OC.13.3: Metformin Inhibits Proliferation, Enhances Apoptosis and Down-Regulates Epithelial to Mesenchymal Transition (EMT) in Human Cholangiocarcinoma (CCA): A Study on Human Primary Cell Cultures

CCA is a very aggressive cancer with marked resistance to chemotherapeutics. We have previously demonstrated that CCA is enriched of cancer stem cells expressing EMT traits, these features being associated with aggressiveness and drug resistance. We have recently established primary cell cultures from human intrahepatic CCA subtypes (i.e., mucin and mixed). Treatment with the anti-diabetic drug metformin has been recently associated with reduced cancer incidence. Furthermore, in immortalized cancer cell lines, metformin showed inhibitory effects on EMT by up-regulating Foxo3a signaling in an Akt-dependent manner. We aimed to evaluate the effects of metformin on proliferation, apoptosis, cell migration and the expression of EMT traits in primary cultures of CCA subtypes. Primary CCA cell cultures were treated with increasing metformin concentrations (from 5 to 1000 µM, for 1-4 days). Then we evaluated: (i) proliferation by MTS assay; (ii) apoptosis by flow cytometry analysis of Annexin VFITC/Propidium Iodide (PI); and (iii) cell migration by wound-healing assay. The expression of Vimentin, E-Cadherin, SNAIL1, SNAIL2, TWIST1, Cytokeratin19(CK19), FOXO3a and AMPK genes were analyzed by RT-qPCR, whereas ck19, Vimentin, E-Cadherin and Foxo3a were analyzed by Immunofluorescence Assay. Metformin inhibited cell proliferation (MTS assay, population doubling and population doubling time) and induced apoptosis in primary cultures of mucin- and mixed-CCA; the effects were dose- and time-dependent (p<0.05 vs. controls). The migration of primary human CCA cells, from both mucin and mixed CCA subtypes, was also significantly reduced by treatment with metformin at different concentrations, from 5 to 1000 µM. The effects of metformin were associated with enhanced gene expression of the epithelial marker E-Cadherin and decreased expression of Vimentin and EMT specific genes, SNAIL1, SNAIL2 and TWIST1. Metformin also increased the AMPK and Foxo3a mRNA levels. FOXO3a gene expression was negatively correlated with the expression of SNAIL1 and Vimentin genes. Metformin exerted similar effects in mucin- and mixed-CCA human primary cell cultures. In conclusion, we demonstrated that metformin inhibits cell proliferation, enhances apoptosis and impairs the expression of EMT traits by upregulating Foxo3a gene in primary cultures of human CCA. Therefore, metformin could play anticancer effects against human CCAs with relevant therapeutic implications.