ITF2357体外对急性髓系白血病细胞增殖的抑制作用及机制探讨

The role of ITF2357 on inhibition of myeloid leukemic cell lines cells proliferation in vitro and its mechanism

目的探讨新型组蛋白去乙酰化酶（HDAC）抑制剂ITF2357对急性髓系白血病（AML）细胞的增殖抑制及诱导分化和促凋亡作用及其机制。方法以AML细胞系Kasumi一1细胞（AML1-ETO阳性）为模型，应用MTr比色法观察ITF2357对白血病细胞的增殖抑制作用，并与THP1细胞（AML1-ETO阴性）进行比较；用流式细胞术分析细胞周期和分化抗原的表达；用AnnexinV标记和流式细胞术分析细胞凋亡；Westernblot法检测AML1-ETO及乙酰化组蛋白、easpase蛋白水平。结果0．5μm0L／LITF2357即能明显抑制Kasumi-1细胞增殖，48h半数抑制浓度（IC50）为0．1μmoL／L，然而对于AML1-ETO阴性的THP1细胞系的起始抑制浓度为5．0μm0L／L；ITF2357诱导Kasumi-1细胞凋亡呈时间、剂量依赖性，1TF2357处理24h早期凋亡细胞由（1．44±1．52）％增加至（24．51±5．79）％，48h晚期凋亡细胞由（2．37±2．80）％增加至（63．66±1．56）％。0．25μmoL／LITF2357即可诱导Kasu-mi-1细胞髓系分化抗原CD13及CD15表达增加，并呈剂量和时间依赖性。经5．0μmoL／LITF2357作用后细胞阻滞于G0／G1期，G0／G1期细胞由（39．69±6．56）％增加至（79．20±6．51）％，伴有s期细胞减少，由（60．12±3．29）％降低至（18．97±6．62）％。Westernblot检测发现0．5μmoL／LITF2357可降低AMLl-ETO蛋白水平，处理24h开始减少，处理96h后AML1-ETO基本消失，并依赖于easpase途径。经ITF2357处理后，组蛋白H4及H3的乙酰化水平增加。结论低剂量HDAC抑制剂ITF2357能有效抑制AML细胞的增殖，对于AML1-ETO阳性AML细胞尤为有效，通过降解AML1-ETO蛋白抑制Kasumi-1细胞增殖，使细胞阻滞于G0／G1期，诱导其发生凋亡及分化。

Objective To explore the effect of ITF2357, a novel histone deacetylase（HDAC） inhibi- tor, on the growth, differentiation and apoptosis of acute myeloid leukemic （AML） cells and its mechanism. Methods AML cell lines kasumi-1 cells as a model for AMLI-ETO positive, and THP1 cells for AML1-ETO negative, the leukemic cells proliferation was analyzed by MTT assay, expression of myeloid-specific differenti- ation antigen and cell cycle by flow cytometry, cell apoptosis by annexin V staining and flow cytometry. AML1-ETO, acetyl-histone, and caspase protein was analyzed by Western blot. Results 0. 5 μmol/L 1TF2357 treatment significantly inhibited kasumi-1 cells proliferation, with the 48 h half inhibitory concentra- tion （ IC50 ） of 0. 1 μmol/L. The initial inhibitory concentration of THP1 cell line AML1-ETO negative was 5 μmol/L. ITF 2357 induced apoptosis of kasumi-1 cells in a time- and dose dependent manner. A dose-de- pendent increase in early apoptosis oceured at 24 hours treatment and in late apoptosis at 48 hours treatment by ITF2357. Early apoptosis cells increased from （1.44 ± 1.52）% to （24.51 ±5.79）%. Late apoptosis cells increased from （2.37 ± 2.80） % to （63.66 ± 1.56） %. ITF2357 induced AML1-ETO degradation by caspase-dependent pathway. 0.25 μmol/L ITF2357 induced a time- and dose-dependent increase in expres- sion of myeloid cell surface protein CD13 and CD15. 5.0 p.moL/L ITF2357 blocked the cells at G0/G1 phase, G0/G1 cells increased from （39.69 ± 6.56）% to （79.20±6.51 ）% and S-phase ceils declined from（60.12 ± 3.29） % to （ 18.97 ± 6.62） %. Kasumi-1 cells incubated with 0.5μmol/L of ITF2357, AML1- ETO protein began to decrease at 24 hours and could hardly be detected at 96 hours. ITF2357 induced AML1/ETO degradation through a caspase-dependent mechanism. At the same time, acetylated H3 and H4 increased. Conclusion Low-dose HDAC inhibitor ITF2357 can effectively inhibit the AML cells prolifera- tion, especially for AML1-ETO positive AML cells. It inhibits Kasumi-1 cells proliferation degradation of AML1-ETO protein expression ,blocks the cells at G0/G1 phase, and induces apoptosis and differentiation of the cells.