| ObjectiveThe effects of arsenic trioxide combined with dihydroartemisinin on proliferation inhibition,cell cycle distribution and apoptosis of human acute monocytic leukemia cell lines THP-1 and SHI-1 were investigated,and the mechanism was discussed.The therapeutic effect of arsenic trioxide combined with dihydroartemisinin on acute monocytic leukemia was investigated by establishing an animal model of acute monocytic leukemia.Methods1.Proliferation inhibition of THP-1 cells treated by arsenic trioxide combined with dihydroartemisininThe effect of arsenic trioxide,dihydroartemisinin and arsenic trioxide combined with dihydroartemisinin on the proliferation of THP-1 cells was detected by MTT method,and the proliferation inhibition rate was calculated.2.Effects of arsenic trioxide combined with dihydroartemisinin on THP-1 cell cycle distribution and apoptosisTHP-1 cells were treated with arsenic trioxide,dihydroartemisinin and arsenic trioxide combined with dihydroartemisinin for 48 h and then cell proliferation cycle distribution and apoptosis were detected by flow cytometry.3.Effect of arsenic trioxide combined with dihydroartemisinin on apoptosis-related proteins expression of THP-1 cells.THP-1 cells were treated with arsenic trioxide,dihydroartemisinin and arsenic trioxide combined with dihydroartemisinin for 48 h and then the expression of six apoptosis-related proteins including Bax,Bcl-2,Caspase-3,Cleaved Caspase-3,Caspase-8 and Caspase-9 were detected by Western Boltting.4.Proliferation inhibition of SHI-1 cells treated by arsenic trioxide and dihydroartemisininThe effect of arsenic trioxide,dihydroartemisinin and arsenic trioxide combined with dihydroartemisinin on the proliferation of SHI-1 cells was detected by MTT method,and the proliferation inhibition rate was calculated.5.Effects of arsenic trioxide and dihydroartemisinin on SHI-1 cell apoptosisSHI-1 cells were treated with arsenic trioxide and dihydroartemisinin for 48 h and then cell apoptosis were detected by flow cytometry.6.Establishment of an acute monocytic leukemia modelAfter three-day quarantine adjustment,BALB/c female nude mice,4-5 weeks old,were divided into five groups:control group,subcutaneous injection of THP-1 cells group,intraperitoneal injection of THP-1 cells group,subcutaneous injection of SHI-1 cells group,intraperitoneal injection of THP-1 cells group.The growth of acute monocytic leukemia cells in nude mice was observed.Acute monocytic leukemia cells in nude mice were got to injected subcutaneously into new mice and observe their growth.7.To investigate the therapeutic effect of arsenic trioxide combined with dihydroartemisinin on acute monocytic leukemia based on nude mouse tumor modelModeling:subcutaneous injection of SHI-1 cells into BALB/c nude mice.Grouping:the nude mice were randomly divided into 10 groups according to the tumor size,containing control group,model group,low ATO group,high ATO group,low DHA group,high DHA group,low ATO+low DHA group,low ATO+high DHA group,high ATO+low DHA group,high ATO+high DHA group.They were administered by arsenic trioxide and/or dihydroartemisinin for 14 days.The growth status of nude mice were observed,the tumor size was measured,and the pathological changes of viscera were detected.Results1.Proliferation inhibition of THP-1 cells treated by arsenic trioxide combined with dihydroartemisininArsenic trioxide and dihydroartemisinin at different concentrations significantly inhibited the proliferation of THP-1 cells(P<0.001)and showed dose-time dependence.The effect of arsenic trioxide combined with dihydroartemisinin was more significant than that of arsenic trioxide and dihydroartemisinin(P<0.001).2.Effects of arsenic trioxide combined with dihydroartemisinin on THP-1 cell cycle distribution and apoptosisCompared with the control group,the G2 and S phase cells were significantly reduced and the proportion of G1 phase cells was increased in each drug group(P<0.001),and the largest change showed in arsenic trioxide combined with dihydroartemisinin group.Compared with the control group,the apoptosis rate of each group increased,and the apoptosis rate of the arsenic trioxide combined with dihydroartemisinin group was 71.4%higher than that of the control group,which was significantly higher than that of the arsenic trioxide group and the dihydroartemisinin group(P<0.001).3.Effect of arsenic trioxide combined with dihydroartemisinin on apoptosis-related protein expression of THP-1 cellsCompared with the control group,the expression levels of Bcl-2(P<0.05),Caspase-3(P<0.001)and Caspase-8(P<0.001)were significantly down-regulated,the expression level of Cleaved Caspase-3(P<0.05)was significantly up-regulated,and the expression levels of Bax and Caspase-9 were not significantly changed.4.Proliferation inhibition of SHI-1 cells treated by arsenic trioxide and dihydroartemisininArsenic trioxide and dihydroartemisinin at different concentrations inhibited the proliferation of SHI-1 cells.Compared with the control group,the proliferation inhibition rate of SHI-1 cells increased with the increase of drug concentration(P<0.05),and showed dose-time dependence.However,when arsenic trioxide of 1,2 or 3μM combined with dihydroartemisinin at different concentrations,there was no significant difference in the inhibition rate(P<0.05).5.Effects of arsenic trioxide and dihydroartemisinin on SHI-1 cell apoptosisCompared with the control group,the apoptosis rate of SHI-1 cells increased significantly with the increase of arsenic trioxide concentration.Compared with the control group,the apoptosis of SHI-1 cells doesn’t show significant increase.Compared with arsenic or dihydroartemisinin at the same concentration,arsenic trioxide combined with dihydroartemisinin did not increase the apoptosis rate of SHI-1 cells.6.Successful establishment of tumor model of acute monocytic leukemiaSHI-1 cells and THP-1 cells cultured in vitro and intraperitoneally injected into nude mice showed no significant changes.THP-1 cells were cultured in vitro and injected subcutaneously into the right scapula of nude mice.The tumor could grow in nude mice 25%.SHI-1 cells were cultured in vitro and injected subcutaneously into the right scapula of nude mice.The tumor could grow in nude mice 100%.The tumor model of nude mice with acute monocytic leukemia was successfully established.7.The therapeutic effect of arsenic trioxide combined with dihydroartemisinin on acute monocytic leukemia based on nude mice tumor modelCompared with the model group,the growth of tumor volume in other groups decreased significantly after administrated by drugs for 7 days,and the decrease was most obvious in the low ATO+low DHA group,with an average increase of only 6.06 mm3.During 7-13 days after administrated by drugs,the increase of tumor volume was significantly higher than that in the previous 7 days,but the increase of value in the low ATO+low DHA group(120.25 mm3)was the lowest compared with that in the model group(292.65 mm3).Compared with the model group,the average tumor weight in the low ATO+low DHA group,high ATO+low DHA group and high ATO+high DHA group were significantly reduced,and the low ATO+low DHA group showed the most significant reduction,with the tumor inhibition rate of 46.47%.Conclusion1.Arsenic trioxide and dihydroartemisinin can inhibit the proliferation of THP-1 cells and showed dose-time dependent.Arsenic trioxide combined with dihydroartemisinin showed more obvious inhibition than that of their single use.2.Cell cycle arrest and apoptosis induction are one of the possible mechanisms of the proliferation inhibition of THP-1 cells treated by arsenic trioxide combined with dihydroartemisinin.3.Arsenic trioxide combined with dihydroartemisinin induces apoptosis of THP-1 cells by activating Caspase-3,down-regulating Bcl-2 and Caspase-8.4.Arsenic trioxide and dihydroartemisinin can inhibit SHI-1 cell proliferation and showed dose-time dependence.There is no synergy between arsenic trioxide and dihydroartemisinin.5.Cell apoptosis induction is one of the possible mechanisms of the proliferation inhibition of SHI-1 cells treated by arsenic trioxide combined.Dihydroartemisinin inhibits SHI-1 cells proliferation without inducing apoptosis.6.The nude mouse tumor model of acute monocytic leukemia can be established by the way of SHI-1 cells cultured in vitro and injected subcutaneously into the right scapula of nude mice.7.2 mg/kg ATO combined with 50 mg/kg DHA could significantly inhibit tumor growth in nude mouse tumor model of acute monocytic leukemia,with a tumor inhibition rate of 46.47%. |
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