Experimental Research On The Effects Of Combination Treatment Of Rapamycin And Adriamycin On Inhibition Of Human Osteosarcoma Mg-63Cell And Their Mechanisms

Objective To investigate the combination effects of rapamycin (RAPA)and adriamycin(ADM) on the inhibition ratio of human osteosarcomaMG-63cell and prove the combination treatment of two agents havesynergistic effect on inhibiting human osteosarcoma MG-63cell,it can alsoimprove sensitivity of Adriamycin to osteosarcoma cell line MG-63, andfurthermore analyze the possible mechanisms.Methods Scratch assay to test the ability of cell migration;humanosteosarcoma MG-63cell in logarithmic growth phase was treated WithADM solution of different concentrations（0.125mg/L,0.25mg/L,0.5mg/L,1mg/L,2mg/L）alone and with RAPA solution（10nmol/L,20nmol/L,50nmol/L,100nmol/L,200nmol/L）alone, the cell inhibition ratedetermined by MTT method after24hours、48hours、72hours;furthermore,ADM solution of different concentrations（0.125mg/L,0.25mg/L,0.5mg/L,1mg/L,2mg/L）in combination with RAPA solution of different concentrations（20nmol/L,50nmol/L,100nmol/L）,the cell inhibition ratedetermined by MTT method after48hours，and draw inhibition rate andconcentration histogram, Coefficient of druginteraction（CDI）to evaluatethe nature of the interaction between the two drugs; According to theoptimal combination concentration,to randomly divided into five groups:blank control group, dissolvent group, ADM group, RAPA group andcombination group, the cell proliferation in5days was detected by MTT;To observe the inhibiting effect of rapamycin, doxorubicin alone and withthe combined application on the MG-63cell line after48hours under theinverted phase contrast microscope;The expression levels of HIF-1α,VEGF, MMP-2mRNA were analyzed by Real time PCR assay.Results Scratch experiments showed that MG-63cell invasion andmetastasis occurred significantly after48h, indicating that its migratorydynamic is good. RAPA and ADM each individual drug group, With higherdrug concentration, the inhibition enhanced gradually, showing a significantdose-dependent effect, Analysis of variance between various concentrationgroup and the comparison with the control group were statisticallysignificant (P <0.05); The proliferation of MG-63cell treated with100nmol/L RAPA combined with0.5mg/L ADM was significantly inhibited,and have the most obvious synergy;From the3rd day to the6th day of thecell culture, the cell proliferation rate in combination group was lower thanthose in control, dissolvent, ADM and RAPA groups,the difference wasstatistically significant（P<0.05）; The number of cells in the combinationgroup was least under inverted phase contrast microscope after48h;Theexpression levels of HIF-1α, VEGF, MMP–2mRNA in combinationgroup was lower than those in control, dissolvent, ADM and RAPA groups, the difference was statistically significant（P<0.01）.Conclusion Combination treatment of RAPA and ADM Has obvioussynergies in improve osteosarcoma MG-63cell inhibition rate，it can alsoimprove the sensitivity of human osteosarcoma MG-63cell to ADM, themechanism perhaps lies in the combination of the two drugs can reduce theexpressions of HIF-1α, VEGF, MMP–2mRNA.