| Objective: The aim of this study was to investigate the effect of a novel Aurora A kinase inhibitor alisertib(ALS)and a selective Bcl-2 inhibitor ABT-199,singly and in combination,on the proliferation,cycle and apoptosis of double-hit lymphoma(DHL)cell lines and to explore its underlying mechanisms,in order to provide a theoretical basis for a combinatorial targeted treatment strategy against DHL,and thus improving the prognosis of DHL patients.Methods: 1.ROS-50、DOHH2 and VAL cells were treated by different concentrations of ALS or ABT-199(0μM,0.01μM,0.1μM,1μM,10μM)for 24 h,48h and 72 h.Cell viability was assessed by MTS assay.2.The values of IC50 of the two agents in three different DHL cell lines were evaluated by Graph Pad Prism 5.0 software.3.Cells were divided into four groups for synergistic experiments: control group,ALS group(1/5,2/5,3/5,4/5,5/5 IC50),ABT-199 group(1/5,2/5,3/5,4/5,5/5 IC50)and co-treatment group(concentrations were consistent with monotherapy group).Cell viability was detected by MTS assay after 48-h incubation.4.The synergistic effect of drug interaction was analyzed using Combination Index(CI)by Compu Syn software.5.Cells were treated with ALS or/and ABT-199 at 2/5 IC50 for 48 h,then PI staining and flow cytometry were performed to assay the cell cycle.6.Cells were treated with ALS or/and ABT-199 at 3/5 IC50 for 48 h and apoptotic rates were detected by Annexin V/PI staining and flow cytometry.7.Western blot was performed to determine alterations in drug-targeted protein(MYC,Bcl-2,Aurka,p-Aurka)expression,cell cycle regulatory protein(Cyclin B1,CDK1,p21)and apoptosis-related protein expression(Caspase-3,PARP);8.Differences were analyzed by one-way ANOVA,using SPSS Statistics 20 software.Results: 1.Exposed to 0μM,0.01μM,0.1μM,1μM,10μM of ALS for 24 h,the inhibitory rates in three DHL cells were 0%,4.20%~10.95%,20.10%~35.87%,38.13%~48.50%,51.87%~71.37%,respectively;the inhibitory rates for 48 h were 0%,14.70%~18.43%,48.80%~69.03%,67.70%~85.63%,79.70%~95.13%;the inhibitory rates for 72 h were 0%,23.97%~31.20%,73.77%~83.33%,86.57%~94.43%,94.50%~97.03%.Exposed to 0μM,0.01μM,0.1μM,1μM,10μM of ABT-199 for 24 h,the inhibitory rates in three DHL cells were 0%,11.62%~18.37%,40.70%~54.33%,55.27%~83.41%,64.53%~95.25%,respectively;the inhibitory rates for 48 h were 0%,24.77%~32.60%,55.73%~61.17%,77.53%~89.23%,92.57%~96.48%;the inhibitory rates for 72 h were 0%,31.47%~43.80%,65.93%~76.12%,86.13%~97.67%,94.90%~101.37%;2.After treatment of ALS single-agent for 24 h in ROS-50,DOHH2 and VAL cells,the IC50 values were: 3082 n M,1280 n M,1452 n M,respectively;the IC50 values for 48 h were:49n M,124 n M,108 n M;the IC50 values for 72 h were: 22 n M,28 n M,16 n M.After treatment of ABT-199 for 24 h in ROS-50,DOHH2 and VAL cells,the IC50 values were: 408 n M,152 n M,120 n M;the IC50 values for 48 h were: 72 n M,60 n M,43 n M;the IC50 values for 72 h were: 28 n M,17 n M,13 n M;3.Cells were treated with ALS and/or ABT-199 at a panel of concentrations(1/5,2/5,3/5,4/5,5/5 IC50)for 48 h,the inhibitory rates of combined treatment in ROS-50 cells were:(43.50±3.54)%,(55.50±2.12)%,(69.53±5.18)%,(82.35±0.92)%,(86.74±1.77)%;in DOHH2 cells were:(36.25±3.85)%,(52.75±2.05)%,(71.95±4.31)%,(82.65±3.61)%,(92.40±1.70)%;in VAL cells were:(38.45±3.18)%,(56.55±2.89)%,(68.40±2.41)%,(76.34±3.18)%,(82.57±2.76)%;4.ALS/ABT-199 co-treatment exhibited synergism in inhibiting DHL cell viability by Compu Syn software(CI < 1);5.Co-exposure to ALS and ABT-199 synergistically arrested cell cycle in G2/M phase.The ratios of ROS-50 cells in G2/M phase of control group,ALS group,ABT-199 group and ALS/ABT-199 combination group were:(13.09±2.94)%,(23.98±1.11)%,(15.95±3.48)%,(37.59±3.96)%;the ratios of DOHH2 cells in G2/M phase were:(17.62±4.62)%,(22.53±1.91)%,(19.04±4.05)%,(35.63±2.68)%;the ratios of VAL cells in G2/M phase were:(11.67±1.20)%,(22.94±1.66)%,(16.09±3.19)%,(30.94±3.14)%,p<0.05;6.Co-administration of ALS and ABT-199 synergistically induced apoptosis in DHL cells: the apoptotic rates of control group,ALS group,ABT-199 group and ALS/ABT-199 combination group in ROS-50 cells were:(3.03±1.04)%,(15.39±1.96)%,(16.32±2.59)%,(51.98±2.73)%;in DOHH2 cells were:(2.23±0.67)%,(16.33±2.19)%,(17.33±4.29)%,(47.50±5.86)%;in VAL cells were:(3.27±0.93)%,(16.47±2.33)%,(15.06±1.05)%,(43.70±6.16)%,P<0.01;7.Western blot revealed an unchanged expression of Bcl-2 protein after drug exposure.The expression of p-Aurka in combination group was dramatically decreased than single agent group,while the alterations of the total Aurka expression were not apparent among groups.MYC protein levels were downmodulated upon treatment with ALS in DOHH2 cells.ALS and ABT-199 downregulated Cyclin B1 and CDK1 expression and upregulated p21 expression.ALS in combination with ABT-199 cooperatively contributed to Caspase-3 activation and PARP cleavage.Conclusions: 1.ALS or ABT-199 as a single agent had modest cytotoxic activity against DHL cells in a concentration-and time-dependent manner.2.Combined exposure of ALS and ABT-199 synergistically suppressed cell viability of MYC/Bcl-2 DHL cells compared with either drug alone.3.Low concentrations(2/5 IC50)of ALS cooperated with ABT-199 to arrest cell cycle in G2-M phase(p<0.05).4.At higher concentrations(3/5 IC50)a significant increase in apoptotic cells was consistently observed when combined(P<0.01).5.Combination with ALS and ABT-199 remarkably reduced p-Aurka expression,while total-Aurka and Bcl-2 protein expression remained unchanged.6.ALS and ABT-199 accumulate cells in G2-M phase by downregulating Cyclin B1 and CDK1 and upregulating p21 expression.7.ALS in combination with ABT-199 induced cell apoptosis via intrinsic apoptotic pathway.The mechanisms of synergistic cytotoxicity of ALS and ABT-199 include the cooperating induction of G2/M phase arrest,activation of intrinsic apoptotic signaling pathway,and reduction in p-Aurka protein expression. |
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