甲氨蝶呤对单核细胞样细胞系U937的生长影响及其促凋亡作用

Effect of Methotrexate on Cell Growth of Human Monocyte-like Cell Line U937 and Its Induction of Apoptosis

一般认为急性髓细胞白血病 (AML)是对甲氨蝶呤 (MTX)原发耐药的恶性肿瘤。AML细胞除急性单核细胞白血病 (AML M5)细胞与MTX孵育时 ,由于不能象急性淋巴细胞白血病 (ALL)细胞一样形成足够量的长链聚谷氨酸盐甲氨蝶呤 (MTXPG) ,而被认为对MTX不敏感。为了开发对AML M5新的治疗 ,本实验采用了具有单核细胞特征的U937细胞系进行研究。细胞分别通过不同浓度 (1nmol/L - 10 0 μmol/L)MTX孵育 2 4小时或 4 8小时后 ,通过XTT法评估细胞生长抑制情况。 2 4小时的半数抑制浓度 (IC50 )为 0 .0 4 μmol/L ;4 8小时的IC50 为 0 .0 37μmol/L ,90 %细胞抑制浓度 (IC90 )为 0 .39μmol/L。为了解MTX细胞毒作用的发生机制 ,进一步分析了细胞的死亡过程。采用了系列实验 ,包括台盼蓝拒染法、流式细胞术、显微镜 (瑞氏染色法 )及DNA片段分析进行研究。结果在MTX作用后短时间内 (4或 6小时 )无明显的细胞凋亡特征出现。随 5nmol/L- 10 μmol/LMTX作用 8小时后 ,亚G1(凋亡 )峰的比率从 0 .1μmol/L的 3 2 %增加到 5 0 μmol/L的 18 2 %,S期的比率从 0 0 1μmol/L的 4 1 2 %到 10 μmol/L的 19 1%。可见到DNA片段电泳后细胞凋亡的特征性改变。MTX所引发U937细胞的生长阻滞和凋亡特征 ,提示它可用于AML

Acute myeloid leukemia (AML) is considered to be a malignancy that is intrinsically resistant to methotre- xate (MTX). As compared to acute lymphoblastic leukemia (ALL) blasts, AML blasts, except those of acute monocytic leukemia (AML-M 5), form fewer amounts of long chain polyglutamate of MTX (MTXPG), when incubated with MTX, thus providing an explanation for their lack of responsiveness to MTX. To explore the novel approach of treatment in patients with AML-M 5, the U937 cell line, which has the monocytic characters, was used. Cell growth inhibition was mearsured by XTT assay after 24 and 48 hours in the continuous presence of various concentrations of MTX ranging from 1?nmol/L to 100?μmol/L. After 24 hours MTX treatment, the IC 50 value for U937 cells was 0.04?μmol/L. After 48 hours treatment, the IC 50 was 0.037?μmol/L and IC 90 was 0.39?μmol/L. To understand the mechanism of MTX cytotoxicity, the process of cell death was analyzed. A variety of assays, including trypan blue exclusion, flow cytometry, light microscopy (Wright′s staining) and DNA fragment electrophoresis, were performed. There were no significant apoptotie changes after shorter exposure of MTX (4 and 6 hours). After 8 hours at various concentrations of MTX treatment ranging from 5?nmol/L to 10?μmol/L, the percentage of the cells in the pre-G 1 (apoptotic) was 3.2% at 0.1?μmol/L and it reached a peak of 18.2% at 5.0?μmol/L. The DNA synthesis in S-phase was inhibited from 41.2% (0.01?μmol/L) to 19.1% (10?μmol/L). DNA ladder band, a feature of apoptosis, was observed. The arrest of cell growth and apoptotic properties induced by MTX have lead to its evaluation as a potentially therapeutic agent in the treatment of AML-M 5.