Detachment of esophageal carcinoma cells from extracellular matrix causes relocalization of death receptor 5 and apoptosis.
OBJECTIVE
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.


CONCLUSIONS
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.
Liu
Guang-Chao
GC
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.
Zhang
Jun
J
Liu
Shi-Gui
SG
Gao
Rong
R
Long
Zhang-Fu
ZF
Tao
Ke
K
Ma
Yuan-Fang
YF
D017209:Apoptosis; D020126:Brefeldin A / Q000627:therapeutic use; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D053181:Caspase 8 / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D004938:Esophageal Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D005109:Extracellular Matrix / Q000378:metabolism / Q000473:pathology*; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism*
D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D020126:Brefeldin A; D053181:Caspase 8