The Role And Mechanism Of Decitabine On Molt-4Cell Line And HL-60Cell Line

Objective:To explore the effect and possible mechanism of DAC on acute myeloid leukemia cell line HL-60and acute lymphoid leukemia cell line Molt-4.Methods:HL-60and Molt-4cell lines treated with different concentrations of DAC at different times(24h,48,72h). Cell proliferation was analyzed by MTT assay. The cell apoptosis rate was examined by Annexin V-FITC/PI double stained and cell cycle was analyzed with PI single stained by flow cytometry; The expression of ID4\P15、DNMT1、DNMT3A、DNMT3B mRNA was detected by RT-PCR.Result:(1) The MTT results show that DAC in a time-and dose-dependent manner to inhibit the proliferation of HL-60and Molt-4cells, and the cell inhibition in HL-60is more stronger than Molt-4. The50%inhibitory concentration (IC50) at different time point on these two cell lines were respectively:5.78×104、44.86、0.614on HL-60and3.07×106、6.30×103、3.06×102.(2) The Annexin V-FITC/PI double staining to detect apoptotic cells show that DAC play a pro-apoptotic and necrotic effects in both HL-60and Molt-4cells with the increase of drug concentration and the time of duration, especially the effect on promote cell apoptotic and it is more stronger in HL-60cell.(3) The PI single staining flow cytometry results show that after treated these two cell lines with DAC,the cell ratio in G2/M phase increased in HL-60cell, while in Molt-4cell it is increased in S phase.(4) The results of RT-PCR show that the basic expression levels of P15and IER3were low in both HL-60and Molt-4cells. After treated with DAC,the mRNA expression of P15and IER3were increade in parallel with the DAC concentrations in both cell lines. Especially at24h,they increased greatest. But they have different features:(1)At the time point of24h and the concentration of0.5umol/L, the expression of P15and IER3were maximum, respectively,1.377±0.095,1.113±0.002in HL-60。(2) The maximum expression of P15and IER3mRNA in Molt-4cell appeared at the time point of24h with the maximum concentration of200umol/L.(5) After treated these two cell lines with DAC show that:(1)The expression of DNMTl mRNA reduced in both two cell lines;(2)There was no significant effect on the expression of DNMT3A mRNA in both two cell lines;(3)The expression of DNMT3B mRNA reduced in HL-60cell and had no significant effect in Molt-4cell.Conclusion:(1) DAC in a time-and dose-dependent manner to inhibit the proliferation of HL-60and Molt-4cells, arrested the HL-60cell cycle in G2/M phase, while arrested the Molt-4cell cycle in S phase, induced apoptosis of HL-60and Molt-4cells. The cell inhibition and induce apoptosis of HL-60are more stronger than Molt-4.(2) DAC may primarily inhibits the activity of DNMTl to reduce the level of P15and IER3methylation,and then upregulate the expression of P15and IER3genes.(3) IER3may be as a tumor suppressor gene play an important role in the formation of hematological malignancies,with the in-depth study of the role of epigenetic mechanisms and changes to IER3gene,it may become a new target for therapy of hematological malignancies.(4) The results of this experiment show that myeloid cell line is more sensitive to DAC than lymphoid cell line,which suggests us to use it in the clinical exploration and research of myeloid leukemia priority.