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Annals of Surgical Oncology
Published in: Annals of Surgical Oncology 12/2007

01-12-2007 | Laboratory Research

Development and Characterization of Gemcitabine-Resistant Pancreatic Tumor Cells

Authors: Ami N. Shah, Justin M. Summy, Jing Zhang, Serk In Park, Nila U. Parikh, Gary E. Gallick

Published in: Annals of Surgical Oncology | Issue 12/2007

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Abstract

Background

Pancreatic cancer is an exceptionally lethal disease with an annual mortality nearly equivalent to its annual incidence. This dismal rate of survival is due to several factors including late presentation with locally advanced, unresectable tumors, early metastatic disease, and rapidly arising chemoresistance. To study the mechanisms of chemoresistance in pancreatic cancer we developed two gemcitabine-resistant pancreatic cancer cell lines.

Methods

Resistant cells were obtained by culturing L3.6pl and AsPC-1 cells in serially increasing concentrations of gemcitabine. Stable cultures were obtained that were 40- to 50-fold increased in resistance relative to parental cells. Immunofluorescent staining was performed to examine changes in β-catenin and E-cadherin localization. Protein expression was determined by immunoblotting. Migration and invasion were determined by modified Boyden chamber assays. Fluorescence-activated cell sorting (FACS) analyses were performed to examine stem cell markers.

Results

Gemcitabine-resistant cells underwent distinct morphological changes, including spindle-shaped morphology, appearance of pseudopodia, and reduced adhesion characteristic of transformed fibroblasts. Gemcitabine-resistant cells were more invasive and migratory. Gemcitabine-resistant cells were increased in vimentin and decreased in E-cadherin expression. Immunofluorescence and immunoblotting revealed increased nuclear localization of total β-catenin. These alterations are hallmarks of epithelial-to-mesenchymal transition (EMT). Resistant cells were activated in the receptor protein tyrosine kinase, c-Met and increased in expression of the stem cell markers CD (cluster of differentiation)24, CD44, and epithelial-specific antigen (ESA).

Conclusions

Gemcitabine-resistant pancreatic tumor cells are associated with EMT, a more-aggressive and invasive phenotype in numerous solid tumors. The increased phosphorylation of c-Met may also be related to chemoresistance and EMT and presents as an attractive adjunctive chemotherapeutic target in pancreatic cancer.
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Metadata
Title
Development and Characterization of Gemcitabine-Resistant Pancreatic Tumor Cells
Authors
Ami N. Shah
Justin M. Summy
Jing Zhang
Serk In Park
Nila U. Parikh
Gary E. Gallick
Publication date
01-12-2007
Publisher
Springer-Verlag
Published in
Annals of Surgical Oncology / Issue 12/2007
Print ISSN: 1068-9265
Electronic ISSN: 1534-4681
DOI
https://doi.org/10.1245/s10434-007-9583-5

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