Development and characterization of gemcitabine-resistant pancreatic tumor cells.
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 beta-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 beta-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.
Shah
Ami N
AN
Department of Surgical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
Summy
Justin M
JM
Zhang
Jing
J
Park
Serk In
SI
Parikh
Nila U
NU
Gallick
Gary E
GE
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D015820:Cadherins / Q000378:metabolism; D002448:Cell Adhesion; D002465:Cell Movement; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D015151:Immunoblotting; D008565:Membrane Proteins / Q000378:metabolism; D008648:Mesoderm / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009361:Neoplasm Invasiveness; D010190:Pancreatic Neoplasms / Q000188:drug therapy*; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects; D051176:beta Catenin / Q000378:metabolism; D000093542:Gemcitabine
D000964:Antimetabolites, Antineoplastic; D015820:Cadherins; D018960:Hyaluronan Receptors; D008565:Membrane Proteins; D051176:beta Catenin; C106354:flotillins; D003841:Deoxycytidine; D019859:Proto-Oncogene Proteins c-met; D000093542:Gemcitabine