Establishment And Genes Expression Profiling Variation Of Drug-resistant Human Colon Cancer HT-29/5-Fu Cell Line

Objectives:To establish drug-resistant human colon cancer cell line in vitro with its parent cell line HT-29 by continuously exposing the HT-29 cells to ascending drug doses, and observe them biological characterization, with the aim to analyze the genes expressing differently between them by Human Gene Expression Array for exploring the possible resistance associated genes and clarifying the mechanisms of tumor drug resistance as well as identifying effect molecular target.Methods:1.The HT-29/5-Fu cell line was established by continuously exposing the HT-29 cel s to ascending doses of 5-Fu.2.The biological properties of HT-29/5-Fu cell line was observed. The morphology and colony formation rate were detected. The growth curve and t he chemosensitivity of HT-29/5-Fu cell line were determined by MTT assay. Western blotting was used to analyze the expression of thymidylate synthase(TS) and dihydropyrimidine dehydrogenase(DPD). Cell cycle test and apoptosis assays were measured by flow cytometry.3.The differentially expressed genes between the drug resistant cell line and their parental cell line were identified by using m RNA expression profiling microarray. The GO and K EGG pathway analysis of the commonly differentially expressed genes were performed for investigating genes related to drug resistance. Results:1.Through induced in 10 months,we established the resistant cell line HT-29/5-Fu,which showed exponential growth in 1μg/m L 5-Fu.2.The biological characteristics of 5- fluorouracil- resistant human colon cancer HT-29/5-Fu cell line altered. The resistance of HT-29/5-Fu cells to 5-Fu was 3.59 fold greater than that of HT-29 cells. Its morphology differed from that of HT-29 cells. Compared with HT-29 cells, HT-29/5-Fu cells showed remarkable reduction of cell proliferation and colony formation, higher expression of TS and DPD, higher percentage of cells in the S phase, and stronger ability of resistance to apoptosis induced by 5-Fu.3.Of the 211 dysregulated genes detected in HT-29/5-Fu cell line when compared to HT-29 cells, 115 genes were up-regulated(fold change≥2) and 96 genes were down-regulated(fold change≤0.5).O f these differentially expressed genes, 17 genes related to chemoresistance have been reported; TYMS, drug transporters ABCG2, YES1 and MDK were strongly linked to 5-fluorouracil resistance. Moreover, certain dysregulated genes involved in multidrug resistance, EMT(epithelial-to- mesenchymal transition),the proliferatio n and invasion ability of human cancer cell, anti-apoptosis and cell cycle arrest. GO analysis(molecular function)of the differentially expressed genes revealed that transmembrane transporter activity, signal transducer,nucleic acid binding and transcriptional factor activity were the 4 most prominent terms. Focal adhesion,tight junction,ABC transporters,p53 signaling pathway and cell adhesion molecules(CAMs) were the top 5 over-represented terms,which were related to cancer in 25 pathways selected from the KEGG pathway analysis. Conclusions:1.The HT-29/5-Fu cell line was established in this research, its biological characters different from the parent cell line HT-29.2.We found certain changes of the m RNA expression profiles between the drug resistant cell line and its parental HT-29 cell line, which could provide preliminary work and experimental evidence for the selection research of novel chemotherapy resistant biomarkers.