Experimental Study Of Bufalin Combined With Ciplatin On Apoptosis Of Osteosarcoma 143B Cells

Objectives:The effects of osteosarcoma 143B cells on the proliferation and apoptosis of osteosarcoma 143B cells were investigated by using 143B cells of osteosarcoma,and the possible mechanisms were explored.Methods:Using logarithmic growth of osteosarcoma 143B cell line,the concentration of bufalin?0,6.25,12.5,25,50,100nmol?and cisplatin?0,50,100,200,400ng/ml?were used to 143B cells at 24,48,72h by MTT method,calculate the IC₅₀0 values of the half-inhibitory concentration of bufalin and cisplatin respectively;Selecting 1/4IC_50,1/2IC₅₀0 and IC₅₀0 values and the same level of IC₅₀0 values of bufalin and cisplatin were combined individually,after interfering with 143B cells for 48 hours,the effect of combination was calculated by the Jin's formula,and the subsequent experiments were grouped.The cells of each group were observed by inverted microscope for 48hours morphological changes were observed.Morphological changes of cells in each group stained with Hoechst 33258 after 48h intervention by fluorescence inverted microscope.The apoptosis of Annexin V-FITC/PI double stained cells was detected by cell sorting instrument.Flow cytometry was used to detect the cell cycle arrest of PI staining cells in each group after 48 hours of intervention.The expression of Bcl-2,Bax and Caspase-3 apoptosis-related proteins in each group were detected by Western blot.Results:1.MTT results showed that 143B cells were intervened in the bufalin drug concentration group?0,6.25,12.5,25,50,100 nmol?and cisplatin drug concentration group?0,50,100,200,400 ng/ml?.The cell proliferation inhibition rate increased,and the cell proliferation inhibition rate increased with the prolongation of the intervention time,and the IC₅₀0 value of bufalin was 15nmol and the IC₅₀0 value of cisplatin was 150ng/ml.2.Jin's formula evaluates the effect of combination therapy:the concentration of bufalin alonely is 3.75nmol,7.5nmol,15nmol group and cisplatin alonely concentration is 37.5ng/ml,75ng/ml,150ng/ml group,and combined with the same level IC₅₀0 concentration group was?3.75nmol+37.5ng/ml?,?7.5nmol+75ng/ml?and?15nmol+150ng/ml?respectively.The inhibition rate was calculated after 48 hours of intervention.The bufalin was combined with cisplatin only?15nmoL+150ng/ml?group,it is calculated the q value to be 1.29 greater than 1.15 by the Jin's formula,and the combination of the two drugs had a synergistic effect.Therefore,the experiments were divided into:blank control group,bufalin?15 nmol?group,cisplatin?150 ng/ml?group and bufalin combined with cisplatin?15 nmol+150 ng/ml?group.3.Observed under an inverted microscope:143B cells in the blank control group grew well,and the growth rate of 143B cells was slowed down in the bufalin group,cisplatin group and the combined group.The exfoliated cells and adherence cells were more obvious than the single drug group,and the cell spacing was also significantly increased.4.Hoechst33258 staining fluorescence detection:143B cells in the blank control group showed light blue fluorescence,and the amount of fluorescence in the bufalin group and cisplatin group 143B increased densely,and the combined group was more obvious.5.Apoptosis double staining assay of cell sorting instrument Annexin V-FITC/PI:The apoptosis rates of the combination group,bufalin group and cisplatin group were:?62.75±4.26?%,?43.35±4.27?%,?43.51±3.01?%,significantly higher than the control group?17.80±2.36?%,the combination group was more obvious,the difference was statistically significant?P<0.01?.6.Flow cytometry PI cell cycle assay:Compared with the control group,the bufalin group ratio of 143B cells in S phase and G₂ phase increased?P<0.05?,and the ratio of cisplatin group in S phase and G₂ phase increased,?P<0.01?,the combined group was more significant than the control group in S phase and G₂ phase increased,the difference was statistically significant?P<0.01?.7.Western blot analysis showed that the expression of Bax and Caspase-3 in bufalin group,cisplatin group and combination group increased significantly,Compared with the control group,the expression of Bcl-2 in each group decreased,which was statistically significant?P<0.01?.The combined group was more obvious?P<0.01?.Conclusions:1.Both bufalin and cisplatin can inhibit the proliferation of osteosarcoma 143B cells in a time-concentration-dependent manner,and the two drugs has synergistic effects,and the combined group effect is more obvious.2.Bufalin and cisplatin can cause changes in cell morphology and apoptosis morphology of osteosarcoma 143B cells,and the combined group effect is more obvious.3.Both bufalin and cisplatin can increase the apoptotic rate of osteosarcoma143B cells,the combination group is higher than the single drug group,and the cell cycle is blocked in the S phase and G₂ phase,and the combined group effect is more obvious.4.Bufalin combined with cisplatin induced apoptosis of osteosarcoma 143B cells,which may be related to the up-regulation of Bax,Caspase-3 protein and down-regulation of Bcl-2 protein expression.