Original Articles
Copyright ©2009 The WJG Press and Baishideng. All rights reserved.
World J Gastroenterol. Feb 21, 2009; 15(7): 836-844
Published online Feb 21, 2009. doi: 10.3748/wjg.15.836
Detachment of esophageal carcinoma cells from extracellular matrix causes relocalization of death receptor 5 and apoptosis
Guang-Chao Liu, Jun Zhang, Shi-Gui Liu, Rong Gao, Zhang-Fu Long, Ke Tao, Yuan-Fang Ma
Guang-Chao Liu, Shi-Gui Liu, Rong Gao, Zhang-Fu Long, Ke Tao, Key Laboratory of Bio-resource and Eco-environment of China Ministry of Education, College of Life Sciences of Sichuan University, Chengdu 610064, Sichuan Province, China
Guang-Chao Liu, Jun Zhang, Yuan-Fang Ma, Henan Provincial Key Laboratory for Natural Drug and Immune Engineering, Henan University, Kaifeng 475004, Henan Province, China
Guang-Chao Liu, Jun Zhang, Yuan-Fang Ma, Institute of Immunology, Medical College, Henan University, Kaifeng 475004, Henan Province, China
Author contributions: Liu GC, Ma YF and Liu SG designed the research; Liu GC and staff of the research group performed the research; Gao R and Long ZF contributed to the analytic tools; Zhang J and Tao K analyzed the data and revised the manuscript; Liu GC wrote the paper; All authors read and approved the final manuscript.
Correspondence to: Dr. Yuan-Fang Ma, Medical College, Henan University, Jinming Dadao, Kaifeng 475004, Henan Province, China. mayf@henu.edu.cn
Telephone: +86-378-3880398
Fax: +86-378-3880398
Received: September 10, 2008
Revised: December 13, 2008
Accepted: December 20, 2008
Published online: February 21, 2009
Abstract

AIM: To investigate the effect of detachment of esophageal cancer cells from extracellular matrix on the localization of death receptor 5 (DR5) and apoptosis.

METHODS: Anchorage-dependent EC9706 cells of esophageal squamous cell carcinoma were pretreated or not treated with brefeldin A. Detached cells were harvested by ethylenediaminetetraacetic acid digestion. Expression and localization of DR5 in these cells were determined by immunocytochemical and immunofluorescence assays, as well as flow cytometry analysis. Apoptosis of EC9706 cells was detected by flow cytometry after stained with fluorescein isothiocyanate-labeled annexin V/propidium iodide. Activation of caspase 8 was detected by Western blot analysis.

RESULTS: Immunocytochemical assay indicated that DR5 was predominantly perinuclear in adherent cells but was mainly localized in cell membrane in detached cells. In addition, immunofluorescence assay also confirmed the above-mentioned results, and further demonstrated that DR5 was present in the form of coarse granules in detached cells, but in the form of fine granules in adherent cells. Cytometry analysis revealed higher levels of DR5 expression on the surfaces of brefeldin-A-untreated cells than on the surfaces of brefeldin-A-treated cells, but brefeldin A treatment did not affect the total DR5 expression levels. Moreover, nocodazole did not influence the extracelluar DR5 expression levels in EC9706 cells. Apoptosis assay revealed that detached cells were more sensitive to DR5 antibody-induced apoptosis than adherent cells. Western blotting showed that caspase 8 was activated in temporarily detached cells 4 h earlier than in adherent cells.

CONCLUSION: Progress from adhesion to detachment of EC9706 cells causes DR5 relocalization, and promotes cytoplasmic translocation of DR5 to cell surfaces via a Golgi-dependent pathway. Moreover, it might also result in DR5 aggregation to render apoptosis of detached cells.

Keywords: Translocation of death receptor 5; Cell detachment; Esophageal carcinoma; Anoikis; Apoptosis