Bosnjak Mihajlo, Ristic Biljana, Arsikin Katarina, Mircic Aleksandar, Suzin-Zivkovic Violeta, Perovic Vladimir, Bogdanovic Andrija, Paunovic Verica, Markovic Ivanka, Bumbasirevic Vladimir, Trajkovic Vladimir, Harhaji-Trajkovic Ljubica
Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade, Serbia.
Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, Belgrade, Serbia.
PLoS One. 2014 Apr 8;9(4):e94374. doi: 10.1371/journal.pone.0094374. eCollection 2014.
The present study investigated the role of autophagy, a cellular self-digestion process, in the cytotoxicity of antileukemic drug cytarabine towards human leukemic cell lines (REH, HL-60, MOLT-4) and peripheral blood mononuclear cells from leukemic patients. The induction of autophagy was confirmed by acridine orange staining of intracellular acidic vesicles, electron microscopy visualization of autophagic vacuoles, as well as by the increase in autophagic proteolysis and autophagic flux, demonstrated by immunoblot analysis of p62 downregulation and LC3-I conversion to autophagosome-associated LC3-II in the presence of proteolysis inhibitors, respectively. Moreover, the expression of autophagy-related genes Atg4, Atg5 and Atg7 was stimulated by cytarabine in REH cells. Cytarabine reduced the phosphorylation of the major negative regulator of autophagy, mammalian target of rapamycin (mTOR), and its downstream target p70S6 kinase in REH cells, which was associated with downregulation of mTOR activator Akt and activation of extracellular signal- regulated kinase. Cytarabine had no effect on the activation of mTOR inhibitor AMP-activated protein kinase. Leucine, an mTOR activator, reduced both cytarabine-induced autophagy and cytotoxicity. Accordingly, pharmacological downregulation of autophagy with bafilomycin A1 and chloroquine, or RNA interference-mediated knockdown of LC3β or p62, markedly increased oxidative stress, mitochondrial depolarization, caspase activation and subsequent DNA fragmentation and apoptotic death in cytarabine-treated REH cells. Cytarabine also induced mTOR-dependent cytoprotective autophagy in HL-60 and MOLT-4 leukemic cell lines, as well as primary leukemic cells, but not normal leukocytes. These data suggest that the therapeutic efficiency of cytarabine in leukemic patients could be increased by the inhibition of the mTOR-dependent autophagic response.
本研究调查了自噬（一种细胞自我消化过程）在抗白血病药物阿糖胞苷对人白血病细胞系（REH、HL - 60、MOLT - 4）以及白血病患者外周血单个核细胞的细胞毒性中的作用。通过细胞内酸性囊泡的吖啶橙染色、自噬泡的电子显微镜观察，以及分别通过在蛋白酶解抑制剂存在下p62下调的免疫印迹分析和LC3 - I转化为自噬体相关的LC3 - II来证明自噬蛋白水解和自噬通量的增加，从而证实了自噬的诱导。此外，阿糖胞苷刺激了REH细胞中自噬相关基因Atg4、Atg5和Atg7的表达。阿糖胞苷降低了自噬主要负调节因子哺乳动物雷帕霉素靶蛋白（mTOR）及其在REH细胞中的下游靶标p70S6激酶的磷酸化作用，这与mTOR激活剂Akt的下调和细胞外信号调节激酶的激活相关。阿糖胞苷对mTOR抑制剂AMP激活的蛋白激酶的激活没有影响。亮氨酸（一种mTOR激活剂）降低了阿糖胞苷诱导的自噬和细胞毒性。因此，用巴弗洛霉素A1和氯喹进行自噬的药理学下调，或RNA干扰介导的LC3β或p62敲低，显著增加了阿糖胞苷处理的REH细胞中的氧化应激、线粒体去极化、半胱天冬酶激活以及随后的DNA片段化和凋亡死亡。阿糖胞苷还在HL - 60和MOLT - 4白血病细胞系以及原代白血病细胞中诱导了mTOR依赖性的细胞保护性自噬，但在正常白细胞中未诱导。这些数据表明，通过抑制mTOR依赖性自噬反应可以提高阿糖胞苷在白血病患者中的治疗效果。
