铜离子对肺癌细胞增殖的影响及相关基因的临床意义

The effects of copper ion on cell proliferation and the clinical significance of copper ion in lung cancer

背景与目的:破坏铜离子在体内的平衡可能会促进癌症的发生、发展。研究铜离子对肺癌细胞增殖的影响,分析铜离子及铜伴侣蛋白编码基因在肺癌及正常肺组织中的表达差异及其临床意义。方法:以肺癌细胞和肺正常细胞系为细胞模型,通过细胞增殖实验检测铜离子对细胞增殖能力的影响,通过蛋白质印迹法(Western blot)实验检测不同浓度铜离子溶液处理细胞后对细胞外调节蛋白激酶(extracellular regulated protein kinase,ERK)/磷酸化ERK(phosphorylated ERK,p-ERK)信号转导通路的影响。以烟草致癌物4-甲基亚硝铵-1-(3-吡啶基)-1-丁酮[4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone,NNK]诱发小鼠肺癌,用电感耦合等离子体质谱仪(inductively coupled plasma-mass spectrometry,ICP-MS)分析小鼠肺癌组织和正常肺组织中铜的含量差异。利用癌症基因组图谱(The Cancer Genome Atlas,TCGA)、Oncomine和Kaplan-Meier Plotter网站等多种数据库分析铜伴侣蛋白超氧化物歧化酶(copper chaperone for superoxide dismutase,CCS)、细胞色素C氧化酶铜伴侣蛋白17(cytochrome C oxidase copper chaperone 17,COX17)和抗氧化剂1铜伴侣蛋白(antioxidant 1 copper chaperone,ATOX1)的mRNA表达水平与肺癌患者预后的相关性。结果:体外实验结果表明,5μmol/L的铜离子浓度能显著提高肺癌细胞的增殖能力,且能诱导细胞内与细胞增殖相关的信号通路如ERK信号转导通路的激活。在NNK诱发的小鼠肺癌中,肺癌组织中的铜含量显著高于肺正常组织。TCGA和Oncomine等数据库分析结果表明,铜伴侣蛋白编码基因CCS、COX17和ATOX1在肺癌组织中的表达水平比癌旁正常组织的表达水平显著增高,其表达水平与肺癌患者的预后呈负相关。结论:在一定浓度范围内,铜离子能促进细胞增殖,肺癌患者铜伴侣蛋白编码基因有可能用于判断患者临床疗效和评估预后。

Background and purpose:Destroying the balance of copper ions in the body may promote the occurrence and development of cancer.This study aimed to investigate the effects of copper ion on cell proliferation and elucidate the clinical significance of copper chaperone genes in lung cancer.Methods:The effects of copper ion on cell proliferation were determined by trypan blue exclusion experiment.Western blot assays were performed to test the effects of copper on the expressions of extracellular regulated protein kinase(ERK)and phosphorylated ERK(p-ERK).Tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)was used to induce lung cancer in mice,and the copper content in lung cancer tissues and normal lung tissues was detected by inductively coupled plasma mass spectrometry(ICP-MS).Databases including The Cancer Genome Atlas(TCGA),Oncomine and the Kaplan-Meier Plotter website containing the microarray data of patients with non-small cell lung cancer(NSCLC)were used to analyze the correlation between the mRNA expression levels of copper chaperone for superoxide dismutase(CCS),cytochrome C oxidase copper chaperone(COX17)and antioxidant 1 copper chaperone(ATOX1)and the prognosis of lung cancer patients.Results:In vitro experimental results showed that copper ions at a concentration of 5μmol/L was able to significantly promote the proliferation of lung cancer cells and normal lung epithelia cells,and induced the activation of intracellular ERK signaling pathways.In the NNK-induced lung cancer mouse model,the concentration of copper ion was significantly higher in cancer tissues than in normal lung tissues.In TCGA and Oncomine databases,the expression levels of CCS,COX17 and ATOX1 were significantly higher in cancer tissues than in counterpart normal controls.The expression levels were inversely associated with prognosis of the patients.Conclusion:Under specific concentration range,copper ions can promote cell proliferation.The copper chaperone genes could potentially be used as biomarkers to predict the prognosis of lung cancer patients.