NGAL蛋白诱导食管癌细胞发生自噬  

Autophagy Induced by NGAL Protein in Esophageal Carcinoma Cells

作　　者：张丕显[1] 方王楷[1] 许丽艳[2] 蒋纪恺[1] 沈忠英[2] 杜则澎[1] 陆晓峰[2] 周飞[1] 谢剑君[1] 吴炳礼[1] 崔有宏[3] 谢东[1,4] 李恩民[1] 

机构地区：[1]汕头大学医学院生物化学与分子生物学教研室,汕头515041 [2]汕头大学医学院肿瘤病理研究室,汕头515041 [3]汕头大学医学院病理生理教研室,汕头515041 [4]中国科学院上海生命科学院营养科学研究所,上海200031

出　　处：《生物化学与生物物理进展》2009年第8期978-986,共9页Progress In Biochemistry and Biophysics

基　　金：国家高技术研究发展计划(863)资助项目(2006AA02A403);国家自然科学基金资助项目(30370641;30570829;30672376);广东省自然科学基金重点项目(5104541;7118419);教育部高等学校博士点重点学科专项基金项目(20050560002;20050560003)~~

摘　　要：以往研究证明,中性粒细胞明胶酶相关载脂蛋白NGAL(neutrophil gelatinase-associated lipocalin)与食管癌密切相关,改变NGAL表达能够对癌细胞的形态结构产生明显影响,但确切的作用机制不明.在酵母细胞中表达NGAL蛋白,柱层析分离纯化.筛选NGALR(NGAL receptor)高表达与弱表达的人食管癌细胞系EC1.71和EC109作为实验细胞模型.5-FAM标记NGAL蛋白,加入到细胞培养上清中,对比研究NGAL蛋白入胞情况、细胞形态学改变、细胞自噬体产生、自噬相关基因表达、细胞内铁离子与铁蛋白水平以及相关细胞信号转导激酶的活性等.结果表明,NGAL蛋白可经由内吞途径进入食管癌细胞发挥作用,致使细胞发生典型的自噬性形态结构变化,自噬体大量产生,自噬相关基因的表达发生相应变化,ERK被激活,但细胞内的铁离子与铁蛋白未受明显影响.上述结果提示,诱导细胞发生自噬是外源性NGAL蛋白经由内吞途径进入食管癌细胞发挥作用的机制之一,与NGAL蛋白跨膜转铁未见直接关联,而ERK信号转导途径可能参与了这一过程.Previous studies suggest that NGAL （neutro phil gelatinase-associated lipocalin） is involved in the transformation and development of esophageal carcinoma. Alteration of NGAL expression can trigger the change of cellular morphology in esophageal carcinoma cells. However, the mechanisms remain unclear. To get a better understanding of NGAL function in esophageal carcinoma, NGAL protein was expressed in methylotrophic yeast, Pichia pastoris, and purified by chromatography. EC1.71 cells expressed high levels ofNGALR （NGAL receptor） and EC109 cells expressed low levels of NGALR were used as cells model. The trafficking and the possible function of NGAL protein were then analyzed in the esophageal carcinoma cells. The results showed that 5-FAM-labeled recombinant NGAL protein could internalize into the EC1.71 and EC109 cells. Furthermore, the internalized NGAL protein could induce the alteration of cellular morphology, resulting in generation of autophagosome, transcriptional up-regulation of genes associated with autophagy and increase of phospho-ERK1/2 （p-ERK1/2）. Interestingly, the treatment with the NGAL protein did not affect the intracellular iron level. These data indicate that induced autophagy by exogenous NGAL protein is a mechanism that internalized NGAL plays important roles in esophageal carcinoma cells, independent with NGAL-mediated iron transport process, while ERK1/2 signal pathway is involved in activation of autophagy by exogenous NGAL protein.

关 键 词：NGAL NGALR 自噬 细胞周期 蛋白质内吞 食管癌细胞 

分 类 号：Q781[生物学—分子生物学] R735[医药卫生—肿瘤]