Metformin arrests the 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. Methods: 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, foxo3a genes was analyzed by RT-qPCR, whereas Cytokeratin19 and Vimentin were also analyzed by Immunofluorescence Assay. Results: Metformin inhibited cell proliferation (MTS assay, population doubling and population doubling time) of primary cultures from mucin- and mixed CCA; the effect was dose and time dependent starting at a concentration of 5 µM and reaching a plateau at 500 µM. At 100 µM, metformin induced apoptosis with a 50% increase of the number of Annexin V/PI positive cells (p<0.05 vs. controls). The migration of primary human CCA cells, from both mucin and mixed CCA subtypes, was significantly reduced by treatment with metformin at different concentrations (from 5 to 1000 µM). The effects of metformin were associated with enhanced expression of the epithelial marker e-cadherin gene and decreased expression of vimentin and of the EMT specific genes, snail1, snail2, and twist1. We also found that metformin increased mRNA expression of fox3a, an effect significantly 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, in primary cultures of human CCA, metformin inhibits cell proliferation, enhances apoptosis and impairs the expression of EMT traits by upregulating foxo2a gene. Therefore, metformin could play anticancer effects against human CCAs with relevant therapeutic implications.