Effect Of Mitoxantrone On Proliferation,differentiation And Apoptosis Of Multiple Myeloma Cell Lines RPMI8226

Objective:The study investigated the effects of mitoxantrone on proliferation,differentiation and apoptosis of human multiple myeloma cell lines RPMI-8226 in vitro and try to explore the possible mechanism.Methods:1.CCK-8 kit detected that proliferation inhibition rates of RPMI8226 cells exposed to different concentrations Mit(0,0.2ug/ml,0.4ug/ml,0.6ug/ml,0.8ug/ml,1.0u g/ml,2.0ug/ml)for 24 h and 48 h.2.Inverted phase-contrast microscope observed that morphological changes of RPMI8226 cells exposed to different concentrations Mit(0,0.2ug/ml,0.6ug/ml,1.0ug/ml,) for 48 h. Apoptosis morphological changes of RPMI8226 cells after AO/EB staining wered observed by fluorescence microscope.3.Flow cytometry detected that surface antigen CD138,CD38,CD56,CD49 e expression of RPMI8226 cells exposed to different concentrations Mit(0,0.2ug/ml,0.6ug/ml,1.0ug/ml,) for 24 h and 48 h.4.Exposed to different concentrations Mit(0,0.2ug/ml,0.6ug/ml,1.0ug/ml) for 48 h, the RPMI8226 cells cell cycle was detected by PI staining and flow cytomet ryanalysis.5.Exposed to different concentrations Mit(0, 0.2ug/ml,0.6ug/ml,1.0ug/ml) for24 h and 48 h, the RPMI8226 cells early-stage apoptotic by Annexin V-FITC/PI double staining with flow cytometry.6.Flow cytometry detected that anti-apoptosis related protein Bcl-2 expression of RPMI8226 cells exposed different concentrations of Mit(0,0.2ug/ml,0.6ug/ml,1.0ug/ml) for 24 h and 48 h.7.All experimental results was analysed by statistical software(version, 17.0).Results:1.CCK-8 method detected that proliferation inhibition rates of RPMI8226 cells exposed to different concentrations Mit(0-2.0ug/ml) was time and dose dependent.They were 9.57±2.07%,14.58±3.05%,27.66±1.74%,34.78±2.90%,42.61±1.78%,59.78±1.92% for 24 h,and the proliferation inhibition were 19.41±1.98%,32.52±2.57%,39.41±2.31%,57.93±2.73%,80.06±2.06%,96.73±2.39% for 48 h.2.Inverted phase contrast microscopic and fluorescence microscope showed that RPMI8226 cells presented typical apoptosis morphological changes after 48 h ofdrug action.3.On the surface of RPMI226 cells were highly expressed CD49 e, CD138,CD38, CD56 molecules. Compared with the control group, the expression rates of CD49 e, CD138, CD38, CD56 were not statistical significantce(P> 0.05).4.Flow cytometry analysised that cells cycle main block in G2/M phase of RPMI8226 cells exposed to different concentrations of Mit(0.2ug/ml,0.6ug/ml,1.0ug/ml) for 48 h,and the proportion of cells in G2/M phase were(13.55±1.08)%,(27.77±1.82)%,(62.81±1.67)%.5.The early-stage apoptotic rates of RPMI8226 cells exposed to different concentrations of Mit(0.2ug/ml,0.6ug/ml,1.0ug/ml) for 24 h were(20.46±2.46)%,(40.56±1.58)%,(53.35±1.49)%;for 48 h early-stage apoptotic rates were(34.23±1.63)%,(57.92±2.94)%,(74.50±2.47)%,early apoptosis rates were significantly higher than the control group, the results were statistically significant(P<0.01).6.The protein Bcl-2 expression rates were(54.97±1.26)%,(45.14±1.79)%,(41.06±1.72)%,(34.96±1.03)%,(47.60±1.29)%,(34.26±1.04)%,(11.71±1.32)%,(5.36±1.41)%of RPMI8226 cells which exposed to different concentrations of Mit(0.2ug/ml,0.6u g/ml,1.0ug/ml) for 24 h and 48 h,with the increase of drug concentration and acti on time,Bcl-2 protein expression was decreased(P<0.05).Conclusion:A certain concentrations of Mitoxantrone could inhibit the proliferation of RPMl8226 cell lines significance,Mitoxantrone of RPMI8226 cells without differentiation, without promoting the transformation of cells to the malignant degree,Mitoxantrone can induce RPMI8226 cell apoptosis,its mechanism of action is through the cell cycle arrest and cut control the Bcl- 2 protein expression.