Unidad de Bioquímica y Biología Molecular, Departamento de Biología de Sistemas, Facultad de Medicina y Ciencias de la Salud, Campus Universitario, Universidad de Alcalá, 28871 Alcalá de Henares (Madrid), Spain.
Unidad de Bioquímica y Biología Molecular, Departamento de Biología de Sistemas, Facultad de Medicina y Ciencias de la Salud, Campus Universitario, Universidad de Alcalá, 28871 Alcalá de Henares (Madrid), Spain.
Chem Biol Interact. 2014 Sep 5;220:129-39. doi: 10.1016/j.cbi.2014.06.021. Epub 2014 Jul 1.
Acute promyelocytic leukemia (APL) is a type of cancer, in which immature cells called promyelocytes proliferate abnormally. Human NB4 cell line appears to be a suitable in vitro model to express the characteristics of APL. In this work, we have investigated the effects of esculetin, a coumarin derivative with antioxidant properties, on the viability, the induction of apoptosis and the expression of apoptotic factors in NB4 cells. Cells treated with esculetin at several concentrations (20-500 μM) and for different times (5-24 h) showed a concentration- and time-dependent viability decrease with increased subdiploid DNA production. Esculetin inhibited cell cycle progression and induced DNA fragmentation. Moreover, annexin-V-FITC cytometry assays suggested that increased toxicity is due to both early and late apoptosis. This apoptosis process is be mediated by activation of caspase-3 and caspase-9. Treatments with progressively increasing concentrations (from 100 μM to 500 μM) of esculetin produced a reduction of Bcl2/Bax ratio in NB4 cells at 19 h, without affecting p53 levels. Proapoptotic action of esculetin involves the ERK MAP kinase cascade since increased levels of phosphorylated ERK were observed after those treatments. Increments in the levels of phosphorylated-Akt were also observed. Additionally, esculetin induced the loss of mitochondrial membrane potential with a release of cytochrome c into the cytosol which starts at 6 h of treatment with esculetin and increases up to 24 h. Esculetin induced an increase in superoxide anion at long times of treatment and a reduction of peroxides at short times (1 h) with an observed increase at 2-4 h of treatment. No significant changes in NO production was observed. Esculetin reduced the GSH levels in a time-dependent manner. In summary, the present work shows the cytotoxic action of esculetin as an efficient tool to study apoptosis mechanism induction on NB4 cell line used as a relevant model of APL disease.
急性早幼粒细胞白血病 (APL) 是一种癌症，其中称为早幼粒细胞的未成熟细胞异常增殖。人类 NB4 细胞系似乎是表达 APL 特征的合适体外模型。在这项工作中，我们研究了具有抗氧化特性的香豆素衍生物秦皮乙素对 NB4 细胞活力、诱导凋亡和凋亡因子表达的影响。用秦皮乙素在几个浓度（20-500 μM）和不同时间（5-24 h）处理的细胞显示出浓度和时间依赖性活力降低，同时亚二倍体 DNA 产量增加。秦皮乙素抑制细胞周期进程并诱导 DNA 片段化。此外， Annexin-V-FITC 细胞术分析表明，增加的毒性是由于早期和晚期凋亡。这个凋亡过程是通过激活 caspase-3 和 caspase-9 介导的。用逐渐增加浓度（从 100 μM 增加到 500 μM）的秦皮乙素处理 NB4 细胞 19 小时，会导致 Bcl2/Bax 比率降低，但不影响 p53 水平。秦皮乙素的促凋亡作用涉及 ERK MAP 激酶级联，因为在用那些药物处理后观察到 ERK 的磷酸化水平增加。还观察到磷酸化-Akt 水平增加。此外，秦皮乙素诱导线粒体膜电位丧失，同时伴有细胞色素 c 向细胞质中的释放，这种释放始于用秦皮乙素处理 6 小时，并持续增加至 24 小时。秦皮乙素诱导超氧阴离子的产生在长时间处理时增加，而在短时间（1 小时）处理时减少，在用秦皮乙素处理 2-4 小时时观察到增加。NO 产生没有明显变化。秦皮乙素以时间依赖性方式降低 GSH 水平。总之，本研究表明秦皮乙素的细胞毒性作用是研究 NB4 细胞系凋亡机制诱导的有效工具，该细胞系可用作 APL 疾病的相关模型。
