Studies On Effects Of The Combination Of Emodin And AZT On Proliferation Inhibition And Apoptosis Induction In Leukemia KG-1a Cells And Related Role Of Egr-1

OBJECTIVE: To investigate the effects of combination of emodin and3ˊ-azido-3ˊ-deoxythymidine (AZT) on proliferation inhibition and apoptosis induction in leukemia cellline KG-1a cells and related role of Egr-1gene plays therein, so as to provide usefulexperiment information for further in vivo study and clinical treatment on leukemia.METHODS: The optimized concentration of emodin and AZT was screened forproliferation-inhibited effect on KG-1a cells. Proliferation inhibition rates of KG-1a cells byemodin at final concentration of10μmol/L, AZT and emodin combined with AZT at differentfinal AZT concentrations of9600μmol/L,6400μmol/L,4800μmol/L,3200μmol/L,2400μmol/L,1600μmol/L,1200μmol/L and800μmol/L were detected with MTT method；synergistic effects between emodin and AZT at different concentrations on proliferation ofKG-1a cells were analyzed by calculation of half inhibitory concentrations (IC50) andcombination indexes (CI) at24、48、72and96hours through Calcusyn2.0software.The expressions of Egr-1, PTEN and P53mRNA in cells before and0,0.5,1,2,4and8hours after treated by the combination of emodin with a final concentration of10μmol/L andAZT with a final concentration of3200μmol/L were assessed by q-PCR.KG-1a cells were transfected with Egr-1siRNA and divided into normal cell control(KG-1a), nonspecific control (transfected with nonspecific sequence, KG-1a/NC) andEgr-1siRNA group (transfected with Egr-1siRNA, KG-1a/siRNA). Transfection efficiencywas observed through fluorescence microscope and flow cytometer and transfection effect ofEgr-1and the expression changes of PTEN and P53mRNA were detected with qPCR.Proliferation rates of cells were detected with MTT method. After the cells were treated with10μmol/L of emodin,3200or1600μmol/L of AZT and their combinations, the proliferationinhibition rates and the apoptosis rates of cells in3groups were detected with MTT methodand FCM, respectively；half inhibitory concentration (IC50) and combination index (CI) ofcells in3groups were calculated through Calcusyn2.0software.RESULTS: Proliferation inhibition effects of KG-1a cells by emodin, AZT or thecombination of them all appeared dosage and time dependence. The inhibitory rates onproliferation of KG-1a cells treated by emodin, AZT or the combination of emodin and AZTwith a fixed final emodin concentration of10μmol/L and different final AZT concentrationsof800μmol/L,1200μmol/L,1600μmol/L,2400μmol/L,3200μmol/L,4800μmol/L,6400μmol/L and9600μmol/L at24,48,72and96hours all increased with the increase of drug concentrations; the overall trend of inhibitory rates is to increase with the increase oftreated time and the inhibitory rates by AZT and the combination of emodin and AZT reachedpeak at48and72hours, respectively. Although, the inhibitory effect of emodin alone with arelatively low final concentration of10μmol/L on proliferation of KG-1a cells was notobvious, the proliferation inhibitory rates of cells treated with combination of emodin andAZT with a fixed final emodin concentration of10μmol/L and different final AZTconcentrations were all significantly greater than those by emodin or AZT alone with sameconcentrations and the differences between them were statistically significant (p <0.01).IC50values of the combination at24,48,72and96h were about ([emodin]/[AZT])10/9600,10/3200,10/1600and10/4800μmol/L, respectively, significantly lower than those ofemodin alone (all greater than10μmol/L) and AZT alone (all close to or great than9600μmol/L).Calculated by CalcuSyn2.0statistical software, Apart from the combination of emodinwith a low concentrations of AZT (final concentration800μmol/L) for24hours, the CI valuesof the combination of emodin and AZT with a fixed final emodin concentration of10μmol/Land different final AZT concentrations of800μmol/L,1200μmol/L,1600μmol/L,2400μmol/L,3200μmol/L,4800μmol/L,6400μmol/L and9600μmol/L on KG-1a cell linestreated for24,48,72and96hours were all less than1, which indicate synergistic effectbetween emodin andAZT on proliferation inhibition of KG-1a cells.Though the treatment of emodin alone with a final concentration of10μmol/L on KG-1acells only resulted in an apoptosis rate of1%at72h, the apoptosis rates in KG-1a cellstreated with the combination of emodin and AZT with a fixed final emodin concentration of10μmol/L and different final AZT concentrations of1600μmol/L and3200μmol/L werehigher than those of in control and single AZT group and the differences were statisticallysignificant (p<0.05), which indicate synergistic effect between emodin and AZT on apoptosisinduction of KG-1a cells. The apoptosis rates increased with the concentration ofAZT.It was found by the q-PCR that expression levels of Egr-1in KG-1a cells treated withemodin, AZT or a combination of them increased gradually over time at first, rose to thehighest at2h, and then declined gradually to a minimum at8h, close to the untreated levels.The changes of expression levels of PTEN and P53over time were similar to that of Egr-1,except for their highest expression at4h.More than59.21%of KG-1a cells were transfected with Egr-1siRNA by electrotransfection. Compared with cells in controls, cells in Egr-1siRNA group were observed an apparent increase in proliferation rate and a significant reduction in expression of Egr-1mRNA (p<0.01). The significantly increased inhibitory rates of the combination of emodinand AZT on proliferation of cells in normal cell control and nonspecific control than those ofemodin or AZT alone indicated synergistic effect between emodin and AZT. However,difference between the inhibitory rates of the combination and AZT alone on proliferation ofcells in Egr-1siRNA group was not significant (p＞0.05). After cells being treated with thecombination of emodin and AZT for72h, apoptosis rate of cells in Egr-1siRNA group wassignificantly lower than that in the normal cell and nonspecific controls (p<0.01).CONCLUSION: The combination of emodin and AZT showed significant synergisticeffect on proliferation inhibition and apoptosis induction of KG-1a cells, which can be berelated to Egr-1. It is suggested that the combination of emodin and AZT can up-regulateexpression of Egr-1, leading to up-regulation of downstream gene PTEN and P53, thusresulting in proliferation inhibition and apoptosis induction of KG-1a cells.