Increase of ceramide and induction of mixed apoptosis necrosis by N-(4-hydroxyphenyl)-retinamide in neuroblastoma cell lines
Increase of ceramide and induction of mixed apoptosis necrosis by N-(4-hydroxyphenyl)-retinamide in neuroblastoma cell lines
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DOI：
10.1093/jnci/91.13.1138
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发表时间：
1999-07-07
影响因子：
10.3
通讯作者：
Reynolds, CP
中科院分区：
文献类型：
--
作者：
Maurer, BJ;Metelitsa, LS;Reynolds, CP
背景资料：合成的维甲酸类化合物N-（4-羟基苯基）维A酰胺（4-HPR或芬维A胺）对髓性白血病和宫颈癌细胞系具有毒性，可能部分是由于其增加活性氧（ROS）水平的能力。由于神经母细胞瘤通常在骨髓中复发，缺氧组织隔室，和许多化疗药物被缺氧拮抗，我们的目的是研究在这些细胞系中影响4-HPR诱导的细胞毒性的几个因素，包括诱导的ROS水平，生理性缺氧和抗氧化剂的影响，神经酰胺的水平，和细胞死亡的机制。方法：用羧基二氯荧光素二乙酸酯荧光法测定活性氧的产生。神经酰胺通过放射性标记和薄层色谱法定量，免疫印迹法用于评估p53蛋白水平。凋亡通过核形态学和核小体间DNA片段化模式分析程序性细胞死亡和坏死。在存在或不存在泛胱天蛋白酶抑制剂BOC-d-fetrin的情况下，通过采用数字成像显微镜的基于荧光的测定法测量细胞毒性。统计学检验为双侧，结果/结论：除了增加ROS外，4-HPR（2.5-10 μ M）在统计学上显著增加细胞内神经酰胺的水平（高达约10倍; P
Background: The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR or fenretinide) is toxic to myeloid leukemia and cervical carcinoma cell lines, probably in part due to its ability to increase levels of reactive oxygen species (ROS), We have studied the effects of 4-HPR on neuroblastoma cell lines. Since neuroblastomas commonly relapse in bone marrow, a hypoxic tissue compartment, and many chemotherapeutic agents are antagonized by hypoxia, our purpose was to study in these cell lines several factors influencing 4-HPR-induced cytotoxicity, including induced levels of ROS, effects of physiologic hypoxia and antioxidants, levels of ceramide, and the mechanism of cell death. Methods: ROS generation was measured by carboxy-dichlorofluorescein diacetate fluorescence. Ceramide was quantified by radiolabeling and thin-layer chromatography, Immunoblotting was used to assess p53 protein levels. Apoptosis (programmed cell death) and necrosis were analyzed by nuclear morphology and internucleosomal DNA fragmentation patterns, Cytotoxicity was measured by a fluorescence-based assay employing digital imaging microscopy in the presence or absence of the pancaspase enzyme inhibitor BOC-d-fmk, Statistical tests were two-sided, Results/ Conclusions: In addition to increasing ROS, 4-HPR (2.5-10 mu M) statistically significantly increased the level of intracellular ceramide (up to approximately 10-fold; P