The FXR agonist obeticholic acid inhibits the cancerogenic potential of human cholangiocarcinoma

Cholangiocarcinoma (CCA) is an aggressive cancer with high resistance to chemotherapeutics. CCA is enriched in cancer stem cells, which correlate with aggressiveness and prognosis. FXR, a member of the metabolic nuclear receptor family, is markedly down-regulated in human CCA. Our aim was to evaluate, in primary cultures of human intrahepatic CCA (iCCA), the effects of the FXR agonist obeticholic acid (OCA), a semisynthetic bile acid derivative, on their cancerogenic potential. Primary human iCCA cell cultures were prepared from surgical specimens of mucinous or mixed iCCA subtypes. Increasing concentrations (0–2.5 μM) of OCA were added to culture media and, after 3–10 days, effects on proliferation (MTS assay, cell population doubling time), apoptosis (annexin V-FITC/propidium iodide), cell migration and invasion (wound healing response and Matrigel invasion assay), and cancerogenic potential (spheroid formation, clonogenic assay, colony formation capacity) were evaluated. Results: FXR gene expression was downregulated (RT-qPCR) in iCCA cells vs normal human biliary tree stem cells (p < 0.05) and in mucinous iCCA vs mixed iCCA cells (p < 0.05) but was upregulated by addition of OCA. OCA significantly (p < 0.05) inhibited proliferation of both mucinous and mixed iCCA cells, starting at a concentration as low as 0.05 μM. Also, CDCA (but not UDCA) inhibited cell proliferation, although to a much lower extent than OCA, consistent with its different affinity for FXR. OCA significantly induced apoptosis of both iCCA subtypes and decreased their in vitro cancerogenic potential, as evaluated by impairment of colony and spheroid formation capacity and delayed wound healing and Matrigel invasion. In general, these effects were more evident in mixed than mucinous iCCA cells. When tested together with Gemcitabine and Cisplatin, OCA potentiated the anti-proliferative and pro-apoptotic effects of these chemotherapeutics, but mainly in mixed iCCA cells. OCA abolished the capacity of both mucinous and mixed iCCA cells to form colonies when administered together with Gemcitabine and Cisplatin. In subcutaneous xenografts of mixed iCCA cells, OCA alone or combined with Gemcitabine or Cisplatin markedly reduced the tumor size after 5 weeks of treatment by inducing necrosis of tumor mass and inhibiting cell proliferation. In conclusion, FXR is down-regulated in iCCA cells, and its activation by OCA results in anti-cancerogenic effects against mucinous and mixed iCCA cells, both in vitro and in vivo. The effects of OCA predominated in mixed iCCA cells, consistent with the lower aggressiveness and the higher FXR expression in this CCA subtype. These results, showing the FXR-mediated capacity of OCA to inhibit cholangiocarcinogenesis, represent the basis for testing OCA in clinical trials of CCA patients

Immunohistochemical study of doublecortin and nucleostemin in canine brain

Finding a marker of neural stem cells remains a medical research priority. It was reported that the proteins doublecortin and nucleostemin were related with stem/progenitor cells in central nervous system. The aim of the present immunohistochemical study was to evaluate the expression of these proteins and their pattern of distribution in canine brain, including age-related changes, and in non-nervous tissues. We found that doublecortin had a more specific expression pattern, related with neurogenesis and neuronal migration, while nucleostemin was expressed in most cells of almost every tissue studied. The immunolabeling of both proteins decreased with age. We may conclude that nucleostemin is not a specific marker of stem/progenitor cells in the dog. Doublecortin, however, is not an exclusive marker of neural stem cells, but also of neuronal precursors

Canine Intracranial Neoplasia: Epidemiologic and Clinicopathologic Characterization in an European Canine Population

No abstract available

Canine Intracranial Neoplasia: Epidemiologic and Clinicopathologic Characterization in an European Canine Population

No abstract available

Immunohistochemical study of doublecortin and nucleostemin in canine brain

Finding a marker of neural stem cells remains a medical research priority. It was reported that the proteins doublecortin and nucleostemin were related with stem/progenitor cells in central nervous system. The aim of the present immunohistochemical study was to evaluate the expression of these proteins and their pattern of distribution in canine brain, including age-related changes, and in non-nervous tissues. We found that doublecortin had a more specific expression pattern, related with neurogenesis and neuronal migration, while nucleostemin was expressed in most cells of almost every tissue studied. The immunolabeling of both proteins decreased with age. We may conclude that nucleostemin is not a specific marker of stem/progenitor cells in the dog. Doublecortin, however, is not an exclusive marker of neural stem cells, but also of neuronal precursors

Metformin exerts anti-cancerogenic effects and reverses epithelial-to-mesenchymal transition trait in primary human intrahepatic cholangiocarcinoma cells

Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive cancer with marked resistance to chemotherapeutics without therapies. The tumour microenvironment of iCCA is enriched of Cancer-Stem-Cells expressing Epithelial-to-Mesenchymal Transition (EMT) traits, being these features associated with aggressiveness and drug resistance. Treatment with the anti-diabetic drug Metformin, has been recently associated with reduced incidence of iCCA. We aimed to evaluate the anti-cancerogenic effects of Metformin in vitro and in vivo on primary cultures of human iCCA. Our results showed that Metformin inhibited cell proliferation and induced dose- and time-dependent apoptosis of iCCA. The migration and invasion of iCCA cells in an extracellular bio-matrix was also significantly reduced upon treatments. Metformin increased the AMPK and FOXO3 and induced phosphorylation of activating FOXO3 in iCCA cells. After 12&nbsp;days of treatment, a marked decrease of mesenchymal and EMT genes and an increase of epithelial genes were observed. After 2&nbsp;months of treatment, in order to simulate chronic administration, Cytokeratin-19 positive cells constituted the majority of cell cultures paralleled by decreased Vimentin protein expression. Subcutaneous injection of iCCA cells previously treated with Metformin, in Balb/c-nude mice failed to induce tumour development. In conclusion, Metformin reverts the mesenchymal and EMT traits in iCCA by activating AMPK-FOXO3 related pathways suggesting it might have therapeutic implications

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

DCLK1, a putative novel stem cell marker in human cholangiocarcinoma

Background &amp; aims: Cholangiocarcinoma (CCA) is a very aggressive cancer showing high cancer stem cells (CSCs) presence. Doublecortin-like kinase1 (DCLK1) has been demonstrated as a CSC marker in different gastroenterological solid tumours. Our aim was to evaluate in vitro the expression and the biological function of DCLK1 in intrahepatic CCA (iCCA) and perihilar CCA (pCCA). Approach &amp; results: Specimens surgically resected of human CCA were enzymatically digested, submitted to immunosorting for specific CSC markers (LGR5, CD90, EpCAM, CD133, CD13) and primary cell cultures were prepared. DCLK1 expression was analysed in CCA cell cultures by real-time quantitative polymerase chain reaction (RT-qPCR), Western Blot and immunofluorescence. Functional studies have been performed by evaluating the effects of selective DCLK1 inhibitor (LRRK2-IN-1) on cell proliferation (MTS-Assay, cell population doubling time), apoptosis and colony formation capacity. DCLK1 was investigated in situ by immunohistochemistry and RT-qPCR. DCLK1 serum concentration was analysed by enzyme-linked immunosorbent assay (ELISA). We describe DCLK1 in CCA with an increased gene and protein DCLK1 expression in pCCALGR5+ and in iCCACD133+ cells compared to unsorted cells. LRRK2-IN-1 showed an anti-proliferative effect in dose-dependent manner. LRRK2-IN-1 markedly impaired cell proliferation, induced apoptosis, decreased colony formation capacity and colony size in both iCCA and pCCA compared to untreated cells. In situ analysis confirm that DCLK1 is present only in tumours, but not in healthy tissue. Interestingly, DCLK1 was detected in the human serum samples of iCCA (high), pCCA (high), HCC (low) and cirrhotic (low) patients, but it was almost undetectable in healthy controls. Conclusion: DCLK1 characterizes a specific CSC-subpopulation of iCCACD133+ and pCCALGR5+ and its inhibition exerts anti-neoplastic effects in primary CCA cell cultures. Human DCLK1 serum might represent a serum biomarker for the early CCA diagnosis