The Effect Of ATO On The Proliferation Of K562 Cells And Its Mechanism Research By Down-regulating CD44

Objective: To explore the possible mechanism of cell growth inhibition induced by ATO in K562 cells, the expression of CD44, β-catenin and Cyclin D1 were investigated. This study intend to explore the new targets for CML therapy.Methods: Human CML cell line K562 cells were cultured in vitro.The cells in experimental group were collected after incubation with ATO in different concentrations for 24 h, 48 h,and 72 h. Cell morphology was observed by optical microscope; cell proliferation was detected by MTT; the cell cycle, the cell apoptosis and the expression of CD44 were detected by flow cytometry(FCM); the expression of β-catenin and Cyclin D1 were measured by reverse transcription polymerase chain reaction(RT-PCR).Results:1 Effect of ATO on K562 cell proliferation: MTT assay displays that the proliferation of K562 cells was significant inhibited by 2μmol/L or higher concentrated of ATO after 24-72 h, and the inhibition rate was time-dependent and dose-dependent. Statistical analysis showed that the comparison between the same concentration of each time point,there was no statistical difference between the 1μmol/L groups after treatment for 24 h, 48 h and 72h(P>0.05). The difference has statistical significance in other dose groups(P<0.05),and the inhibition rate increased with the extension of time; the comparison between the same time point of each dose group, 24 h among 1, 2, 2.5μmol/L groups, there was no significant difference(P>0.05), there was significant difference between the other dose groups(P<0.05), there were statistical differences beween 48 h and 72 h in each dose group(P=0.016), and the inhibition rate increased with the drug concentration increasing.2 Effect of ATO on the expression CD44 in K562 cells: in the control group, the K562 cells were cultured for 24h、48h、72h, the expression of CD44 were(83.47±1.28)%、(82.63±0.62)%、(82.32±0.86)%, respectively. The K562 cells treated with different concentration of ATO after 24h、48h、72h, the percentage of CD44 expression was shown at table 2. Statistical analysis showed that, the same treatment groups with time extending, the expression of CD44 decreased gradually, there were significant differences between different time points(P<0.05); the same time, the comparison between the treatment groups, except there was no statistical difference between the 1,2μmol/L groups 24h(P>0.05), the other treatment groups with the drug concentration increasing, the expression rate of CD44 decreased gradually, there was statistical difference(P=0.003).3 Effect of ATO on apoptosis of K562 cells: In the control group,the K562 cells were treated with 1、2、2.5、5、10μmol/l ATO for 48 h, and the apoptosis rate were(2.09±1.42)% 、(2.13±1.57)% 、(2.41±1.48)% 、(5.61±2.03)%、(11.84±2.69)%、(30.98±4.16)%, respectively. Compared with the control group, the different had no statistically significant in 1、2μmol/l ATO treatment groups(P>0.05); The apoptosis rate of K562 cells in 2.5、5、10μmol/l ATO treatment groups increased significantly compared with the control group, there was statistical difference(P<0.05). So in a certain range, ATO inhibited the proliferation of K562 cells in a concentration-dependent manner, and the apoptosis rate increased with the drug concentration increasing.4 Effect of ATO on K562 cell cycle distribution: The result of the distribution of cell cycle by flow cytometry showed that, in the control group, K562 cells treated with 1、2、2.5、5、10μmol/l ATO for 48 h, the cell proportion of G0/G1 phase, S phase and G2/M phase were shown at table 4. Different concentrations of ATO after 48 h, the K562 cells arrest in G0/G1 phase cells increased significantly with the drug concentration increasing, the proportion of S phase and G2/M phase cells decreased, the difference was statistically significant(P<0.05). It suggested that ATO might arrest K562 cells in G0/G1 phase by a certain mechanism.5 Effect of ATO on the expression of β-catenin in K562 cells: RT-PCR test results showed that the indexes of each group with blank control RQ value is 1, the K562 cells treated with different concentration ATO after 48 h, the β-catenin RQ vaules were shown at table 5. Statistical analysis showed that different concentration ATO significantly influenced the expression of β-catenin in K562 cells(P<0.05), and with the drug concentration increasing, the expression of β-catenin decreased. The scatter diagram and liner correlation analysis indicated that, at the same time, with the chaging of drug concentration, the change trend of CD44 and β-catenin were positively correlated,correlation analysis of two factors indicated that, the cells treated with drug for 48 h, the correlation coefficient between them was r=0.973, there was statistical significance(P<0.001).6 Effect of ATO on the expression of Cyclin D1 in K562 cells: RT-PCR test results showed that the indexes of each group with blank control RQ value is 1, the K562 cells treated with different concentration ATO after 48 h, the Cyclin D1 RQ vaules were shown at table 5. Statistical analysis showed that different concentration ATO significantly influenced the expression of Cyclin D1 in K562 cells(P<0.05), and with the drug concentration increasing, the expression of β-catenin and Cyclin D1 decreased.Conclusions:1 In a certain range of concentration, ATO could inhibit the proliferation of K562 cells in a dose-and time-dependent pattern.2 The expression of CD44 was inhibited by ATO in a dose-and time-dependent pattern.3 ATO down-regulating CD44 could arrest K562 cells at G0/G1 phase. This may be the main cause of K562 cells growth inhibition.4 The expression of Cyclin D1 were down-regulated by ATO treatment. This may be the molecular mechanism of k562 cells arrest at G0/G1 phase.5 There was positive correlation between the expression level of CD44 and β-catenin. Down-regulating CD44 could induce the expression level of β-catenin decrease. Inhibited the activation of Wnt/β-catenin signaling pathway, thus inhibited the proliferation of K562 cells.