摘要
目的研究手霉素对HL-60白血病细胞的作用,探讨其作用机制,主要是线粒体的改变。方法使用人的白血病细胞株HL-60细胞。MTT细胞毒性测定评价对白血病细胞的作用,使用Annexin V和一氧化氮(nitric oxide,NO)染料标记细胞,应用流式细胞仪术检测细胞内NO生成和细胞凋亡。细胞内超氧阴离子通过二氢乙啶(dihydroethidium,DHE)测定。应用化学发光法测定超氧歧化酶(superoxide dismutase,SOD)活性。采用荧光法测定谷胱甘肽(glutathi-one,GSH),萤光素-萤光素酶发光法测定ATP含量。免疫印迹技术检测细胞色素C和Mn-SOD的表达。结果手霉素引起HL-60细胞活性的下降,且呈剂量依赖性。手霉素诱导产生反应性氧基(reactive oxygen species,ROS):NO和超氧阴离子,降低GSH,但不影响SOD。手霉素诱导线粒体降低细胞ATP的含量,线粒体肿胀和细胞色素C从线粒体释放到细胞质。手霉素诱导的凋亡与ROS的增加有关。用N-乙酰基-L-半胱氨酸(N-acetyl-L-cysteine,NAC)抑制ROS可保护HL-60细胞逃避手霉素的细胞毒作用和避免手霉素诱导的凋亡。结论细胞内ROS的产生对手霉素的细胞毒作用起非常重要的作用。手霉素通过包括上游ROS产生,线粒体形态改变和细胞色素C释放的线粒体途径,诱导白血病细胞凋亡。
Aim To investigate the effects of Manumycin on HL-60 myeloid leukemia cell line, and to explore the mechanism, major in investigating changes in the mitochondria of leukemia cell line in response to Manumycin. Methods Human myeloid leukemia cells HL-60 were used. The cytotoxicity was analyzed by MTT assay. Apoptosis and cellular nitric oxide (NO) were detected by flow cytometry using Annexin V and NO sensor dye. Superoxide anion was measured with a fluorescent plate reader by DHE. GSH was assayed by fluorescent Monochlorobimane. The SOD activities were assayed by colorimetric methods using WST. ATP content was measured by luciferin-luciferase bioluminescence assay. The cytosolic proteins were extracted from the cells using a digitonin buffer. The protein expres- sion of cytochrome C and Mn-SOD were determined by Western blot. Results Manumycin resulted in viability decrease in a dose-dependent manner, and induced the generation of ROS:NO and superoxide anions. Manumycin reduced intracellular glutathione. Manumycin induced mitochondria swollen, intracellular ATP content decrease and cytochrome C release from mitochondria to cytosol. Manumycin-induced apoptosis correlated with increase in ROS. Quenching of ROS with N-acetyl- L-cysteine protected leukemia cells from the cytotoxicity of Manumycin and prevented apoptosis induction by Manumycin. Cellular ROS generation plays an important role in the cytotoxic effect of Manu- mycin. Manumycin induced apoptosis through mitochondria-mediated pathway that required upstream ROS generation, change of mitochondria, and cytochrome C release.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2008年第6期772-777,共6页
Chinese Pharmacological Bulletin
基金
广东省自然科学基金资助项目(No7001196)
广东省医学科研基金资助项目(NoA2006020)
关键词
白血病
凋亡
手霉素
反应性氧基
一氧化氮
超氧
阴离子
线粒体
超氧歧化酶
leukemia
apoptosis
Manumycin
reactiveoxygen species
nitric oxide
superoxide anion
mito-chondria
superoxide dismutase