Establishment and characterization of gemcitabine-resistant human cholangiocarcinoma cell lines with multidrug resistance and enhanced invasiveness.
Int J Oncol
; 47(1): 398-410, 2015 Jul.
Article
em En
| MEDLINE
| ID: mdl-25998688
ABSTRACT
To establish and characterize the gemcitabine-resistant cholangiocarcinoma (CCA) cell lines, CCA KKUM139 and KKUM214 cell lines were exposed stepwisely to increasing gemcitabine (GEM). The resultant drug-resistant cell lines, KKUM139/GEM and KKUM214/GEM, retained the resistant phenotype in drug-free medium at least for 2 months. Sulforhodamine B assay demonstrated that KKUM139/GEM and KKUM214/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 KKUM214/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.
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Base de dados:
MEDLINE
Assunto principal:
Neoplasias dos Ductos Biliares
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Regulação Neoplásica da Expressão Gênica
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Colangiocarcinoma
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Resistência a Múltiplos Medicamentos
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Resistencia a Medicamentos Antineoplásicos
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Desoxicitidina
Idioma:
En
Ano de publicação:
2015
Tipo de documento:
Article