantly, indicating that cisplatin and carboplatin on osteosarcoma cell lines significantly inhibited proliferation.6. Apoptosis is genetically controlled form of cell death, Bcl-2 gene is the first found in apoptosis suppressor gene, Bax can inhibit Bcl-2, promoting apoptosis, Bcl-2 and Bax between decide the proportion between the fate of the cells, if the majority of Bax, while Bcl-2 was inhibited, apoptosis was induced, cell death; otherwise inhibited Bax, the cell to survive In this study, cisplatin and carboplatin group Bax expression increased significantly compared with control group, Bcl-2 expression slightly decreased .under the combination of inverted microscope, Indicated the existence of apoptosis after interaction between cells and drugs, However, the lack of apoptotic peak, which may be associated with platinum-based compounds to be able to grow in any of the cell cycle.