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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
Abstracts of the 23rd National Congress of Digestive Diseases / Digestive and Liver Disease 49S2 (2017) e73–e223
between January 2014 and December 2015: 412 patients had a diagnosis of PBC according to the ICD9 code 571.6. In 2015, the prevalence was 27.9/100,000 while the incidence was 5.3/100,000/year. The female to male ratio was 8:2 and the mean ± SD age was 64.6±14.3 years. There was no statistically significant difference between incident and prevalent cases or among sexes. Three-hundred sixty-four patients (88.4%) received a pharmacologic treatment for PBC: of those 334 (92%) were treated with UDCA, mostly in monotherapy (281–77.2%). A significantly lower number of newly diagnosed patients were treated compared to prevalent cases (76 vs. 94%; p<0.001). Conclusions: The estimated prevalence of PBC, based on a real world database analysis, is consistent with a previous Italian report based on administrative databases and with Orphanet data, while the incidence is slightly higher. Despite limitations, data collected through the IMS longitudinal database provides a unique and valuable insight into the current epidemiology of PBC in Italy and on its management.
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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 mucinand mixed-CCA human primary cell cultures. Conclusions: 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.
OC.13.4 THROMBOELASTOGRAPHY-GUIDED TRANSFUSIONS REDUCES THE USE OF BLOOD PRODUCTS IN CIRRHOTIC PATIENTS UNDERGOING INVASIVE ENDOSCOPIC PROCEDURES M. Marocchi ∗ , M. Bianchini, L. De Pietri, G. Bolondi, A. Merighi, G. Olivetti, V. Boarino, A. Bertani, E. Villa Azienda Ospedaliero Universitaria, Modena, Italy
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 S. Di Matteo ∗,1 , A.M. Lustri 1 , D. Costantini 1 , L. Nevi 1 , J. Faccioli 1 , C. Napoletano 1 , A. Cantafora 1 , E. Manzi 2 , F. Melandro 1 , A.M. De Rose 3 , M.C. Bragazzi 1 , G.L. Grazi 2 , P.B. Berloco 1 , F. Giuliante 3 , V. Cardinale 1 , G. Carpino 1 , D. Alvaro 1 University of Rome, Rome, Italy; 2 Regina Elena National Cancer Institute, Rome, Italy; 3 Catholic University of the Sacred Heart School of Medicine, Rome, Italy 1 Sapienza
Background and aim: 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. Material and 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 (CK19), FOXO3a and AMPK genes were analyzed by RT-qPCR, whereas ck19, Vimentin, E-Cadherin and Foxo3a were analyzed by Immunofluorescence Assay. Results: 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
Background and aim: Cirrhotic patients are usually labelled as at high risk of bleeding because of prolonged prothrombin time and reduced platelet count. However, cirrhotics present indeed a rebalanced hemostasis, with parallel reduction of pro- and anticoagulant factors and routinary coagulation tests (i.e. international normalized ratio [INR] and platelet count) cannot adequately evaluate the coagulation status in this cohort of patients. Thus, the management of blood products use in cirrhotic patients before endoscopic invasive procedures is still controversial. Nowadays no clear guidelines define how to monitor adequately the coagulation status of cirrhotics before invasive procedures, and there are not sufficient evidences to avoid coagulation control in these patients. In case of significant coagulopathy (arbitrarily defined as INR>1.8 and/or platelet count <50×109 /L) international guidelines still recommend transfusion of fresh frozen plasma (FFP) or platelets (PLT) before invasive procedures. Thromboelastography (TEG) instead provides a more comprehensive global coagulation assessment, and showed to be safe, reducing the use of blood products before different invasive procedures. Material and methods: Thirty cirrhotic patients with INR>1.8 and platelet count <50×109 /L undergoing endoscopic invasive procedures (variceal band ligation, 22 pts and polypectomy, 8 pts) were randomly allocated to TEG-guided transfusion strategy or standard of care (SOC). The TEG group received FFP if the reaction time (r) was >40 min and/or PLT if maximum amplitude (MA) was <30 mm. All SOC patients received FFP and/or PLT according to hospital guidelines. Results: Baseline characteristics of the two groups were similar. Per protocol, all subjects in the SOC group received blood product transfusions versus 2 in the TEG group (93.3% vs. 13.3%; P<0.0001). Two SOC (13.3%) received FFP, 8 (53.3%) received PLT, and 4 (28.6%) both FFP and PLT. In the TEG group, none received FFP alone (P<0.0001 vs. SOC) and only 2 received PLT (13.3%; P<0.0001 vs. SOC). No post-procedural bleeding occurred; only 3 patients (2 in TEG group and 1 in SOC group) required blood transfusion due to moderate aneamia not related to the endoscopic procedure. Conclusions: In patients with cirrhosis and significant coagulopathy TEG-guided transfusion strategy before endoscopic invasive procedures leads to a significantly lower use of blood products compared to SOC, without an increase in bleeding complications.
between January 2014 and December 2015: 412 patients had a diagnosis of PBC according to the ICD9 code 571.6. In 2015, the prevalence was 27.9/100,000 while the incidence was 5.3/100,000/year. The female to male ratio was 8:2 and the mean ± SD age was 64.6±14.3 years. There was no statistically significant difference between incident and prevalent cases or among sexes. Three-hundred sixty-four patients (88.4%) received a pharmacologic treatment for PBC: of those 334 (92%) were treated with UDCA, mostly in monotherapy (281–77.2%). A significantly lower number of newly diagnosed patients were treated compared to prevalent cases (76 vs. 94%; p<0.001). Conclusions: The estimated prevalence of PBC, based on a real world database analysis, is consistent with a previous Italian report based on administrative databases and with Orphanet data, while the incidence is slightly higher. Despite limitations, data collected through the IMS longitudinal database provides a unique and valuable insight into the current epidemiology of PBC in Italy and on its management.
e113
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 mucinand mixed-CCA human primary cell cultures. Conclusions: 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.
OC.13.4 THROMBOELASTOGRAPHY-GUIDED TRANSFUSIONS REDUCES THE USE OF BLOOD PRODUCTS IN CIRRHOTIC PATIENTS UNDERGOING INVASIVE ENDOSCOPIC PROCEDURES M. Marocchi ∗ , M. Bianchini, L. De Pietri, G. Bolondi, A. Merighi, G. Olivetti, V. Boarino, A. Bertani, E. Villa Azienda Ospedaliero Universitaria, Modena, Italy
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 S. Di Matteo ∗,1 , A.M. Lustri 1 , D. Costantini 1 , L. Nevi 1 , J. Faccioli 1 , C. Napoletano 1 , A. Cantafora 1 , E. Manzi 2 , F. Melandro 1 , A.M. De Rose 3 , M.C. Bragazzi 1 , G.L. Grazi 2 , P.B. Berloco 1 , F. Giuliante 3 , V. Cardinale 1 , G. Carpino 1 , D. Alvaro 1 University of Rome, Rome, Italy; 2 Regina Elena National Cancer Institute, Rome, Italy; 3 Catholic University of the Sacred Heart School of Medicine, Rome, Italy 1 Sapienza
Background and aim: 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. Material and 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 (CK19), FOXO3a and AMPK genes were analyzed by RT-qPCR, whereas ck19, Vimentin, E-Cadherin and Foxo3a were analyzed by Immunofluorescence Assay. Results: 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
Background and aim: Cirrhotic patients are usually labelled as at high risk of bleeding because of prolonged prothrombin time and reduced platelet count. However, cirrhotics present indeed a rebalanced hemostasis, with parallel reduction of pro- and anticoagulant factors and routinary coagulation tests (i.e. international normalized ratio [INR] and platelet count) cannot adequately evaluate the coagulation status in this cohort of patients. Thus, the management of blood products use in cirrhotic patients before endoscopic invasive procedures is still controversial. Nowadays no clear guidelines define how to monitor adequately the coagulation status of cirrhotics before invasive procedures, and there are not sufficient evidences to avoid coagulation control in these patients. In case of significant coagulopathy (arbitrarily defined as INR>1.8 and/or platelet count <50×109 /L) international guidelines still recommend transfusion of fresh frozen plasma (FFP) or platelets (PLT) before invasive procedures. Thromboelastography (TEG) instead provides a more comprehensive global coagulation assessment, and showed to be safe, reducing the use of blood products before different invasive procedures. Material and methods: Thirty cirrhotic patients with INR>1.8 and platelet count <50×109 /L undergoing endoscopic invasive procedures (variceal band ligation, 22 pts and polypectomy, 8 pts) were randomly allocated to TEG-guided transfusion strategy or standard of care (SOC). The TEG group received FFP if the reaction time (r) was >40 min and/or PLT if maximum amplitude (MA) was <30 mm. All SOC patients received FFP and/or PLT according to hospital guidelines. Results: Baseline characteristics of the two groups were similar. Per protocol, all subjects in the SOC group received blood product transfusions versus 2 in the TEG group (93.3% vs. 13.3%; P<0.0001). Two SOC (13.3%) received FFP, 8 (53.3%) received PLT, and 4 (28.6%) both FFP and PLT. In the TEG group, none received FFP alone (P<0.0001 vs. SOC) and only 2 received PLT (13.3%; P<0.0001 vs. SOC). No post-procedural bleeding occurred; only 3 patients (2 in TEG group and 1 in SOC group) required blood transfusion due to moderate aneamia not related to the endoscopic procedure. Conclusions: In patients with cirrhosis and significant coagulopathy TEG-guided transfusion strategy before endoscopic invasive procedures leads to a significantly lower use of blood products compared to SOC, without an increase in bleeding complications.