The Recombinant Expression, Purification And Induction Of NGAL Protein To Esophageal Carcinoma Cells In Vitro

Neutrophil gelatinase-associated lipocalin (NGAL) is a 25KD glucoprotein and a new member of lipocalin family with a typicalβ-barrel structure of the family. Based on the recent research, the function of NGAL protein may be is to protect and regulate the activity of matrix metalloproteinase-9(MMP-9) and to involve in inflammation and innate immunity reaction. NGAL protein also is a novel iron transporter that mediates a new iron delivery pathway.Since 2000, our group found that NGAL overexpression played an important role in the process of malignant transformation of human immortalized esophageal epithelial cells and was involved in the invasion of esophageal carcinoma cells via protecting the activity of MMP-9 from degradation. We also demonstrated that down regulation of NGAL gene altered the distribution of F-actin, a microfilament cytoskeleton, and proliferated differentiation of esophageal carcinoma cells, resulting in that main structure characteristics of the cancer cells tended to consist with that of immortalized cells. These data indicated that NGAL may be an important human oncoprotein which is significantly correlated with esophageal carcinoma and other cancers. However, the functional mechanism of NGAL protein is unknown in the esophageal carcinoma cells, including which receptor mediated NGAL protein across membrane; what pathway is involved in NGAL intracellular signaling; which genes responded to NGAL protein stimulation and so on. In present study, we focused on the induction of recombinant NGAL protein to esophageal carcinoma cells in vitro. Results showed that the biological effect of recombinant NGAL protein was different from that of NGAL gene having been stable transfected and forced expressed. The former could internalize into the esophageal carcinoma cells and induce the alteration of cellular morphology, resulting in generation of autophagosome, but not apoptosis; the latter could promote the invasion and metabasis of the cells and result in the cachexia of model animals. The detailed data about recombinant NGAL protein inducing the autophagy of esophageal carcinoma cells were described as follows:Human NGAL protein was expressed in methylotrophic yeast, Pichia pastoris and purified by column chromatography. EC1.71 cells expressing high levels of NGAL receptor (NGALR) and EC109 cells expressing low levels of NGALR were used as cells model. EC1.71 or EC109 cells were treated with various concentrations of recombinant NGAL protein for different time intervals and assessed the trafficking and the possible function of the NGAL on the esophageal carcinoma cells by protein labeling, immunofluorescence, western blot, RT-PCR and ferrozine staining. Result showed that 5-FAM-labeled recombinant NGAL protein could internalize into the EC1.71 and EC109 cells, but the uptaking capacity of EC1.71 cells was evidently stronger than that of EC109 cells. After the treatment with various concentrations of the NGAL protein, especially up to 50μg/ml, the internalized NGAL protein could induce the alteration of cellular morphology, including that the filopodia on cellular surface was lost and smoothed out, cellular numbers were reduced and the adherence ability of the cells was decreased. Moreover, EC1.71 cells were more sensitive to stimulation of NGAL proein than EC109 cells. MDC staining which was used to label the autophagic vacuoles showed that the treatment with various concentrations of the NGAL protein, especially from 5 to 50μg/ml, resulted in generation of autophagosome in EC1.71 and EC109 cells. The signaling of the autophagy was stronger in EC1.71 cells than that in EC109 cells, indicating that maybe is mediated by NGALR or other molecules. To further confirm the function of the NGAL protein, EC1.71 and EC109 was transient transfected by pLC3-GFP vector for 24h and treated with recombinant NGAL protein (50μg/ml) for another 24h, we found that the LC3 as a autophagy marker appeared translocation from the plasma to membrane. This indicated that the NGAL protein induced a generation of autophagy. Interestingly, ferrozine assays and western blot analysis showed that the treatment with the NGAL protein did not affect the intracellular iron level in EC1.71 and EC109 cells. However, RT-PCR and western blot assay showed that when EC1.71 and EC109 cells were treated with the NGAL protein (50μg/ml) for different time intervals, phospho-ERK1/2 (p-ERK1/2) was increased and genes associated with autophagy, such as atg5, beclin1and DAPK, were transcriptional up-regulated in time-dependent manner, peaking at 5 min and lasting for ~24 h; moreover, the signaling pathway of p38 and JNK also was activated a little.In conclusion, exogenous NGAL protein could be internalized into esophageal carcinoma cells to induce a generation of autophagy. ERK1/2 signal pathway is involved in activation of autophagy by exogenous NGAL protein, but not NGAL-mediated iron transport process.