title: Establishment and characterization of gemcitabine-resistant human cholangiocarcinoma cell lines with multidrug resistance and enhanced invasiveness.
abstract: To establish and characterize the gemcitabine-resistant cholangiocarcinoma (CCA) cell lines, CCA KKU‑M139 and KKU‑M214 cell lines were exposed stepwisely to increasing gemcitabine (GEM). The resultant drug-resistant cell lines, KKU‑M139/GEM and KKU‑M214/GEM, retained the resistant phenotype in drug-free medium at least for 2 months. Sulforhodamine B assay demonstrated that KKU‑M139/GEM and KKU‑M214/GEM were 25.88- and 62.31-fold more resistant to gemcitabine than their parental cells. Both gemcitabine-resistant cell lines were cross-resistant to 5-fluorouracil (5-FU), doxorubicin and paclitaxel indicating their multidrug-resistant nature. Using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR and western blot analyses, gemcitabine-resistant cells showed upregulation of RRM1 and downregulation of hENT1 and dCK. In relation to multidrug resistance, these cell lines showed upregulation of multidrug resistance protein 1 (MRP1) leading to an increase of drug efflux. Using cell adhesion and Boyden chamber transwell assays, these cell lines also showed higher cell adhesion, migration and invasion capabilities via the activations of protein kinase C (PKC), focal adhesion kinase (FAK), extracellular signal-regulated kinase-1/2 (ERK1/2) and nuclear factor-κB (NF-κB). Higher activity of matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator (uPA) was also observed by a gelatin zymography assay and a casein-plasminogen zymography assay. Flow cytometry analysis indicated the G2/M arrest regulated by downregulation of cyclin B1 and cyclin-dependent kinase 1 (Cdk1) resulted in an extended population doubling time. Using Annexin V/propidium iodide staining, evasion of apoptosis via an intrinsic pathway was observed in both cell lines in association with upregulation of Bcl-2 and downregulation of Bax. Interestingly, Fas was additionally downregulated in KKU‑M214/GEM supporting the view of its higher GEM resistant characteristics. These findings indicate that long-term exposure of CCA cell lines to gemcitabine induce not only multidrug resistance but also enhance their invasiveness.
authors.affiliation: Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Wareeporn
authors.initials: W
authors.lastname: Wattanawongdon
authors.affiliation: Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Chariya
authors.initials: C
authors.lastname: Hahnvajanawong
authors.affiliation: Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Nisana
authors.initials: N
authors.lastname: Namwat
authors.affiliation: Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Sirimas
authors.initials: S
authors.lastname: Kanchanawat
authors.affiliation: Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Thidarut
authors.initials: T
authors.lastname: Boonmars
authors.affiliation: Center for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Patcharee
authors.initials: P
authors.lastname: Jearanaikoon
authors.affiliation: Center for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Chanwit
authors.initials: C
authors.lastname: Leelayuwat
authors.affiliation: Center for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Anchalee
authors.initials: A
authors.lastname: Techasen
authors.affiliation: Department of Forensic Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Wunchana
authors.initials: W
authors.lastname: Seubwai
mesh_terms: D001650:Bile Duct Neoplasms / Q000473:pathology*; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002465:Cell Movement; D018281:Cholangiocarcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D005472:Fluorouracil / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D009361:Neoplasm Invasiveness; D017239:Paclitaxel / Q000494:pharmacology; D000093542:Gemcitabine
chemical_list: D003841:Deoxycytidine; D004317:Doxorubicin; D017239:Paclitaxel; D005472:Fluorouracil; D000093542:Gemcitabine