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BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is characterized by high resistance to chemotherapy and poor prognosis. Several oncogenic pathways converge on activation of extracellular signal-regulated kinase 5 (ERK5), whose role in CCA has not been explored. The aim of this study was to investigate the role of ERK5 in the biology of CCA. APPROACH AND RESULTS: ERK5 expression was detected in two established (HuCCT-1 and CCLP-1) and two primary human intrahepatic CCA cell lines (iCCA58 and iCCA60). ERK5 phosphorylation was increased in CCA cells exposed to soluble mediators. In both HuCCT-1 and CCLP-1 cells, ERK5 was localized in the nucleus, and exposure to fetal bovine serum (FBS) further increased the amount of nuclear ERK5. In human CCA specimens, ERK5 mRNA expression was increased in tumor cells and positively correlated with portal invasion. ERK5 protein levels were significantly associated with tumor grade. Growth, migration, and invasion of CCA cells were decreased when ERK5 was silenced using specific short hairpin RNA (shRNA). The inhibitory effects on CCA cell proliferation, migration and invasion were recapitulated by treatment with small molecule inhibitors targeting ERK5. In addition, expression of the angiogenic factors VEGF and angiopoietin 1 was reduced after ERK5 silencing. Conditioned medium from ERK5-silenced cells had a lower ability to induce tube formation by human umbilical vein endothelial cells and to induce migration of myofibroblasts and monocytes/macrophages. In mice, subcutaneous injection of CCLP-1 cells silenced for ERK5 resulted in less frequent tumor development and smaller size of xenografts compared with cells transfected with nontargeting shRNA. CONCLUSIONS: ERK5 is a key mediator of growth and migration of CCA cells and supports a protumorigenic crosstalk between the tumor and the microenvironment.
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Neoplasias dos Ductos Biliares/genética , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma/genética , Proteína Quinase 7 Ativada por Mitógeno/genética , Animais , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Meios de Cultivo Condicionados , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Macrófagos , Camundongos , Monócitos , Miofibroblastos , Gradação de Tumores , Invasividade Neoplásica , Transplante de Neoplasias , Neovascularização Patológica/genética , Fenótipo , RNA Mensageiro/metabolismoRESUMO
BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a form of primary liver cancer with limited therapeutic options. Recently, cancer stem cells (CSCs) have been proposed as a driving force of tumour initiation and dissemination, thus representing a crucial therapeutic target. The protease inhibitor SerpinB3 (SB3) has been identified in several malignancies including hepatocellular carcinoma. SB3 has been involved in the early events of hepatocarcinogenesis and is highly expressed in hepatic progenitor cells and in a mouse model of liver progenitor cell activation. However, only limited information on the possible role of SB3 in CCA stem-like compartment is available. METHODS: Enrichment of CCA stem-like subset was performed by sphere culture (SPH) in CCA cell lines (CCLP1, HUCCT1, MTCHC01 and SG231). Quantitative RT-PCR and Western blotting were used to detect SB3 in both SPH and parental monolayer (MON) cells. Acquired CSC-like features were analysed using an endogenous and a paracrine in vitro model, with transfection of SB3 gene or addition of recombinant SB3 to cell medium respectively. SB3 tumorigenic role was explored in an in vivo mouse model of CCA by subcutaneous injection of SB3-transfected MON (MONSB3+ ) cells in immune-deficient NOD-SCID/IL2Rgnull (NSG) mice. SB3 expression in human CCA sections was investigated by immunohistochemistry. Overall survival (OS) and time to recurrence (TTR) analyses were carried out from a transcriptome database of 104 CCA patients. RESULTS: SB3, barely detected in parental MON cells, was overexpressed in the same CCA cells grown as 3D SPH. Notably, MONSB3+ showed significant overexpression of genes associated with stemness (CD24, CD44, CD133), pluripotency (c-MYC, NOTCH1, STAT3, YAP, NANOG, BMI1, KLF4, OCT4, SOX2), epithelial mesenchymal transition (ß-catenin, SLUG) and extracellular matrix remodelling (MMP1, MMP7, MMP9, ADAM9, ADAM10, ADAM17, ITGB3). SB3-overexpressing cells showed superior spherogenic capacity and invasion ability compared to control. Importantly, MONSB3+ exhibited activation of MAP kinases (ERK1/2, p38, JNK) as well as phosphorylation of NFκB (p65) in addition to up-regulation of the proto-oncogene ß-catenin. All these effects were reversed after transient silencing of SB3. According to the in vitro finding, MONSB3+ cells retained high tumorigenic potential in NSG mice. SB3 overexpression was observed in human CCA tissues and analysis of OS as well as TTR indicated a worse prognosis in SB3+ CCA patients. CONCLUSION: These findings indicate a SB3 role in mediating malignant phenotype of CCA and identify a new therapeutic target.
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Neoplasias dos Ductos Biliares , Colangiocarcinoma , Neoplasias Hepáticas , Proteínas ADAM/metabolismo , Animais , Antígenos de Neoplasias , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/patologia , Linhagem Celular Tumoral , Colangiocarcinoma/patologia , Transição Epitelial-Mesenquimal/genética , Humanos , Neoplasias Hepáticas/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/patologia , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases , SerpinasRESUMO
BACKGROUND & AIMS: Little is known about the metabolic regulation of cancer stem cells (CSCs) in cholangiocarcinoma (CCA). We analyzed whether mitochondrial-dependent metabolism and related signaling pathways contribute to stemness in CCA. METHODS: The stem-like subset was enriched by sphere culture (SPH) in human intrahepatic CCA cells (HUCCT1 and CCLP1) and compared to cells cultured in monolayer. Extracellular flux analysis was examined by Seahorse technology and high-resolution respirometry. In patients with CCA, expression of factors related to mitochondrial metabolism was analyzed for possible correlation with clinical parameters. RESULTS: Metabolic analyses revealed a more efficient respiratory phenotype in CCA-SPH than in monolayers, due to mitochondrial oxidative phosphorylation. CCA-SPH showed high mitochondrial membrane potential and elevated mitochondrial mass, and over-expressed peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α, a master regulator of mitochondrial biogenesis. Targeting mitochondrial complex I in CCA-SPH using metformin, or PGC-1α silencing or pharmacologic inhibition (SR-18292), impaired spherogenicity and expression of markers related to the CSC phenotype, pluripotency, and epithelial-mesenchymal transition. In mice with tumor xenografts generated by injection of CCA-SPH, administration of metformin or SR-18292 significantly reduced tumor growth and determined a phenotype more similar to tumors originated from cells grown in monolayer. In patients with CCA, expression of PGC-1α correlated with expression of mitochondrial complex II and of stem-like genes. Patients with higher PGC-1α expression by immunostaining had lower overall and progression-free survival, increased angioinvasion and faster recurrence. In GSEA analysis, patients with CCA and high levels of mitochondrial complex II had shorter overall survival and time to recurrence. CONCLUSIONS: The CCA stem-subset has a more efficient respiratory phenotype and depends on mitochondrial oxidative metabolism and PGC-1α to maintain CSC features. LAY SUMMARY: The growth of many cancers is sustained by a specific type of cells with more embryonic characteristics, termed 'cancer stem cells'. These cells have been described in cholangiocarcinoma, a type of liver cancer with poor prognosis and limited therapeutic approaches. We demonstrate that cancer stem cells in cholangiocarcinoma have different metabolic features, and use mitochondria, an organelle located within the cells, as the major source of energy. We also identify PGC-1α, a molecule which regulates the biology of mitochondria, as a possible new target to be explored for developing new treatments for cholangiocarcinoma.
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Neoplasias dos Ductos Biliares/metabolismo , Colangiocarcinoma/metabolismo , Mitocôndrias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fosforilação Oxidativa , Fenótipo , Transdução de Sinais/genética , Animais , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/patologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Linhagem Celular Tumoral , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/patologia , Complexo II de Transporte de Elétrons/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Inativação Gênica , Humanos , Indóis/administração & dosagem , Masculino , Metformina/administração & dosagem , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação Oxidativa/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/antagonistas & inibidores , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Intervalo Livre de Progressão , Propanóis/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Transfecção , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND AND AIMS: The proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in cholesterol homeostasis, and its inhibition represents an effective therapy to lower low-density lipoprotein cholesterol (LDL-C) levels. In this study, we examined the impact of the PCSK9 rs11591147 loss-of-function (LOF) variant on liver damage in a multicenter collection of patients at risk of nonalcoholic steatohepatitis (NASH), in clinical samples and experimental models. METHODS: We considered 1874 consecutive individuals at risk of NASH as determined by histology. The SNP rs11591147, encoding for the p.R46L variant of PCSK9, was genotyped by TaqMan assays. We also evaluated 1) PCSK9 mRNA hepatic expression in human liver, and 2) the impact of a NASH-inducing diet in mice with hepatic overexpression of human PCSK9. RESULTS: Carriers of PCSK9 rs11591147 had lower circulating LDL-C levels and were protected against nonalcoholic fatty liver disease (NAFLD) (OR: 0.42; 95% CI: 0.22-0.81; P = .01), NASH (OR: 0.48; 95% CI: 0.26-0.87; P = .01) and more severe fibrosis (OR: 0.55; 95% CI: 0.32-0.94; P = .03) independently of clinical, metabolic and genetic confounding factors. PCSK9 hepatic expression was directly correlated with liver steatosis (P = .03). Finally, liver-specific overexpression of human PCSK9 in male mice drives NAFLD and fibrosis upon a dietary challenge. CONCLUSIONS: In individuals at risk of NASH, PCSK9 was induced with hepatic fat accumulation and PCSK9 rs11591147 LOF variant was protective against liver steatosis, NASH and fibrosis, suggesting that PCSK9 inhibition may be a new therapeutic strategy to treat NASH.
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Hepatopatia Gordurosa não Alcoólica , Pró-Proteína Convertase 9 , Animais , LDL-Colesterol , Humanos , Fígado , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Pró-Proteína Convertase 9/genéticaRESUMO
GEM Premier ChemSTAT is a whole-blood analyzer designed for providing a rapid basic metabolic panel, inclusive of creatinine and blood urea nitrogen, with the unique characteristic of providing measured bicarbonate (HCO3-) levels. The aim of this work was to evaluate the clinical performance of HCO3- assessment with this analyser in a real-life hemodialysis setting. Imprecision was calculated at different HCO3- levels, along with assay comparison with Gem Premier 4000 analysers. GEM Premier ChemSTAT displayed an imprecision and a bias (in comparison to GEM Premier 4000) for HCO3- of 0.4% and 37.3% at 20.8 mmol/L, 1.2% and 25.6% at 16.4 mmol/L, and 2.1% and 11.6% at 11.5 mmol/L, respectively, using three levels of HCO3- quality control sample ChemSTAT System Evaluator. At direct comparison with the GEM Premier 4000 in the hemodialysis setting, Bland-Altman analysis of HCO3- levels evidenced a bias (µ) of -4.9 (95% CI, -5.2 to -4.7) mmol/L. Such difference was attenuated by recalculating the GEM ChemSTAT expected HCO3- values from pH and pCO2 using the Henderson Hasselbach equation, µ=-0.07 (95%CI, -0.19 to 0.05) mmol/L (p = .24). In conclusion, our results show a remarkable difference between the HCO3- values reported by GEM ChemSTAT or GEM 4000. New reference values for GEM ChemSTAT HCO3- shall hence be defined according to our findings. We suggest that further investigation and a re-evaluation of the reference range should be made before extending the clinical use of this device.
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Bicarbonatos/sangue , Diálise Renal , Humanos , Valores de ReferênciaRESUMO
LMW-PTP has been associated with the development of colorectal cancer (CRC) and with the resistance to chemotherapy in cancer cells. To clarify its role in vivo, we studied LMW-PTP expression in Pirc rats (F344/NTac-Apc am1137 ), genetically prone to CRC and resistant to apoptosis. In the morphologically normal mucosa (NM) of Pirc rats, a dramatic over-expression of LMW-PTP was found compared to wt rats (about 60 times higher). Moreover, LMW-PTP levels further increase in spontaneously developed Pirc colon tumors. To understand if and how LMW-PTP affects resistance to apoptosis, we studied CRC cell lines, sensitive (HT29 and HCT-116), or resistant (HT29R, HCT116R) to 5-Fluorouracil (5-FU): resistant cells over-express LMW-PTP. When resistant cells were challenged with morin, a polyphenol inhibiting LMW-PTP, a fast and dose-related down-regulation of LMW-PTP was observed. 5-FU and morin co-treatment dramatically decreased cell viability, increased apoptosis, and significantly impaired self-renewal ability of all the cancer cell lines we have studied. Similarly, we observed that, in Pirc rats, one-week morin administration (50 mg/kg) down-regulated LMW-PTP and restored the apoptotic response to 5-FU in the NM. Finally, administration of morin for a longer period led to a significant reduction in colon precancerous lesions, together with a down-regulation of LMW-PTP. Taken together, these results document the involvement of LMW-PTP in the process of CRC in vitro and in vivo. Morin treatment may be envisaged as a system to increase the sensitivity to chemotherapy and to prevent carcinogenesis.
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Carcinogênese/patologia , Colo/patologia , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Flavonoides/farmacologia , Genes APC , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Animais , Antioxidantes/farmacologia , Carcinogênese/induzido quimicamente , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Colo/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/etiologia , Técnicas In Vitro , Masculino , Proteínas Tirosina Fosfatases/genética , Proteínas Proto-Oncogênicas/genética , Ratos , Ratos Endogâmicos F344RESUMO
BACKGROUND: Low molecular weight protein tyrosine phosphatase (LMW-PTP) is overexpressed in different cancer types and its expression is related to more aggressive disease, reduced survival rate and drug resistance. Morin is a natural polyphenol which negatively modulates, among others, the activity of LMW-PTP, leading to the potentiation of the effects of different antitumoral drugs, representing a potential beneficial treatment against cancer. METHODS: LMW-PTP levels were measured by immunoblot analysis both in CLL cells from patients and in chronic lymphocytic leukemia (CLL)-derived Mec-1 cells. Cell viability was assessed in Mec-1 cells treated with morin alone or in combination with either fludarabine or ibrutinib or following siRNA-mediated LMW-PTP knockdown. Furthermore, the expression levels of VLA-4 and CXCR4 were assessed by both qRT-PCR and flow cytometry and both adhesion to fibronectin-coated plates and migration toward CXCL12 were analyzed in Mec-1 cells treated with morin alone or in combination with fludarabine or ibrutinib. RESULTS: We observed that LMW-PTP is highly expressed in Mec-1 cells as well as in leukemic B lymphocytes purified from CLL patients compared to normal B lymphocytes. Morin treatment strongly decreased LMW-PTP expression levels in Mec-1 cells and potentiated the anticancer properties of both fludarabine and ibrutinib by increasing their apoptotic effects on leukemic cells. Moreover, morin negatively regulates adhesion and CXCL12-dependent migration of Mec-1 cells by affecting VLA-4 integrin expression and CXCR4 receptor recycling. CONCLUSIONS: Morin treatment in CLL-derived Mec-1 cell line synergizes with conventional anticancer drugs currently used in CLL therapy by affecting leukemic cell viability and trafficking.
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Protein tyrosine phosphatase 1B (PTP1B) acts as a negative regulator of insulin and leptin signalling and is crucially involved in the development of type 2 diabetes mellitus, obesity, cancer and neurodegenerative diseases. Pursuing our efforts to identify PTP1B inhibitors endowed with drug-like properties, we designed and evaluated 3-aryl-5-arylidene-2-thioxo-4-imidazolidinones (7) as a novel class of non-carboxylate PTP1B inhibitors. In agreement with our design, kinetic studies demonstrated that selected compounds 7 act as reversible, non-competitive inhibitors of the target enzyme at low micromolar concentrations. Accordingly, molecular docking experiments suggested that these inhibitors can fit an allosteric site of PTP1B that we previously individuated. Moreover, cellular assays demonstrated that compound 7e acts as a potent insulin-sensitizing agent in human liver HepG2 cells. Taken together, our results showed that these non-competitive PTP1B inhibitors can be considered promising lead compounds aimed to enhance druggability of the target enzyme and identify novel antidiabetic drugs.
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Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Hipoglicemiantes/farmacologia , Imidazolidinas/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Células Hep G2 , Humanos , Hipoglicemiantes/síntese química , Hipoglicemiantes/química , Imidazolidinas/síntese química , Imidazolidinas/química , Estrutura Molecular , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Designed multiple ligands (DMLs), developed to modulate simultaneously a number of selected targets involved in etiopathogenetic mechanisms of a multifactorial disease, such as diabetes mellitus (DM), are considered a promising alternative to combinations of drugs, when monotherapy results to be unsatisfactory. In this work, compounds 1-17 were synthesized and in vitro evaluated as DMLs directed to aldose reductase (AR) and protein tyrosine phosphatase 1B (PTP1B), two key enzymes involved in different events which are critical for the onset and progression of type 2 DM and related pathologies. Out of the tested 4-thiazolidinone derivatives, compounds 12 and 16, which exhibited potent AR inhibitory effects along with interesting inhibition of PTP1B, can be assumed as lead compounds to further optimize and balance the dual inhibitory profile. Moreover, several structural portions were identified as features that could be useful to achieve simultaneous inhibition of both human AR and PTP1B through binding to non-catalytic regions of both target enzymes.
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Aldeído Redutase/antagonistas & inibidores , Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Tiazolidinas/química , Tiazolidinas/farmacologia , Aldeído Redutase/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Ligantes , Simulação de Acoplamento Molecular , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Relação Estrutura-AtividadeRESUMO
Resistance to anticancer therapy still represents one of the main obstacles to cancer treatment. Numerous components of the tumor microenvironment (TME) contribute significantly to the acquisition of drug resistance. Microenvironmental pressures arising during cancer evolution foster tumor heterogeneity (TH) and facilitate the emergence of drug-resistant clones. In particular, metabolic pressures arising in the TME may favor epigenetic adaptations supporting the acquisition of persistence features in tumor cells. Tumor-persistent cells (TPCs) are characterized by high phenotypic and metabolic plasticity, representing a noticeable advantage in chemo- and radio-resistance. Understanding the crosslink between the evolution of metabolic pressures in the TME, epigenetics, and TPC evolution is significant for developing novel therapeutic strategies specifically targeting TPC vulnerabilities to overcome drug resistance.
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Neoplasias , Humanos , Neoplasias/patologia , Microambiente TumoralRESUMO
Primary liver cancers represent the third-most-common cause of cancer-related mortality worldwide, with an incidence of 80-90% for hepatocellular carcinoma (HCC) and 10-15% for cholangiocarcinoma (CCA), and an increasing morbidity and mortality rate. Although HCC and CCA originate from independent cell populations (hepatocytes and biliary epithelial cells, respectively), they develop in chronically inflamed livers. Evidence obtained in the last decade has revealed a role for cytokines of the IL-6 family in the development of primary liver cancers. These cytokines operate through the receptor subunit gp130 and the downstream Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways. Oncostatin M (OSM), a member of the IL-6 family, plays a significant role in inflammation, autoimmunity, and cancer, including liver tumors. Although, in recent years, therapeutic approaches for the treatment of HCC and CCA have been implemented, limited treatment options with marginal clinical benefits are available. We discuss how OSM-related pathways can be selectively inhibited and therapeutically exploited for the treatment of liver malignancies.
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Cholangiocarcinoma (CCA), a heterogeneous tumor with poor prognosis, can arise at any level in the biliary tree. It may derive from epithelial cells in the biliary tracts and peribiliary glands and possibly from progenitor cells or even hepatocytes. Several risk factors are responsible for CCA onset, however an inflammatory milieu nearby the biliary tree represents the most common condition favoring CCA development. Chemokines play a key role in driving the immunological response upon liver injury and may sustain tumor initiation and development. Chemokine receptor-dependent pathways influence the interplay among various cellular components, resulting in remodeling of the hepatic microenvironment towards a pro-inflammatory, pro-fibrogenic, pro-angiogenic and pre-neoplastic setting. Moreover, once tumor develops, chemokine signaling may influence its progression. Here we review the role of chemokines in the regulation of CCA development and progression, and the modulation of angiogenesis, metastasis and immune control. The potential role of chemokines and their receptors as possible biomarkers and/or therapeutic targets for hepatobiliary cancer is also discussed.
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Cholangiocarcinoma (CCA) is a deadly tumor without an effective therapy. Unique metabolic and bioenergetics features are important hallmarks of tumor cells. Metabolic plasticity allows cancer cells to survive in poor nutrient environments and maximize cell growth by sustaining survival, proliferation, and metastasis. In recent years, an increasing number of studies have shown that specific signaling networks contribute to malignant tumor onset by reprogramming metabolic traits. Several evidences demonstrate that numerous metabolic mediators represent key-players of CCA progression by regulating many signaling pathways. Besides the well-known Warburg effect, several other different pathways involving carbohydrates, proteins, lipids, and nucleic acids metabolism are altered in CCA. The goal of this review is to highlight the main metabolic processes involved in the cholangio-carcinogeneis that might be considered as potential novel druggable candidates for this disease.
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Neoplasias dos Ductos Biliares/genética , Plasticidade Celular/genética , Colangiocarcinoma/genética , Neoplasias dos Ductos Biliares/fisiopatologia , Proliferação de Células , Colangiocarcinoma/fisiopatologia , Humanos , Mitocôndrias/metabolismoRESUMO
Recent lines of evidence highlight the involvement of myeloid-epithelial-reproductive tyrosine kinase (MerTK) in metabolic disease associated with liver damage. MerTK is mainly expressed in anti-inflammatory M2 macrophages where it mediates transcriptional changes including suppression of proinflammatory cytokines and enhancement of inflammatory repressors. MerTK is regulated by metabolic pathways through nuclear sensors including LXRs, PPARs, and RXRs, in response to apoptotic bodies or to other sources of cholesterol. Nonalcoholic fatty liver disease (NAFLD) is one of the most serious public health problems worldwide. It is a clinicopathological syndrome closely related to obesity, insulin resistance, and oxidative stress. It includes a spectrum of conditions ranging from simple steatosis, characterized by hepatic fat accumulation with or without inflammation, to nonalcoholic steatohepatitis (NASH), defined by hepatic fat deposition with hepatocellular damage, inflammation, and accumulating fibrosis. Several studies support an association between NAFLD and the incidence of cardiovascular diseases including atherosclerosis, a major cause of death worldwide. This pathological condition consists in a chronic and progressive inflammatory process in the intimal layer of large- and medium-sized arteries. The complications of advanced atherosclerosis include chronic or acute ischemic damage in the tissue perfused by the affected artery, leading to cellular death. By identifying specific targets influencing lipid metabolism and cardiovascular-related diseases, the present review highlights the role of MerTK in NAFLD-associated atherosclerotic lesions as a potential innovative therapeutic target. Therapeutic advantages might derive from the use of compounds selective for nuclear receptors targeting PPARs rather than LXRs regulating macrophage lipid metabolism and macrophage mediated inflammation, by favoring the expression of MerTK, which mediates an immunoregulatory action with a reduction in inflammation and in atherosclerosis.
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Development of cholangiocarcinoma (CCA) is dependent on a cross-talk with stromal cells, which release different chemokines including CXCL12, that interacts with two different receptors, CXCR4 and CXCR7. The aim of the present study was to investigate the role of CXCR7 in CCA cells. CXCR7 is overexpressed by different CCA cell lines and in human CCA specimens. Knock-down of CXCR7 in HuCCT-1 cells reduced migration, invasion, and CXCL12-induced adhesion to collagen I. Survival of CCA was also reduced in CXCR7-silenced cells. The ability of CXCL12 to induce cell migration and survival was also blocked by CCX733, a CXCR7 antagonist. Similar effects of CXCR7 activation were observed in CCLP-1 cells and in primary iCCA cells. Enrichment of tumor stem-like cells by a 3D culture system resulted in increased CXCR7 expression compared to cells grown in monolayers, and genetic knockdown of CXCR7 robustly reduced sphere formation both in HuCCT-1 and in CCLP-1 cells. In HuCCT-1 cells CXCR7 was found to interact with ß-arrestin 2, which was necessary to mediate CXCL12-induced migration, but not survival. In conclusion, CXCR7 is widely expressed in CCA, and contributes to the aggressive phenotype of CCA cells, inducing cell migration, invasion, adhesion, survival, growth and stem cell-like features. Cell migration induced by CXCR7 requires interaction with ß-arrestin 2.
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Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/patologia , Receptores CXCR/metabolismo , Neoplasias dos Ductos Biliares/metabolismo , Movimento Celular , Sobrevivência Celular , Quimiocina CXCL12/metabolismo , Colangiocarcinoma/metabolismo , Feminino , Humanos , Fígado/metabolismo , Fígado/patologia , Masculino , Fenótipo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores CXCR/antagonistas & inibidores , Receptores CXCR/genética , Células Tumorais Cultivadas , beta-Arrestina 2/metabolismoRESUMO
BACKGROUND: Low Molecular Weight Phosphotyrosine Protein Phosphatase (LMW-PTP) is an enzyme involved not only in tumor onset and progression but also in type 2 diabetes. A recent review shows that LMW-PTP acts on several RTK (receptor tyrosine kinase) such as PDGFR, EGFR, EphA2, Insulin receptor. It is well described also its interaction with cSrc. It is noteworthy that most of these conclusions are based on the use of cell lines expressing low levels of LMW-PTP. The aim of the present study was to discover new LMW-PTP substrates in aggressive human tumors where the over-expression of this phosphatase is a common feature. METHODS: We investigated, by proteomic analysis, the protein phosphorylation pattern of A375 human melanoma cells silenced for LMW-PTP. Two-dimensional electrophoresis (2-DE) analysis, followed by western blot was performed using anti-phosphotyrosine antibodies, in order to identify differentially phosphorylated proteins. RESULTS: Proteomic analysis pointed out that most of the identified proteins belong to the glycolytic metabolism, such as α-enolase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase, suggesting an involvement of LMW-PTP in glucose metabolism. Assessment of lactate production and oxygen consumption demonstrated that LMW-PTP silencing enhances glycolytic flux and slow down the oxidative metabolism. In particular, LMW-PTP expression affects PKM2 tyrosine-phosphorylation and nuclear localization, modulating its activity. CONCLUSION: All these findings propose that tumor cells are subjected to metabolic reprogramming after LMW-PTP silencing, enhancing glycolytic flux, probably to compensate the inhibition of mitochondrial metabolism. GENERAL SIGNIFICANCE: Our results highlight the involvement of LMW-PTP in regulating glucose metabolism in A375 melanoma cells.
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Proteínas de Fluorescência Verde/metabolismo , Concentração de Íons de Hidrogênio , Neoplasias/metabolismo , Fluorescência , Humanos , Peso Molecular , Neoplasias/patologiaRESUMO
Tumor resistance to apoptosis is one the main causes of anticancer treatment failure. Previous studies showed that LMW-PTP overexpression enhances resistance of cancer cells to traditional anticancer drugs. Today, the role of LMW-PTP in inducing resistance to apoptosis in melanoma cells remains to be elucidated. Experimental setting include MTT assay, Annexin V/Pi method, and colony assay to assess whether silencing of LMW-PTP improves the sensitivity of A375 to dacarbazine, 5-FU, and radiotherapy. Pharmacological targeting of LMW-PTP was obtained using Morin, a LMW-PTP inhibitor. The ability of Morin to improve the effectiveness of anticancer drugs and radiotherapy was also studied. Moreover, PC3 cells were used as an alternative cellular model to confirm the data obtained with melanoma cells. We found that LMW-PTP silencing improves the effectiveness of dacarbazine, 5-FU, and radiotherapy. Identical results were obtained in vivo when Morin was used to target LMW-PTP. We demonstrated that Morin synergizes with dacarbazine, improving its cytotoxic activity. However, we showed that the combined treatment, Morin-anticancer drug, does not affect the viability of noncancerous cells. Knockdown of LMW-PTP sensitizes also PC3 cells to docetaxel and radiotherapy. In conclusion, we showed that LMW-PTP targeting improves effectiveness of anticancer drugs used for treatment of melanoma. Moreover, our results suggest that Morin could be used as adjuvant to improve the outcome of patients affected by metastatic melanoma.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Flavonoides/farmacologia , Melanoma/terapia , Proteínas Tirosina Fosfatases/genética , Proteínas Proto-Oncogênicas/genética , Tolerância a Radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dacarbazina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Tratamento Farmacológico , Fluoruracila , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Melanoma/genética , Terapia de Alvo Molecular , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Tolerância a Radiação/efeitos dos fármacos , Radioterapia , Regulação para Cima/efeitos dos fármacosRESUMO
This work presents the synthesis of two hybrid compounds (1 and 2) with thiazolidine-2,4-dione structure as a central scaffold which were further screened in combo (in vitro as PTP-1B inhibitors, in vivo antihyperglycemic activity, in silico toxicological profile and molecular docking). Compound 1 was tested in the enzymatic assay showing an IC50 = 9.6 ± 0.5 µM and compound 2 showed about a 50% of inhibition of PTP-1B at 20 µM. Therefore, compound 1 was chosen to test its antihyperglycemic effect in a rat model for non-insulin-dependent diabetes mellitus (NIDDM), which was determined at 50 mg/kg in a single dose. The results indicated that compound showed a significant decrease of plasma glucose levels that reached 34%, after a 7 h post-administration. Molecular docking was employed to study the inhibitory properties of thiazolidine-2,4-dione derivatives against Protein Tyrosine Phosphatase 1B (PDB ID: 1c83). Concerning to the two binding sites in this enzyme (sites A and B), compound 1 has shown the best docking score, which indicates the highest affinity. Finally, compounds 1 and 2 have demonstrated an in silico satisfactory pharmacokinetic profile. This shows that it could be a very good candidate or leader for new series of compounds with this central scaffold.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/síntese química , Hipoglicemiantes/uso terapêutico , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Tiazolidinedionas/síntese química , Tiazolidinedionas/uso terapêutico , Animais , Simulação por Computador , Relação Dose-Resposta a Droga , Escherichia coli/enzimologia , Hipoglicemiantes/química , Masculino , Simulação de Acoplamento Molecular , Ratos Wistar , Estreptozocina , Tiazolidinedionas/químicaRESUMO
New 4-{[5-arylidene-2-(4-fluorophenylimino)-4-oxothiazolidin-3-yl]methyl}benzoic acids (5) and 2-thioxo-4-thiazolidinone analogues (6) were synthesised as a part of a continuing search for new inhibitors of protein tyrosine phosphatase 1B (PTP1B), an enzyme which is implicated in metabolic disorders and inflammatory signaling. Most of the tested compounds were shown to be potent PTP1B inhibitors. Moreover, their inhibition mechanism was markedly influenced by the substituents in the positions 2 and 5, as kinetic studies indicated. Docking experiments suggested that certain derivatives 5 and 6 may efficiently fit into an allosteric site positioned between the ß-sheet including Leu71 and Lys73 and a lipophilic pocket closed by the loop consisting of Pro210 to Leu 204. In cellular assays, several of these new 4-thiazolidinone derivatives showed insulinomimetic and anti-inflammatory properties. Out of them, compound 5b exhibited the most promising profile, being able to promote the activation of both insulin receptor and downstream Akt protein as well as to increase 2-deoxyglucose cellular uptake. Interestingly, compound 5b was also able to interrupt critical events in inflammatory signaling.