丁硫氨酸亚砜胺和胡椒基丁醚协同诱导 CML 细胞凋亡与 AIF 和 GSH-ROS-JNK-ERK-iNOS 通路的激活有关。
Synergistic apoptosis of CML cells by buthionine sulfoximine and hydroxychavicol correlates with activation of AIF and GSH-ROS-JNK-ERK-iNOS pathway.
机构信息
Division of Cancer Biology and Inflammatory Disorder, Council of Scientific and Industrial Research (CSIR)-Indian Institute of Chemical Biology (IICB), Kolkata, India.
出版信息
PLoS One. 2013 Sep 9;8(9):e73672. doi: 10.1371/journal.pone.0073672. eCollection 2013.
BACKGROUND
Hydroxychavicol (HCH), a constituent of Piper betle leaf has been reported to exert anti-leukemic activity through induction of reactive oxygen species (ROS). The aim of the study is to optimize the oxidative stress -induced chronic myeloid leukemic (CML) cell death by combining glutathione synthesis inhibitor, buthionine sulfoximine (BSO) with HCH and studying the underlying mechanism.
MATERIALS AND METHODS
Anti-proliferative activity of BSO and HCH alone or in combination against a number of leukemic (K562, KCL22, KU812, U937, Molt4), non-leukemic (A549, MIA-PaCa2, PC-3, HepG2) cancer cell lines and normal cell lines (NIH3T3, Vero) was measured by MTT assay. Apoptotic activity in CML cell line K562 was detected by flow cytometry (FCM) after staining with annexin V-FITC/propidium iodide (PI), detection of reduced mitochondrial membrane potential after staining with JC-1, cleavage of caspase- 3 and poly (ADP)-ribose polymerase proteins by western blot analysis and translocation of apoptosis inducing factor (AIF) by confocal microscopy. Intracellular reduced glutathione (GSH) was measured by colorimetric assay using GSH assay kit. 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) were used as probes to measure intracellular increase in ROS and nitric oxide (NO) levels respectively. Multiple techniques like siRNA transfection and pharmacological inhibition were used to understand the mechanisms of action.
RESULTS
Non-apoptotic concentrations of BSO significantly potentiated HCH-induced apoptosis in K562 cells. BSO potentiated apoptosis-inducing activity of HCH in CML cells by caspase-dependent as well as caspase-independent but apoptosis inducing factor (AIF)-dependent manner. Enhanced depletion of intracellular GSH induced by combined treatment correlated with induction of ROS. Activation of ROS- dependent JNK played a crucial role in ERK1/2 activation which subsequently induced the expression of inducible nitric oxide synthase (iNOS). iNOS- mediated production of NO was identified as an effector molecule causing apoptosis of CML cells.
CONCLUSION/SIGNIFICANCE: BSO synergizes with HCH in inducing apoptosis of CML cells through the GSH-ROS-JNK-ERK-iNOS pathway.
背景
已报道胡椒科胡椒属植物叶中的胡椒基甲酮（HCH）通过诱导活性氧（ROS）发挥抗白血病活性。本研究的目的是通过联合使用谷胱甘肽合成抑制剂丁硫氨酸亚砜（BSO）与 HCH 来优化氧化应激诱导的慢性髓性白血病（CML）细胞死亡，并研究其潜在机制。
材料和方法
通过 MTT 测定法测量 BSO 和 HCH 单独或联合对多种白血病（K562、KCL22、KU812、U937、Molt4）、非白血病（A549、MIA-PaCa2、PC-3、HepG2）癌细胞系和正常细胞系（NIH3T3、Vero）的增殖活性。用 Annexin V-FITC/PI 染色后通过流式细胞术（FCM）检测 CML 细胞系 K562 的凋亡活性，用 JC-1 染色检测线粒体膜电位降低，用 Western blot 分析检测 caspase-3 和多聚（ADP-核糖）聚合酶蛋白的裂解，用共聚焦显微镜检测凋亡诱导因子（AIF）的易位。用 GSH 测定试剂盒通过比色法测定细胞内还原型谷胱甘肽（GSH）。2'，7'-二氯二氢荧光素二乙酸酯（DCF-DA）和 4-氨基-5-甲基氨基-2'，7'-二氟荧光素（DAF-FM）分别用作探针以测量细胞内 ROS 和一氧化氮（NO）水平的增加。使用 siRNA 转染和药理学抑制等多种技术来了解作用机制。
结果
非凋亡浓度的 BSO 显著增强了 K562 细胞中 HCH 诱导的细胞凋亡。BSO 通过 caspase 依赖性和非依赖性（但凋亡诱导因子（AIF）依赖性）方式增强了 HCH 在 CML 细胞中的诱导凋亡活性。联合治疗引起的细胞内 GSH 耗竭增强与 ROS 诱导相关。ROS 依赖性 JNK 的激活在 ERK1/2 激活中起关键作用，随后诱导诱导型一氧化氮合酶（iNOS）的表达。鉴定 iNOS 介导的 NO 产生是导致 CML 细胞凋亡的效应分子。
结论/意义：BSO 通过 GSH-ROS-JNK-ERK-iNOS 途径与 HCH 协同诱导 CML 细胞凋亡。
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