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Establishment and characterization of fluorouracil and gemcitabine resistant cholangiocarcinoma cell lines

ABSTRACT: Resistance to commonly used chemotherapeutic drugs is a major problem of cholangiocarcinoma (CCA) therapy, therefore more effective treatment strategy is urgently needed. Drug resistant cell lines have been used for elucidating drug resistance mechanisms in various cancers leading to the discovery of novel treatments. Here in, we established a 5-Fluorouracil and Gemcitabine resistant CCA cell lines. The resistant sublines were selected based on constant exposure of KKU-213B parental cells to 5-Fluorouracil and gemcitabine in a stepwise dose incremental strategy starting from below IC25 concentration). The cells that grown normally in the presence of 5-FU 7 and Gem 3 μM were used for subsequence experiments and weredesignated as KKU-213B-FR and KKU-213B-GR. The resultant cell lines were confirmed to be resistant to chemotherapeutic drug and also increased cell migration and invasion activity. In addition, the quantitative LC-MS/MS were utilized for the analysis of proteins involved in drug resistance mechanisms.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Sumalee Obchoei

PROVIDER: PXD049450 | JPOST Repository | Tue Feb 18 00:00:00 GMT 2025

REPOSITORIES: jPOST

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Project description:In pancreatic cancer the survival rate is low, as the available treatment options usually only extend survival and seldom produce a cure. Drug resistance and disease reoccurrence is the typical reason for death after cancer diagnosis. 5-Fluorouracil (5-FU) is the main chemostatic used in first line therapy. However the majority of the tumors become resistant to treatment. To investigate acquired 5-FU resistance in pancreatic adenocarcinoma, we established chemoresistant monoclonal cell lines from the Panc03.27 cell line by long-term exposure to 5-FU. In addition to increased expression of markers associated with multidrug resistance, the 5-FU resistant clones showed alterations typical of the process of epithelial-to-mesenchymal transition (EMT), including upregulation of mesenchymal markers and increased invasiveness. Microarray analysis revealed the L1CAM pathway as one of the most upregulated pathways in the chemoresistant clones, which was confirmed on RNA and protein levels. Expression of the adhesion molecule L1CAM is associated with a chemoresistant and migratory phenotype of pancreatic cancer. Using esiRNA targeting L1CAM, or by blocking the extracellular part of L1CAM with monoclonal antibodies, we discovered that the increased invasiveness observed in the chemoresistant cells depends on L1CAM. Using esiRNA targeting β-catenin and/or Slug, we discovered that L1CAM expression depends on Slug rather than β-catenin in the 5-FU resistant cells. We demonstrate a functional link between Slug and the expression level of L1CAM in pancreatic cancer cells having undergone EMT following long-term exposure to 5-FU. Our findings provide further insight into the molecular mechanisms leading to a chemoresistant and migratory phenotype in pancreatic cancer cells and indicate the importance of Slug-induced L1CAM in refractory pancreatic cancer. Examination of expression of 5-Fluorouracil (5-FU) Panc03.27 cell line resistant clone versus expression of 5-FU sensitive clones (NT) in 4 replicates per cell lines

Project description:Background: Current protocols for the treatment of ovarian cancer include combination chemotherapy with a platinating agent and a taxane. However, many patients experience relapse of their cancer and the development of drug resistance is not uncommon, making successful second line therapy difficult to achieve. The objective of this study was to develop a cell line resistant to both carboplatin and docetaxel (dual drug resistant ovarian cell line A2780CBNDXL), along with single agent resistant lines (docetaxel resistant A2780DXL and carboplatin resistant A2780CBN), to investigate the mechanisms which underlie the development of dual drug resistance. Methods: The A2780 epithelial ovarian cancer cell line was used to select for isogenic carboplatin, docetaxel and dual drug resistant cell lines. A selection method of gradually increasing drug doses was implemented to avoid clonal selection. Resistance was confirmed using a clonogenic assay. Changes in gene expression associated with the development of drug resistance were determined by microarray analysis compared to parental co-culture control A2780CC. Changes in selected genes were validated by QPCR and immunoblotting. Results: Three isogenic cell lines were developed and resistance to each drug or the combination of drugs was confirmed. Development of resistance was accompanied by a reduced growth rate. The microarray and QPCR analyses showed that unique changes in gene expression occurred in the dual drug resistant cell line and that genes known to be involved in resistance could be identified in all cell lines. Conclusions: Novel changes in gene expression can occur in the development of dual drug resistance, indicating that dual drug resistance is not a simple combination of the changes occurring in single agent resistance Carboplatin, docetaxel (GSE26129) and carboplatin/docetaxel dual resistant cell lines of ovarian A2780 were generated for gene expression profilling. Two colour microarray of Agilent whole human genome nucleotide arrays was conducted with two replicate of both forward and reverse labellings for each cell line. Four arrays were used for this experiments. And it was four replicate of both forward and reverse labellings for A2780 cells. Eight arrays were used for this experiments

			Action	DRS
	KKU-214FR-1_1.mzXML	Mzxml
	KKU-214FR-1_2.mzXML	Mzxml
	KKU-214FR-1_3.mzXML	Mzxml
	KKU-214FR-2_1.mzXML	Mzxml
	KKU-214FR-2_2.mzXML	Mzxml

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Establishment and characterization of fluorouracil and gemcitabine resistant cholangiocarcinoma cell lines

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