College of Life Sciences, Shenzhen University, Shenzhen 518060, China.
J Trace Elem Med Biol. 2011 Jul;25(3):130-7. doi: 10.1016/j.jtemb.2011.06.001. Epub 2011 Jul 20.
Sodium selenite can induce the apoptosis of cancer cells, however its mechanism has seldom been studied via proteomics. In this paper, human cervical cancer HeLa cells were investigated by MTT assay and morphological observation to get appropriate selenite concentrations for proteomic study. Results showed that selenite at concentrations larger than 10 μmol/L significantly inhibited the viability of HeLa cells. 40 μmol/L selenite was in the appropriate range for proteomic study. After 24 h treatment with 40 μmol/L selenite, total proteins were extracted from the cells and applied to two-dimensional gel electrophoresis (2DE). Those proteins with their expression levels altered at least 2-fold comparing to the control were picked up for protein identification via MALDI-TOF mass spectrometry and further confirmed by Western blot analysis. About 1000 spots were detected by the software in each 2DE gel, among which 13 differentially expressed proteins were identified by mass spectrometry and most of them are relevant to oxidative stress, such as peroxiredoxins, superoxide dismutase, quinolinate phosphoribosyl transferase, and D-dopachrome tautomerase. Meanwhile, reactive oxygen species (ROS) and mitochondrial membrane potential were also detected by flow cytometry and laser confocal scanning microscope. An increase in ROS generation and a decrease in mitochondrial membrane potential were detected in the selenite-treated cells compared with the control, which are consistent with the down-expression of antioxidative proteins in proteomics. Those results indicate that selenite induces the apoptosis of HeLa cells via ROS-mediated mitochondrial pathway. The present study also implies the potentiality of selenium in cervical cancer treatment.
亚硒酸钠可以诱导癌细胞凋亡，但其通过蛋白质组学的机制研究甚少。本文采用 MTT 检测和形态学观察法，研究了人宫颈癌 HeLa 细胞，以获得适合蛋白质组学研究的亚硒酸钠浓度。结果表明，亚硒酸钠浓度大于 10 μmol/L 时明显抑制 HeLa 细胞活力。40 μmol/L 亚硒酸钠适用于蛋白质组学研究。用 40 μmol/L 亚硒酸钠处理 24 h 后，从细胞中提取总蛋白，并应用二维凝胶电泳（2DE）。与对照组相比，表达水平至少变化 2 倍的蛋白质被挑选出来，通过 MALDI-TOF 质谱进行蛋白质鉴定，并通过 Western blot 分析进一步确认。每张 2DE 凝胶中软件可检测到约 1000 个斑点，其中 13 个差异表达蛋白通过质谱鉴定，大多数与氧化应激有关，如过氧化物酶、超氧化物歧化酶、喹啉酸磷酸核糖基转移酶和 D-多巴色素互变异构酶。同时，通过流式细胞术和激光共聚焦扫描显微镜检测活性氧（ROS）和线粒体膜电位。与对照组相比，亚硒酸钠处理的细胞中 ROS 生成增加，线粒体膜电位降低，与蛋白质组学中抗氧化蛋白的下调一致。这些结果表明，亚硒酸钠通过 ROS 介导的线粒体途径诱导 HeLa 细胞凋亡。本研究还暗示了硒在宫颈癌治疗中的潜力。
