Study Of The Effects Of Chidamide Alone Or Combined With Decitabine On Proliferation And Apoptosis Of Leukemia Cell Lines

Objective:To investigate the inhibitory effect of chidamide alone or in combination with decitabine on leukemia cells and its possible molecular mechanism.Methods:The proliferation inhibition of chidamide alone or combined with decitabine by different concentrations on HL60 and NB4 cells was assessed by cell counting kit-8(CCK8).The combination index(CI)value was determined from the fraction-affected value of each combination according to the Chou-Talalay method by using CompuSyn software.The morphological changes were observed by inverted microscope.Cell-cycle kinetics and apoptosis were analyzed by flow cytometry(FCM).Histone deacetylases(HDAC)activity was detected as described in the Colorimetric HDAC Activity Assay kit.The mRNA expression levels of Bax,Bcl-2,caspase-3 were measured with RT-PCR.The protein levels of p21,Procaspase-3,Bcl-2,Bax and Acety-histone H3 were determined by Western Blot.All experiments were repeated 3 times and the results were expressed with((?)±s,%),P<0.05 was considered statistically significant.Results:1.The proliferation of HL60 and NB4 cells were significantly inhibited by chidamide and decitabine alone or combined.In the concentration range of 0.25 to 8μmol/L,the inhibition was dose and time dependent(P<0.05);the inhibition of decitabine was also dose and time dependent at the concentration range of 0.5 to 100μmol/L(P<0.05).Chidamide combined with decitabine significantly increased the inhibitory effect on leukemia cell lines HL60 and NB4,the combined effects of two drugs was stronger than that of single drug(P<0.05).CompuSyn analysis showed that there was a significant synergistic anti leukemia effect among the two drugs.2.After treatment with chidamide alone or combination with decitabine,the apoptotic changes of HL60 and NB4 cells were observed under inverted microscope.3.Chidmide increased the levels of acetylated histone H3 in both HL60 and NB4 cells by effectively inhibiting HD AC enzymatic activities.4.The cells were blocked in G0/G1 phase and G2/M phases by chidamide and decitabine,respectively.But the cells were induced G2/M cell cycle arrest by chidamide in combination with decitabine.Western Blot assay showed that the expression level of cell cycle protein p21 was significantly up-regulated.5.The apoptotic rate of the chidamide,decitabine alone and combination group was significantly higher than the control group(P<0.05).The expression level of Bax and Caspase-3 protein was increased and the expression level of Bcl-2 protein was decreased in the monotherapy group and the combined group by RT-PCR and Western Blot.Conclusions:1.The proliferation of HL60 and NB4 cells were inhibited by chidamide or decitabine in a concentration-and time-dependent manners.The combination of two drugs significantly inhibited the proliferation of HL60 and NB4 cells and showed synergistic effects.2.The inhibition effects were reached through cell apoptosis and cell cycle arrest in vitro by chidamide combined with decitabine on HL60 and NB4 cells.3.Chidmide plays a role by inhibiting histone deacetylase activity and upregulating acetylated histone H3 levels.4.The possible mechanism of the effect of chidamide,decitabine alone and combination group is to directly or indirectly increase the protein expression of p21,caspase-3,regulate the expression of Bcl-2 family protein to promote cell apoptosis.