Research On The Effect And Mechanism Of AML Cells With Arsenic Trioxide Alone Or Combined With Decitabine

Objective:To explore the the effect and possible mechanism of Arsenic tri oxide alone or combined with Decitabine on acute myeloid leukemia cell line HL-60, in order to provide a theoretical basis for the clinical use of AML.Methods:HL-60cells were cultured through cell lines subculture and treated with different concentrations of As2O3single or combined with Decitabine at different time(24h,48,72h). Cells 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、 DNMT-3A、DNMT3B mRNA was detected by RT-PCR.Result:1、MTT results showed that different concentrations of As2O3at different time have a certain degree of inhibition to the HL-60cell; At the same time point As2O3on cell growth inhibition in a dose-dependent manner, single As2O312umol/1effect reached the peak at the72h,the cell growth inhibition was78.92%±2.17%; As2O3can collaborate with the DAC associated with increased proliferation efficiency when As2O3+DAC concentration was6umol/1+5umol/1for72h on HL-60cell proliferation most obvious, up to74.04%±1.2%, significantly higher than As2O3monotherapy6umol/1for72hours of high,P<0.05.2、Annexin V-FITC/PI double stained results showed that As2O3could promote apoptosis effect on HL-60cell line, the effect showed a dose-and time-dependent manner; And with the increase in drug concentration and time, and necrotic cells gradually increased; As2O3combined with Decitabine synergistic enhancement on apoptosis of HL-60cells,it aslo showed a dose-and time-dependent manner; But two drugs pro-necrotic effect of HL-60cells is not obvious.3、The cell cycle results showed that As2O3mainly arrest HL-60cells at G1/G0phase, the effect showed a dose-and time-dependent manner; But As2O3combined with DAC have an effect on the cell cycle, but there was no significant regularity.4、RT-PCR test results showed, HL-60cells has a low expression experession of ID4and P15mRNA in HL-60cell line, and it maybe re-experssion after the role of the two drugs. With the increase of drug concentration and time, its expression increased; At the same time, decreased in HL-60high expression of DNMT-1, DNMT-3A, DNMT-3B expression after effects of the two drugs; As2O3combined DAC with a synergistic increase in mRNA expression ID4and P15and collaborative reduce DNMT-1, DNMT-3A, DNMT-3B mRNA expression.Conclusion1. As2O3alone inhibited HL-60cell proliferation and promote apoptosis or necrosis, the role of cells in G0/G1cycle arrest, the effect showed a dose-and time-dependent manner.2. As2O3combined DAC with a synergistic increased HL-60cell proliferation and apoptosis, but HL-60cell cycle arrest is not significant regularity;3. As2O3combined DAC with a synergistic increase in tumor suppressor genes ID4and P15mRNA expression and collaborative reduce DNMT-1, DNMT-3A, DNMT-3B mRNA expression. Mechanism we are considering is to inhibit DNMT-1, DNMT-3A, DNMT-3B activity, reducing the ID4and P15gene methylation level.