ABSTRACT
BACKGROUND: Pancreatic cancer stromal cells produce various protein factors, which presumably provide cancer cells with drug resistance and may influence their ability to form metastasis via induction of epithelial-mesenchymal transition (ÐÐТ). The goal of our project was to study the effects of IGF-I on expression of protein markers of epithelial and mesenchymal differentiation, and on expression of transcriptional regulators of EMT in pancreatic cancer cell lines. METHODS: We used Western blot analysis to study the expression patterns of epithelial and mesenchymal protein markers in pancreatic cancer cell lines, which have been stimulated with IGF-I for various periods of time. The ELISA technique was employed to determine the concentration of IGF-I in conditioned media. Additionally, the effect of IGF-I on proliferation of pancreatic cancer cells was measured via MTS technique. RESULTS: We investigated the effect of IGF/IGF-IR signaling pathway activation on expression levels of cell differentiation markers in five pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-2, MiaPaCa-2 and Panc1). The IGF-I stimulation led to phosphorylation of IGF-IR and activation of PI-3K/Akt signaling cascade. At the same time our results reveal that the activation of IGF/IGF-IR signaling pathway in pancreatic cancer cells does not induce a significant shift in cell phenotype towards mesenchymal differentiation and does not induce a decrease in expression levels of epithelial protein markers. CONCLUSIONS: Our results demonstrate that IGF-I does not function as an effective inductor of EMT in pancreatic cancer cell lines and that stimulation of IGF-I/IGF-IR signaling pathway does not lead to EMT associated changes in cell differentiation.
Subject(s)
Epithelial-Mesenchymal Transition/physiology , Insulin-Like Growth Factor I/metabolism , Pancreatic Neoplasms/metabolism , Receptor, IGF Type 1/metabolism , Biomarkers, Tumor , Cell Differentiation , Cell Line, Tumor , Gene Expression Regulation, Neoplastic/drug effects , Humans , Insulin-Like Growth Factor I/pharmacology , Transforming Growth Factor beta2/pharmacologyABSTRACT
Hybrid therapeutic gene FCU1 gene was cloned into a lentiviral expression vector and the therapeutic effect of its expression was studied in three pancreatic cancer cell lines. Expression of FCU1 gene sensitized cells of two of three studied pancreatic cancer cell lines to 5-fluorocytosine. In addition, uracil phosphoribosyl transferase activity of the hybrid FCU1 protein increased sensitivity of transfected cells of all three studied pancreatic cancer cell lines to 5-fluorouracil, a standard chemotherapeutic agent.
Subject(s)
Antineoplastic Agents/pharmacology , Cytosine Deaminase/genetics , Insulin-Secreting Cells/drug effects , Pentosyltransferases/genetics , Recombinant Fusion Proteins/genetics , Cell Line, Tumor , Cytosine Deaminase/metabolism , Drug Resistance, Neoplasm , Flucytosine/metabolism , Flucytosine/pharmacology , Fluorouracil/pharmacology , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/pathology , Lentivirus/genetics , Lentivirus/metabolism , Pentosyltransferases/metabolism , Plasmids/chemistry , Plasmids/metabolism , Recombinant Fusion Proteins/metabolism , Transduction, GeneticABSTRACT
We show characteristic morphological changes corresponding to epithelial-mesenchymal transition (EMT) program fulfillment in PANC1 cell line stimulated with TGFß1. Our results support downregulation of E-cadherin protein. We show 5- and 28-fold increase in SNAI1 and SNAI2 expression levels and 25- and 15-fold decrease in CDH1 and KRT8 expression levels, respectively, which confirms the EMT-program fulfillment. We demonstrate downregulation of expression of pancreatic master genes SOX9, FOXA2, and GATA4 (2-, 5-, and 4-fold, respectively) and absence of significant changes in HES1, NR5A2, and GATA6 expression levels in the cells stimulated with TGFß1. Our results indicate the absence of induction of expression of PTF1A, PDX1, HNF1b, NEUROG3, RPBJL, NKX6.1, and ONECUT1 genes, which are inactive in PANC1 cell line after the EMT stimulated by TGFß1.
