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  1. Genetic Nrf2 Overactivation Inhibits the Deleterious Effects Induced by Hepatocyte-Specific c-met Deletion during the Progression of NASH

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    Pierluigi Ramadori

    2017-01-01

    Full Text Available We have recently shown that hepatocyte-specific c-met deficiency accelerates the progression of nonalcoholic steatohepatitis in experimental murine models resulting in augmented production of reactive oxygen species and accelerated development of fibrosis. The aim of this study focuses on the elucidation of the underlying cellular mechanisms driven by Nrf2 overactivation in hepatocytes lacking c-met receptor characterized by a severe unbalance between pro-oxidant and antioxidant functions. Control mice (c-metfx/fx, single c-met knockouts (c-metΔhepa, and double c-met/Keap1 knockouts (met/Keap1Δhepa were then fed a chow or a methionine-choline-deficient (MCD diet, respectively, for 4 weeks to reproduce the features of nonalcoholic steatohepatitis. Upon MCD feeding, met/Keap1Δhepa mice displayed increased liver mass albeit decreased triglyceride accumulation. The marked increase of oxidative stress observed in c-metΔhepa was restored in the double mutants as assessed by 4-HNE immunostaining and by the expression of genes responsible for the generation of free radicals. Moreover, double knockout mice presented a reduced amount of liver-infiltrating cells and the exacerbation of fibrosis progression observed in c-metΔhepa livers was significantly inhibited in met/Keap1Δhepa. Therefore, genetic activation of the antioxidant transcription factor Nrf2 improves liver damage and repair in hepatocyte-specific c-met-deficient mice mainly through restoring a balance in the cellular redox homeostasis.

  2. Aptamers Binding to c-Met Inhibiting Tumor Cell Migration.

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    Birgit Piater

    Full Text Available The human receptor tyrosine kinase c-Met plays an important role in the control of critical cellular processes. Since c-Met is frequently over expressed or deregulated in human malignancies, blocking its activation is of special interest for therapy. In normal conditions, the c-Met receptor is activated by its bivalent ligand hepatocyte growth factor (HGF. Also bivalent antibodies can activate the receptor by cross linking, limiting therapeutic applications. We report the generation of the RNA aptamer CLN64 containing 2'-fluoro pyrimidine modifications by systematic evolution of ligands by exponential enrichment (SELEX. CLN64 and a previously described single-stranded DNA (ssDNA aptamer CLN3 exhibited high specificities and affinities to recombinant and cellular expressed c-Met. Both aptamers effectively inhibited HGF-dependent c-Met activation, signaling and cell migration. We showed that these aptamers did not induce c-Met activation, revealing an advantage over bivalent therapeutic molecules. Both aptamers were shown to bind overlapping epitopes but only CLN3 competed with HGF binding to cMet. In addition to their therapeutic and diagnostic potential, CLN3 and CLN64 aptamers exhibit valuable tools to further understand the structural and functional basis for c-Met activation or inhibition by synthetic ligands and their interplay with HGF binding.

  3. Hepatocyte Growth Factor/c-Met Signaling in Head and Neck Cancer and Implications for Treatment

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    Natalie J. Rothenberger

    2017-04-01

    Full Text Available Aberrant signaling of the hepatocyte growth factor (HGF/c-Met pathway has been identified as a promoter of tumorigenesis in several tumor types including head and neck squamous cell carcinoma (HNSCC. Despite a relatively low c-Met mutation frequency, overexpression of HGF and its receptor c-Met has been observed in more than 80% of HNSCC tumors, with preclinical and clinical studies linking overexpression with cellular proliferation, invasion, migration, and poor prognosis. c-Met is activated by HGF through a paracrine mechanism to promote cellular morphogenesis enabling cells to acquire mesenchymal phenotypes in part through the epithelial-mesenchymal transition, contributing to metastasis. The HGF/c-Met pathway may also act as a resistance mechanism against epidermal growth factor receptor (EGFR inhibition in advanced HNSCC. Furthermore, with the identification of a biologically distinct subset of HNSCC tumors acquired from human papillomavirus (HPV infection that generally portends a good prognosis, high expression of HGF or c-Met in HPV-negative tumors has been associated with worse prognosis. Dysregulated HGF/c-Met signaling results in an aggressive HNSCC phenotype which has led to clinical investigations for targeted inhibition of this pathway. In this review, HGF/c-Met signaling, pathway alterations, associations with clinical outcomes, and preclinical and clinical therapeutic strategies for targeting HGF/c-Met signaling in HNSCC are discussed.

  4. The EGFR/ErbB3 Pathway Acts as a Compensatory Survival Mechanism upon c-Met Inhibition in Human c-Met+ Hepatocellular Carcinoma.

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    Steven N Steinway

    Full Text Available c-Met, a high-affinity receptor for Hepatocyte Growth Factor (HGF, plays a critical role in tumor growth, invasion, and metastasis. Hepatocellular carcinoma (HCC patients with activated HGF/c-Met signaling have a significantly worse prognosis. Targeted therapies using c-Met tyrosine kinase inhibitors are currently in clinical trials for HCC, although receptor tyrosine kinase inhibition in other cancers has demonstrated early success. Unfortunately, therapeutic effect is frequently not durable due to acquired resistance.We utilized the human MHCC97-H c-Met positive (c-Met+ HCC cell line to explore the compensatory survival mechanisms that are acquired after c-Met inhibition. MHCC97-H cells with stable c-Met knockdown (MHCC97-H c-Met KD cells were generated using a c-Met shRNA vector with puromycin selection and stably transfected scrambled shRNA as a control. Gene expression profiling was conducted, and protein expression was analyzed to characterize MHCC97-H cells after blockade of the c-Met oncogene. A high-throughput siRNA screen was performed to find putative compensatory survival proteins, which could drive HCC growth in the absence of c-Met. Findings from this screen were validated through subsequent analyses.We have previously demonstrated that treatment of MHCC97-H cells with a c-Met inhibitor, PHA665752, results in stasis of tumor growth in vivo. MHCC97-H c-Met KD cells demonstrate slower growth kinetics, similar to c-Met inhibitor treated tumors. Using gene expression profiling and siRNA screening against 873 kinases and phosphatases, we identified ErbB3 and TGF-α as compensatory survival factors that are upregulated after c-Met inhibition. Suppressing these factors in c-Met KD MHCC97-H cells suppresses tumor growth in vitro. In addition, we found that the PI3K/Akt signaling pathway serves as a negative feedback signal responsible for the ErbB3 upregulation after c-Met inhibition. Furthermore, in vitro studies demonstrate that

  5. Cytotoxic activity of tivantinib (ARQ 197) is not due solely to c-MET inhibition.

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    Katayama, Ryohei; Aoyama, Aki; Yamori, Takao; Qi, Jie; Oh-hara, Tomoko; Song, Youngchul; Engelman, Jeffrey A; Fujita, Naoya

    2013-05-15

    The receptor tyrosine kinase c-MET is the high-affinity receptor for the hepatocyte growth factor (HGF). The HGF/c-MET axis is often dysregulated in tumors. c-MET activation can be caused by MET gene amplification, activating mutations, and auto- or paracrine mechanisms. Thus, c-MET inhibitors are under development as anticancer drugs. Tivantinib (ARQ 197) was reported as a small-molecule c-MET inhibitor and early clinical studies suggest antitumor activity. To assess whether the antitumor activity of tivantinib was due to inhibition of c-MET, we compared the activity of tivantinib with other c-MET inhibitors in both c-MET-addicted and nonaddicted cancer cells. As expected, other c-MET inhibitors, crizotinib and PHA-665752, suppressed the growth of c-MET-addicted cancers, but not the growth of cancers that are not addicted to c-MET. In contrast, tivantinib inhibited cell viability with similar potency in both c-MET-addicted and nonaddicted cells. These results suggest that tivantinib exhibits its antitumor activity in a manner independent of c-MET status. Tivantinib treatment induced a G(2)-M cell-cycle arrest in EBC1 cells similarly to vincristine treatment, whereas PHA-665752 or crizotinib treatment markedly induced G(0)-G(1) cell-cycle arrest. To identify the additional molecular target of tivantinib, we conducted COMPARE analysis, an in silico screening of a database of drug sensitivities across 39 cancer cell lines (JFCR39), and identified microtubule as a target of tivantinib. Tivantinib-treated cells showed typical microtubule disruption similar to vincristine and inhibited microtubule assembly in vitro. These results suggest that tivantinib inhibits microtubule polymerization in addition to inhibiting c-MET. ©2013 AACR.

  6. Inhibition of c-Met as a Therapeutic Strategy for Esophageal Adenocarcinoma

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    Gregory A. Watson

    2006-11-01

    Full Text Available The hepatocyte growth factor (HGF receptor c-Met is a tyrosine kinase receptor with established oncogenic properties. We have previously shown that c-Met is usually overexpressed in esophageal adenocarcinoma (EA, yet the implications of c-Met inhibition in EA remain unknown. Three c-Met-overexpressiog EA cell lines (Seg-1, Bic-1, Flo-1 were used to examine the effects of a c-Met-specific small molecule inhibitor (PHA665752 on cell viability, apoptosis, motility, invasion, downstream signaling pathways. PHA665752 demonstrated dose-dependent inhibition of constitutive and/or HGF-induced phosphorylation of c-Met, which correlated with reduced cell viability and inhibition of extracellular regulated kinase 1/2 phosphorylation in all three EA cell lines. In contrast, PHA665752 induced apoptosis and reduced motility and invasion in only one EA cell line, Flo-1. Interestingly, Flo-1 was the only cell line in which phosphatidylinositol 3-kinase (PI3K/Akt was induced following HGF stimulation. The PI3K inhibitor LY294002 produced effects equivalent to those of PHA665752 in these cells. We conclude that inhibition of c-Met may be a useful therapeutic strategy for EA. Factors other than receptor overexpression, such as c-Met-dependent PI3K/Akt signaling, may be predictive of an individual tumor's response to c-Met inhibition.

  7. Clinical Development of c-MET Inhibition in Hepatocellular Carcinoma

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    Joycelyn J. X. Lee

    2015-10-01

    Full Text Available Hepatocellular carcinoma (HCC is one of the leading causes of cancer death. In patients with advanced or unresectable HCC, there are few treatment options. Conventional chemotherapy has limited benefits. Sorafenib, a multi-kinase inhibitor, improves survival, but options for patients intolerant of or progressing on sorafenib are limited. There has been much interest in recent years in molecular therapeutic targets and drug development for HCC. One of the more promising molecular targets in HCC is the cellular-mesenchymal-epithelial transition (c-MET factor receptor. Encouraging phase II data on two c-MET inhibitors, tivantinib and cabozantinib, has led to phase III trials. This review describes the c-MET/hepatocyte growth factor (HGF signalling pathway and its relevance to HCC, and discusses the preclinical and clinical trial data for inhibitors of this pathway in HCC.

  8. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product

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    Bottaro, D.P.; Rubin, J.S.; Chan, A.M.L.; Aaronson, S.A. (National Cancer Institute, Bethesda, MD (United States)); Faletto, D.L.; Kmiecik, T.E.; Vande Woude, G.F. (NCI-Frederick Cancer Research and Development Center, MD (United States))

    1991-02-15

    Hepatocyte growth factor (HGF) is a plasminogen-like protein thought to be a humoral mediator of liver regeneration. A 145-kilodalton tyrosyl phosphoprotein observed in rapid response to HGF treatment of intact target cells was identified by immunoblot analysis as the {beta} subunit of the c-met proto-oncogene product, a membrane-spanning tyrosine kinase. Covalent cross-linking of {sup 125}I-labeled ligand to cellular proteins of appropriate size that were recognized by antibodies to c-met directly established the c-met product as the cell-surface receptor for HGF.

  9. The c-Met Inhibitor MSC2156119J Effectively Inhibits Tumor Growth in Liver Cancer Models

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    Bladt, Friedhelm, E-mail: Friedhelm.Bladt@merckgroup.com; Friese-Hamim, Manja; Ihling, Christian; Wilm, Claudia; Blaukat, Andree [EMD Serono, and Merck Serono Research and Development, Merck KGaA, Darmstadt 64293 (Germany)

    2014-08-19

    The mesenchymal-epithelial transition factor (c-Met) is a receptor tyrosine kinase with hepatocyte growth factor (HGF) as its only high-affinity ligand. Aberrant activation of c-Met is associated with many human malignancies, including hepatocellular carcinoma (HCC). We investigated the in vivo antitumor and antimetastatic efficacy of the c-Met inhibitor MSC2156119J (EMD 1214063) in patient-derived tumor explants. BALB/c nude mice were inoculated with MHCC97H cells or with tumor fragments of 10 patient-derived primary liver cancer explants selected according to c-Met/HGF expression levels. MSC2156119J (10, 30, and 100 mg/kg) and sorafenib (50 mg/kg) were administered orally as single-agent treatment or in combination, with vehicle as control. Tumor response, metastases formation, and alpha fetoprotein (AFP) levels were measured. MSC2156119J inhibited tumor growth and induced complete regression in mice bearing subcutaneous and orthotopic MHCC97H tumors. AFP levels were undetectable after 5 weeks of MSC2156119J treatment, and the number of metastatic lung foci was reduced. Primary liver explant models with strong c-Met/HGF activation showed increased responsiveness to MSC2156119J, with MSC2156119J showing similar or superior activity to sorafenib. Tumors characterized by low c-Met expression were less sensitive to MSC2156119J. MSC2156119J was better tolerated than sorafenib, and combination therapy did not improve efficacy. These findings indicate that selective c-Met/HGF inhibition with MSC2156119J is associated with marked regression of c-Met high-expressing tumors, supporting its clinical development as an antitumor treatment for HCC patients with active c-Met signaling.

  10. Hepatocyte growth factor and c-MET are expressed in rat prepuberal testis.

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    Catizone, A; Ricci, G; Arista, V; Innocenzi, A; Galdieri, M

    1999-07-01

    The hepatocyte growth factor (HGF) receptor (c-MET) is present in different mammalian tissues and transduces multiple biological effects. The HGF is known to regulate many fundamental cellular functions, such as cell growth, movement and differentiation, and is involved in embryonal morphogenesis. We have studied HGF and c-MET expression in prepuberal rat testis. c-MET gene expression was found in total testis and in homogeneous cell populations, as demonstrated by Northern blotting. In the seminiferous tubules, c-MET gene was only expressed in the myoid cells. In these cells, c-MET was detectable and constantly expressed for at least six days of culture. The interstitial tissue was also c-MET positive. The protein encoded by the MET proto-oncogene was detected in myoid cells, and HGF administration to these cells induced morphological changes in the cells. HGF expression was not detected by Northern blotting using RNA extracted from total testis. By contrast, when homogenous cell populations were used, HGF expression was detectable and exclusively localized in myoid cells. Myoid cell-conditioned medium was able to induce scattering of canine kidney epithelial (MDCK) cells, and the scatter effect of a 3-days conditioned medium was evident even after 7-fold dilution of the medium. Our findings demonstrate that HGF and its receptor are present in rat prepuberal testis. The coexpression of factor and receptor in the myoid cells suggests a new role for HGF as autocrine regulator of myoid cell function and, possibly, as regulator of mammalian testicular function.

  11. Expression of hepatocyte growth factor and the proto-oncogenic receptor c-Met in canine osteosarcoma

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    Fieten, H; Spee, B; Ijzer, J; Kik, M J; Penning, L C; Kirpensteijn, J

    Hepatocyte growth factor (HGF) and the proto-oncogenic receptor c-Met are implicated in growth, invasion, and metastasis in human cancer. Little information is available on the expression and role of both gene products in canine osteosarcoma. We hypothesized that the expression of c-Met is

  12. Expression and functional role of hepatocyte growth factor receptor (C-MET) during postnatal rat testis development.

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    Catizone, A; Ricci, G; Galdieri, M

    2001-05-01

    The met protooncogene encodes the hepatocyte growth factor receptor (HGFR, c-met). C-met, a tyrosine kinase receptor protein, is widely expressed in different cell types including the male reproductive tract. As we recently demonstrated, both c-met messenger RNA and protein are expressed in prebuberal rat testis. The aim of this work was to detect the expression of c-met during postnatal testis development and to study its functional role. Our findings show that in total rat testis c-met is expressed during postnatal life until the sexual maturation of the animals. To evaluate the receptor expression in the different cell types in the testis, homogeneous cell populations of Sertoli and peritubular myoid cells were isolated from the seminiferous tubules of 10- and 35-day-old animals. c-met gene is expressed in myoid cells at the ages considered and its expression decreases with increasing age. By contrast, in Sertoli cells c-met expression is first detectable at 25 days of life and its expression increases with the increasing age being well evident at 35 days of age. C-met protein was detected by immunocytochemistry and its expression correlates with gene expression. The receptor is functionally active because HGF administration induces morphological changes in myoid cells and in c-met-expressing Sertoli cells. As a consequence of HGF addition, Sertoli cells cultured on reconstituted basement membrane reorganize into cord-like structures that resemble testicular seminiferous cords. The data here reported demonstrate for the first time that in Sertoli cells c-met expression is developmentally regulated being present and functionally active in postpuberal Sertoli cells. Given that c-met expression persists in myoid cells during postnatal testis development and that in Sertoli cells its expression correlates over time with germ cell differentiation and lumen formation, we conclude that the c-met/HGF system is involved in testis development and function.

  13. Hepatocyte Growth Factor/C-Met Axis in Thyroid Cancer: From Diagnostic Biomarker to Therapeutic Target

    National Research Council Canada - National Science Library

    Maria. Trovato; Alfredo. CampennÃ; Salvatore. Giovinazzo; Massimiliano. Siracusa; Rosaria Maddalena. Ruggeri

    2017-01-01

    ... therapies with HGF/c-met inhibitors or antagonists in thyroid tumour, as well as in other malignancies. This may be relevant for iodine-refractory cancers, the treatment of which is still a major challenge. With this in mind, HGF/c-met expression in thyroid cancer tissue may be useful for prognostic and therapeutic stratification of patients.

  14. Non-agonistic bivalent antibodies that promote c-MET degradation and inhibit tumor growth and others specific for tumor related c-MET.

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    Sameer A Greenall

    Full Text Available The c-MET receptor has a function in many human cancers and is a proven therapeutic target. Generating antagonistic or therapeutic monoclonal antibodies (mAbs targeting c-MET has been difficult because bivalent, intact anti-Met antibodies frequently display agonistic activity, necessitating the use of monovalent antibody fragments for therapy. By using a novel strategy that included immunizing with cells expressing c-MET, we obtained a range of mAbs. These c-MET mAbs were tested for binding specificity and anti-tumor activity using a range of cell-based techniques and in silico modeling. The LMH 80 antibody bound an epitope, contained in the small cysteine-rich domain of c-MET (amino acids 519-561, that was preferentially exposed on the c-MET precursor. Since the c-MET precursor is only expressed on the surface of cancer cells and not normal cells, this antibody is potentially tumor specific. An interesting subset of our antibodies displayed profound activities on c-MET internalization and degradation. LMH 87, an antibody binding the loop connecting strands 3d and 4a of the 7-bladed β-propeller domain of c-MET, displayed no intrinsic agonistic activity but promoted receptor internalization and degradation. LMH 87 inhibited HGF/SF-induced migration of SK-OV-3 ovarian carcinoma cells, the proliferation of A549 lung cancer cells and the growth of human U87MG glioma cells in a mouse xenograft model. These results indicate that c-MET antibodies targeting epitopes controlling receptor internalization and degradation provide new ways of controlling c-MET expression and activity and may enable the therapeutic targeting of c-MET by intact, bivalent antibodies.

  15. Structural Basis for Selective Small Molecule Kinase Inhibition of Activated c-Met

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    Rickert, Keith W.; Patel, Sangita B.; Allison, Timothy J.; Byrne, Noel J.; Darke, Paul L.; Ford, Rachael E.; Guerin, David J.; Hall, Dawn L.; Kornienko, Maria; Lu, Jun; Munshi, Sanjeev K.; Reid, John C.; Shipman, Jennifer M.; Stanton, Elizabeth F.; Wilson, Kevin J.; Young, Jonathon R.; Soisson, Stephen M.; Lumb, Kevin J. (Merck)

    2012-03-15

    The receptor tyrosine kinase c-Met is implicated in oncogenesis and is the target for several small molecule and biologic agents in clinical trials for the treatment of cancer. Binding of the hepatocyte growth factor to the cell surface receptor of c-Met induces activation via autophosphorylation of the kinase domain. Here we describe the structural basis of c-Met activation upon autophosphorylation and the selective small molecule inhibiton of autophosphorylated c-Met. MK-2461 is a potent c-Met inhibitor that is selective for the phosphorylated state of the enzyme. Compound 1 is an MK-2461 analog with a 20-fold enthalpy-driven preference for the autophosphorylated over unphosphorylated c-Met kinase domain. The crystal structure of the unbound kinase domain phosphorylated at Tyr-1234 and Tyr-1235 shows that activation loop phosphorylation leads to the ejection and disorder of the activation loop and rearrangement of helix {alpha}C and the G loop to generate a viable active site. Helix {alpha}C adopts a orientation different from that seen in activation loop mutants. The crystal structure of the complex formed by the autophosphorylated c-Met kinase domain and compound 1 reveals a significant induced fit conformational change of the G loop and ordering of the activation loop, explaining the selectivity of compound 1 for the autophosphorylated state. The results highlight the role of structural plasticity within the kinase domain in imparting the specificity of ligand binding and provide the framework for structure-guided design of activated c-Met inhibitors.

  16. Expression and functional role of hepatocyte growth factor and its receptor (c-met) during fetal mouse testis development.

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    Ricci, G; Catizone, A; Galdieri, M

    2006-12-01

    The hepatocyte growth factor (HGF) is a pleiotropic cytokine able to regulate different cellular functions. HGF action is mediated by its receptor, c-met, a glycoprotein with tyrosine kinase activity. We previously demonstrated that c-met is expressed in the newly formed seminiferous cords of the mice embryonic testes and that HGF acts as a morphogenetic factor. In this paper, we report that at 15.5 days post-coitum (dpc) c-met is expressed in the testicular cords, whereas at 18.5 dpc c-met expression is almost exclusively localized in the interstitial tissue of the testis in particular in the fetal Leydig cells. In addition, we demonstrate that HGF gene is expressed during the fetal period of testis development, heavily detectable in the interstitial compartment of 18.5 dpc testes. Interestingly, HGF is not expressed in the Leydig cells that, as above reported, express the HGF receptor. Looking for the functional role of HGF on Leydig cells, we evaluated the amount of testosterone secreted by testes isolated from 18.5 dpc embryos and cultured in the presence of HGF. The results of the in vitro organ culture show that, at this age, HGF increases the amount of testosterone secreted in the culture medium. On the contrary, HGF does not modulate the amount of testosterone secreted by testes isolated from 15.5 dpc embryos. In conclusion, we report that HGF is produced in the interstitial compartment of the developing testis but not by the Leydig cells. Conversely, the HGF receptor c-met is expressed in the Leydig cells and HGF modulates Leydig cell function during the late period of prenatal development.

  17. Overexpression of MACC1 and the association with hepatocyte growth factor/c-Met in epithelial ovarian cancer.

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    Li, Hongyu; Zhang, Hui; Zhao, Shujun; Shi, Yun; Yao, Junge; Zhang, Yanyan; Guo, Huanhuan; Liu, Xingsuo

    2015-05-01

    Metastasis-associated in colon cancer-1 (MACC1) is a gene that has been newly identified by a genome-wide search for differentially expressed genes in human colon cancer tissues, metastases and normal tissues. MACC1 exerts an important role in colon cancer metastasis through upregulation of the c-Met proto-oncogene. The tyrosine kinase receptor encoded by the c-Met oncogene exhibits the unusual property of mediating the invasive growth of epithelial cells upon binding with the hepatocyte growth factor (HGF). MACC1 has been investigated with regard to colon carcinoma and MACC1 expression is associated with metastasis in various types of human cancer. However, the value of MACC1 as a potential biomarker for ovarian cancer remains unknown, although the c-Met/HGF receptor has been shown to be overexpressed in epithelial ovarian cancer tissues. To investigate the role of MACC1 in epithelial ovarian tumors, the expression levels of MACC1 mRNA in ovarian tumor specimens were analyzed together with the prognostic significance. MACC1 protein expression was also detected in the epithelial ovarian tissue specimens, and the effects of MACC1 overexpression on ovarian cancer migration, invasion and prognosis were evaluated. Due to the close association between MACC1 and c-Met expression levels in colon cancer, the expression levels of HGF/c-Met in the ovarian specimens were also examined to determine whether such a correlation is also present in epithelial ovarian cancer. A total of 92 epithelial ovarian tissue samples were used to assess the expression levels of MACC1 mRNA and protein using reverse transcription-polymerase chain reaction and immunohistochemical methods, respectively. The serum levels of MACC1 protein expression in patients with epithelial ovarian cancer were detected by enzyme-linked immunosorbent assay. The results indicated that MACC1 may be important in the malignant progression of epithelial ovarian tumors, in particular for early stage patients. Thus, MACC

  18. Host-pathogen systems biology: logical modelling of hepatocyte growth factor and Helicobacter pylori induced c-Met signal transduction

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    Kähne Thilo

    2008-01-01

    Full Text Available Abstract Background The hepatocyte growth factor (HGF stimulates mitogenesis, motogenesis, and morphogenesis in a wide range of tissues, including epithelial cells, on binding to the receptor tyrosine kinase c-Met. Abnormal c-Met signalling contributes to tumour genesis, in particular to the development of invasive and metastatic phenotypes. The human microbial pathogen Helicobacter pylori can induce chronic gastritis, peptic ulceration and more rarely, gastric adenocarcinoma. The H. pylori effector protein cytotoxin associated gene A (CagA, which is translocated via a type IV secretion system (T4SS into epithelial cells, intracellularly modulates the c-Met receptor and promotes cellular processes leading to cell scattering, which could contribute to the invasiveness of tumour cells. Using a logical modelling framework, the presented work aims at analysing the c-Met signal transduction network and how it is interfered by H. pylori infection, which might be of importance for tumour development. Results A logical model of HGF and H. pylori induced c-Met signal transduction is presented in this work. The formalism of logical interaction hypergraphs (LIH was used to construct the network model. The molecular interactions included in the model were all assembled manually based on a careful meta-analysis of published experimental results. Our model reveals the differences and commonalities of the response of the network upon HGF and H. pylori induced c-Met signalling. As another important result, using the formalism of minimal intervention sets, phospholipase Cγ1 (PLCγ1 was identified as knockout target for repressing the activation of the extracellular signal regulated kinase 1/2 (ERK1/2, a signalling molecule directly linked to cell scattering in H. pylori infected cells. The model predicted only an effect on ERK1/2 for the H. pylori stimulus, but not for HGF treatment. This result could be confirmed experimentally in MDCK cells using a specific

  19. Targeting of Both the c-Met and EGFR Pathways Results in Additive Inhibition of Lung Tumorigenesis in Transgenic Mice

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    Stabile, Laura P. [Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Lung and Thoracic Malignancy Program, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Rothstein, Mary E. [Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Keohavong, Phouthone [Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Lung and Thoracic Malignancy Program, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Lenzner, Diana [Department of Biostatistics, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Land, Stephanie R. [Department of Biostatistics, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Lung and Thoracic Malignancy Program, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Gaither-Davis, Autumn L. [Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Lung and Thoracic Malignancy Program, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Kim, K. Jin [Galaxy Biotech, LLC, Sunnyvale, CA 94089 (United States); Kaminski, Naftali [Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Lung and Thoracic Malignancy Program, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Siegfried, Jill M., E-mail: siegfriedjm@upmc.edu [Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Lung and Thoracic Malignancy Program, University of Pittsburgh, Pittsburgh, PA 15213 (United States)

    2010-12-22

    EGFR and c-Met are both overexpressed in lung cancer and initiate similar downstream signaling, which may be redundant. To determine how frequently ligands that initiate signaling of both pathways are found in lung cancer, we analyzed serum for hepatocyte growth factor (HGF), transforming growth factor-alpha, and amphiregulin (AREG) in lung cancer cases and tobacco-exposed controls. HGF and AREG were both significantly elevated in cases compared to controls, suggesting that both HGF/c-Met and AREG/EGFR pathways are frequently active. When both HGF and AREG are present in vitro, downstream signaling to MAPK and Akt in non-small cell lung cancer (NSCLC) cells can only be completely inhibited by targeting both pathways. To test if dual blockade of the pathways could better suppress lung tumorigenesis in an animal model than single blockade, mice transgenic for airway expression of human HGF were treated with inhibitors of both pathways alone and in combination after exposure to a tobacco carcinogen. Mean tumor number in the group using both the HGF neutralizing antibody L2G7 and the EGFR inhibitor gefitinib was significantly lower than with single agents. A higher tumor K-ras mutation rate was observed with L2G7 alone compared to controls, suggesting that agents targeting HGF may be less effective against mutated K-ras lung tumors. This was not observed with combination treatment. A small molecule c-Met inhibitor decreased formation of both K-ras wild-type and mutant tumors and showed additive anti-tumor effects when combined with gefitinib. Dual targeting of c-Met/EGFR may have clinical benefit for lung cancer.

  20. Hepatocyte growth factor increases vascular endothelial growth factor-A production in human synovial fibroblasts through c-Met receptor pathway.

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    Yu-Min Lin

    Full Text Available BACKGROUND: Angiogenesis is essential for the progression of osteoarthritis (OA. Hepatocyte growth factor (HGF is an angiogenic mediator, and it shows elevated levels in regions of OA. However, the relationship between HGF and vascular endothelial growth factor (VEGF-A in OA synovial fibroblasts (OASFs is mostly unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we found that stimulation of OASFs with HGF induced concentration- and time-dependent increases in VEGF-A expression. Pretreatment with PI3K inhibitor (Ly294002, Akt inhibitor, or mTORC1 inhibitor (rapamycin blocked the HGF-induced VEGF-A production. Treatment of cells with HGF also increased PI3K, Akt, and mTORC1 phosphorylation. Furthermore, HGF increased the stability and activity of HIF-1 protein. Moreover, the use of pharmacological inhibitors or genetic inhibition revealed that c-Met, PI3K, Akt, and mTORC1 signaling pathways were potentially required for HGF-induced HIF-1α activation. CONCLUSIONS/SIGNIFICANCE: Taken together, our results provide evidence that HGF enhances VEGF-A expression in OASFs by an HIF-1α-dependent mechanism involving the activation of c-Met/PI3K/Akt and mTORC1 pathways.

  1. Curcumin inhibited HGF-induced EMT and angiogenesis through regulating c-Met dependent PI3K/Akt/mTOR signaling pathways in lung cancer

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    Demin Jiao

    2016-01-01

    Full Text Available The epithelial-mesenchymal transition (EMT and angiogenesis have emerged as two pivotal events in cancer progression. Curcumin has been extensively studied in preclinical models and clinical trials of cancer prevention due to its favorable toxicity profile. However, the possible involvement of curcumin in the EMT and angiogenesis in lung cancer remains unclear. This study found that curcumin inhibited hepatocyte growth factor (HGF-induced migration and EMT-related morphological changes in A549 and PC-9 cells. Moreover, pretreatment with curcumin blocked HGF-induced c-Met phosphorylation and downstream activation of Akt, mTOR, and S6. These effects mimicked that of c-Met inhibitor SU11274 or PI3 kinase inhibitor LY294002 or mTOR inhibitor rapamycin treatment. c-Met gene overexpression analysis further demonstrated that curcumin suppressed lung cancer cell EMT by inhibiting c-Met/Akt/mTOR signaling pathways. In human umbilical vein endothelial cells (HUVECs, we found that curcumin also significantly inhibited PI3K/Akt/mTOR signaling and induced apoptosis and reduced migration and tube formation of HGF-treated HUVEC. Finally, in the experimental mouse model, we showed that curcumin inhibited HGF-stimulated tumor growth and induced an increase in E-cadherin expression and a decrease in vimentin, CD34, and vascular endothelial growth factor (VEGF expression. Collectively, these findings indicated that curcumin could inhibit HGF-promoted EMT and angiogenesis by targeting c-Met and blocking PI3K/Akt/mTOR pathways.

  2. Reduction of stromal fibroblast-induced mammary tumor growth, by retroviral ribozyme transgenes to hepatocyte growth factor/scatter factor and its receptor, c-MET.

    Science.gov (United States)

    Jiang, Wen G; Grimshaw, David; Martin, Tracey A; Davies, Gaynor; Parr, Christian; Watkins, Gareth; Lane, Jane; Abounader, Roger; Laterra, John; Mansel, Robert E

    2003-09-15

    Hepatocyte growth factor/scatter factor (HGF/SF) is known to increase the invasiveness and migration of cancer cells in vitro and induce angiogenesis. This study examined if inhibition of HGF/SF receptor expression by cancer cells and HGF/SF expression by stromal fibroblasts affects the growth of mammary cancer. Transgenes encoding ribozymes to specifically target human HGF/SF receptor (pLXSN-MET) or HGF/SF (pLXSN-HGF) were constructed using a pLXSN retroviral vector. Human mammary cancer cells MDA MB 231 was transduced with pLXSN-MET (MDA(+/+)). A human fibroblast cell line MRC5, which produces bioactive HGF/SF, was transduced with pLXSN-HGF (MRC5(+/+)). These cells were used in a nude mice breast tumor model. HGF receptor in MDA(+/+) cells and HGF in MRC5(+/+)cells were successfully removed with respective ribozymes as shown by reverse transcription-PCR and Western blotting, respectively. MDA(+/+) was found to have reduced invasiveness when stimulated with HGF/SF. MRC5(+/+) exhibited a significant reduction in HGF/SF production. When injected into athymic nude mice, MDA(+/+) exhibited a slower rate of growth, compared with the wild type (MDA(-/-)), and the cells transduced with control viral vector (MDA(+/-)). The growth of MDA(-/-) tumor was significantly enhanced when coimplanted with wild-type MRC5 (MRC5(-/-)), and the stimulatory effect was reduced when MRC5(+/+) cells were coimplanted instead of MRC5(-/-). The reduction of tumor growth was accompanied by reduction of angiogenesis, as demonstrated by the staining of VE-cadherin in primary tumor tissues. Retroviral ribozyme transgenes targeting HGF/SF in fibroblasts or its receptor cMET in mammary cancer cells can reduce the growth of mammary cancer and associated angiogenesis by inhibiting paracrine stromal-tumor cell interactions.

  3. Tumor-stromal cell interaction under hypoxia increases the invasiveness of pancreatic cancer cells through the hepatocyte growth factor/c-Met pathway.

    Science.gov (United States)

    Ide, Takao; Kitajima, Yoshihiko; Miyoshi, Atsushi; Ohtsuka, Takao; Mitsuno, Mayumi; Ohtaka, Kazuma; Koga, Yasuo; Miyazaki, Kohji

    2006-12-15

    The hypoxic environment in tumor is reported to play an important role in pancreatic cancer progression. The interaction between stromal and cancer cells also contributes to the malignant behavior of pancreatic cancer. In the present study, we investigated whether hypoxic stimulation affects stromal as well as pancreatic cancer cells. Our findings demonstrated that hypoxia remarkably elevated the HIF-1alpha expression in both pancreatic cancer (PK8) and fibroblast cells (MRC5). Hypoxic stimulation accelerated the invasive activity of PK8 cells, and invasiveness was thus further accelerated when the hypoxic PK8 cells were cultured with conditioned medium prepared from hypoxic MRC5 cells (hypoxic conditioned medium). MMP-2, MMP-7, MT1-MMP and c-Met expressions were increased in PK8 cells under hypoxia. Hypoxic stimulation also increased the hepatocyte growth factor (HGF) secretion from MRC5 cells, which led to an elevation of c-Met phosphorylation in PK8 cells. Conversely, the elevated cancer invasion, MMP activity and c-Met phosphorylation of PK8 cells were reduced by the removal of HGF from hypoxic conditioned medium. In immunohistochemical study, the HIF-1alpha expression was observed in surrounding stromal as well as pancreatic cancer cells, thus indicating hypoxia exists in both of cancer and stromal cells. Moreover, the stromal HGF expression was found to significantly correlate with not only the stromal HIF-1alpha expression but also the c-Met expression in cancer cells. These results indicate that the hypoxic environment within stromal as well as cancer cells activates the HGF/c-Met system, thereby contributing to the aggressive invasive features of pancreatic cancer. Copyright 2006 Wiley-Liss, Inc.

  4. Reversal of c-MET-mediated Resistance to Cytotoxic Anticancer Drugs by a Novel c-MET Inhibitor TAS-115.

    Science.gov (United States)

    Kunii, Eiji; Ozasa, Hiroaki; Oguri, Tetsuya; Maeno, Ken; Fukuda, Satoshi; Uemura, Takehiro; Takakuwa, Osamu; Ohkubo, Hirotsugu; Takemura, Masaya; Niimi, Akio

    2015-10-01

    The cellular N-methyl-N'-nitroso-guanidine human osteosarcoma transforming gene (c-MET) protein is the receptor tyrosine kinase for hepatocyte growth factor. We recently found that c-MET protein expression and activation were enhanced in the majority of small cell lung cancer cell lines with cytotoxic anticancer drug resistance, and that down-regulation of c-MET reduced resistance to these drugs. Expression of c-MET was studied in three non-small cell lung cancer (NSCLC) cell lines, including six resistant cell strains to cytotoxic anticancer drugs. To assess the effect of c-MET activation on drug resistance, we studied drug sensitivity in the presence of a novel c-MET inhibitor TAS-115. c-MET expression and activation are also enhanced in some cytotoxic anticancer drug-resistant NSCLC cell lines, and inhibition of c-MET activation by TAS-115 reduced resistance of these cell lines to anticancer drugs. The mechanism of cellular resistance to anticancer drugs via hepatocyte growth factor/c-MET signal activation is not restricted to small cell lung cancer cell lines, and TAS-115 might be able to reverse the drug resistance of these cancer cells. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  5. Discovery of a Novel Mode of Protein Kinase Inhibition Characterized by the Mesenchymal-epithelial Transition Factor (c-Met) Protein Autophosphorylation by ARQ 197

    Energy Technology Data Exchange (ETDEWEB)

    S Eathiraj; R Palma; E Volckova; M Hirschi; D France; M Ashwell; T Chan

    2011-12-31

    A number of human malignancies exhibit sustained stimulation, mutation, or gene amplification of the receptor tyrosine kinase human mesenchymal-epithelial transition factor (c-Met). ARQ 197 is a clinically advanced, selective, orally bioavailable, and well tolerated c-Met inhibitor, currently in Phase 3 clinical testing in non-small cell lung cancer patients. Herein, we describe the molecular and structural basis by which ARQ 197 selectively targets c-Met. Through our analysis we reveal a previously undisclosed, novel inhibitory mechanism that utilizes distinct regulatory elements of the c-Met kinase. The structure of ARQ 197 in complex with the c-Met kinase domain shows that the inhibitor binds a conformation that is distinct from published kinase structures. ARQ 197 inhibits c-Met autophosphorylation and is highly selective for the inactive or unphosphorylated form of c-Met. Through our analysis of the interplay between the regulatory and catalytic residues of c-Met, and by comparison between the autoinhibited canonical conformation of c-Met bound by ARQ 197 to previously described kinase domains of type III receptor tyrosine kinases, we believe this to be the basis of a powerful new in silico approach for the design of similar inhibitors for other protein kinases of therapeutic interest.

  6. Detection of hepatocyte growth factor/scatter factor receptor (c-Met in axillary clearance after mastectomy for breast cancer using reverse transcriptase-polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    El-Refaey H.K.

    2007-06-01

    Full Text Available The diverse biological effects of hepatocyte growth factor/scatter factor (HGF/SF are mediated by c-Met which is preferentially expressed on epithelial cells. Met signaling has a role in normal cellular activities, and may be associated with development and progression of malignant processes. In this study presence of Met in the axillary drainage from patients who underwent conservative operations for breast cancer, and its prognostic significance was examined. Sixty-two consecutive patients with invasive ductal carcinoma of the breast which were suitable for breast-conserving treatment participated in the study. The output of the drain that had been placed in the axilla during the operation was collected, and the presence of Met and β-actin were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR assays. The data were compared with the pathological features of the tumor and the axillary lymph nodes, and with the estrogen and progesterone receptors status.RT-PCR of the axillary lymphatic drainage was positive for Met in 46 (74.2% of the patients and positive assays were correlated with increase in tumor size and grade of capillary and lymphatic invasion, as well as with lymph node metastasis (P < 0.02, for all comparisons. All 24 patients with axillary lymph node metastases in comparison with those without lymph node (57.9% metastases had positive assays for Met. While all ten patients with tumor involvement in the margins of the resection had positive assays for Met in their lymphatic fluid, only 36 out of 52 patients (69.2% were positive for met assay. Finally, Met showed negative correlations with positive estrogen and progesterone receptor assays (P<0.02.From the results of this study it may concluded that Met can be detected in the axillary fluids of patients with breast cancer and its expression in the axillary drainage may be a potential prognostic factor. This finding might be useful in therapeutic considerations since a

  7. Expression of the proto-oncogenes c-met and c-kit and their ligands, hepatocyte growth factor/scatter factor and stem cell factor, in SCLC cell lines and xenografts

    DEFF Research Database (Denmark)

    Rygaard, K; Nakamura, T; Spang-Thomsen, M

    1993-01-01

    We examined a panel of 25 small cell lung cancer (SCLC) cell lines and nude mouse xenografts for expression of the proto-oncogenes c-met and c-kit, and for expression of the corresponding ligands, hepatocyte growth factor (HGF) (also known as scatter factor (SF)), and stem cell factor (SCF......), respectively. Expression of mRNA was detected by Northern blotting, and c-met and c-kit protein expression was detected by Western blotting and immunocytochemistry. c-met and c-kit mRNA was expressed in 22 of the examined cell lines or xenografts, and coexpression of the two proto-oncogenes was observed in 20...... tumours. Expression of c-met and c-kit protein paralleled in the mRNA expression. HGF/SF mRNA was expressed in two of the examined tumours, and only one of these also expressed the c-met proto-oncogene. SCF mRNA was expressed in 19 of the examined tumours, and in 18 of these coexpression of c-kit and SCF...

  8. Discovery of a novel mode of protein kinase inhibition characterized by the mechanism of inhibition of human mesenchymal-epithelial transition factor (c-Met) protein autophosphorylation by ARQ 197.

    Science.gov (United States)

    Eathiraj, Sudharshan; Palma, Rocio; Volckova, Erika; Hirschi, Marscha; France, Dennis S; Ashwell, Mark A; Chan, Thomas C K

    2011-06-10

    A number of human malignancies exhibit sustained stimulation, mutation, or gene amplification of the receptor tyrosine kinase human mesenchymal-epithelial transition factor (c-Met). ARQ 197 is a clinically advanced, selective, orally bioavailable, and well tolerated c-Met inhibitor, currently in Phase 3 clinical testing in non-small cell lung cancer patients. Herein, we describe the molecular and structural basis by which ARQ 197 selectively targets c-Met. Through our analysis we reveal a previously undisclosed, novel inhibitory mechanism that utilizes distinct regulatory elements of the c-Met kinase. The structure of ARQ 197 in complex with the c-Met kinase domain shows that the inhibitor binds a conformation that is distinct from published kinase structures. ARQ 197 inhibits c-Met autophosphorylation and is highly selective for the inactive or unphosphorylated form of c-Met. Through our analysis of the interplay between the regulatory and catalytic residues of c-Met, and by comparison between the autoinhibited canonical conformation of c-Met bound by ARQ 197 to previously described kinase domains of type III receptor tyrosine kinases, we believe this to be the basis of a powerful new in silico approach for the design of similar inhibitors for other protein kinases of therapeutic interest.

  9. c-MET receptor tyrosine kinase as a molecular target in advanced hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Granito A

    2015-04-01

    Full Text Available Alessandro Granito,1 Elena Guidetti,1 Laura Gramantieri2,3 1Dipartimento di Scienze Mediche e Chirurgiche Università di Bologna, Bologna, Italy; 2Dipartimento dell'Apparato Digerente, Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; 3Centro di Ricerca Biomedica Applicata (CRBA, Azienda Ospedaliero-Universitaria Policlinico S Orsola-Malpighi e Università di Bologna, Bologna, Italy Abstract: c-MET is the membrane receptor for hepatocyte growth factor (HGF, also known as scatter factor or tumor cytotoxic factor, a mitogenic growth factor for hepatocytes. HGF is mainly produced by cells of mesenchymal origin and it mainly acts on neighboring epidermal and endothelial cells, regulating epithelial growth and morphogenesis. HGF/MET signaling has been identified among the drivers of tumorigenesis in human cancers. As such, c-MET is a recognized druggable target, and against it, targeted agents are currently under clinical investigation. c-MET overexpression is a common event in a wide range of human malignancies, including gastric, lung, breast, ovary, colon, kidney, thyroid, and liver carcinomas. Despite c-MET overexpression being reported by a large majority of studies, no evidence for a c-MET oncogenic addiction exists in hepatocellular carcinoma (HCC. In particular, c-MET amplification is a rare event, accounting for 4%–5% of cases while no mutation has been identified in c-MET oncogene in HCC. Thus, the selection of patient subgroups more likely to benefit from c-MET inhibition is challenging. Notwithstanding, c-MET overexpression was reported to be associated with increased metastatic potential and poor prognosis in patients with HCC, providing a rationale for its therapeutic inhibition. Here we summarize the role of activated HGF/MET signaling in HCC, its prognostic relevance, and the implications for therapeutic approaches in HCC. Keywords: hepatocellular carcinoma, c-MET, clinical trials

  10. Regulation of HGF Expression by ΔEGFR-Mediated c-Met Activation in Glioblastoma Cells

    Directory of Open Access Journals (Sweden)

    Jeannine Garnett

    2013-01-01

    Full Text Available The hepatocyte growth factor receptor (c-Met and a constitutively active mutant of the epidermal growth factor receptor (ΔEGFR/EGFRvIII are frequently overexpressed in glioblastoma (GBM and promote tumorigenesis. The mechanisms underlying elevated hepatocyte growth factor (HGF production in GBM are not understood. We found higher, coordinated mRNA expression levels of HGF and c-Met in mesenchymal (Mes GBMs, a subtype associated with poor treatment response and shorter overall survival. In an HGF/c-Met–dependent GBM cell line, HGF expression declined upon silencing of c-Met using RNAi or by inhibiting its activity with SU11274. Silencing c-Met decreased anchorage-independent colony formation and increased the survival of mice bearing intracranial GBM xenografts. Consistent with these findings, c-Met activation by ΔEGFR also elevated HGF expression, and the inhibition of ΔEGFR with AG1478 reduced HGF levels. Interestingly, c-Met expression was required for ΔEGFR-mediated HGF production, anchorage-independent growth, and in vivo tumorigenicity, suggesting that these pathways are coupled. Using an unbiased mass spectrometry–based screen, we show that signal transducer and activator of transcription 3 (STAT3 Y705 is a downstream target of c-Met signaling. Suppression of STAT3 phosphorylation with WP1193 reduced HGF expression in ΔEGFR-expressing GBM cells, whereas constitutively active STAT3 partially rescued HGF expression and colony formation in c-Met knockdown cells expressing ΔEGFR. These results suggest that the c-Met/HGF signaling axis is enhanced by ΔEGFR through increased STAT3-dependent HGF expression and that targeting c-Met in Mes GBMs may be an important strategy for therapy.

  11. (-)-Oleocanthal as a c-Met inhibitor for the control of metastatic breast and prostate cancers.

    Science.gov (United States)

    Elnagar, Ahmed Y; Sylvester, Paul W; El Sayed, Khalid A

    2011-07-01

    The proto-oncogene receptor tyrosine kinase c-Met encodes the high-affinity receptor for hepatocyte growth factor (HGF). Dysregulation of the HGF-c-Met pathway plays a significant oncogenic role in many tumors. Overexpression of c-Met is a prognostic indicator for some transitional cell carcinomas. Extra-virgin olive oil (EVOO) provides a variety of minor phenolic compounds with beneficial properties. (-)-Oleocanthal (1) is a naturally occurring minor secoiridoid isolated from EVOO, which showed potent anti-inflammatory activity via its ability to inhibit COX-1 and COX-2. It altered the structure of neurotoxic proteins believed to contribute to the debilitating effects of Alzheimer's disease. Computer-Assisted Molecular Design (CAMD) identified 1 as a potential virtual c-Met inhibitor hit. Oleocanthal inhibited the proliferation, migration, and invasion of the epithelial human breast and prostate cancer cell lines MCF7, MDA-MB-231, and PC-3, respectively, with an IC (50) range of 10-20 µM, and demonstrated anti-angiogenic activity via downregulating the expression of the microvessel density marker CD31 in endothelial colony forming cells with an IC (50) of 4.4 µM. It inhibited the phosphorylation of c-Met kinase IN VITRO in the Z'-LYTE™ assay, with an IC (50) value of 4.8 µM. (-)-Oleocanthal and EVOO can have potential therapeutic use for the control of c-Met-dependent malignancies. © Georg Thieme Verlag KG Stuttgart · New York.

  12. (89)Zr-Onartuzumab PET imaging of c-MET receptor dynamics

    NARCIS (Netherlands)

    Pool, Martin; van Scheltinga, Anton G. T. Terwisscha; Kol, Arjan; Giesen, Danique; de Vries, Elisabeth G. E.; Lub-de Hooge, Marjolijn N.

    PURPOSE: c-MET and its ligand hepatocyte growth factor are often dysregulated in human cancers. Dynamic changes in c-MET expression occur and might predict drug efficacy or emergence of resistance. Noninvasive visualization of c-MET dynamics could therefore potentially guide c-MET-directed

  13. Isolation and characterization of anti c-met single chain fragment variable (scFv) antibodies.

    Science.gov (United States)

    Qamsari, Elmira Safaie; Sharifzadeh, Zahra; Bagheri, Salman; Riazi-Rad, Farhad; Younesi, Vahid; Abolhassani, Mohsen; Ghaderi, Sepideh Safaei; Baradaran, Behzad; Somi, Mohammad Hossein; Yousefi, Mehdi

    2017-12-01

    The receptor tyrosine kinase (RTK) Met is the cell surface receptor for hepatocyte growth factor (HGF) involved in invasive growth programs during embryogenesis and tumorgenesis. There is compelling evidence suggesting important roles for c-Met in colorectal cancer proliferation, migration, invasion, angiogenesis, and survival. Hence, a molecular inhibitor of an extracellular domain of c-Met receptor that blocks c-Met-cell surface interactions could be of great thera-peutic importance. In an attempt to develop molecular inhibitors of c-Met, single chain variable fragment (scFv) phage display libraries Tomlinson I + J against a specific synthetic oligopeptide from the extracellular domain of c-Met receptor were screened; selected scFv were then characterized using various immune techniques. Three c-Met specific scFv (ES1, ES2, and ES3) were selected following five rounds of panning procedures. The scFv showed specific binding to c-Met receptor, and significantly inhibited proliferation responses of a human colorectal carcinoma cell line (HCT-116). Moreover, anti- apoptotic effects of selected scFv antibodies on the HCT-116 cell line were also evaluated using Annexin V/PI assays. The results demonstrated rates of apoptotic cell death of 46.0, 25.5, and 37.8% among these cells were induced by use of ES1, ES2, and ES3, respectively. The results demonstrated ability to successfully isolate/char-acterize specific c-Met scFv that could ultimately have a great therapeutic potential in immuno-therapies against (colorectal) cancers.

  14. Extracellular superoxide dismutase inhibits hepatocyte growth factor-mediated breast cancer-fibroblast interactions.

    Science.gov (United States)

    Golden, Briana Ormsbee; Griess, Brandon; Mir, Shakeel; Fitzgerald, Matthew; Kuperwasser, Charlotte; Domann, Frederick; Teoh-Fitzgerald, Melissa

    2017-12-08

    We have previously shown tumor suppressive effects of extracellular superoxide dismutase, EcSOD in breast cancer cells. In this study, an RTK signaling array revealed an inhibitory effect of EcSOD on c-Met phosphorylation and its downstream kinase c-Abl in MDA-MB231 cells. Moreover, an extracellular protein array showed that thrombospondin 1 (TSP-1), a scavenger of the c-Met ligand, hepatocyte growth factor (HGF) is significantly up-regulated in EcSOD overexpressing cells (Ec.20). We further determined the effects of EcSOD on HGF/c-Met-mediated cancer-fibroblast interactions by co-culturing normal fibroblasts (RMF) or RMF which overexpresses HGF (RMF-HGF) with MDA-MB231 cells. We observed that while RMF-HGF significantly promoted Matrigel growth of MDA-MB231, overexpression of EcSOD inhibited the HGF-stimulated growth. Similarly, a SOD mimetic, MnTE-2-PyP, inhibited HGF-induced growth and invasion of MDA-MB231. In addition, a long-term heterotypic co-culture study not only showed that Ec.20 cells are resistant to RMF-HGF-induced invasive stimulation but RMF-HGF that were co-cultured with Ec.20 cells showed an attenuated phenotype, suggesting an oxidative-mediated reciprocal interaction between the two cell types. In addition, we demonstrated that RMF-HGF showed an up-regulation of an ROS-generating enzyme, NADPH oxidase 4 (Nox4). Targeting this pro-oxidant significantly suppressed the activated phenotype of RMF-HGF in a collagen contraction assay, suggesting that RMF-HGF contributes to the oxidative tumor microenvironment. We have further shown that scavenging ROS with EcSOD significantly inhibited RMF-HGF-stimulated orthotopic tumor growth of MDA-MB231. This study suggests the loss of EcSOD in breast cancer plays a pivotal role in promoting the HGF/c-Met-mediated cancer-fibroblast interactions.

  15. Homocysteine inhibits hepatocyte proliferation via endoplasmic reticulum stress.

    Directory of Open Access Journals (Sweden)

    Xue Yu

    Full Text Available Homocysteine is an independent risk factor for coronary, cerebral, and peripheral vascular diseases. Recent studies have shown that levels of homocysteine are elevated in patients with impaired hepatic function, but the precise role of homocysteine in the development of hepatic dysfunction is unclear. In this study, we examined the effect of homocysteine on hepatocyte proliferation in vitro. Our results demonstrated that homocysteine inhibited hepatocyte proliferation by up-regulating protein levels of p53 as well as mRNA and protein levels of p21(Cip1 in primary cultured hepatocytes. Homocysteine induced cell growth arrest in p53-positive hepatocarcinoma cell line HepG2, but not in p53-null hepatocarcinoma cell line Hep3B. A p53 inhibitor pifithrin-α inhibited the expression of p21(Cip1 and attenuated homocysteine-induced cell growth arrest. Homocysteine induced TRB3 expression via endoplasmic reticulum stress pathway, resulting in Akt dephosphorylation. Knock-down of endogenous TRB3 significantly suppressed the inhibitory effect of homocysteine on cell proliferation and the phosphorylation of Akt. LiCl reversed homocysteine-mediated cell growth arrest by inhibiting TRB3-mediated Akt dephosphorylation. These results demonstrate that both TRB3 and p21(Cip1 are critical molecules in the homocysteine signaling cascade and provide a mechanistic explanation for impairment of liver regeneration in hyperhomocysteinemia.

  16. Research progress in c-Met and hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    WANG Changqing

    2015-06-01

    Full Text Available c-Met plays a pivotal role in the development and progression of hepatocellular carcinoma (HCC, which can lead to proliferation, survival, cytoskeleton reorganization, separation and diffusion, and angiogenesis of tumor cells. Moreover, c-Met is an important prognostic factor for HCC. In HCC, c-Met acts as an activator of a series of signaling pathways, including PI3K/AKT/mTOR, ERK/MAPK, and Rac-Pak. In recent years, it has been reported that small-molecule kinase inhibitors can abolish phosphorylation at the intracellular carboxyl terminal of c-Met, and then inhibit the recruitment of signal convertors and downstream signaling pathways, which finally achieve anti-tumor activities. Based on the carcinogenic activity of c-Met in HCC, this paper points out that selective inhibitors of c-Met hold promise for targeted therapies for HCC.

  17. Antitumor activity, pharmacokinetics, tumor-homing effect, and hepatotoxicity of a species cross-reactive c-Met antibody.

    Science.gov (United States)

    Park, Hyunkyu; Kim, Donggeon; Son, Eunju; Shin, Sunhwa; Sa, Jason K; Kim, Seok-Hyung; Yoon, Yeup; Nam, Do-Hyun

    2017-12-09

    The receptor tyrosine kinase c-Met plays critical roles in promoting tumor growth, invasion, metastasis, and angiogenesis in various types of cancer and is a promising therapeutic target. The development of a species cross-reactive therapeutic antibody could provide useful to comprehensive preclinical assessment in animal models. Towards this goal, we developed human/mouse cross-reactive c-Met antibodies using an antibody phage library. IRCR201, a c-Met antibody with species cross-reactivity, successfully inhibited the HGF/c-Met signaling pathway via degradation of c-Met and disruption of the binding with its partners, and demonstrated strong in vivo antitumor activity. In pharmacokinetic analysis, IRCR201 exhibited a nonlinear pharmacokinetic profile and showed rapid serum clearance at low dosage. Ex vivo fluorescence imaging and immunohistochemistry demonstrated strong tumor accumulation of IRCR201. Hepatotoxicity analysis revealed that IRCR201 does not significantly affect primary human and mouse hepatocytes. Serum chemistry analysis demonstrated that the alanine aminotransferase serum level was elevated in mice treated with 30 mg/kg IRCR201 than in PBS-treated mice, whereas the levels of aspartate aminotransferase and blood urea nitrogen did not significantly differ. Thus, IRCR201 is a potent therapeutic antibody that can disrupt the HGF/c-Met signaling axis and its species cross-reactivity would enable to evaluate precise biological activity in animal models. Copyright © 2017. Published by Elsevier Inc.

  18. Profiling the dynamics of a human phosphorylome reveals new components in HGF/c-Met signaling.

    Directory of Open Access Journals (Sweden)

    Crystal L Woodard

    Full Text Available Protein phosphorylation is a dynamic and reversible event that greatly influences cellular function. Identifying the key regulatory elements that determine cellular phenotypes during development and oncogenesis requires the ability to dynamically monitor proteome-wide events. Here, we report the development of a new strategy to monitor dynamic changes of protein phosphorylation in cells and tissues using functional protein microarrays as the readout. To demonstrate this technology's ability to identify condition-dependent phosphorylation events, human protein microarrays were incubated with lysates from cells or tissues under activation or inhibition of c-Met, a receptor tyrosine kinase involved in tissue morphogenesis and malignancy. By comparing the differences between the protein phosphorylation profiles obtained using the protein microarrays, we were able to recover many of the proteins that are known to be specifically activated (i.e., phosphorylated upon c-Met activation by the hepatocyte growth factor (HGF. Most importantly, we discovered many proteins that were differentially phosphorylated by lysates from cells or tissues when the c-Met pathway was active. Using phosphorylation-specific antibodies, we were able to validate several candidate proteins as new downstream components of the c-Met signaling pathway in cells. We envision that this new approach, like its DNA microarray counterpart, can be further extended toward profiling dynamics of global protein phosphorylation under many different physiological conditions both in cellulo and in vivo in a high-throughput and cost-effective fashion.

  19. Pioglitazone inhibits mitochondrial pyruvate metabolism and glucose production in hepatocytes.

    Science.gov (United States)

    Shannon, Christopher E; Daniele, Giuseppe; Galindo, Cynthia; Abdul-Ghani, Muhammad A; DeFronzo, Ralph A; Norton, Luke

    2017-02-01

    Pioglitazone is used globally for the treatment of type 2 diabetes mellitus (T2DM) and is one of the most effective therapies for improving glucose homeostasis and insulin resistance in T2DM patients. However, its mechanism of action in the tissues and pathways that regulate glucose metabolism are incompletely defined. Here we investigated the direct effects of pioglitazone on hepatocellular pyruvate metabolism and the dependency of these observations on the purported regulators of mitochondrial pyruvate transport, MPC1 and MPC2. In cultured H4IIE hepatocytes, pioglitazone inhibited [2-14 C]-pyruvate oxidation and pyruvate-driven oxygen consumption and, in mitochondria isolated from both hepatocytes and human skeletal muscle, pioglitazone selectively and dose-dependently inhibited pyruvate-driven ATP synthesis. Pioglitazone also suppressed hepatocellular glucose production (HGP), without influencing the mRNA expression of key HGP regulatory genes. Targeted siRNA silencing of MPC1 and 2 caused a modest inhibition of pyruvate oxidation and pyruvate-driven ATP synthesis, but did not alter pyruvate-driven HGP and, importantly, it did not influence the actions of pioglitazone on either pathway. In summary, these findings outline a novel mode of action of pioglitazone relevant to the pathogenesis of T2DM and suggest that targeting pyruvate metabolism may lead to the development of effective new T2DM therapies. © 2016 Federation of European Biochemical Societies.

  20. A novel SND1-BRAF fusion confers resistance to c-Met inhibitor PF-04217903 in GTL16 cells through [corrected] MAPK activation.

    Directory of Open Access Journals (Sweden)

    Nathan V Lee

    Full Text Available Targeting cancers with amplified or abnormally activated c-Met (hepatocyte growth factor receptor may have therapeutic benefit based on nonclinical and emerging clinical findings. However, the eventual emergence of drug resistant tumors motivates the pre-emptive identification of potential mechanisms of clinical resistance. We rendered a MET amplified gastric cancer cell line, GTL16, resistant to c-Met inhibition with prolonged exposure to a c-Met inhibitor, PF-04217903 (METi. Characterization of surviving cells identified an amplified chromosomal rearrangement between 7q32 and 7q34 which overexpresses a constitutively active SND1-BRAF fusion protein. In the resistant clones, hyperactivation of the downstream MAPK pathway via SND1-BRAF conferred resistance to c-Met receptor tyrosine kinase inhibition. Combination treatment with METi and a RAF inhibitor, PF-04880594 (RAFi inhibited ERK activation and circumvented resistance to either single agent. Alternatively, treatment with a MEK inhibitor, PD-0325901 (MEKi alone effectively blocked ERK phosphorylation and inhibited cell growth. Our results suggest that combination of a c-Met tyrosine kinase inhibitor with a BRAF or a MEK inhibitor may be effective in treating resistant tumors that use activated BRAF to escape suppression of c-Met signaling.

  1. Curcumin inhibits activation of TRPM2 channels in rat hepatocytes

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    E. Kheradpezhouh

    2016-04-01

    Full Text Available Oxidative stress is a hallmark of many liver diseases including viral and drug-induced hepatitis, ischemia-reperfusion injury, and non-alcoholic steatohepatitis. One of the consequences of oxidative stress in the liver is deregulation of Ca2+ homeostasis, resulting in a sustained elevation of the free cytosolic Ca2+ concentration ([Ca2+]c in hepatocytes, which leads to irreversible cellular damage. Recently it has been shown that liver damage induced by paracetamol and subsequent oxidative stress is, in large part, mediated by Ca2+ entry through Transient Receptor Potential Melastatin 2 (TRPM2 channels. Involvement of TRPM2 channels in hepatocellular damage induced by oxidative stress makes TRPM2 a potential therapeutic target for treatment of a range of oxidative stress-related liver diseases. We report here the identification of curcumin ((1E,6E-1,7-bis(4-hydroxy-3-methoxyphenyl-1,6-heptadiene-3,5-dione, a natural plant-derived polyphenol in turmeric spice, as a novel inhibitor of TRPM2 channel. Presence of 5 µM curcumin in the incubation medium prevented the H2O2- and paracetamol-induced [Ca2+]c rise in rat hepatocytes. Furthermore, in patch clamping experiments incubation of hepatocytes with curcumin inhibited activation of TRPM2 current by intracellular ADPR with IC50 of approximately 50 nM. These findings enhance understanding of the actions of curcumin and suggest that the known hepatoprotective properties of curcumin are, at least in part, mediated through inhibition of TRPM2 channels.

  2. c-Met Confers Protection Against Chronic Liver Tissue Damage and Fibrosis Progression After Bile Duct Ligation in Mice.

    NARCIS (Netherlands)

    Giebeler, A.; Boekschoten, M.V.; Klein, C.; Borowiak, M.; Birchmeier, C.; Gassler, N.; Wasmuth, H.E.; Müller, M.R.; Trautwein, C.; Streetz, K.L.

    2009-01-01

    Background & Aims The hepatocyte growth factor (HGF)/mesenchymal-epithelial transition factor (c-Met) system is an essential inducer of hepatocyte growth and proliferation. Although a fundamental role for the HGF receptor c-Met has been shown in acute liver regeneration, its cell-specific role

  3. Efficacy of c-Met inhibitor for advanced prostate cancer

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    Christensen James G

    2010-10-01

    Full Text Available Abstract Background Aberrant expression of HGF/SF and its receptor, c-Met, often correlates with advanced prostate cancer. Our previous study showed that expression of c-Met in prostate cancer cells was increased after attenuation of androgen receptor (AR signalling. This suggested that current androgen ablation therapy for prostate cancer activates c-Met expression and may contribute to development of more aggressive, castration resistant prostate cancer (CRPC. Therefore, we directly assessed the efficacy of c-Met inhibition during androgen ablation on the growth and progression of prostate cancer. Methods We tested two c-Met small molecule inhibitors, PHA-665752 and PF-2341066, for anti-proliferative activity by MTS assay and cell proliferation assay on human prostate cancer cell lines with different levels of androgen sensitivity. We also used renal subcapsular and castrated orthotopic xenograft mouse models to assess the effect of the inhibitors on prostate tumor formation and progression. Results We demonstrated a dose-dependent inhibitory effect of PHA-665752 and PF-2341066 on the proliferation of human prostate cancer cells and the phosphorylation of c-Met. The effect on cell proliferation was stronger in androgen insensitive cells. The c-Met inhibitor, PF-2341066, significantly reduced growth of prostate tumor cells in the renal subcapsular mouse model and the castrated orthotopic mouse model. The effect on cell proliferation was greater following castration. Conclusions The c-Met inhibitors demonstrated anti-proliferative efficacy when combined with androgen ablation therapy for advanced prostate cancer.

  4. Targeting c-Met receptor overcomes TRAIL-resistance in brain tumors

    National Research Council Canada - National Science Library

    Du, Wanlu; Uslar, Liubov; Sevala, Sindhura; Shah, Khalid

    2014-01-01

    .... We show that the knock down c-Met protein, but not inhibition, sensitized brain tumor cells to TRAIL-mediated apoptosis by interrupting the interaction between c-Met and TRAIL cognate death receptor (DR) 5...

  5. Targeting c-Met Receptor Overcomes TRAIL-Resistance in Brain Tumors: e95490

    National Research Council Canada - National Science Library

    Wanlu Du; Liubov Uslar; Sindhura Sevala; Khalid Shah

    2014-01-01

    .... We show that the knock down c-Met protein, but not inhibition, sensitized brain tumor cells to TRAIL-mediated apoptosis by interrupting the interaction between c-Met and TRAIL cognate death receptor (DR) 5...

  6. A novel rabbit anti-hepatocyte growth factor monoclonal neutralizing antibody inhibits tumor growth in prostate cancer cells and mouse xenografts

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    Yu, Yanlan; Chen, Yicheng; Ding, Guoqing; Wang, Mingchao; Wu, Haiyang; Xu, Liwei; Rui, Xuefang; Zhang, Zhigen, E-mail: srrshurology@163.com

    2015-08-14

    The hepatocyte growth factor and its receptor c-Met are correlated with castration-resistance in prostate cancer. Although HGF has been considered as an attractive target for therapeutic antibodies, the lack of cross-reactivity of monoclonal antibodies with human/mouse HGFs is a major obstacle in preclinical developments. We generated a panel of anti-HGF RabMAbs either blocking HGF/c-Met interaction or inhibiting c-Met phosphorylation. We selected one RabMAb with mouse cross-reactivity and demonstrated that it blocked HGF-stimulated downstream activation in PC-3 and DU145 cells. Anti-HGF RabMAb inhibited not only the growth of PC-3 cells but also HGF-dependent proliferation in HUVECs. We further demonstrated the efficacy and potency of the anti-HGF RabMAb in tumor xenograft mice models. Through these in vitro and in vivo experiments, we explored a novel therapeutic antibody for advanced prostate cancer. - Highlights: • HGF is an attractive target for castration-refractory prostate cancer. • We generated and characterized a panel of anti-HGF rabbit monoclonal antibodies. • More than half of these anti-HGF RabMAbs was cross-reactive with mouse HGF. • Anti-HGF RabMAb blocks HGF-stimulated phosphorylation and cell growth in vitro. • Anti-HGF RabMAb inhibits tumor growth and angiogenesis in xenograft mice.

  7. Epigenetic upregulation of HGF and c-Met drives metastasis in hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Olorunseun O Ogunwobi

    Full Text Available Hepatocyte growth factor (HGF and its receptor, c-Met, are important regulators of growth and differentiation of healthy hepatocytes. However, upregulation of HGF and c-Met have been associated with tumor progression and metastasis in hepatocellular carcinoma (HCC. Hematogenous dissemination is the most common route for cancer metastasis, but the role of HGF and c-Met in circulating tumor cells (CTCs is unknown. We have isolated and established a circulating tumor cell line from the peripheral blood of a mouse HCC model. Our studies show that these CTCs have increased expression of HGF and c-Met in comparison to the primary tumor cells. The CTCs display phenotypic evidence of epithelial-mesenchymal transition (EMT and the EMT appears to be inducible by HGF. Epigenetic analysis of the c-Met promoter identified significant loss of DNA methylation in CTCs which correlated with overexpression of c-Met and increased expression of HGF. Six specific CpG sites of c-Met promoter demethylation were identified. CTCs show significantly increased tumorigenicity and metastatic potential in a novel orthotopic syngeneic model of metastatic HCC. We conclude that during hematogenous dissemination in HCC, CTCs undergo EMT under the influence of increased HGF. This process also involves up regulation of c-Met via promoter demethylation at 6 CpG sites. Consequently, targeting HGF and c-Met expression by CTCs may be a novel non-invasive approach with potential clinical applications in HCC management.

  8. Impact of Cell-surface Antigen Expression on Target Engagement and Function of an Epidermal Growth Factor Receptor × c-MET Bispecific Antibody.

    Science.gov (United States)

    Jarantow, Stephen W; Bushey, Barbara S; Pardinas, Jose R; Boakye, Ken; Lacy, Eilyn R; Sanders, Renouard; Sepulveda, Manuel A; Moores, Sheri L; Chiu, Mark L

    2015-10-09

    The efficacy of engaging multiple drug targets using bispecific antibodies (BsAbs) is affected by the relative cell-surface protein levels of the respective targets. In this work, the receptor density values were correlated to the in vitro activity of a BsAb (JNJ-61186372) targeting epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-MET). Simultaneous binding of the BsAb to both receptors was confirmed in vitro. By using controlled Fab-arm exchange, a set of BsAbs targeting EGFR and c-MET was generated to establish an accurate receptor quantitation of a panel of lung and gastric cancer cell lines expressing heterogeneous levels of EGFR and c-MET. EGFR and c-MET receptor density levels were correlated to the respective gene expression levels as well as to the respective receptor phosphorylation inhibition values. We observed a bias in BsAb binding toward the more highly expressed of the two receptors, EGFR or c-MET, which resulted in the enhanced in vitro potency of JNJ-61186372 against the less highly expressed target. On the basis of these observations, we propose an avidity model of how JNJ-61186372 engages EGFR and c-MET with potentially broad implications for bispecific drug efficacy and design. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Recent advances in the discovery of small molecule c-Met Kinase inhibitors.

    Science.gov (United States)

    Parikh, Palak K; Ghate, Manjunath D

    2018-01-01

    c-Met is a prototype member of a subfamily of heterodimeric receptor tyrosine kinases (RTKs) and is the receptor for hepatocyte growth factor (HGF). Binding of HGF to its receptor c-Met, initiates a wide range of cellular signalling, including those involved in proliferation, motility, migration and invasion. Importantly, dysregulated HGF/c-Met signalling is a driving factor for numerous malignancies and promotes tumour growth, invasion, dissemination and/or angiogenesis. Dysregulated HGF/c-Met signalling has also been associated with poor clinical outcomes and resistance acquisition to some approved targeted therapies. Thus, c-Met kinase has emerged as a promising target for cancer drug development. Different therapeutic approaches targeting the HGF/c-Met signalling pathway are under development for targeted cancer therapy, among which small molecule inhibitors of c-Met kinase constitute the largest effort within the pharmaceutical industry. The review is an effort to summarize recent advancements in medicinal chemistry development of small molecule c-Met kinase inhibitors as potential anti-cancer agents which would certainly help future researchers to bring further developments in the discovery of small molecule c-Met kinase inhibitors. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  10. c-Met must translocate to the nucleus to initiate calcium signals.

    Science.gov (United States)

    Gomes, Dawidson A; Rodrigues, Michele A; Leite, M Fatima; Gomez, Marcus V; Varnai, Peter; Balla, Tamas; Bennett, Anton M; Nathanson, Michael H

    2008-02-15

    Hepatocyte growth factor (HGF) is important for cell proliferation, differentiation, and related activities. HGF acts through its receptor c-Met, which activates downstream signaling pathways. HGF binds to c-Met at the plasma membrane, where it is generally believed that c-Met signaling is initiated. Here we report that c-Met rapidly translocates to the nucleus upon stimulation with HGF. Ca(2+) signals that are induced by HGF result from phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate formation within the nucleus rather than within the cytoplasm. Translocation of c-Met to the nucleus depends upon the adaptor protein Gab1 and importin beta1, and formation of Ca(2+) signals in turn depends upon this translocation. HGF may exert its particular effects on cells because it bypasses signaling pathways in the cytoplasm to directly activate signaling pathways in the nucleus.

  11. Fibulin-3 negatively regulates ALDH1 via c-MET suppression and increases γ-radiation-induced sensitivity in some pancreatic cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Kim, In-Gyu, E-mail: igkim@kaeri.re.kr [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, 989-111 Daedeok-daero, Yuseong-gu, Daejeon 305-353 (Korea, Republic of); Department of Radiation Biotechnology and Applied Radioisotope, Korea University of Science and Technology (UST), 989-111 Daedeok-daero, Yusong-gu, Daejeon 305-353 (Korea, Republic of); Lee, Jae-Ha [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, 989-111 Daedeok-daero, Yuseong-gu, Daejeon 305-353 (Korea, Republic of); Department of Radiation Biotechnology and Applied Radioisotope, Korea University of Science and Technology (UST), 989-111 Daedeok-daero, Yusong-gu, Daejeon 305-353 (Korea, Republic of); Kim, Seo-Yoen; Kim, Jeong-Yul [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, 989-111 Daedeok-daero, Yuseong-gu, Daejeon 305-353 (Korea, Republic of); Cho, Eun-Wie [Epigenomics Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of)

    2014-11-21

    Highlights: • FBLN-3 gene was poorly expressed in some pancreatic cancer lines. • FBLN-3 promoter region was highly methylated in some pancreatic cancer cell lines. • FBLN-3 inhibited c-MET activation and expression and reduced cellular level of ALDH1. • FBLN-3/c-Met/ALDH1 axis modulates stemness and EMT in pancreatic cancer cells. - Abstract: Fibulin-3 (FBLN-3) has been postulated to be either a tumor suppressor or promoter depending on the cell type, and hypermethylation of the FBLN-3 promoter is often associated with human disease, especially cancer. We report that the promoter region of the FBLN-3 was significantly methylated (>95%) in some pancreatic cancer cell lines and thus FBLN-3 was poorly expressed in pancreatic cancer cell lines such as AsPC-1 and MiaPaCa-2. FBLN-3 overexpression significantly down-regulated the cellular level of c-MET and inhibited hepatocyte growth factor-induced c-MET activation, which were closely associated with γ-radiation resistance of cancer cells. Moreover, we also showed that c-MET suppression or inactivation decreased the cellular level of ALDH1 isozymes (ALDH1A1 or ALDH1A3), which serve as cancer stem cell markers, and subsequently induced inhibition of cell growth in pancreatic cancer cells. Therefore, forced overexpression of FBLN-3 sensitized cells to cytotoxic agents such as γ-radiation and strongly inhibited the stemness and epithelial to mesenchymal transition (EMT) property of pancreatic cancer cells. On the other hand, if FBLN3 was suppressed in FBLN-3-expressing BxPC3 cells, the results were opposite. This study provides the first demonstration that the FBLN-3/c-MET/ALDH1 axis in pancreatic cancer cells partially modulates stemness and EMT as well as sensitization of cells to the detrimental effects of γ-radiation.

  12. HGF and c-Met Interaction Promotes Migration in Human Chondrosarcoma Cells

    Science.gov (United States)

    Tsou, Hsi-Kai; Chen, Hsien-Te; Hung, Ya-Huey; Chang, Chia-Hao; Li, Te-Mao; Fong, Yi-Chin; Tang, Chih-Hsin

    2013-01-01

    Chondrosarcoma is a type of highly malignant tumor with a potent capacity for local invasion and causing distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Hepatocyte growth factor (HGF) has been demonstrated to stimulate cancer proliferation, migration, and metastasis. However, the effect of HGF on migration activity of human chondrosarcoma cells is not well known. Here, we found that human chondrosarcoma tissues demonstrated significant expression of HGF, which was higher than that in normal cartilage. We also found that HGF increased the migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells. c-Met inhibitor and siRNA reduced HGF-increased cell migration and MMP-2 expression. HGF treatment resulted in activation of the phosphatidylinositol 3′-kinase (PI3K)/Akt/PKCδ/NF-κB pathway, and HGF-induced expression of MMP-2 and cell migration was inhibited by specific inhibitors or siRNA-knockdown of PI3K, Akt, PKCδ, and NF-κB cascades. Taken together, our results indicated that HGF enhances migration of chondrosarcoma cells by increasing MMP-2 expression through the c-Met receptor/PI3K/Akt/PKCδ/NF-κB signal transduction pathway. PMID:23320110

  13. HGF and c-Met interaction promotes migration in human chondrosarcoma cells.

    Directory of Open Access Journals (Sweden)

    Hsi-Kai Tsou

    Full Text Available Chondrosarcoma is a type of highly malignant tumor with a potent capacity for local invasion and causing distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Hepatocyte growth factor (HGF has been demonstrated to stimulate cancer proliferation, migration, and metastasis. However, the effect of HGF on migration activity of human chondrosarcoma cells is not well known. Here, we found that human chondrosarcoma tissues demonstrated significant expression of HGF, which was higher than that in normal cartilage. We also found that HGF increased the migration and expression of matrix metalloproteinase (MMP-2 in human chondrosarcoma cells. c-Met inhibitor and siRNA reduced HGF-increased cell migration and MMP-2 expression. HGF treatment resulted in activation of the phosphatidylinositol 3'-kinase (PI3K/Akt/PKCδ/NF-κB pathway, and HGF-induced expression of MMP-2 and cell migration was inhibited by specific inhibitors or siRNA-knockdown of PI3K, Akt, PKCδ, and NF-κB cascades. Taken together, our results indicated that HGF enhances migration of chondrosarcoma cells by increasing MMP-2 expression through the c-Met receptor/PI3K/Akt/PKCδ/NF-κB signal transduction pathway.

  14. Olive phenolics as c-Met inhibitors: (--Oleocanthal attenuates cell proliferation, invasiveness, and tumor growth in breast cancer models.

    Directory of Open Access Journals (Sweden)

    Mohamed R Akl

    Full Text Available Dysregulation of the Hepatocyte growth factor (HGF/c-Met signaling axis upregulates diverse tumor cell functions, including cell proliferation, survival, scattering and motility, epithelial-to-mesenchymal transition (EMT, angiogenesis, invasion, and metastasis. (--Oleocanthal is a naturally occurring secoiridoid from extra-virgin olive oil, which showed antiproliferative and antimigratory activity against different cancer cell lines. The aim of this study was to characterize the intracellular mechanisms involved in mediating the anticancer effects of (--oleocanthal treatment and the potential involvement of c-Met receptor signaling components in breast cancer. Results showed that (--oleocanthal inhibits the growth of human breast cancer cell lines MDA-MB-231, MCF-7 and BT-474 while similar treatment doses were found to have no effect on normal human MCF10A cell growth. In addition, (--oleocanthal treatment caused a dose-dependent inhibition of HGF-induced cell migration, invasion and G1/S cell cycle progression in breast cancer cell lines. Moreover, (--oleocanthal treatment effects were found to be mediated via inhibition of HGF-induced c-Met activation and its downstream mitogenic signaling pathways. This growth inhibitory effect is associated with blockade of EMT and reduction in cellular motility. Further results from in vivo studies showed that (--oleocanthal treatment suppressed tumor cell growth in an orthotopic model of breast cancer in athymic nude mice. Collectively, the findings of this study suggest that (--oleocanthal is a promising dietary supplement lead with potential for therapeutic use to control malignancies with aberrant c-Met activity.

  15. Higher levels of c-Met expression and phosphorylation identify cell lines with increased sensitivity to AMG-458, a novel selective c-Met inhibitor with radiosensitizing effects.

    Science.gov (United States)

    Li, Bo; Torossian, Artour; Sun, Yunguang; Du, Ruihong; Dicker, Adam P; Lu, Bo

    2012-11-15

    c-Met is overexpressed in some non-small cell lung cancer (NSCLC) cell lines and tissues. Cell lines with higher levels of c-Met expression and phosphorylation depend on this receptor for survival. We studied the effects of AMG-458 on 2 NSCLC cell lines. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays assessed the sensitivities of the cells to AMG-458. Clonogenic survival assays illustrated the radiosensitizing effects of AMG-458. Western blot for cleaved caspase 3 measured apoptosis. Immunoblotting for c-Met, phospho-Met (p-Met), Akt/p-Akt, and Erk/p-Erk was performed to observe downstream signaling. AMG-458 enhanced radiosensitivity in H441 but not in A549. H441 showed constitutive phosphorylation of c-Met. A549 expressed low levels of c-Met, which were phosphorylated only in the presence of exogenous hepatocyte growth factor. The combination of radiation therapy and AMG-458 treatment was found to synergistically increase apoptosis in the H441 cell line but not in A549. Radiation therapy, AMG-458, and combination treatment were found to reduce p-Akt and p-Erk levels in H441 but not in A549. H441 became less sensitive to AMG-458 after small interfering RNA knockdown of c-Met; there was no change in A549. After overexpression of c-Met, A549 became more sensitive, while H441 became less sensitive to AMG-458. AMG-458 was more effective in cells that expressed higher levels of c-Met/p-Met, suggesting that higher levels of c-Met and p-Met in NSCLC tissue may classify a subset of tumors that are more sensitive to molecular therapies against this receptor. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Higher Levels of c-Met Expression and Phosphorylation Identify Cell Lines With Increased Sensitivity to AMG-458, a Novel Selective c-Met Inhibitor With Radiosensitizing Effects

    Energy Technology Data Exchange (ETDEWEB)

    Li Bo; Torossian, Artour [Department of Radiation Oncology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee (United States); Sun, Yunguang [Department of Radiation Oncology, Kimmel Cancer Center, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania (United States); Du, Ruihong [Department of Radiation Oncology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee (United States); Dicker, Adam P. [Department of Radiation Oncology, Kimmel Cancer Center, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania (United States); Lu Bo, E-mail: bo.lu@jefferson.edu [Department of Radiation Oncology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee (United States); Department of Radiation Oncology, Kimmel Cancer Center, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania (United States)

    2012-11-15

    Purpose: c-Met is overexpressed in some non-small cell lung cancer (NSCLC) cell lines and tissues. Cell lines with higher levels of c-Met expression and phosphorylation depend on this receptor for survival. We studied the effects of AMG-458 on 2 NSCLC cell lines. Methods and Materials: 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H-tetrazolium assays assessed the sensitivities of the cells to AMG-458. Clonogenic survival assays illustrated the radiosensitizing effects of AMG-458. Western blot for cleaved caspase 3 measured apoptosis. Immunoblotting for c-Met, phospho-Met (p-Met), Akt/p-Akt, and Erk/p-Erk was performed to observe downstream signaling. Results: AMG-458 enhanced radiosensitivity in H441 but not in A549. H441 showed constitutive phosphorylation of c-Met. A549 expressed low levels of c-Met, which were phosphorylated only in the presence of exogenous hepatocyte growth factor. The combination of radiation therapy and AMG-458 treatment was found to synergistically increase apoptosis in the H441 cell line but not in A549. Radiation therapy, AMG-458, and combination treatment were found to reduce p-Akt and p-Erk levels in H441 but not in A549. H441 became less sensitive to AMG-458 after small interfering RNA knockdown of c-Met; there was no change in A549. After overexpression of c-Met, A549 became more sensitive, while H441 became less sensitive to AMG-458. Conclusions: AMG-458 was more effective in cells that expressed higher levels of c-Met/p-Met, suggesting that higher levels of c-Met and p-Met in NSCLC tissue may classify a subset of tumors that are more sensitive to molecular therapies against this receptor.

  17. Reactive Neutrophil Responses Dependent on the Receptor Tyrosine Kinase c-MET Limit Cancer Immunotherapy.

    Science.gov (United States)

    Glodde, Nicole; Bald, Tobias; van den Boorn-Konijnenberg, Debby; Nakamura, Kyohei; O'Donnell, Jake S; Szczepanski, Sabrina; Brandes, Maria; Eickhoff, Sarah; Das, Indrajit; Shridhar, Naveen; Hinze, Daniel; Rogava, Meri; van der Sluis, Tetje C; Ruotsalainen, Janne J; Gaffal, Evelyn; Landsberg, Jennifer; Ludwig, Kerstin U; Wilhelm, Christoph; Riek-Burchardt, Monika; Müller, Andreas J; Gebhardt, Christoffer; Scolyer, Richard A; Long, Georgina V; Janzen, Viktor; Teng, Michele W L; Kastenmüller, Wolfgang; Mazzone, Massimiliano; Smyth, Mark J; Tüting, Thomas; Hölzel, Michael

    2017-10-17

    Inhibitors of the receptor tyrosine kinase c-MET are currently used in the clinic to target oncogenic signaling in tumor cells. We found that concomitant c-MET inhibition promoted adoptive T cell transfer and checkpoint immunotherapies in murine cancer models by increasing effector T cell infiltration in tumors. This therapeutic effect was independent of tumor cell-intrinsic c-MET dependence. Mechanistically, c-MET inhibition impaired the reactive mobilization and recruitment of neutrophils into tumors and draining lymph nodes in response to cytotoxic immunotherapies. In the absence of c-MET inhibition, neutrophils recruited to T cell-inflamed microenvironments rapidly acquired immunosuppressive properties, restraining T cell expansion and effector functions. In cancer patients, high serum levels of the c-MET ligand HGF correlated with increasing neutrophil counts and poor responses to checkpoint blockade therapies. Our findings reveal a role for the HGF/c-MET pathway in neutrophil recruitment and function and suggest that c-MET inhibitor co-treatment may improve responses to cancer immunotherapy in settings beyond c-MET-dependent tumors. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Antitumor activity of miR-34a in peritoneal mesothelioma relies on c-MET and AXL inhibition: persistent activation of ERK and AKT signaling as a possible cytoprotective mechanism

    Directory of Open Access Journals (Sweden)

    Rihan El Bezawy

    2017-01-01

    Full Text Available Abstract Background The value of microRNAs (miRNAs as novel targets for cancer therapy is now widely recognized. However, no information is currently available on the expression/functional role of miRNAs in diffuse malignant peritoneal mesothelioma (DMPM, a rapidly lethal disease, poorly responsive to conventional treatments, for which the development of new therapeutic strategies is urgently needed. Here, we evaluated the expression and biological effects of miR-34a—one of the most widely deregulated miRNAs in cancer and for which a lipid-formulated mimic is already clinically available—in a large cohort of DMPM clinical samples and a unique collection of in house-developed preclinical models, with the aim to assess the potential of a miR-34a-based approach for disease treatment. Methods miR-34a expression was determined by qRT-PCR in 45 DMPM and 7 normal peritoneum specimens as well as in 5 DMPM cell lines. Following transfection with miR-34a mimic, the effects on DMPM cell phenotype, in terms of proliferative potential, apoptotic rate, invasion ability, and cell cycle distribution, were assessed. In addition, three subcutaneous and orthotopic DMPM xenograft models were used to examine the effect of miR-34a on tumorigenicity. The expression of miRNA targets and the activation status of relevant pathways were investigated by western blot. Results miR-34a was found to be down-regulated in DMPM clinical specimens and cell lines compared to normal peritoneal samples. miR-34a reconstitution in DMPM cells significantly inhibited proliferation and tumorigenicity, induced an apoptotic response, and declined invasion ability, mainly through the down-regulation of c-MET and AXL and the interference with the activation of downstream signaling. Interestingly, a persistent activation of ERK1/2 and AKT in miR-34a-reconstituted cells was found to counteract the antiproliferative and proapoptotic effects of miRNA, yet not affecting its anti

  19. Phase I trial of a selective c-MET inhibitor ARQ 197 incorporating proof of mechanism pharmacodynamic studies.

    Science.gov (United States)

    Yap, Timothy A; Olmos, David; Brunetto, Andre T; Tunariu, Nina; Barriuso, Jorge; Riisnaes, Ruth; Pope, Lorna; Clark, Jeremy; Futreal, Andrew; Germuska, Michael; Collins, David; deSouza, Nandita M; Leach, Martin O; Savage, Ronald E; Waghorne, Carol; Chai, Feng; Garmey, Edward; Schwartz, Brian; Kaye, Stan B; de Bono, Johann S

    2011-04-01

    The hepatocyte growth factor/c-MET axis is implicated in tumor cell proliferation, survival, and angiogenesis. ARQ 197 is an oral, selective, non-adenosine triphosphate competitive c-MET inhibitor. A phase I trial of ARQ 197 was conducted to assess safety, tolerability, and target inhibition, including intratumoral c-MET signaling, apoptosis, and angiogenesis. Patients with solid tumors amenable to pharmacokinetic and pharmacodynamic studies using serial biopsies, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), and circulating endothelial cell (CEC) and circulating tumor cell (CTC) enumeration were enrolled. Fifty-one patients received ARQ 197 at 100 to 400 mg twice per day. ARQ 197 was well tolerated, with the most common toxicities being grade 1 to 2 fatigue, nausea, and vomiting. Dose-limiting toxicities included grade 3 fatigue (200 mg twice per day; n = 1); grade 3 mucositis, palmar-plantar erythrodysesthesia, and hypokalemia (400 mg twice per day; n = 1); and grade 3 to 4 febrile neutropenia (400 mg twice per day, n = 2; 360 mg twice per day, n = 1). The recommended phase II dose was 360 mg twice per day. ARQ 197 systemic exposure was dose dependent and supported twice per day oral dosing. ARQ 197 decreased phosphorylated c-MET, total c-MET, and phosphorylated focal adhesion kinase and increased terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) staining in tumor biopsies (n = 15). CECs decreased in 25 (58.1%) of 43 patients, but no significant changes in DCE-MRI parameters were observed after ARQ 197 treatment. Of 15 patients with detectable CTCs, eight (53.3%) had ≥ 30% decline in CTCs after treatment. Stable disease, as defined by Response Evaluation Criteria in Solid Tumors (RECIST), ≥ 4 months was observed in 14 patients, with minor regressions in gastric and Merkel cell cancers. ARQ 197 safely inhibited intratumoral c-MET signaling. Further clinical evaluation focusing on

  20. Breast cancer-derived bone metastasis can be effectively reduced through specific c-MET inhibitor tivantinib (ARQ 197) and shRNA c-MET knockdown.

    Science.gov (United States)

    Previdi, Sara; Abbadessa, Giovanni; Dalò, Francesca; France, Dennis S; Broggini, Massimo

    2012-01-01

    Breast cancer exhibits a propensity to metastasize to bone, resulting in debilitating skeletal complications associated with significant morbidity and poor prognosis. The cross-talk between metastatic cancer cells and bone is critical to the development and progression of bone metastases. We have shown the involvement of the HGF/c-MET system in tumor-bone interaction contributing to human breast cancer metastasis. Therefore, disruption of HGF/c-MET signaling is a potential targeted approach to treating metastatic bone disease. In this study, we evaluated the effects of c-MET inhibition by both an oral, selective, small-molecule c-MET inhibitor, tivantinib, and a specific short hairpin RNA (shRNA) against c-MET in a mouse model of human breast cancer. Tivantinib exhibited dose-dependent antimetastatic activity in vivo, and the 120 mg/kg dose, proven to be suboptimal in reducing subcutaneous tumor growth, induced significant inhibition of metastatic growth of breast cancer cells in bone and a noteworthy reduction of tumor-induced osteolysis. shRNA-mediated c-MET silencing did not affect in vitro proliferation of bone metastatic cells, but significantly reduced their migration, and this effect was further enhanced by tivantinib. Both observations were confirmed in vivo. Indeed, more pronounced tumor growth suppression with concomitant marked decreases of lytic lesions and prolongation of survival were achieved by dual c-MET inhibition using both tivantinib and RNA interference strategies. Overall, our findings highlighted the effectiveness of c-MET inhibition in delaying the onset and progression of bone metastases and strongly suggest that targeting c-MET may have promising therapeutic value in the treatment of bone metastases from breast cancer. ©2011 AACR.

  1. Activated HGF-c-Met Axis in Head and Neck Cancer

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    Levi Arnold

    2017-12-01

    Full Text Available Head and neck squamous cell carcinoma (HNSCC is a highly morbid disease. Recent developments including Food and Drug Administration (FDA approved molecular targeted agent’s pembrolizumab and cetuximab show promise but did not improve the five-year survival which is currently less than 40%. The hepatocyte growth factor receptor; also known as mesenchymal–epithelial transition factor (c-Met and its ligand hepatocyte growth factor (HGF are overexpressed in head and neck squamous cell carcinoma (HNSCC; and regulates tumor progression and response to therapy. The c-Met pathway has been shown to regulate many cellular processes such as cell proliferation, invasion, and angiogenesis. The c-Met pathway is involved in cross-talk, activation, and perpetuation of other signaling pathways, curbing the cogency of a blockade molecule on a single pathway. The receptor and its ligand act on several downstream effectors including phospholipase C gamma (PLCγ, cellular Src kinase (c-Src, phosphotidylinsitol-3-OH kinase (PI3K alpha serine/threonine-protein kinase (Akt, mitogen activate protein kinase (MAPK, and wingless-related integration site (Wnt pathways. They are also known to cross-talk with other receptors; namely epidermal growth factor receptor (EGFR and vascular endothelial growth factor receptor (VEGFR and specifically contribute to treatment resistance. Clinical trials targeting the c-Met axis in HNSCC have been undertaken because of significant preclinical work demonstrating a relationship between HGF/c-Met signaling and cancer cell survival. Here we focus on HGF/c-Met impact on cellular signaling in HNSCC to potentiate tumor growth and disrupt therapeutic efficacy. Herein we summarize the current understanding of HGF/c-Met signaling and its effects on HNSCC. The intertwining of c-Met signaling with other signaling pathways provides opportunities for more robust and specific therapies, leading to better clinical outcomes.

  2. Mechanism of c-MET in Non-small Cell Lung Cancer and Its Treatment and Testing

    Directory of Open Access Journals (Sweden)

    Hongge LIANG

    2015-12-01

    Full Text Available Hepatocyte growth factor/c-MET (HGF/c-MET signaling pathway can be abnormal activated by many mechanisms such as c-MET mutation, amplification and the overexpression of HGF, and it plays an important role in the development of non-small cell lung cancer (NSCLC, as well as in the tolerance of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI in NSCLC. Therefore, c-MET is a new molecular target for the therapy of NSCLC since EGFR and ALK. At present, although the c-MET inhibitors have shown a potential prospect in some clinical trials, its assessment of safety and effectiveness in clinical applications, and the choice of testing methods and standards still need a further discussion. In this paper, we summarized the mechanism of c-MET in NSCLC, as well as its treatment prospect and selection of testing methods.

  3. {sup 89}Zr-Onartuzumab PET imaging of c-MET receptor dynamics

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    Pool, Martin; Kol, Arjan; Giesen, Danique; Vries, Elisabeth G.E. de [University of Groningen, Department of Medical Oncology, University Medical Center Groningen, Groningen (Netherlands); Terwisscha van Scheltinga, Anton G.T. [University of Groningen, Department of Clinical Pharmacy and Pharmacology, University Medical Center Groningen, Groningen (Netherlands); Lub-de Hooge, Marjolijn N. [University of Groningen, Department of Clinical Pharmacy and Pharmacology, University Medical Center Groningen, Groningen (Netherlands); University of Groningen, Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, Groningen (Netherlands)

    2017-08-15

    c-MET and its ligand hepatocyte growth factor are often dysregulated in human cancers. Dynamic changes in c-MET expression occur and might predict drug efficacy or emergence of resistance. Noninvasive visualization of c-MET dynamics could therefore potentially guide c-MET-directed therapies. We investigated the feasibility of {sup 89}Zr-labelled one-armed c-MET antibody onartuzumab PET for detecting relevant changes in c-MET levels induced by c-MET-mediated epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib resistance or heat shock protein-90 (HSP90) inhibitor NVP-AUY-922 treatment in human non-small-cell lung cancer (NSCLC) xenografts. In vitro membrane c-MET levels were determined by flow cytometry. HCC827ErlRes, an erlotinib-resistant clone with c-MET upregulation, was generated from the exon-19 EGFR-mutant human NSCLC cell line HCC827. Mice bearing HCC827 and HCC827ErlRes tumours in opposite flanks underwent {sup 89}Zr-onartuzumab PET scans. The HCC827-xenografted mice underwent {sup 89}Zr-onartuzumab PET scans before treatment and while receiving biweekly intraperitoneal injections of 100 mg/kg NVP-AUY-922 or vehicle. Ex vivo, tumour c-MET immunohistochemistry was correlated with the imaging results. In vitro, membrane c-MET was upregulated in HCC827ErlRes tumours by 213 ± 44% in relation to the level in HCC827 tumours, while c-MET was downregulated by 69 ± 9% in HCC827 tumours following treatment with NVP-AUY-922. In vivo, {sup 89}Zr-onartuzumab uptake was 26% higher (P < 0.05) in erlotinib-resistant HCC827ErlRes than in HCC827 xenografts, while HCC827 tumour uptake was 33% lower (P < 0.001) following NVP-AUY-922 treatment. The results show that {sup 89}Zr-onartuzumab PET effectively discriminates relevant changes in c-MET levels and could potentially be used clinically to monitor c-MET status. (orig.)

  4. Blood vessel endothelium-directed tumor cell streaming in breast tumors requires the HGF/C-Met signaling pathway.

    Science.gov (United States)

    Leung, E; Xue, A; Wang, Y; Rougerie, P; Sharma, V P; Eddy, R; Cox, D; Condeelis, J

    2017-05-11

    During metastasis to distant sites, tumor cells migrate to blood vessels. In vivo, breast tumor cells utilize a specialized mode of migration known as streaming, where a linear assembly of tumor cells migrate directionally towards blood vessels on fibronectin-collagen I-containing extracellular matrix (ECM) fibers in response to chemotactic signals. We have successfully reconstructed tumor cell streaming in vitro by co-plating tumors cells, macrophages and endothelial cells on 2.5 μm thick ECM-coated micro-patterned substrates. We found that tumor cells and macrophages, when plated together on the micro-patterned substrates, do not demonstrate sustained directional migration in only one direction (sustained directionality) but show random bi-directional walking. Sustained directionality of tumor cells as seen in vivo was established in vitro when beads coated with human umbilical vein endothelial cells were placed at one end of the micro-patterned 'ECM fibers' within the assay. We demonstrated that these endothelial cells supply the hepatocyte growth factor (HGF) required for the chemotactic gradient responsible for sustained directionality. Using this in vitro reconstituted streaming system, we found that directional streaming is dependent on, and most effectively blocked, by inhibiting the HGF/C-Met signaling pathway between endothelial cells and tumor cells. Key observations made with the in vitro reconstituted system implicating C-Met signaling were confirmed in vivo in mammary tumors using the in vivo invasion assay and intravital multiphoton imaging of tumor cell streaming. These results establish HGF/C-Met as a central organizing signal in blood vessel-directed tumor cell migration in vivo and highlight a promising role for C-Met inhibitors in blocking tumor cell streaming and metastasis in vivo, and for use in human trials.

  5. The HGF Receptor c-Met Is Overexpressed in Esophageal Adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Luis J. Herrera

    2005-01-01

    Full Text Available The hepatocyte growth factor (HGF receptor, Met, has established oncogenic properties; however, its expression and function in esophageal adenocarcinoma (EA remain poorly understood. We aimed to determine the expression and potential alterations in Met expression in EA. Met expression was investigated in surgical specimens of EA, Barrett's esophagus (BE, and normal esophagus (NE using immunohistochemistry (IHC and quantitative reverse transcriptase polymerase chain reaction. Met expression, phosphorylation, and the effect of COX-2 inhibition on expression were examined in EA cell lines. IHC demonstrated intense Met immunoreactivity in all (100% EA and dysplastic BE specimens. In contrast, minimal immunostaining was observed in BE without dysplasia or NE specimens. Met mRNA and protein levels were increased in three EA cell lines, and Met protein was phosphorylated in the absence of serum. Sequence analysis found the kinase domain of c-met to be wild type in all three EA cell lines. HGF mRNA expression was identified in two EA cell lines. In COX-2-overexpressing cells, COX-2 inhibition decreased Met expression. Met is consistently overexpressed in EA surgical specimens and in three EA cell lines. Met dysregulation occurs early in Barrett's dysplasia to adenocarcinoma sequence. Future study of Met inhibition as a potential biologic therapy for EA is warranted.

  6. Negative control of the HGF/c-MET pathway by TGF-β: a new look at the regulation of stemness in glioblastoma.

    Science.gov (United States)

    Papa, Eleanna; Weller, Michael; Weiss, Tobias; Ventura, Elisa; Burghardt, Isabel; Szabó, Emese

    2017-12-13

    Multiple target inhibition has gained considerable interest in combating drug resistance in glioblastoma, however, understanding the molecular mechanisms of crosstalk between signaling pathways and predicting responses of cancer cells to targeted interventions has remained challenging. Despite the significant role attributed to transforming growth factor (TGF)-β family and hepatocyte growth factor (HGF)/c-MET signaling in glioblastoma pathogenesis, their functional interactions have not been well characterized. Using genetic and pharmacological approaches to stimulate or antagonize the TGF-β pathway in human glioma-initiating cells (GIC), we observed that TGF-β exerts an inhibitory effect on c-MET phosphorylation. Inhibition of either mitogen-activated protein kinase (MAPK)/ extracellular signal-regulated kinase (ERK) or phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT) signaling pathway attenuated this effect. A comparison of c-MET-driven and c-MET independent GIC models revealed that TGF-β inhibits stemness in GIC at least in part via its negative regulation of c-MET activity, suggesting that stem cell (SC) maintenance may be controlled by the balance between these two oncogenic pathways. Importantly, immunohistochemical analyses of human glioblastoma and ex vivo single-cell gene expression profiling of TGF-β and HGF confirm the negative interaction between both pathways. These novel insights into the crosstalk of two major pathogenic pathways in glioblastoma may explain some of the disappointing results when targeting either pathway alone in human glioblastoma patients and inform on potential future designs on targeted pharmacological or genetic intervention.

  7. Nitric oxide inhibits glycogen synthesis in isolated rat hepatocytes

    NARCIS (Netherlands)

    Sprangers, F.; Sauerwein, H. P.; Romijn, J. A.; van Woerkom, G. M.; Meijer, A. J.

    1998-01-01

    There is increasing evidence for the existence of intrahepatic regulation of glucose metabolism by Kupffer cell products. Nitric oxide (NO) is known to inhibit gluconeogenic flux through pyruvate carboxylase and phosphoenolpyruvate carboxykinase. However, NO may also influence glucose metabolism at

  8. Expression of HGF and c-Met Proteins in Human Keratoconus Corneas

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    Jingjing You

    2015-01-01

    Full Text Available Keratoconus (KC is a progressive degenerative inflammatory-related disease of the human cornea leading to decreased visual function. The pathogenesis of KC remains to be understood. Recent genetic studies indicate that gene variants of an inflammation-related molecule, hepatocyte growth factor (HGF, are associated with an increased susceptibility for developing KC. However HGF protein expression in KC has not been explored. In this initial study, we investigated late-stage KC and control corneas for the expression of HGF and its receptor mesenchymal-epithelial transition factor (c-Met/Met. KC buttons (~8 mm diameter (n=10 and whole control corneas (n=6 were fixed in 10% formalin or 2% paraformaldehyde, paraffin embedded and sectioned. Sections were immunolabelled with HGF and c-Met antibodies, visualised using immunofluorescence, and examined with scanning laser confocal microscopy. Semiquantitative grading was used to compare HGF and c-Met immunostaining in KC and control corneas. Overall, KC corneas showed increased HGF and c-Met immunostaining compared to controls. KC corneal epithelium displayed heterogeneous moderate-to-strong immunoreactivity for HGF and c-Met, particularly in the basal epithelium adjacent to the cone area. Taken together with the recent genetic studies, our results further support a possible role for HGF/c-Met in the pathogenesis of KC.

  9. Expression of HGF and c-Met Proteins in Human Keratoconus Corneas

    Science.gov (United States)

    You, Jingjing; Wen, Li; Roufas, Athena; Hodge, Chris; Sutton, Gerard; Madigan, Michele C.

    2015-01-01

    Keratoconus (KC) is a progressive degenerative inflammatory-related disease of the human cornea leading to decreased visual function. The pathogenesis of KC remains to be understood. Recent genetic studies indicate that gene variants of an inflammation-related molecule, hepatocyte growth factor (HGF), are associated with an increased susceptibility for developing KC. However HGF protein expression in KC has not been explored. In this initial study, we investigated late-stage KC and control corneas for the expression of HGF and its receptor mesenchymal-epithelial transition factor (c-Met/Met). KC buttons (~8 mm diameter) (n = 10) and whole control corneas (n = 6) were fixed in 10% formalin or 2% paraformaldehyde, paraffin embedded and sectioned. Sections were immunolabelled with HGF and c-Met antibodies, visualised using immunofluorescence, and examined with scanning laser confocal microscopy. Semiquantitative grading was used to compare HGF and c-Met immunostaining in KC and control corneas. Overall, KC corneas showed increased HGF and c-Met immunostaining compared to controls. KC corneal epithelium displayed heterogeneous moderate-to-strong immunoreactivity for HGF and c-Met, particularly in the basal epithelium adjacent to the cone area. Taken together with the recent genetic studies, our results further support a possible role for HGF/c-Met in the pathogenesis of KC. PMID:26697215

  10. In Vivo Detection of c-MET Expression in a Rat Hepatocarcinogenesis Model Using Molecularly Targeted Magnetic Resonance Imaging

    Directory of Open Access Journals (Sweden)

    Rheal A. Towner

    2007-01-01

    Full Text Available The multifunctional growth factor scatter factor/hepatocyte growth factor and its tyrosine kinase receptor, c-MET, have been implicated in the genesis and malignant progression of numerous human malignancies, including hepatocellular carcinomas. The incidence of hepatocellular carcinomas in the United States has increased noticeably over the past two decades and is listed as the fifth major cancer in men worldwide. In this study, we used a choline-deficient l-amino acid (CDAA-defined rat hepatocarcinogenesis model to visualize increased in vivo expression of the c-MET antigen in neoplastic lesion formation with the use of a super paramagnetic iron oxide (SPIO–anti-c-MET molecularly targeted magnetic resonance imaging (MRI contrast agent. SPIO–anti-c-MET was used for the first time to detect overexpression of c-MET in neoplastic nodules and tumors within the livers of CDAA-treated rats, as determined by a decrease in MRI signal intensity and a decrease in regional T2 values. Specificity for the binding of the molecularly targeted anti-c-MET contrast agent was determined using rat hepatoma (H4-II-E-C3 cell cultures and immunofluorescence microscopic imaging of the targeting agents within neoplastic liver tissue 1 to 2 hours following intravenous administration of SPIO–anti-c-MET and MRI investigation. This method has the ability to visualize in vivo the overexpression of c-MET at early developmental stages of tumor formation.

  11. HCV core protein promotes hepatocyte proliferation and chemoresistance by inhibiting NR4A1

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    Tan, Yongsheng, E-mail: yongshengtanwhu@126.com; Li, Yan, E-mail: liyansd2@163.com

    2015-10-23

    This study investigated the effect of HCV core protein on the proliferation of hepatocytes and hepatocellular carcinoma cells (HCC), the influence of HCV core protein on HCC apoptosis induced by the chemotherapeutic agent cisplatin, and the mechanism through which HCV core protein acts as a potential oncoprotein in HCV-related HCC by measuring the levels of NR4A1 and Runt-related transcription factor 3 (RUNX3), which are associated with tumor suppression and chemotherapy resistance. In the present study, PcDNA3.1-core and RUNX3 siRNA were transfected into LO2 and HepG2 cells using Lipofectamine 2000. LO2-core, HepG2-core, LO2-RUNX3 {sup low} and control cells were treated with different concentrations of cisplatin for 72 h, and cell proliferation and apoptosis were assayed using the CellTiter 96{sup ®}Aqueous Non-Radioactive Cell Proliferation Assay Kit. Western blot and real time PCR analyses were used to detect NR4A1, RUNX3, smad7, Cyclin D1 and BAX. Confocal microscopy was used to determine the levels of NR4A1 in HepG2 and HepG2-core cells. The growth rate of HepG2-core cells was considerably greater than that of HepG2 cells. HCV core protein increased the expression of cyclin D1 and decreased the expressions of NR4A1 and RUNX3. In LO2 – RUNX3 {sup low}, the rate of cell proliferation and the level of cisplatin resistance were the same as in the LO2 -core. These results suggest that HCV core protein decreases the sensitivity of hepatocytes to cisplatin by inhibiting the expression of NR4A1 and promoting the expression of smad7, which negatively regulates the TGF-β pathway. This effect results in down regulation of RUNX3, a target of the TGF-β pathway. Taken together, these findings indicate that in hepatocytes, HCV core protein increases drug resistance and inhibits cell apoptosis by inhibiting the expressions of NR4A1 and RUNX3. - Highlights: • HCV core protein inhibits HepG2 cell sensitivity to cisplatin. • Core expression in HepG2 decreases

  12. Mutant IDH inhibits HNF-4α to block hepatocyte differentiation and promote biliary cancer.

    Science.gov (United States)

    Saha, Supriya K; Parachoniak, Christine A; Ghanta, Krishna S; Fitamant, Julien; Ross, Kenneth N; Najem, Mortada S; Gurumurthy, Sushma; Akbay, Esra A; Sia, Daniela; Cornella, Helena; Miltiadous, Oriana; Walesky, Chad; Deshpande, Vikram; Zhu, Andrew X; Hezel, Aram F; Yen, Katharine E; Straley, Kimberly S; Travins, Jeremy; Popovici-Muller, Janeta; Gliser, Camelia; Ferrone, Cristina R; Apte, Udayan; Llovet, Josep M; Wong, Kwok-Kin; Ramaswamy, Sridhar; Bardeesy, Nabeel

    2014-09-04

    Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly liver cancer. Mutant IDH proteins in IHCC and other malignancies acquire an abnormal enzymatic activity allowing them to convert α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple αKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extracellular matrix maturation. However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF-4α, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models expressing mutant IDH in the adult liver show an aberrant response to hepatic injury, characterized by HNF-4α silencing, impaired hepatocyte differentiation, and markedly elevated levels of cell proliferation. Moreover, IDH and Kras mutations, genetic alterations that co-exist in a subset of human IHCCs, cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional link between IDH mutations, hepatic cell fate, and IHCC pathogenesis, and present a novel genetically engineered mouse model of IDH-driven malignancy.

  13. Cytotoxic activity of Tivantinib (ARQ 197) is not due solely to MET inhibition

    Science.gov (United States)

    Katayama, Ryohei; Aoyama, Aki; Yamori, Takao; Qi, Jie; Oh-hara, Tomoko; Song, Youngchul; Engelman, Jeffrey A.; Fujita, Naoya

    2013-01-01

    The receptor tyrosine kinase c-MET is the high-affinity receptor for the hepatocyte growth factor (HGF). The HGF/c-MET axis is often dysregulated in tumors. c-MET activation can be caused by MET gene amplification, activating mutations, and auto- or paracrine mechanisms. Thus, c-MET inhibitors are under development as anti-cancer drugs. Tivantinib (ARQ 197) was reported as a small molecule c-MET inhibitor and early clinical studies suggest anti-tumor activity. To assess if the anti-tumor activity of tivantinib was due to inhibition of c-MET, we compared the activity of tivantinib to other c-MET inhibitors in both c-MET addicted and non-addicted cancer cells. As expected, other c-MET inhibitors, crizotinib and PHA-665752, suppressed the growth of c-MET addicted cancers, but not the growth of cancers that are not addicted to c-MET. In contrast, tivantinib inhibited cell viability with similar potency in both c-MET addicted and non-addicted cells. These results suggest that tivantinib exhibits its antitumor activity in a manner independent of c-MET status. Tivantinib treatment induced a G2/M cell cycle arrest in EBC1 cells similarly to vincristine treatment, whereas PHA-665752 or crizotinib treatment markedly induced G0/G1 cell cycle arrest. To identify the additional molecular target of tivantinib, we performed COMPARE analysis, an in silico screening of a database of drug sensitivities across 39 cancer cell lines (JFCR39), and identified microtubule as a target of tivantinib. Tivantinib treated cells demonstrated typical microtubule disruption similar to vincristine and inhibited microtubule assembly in vitro. These results suggest that tivantinib inhibits microtubule polymerization in addition to inhibiting c-MET. PMID:23598276

  14. cMET in NSCLC: Can We Cut off the Head of the Hydra? From the Pathway to the Resistance

    Energy Technology Data Exchange (ETDEWEB)

    Van Der Steen, Nele [Center for Oncological Research Antwerp, University of Antwerp, Universiteitsplein 1, Wilrijk 2610 (Belgium); Pauwels, Patrick [Center for Oncological Research Antwerp, University of Antwerp, Universiteitsplein 1, Wilrijk 2610 (Belgium); Molecular Pathology Unit, Pathology Department, Antwerp University Hospital, Wilrijkstraat 10, Edegem 2650 (Belgium); Gil-Bazo, Ignacio [Department of Oncology, Clínica Universidad de Navarra, Pamplona 31008 (Spain); Castañon, Eduardo [Department of Oncology, Clínica Universidad de Navarra, Pamplona 31008 (Spain); Phase I-Early Clinical Trials Unit, Oncology Department, Antwerp University Hospital, Wilrijkstraat 10, Edegem 2650 (Belgium); Raez, Luis [Thoracic Oncology Program, Memorial Cancer Institute, Memorial Health Care System, Pembroke Pines, FL 33024 (United States); Cappuzzo, Federico [4Thoracic Oncology Program, Memorial Cancer Institute, Memorial Health Care System, Pembroke Pines, FL 33024 (United States); Rolfo, Christian, E-mail: Christian.Rolfo@uza.be [Center for Oncological Research Antwerp, University of Antwerp, Universiteitsplein 1, Wilrijk 2610 (Belgium); Phase I-Early Clinical Trials Unit, Oncology Department, Antwerp University Hospital, Wilrijkstraat 10, Edegem 2650 (Belgium)

    2015-03-25

    In the last decade, the tyrosine kinase receptor cMET, together with its ligand hepatocyte growth factor (HGF), has become a target in non-small cell lung cancer (NSCLC). Signalization via cMET stimulates several oncological processes amongst which are cell motility, invasion and metastasis. It also confers resistance against several currently used targeted therapies, e.g., epidermal growth factor receptor (EGFR) inhibitors. In this review, we will discuss the basic structure of cMET and the most important signaling pathways. We will also look into aberrations in the signaling and the effects thereof in cancer growth, with the focus on NSCLC. Finally, we will discuss the role of cMET as resistance mechanism.

  15. cMET in NSCLC: Can We Cut off the Head of the Hydra? From the Pathway to the Resistance

    Directory of Open Access Journals (Sweden)

    Nele Van Der Steen

    2015-03-01

    Full Text Available In the last decade, the tyrosine kinase receptor cMET, together with its ligand hepatocyte growth factor (HGF, has become a target in non-small cell lung cancer (NSCLC. Signalization via cMET stimulates several oncological processes amongst which are cell motility, invasion and metastasis. It also confers resistance against several currently used targeted therapies, e.g., epidermal growth factor receptor (EGFR inhibitors. In this review, we will discuss the basic structure of cMET and the most important signaling pathways. We will also look into aberrations in the signaling and the effects thereof in cancer growth, with the focus on NSCLC. Finally, we will discuss the role of cMET as resistance mechanism.

  16. Development of antibody-based c-Met inhibitors for targeted cancer therapy

    Directory of Open Access Journals (Sweden)

    Lee D

    2015-02-01

    Full Text Available Dongheon Lee, Eun-Sil Sung, Jin-Hyung Ahn, Sungwon An, Jiwon Huh, Weon-Kyoo You Hanwha Chemical R&D Center, Biologics Business Unit, Daejeon, Republic of Korea Abstract: Signaling pathways mediated by receptor tyrosine kinases (RTKs and their ligands play important roles in the development and progression of human cancers, which makes RTK-mediated signaling pathways promising therapeutic targets in the treatment of cancer. Compared with small-molecule compounds, antibody-based therapeutics can more specifically recognize and bind to ligands and RTKs. Several antibody inhibitors of RTK-mediated signaling pathways, such as human epidermal growth factor receptor 2, vascular endothelial growth factor, epidermal growth factor receptor or vascular endothelial growth factor receptor 2, have been developed and are widely used to treat cancer patients. However, since the therapeutic options are still limited in terms of therapeutic efficacy and types of cancers that can be treated, efforts are being made to identify and evaluate novel RTK-mediated signaling pathways as targets for more efficacious cancer treatment. The hepatocyte growth factor/c-Met signaling pathway has come into the spotlight as a promising target for development of potent cancer therapeutic agents. Multiple antibody-based therapeutics targeting hepatocyte growth factor or c-Met are currently in preclinical or clinical development. This review focuses on the development of inhibitors of the hepatocyte growth factor/c-Met signaling pathway for cancer treatment, including critical issues in clinical development and future perspectives for antibody-based therapeutics. Keywords: hepatocyte growth factor, ligands, receptor tyrosine kinase, signaling pathway, therapeutic agent

  17. Immunohistochemical expression of HGF, c-MET and transcription factor STAT3 in colorectal tumors

    Directory of Open Access Journals (Sweden)

    M Trovato

    2009-06-01

    Full Text Available By immunohistochemistry, we have investigated the expression of hepatocyte growth factor (HGF, HGF-R or c-met and the transcritor factor STAT3 in a series of 80 colorectal tumours (40 adenomas and 40 adenocarcinomas. The expression of HGF, c-met and STAT3 was revealed in 40/40 (100% of adenomas and in 26/40 (65% of adenocarcinomas; the remaining 14/40 (35% carcinomas expressed cmet but failed to express HGF and STAT3. Positive immunoreaction score was defined through the number of stained cells: low (1-10%, moderate (11-50% and high (>51%. In adenomas, the HGF immunoreaction was high in 33 (82.5% and moderate in 7 (17.5%; the c-met staining was high in 3 (7.5% and moderate in 37 (92.5%; and the STAT3 reactivity was high in 25 (62.5% and moderate in 15(37.5%. In carcinomas, the HGF immunoreaction was moderate in 21 (80.7% and low in 5 (19.2%; the c-met staining was high in 14 (35%, moderate in 25 (62.5 and low in 1 (2.5%; and the STAT3 reactivity was moderate in 17 (65.3% and low in 9 (34.6%. In both type of lesions, HGF and c-met showed a membranous and cytoplasmic location. In adenomas, STAT3 was detected in cytoplasm and nucleus and in carcinomas it was limited to cytoplasm. While the HGF/c-met/STAT3 expression in adenomas was significantly different from carcinomas (c2 = 17, p < 0.0001, no correlation was found among HGF, c-met, or STAT3 immunostaining with histotype or degree of dysplasia in adenomas and the same for histotype, grading or staging in carcinomas. These features, suggesting a role of the HGF/c-met/STAT3 signal in colon tumorigenesis, indicate that a reduced expression of HGF and c-met is associated to progression of adenoma into carcinoma.

  18. Efficacy of c-Met inhibitor for advanced prostate cancer

    OpenAIRE

    Tu, William H; Zhu, Chunfang; Clark, Curtis; Christensen, James G; Sun, Zijie

    2010-01-01

    Abstract Background Aberrant expression of HGF/SF and its receptor, c-Met, often correlates with advanced prostate cancer. Our previous study showed that expression of c-Met in prostate cancer cells was increased after attenuation of androgen receptor (AR) signalling. This suggested that current androgen ablation therapy for prostate cancer activates c-Met expression and may contribute to development of more aggressive, castration resistant prostate cancer (CRPC). Therefore, we directly asses...

  19. Targeting c-Met receptor overcomes TRAIL-resistance in brain tumors.

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    Wanlu Du

    Full Text Available Tumor necrosis factor related apoptosis-inducing ligand (TRAIL induced apoptosis specifically in tumor cells. However, with approximately half of all known tumor lines being resistant to TRAIL, the identification of TRAIL sensitizers and their mechanism of action become critical to broadly use TRAIL as a therapeutic agent. In this study, we explored whether c-Met protein contributes to TRAIL sensitivity. We found a direct correlation between the c-Met expression level and TRAIL resistance. We show that the knock down c-Met protein, but not inhibition, sensitized brain tumor cells to TRAIL-mediated apoptosis by interrupting the interaction between c-Met and TRAIL cognate death receptor (DR 5. This interruption greatly induces the formation of death-inducing signaling complex (DISC and subsequent downstream apoptosis signaling. Using intracranially implanted brain tumor cells and stem cell (SC lines engineered with different combinations of fluorescent and bioluminescent proteins, we show that SC expressing a potent and secretable TRAIL (S-TRAIL have a significant anti-tumor effect in mice bearing c-Met knock down of TRAIL-resistant brain tumors. To our best knowledge, this is the first study that demonstrates c-Met contributes to TRAIL sensitivity of brain tumor cells and has implications for developing effective therapies for brain tumor patients.

  20. c-Met inhibitor SU11274 enhances the response of the prostate cancer cell line DU145 to ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Hongliang; Li, Xiaoying; Sun, Shaoqian [Department of Radiation Oncology, Peking University First Hospital, Peking University, Beijing (China); Gao, Xianshu, E-mail: xsgao777@hotmail.com [Department of Radiation Oncology, Peking University First Hospital, Peking University, Beijing (China); Zhou, Demin, E-mail: deminzhou@bjmu.edu.cn [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing (China)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer c-Met inhibition could significantly enhance the radiosensitivity of DU145 cells. Black-Right-Pointing-Pointer The mechanisms of the radiosensitization effect of c-Met inhibition on DU145 cells were also presented in this paper. Black-Right-Pointing-Pointer This is the first study demonstrating the effectiveness of c-Met inhibition on treating HRPC cells with radiotherapy. -- Abstract: Hormone-refractory prostate cancer shows substantial resistance to most conventional therapies including radiotherapy, constitutes a key impediment to curing patients with the disease. c-Met overexpression plays a key role in prostate cancer tumorigenesis and disease progression. Here, we demonstrate that c-Met inhibition by SU11274 could significantly suppress cell survival and proliferation as well as enhance the radiosensitivity of DU145 cells. The underlying mechanisms of the effects of SU11274 on DU145 cells may include the inhibition of c-Met signaling, depolarization of the mitochondrial membrane potential, impairment of DNA repair function, abrogation of cell cycle arrest, and enhancement of cell death. Our study is the first to show the effectiveness of combining c-Met inhibition with ionizing radiation to cure hormone-refractory prostate cancer.

  1. Activation of c-MET induces a stem-like phenotype in human prostate cancer.

    Directory of Open Access Journals (Sweden)

    Geert J L H van Leenders

    Full Text Available Prostate cancer consists of secretory cells and a population of immature cells. The function of immature cells and their mutual relation with secretory cells are still poorly understood. Immature cells either have a hierarchical relation to secretory cells (stem cell model or represent an inducible population emerging upon appropriate stimulation of differentiated cells. Hepatocyte Growth Factor (HGF receptor c-MET is specifically expressed in immature prostate cells. Our objective is to determine the role of immature cells in prostate cancer by analysis of the HGF/c-MET pathway.Gene-expression profiling of DU145 prostate cancer cells stimulated with HGF revealed induction of a molecular signature associated with stem cells, characterized by up-regulation of CD49b, CD49f, CD44 and SOX9, and down-regulation of CD24 ('stem-like signature'. We confirmed the acquisition of a stem-like phenotype by quantitative PCR, FACS analysis and Western blotting. Further, HGF led to activation of the stem cell related Notch pathway by up-regulation of its ligands Jagged-1 and Delta-like 4. Small molecules SU11274 and PHA665752 targeting c-MET activity were both able to block the molecular and biologic effects of HGF. Knock-down of c-MET by shRNA infection resulted in significant reduction and delay of orthotopic tumour-formation in male NMRI mice. Immunohistochemical analysis in prostatectomies revealed significant enrichment of c-MET positive cells at the invasive front, and demonstrated co-expression of c-MET with stem-like markers CD49b and CD49f.In conclusion, activation of c-MET in prostate cancer cells induced a stem-like phenotype, indicating a dynamic relation between differentiated and stem-like cells in this malignancy. Its mediation of efficient tumour-formation in vivo and predominant receptor expression at the invasive front implicate that c-MET regulates tumour infiltration in surrounding tissues putatively by acquisition of a stem-like phenotype.

  2. Prognostic Value of MACC1 and c-met Expressions in Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Xingsheng HU

    2012-07-01

    Full Text Available Background and objective It has been proven that metastasis-associated in colon cancer 1 (MACC1 is a new gene that is related to the invasion and metastasis of tumors. MACC1 also regulates c-met expression. The aim of this study is to explore the expressions of MACC1 and hepatocyte growth factor receptor (c-met, and its relationship with invasion, metastasis, and prognosis of non-small cell lung cancer (NSCLC. Methods MACC1 and c-met expressions were detected in 103 cases of NSCLC and 40 cases of neighboring normal lung cancer tissue using immunohistochemistry. Results MACC1 and c-met expressions were significantly higher in lung cancer tissues than that in neighboring normal tissue (P<0.001. MACC1 and c-met expressions were associated with poor differentiation, advanced T stages, lymph node metastasis, and advanced TNM stages (P<0.05 of NSCLC, but not with sex, age, smoking, and histological classification (P>0.05. In addition, a positive correlation between MACC1 and c-met expressions was observed (r=0.403, P<0.001. The result from the Kaplan-Meier survival analysis showed that the five-year survival rate in patients with positive MACC1 and c-met expressions was remarkanly lower than that in patients with negative expressions (P<0.05. The result from the Cox regression analysis showed that MACC1 expression was an independent prognostic factor for NSCLC (P=0.026. Conclusion MACC1 and c-met have an important function in the differentiation, invasion, and metastasis of NSCLC. MACC1 and c-met have poor prognosis in patients with NSCLC. Moreover, MACC1 expression is an independent prognostic factor for NSCLC.

  3. 6-Phosphogluconate dehydrogenase regulates tumor cell migration in vitro by regulating receptor tyrosine kinase c-Met

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Barden, E-mail: cchan@bidmc.harvard.edu [Division of Interdisciplinary Medicine and Biotechnology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215 (United States); VanderLaan, Paul A. [Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215 (United States); Sukhatme, Vikas P. [Division of Interdisciplinary Medicine and Biotechnology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215 (United States)

    2013-09-20

    Highlights: •Expression of 6PGD positively correlates with advancing stage of lung carcinoma. •Knockdown of 6PGD by shRNA potently inhibits c-Met tyrosine phosphorylation. •Exogenous HGF fails to restore c-Met phosphorylation in cells with 6PGD knocked down. •6PGD knockdown results in inhibition of cell migration in vitro. •Constitutively active TPR-cMet significantly restores migration of cells without 6PGD. -- Abstract: 6-Phosphogluconate dehydrogenase (6PGD) is the third enzyme in the oxidative pentose phosphate pathway (PPP). Recently, we reported that knockdown of 6PGD inhibited lung tumor growth in vitro and in a xenograft model in mice. In this study, we continued to examine the functional role of 6PGD in cancer. We show that 6PGD expression positively correlates with advancing stage of lung carcinoma. In search of functional signals related to 6PGD, we discovered that knockdown of 6PGD significantly inhibited phosphorylation of c-Met at tyrosine residues known to be critical for activity. This downregulation of c-Met phosphorylation correlated with inhibition of cell migration in vitro. Overexpression of a constitutively active c-Met specifically rescued the migration but not proliferation phenotype of 6PGD knockdown. Therefore, 6PGD appears to be required for efficient c-Met signaling and migration of tumor cells in vitro.

  4. Chinese Herbal Preparation Xuebijing Potently Inhibits Inflammasome Activation in Hepatocytes and Ameliorates Mouse Liver Ischemia-Reperfusion Injury.

    Directory of Open Access Journals (Sweden)

    Xiqiang Liu

    Full Text Available The Chinese herb preparation Xuebijing injection (XBJ has been widely used in the management of various septic disorders or inflammation-related conditions, however the molecular mechanism of its anti-inflammatory effect remains largely elusive. In the current study, we found that XBJ treatment potently ameliorated mouse hepatic ischemia-reperfusion (IR injury, manifested as decreased liver function tests (LDH, ALT, AST, improved inflammation and less hepatocyte apoptosis. Notably, XBJ markedly inhibited inflammasome activation and IL-1 production in mouse livers subjected to IRI, even in the absence of Kupffer cells, suggesting Kupffer cells are not necessary for hepatic inflammasome activation upon Redox-induced sterile inflammation. This finding led us to investigate the role of XBJ on hepatocyte apoptosis and inflammasome activation using an in vitro hydrogen peroxide (H2O2-triggered hepatocyte injury model. Our data clearly demonstrated that XBJ potently inhibited apoptosis, as well as caspase-1 cleavage and IL-1β production in a time- and dose-dependent manner in isolated hepatocytes, suggesting that in addition to its known modulatory effect on NF-κB-dependent inflammatory gene expression, it also has a direct impact on hepatocyte inflammasome activation. The current study not only deepens our understanding of how XBJ ameliorates inflammation and apoptosis, but also has immediate practical significance in many clinical situations such as partial hepatectomy, liver transplantation, etc.

  5. ARQ-197, an oral small-molecule inhibitor of c-Met for the treatment of solid tumors.

    Science.gov (United States)

    Bagai, Rakesh; Fan, Weiwen; Ma, Patrick C

    2010-06-01

    ARQ-197 is an oral, selective c-Met inhibitor under development by ArQule Inc, in partnership with Daiichi Sankyo Co Ltd and Asian licensee Kyowa Hakko Kirin Co Ltd, for the potential treatment of solid tumors, including NSCLC, hepatocellular carcinoma and pancreatic cancer, as well as microphthalmia transcription factor-driven tumors. c-Met, a key cell surface receptor tyrosine kinase involved in diverse regulatory functions, is often aberrantly activated in human cancers. While the precise mechanism of action of ARQ-197 remains undefined, data from preclinical studies have demonstrated that ARQ-197 inhibits c-Met activation in numerous human tumor cell lines and specifically targets c-Met in various cancer types; uniquely, ARQ-197 inhibits c-Met in a non-ATP-competitive manner. Phase I/II clinical trials demonstrated promise in terms of both tolerability and tumor response. Intriguingly, dose-limiting adverse effects were hematological in nature. Combinational trials are also ongoing to take advantage of the signaling crosstalk between c-Met and other oncogenic signaling systems. Prioritization of the clinical development of c-Met inhibitors, such as ARQ-197, among different tumor disease types is a key challenge at present; an improved understanding of the prediction of molecular determinants in tumors with respect to c-Met kinase as the driver oncogenic receptor, and of the prediction of tumor response, is still urgently needed.

  6. Tivantinib (ARQ 197) affects the apoptotic and proliferative machinery downstream of c-MET: role of Mcl-1, Bcl-xl and Cyclin B1.

    Science.gov (United States)

    Lu, Shuai; Török, Helga-Paula; Gallmeier, Eike; Kolligs, Frank T; Rizzani, Antonia; Arena, Sabrina; Göke, Burkhard; Gerbes, Alexander L; De Toni, Enrico N

    2015-09-08

    Tivantinib, a c-MET inhibitor, is investigated as a second-line treatment of HCC. It was shown that c-MET overexpression predicts its efficacy. Therefore, a phase-3 trial of tivantinib has been initiated to recruit "c-MET-high" patients only. However, recent evidence indicates that the anticancer activity of tivantinib is not due to c-MET inhibition, suggesting that c-MET is a predictor of response to this compound rather than its actual target. By assessing the mechanisms underlying the anticancer properties of tivantinib we showed that this agent causes apoptosis and cell cycle arrest by inhibiting the anti-apoptotic molecules Mcl-1 and Bcl-xl, and by increasing Cyclin B1 expression regardless of c-MET status. However, we found that tivantinib might antagonize the antiapoptotic effects of c-MET activation since HGF enhanced the expression of Mcl-1 and Bcl-xl. In summary, we show that the activity of tivantinib is independent of c-MET and describe Mcl-1, Bcl-xl and Cyclin B1 as effectors of its antineoplastic effects in HCC cells. We suggest that the predictive effect of c-MET expression in part reflects the c-MET-driven overexpression of Mcl-1 and Bcl-xl in c-MET-high patients and that these molecules are considered as possible response predictors.

  7. HGF/c-MET Axis in Tumor Microenvironment and Metastasis Formation

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    Anna Spina

    2015-01-01

    Full Text Available Tumor metastases are responsible for approximately 90% of all cancer-related deaths. Metastasis formation is a multistep process that requires acquisition by tumor cells of a malignant phenotype that allows them to escape from the primary tumor site and invade other organs. Each step of this mechanism involves a deep crosstalk between tumor cells and their microenvironment where the host cells play a key role in influencing metastatic behavior through the release of many secreted factors. Among these signaling molecules, Hepatocyte Growth Factor (HGF is released by many cell types of the tumor microenvironment to target its receptor c-MET within the cells of the primary tumor. Many studies reveal that HGF/c-MET axis is implicated in various human cancers, and genetic and epigenetic gain of functions of this signaling contributes to cancer development through a variety of mechanisms. In this review, we describe the specific types of cells in the tumor microenvironment that release HGF in order to promote the metastatic outgrowth through the activation of extracellular matrix remodeling, inflammation, migration, angiogenesis, and invasion. We dissect the potential use of new molecules that interfere with the HGF/c-MET axis as therapeutic targets for future clinical trials in cancer disease.

  8. Evaluation of c-Met, HGF, and HER-2 expressions in gastric carcinoma and their association with other clinicopathological factors

    Directory of Open Access Journals (Sweden)

    Yıldız Y

    2016-09-01

    Full Text Available Yetkin Yıldız,1 Cenk Sokmensuer,2 Suayib Yalcin1 1Department of Medical Oncology, 2Department of Pathology, Hacettepe University, Ankara, Turkey Background: Met and HER-2 are proto-oncogenes encoding receptor tyrosine kinase c-Met and HER-2, respectively. Hepatocyte growth factor (HGF is a ligand of c-Met. The frequency of c-Met, HGF, and HER-2 expressions in gastric cancer and their association with other clinicopathological factors have not been fully understood. Patients and methods: Patients with stage 1–4 disease were analyzed. Expressions of c-Met, HGF, and HER-2 were examined using immunohistochemistry. Results: A total of 143 patients, 97 males and 46 females, were included. C-Met scores were 3(+ in 31.5%, 2(+ in 27.3%, and 1(+ in 10.5% of the patients. There was no statistically significant difference in age, sex, tumor location, differentiation, Lauren classification, TNM staging, presence of distant metastasis, depth of tumor invasion (T, lymphovascular invasion, and survival between c-Met subgroups. Overall HGF positivity was 20.6%. HER-2 scores were 3(+ in 9.1%, 2(+ in 9.8%, and 1(+ in 16.1% of the patients. HER-2 overexpression was associated with better differentiation, intestinal subtype, and advanced stage. C-Met overexpressions were 84.6% in the HER-2-overexpression-positive group and 56.2% in the HER-2-overexpression-negative group. There were no statistically significant differences in survival between the high c-Met-expression-positive and -negative stage 3 and stage 4 patients and between the HGF-positive and -negative groups. The mean survival was 11.6±6.3 months in the HER-2-overexpression-positive stage 4 group and 11.9±6.8 months in the HER-2-overexpression-negative stage 4 group. There were no statistically significant differences in survival between the two groups. Conclusion: c-Met was not associated with any prognostic factors in gastric cancer. HER-2 was associated with better differentiation, intestinal

  9. ARQ 197, a novel and selective inhibitor of the human c-Met receptor tyrosine kinase with antitumor activity.

    Science.gov (United States)

    Munshi, Neru; Jeay, Sébastien; Li, Youzhi; Chen, Chang-Rung; France, Dennis S; Ashwell, Mark A; Hill, Jason; Moussa, Magdi M; Leggett, David S; Li, Chiang J

    2010-06-01

    The met proto-oncogene is functionally linked with tumorigenesis and metastatic progression. Validation of the receptor tyrosine kinase c-Met as a selective anticancer target has awaited the emergence of selective c-Met inhibitors. Herein, we report ARQ 197 as the first non-ATP-competitive small molecule that selectively targets the c-Met receptor tyrosine kinase. Exposure to ARQ 197 resulted in the inhibition of proliferation of c-Met-expressing cancer cell lines as well as the induction of caspase-dependent apoptosis in cell lines with constitutive c-Met activity. These cellular responses to ARQ 197 were phenocopied by RNAi-mediated c-Met depletion and further demonstrated by the growth inhibition of human tumors following oral administration of ARQ 197 in multiple mouse xenograft efficacy studies. Cumulatively, these data suggest that ARQ 197, currently in phase II clinical trials, is a promising agent for targeting cancers in which c-Met-driven signaling is important for their survival and proliferation.

  10. Madecassoside suppresses proliferation and invasiveness of HGF-induced human hepatocellular carcinoma cells via PKC-cMET-ERK1/2-COX-2-PGE2 pathway.

    Science.gov (United States)

    Li, Zexin; You, Kun; Li, Jian; Wang, Ying; Xu, Hongwei; Gao, Baoqin; Wang, Jianguo

    2016-04-01

    Recent studies showed that Madecassoside (MAD), a pentacyclic triterpene isolated from Centella asitica (L.), was used as a therapeutic agent in wound healing and also as an anti-inflammatory, anti-oxidative activities and anti-aging agent. However, its role in cancer has not been elucidated. In our present study, hepatocyte growth factor (HGF) induced the phosphorylation of its corresponding receptor cMET, increased expression of cyclo-oxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in human hepatocellular carcinoma (HCC) cells lines (HepG2 and SMMC-77), and this effect was inhibited by MAD in a dose-dependent manner. In addition, MAD exhibited significant anti-proliferative and anti-invasive effect in HGF-induced HepG2 and SMMC-77 cells. Moreover, MAD inhibited the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and the protein kinase C (PKC) activity in HGF-induced HepG2 and SMMC-77 cells. This conclusion was consistent with the effect of selective COX-2 inhibitor (NS-398) and knockdown of COX-2 by siRNA on attenuating the proliferation and invasiveness potential, and over-expression of COX-2 on abolishing the effects of MAD on proliferation and invasiveness potential, and was also in parallel with the effect of PKC inhibitor (Bisindolylmaleimide) on inhibiting PKC activity, MEK/ERK1/2 inhibitor (PD98059) inhibited MEK/ERK1/2 pathways in HGF-induced HepG2 and SMMC-77 cells. Collectively, MAD could inhibit the HGF-activated proliferation and invasiveness of HCC cells via regulating the activation of cMET-PKC-ERK1/2-COX-2-PGE2 cascade, which indicated that MAD might help control HGF-linked HCC. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Cholesterol Sulfate and Cholesterol Sulfotransferase Inhibit Gluconeogenesis by Targeting Hepatocyte Nuclear Factor 4α

    Science.gov (United States)

    Shi, Xiongjie; Cheng, Qiuqiong; Xu, Leyuan; Yan, Jiong; Jiang, Mengxi; He, Jinhan; Xu, Meishu; Stefanovic-Racic, Maja; Sipula, Ian; O'Doherty, Robert Martin; Ren, Shunlin

    2014-01-01

    Sulfotransferase (SULT)-mediated sulfation represents a critical mechanism in regulating the chemical and functional homeostasis of endogenous and exogenous molecules. The cholesterol sulfotransferase SULT2B1b catalyzes the sulfoconjugation of cholesterol to synthesize cholesterol sulfate (CS). In this study, we showed that the expression of SULT2B1b in the liver was induced in obese mice and during the transition from the fasted to the fed state, suggesting that the regulation of SULT2B1b is physiologically relevant. CS and SULT2B1b inhibited gluconeogenesis by targeting the gluconeogenic factor hepatocyte nuclear factor 4α (HNF4α) in both cell cultures and transgenic mice. Treatment of mice with CS or transgenic overexpression of the CS-generating enzyme SULT2B1b in the liver inhibited hepatic gluconeogenesis and alleviated metabolic abnormalities both in mice with diet-induced obesity (DIO) and in leptin-deficient (ob/ob) mice. Mechanistically, CS and SULT2B1b inhibited gluconeogenesis by suppressing the expression of acetyl coenzyme A (acetyl-CoA) synthetase (Acss), leading to decreased acetylation and nuclear exclusion of HNF4α. Our results also suggested that leptin is a potential effector of SULT2B1b in improving metabolic function. We conclude that SULT2B1b and its enzymatic by-product CS are important metabolic regulators that control glucose metabolism, suggesting CS as a potential therapeutic agent and SULT2B1b as a potential therapeutic target for metabolic disorders. PMID:24277929

  12. Dual regulation of receptor tyrosine kinase genes EGFR and c-Met by the tumor-suppressive microRNA-23b/27b cluster in bladder cancer.

    Science.gov (United States)

    Chiyomaru, Takeshi; Seki, Naohiko; Inoguchi, Satoru; Ishihara, Tomoaki; Mataki, Hiroko; Matsushita, Ryosuke; Goto, Yusuke; Nishikawa, Rika; Tatarano, Shuichi; Itesako, Toshihiko; Nakagawa, Masayuki; Enokida, Hideki

    2015-02-01

    Recent clinical trials of chemotherapeutics for advanced bladder cancer (BC) have shown limited benefits. Therefore, new prognostic markers and more effective treatment strategies are required. One approach to achieve these goals is through the analysis of RNA networks. Our recent studies of microRNA (miRNA) expression signatures revealed that the microRNA-23b/27b (miR-23b/27b) cluster is frequently downregulated in various types of human cancers. However, the functional role of the miR-23b/27b cluster in BC cells is still unknown. Thus, the aim of the present study was to investigate the functional significance of the miR-23b/27b cluster and its regulated molecular targets, with an emphasis on its contributions to BC oncogenesis and metastasis. The expression levels of the miR-23b/27b cluster were significantly reduced in BC clinical specimens. Restoration of mature miR-23b or miR-27b miRNAs significantly inhibited cancer cell migration and invasion, suggesting that these clustered miRNAs function as tumor suppressors. Gene expression data and in silico analysis demonstrated that the genes coding for the epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-Met) were potential targets of the miR-23b/27b cluster. Luciferase reporter assays and western blotting demonstrated that EGFR and c-Met receptor trypsine kinases were directly regulated by these clustered miRNAs. We conclude that the decreased expression of the tumor-suppressive miR-23b/27b cluster enhanced cancer cell proliferation, migration and invasion in BC through direct regulation of EGFR and c-Met signaling pathways. Our data on RNA networks regulated by tumor-suppressive miR-23b/27b provide new insights into the potential mechanisms of BC oncogenesis and metastasis.

  13. c-Met Signaling Pathway Participating in the Gefitinib Resistance of Different Gene Types of Non-small Cell Lung Cancer Cells Induced by HGF In Vitro

    Directory of Open Access Journals (Sweden)

    Xianglan XUAN

    2013-09-01

    Full Text Available Background and objective It has been known that hepatocyte growth factor (HGF induces gefitinib resistance in non-small cell lung cancer (NSCLC cells. The possible mechanism may be related to the activation of the HGF receptor c-Met. The aim of this study is to investigate the involvement of c-Met and its downstream signaling pathway in the HGF-induced gefitinib resistance of NSCLC cells with different epidermal growth factor receptor (EGFR gene types. Methods NSCLC cell lines with different EGFR genes (PC-9, PC9/R, H292, and A549 were selected and induced by HGF. Cell survival was determined by MTT assay and the expression of Met and downstream signaling proteins were examined by Western blot. Results Gefitinib inhibited the cell growth of PC9, H292, and A549 cell lines in a dose-dependent manner. The concentration-survival curve notably shifted to the right when induced by HGF. The apoptotic rate was lower when the cells were treated with HGF and gefitinib than when these cells were treated with gefitinib alone (P<0.05, particularly in PC9, H292, and A549 cells, but not in PC9/R. HGF stimulated the phosphorylation of Met and downstream signaling proteins in PC9, H292, PC9/R, and A549 cell lines. p-Met, p-Akt, p-Stat3, and p-Erk1/2 expressions were higher when the cells were treated with HGF and gefitinib than when these cells were treated with gefitinib alone, particularly in PC9, H292, and A549 cells, but not in PC9/R. Conclusion c-Met and its downstream signaling pathway possibly participated in the HGF-induced gefitinib resistance in NSCLC cells with different EGFR gene types.

  14. CAF-derived HGF promotes cell proliferation and drug resistance by up-regulating the c-Met/PI3K/Akt and GRP78 signalling in ovarian cancer cells.

    Science.gov (United States)

    Deying, Wei; Feng, Geng; Shumei, Liang; Hui, Zhao; Ming, Liu; Hongqing, Wang

    2017-04-28

    The tumour microenvironment is a highly heterogeneous entity that plays crucial roles in cancer progression. As the most prominent stromal cell types, cancer-associated fibroblasts (CAFs) produce a variety of factors into the tumour microenvironment. In the present study, we firstly isolated CAFs from tumour tissues of the patients with ovarian cancer and demonstrated that the hepatocyte growth factor (HGF) was highly expressed in the supernatants of CAFs. CAF-derived HGF or human recombinant HGF promoted cell proliferation in human ovarian cell lines SKOV3 and HO-8910 cells. Western blotting analysis also showed that CAF-derived HGF or recombinant HGF activated c-Met/phosphoinositide 3-kinase (PI3K)/Akt and glucose-regulated protein 78 (GRP78) signalling pathways in ovarian cancer cells, and these effects could be abrogated by anti-HGF and c-Met inhibitor INCB28060. Moreover, HGF in CAF matrix attenuated paclitaxel (PAC)-caused inhibition of cell proliferation and increase in cell apoptosis through activating c-Met/PI3K/Akt and GRP78 pathways in SKOV3 and HO-8910 cells. The results in vitro were further validated in nude mice. These findings suggest that CAF-derived HGF plays crucial roles in cell proliferation and drug resistance in ovarian cancer cells. © 2017 The Author(s).

  15. The indole alkaloid meleagrin, from the olive tree endophytic fungus Penicillium chrysogenum, as a novel lead for the control of c-Met-dependent breast cancer proliferation, migration and invasion.

    Science.gov (United States)

    Mady, Mohamed S; Mohyeldin, Mohamed M; Ebrahim, Hassan Y; Elsayed, Heba E; Houssen, Wael E; Haggag, Eman G; Soliman, Randa F; El Sayed, Khalid A

    2016-01-15

    Fungi of the genus Penicillium produce unique and chemically diverse biologically active secondary metabolites, including indole alkaloids. The role of dysregulated hepatocyte growth factor (HGF) and its receptor, c-Met, in the development and progression of breast carcinoma is documented. The goal of this work is to explore the chemistry and bioactivity of the secondary metabolites of the endophytic Penicillium chrysogenum cultured from the leaf of the olive tree Olea europea, collected in its natural habitat in Egypt. This fungal extract showed good inhibitory activities against the proliferation and migration of several human breast cancer lines. The CH2Cl2 extract of P. chrysogenum mycelia was subjected to bioguided chromatographic separation to afford three known indole alkaloids; meleagrin (1), roquefortine C (2) and DHTD (3). Meleagrin inhibited the growth of the human breast cancer cell lines MDA-MB-231, MDA-468, BT-474, SK BR-3, MCF7 and MCF7-dox, while similar treatment doses were found to have no effect on the growth and viability of the non-tumorigenic human mammary epithelial cells MCF10A. Meleagrin also showed excellent ATP competitive c-Met inhibitory activity in Z-Lyte assay, which was further confirmed via molecular docking studies and Western blot analysis. In addition, meleagrin treatment caused a dose-dependent inhibition of HGF-induced cell migration, and invasion of breast cancer cell lines. Meleagrin treatment potently suppressed the invasive triple negative breast tumor cell growth in an orthotopic athymic nude mice model, promoting this unique natural product from hit to a lead rank. The indole alkaloid meleagrin is a novel lead c-Met inhibitory entity useful for the control of c-Met-dependent metastatic and invasive breast malignancies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. YAP Inhibition Restores Hepatocyte Differentiation in Advanced HCC, Leading to Tumor Regression

    Directory of Open Access Journals (Sweden)

    Julien Fitamant

    2015-03-01

    Full Text Available Defective Hippo/YAP signaling in the liver results in tissue overgrowth and development of hepatocellular carcinoma (HCC. Here, we uncover mechanisms of YAP-mediated hepatocyte reprogramming and HCC pathogenesis. YAP functions as a rheostat in maintaining metabolic specialization, differentiation, and quiescence within the hepatocyte compartment. Increased or decreased YAP activity reprograms subsets of hepatocytes to different fates associated with deregulation of the HNF4A, CTNNB1, and E2F transcriptional programs that control hepatocyte quiescence and differentiation. Importantly, treatment with small interfering RNA-lipid nanoparticles (siRNA-LNPs targeting YAP restores hepatocyte differentiation and causes pronounced tumor regression in a genetically engineered mouse HCC model. Furthermore, YAP targets are enriched in an aggressive human HCC subtype characterized by a proliferative signature and absence of CTNNB1 mutations. Thus, our work reveals Hippo signaling as a key regulator of the positional identity of hepatocytes, supports targeting of YAP using siRNA-LNPs as a paradigm of differentiation-based therapy, and identifies an HCC subtype that is potentially responsive to this approach.

  17. Low c-Met expression levels are prognostic for and predict the benefits of temozolomide chemotherapy in malignant gliomas.

    Science.gov (United States)

    Li, Ming-Yang; Yang, Pei; Liu, Yan-Wei; Zhang, Chuan-Bao; Wang, Kuan-Yu; Wang, Yin-Yan; Yao, Kun; Zhang, Wei; Qiu, Xiao-Guang; Li, Wen-Bin; Peng, Xiao-Xia; Wang, Yong-Zhi; Jiang, Tao

    2016-02-16

    Aberrant c-Met has been implicated in the development of many cancers. The objective of this study was to identify an unfavorable prognostic marker that might guide decisions regarding clinical treatment strategies for high-grade gliomas. C-Met expression was measured using immunohistochemistry in 783 gliomas, and we further analyzed c-Met mRNA levels using the Agilent Whole Genome mRNA Microarray in 286 frozen samples. In vitro, we performed cell migration and invasion assays. Cell sensitivity to temozolomide (TMZ) chemotherapy was determined using MTT assays. Both mRNA and protein levels of c-Met were significantly associated with tumor grade progression and inversely correlated with overall and progression-free survival in high-grade gliomas (all P DNA methyltransferase (MGMT) promoter methylation status. Further analysis in vitro revealed that downregulating the expression of c-Met dramatically inhibited cell migration and invasion capacities, enhanced sensitivity to TMZ chemotherapy in H4 and U87 glioma cells. Our results suggest that c-Met may serve as a potential predictive maker for clinical decision making.

  18. Inhibition of hepatitis B viral entry by nucleic acid polymers in HepaRG cells and primary human hepatocytes.

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    Clément Guillot

    Full Text Available Hepatitis B virus (HBV infection remains a major public health concern worldwide with 240 million individuals chronically infected and at risk of developing cirrhosis and hepatocellular carcinoma. Current treatments rarely cure chronic hepatitis B infection, highlighting the need for new anti-HBV drugs. Nucleic acid polymers (NAPs are phosphorothioated oligonucleotides that have demonstrated a great potential to inhibit infection with several viruses. In chronically infected human patients, NAPs administration lead to a decline of blood HBsAg and HBV DNA and to HBsAg seroconversion, the expected signs of functional cure. NAPs have also been shown to prevent infection of duck hepatocytes with the Avihepadnavirus duck hepatitis B virus (DHBV and to exert an antiviral activity against established DHBV infection in vitro and in vivo. In this study, we investigated the specific anti-HBV antiviral activity of NAPs in the HepaRG human hepatoma cell line and primary cultures of human hepatocytes. NAPs with different chemical features (phosphorothioation, 2'O-methyl ribose, 5-methylcytidine were assessed for antiviral activity when provided at the time of HBV inoculation or post-inoculation. NAPs dose-dependently inhibited HBV entry in a phosphorothioation-dependent, sequence-independent and size-dependent manner. This inhibition of HBV entry by NAPs was impaired by 2'O-methyl ribose modification. NAP treatment after viral inoculation did not elicit any antiviral activity.

  19. Inhibition of fatty acid synthesis in rat hepatocytes by exogenous polyunsaturated fatty acids is caused by lipid peroxidation

    DEFF Research Database (Denmark)

    Mikkelsen, L.; Hansen, Harald S.; Grunnet, N.

    1993-01-01

    between LDH-leakage and inhibition of fatty acid synthesis. Lipid peroxidation, measured as thiobarbituric acid-reactive substances, also showed a significant correlation with the degree of inhibition of fatty acid synthesis. Furthermore, PUFA had no inhibitory effect on fatty acid synthesis when...... peroxidation was minimized by the use of proper antioxidants. These data indicate that PUFA in vitro inhibit the insulin-induced de novo fatty acid synthesis in hepatocytes from starved rats, due to cytotoxic effects caused by lipid peroxidation....... by the peroxidized PUFA. Arachidonic acid and eicosapentaenoic acid showed a dose- and time-dependent cytotoxicity. Two other antioxidants: 50 µM a-tocopherol acid succinate and 1 µM N,N'-diphenyl-1,4-phenylenediamine, both proved more efficient than a-tocopherol phosphate. There was a significant correlation...

  20. Dicer-dependent production of microRNA221 in hepatocytes inhibits p27 and is required for liver regeneration in mice.

    Science.gov (United States)

    Oya, Yuki; Masuzaki, Ryota; Tsugawa, Daisuke; Ray, Kevin C; Dou, Yongchao; Karp, Seth J

    2017-05-01

    Dicer processes microRNAs (miRs) into active forms in a wide variety of tissues, including the liver. To determine the role of Dicer in liver regeneration, we performed a series of in vivo and in vitro studies in a murine 2/3 hepatectomy model. Dicer was downregulated after 2/3 hepatectomy, and loss of Dicer inhibited liver regeneration associated with decreased cyclin A2 and miR-221, as well as increased levels of the cell cycle inhibitor p27. In vitro, miR-221 inhibited p27 production in primary hepatocytes and increased hepatocyte proliferation. Specific reconstitution of miR-221 in hepatocyte-specific Dicer-null mice inhibited p27 and restored liver regeneration. In wild type mice, targeted inhibition of miR-221 using a cholesterol-conjugated miR-221 inhibited hepatocyte proliferation after 2/3 hepatectomy. These results identify Dicer production of miR-221 as an essential component of a miRNA-dependent mechanism for suppression of p27 that controls the rate of hepatocyte proliferation after partial hepatectomy. NEW & NOTEWORTHY Our findings demonstrate a direct role for microRNAs in controlling the rate of liver regeneration after injury. By deleting Dicer, an enzyme responsible for processing microRNAs into mature forms, we determined miR-221 is a critical microRNA in the physiological process of restoration of liver mass after injury. miR-221 suppresses p27, releasing its inhibitory effects on hepatocyte proliferation. Pharmaceuticals based on miR-221 may be useful to modulate hepatocyte proliferation in the setting of liver injury. Copyright © 2017 the American Physiological Society.

  1. Downregulation of mPGES-1 Expression via EGR1 Plays an Important Role in Inhibition of Caffeine on PGE2 Synthesis of HBx(+) Hepatocytes.

    Science.gov (United States)

    Ma, Yan; Wang, Xiaoqian; Tang, Nanhong

    2015-01-01

    We investigated the mechanism of caffeine in influencing HBx(+) hepatocytes to synthesize PGE2. The inhibitory effect of caffeine on hepatocyte proliferation increased with increasing caffeine concentrations (200-800 μM) and treatment times (1-7 days), which was first observed at the second test time point (caffeine treatment for 4 days). The inhibition of caffeine on the growth of HL7702-HBx and HepG2-HBx cells was most obvious at 800 μM caffeine and at caffeine treatment for 7 days. The PGE2 secretion and the expression of mPGES-1 and EGR1 were downregulated, whereas PPARγ expression was upregulated. The mPGES-1 promoter activity of HBx(+) hepatocytes decreased more significantly than that of HBx(-) hepatocytes. Moreover, the expression of EGR1 and PPARγ changed more significantly in HBx(+) hepatocytes cultured for 12 to 24 hours in the presence of 5 mM caffeine. This limited success may be attributed to caffeine releasing the binding of HBx and PPARγ and furthermore affecting the mPGES-1 expression by EGR1 in HBx(+) hepatocytes. The results indicate that caffeine could effectively reduce PGE2 synthesis in HBx(+) hepatocytes by specifically blocking the PPARγ-EGR1-mPGES-1 pathway, thereby providing a new evidence of molecular biology for the hypothesis that drinking coffee is beneficial to HBV-infected patients.

  2. Downregulation of mPGES-1 Expression via EGR1 Plays an Important Role in Inhibition of Caffeine on PGE2 Synthesis of HBx(+ Hepatocytes

    Directory of Open Access Journals (Sweden)

    Yan Ma

    2015-01-01

    Full Text Available We investigated the mechanism of caffeine in influencing HBx(+ hepatocytes to synthesize PGE2. The inhibitory effect of caffeine on hepatocyte proliferation increased with increasing caffeine concentrations (200–800 μM and treatment times (1–7 days, which was first observed at the second test time point (caffeine treatment for 4 days. The inhibition of caffeine on the growth of HL7702-HBx and HepG2-HBx cells was most obvious at 800 μM caffeine and at caffeine treatment for 7 days. The PGE2 secretion and the expression of mPGES-1 and EGR1 were downregulated, whereas PPARγ expression was upregulated. The mPGES-1 promoter activity of HBx(+ hepatocytes decreased more significantly than that of HBx(− hepatocytes. Moreover, the expression of EGR1 and PPARγ changed more significantly in HBx(+ hepatocytes cultured for 12 to 24 hours in the presence of 5 mM caffeine. This limited success may be attributed to caffeine releasing the binding of HBx and PPARγ and furthermore affecting the mPGES-1 expression by EGR1 in HBx(+ hepatocytes. The results indicate that caffeine could effectively reduce PGE2 synthesis in HBx(+ hepatocytes by specifically blocking the PPARγ-EGR1-mPGES-1 pathway, thereby providing a new evidence of molecular biology for the hypothesis that drinking coffee is beneficial to HBV-infected patients.

  3. Signal transduction and downregulation of C-MET in HGF stimulated low and highly metastatic human osteosarcoma cells

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    Husmann, Knut, E-mail: khusmann@research.balgrist.ch [Laboratory for Orthopedic Research, Department of Orthopedics, Balgrist University Hospital, University of Zurich, Zurich (Switzerland); Ducommun, Pascal [Laboratory for Orthopedic Research, Department of Orthopedics, Balgrist University Hospital, University of Zurich, Zurich (Switzerland); Division of Plastic Surgery and Hand Surgery, Department of Surgery, University Hospital Zurich, Zurich (Switzerland); Sabile, Adam A.; Pedersen, Else-Marie; Born, Walter; Fuchs, Bruno [Laboratory for Orthopedic Research, Department of Orthopedics, Balgrist University Hospital, University of Zurich, Zurich (Switzerland)

    2015-09-04

    The poor outcome of osteosarcoma (OS), particularly in patients with metastatic disease and a five-year survival rate of only 20%, asks for more effective therapeutic strategies targeting malignancy-promoting mechanisms. Dysregulation of C-MET, its ligand hepatocyte growth factor (HGF) and the fusion oncogene product TPR-MET, first identified in human MNNG-HOS OS cells, have been described as cancer-causing factors in human cancers. Here, the expression of these molecules at the mRNA and the protein level and of HGF-stimulated signaling and downregulation of C-MET was compared in the parental low metastatic HOS and MG63 cell lines and the respective highly metastatic MNNG-HOS and 143B and the MG63-M6 and MG63-M8 sublines. Interestingly, expression of TPR-MET was only observed in MNNG-HOS cells. HGF stimulated the phosphorylation of Akt and Erk1/2 in all cell lines investigated, but phospho-Stat3 remained at basal levels. Downregulation of HGF-stimulated Akt and Erk1/2 phosphorylation was much faster in the HGF expressing MG63-M8 cells than in HOS cells. Degradation of HGF-activated C-MET occurred predominantly through the proteasomal and to a lesser extent the lysosomal pathway in the cell lines investigated. Thus, HGF-stimulated Akt and Erk1/2 signaling as well as proteasomal degradation of HGF activated C-MET are potential therapeutic targets in OS. - Highlights: • Expression of TPR-MET was only observed in MNNG-HOS cells. • HGF stimulated the phosphorylation of Akt and Erk1/2 but not of Stat3 in osteosarcoma cell lines. • Degradation of HGF-activated C-MET occurred predominantly through the proteasomal pathway.

  4. Inhibition of glucose-6-phosphate dehydrogenase protects hepatocytes from aluminum phosphide-induced toxicity.

    Science.gov (United States)

    Salimi, Ahmad; Paeezi, Maryam; Yousefsani, Bahareh Sadat; Shadnia, Shahin; Hassanian-Moghaddam, Hossein; Zamani, Nasim; Pourahmad, Jalal

    2017-11-01

    Aluminum phosphide (AlP) poisoning is a severe toxicity with 30-70% mortality rate. However, several case reports presented AlP-poisoned patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency and extensive hemolysis who survived the toxicity. This brought to our mind that maybe G6PD deficiency could protect the patients from severe fatal poisoning by this pesticide. In this research, we investigated the protective effect of 6-aminonicotinamide (6-AN)- as a well-established inhibitor of the NADP+- dependent enzyme 6-phosphogluconate dehydrogenase- on isolated rat hepatocytes in AlP poisoning. Hepatocytes were isolated by collagenase perfusion method and incubated into three different flasks: control, AlP, and 6-AN+ALP. Cellar parameters such as cell viability, reactive oxygen species (ROS) formation, mitochondria membrane potential collapse (MMP), lysosomal integrity, content of reduced (GSH) and oxidized glutathione (GSSG) and lipid peroxidation were assayed at intervals. All analyzed cellular parameters significantly decreased in the third group (6-AN+AlP) compared to the second group (AlP), showing the fact that G6PD deficiency induced by 6-AN had a significant protective effect on the hepatocytes. It was concluded that G6PD deficiency significantly reduced the hepatotoxicity of AlP. Future drugs with the power to induce such deficiency may be promising in treatment of AlP poisoning. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Sab (Sh3bp5) dependence of JNK mediated inhibition of mitochondrial respiration in palmitic acid induced hepatocyte lipotoxicity.

    Science.gov (United States)

    Win, Sanda; Than, Tin Aung; Le, Bao Han Allison; García-Ruiz, Carmen; Fernandez-Checa, Jose C; Kaplowitz, Neil

    2015-06-01

    Sustained c-Jun N-terminal kinase (JNK) activation by saturated fatty acids plays a role in lipotoxicity and the pathogenesis of non-alcoholic steatohepatitis (NASH). We have reported that the interaction of JNK with mitochondrial Sab leads to inhibition of respiration, increased reactive oxygen species (ROS), cell death and hepatotoxicity. We tested whether this pathway underlies palmitic acid (PA)-induced lipotoxicity in hepatocytes. Primary mouse hepatocytes (PMH) from adeno-shlacZ or adeno-shSab treated mice and HuH7 cells were used. In PMH, PA dose-dependently up to 1mM stimulated oxygen consumption rate (OCR) due to mitochondrial β-oxidation. At ⩾1.5mM, PA gradually reduced OCR, followed by cell death. Inhibition of JNK, caspases or treatment with antioxidant butylated hydroxyanisole (BHA) protected PMH against cell death. Sab knockdown or a membrane permeable Sab blocking peptide prevented PA-induced mitochondrial impairment, but inhibited only the late phase of both JNK activation (beyond 4h) and cell death. In PMH, PA increased p-PERK and its downstream target CHOP, but failed to activate the IRE-1α arm of the UPR. However, Sab silencing did not affect PA-induced PERK activation. Conversely, specific inhibition of PERK prevented JNK activation and cell death, indicating a major role upstream of JNK activation. The effect of p-JNK on mitochondria plays a key role in PA-mediated lipotoxicity. The interplay of p-JNK with mitochondrial Sab leads to impaired respiration, ROS production, sustained JNK activation, and apoptosis. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  6. Lysophosphatidic Acid Inhibits Insulin Signaling in Primary Rat Hepatocytes via the LPA3 Receptor Subtype and is Increased in Obesity

    Directory of Open Access Journals (Sweden)

    Susann Fayyaz

    2017-09-01

    Full Text Available Background/Aims: Obesity is a main risk factor for the development of hepatic insulin resistance and it is accompanied by adipocyte hypertrophy and an elevated expression of different adipokines such as autotaxin (ATX. ATX converts lysophosphatidylcholine to lysophosphatidic acid (LPA and acts as the main producer of extracellular LPA. This bioactive lipid regulates a broad range of physiological and pathological responses by activation of LPA receptors (LPA1-6. Methods: The activation of phosphatidylinositide 3-kinases (PI3K signaling (Akt and GSK-3ß was analyzed via western blotting in primary rat hepatocytes. Incorporation of glucose into glycogen was measured by using radio labeled glucose. Real-time PCR analysis and pharmacological modulation of LPA receptors were performed. Human plasma LPA levels of obese (BMI > 30, n = 18 and normal weight individuals (BMI 18.5-25, n = 14 were analyzed by liquid chromatography tandem-mass spectrometry (LC-MS/MS. Results: Pretreatment of primary hepatocytes with LPA resulted in an inhibition of insulin-mediated Gck expression, PI3K activation and glycogen synthesis. Pharmacological approaches revealed that the LPA3-receptor subtype is responsible for the inhibitory effect of LPA on insulin signaling. Moreover, human plasma LPA concentrations (16: 0 LPA of obese participants (BMI > 30 are significantly elevated in comparison to normal weight individuals (BMI 18.5-25. Conclusion: LPA is able to interrupt insulin signaling in primary rat hepatocytes via the LPA3 receptor subtype. Moreover, the bioactive lipid LPA (16: 0 is increased in obesity.

  7. Lysophosphatidic Acid Inhibits Insulin Signaling in Primary Rat Hepatocytes via the LPA3 Receptor Subtype and is Increased in Obesity.

    Science.gov (United States)

    Fayyaz, Susann; Japtok, Lukasz; Schumacher, Fabian; Wigger, Dominik; Schulz, Tim Julius; Haubold, Kathrin; Gulbins, Erich; Völler, Heinz; Kleuser, Burkhard

    2017-01-01

    Obesity is a main risk factor for the development of hepatic insulin resistance and it is accompanied by adipocyte hypertrophy and an elevated expression of different adipokines such as autotaxin (ATX). ATX converts lysophosphatidylcholine to lysophosphatidic acid (LPA) and acts as the main producer of extracellular LPA. This bioactive lipid regulates a broad range of physiological and pathological responses by activation of LPA receptors (LPA1-6). The activation of phosphatidylinositide 3-kinases (PI3K) signaling (Akt and GSK-3ß) was analyzed via western blotting in primary rat hepatocytes. Incorporation of glucose into glycogen was measured by using radio labeled glucose. Real-time PCR analysis and pharmacological modulation of LPA receptors were performed. Human plasma LPA levels of obese (BMI > 30, n = 18) and normal weight individuals (BMI 18.5-25, n = 14) were analyzed by liquid chromatography tandem-mass spectrometry (LC-MS/MS). Pretreatment of primary hepatocytes with LPA resulted in an inhibition of insulin-mediated Gck expression, PI3K activation and glycogen synthesis. Pharmacological approaches revealed that the LPA3-receptor subtype is responsible for the inhibitory effect of LPA on insulin signaling. Moreover, human plasma LPA concentrations (16: 0 LPA) of obese participants (BMI > 30) are significantly elevated in comparison to normal weight individuals (BMI 18.5-25). LPA is able to interrupt insulin signaling in primary rat hepatocytes via the LPA3 receptor subtype. Moreover, the bioactive lipid LPA (16: 0) is increased in obesity. © 2017 The Author(s). Published by S. Karger AG, Basel.

  8. Novel Roles of c-Met in the Survival of Renal Cancer Cells through the Regulation of HO-1 and PD-L1 Expression*

    Science.gov (United States)

    Balan, Murugabaskar; Mier y Teran, Eduardo; Waaga-Gasser, Ana Maria; Gasser, Martin; Choueiri, Toni K.; Freeman, Gordon; Pal, Soumitro

    2015-01-01

    The receptor tyrosine kinase c-Met is overexpressed in renal cancer cells and can play major role in the growth and survival of tumor. We investigated how the c-Met-mediated signaling through binding to its ligand hepatocyte growth factor (HGF) can modulate the apoptosis and immune escape mechanism(s) of renal cancer cells by the regulations of novel molecules heme oxygenase-1 (HO-1) and programmed death-1 ligand 1 (PD-L1). We found that HGF/c-Met-mediated signaling activated the Ras/Raf pathway and down-regulated cancer cell apoptosis; and it was associated with the overexpression of cytoprotective HO-1 and anti-apoptotic Bcl-2/Bcl-xL. c-Met-induced HO-1 overexpression was regulated at the transcriptional level. Next, we observed that c-Met induction markedly up-regulated the expression of the negative co-stimulatory molecule PD-L1, and this can be prevented following treatment of the cells with pharmacological inhibitors of c-Met. Interestingly, HGF/c-Met-mediated signaling could not induce PD-L1 at the optimum level when either Ras or HO-1 was knocked down. To study the functional significance of c-Met-induced PD-L1 expression, we performed a co-culture assay using mouse splenocytes (expressing PD-L1 receptor PD-1) and murine renal cancer cells (RENCA, expressing high PD-L1). We observed that the splenocyte-mediated apoptosis of cancer cells during co-culture was markedly increased in the presence of either c-Met inhibitor or PD-L1 neutralizing antibody. Finally, we found that both c-Met and PD-L1 are significantly up-regulated and co-localized in human renal cancer tissues. Together, our study suggests a novel mechanism(s) by which c-Met can promote increased survival of renal cancer cells through the regulation of HO-1 and PD-L1. PMID:25645920

  9. Novel roles of c-Met in the survival of renal cancer cells through the regulation of HO-1 and PD-L1 expression.

    Science.gov (United States)

    Balan, Murugabaskar; Mier y Teran, Eduardo; Waaga-Gasser, Ana Maria; Gasser, Martin; Choueiri, Toni K; Freeman, Gordon; Pal, Soumitro

    2015-03-27

    The receptor tyrosine kinase c-Met is overexpressed in renal cancer cells and can play major role in the growth and survival of tumor. We investigated how the c-Met-mediated signaling through binding to its ligand hepatocyte growth factor (HGF) can modulate the apoptosis and immune escape mechanism(s) of renal cancer cells by the regulations of novel molecules heme oxygenase-1 (HO-1) and programmed death-1 ligand 1 (PD-L1). We found that HGF/c-Met-mediated signaling activated the Ras/Raf pathway and down-regulated cancer cell apoptosis; and it was associated with the overexpression of cytoprotective HO-1 and anti-apoptotic Bcl-2/Bcl-xL. c-Met-induced HO-1 overexpression was regulated at the transcriptional level. Next, we observed that c-Met induction markedly up-regulated the expression of the negative co-stimulatory molecule PD-L1, and this can be prevented following treatment of the cells with pharmacological inhibitors of c-Met. Interestingly, HGF/c-Met-mediated signaling could not induce PD-L1 at the optimum level when either Ras or HO-1 was knocked down. To study the functional significance of c-Met-induced PD-L1 expression, we performed a co-culture assay using mouse splenocytes (expressing PD-L1 receptor PD-1) and murine renal cancer cells (RENCA, expressing high PD-L1). We observed that the splenocyte-mediated apoptosis of cancer cells during co-culture was markedly increased in the presence of either c-Met inhibitor or PD-L1 neutralizing antibody. Finally, we found that both c-Met and PD-L1 are significantly up-regulated and co-localized in human renal cancer tissues. Together, our study suggests a novel mechanism(s) by which c-Met can promote increased survival of renal cancer cells through the regulation of HO-1 and PD-L1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Niacin inhibits fat accumulation, oxidative stress, and inflammatory cytokine IL-8 in cultured hepatocytes: Impact on non-alcoholic fatty liver disease.

    Science.gov (United States)

    Ganji, Shobha H; Kashyap, Moti L; Kamanna, Vaijinath S

    2015-09-01

    Non-alcoholic fatty liver disease (NAFLD) is a common disorder characterized by excessive hepatic fat accumulation, production of reactive oxygen species (ROS), inflammation and potentially resulting in non-alcoholic steatohepatitis (NASH), cirrhosis and end-stage liver disease. Recently, we have shown that niacin significantly prevented hepatic steatosis and regressed pre-existing steatosis in high-fat fed rat model of NAFLD. To gain further insight into the cellular mechanisms, this study investigated the effect of niacin on human hepatocyte fat accumulation, ROS production, and inflammatory mediator IL-8 secretion. Human hepatoblastoma cell line HepG2 or human primary hepatocytes were first stimulated with palmitic acid followed by treatment with niacin or control for 24 h. The data indicated that niacin (at 0.25 and 0.5 mmol/L doses) significantly inhibited palmitic acid-induced fat accumulation in human hepatocytes by 45-62%. This effect was associated with inhibition of diacylglycerol acyltransferase 2 (DGAT2) mRNA expression without affecting the mRNA expression of fatty acid synthase (FAS) and carnitine palmitoyltransferase 1 (CPT1). Niacin attenuated hepatocyte ROS production and it also inhibited NADPH oxidase activity. Niacin reduced palmitic acid-induced IL-8 levels. These findings suggest that niacin, through inhibiting hepatocyte DGAT2 and NADPH oxidase activity, attenuates hepatic fat accumulation and ROS production respectively. Decreased ROS production, at least in part, may have contributed to the inhibition of pro-inflammatory IL-8 levels. These mechanistic studies may be useful for the clinical development of niacin and niacin-related compounds for the treatment of NAFLD/NASH and its complications. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Downregulation of mPGES-1 Expression via EGR1 Plays an Important Role in Inhibition of Caffeine on PGE2 Synthesis of HBx(+) Hepatocytes

    OpenAIRE

    Yan Ma; Xiaoqian Wang; Nanhong Tang

    2015-01-01

    We investigated the mechanism of caffeine in influencing HBx(+) hepatocytes to synthesize PGE2. The inhibitory effect of caffeine on hepatocyte proliferation increased with increasing caffeine concentrations (200?800??M) and treatment times (1?7 days), which was first observed at the second test time point (caffeine treatment for 4 days). The inhibition of caffeine on the growth of HL7702-HBx and HepG2-HBx cells was most obvious at 800??M caffeine and at caffeine treatment for 7 days. The PGE...

  12. Activation of the constitutive androstane receptor inhibits gluconeogenesis without affecting lipogenesis or fatty acid synthesis in human hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lynch, Caitlin; Pan, Yongmei; Li, Linhao [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States); Heyward, Scott; Moeller, Timothy [Bioreclamation In Vitro Technologies, Baltimore, MD 21227 (United States); Swaan, Peter W. [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States); Wang, Hongbing, E-mail: hwang@rx.umaryland.edu [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States)

    2014-08-15

    Objective: Accumulating evidence suggests that activation of mouse constitutive androstane receptor (mCAR) alleviates type 2 diabetes and obesity by inhibiting hepatic gluconeogenesis, lipogenesis, and fatty acid synthesis. However, the role of human (h) CAR in energy metabolism is largely unknown. The present study aims to investigate the effects of selective hCAR activators on hepatic energy metabolism in human primary hepatocytes (HPH). Methods: Ligand-based structure–activity models were used for virtual screening of the Specs database ( (www.specs.net)) followed by biological validation in cell-based luciferase assays. The effects of two novel hCAR activators (UM104 and UM145) on hepatic energy metabolism were evaluated in HPH. Results: Real-time PCR and Western blotting analyses reveal that activation of hCAR by UM104 and UM145 significantly repressed the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two pivotal gluconeogenic enzymes, while exerting negligible effects on the expression of genes associated with lipogenesis and fatty acid synthesis. Functional experiments show that UM104 and UM145 markedly inhibit hepatic synthesis of glucose but not triglycerides in HPH. In contrast, activation of mCAR by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, a selective mCAR activator, repressed the expression of genes associated with gluconeogenesis, lipogenesis, and fatty acid synthesis in mouse primary hepatocytes, which were consistent with previous observations in mouse model in vivo. Conclusion: Our findings uncover an important species difference between hCAR and mCAR in hepatic energy metabolism, where hCAR selectively inhibits gluconeogenesis without suppressing fatty acid synthesis. Implications: Such species selectivity should be considered when exploring CAR as a potential therapeutic target for metabolic disorders. - Highlights: • Novel hCAR activators were identified by computational and biological approaches. • The role

  13. MiR-375 inhibits the hepatocyte growth factor-elicited migration of mesenchymal stem cells by downregulating Akt signaling.

    Science.gov (United States)

    He, Lihong; Wang, Xianyao; Kang, Naixin; Xu, Jianwei; Dai, Nan; Xu, Xiaojing; Zhang, Huanxiang

    2018-01-10

    The migration of mesenchymal stem cells (MSCs) is critical for their use in cell-based therapies. Accumulating evidence suggests that microRNAs are important regulators of MSC migration. Here, we report that the expression of miR-375 was downregulated in MSCs treated with hepatocyte growth factor (HGF), which strongly stimulates the migration of these cells. Overexpression of miR-375 decreased the transfilter migration and the migration velocity of MSCs triggered by HGF. In our efforts to determine the mechanism by which miR-375 affects MSC migration, we found that miR-375 significantly inhibited the activation of Akt by downregulating its phosphorylation at T308 and S473, but had no effect on the activity of mitogen-activated protein kinases. Further, we showed that 3'phosphoinositide-dependent protein kinase-1 (PDK1), an upstream kinase necessary for full activation of Akt, was negatively regulated by miR-375 at the protein level. Moreover, miR-375 suppressed the phosphorylation of focal adhesion kinase (FAK) and paxillin, two important regulators of focal adhesion (FA) assembly and turnover, and decreased the number of FAs at cell periphery. Taken together, our results demonstrate that miR-375 inhibits HGF-elicited migration of MSCs through downregulating the expression of PDK1 and suppressing the activation of Akt, as well as influencing the tyrosine phosphorylation of FAK and paxillin and FA periphery distribution.

  14. Prognostic value of c-MET in head and neck cancer: A systematic review and meta-analysis of aggregate data.

    Science.gov (United States)

    Szturz, Petr; Budíková, Marie; Vermorken, Jan B; Horová, Ivana; Gál, Břetislav; Raymond, Eric; de Gramont, Armand; Faivre, Sandrine

    2017-11-01

    The hepatocyte growth factor (HGF)/mesenchymal-epithelial transition factor (c-MET) ligand/receptor axis has been implicated in pathogenesis of malignant diseases including squamous cell carcinoma of the head and neck (SCCHN). Overexpression of c-MET has been reported as a common molecular abnormality in SCCHN, although its prognostic and predictive value remains to be validated. We systematically searched literature for studies evaluating c-MET expression on immunohistochemistry in newly diagnosed, non-metastatic SCCHN. The c-MET expressing cases were classified into three categories according to predefined cut-off values for positivity. Our aim was to assess the prevalence of c-MET expression and its relationship with selected clinicopathological variables. Twenty-eight studies with 2019 cases were included. Relative frequencies of c-MET expression above cut-off levels I, II, and III were 81.8%, 63.8%, and 46.2%, respectively. Differences between these three values were statistically significant (pc-MET positivity was associated with worse overall survival (p=4.0×10(-6)), positive nodal status (p=1.0×10(-4)), higher disease stage (p=7.0×10(-4)), older age (p=2.1×10(-3)), disease recurrence (p=2.0×10(-2)), and primary tumour localization in the oral cavity (p=2.3×10(-2)). Above cut-off level III, c-MET positivity was associated with worse disease-free or progression-free survival (p=9.0×10(-6)), p16 negativity (p=2.4×10(-4)), worse overall survival (p=4.0×10(-4)), positive epidermal growth factor receptor (EGFR) status (p=7.2×10(-4)), and larger primary tumours (p=4.6×10(-3)). In SCCHN, immunohistochemical overexpression of c-MET above cut-off levels III and particularly II was associated with inferior survival outcomes and advanced disease. Moreover, it represents a promising predictive biomarker for c-MET targeting, yet the optimal scoring method remains to be defined. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Dynamic expression and localization of c-MET isoforms in the developing rat pancreas.

    Science.gov (United States)

    Wu, Yulong; Cheng, Mei; Shi, Zhen; Feng, Zhenqing; Guan, Xiaohong

    2014-01-01

    Pancreata from Sprague Dawley rats of different developmental stages were studied to determine the expression and cellular localization of different c-MET isoforms in the developing rat pancreas. Pancreatic mRNA and protein expression levels of c-MET at different developmental stages from embryo to adult were detected by reverse transcription-polymerase chain reaction and by western blotting. To identify the cellular localization of c-MET protein in the developing rat pancreas, double immunofluorescent staining was performed using antibodies for cell type-specific markers and for c-MET. The expression of two isoforms of c-MET (190 kDa and 170 kDa) coincided with the development of the pancreas. The 190 kDa isoform of c-MET is expressed during embryonic stages, and its expression is replaced by the expression of the 170 kDa isoform as the pancreas develops. Only the 170 kDa isoform is expressed in the adult rat pancreas. Throughout all stages of pancreatic development, c-MET is expressed by vimentin-positive cells. In contrast, c-MET staining was stronger in rat pancreata from newborn to adult stages and overlapped with insulin-positive beta-cells. The dynamic expression and localization of different c-MET isoforms in the rat pancreas during different developmental stages indicates that distinct c-MET isoform might be involved in different aspects of pancreatic development.

  16. Competitive inhibition of the uptake of demethylphalloin by cholic acid in isolated hepatocytes. Evidence for a transport competition rather than a binding competition.

    Science.gov (United States)

    Petzinger, E

    1981-07-01

    Cholic acid inhibits the uptake of demethylphalloin (DMP), in a competitive manner. The bile acid increases the Michaelis constant but not Vmax of the inward transport. The inhibition constant Ki for cholate was found to be 8 microM. Cholate competes for the transport system but not for intracellular binding of phallotoxins. Various experimental data presented in this paper exclude an accumulation of phallotoxins in hepatocytes by intracellular binding only. Preincubation of hepatocytes with small concentration of either (3H)-demethylphalloin or (14C)-cholate and subsequent treatment with high concentrations of the non-labelled compounds reduces the intracellular concentration of both radioactive substrates. In accordance with earlier findings the above results suggest a common component needed for the transport of both phallotoxins and cholic acid.

  17. Citreoviridin induces triglyceride accumulation in hepatocytes through inhibiting PPAR-α in vivo and in vitro.

    Science.gov (United States)

    Feng, Chang; Li, Dandan; Jiang, Liping; Liu, Xiaofang; Li, Qiujuan; Geng, Chengyan; Sun, Xiance; Yang, Guang; Yao, Xiaofeng; Chen, Min

    2017-08-01

    Citreoviridin (CIT) is a mycotoxin produced by Penicillum citreonigrum, Aspergillus terreus and Eupenicillium ochrosalmoneum. CIT occurs naturally in moldy rice and corn. CIT is associated with the development of atherosclerosis in the general population. Alteration in hepatic lipid metabolism is a pathogenic factor in atherosclerosis. However the effect and the underlying mechanism of CIT on hepatic lipid metabolism are largely unknown. In this study, we reported that CIT induced triglyceride accumulation in mice liver and human liver HepG2 cells as shown in oil red O staining. CIT (0.1 mg/kg-0.3 mg/kg) for 6 weeks elevated liver triglyceride contents in mice. CIT inhibited the transactivation activity of peroxisome proliferator-activated receptor-α (PPAR-α) in hepatocyte in vivo and in vitro, as shown by the reduced mRNA levels of PPAR-α target genes which play key roles in lipid metabolism in various aspects. PPAR-α agonist fenofibrate attenuated CIT-induced triglyceride accumulation in HepG2 cells. Furthermore, CIT increased serum total cholesterol/high-density lipoprotein cholesterol ratio, a strong risk factor for cardiovascular disease. In summary, we reported that CIT induced PPAR-α-dependent hepatic triglyceride accumulation and dyslipidemia. Our data will provide new mechanistic insights into CIT-induced lipid alterations. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Lipopolysaccharide preconditioning protects hepatocytes from ischemia/reperfusion injury (IRI through inhibiting ATF4-CHOP pathway in mice.

    Directory of Open Access Journals (Sweden)

    Jianhua Rao

    consistent with results of LPS preconditioning in macrophages. CONCLUSIONS: This work is the first time to provide evidence for LPS preconditioning protects hepatocytes from IRI through inhibiting ATF4-CHOP pathway, which may be critical to reducing related apoptosis molecules and modulating innate inflammation.

  19. Phase II study of the c-MET inhibitor tivantinib (ARQ 197) in patients with relapsed or relapsed/refractory multiple myeloma.

    Science.gov (United States)

    Baljevic, Muhamed; Zaman, Shadia; Baladandayuthapani, Veerabhadran; Lin, Yan Heather; de Partovi, Claudia Morales; Berkova, Zuzana; Amini, Behrang; Thomas, Sheeba K; Shah, Jatin J; Weber, Donna M; Fu, Min; Cleeland, Charles S; Wang, Xin Shelley; Stellrecht, Christine M; Davis, Richard E; Gandhi, Varsha; Orlowski, Robert Z

    2017-06-01

    The hepatocyte growth factor/c-MET pathway has been implicated in the pathobiology of multiple myeloma, and c-MET inhibitors induce myeloma cell apoptosis, suggesting that they could be useful clinically. We conducted a phase II study with the c-MET inhibitor tivantinib in patients with relapsed, or relapsed and refractory myeloma whose disease had progressed after one to four prior therapies. Tivantinib, 360 mg orally per dose, was administered twice daily continuously over a 4-week treatment cycle without a cap on the number of allowed cycles, barring undue toxicities or disease progression. Primary objectives were to determine the overall response rate and the toxicities of tivantinib in this patient population. Sixteen patients were enrolled in a two-stage design. Notable grade 3 and 4 hematological adverse events were limited to neutropenia in five and four patients, respectively. Nonhematological adverse events of grade 3 or higher included hypertension (in four patients); syncope, infection, and pain (two each); and fatigue, cough, and pulmonary embolism (one each). Four of 11 evaluable patients (36%) had stable disease as their best response, while the remainder showed disease progression. Overall, tivantinib as a single agent did not show promise for unselected relapsed/refractory myeloma patients. However, the ability to achieve stable disease does suggest that combination regimens incorporating targeted inhibitors in patients with c-MET pathway activation could be of interest.

  20. Glycochenodeoxycholate (GCDC) inhibits cytokine induced iNOS expression in rat hepatocytes.

    Science.gov (United States)

    Bucher, Brian T; Feng, Xuesheng; Jeyabalan, Geetha; Zhang, Baochun; Shao, Lifang; Guo, Zhong; Geller, David A

    2007-03-01

    Although the accumulation of hydrophobic bile acid (e.g., glycine conjugated chenodeoxycholic acid, GCDC) is considered to be an important factor contributing to cholestatic liver dysfunction, its pathogenesis is poorly understood. The purpose of this study was to examine the effect of the bile salt GCDC on the regulation of iNOS expression, a key immune modulator during liver inflammation. GCDC significantly decreased cytokine-stimulated iNOS promoter activity, and both iNOS mRNA and protein expression. GCDC decreased iNOS promoter activity by preventing IkappaB degradation and inhibiting NF-kappaB DNA-binding activity. To explore the role of iNOS in bile salt induced apoptosis, we also examined the effect of NO on caspase-3 activity. GCDC strongly induced caspase-3 activity, and this increase was abrogated by both exogenous NO exposure and endogenous NO synthesis. Furthermore, adenoviral iNOS (AdiNOS) pre-treatment decreased acute cholestatic-induced liver injury in a rat bile duct ligation model. These findings indicate a novel signaling pathway where potentially toxic bile salts down-regulate hepatic iNOS expression. This blockade of the iNOS mediated antiapoptotic phenotype may have important implications in certain liver disorders.

  1. Cyclooxygenase-2 inhibition inhibits c-Met kinase activity and wnt activity in colon cancer

    NARCIS (Netherlands)

    Tuynman, Jurriaan B.; Vermeulen, Louis; Boon, Elles M.; Kemper, Kristel; Zwinderman, Aeilko H.; Peppelenbosch, Maikel P.; Richel, Dirk J.

    2008-01-01

    Activity of receptor tyrosine kinases (RTK) in colorectal cancer (CRC) is associated with enhanced tumor growth and a poorer prognosis. In addition, cyclooxygenase-2 (COX-2) expression contributes to tumor growth and invasion. COX-2 inhibitors exhibit important anticarcinogenic potential against

  2. The expression of c-Met pathway components in unclassified pleomorphic sarcoma/malignant fibrous histiocytoma (UPS/MFH): a tissue microarray study.

    Science.gov (United States)

    Lahat, Guy; Zhang, Pingyu; Zhu, Quan-Sheng; Torres, Keila; Ghadimi, Markus; Smith, Kerrington D; Wang, Wei-Lien; Lazar, Alexander J; Lev, Dina

    2011-09-01

    Subclassification of undifferentiated pleomorphic sarcoma/malignant fibrous histiocytoma (UPS/MFH) into distinct biological cohorts based on the expression patterns of molecular markers can identify patient subsets with especially unfavourable clinical outcomes. Identification of molecular prognosticators amenable for drug targeting can facilitate rational development of UPS/MFH tailored therapies. The aim was to evaluate expression of c-Met pathway components in a large cohort of UPS/MFH samples. An immunohistochemical analysis for hepatocyte growth factor (HGF), c-Met, phospho-c-Met (pc-Met), phospho-mitogen-activated protein kinase kinase (MAPKK) also known as mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (p-MEK) and phospho-protein kinase B (p-AKT) was performed on a clinically annotated tissue microarray of 158 UPS/MFH samples. Univariable and multivariable analyses were conducted to evaluate the correlation of molecular variables with UPS/MFH disease specific survival. All evaluated markers were expressed in UPS/MFH to varying levels. Most importantly, strong HGF, pc-Met, p-MEK and p-AKT expression correlated significantly with dismal patient outcome on univariable statistical analysis. Expression of p-MEK and p-AKT remained statistically significant independent prognosticators on multivariable analysis. c-Met pathway components and especially p-MEK and p-AKT are potential prognostic biomarkers for UPS/MFH; their inclusion in future molecular-based staging systems should be evaluated. Furthermore, novel approaches targeting HGF, c-Met, MEK/extracellular-regulated kinase (ERK) and/or AKT should be considered for a subset of UPS/MFH patients. © 2011 Blackwell Publishing Limited.

  3. Phosphatidylinositol 3-Kinase and Protein Kinase C Contribute to the Inhibition by Interleukin 6 of Phosphoenolpyruvate Carboxykinase Gene Expression in Cultured Rat Hepatocytes

    DEFF Research Database (Denmark)

    Christ, Bruno; Yazici, Emine; Nath, Annegret

    2000-01-01

    Gluconeogenesis, hepatocytes, interleukin 6, liver, phosphoinositide 3-kinase, phosphoenolpyruvate carboxykinase......Gluconeogenesis, hepatocytes, interleukin 6, liver, phosphoinositide 3-kinase, phosphoenolpyruvate carboxykinase...

  4. Hepatocyte Growth Factor from a Clinical Perspective: A Pancreatic Cancer Challenge

    Energy Technology Data Exchange (ETDEWEB)

    Rizwani, Wasia [Department of Biochemistry, Osmania University, Hyderabad, Telangana 500007 (India); Allen, Amanda E.; Trevino, Jose G., E-mail: Jose.Trevino@surgery.ufl.edu [Department of Surgery, University of Florida, 1600 SW Archer Rd, Rm 6175, P.O. Box 100109, Gainesville, FL 32610 (United States)

    2015-09-03

    Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States and incidence rates are rising. Both detection and treatment options for pancreatic cancer are limited, providing a less than 5% five-year survival advantage. The need for new biomarkers for early detection and treatment of pancreatic cancer demands the efficient translation of bench knowledge to provide clinical benefit. One source of therapeutic resistance is the pancreatic tumor microenvironment, which is characterized by desmoplasia and hypoxia making it less conducive to current therapies. A major factor regulating desmoplasia and subsequently promoting chemoresistance in pancreatic cancer is hepatocyte growth factor (HGF), the sole ligand for c-MET (mesenchymal-epithelial transition), an epithelial tyrosine kinase receptor. Binding of HGF to c-MET leads to receptor dimerization and autophosphorylation resulting in the activation of multiple cellular processes that support cancer progression. Inhibiting activation of c-MET in cancer cells, in combination with other approaches for reducing desmoplasia in the tumor microenvironment, might significantly improve the success of chemotherapy. Therefore, HGF makes a potent novel target for developing therapeutic strategies in combination with existing drugs for treating pancreatic adenocarcinoma. This review provides a comprehensive analysis of HGF and its promising potential as a chemotherapeutic target for pancreatic cancer.

  5. Hepatocyte Growth Factor from a Clinical Perspective: A Pancreatic Cancer Challenge

    Directory of Open Access Journals (Sweden)

    Wasia Rizwani

    2015-09-01

    Full Text Available Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States and incidence rates are rising. Both detection and treatment options for pancreatic cancer are limited, providing a less than 5% five-year survival advantage. The need for new biomarkers for early detection and treatment of pancreatic cancer demands the efficient translation of bench knowledge to provide clinical benefit. One source of therapeutic resistance is the pancreatic tumor microenvironment, which is characterized by desmoplasia and hypoxia making it less conducive to current therapies. A major factor regulating desmoplasia and subsequently promoting chemoresistance in pancreatic cancer is hepatocyte growth factor (HGF, the sole ligand for c-MET (mesenchymal-epithelial transition, an epithelial tyrosine kinase receptor. Binding of HGF to c-MET leads to receptor dimerization and autophosphorylation resulting in the activation of multiple cellular processes that support cancer progression. Inhibiting activation of c-MET in cancer cells, in combination with other approaches for reducing desmoplasia in the tumor microenvironment, might significantly improve the success of chemotherapy. Therefore, HGF makes a potent novel target for developing therapeutic strategies in combination with existing drugs for treating pancreatic adenocarcinoma. This review provides a comprehensive analysis of HGF and its promising potential as a chemotherapeutic target for pancreatic cancer.

  6. Clinical and Prognostic Value of the C-Met/HGF Signaling Pathway in Cervical Cancer.

    Science.gov (United States)

    Boromand, Nadia; Hasanzadeh, Malihe; Sales, Soodabeh Shahid; Farazestanian, Marjaneh; Gharib, Masoumeh; Fiuji, Hamid; Behboodi, Negin; Ghobadi, Niloofar; Hassanian, Seyed Mahdi; Ferns, Gordon A; Avan, Amir

    2017-10-23

    Aberrant activation of the HGF/c-Met signalling pathway is reported to be associated with cell proliferation, progression, and metastasis features of several tumor types, including cervical cancer, suggesting that it may be of potential value as a novel therapeutic target. Furthermore, HPV-positive patients had a higher serum level of HGF or c-Met protein, compared with HPV-negative patients. c-Met or HGF overexpression in lesions of cervical cancer is reported to be related to a poorer prognosis, and hence this may be of value as a prognostic and predictive biomarker. Several approaches have been developed for targeting HGF and/or c-Met. One of these is crizotinib (a dual c-Met/ALK inhibitor). This has been approved by FDA for the treatment of lung-cancer. Further investigations are required to evaluate and optimize the use of c-Met inhibitors in cervical cancer or parallel targeting signalling pathway associated/activated via MET/HGF pathway. The main aim of current review was to give an overview of the potential of the c-Met/HGF pathway as a prognostic, or predictive biomarker in cervical cancer. This article isprotected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  7. Insulin inhibits AMPK activity and phosphorylates AMPK Ser⁴⁸⁵/⁴⁹¹ through Akt in hepatocytes, myotubes and incubated rat skeletal muscle.

    Science.gov (United States)

    Valentine, Rudy J; Coughlan, Kimberly A; Ruderman, Neil B; Saha, Asish K

    2014-11-15

    Recent studies have highlighted the importance of an inhibitory phosphorylation site, Ser(485/491), on the α-subunit of AMP-activated protein kinase (AMPK); however, little is known about the regulation of this site in liver and skeletal muscle. We examined whether the inhibitory effects of insulin on AMPK activity may be mediated through the phosphorylation of this inhibitory Ser(485/491) site in hepatocytes, myotubes and incubated skeletal muscle. HepG2 and C2C12 cells were stimulated with or without insulin for 15-min. Similarly, rat extensor digitorum longus (EDL) muscles were treated +/- insulin for 10-min. Insulin significantly increased Ser(485/491) p-AMPK under all conditions, resulting in a subsequent reduction in AMPK activity, ranging from 40% to 70%, despite no change in p-AMPK Thr(172). Akt inhibition both attenuated the increase in Ser(485/491) p-AMPK caused by insulin, and prevented the decrease in AMPK activity. Similarly, the growth factor IGF-1 stimulated Ser(485/491) AMPK phosphorylation, and this too was blunted by inhibition of Akt. Inhibition of the mTOR pathway with rapamycin, however, had no effect on insulin-stimulated Ser(485/491) p-AMPK. These data suggest that insulin and IGF-1 diminish AMPK activity in hepatocytes and muscle, most likely through Akt activation and the inhibitory phosphorylation of Ser(485/491) on its α-subunit. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Carbon monoxide blocks oxidative stress-induced hepatocyte apoptosis via inhibition of the p54 JNK isoform

    NARCIS (Netherlands)

    Conde de la Rosa, Laura; Vrenken, Titia E.; Hannivoort, Rebekka A.; Buist-Homan, Manon; Havinga, Rick; Slebos, Dirk-Jan; Kauffman, Henk F.; Faber, Klaas Nico; Jansen, Peter L. M.; Moshage, Han

    2008-01-01

    Most chronic liver diseases are accompanied by oxidative stress, which may induce apoptosis in hepatocytes and liver injury. Oxidative stress induces heme oxygenase-1 (HO-1) expression. This stress-responsive cytoprotective protein is responsible for heme degradation into carbon monoxide (CO), free

  9. DIFFERENCES IN PROPIONATE-INDUCED INHIBITION OF CHOLESTEROL AND TRIACYLGLYCEROL SYNTHESIS BETWEEN HUMAN AND RAT HEPATOCYTES IN PRIMARY CULTURE

    NARCIS (Netherlands)

    LIN, YG; VONK, RJ; SLOOFF, MJH; KUIPERS, F; SMIT, MJ

    Propionate is a short-chain fatty acid formed in the colon and supposedly involved in the cholesterol-lowering effect of soluble fibre. To explore the underlying mechanism(s) of this fibre action, we have used human hepatocytes in primary culture to study the effects of propionate on hepatic lipid

  10. Carbon monoxide blocks oxidative stress-induced hepatocyte apoptosis via inhibition of the p54 JNK isoform.

    NARCIS (Netherlands)

    Rosa, L Conde de la; Woudenberg-Vrenken, T.E.; Hannivoort, R.A.; Buist-Homan, M.; Havinga, R.; Slebos, D.J.; Kauffman, H.F.; Faber, K.N.; Jansen, P.L.; Moshage, H.

    2008-01-01

    Most chronic liver diseases are accompanied by oxidative stress, which may induce apoptosis in hepatocytes and liver injury. Oxidative stress induces heme oxygenase-1 (HO-1) expression. This stress-responsive cytoprotective protein is responsible for heme degradation into carbon monoxide (CO), free

  11. Association of integrin beta1 and c-MET in mediating EGFR TKI gefitinib resistance in non-small cell lung cancer

    Directory of Open Access Journals (Sweden)

    Ju Lixia

    2013-02-01

    Full Text Available Abstract Although some patients are initially sensitive to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs, resistance invariably develops. Therefore, it’s very important to study the molecular mechanism of this resistance. In our previous study we found that integrin beta1 can induce EGFR TKIs resistance in non-small cell lung cancer (NSCLC cells. Here we analyzed the association of integrin beta1 and c-MET that is a recognized mechanism of EGFR TKIs resistance in NSCLC to demonstrate the mechanism of integrin beta1 related EGFR TKIs resistance. We found that the ligands of integrin beta1 and c-MET could synergistically promote cell proliferation and their inhibitors could synergistically improve the sensitivity to gfitinib, increase apoptosis, and inhibit the downstream signal transduction: focal adhesion kinase (FAK and AKT. On the other hand, ligand-dependent activation of integrin beta1 could induce EGFR TKIs resistance through activating c-MET and its downstream signals. Thus, it can be concluded that there is crosstalk between integrin beta1 and c-MET and integrin beta1 mediates EGFR TKI resistance associating with c-MET signaling pathway in non-small cell lung cancer.

  12. Obesity-Mediated Regulation of HGF/c-Met Is Associated with Reduced Basal-Like Breast Cancer Latency in Parous Mice

    Science.gov (United States)

    Sundaram, Sneha; Freemerman, Alex J.; Galanko, Joseph A.; McNaughton, Kirk K.; Bendt, Katharine M.; Darr, David B.; Troester, Melissa A.; Makowski, Liza

    2014-01-01

    It is widely thought that pregnancy reduces breast cancer risk, but this lacks consideration of breast cancer subtypes. While a full term pregnancy reduces risk for estrogen receptor positive (ER+) and luminal breast cancers, parity is associated with increased risk of basal-like breast cancer (BBC) subtype. Basal-like subtypes represent less than 10% of breast cancers and are highly aggressive, affecting primarily young, African American women. Our previous work demonstrated that high fat diet-induced obesity in nulliparous mice significantly blunted latency in C3(1)-TAg mice, a model of BBC, potentially through the hepatocyte growth factor (HGF)/c-Met oncogenic pathway. Experimental studies have examined parity and obesity individually, but to date, the joint effects of parity and obesity have not been studied. We investigated the role of obesity in parous mice on BBC. Parity alone dramatically blunted tumor latency compared to nulliparous controls with no effects on tumor number or growth, while obesity had only a minor role in further reducing latency. Obesity-associated metabolic mediators and hormones such as insulin, estrogen, and progesterone were not significantly regulated by obesity. Plasma IL-6 was also significantly elevated by obesity in parous mice. We have previously reported a potential role for stromal-derived hepatocyte growth factor (HGF) via its cognate receptor c-Met in the etiology of obesity-induced BBC tumor onset and in both human and murine primary coculture models of BBC-aggressiveness. Obesity-associated c-Met concentrations were 2.5-fold greater in normal mammary glands of parous mice. Taken together, our studies demonstrate that, parity in C3(1)-TAg mice dramatically reduced BBC latency compared to nulliparous mice. In parous mice, c-Met is regulated by obesity in unaffected mammary gland and is associated with tumor onset. C3(1)-TAg mice recapitulate epidemiologic findings such that parity drives increased BBC risk and potential

  13. Obesity-mediated regulation of HGF/c-Met is associated with reduced basal-like breast cancer latency in parous mice.

    Science.gov (United States)

    Sundaram, Sneha; Freemerman, Alex J; Galanko, Joseph A; McNaughton, Kirk K; Bendt, Katharine M; Darr, David B; Troester, Melissa A; Makowski, Liza

    2014-01-01

    It is widely thought that pregnancy reduces breast cancer risk, but this lacks consideration of breast cancer subtypes. While a full term pregnancy reduces risk for estrogen receptor positive (ER+) and luminal breast cancers, parity is associated with increased risk of basal-like breast cancer (BBC) subtype. Basal-like subtypes represent less than 10% of breast cancers and are highly aggressive, affecting primarily young, African American women. Our previous work demonstrated that high fat diet-induced obesity in nulliparous mice significantly blunted latency in C3(1)-TAg mice, a model of BBC, potentially through the hepatocyte growth factor (HGF)/c-Met oncogenic pathway. Experimental studies have examined parity and obesity individually, but to date, the joint effects of parity and obesity have not been studied. We investigated the role of obesity in parous mice on BBC. Parity alone dramatically blunted tumor latency compared to nulliparous controls with no effects on tumor number or growth, while obesity had only a minor role in further reducing latency. Obesity-associated metabolic mediators and hormones such as insulin, estrogen, and progesterone were not significantly regulated by obesity. Plasma IL-6 was also significantly elevated by obesity in parous mice. We have previously reported a potential role for stromal-derived hepatocyte growth factor (HGF) via its cognate receptor c-Met in the etiology of obesity-induced BBC tumor onset and in both human and murine primary coculture models of BBC-aggressiveness. Obesity-associated c-Met concentrations were 2.5-fold greater in normal mammary glands of parous mice. Taken together, our studies demonstrate that, parity in C3(1)-TAg mice dramatically reduced BBC latency compared to nulliparous mice. In parous mice, c-Met is regulated by obesity in unaffected mammary gland and is associated with tumor onset. C3(1)-TAg mice recapitulate epidemiologic findings such that parity drives increased BBC risk and potential

  14. Scatter factor and the c-met receptor: a paradigm for mesenchymal/epithelial interaction

    OpenAIRE

    1994-01-01

    Epithelia and mesenchyme interact during various physiologic and pathologic processes. Scatter factor is a mesenchyme-derived cytokine that stimulates motility, proliferation, and morphogenesis of epithelia. Recent studies suggest that scatter factor and its receptor (c-met) mediate mesenchyme/epithelia signalling and even interconversion. In this mini-review, we will discuss how scatter factor and c-met may mediate interactions between mesenchyme and epithelia during embryogenesis, organ rep...

  15. c-MET regulates myoblast motility and myocyte fusion during adult skeletal muscle regeneration.

    Science.gov (United States)

    Webster, Micah T; Fan, Chen-Ming

    2013-01-01

    Adult muscle stem cells, satellite cells (SCs), endow skeletal muscle with tremendous regenerative capacity. Upon injury, SCs activate, proliferate, and migrate as myoblasts to the injury site where they become myocytes that fuse to form new muscle. How migration is regulated, though, remains largely unknown. Additionally, how migration and fusion, which both require dynamic rearrangement of the cytoskeleton, might be related is not well understood. c-MET, a receptor tyrosine kinase, is required for myogenic precursor cell migration into the limb for muscle development during embryogenesis. Using a genetic system to eliminate c-MET function specifically in adult mouse SCs, we found that c-MET was required for muscle regeneration in response to acute muscle injury. c-MET mutant myoblasts were defective in lamellipodia formation, had shorter ranges of migration, and migrated slower compared to control myoblasts. Surprisingly, c-MET was also required for efficient myocyte fusion, implicating c-MET in dual functions of regulating myoblast migration and myocyte fusion.

  16. Hepatocyte specific deletion of c-Met leads to the development of severe non-alcoholic steatohepatitis in mice

    NARCIS (Netherlands)

    Kroy, D.C.; Schumacher, F.; Ramadori, P.; Hatting, M.; Bergheim, I.; Gassler, N.; Boekschoten, M.V.; Müller, M.R.; Streetz, K.L.; Trautwein, C.

    2014-01-01

    Background & Aims Non-alcoholic-fatty-liver disease (NAFLD) is part of the metabolic syndrome. The spectrum of NAFLD includes NASH (non-alcoholic steatohepatitis), which is characterised by progressive inflammation associated with oxidative stress and apoptosis, finally triggering liver

  17. Melatonin protects against lipid-induced mitochondrial dysfunction in hepatocytes and inhibits stellate cell activation during hepatic fibrosis in mice.

    Science.gov (United States)

    Das, Nabanita; Mandala, Ashok; Naaz, Shamreen; Giri, Suresh; Jain, Mukul; Bandyopadhyay, Debasish; Reiter, Russel J; Roy, Sib Sankar

    2017-05-01

    Lipid generates reactive oxygen species (ROS) in consequence to mitochondrial fission followed by inflammation in propagating hepatic fibrosis. The interaction of SIRT1/Mitofusin2 is critical for maintaining mitochondrial integrity and functioning, which is disrupted upon excess lipid infiltration during the progression of steatohepatitis. The complex interplay between hepatic stellate cells and steatotic hepatocytes is critically regulated by extracellular factors including increased circulating free fatty acids during fibrogenesis. Melatonin, a potent antioxidant, protects against lipid-mediated mitochondrial ROS generation. Lipotoxicity induces disruption of SIRT1 and Mitofusin2 interaction leading to mitochondrial morphological disintegration in hepatocytes. Further, fragmented mitochondria leads to mitochondrial permeability transition pore opening, cell cycle arrest and apoptosis and melatonin protects against all these lipotoxicity-mediated dysfunctions. These impaired mitochondrial dynamics also enhances the cellular glycolytic flux and reduces mitochondrial oxygen consumption rate that potentiates ROS production. High glycolytic flux generates metabolically unfavorable milieu in hepatocytes leading to inflammation, which is abrogated by melatonin. The melatonin-mediated protection against mitochondrial dysfunction was also observed in high-fat diet (HFD)-fed mice through restoration of enzymatic activities associated with respiratory chain and TCA cycle. Subsequently, melatonin reduces hepatic fat deposition and inflammation in HFD-fed mice. Thus, melatonin disrupts the interaction between steatotic hepatocyte and stellate cells, leading to the activation of the latter to abrogate collagen deposition. Altogether, the results of the current study document that the pharmacological intervention with low dose of melatonin could abrogate lipotoxicity-mediated hepatic stellate cell activation and prevent the fibrosis progression. © 2017 John Wiley & Sons A

  18. 11C-MET PET/MRI for detection of recurrent glioma.

    Science.gov (United States)

    Deuschl, C; Kirchner, J; Poeppel, T D; Schaarschmidt, B; Kebir, S; El Hindy, N; Hense, J; Quick, H H; Glas, M; Herrmann, K; Umutlu, L; Moenninghoff, C; Radbruch, A; Forsting, M; Schlamann, M

    2017-12-28

    Radiological assessment of brain tumors is widely based on the Radiology Assessment of Neuro-Oncology (RANO) criteria that consider non-specific T1 and T2 weighted images. Limitation of the RANO criteria is that they do not include metabolic imaging techniques that have been reported to be helpful to differentiate treatment related changes from true tumor progression. In the current study, we assessed if the combined use of MRI and PET with hybrid 11C-MET PET/MRI can improve diagnostic accuracy and diagnostic confidence of the readers to differentiate treatment related changes from true progression in recurrent glioma. Fifty consecutive patients with histopathologically proven glioma were prospectively enrolled for a hybrid 11C-MET PET/MRI to differentiate recurrent glioma from treatment induced changes. Sole MRI data were analyzed based on RANO. Sole PET data and in a third evaluation hybrid 11C-MET-PET/MRI data were assessed for metabolic respectively metabolic and morphologic glioma recurrence. Diagnostic performance and diagnostic confidence of the reader were calculated for the different modalities, and the McNemar test and Mann-Whitney U Test were applied for statistical analysis. Hybrid 11C-MET PET/MRI was successfully performed in all 50 patients. Glioma recurrence was diagnosed in 35 of the 50 patients (70%). Sensitivity and specificity were calculated for MRI (86.11% and 71.43%), for 11C-MET PET (96.77% and 73.68%), and for hybrid 11C-MET-PET/MRI (97.14% and 93.33%). For diagnostic accuracy hybrid 11C-MET-PET/MRI (96%) showed significantly higher values than MRI alone (82%), whereas no significant difference was found for 11C-MET PET (88%). Furthermore, by rating on a five-point Likert scale significantly higher scores were found for diagnostic confidence when comparing 11C-MET PET/MRI (4.26 ± 0,777) to either PET alone (3.44 ± 0.705) or MRI alone (3.56 ± 0.733). This feasibility study showed that hybrid PET/MRI might strengthen RANO

  19. Sequence-defined cMET/HGFR-targeted Polymers as Gene Delivery Vehicles for the Theranostic Sodium Iodide Symporter (NIS) Gene.

    Science.gov (United States)

    Urnauer, Sarah; Morys, Stephan; Krhac Levacic, Ana; Müller, Andrea M; Schug, Christina; Schmohl, Kathrin A; Schwenk, Nathalie; Zach, Christian; Carlsen, Janette; Bartenstein, Peter; Wagner, Ernst; Spitzweg, Christine

    2016-08-01

    The sodium iodide symporter (NIS) as well-characterized theranostic gene represents an outstanding tool to target different cancer types allowing noninvasive imaging of functional NIS expression and therapeutic radioiodide application. Based on its overexpression on the surface of most cancer types, the cMET/hepatocyte growth factor receptor serves as ideal target for tumor-selective gene delivery. Sequence-defined polymers as nonviral gene delivery vehicles comprising polyethylene glycol (PEG) and cationic (oligoethanoamino) amide cores coupled with a cMET-binding peptide (cMBP2) were complexed with NIS-DNA and tested for receptor-specificity, transduction efficiency, and therapeutic efficacy in hepatocellular cancer cells HuH7. In vitro iodide uptake studies demonstrated high transduction efficiency and cMET-specificity of NIS-encoding polyplexes (cMBP2-PEG-Stp/NIS) compared to polyplexes without targeting ligand (Ala-PEG-Stp/NIS) and without coding DNA (cMBP2-PEG-Stp/Antisense-NIS). Tumor recruitment and vector biodistribution were investigated in vivo in a subcutaneous xenograft mouse model showing high tumor-selective iodide accumulation in cMBP2-PEG-Stp/NIS-treated mice (6.6 ± 1.6% ID/g (123)I, biological half-life 3 hours) by (123)I-scintigraphy. Therapy studies with three cycles of polyplexes and (131)I application resulted in significant delay in tumor growth and prolonged survival. These data demonstrate the enormous potential of cMET-targeted sequence-defined polymers combined with the unique theranostic function of NIS allowing for optimized transfection efficiency while eliminating toxicity.

  20. Japanese Kampo Saireito Has a Liver-Protective Effect Through the Inhibition of Inducible Nitric Oxide Synthase Induction in Primary Cultured Rat Hepatocytes.

    Science.gov (United States)

    Miki, Hirokazu; Tokuhara, Katsuji; Oishi, Masaharu; Nakatake, Richi; Tanaka, Yoshito; Kaibori, Masaki; Nishizawa, Mikio; Okumura, Tadayoshi; Kon, Masanori

    2016-09-01

    Japanese herbal medicine, Kampo saireito, is used for treatments in patients with digestive diseases, including chronic hepatitis and cirrhosis. However, few studies demonstrate scientific evidence for liver-protective effects of saireito. In inflamed liver, proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β stimulate the induction of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production. Excessive levels of NO synthesized by iNOS have been implicated as one of the factors in liver injury, so it is essential to reduce the induction of iNOS for the prevention of liver injury. In this study, we examined IL-1β-stimulated hepatocytes as a simple "in vitro injury model" to investigate liver-protective effects of saireito. Primary cultured rat hepatocytes were treated with IL-1β in the presence or absence of saireito. The induction of NO production and iNOS and its signaling pathway were analyzed. Saireito inhibited the production of NO dose and time dependently and reduced the expression of iNOS messenger RNA (mRNA) and its protein. Saireito had no effect on IκB degradation but inhibited the translocation of nuclear factor (NF)-κB to the nucleus and its DNA binding. Saireito also inhibited the activation of Akt, resulting in the reduction of type I IL-1 receptor (IL-1RI) mRNA and protein expression. These findings demonstrate that saireito suppresses iNOS gene expression through the inhibition of NF-κB and IL-1RI-dependent pathways, leading to the reduction of NO production. Saireito may have therapeutic potential for organ injuries, including liver. © 2015 American Society for Parenteral and Enteral Nutrition.

  1. Deguelin action involves c-Met and EGFR signaling pathways in triple negative breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Rajeshwari Mehta

    Full Text Available Treatment of breast cancer patients with antiestrogens and aromatase inhibitor(s or Herceptin have shown significant success in steroid receptor positive or Her-2+ breast cancers respectively. However, choice of treatments for breast cancer patients with negative status for estrogen, progesterone receptors and HER2/neu is limited. As a result, search for appropriate therapy regimen for these triple negative breast cancers (TNBC has become a major focus of investigations for many laboratories. Recently, Deguelin, a natural product isolated from African plant Mundulea sericea (Leguminossae has shown both antiproliferative actions in various cancers including breast as well as chemoprenventive activity against carcinogen induced experimental cancers. In this report we evaluated efficacy and mechanism of action of Deguelin in triple negative breast cancer cell lines.In vitro, Deguelin in a dose and time dependent manner inhibited the growth of MDA-MB-231, MDA-MB-468, BT-549 and BT-20 cells. Deguelin (2 or 4 mg/kg body weight, when injected intraperitoneally, reduced the in vivo tumor growth of MDA-MB-231 cells transplanted subcutaneously in athymic mice. Moreover it was nontoxic as evident from daily observations on mobility, food and water consumption and comparison of bodyweight and other visceral organ weights with those in control animals at the termination of the study. The western blot analyses and immunostaining studies indicated that the deguelin effects may be mediated through EGFR-PAKT/c-Met p-ERK and NF-κB by down regulating their downstream targets such as p-STAT3, c-Myc, Survivin.These results suggest that Deguelin may have a significant therapeutic value for the treatment of TNBC patients.

  2. Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition

    Directory of Open Access Journals (Sweden)

    Laura Ordovás

    2015-11-01

    Full Text Available Tools for rapid and efficient transgenesis in “safe harbor” loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs. We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.

  3. YB-1 regulates tumor growth by promoting MACC1/c-Met pathway in human lung adenocarcinoma

    Science.gov (United States)

    Xue, Xiaoyuan; Zhang, Yan; Yang, Mengying; Li, Nan; Li, Zhuoshi; Xu, Lingzhi; Jiang, Lei; Zhao, Lei; Ma, Patrick C.; Rosell, Rafael; Li, Jinxiu; Gu, Chundong

    2017-01-01

    Aberrant overexpression of the transcription/translation factor Y-box-binding protein (YB-1) is associated with poor prognosis of lung adenocarcinoma, however the underlying mechanism by which YB-1 acts has not been fully elucidated. Here, we reported that inhibition of YB-1 diminished proliferation, migration and invasion of lung adenocarcinoma cells. Interestingly, we identified metastasis associated in colon cancer-1 (MACC1) as a target of YB-1. Depletion of YB-1 markedly decreased MACC1 promoter activity and suppressed the MACC1/c-Met signaling pathway in lung adenocarcinoma cells. Additionally, chromatin immunoprecipitation (ChIP) assay demonstrated that YB-1 bound to the MACC1 promoter. Moreover, YB-1 was positively correlated with MACC1, and both proteins were over-expressed in lung adenocarcinoma tissues. The Cox-regression analysis indicated that high YB-1 expression was an independent risk factor for prognosis in enrolled patients. Furthermore, depletion of YB-1 attenuated tumorigenesis in a xenograft mouse model and reduced MACC1 expression in tumor tissues. Collectively, our data suggested that targeting YB-1 suppressed lung adenocarcinoma progression through the MACC1/c-Met pathway and that the high expression of YB-1/MACC1 is a potential prognostic marker in lung adenocarcinoma. PMID:28624808

  4. High levels of c-Met is associated with poor prognosis in glioblastoma

    DEFF Research Database (Denmark)

    Petterson, Stine Asferg; Dahlrot, Rikke Hedegaard; Hermansen, Simon Kjær

    2015-01-01

    . Measurements of high c-Met intensity correlated with high WHO grade (p = 0.006) but no association with survival was observed in patients with WHO grade II (p = 0.09) or III (p = 0.17) tumors. High expression of c-Met was associated with shorter overall survival in patients with glioblastoma multiforme (p = 0...... and the clinical variables age (HR 1.01, 95 % CI 0.99-1.03, p = 0.30), performance status (HR 1.34, 95 % CI 1.17-1.53, p

  5. Novel germline c-MET mutation in a family with hereditary papillary renal carcinoma

    DEFF Research Database (Denmark)

    Wadt, Karin; Gerdes, Anne-Marie; Hansen, Thomas V O

    2012-01-01

    Hereditary papillary renal carcinoma (HPRC) is a highly penetrant hereditary renal cancer syndrome caused by germline missense mutations in the c-MET proto-oncogene. HPRC is clinically characterized by multiple bilateral papillary renal-cell carcinomas. Here we report a family with a novel missense...... mutation in c-MET. The original pathology report of four primary kidney cancers (1988-1997) revealed renal-cell carcinoma. A revised report described multiple adenomas and papillary renal-cell carcinomas with focal clear cells and a mixture of type 1 and type 2 pattern, emphasizing the importance...

  6. Ghrelin protects against palmitic acid or lipopolysaccharide-induced hepatocyte apoptosis through inhibition of MAPKs/iNOS and restoration of Akt/eNOS pathways.

    Science.gov (United States)

    Mao, Yuqing; Wang, Jianbo; Yu, Fujun; Li, Zhengyang; Li, Huanqing; Guo, Chuanyong; Fan, Xiaoming

    2016-12-01

    Ghrelin has been shown to exert various biological functions. However, the effect and mechanism of ghrelin on PA- or LPS-induced liver injury remains unknown. Normal human hepatocyte lines (LO2 and 7701) were pretreated with ghrelin (10 -8 M) for 30min before stimulation with lipopolysaccharide (LPS) or palmitic acid (PA). The proliferation and apoptosis of cells were detected with CCK8, Hoechst staining and flow cytometric analysis. Levels of NO of cell supernatants were examined by enzyme-linked immunosorbent assay (ELISA). The protein levels and mRNA of target genes of endothelial NOS (eNOS) and inducible NOS (iNOS) were measured by western blotting, immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR). The expression of Bax, Bcl2, caspase 3, p-Akt, p-P38 and p-JNK were detected by western blotting. Results of CCK8, Hoechst staining and flow cytometric analysis showed that ghrelin-pretreatment attenuated LPS- or PA- induced cellular proliferation inhibition and apoptosis induction. ELISA results revealed that ghrelin pretreatment reduced levels of NO of cell supernatants (Pghrelin- pretreated group were significantly reduced compared with LPS- or PA- treated group, while protein levels of eNOS were restored by ghrelin pretreatment. Results of qRT-PCR showed that mRNA levels of Bax, iNOS were reduced by ghrelin pretreatment, while levels of mRNA of Bcl2 and eNOS were increased (Pghrelin pretreatment, while the protein levels of p-JNK, p-P38 and caspase 3 were reduced. The restoration of eNOS could be reversed by an Akt inhibitor. Ghrelin pretreatment attenuated LPS- or PA-induced hepatocyte apoptosis, which may least partly via inhibition of mitogen-activated protein kinases (MAPKs)/iNOS and restoration of Akt/eNOS pathways. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  7. E-cadherin and c-Met expression in actinic cheilits and lip squamous cell carcinoma

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    A Martínez

    2011-12-01

    Full Text Available Objective: The aim of this study was to assess epithelial expression of E-cadherin and c-Met in normal lip, in actinic cheilitis and lip squamous cell carcinoma. Study Design: Biopsies of normal lip vermillion (NL, n=18, actinic cheilitis (AC, n=37, and lip SCC (n=22 were processed for E-cadherin and c-Met immunodetection. Epithelial and tumor cell expression was scored for each sample considering staining intensity and percentage. Results: E-cadherin expression was significantly reduced in AC and lip SCC as compared to normal lip (P<0.05, with a significant reduction in lip SCC as compared to AC (P=0.003. Expression of c-Met was significantly higher in AC and lip SCC as compared to NL (P<0.05, with a significant increase in lip SCC as compared to AC (P<0.0001. Conclusion: The results showed that epithelial E-cadherin expression is reduced and c-Met expression is increased as lip carcinogenesis progresses, suggesting that these proteins may be useful markers of malignant transformation.

  8. Expression and significance of CD44, CD47 and c-met in ovarian clear cell carcinoma.

    Science.gov (United States)

    Wang, Huimin; Tan, Mingzi; Zhang, Song; Li, Xiao; Gao, Jian; Zhang, Danye; Hao, Yingying; Gao, Song; Liu, Juanjuan; Lin, Bei

    2015-02-04

    The aim of the present study is to investigate the differential expression of CD44, CD47 and c-met in ovarian clear cell carcinoma (OCCC), the correlation in their expression and their relationship with the biological behavior of OCCC. We used immunohistochemistry to examine the expression of CD44, CD47 and c-met in OCCC (86 cases) and investigated the effects of the expression and interaction of these molecules on the development of OCCC. CD44, CD47 and c-met expression was significantly high in OCCC. Expression of CD44 and CD47 correlated with patient surgical stage, chemotherapy resistance and prognosis (all p0.05). The surgical stage, CD44, CD47 and c-met expression were independent risk factors for OCCC prognosis (all pCD47 and c-met showed better survival than those with high levels (all pCD47) and c-met, as well as between CD44 and CD47 (the Spearman correlation coefficient rs was 0.783, 0.776 and 0.835, respectively, all pCD47, CD44/c-met and CD47/c-met were correlated with patient surgical stage, chemotherapy resistance and prognosis (all p0.05). Expression of CD44, CD47 and c-met was upregulated in OCCC and pairwise correlation. CD44, CD47 and c-met may have synergistic effects on the development of OCCC and are prognostic factors for ovarian cancer.

  9. Irisin inhibits hepatic gluconeogenesis and increases glycogen synthesis via the PI3K/Akt pathway in type 2 diabetic mice and hepatocytes.

    Science.gov (United States)

    Liu, Tong-Yan; Shi, Chang-Xiang; Gao, Run; Sun, Hai-Jian; Xiong, Xiao-Qing; Ding, Lei; Chen, Qi; Li, Yue-Hua; Wang, Jue-Jin; Kang, Yu-Ming; Zhu, Guo-Qing

    2015-11-01

    Increased glucose production and reduced hepatic glycogen storage contribute to metabolic abnormalities in diabetes. Irisin, a newly identified myokine, induces the browning of white adipose tissue, but its effects on gluconeogenesis and glycogenesis are unknown. In the present study, we investigated the effects and underlying mechanisms of irisin on gluconeogenesis and glycogenesis in hepatocytes with insulin resistance, and its therapeutic role in type 2 diabetic mice. Insulin resistance was induced by glucosamine (GlcN) or palmitate in human hepatocellular carcinoma (HepG2) cells and mouse primary hepatocytes. Type 2 diabetes was induced by streptozotocin/high-fat diet (STZ/HFD) in mice. In HepG2 cells, irisin ameliorated the GlcN-induced increases in glucose production, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) expression, and glycogen synthase (GS) phosphorylation; it prevented GlcN-induced decreases in glycogen content and the phosphoinositide 3-kinase (PI3K) p110α subunit level, and the phosphorylation of Akt/protein kinase B, forkhead box transcription factor O1 (FOXO1) and glycogen synthase kinase-3 (GSK3). These effects of irisin were abolished by the inhibition of PI3K or Akt. The effects of irisin were confirmed in mouse primary hepatocytes with GlcN-induced insulin resistance and in human HepG2 cells with palmitate-induced insulin resistance. In diabetic mice, persistent subcutaneous perfusion of irisin improved the insulin sensitivity, reduced fasting blood glucose, increased GSK3 and Akt phosphorylation, glycogen content and irisin level, and suppressed GS phosphorylation and PEPCK and G6Pase expression in the liver. Irisin improves glucose homoeostasis by reducing gluconeogenesis via PI3K/Akt/FOXO1-mediated PEPCK and G6Pase down-regulation and increasing glycogenesis via PI3K/Akt/GSK3-mediated GS activation. Irisin may be regarded as a novel therapeutic strategy for insulin resistance and type 2 diabetes. © 2015

  10. TGF-β Negatively Regulates CXCL1 Chemokine Expression in Mammary Fibroblasts through Enhancement of Smad2/3 and Suppression of HGF/c-Met Signaling Mechanisms.

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    Wei Bin Fang

    Full Text Available Fibroblasts are major cellular components of the breast cancer stroma, and influence the growth, survival and invasion of epithelial cells. Compared to normal tissue fibroblasts, carcinoma associated fibroblasts (CAFs show increased expression of numerous soluble factors including growth factors and cytokines. However, the mechanisms regulating expression of these factors remain poorly understood. Recent studies have shown that breast CAFs overexpress the chemokine CXCL1, a key regulator of tumor invasion and chemo-resistance. Increased expression of CXCL1 in CAFs correlated with poor patient prognosis, and was associated with decreased expression of TGF-β signaling components. The goal of these studies was to understand the role of TGF-β in regulating CXCL1 expression in CAFs, using cell culture and biochemical approaches. We found that TGF-β treatment decreased CXCL1 expression in CAFs, through Smad2/3 dependent mechanisms. Chromatin immunoprecipitation and site-directed mutagenesis assays revealed two new binding sites in the CXCL1 promoter important for Smad2/3 modulation of CXCL1 expression. Smad2/3 proteins also negatively regulated expression of Hepatocyte Growth Factor (HGF, which was found to positively regulate CXCL1 expression in CAFs through c-Met receptor dependent mechanisms. HGF/c-Met signaling in CAFs was required for activity of NF-κB, a transcriptional activator of CXCL1 expression. These studies indicate that TGF-β negatively regulates CXCL1 expression in CAFs through Smad2/3 binding to the promoter, and through suppression of HGF/c-Met autocrine signaling. These studies reveal novel insight into how TGF-β and HGF, key tumor promoting factors modulate CXCL1 chemokine expression in CAFs.

  11. Fullerene inhibits benzo(a)pyrene Efflux from Cyprinus carpio hepatocytes by affecting cell membrane fluidity and P-glycoprotein expression.

    Science.gov (United States)

    Chen, Qiqing; Hu, Xialin; Wang, Rui; Yuan, Jin; Yin, Daqiang

    2016-05-01

    P-Glycoprotein (P-gp) can protect cells by pumping out toxic compounds, and has been found widely expressed in fish tissues. Here, we illustrate the P-gp efflux ability for benzo(a)pyrene (BaP) in the hepatocytes of common carp (Cyprinus carpio) after exposing to fullerene aqueous suspension (nC60). The results revealed that nC60 increased the membrane fluidity by decreasing the ratio of saturated to unsaturated fatty acids, and increased the cholesterol contents. These findings, combined with 10-38% and 70-75% down-regulation of P-gp mRNA and protein respectively, suggested that nC60 caused inhibition on P-gp efflux transport system. Therefore, we further investigated the cellular efflux ability for BaP. Results showed unequivocally that nC60 is a potent P-gp inhibitor. The retaining BaP amounts after efflux were elevated by 1.7-2.8 fold during the 10 day exposure. Meanwhile, 5mg/L humic acid (one of the important fractions of natural organic matter, which is ubiquitous in aquatic environment) alleviated the nC60 damage to hepatocytes in terms of oxidative damage, cholesterol increment, and P-gp content reduction; and finally attenuated the suppressed P-gp efflux ability. Collectively, this study provides the first evidence of nC60 toxicity to P-gp functionality in fish and illustrates the possible mechanism of the suppressed P-gp efflux ability for BaP. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Hepatocyte and keratinocyte growth factors and their receptors in human lung emphysema

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    Marchal Joëlle

    2005-10-01

    Full Text Available Abstract Background Hepatocyte and keratinocyte growth factors are key growth factors in the process of alveolar repair. We hypothesized that excessive alveolar destruction observed in lung emphysema involves impaired expression of hepatocyte and keratinocyte growth factors or their respective receptors, c-met and keratinocyte growth factor receptor. The aim of our study was to compare the expression of hepatocyte and keratinocyte growth factors and their receptors in lung samples from 3 groups of patients: emphysema; smokers without emphysema and non-smokers without emphysema. Methods Hepatocyte and keratinocyte growth factor proteins were analysed by immunoassay and western blot; mRNA expression was measured by real time quantitative polymerase chain reaction. Results Hepatocyte and keratinocyte growth factors, c-met and keratinocyte growth factor receptor mRNA levels were similar in emphysema and non-emphysema patients. Hepatocyte growth factor mRNA correlated negatively with FEV1 and the FEV1/FVC ratio both in emphysema patients and in smokers with or without emphysema. Hepatocyte and keratinocyte growth factor protein concentrations were similar in all patients' groups. Conclusion The expression of hepatocyte and keratinocyte growth factors and their receptors is preserved in patients with lung emphysema as compared to patients without emphysema. Hepatocyte growth factor mRNA correlates with the severity of airflow obstruction in smokers.

  13. Hepatocyte growth factor enhances death receptor-induced apoptosis by up-regulating DR5

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    Goodwin C Rory

    2008-11-01

    Full Text Available Abstract Background Hepatocyte growth factor (HGF and its receptor c-MET are commonly expressed in malignant gliomas and embryonic neuroectodermal tumors including medulloblastoma and appear to play an important role in the growth and dissemination of these malignancies. Dependent on cell context and the involvement of specific downstream effectors, both pro- and anti-apoptotic effects of HGF have been reported. Methods Human medulloblastoma cells were treated with HGF for 24–72 hours followed by death receptor ligand TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand for 24 hours. Cell death was measured by MTT and Annexin-V/PI flow cytometric analysis. Changes in expression levels of targets of interest were measured by Northern blot analysis, quantitative reverse transcription-PCR, Western blot analysis as well as immunoprecipitation. Results In this study, we show that HGF promotes medulloblastoma cell death induced by TRAIL. TRAIL alone triggered apoptosis in DAOY cells and death was enhanced by pre-treating the cells with HGF for 24–72 h prior to the addition of TRAIL. HGF (100 ng/ml enhanced TRAIL (10 ng/ml induced cell death by 36% (P Conclusion Taken together, these and previous findings indicate that HGF:c-Met pathway either promotes or inhibits medulloblastoma cell death via pathway and context specific mechanisms.

  14. In vitro oxidative metabolism of cajaninstilbene Acid by human liver microsomes and hepatocytes: involvement of cytochrome p450 reaction phenotyping, inhibition, and induction studies.

    Science.gov (United States)

    Hua, Xin; Peng, Xiao; Tan, Shengnan; Li, Chunying; Wang, Wei; Luo, Meng; Fu, Yujie; Zu, Yuangang; Smyth, Hugh

    2014-10-29

    Cajaninstilbene acid (CSA, 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid), an active constituent of pigeonpea leaves, an important tropical crop, is known for its clinical effects in the treatment of diabetes, hepatitis, and measles and its potential antitumor effect. In this study, the effect of the cytochrome P450 isozymes on the activity of CSA was investigated. Two hydroxylation metabolites were identified in the study. The reaction phenotype study showed that CYP3A4, CYP2C9, and CYP1A2 were the major cytochrome P450 isozymes in the metabolism of CSA. The metabolic food-drug interaction potential was also evaluated in vitro. The effect of CSA inhibition/induction of enzymatic activities of seven drug-metabolizing CYP450 isozymes in vitro was estimated by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analytical techniques. CSA showed different inhibitory effects on different isozymes. CSA reversibly inhibited CYP3A4 and CYP2C9 activities in human liver microsomes with IC50 values of 28.3 and 31.3 μM, respectively, but exhibited no inhibition activities to CYP1A2, CYP2A6, CYP2C19, CYP2D6, and CYP2E1. CSA showed a weak effect on CYP450 enzymes in a time-dependent manner. CSA did not substantially induce CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C9, CYP2C19, CYP2D6, or CYP3A4 at concentrations up to 30 μM in primary human hepatocytes. The results of our experiments may be helpful to predict clinically significant food-drug interactions when other drugs are administered in combination with CSA.

  15. Structure Based Drug Design of Crizotinib (PF-02341066), a Potent and Selective Dual Inhibitor of Mesenchymal-Epithelial Transition Factor (c-MET) Kinase and Anaplastic Lymphoma Kinase (ALK)

    Energy Technology Data Exchange (ETDEWEB)

    Cui, J Jean; Tran-Dube,; #769; Michelle,; Shen, Hong; Nambu, Mitchell; Kung, Pei-Pei; Pairish, Mason; Jia, Lei; Meng, Jerry; Funk, Lee; Botrous, Iriny; McTigue, Michele; Grodsky, Neil; Ryan, Kevin; Padrique, Ellen; Alton, Gordon; Timofeevski, Sergei; Yamazaki, Shinji; Li, Qiuhua; Zou, Helen; Christensen, James; Mroczkowski, Barbara; Bender, Steve; Kania, Robert S; Edwards, Martin P [Pfizer

    2011-08-03

    Because of the critical roles of aberrant signaling in cancer, both c-MET and ALK receptor tyrosine kinases are attractive oncology targets for therapeutic intervention. The cocrystal structure of 3 (PHA-665752), bound to c-MET kinase domain, revealed a novel ATP site environment, which served as the target to guide parallel, multiattribute drug design. A novel 2-amino-5-aryl-3-benzyloxypyridine series was created to more effectively make the key interactions achieved with 3. In the novel series, the 2-aminopyridine core allowed a 3-benzyloxy group to reach into the same pocket as the 2,6-dichlorophenyl group of 3 via a more direct vector and thus with a better ligand efficiency (LE). Further optimization of the lead series generated the clinical candidate crizotinib (PF-02341066), which demonstrated potent in vitro and in vivo c-MET kinase and ALK inhibition, effective tumor growth inhibition, and good pharmaceutical properties.

  16. Expressions of CD44,CD47,and c-met in Ovarian Clear Cell Carcinoma and Their Clinical Significance.

    Science.gov (United States)

    Wang, Hui-Min; Tan, Ming-Zi; Zhang, Song; Li, Xiao; Gao, Jian; Zhang, Dan-Ye; Hao, Ying-Ying; Gao, Song; Liu, Juan-Juan; Lin, Bei

    2016-12-20

    Objective To investigate the expressions of CD44,CD47,and c-met in ovarian clear cell carcinoma (OCCC) tissue and their correlations with clinical variables and prognosis. Methods Immunohistochemical method was used to investigate the expressions of CD44,CD47,and c-met in tissues from 86 OCCC patients and the relationships of their expressions with the clinicopathological factors of OCCC were analyzed. Results The expressions of CD44,CD47,and c-met were significantly high in OCCC tissues (90.7%,91.9%,and 94.2%,respectively). The strong positive expressions of CD44 and CD47 were significantly correlated with advanced International Federation of Gynecology and Obstetrics stages,chemotherapeutic resistance,and poor prognosis (all PCD47,and c-met and the lymphatic node metastasis. COX survival analysis revealed that advanced International Federation of Gynecology and Obstetrics stages and high expressions of CD44,CD47 and c-met were independent risk factors for poor prognosis (PCD47) and c-met and between CD44 and CD47 (the Spearman correlation coefficient rs was 0.783,0.776,and 0.835,respectively,all PCD47,and c-met increase in OCCC tissues and are correlated with each other. High expressions of CD44,CD47,and c-met are independent factors for poor prognosis.

  17. Curcumin Ameliorates Furazolidone-Induced DNA Damage and Apoptosis in Human Hepatocyte L02 Cells by Inhibiting ROS Production and Mitochondrial Pathway

    Directory of Open Access Journals (Sweden)

    Chongshan Dai

    2016-08-01

    Full Text Available Furazolidone (FZD, a synthetic nitrofuran derivative, has been widely used as an antibacterial and antiprotozoal agent. Recently, the potential toxicity of FZD has raised concerns, but its mechanism is still unclear. This study aimed to investigate the protective effect of curcumin on FZD-induced cytotoxicity and the underlying mechanism in human hepatocyte L02 cells. The results showed that curcumin pre-treatment significantly ameliorated FZD-induced oxidative stress, characterized by decreased reactive oxygen species (ROS and malondialdehyde formation, and increased superoxide dismutase, catalase activities and glutathione contents. In addition, curcumin pre-treatment significantly ameliorated the loss of mitochondrial membrane potential, the activations of caspase-9 and -3, and apoptosis caused by FZD. Alkaline comet assay showed that curcumin markedly reduced FZD-induced DNA damage in a dose-dependent manner. Curcumin pre-treatment consistently and markedly down-regulated the mRNA expression levels of p53, Bax, caspase-9 and -3 and up-regulated the mRNA expression level of Bcl-2. Taken together, these results reveal that curcumin protects against FZD-induced DNA damage and apoptosis by inhibiting oxidative stress and mitochondrial pathway. Our study indicated that curcumin may be a promising combiner with FZD to reduce FZD-related toxicity in clinical applications.

  18. Combination Therapy with c-Met and Src Inhibitors Induces Caspase-Dependent Apoptosis of Merlin-Deficient Schwann Cells and Suppresses Growth of Schwannoma Cells.

    Science.gov (United States)

    Fuse, Marisa A; Plati, Stephani Klingeman; Burns, Sarah S; Dinh, Christine T; Bracho, Olena; Yan, Denise; Mittal, Rahul; Shen, Rulong; Soulakova, Julia N; Copik, Alicja J; Liu, Xue Zhong; Telischi, Fred F; Chang, Long-Sheng; Franco, Maria Clara; Fernandez-Valle, Cristina

    2017-11-01

    Neurofibromatosis type 2 (NF2) is a nervous system tumor disorder caused by inactivation of the merlin tumor suppressor encoded by the NF2 gene. Bilateral vestibular schwannomas are a diagnostic hallmark of NF2. Mainstream treatment options for NF2-associated tumors have been limited to surgery and radiotherapy; however, off-label uses of targeted molecular therapies are becoming increasingly common. Here, we investigated drugs targeting two kinases activated in NF2-associated schwannomas, c-Met and Src. We demonstrated that merlin-deficient mouse Schwann cells (MD-MSC) treated with the c-Met inhibitor, cabozantinib, or the Src kinase inhibitors, dasatinib and saracatinib, underwent a G 1 cell-cycle arrest. However, when MD-MSCs were treated with a combination of cabozantinib and saracatinib, they exhibited caspase-dependent apoptosis. The combination therapy also significantly reduced growth of MD-MSCs in an orthotopic allograft mouse model by greater than 80% of vehicle. Moreover, human vestibular schwannoma cells with NF2 mutations had a 40% decrease in cell viability when treated with cabozantinib and saracatinib together compared with the vehicle control. This study demonstrates that simultaneous inhibition of c-Met and Src signaling in MD-MSCs triggers apoptosis and reveals vulnerable pathways that could be exploited to develop NF2 therapies. Mol Cancer Ther; 16(11); 2387-98. ©2017 AACR . ©2017 American Association for Cancer Research.

  19. Hepatocyte growth factor (HGF) receptor expression and role of HGF during embryonic mouse testis development.

    Science.gov (United States)

    Ricci, G; Catizone, A; Innocenzi, A; Galdieri, M

    1999-12-01

    The hepatocyte growth factor (HGF) receptor, c-met, transduces the HGF multiple biological activities. During embryonic development the system HGF/c-met regulates the morphogenesis of different organs and tissues. In this study we examined c-met gene expression during mouse testis development and, by means of Northern blot and in situ hybridization, we report the receptor expression pattern. C-met expression is not detectable in male genital ridges isolated from embryos at 11.5 days postcoitum (dpc). In testes isolated from 12.5 and 13.5 dpc, c-met expression is detectable and essentially localized in the developing cords. Male genital ducts do not express c-met at the reported ages, whereas female ducts appear c-met positive. Moreover, we report that HGF is able to induce testicular morphogenesis in vitro. Male genital ridges isolated from embryos at 11.5 dpc are morphologically nonorganized. Culturing 11.5 dpc urogenital ridges in the presence of HGF we obtained testis organization and testicular cord formation. Our data demonstrate that c-met is expressed during the beginning period of testis differentiation and that HGF is able to support testicular differentiation in vitro. All these data indicate that this growth factor, besides its role as mitogenic factor, plays a fundamental role during testicular cord formation probably inducing cell migration and/or cell differentiation. Copyright 1999 Academic Press.

  20. Pleiotropic activities of HGF/c-Met system in testicular physiology: paracrine and endocrine implications

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    Giulia eRicci

    2014-04-01

    Full Text Available In the last decades a growing body of evidences has been reported concerning the expression and functional role of Hepathocyte Growth Factor (HGF on different aspects of testicular physiology. This review has the aim to summarize what is currently known regarding this topic. From early embryonic development to adult age, HGF and its receptor c-Met appeared to be clearly detectable in the testis. These molecules acquire different distribution patterns and roles depending on the developmental stage or the post-natal age considered. HGF acts as a paracrine modulator of testicular functions promoting the ephitelium-mesenchyme cross-talk as described even in other organs. Interestingly, it has been reported that testicular HGF acts even as an autocrine factor and that its receptor might be modulated by endocrine signals that change at puberty: HGF receptor expressed by Sertoli cells, in fact, is up-regulated by FSH administration. HGF is in turn able to modify endocrine state of the organism being able to increase testosterone secretion of both fetal and adult Leydig cells. Moreover c-Met is expressed in mitotic and meiotic male germ cells as well as in spermatozoa. The distribution pattern of c-Met on sperm cell membrane changes in the caput and cauda epididymal sperms and HGF is able to maintain epididymal sperm motility in vitro suggesting a physiological role of this growth factor in the acquisition of sperm motility. Noteworthy changes in HGF concentration in seminal plasma have been reported in different andrological diseases. All together these data indicate that HGF has a role in the control of spermatogenesis and sperm quality either directly, acting on male germ cells, or indirectly acting on tubular and interstitial somatic cells of the testis.

  1. Activation of RAS/ERK alone is insufficient to inhibit RXRα function and deplete retinoic acid in hepatocytes

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    Wang, Ai-Guo, E-mail: wangaiguotl@hotmail.com; Song, Ya-Nan; Chen, Jun; Li, Hui-Ling; Dong, Jian-Yi; Cui, Hai-Peng; Yao, Liang; Li, Xue-Feng; Gao, Wen-Ting; Qiu, Ze-Wen; Wang, Fu-Jin; Wang, Jing-Yu, E-mail: wangjingyus@163.com

    2014-09-26

    Highlights: • The activation of RAS/ERK is insufficient to inhibit RXRα function and deplete RA. • The retinoid metabolism-related genes are down-regulated by ras oncogene. • The atRA has no effect on preventing hepatic tumorigenesis or curing the developed hepatic nodules. - Abstract: Activation of RAS/ERK signaling pathway, depletion of retinoid, and phosphorylation of retinoid X receptor alpha (RXRα) are frequent events found in liver tumors and thought to play important roles in hepatic tumorigenesis. However, the relationships among them still remained to be elucidated. By exploring the transgenic mouse model of hepatic tumorigenesis induced by liver-specific expression of H-ras12V oncogene, the activation of RAS/ERK, the mRNA expression levels of retinoid metabolism-related genes, the contents of retinoid metabolites, and phosphorylation of RXRα were determined. RAS/ERK signaling pathway was gradually and significantly activated in hepatic tumor adjacent normal liver tissues (P) and hepatic tumor tissues (T) of H-ras12V transgenic mice compared with normal liver tissues (Wt) of wild type mice. On the contrary, the mRNA expression levels of retinoid metabolism-related genes were significantly reduced in P and T compared with Wt. Interestingly, the retinoid metabolites 9-cis-retinoic acid (9cRA) and all-trans-retinoic acid (atRA), the well known ligands for nuclear transcription factor RXR and retinoic acid receptor (RAR), were significantly decreased only in T compared with Wt and P, although the oxidized polar metabolite of atRA, 4-keto-all-trans-retinoic-acid (4-keto-RA) was significantly decreased in both P and T compared with Wt. To our surprise, the functions of RXRα were significantly blocked only in T compared with Wt and P. Namely, the total protein levels of RXRα were significantly reduced and the phosphorylation levels of RXRα were significantly increased only in T compared with Wt and P. Treatment of H-ras12V transgenic mice at 5-week

  2. Targeting hepatocyte growth factor receptor (Met) positive tumor cells using internalizing nanobody-decorated albumin nanoparticles

    NARCIS (Netherlands)

    Heukers, Raimond|info:eu-repo/dai/nl/325788103; Altintas, Isil|info:eu-repo/dai/nl/341537160; Raghoenath, Smiriti; De Zan, Erica; Pepermans, Richard; Roovers, Rob C.|info:eu-repo/dai/nl/205435599; Haselberg, Rob|info:eu-repo/dai/nl/304822647; Hennink, Wim E.|info:eu-repo/dai/nl/070880409; Schiffelers, Raymond M.|info:eu-repo/dai/nl/212909509; Kok, Robbert J.|info:eu-repo/dai/nl/170678326; Van Bergen en Henegouwen, Paul M P|info:eu-repo/dai/nl/071919481

    2014-01-01

    The hepatocyte growth factor receptor (HGFR, c-Met or Met) is a receptor tyrosine kinase that is involved in embryogenesis, tissue regeneration and wound healing. Abnormal activation of this proto-oncogene product is implicated in the development, progression and metastasis of many cancers. Current

  3. Research Progress of HGF/c-MET Inhibitor in the Treatment 
of Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Tao JIANG

    2015-04-01

    Full Text Available Molecular targeted therapy has become more and more important in the treatment of non-small cell lung cancer (NSCLC. HGF/c-MET plays the pivotal role in the growth, development and tolerance to epidermal growth factor receptor tyrosine kinase inhibitor of NSCLC. Moreover it has become another heat point in the molecular targeted therapy of NSCLC. c-MET amplification or high expression was deemed to another significant gene modification beyond EGFR and ALK. In the preclinical studies, HGF/c-MET inhibitors have showed the promising anti-tumor effect. Recently, some phase II/III clinical trials have proved that these inhibitors could improve the survival of patients with NSCLC. Hence we performed this review to elaborate the research progress of c-MET inhibitor in the treatment of NSCLC.

  4. Expression and Significance of CD44, CD47 and c-met in Ovarian Clear Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Huimin Wang

    2015-02-01

    Full Text Available Aims: The aim of the present study is to investigate the differential expression of CD44, CD47 and c-met in ovarian clear cell carcinoma (OCCC, the correlation in their expression and their relationship with the biological behavior of OCCC. Methods: We used immunohistochemistry to examine the expression of CD44, CD47 and c-met in OCCC (86 cases and investigated the effects of the expression and interaction of these molecules on the development of OCCC. Results: CD44, CD47 and c-met expression was significantly high in OCCC. Expression of CD44 and CD47 correlated with patient surgical stage, chemotherapy resistance and prognosis (all p < 0.05, and expression of c-met correlated with chemotherapy resistance and prognosis (all p < 0.05, but did not correlate with lymph node metastasis (all p > 0.05. The surgical stage, CD44, CD47 and c-met expression were independent risk factors for OCCC prognosis (all p < 0.05. Patients with low levels of CD44, CD47 and c-met showed better survival than those with high levels (all p < 0.05. There was a positive correlation between CD44 (or CD47 and c-met, as well as between CD44 and CD47 (the Spearman correlation coefficient rs was 0.783, 0.776 and 0.835, respectively, all p < 0.01. Additionally, pairwise correlation analysis of these three markers shows that the high expression of CD44/CD47, CD44/c-met and CD47/c-met were correlated with patient surgical stage, chemotherapy resistance and prognosis (all p < 0.05, but did not correlate with lymph node metastasis (all p > 0.05. Conclusions: Expression of CD44, CD47 and c-met was upregulated in OCCC and pairwise correlation. CD44, CD47 and c-met may have synergistic effects on the development of OCCC and are prognostic factors for ovarian cancer.

  5. C-Met as a potential target for the treatment of gastrointestinal cancer: Current status and future perspectives.

    Science.gov (United States)

    Bahrami, Afsane; Shahidsales, Soodabeh; Khazaei, Majid; Ghayour-Mobarhan, Majid; Maftouh, Mina; Hassanian, Seyed Mahdi; Avan, Amir

    2017-10-01

    Aberrant activation of the HGF/c-Met signalling pathways is shown to be related with cell proliferation, progression, metastasis, and worse prognosis in several tumor types, including gastrointestinal cancers, suggesting its value as a stimulating-target for cancer-therapy. Several approaches have been developed for targeting HGF and/or c-Met, and one of them, crizotinib (dual c-Met/ALK inhibitor), is recently been approved by FDA for lung-cancers with ALK-rearrangement. The main aim of current review is to give an overview on the role of c-Met/HGF pathway in gastrointestinal cancer, in preclinical and clinical trials. Although several important matters is still remained to be elucidated on the molecular pathways underlying the antitumor effects of this therapy in gastrointestinal-cancers. Further investigations are warranted to recognize the main determinants of the activity of c-Met inhibitors, for parallel targeting signalling pathway associated/activated via MET/HGF pathway or in response to the cell resistance to anti-c-Met agents. Additionally, identification of patients that might benefit from therapy could help to increase the selectivity and efficacy of the therapy. © 2017 Wiley Periodicals, Inc.

  6. Convergent RANK- and c-Met-mediated signaling components predict survival of patients with prostate cancer: an interracial comparative study.

    Science.gov (United States)

    Hu, Peizhen; Chung, Leland W K; Berel, Dror; Frierson, Henry F; Yang, Hua; Liu, Chunyan; Wang, Ruoxiang; Li, Qinlong; Rogatko, Andre; Zhau, Haiyen E

    2013-01-01

    We reported (PLoS One 6 (12):e28670, 2011) that the activation of c-Met signaling in RANKL-overexpressing bone metastatic LNCaP cell and xenograft models increased expression of RANK, RANKL, c-Met, and phosphorylated c-Met, and mediated downstream signaling. We confirmed the significance of the RANK-mediated signaling network in castration resistant clinical human prostate cancer (PC) tissues. In this report, we used a multispectral quantum dot labeling technique to label six RANK and c-Met convergent signaling pathway mediators simultaneously in formalin fixed paraffin embedded (FFPE) tissue specimens, quantify the intensity of each expression at the sub-cellular level, and investigated their potential utility as predictors of patient survival in Caucasian-American, African-American and Chinese men. We found that RANKL and neuropilin-1 (NRP-1) expression predicts survival of Caucasian-Americans with PC. A Gleason score ≥ 8 combined with nuclear p-c-Met expression predicts survival in African-American PC patients. Neuropilin-1, p-NF-κB p65 and VEGF are predictors for the overall survival of Chinese men with PC. These results collectively support interracial differences in cell signaling networks that can predict the survival of PC patients.

  7. Convergent RANK- and c-Met-mediated signaling components predict survival of patients with prostate cancer: an interracial comparative study.

    Directory of Open Access Journals (Sweden)

    Peizhen Hu

    Full Text Available We reported (PLoS One 6 (12:e28670, 2011 that the activation of c-Met signaling in RANKL-overexpressing bone metastatic LNCaP cell and xenograft models increased expression of RANK, RANKL, c-Met, and phosphorylated c-Met, and mediated downstream signaling. We confirmed the significance of the RANK-mediated signaling network in castration resistant clinical human prostate cancer (PC tissues. In this report, we used a multispectral quantum dot labeling technique to label six RANK and c-Met convergent signaling pathway mediators simultaneously in formalin fixed paraffin embedded (FFPE tissue specimens, quantify the intensity of each expression at the sub-cellular level, and investigated their potential utility as predictors of patient survival in Caucasian-American, African-American and Chinese men. We found that RANKL and neuropilin-1 (NRP-1 expression predicts survival of Caucasian-Americans with PC. A Gleason score ≥ 8 combined with nuclear p-c-Met expression predicts survival in African-American PC patients. Neuropilin-1, p-NF-κB p65 and VEGF are predictors for the overall survival of Chinese men with PC. These results collectively support interracial differences in cell signaling networks that can predict the survival of PC patients.

  8. The c-Met tyrosine kinase inhibitor JNJ-38877605 causes renal toxicity through species-specific insoluble metabolite formation

    NARCIS (Netherlands)

    M.P. Lolkema (Martijn); H.H. Bohets (Hilde H.); H.-T. Arkenau (H.); A. Lampo (Ann); E. Barale (Erio); M.J.A. de Jonge (Maja); L. van Doorn (Leni); P. Hellemans (Peter); J.S. de Bono (Johann); F.A.L.M. Eskens (Ferry)

    2015-01-01

    textabstractPurpose: The receptor tyrosine kinase c-Met plays an important role in tumorigenesis and is a novel target for anticancer treatment. This phase I, first-in-human trial, explored safety, pharmacokinetics, pharmacodynamics, and initial antitumor activity of JNJ-38877605, a potent and

  9. Multiplexed quantum dot labeling of activated c-Met signaling in castration-resistant human prostate cancer.

    Directory of Open Access Journals (Sweden)

    Peizhen Hu

    Full Text Available The potential application of multiplexed quantum dot labeling (MQDL for cancer detection and prognosis and monitoring therapeutic responses has attracted the interests of bioengineers, pathologists and cancer biologists. Many published studies claim that MQDL is effective for cancer biomarker detection and useful in cancer diagnosis and prognosis, these studies have not been standardized against quantitative biochemical and molecular determinations. In the present study, we used a molecularly characterized human prostate cancer cell model exhibiting activated c-Met signaling with epithelial to mesenchymal transition (EMT and lethal metastatic progression to bone and soft tissues as the gold standard, and compared the c-Met cell signaling network in this model, in clinical human prostate cancer tissue specimens and in a castration-resistant human prostate cancer xenograft model. We observed c-Met signaling network activation, manifested by increased phosphorylated c-Met in all three. The downstream survival signaling network was mediated by NF-κB and Mcl-1 and EMT was driven by receptor activator of NF-κB ligand (RANKL, at the single cell level in clinical prostate cancer specimens and the xenograft model. Results were confirmed by real-time RT-PCR and western blots in a human prostate cancer cell model. MQDL is a powerful tool for assessing biomarker expression and it offers molecular insights into cancer progression at both the cell and tissue level with high degree of sensitivity.

  10. Araguspongine C Induces Autophagic Death in Breast Cancer Cells through Suppression of c-Met and HER2 Receptor Tyrosine Kinase Signaling

    Directory of Open Access Journals (Sweden)

    Mohamed R. Akl

    2015-01-01

    Full Text Available Receptor tyrosine kinases are key regulators of cellular growth and proliferation. Dysregulations of receptor tyrosine kinases in cancer cells may promote tumorigenesis by multiple mechanisms including enhanced cell survival and inhibition of cell death. Araguspongines represent a group of macrocyclic oxaquinolizidine alkaloids isolated from the marine sponge Xestospongia species. This study evaluated the anticancer activity of the known oxaquinolizidine alkaloids araguspongines A, C, K and L, and xestospongin B against breast cancer cells. Araguspongine C inhibited the proliferation of multiple breast cancer cell lines in vitro in a dose-dependent manner. Interestingly, araguspongine C-induced autophagic cell death in HER2-overexpressing BT-474 breast cancer cells was characterized by vacuole formation and upregulation of autophagy markers including LC3A/B, Atg3, Atg7, and Atg16L. Araguspongine C-induced autophagy was associated with suppression of c-Met and HER2 receptor tyrosine kinase activation. Further in-silico docking studies and cell-free Z-LYTE assays indicated the potential of direct interaction between araguspongine C and the receptor tyrosine kinases c-Met and HER2 at their kinase domains. Remarkably, araguspongine C treatment resulted in the suppression of PI3K/Akt/mTOR signaling cascade in breast cancer cells undergoing autophagy. Induction of autophagic death in BT-474 cells was also associated with decreased levels of inositol 1,4,5-trisphosphate receptor upon treatment with effective concentration of araguspongine C. In conclusion, results of this study are the first to reveal the potential of araguspongine C as an inhibitor to receptor tyrosine kinases resulting in the induction of autophagic cell death in breast cancer cells.

  11. Characterization, biodistribution and small-animal SPECT of I-125-labeled c-Met binding peptide in mice bearing c-Met receptor tyrosine kinase-positive tumor xenografts

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Eun-Mi; Park, Eun-Hye; Cheong, Su-Jin; Lee, Chang-Moon; Kim, Dong Wook [Department of Nuclear Medicine, Chonbuk National University Medical School and Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Research Institute of Clinical Medicine, Chonbuk National University Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Cyclotron Research Center, Chonbuk National University Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Jeong, Hwan-Jeong [Department of Nuclear Medicine, Chonbuk National University Medical School and Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Research Institute of Clinical Medicine, Chonbuk National University Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Cyclotron Research Center, Chonbuk National University Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of)], E-mail: jayjeong@chonbuk.ac.kr; Lim, Seok Tae; Sohn, Myung-Hee [Department of Nuclear Medicine, Chonbuk National University Medical School and Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Research Institute of Clinical Medicine, Chonbuk National University Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Cyclotron Research Center, Chonbuk National University Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Kim, Kisu; Chung, Junho [Cancer Research Institute, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

    2009-05-15

    c-Met is a receptor tyrosine kinase involved in tumor cell growth, invasion, metastases and angiogenesis. Overexpression of c-Met is frequently observed in several tumor types. Here, we report the in vitro cell-binding properties and biodistribution and SPECT/CT imaging in glioma (U87MG) xenograft-bearing mice of {sup 125}I-labeled c-Met-binding peptides (cMBPs) including analogs conjugated to amino acid and aliphatic carbon linkers. In vitro assays showed that the peptide without any linker and those with GGG and 8-aminooctanoic acid linkers had low cellular internalization and that IC{sub 50} values of peptides were 1.5 {mu}M, 65 nM and 85.3 nM, respectively. Biodistribution studies showed the GGG-containing peptide had higher tumor uptake and a higher tumor-to-blood activity concentration ratio than other receptor-binding ligands. SPECT/CT studies with a dedicated small-animal imaging system were performed in U87MG-bearing athymic mice. Although U87MG tumor xenografts could be visualized by SPECT/micro-CT using the various {sup 125}I labeled cMBPs, image contrast and overall quality were unremarkable.

  12. Up-regulated expression of Ezrin and c-Met proteins are related to the metastasis and prognosis of gastric carcinomas

    OpenAIRE

    Zhao, Jing; Zhang, Xiaoxiao; Xin, Yang

    2011-01-01

    Recent publications demonstrated that abnormal expression of Ezrin and c-Met proteins were related to carcinogenesis, metastasis and prognosis of various sorts of tumors. In this study we detected the expressions of Ezrin and c-Met proteins in normal gastric mucosa, chronic atrophic gastritis, intestinal metaplasia, dysplasia and gastric carcinoma and analyzed the correlations with metastasis and prognosis of gastric carcinomas. The results demonstrated that both Ezrin and c-Met overexpressio...

  13. Biochemical characterization of AMG 102: a neutralizing, fully human monoclonal antibody to human and nonhuman primate hepatocyte growth factor.

    Science.gov (United States)

    Burgess, Teresa L; Sun, Jan; Meyer, Susanne; Tsuruda, Trace S; Sun, Jilin; Elliott, Gary; Chen, Qing; Haniu, Mitsuru; Barron, Will F; Juan, Todd; Zhang, Ke; Coxon, Angela; Kendall, Richard L

    2010-02-01

    AMG 102 is a fully human monoclonal antibody that selectively targets and neutralizes hepatocyte growth factor/scatter factor (HGF/SF). A detailed biochemical and functional characterization of AMG 102 was done to support its clinical development for the treatment of cancers dependent on signaling through the HGF/SF:c-Met pathway. In competitive equilibrium binding experiments, AMG 102 bound to human and cynomolgus monkey HGF with affinities of approximately 19 pmol/L and 41 pmol/L, respectively. However, AMG 102 did not detect mouse or rabbit HGF on immunoblots. Immunoprecipitation experiments showed that AMG 102 preferentially bound to the mature, active form of HGF, and incubation of AMG 102/HGF complexes with kallikrein protease indicated that AMG 102 had no apparent effect on proteolytic processing of the inactive HGF precursor. AMG 102 inhibited human and cynomolgus monkey HGF-induced c-Met autophosphorylation in PC3 cells with IC(50) values of 0.12 nmol/L and 0.24 nmol/L, respectively. AMG 102 also inhibited cynomolgus monkey HGF-induced migration of human MDA-MB-435 cells but not rat HGF-induced migration of mouse 4T1 cells. Epitope-mapping studies of recombinant HGF molecules comprising human/mouse chimeras and human-to-mouse amino acid substitutions showed that amino acid residues near the NH(2)-terminus of the beta-chain are critical for AMG 102 binding. Bound AMG 102 protected one trypsin protease cleavage site near the NH(2)-terminus of the beta-chain of human HGF, further substantiating the importance of this region for AMG 102 binding. Currently, AMG 102 is in phase II clinical trials in a variety of solid tumor indications. Mol Cancer Ther; 9(2); 400-9.

  14. The c-MET Network as Novel Prognostic Marker for Predicting Bladder Cancer Patients with an Increased Risk of Developing Aggressive Disease.

    Directory of Open Access Journals (Sweden)

    Young-Won Kim

    Full Text Available Previous studies have shown that c-MET is overexpressed in cases of aggressive bladder cancer (BCa. Identification of crosstalk between c-MET and other RTKs such as AXL and PDGFR suggest that c-MET network genes (c-MET-AXL-PDGFR may be clinically relevant to BCa. Here, we examine whether expression of c-MET network genes can be used to identify BCa patients at increased risk of developing aggressive disease. In vitro analysis, c-MET knockdown suppressed cell proliferation, invasion, and migration, and increased sensitivity to cisplatin-induced apoptosis. In addition, c-MET network gene (c-MET, AXL, and PDGFR expression allowed discrimination of BCa tissues from normal control tissues and appeared to predict poor disease progression in non-muscle invasive BCa patients and poor overall survival in muscle invasive BCa patients. These results suggest that c-MET network gene expression is a novel prognostic marker for predicting which BCa patients have an increased risk of developing aggressive disease. These genes might be a useful marker for co-targeting therapy, and are expected to play an important role in improving both response to treatment and survival of BCa patients.

  15. Response assessment of bevacizumab therapy in GBM with integrated 11C-MET-PET/MRI: a feasibility study

    Energy Technology Data Exchange (ETDEWEB)

    Deuschl, Cornelius [University Hospital Essen, Institute of Diagnostic and Interventional Radiology and Neuroradiology, Essen (Germany); University of Duisburg-Essen, Erwin L. Hahn Institute for Magnetic Resonance Imaging, Duisburg (Germany); Moenninghoff, Christoph; Goericke, Sophia; Forsting, Michael; Umutlu, Lale [University Hospital Essen, Institute of Diagnostic and Interventional Radiology and Neuroradiology, Essen (Germany); Kirchner, Julian [University Hospital Duesseldorf, Institute of Diagnostic and Interventional Radiology, Duesseldorf (Germany); Koeppen, Susanne [University Hospital Essen, Department of Neurology, Essen (Germany); Binse, Ina; Poeppel, Thorsten D.; Herrmann, Ken [University Hospital Essen, Department of Nuclear Medicine, Essen (Germany); Quick, Harald H. [University of Duisburg-Essen, Erwin L. Hahn Institute for Magnetic Resonance Imaging, Duisburg (Germany); University Hospital Essen, High Field and Hybrid MR Imaging, Essen (Germany); Hense, Joerg [University Hospital Essen, Department of Medical Oncology, West German Cancer Center, Essen (Germany); Schlamann, Marc [University Hospital Essen, Institute of Diagnostic and Interventional Radiology and Neuroradiology, Essen (Germany); University Hospital Giessen, Department of Neuroradiology, Essen (Germany)

    2017-08-15

    The objective of this study was to evaluate the potential of integrated 11C-MET PET/MR for response assessment of relapsed glioblastoma (GBM) receiving bevacizumab treatment. Eleven consecutive patients with relapsed GBM were enrolled for an integrated 11C-MET PET/MRI at baseline and at follow-up. Treatment response for MRI was evaluated according to Response Assessment in Neuro-oncology (RANO) criteria and integrated 11C-MET PET was assessed by the T/N ratio. MRI showed no patient with complete response (CR), six of 11 patients with PR, four of 11 patients with SD, and one of 11 patients with progressive disease (PD). PET revealed metabolic response in five of the six patients with partial response (PR) and in two of the four patients with stable disease (SD), whereas metabolic non-response was detected in one of the six patients with PR, in two of the four patients with SD, and in the one patient with PD. Morphological imaging was predictive for PFS and OS when response was defined as CR, PR, SD, and non-response as PD. Metabolic imaging was predictive when using T/N ratio reduction of >25 as discriminator. Based on the morphologic and metabolic findings of this study a proposal for applying integrated PET/MRI for treatment response in relapsed GBM was developed, which was significantly predictive for PFS and OS (P = 0.010 respectively 0,029, log). This study demonstrates the potential of integrated 11C-MET-PET/MRI for response assessment of GBM and the utility of combined assessment of morphologic and metabolic information with the proposal for assessing relapsed GBM. (orig.)

  16. Tumor budding in colorectal carcinoma assessed by cytokeratin immunostaining and budding areas: possible involvement of c-Met.

    Science.gov (United States)

    Satoh, Keisuke; Nimura, Satoshi; Aoki, Mikiko; Hamasaki, Makoto; Koga, Kaori; Iwasaki, Hiroshi; Yamashita, Yuichi; Kataoka, Hiroaki; Nabeshima, Kazuki

    2014-11-01

    Tumor budding/sprouting has been shown to be an independent adverse prognostic factor in T1 and T3N0 colorectal carcinomas, however, its assessment could be improved by more accurate identification of budding carcinoma cells and consideration of budding areas. Moreover, tumor budding mechanisms are yet to be defined. In this study, we evaluated the identification of budding tumor cells by either H&E staining alone or H&E with immunohistochemistry and developed a scoring system based on budding grades and areas. We examined whether the budding score correlated with clinicopathologic features and prognosis and the association between tumor budding/sprouting and c-Met protein expression and phosphorylation and MET gene copy numbers because c-Met is known to play an important role in colorectal carcinoma tumorigenesis. Cytokeratin immunohistochemistry could identify tumors with shorter disease-free survival (DFS) from the low-grade budding group assessed with H&E alone. High budding scores based on budding grade and area were more significantly correlated with DFS than scores obtained using the budding grade alone. In tumors with a high budding score, c-Met expression and phosphorylation levels and MET gene copy numbers were significantly increased at the invasive front compared with those in superficial tumor portions. This study showed for the first time that high levels of phospho-c-Met at the invasive front were significantly associated with a high budding score and shorter DFS. In conclusion, a budding score assessed by budding grades and budding-positive areas correlates highly with clinicopathologic aggressive features of colorectal carcinoma. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  17. Fatty Acid Synthase Activity as a Target for c-Met Driven Prostate Cancer

    Science.gov (United States)

    2013-07-01

    cancer potentially due to increased fecal fat excretion. In addition, several families of plant-derived flavonoid compounds including...Apoptosis by Flavonoids Is Associated with Their Ability to Inhibit Fatty Acid Synthase Activity. J. Biol. Chem., 2005. 280(7): p. 5636-5645. 156... flavonoids , represent a source of relatively nontoxic, orally available and affordable compounds that are known to affect a number of different

  18. c-Met Expression Is a Marker of Poor Prognosis in Patients With Locally Advanced Head and Neck Squamous Cell Carcinoma Treated With Chemoradiation

    Energy Technology Data Exchange (ETDEWEB)

    Baschnagel, Andrew M. [Department of Radiation Oncology, William Beaumont Hospital, Royal Oak, Michigan (United States); Williams, Lindsay [Department of Pathology, William Beaumont Hospital, Royal Oak, Michigan (United States); Hanna, Alaa; Chen, Peter Y.; Krauss, Daniel J. [Department of Radiation Oncology, William Beaumont Hospital, Royal Oak, Michigan (United States); Pruetz, Barbara L. [Beaumont BioBank, William Beaumont Hospital, Royal Oak, Michigan (United States); Akervall, Jan [Beaumont BioBank, William Beaumont Hospital, Royal Oak, Michigan (United States); Department of Otolaryngology, William Beaumont Hospital, Royal Oak, Michigan (United States); Wilson, George D., E-mail: George.Wilson@Beaumont.edu [Department of Radiation Oncology, William Beaumont Hospital, Royal Oak, Michigan (United States); Beaumont BioBank, William Beaumont Hospital, Royal Oak, Michigan (United States)

    2014-03-01

    Purpose: To examine the prognostic significance of c-Met expression in relation to p16 and epidermal growth factor receptor (EGFR) in patients with locally advanced head and neck squamous cell carcinoma (HNSCC) treated with definitive concurrent chemoradiation. Methods and Materials: Archival tissue from 107 HNSCC patients treated with chemoradiation was retrieved, and a tissue microarray was assembled. Immunohistochemical staining of c-Met, p16, and EGFR was performed. c-Met expression was correlated with p16, EGFR, clinical characteristics, and clinical endpoints including locoregional control (LRC), distant metastasis (DM), disease-free survival (DFS), and overall survival (OS). Results: Fifty-one percent of patients were positive for p16, and 53% were positive for EGFR. Both p16-negative (P≤.001) and EGFR-positive (P=.019) status predicted for worse DFS. Ninety-three percent of patients stained positive for c-Met. Patients were divided into low (0, 1, or 2+ intensity) or high (3+ intensity) c-Met expression. On univariate analysis, high c-Met expression predicted for worse LRC (hazard ratio [HR] 2.27; 95% CI, 1.08-4.77; P=.031), DM (HR 4.41; 95% CI, 1.56-12.45; P=.005), DFS (HR 3.00; 95% CI, 1.68-5.38; P<.001), and OS (HR 4.35; 95% CI, 2.13-8.88; P<.001). On multivariate analysis, after adjustment for site, T stage, smoking history, and EGFR status, only high c-Met expression (P=.011) and negative p16 status (P=.003) predicted for worse DFS. High c-Met expression was predictive of worse DFS in both EGFR-positive (P=.032) and -negative (P=.008) patients. In the p16-negative patients, those with high c-Met expression had worse DFS (P=.036) than did those with low c-Met expression. c-Met expression was not associated with any outcome in the p16-positive patients. Conclusions: c-Met is expressed in the majority of locally advanced HNSCC cases, and high c-Met expression predicts for worse clinical outcomes. High c-Met expression predicted for worse DFS in p16

  19. c-met is overexpressed in type I ovarian cancer: Results of an investigative analysis in a cohort of consecutive ovarian cancer patients

    Science.gov (United States)

    Battista, Marco Johannes; Schmidt, Marcus; Jakobi, Sina; Cotarelo, Cristina; Almstedt, Katrin; Heimes, Anne-Sophie; Makris, Georgios-Marios; Weyer, Veronika; Lebrecht, Antje; Hoffmann, Gerald; Eichbaum, Michael

    2016-01-01

    The tyrosine kinase c-met alters signaling cascades such as the BRAF-MAPK and PI3K-PKB pathways. These alterations are involved in the carcinogenesis of type I but not type II ovarian cancer (OC). Therefore, the present study investigated the patterns of c-met expression in a cohort of consecutive patients with OC. c-met expression was determined by immunohistochemical analysis. Differences in c-met overexpression among subgroups of established clinicopathological features, including age, histological subtype, tumor stage, histological grading, post-operative tumor burden and completeness of chemotherapy, were determined by χ2 test. Cox regression analyses were performed to determine the prognostic effect of c-met. Survival rates were estimated using the Kaplan-Meier method. A total of 106 patients were enrolled into the study. c-met was overexpressed in 20.8% of the entire cohort; 35.7% of patients with type I OC and 8.6% of patients with type II OC showed overexpression (P=0.001). However, c-met overexpression was not associated with any other established clinicopathological features (all P-values >0.05). Univariate Cox regression analysis showed that overexpression of c-met was associated neither with progression-free survival (PFS) nor with disease-specific survival (DSS) (P=0.835 and P=0.414, respectively). Kaplan-Meier plots also failed to demonstrate an effect of c-met on the 5-year PFS and DSS rates (P=0.938 and P=0.412, respectively). These findings support the hypotheses that the overexpression of c-met is associated with type I but not type II OC, and that overexpression of c-met does not affect the prognosis of OC. PMID:27602128

  20. Marine Bromophenol Derivative 3,4-Dibromo-5-(2-bromo-3,4-dihydroxy-6-isopropoxymethyl benzylbenzene-1,2-diol Protects Hepatocytes from Lipid-Induced Cell Damage and Insulin Resistance via PTP1B Inhibition

    Directory of Open Access Journals (Sweden)

    Jiao Luo

    2015-07-01

    Full Text Available 3,4-Dibromo-5-(2-bromo-3,4-dihydroxy-6-isopropoxymethyl benzylbenzene-1,2-diol (HPN is a bromophenol derivative from the marine red alga Rhodomela confervoides. We have previously found that HPN exerted an anti-hyperglycemic property in db/db mouse model. In the present study, we found that HPN could protect HepG2 cells against palmitate (PA-induced cell death. Data also showed that HPN inhibited cell death mainly by blocking the cell apoptosis. Further studies demonstrated that HPN (especially at 1.0 μM significantly restored insulin-stimulated tyrosine phosphorylation of IR and IRS1/2, and inhibited the PTP1B expression level in HepG2 cells. Furthermore, the expression of Akt was activated by HPN, and glucose uptake was significantly increased in PA-treated HepG2 cells. Our results suggest that HPN could protect hepatocytes from lipid-induced cell damage and insulin resistance via PTP1B inhibition. Thus, HPN can be considered to have potential for the development of anti-diabetic agent that could protect both hepatic cell mass and function.

  1. Hepatocyte growth factor-modulated rat Leydig cell functions.

    Science.gov (United States)

    Del Bravo, Jessica; Catizone, Angela; Ricci, Giulia; Galdieri, Michela

    2007-01-01

    Hepatocyte growth factor (HGF) regulates many cellular functions acting through c-Met, its specific tyrosine kinase receptor. We previously reported that in prepuberal rats HGF is secreted by the peritubular myoid cells during the entire postnatal testicular development and by the Sertoli cells only at puberty. We have also demonstrated that germ cells at different stages of development express c-Met and that HGF modulates germ cell proliferation and apoptosis. In the present article, we extend our study to the interstitial compartment of the testis and demonstrate that the c-Met protein is present on Leydig cells. The receptor is functionally active as demonstrated by the detected effects of HGF. We report in this article that HGF significantly increases the amount of testosterone secreted by the Leydig cells and decreases the number of Leydig cells undergoing apoptosis. The antiapoptotic effect of HGF is mediated by caspase-3 activity because the amount of the active fragment of the enzyme is decreased in Leydig cells cultured in the presence of HGF. However, treatment with the growth factor does not modify the expression levels of caspase-3 mRNA. These data indicate that HGF regulates the functional activities of Leydig cells. Interestingly, the steroidogenetic activity of the cells is increased by HGF in cultured explants of testicular tissues as well as the antiapoptotic effect of HGF. Therefore, our data indicate that HGF has a crucial role in the regulation of male fertility.

  2. Preclinical trials for prevention of tumor progression of hepatocellular carcinoma by LZ-8 targeting c-Met dependent and independent pathways.

    Directory of Open Access Journals (Sweden)

    Jia-Ru Wu

    Full Text Available Hepatocellular carcinoma (HCC is among the most lethal cancers. Mounting studies highlighted the essential role of the HGF/c-MET axis in driving HCC tumor progression. Therefore, c-Met is a potential therapeutic target for HCC. However, several concerns remain unresolved in c-Met targeting. First, the status of active c-Met in HCC must be screened to determine patients suitable for therapy. Second, resistance and side effects have been observed frequently when using conventional c-Met inhibitors. Thus, a preclinical system for screening the status of c-Met signaling and identifying efficient and safe anti-HCC agents is urgently required. In this study, immunohistochemical staining of phosphorylated c-Met (Tyr1234 on tissue sections indicated that HCCs with positive c-Met signaling accounted for approximately 46% in 26 cases. Second, many patient-derived HCC cell lines were established and characterized according to motility and c-Met signaling status. Moreover, LZ8, a medicinal peptide purified from the herb Lingzhi, featuring immunomodulatory and anticancer properties, was capable of suppressing cell migration and slightly reducing the survival rate of both c-Met positive and negative HCCs, HCC372, and HCC329, respectively. LZ8 also suppressed the intrahepatic metastasis of HCC329 in SCID mice. On the molecular level, LZ8 suppressed the expression of c-Met and phosphorylation of c-Met, ERK and AKT in HCC372, and suppressed the phosphorylation of JNK, ERK, and AKT in HCC329. According to receptor array screening, the major receptor tyrosine kinase activated in HCC329 was found to be the epidermal growth factor receptor (EGFR. Moreover, tyrosine-phosphorylated EGFR (the active EGFR was greatly suppressed in HCC329 by LZ8 treatment. In addition, LZ8 blocked HGF-induced cell migration and c-Met-dependent signaling in HepG2. In summary, we designed a preclinical trial using LZ8 to prevent the tumor progression of patient-derived HCCs with c-Met

  3. Synergistic effects of various Her inhibitors in combination with IGF-1R, C-MET and Src targeting agents in breast cancer cell lines.

    Science.gov (United States)

    Stanley, Aryan; Ashrafi, G Hossein; Seddon, Alan M; Modjtahedi, Helmout

    2017-06-21

    Overexpression of HER2 has been reported in around 25% of human breast cancers. Despite recent advances in HER2 targeted therapy, many patients still experience primary and secondary resistance to such treatments, the mechanisms for which are poorly understood. Here, we investigated the sensitivity of a panel of breast cancer cell lines to treatment with various types of HER-family inhibitors alone or in combination with other tyrosine kinase inhibitors or chemotherapeutic agents. We found that treatment with the second-generation irreversible HER-family inhibitors, particularly afatinib and neratinib, were more effective than treatment with the first-generation reversible inhibitors in inhibiting growth, migration and downstream cell signalling in breast cancer cells. Of the three HER2 overexpressing cell lines in this panel, SKBr3 and BT474 were highly sensitive to treatment with HER-family inhibitors, while MDA-MB-453 was comparatively resistant. Combinations of HER-family inhibitors with NVP-AEW541, dasatinib or crizotinib (inhibitors of IGF-1R, Src and c-Met/ALK, respectively) led to synergistic effects in some of the cell lines examined. In particular, treatment with a combination of Src and HER-family member inhibitors resulted in synergistic growth inhibition of MDA-MB453 cells, implicating Src as a mediator of resistance to HER2-targeting agents. Our results suggest that combining HER-family inhibitors with other TKIs such as dasatinib may have therapeutic advantages in certain breast cancer subtypes and warrants further investigation.

  4. Induction and inhibition of cytochrome P450 1A1 and ethoxyresorufin-O-deethylation activity by polybrominated diphenyl ethers (PBDEs) in cynomolgus monkey primary hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Peters, L.; Sanderson, J.T.; Berg, M. van den [Utrecht Univ. (Netherlands). Inst. for Risk Assessment Sciences; Bergman, A. [Stockholm Univ. (Sweden). Dept. of Environmental Chemistry

    2004-09-15

    Brominated flame retardants (BFRs) make up for 39% of the worldwide flame-retardants market. One groups of BFR, Polybrominated diphenylethers (PBDEs) are used as additive flameretardants in plastic materials, paints, and textile fabrics. Some PBDEs have been found to be lipophilic and persistent, and consequently bioaccumulate. Recently, levels of some PBDEs have been increasing in fish, wildlife, and in human tissue. The structural similarity of certain PBDE congeners to other polyhalogenated aromatic hydrocarbons such as polychlorinated biphenyls (PCBs) has raised concerns that these compounds might act as agonists for the aryl hydrocarbon receptor (AhR). If some of these PBDEs were to act as Ah receptor agonists, they would warrant inclusion in the toxic equivalence factor (TEF) concept. CYP1A1 is a cytochrome P450 (CYP) enzyme that is involved in phase 1 biotransformation of xenobiotics and endogenous compounds such as estrogens. Many CYP enzymes detoxify xenobiotics or bioactivate xenobiotics to reactive intermediates. Although CYP1A1 is expressed in all mammals, there are differences in expression levels among species and tissues. To study the possible dioxin-like effects of environmentally most relevant PBDEs (BDE47, 77, 99, 100, 153, 154, 183, 209), the Ah receptor-mediated induction CYP1A1 was studied in cynomolgus monkey (Macaca fascicularis) primary hepatocytes. CYP 1A1 is the major enzyme that catalyses the deethylation of 7-ethoxyresorufin to resorufin. This ethoxyresorufin-Odeethylation (EROD) activity was used as a marker for CYP1A1 activity.

  5. Syndecan-1 Attenuates Lung Injury during Influenza Infection by Potentiating c-Met Signaling to Suppress Epithelial Apoptosis.

    Science.gov (United States)

    Brauer, Rena; Ge, Lingyin; Schlesinger, Saundra Y; Birkland, Timothy P; Huang, Ying; Parimon, Tanyalak; Lee, Vivian; McKinney, Bonnie L; McGuire, John K; Parks, William C; Chen, Peter

    2016-08-01

    Syndecan-1 is a cell surface heparan sulfate proteoglycan primarily expressed in the lung epithelium. Because the influenza virus is tropic to the airway epithelium, we investigated the role of syndecan-1 in influenza infection. To determine the mechanism by which syndecan-1 regulates the lung mucosal response to influenza infection. Wild-type (WT) and Sdc1(-/-) mice were infected with a H1N1 virus (PR8) as an experimental model of influenza infection. Human and murine airway epithelial cell cultures were also infected with PR8 to study the mechanism by which syndecan-1 regulates the inflammatory response. We found worsened outcomes and lung injury in Sdc1(-/-) mice compared with WT mice after influenza infection. Our data demonstrated that syndecan-1 suppresses bronchial epithelial apoptosis during influenza infection to limit widespread lung inflammation. Furthermore, we determined that syndecan-1 attenuated apoptosis by crosstalking with c-Met to potentiate its cytoprotective signals in airway epithelial cells during influenza infection. Our work shows that cell-associated syndecan-1 has an important role in regulating lung injury. Our findings demonstrate a novel mechanism in which cell membrane-associated syndecan-1 regulates the innate immune response to influenza infection by facilitating cytoprotective signals through c-Met signaling to limit bronchial epithelial apoptosis, thereby attenuating lung injury and inflammation.

  6. c-Met PET Imaging Detects Early-Stage Locoregional Recurrence of Basal-Like Breast Cancer.

    Science.gov (United States)

    Arulappu, Appitha; Battle, Mark; Eisenblaetter, Michel; McRobbie, Graeme; Khan, Imtiaz; Monypenny, James; Weitsman, Gregory; Galazi, Myria; Hoppmann, Susan; Gazinska, Patrycja; Wulaningsih, Wulan; Dalsgaard, Grethe Tang; Macholl, Sven; Ng, Tony

    2016-05-01

    Locoregional recurrence of breast cancer poses significant clinical problems because of frequent inoperability once the chest wall is involved. Early detection of recurrence by molecular imaging agents against therapeutically targetable receptors, such as c-Met, would be of potential benefit. The aim of this study was to assess (18)F-AH113804, a peptide-based molecular imaging agent with high affinity for human c-Met, for the detection of early-stage locoregional recurrence in a human basal-like breast cancer model, HCC1954. HCC1954 tumor-bearing xenograft models were established, and (18)F-AH113804 was administered. Distribution of radioactivity was determined via PET at 60 min after radiotracer injection. PET and CT images were acquired 10 d after tumor inoculation, to establish baseline distribution and uptake, and then on selected days after surgical tumor resection. CT images and caliper were used to determine the tumor volume. Radiotracer uptake was assessed by (18)F-AH113804 PET imaging. c-Met expression was assessed by immunofluorescence imaging of tumor samples and correlated with (18)F-AH113804 PET imaging results. Baseline uptake of (18)F-AH113804, determined in tumor-bearing animals after 10 d, was approximately 2-fold higher in the tumor than in muscle tissue or the contralateral mammary fat pad. The tumor growth rate, determined from CT images, was comparable between the animals with recurrent tumors, with detection of tumors of low volume (tumor resection. (18)F-AH113804 PET detected local tumor recurrence as early as 6 d after surgery in the recurrent tumor-bearing animals and exhibited significantly higher (18)F-AH113804 uptake (in comparison to mammary fatty tissue), with a target-to-background (muscle) ratio of approximately 3:1 (P tumor samples, determined by immunofluorescence, correlated with the respective (18)F-AH113804 imaging signals (r = 0.82, P tumor and has potential utility for the detection of locoregional recurrence from an early

  7. Gene signatures derived from a c-MET-driven liver cancer mouse model predict survival of patients with hepatocellular carcinoma.

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    Irena Ivanovska

    Full Text Available Biomarkers derived from gene expression profiling data may have a high false-positive rate and must be rigorously validated using independent clinical data sets, which are not always available. Although animal model systems could provide alternative data sets to formulate hypotheses and limit the number of signatures to be tested in clinical samples, the predictive power of such an approach is not yet proven. The present study aims to analyze the molecular signatures of liver cancer in a c-MET-transgenic mouse model and investigate its prognostic relevance to human hepatocellular carcinoma (HCC. Tissue samples were obtained from tumor (TU, adjacent non-tumor (AN and distant normal (DN liver in Tet-operator regulated (TRE human c-MET transgenic mice (n = 21 as well as from a Chinese cohort of 272 HBV- and 9 HCV-associated HCC patients. Whole genome microarray expression profiling was conducted in Affymetrix gene expression chips, and prognostic significances of gene expression signatures were evaluated across the two species. Our data revealed parallels between mouse and human liver tumors, including down-regulation of metabolic pathways and up-regulation of cell cycle processes. The mouse tumors were most similar to a subset of patient samples characterized by activation of the Wnt pathway, but distinctive in the p53 pathway signals. Of potential clinical utility, we identified a set of genes that were down regulated in both mouse tumors and human HCC having significant predictive power on overall and disease-free survival, which were highly enriched for metabolic functions. In conclusions, this study provides evidence that a disease model can serve as a possible platform for generating hypotheses to be tested in human tissues and highlights an efficient method for generating biomarker signatures before extensive clinical trials have been initiated.

  8. Activation of the c-Met pathway mobilizes an inflammatory network in the brain microenvironment to promote brain metastasis of breast cancer

    Science.gov (United States)

    Xing, Fei; Liu, Yin; Sharma, Sambad; Wu, Kerui; Chan, Michael D.; Lo, Hui-Wen; Carpenter, Richard L.; Metheny-Barlow, Linda J.; Zhou, Xiaobo; Qasem, Shadi A.; Pasche, Boris; Watabe, Kounosuke

    2016-01-01

    Brain metastasis is one of the chief causes of mortality in breast cancer patients, but the mechanisms that drive this process remains poorly understood. Here we report that brain metastatic cells expressing high levels of c-Met promote the metastatic process via inflammatory cytokine upregulation and vascular reprogramming. Activated c-Met signaling promoted adhesion of tumor cells to brain endothelial cells and enhanced neovascularization by inducing the secretion of IL-8 and CXCL1. Additionally, stimulation of IL1β secretion by activation of c-Met induced tumor-associated astrocytes to secrete the c-Met ligand HGF. Thus, a feed-forward mechanism of cytokine release initiated and sustained by c-Met fed a vicious cycle which generated a favorable microenvironment for metastatic cells. Reinforcing our results, we found that pterostilbene, a compound that penetrates the blood-brain barrier, could suppress brain metastasis by targeting c-Met signaling. These findings suggest a potential utility of this natural compound for chemoprevention. PMID:27364556

  9. Hepatocyte growth factor modulates in vitro survival and proliferation of germ cells during postnatal testis development.

    Science.gov (United States)

    Catizone, A; Ricci, G; Del Bravo, J; Galdieri, M

    2006-04-01

    The hepatocyte growth factor (HGF) is a pleiotropic cytokine that influences mitogenesis, motility and differentiation of many different cell types by its tyrosine kinase receptor c-Met. We previously demonstrated that the c-Met/HGF system is present and functionally active during postnatal testis development. We found also that spermatozoa express c-Met and that HGF has a positive effect on the maintenance of sperm motility. In the present paper, we extend our study on the germ cells at different stages of differentiation during the postnatal development of the testis. We demonstrate that c-met is present in rat spermatogonia, pachytene spermatocytes and round spermatids and that HGF significantly increases spermatogonial proliferation in 8- to 10-day-old pre-pubertal rats. At this age HGF does not affect Sertoli cells and peritubular myoid cells proliferation. In addition, we studied the effect of the factor on germ cell apoptosis and we show that HGF prevents the germ cell apoptotic process. We also studied the effect of HGF on 18- to 20-day-old and 28- to 30-day-old rat testes. At these ages also the factor significantly increases germ cell duplication and decreases the number of apoptotic cells. However, the effect on programmed cell death is higher in the 8- to 10-day-old rats and declines in the older animals. In conclusion, we report that rat germ cells (spermatogonia, pachytene spermatocytes and round spermatids) express c-met and that HGF modulates germ cell proliferating activity and apoptosis in vitro. These data indicate that the c-Met/HGF system is involved in male germ cell homeostasis and, consequently, has a role in male fertility.

  10. Functional role of hepatocyte growth factor receptor during sperm maturation.

    Science.gov (United States)

    Catizone, A; Ricci, G; Galdieri, M

    2002-01-01

    Mammalian spermatozoa acquire motility and fertilizing capacity during their transit through the epididymis. Hepatocyte growth factor (HGF) is a pleiotropic cytokine with potent motogenic capacities that has been identified in different organs, including the mammalian male genital tract. In mice, HGF is present in the testis and, in large amounts, in the distal part of the epididymis. In prepuberal rats, we have demonstrated that HGF is synthesized by the peritubular myoid cells and in men, HGF is present in significant quantities in seminal plasma. It has been suggested that in mice, HGF has a role in initiating sperm motility, whereas in men, no significant correlations between HGF concentration and sperm motility have been found. In the present paper we report that in rats, HGF receptor, c-met, is expressed in testicular and epididymal spermatozoa. Through immunocytochemistry, we have found that c-met is exclusively localized on the head in testicular sperm. A different localization of c-met has been found in sperm isolated from caput and cauda epididymidis. Cells isolated from epididymal caput show a c-met localization exclusively restricted to the head in most cells. In a minority of caput epididymis spermatozoa the receptor is localized both in the cell head and along the flagellum. Spermatozoa isolated from the epididymal cauda were quite homogeneous, showing the receptor localized along the entire cell surface. We also report that HGF is synthesized and secreted by the rat epididymis as indicated by the scatter effect of epididymal cell homogenate and culture medium on MDCK cells. To clarify whether HGF is involved in the acquisition of sperm motility in the epididymis, its maintenance, or both, spermatozoa isolated from caput epididymidis have been cultured in medium alone or supplemented with HGF. The results obtained indicated that HGF has a positive effect on the maintenance of sperm motility which, in the absence of HGF, significantly decreases during

  11. Hepatic Stellate Cell Coculture Enables Sorafenib Resistance in Huh7 Cells through HGF/c-Met/Akt and Jak2/Stat3 Pathways

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    Weibo Chen

    2014-01-01

    Full Text Available Purpose. Tumor microenvironment confers drug resistance to kinase inhibitors by increasing RKT ligand levels that result in the activation of cell-survival signaling including PI3K and MAPK signals. We assessed whether HSC-LX2 coculture conferred sorafenib resistance in Huh7 and revealed the mechanism underlying the drug resistance. Experimental Design. The effect of LX2 on sorafenib resistance was determined by coculture system with Huh7 cells. The rescue function of LX2 supernatants was assessed by MTT assay and fluorescence microscopy. The underlying mechanism was tested by administration of pathway inhibitors and manifested by Western blotting. Results. LX2 coculture significantly induced sorafenib resistance in Huh7 by activating p-Akt that led to reactivation of p-ERK. LX2 secreted HGF into the culture medium that triggered drug resistance, and exogenous HGF could also induce sorafenib resistance. The inhibition of p-Akt blocked sorafenib resistance caused by LX2 coculture. Increased phosphorylation of Jak2 and Stat3 was also detected in LX2 cocultured Huh7 cells. The Jak inhibitor tofacitinib reversed sorafenib resistance by blocking Jak2 and Stat3 activation. The combined administration of sorafenib and p-Stat3 inhibitor S3I-201 augmented induced apoptosis even in the presence of sorafenib resistance. Conclusions. HSC-LX2 coculture induced sorafenib resistance in Huh7 through multiple pathways: HGF/c-Met/Akt pathway and Jak2/Stat3 pathway. A combined administration of sorafenib and S3I-201 was able to augment sorafenib-induced apoptosis even in the presence of LX2 coculture.

  12. Hepatocyte growth factor as a downstream mediator of vascular endothelial growth factor-dependent preservation of growth in the developing lung.

    Science.gov (United States)

    Seedorf, Gregory; Metoxen, Alexander J; Rock, Robert; Markham, Neil; Ryan, Sharon; Vu, Thiennu; Abman, Steven H

    2016-06-01

    Impaired vascular endothelial growth factor (VEGF) signaling contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). We hypothesized that the effects of VEGF on lung structure during development may be mediated through its downstream effects on both endothelial nitric oxide synthase (eNOS) and hepatocyte growth factor (HGF) activity, and that, in the absence of eNOS, trophic effects of VEGF would be mediated through HGF signaling. To test this hypothesis, we performed an integrative series of in vitro (fetal rat lung explants and isolated fetal alveolar and endothelial cells) and in vivo studies with normal rat pups and eNOS(-/-) mice. Compared with controls, fetal lung explants from eNOS(-/-) mice had decreased terminal lung bud formation, which was restored with recombinant human VEGF (rhVEGF) treatment. Neonatal eNOS(-/-) mice were more susceptible to hyperoxia-induced inhibition of lung growth than controls, which was prevented with rhVEGF treatment. Fetal alveolar type II (AT2) cell proliferation was increased with rhVEGF treatment only with mesenchymal cell (MC) coculture, and these effects were attenuated with anti-HGF antibody treatment. Unlike VEGF, HGF directly stimulated isolated AT2 cells even without MC coculture. HGF directly stimulates fetal pulmonary artery endothelial cell growth and tube formation, which is attenuated by treatment with JNJ-38877605, a c-Met inhibitor. rHGF treatment preserves alveolar and vascular growth after postnatal exposure to SU-5416, a VEGF receptor inhibitor. We conclude that the effects of VEGF on AT2 and endothelial cells during lung development are partly mediated through HGF-c-Met signaling and speculate that reciprocal VEGF-HGF signaling between epithelia and endothelia is disrupted in infants who develop BPD. Copyright © 2016 the American Physiological Society.

  13. Toward operative in vivo fluorescence imaging of the c-Met proto-oncogene for personalization of therapy in ovarian cancer.

    Science.gov (United States)

    Liu, Shujuan; Zheng, Yong; Volpi, Davide; El-Kasti, Muna; Klotz, Daniel; Tullis, Iain; Henricks, Andrea; Campo, Leticia; Myers, Kevin; Laios, Alex; Thomas, Peter; Ng, Tony; Dhar, Sunanda; Becker, Christian; Vojnovic, Borivoj; Ahmed, Ahmed Ashour

    2015-01-15

    Standard biomarker testing of a single macroscopic disease site is unlikely to be sufficient because of tumor heterogeneity. A focus on examining global biomarker expression or activity, particularly in microscopic residual chemotherapy-resistant disease, is needed for the appropriate selection of targeted therapies. This study was aimed at establishing a technique for the assessment of biomarkers of ovarian cancer peritoneal spread. An in-house developed fluorescent imaging device was used to detect the expression of the c-Met oncogene in ovarian cancer. A modified cyanine 5-tagged peptide, GE137, with a high in vitro affinity for the human c-Met protein, was tested in a panel of ovarian cancer cell lines. Finally, the feasibility of detecting submillimeter ovarian cancer cell peritoneal metastases in vivo was tested through the intravenous injection of GE137 into mice with tumor xenografts. Using optical imaging it was possible to detect c-Met expression in submillimeter peritoneal metastases that were freshly excised from a human high-grade serous ovarian cancer. GE137 selectively bound to the c-Met tyrosine kinase without activating survival signaling pathways (AKT or extracellular signal-regulated kinase phosphorylation) downstream of c-Met. GE137 specifically accumulated in SKOv3 ovarian cancer cells expressing c-Met via clathrin-mediated endocytosis and emitted a fluorescent signal that lasted for at least 8 hours in tumor xenografts in vivo with a sustained high signal-to-noise ratio. Our results suggest that intraoperative optical imaging could provide a new paradigm for selecting cancer patients for appropriate targeted therapies, particularly after initial chemotherapy. © 2014 American Cancer Society.

  14. Transcriptional activation of the Axl and PDGFR-α by c-Met through a ras- and Src-independent mechanism in human bladder cancer

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    Tseng Vincent S

    2011-04-01

    Full Text Available Abstract Background A cross-talk between different receptor tyrosine kinases (RTKs plays an important role in the pathogenesis of human cancers. Methods Both NIH-Met5 and T24-Met3 cell lines harboring an inducible human c-Met gene were established. C-Met-related RTKs were screened by RTK microarray analysis. The cross-talk of RTKs was demonstrated by Western blotting and confirmed by small interfering RNA (siRNA silencing, followed by elucidation of the underlying mechanism. The impact of this cross-talk on biological function was demonstrated by Trans-well migration assay. Finally, the potential clinical importance was examined in a cohort of 65 cases of locally advanced and metastatic bladder cancer patients. Results A positive association of Axl or platelet-derived growth factor receptor-alpha (PDGFR-α with c-Met expression was demonstrated at translational level, and confirmed by specific siRNA knock-down. The transactivation of c-Met on Axl or PDGFR-α in vitro was through a ras- and Src-independent activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK pathway. In human bladder cancer, co-expression of these RTKs was associated with poor patient survival (p p Conclusions In addition to c-Met, the cross-talk with Axl and/or PDGFR-α also contributes to the progression of human bladder cancer. Evaluation of Axl and PDGFR-α expression status may identify a subset of c-Met-positive bladder cancer patients who may require co-targeting therapy.

  15. The role of hepatocyte nuclear factor 4 alpha in development and progression of liver diseases

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    YANG Jinlian

    2016-02-01

    Full Text Available Hepatocyte nuclear factor 4 alpha (HNF4α, a member of the nuclear receptor superfamily, has a high expression level in mature hepatocytes. HNF4α can regulate hepatocyte-specific gene expression at a transcriptional level, promote hepatocyte development and differentiation, participate in establishment and maintenance of hepatocyte polarity, and enhance the synthetic, metabolic, and detoxifying functions of the liver. Through inhibiting the activation of hepatic stellate cells, reversing epithelial-mesenchymal transition, and inhibiting the proliferation, invasion, and metastasis of hepatoma cells, HNF4α may be involved in the development and progression of various liver diseases including liver fibrosis, liver cirrhosis, and hepatocellular carcinoma. This paper elaborates on the biological functions of HNF4α, and summarizes and analyzes the research advances in the mechanisms of action of HNF4α in the pathological process of liver diseases, in order to provide references for further investigation of the potential targeted therapies for liver diseases.

  16. Intracellular changes in rat hepatocytes after intratracheal administration of highly dispersed silicon dioxide and uridine effects on these changes.

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    Lebkova, N P; Baranov, V I

    2006-05-01

    Rat hepatocytes were examined under electron microscope at early terms after intratracheal administration of highly dispersed silicon dioxide powder against the background of uridine treatment. Penetration of powder particles into hepatocyte cytoplasm, nuclei, mitochondria, and peroxisomes and development of bacteria in these cells were observed. Uridine reduced the destructive effect of powder on the organelles, increased glycogen content in hepatocytes, and inhibited the formation of capsulated bacterial forms in these cells.

  17. Fetal liver-derived mesenchymal stromal cells augment engraftment of transplanted hepatocytes.

    Science.gov (United States)

    Joshi, Meghnad; B Patil, Pradeep; He, Zhong; Holgersson, Jan; Olausson, Michael; Sumitran-Holgersson, Suchitra

    2012-07-01

    One important problem commonly encountered after hepatocyte transplantation is the low numbers of transplanted cells found in the graft. If hepatocyte transplantation is to be a viable therapeutic approach, significant liver parenchyma repopulation is required. Mesenchymal stromal cells (MSC) produce high levels of various growth factors, cytokines and metalloproteinases, and have immunomodulatory effects. We therefore hypothesized that co-transplantation of MSC with human fetal hepatocytes (hFH) could augment in vivo expansion after transplantation. We investigated the ability of human fetal liver MSC (hFLMSC) to augment expansion of phenotypically and functionally well-characterized hFH. Two million hFH (passage 6) were either transplanted alone or together (1:1 ratio) with green fluorescence protein-expressing hFLMSC into the spleen of C57BL/6 nude mice with retrorsine-induced liver injury. After 4 weeks, engraftment of cells was detected by fluorescence in situ hybridization using a human-specific DNA probe. Significantly higher numbers of cells expressing human cytokeratin (CK)8, CK18, CK19, Cysteine-rich MNNG HOS Transforming gene (c-Met), alpha-fetoprotein (AFP), human nuclear antigen, mitochondrial antigen, hepatocyte-specific antigen and albumin (ALB) were present in the livers of recipient animals co-transplanted with hFLMSC compared with those without. Furthermore, expression of human hepatocyte nuclear factor (HNF)-4α and HNF-1β, and cytochrome P450 (CYP) 3A7 mRNA was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) in these animals. In addition, significantly increased amounts of human ALB were detected. Importantly, hFLMSC did not transdifferentiate into hepatocytes. Our study reports the use of a novel strategy for enhanced liver repopulation and thereby advances this experimental procedure closer to clinical liver cell therapy.

  18. Phase II study evaluating 2 dosing schedules of oral foretinib (GSK1363089, cMET/VEGFR2 inhibitor, in patients with metastatic gastric cancer.

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    Manish A Shah

    Full Text Available PURPOSE: The receptors for hepatocyte and vascular endothelial cell growth factors (MET and VEGFR2, respectively are critical oncogenic mediators in gastric adenocarcinoma. The purpose is to examine the safety and efficacy of foretinib, an oral multikinase inhibitor targeting MET, RON, AXL, TIE-2, and VEGFR2 receptors, for the treatment of metastatic gastric adenocarcinoma. PATIENTS AND METHODS: Foretinib safety and tolerability, and objective response rate (ORR were evaluated in patients using intermittent (240 mg/day, for 5 days every 2 weeks or daily (80 mg/day dosing schedules. Thirty evaluable patients were required to achieve alpha = 0.10 and beta = 0.2 to test the alternative hypothesis that single-agent foretinib would result in an ORR of ≥ 25%. Up to 10 additional patients could be enrolled to ensure at least eight with MET amplification. Correlative studies included tumor MET amplification, MET signaling, pharmacokinetics and plasma biomarkers of foretinib activity. RESULTS: From March 2007 until October 2009, 74 patients were enrolled; 74% male; median age, 61 years (range, 25-88; 93% had received prior therapy. Best response was stable disease (SD in 10 (23% patients receiving intermittent dosing and five (20% receiving daily dosing; SD duration was 1.9-7.2 months (median 3.2 months. Of 67 patients with tumor samples, 3 had MET amplification, one of whom had SD. Treatment-related adverse events occurred in 91% of patients. Rates of hypertension (35% vs. 15% and elevated aspartate aminotransferase (23% vs. 8% were higher with intermittent dosing. In both patients with high baseline tumor phospho-MET (pMET, the pMET:total MET protein ratio decreased with foretinib treatment. CONCLUSION: These results indicate that few gastric carcinomas are driven solely by MET and VEGFR2, and underscore the diverse molecular oncogenesis of this disease. Despite evidence of MET inhibition by foretinib, single-agent foretinib lacked efficacy in

  19. Disappearance of GFP-positive hepatocytes transplanted into the liver of syngeneic wild-type rats pretreated with retrorsine.

    Science.gov (United States)

    Maeda, Hiromichi; Shigoka, Masatoshi; Wang, Yongchun; Fu, Yingxin; Wesson, Russell N; Lin, Qing; Montgomery, Robert A; Enzan, Hideaki; Sun, Zhaoli

    2014-01-01

    Green fluorescent protein (GFP) is a widely used molecular tag to trace transplanted cells in rodent liver injury models. The differing results from various previously reported studies using GFP could be attributed to the immunogenicity of GFP. Hepatocytes were obtained from GFP-expressing transgenic (Tg) Lewis rats and were transplanted into the livers of wild-type Lewis rats after they had undergone a partial hepatectomy. The proliferation of endogenous hepatocytes in recipient rats was inhibited by pretreatment with retrorsine to enhance the proliferation of the transplanted hepatocytes. Transplantation of wild-type hepatocytes into GFP-Tg rat liver was also performed for comparison. All biopsy specimens taken seven days after transplantation showed engraftment of transplanted hepatocytes, with the numbers of transplanted hepatocytes increasing until day 14. GFP-positive hepatocytes in wild-type rat livers were decreased by day 28 and could not be detected on day 42, whereas the number of wild-type hepatocytes steadily increased in GFP-Tg rat liver. Histological examination showed degenerative change of GFP-positive hepatocytes and the accumulation of infiltrating cells on day 28. PCR analysis for the GFP transgene suggested that transplanted hepatocytes were eliminated rather than being retained along with the loss of GFP expression. Both modification of the immunological response using tacrolimus and bone marrow transplantation prolonged the survival of GFP-positive hepatocytes. In contrast, host immunization with GFP-positive hepatocytes led to complete loss of GFP-positive hepatocytes by day 14. GFP-positive hepatocytes isolated from GFP-Tg Lewis rats did not survive long term in the livers of retrorsine-pretreated wild-type Lewis rats. The mechanism underlying this phenomenon most likely involves an immunological reaction against GFP. The influence of GFP immunogenicity on cell transplantation models should be considered in planning in vivo experiments

  20. Ethanol stimulates phosphoinositide-specific phospholipase C in rat hepatocytes

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    Rubin, R.; Thomas, A.; Hoek, J.B.

    1986-05-01

    Ethanol (50-300 mM) causes a transient mobilization of calcium from hormone-sensitive pools in rat hepatocytes. They have studied the action of ethanol on polyphosphoinositide and inositol phosphate levels in these cells. Addition of ethanol to isolated hepatocytes prelabelled with /sup 32/Pi resulted in a small (3-5%) decrease in the level of (/sup 32/P)-phosphatidylinositol 4,5-bisphosphate and a 10-15% increase in (/sup 32/P) phosphatidylinositol 4-phosphate and (/sup 32/P) phosphatidic acid. Maximal changes were seen within 30 sec after ethanol. (/sup 32/P) phosphatidylcholine was unaffected. The changes were concentration dependent over a range of 50-500 mM ethanol. Phorbol esters, which inhibit ethanol-induced calcium mobilization, also inhibited these effects of ethanol on polyphosphoinositides. In intact hepatocytes labeled with myo-(2-/sup 3/H)inositol a significant ethanol-induced increase in (/sup 3/H)inositol phosphates could not be detected. After permeabilization of the cells with digitonin, however, ethanol caused a time and concentration dependent release of (/sup 3/H) inositol trisphosphate and (/sup 3/H)inositol bisphosphate. These results indicate that ethanol-induced calcium mobilization in hepatocytes is due to the activation by ethanol of the phosphoinositide-specific phospholipase C.

  1. Glioma survival prediction with the combined analysis of in vivo 11C-MET-PET, ex vivo and patient features by supervised machine learning.

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    Papp, Laszlo; Poetsch, Nina; Grahovac, Marko; Schmidbauer, Victor; Woehrer, Adelheid; Preusser, Matthias; Mitterhauser, Markus; Kiesel, Barbara; Wadsak, Wolfgang; Beyer, Thomas; Hacker, Marcus; Traub-Weidinger, Tatjana

    2017-11-24

    Gliomas are the most common types of tumors in the brain. While the definite diagnosis is routinely made ex vivo by histopathologic and molecular examination, diagnostic work-up of patients with suspected glioma is mainly done by using magnetic resonance imaging (MRI). Nevertheless, L-S-methyl-11C-methionine (11C-MET) Positron Emission Tomography (PET) holds a great potential in characterization of gliomas. The aim of this study was to establish machine learning (ML) driven survival models for glioma built on 11C-MET-PET, ex vivo and patient characteristics. Methods: 70 patients with a treatment naïve glioma, who had a positive 11C-MET-PET and histopathology-derived ex vivo feature extraction, such as World Health Organization (WHO) 2007 tumor grade, histology and isocitrate dehydrogenase (IDH1-R132H) mutation status were included. The 11C-MET-positive primary tumors were delineated semi-automatically on PET images followed by the feature extraction of tumor-to-background ratio based general and higher-order textural features by applying five different binning approaches. In vivo and ex vivo features, as well as patient characteristics (age, weight, height, body-mass-index, Karnofsky-score) were merged to characterize the tumors. Machine learning approaches were utilized to identify relevant in vivo, ex vivo and patient features and their relative weights for 36 months survival prediction. The resulting feature weights were used to establish three predictive models per binning configuration based on a combination of: in vivo/ex vivo and clinical patient information (M36IEP), in vivo and patient-only information (M36IP), and in vivo only (M36I). In addition a binning-independent ex vivo and patient-only (M36EP) model was created. The established models were validated in a Monte Carlo (MC) cross-validation scheme. Results: Most prominent ML-selected and -weighted features were patient and ex vivo based followed by in vivo features. The highest area under the curve

  2. The coexpression and prognostic significance of c-MET, fibroblast growth factor receptor 2, and human epidermal growth factor receptor 2 in resected gastric cancer: a retrospective study

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    Jia YX

    2016-09-01

    Full Text Available Yong-Xu Jia,1,2,* Teng-Fei Li,3,* Dan-Dan Zhang,4 Zong-Min Fan,5 Hui-Jie Fan,1,2 Jie Yan,1,2 Li-Juan Chen,6 Hong Tang,6 Yan-Ru Qin,1,2 Xing-Ya Li1 1Department of Oncology, The First Affiliated Hospital of Zhengzhou University, 2Esophageal and Gastric Cancer Center of Henan Province, 3Department of Interventional Radiology, 4Department of Pathology, 5Henan Key Laboratory for Esophageal Cancer Research, The First Affiliated Hospital of Zhengzhou University, 6Department of Oncology, Tumor Hospital of Henan Province, Zhengzhou, Henan, People’s Republic of China *These authors contributed equally to this work Abstract: Molecular-targeted therapy against tyrosine kinase receptors (RTKs plays an important role in gastric cancer (GC treatment. Understanding the correlation between RTK coexpression could better guide clinical drug use. In the present study, the coexpression status of c-MET, fibroblast growth factor receptor 2 (FGFR2, and human epidermal growth factor receptor 2 (HER2 in human GC and their clinical significance in clinical therapy were explored. Immunohistochemical (IHC staining, quantitative real-time polymerase chain reaction and fluorescent in situ hybridization were performed in 143 cases of GC who had undergone gastrectomy without preoperative chemoradiotherapy. Their association with clinicopathological features and clinical prognosis was analyzed. The frequencies of c-MET, FGFR2, and HER2 overexpression were 47.6% (68/143, 34.3% (49/143, and 10.5% (15/143, respectively. In the RTK coexpression study, 30.1% of patients (43/143 were positive for only one RTK, 25.8% (37/143 were positive for two RTKs, 3.5% (5/143 had triple-positive status, and 40.6% (58/143 had triple-negative status. In survival analysis, the overexpression of c-MET, FGFR2, and HER2 were significantly associated with overall survival (OS (P=0.018, 0.004, and 0.049, respectively. In coexpression analysis, patients with triple-positive GC had the poorest OS (P=0

  3. The ERK1/2-Hepatocyte Nuclear Factor 4α Axis Regulates Human ABCC6 Gene Expression in Hepatocytes*

    OpenAIRE

    de Boussac, Hugues; Ratajewski, Marcin; Sachrajda, Iwona; Köblös, Gabriella; Tordai, Attila; Pulaski, Lukasz; Buday, László; Váradi, András; Arányi, Tamás

    2010-01-01

    ABCC6 mutations are responsible for the development of pseudoxanthoma elasticum, a rare recessive disease characterized by calcification of elastic fibers. Although ABCC6 is mainly expressed in the liver the disease has dermatologic, ocular, and cardiovascular symptoms. We investigated the transcriptional regulation of the gene and observed that hepatocyte growth factor (HGF) inhibits its expression in HepG2 cells via the activation of ERK1/2. Similarly, other factors activating the cascade a...

  4. The Mechanism of Gefitinib Resistance Induced by Hepatocyte Growth Factor 
in Sensitive Non-small Cell Lung Cancer Cells in Vitro

    Directory of Open Access Journals (Sweden)

    Xianglan XUAN

    2013-01-01

    Full Text Available Background and objective Previous studies have reported that Met might be related to gefitinib resistance in non-small cell lung cancer (NSCLC. The present study aims to explore the mechanism of hepatocyte growth factor (HGF-induced gefitinib resistance in different gene types of sensitive NSCLC in vitro. Methods The PC-9 and H292 cell lines were chosen and induced by HGF. The cell survival was measured using MTT assay, the cell cycle distribution was measured using PI assay, and cell apoptosis with an Annexin V-PE assay, respectively. The c-Met and p-Met protein expression was determined via Western blot analysis. Results Gefitinib inhibited the growth of PC-9 and H292 cells in a dose-dependent manner. The concentration-survival curves of both cell lines shifted to the right when induced with HGF. HGF did not affect PC-9 and H292 cell proliferation. The cell also had a higher cell survival rate when treated with HGF and gefitinib compared with that under gefitinib alone (P<0.05. The apoptotic rate and cell cycle progression showed no significant difference between the HG and G group (P>0.05. HGF stimulated Met phosphorylation in the PC-9 and H292 cells. Gefitinib inhibited the HGF-induced Met phosphorylation in PC-9 cells, but not in H292 cells. Conclusion HGF induces gefitinib resistance in PC-9 and H292 cells. HGF-induced Met phosphorylation may be an important mechanism of gefitinib resistance in sensitive NSCLC.

  5. Hepatocyte growth factor (HGF) modulates Leydig cell extracellular matrix components.

    Science.gov (United States)

    Catizone, A; Ricci, G; Tufano, M A; Perfetto, B; Canipari, R; Galdieri, M

    2010-01-01

    Hepatocyte growth factor (HGF) is a pleiotropic factor that plays multiple roles during mammalian development. We previously demonstrated that in the postnatal testes, the HGF receptor, c-met, is expressed by Leydig cells and HGF increases the steroidogenetic activity of the cells. In the present article, we report that HGF modifies the composition of the extracellular matrix of cultured Leydig cells. We show that HGF increases the metabolic activity of isolated Leydig cells; in particular, the factor increases urokinase plasminogen activator and matrix metalloproteinase 2 secretion. We have also shown that the levels of active transforming growth factor beta are increased by HGF. On the contrary, using the Western blotting technique, a strong reduction in the amount of fibronectin present in the culture medium of cells cultured in the presence of HGF has been detected. The presented data demonstrate that HGF modulates several functional activities of Leydig cells, further supporting the hypothesis that this factor has a relevant role in the regulation of mammalian spermatogenesis.

  6. Antiglucocorticoid activity of hepatocyte nuclear factor-6.

    Science.gov (United States)

    Pierreux, C E; Stafford, J; Demonte, D; Scott, D K; Vandenhaute, J; O'Brien, R M; Granner, D K; Rousseau, G G; Lemaigre, F P

    1999-08-03

    Glucocorticoids exert their effects on gene transcription through ubiquitous receptors that bind to regulatory sequences present in many genes. These glucocorticoid receptors are present in all cell types, yet glucocorticoid action is controlled in a tissue-specific way. One mechanism for this control relies on tissue-specific transcriptional activators that bind in the vicinity of the glucocorticoid receptor and are required for receptor action. We now describe a gene-specific and tissue-specific inhibitory mechanism through which glucocorticoid action is repressed by a tissue-restricted transcription factor, hepatocyte nuclear factor-6 (HNF-6). HNF-6 inhibits the glucocorticoid-induced stimulation of two genes coding for enzymes of liver glucose metabolism, namely 6-phosphofructo-2-kinase and phosphoenolpyruvate carboxykinase. Binding of HNF-6 to DNA is required for inhibition of glucocorticoid receptor activity. In vitro and in vivo experiments suggest that this inhibition is mediated by a direct HNF-6/glucocorticoid receptor interaction involving the amino-terminal domain of HNF-6 and the DNA-binding domain of the receptor. Thus, in addition to its known property of stimulating transcription of liver-expressed genes, HNF-6 can antagonize glucocorticoid-stimulated gene transcription.

  7. DNA modification and repair by 2-nitropropane is extensive in hepatocytes of rats compared to those of humans and mice.

    Science.gov (United States)

    Davies, J E; Mynett, K; Gescher, A; Chipman, J K

    1993-06-01

    Hepatocytes from rats, mice or humans were exposed to either 2-nitropropane or propane 2-nitronate and the extent of unscheduled DNA synthesis (UDS) was measured. At a concentration of 2-nitropropane (0.1 mM) that produced a marked induction of UDS in rat hepatocytes, a negative or minimal response was seen in hepatocytes from mice and humans. A 10-fold higher concentration of 2-nitropropane was required in mouse hepatocytes for an equivalent UDS response to that seen in rat-liver cells. All three species showed UDS after exposure of hepatocytes to propane 2-nitronate at 0.1 mM although the effect in human cells was variable. In rat hepatocytes the induction of UDS by 2-nitropropane was not inhibited by the antioxidant dimethyl sulphoxide (1%). In these cells, 2-nitropropane produced an electrochemically active modified deoxynucleoside, similar to that reported to be formed in livers of rats which had received 2-nitropropane in vivo. A smaller but significant production of this altered deoxynucleoside was found in mouse hepatocytes but not in human hepatocytes treated identically. The level of 8-oxo-7,8-dihydrode-oxyguanosine was not increased in hepatocytes from any of the three species exposed to 2-NP under the same conditions. It is suggested that the UDS caused by 2-nitropropane may be in response to damage other than that produced by oxygen radicals.

  8. Mechanism of c-Met and EGFR tyrosine kinase inhibitor resistance through epithelial mesenchymal transition in non-small cell lung cancer

    Energy Technology Data Exchange (ETDEWEB)

    Rastogi, Ichwaku; Rajanna, Supriya; Webb, Andrew; Chhabra, Gagan; Foster, Brad [Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, Illinois (United States); Webb, Brian [Thermo Fisher Scientific, Rockford, Illinois (United States); Puri, Neelu, E-mail: neelupur@uic.edu [Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, Illinois (United States)

    2016-09-02

    According to currently available estimates from Cancer Research UK, 14.1 million new lung cancer cases were diagnosed and a staggering 8.2 million people worldwide died from lung cancer in 2012. EGFR and c-Met are two tyrosine kinase receptors most commonly overexpressed or mutated in Non-small Cell Lung Cancer (NSCLC) resulting in increased proliferation and survival of lung cancer cells. Tyrosine kinase inhibitors (TKIs), such as erlotinib, approved by the FDA as first/second line therapy for NSCLC patients have limited clinical efficacy due to acquired resistance. In this manuscript, we investigate and discuss the role of epithelial mesenchymal transition (EMT) in the development of resistance against EGFR and c-Met TKIs in NSCLC. Our findings show that Zeb-1, a transcriptional repressor of E-Cadherin, is upregulated in TKI-resistant cells causing EMT. We observed that TKI-resistant cells have increased gene and protein expression of EMT related proteins such as Vimentin, N-Cadherin, β-Catenin and Zeb-1, while expression of E-Cadherin, an important cell adhesion molecule, was suppressed. We also confirmed that TKI-resistant cells display mesenchymal cell type morphology, and have upregulation of β-Catenin which may regulate expression of Zeb-1, a transcriptional repressor of E-Cadherin in TKI-resistant NSCLC cells. Finally, we show that down-regulating Zeb-1 by inducing miR-200a or β-Catenin siRNA can increase drug sensitivity of TKI-resistant cells. - Highlights: • Resistance to TKIs in NSCLC cells is mediated via modulation in EMT related proteins. • EMT may induce c-Met mediated TKI resistance, similar to EGFR TKI resistance. • Role of β-catenin and cadherins in TKI resistance was validated by FACS and qPCR. • Knockdown of β-catenin or Zeb-1 can increase TKI sensitivity in TKI-resistant cells. • Targeting key EMT related proteins may overcome TKI resistance in NSCLC.

  9. Cancer-Associated Fibroblasts Regulate Tumor-Initiating Cell Plasticity in Hepatocellular Carcinoma through c-Met/FRA1/HEY1 Signaling

    Directory of Open Access Journals (Sweden)

    Eunice Yuen Ting Lau

    2016-05-01

    Full Text Available Like normal stem cells, tumor-initiating cells (T-ICs are regulated extrinsically within the tumor microenvironment. Because HCC develops primarily in the context of cirrhosis, in which there is an enrichment of activated fibroblasts, we hypothesized that cancer-associated fibroblasts (CAFs would regulate liver T-ICs. We found that the presence of α-SMA(+ CAFs correlates with poor clinical outcome. CAF-derived HGF regulates liver T-ICs via activation of FRA1 in an Erk1,2-dependent manner. Further functional analysis identifies HEY1 as a direct downstream effector of FRA1. Using the STAM NASH-HCC mouse model, we find that HGF-induced FRA1 activation is associated with the fibrosis-dependent development of HCC. Thus, targeting the CAF-derived, HGF-mediated c-Met/FRA1/HEY1 cascade may be a therapeutic strategy for the treatment of HCC.

  10. Increased NQO1 but Not c-MET and Survivin Expression in Non-Small Cell Lung Carcinoma with KRAS Mutations

    Directory of Open Access Journals (Sweden)

    Ahmet Yilmaz

    2014-09-01

    Full Text Available Cigarette smoking is one of the most significant public health issues and the most common environmental cause of preventable cancer deaths worldwide. EGFR (Epidermal Growth Factor Receptor-targeted therapy has been used in the treatment of LC (lung cancer, mainly caused by the carcinogens in cigarette smoke, with variable success. Presence of mutations in the KRAS (Kirsten rat sarcoma viral oncogene homolog driver oncogene may confer worse prognosis and resistance to treatment for reasons not fully understood. NQO1 (NAD(PH:quinone oxidoreductase, also known as DT-diaphorase, is a major regulator of oxidative stress and activator of mitomycins, compounds that have been targeted in over 600 pre-clinical trials for treatment of LC. We sequenced KRAS and investigated expression of NQO1 and five clinically relevant proteins (DNMT1, DNMT3a, ERK1/2, c-MET, and survivin in 108 patients with non-small cell lung carcinoma (NSCLC. NQO1, ERK1/2, DNMT1, and DNMT3a but not c-MET and survivin expression was significantly more frequent in patients with KRAS mutations than those without, suggesting the following: (1 oxidative stress may play an important role in the pathogenesis, worse prognosis, and resistance to treatment reported in NSCLC patients with KRAS mutations, (2 selecting patients based on their KRAS mutational status for future clinical trials may increase success rate, and (3 since oxidation of nucleotides also specifically induces transversion mutations, the high rate of KRAS transversions in lung cancer patients may partly be due to the increased oxidative stress in addition to the known carcinogens in cigarette smoke.

  11. The effect of hepatocyte growth factor on the initial stages of mouse follicle development.

    Science.gov (United States)

    Guglielmo, M C; Ricci, G; Catizone, A; Barberi, M; Galdieri, M; Stefanini, M; Canipari, R

    2011-02-01

    Interactions between theca and granulosa cells of the follicle are critical for the coordination of ovarian follicle development. The cell-cell interactions are mediated through the local production and actions of a variety of factors. The current study is designed to investigate the expression of Hgf and its receptor, c-Met, in the mouse ovary during in vivo folliculogenesis. We found that Hgf and c-Met mRNAs were already expressed in 2-day-old ovaries, and that, while c-Met levels remained constant until 22-day-old, Hgf levels slightly but not significantly increased with age. The expression of Hgf mRNA in theca/interstitial cells was higher than in granulosa cells in 22-day-old ovaries. Immunohistochemistry analysis confirmed the expression pattern demonstrated by RT-PCR. We investigated the role of hepatocyte growth factor (HGF) at the beginning of mouse folliculogenesis and its possible interaction with kit ligand (KL). Interestingly, both KL and HGF were able to increase the expression of each other, creating a positive feedback loop. In the presence of HGF, we observed an increase of granulosa cell proliferation and an increase in the number of pre-antral and early antral follicles in ovary organ cultures. We also observed a significant increase in the diameters of follicles in individual follicle cultures. Moreover, HGF stimulated the expression of the FSH receptors, both in the whole ovary and in isolated pre-antral follicle cultures. Based on the data presented, we concluded that HGF exerts multiple levels of control over follicular cell functions, which collectively enable the progression of follicular development. © 2010 Wiley-Liss, Inc.

  12. Decorin is down-regulated in multiple myeloma and MGUS bone marrow plasma and inhibits HGF-induced myeloma plasma cell viability and migration

    DEFF Research Database (Denmark)

    Kristensen, Ida Bruun; Pedersen, Lise Mariager; Rø, Torstein Baade

    2013-01-01

    OBJECTIVES: Decorin is a stromal-produced small leucine-rich proteoglycan known to attenuate tumour pro-survival, migration, proliferation and angiogenic signalling pathways. Recent studies have shown that decorin interacts with the hepatocyte growth factor (HGF) receptor c-Met, a potential key...

  13. Hepatocyte growth factor is a mouse fetal Leydig cell terminal differentiation factor.

    Science.gov (United States)

    Ricci, Giulia; Guglielmo, Maria Cristina; Caruso, Maria; Ferranti, Francesca; Canipari, Rita; Galdieri, Michela; Catizone, Angela

    2012-06-01

    The hepatocyte growth factor (HGF) is a pleiotropic cytokine and a well-known regulator of mouse embryonic organogenesis. In previous papers, we have shown the expression pattern of HGF and its receptor, C-MET, during the different stages of testis prenatal development. We demonstrated that C-MET is expressed in fetal Leydig cells (FLCs) and that HGF stimulates testosterone secretion in organ culture of late fetal testes. In the present study, we analyzed the proliferation rate, apoptotic index, and differentiation of FLCs in testicular organ culture of 17.5 days postcoitum (17.5 dpc) embryos to clarify the physiological role of HGF in late testis organogenesis. Based on our data, we conclude the following: 1) HGF acts as an antiapoptotic factor that is able to reduce the number of apoptotic FLCs and testicular caspase-3 active fragment; 2) HGF does not affect FLC proliferation; 3) HGF significantly increases expression of insulin-like 3 (INSL3), a marker of Leydig cell terminal differentiation, without affecting 3beta-hydroxysteroid dehydrogenase (3betaHSD) expression; 4) HGF significantly decreases the expression of nestin, a marker of Leydig cell progenitors; and 5) HGF significantly increases the number of fully developed FLCs. Taken together, these observations demonstrate that HGF is able to act in vitro as a survival and differentiation factor in FLC population.

  14. Hepatocyte growth factor mediates mesenchymal stem cell–induced recovery in multiple sclerosis models.

    Science.gov (United States)

    Bai, Lianhua; Lennon, Donald P; Caplan, Arnold I; DeChant, Anne; Hecker, Jordan; Kranso, Janet; Zaremba, Anita; Miller, Robert H

    2012-06-01

    Mesenchymal stem cells (MSCs) have emerged as a potential therapy for a range of neural insults. In animal models of multiple sclerosis, an autoimmune disease that targets oligodendrocytes and myelin, treatment with human MSCs results in functional improvement that reflects both modulation of the immune response and myelin repair. Here we demonstrate that conditioned medium from human MSCs (MSC-CM) reduces functional deficits in mouse MOG35–55-induced experimental autoimmune encephalomyelitis (EAE) and promotes the development of oligodendrocytes and neurons. Functional assays identified hepatocyte growth factor (HGF) and its primary receptor cMet as critical in MSC-stimulated recovery in EAE, neural cell development and remyelination. Active MSC-CM contained HGF, and exogenously supplied HGF promoted recovery in EAE, whereas cMet and antibodies to HGF blocked the functional recovery mediated by HGF and MSC-CM. Systemic treatment with HGF markedly accelerated remyelination in lysolecithin-induced rat dorsal spinal cord lesions and in slice cultures. Together these data strongly implicate HGF in mediating MSC-stimulated functional recovery in animal models of multiple sclerosis.

  15. Protective effect of allicin against acrylamide-induced hepatocyte damage in vitro and in vivo.

    Science.gov (United States)

    Zhang, Lulu; Zhang, Huanjie; Miao, Yutian; Wu, Sijia; Ye, Haiqing; Yuan, Yuan

    2012-09-01

    Acrylamide (AA) is known to be a neurotoxic, genotoxic, and carcinogenic compound. The presence of AA in foods causes public health concerns. In our previous study, we found that allicin can effectively reduce AA content in Maillard model system. However, there has been no report on whether allicin can reduce AA-induced toxicity in vitro and in vivo. In our present study, we evaluated the protective effect of allicin against AA-induced hepatocyte damage in cultured mouse primary hepatocytes and mouse liver. Our date suggested that allicin significantly decreased the levels of maleic dialdehyde (MDA) and 8-hydroxy-desoxyguanosine (8-OHdG) both in vitro and in vivo study. Allicin also markedly increased the activity of total superoxide dismutase (SOD) and level of glutathione (GSH). The in vitro study revealed that 15 μM allicin was the optimum concentration for inhibiting AA-induced hepatocyte damage. The in vivo study revealed that 20mg/kg b.w./day allicin was the optimum dose for inhibiting AA-induced hepatocyte damage. The protective effects of allicin against AA-induced hepatocyte damage may be due to its ability to scavenge free radicals and its effective recovery of the antioxidative defense system, and its ability to block the epoxidation process of AA to GA by inhibiting P450 enzyme. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Malaria parasite liver stages render host hepatocytes susceptible to mitochondria-initiated apoptosis.

    Science.gov (United States)

    Kaushansky, A; Metzger, P G; Douglass, A N; Mikolajczak, S A; Lakshmanan, V; Kain, H S; Kappe, S Hi

    2013-08-08

    Intracellular eukaryotic parasites and their host cells constitute complex, coevolved cellular interaction systems that frequently cause disease. Among them, Plasmodium parasites cause a significant health burden in humans, killing up to one million people annually. To succeed in the mammalian host after transmission by mosquitoes, Plasmodium parasites must complete intracellular replication within hepatocytes and then release new infectious forms into the blood. Using Plasmodium yoelii rodent malaria parasites, we show that some liver stage (LS)-infected hepatocytes undergo apoptosis without external triggers, but the majority of infected cells do not, and can also resist Fas-mediated apoptosis. In contrast, apoptosis is dramatically increased in hepatocytes infected with attenuated parasites. Furthermore, we find that blocking total or mitochondria-initiated host cell apoptosis increases LS parasite burden in mice, suggesting that an anti-apoptotic host environment fosters parasite survival. Strikingly, although LS infection confers strong resistance to extrinsic host hepatocyte apoptosis, infected hepatocytes lose their ability to resist apoptosis when anti-apoptotic mitochondrial proteins are inhibited. This is demonstrated by our finding that B-cell lymphoma 2 family inhibitors preferentially induce apoptosis in LS-infected hepatocytes and significantly reduce LS parasite burden in mice. Thus, targeting critical points of susceptibility in the LS-infected host cell might provide new avenues for malaria prophylaxis.

  17. CD163 and c-Met expression in the lymph node and the correlations between elevated levels of serum free light chain and the different clinicopathological parameters of advanced classical Hodgkin's lymphoma.

    Science.gov (United States)

    Bedewy, Magdy; El-Maghraby, Shereen; Bedewy, Ahmed

    2013-06-01

    Advances in the understanding of Hodgkin's lymphoma (HL) show various functions of infiltrating immune cells and cytokines in relation to clinical outcomes. The expression of CD163 and c-Met has been suggested to have a role in lymphoid malignancy. Thus, we evaluated the expressions of CD163, c-Met, and serum free light chain (sFLC) in relation to the clinicopathological features of patients with advanced classical HL (cHL). We assessed the expression of CD163 and c-Met in 34 patients with cHL through immunohistochemistry on the lymph node biopsy sections and the levels of pretreatment sFLC were estimated using ELISA. High CD163 expression correlated with increased age, B symptoms, International Prognostic Score (IPS) ≥3, mixed cellularity subtype, and low response to treatment. Further, high c-Met expression correlated with increased age at diagnosis, leukocytosis, B symptoms, and lower chance to achieve complete remission. The sFLC levels correlated with increased age at diagnosis, lymphopenia, IPS ≥3, B symptoms, and lower complete remission rates. In advanced cHL, increased expression of CD163 and c-Met showed a significant association with adverse prognostic parameters and poor response to treatment. Pretreatment high sFLC level also correlated with poor risk factors, suggesting its use as a candidate prognostic marker. A comprehensive approach for prognostic markers might represent a step towards developing a tailored therapeutic approach for HL.

  18. Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Yeom, Chul-gon; Kim, Dong-il; Park, Min-jung; Choi, Joo-hee [College of Veterinary Medicine, Chonnam National University, Gwangju 500-757 (Korea, Republic of); Jeong, Jieun; Wi, Anjin; Park, Whoashig [Jeollanamdo Forest Resources Research Institute, Naju 520-833 (Korea, Republic of); Han, Ho-jae [College of Veterinary Medicine, Seoul National University, Seoul 151-741 (Korea, Republic of); Park, Soo-hyun, E-mail: parksh@chonnam.ac.kr [College of Veterinary Medicine, Chonnam National University, Gwangju 500-757 (Korea, Republic of)

    2015-06-05

    Previously, we reported that CARM1 undergoes ubiquitination-dependent degradation in renal podocytes. It was also reported that CARM1 is necessary for fasting-induced hepatic gluconeogenesis. Based on these reports, we hypothesized that treatment with insulin, a hormone typically present under the ‘fed’ condition, would inhibit gluconeogenesis via CARM1 degradation. HepG2 cells, AML-12 cells, and rat primary hepatocytes were treated with insulin to confirm CARM1 downregulation. Surprisingly, insulin treatment increased CARM1 expression in all cell types examined. Furthermore, treatment with insulin increased histone 3 methylation at arginine 17 and 26 in HepG2 cells. To elucidate the role of insulin-induced CARM1 upregulation, the HA-CARM1 plasmid was transfected into HepG2 cells. CARM1 overexpression did not increase the expression of lipogenic proteins generally increased by insulin signaling. Moreover, CARM1 knockdown did not influence insulin sensitivity. Insulin is known to facilitate hepatic proliferation. Like insulin, CARM1 overexpression increased CDK2 and CDK4 expression. In addition, CARM1 knockdown reduced the number of insulin-induced G2/M phase cells. Moreover, GFP-CARM1 overexpression increased the number of G2/M phase cells. Based on these results, we concluded that insulin-induced CARM1 upregulation facilitates hepatocyte proliferation. These observations indicate that CARM1 plays an important role in liver pathophysiology. - Highlights: • Insulin treatment increases CARM1 expression in hepatocytes. • CARM1 overexpression does not increase the expression of lipogenic proteins. • CARM1 knockdown does not influence insulin sensitivity. • Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation.

  19. A phase II trial of a selective c-Met inhibitor tivantinib (ARQ 197) monotherapy as a second- or third-line therapy in the patients with metastatic gastric cancer.

    Science.gov (United States)

    Kang, Yoon-Koo; Muro, Kei; Ryu, Min-Hee; Yasui, Hirofumi; Nishina, Tomohiro; Ryoo, Baek-Yeol; Kamiya, Yukimasa; Akinaga, Shiro; Boku, Narikazu

    2014-04-01

    Tivantinib is a selective, non-ATP competitive, small-molecule inhibitor of c-Met and is under development in several cancers including non-small cell lung and hepatocellular carcinoma. Activation of c-Met has been frequently found in metastatic gastric cancer (MGC) and is associated with poor prognosis. In this single-arm study, we evaluated the efficacy of tivantinib monotherapy in Asian patients with previously treated MGC. This is the first clinical report from the trials evaluating the efficacy of a selective c-Met inhibitor for MGC. Eligibility criteria included: MGC with at least one measurable lesion; 1 or 2 prior chemotherapy regimens; and ECOG PS 0 or 1. Tivantinib was daily administered orally. The primary endpoint was the disease control rate (DCR). Pre-treatment tumor tissue was collected to evaluate the biomarkers related to efficacy. Thirty patients, including 12 patients with prior gastrectomy, received tivantinib: median age 62.5 years; ECOG PS 0/1 (8/22); 1/2 prior regimen (16/14). No objective response was observed, and DCR was 36.7 %. Median progression-free survival was 43 days (95 % CI: 29.0-92.0). Grade 3 or 4 adverse events occurred in 13 patients (43.3 %), in whom neutropenia (N = 4) and anemia (N = 4) were recognized as drug-related. c-Met gene amplification was observed in 2 patients (6.9 %). No obvious relationship was identified between efficacy and biomarkers including gene amplification of c-Met, expression of c-Met, p-Met and HGF. Tivantinib as a monotherapy showed a modest efficacy in previously treated MGC, and further studies taking account of predictive biomarkers and/or combination with other chemotherapy may be needed in MGC.

  20. Immortalized hepatocytes using human artificial chromosome.

    Science.gov (United States)

    Ito, Masahiro; Ito, Ryoutaro; Yoshihara, Daisuke; Ikeno, Masashi; Kamiya, Megumi; Suzuki, Nobutaka; Horiguchi, Akihiko; Nagata, Hideo; Yamamoto, Toshiyuki; Kobayashi, Naoya; Fox, Ira J; Okazaki, Tsuneko; Miyakama, Syuichi

    2008-01-01

    The shortage of organ donors has impeded the development of human hepatocyte transplantation. Immortalized hepatocytes could provide an unlimited supply of transplantable cells. To determine whether immortalized hepatocytes could provide global metabolic support in end-stage liver disease, rat hepatocyte clones were developed by transduction with the gene encoding the Simian virus 40 T antigen (SVT) using the human artificial minichromosome (HAC). The SVLT sequence was excised by FRT recombination. Following HAC infusion, the transduced hepatocytes express SVT, blasticidine resistance (BS), and the PGK promoter TK gene. Forty-six cell clones were obtained and at least partially characterized, as previously described, for albumin, alpha-1-antitrypsin, glucose-6-phosphatase (G6Pase), dipeptidylpeptidase 4 (Dpp4), gamma-glutamyltransferase 1 (Ggt), SVT, and beta-actin expression using RT-PCR. Clones were also assessed for albumin secretion into the culture medium using ELISA. All of the cell line secreted approximately 10 mg/dl of albumin, which is equivalent to the amount secreted by primary hepatocytes. In further experiments, this cell line will be used for transplantable cells or artificial organ using HAC. These results represent an important step toward the development of immortalized hepatocytes.

  1. Comparative investigations on the uptake of phallotoxins, bile acids, bovine lactoperoxidase and horseradish peroxidase into rat hepatocytes in suspension and in cell cultures.

    Science.gov (United States)

    Petzinger, E; Frimmer, M

    1988-01-13

    Two alternative uptake mechanisms for phallotoxins by liver cells are debated: carrier-mediated uptake and receptor-mediated endocytosis. We have compared the properties of hepatocellular uptake of the phallotoxins, phalloidin and demethylphalloin, with the uptake of cholate as a substrate for carrier-mediated uptake and compared with iodinated bovine lactoperoxidase or iodinated horseradish peroxidase, as the latter are known to be taken up by vesicular endocytosis. Uptake of phallotoxins and [14C]cholate uptake into isolated hepatocytes is independent of extracellular calcium but inhibited by A23187 or by monensin. Uptake of bovine lactoperoxidase strictly depends on external Ca2+, was insensitive to A23197 and was not inhibited by monensin. No mutual uptake inhibition between phalloidin or cholate and peroxidases was seen, indicating independent permeation pathways in hepatocytes. However, high concentrations of cytochalasin B inhibited the uptake of either phalloidin, cholate or bovine lactoperoxidase. Horseradish peroxidase uptake, which was taken as an indicator for fluid pinocytosis, was low in isolated hepatocytes and could not account for the amount of phalloidin or cholate taken up. In cultured rat hepatocytes, uptake of phallotoxins decreased within 1 day to 10% of the uptake seen in freshly isolated hepatocytes. The results indicate different mechanisms for hepatocellular phallotoxin/bile-acid uptake and peroxidase internalization. As monolayer cultures of hepatocytes rapidly lost the carrier-mediated uptake of phallotoxins and bile acids, freshly isolated hepatocytes might be a more suitable experimental model than cultured cells for kinetic studies on this transport system.

  2. Exosomes derived from alcohol-treated hepatocytes horizontally transfer liver specific miRNA-122 and sensitize monocytes to LPS

    Science.gov (United States)

    Momen-Heravi, Fatemeh; Bala, Shashi; Kodys, Karen; Szabo, Gyongyi

    2015-01-01

    Hepatocyte damage and inflammation in monocytes/macrophages are central to the pathogenesis of alcoholic hepatitis (AH). MicroRNAs (miRNAs) regulate all of these processes. MiRNA-122 is abundantly expressed in hepatocytes while monocytes/macrophages have low levels. The role of exosomes in AH and possible cross talk between hepatocyte-derived exosomes and immune cells is not explored yet. Here, we show that the number of exosomes significantly increases in the sera of healthy individuals after alcohol binge drinking and in mice after binge or chronic alcohol consumption. Exosomes isolated from sera after alcohol consumption or from in vitro ethanol-treated hepatocytes contained miRNA-122. Exosomes derived from ethanol-treated Huh7.5 cells were taken up by the recipients THP1 monocytes and horizontally transferred a mature form of liver-specific miRNA-122. In vivo, liver mononuclear cells and Kupffer cells from alcohol-fed mice had increased miRNA-122 levels. In monocytes, miRNA-122 transferred via exosomes inhibited the HO-1 pathway and sensitized to LPS stimulation and increased levels of pro-inflammatory cytokines. Finally, inflammatory effects of exosomes from ethanol-treated hepatocytes were prevented by using RNA interference via exosome-mediated delivery of a miRNA-122 inhibitor. These results demonstrate that first, exosomes mediate communication between hepatocytes and monocytes/macrophages and second, hepatocyte-derived miRNA-122 can reprogram monocytes inducing sensitization to LPS. PMID:25973575

  3. Cold storage of rat hepatocyte spheroids.

    Science.gov (United States)

    Liu, Hongling; Yu, Yue; Glorioso, Jaime; Mao, Shennen; Rodysil, Brian; Amiot, Bruce P; Rinaldo, Piero; Nyberg, Scott L

    2014-01-01

    Cell-based therapies for liver disease rely on a high-quality supply of hepatocytes and a means for storage during transportation from site of isolation to site of usage. Unfortunately, frozen cryopreservation is associated with unacceptable loss of hepatocyte viability after thawing. The purpose of this study was to optimize conditions for cold storage of rat hepatocyte spheroids without freezing. Rat hepatocytes were isolated by a two-step perfusion method; hepatocyte spheroids were formed during 48 h of rocked culture in serum-free medium (SFM). Spheroids were then maintained in rocked culture at 37 °C (control condition) or cold stored at 4 °C for 24 or 48 h in six different cold storage solutions: SFM alone; SFM + 1 mM deferoxamine (Def); SFM + 1 μM cyclosporin A (CsA); SFM + 1 mM Def + 1 μM CsA, University of Wisconsin (UW) solution alone, UW + 1 mM Def. Performance metrics after cold storage included viability, gene expression, albumin production, and functional activity of cytochrome P450 enzymes and urea cycle proteins. We observed that cold-induced injury was reduced significantly by the addition of the iron chelator (Def) to both SFM and UW solution. Performance metrics (ammonia detoxification, albumin production) of rat hepatocyte spheroids stored in SFM + Def for 24 h were significantly increased from SFM alone and approached those in control conditions, while performance metrics after cold storage in SFM alone or cold storage for 48 h were both significantly reduced. A serum-free medium supplemented with Def allowed hepatocyte spheroids to tolerate 24 h of cold storage with less than 10% loss in viability and functionality. Further research is warranted to optimize a solution for extended cold storage of hepatocyte spheroids.

  4. Silencing of phospholipase C gamma 2 promotes proliferation of rat hepatocytes in vitro.

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    Chen, Xiaoguang; Zhu, Xuemin; Liu, Yumei; Lv, Qiongxia; Ma, Jun

    2017-12-13

    The management of hepatic failure is undoubtedly difficult, and poor results have led to the search for novel therapeutic approaches. Nowadays, anti-apoptotic gene therapy is considered as an ideal approach. It has been proved that phospholipase Cγ2 (PLCγ2) is involved in the apoptosis of immune cells and tumor cells; however, whether this gene is related to hepatocyte death is still unclear. This study examined the role of PLCγ2 by inhibiting its expression in rat hepatocytes with siRNA. We also further analyzed the cellular mechanism by which the expression inhibition of PLCγ2 induces cell death. Silencing PLCγ2 gene by adenovirus vector expressing PLCγ2-targeted siRNA caused the great decline in the number of G1- and G2/M phase cells, the significant increase in the number of S phase cells, and the obvious reduction in apoptosis index. In addition, silencing PLCγ2 gene relieved the rat hepatocyte damage, such as the cell shrinkage and chromatin condensation, nuclear fragmentation. Further analysis of Ad-PLCγ2 siRNA-transfected hepatocytes demonstrated that suppression of PLCγ2 gene expression could cause the caspase dependent cell death by inhibiting the signal pathway MEKK1/MKK4/JNK1/2/c-Jun. In conclusion, these findings suggest that interference with PLCγ2 expression could relieve the inhibitory effect of PLCγ2 on hepaocyte apoptosis, thus, promote proliferation through inactivating PKCδ-mediated JNK1/2 signaling pathway. © 2017 Wiley Periodicals, Inc.

  5. Metformin protects rat hepatocytes against bile acid-induced apoptosis.

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    Titia E Woudenberg-Vrenken

    Full Text Available BACKGROUND: Metformin is used in the treatment of Diabetes Mellitus type II and improves liver function in patients with non-alcoholic fatty liver disease (NAFLD. Metformin activates AMP-activated protein kinase (AMPK, the cellular energy sensor that is sensitive to changes in the AMP/ATP-ratio. AMPK is an inhibitor of mammalian target of rapamycin (mTOR. Both AMPK and mTOR are able to modulate cell death. AIM: To evaluate the effects of metformin on hepatocyte cell death. METHODS: Apoptotic cell death was induced in primary rat hepatocytes using either the bile acid glycochenodeoxycholic acid (GCDCA or TNFα in combination with actinomycin D (actD. AMPK, mTOR and phosphoinositide-3 kinase (PI3K/Akt were inhibited using pharmacological inhibitors. Apoptosis and necrosis were quantified by caspase activation, acridine orange staining and Sytox green staining respectively. RESULTS: Metformin dose-dependently reduces GCDCA-induced apoptosis, even when added 2 hours after GCDCA, without increasing necrotic cell death. Metformin does not protect against TNFα/ActD-induced apoptosis. The protective effect of metformin is dependent on an intact PI3-kinase/Akt pathway, but does not require AMPK/mTOR-signaling. Metformin does not inhibit NF-κB activation. CONCLUSION: Metformin protects against bile acid-induced apoptosis and could be considered in the treatment of chronic liver diseases accompanied by inflammation.

  6. Metformin Protects Rat Hepatocytes against Bile Acid-Induced Apoptosis

    Science.gov (United States)

    Woudenberg-Vrenken, Titia E.; Conde de la Rosa, Laura; Buist-Homan, Manon; Faber, Klaas Nico; Moshage, Han

    2013-01-01

    Background Metformin is used in the treatment of Diabetes Mellitus type II and improves liver function in patients with non-alcoholic fatty liver disease (NAFLD). Metformin activates AMP-activated protein kinase (AMPK), the cellular energy sensor that is sensitive to changes in the AMP/ATP-ratio. AMPK is an inhibitor of mammalian target of rapamycin (mTOR). Both AMPK and mTOR are able to modulate cell death. Aim To evaluate the effects of metformin on hepatocyte cell death. Methods Apoptotic cell death was induced in primary rat hepatocytes using either the bile acid glycochenodeoxycholic acid (GCDCA) or TNFα in combination with actinomycin D (actD). AMPK, mTOR and phosphoinositide-3 kinase (PI3K)/Akt were inhibited using pharmacological inhibitors. Apoptosis and necrosis were quantified by caspase activation, acridine orange staining and Sytox green staining respectively. Results Metformin dose-dependently reduces GCDCA-induced apoptosis, even when added 2 hours after GCDCA, without increasing necrotic cell death. Metformin does not protect against TNFα/ActD-induced apoptosis. The protective effect of metformin is dependent on an intact PI3-kinase/Akt pathway, but does not require AMPK/mTOR-signaling. Metformin does not inhibit NF-κB activation. Conclusion Metformin protects against bile acid-induced apoptosis and could be considered in the treatment of chronic liver diseases accompanied by inflammation. PMID:23951244

  7. Selective insulin resistance in hepatocyte senescence

    Energy Technology Data Exchange (ETDEWEB)

    Aravinthan, Aloysious [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Challis, Benjamin [Institute of Metabolic Sciences, University of Cambridge, Cambridge (United Kingdom); Shannon, Nicholas [Cancer Research UK Cambridge Institute, Cambridge (United Kingdom); Hoare, Matthew [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Cancer Research UK Cambridge Institute, Cambridge (United Kingdom); Heaney, Judith [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Foundation for Liver Research, Institute of Hepatology, London (United Kingdom); Alexander, Graeme J.M., E-mail: gja1000@doctors.org.uk [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom)

    2015-02-01

    Insulin resistance has been described in association with chronic liver disease for decades. Hepatocyte senescence has been demonstrated in chronic liver disease and as many as 80% of hepatocytes show a senescent phenotype in advanced liver disease. The aim of this study was to understand the role of hepatocyte senescence in the development of insulin resistance. Senescence was induced in HepG2 cells via oxidative stress. The insulin metabolic pathway was studied in control and senescent cells following insulin stimulation. GLUT2 and GLUT4 expressions were studied in HepG2 cells and human liver tissue. Further, GLUT2 and GLUT4 expressions were studied in three independent chronic liver disease cohorts. Signalling impairment distal to Akt in phosphorylation of AS160 and FoxO1 was evident in senescent HepG2 cells. Persistent nuclear localisation of FoxO1 was demonstrated in senescent cells despite insulin stimulation. Increased GLUT4 and decreased GLUT2 expressions were evident in senescent cells, human cirrhotic liver tissue and publically available liver disease datasets. Changes in GLUT expressions were associated with a poor clinical prognosis. In conclusion, selective insulin resistance is evident in senescent HepG2 cells and changes in GLUT expressions can be used as surrogate markers of hepatocyte senescence. - Highlights: • Senescent hepatocytes demonstrate selective insulin resistance. • GLUT changes act as markers of hepatocyte senescence and have prognostic value. • Study offers insight into long noticed intimacy of cirrhosis and insulin resistance.

  8. Hepatic heme catabolism in cultured hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lincoln, B.C.; Bonkovsky, H.L.

    1987-05-01

    Uncertainty persists concerning the role and importance of heme oxygenase in the catabolism of heme by hepatocytes. The products of heme oxygenase catalyzed heme catabolism are equimolar amounts of biliverdin IX..cap alpha.., CO, and iron. Previous reports from studies with rodent hepatocyte cultures have suggested the possibility that non-heme oxygenase pathway(s) predominate in the breakdown of hepatic hemoprotein heme. The authors have studied this question in cultured chick embryo hepatocytes, which retain normal regulation of heme metabolism and levels of cytochromes P-450 as in intact animals. Exogenous heme added to the culture medium with control chick embryo hepatocyte cultures was quantitatively converted to biliverdin IX..cap alpha... To study endogenous heme breakdown, cellular heme was labelled by exposing cultured cells to (5-/sup 14/C) 5-aminolevulinic acid (ALA). The hepatocytes were also treated with mephenytoin that increases cytochrome P-450, total hepatic heme and heme oxygenase. At various times after labelling heme, biliverdin, and CO were isolated and counted. For at least 8 hrs, the increase in CO radioactivity corresponded to the loss of radioactivity in heme. Beyond 1 h biliverdin was unstable in culture medium, but for 1 h after labelling (dpm BVIX..cap alpha.. + dpm CO) ..delta..dpm heme. All BV detected was the ..cap alpha.. isomer. They conclude that heme oxygenase accounts for both endogenous and exogenous heme breakdown by hepatocytes.

  9. Hepatocyte Growth Factor Effects on Mesenchymal Stem Cells Derived from Human Arteries: A Novel Strategy to Accelerate Vascular Ulcer Wound Healing

    Directory of Open Access Journals (Sweden)

    Sabrina Valente

    2016-01-01

    Full Text Available Vascular ulcers are a serious complication of peripheral vascular disease, especially in diabetics. Several approaches to treat the wounds are proposed but they show poor outcomes and require long healing times. Hepatocyte Growth Factor/Scatter Factor (HGF/SF is a pleiotropic cytokine exerting many biological activities through the c-Met receptor. This study was aimed at verifying whether HGF/SF influences proliferation, migration, and angiogenesis on mesenchymal stem cells isolated from human arteries (hVW-MSCs. hVW-MSCs were exposed to NIBSC HGF/SF (2.5, 5, 10, and 70 ng/mL from 6 hrs to 7 days. HGF and c-MET mRNA and protein expression, cell proliferation (Alamar Blue and Ki–67 assay, migration (scratch and transwell assays, and angiogenesis (Matrigel were investigated. hVW-MSCs displayed stemness features and expressed HGF and c-MET. HGF/SF did not increase hVW-MSC proliferation, whereas it enhanced the cell migration, the formation of capillary-like structures, and the expression of angiogenic markers (vWF, CD31, and KDR. The HGF/SF effects on hVW-MSC migration and angiogenic potential are of great interest to accelerate wound healing process. Local delivery of HGF/SF could therefore improve the healing of unresponsive vascular ulcers.

  10. Endothelial cell-derived matrix promotes the metabolic functional maturation of hepatocyte via integrin-Src signalling.

    Science.gov (United States)

    Guo, Xinyue; Li, Weihong; Ma, Minghui; Lu, Xin; Zhang, Haiyan

    2017-11-01

    The extracellular matrix (ECM) microenvironment is involved in the regulation of hepatocyte phenotype and function. Recently, the cell-derived extracellular matrix has been proposed to represent the bioactive and biocompatible materials of the native ECM. Here, we show that the endothelial cell-derived matrix (EC matrix) promotes the metabolic maturation of human adipose stem cell-derived hepatocyte-like cells (hASC-HLCs) through the activation of the transcription factor forkhead box protein A2 (FOXA2) and the nuclear receptors hepatocyte nuclear factor 4 alpha (HNF4α) and pregnane X receptor (PXR). Reducing the fibronectin content in the EC matrix or silencing the expression of α5 integrin in the hASC-HLCs inhibited the effect of the EC matrix on Src phosphorylation and hepatocyte maturation. The inhibition of Src phosphorylation using the inhibitor PP2 or silencing the expression of Src in hASC-HLCs also attenuated the up-regulation of the metabolic function of hASC-HLCs in a nuclear receptor-dependent manner. These data elucidate integrin-Src signalling linking the extrinsic EC matrix signals and metabolic functional maturation of hepatocyte. This study provides a model for studying the interaction between hepatocytes and non-parenchymal cell-derived matrix. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  11. A phase I dose escalation study of oral c-MET inhibitor tivantinib (ARQ 197) in combination with gemcitabine in patients with solid tumors.

    Science.gov (United States)

    Pant, S; Saleh, M; Bendell, J; Infante, J R; Jones, S; Kurkjian, C D; Moore, K M; Kazakin, J; Abbadessa, G; Wang, Y; Chen, Y; Schwartz, B; Camacho, L H

    2014-07-01

    Tivantinib (ARQ 197) is an orally available, non-adenosine triphosphate competitive, selective c-MET inhibitor. The primary objective of this study was to evaluate the safety, tolerability and to establish the recommended phase II dose (RP2D) of tivantinib and gemcitabine combination. Patients with advanced or metastatic solid tumors were treated with escalating doses of tivantinib (120-360 mg capsules) in combination with gemcitabine (1000 mg/m(2) weekly for 3 of 4 weeks). Different schedules of administration were tested and modified based on emerging preclinical data. Tivantinib was given continuously, twice a day (b.i.d.) for 2, 3 or 4 weeks of a 28-day cycle or on a 5-day on, 2-day off schedule (the day before and day of gemcitabine administration). Twenty-nine patients were treated with gemcitabine and escalating doses of tivantinib: 120 mg b.i.d. (n = 4), 240 mg b.i.d. (n = 6) and 360 mg b.i.d. (n = 19). No dose-limiting toxicities were observed in escalation. The RP2D was 360 mg b.i.d. daily, and 45 additional patients were enrolled in the expansion cohort. Grade ≥3 treatment-related toxicities were observed in 54 of 74 (73%) patients with the most common being neutropenia (43%), anemia (30%), thrombocytopenia (28%) and fatigue (15%). There was one treatment-related death due to neutropenia. Administration of gemcitabine did not affect tivantinib concentration. Fifty-six patients were assessable for response. Eleven (20%) patients achieved a partial response and 26 (46%) had stable disease (SD), including 15 (27%) who achieved SD for over 4 months. Ten of 37 patients with clinical benefit had prior exposure to gemcitabine. The combination of tivantinib at its monotherapy dose and standard dose gemcitabine was safe and tolerable. Early signs of antitumor activity may warrant further development of this combination in nonsmall-cell lung cancer, ovarian, pancreatic and cholangiocarcinoma. NCT00874042. © The Author 2014. Published by Oxford University Press

  12. Citotoxicity of Fipronil on Hepatocytes Isolated from Rat and Effects of Its Biotransformation

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    Marieli Guelfi

    2015-12-01

    Full Text Available ABSTRACT The aim of this study was to characterize the mechanism of toxicity of fipronil on hepatocytes isolated from the rat and the effect of its biotransformation on the toxicological potential. The toxicity of fipronil was assessed by monitoring the oxygen consumption and mitochondrial membrane potential, intracellular ATP concentration, Ca2+ homeostasis and cell viability. The cell viability was evaluated by trypan blue exclusion in hepatocytes that were isolated from the normal rats and by the release of the enzymes alanine transaminase and aspartate transaminase in hepatocytes that were isolated from the normal rats or proadifen-pretreated rats. Fipronil reduced mitochondrial respiration in the cells that were energized with glutamate plus malate in a dose-dependent manner and dissipated the mitochondrial membrane potential that was accompanied by a reduction in ATP concentration and a disruption of intracellular Ca2+ homeostasis. The cell viability was affected by fipronil with higher potency in hepatocytes that were isolated from the normal rats, which indicated that the metabolism of this insecticide increased its toxicological potential. The results of this study indicated that the toxicity of fipronil to the hepatocytes was related to the inhibition of mitochondrial activity, which led to decreased ATP synthesis and a consequent alteration in intracellular Ca2+ homeostasis and ultimately resulted in cell death.

  13. Glutamic Acid as Enhancer of Protein Synthesis Kinetics in Hepatocytes from Old Rats.

    Science.gov (United States)

    Brodsky, V Y; Malchenko, L A; Butorina, N N; Lazarev Konchenko, D S; Zvezdina, N D; Dubovaya, T K

    2017-08-01

    Dense cultures of hepatocytes from old rats (~2 years old, body weight 530-610 g) are different from similar cultures of hepatocytes from young rats by the low amplitude of protein synthesis rhythm. Addition of glutamic acid (0.2, 0.4, or 0.6 mg/ml) into the culture medium with hepatocytes of old rats resulted in increase in the oscillation amplitudes of the protein synthesis rhythm to the level of young rats. A similar action of glutamic acid on the protein synthesis kinetics was observed in vivo after feeding old rats with glutamic acid. Inhibition of metabotropic receptors of glutamic acid with α-methyl-4-carboxyphenylglycine (0.01 mg/ml) abolished the effect of glutamic acid. The amplitude of oscillation of the protein synthesis rhythm in a cell population characterizes synchronization of individual oscillations caused by direct cell-cell communications. Hence, glutamic acid, acting as a receptor-dependent transmitter, enhanced direct cell-cell communications of hepatocytes that were decreased with aging. As differentiated from other known membrane signaling factors (gangliosides, norepinephrine, serotonin, dopamine), glutamic acid can penetrate into the brain and thus influence the communications and protein synthesis kinetics that are disturbed with aging not only in hepatocytes, but also in neurons.

  14. Hepatitis B virus evades innate immunity of hepatocytes but activates cytokine production by macrophages.

    Science.gov (United States)

    Cheng, Xiaoming; Xia, Yuchen; Serti, Elisavet; Block, Peter Daniel; Chung, Michelle; Chayama, Kazuaki; Rehermann, Barbara; Liang, T Jake

    2017-12-01

    Hepatitis B virus (HBV) infects hepatocytes specifically and causes immune-mediated liver damage. How HBV interacts with the innate immunity at the early phase of infection, either with hepatocytes or other cells in the liver, remains controversial. To address this question, we utilized various human cell-culture models and humanized Alb-uPA/SCID mice. All these models were unable to mount an interferon (IFN) response despite robust HBV replication. To elucidate the mechanisms involved in the lack of IFN response, we examined whether HBV actively inhibits innate immune functions of hepatocytes. By treating HBV-infected cells with known inducers of the IFN signaling pathway, we observed no alteration of either sensing or downstream IFN response by HBV. We showed that the DNA innate sensing pathways are poorly active in hepatocytes, consistent with muted innate immune recognition of HBV. Upon exposure to high-level HBV, human macrophages could be activated with increased inflammatory cytokine expressions. HBV behaves like a "stealth" virus and is not sensed by, nor actively interferes with, the intrinsic innate immunity of infected hepatocytes. Macrophages are capable of sensing HBV, but require exposure to high HBV titers, potentially explaining the long "window period" during acute infection and HBV's propensity to chronic infection. (Hepatology 2017;66:1779-1793). © 2017 by the American Association for the Study of Liver Diseases. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

  15. Dynamic regulation of EZH2 from HPSc to hepatocyte-like cell fate.

    Science.gov (United States)

    Pistoni, Mariaelena; Helsen, Nicky; Vanhove, Jolien; Boon, Ruben; Xu, Zhuofei; Ordovas, Laura; Verfaillie, Catherine M

    2017-01-01

    Currently, drug metabolization and toxicity studies rely on the use of primary human hepatocytes and hepatoma cell lines, which both have conceivable limitations. Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) are an alternative and valuable source of hepatocytes that can overcome these limitations. EZH2 (enhancer of zeste homolog 2), a transcriptional repressor of the polycomb repressive complex 2 (PRC2), may play an important role in hepatocyte development, but its role during in vitro hPSC-HLC differentiation has not yet been assessed. We here demonstrate dynamic regulation of EZH2 during hepatic differentiation of hPSC. To enhance EZH2 expression, we inducibly overexpressed EZH2 between d0 and d8, demonstrating a significant improvement in definitive endoderm formation, and improved generation of HLCs. Despite induction of EZH2 overexpression until d8, EZH2 transcript and protein levels decreased from d4 onwards, which might be caused by expression of microRNAs predicted to inhibit EZH2 expression. In conclusion, our studies demonstrate that EZH2 plays a role in endoderm formation and hepatocyte differentiation, but its expression is tightly post-transcriptionally regulated during this process.

  16. Stimulation of fibrinogen synthesis in cultured rat hepatocytes by fibrinogen degradation product fragment D

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    LaDuca, F.M.; Tinsley, L.A.; Dang, C.V.; Bell, W.R. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (USA))

    1989-11-01

    The direct stimulation of fibrinogen biosynthesis by fibrinogen degradation produces (FDPs) was studied in rat hepatocyte cultures. Pure rat FDP fragment D (FDP-D) (Mr 90,000) and FDP fragment E (FDP-E) (Mr 40,000) and mixtures of the two (FDP-DE) were added to rat hepatocytes cultured in serum-free hormonally defined medium. Hydrocortisone (20 microM) significantly increased synthesis of fibrinogen, as determined by incorporation of (35S)methionine. FDP-D and FDP-E did not increase fibrinogen synthesis in the presence of hydrocortisone. However, hepatocytes cultured without hydrocortisone displayed increased fibrinogen synthesis (2.0- to 2.8-fold) with FDP-D (2.6-6.7 microM) but not with FDP-E (5.7 microM). At these FDP concentrations the synthesis of albumin, haptoglobin, and transferrin was not increased. FDP-D-induced fibrinogen synthesis was inhibited (greater than 90%) by actinomycin D and cycloheximide, indicating that the increase in (35S)methionine incorporation was from de novo protein synthesis. The role of FDP-D was further substantiated by showing that FDP-D, but not FDP-E, bound to the hepatocytes. These data indicate that FDP-D, but not FDP-E, directly and specifically stimulates fibrinogen synthesis in rat hepatocytes; this stimulation does not require any additional serum or protein cofactors.

  17. Regulation of hepatocyte-specific gene expression in cultures of human embryonic hepatocytes

    NARCIS (Netherlands)

    van Roon, M. A.; Zonneveld, D.; de Boer, P. A.; Moorman, A. F.; Charles, R.; Lamers, W. H.

    1990-01-01

    The aim of this study was to see whether the rat embryo can serve as a model system for hepatocyte-specific gene expression in the human embryo. Carbamoylphosphate synthetase was used as a hepatocyte-specific marker molecule. Despite the earlier developmental appearance of this enzyme in human than

  18. Modulation of Insulin Sensitivity of Hepatocytes by the Pharmacological Downregulation of Phospholipase D

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    Nataliya A. Babenko

    2015-01-01

    Full Text Available Background. The role of phospholipase D (PLD as a positive modulator of glucose uptake activation by insulin in muscle and adipose cells has been demonstrated. The role of PLD in the regulation of glucose metabolism by insulin in the primary hepatocytes has been determined in this study. Methods. For this purpose, we studied effects of inhibitors of PLD on glucose uptake and glycogen synthesis stimulation by insulin. To determine the PLD activity, the method based on determination of products of transphosphatidylation reaction, phosphatidylethanol or phosphatidylbutanol, was used. Results. Inhibition of PLD by a general antagonist (1-butanol or specific inhibitor, halopemide, or N-hexanoylsphingosine, or by cellular ceramides accumulated in doxorubicin-treated hepatocytes decreased insulin-stimulated glucose metabolism. Doxorubicin-induced hepatocytes resistance to insulin action could be abolished by inhibition of ceramide production. Halopemide could nullify this effect. Addition of propranolol, as well as inhibitors of phosphatidylinositol 3-kinase (PI3-kinase (wortmannin, LY294002 or suppressors of Akt phosphorylation/activity, luteolin-7-O-glucoside or apigenin-7-O-glucoside, to the culture media could block cell response to insulin action. Conclusion. PLD plays an important role in the insulin signaling in the hepatocytes. PLD is activated downstream of PI3-kinase and Akt and is highly sensitive to ceramide content in the liver cells.

  19. Lipid-Induced Signaling Causes Release of Inflammatory Extracellular Vesicles From Hepatocytes.

    Science.gov (United States)

    Hirsova, Petra; Ibrahim, Samar H; Krishnan, Anuradha; Verma, Vikas K; Bronk, Steven F; Werneburg, Nathan W; Charlton, Michael R; Shah, Vijay H; Malhi, Harmeet; Gores, Gregory J

    2016-04-01

    Hepatocyte cellular dysfunction and death induced by lipids and macrophage-associated inflammation are characteristics of nonalcoholic steatohepatitis (NASH). The fatty acid palmitate can activate death receptor 5 (DR5) on hepatocytes, leading to their death, but little is known about how this process contributes to macrophage-associated inflammation. We investigated whether lipid-induced DR5 signaling results in the release of extracellular vesicles (EVs) from hepatocytes, and whether these can induce an inflammatory macrophage phenotype. Primary mouse and human hepatocytes and Huh7 cells were incubated with palmitate, its metabolite lysophosphatidylcholine, or diluent (control). The released EV were isolated, characterized, quantified, and applied to macrophages. C57BL/6 mice were placed on chow or a diet high in fat, fructose, and cholesterol to induce NASH. Some mice also were given the ROCK1 inhibitor fasudil; 2 weeks later, serum EVs were isolated and characterized by immunoblot and nanoparticle-tracking analyses. Livers were collected and analyzed by histology, immunohistochemistry, and quantitative polymerase chain reaction. Incubation of primary hepatocytes and Huh7 cells with palmitate or lysophosphatidylcholine increased their release of EVs, compared with control cells. This release was reduced by inactivating mediators of the DR5 signaling pathway or rho-associated, coiled-coil-containing protein kinase 1 (ROCK1) inhibition. Hepatocyte-derived EVs contained tumor necrosis factor-related apoptosis-inducing ligand and induced expression of interleukin 1β and interleukin 6 messenger RNAs in mouse bone marrow-derived macrophages. Activation of macrophages required DR5 and receptor-interacting protein kinase 1. Administration of the ROCK1 inhibitor fasudil to mice with NASH reduced serum levels of EVs; this reduction was associated with decreased liver injury, inflammation, and fibrosis. Lipids, which stimulate DR5, induce release of hepatocyte EVs, which

  20. Three different hepatocyte transplantation techniques for enzyme deficiency disease and acute hepatic failure

    NARCIS (Netherlands)

    te Velde, A. A.; Bosman, D. K.; Oldenburg, J.; Sala, M.; Maas, M. A.; Chamuleau, R. A.

    1992-01-01

    The effects of three different techniques of hepatocyte transplantation were investigated: transplantation of free hepatocytes into the spleen and intraperitoneal transplantation of microcarrier-attached hepatocytes or of microencapsulated hepatocytes. The liver-supportive functions of these

  1. Activation of CD40 with platelet derived CD154 promotes reactive oxygen species dependent death of human hepatocytes during hypoxia and reoxygenation.

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    Ricky H Bhogal

    Full Text Available BACKGROUND: Hypoxia and hypoxia-reoxygenation (H-R are pathogenic factors in many liver diseases that lead to hepatocyte death as a result of reactive oxygen species (ROS accumulation. The tumor necrosis factor super-family member CD154 can also induce hepatocyte apoptosis via activation of its receptor CD40 and induction of autocrine/paracrine Fas Ligand/CD178 but the relationship between CD40 activation, ROS generation and apoptosis is poorly understood. We hypothesised that CD40 activation and ROS accumulation act synergistically to drive human hepatocyte apoptosis. METHODS: Human hepatocytes were isolated from liver tissue and exposed to an in vitro model of hypoxia and H-R in the presence or absence of CD154 and/or various inhibitors. Hepatocyte ROS production, apoptosis and necrosis were determined by labelling cells with 2',7'-dichlorofluorescin, Annexin-V and 7-AAD respectively in a three-colour reporter flow cytometry assay. RESULTS: Exposure of human hepatocytes to recombinant CD154 or platelet-derived soluble CD154 augments ROS accumulation during H-R resulting in NADPH oxidase-dependent apoptosis and necrosis. The inhibition of c-Jun N-terminal Kinase and p38 attenuated CD154-mediated apoptosis but not necrosis. CONCLUSIONS: CD154-mediated apoptosis of hepatocytes involves ROS generation that is amplified during hypoxia-reoxygenation. This finding provides a molecular mechanism to explain the role of platelets in hepatocyte death during ischemia-reperfusion injury.

  2. Activation of CD40 with platelet derived CD154 promotes reactive oxygen species dependent death of human hepatocytes during hypoxia and reoxygenation.

    Science.gov (United States)

    Bhogal, Ricky H; Weston, Christopher J; Curbishley, Stuart M; Adams, David H; Afford, Simon C

    2012-01-01

    Hypoxia and hypoxia-reoxygenation (H-R) are pathogenic factors in many liver diseases that lead to hepatocyte death as a result of reactive oxygen species (ROS) accumulation. The tumor necrosis factor super-family member CD154 can also induce hepatocyte apoptosis via activation of its receptor CD40 and induction of autocrine/paracrine Fas Ligand/CD178 but the relationship between CD40 activation, ROS generation and apoptosis is poorly understood. We hypothesised that CD40 activation and ROS accumulation act synergistically to drive human hepatocyte apoptosis. Human hepatocytes were isolated from liver tissue and exposed to an in vitro model of hypoxia and H-R in the presence or absence of CD154 and/or various inhibitors. Hepatocyte ROS production, apoptosis and necrosis were determined by labelling cells with 2',7'-dichlorofluorescin, Annexin-V and 7-AAD respectively in a three-colour reporter flow cytometry assay. Exposure of human hepatocytes to recombinant CD154 or platelet-derived soluble CD154 augments ROS accumulation during H-R resulting in NADPH oxidase-dependent apoptosis and necrosis. The inhibition of c-Jun N-terminal Kinase and p38 attenuated CD154-mediated apoptosis but not necrosis. CD154-mediated apoptosis of hepatocytes involves ROS generation that is amplified during hypoxia-reoxygenation. This finding provides a molecular mechanism to explain the role of platelets in hepatocyte death during ischemia-reperfusion injury.

  3. Early morphological and functional alterations in canine hepatocytes due to alpha-amanitin, a major toxin of Amanita phalloides.

    Science.gov (United States)

    Magdalan, Jan; Ostrowska, Alina; Podhorska-Okołów, Marzena; Piotrowska, Aleksandra; Izykowska, Ilona; Nowak, Marcin; Dolińska-Krajewska, Barbara; Zabel, Maciej; Szelag, Adam; Dziegiel, Piotr

    2009-01-01

    The toadstool death cap (Amanita phalloides) and its subspecies, destroying angel (A. virosa) and death angel (A. verna) are responsible for nearly 95% of all fatal mushroom poisonings. High mortality rate in A. phalloides intoxications is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amanitins, the major toxins of this mushroom. This study evaluated early morphological and functional alterations in hepatocytes exposed to different concentrations of alpha-amanitin (alpha-AMA). All experiments were performed on cultured canine hepatocytes since intoxicated with A. phalloides dogs have clinical course and pathological findings similar to those seen in humans. The overall functional integrity and viability of cultured hepatocytes were assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and by measurements of lactate dehydrogenase (LDH), total protein, and urea levels. Our results showed that the course of alpha-AMA toxicity in cultured dog hepatocytes is divided into two phases. The first phase comprises functional cell impairments expressed by significant increase of LDH activity and inhibition of protein and urea synthesis when compared with the control group. This is followed by discrete changes in hepatocyte ultrastructure, including marginalization and condensation of nuclear chromatin, as well as formation of the foamlike cytoplasm. The second stage is lethal and is characterized by ongoing necrosis, and/or apoptosis. This may be related to dose of toxin and time of exposure.

  4. Resveratrol differentially regulates NAMPT and SIRT1 in Hepatocarcinoma cells and primary human hepatocytes.

    Directory of Open Access Journals (Sweden)

    Susanne Schuster

    Full Text Available Resveratrol is reported to possess chemotherapeutic properties in several cancers. In this study, we wanted to investigate the molecular mechanisms of resveratrol-induced cell cycle arrest and apoptosis as well as the impact of resveratrol on NAMPT and SIRT1 protein function and asked whether there are differences in hepatocarcinoma cells (HepG2, Hep3B cells and non-cancerous primary human hepatocytes. We found a lower basal NAMPT mRNA and protein expression in hepatocarcinoma cells compared to primary hepatocytes. In contrast, SIRT1 was significantly higher expressed in hepatocarcinoma cells than in primary hepatocytes. Resveratrol induced cell cycle arrest in the S- and G2/M- phase and apoptosis was mediated by activation of p53 and caspase-3 in HepG2 cells. In contrast to primary hepatocytes, resveratrol treated HepG2 cells showed a reduction of NAMPT enzymatic activity and increased p53 acetylation (K382. Resveratrol induced NAMPT release from HepG2 cells which was associated with increased NAMPT mRNA expression. This effect was absent in primary hepatocytes where resveratrol was shown to function as NAMPT and SIRT1 activator. SIRT1 inhibition by EX527 resembled resveratrol effects on HepG2 cells. Furthermore, a SIRT1 overexpression significantly decreased both p53 hyperacetylation and resveratrol-induced NAMPT release as well as S-phase arrest in HepG2 cells. We could show that NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and primary hepatocytes and that resveratrol did not act as a SIRT1 activator in hepatocarcinoma cells.

  5. Resveratrol Differentially Regulates NAMPT and SIRT1 in Hepatocarcinoma Cells and Primary Human Hepatocytes

    Science.gov (United States)

    Schuster, Susanne; Penke, Melanie; Gorski, Theresa; Petzold-Quinque, Stefanie; Damm, Georg; Gebhardt, Rolf; Kiess, Wieland; Garten, Antje

    2014-01-01

    Resveratrol is reported to possess chemotherapeutic properties in several cancers. In this study, we wanted to investigate the molecular mechanisms of resveratrol-induced cell cycle arrest and apoptosis as well as the impact of resveratrol on NAMPT and SIRT1 protein function and asked whether there are differences in hepatocarcinoma cells (HepG2, Hep3B cells) and non-cancerous primary human hepatocytes. We found a lower basal NAMPT mRNA and protein expression in hepatocarcinoma cells compared to primary hepatocytes. In contrast, SIRT1 was significantly higher expressed in hepatocarcinoma cells than in primary hepatocytes. Resveratrol induced cell cycle arrest in the S- and G2/M- phase and apoptosis was mediated by activation of p53 and caspase-3 in HepG2 cells. In contrast to primary hepatocytes, resveratrol treated HepG2 cells showed a reduction of NAMPT enzymatic activity and increased p53 acetylation (K382). Resveratrol induced NAMPT release from HepG2 cells which was associated with increased NAMPT mRNA expression. This effect was absent in primary hepatocytes where resveratrol was shown to function as NAMPT and SIRT1 activator. SIRT1 inhibition by EX527 resembled resveratrol effects on HepG2 cells. Furthermore, a SIRT1 overexpression significantly decreased both p53 hyperacetylation and resveratrol-induced NAMPT release as well as S-phase arrest in HepG2 cells. We could show that NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and primary hepatocytes and that resveratrol did not act as a SIRT1 activator in hepatocarcinoma cells. PMID:24603648

  6. Angiotensin II Induces C-Reactive Protein Expression via AT1-ROS-MAPK-NF-κB Signal Pathway in Hepatocytes

    Directory of Open Access Journals (Sweden)

    Jingjing Zhao

    2013-09-01

    Full Text Available Background: C-reactive protein (CRP participates in development of inflammatory diseases. Hepatocytes are a major contributor of circulating CRP. Although angiotensin II (Ang II is known to evoke inflammatory response, it remains unknown whether Ang II induces CRP expression in hepatocytes. The present study observed effect of Ang II on CRP expression and the related signal pathway in hepatocytes. Methods: mRNA and protein expressions in human hepatocytes were determined with RT-PCR and Western blot respectively. Reactive oxygen species (ROS was measured using a fluorescence probe. CRP in liver and serum of rats was determined by immunohistochemistry and ELISA respectively. Results: Ang II induced mRNA and protein expression of CRP in hepatocytes and increased CRP production in liver and CRP level in serum. Losartan reduced Ang II- induced CRP expression in hepatocytes. Losartan and thenoyltrifluoroacetone decreased Ang II-stimulated ROS production. N-acetylcysteine antagonized Ang II-induced CRP expression. Losartan and N-acetylcysteine inhibited Ang II-activated ERK1/2. Unlike ERK1/2, only losartan inhibited Ang II-activated JNK. Furthermore, pyrrolidine dithiocarbamate abolished Ang II-induced CRP expression. Conclusion: Ang II has ability to induce CRP expression in hepatocytes in vitro and in vivo through AT1 receptor followed by ROS, MAPK and NF-κB signal pathway.

  7. The Hepatitis B Virus (HBV) HBx Protein Activates AKT To Simultaneously Regulate HBV Replication and Hepatocyte Survival

    Science.gov (United States)

    Rawat, Siddhartha

    2014-01-01

    ABSTRACT Chronic infection with hepatitis B virus (HBV) is a risk factor for developing liver diseases such as hepatocellular carcinoma (HCC). HBx is a multifunctional protein encoded by the HBV genome; HBx stimulates HBV replication and is thought to play an important role in the development of HBV-associated HCC. HBx can activate the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway in some cell lines; however, whether HBx regulates PI3K/AKT signaling in normal hepatocytes has not been evaluated. In studies described here, we assessed HBx activation of PI3K/AKT signaling in an ex vivo model of cultured primary hepatocytes and determined how this HBx activity affects HBV replication. We report that HBx activates AKT in primary hepatocytes and that the activation of AKT decreases HBV replication and HBV mRNA and core protein levels. We show that the transcription factor hepatocyte nuclear factor 4α (HNF4α) is a target of HBx-regulated AKT, and we link HNF4α to HBx-regulated AKT modulation of HBV transcription and replication. Although we and others have shown that HBx stimulates and is likely required for HBV replication, we now report that HBx also activates signals that can diminish the overall level of HBV replication. While this may seem counterintuitive, we show that an important effect of HBx activation of AKT is inhibition of apoptosis. Consequently, our studies suggest that HBx balances HBV replication and cell survival by stimulating signaling pathways that enhance hepatocyte survival at the expense of higher levels of HBV replication. IMPORTANCE Chronic hepatitis B virus (HBV) infection is a common cause of the development of liver cancer. Regulation of cell signaling pathways by the HBV HBx protein is thought to influence the development of HBV-associated liver cancer. HBx stimulates, and may be essential for, HBV replication. We show that HBx activates AKT in hepatocytes to reduce HBV replication. While this seems contradictory to an

  8. Transforming growth factor-beta and epidermal growth factor modulate basal and interleukin-6-induced amino acid uptake and acute phase protein synthesis in cultured rat hepatocytes.

    Science.gov (United States)

    Bereta, J; Szuba, K; Fiers, W; Gauldie, J; Koj, A

    1990-06-18

    Rat hepatocytes cultured for 2 days with interleukin-6 show increased synthesis of acute phase proteins and enhanced accumulation of 14C-labelled alpha-aminoisobutyric acid. Transforming growth factor-beta 1 (0.1-10 ng/ml) inhibits whereas epidermal growth factor (1-100 ng/ml) enhances both basal and interleukin-6-induced amino acid uptake by rat hepatocytes with only a slight alteration of acute phase protein synthesis.

  9. Hepatocyte Growth Factor Reduces Free Cholesterol-Mediated Lipotoxicity in Primary Hepatocytes by Countering Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Mayra Domínguez-Pérez

    2016-01-01

    Full Text Available Cholesterol overload in the liver has shown toxic effects by inducing the aggravation of nonalcoholic fatty liver disease to steatohepatitis and sensitizing to damage. Although the mechanism of damage is complex, it has been demonstrated that oxidative stress plays a prominent role in the process. In addition, we have proved that hepatocyte growth factor induces an antioxidant response in hepatic cells; in the present work we aimed to figure out the protective effect of this growth factor in hepatocytes overloaded with free cholesterol. Hepatocytes from mice fed with a high-cholesterol diet were treated or not with HGF, reactive oxygen species present in cholesterol overloaded hepatocytes significantly decreased, and this effect was particularly associated with the increase in glutathione and related enzymes, such as γ-gamma glutamyl cysteine synthetase, GSH peroxidase, and GSH-S-transferase. Our data clearly indicate that HGF displays an antioxidant response by inducing the glutathione-related protection system.

  10. Sphingosine Kinase 1 Protects Hepatocytes from Lipotoxicity via Down-regulation of IRE1α Protein Expression*

    Science.gov (United States)

    Qi, Yanfei; Wang, Wei; Chen, Jinbiao; Dai, Lan; Kaczorowski, Dominik; Gao, Xin; Xia, Pu

    2015-01-01

    Aberrant deposition of fat including free fatty acids in the liver often causes damage to hepatocytes, namely lipotoxicity, which is a key pathogenic event in the development and progression of fatty liver diseases. This study demonstrates a pivotal role of sphingosine kinase 1 (SphK1) in protecting hepatocytes from lipotoxicity. Exposure of primary murine hepatocytes to palmitate resulted in dose-dependent cell death, which was enhanced significantly in Sphk1-deficient cells. In keeping with this, expression of dominant-negative mutant SphK1 also markedly promoted palmitate-induced cell death. In contrast, overexpression of wild-type SphK1 profoundly protected hepatocytes from lipotoxicity. Mechanistically, the protective effect of SphK1 is attributable to suppression of ER stress-mediated pro-apoptotic pathways, as reflected in the inhibition of IRE1α activation, XBP1 splicing, JNK phosphorylation, and CHOP induction. Of note, SphK1 inhibited the IRE1α pathway by reducing IRE1α expression at the transcriptional level. Moreover, S1P mimicked the effect of SphK1, suppressing IRE1α expression in a receptor-dependent manner. Furthermore, enforced overexpression of IRE1α significantly blocked the protective effect of SphK1 against lipotoxicity. Therefore, this study provides new insights into the role of SphK1 in hepatocyte survival and uncovers a novel mechanism for protection against ER stress-mediated cell death. PMID:26240153

  11. Swelling of rat hepatocytes stimulates glycogen synthesis

    NARCIS (Netherlands)

    Baquet, A.; Hue, L.; Meijer, A. J.; van Woerkom, G. M.; Plomp, P. J.

    1990-01-01

    In hepatocytes from fasted rats, several amino acids are known to stimulate glycogen synthesis via activation of glycogen synthase. The hypothesis that an increase in cell volume resulting from amino acid uptake may be involved in the stimulation of glycogen synthesis is supported by the following

  12. Decreased phalloidin response, phallotoxin uptake and bile acid transport in hepatocytes prepared from wistar rats treated chronically with diethylnitrosamine.

    Science.gov (United States)

    Ziegler, K; Petzinger, E; Frimmer, M

    1980-01-01

    Isolated hepatocytes prepared from rats pretreated with diethylnitrosamine (0.5 mg/kg DENA/DAY P.O.) are less sensitive to phalloidin poisoning. They take up lower amounts of both phallotoxins and bile acids than controls. The degree of inhibition depends on the period of pretreatment.

  13. Defense mechanisms of hepatocytes against Burkholderia pseudomallei

    Directory of Open Access Journals (Sweden)

    Antje eBast

    2012-01-01

    Full Text Available The gram-negative facultative intracellular rod Burkholderia pseudomallei causes melioidosis, an infectious disease with a wide range of clinical presentations. Among the observed visceral abscesses, the liver is commonly affected. However, neither this organotropism of B. pseudomallei nor local hepatic defense mechanisms have been thoroughly investigated so far. Own previous studies using electron microscopy of the murine liver after systemic infection of mice indicated that hepatocytes might be capable of killing B. pseudomallei. Therefore, the aim of this study was to further elucidate the interaction of B. pseudomallei with these cells and to analyse the role of hepatocytes in anti-B. pseudomallei host defense. In vitro studies using the human hepatocyte cell line HepG2 revealed that B. pseudomallei can invade these cells. Subsequently, B. pseudomallei is able to escape from the vacuole, to replicate within the cytosol of HepG2 cells involving its type 3 and type 6 secretion systems, and to induce actin tail formation. Furthermore, stimulation of HepG2 cells showed that IFNgamma can restrict growth of B. pseudomallei in the early and late phase of infection whereas the combination of IFNgamma, IL-1beta and TNFalpha is required for the maximal antibacterial activity. This anti-B. pseudomallei defense of HepG2 cells did not seem to be mediated by iNOS-derived nitric oxide or NADPH oxidase-derived superoxide. In summary, this is the first study describing B. pseudomallei intracellular life cycle characteristics in hepatocytes and showing that IFNgamma-mediated, but nitric oxide- and reactive oxygen species-independent, effector mechanisms are important in anti-B. pseudomallei host defense of hepatocytes.

  14. Metformin action in human hepatocytes: coactivation of atypical protein kinase C alters 5'-AMP-activated protein kinase effects on lipogenic and gluconeogenic enzyme expression.

    Science.gov (United States)

    Sajan, Mini P; Ivey, Robert A; Farese, Robert V

    2013-11-01

    Atypical protein kinase C (aPKC) levels and activity are elevated in hepatocytes of individuals with type 2 diabetes and cause excessive increases in the levels of lipogenic and gluconeogenic enzymes; aPKC inhibitors largely correct these aberrations. Metformin improves hepatic gluconeogenesis by activating 5'-AMP-activated protein kinase (AMPK). However, metformin also activates aPKC in certain tissues; in the liver, this activation could amplify diabetic aberrations and offset the positive effects of AMPK. In this study, we examined whether metformin activates aPKC in human hepatocytes and the metabolic consequences of any such activation. We compared protein kinase activities and alterations in lipogenic and gluconeogenic enzyme levels during activity of the AMPK activators metformin and AICAR, relative to those of an aPKC-ι inhibitor, in hepatocytes from non-diabetic and type 2 diabetic human organ donors. Metformin and 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) activated aPKC at concentrations comparable with those required for AMPK activation. Moreover, both agents increased lipogenic enzyme levels by an aPKC-dependent mechanism. Thus, whereas insulin- and diabetes-dependent increases in lipogenic enzyme levels were reversed by aPKC inhibition, such levels were increased in hepatocytes from non-diabetic donors and remained elevated in hepatocytes from diabetic donors following metformin and AICAR treatment. In addition, whereas aPKC inhibition diminished gluconeogenic enzyme levels in the absence and presence of insulin in hepatocytes from both non-diabetic and diabetic donors, metformin and AICAR increased gluconeogenic enzyme levels in hepatocytes from non-diabetic individuals, but nevertheless diminished gluconeogenic enzyme levels in insulin-treated hepatocytes from diabetic donors. Metformin and AICAR activate aPKC together with AMPK in human hepatocytes. Activation of aPKC increases lipogenic enzyme levels and alters gluconeogenic

  15. Effects of dehydroepiandrosterone (DHEA) on glucose metabolism in isolated hepatocytes from Zucker rats

    Energy Technology Data Exchange (ETDEWEB)

    Finan, A.; Cleary, M.P.

    1986-03-05

    DHEA has been shown to competitively inhibit the pentose phosphate shunt (PPS) enzyme glucose-6-phosphate dehydrogenase (G6PD) when added in vitro to supernatants or homogenates prepared from mammalian tissues. However, no consistent effect on G6PD activity has been determined in tissue removed from DHEA-treated rats. To explore the effects of DHEA on PPS, glucose utilization was measured in hepatocytes from lean and obese male Zucker rats (8 wks of age) following 1 wk of DHEA treatment (0.6% in diet). Incubation of isolated hepatocytes from treated lean Zucker rats with either (1-/sup 14/C) glucose or (6-/sup 14/C) glucose resulted in significant decreases in CO/sub 2/ production and total glucose utilization. DHEA-lean rats also had lowered fat pad weights. In obese rats, there was no effect of 1 wk of treatment on either glucose metabolism or fat pad weight. The calculated percent contribution of the PPS to glucose metabolism in hepatocytes was not changed for either DHEA-lean or obese rats when compared to control rats. In conclusion, 1 wk of DHEA treatment lowered overall glucose metabolism in hepatocytes of lean Zucker rats, but did not selectively affect the PPS. The lack of an effect of short-term treatment in obese rats may be due to differences in their metabolism or storage/release of DHEA in tissues in comparison to lean rats.

  16. The Expression of the Hepatocyte SLAMF3 (CD229) Receptor Enhances the Hepatitis C Virus Infection

    Science.gov (United States)

    Cartier, Flora; Marcq, Ingrid; Douam, Florian; Ossart, Christèle; Regnier, Aline; Debuysscher, Véronique; Lavillette, Dimitri; Bouhlal, Hicham

    2014-01-01

    Hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer worldwide. We recently characterized for the first time the expression of Signaling Lymphocyte Activating Molecule 3 (SLAMF3) in human hepatocytes and here, we report that SLAMF3 interacts with the HCV viral protein E2 and is implicated in HCV entry process. We found a strong correlation between SLAMF3 expression level and hepatocyte susceptibility to HCV infection. The use of specific siRNAs to down-modulate SLAMF3 expression and SLAMF3-blocking antibodies both decreased the hepatocytes susceptibility to HCV infection. Moreover, SLAMF3 over-expression significantly increased susceptibility to HCV infection. Interestingly, experiments with peptides derived from each SLAMF3 domain showed that the first N-terminal extracellular domain is essential for interaction with HCV particles. Finally, we showed that recombinant HCV envelop protein E2 can bind SLAMF3 and that anti-SLAMF3 antibodies inhibited specifically this interaction. Overall, our results revealed that SLAMF3 plays a role during HCV entry, likely by enhancing entry of viral particle within hepatocytes. PMID:24927415

  17. The expression of the hepatocyte SLAMF3 (CD229 receptor enhances the hepatitis C virus infection.

    Directory of Open Access Journals (Sweden)

    Flora Cartier

    Full Text Available Hepatitis C virus (HCV is a leading cause of cirrhosis and liver cancer worldwide. We recently characterized for the first time the expression of Signaling Lymphocyte Activating Molecule 3 (SLAMF3 in human hepatocytes and here, we report that SLAMF3 interacts with the HCV viral protein E2 and is implicated in HCV entry process. We found a strong correlation between SLAMF3 expression level and hepatocyte susceptibility to HCV infection. The use of specific siRNAs to down-modulate SLAMF3 expression and SLAMF3-blocking antibodies both decreased the hepatocytes susceptibility to HCV infection. Moreover, SLAMF3 over-expression significantly increased susceptibility to HCV infection. Interestingly, experiments with peptides derived from each SLAMF3 domain showed that the first N-terminal extracellular domain is essential for interaction with HCV particles. Finally, we showed that recombinant HCV envelop protein E2 can bind SLAMF3 and that anti-SLAMF3 antibodies inhibited specifically this interaction. Overall, our results revealed that SLAMF3 plays a role during HCV entry, likely by enhancing entry of viral particle within hepatocytes.

  18. Toll-like receptor 3-activated macrophages confer anti-HCV activity to hepatocytes through exosomes.

    Science.gov (United States)

    Zhou, Yu; Wang, Xu; Sun, Li; Zhou, Li; Ma, Tong-Cui; Song, Li; Wu, Jian-Guo; Li, Jie-Liang; Ho, Wen-Zhe

    2016-12-01

    Exosomes are a class of cell-released small vesicles that mediate intercellular communication by delivering functional factors to recipient cells. During hepatitis C virus (HCV) infection, the interaction between liver resident macrophages and hepatocytes is a key component in liver innate immunity. In this study, we explored the role of exosomes in the delivery of innate anti-HCV factors to hepatocytes from macrophages. We showed that supernatant from TLR3-activated macrophage cultures could efficiently inhibit HCV replication in Huh7 cells. This macrophage-mediated anti-HCV activity was through exosomes because inhibiting exosomes could abrogate the action of macrophages. Further analyses demonstrated that TLR3-activated macrophages release exosomes that contain anti-HCV microRNA (miRNA)-29 family members. Inhibiting miRNA29 could restore HCV replication. These findings suggest a novel antiviral mechanism in liver innate immunity against HCV infection and provide insights to support further studies on developing exosome-based delivery system for disease treatment.-Zhou, Y., Wang, X., Sun, L., Zhou, L., Ma, T.-C., Song, L., Wu, J.-G., Li, J.-L., Ho, W.-Z. Toll-like receptor 3-activated macrophages confer anti-HCV activity to hepatocytes through exosomes. © FASEB.

  19. Mixed Lineage Kinase 3 Mediates the Induction of CXCL10 by a STAT1-Dependent Mechanism During Hepatocyte Lipotoxicity.

    Science.gov (United States)

    Tomita, Kyoko; Kabashima, Ayano; Freeman, Brittany L; Bronk, Steven F; Hirsova, Petra; Ibrahim, Samar H

    2017-10-01

    Saturated fatty acids (SFA) and their toxic metabolites contribute to hepatocyte lipotoxicity in nonalcoholic steatohepatitis (NASH). We previously reported that hepatocytes, under lipotoxic stress, express the potent macrophage chemotactic ligand C-X-C motif chemokine 10 (CXCL10), and release CXCL10-enriched extracellular vesicles (EV) by a mixed lineage kinase (MLK) 3-dependent mechanism. In the current study, we sought to examine the signaling pathway responsible for CXCL10 induction during hepatocyte lipotoxicity. Here, we demonstrate a role for signal transducer and activator of transcription (STAT) 1 in regulating CXCL10 expression. Huh7 and HepG2 cells were treated with lysophosphatidylcholine (LPC), the toxic metabolite of the SFA palmitate. In LPC-treated hepatocytes, CXCL10 induction is mediated by a mitogen activated protein kinase (MAPK) signaling cascade consisting of a relay kinase module of MLK3, MKK3/6, and p38. P38 in turn induces STAT1 Ser727 phosphorylation and CXCL10 upregulation in hepatocytes, which is reduced by genetic or pharmacological inhibition of this MAPK signaling cascade. The binding and activity of STAT1 at the CXCL10 gene promoter were identified by chromatin immunoprecipitation and luciferase gene expression assays. Promoter activation was attenuated by MLK3/STAT1 inhibition or by deletion of the consensus STAT1 binding sites within the CXCL10 promoter. In lipotoxic hepatocytes, MLK3 activates a MAPK signaling cascade, resulting in the activating phosphorylation of STAT1, and CXCL10 transcriptional upregulation. Hence, this kinase relay module and/or STAT1 inhibition may serve as a therapeutic target to reduce CXCL10 release, thereby attenuating NASH pathogenesis. J. Cell. Biochem. 118: 3249-3259, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  20. Cdc42 and phosphoinositide 3-kinase drive Rac-mediated actin polymerization downstream of c-Met in distinct and common pathways

    DEFF Research Database (Denmark)

    Bosse, Tanja; Ehinger, Julia; Czuchra, Aleksandra

    2007-01-01

    impaired but not abrogated upon genetic removal of either Cdc42 or pharmacological inhibition of phosphoinositide 3-kinase (PI3-kinase). While loss of Cdc42 or PI3-kinase function correlated with reduced HGF- and InlB-triggered Rac activation, complete abolishment of actin reorganization and Rac activation...... required the simultaneous inactivation of both Cdc42 and PI3-kinase signaling. Moreover, Cdc42 activation was fully independent of PI3-kinase activity, whereas the latter partly depended on Cdc42. Finally, Cdc42 function did not require its interaction with the actin nucleation-promoting factor N......-WASP. Instead, actin polymerization was driven by Arp2/3 complex activation through the WAVE complex downstream of Rac. Together, our data establish an intricate signaling network comprising as key molecules Cdc42 and PI3-kinase, which converge on Rac-mediated actin reorganization essential for Listeria...

  1. Effects of dexamethasone and insulin on the acute phase response of Morris hepatoma cells and of rat hepatocytes in culture.

    Science.gov (United States)

    Magielska-Zero, D; Guzdek, A; Bereta, J; Kurdowska, A; Cieszka, K; Koj, A

    1988-01-01

    Dexamethasone and insulin stimulate production of several plasma proteins in primary cultures of adult rat hepatocytes but inhibit their production in primary cultures of Morris hepatoma cell line 7777W. The acute phase response elicited in cultured cells by crude cytokines from activated rat peritoneal macrophages is considerably higher in hepatocytes in the presence of hormones, and especially of dexamethasone. In hepatoma cells the hormones enhance the cytokine-induced formation of fibrinogen and cysteine proteinase inhibitor but are without significant effect on suppression of albumin and alpha-fetoprotein synthesis by macrophage supernatants.

  2. Regulation of pyruvate kinase in isolated hepatocytes by metabolites arising from the glycolysis of fructose and other related substrates

    OpenAIRE

    Mapungwana, Sibusisiwe Mavis

    1982-01-01

    It has been reported that fructose is rapidly converted to lactate by the liver. The only regulatory enzyme involved in this conversion is L-type pyruvate kinase. Thus the effects of fructose and other related substrates which enter the glycolytic sequence at the triose phosphate level are of physiological importance. Incubation of isolated hepatocytes with fructose at high concentrations and with glycerol caused an apparent inhibition of pyruvate kinase. This inhibition was correlated to the...

  3. Prolyl hydroxylase-1 regulates hepatocyte apoptosis in an NF-κB-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Fitzpatrick, Susan F.; Fábián, Zsolt; Schaible, Bettina; Lenihan, Colin R.; Schwarzl, Thomas [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Rodriguez, Javier [Systems Biology Ireland, University College Dublin, Dublin 4 (Ireland); Zheng, Xingnan; Li, Zongwei [Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC (United States); Tambuwala, Murtaza M. [School of Pharmacy and Pharmaceutical Sciences, Ulster University, Coleraine, BT52 1SA, Northern Ireland (United Kingdom); Higgins, Desmond G.; O' Meara, Yvonne [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Slattery, Craig [School of Biomolecular and Biomedical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Manresa, Mario C. [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Fraisl, Peter; Bruning, Ulrike [Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, University of Leuven, Vesalius Research Center, VIB, B-3000 (Belgium); Baes, Myriam [Laboratory for Cell Metabolism, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven (Belgium); Carmeliet, Peter; Doherty, Glen [Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, University of Leuven, Vesalius Research Center, VIB, B-3000 (Belgium); Kriegsheim, Alex von [Systems Biology Ireland, University College Dublin, Dublin 4 (Ireland); Cummins, Eoin P. [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); and others

    2016-06-03

    Hepatocyte death is an important contributing factor in a number of diseases of the liver. PHD1 confers hypoxic sensitivity upon transcription factors including the hypoxia inducible factor (HIF) and nuclear factor-kappaB (NF-κB). Reduced PHD1 activity is linked to decreased apoptosis. Here, we investigated the underlying mechanism(s) in hepatocytes. Basal NF-κB activity was elevated in PHD1{sup −/−} hepatocytes compared to wild type controls. ChIP-seq analysis confirmed enhanced binding of NF-κB to chromatin in regions proximal to the promoters of genes involved in the regulation of apoptosis. Inhibition of NF-κB (but not knock-out of HIF-1 or HIF-2) reversed the anti-apoptotic effects of pharmacologic hydroxylase inhibition. We hypothesize that PHD1 inhibition leads to altered expression of NF-κB-dependent genes resulting in reduced apoptosis. This study provides new information relating to the possible mechanism of therapeutic action of hydroxylase inhibitors that has been reported in pre-clinical models of intestinal and hepatic disease. -- Highlights: •Genetic ablation of PHD1 upregulates NF-kappaB (NF-κB) in hepatocytes. •Activation of NF-κB leads to differential DNA-binding of p50/p65 and results in differential regulation of apoptotic genes. •We identified proline 191 in the beta subunit of the I-kappaB kinase as a target for PHD1-mediated hydroxylation. •Blockade of prolyl-4-hydroxylases has been found cytoprotective in liver cells.

  4. A phase-I study of lapatinib in combination with foretinib, a c-MET, AXL and vascular endothelial growth factor receptor inhibitor, in human epidermal growth factor receptor 2 (HER-2)-positive metastatic breast cancer.

    Science.gov (United States)

    Chia, Stephen K; Ellard, Susan L; Mates, Mihaela; Welch, Stephen; Mihalcioiu, Catalin; Miller, Wilson H; Gelmon, Karen; Lohrisch, Caroline; Kumar, Vikaash; Taylor, Sara; Hagerman, Linda; Goodwin, Rachel; Wang, Tao; Sakashita, Shingo; Tsao, Ming S; Eisenhauer, Elizabeth; Bradbury, Penelope

    2017-05-02

    The mechanisms of resistance to anti-human epidermal growth factor receptor 2 (HER 2) therapies are unclear but may include the tyrosine-protein kinase Met (c-Met), vascular endothelial growth factor (VEGF) and AXL pathways. Foretinib is an inhibitor of c-Met, VEGF receptor 2 (VEGFR-2), platelet-derived growth factor receptor beta (PDGFRB), AXL, Fms-like tyrosine kinase 3 (FLT3), angiopoiten receptor (TIE-2), RET and RON kinases. This phase Ib study sought to establish the associated toxicities, pharmacokinetics (PK) and recommended phase II doses (RP2D) of foretinib and lapatinib in a cohort of HER-2-positive patients with metastatic breast cancer (MBC). Women with HER-2 positive MBC, Performance status (PS 0-2), and no limit on number of prior chemotherapies or lines of anti-HER-2 therapies were enrolled. A 3 + 3 dose escalation design was utilized. Four dose levels were intended with starting doses of foretinib 30 mg and lapatinib 750 mg orally once a day (OD) on a 4-weekly cycle. Assessment of c-MET status from the primary archival tissue was performed. We enrolled 19 patients, all evaluable for toxicity assessment and for response evaluation. Median age was 60 years (34-86 years), 95% were PS 0-1, 53% were estrogen receptor-positive and 95% had at least one prior anti-HER-2-based regimen. The fourth dose level was reached (foretinib 45 mg/lapatinib 1250 mg) with dose-limiting toxicities of grade-3 diarrhea and fatigue. There was only one grade-4 non-hematological toxicity across all dose levels. There were no PK interactions between the agents. A median of two cycles was delivered across the dose levels (range 1-20) with associated progression-free survival of 3.2 months (95% CI 1.61-4.34 months). By immunohistochemical assessment with a specified cutoff, none of the 17 samples tested were classified as positive for c-Met. The RP2D of the combined foretinib and lapatinib is 45 mg and 1000 mg PO OD, respectively. Limited activity was seen with this

  5. Regulation of hepatocyte identity and quiescence.

    Science.gov (United States)

    Berasain, Carmen; Avila, Matías A

    2015-10-01

    The liver is a highly differentiated organ with a central role in metabolism, detoxification and systemic homeostasis. To perform its multiple tasks, liver parenchymal cells, the hepatocytes, express a large complement of enabling genes defining their complex phenotype. This phenotype is progressively acquired during fetal development and needs to be maintained in adulthood to guarantee the individual's survival. Upon injury or loss of functional mass, the liver displays an extraordinary regenerative response, mainly based on the proliferation of hepatocytes which otherwise are long-lived quiescent cells. Increasing observations suggest that loss of hepatocellular differentiation and quiescence underlie liver malfunction in chronic liver disease and pave the way for hepatocellular carcinoma development. Here, we briefly review the essential mechanisms leading to the acquisition of liver maturity. We also identify the key molecular factors involved in the preservation of hepatocellular homeostasis and finally discuss potential strategies to preserve liver identity and function.

  6. Lipid-induced toxicity stimulates hepatocytes to release angiogenic microparticles that require Vanin-1 for uptake by endothelial cells

    Science.gov (United States)

    Povero, Davide; Eguchi, Akiko; Niesman, Ingrid R.; Andronikou, Nektaria; de Mollerat du Jeu, Xavier; Mulya, Anny; Berk, Michael; Lazic, Milos; Thapaliya, Samjana; Parola, Maurizio; Patel, Hemal H.; Feldstein, Ariel E.

    2014-01-01

    Angiogenesis is a key pathological feature of experimental and human steatohepatitis, a common chronic liver disease that is associated with obesity. We demonstrated that hepatocytes generated a type of membrane-bound vesicle, microparticles, in response to conditions that mimicked the lipid accumulation that occurs in the liver in some forms of steatohepatitis and that these microparticles promoted angiogenesis. When applied to an endothelial cell line, medium conditioned by murine hepatocytes or a human hepatocyte cell line exposed to saturated free fatty acids induced migration and tube formation, two processes required for angiogenesis. Medium from hepatocytes in which caspase 3 was inhibited or medium in which the microparticles were removed by ultracentrifugation lacked proangiogenic activity. Isolated hepatocyte-derived microparticles induced migration and tube formation of an endothelial cell line in vitro and angiogenesis in mice, processes that depended on internalization of microparticles. Microparticle internalization required the interaction of the ectoenzyme Vanin-1 (VNN1), an abundant surface protein on the microparticles, with lipid raft domains of endothelial cells. Large quantities of hepatocyte-derived microparticles were detected in the blood of mice with diet-induced steatohepatitis, and microparticle quantity correlated with disease severity. Genetic ablation of caspase 3 or RNA interference directed against VNN1 protected mice from steatohepatitis-induced pathological angiogenesis in the liver and resulted in a loss of the proangiogenic effects of microparticles. Our data identify hepatocyte-derived microparticles as critical signals that contribute to angiogenesis and liver damage in steatohepatitis and suggest a therapeutic target for this condition. PMID:24106341

  7. Cardiac concentric hypertrophy promoted by activated Met receptor is mitigated in vivo by inhibition of Erk1,2 signalling with Pimasertib.

    Science.gov (United States)

    Sala, Valentina; Gallo, Simona; Gatti, Stefano; Medico, Enzo; Vigna, Elisa; Cantarella, Daniela; Fontani, Lara; Natale, Massimo; Cimino, James; Morello, Mara; Comoglio, Paolo Maria; Ponzetto, Antonio; Crepaldi, Tiziana

    2016-04-01

    Cardiac hypertrophy is a major risk factor for heart failure. Hence, its attenuation represents an important clinical goal. Erk1,2 signalling is pivotal in the cardiac response to stress, suggesting that its inhibition may be a good strategy to revert heart hypertrophy. In this work, we unveiled the events associated with cardiac hypertrophy by means of a transgenic model expressing activated Met receptor. c-Met proto-oncogene encodes for the tyrosine kinase receptor of Hepatocyte growth factor and is a strong inducer of Ras-Raf-Mek-Erk1,2 pathway. We showed that three weeks after the induction of activated Met, the heart presents a remarkable concentric hypertrophy, with no signs of congestive failure and preserved contractility. Cardiac enlargement is accompanied by upregulation of growth-regulating transcription factors, natriuretic peptides, cytoskeletal proteins, and Extracellular Matrix remodelling factors (Timp1 and Pai1). At a later stage, cardiac hypertrophic remodelling results into heart failure with preserved systolic function. Prevention trial by suppressing activated Met showed that cardiac hypertrophy is reversible, and progression to heart failure is prevented. Notably, treatment with Pimasertib, Mek1 inhibitor, attenuates cardiac hypertrophy and remodelling. Our results suggest that modulation of Erk1.2 signalling may constitute a new therapeutic approach for treating cardiac hypertrophies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Duck hepatitis B virus covalently closed circular DNA appears to survive hepatocyte mitosis in the growing liver

    Energy Technology Data Exchange (ETDEWEB)

    Reaiche-Miller, Georget Y.; Thorpe, Michael; Low, Huey Chi; Qiao, Qiao; Scougall, Catherine A. [School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005 (Australia); Mason, William S.; Litwin, Samuel [Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111 (United States); Jilbert, Allison R., E-mail: allison.jilbert@adelaide.edu.au [School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005 (Australia)

    2013-11-15

    Nucleos(t)ide analogues that inhibit hepatitis B virus (HBV) DNA replication are typically used as monotherapy for chronically infected patients. Treatment with a nucleos(t)ide analogue eliminates most HBV DNA replication intermediates and produces a gradual decline in levels of covalently closed circular DNA (cccDNA), the template for viral RNA synthesis. It remains uncertain if levels of cccDNA decline primarily through hepatocyte death, or if loss also occurs during hepatocyte mitosis. To determine if cccDNA survives mitosis, growing ducklings infected with duck hepatitis B virus (DHBV) were treated with the nucleoside analogue, Entecavir. Viremia was suppressed at least 10{sup 5}-fold, during a period when average liver mass increased 23-fold. Analysis of the data suggested that if cccDNA synthesis was completely inhibited, at least 49% of cccDNA survived hepatocyte mitosis. However, there was a large duck-to-duck variation in cccDNA levels, suggesting that low level cccDNA synthesis may contribute to this apparent survival through mitosis. - Highlights: • The hepatitis B virus nuclear template is covalently closed circular DNA (cccDNA). • cccDNA was studied during liver growth in duck hepatitis B virus infected ducks. • Virus DNA replication and new cccDNA synthesis were inhibited with Entecavir. • At least 49% of cccDNA appeared to survive hepatocyte mitosis. • Low level virus DNA synthesis may contribute to survival of cccDNA through mitosis.

  9. Sirtuin 3 acts as a negative regulator of autophagy dictating hepatocyte susceptibility to lipotoxicity.

    Science.gov (United States)

    Li, Songtao; Dou, Xiaobing; Ning, Hua; Song, Qing; Wei, Wei; Zhang, Ximei; Shen, Chen; Li, Jiaxin; Sun, Changhao; Song, Zhenyuan

    2017-09-01

    Lipotoxicity induced by saturated fatty acids (SFAs) plays a central role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); however, the exact mechanisms remain to be fully elucidated. Sirtuin 3 (SIRT3) is a nicotinamide adenine dinucleotide-dependent deacetylase located primarily inside mitochondria. In this study, we demonstrated that an SFA-rich high-fat diet (HFD) was more detrimental to the liver than an isocaloric unsaturated HFD rich in fatty acids. Unexpectedly, SIRT3 expression and activity were significantly elevated in the livers of mice exposed to the SFA-rich HFD. Using cultured HepG2 and AML-12 hepatocytes, we demonstrated that unlike monounsaturated fatty acids, SFAs up-regulate SIRT3 expression and activity. SIRT3 overexpression renders both the liver and hepatocytes susceptible to palmitate-induced cell death, which can be alleviated by SIRT3 small interfering RNA (siRNA) transfection. In contrast, SIRT3 suppression protects hepatocytes from palmitate cytotoxicity. Further studies revealed that SIRT3 acts as a negative regulator of autophagy, thereby enhancing the susceptibility of hepatocytes to SFA-induced cytotoxicity. Mechanistic investigations revealed that SIRT3 overexpression causes manganese superoxide dismutase deacetylation and activation, which depleted intracellular superoxide contents, leading to adenosine monophosphate-activated protein kinase (AMPK) inhibition and mammalian target of rapamycin C1 activation, resulting in autophagy suppression. In contrast, SIRT3 siRNA gene silencing enhanced autophagy flux. A similar result was observed in the liver tissue of SIRT3 knockout mice. Our data indicate that SIRT3 is a negative regulator of autophagy whose activation by SFAs contributes to lipotoxicity in hepatocytes and suggest that restraining SIRT3 overactivation can be a potential therapeutic choice for the treatment of NAFLD as well as other metabolic disorders, with lipotoxicity being the principal pathomechanism

  10. beta-Carotene exhibits antioxidant and anti-apoptotic properties to prevent ethanol-induced cytotoxicity in isolated rat hepatocytes.

    Science.gov (United States)

    Peng, Hsiang-Chi; Chen, Jiun-Rong; Chen, Ya-Ling; Yang, Suh-Ching; Yang, Sien-Sing

    2010-06-01

    The study was designed to evaluate the effects of 1 microM beta-carotene on antioxidant status in ethanol-treated rat hepatocytes and investigate possible anti-apoptotic mechanisms of beta-carotene in protecting ethanol-induced cytotoxicity. The isolated rat hepatocytes were incubated for 48 h in a medium with or without alcohol (100 mM) and mu-carotene (1 microM) using the following groups: the control (C), beta-carotene (CB), ethanol (E), and ethanol + beta-carotene (EB) groups. The cell viability, antioxidative status, cytochrome P450 2E1 (CYP2E1) and caspase expressions in hepatocytes were measured. The E group demonstrated lower cell viability, glutathione (GSH) levels, and lipid peroxide accumulation in rat hepatocytes; meanwhile, CYP2E1, caspase-3, and caspase-9 expressions increased. In contrast, cell viability, GSH levels, and glutathione reductase (GRD) activity significantly increased while lipid peroxides and expressions of CYP2E1, casapse-3, and caspase-9 decreased in the EB group. The results suggest that ethanol treatment decreases cell viability in rat hepatocytes via induced oxidative stress. 1 muM beta-carotene decreased oxidative stress and prevented ethanol-induced cell death by inhibiting caspase-9 and caspase-3 expression.

  11. Reduction of Oxidative Stress Attenuates Lipoapoptosis Exacerbated by Hypoxia in Human Hepatocytes

    Directory of Open Access Journals (Sweden)

    Sang Youn Hwang

    2015-02-01

    Full Text Available Chronic intermittent hypoxia, a characteristic of obstructive sleep apnea (OSA, is associated with the progression of simple hepatic steatosis to necroinflammatory hepatitis. We determined whether inhibition of a hypoxia-induced signaling pathway could attenuate hypoxia-exacerbated lipoapoptosis in human hepatocytes. The human hepatocellular carcinoma cell line (HepG2 was used in this study. Palmitic acid (PA-treated groups were used for two environmental conditions: Hypoxia (1% O2 and normoxia (20% O2. Following the treatment, the cell viability was determined by the 3,4-(5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium salt (MTS assay, and the mechanism of lipoapoptosis was evaluated by Western blotting. Hypoxia exacerbated the suppression of hepatocyte growth induced by palmitic acid via activation of mitochondrial apoptotic pathways as a result of endoplasmic reticulum (ER and oxidative stresses. Ammonium pyrrolidine dithiocarbamate, a scavenger of reactive oxygen species, attenuated the hypoxia-exacerbated lipoapoptosis in hepatocytes, whereas glycerol, which reduces ER stress, did not. This may have been because inhibition of oxidative stress decreases the expression of pro-apoptotic proteins, such as caspase 9 and cytochrome c. These results suggested that modulation of apoptotic signaling pathways activated by oxidative stress can aid in identifying novel therapeutic strategies for the treatment of nonalcoholic steatohepatitis (NASH with OSA. Further in vivo studies are necessary to understand the pathophysiologic mechanism of NASH with OSA and to prove the therapeutic effect of the modulation of the signaling pathways.

  12. Optimized Hepatocyte-Like Cells with Functional Drug Transporters Directly-Reprogrammed from Mouse Fibroblasts and their Potential in Drug Disposition and Toxicology

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    Zhi-Tao Wu

    2016-05-01

    Full Text Available Background/Aims: To develop a suitable hepatocyte-like cell model that could be a substitute for primary hepatocytes with essential transporter expression and functions. Induced hepatocyte-like (iHep cells directly reprogrammed from mice fibroblast cells were fully characterized. Methods: Naïve iHep cells were transfected with nuclear hepatocyte factor 4 alpha (Hnf4α and treated with selected small molecules. Sandwich cultured configuration was applied. The mRNA and protein expression of transporters were determined by Real Time PCR and confocal. The functional transporters were estimated by drug biliary excretion measurement. The inhibition of bile acid efflux transporters by cholestatic drugs were assessed. Results: The expression and function of p-glycoprotein (P-gp, bile salt efflux pump (Bsep, multidrug resistance-associated protein 2 (Mrp2, Na+-dependent taurocholate cotransporting polypeptide (Ntcp, and organic anion transporter polypedtides (Oatps in iHep cells were significantly improved after transfection of hepatocyte nuclear factor 4 alpha (Hnf4α and treatment with selected inducers. In vitro intrinsic biliary clearances (CLb,int of optimized iHep cells for rosuvastatin, methotrexate, d8-TCA (deuterium-labeled sodium taurocholate acid and DPDPE ([D-Pen2,5] enkephalin hydrate correlated well with that of sandwich-cultured primary mouse hepatocytes (SCMHs (r2 = 0.984. Cholestatic drugs were evaluated and the results were compared well with primary mice hepatocytes. Conclusion: The optimized iHep cells expressed functional drug transporters and were comparable to primary mice hepatocytes. This study suggested direct reprogramming could provide a potential alternative to primary hepatocytes for drug candidate hepatobiliary disposition and hepatotoxicity screening.

  13. Glu-Phe from onion (Allium Cepa L.) attenuates lipogenesis in hepatocytes.

    Science.gov (United States)

    Lee, Yu Geon; Cho, Jeong-Yong; Hwang, Eom Ji; Jeon, Tae-Il; Moon, Jae-Hak

    2017-07-01

    A Glu-Phe (EF) was isolated from onion (Allium cepa L. cv. Sunpower). The chemical structure of EF was determined by nuclear magnetic resonance and electrospray ionization-mass (ESI-MS) spectroscopy. We showed that EF reduced lipid accumulation in mouse hepatocytes by inhibiting the expression of sterol regulatory element-binding protein-1c (SREBP-1c) and its lipogenic target genes. We also found that AMP-activated protein kinase (AMPK) was required for the inhibitory effect of EF on lipid accumulation in mouse hepatocytes. Furthermore, EF was qualified in nine onion cultivars by selective multiple reaction-monitoring detection of liquid chromatography-ESI-MS. These results suggest that EF could contribute to the beneficial effect of onion supplement in maintaining hepatic lipid homeostasis.

  14. Heme oxygenase-1 prevents non-alcoholic steatohepatitis through suppressing hepatocyte apoptosis in mice

    Directory of Open Access Journals (Sweden)

    Fu Na

    2010-10-01

    Full Text Available Abstract Objective Heme oxygenase-1 (HO-1, the rate-limiting enzyme in heme catabolism, has been reported to have potential antioxidant properties. However, the role of HO-1 on hepatocyte apoptosis remains unclear. We aim to elucidate the effects of HO-1 on oxidative stress related hepatocellular apoptosis in nutritional steatohepatitis in mice. Methods C57BL/6J mice were fed with methionine-choline deficient (MCD diet for four weeks to induce hepatic steatohepatitis. HO-1 chemical inducer (hemin, HO-1 chemical inhibitor zinc protoporphyrin IX (ZnPP-IX and/or adenovirus carrying HO-1 gene (Ad-HO-1 were administered to mice, respectively. Hepatocyte apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL assay, the mRNA and protein expression of apoptosis related genes were assayed by quantitative real-time PCR and Western blot. Results Hepatocyte signs of oxidative related apoptotic injury were presented in mice fed with MCD diet for 4 weeks. Induction of HO-1 by hemin or Ad-HO-1 significantly attenuated the severity of liver histology, which was associated with decreased hepatic lipid peroxidation content, reduced number of apoptotic cells by TUNEL staining, down-regulated expression of pro-apoptosis related genes including Fas/FasL, Bax, caspase-3 and caspase-9, reduced expression of cytochrome p4502E1 (CYP2E1, inhibited cytochrome c (Cyt-c release, and up-regulated expression of anti-apoptosis gene Bcl-2. Whereas, inhibition of HO-1 by ZnPP-IX caused oxidative stress related hepatic injury, which concomitant with increased number of TUNEL positive cells and up-regulated expression of pro-apoptosis related genes. Conclusions The present study provided evidences for the protective role of HO-1 in preventing nutritional steatohepatitis through suppressing hepatocyte apoptosis in mice.

  15. Engraftment and Repopulation Potential of Late Gestation Fetal Rat Hepatocytes.

    Science.gov (United States)

    Boylan, Joan M; Francois-Vaughan, Heather; Gruppuso, Philip A; Sanders, Jennifer A

    2017-10-01

    The limited availability of donor organs has led to a search for alternatives to liver transplantation to restore liver function and bridge patients to transplantation. We have shown that the proliferation of late gestation (embryonic day 19) fetal rat hepatocytes is mitogen-independent and that mechanisms regulating mRNA translation, cell cycle progression, and gene expression differ from those of adult rat hepatocytes. In the present study, we investigated whether E19 fetal hepatocytes can engraft and repopulate an injured adult liver. Fetal hepatocytes were isolated using a monoclonal antibody against a hepatic surface protein, leucine amino peptidase (LAP). LAP+ and LAP- fractions were analyzed by immunofluorescence and microarray. Immunopurified E19 liver cells from DPPIV+ rats were transplanted via splenic injection into partial hepatectomized DPPIV- rats that had been pretreated with mitomycin C. More than a third of LAP+ fetal hepatocytes expressed ductal markers. Transcriptomic analysis revealed that these dual-expressing cells represent a population of less well-differentiated hepatocytes. Upon transplantation, LAP+ late gestation fetal hepatocytes formed hepatic, endothelial, and ductal colonies within 1 month. By 10 months, colonies derived from LAP+ cells increased so that up to 35% of the liver was repopulated by donor-derived cells. Late gestation fetal hepatocytes, despite being far along in the differentiation process, possess the capacity for extensive liver repopulation. This is likely related to the unexpected presence of a significant proportion of hepatocyte marker-positive cells maintaining a less well-differentiated phenotype.

  16. Hepatocyte growth factor promotes proliferation, invasion, and metastasis of myeloid leukemia cells through PI3K-AKT and MAPK/ERK signaling pathway.

    Science.gov (United States)

    Guo, Jiang-Rui; Li, Wei; Wu, Yong; Wu, Lin-Qing; Li, Xin; Guo, Ya-Fei; Zheng, Xiao-Hui; Lian, Xiao-Lan; Huang, Hui-Fang; Chen, Yuan-Zhong

    2016-01-01

    This study aims to investigate effects of HGF expression on biological behaviors of Kasumi-1 and HL60. Expression of HGF and c-Met gene were detected using qRT-PCR. Short hairpin RNA (shRNA) was used to reduce HGF expression. Silencing effect of shRNA was verified by qRT-PCR and western blot. Cell reproductive capacity, cell clonality and cell cycle (apoptosis) were detected by CCK-8, clone formation, flow cytometry (FCM), respectively. Cell adhesion, cell invasion ability and cell proliferation were also examined. Changes of PI3K-AKT, MAPK/ERK signaling factors were detected by western blot. HGF and c-Met expression in first-vist AML group was significantly higher than in AML-relief and normal control group. HGF shRNA can inhibit cell proliferation, inhibit cloning ability. Compared with control group, apoptosis ratios of Kasumi-1 and HL60 cell in interference groups were significantly higher. After shRNA interference, the number of adherent cells and transmembrane cells were significantly decreased compared with control group. Meanwhile, shRNA also down-regulated Bad, Bcl-XL, Bcl-2, CDK1, Cyclin B, MMP2, MMP9, and up-regulated cleaved caspase9, cleaved caspase3, cleaved PARP, Bax, and P21. Moreover, phosphorylated c-Met, AKT, Erk, and mTOR were also reduced. In conclusion, HGF and c-Met gene highly expressed among first-visit AML patients, but decreased after relief treatment. HGF may promote proliferation, invasion, and metastasis of AML cells through PI3K-AKT and MAPK/ERK signaling pathway. Therefore, proliferation and invasion ability of AML cell can be inhibited by down-regulating HGF gene to retardate cell in G2/M stage.

  17. Functional activity of isolated pig hepatocytes attached to different extracellular matrix substrates. Implication for application of pig hepatocytes in a bioartificial liver

    NARCIS (Netherlands)

    te Velde, A. A.; Ladiges, N. C.; Flendrig, L. M.; Chamuleau, R. A.

    1995-01-01

    For the manufacture of a bioartificial liver for human application, large amounts of viable and active hepatocytes are needed. Pig hepatocytes are considered to be the best alternative to scarce human hepatocytes. In vitro hepatocyte functions have so far been tested under different circumstances,

  18. Deficiency of G1 regulators P53, P21Cip1 and/or pRb decreases hepatocyte sensitivity to TGFβ cell cycle arrest

    Directory of Open Access Journals (Sweden)

    Harrison David J

    2007-11-01

    Full Text Available Abstract Background TGFβ is critical to control hepatocyte proliferation by inducing G1-growth arrest through multiple pathways leading to inhibition of E2F transcription activity. The retinoblastoma protein pRb is a key controller of E2F activity and G1/S transition which can be inhibited in viral hepatitis. It is not known whether the impairment of pRb would alter the growth inhibitory potential of TGFβ in disease. We asked how Rb-deficiency would affect responses to TGFβ-induced cell cycle arrest. Results Primary hepatocytes isolated from Rb-floxed mice were infected with an adenovirus expressing CRE-recombinase to delete the Rb gene. In control cells treatment with TGFβ prevented cells to enter S phase via decreased cMYC activity, activation of P16INK4A and P21Cip and reduction of E2F activity. In Rb-null hepatocytes, cMYC activity decreased slightly but P16INK4A was not activated and the great majority of cells continued cycling. Rb is therefore central to TGFβ-induced cell cycle arrest in hepatocytes. However some Rb-null hepatocytes remained sensitive to TGFβ-induced cell cycle arrest. As these hepatocytes expressed very high levels of P21Cip1 and P53 we investigated whether these proteins regulate pRb-independent signaling to cell cycle arrest by evaluating the consequences of disruption of p53 and p21Cip1. Hepatocytes deficient in p53 or p21Cip1 showed diminished growth inhibition by TGFβ. Double deficiency had a similar impact showing that in cells containing functional pRb; P21Cip and P53 work through the same pathway to regulate G1/S in response to TGFβ. In Rb-deficient cells however, p53 but not p21Cip deficiency had an additive effect highlighting a pRb-independent-P53-dependent effector pathway of inhibition of E2F activity. Conclusion The present results show that otherwise genetically normal hepatocytes with disabled p53, p21Cip1 or Rb genes respond less well to the antiproliferative effects of TGFβ. As the function of

  19. Inhibitory effect of TNF-α on malaria pre-erythrocytic stage development: influence of host hepatocyte/parasite combinations.

    Directory of Open Access Journals (Sweden)

    Nadya Depinay

    Full Text Available BACKGROUND: The liver stages of malaria parasites are inhibited by cytokines such as interferon-γ or Interleukin (IL-6. Binding of these cytokines to their receptors at the surface of the infected hepatocytes leads to the production of nitric oxide (NO and radical oxygen intermediates (ROI, which kill hepatic parasites. However, conflicting results were obtained with TNF-α possibly because of differences in the models used. We have reassessed the role of TNF-α in the different cellular systems used to study the Plasmodium pre-erythrocytic stages. METHODS AND FINDINGS: Human or mouse TNF-α were tested against human and rodent malaria parasites grown in vitro in human or rodent primary hepatocytes, or in hepatoma cell lines. Our data demonstrated that TNF-α treatment prevents the development of malaria pre-erythrocytic stages. This inhibitory effect however varies with the infecting parasite species and with the nature and origin of the cytokine and hepatocytes. Inhibition was only observed for all parasite species tested when hepatocytes were pre-incubated 24 or 48 hrs before infection and activity was directed only against early hepatic parasite. We further showed that TNF-α inhibition was mediated by a soluble factor present in the supernatant of TNF-α stimulated hepatocytes but it was not related to NO or ROI. Treatment TNF-α prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified. CONCLUSIONS: Treatment TNF-α prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified. However, the nature of the cytokine-host cell-parasite combination must be carefully considered for extrapolation to the human infection.

  20. Factor VIIa binding and internalization in hepatocytes

    DEFF Research Database (Denmark)

    Hjortoe, G; Sorensen, B B; Petersen, L C

    2005-01-01

    The liver is believed to be the primary clearance organ for coagulation proteases, including factor VIIa (FVIIa). However, at present, clearance mechanisms for FVIIa in liver are unknown. To obtain information on the FVIIa clearance mechanism, we investigated the binding and internalization...... of FVIIa in liver cells using a human hepatoma cell line (HEPG2), and primary rat and human hepatocytes as cell models. 125I-FVIIa bound to HEPG2 cells in a time- and dose-dependent manner. Anti-tissue factor antibodies reduced the binding by about 25%, whereas 50-fold molar excess of unlabeled FVIIa had...

  1. Evaluation of D-18F-FMT, 18F-FDG, L-11C-MET, and 18F-FLT for monitoring the response of tumors to radiotherapy in mice.

    Science.gov (United States)

    Murayama, Chieko; Harada, Norihiro; Kakiuchi, Takeharu; Fukumoto, Dai; Kamijo, Akemi; Kawaguchi, Akira T; Tsukada, Hideo

    2009-02-01

    O-18F-fluoromethyl-D-tyrosine (D-18F-FMT) is a promising novel agent for tumor imaging by PET. The aim of this study was to evaluate the potential of D-18F-FMT and the other conventional ligands used for tumor imaging, namely, 18F-FDG, L-11C-methionine (L-11C-MET), and 3'-deoxy-3'-18F-fluorothymidine (18F-FLT), as a PET ligand for monitoring early responses to radiotherapy in tumor-bearing mice. C3H/HeN mice inoculated with murine squamous cell carcinomas were treated with a single dose of x-ray irradiation at 2, 6, 20, or 60 Gy. Tumor uptake of each ligand was examined 1, 3, and 7 d after the irradiation. Tumor uptake of D-18F-FMT was decreased on day 1 after irradiation at 6, 20, or 60 Gy, and the decrease persisted until day 7. Tumor uptake of 18F-FDG was elevated on days 1 and 3 after irradiation at 2, 6, or 20 Gy, followed by a decrease in uptake on day 7 in mice irradiated at 20 or 60 Gy. Decreased tumor uptake of L-11C-MET was observed only on day 3 after the irradiation. Decreased tumor uptake of 18F-FLT was detected on day 1 after irradiation at 2, 6, 20, or 60 Gy; thereafter, the dose-dependent decrease in uptake was no longer seen. Only for D-18F-FMT were significant positive correlations found between ligand uptake at all the time points examined and tumor volume on day 14 after various doses of irradiation. The findings suggest that D-18F-FMT is a promising PET ligand for early-phase detection and prediction of the effects of radiation therapy.

  2. Hepatocyte nuclear factor 4A improves hepatic differentiation of immortalized adult human hepatocytes and improves liver function and survival.

    Science.gov (United States)

    Hang, Hua-Lian; Liu, Xin-Yu; Wang, Hai-Tian; Xu, Ning; Bian, Jian-Min; Zhang, Jian-Jun; Xia, Lei; Xia, Qiang

    2017-11-15

    Immortalized human hepatocytes (IHH) could provide an unlimited supply of hepatocytes, but insufficient differentiation and phenotypic instability restrict their clinical application. This study aimed to determine the role of hepatocyte nuclear factor 4A (HNF4A) in hepatic differentiation of IHH, and whether encapsulation of IHH overexpressing HNF4A could improve liver function and survival in rats with acute liver failure (ALF). Primary human hepatocytes were transduced with lentivirus-mediated catalytic subunit of human telomerase reverse transcriptase (hTERT) to establish IHH. Cells were analyzed for telomerase activity, proliferative capacity, hepatocyte markers, and tumorigenicity (c-myc) expression. Hepatocyte markers, hepatocellular functions, and morphology were studied in the HNF4A-overexpressing IHH. Hepatocyte markers and karyotype analysis were completed in the primary hepatocytes using shRNA knockdown of HNF4A. Nuclear translocation of β-catenin was assessed. Rat models of ALF were treated with encapsulated IHH or HNF4A-overexpressing IHH. A HNF4A-positive IHH line was established, which was non-tumorigenic and conserved properties of primary hepatocytes. HNF4A overexpression significantly enhanced mRNA levels of genes related to hepatic differentiation in IHH. Urea levels were increased by the overexpression of HNF4A, as measured 24h after ammonium chloride addition, similar to that of primary hepatocytes. Chromosomal abnormalities were observed in primary hepatocytes transfected with HNF4A shRNA. HNF4α overexpression could significantly promote β-catenin activation. Transplantation of HNF4A overexpressing IHH resulted in better liver function and survival of rats with ALF compared with IHH. HNF4A improved hepatic differentiation of IHH. Transplantation of HNF4A-overexpressing IHH could improve the liver function and survival in a rat model of ALF. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Liver regeneration studies with rat hepatocytes in primary culture.

    Science.gov (United States)

    Michalopoulos, G; Cianciulli, H D; Novotny, A R; Kligerman, A D; Strom, S C; Jirtle, R L

    1982-11-01

    Adult rat parenchymal hepatocytes in primary culture can be induced to enter into DNA synthesis and mitosis. The optimal conditions for hepatocyte replication are low plating density (less than 10,000 cells/sq cm) and 50% serum from two-thirds partially hepatectomized rats (48 hr after hepatectomy). Approximately 80% of the hepatocytes enter the cell cycle, and most of these cells go through mitosis. The replicating hepatocytes remain positive for glucose-6-phosphatase and negative for gamma-glutamyl transpeptidase, and they accumulate fat, in analogy to regenerating liver. Most of the replicating hepatocytes enter into multiple consecutive rounds of DNA synthesis. Dose-response studies between control animal serum and hepatocyte labeling index indicate that in unoperated animals the serum contains substances stimulatory as well as inhibitory for hepatic growth, with the inhibitory effect prevailing at high concentrations. After partial hepatectomy, the inhibitory activity disappears whereas the hepatopoietin activity reaches almost 90% of maximal biological effectiveness at 25% serum concentration. Addition of hormones to the system shows that the hepatopoietin activity is not identical to epidermal growth factor, platelet-derived growth factor, thyroxine, glucagon, or hydrocortisone. Norepinephrine abolishes the difference between control and hepatectomized serum but does not restore hepatopoietin activity when added to heat-inactivated serum. The results show that this system of replicating hepatocytes can be used to investigate the trophic factors that control growth of normal and neoplastic hepatocytes.

  4. Cell swelling and glycogen metabolism in hepatocytes from fasted rats

    NARCIS (Netherlands)

    Gustafson, L. A.; Jumelle-Laclau, M. N.; van Woerkom, G. M.; van Kuilenburg, A. B.; Meijer, A. J.

    1997-01-01

    Cell swelling is known to increase net glycogen production from glucose in hepatocytes from fasted rats by activating glycogen synthase. Since both active glycogen synthase and phosphorylase are present in hepatocytes, suppression of flux through phosphorylase may also contribute to the net increase

  5. Hepatic Stellate Cell-Derived Microvesicles Prevent Hepatocytes from Injury Induced by APAP/H2O2

    Directory of Open Access Journals (Sweden)

    Renwei Huang

    2016-01-01

    Full Text Available Hepatic stellate cells (HSCs, previously described for liver-specific mesenchymal stem cells (MSCs, appear to contribute to liver regeneration. Microvesicles (MVs are nanoscale membrane fragments, which can regulate target cell function by transferring contents from their parent cells. The aim of this study was to investigate the effect of HSC-derived MVs on xenobiotic-induced liver injury. Rat and human hepatocytes, BRL-3A and HL-7702, were used to build hepatocytes injury models by n-acetyl-p-aminophenol n-(APAP or H2O2 treatment. MVs were prepared from human and rat HSCs, LX-2, and HST-T6 and, respectively, added to injured BRL-3A and HL-7702 hepatocytes. MTT assay was utilized to determine cell proliferation. Cell apoptosis was analyzed by flow cytometry and hoechst33258 staining. Western blot was used for analyzing the expression of activated caspase-3. Liver injury indicators, alanine aminotransferase (ALT, aspartate aminotransferase (AST, and lactate dehydrogenase (LDH in culture medium were also assessed. Results showed that (1 HSC-MVs derived from LX-2 and HST-T6 were positive to CD90 and annexin V surface markers; (2 HSC-MVs dose-dependently improved the viability of hepatocytes in both injury models; (3 HSC-MVs dose-dependently inhibited the APAP/H2O2 induced hepatocytes apoptosis and activated caspase-3 expression and leakage of LDH, ALT, and AST. Our results demonstrate that HSC-derived MVs protect hepatocytes from toxicant-induced injury.

  6. Hepatitis B virus X protein activates the p38 mitogen-activated protein kinase pathway in dedifferentiated hepatocytes.

    Science.gov (United States)

    Tarn, Chi; Zou, Lin; Hullinger, Ronald L; Andrisani, Ourania M

    2002-10-01

    Hepatitis B virus X protein (pX) is implicated in hepatocarcinogenesis by an unknown mechanism. Employing a cellular model linked to pX-mediated transformation, we investigated the role of the previously reported Stat3 activation by pX in hepatocyte transformation. Our model is composed of a differentiated hepatocyte (AML12) 3pX-1 cell line that undergoes pX-dependent transformation and a dedifferentiated hepatocyte (AML12) 4pX-1 cell line that does not exhibit transformation by pX. We report that pX-dependent Stat3 activation occurs only in non-pX-transforming 4pX-1 cells and conclude that Stat3 activation is not linked to pX-mediated transformation. Maximum Stat3 transactivation requires Ser727 phosphorylation, mediated by mitogenic pathway activation. Employing dominant negative mutants and inhibitors of mitogenic pathways, we demonstrate that maximum, pX-dependent Stat3 transactivation is inhibited by the p38 mitogen-activated protein kinase (MAPK)-specific inhibitor SB 203580. Using transient-transreporter and in vitro kinase assays, we demonstrate for the first time that pX activates the p38 MAPK pathway only in 4pX-1 cells. pX-mediated Stat3 and p38 MAPK activation is Ca(2+) and c-Src dependent, in agreement with the established cellular action of pX. Importantly, pX-dependent activation of p38 MAPK inactivates Cdc25C by phosphorylation of Ser216, thus initiating activation of the G(2)/M checkpoint, resulting in 4pX-1 cell growth retardation. Interestingly, pX expression in the less differentiated hepatocyte 4pX-1 cells activates signaling pathways known to be active in regenerating hepatocytes. These results suggest that pX expression in the infected liver effects distinct mitogenic pathway activation in less differentiated versus differentiated hepatocytes.

  7. Novel Regulation of Ski Protein Stability and Endosomal Sorting by Actin Cytoskeleton Dynamics in Hepatocytes*

    Science.gov (United States)

    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R.; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A.; Macías-Silva, Marina

    2015-01-01

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration. PMID:25561741

  8. Novel regulation of Ski protein stability and endosomal sorting by actin cytoskeleton dynamics in hepatocytes.

    Science.gov (United States)

    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A; Macías-Silva, Marina

    2015-02-13

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Property of hepatitis B virus replication in Tupaia belangeri hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Sanada, Takahiro [Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506 (Japan); Tsukiyama-Kohara, Kyoko, E-mail: kkohara@vet.kagoshima-u.ac.jp [Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24, Korimoto, Kagoshima-city, Kagoshima 890-0065 (Japan); Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24, Korimoto, Kagoshima, Kagoshima 890-0065 (Japan); Yamamoto, Naoki [Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506 (Japan); Ezzikouri, Sayeh; Benjelloun, Soumaya [Viral Hepatitis Laboratory, Virology Unit, Institut Pasteur du Maroc, 1, Louis Pasteur, Casablanca 20360 (Morocco); Murakami, Shuko; Tanaka, Yasuhito [Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Kawasumi 1, Mizuho-ku, Nagoya, Aichi 467-8601 (Japan); Tateno, Chise [PhoenixBio Co. Ltd., 3-4-1, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046 (Japan); Kohara, Michinori, E-mail: kohara-mc@igakuken.or.jp [Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506 (Japan)

    2016-01-08

    The northern treeshrew (Tupaia belangeri) has been reported to be an effective candidate for animal infection model with hepatitis B virus (HBV). The objective of our study was to analyze the growth characteristics of HBV in tupaia hepatocytes and the host response to HBV infection. We established primary tupaia hepatocytes (3–6-week old tupaia) and infected them with HBV genotypes A, B and C, and all the genotypes proliferated as well as those in human primary hepatocytes (>10{sup 5} copies/ml in culture supernatant). We next generated a chimeric mouse with tupaia liver by transplantation of tupaia primary hepatocytes to urokinase-type plasminogen activator cDNA (cDNA-uPA)/severe combined immunodeficient (SCID) mice and the replacement ratio with tupaia hepatocytes was found to be more than 95%. Infection of chimeric mice with HBV (genotypes B, C, and D) resulted in HBV-DNA level of 10{sup 4}-10{sup 6} copies/ml after 8 weeks of infection, which were almost similar to that in humanized chimeric mouse. In contrast, serum HBV level in adult tupaia (1-year-old tupaia) was quite low (<10{sup 3} copies/ml). Understanding the differences in the response to HBV infection in primary tupaia hepatocytes, chimeric mouse, and adult tupaia will contribute to elucidating the mechanism of persistent HBV infection and viral eradication. Thus, T. belangeri was found to be efficient for studying the host response to HBV infection, thereby providing novel insight into the pathogenesis of HBV. - Highlights: • Primary hepatocytes were established from tupaia that is a novel HBV infection model. • Tupaia primary hepatocytes were susceptible for HBV infection. • The immunodeficient chimeric mice with tupaia hepatocytes were established. • The chimeric mice with tupaia hepatocytes were susceptible for HBV infection.

  10. Nicotinamide protects hepatocytes against palmitate-induced lipotoxicity via SIRT1-dependent autophagy induction.

    Science.gov (United States)

    Shen, Chen; Dou, Xiaobing; Ma, Yue; Ma, Wang; Li, Songtao; Song, Zhenyuan

    2017-04-01

    Lipotoxicity induced by saturated fatty acids (SFAs) plays a pathological role in the development of non-alcoholic fatty liver disease (NAFLD); however, the exact mechanism remains to be clearly elucidated. Palmitate is the most abundant SFA in the circulation and major lipotoxic inducer. Accumulating evidence supports that autophagy induction is protective against palmitate-induced cell death in a variety of cell types, including hepatocytes. Nicotinamide is the amide form of nicotinic acid (vitamin B3, Niacin) and a dietary supplementation as a source of vitamin B3. We previously reported that nicotinamide endowed hepatocytes resistance to palmitate-induced ER stress via up-regulating SIRT1, with cAMP/PKA/CREB pathway activation being a fundamental mechanism. This study was undertaken to investigate the potential anti-lipotoxic effect of nicotinamide and to elucidate underlying mechanism(s). Our data demonstrated that nicotinamide supplementation protected hepatocytes against palmitate-induced cell death. Mechanistic investigations revealed that nicotinamide supplementation activated autophagy in hepatocytes. Importantly, we showed that the anti-lipotoxic property of nicotinamide was abolished by autophagy inhibitors, suggesting that autophagy induction plays a mechanistic role in nicotinamide's anti-lipotoxic effect. Furthermore, we showed that SIRT1 inhibition blunted autophagy induction in response to nicotinamide supplementation and similarly abrogated the anti-lipotoxic effect conferred by nicotinamide supplementation. In conclusion, our data suggest that nicotinamide protects against palmitate-induced hepatotoxicity via SIRT1-dependent autophagy induction and that nicotinamide supplementation may represent a therapeutic choice for NAFLD. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Cytotoxic effects of hydroxylated fullerenes on isolated rat hepatocytes via mitochondrial dysfunction.

    Science.gov (United States)

    Nakagawa, Yoshio; Suzuki, Toshinari; Ishii, Hidemi; Nakae, Dai; Ogata, Akio

    2011-11-01

    The cytotoxic effects of hydroxylated fullerenes, also termed fullerenols or fullerols [C(60)(OH)( n )], which are known nanomaterials and water-soluble fullerene derivatives, were studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to C(60)(OH)(24) caused not only concentration (0-0.25 mM)- and time (0-3 h)-dependent cell death accompanied by the formation of cell blebs, loss of cellular ATP, reduced glutathione (GSH), and protein thiol levels, but also the accumulation of glutathione disulfide and malondialdehyde, indicating lipid peroxidation. Of the other analogues examined, the cytotoxic effects of C(60)(OH)(12) and fullerene C(60) at a concentration of 0.125 mM were less than those of C(60)(OH)(24). The loss of mitochondrial membrane potential and generation of oxygen radical species in hepatocytes incubated with C(60)(OH)(24) were greater than those with C(60)(OH)(12) and fullerene C(60). In the oxygen consumption of mitochondria isolated from rat liver, the ratios of state-3/state-4 respiration were more markedly decreased by C(60)(OH)(24) and C(60)(OH)(12) compared with C(60). In addition, C(60)(OH)(24) and C(60)(OH)(12) resulted in the induction of the mitochondrial permeability transition (MPT), and the effects of C(60)(OH)(12) were less than those of C(60)(OH)(24). Taken collectively, these results indicate that (a) mitochondria are target organelles for fullerenols, which elicit cytotoxicity through mitochondrial failure related to the induction of the MPT, mitochondrial depolarization, and inhibition of ATP synthesis in the early stage and subsequently oxidation of GSH and protein thiols, and lipid peroxidation through oxidative stress at a later stage; and (b) the toxic effects of fullerenols may depend on the number of hydroxyl groups participating in fullerene in rat hepatocytes.

  12. Hepatitis B Virus Activates Signal Transducer and Activator of Transcription 3 Supporting Hepatocyte Survival and Virus ReplicationSummary

    Directory of Open Access Journals (Sweden)

    Marianna Hösel

    2017-11-01

    Full Text Available Background & Aims: The human hepatitis B virus (HBV is a major cause of chronic hepatitis and hepatocellular carcinoma, but molecular mechanisms driving liver disease and carcinogenesis are largely unknown. We therefore studied cellular pathways altered by HBV infection. Methods: We performed gene expression profiling of primary human hepatocytes infected with HBV and proved the results in HBV-replicating cell lines and human liver tissue using real-time polymerase chain reaction and Western blotting. Activation of signal transducer and activator of transcription (STAT3 was examined in HBV-replicating human hepatocytes, HBV-replicating mice, and liver tissue from HBV-infected individuals using Western blotting, STAT3-luciferase reporter assay, and immunohistochemistry. The consequences of STAT3 activation on HBV infection and cell survival were studied by chemical inhibition of STAT3 phosphorylation and small interfering RNA–mediated knockdown of STAT3. Results: Gene expression profiling of HBV-infected primary human hepatocytes detected no interferon response, while genes encoding for acute phase and antiapoptotic proteins were up-regulated. This gene regulation was confirmed in liver tissue samples of patients with chronic HBV infection and in HBV-related hepatocellular carcinoma. Pathway analysis revealed activation of STAT3 to be the major regulator. Interleukin-6–dependent and –independent activation of STAT3 was detected in HBV-replicating hepatocytes in cell culture and in vivo. Prevention of STAT3 activation by inhibition of Janus tyrosine kinases as well as small interfering RNA–mediated knockdown of STAT3-induced apoptosis and reduced HBV replication and gene expression. Conclusions: HBV activates STAT3 signaling in hepatocytes to foster its own replication but also to prevent apoptosis of infected cells. This very likely supports HBV-related carcinogenesis. Keywords: Hepatitis B Virus Infection, STAT3 Signaling

  13. Sphingosine Kinase 1 Protects Hepatocytes from Lipotoxicity via Down-regulation of IRE1α Protein Expression.

    Science.gov (United States)

    Qi, Yanfei; Wang, Wei; Chen, Jinbiao; Dai, Lan; Kaczorowski, Dominik; Gao, Xin; Xia, Pu

    2015-09-18

    Aberrant deposition of fat including free fatty acids in the liver often causes damage to hepatocytes, namely lipotoxicity, which is a key pathogenic event in the development and progression of fatty liver diseases. This study demonstrates a pivotal role of sphingosine kinase 1 (SphK1) in protecting hepatocytes from lipotoxicity. Exposure of primary murine hepatocytes to palmitate resulted in dose-dependent cell death, which was enhanced significantly in Sphk1-deficient cells. In keeping with this, expression of dominant-negative mutant SphK1 also markedly promoted palmitate-induced cell death. In contrast, overexpression of wild-type SphK1 profoundly protected hepatocytes from lipotoxicity. Mechanistically, the protective effect of SphK1 is attributable to suppression of ER stress-mediated pro-apoptotic pathways, as reflected in the inhibition of IRE1α activation, XBP1 splicing, JNK phosphorylation, and CHOP induction. Of note, SphK1 inhibited the IRE1α pathway by reducing IRE1α expression at the transcriptional level. Moreover, S1P mimicked the effect of SphK1, suppressing IRE1α expression in a receptor-dependent manner. Furthermore, enforced overexpression of IRE1α significantly blocked the protective effect of SphK1 against lipotoxicity. Therefore, this study provides new insights into the role of SphK1 in hepatocyte survival and uncovers a novel mechanism for protection against ER stress-mediated cell death. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Role of hepatocyte S6K1 in palmitic acid-induced endoplasmic reticulum stress, lipotoxicity, insulin resistance and in oleic acid-induced protection.

    Science.gov (United States)

    Pardo, Virginia; González-Rodríguez, Águeda; Muntané, Jordi; Kozma, Sara C; Valverde, Ángela M

    2015-06-01

    The excess of saturated free fatty acids, such as palmitic acid, that induces lipotoxicity in hepatocytes, has been implicated in the development of non-alcoholic fatty liver disease also associated with insulin resistance. By contrast, oleic acid, a monounsaturated fatty acid, attenuates the effects of palmitic acid. We evaluated whether palmitic acid is directly associated with both insulin resistance and lipoapoptosis in mouse and human hepatocytes and the impact of oleic acid in the molecular mechanisms that mediate both processes. In human and mouse hepatocytes palmitic acid at a lipotoxic concentration triggered early activation of endoplasmic reticulum (ER) stress-related kinases, induced the apoptotic transcription factor CHOP, activated caspase 3 and increased the percentage of apoptotic cells. These effects concurred with decreased IR/IRS1/Akt insulin pathway. Oleic acid suppressed the toxic effects of palmitic acid on ER stress activation, lipoapoptosis and insulin resistance. Besides, oleic acid suppressed palmitic acid-induced activation of S6K1. This protection was mimicked by pharmacological or genetic inhibition of S6K1 in hepatocytes. In conclusion, this is the first study highlighting the activation of S6K1 by palmitic acid as a common and novel mechanism by which its inhibition by oleic acid prevents ER stress, lipoapoptosis and insulin resistance in hepatocytes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Phthalazinone Pyrazole Enhances the Hepatic Functions of Human Embryonic Stem Cell-Derived Hepatocyte-Like Cells via Suppression of the Epithelial-Mesenchymal Transition.

    Science.gov (United States)

    Choi, Young-Jun; Kim, Hyemin; Kim, Ji-Woo; Song, Chang-Woo; Kim, Dae-Sung; Yoon, Seokjoo; Park, Han-Jin

    2017-12-13

    During liver development, nonpolarized hepatic progenitor cells differentiate into mature hepatocytes with distinct polarity. This polarity is essential for maintaining the intrinsic properties of hepatocytes. The balance between the epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) plays a decisive role in differentiation of polarized hepatocytes. In this study, we found that phthalazinone pyrazole (PP), a selective inhibitor of Aurora-A kinase (Aurora-A), suppressed the EMT during the differentiation of hepatocyte-like cells (HLCs) from human embryonic stem cells. The differentiated HLCs treated with PP at the hepatoblast stage showed enhanced hepatic morphology and functions, particularly with regard to the expression of drug metabolizing enzymes. Moreover, we found that these effects were mediated though suppression of the AKT pathway, which is involved in induction of the EMT, and upregulation of hepatocyte nuclear factor 4α expression rather than Aurora-A inhibition. In conclusion, these findings provided insights into the regulatory role of the EMT on in vitro hepatic maturation, suggesting that inhibition of the EMT may drive transformation of hepatoblast cells into mature and polarized HLCs.

  16. Pleiotropic activity of hepatocyte growth factor during embryonic mouse testis development.

    Science.gov (United States)

    Ricci, G; Catizone, A; Galdieri, M

    2002-10-01

    The hepatocyte growth factor (HGF) is a pleiotropic cytokine whose action is mediated by c-met, a glycoproteic receptor with tyrosine kinase activity which transduces its multiple biological activities including cell proliferation, motility and differentiation. During embryonic development HGF acts as a morphogenetic factor as previously demonstrated for metanephric and lung development. Recently, culturing male genital ridges, we demonstrated that HGF is able to support in vitro testicular cord formation. In the present paper we report the expression pattern of the HGF gene during embryonic testis development and the multiple roles exerted by this factor during the morphogenesis of this organ. Northern blot analysis reveals a positive signal in urogenital ridges isolated from 11.5 days post coitum (dpc) embryos and in testes isolated from 13.5 and 15.5 dpc male embryos. On the contrary HGF mRNA is undetectable in ovaries isolated from 13.5 and 15.5 dpc embryos. Moreover, we demonstrate that HGF is synthesized and secreted by the male gonad and is biologically active. These data indicate a male specific biological function of HGF during embryonic gonadal development. This hypothesis is supported by the in vitro demonstration that HGF acts as a migratory factor for male mesonephric cells which is a male specific event. In addition we demonstrate that during testicular development, HGF acts as a morphogenetic factor able to reorganize dissociated testicular cells which, under HGF stimulation, form a tridimensional network of cord-like structures. Finally, we demonstrate that HGF induces testicular cell proliferation in this way being responsible for the size increase of the testis. All together the data presented in this paper demonstrate that HGF is expressed during the embryonic development of the testis and clarify the multiple roles exerted by this factor during the morphogenesis of the male gonad.

  17. Toxicogenomics directory of chemically exposed human hepatocytes.

    Science.gov (United States)

    Grinberg, Marianna; Stöber, Regina M; Edlund, Karolina; Rempel, Eugen; Godoy, Patricio; Reif, Raymond; Widera, Agata; Madjar, Katrin; Schmidt-Heck, Wolfgang; Marchan, Rosemarie; Sachinidis, Agapios; Spitkovsky, Dimitry; Hescheler, Jürgen; Carmo, Helena; Arbo, Marcelo D; van de Water, Bob; Wink, Steven; Vinken, Mathieu; Rogiers, Vera; Escher, Sylvia; Hardy, Barry; Mitic, Dragana; Myatt, Glenn; Waldmann, Tanja; Mardinoglu, Adil; Damm, Georg; Seehofer, Daniel; Nüssler, Andreas; Weiss, Thomas S; Oberemm, Axel; Lampen, Alfons; Schaap, Mirjam M; Luijten, Mirjam; van Steeg, Harry; Thasler, Wolfgang E; Kleinjans, Jos C S; Stierum, Rob H; Leist, Marcel; Rahnenführer, Jörg; Hengstler, Jan G

    2014-12-01

    A long-term goal of numerous research projects is to identify biomarkers for in vitro systems predicting toxicity in vivo. Often, transcriptomics data are used to identify candidates for further evaluation. However, a systematic directory summarizing key features of chemically influenced genes in human hepatocytes is not yet available. To bridge this gap, we used the Open TG-GATES database with Affymetrix files of cultivated human hepatocytes incubated with chemicals, further sets of gene array data with hepatocytes from human donors generated in this study, and publicly available genome-wide datasets of human liver tissue from patients with non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular cancer (HCC). After a curation procedure, expression data of 143 chemicals were included into a comprehensive biostatistical analysis. The results are summarized in the publicly available toxicotranscriptomics directory ( http://wiki.toxbank.net/toxicogenomics-map/ ) which provides information for all genes whether they are up- or downregulated by chemicals and, if yes, by which compounds. The directory also informs about the following key features of chemically influenced genes: (1) Stereotypical stress response. When chemicals induce strong expression alterations, this usually includes a complex but highly reproducible pattern named 'stereotypical response.' On the other hand, more specific expression responses exist that are induced only by individual compounds or small numbers of compounds. The directory differentiates if the gene is part of the stereotypical stress response or if it represents a more specific reaction. (2) Liver disease-associated genes. Approximately 20 % of the genes influenced by chemicals are up- or downregulated, also in liver disease. Liver disease genes deregulated in cirrhosis, HCC, and NASH that overlap with genes of the aforementioned stereotypical chemical stress response include CYP3A7, normally expressed in fetal liver; the

  18. Isolation and Primary Culture of Rat Hepatocytes Using Kiwifruit Actinidin

    Directory of Open Access Journals (Sweden)

    Z. Shirvani Farsani

    2007-07-01

    Full Text Available Introduction & Objective: Isolation of cells from different tissues rely on proteolytic enzymes mainly collagenases that selectively digest collagen fibers of extra-cellular matrix. It is important to find new and proper collagenases from plant sources. In the present research actinidin, a cysteine protease abundant in Kiwifruit, was used to isolate and culture of rat hepatocytes. Materials & Methods: Different concentrations of actinidin was used to isolate rat hepatocytes by one or two-step perfusion method. The viability of the separated cells was examined by the trypan blue test. The isolated rat hepatocytes were cultured on collagen coated plates in William´s E medium. The morphology of hepatocytes was examined microscopically after staining with the Papanicolaou method.Results: Actinidin in the concentration of 0.4 mg/ml in two-step perfusion method properly isolated hepatocytes from rat liver. The viability of isolated hepatocytes that successfully cultured in collagen coated plates was 90-95 percent.Conclusion: These results showed that actinidin is a proper protease for isolation of hepatocytes. In addition, purification of this enzyme is simpler than the collagenases.

  19. Protective role of morin, a flavonoid, against high glucose induced oxidative stress mediated apoptosis in primary rat hepatocytes.

    Directory of Open Access Journals (Sweden)

    Radhika Kapoor

    Full Text Available Apoptosis is an early event of liver damage in diabetes and oxidative stress has been linked to accelerate the apoptosis in hepatocytes. Therefore, the compounds that can scavenge ROS may confer regulatory effects on high-glucose induced apoptosis. In the present study, primary rat hepatocytes were exposed to high concentration (40 mM of glucose. At this concentration decreased cell viability and enhanced ROS generation was observed. Depleted antioxidant status of hepatocytes under high glucose stress was also observed as evident from transcriptional level and activities of antioxidant enzymes. Further, mitochondrial depolarisation was accompanied by the loss of mitochondrial integrity and altered expression of Bax and Bcl-2. Increased translocation of apoptotic proteins like AIF (Apoptosis inducing factor & Endo-G (endonuclease-G from its resident place mitochondria to nucleus was also observed. Cyt-c residing in the inter-membrane space of mitochondria also translocated to cytoplasm. These apoptotic proteins initiated caspase activation, DNA fragmentation, chromatin condensation, increased apoptotic DNA content in glucose treated hepatocytes, suggesting mitochondria mediated apoptotic mode of cell death. Morin, a dietary flavonoid from Psidium guajava was effective in increasing the cell viability and decreasing the ROS level. It maintained mitochondrial integrity, inhibited release of apoptotic proteins from mitochondria, prevented DNA fragmentation, chromatin condensation and hypodiploid DNA upon exposure to high glucose. This study confirms the capacity of dietary flavonoid Morin in regulating apoptosis induced by high glucose via mitochondrial mediated pathway through intervention of oxidative stress.

  20. Ploidy reductions in murine fusion-derived hepatocytes.

    Directory of Open Access Journals (Sweden)

    Andrew W Duncan

    2009-02-01

    Full Text Available We previously showed that fusion between hepatocytes lacking a crucial liver enzyme, fumarylacetoacetate hydrolase (FAH, and wild-type blood cells resulted in hepatocyte reprogramming. FAH expression was restored in hybrid hepatocytes and, upon in vivo expansion, ameliorated the effects of FAH deficiency. Here, we show that fusion-derived polyploid hepatocytes can undergo ploidy reductions to generate daughter cells with one-half chromosomal content. Fusion hybrids are, by definition, at least tetraploid. We demonstrate reduction to diploid chromosome content by multiple methods. First, cytogenetic analysis of fusion-derived hepatocytes reveals a population of diploid cells. Secondly, we demonstrate marker segregation using ss-galactosidase and the Y-chromosome. Approximately 2-5% of fusion-derived FAH-positive nodules were negative for one or more markers, as expected during ploidy reduction. Next, using a reporter system in which ss-galactosidase is expressed exclusively in fusion-derived hepatocytes, we identify a subpopulation of diploid cells expressing ss-galactosidase and FAH. Finally, we track marker segregation specifically in fusion-derived hepatocytes with diploid DNA content. Hemizygous markers were lost by >or=50% of Fah-positive cells. Since fusion-derived hepatocytes are minimally tetraploid, the existence of diploid hepatocytes demonstrates that fusion-derived cells can undergo ploidy reduction. Moreover, the high degree of marker loss in diploid daughter cells suggests that chromosomes/markers are lost in a non-random fashion. Thus, we propose that ploidy reductions lead to the generation of genetically diverse daughter cells with about 50% reduction in nuclear content. The generation of such daughter cells increases liver diversity, which may increase the likelihood of oncogenesis.

  1. Peroxisome proliferator-activated receptor alpha acts as a mediator of endoplasmic reticulum stress-induced hepatocyte apoptosis in acute liver failure

    Directory of Open Access Journals (Sweden)

    Li Zhang

    2016-07-01

    Full Text Available Peroxisome proliferator-activated receptor α (PPARα is a key regulator to ameliorate liver injury in cases of acute liver failure (ALF. However, its regulatory mechanisms remain largely undetermined. Endoplasmic reticulum stress (ER stress plays an important role in a number of liver diseases. This study aimed to investigate whether PPARα activation inhibits ER stress-induced hepatocyte apoptosis, thereby protecting against ALF. In a murine model of D-galactosamine (D-GalN- and lipopolysaccharide (LPS-induced ALF, Wy-14643 was administered to activate PPARα, and 4-phenylbutyric acid (4-PBA was administered to attenuate ER stress. PPARα activation ameliorated liver injury, because pre-administration of its specific inducer, Wy-14643, reduced the serum aminotransferase levels and preserved liver architecture compared with that of controls. The protective effect of PPARα activation resulted from the suppression of ER stress-induced hepatocyte apoptosis. Indeed, (1 PPARα activation decreased the expression of glucose-regulated protein 78 (Grp78, Grp94 and C/EBP-homologous protein (CHOP in vivo; (2 the liver protection by 4-PBA resulted from the induction of PPARα expression, as 4-PBA pre-treatment promoted upregulation of PPARα, and inhibition of PPARα by small interfering RNA (siRNA treatment reversed liver protection and increased hepatocyte apoptosis; (3 in vitro PPARα activation by Wy-14643 decreased hepatocyte apoptosis induced by severe ER stress, and PPARα inhibition by siRNA treatment decreased the hepatocyte survival induced by mild ER stress. Here, we demonstrate that PPARα activation contributes to liver protection and decreases hepatocyte apoptosis in ALF, particularly through regulating ER stress. Therefore, targeting PPARα could be a potential therapeutic strategy to ameliorate ALF.

  2. Inhibition of mitochondrial electron transfer in rats by ethanethiol and methanethiol

    NARCIS (Netherlands)

    Vahlkamp, T.; Meijer, A. J.; Wilms, J.; Chamuleau, R. A.

    1979-01-01

    1. We have investigated the effects of ethanethiol, methanethiol and dimethyl sulphide on some metabolic processes of isolated rat hepatocytes, isolated mitochondria from liver and brain and ox-heart submitochondrial particles. 2. Ethanethiol, but not dimethyl sulphide, inhibited both

  3. Metformin stimulates FGF21 expression in primary hepatocytes

    DEFF Research Database (Denmark)

    Nygaard, Eva B; Vienberg, Sara G; Ørskov, Cathrine

    2012-01-01

    Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator of glucose and lipid metabolism; however, the exact mechanism of action and regulation of FGF21 is not fully understood. Metabolic status plays an important role in the regulation of FGF21, and we therefore examined whether...... metformin, an indirect AMPK-activator, regulates FGF21 expression in hepatocytes. FGF21 mRNA and protein expression were determined after incubation of primary cultured rat and human hepatocytes with metformin for 24 hours. To study the role of AMPK in the putative regulation of FGF21, hepatocytes were...... incubated with Compound C (an AMPK inhibitor) in the presence of metformin. A strong dose-dependent increase in FGF21 expression was observed in both rat and human hepatocytes treated with metformin. This effect was blocked by addition of the AMPK-inhibitor Compound C. The study shows that metformin...

  4. Differences in metabolism and isomerization of all-trans-retinoic acid and 9-cis-retinoic acid between human endothelial cells and hepatocytes.

    Science.gov (United States)

    Lansink, M; van Bennekum, A M; Blaner, W S; Kooistra, T

    1997-07-15

    Retinoic acid stimulates the expression of tissue-type plasminogen activator (t-PA) in vascular endothelial cells in vitro and enhances t-PA levels in plasma and tissues in vivo. Compared with the in vivo situation, high retinoic acid concentrations are required to induce optimally t-PA expression in vitro. These findings led us to study retinoic acid metabolism in cultured human endothelial cells. For comparison, these studies were also performed in the human hepatoma cell line, HepG2, and key experiments were repeated with human primary hepatocytes. Both hepatocyte cultures gave very similar results. Human endothelial cells were shown to possess an active retinoic acid metabolizing capacity, which is quantitatively comparable to that of hepatocytes, but different from that of hepatocytes in several qualitative aspects. Our results demonstrate that all-trans-retinoic acid is quickly metabolized by both endothelial cells and hepatocytes. All-trans-retinoic acid induces its own metabolism in endothelial cells but not in hepatocytes. 9-cis-Retinoic acid is degraded slowly by endothelial cells, whereas hepatocytes metabolize 9-cis-retinoic acid very quickly. Furthermore, our data show that hepatocytes, but not endothelial cells, detectably isomerise all-trans-retinoic acid to 9-cis-retinoic acid and vice versa. In both endothelial cells and hepatocytes all-trans-retinoic acid metabolism was inhibitable by the cytochrome P-450 inhibitors liarozole (10 microM) and ketoconazole (10 microM), albeit to different extents and with different specificities. In the presence of the most potent retinoic acid metabolism inhibitor in endothelial cells, liarozole, at least 10-fold lower all-trans-retinoic acid concentrations were required than in the absence of the inhibitor to obtain the same induction of t-PA. In conclusion, our results clearly demonstrate that all-trans-retinoic acid and 9-cis retinoic acid are actively but differently metabolized and isomerised by human

  5. Correction: Magnetic Cell Labeling of Primary and Stem Cell-Derived Pig Hepatocytes for MRI-Based Cell Tracking of Hepatocyte Transplantation

    National Research Council Canada - National Science Library

    Dwayne R Roach; Wesley M Garrett; Glenn Welch; Dorela D Shuboni-Mulligan; Thomas J Caperna; Neil C Talbot; Erik M Shapiro

    2017-01-01

    Roach DR, Garrett WM, Welch G, Caperna TJ, Talbot NC, Shapiro EM (2015) Magnetic Cell Labeling of Primary and Stem Cell-Derived Pig Hepatocytes for MRI-Based Cell Tracking of Hepatocyte Transplantation. (2017) Correction...

  6. Bile acid-induced apoptosis in hepatocytes is caspase-6-dependent.

    Science.gov (United States)

    Rust, Christian; Wild, Nadine; Bernt, Carina; Vennegeerts, Timo; Wimmer, Ralf; Beuers, Ulrich

    2009-01-30

    Apoptosis induced by hydrophobic bile acids is thought to contribute to liver injury during cholestasis. Caspase-6 is an executioner caspase that also appears to have regulatory functions in hematopoetic cell lines. We aimed to elucidate the role of caspase-6 in bile acid-induced apoptosis. The major human hydrophobic bile acid, glycochenodeoxycholic acid (GCDCA, 75 micromol/liter), rapidly induced caspase-6 cleavage in HepG2-Ntcp human hepatoma cells. GCDCA-induced, but not tumor necrosis factor alpha- or etoposide-induced activation of effector caspases-3 and -7 was significantly reduced by 50% in caspase-6-deficient HepG2-Ntcp cells as well as in primary rat hepatocytes pretreated with a caspase-6 inhibitor. Inhibition of caspase-9 reduced GCDCA-induced activation of caspase-6, whereas inhibition of caspase-6 reduced activation of caspase-8 placing caspase-6 between caspase-9 and caspase-8. GCDCA also induced apoptosis in Fas-deficient Hep3B-Ntcp and HuH7-Ntcp hepatoma cells. In addition, GCDCA-induced apoptosis was reduced by 50% in FADD-deficient HepG2-Ntcp cells, whereas apoptosis induced by tumor necrosis factor alpha was reduced by 90%. Collectively, these observations suggest that GCDCA can induce hepatocyte apoptosis in the absence of death receptor signaling, presumably by a compensatory mitochondrial pathway. In conclusion, caspase-6 appears to play an important regulatory role in the promotion of bile acid-induced apoptosis as part of a feedback loop.

  7. Protective effect of Cistanchis A on ethanol-induced damage in primary cultured mouse hepatocytes.

    Science.gov (United States)

    Luo, Huiying; Cao, Rongrong; Wang, Lijuan; Zhu, Lijuan

    2016-10-01

    Cistanoside A (C. A) was one of phenylethanol glycosides isolated from Cistanche deserticola, a tonic in traditional Chinese medicine. In our previous research, we demonstrated that Cistanoside A (C. A) possess the protective activities on CCl4 induced hepatotoxicity in mice, such as increasing free radicals clearing activities, alleviating lipid-overoxidation damage, and improving respiratory chain function in mitochondria. Meanwhile, our previous research also demonstrated C.A possess protective activities on alcohol induced hepatotoxicity in mice, shown in ameliorate the hepatic function indices, lightening steatosis and inflammatory infiltration, increasing free radicals clearing activities, alleviating lipid-overoxidation damage, and alleviating energy metabolism in mitochondria. The aim of this research was to evaluate the effects of Cistanoside A (C. A) on ethanol-induced damage in primary cultured mouse hepatocytes, and probe into the mechanism related. Using fluorescent staining, flow cytometer, immunohistochemistry analysis, and Western blotting, we demonstrated that C.A could enhance the survival rate of the primary cultured hepatocytes, alleviate apoptosis and necrosis, the mechanism was involved with enhance the expression of apoptosis inhibition factor bcl-2, and inhibition the expression of immediate early genes c-fos. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  8. [Oxidative stress promotes hepatocyte apoptosis mediated by glycogen synthase kinase 3β].

    Science.gov (United States)

    Zhang, Xiangying; Guo, Yuanyuan; Zhang, Li; Wen, Tao; Piao, Zhengfu; Shi, Hongbo; Chen, Dexi; Duan, Zhongping; Ren, Feng

    2015-01-01

    To analyze the role of glycogen synthase kinase 3β (GSK3β) in hepatocyte apoptosis induced by oxidative stress. Human HL-7702 hepatoma cells were induced by H₂O₂/antimycin A to establish oxidative stress-induced cell apoptosis models. SB216763, a specific inhibitor of GSK3β, was given to the cells two hours before H₂O₂/antimycin A induction. Cell survival was observed using calcein acetoxymethyl ester/propidium iodide (PI) double staining, and cell apoptosis was detected using annexin V-FITC/PI staining combined with flow cytometry. In the meanwhile, the cell culture supernatant was subjected to lactate dehydrogenase (LDH) assay to evaluate the extent of cell death. The expressions of p-GSK3β, GSK3β, caspase-3, cleaved caspase-3, c-Jun N-terminal kinase (JNK) and cytochrome C (CytC) proteins were examined using Western blotting. Oxidative stress triggered by H₂O₂/antimycin A promoted GSK3β activity; inhibition of GSK3β activity by SB216763 relieved oxidative stress and reduced cell apoptosis induced by oxidative stress. Compared with the model groups, SB216763 intervened group showed that the cell apoptosis rate and the level of LDH were reduced significantly, and that the expressions of cleaved caspase-3, JNK, CytC proteins decreased. GSK3β is an important signaling molecule in the apoptosis pathway induced by oxidative stress. The inhibition on GSK3β may alleviate the oxidative stress-induced hepatocyte apoptosis.

  9. alpha-Amanitin induced apoptosis in primary cultured dog hepatocytes.

    Directory of Open Access Journals (Sweden)

    Adam Szelag

    2010-06-01

    Full Text Available Amatoxin poisoning is caused by mushroom species belonging to the genera Amanita, Galerina and Lepiota with the majority of lethal mushroom exposures attributable to Amanita phalloides. High mortality rate in intoxications with these mushrooms is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amatoxins. A wide variety of amatoxins have been isolated; however, alpha-amanitin (alpha-AMA appears to be the primary toxin. Studies in vitro and in vivo suggest that alpha-AMA does not only cause hepatocyte necrosis, but also may lead to apoptotic cell death. The objective of this study was to evaluate the complex hepatocyte apoptosis in alpha-AMA cytotoxicity. All experiments were performed on primary cultured canine hepatocytes. The cells were incubated for 12 h with alpha-AMA at a final concentration of 1, 5, 10 and 20 microM. Viability test (MTT assay, apoptosis evaluation (TUNEL reaction, detection of DNA laddering and electron microscopy were performed at 6 and 12 h of exposure to alpha-AMA. There was a clear correlation between hepatocyte viability, concentration of alpha-AMA and time of exposure to this toxin. The decline in cultured dog hepatocyte viability during the exposure to alpha-AMA is most likely preceded by enhanced cellular apoptosis. Our results demonstrate that apoptosis might contribute to pathogenesis of the severe liver injury in the course of amanitin intoxication, particularly during the early phase of poisoning.

  10. Biomechanics and functionality of hepatocytes in liver cirrhosis.

    Science.gov (United States)

    Sun, Shan; Song, Zhenyuan; Cotler, Scott J; Cho, Michael

    2014-06-27

    Cirrhosis is a life-threatening condition that is generally attributed to overproduction of collagen fibers in the extracellular matrix that mechanically stiffens the liver. Chronic liver injury due to causes including viral hepatitis, inherited and metabolic liver diseases and external factors such as alcohol abuse can result in the development of cirrhosis. Progression of cirrhosis leads to hepatocellular dysfunction. While extensive studies to understand the complexity underlying liver fibrosis have led to potential application of anti-fibrotic drugs, no such FDA-approved drugs are currently available. Additional studies of hepatic fibrogenesis and cirrhosis primarily have focused on the extracellular matrix, while hepatocyte biomechanics has received limited attention. The role of hepatocyte biomechanics in liver cirrhosis remains elusive, and how the cell stiffness is correlated with biological functions of hepatocytes is also unknown. In this study, we demonstrate that the biomechanical properties of hepatocytes are correlated with their functions (e.g., glucose metabolism), and that hepatic dysfunction can be restored through modulation of the cellular biomechanics. Furthermore, our results indicate the hepatocyte functionality appears to be regulated through a crosstalk between the Rho and Akt signaling. These novel findings may lead to biomechanical intervention of hepatocytes and the development of innovative tissue engineering for clinical treatment to target liver cells rather than exclusively focusing on the extracellular matrix alone in liver cirrhosis. © 2013 Published by Elsevier Ltd.

  11. Force spectroscopy of hepatocytic extracellular matrix components

    Energy Technology Data Exchange (ETDEWEB)

    Yongsunthon, R., E-mail: YongsuntR@Corning.com [Corning Incorporated, SP-FR-01, R1S32D, Corning, NY 14831 (United States); Baker, W.A.; Bryhan, M.D.; Baker, D.E.; Chang, T.; Petzold, O.N.; Walczak, W.J.; Liu, J.; Faris, R.A.; Senaratne, W.; Seeley, L.A.; Youngman, R.E. [Corning Incorporated, SP-FR-01, R1S32D, Corning, NY 14831 (United States)

    2009-07-15

    We present atomic force microscopy and force spectroscopy data of live hepatocytes (HEPG2/C3A liver cell line) grown in Eagle's Minimum Essential Medium, a complex solution of salts and amino acids commonly used for cell culture. Contact-mode imaging and force spectroscopy of this system allowed correlation of cell morphology and extracellular matrix (ECM) properties with substrate properties. Force spectroscopy analysis of cellular 'footprints' indicated that the cells secrete large polymers (e.g., 3.5 {mu}m contour length and estimated MW 1000 kDa) onto their substrate surface. Although definitive identification of the polymers has not yet been achieved, fluorescent-labeled antibody staining has specified the presence of ECM proteins such as collagen and laminin in the cellular footprints. The stretched polymers appear to be much larger than single molecules of known ECM components, such as collagen and heparan sulfate proteoglycan, thus suggesting that the cells create larger entangled, macromolecular structures from smaller components. There is strong evidence which suggests that the composition of the ECM is greatly influenced by the hydrophobicity of the substrate surface, with preferential production and/or adsorption of larger macromolecules on hydrophobic surfaces.

  12. Duck hepatitis B virus covalently closed circular DNA appears to survive hepatocyte mitosis in the growing liver.

    Science.gov (United States)

    Reaiche-Miller, Georget Y; Thorpe, Michael; Low, Huey Chi; Qiao, Qiao; Scougall, Catherine A; Mason, William S; Litwin, Samuel; Jilbert, Allison R

    2013-11-01

    Nucleos(t)ide analogues that inhibit hepatitis B virus (HBV) DNA replication are typically used as monotherapy for chronically infected patients. Treatment with a nucleos(t)ide analogue eliminates most HBV DNA replication intermediates and produces a gradual decline in levels of covalently closed circular DNA (cccDNA), the template for viral RNA synthesis. It remains uncertain if levels of cccDNA decline primarily through hepatocyte death, or if loss also occurs during hepatocyte mitosis. To determine if cccDNA survives mitosis, growing ducklings infected with duck hepatitis B virus (DHBV) were treated with the nucleoside analogue, Entecavir. Viremia was suppressed at least 10(5)-fold, during a period when average liver mass increased 23-fold. Analysis of the data suggested that if cccDNA synthesis was completely inhibited, at least 49% of cccDNA survived hepatocyte mitosis. However, there was a large duck-to-duck variation in cccDNA levels, suggesting that low level cccDNA synthesis may contribute to this apparent survival through mitosis. Crown Copyright © 2013 Published by Elsevier Inc. All rights reserved.

  13. Cold storage of rat hepatocyte suspensions for one week in a customized cold storage solution--preservation of cell attachment and metabolism.

    Directory of Open Access Journals (Sweden)

    Gesine Pless-Petig

    Full Text Available BACKGROUND & AIMS: Primary hepatocytes are of great importance for basic research as well as cell transplantation. However, their stability, especially in suspension, is very low. This feature severely compromises storage and shipment. Based on previous studies with adherent cells, we here assessed cold storage injury in rat hepatocyte suspensions and aimed to find a cold storage solution that preserves viability, attachment ability and functionality of these cells. METHODS: Rat hepatocyte suspensions were stored in cell culture medium, organ preservation solutions and modified TiProtec solutions at 4°C for one week. Viability and cell volume were determined by flow cytometry. Thereafter, cells were seeded and density and metabolic capacity (reductive metabolism, forskolin-induced glucose release, urea production of adherent cells were assessed. RESULTS: Cold storage injury in hepatocyte suspensions became evident as cell death occurring during cold storage or rewarming or as loss of attachment ability. Cell death during cold storage was not dependent on cell swelling and was almost completely inhibited in the presence of glycine and L-alanine. Cell attachment could be greatly improved by use of chloride-poor solutions and addition of iron chelators. Using a chloride-poor, potassium-rich storage solution containing glycine, alanine and iron chelators, cultures with 75% of the density of control cultures and with practically normal cell metabolism could be obtained after one week of cold storage. CONCLUSION: In the solution presented here, cold storage injury of hepatocyte suspensions, differing from that of adherent hepatocytes, was effectively inhibited. The components which acted on the different injurious processes were identified.

  14. A simple isolation and cryopreservation method for adult human hepatocytes.

    Science.gov (United States)

    Hang, Hualian; Shi, Xiaolei; Gu, Guangxiang; Wu, Yafu; Ding, Yitao

    2009-10-01

    The objective of this study was to establish a stable method of isolation, culture and cryopreservation of adult primary hepatocytes to provide potential hepatocyte resources for the treatment of acute and chronic liver diseases by hepatocyte transplantation and bioartificial liver support systems, and for the use of hepatocytes as an in vitro model of the liver. Adult hepatocytes of 20 separate donors were isolated with a two-step extracoporeal collagenase perfusion technique. Seven preincubation time points (2h, 6h, 12h, 24h, 36h, 48h and 72h) were selected, then the hepatocytes were transferred to HepatoZYME-SFM medium containing 10% FBS and 10% DMSO, and were immediately put into an isopropanol progressive freezing container at -80 degrees C overnight and immersed in liquid nitrogen the next day. During the postthawing culture period, viability, plating efficiency, albumin secretion and urea synthesis were analyzed. The viability and plating efficiency of hepatocytes after partial hepatectomy using two-step extracorporeal collagenase perfusion technique were 75.0+/-4.6% and 72.0+/-6.0% respectively. Preincubation at 4? for 12 hours or 24 hours proved to be the optimal time at which the albumin secretion was higher than at other time points (p<0.05). Compared to the immediate cryopreservation groups (IC), we also found significant improvement in viability (61.4+/-4.8%/62.0+/-5.6% vs. 53.4+/-4.2%, p<0.05), plating efficiency (63.2+/-5.8%/62.6+/-3.6% vs. 55.2+/-4.6%, p<0.05), albumin secretion and urea synthesis (p<0.05) at these time points. The two-step extracorporeal collagenase perfusion technique after partial hepatectomy provides a novel, simple, and reliable method for hepatocyte isolation. The results of the present study suggest that recovery of human hepatocytes after isolation preincubation at 4 degrees C for 12 hours to 24 hours prior to cryopreservation can obtain hepatocytes ideal for use in pharmacotoxicology, bioartificial liver and cell therapy

  15. Simultaneous detection of multiple mRNA markers CK19, CEA, c-Met, Her2/neu and hMAM with membrane array, an innovative technique with a great potential for breast cancer diagnosis.

    Science.gov (United States)

    Chen, Chung-Chi; Hou, Ming-Feng; Wang, Jaw-Yuan; Chang, Tsai-Wang; Lai, Dan-Yu; Chen, Yi-Fang; Hung, Sung-Yu; Lin, Shiu-Ru

    2006-08-28

    The objective of this study was mainly to develop and evaluate a membrane array-based method simultaneously detecting the expression levels of a multiple mRNA marker panel in the peripheral blood for used in complementary breast cancer diagnosis. The mRNA markers employed included cytokeratin 19 (CK-19), carcinoembryonic antigen (CEA), c-Met, Her2/neu, and mammaglobin (hMAM). The specimens of peripheral blood were collected from 80 healthy women and 102 female patients with breast cancer. The expression levels of molecular markers were evaluated by real-time Q-PCR and membrane array. Data obtained from real-time Q-PCR and membrane array were subjected to linear regression analysis, revealing that there was a high degree of correlation between the results of these two methods (r=0.979, Pmembrane array assay with a combined panel of five mRNA markers was demonstrated to achieve sensitivity of 80.6%, and specificity of 83.8% for breast cancer detection, much higher than those of analysis of single marker. In addition, we demonstrated that the membrane array method could detect circulating cancer cells at a density as low as five cancer cells per 1 ml of blood. The analysis of correlation between the outcome of membrane array and clinicopathological characteristics indicated that overexpression of the multiple marker panel was significantly correlated with tumor size (P=0.030) and TNM stage (0.009). In conclusion, the detection of circulating cancer cells by means of membrane array simultaneously monitoring five mRNA markers could significantly enhance the sensitivity and specificity for cancer cell detection.

  16. Use of Thymidine Kinase Recombinant Adenovirus and Ganciclovir Mediated Mouse Liver Preconditioning for Hepatocyte Xenotransplantation.

    Science.gov (United States)

    Moreno, Daniel; Neri, Leire; Vicente, Eva; Vales, Africa; Aldabe, Rafael

    2017-01-01

    Hepatocyte transplantation is the best approach to maintain and propagate differentiated hepatocytes from different species. Host liver has to be adapted for transplanted hepatocytes productive engraftment and proliferation being required a chronic liver injury to eliminate host hepatocytes and provide a proliferative advantage to the transplanted hepatocytes. Most valuable mouse models for xenograft hepatocyte transplantation are based on genetically modified animals to cause a chronic liver damage and to limit host hepatocyte regeneration potential. We present a methodology that generates a chronic liver damage and can be applied to any host mouse strain and animal species based on the inoculation of a recombinant adenovirus to express herpes simplex thymidine kinase in host hepatocytes sensitizing them to ganciclovir treatment. This causes a prolonged liver damage that allows hepatocyte transplantation and generation of regenerative nodules in recipient mouse liver integrated by transplanted cells and host sinusoidal. Obtained chimeric animals maintain functional chimeric nodules for several weeks, ready to be used in any study.

  17. Correction of liver disease following transplantation of normal rat hepatocytes into Long-Evans Cinnamon rats modeling Wilson's disease.

    Science.gov (United States)

    Irani, A N; Malhi, H; Slehria, S; Gorla, G R; Volenberg, I; Schilsky, M L; Gupta, S

    2001-03-01

    To establish the efficacy of cell therapy in Wilson's disease, we used the Long-Evans Cinnamon (LEC) rat model with atp7b gene mutation and copper toxicosis. Several groups of LEC rats were established, including animals pretreated with retrorsine to exacerbate copper toxicosis and inhibit proliferation in native hepatocytes followed by partial hepatectomy to promote liver repopulation. Hepatocytes from normal, syngeneic LEA rats were transplanted intrasplenically. Animal survival, biliary copper excretion, and hepatic copper were determined. The magnitude of liver repopulation was demonstrated by measuring serum ceruloplasmin and hepatic atp7b mRNA. Long-term survival in LEC rats treated with retrorsine, partial hepatectomy, and cell transplantation was up to 90%, whereas fewer than 10% of animals pretreated with retrorsine, without cell therapy, survived, P Wilson's disease.

  18. Hepatocyte specific expression of human cloned genes

    Energy Technology Data Exchange (ETDEWEB)

    Cortese, R.

    1986-01-01

    A large number of proteins are specifically synthesized in the hepatocyte. Only the adult liver expresses the complete repertoire of functions which are required at various stages during development. There is therefore a complex series of regulatory mechanisms responsible for the maintenance of the differentiated state and for the developmental and physiological variations in the pattern of gene expression. Human hepatoma cell lines HepG2 and Hep3B display a pattern of gene expression similar to adult and fetal liver, respectively; in contrast, cultured fibroblasts or HeLa cells do not express most of the liver specific genes. They have used these cell lines for transfection experiments with cloned human liver specific genes. DNA segments coding for alpha1-antitrypsin and retinol binding protein (two proteins synthesized both in fetal and adult liver) are expressed in the hepatoma cell lines HepG2 and Hep3B, but not in HeLa cells or fibroblasts. A DNA segment coding for haptoglobin (a protein synthesized only after birth) is only expressed in the hepatoma cell line HepG2 but not in Hep3B nor in non hepatic cell lines. The information for tissue specific expression is located in the 5' flanking region of all three genes. In vivo competition experiments show that these DNA segments bind to a common, apparently limiting, transacting factor. Conventional techniques (Bal deletions, site directed mutagenesis, etc.) have been used to precisely identify the DNA sequences responsible for these effects. The emerging picture is complex: they have identified multiple, separate transcriptional signals, essential for maximal promoter activation and tissue specific expression. Some of these signals show a negative effect on transcription in fibroblast cell lines.

  19. Gender difference in NASH susceptibility: Roles of hepatocyte Ikkβ and Sult1e1

    Science.gov (United States)

    Matsushita, Noriko; Hassanein, Mohamed T.; Martinez-Clemente, Marcos; Lazaro, Raul; French, Samuel W.; Xie, Wen; Lai, Keane; Karin, Michael; Tsukamoto, Hidekazu

    2017-01-01

    Myeloid cell and hepatocyte IKKβ may mediate the genesis of obesity and insulin resistance in mice fed high fat diet. However, their gender-specific roles in the pathogenesis of non-alcoholic steatohepatitis (NASH) are not known. Here we demonstrate myeloid IKKβ deficiency prevents Western diet-induced obesity and visceral adiposity in females but not in males, and attenuates hyperglycemia, global IR, and NASH in both genders. In contrast, all metabolic sequela including NASH are aggravated by hepatocyte IKKβ deficiency (IkbkbΔhep) in male but not female mice. Gene profiling identifies sulfotransferase family 1E (Sult1e1), which encodes a sulfotransferase E1 responsible for inactivation of estrogen, as a gene upregulated in NASH in both genders and most conspicuously in male IkbkbΔhep mice having worst NASH and lowest plasma estradiol levels. LXRα is enriched to LXRE on Sult1e1 promoter in male WT and IkbkbΔhep mice with NASH, and a Sult1e1 promoter activity is increased by LXRα and its ligand and augmented by expression of a S32A mutant of IκBα. These results demonstrate striking gender differences in regulation by IKKβ of high cholesterol saturated fat diet-induced metabolic changes including NASH and suggest hepatocyte IKKβ is protective in male due at least in part to its ability to repress LXR-induced Sult1e1. Our findings also raise a caution for systemic IKK inhibition for the treatment of NASH as it may exacerbate the disease in male patients. PMID:28797077

  20. Gender difference in NASH susceptibility: Roles of hepatocyte Ikkβ and Sult1e1.

    Directory of Open Access Journals (Sweden)

    Noriko Matsushita

    Full Text Available Myeloid cell and hepatocyte IKKβ may mediate the genesis of obesity and insulin resistance in mice fed high fat diet. However, their gender-specific roles in the pathogenesis of non-alcoholic steatohepatitis (NASH are not known. Here we demonstrate myeloid IKKβ deficiency prevents Western diet-induced obesity and visceral adiposity in females but not in males, and attenuates hyperglycemia, global IR, and NASH in both genders. In contrast, all metabolic sequela including NASH are aggravated by hepatocyte IKKβ deficiency (IkbkbΔhep in male but not female mice. Gene profiling identifies sulfotransferase family 1E (Sult1e1, which encodes a sulfotransferase E1 responsible for inactivation of estrogen, as a gene upregulated in NASH in both genders and most conspicuously in male IkbkbΔhep mice having worst NASH and lowest plasma estradiol levels. LXRα is enriched to LXRE on Sult1e1 promoter in male WT and IkbkbΔhep mice with NASH, and a Sult1e1 promoter activity is increased by LXRα and its ligand and augmented by expression of a S32A mutant of IκBα. These results demonstrate striking gender differences in regulation by IKKβ of high cholesterol saturated fat diet-induced metabolic changes including NASH and suggest hepatocyte IKKβ is protective in male due at least in part to its ability to repress LXR-induced Sult1e1. Our findings also raise a caution for systemic IKK inhibition for the treatment of NASH as it may exacerbate the disease in male patients.

  1. Lack of CD47 on donor hepatocytes promotes innate immune cell activation and graft loss: a potential barrier to hepatocyte xenotransplantation.

    Science.gov (United States)

    Navarro-Alvarez, Nalu; Yang, Yong-Guang

    2014-03-01

    We have previously shown that interspecies incompatibility of CD47 plays an important role in triggering rejection of xenogeneic hematopoietic cells by macrophages. However, whether CD47 incompatibility also induces rejection of nonhematopoietic cellular xenografts remains unknown. Herein, we have addressed this question in a mouse model of hepatocyte transplantation in which CD47(-/-) hepatocytes were used to resemble xenografts for CD47 incompatibility. We show that intrasplenic transplantation of CD47(-/-), but not wild-type (WT) hepatocytes, into partially hepatectomized syngeneic WT mice resulted in a rapid increase in Mac-1(+) cells with an activation phenotype (i.e., Mac-1(+)CD14(+) and Mac-1(+)CD16/32(high)), compared to nontransplant controls. In addition, CD47(-/-) hepatocytes were more severely damaged than WT hepatocytes as indicated by the greater AST and ALT serum levels in these mice. Furthermore, long-term donor hepatocyte survival and liver repopulation were observed in mice receiving WT hepatocytes, whereas CD47(-/-) hepatocytes were completely rejected within 2 weeks. These results suggest that CD47 on donor hepatocytes prevents recipient myeloid innate immune cell activation, hence aiding in graft survival after hepatocyte transplantation. Thus, CD47 incompatibility is likely to present an additional barrier to hepatocyte xenotransplantation.

  2. Honokiol activates the LKB1–AMPK signaling pathway and attenuates the lipid accumulation in hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Min Suk; Kim, Jung Hwan; Kim, Hye Jung; Chang, Ki Churl; Park, Sang Won, E-mail: parksw@gnu.ac.kr

    2015-04-15

    Honokiol is a bioactive neolignan compound isolated from the species of Magnolia. This study was designed to elucidate the cellular mechanism by which honokiol alleviates the development of non-alcoholic steatosis. HepG2 cells were treated with honokiol for 1 h, and then exposed to 1 mM free fatty acid (FFA) for 24 h to simulate non-alcoholic steatosis in vitro. C57BL/6 mice were fed with a high-fat diet for 28 days, and honokiol (10 mg/kg/day) was daily treated. Honokiol concentration-dependently attenuated intracellular fat overloading and triglyceride (TG) accumulation in FFA-exposed HepG2 cells. These effects were blocked by pretreatment with an AMP-activated protein kinase (AMPK) inhibitor. Honokiol significantly inhibited sterol regulatory element-binding protein-1c (SREBP-1c) maturation and the induction of lipogenic proteins, stearoyl-CoA desaturase-1 (SCD-1) and fatty acid synthase (FAS) in FFA-exposed HepG2 cells, but these effects were blocked by pretreatment of an AMPK inhibitor. Honokiol induced AMPK phosphorylation and subsequent acetyl-CoA carboxylase (ACC) phosphorylation, which were inhibited by genetic deletion of liver kinase B1 (LKB1). Honokiol stimulated LKB1 phosphorylation, and genetic deletion of LKB1 blocked the effect of honokiol on SREBP-1c maturation and the induction of SCD-1 and FAS proteins in FFA-exposed HepG2 cells. Honokiol attenuated the increases in hepatic TG and lipogenic protein levels and fat accumulation in the mice fed with high-fat diet, while significantly induced LKB1 and AMPK phosphorylation. Taken together, our findings suggest that honokiol has an anti-lipogenic effect in hepatocytes, and this effect may be mediated by the LKB1–AMPK signaling pathway, which induces ACC phosphorylation and inhibits SREBP-1c maturation in hepatocytes. - Highlights: • Honokiol attenuates lipid accumulation induced by free fatty acid in hepatocyte. • Honokiol inhibits the increase in lipogenic enzyme levels induced by free fatty

  3. Lymphocyte traffic through sinusoidal endothelial cells is regulated by hepatocytes.

    Science.gov (United States)

    Edwards, Sarah; Lalor, Patricia F; Nash, Gerard B; Rainger, G Ed; Adams, David H

    2005-03-01

    Crosstalk between hepatic sinusoidal ECs and closely juxtaposed hepatocytes via vascular endothelial growth factor is essential for the maintenance of sinusoidal endothelial growth and differentiation. We propose that paracrine interactions between endothelial cells and hepatocytes also may be responsible for the unique complement of adhesion receptors expressed on sinusoidal endothelium that regulate the recruitment of lymphocytes into the liver. To address this hypothesis, we developed an in vitro model of the hepatic sinusoid in which flowing lymphocytes could interact with hepatic endothelium conditioned by the presence of hepatocytes. Human hepatic sinusoidal endothelial cells cocultured with hepatocytes were activated so that they supported the adhesion of lymphocytes at levels equivalent to those seen on endothelium stimulated with the inflammatory cytokine tumour necrosis factor-beta. Lymphocyte adhesion was supported by intracellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin, with an additional contribution from the novel adhesion receptor VAP-1. In conclusion, we show that interactions between hepatocytes and endothelial cells amplify leukocyte recruitment through the sinusoids by regulating the expression and function of endothelial adhesion molecules. These paracrine interactions may be responsible for the induction of the adhesion molecules that support constitutive lymphocyte recruitment to the liver as well as contributing significantly to the patterns of leukocyte adhesion seen during episodes of hepatic inflammation.

  4. The endocannabinoid N-arachidonoyl dopamine (NADA) selectively induces oxidative stress-mediated cell death in hepatic stellate cells but not in hepatocytes.

    Science.gov (United States)

    Wojtalla, Alexandra; Herweck, Frank; Granzow, Michaela; Klein, Sabine; Trebicka, Jonel; Huss, Sebastian; Lerner, Raissa; Lutz, Beat; Schildberg, Frank Alexander; Knolle, Percy Alexander; Sauerbruch, Tilman; Singer, Manfred Vincenz; Zimmer, Andreas; Siegmund, Sören Volker

    2012-04-15

    The endocannabinoid system is a crucial regulator of hepatic fibrogenesis. We have previously shown that the endocannabinoid anandamide (AEA) is a lipid mediator that blocks proliferation and induces death in hepatic stellate cells (HSCs), the main fibrogenic cell type in the liver, but not in hepatocytes. However, the effects of other endocannabinoids such as N-arachidonoyl dopamine (NADA) have not yet been investigated. The NADA-synthesizing enzyme tyrosine hydroxylase was mainly expressed in sympathetic neurons in portal tracts. Its expression pattern stayed unchanged in normal or fibrotic liver. NADA dose dependently induced cell death in culture-activated primary murine or human HSCs after 2-4 h, starting from 5 μM. Despite caspase 3 cleavage, NADA-mediated cell death showed typical features of necrosis, including ATP depletion. Although the cannabinoid receptors CB1, CB2, or transient receptor potential cation channel subfamily V, member 1 were expressed in HSCs, their pharmacological or genetic blockade failed to inhibit NADA-mediated death, indicating a cannabinoid-receptor-independent mechanism. Interestingly, membrane cholesterol depletion with methyl-β-cyclodextrin inhibited AEA- but not NADA-induced death. NADA significantly induced reactive oxygen species formation in HSCs. The antioxidant glutathione (GSH) significantly decreased NADA-induced cell death. Similar to AEA, primary hepatocytes were highly resistant against NADA-induced death. Resistance to NADA in hepatocytes was due to high levels of GSH, since GSH depletion significantly increased NADA-induced death. Moreover, high expression of the AEA-degrading enzyme fatty acid amide hydrolase (FAAH) in hepatocytes also conferred resistance towards NADA-induced death, since pharmacological or genetic FAAH inhibition significantly augmented hepatocyte death. Thus the selective induction of cell death in HSCs proposes NADA as a novel antifibrogenic mediator.

  5. In vitro culture of functionally active buffalo hepatocytes isolated by using a simplified manual perfusion method.

    Directory of Open Access Journals (Sweden)

    Santanu Panda

    Full Text Available In farm animals, there is no suitable cell line available to understand liver-specific functions. This has limited our understanding of liver function and metabolism in farm animals. Culturing and maintenance of functionally active hepatocytes is difficult, since they survive no more than few days. Establishing primary culture of hepatocytes can help in studying cellular metabolism, drug toxicity, hepatocyte specific gene function and regulation. Here we provide a simple in vitro method for isolation and short-term culture of functionally active buffalo hepatocytes.Buffalo hepatocytes were isolated from caudate lobes by using manual enzymatic perfusion and mechanical disruption of liver tissue. Hepatocyte yield was (5.3 ± 0.66×107 cells per gram of liver tissue with a viability of 82.3 ± 3.5%. Freshly isolated hepatocytes were spherical with well contrasted border. After 24 hours of seeding onto fibroblast feeder layer and different extracellular matrices like dry collagen, matrigel and sandwich collagen coated plates, hepatocytes formed confluent monolayer with frequent clusters. Cultured hepatocytes exhibited typical cuboidal and polygonal shape with restored cellular polarity. Cells expressed hepatocyte-specific marker genes or proteins like albumin, hepatocyte nuclear factor 4α, glucose-6-phosphatase, tyrosine aminotransferase, cytochromes, cytokeratin and α1-antitrypsin. Hepatocytes could be immunostained with anti-cytokeratins, anti-albumin and anti α1-antitrypsin antibodies. Abundant lipid droplets were detected in the cytosol of hepatocytes using oil red stain. In vitro cultured hepatocytes could be grown for five days and maintained for up to nine days on buffalo skin fibroblast feeder layer. Cultured hepatocytes were viable for functional studies.We developed a convenient and cost effective technique for hepatocytes isolation for short-term culture that exhibited morphological and functional characteristics of active hepatocytes

  6. Effects of volatile fatty acids on propionate metabolism and gluconeogenesis in caprine hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Aiello, R.J.; Armentano, L.E.

    1987-12-01

    Isolated caprine hepatocytes were incubated with fatty acids of various chain lengths. Short-chain fatty acids effects on rates of gluconeogenesis and oxidation from (2-/sup 14/C) propionate were determined. Additions of glucose (2.5 mM) had no effect on hepatic (2-/sup 14/C)-propionate metabolism in the presence and absence of amino acids. A complete mixture of amino acids increased label incorporation from (2-/sup 14/C) propionate into (/sup 14/C) glucose by 22%. Butyrate inhibited (2-/sup 14/C) propionate metabolism and increased the apparent Michaelis constant for (2-/sup 14/C) propionate incorporation into (/sup 14/C) glucose from 2.4 +/- 1.5 to 5.6 +/- .9 mM. Butyrate's effects on propionate were similar in the presence and absence of L-carnitine (1 mM). Isobutyrate, 2-methylbutyrate, and valerate (1.25 mM) had no effect on (/sup 14/C) glucose production but decreased /sup 14/CO/sub 2/ production to 57, 61, and 54% of the control (2-/sup 14/C) propionate (1.25 mM). This inhibition on /sup 14/CO/sub 2/ was not competitive. Isovalerate had no effect on either (2-/sup 14/C) propionate incorporation into glucose of CO/sub 2/. An increase in ratio of (/sup 14/C) glucose to /sup 14/CO/sub 2/ from (2-/sup 14/C)-propionate demonstrated that short-chain fatty acids other than butyrate do not inhibit gluconeogenesis from propionate. In addition, fatty acids that generate a net synthesis of intracellular oxaloacetate may partition propionate carbons toward gluconeogenic rather than oxidative pathways in goat hepatocytes.

  7. Alternative Cell Sources to Adult Hepatocytes for Hepatic Cell Therapy.

    Science.gov (United States)

    Pareja, Eugenia; Gómez-Lechón, María José; Tolosa, Laia

    2017-01-01

    Adult hepatocyte transplantation is limited by scarce availability of suitable donor liver tissue for hepatocyte isolation. New cell-based therapies are being developed to supplement whole-organ liver transplantation, to reduce the waiting-list mortality rate, and to obtain more sustained and significant metabolic correction. Fetal livers and unsuitable neonatal livers for organ transplantation have been proposed as potential useful sources of hepatic cells for cell therapy. However, the major challenge is to use alternative cell sources for transplantation that can be derived from reproducible methods. Different types of stem cells with hepatic differentiation potential are eligible for generating large numbers of functional hepatocytes for liver cell therapy to treat degenerative disorders, inborn hepatic metabolic diseases, and organ failure. Clinical trials are designed to fully establish the safety profile of such therapies and to define target patient groups and standardized protocols.

  8. Effect of ethanol on glutathione concentration in isolated hepatocytes.

    Science.gov (United States)

    Viña, J; Estrela, J M; Guerri, C; Romero, F J

    1980-01-01

    1. Ethanol induces a decrease in GSH (reduced glutathione) concentration is isolated hepatocytes. Maximal effects appear at 20 mM-ethanol. The concentration-dependence of this decrease is paralleled by the concentration-dependence of the activity of alcohol dehydrogenase. 2. Pyrazole, a specific inhibitor of alcohol dehydrogenase, prevents the ethanol-induced GSH depletion. 3. Acetaldehyde, above 0.05 mM, also promotes a decrease in GSH concentration in hepatocytes. 4. Disulfiram (0.05 mM), an inhibitor of aldehyde dehydrogenase, potentiates the fall in GSH concentration caused by acetaldehyde. 5. The findings support the hypothesis that acetaldehyde is responsible for the depletion of GSH induced by ethanol. 6. Methionine prevents the effect of alcohol or acetaldehyde on GSH concentration in hepatocytes. PMID:6994718

  9. Early membrane depressions in hepatocytes cultured on Primaria supports.

    Science.gov (United States)

    Griffiths, B J; Evans, P J

    2001-01-01

    The topography of spreading hepatocytes on positively charged Primaria plates was examined using scanning electron microscopy. The cells acquired a uniform morphology within 2 h. The spreading was rapid and the surface of the cells showed early prominent depressions or dips. The hepatocytes had either one or two of these structures which corresponded to the frequency of mononuclear and binuclear cells, respectively. The nuclear origin of the dips was strengthened after 6 h. They contained solid structures, whose number, size and shape were the same as nucleoli. The membrane dipping was independent of cell density and took place under conditions where phenotypic changes can occur. Kidney proximal tubule cells had no dips. Co-cultures of hepatocytes and kidney proximal tubule cells showed that the cell types behave differently.

  10. Intracytoplasmic Crystalline Inclusions in the Hepatocytes of an Antelope

    Directory of Open Access Journals (Sweden)

    Sanjeev Gumber

    2010-01-01

    Full Text Available This case report describes intracytoplasmic crystalline inclusions in the hepatocytes of a 13-year-old female Thomson's gazelle. Histologically, multifocal to coalescing areas of many hepatocytes contained large cytoplasmic vacuoles filled with pale eosinophilic homogeneous material and rare fine basophilic granules. Von Kossa staining showed the presence of calcium within cytoplasm, mainly in the inclusions, of hepatocytes. Transmission electron microscopy, scanning electron microscopy, energy dispersive X-rays analyses, and infrared spectroscopy on the liver showed the hepatocellular material consistent with protein and carbohydrate with secondary accumulation of calcium and phosphorus. It was concluded that crystalline inclusions may have been derived due to failure of normal physiological hepatocellular clearance associated with a severe chronic disease. To the authors' knowledge this is the first reported case of hepatocellular crystalline inclusions in an antelope.

  11. Effect of brain-derived neurotrophic factor (BDNF) on hepatocyte metabolism.

    Science.gov (United States)

    Genzer, Yoni; Chapnik, Nava; Froy, Oren

    2017-07-01

    Brain-derived neurotrophic factor (BDNF) plays crucial roles in the development, maintenance, plasticity and homeostasis of the central and peripheral nervous systems. Perturbing BDNF signaling in mouse brain results in hyperphagia, obesity, hyperinsulinemia and hyperglycemia. Currently, little is known whether BDNF affects liver tissue directly. Our aim was to determine the metabolic signaling pathways activated after BDNF treatment in hepatocytes. Unlike its effect in the brain, BDNF did not lead to activation of the liver AKT pathway. However, AMP protein activated kinase (AMPK) was ∼3 times more active and fatty acid synthase (FAS) ∼2-fold less active, suggesting increased fatty acid oxidation and reduced fatty acid synthesis. In addition, cAMP response element binding protein (CREB) was ∼3.5-fold less active together with its output the gluconeogenic transcript phosphoenolpyruvate carboxykinase (Pepck), suggesting reduced gluconeogenesis. The levels of glycogen synthase kinase 3b (GSK3b) was ∼3-fold higher suggesting increased glycogen synthesis. In parallel, the expression levels of the clock genes Bmal1 and Cry1, whose protein products play also a metabolic role, were ∼2-fold increased and decreased, respectively. In conclusion, BDNF binding to hepatocytes leads to activation of catabolic pathways, such as fatty acid oxidation. In parallel gluconeogenesis is inhibited, while glycogen storage is triggered. This metabolic state mimics that of after breakfast, in which the liver continues to oxidize fat, stops gluconeogenesis and replenishes glycogen stores. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Down-regulation of MiR-127 facilitates hepatocyte proliferation during rat liver regeneration.

    Directory of Open Access Journals (Sweden)

    Chuanyong Pan

    Full Text Available Liver regeneration (LR after partial hepatectomy (PH involves the proliferation and apoptosis of hepatocytes, and microRNAs have been shown to post-transcriptionally regulate genes involved in the regulation of these processes. To explore the role of miR-127 during LR, the expression patterns of miR-127 and its related proteins were investigated. MiR-127 was introduced into a rat liver cell line to examine its effects on the potential target genes Bcl6 and Setd8, and functional studies were undertaken. We discovered that miR-127 was down-regulated and inversely correlated with the expression of Bcl6 and Setd8 at 24 hours after PH, a time at which hypermethylation of the promoter region of the miR-127 gene was detected. Furthermore, in BRL-3A rat liver cells, we observed that overexpression of miR-127 significantly suppressed cell growth and directly inhibited the expression of Bcl6 and Setd8. The results suggest that down-regulation of miR-127 may be due to the rapid methylation of its promoter during the first 24 h after PH, and this event facilitates hepatocyte proliferation by releasing Bcl6 and Setd8. These findings support a miRNA-mediated negative regulation pattern in LR and implicate an anti-proliferative role for miR-127 in liver cells.

  13. Metabolism of oxycodone in human hepatocytes from different age groups and prediction of hepatic plasma clearance

    Directory of Open Access Journals (Sweden)

    Timo eKorjamo

    2012-01-01

    Full Text Available Oxycodone is commonly used to treat severe pain in adults and children. It is extensively metabolized in the liver in adults, but the maturation of metabolism is not well understood. Our aim was to study the metabolism of oxycodone in cryopreserved human hepatocytes from different age groups (3 days, 2 and 5 months, 4 years, adult pool and predict hepatic plasma clearance of oxycodone using these data. Oxycodone (0.1, 1 and 10 µM was incubated with hepatocytes for 4 hours, and 1 µM oxycodone also with CYP3A inhibitor ketoconazole (1 µM. Oxycodone and noroxycodone concentrations were determined at several time points with liquid chromatography-mass spectrometry. In vitro clearance of oxycodone was used to predict hepatic plasma clearance, using the well-stirred model and published physiological parameters. Noroxycodone was the major metabolite in all batches and ketoconazole inhibited the metabolism markedly in most cases. A clear correlation between in vitro oxycodone clearance and CYP3A4 activity was observed. The predicted hepatic plasma clearances were typically much lower than the published median total plasma clearance from pharmacokinetic studies. In general, this in vitro to in vivo extrapolation method provides valuable information on the maturation of oxycodone metabolism that can be utilized in the design of clinical pharmacokinetic studies in infants and young children.

  14. [Influence of specification on chemical composition of dissolution and hepatocytes toxicity of Polygonum multiflorum].

    Science.gov (United States)

    lI, Yu-Meng; Li, Rui-Yu; Niu, Ming; Li, Chun-Yu; Bai, Zhao-Fang; Feng, Wu-Wen; Zhang, Cong-En; Tan, Peng; Huang, Zhi-Pu; Ma, Wei-Guang; Wang, Jia-Bo; Xiao, Xiao-He

    2016-03-01

    According to different toxicities of various aqueous extracts of Polygonum multiflorum on hepatocyte, the impacts of chemical composition on the safety of P. multiforum was studied. In this study, 8 main chemical compositions in aqueous extracts of P. multiflorum were determined by the established HPLC method; at the same time, the inhibition ratios of different aqueous extracts of P. multiflorum on L02 cell were determined. Afterwards, the potential compounds related to the toxicity of P. multiforum were tentatively found through a multiple correlation analysis. The results showed that P. multiforum with different chemical compositions exhibited great differences in dissolution. The hepatocyte toxicity of P. multiflorum powder was much greater than P. multiflorum lumps. In addition, three constituents closely related to toxicity of P. multiflorum were found by multiple correlation analysis. The study revealed that chemical composition of P. multiflorum is closely related to the hepatotoxicity, and the hepatotoxicity of P. multiflorum powder is greater than that of other dosage forms. This study indicates that P. multiflorum with different chemical compositions show varying toxicity, which therefore shall be given high attention. Copyright© by the Chinese Pharmaceutical Association.

  15. A continuous culture of pluripotent fetal hepatocytes derived from the 8-day epiblast of the pig.

    Science.gov (United States)

    Talbot, N C; Rexroad, C E; Powell, A M; Pursel, V G; Caperna, T J; Ogg, S L; Nel, N D

    1994-12-01

    Continuous cultures of pluripotent parenchymal hepatocytes were derived from the epiblasts of 8-day-old pig blastocysts. The cells were polygonal and had phase-contrast dark, granular cytoplasm with prominent nuclei and nucleoli. These feeder-dependent cell cultures differentiated into large, multicellular, secretory, duct-like structures or formed small canaliculi between individual cells. Alternatively, the cells accumulated droplets that stained intensely with Oil Red O, a lipid-specific stain. Alpha-fetoprotein (AFP), albumin, and beta-fibrinogen mRNAs were expressed as the cells differentiated in culture. Serum-free medium that was conditioned by the cells contained transferrin, AFP, and albumin. The growth and viability of the cells were inhibited by transforming growth factor beta 1 (TGF beta 1) at concentrations > or = 1 ng/ml. The cell cultures grew slowly with doubling times of 2 to 3 d. One of the cultures, pig inner cell mass-19 (PICM-19), was passaged continuously for over 2 yr [> 100 population doublings (PD)] and appears to be an infinitely self-renewing cell population. The stem cell characteristics of the epiblast-derived fetal hepatocytes indicate that the cells may be unique for investigations of liver differentiation and organogenesis.

  16. Resistin induces insulin resistance, but does not affect glucose output in rat-derived hepatocytes.

    Science.gov (United States)

    Liu, Feng; Yang, Tao; Wang, Bin; Zhang, Min; Gu, Nan; Qiu, Jie; Fan, Hong-qi; Zhang, Chun-mei; Fei, Li; Pan, Xiao-qing; Guo, Mei; Chen, Rong-hua; Guo, Xi-rong

    2008-01-01

    The aim of the present study was to observe the effects of resistin on insulin sensitivity and glucose output in rat-derived hepatocytes. The rat hepatoma cell line H4IIE was cultured and stimulated with resistin; supernant glucose and glycogen content were detected. The insulin receptor substrate (IRS)-1 and IRS-2, protein kinase B/Akt, glycogen synthase kinase-3beta(GSK-3 beta), the suppressor of cytokine signaling 3 (SOCS-3) protein content, as well as the phosphorylation status were assessed by Western blotting. Specific antisense oligodeoxynucleotides directed against SOCS-3 were used to knockdown SOCS-3. Resistin induced insulin resistance, but did not affect glucose output in rat hepatoma cell line H4IIE. Resistin attenuated multiple effects of insulin, including insulin-stimulated glycogen synthesis and phosphorylation of IRS, protein kinase B/Akt, as well as GSK-3beta. Resistin treatment markedly induced the gene and protein expression of SOCS-3, a known inhibitor of insulin signaling. Furthermore, a specific antisense oligodeoxynucleotide directed against SOCS-3 treatment prevented resistin from antagonizing insulin action. The major function of resistin on liver is to induce insulin resistance. SOCS-3 induction may contribute to the resistin-mediated inhibition of insulin signaling in H4IIE hepatocytes.

  17. Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration

    National Research Council Canada - National Science Library

    Malato, Yann; Naqvi, Syed; Schürmann, Nina; Ng, Raymond; Wang, Bruce; Zape, Joan; Kay, Mark A; Grimm, Dirk; Willenbring, Holger

    2011-01-01

    .... To test these concepts, we generated a hepatocyte fate-tracing model based on timed and specific Cre recombinase expression and marker gene activation in all hepatocytes of adult Rosa26 reporter mice...

  18. No evidence for protective erythropoietin alpha signalling in rat hepatocytes

    Directory of Open Access Journals (Sweden)

    Frede Stilla

    2009-04-01

    Full Text Available Abstract Background Recombinant human erythropoietin alpha (rHu-EPO has been reported to protect the liver of rats and mice from ischemia-reperfusion injury. However, direct protective effects of rHu-EPO on hepatocytes and the responsible signalling pathways have not yet been described. The aim of the present work was to study the protective effect of rHu-EPO on warm hypoxia-reoxygenation and cold-induced injury to hepatocytes and the rHu-EPO-dependent signalling involved. Methods Loss of viability of isolated rat hepatocytes subjected to hypoxia/reoxygenation or incubated at 4°C followed by rewarming was determined from released lactate dehydrogenase activity in the absence and presence of rHu-EPO (0.2–100 U/ml. Apoptotic nuclear morphology was assessed by fluorescence microscopy using the nuclear fluorophores H33342 and propidium iodide. Erythropoietin receptor (EPOR, EPO and Bcl-2 mRNAs were quantified by real time PCR. Activation of JAK-2, STAT-3 and STAT-5 in hepatocytes and rat livers perfused in situ was assessed by Western blotting. Results In contrast to previous in vivo studies on ischemia-reperfusion injury to the liver, rHu-EPO was without any protective effect on hypoxic injury, hypoxia-reoxygenation injury and cold-induced apoptosis to isolated cultured rat hepatocytes. EPOR mRNA was identified in these cells but specific detection of the EPO receptor protein was not possible due to the lack of antibody specificity. Both, in the cultured rat hepatocytes (10 U/ml for 15 minutes and in the rat liver perfused in situ with rHu-EPO (8.9 U/ml for 15 minutes no evidence for EPO-dependent signalling was found as indicated by missing effects of rHu-EPO on phosphorylation of JAK-2, STAT-3 and STAT-5 and on the induction of Bcl-2 mRNA. Conclusion Together, these results indicate the absence of any protective EPO signalling in rat hepatocytes. This implies that the protection provided by rHu-EPO in vivo against ischemia-reperfusion and

  19. Effects of pharmaceuticals on the expression of genes involved in detoxification in a carp primary hepatocyte model.

    Science.gov (United States)

    Corcoran, Jenna; Lange, Anke; Winter, Matthew J; Tyler, Charles R

    2012-06-05

    Fish in many surface freshwaters are exposed to a range of pharmaceuticals via wastewater treatment works effluent discharges. In mammals the pregnane X receptor (PXR) plays a key role in the regulation of a suite of genes involved in drug biotransformation, but information on the role of this response pathway in fish is limited. Here we investigated the effects of exposure of carp (Cyprinus carpio) primary hepatocytes to the human PXR agonist rifampicin (RIF) on expression of target genes involved in phase I (cyp2k, cyp3a) and phase II (gstα, gstπ) drug metabolism and drug transporters mdr1 and mrp2. RIF induced expression of all target genes measured and the PXR antagonist ketoconazole (KET) inhibited responses of cyp2k and cyp3a. Exposure of the primary carp hepatocytes to the pharmaceuticals ibuprofen (IBU), clotrimazole (CTZ), clofibric acid (CFA) and propranolol (PRP), found responses to IBU and CFA, but not CTZ or PRP. This is in contrast with mammals, where CTZ is a potent PXR-agonist. Collectively our data indicate potential PXR involvement in regulating selected genes involved in drug metabolism in fish, but suggest some divergence in the regulation pathways with those in mammals. The carp primary hepatocyte model serves as a useful system for screening for responses in these target genes involved in drug metabolism.

  20. Histone deacetylase inhibitor valproic acid promotes the differentiation of human induced pluripotent stem cells into hepatocyte-like cells.

    Directory of Open Access Journals (Sweden)

    Yuki Kondo

    Full Text Available In this study, we aimed to elucidate the effects and mechanism of action of valproic acid on hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. Human induced pluripotent stem cells were differentiated into endodermal cells in the presence of activin A and then into hepatic progenitor cells using dimethyl sulfoxide. Hepatic progenitor cells were matured in the presence of hepatocyte growth factor, oncostatin M, and dexamethasone with valproic acid that was added during the maturation process. After 25 days of differentiation, cells expressed hepatic marker genes and drug-metabolizing enzymes and exhibited drug-metabolizing enzyme activities. These expression levels and activities were increased by treatment with valproic acid, the timing and duration of which were important parameters to promote differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells into hepatocytes. Valproic acid inhibited histone deacetylase activity during differentiation of human induced pluripotent stem cells, and other histone deacetylase inhibitors also enhanced differentiation into hepatocytes. In conclusion, histone deacetylase inhibitors such as valproic acid can be used to promote hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells.

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  6. File list: Oth.Liv.05.AllAg.Hepatocytes [Chip-atlas[Archive

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    Full Text Available Oth.Liv.05.AllAg.Hepatocytes hg19 TFs and others Liver Hepatocytes SRX530184,SRX530...186 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Liv.05.AllAg.Hepatocytes.bed ...

  7. File list: His.Liv.10.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Liv.10.AllAg.Hepatocytes hg19 Histone Liver Hepatocytes SRX1013892,SRX1013890,S...RX1013888,SRX1013889,SRX1013891 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Liv.10.AllAg.Hepatocytes.bed ...

  8. Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration

    Science.gov (United States)

    Malato, Yann; Naqvi, Syed; Schürmann, Nina; Ng, Raymond; Wang, Bruce; Zape, Joan; Kay, Mark A.; Grimm, Dirk; Willenbring, Holger

    2011-01-01

    Recent evidence has contradicted the prevailing view that homeostasis and regeneration of the adult liver are mediated by self duplication of lineage-restricted hepatocytes and biliary epithelial cells. These new data suggest that liver progenitor cells do not function solely as a backup system in chronic liver injury; rather, they also produce hepatocytes after acute injury and are in fact the main source of new hepatocytes during normal hepatocyte turnover. In addition, other evidence suggests that hepatocytes are capable of lineage conversion, acting as precursors of biliary epithelial cells during biliary injury. To test these concepts, we generated a hepatocyte fate-tracing model based on timed and specific Cre recombinase expression and marker gene activation in all hepatocytes of adult Rosa26 reporter mice with an adenoassociated viral vector. We found that newly formed hepatocytes derived from preexisting hepatocytes in the normal liver and that liver progenitor cells contributed minimally to acute hepatocyte regeneration. Further, we found no evidence that biliary injury induced conversion of hepatocytes into biliary epithelial cells. These results therefore restore the previously prevailing paradigms of liver homeostasis and regeneration. In addition, our new vector system will be a valuable tool for timed, efficient, and specific loop out of floxed sequences in hepatocytes. PMID:22105172

  9. File list: NoD.Liv.05.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Liv.05.AllAg.Hepatocytes mm9 No description Liver Hepatocytes ERX008721,ERX1129...,ERX008753,ERX008747,ERX008746,ERX113010 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Liv.05.AllAg.Hepatocytes.bed ...

  10. Effect of trifluoperazine on toxicity, HIF-1α induction and hepatocyte regeneration in acetaminophen toxicity in mice

    Energy Technology Data Exchange (ETDEWEB)

    Chaudhuri, Shubhra, E-mail: SCHAUDHURI@uams.edu [Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States); Arkansas Children' s Hospital Research Institute, Little Rock, AR (United States); McCullough, Sandra S., E-mail: mcculloughsandras@uams.edu [Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States); Arkansas Children' s Hospital Research Institute, Little Rock, AR (United States); Hennings, Leah, E-mail: lhennings@uams.edu [Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States); Arkansas Children' s Hospital Research Institute, Little Rock, AR (United States); Brown, Aliza T., E-mail: brownalizat@uams.edu [Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States); Arkansas Children' s Hospital Research Institute, Little Rock, AR (United States); Li, Shun-Hwa [Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI (United States); Simpson, Pippa M., E-mail: psimpson@mcw.edu [Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI (United States); Hinson, Jack A., E-mail: hinsonjacka@uams.edu [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, Arkansas Children' s Hospital Research Institute, Little Rock, AR (United States); James, Laura P., E-mail: jameslaurap@uams.edu [Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States); Arkansas Children' s Hospital Research Institute, Little Rock, AR (United States); Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, Arkansas Children' s Hospital Research Institute, Little Rock, AR (United States)

    2012-10-15

    Oxidative stress and mitochondrial permeability transition (MPT) are important mechanisms in acetaminophen (APAP) toxicity. The MPT inhibitor trifluoperazine (TFP) reduced MPT, oxidative stress, and toxicity in freshly isolated hepatocytes treated with APAP. Since hypoxia inducible factor-one alpha (HIF-1α) is induced very early in APAP toxicity, a role for oxidative stress in the induction has been postulated. In the present study, the effect of TFP on toxicity and HIF-1α induction in B6C3F1 male mice treated with APAP was examined. Mice received TFP (10 mg/kg, oral gavage) prior to APAP (200 mg/kg IP) and at 7 and 36 h after APAP. Measures of metabolism (hepatic glutathione and APAP protein adducts) were comparable in the two groups of mice. Toxicity was decreased in the APAP/TFP mice at 2, 4, and 8 h, compared to the APAP mice. At 24 and 48 h, there were no significant differences in toxicity between the two groups. TFP lowered HIF-1α induction but also reduced the expression of proliferating cell nuclear antigen, a marker of hepatocyte regeneration. TFP can also inhibit phospholipase A{sub 2}, and cytosolic and secretory PLA{sub 2} activity levels were reduced in the APAP/TFP mice compared to the APAP mice. TFP also lowered prostaglandin E{sub 2} expression, a known mechanism of cytoprotection. In summary, the MPT inhibitor TFP delayed the onset of toxicity and lowered HIF-1α induction in APAP treated mice. TFP also reduced PGE{sub 2} expression and hepatocyte regeneration, likely through a mechanism involving PLA{sub 2}. -- Highlights: ► Trifluoperazine reduced acetaminophen toxicity and lowered HIF-1α induction. ► Trifluoperazine had no effect on the metabolism of acetaminophen. ► Trifluoperazine reduced hepatocyte regeneration. ► Trifluoperazine reduced phospholipase A{sub 2} activity and prostaglandin E{sub 2} levels.

  11. TGFbeta induces apoptosis and EMT in primary mouse hepatocytes independently of p53, p21Cip1 or Rb status

    Directory of Open Access Journals (Sweden)

    Harrison David J

    2008-07-01

    Full Text Available Abstract Background TGFβ has pleiotropic effects that range from regulation of proliferation and apoptosis to morphological changes and epithelial-mesenchymal transition (EMT. Some evidence suggests that these effects may be interconnected. We have recently reported that P53, P21Cip1 and pRB, three critical regulators of the G1/S transition are variably involved in TGFβ-induced cell cycle arrest in hepatocytes. As these proteins are also involved in the regulation of apoptosis in many circumstances, we investigated their contribution to other relevant TGFβ-induced effects, namely apoptosis and EMT, and examined how the various processes were interrelated. Methods Primary mouse hepatocytes deficient in p53, p21 and/or Rb, singly or in combination were treated with TGFβ for 24 to 96 hours. Apoptosis was quantified according to morphology and by immunostaining for cleaved-capsase 3. Epithelial and mesenchymal marker expression was studied using immunocytochemistry and real time PCR. Results We found that TGFβ similarly induced morphological changes regardless of genotype and independently of proliferation index or sensitivity to inhibition of proliferation by TGFβ. Morphological changes were accompanied by decrease in E-cadherin and increased Snail expression but the mesenchymal markers (N-cadherin, SMAα and Vimentin studied remained unchanged. TGFβ induced high levels of apoptosis in p53-/-, Rb-/-, p21cip1-/- and control hepatocytes although with slight differences in kinetics. This was unrelated to proliferation or changes in morphology and loss of cell-cell adhesion. However, hepatocytes deficient in both p53 and p21cip1were less sensitive to TGFβ-induced apoptosis. Conclusion Although p53, p21Cip1 and pRb are well known regulators of both proliferation and apoptosis in response to a multitude of stresses, we conclude that they are critical for TGFβ-driven inhibition of hepatocytes proliferation, but only slightly modulate TGF

  12. A biochemical and NMR spectroscopic study of hydrazine in the isolated rat hepatocyte

    Energy Technology Data Exchange (ETDEWEB)

    Ghatineh, S.; Morgan, W.; Timbrell, J.A. (London School of Pharmacy (United Kingdom). Dept. of Toxicology); Preece, N.E. (Hunterian Inst., Royal Coll. of Surgeons, London (United Kingdom). Dept. of Biophysics)

    1992-11-01

    Using isolated rat hepatocytes the biochemical effects of hydrazine have been investigated using both conventional assay techniques and high resolution proton NMR. High resolution proton NMR revealed that hydrazine caused a significant increase in alanine and lactate levels in the incubation buffer, whereas levels of [beta]-hydroxybutyrate were decreased. NMR also detected metabolites of hydrazine notably acetylhydrazine and a cyclised hydrazone formed with [alpha]-ketoglutarate. Changes were detected in NADH and NADPH, ATP, succinate dehydrogenase (SDH) and total non-protein sulphydryl groups (TNPSH). However, the changes in pyridine nucleotides occurred at higher concentrations than those affecting succinate dehydrogenase and ATP. Similarly, the depletion of TNPSH occurred at a higher concentration and with a different time course to that seen with ATP depletion and inhibition of succinate dehydrogenase. (orig.).

  13. Insulin infusion reduces hepatocyte growth factor in lean humans

    DEFF Research Database (Denmark)

    de Courten, Barbora; de Courten, Maximilian; Dougherty, Sonia

    2013-01-01

    OBJECTIVE: Plasma Hepatocyte Growth Factor (HGF) is significantly elevated in obesity and may contribute to vascular disease, metabolic syndrome or cancer in obese individuals. The current studies were done to determine if hyperinsulinemia increases plasma HGF. MATERIALS/METHODS: Twenty-two parti...

  14. Metformin Stimulates FGF21 Expression in Primary Hepatocytes

    Directory of Open Access Journals (Sweden)

    Eva B. Nygaard

    2012-01-01

    Full Text Available Fibroblast growth factor 21 (FGF21 is a novel metabolic regulator of glucose and lipid metabolism; however, the exact mechanism of action and regulation of FGF21 is not fully understood. Metabolic status plays an important role in the regulation of FGF21, and we therefore examined whether metformin, an indirect AMPK-activator, regulates FGF21 expression in hepatocytes. FGF21 mRNA and protein expression were determined after incubation of primary cultured rat and human hepatocytes with metformin for 24 hours. To study the role of AMPK in the putative regulation of FGF21, hepatocytes were incubated with Compound C (an AMPK inhibitor in the presence of metformin. A strong dose-dependent increase in FGF21 expression was observed in both rat and human hepatocytes treated with metformin. This effect was blocked by addition of the AMPK-inhibitor Compound C. The study shows that metformin is a potent inducer of hepatic FGF21 expression and that the effect of metformin seems to be mediated through AMPK activation. As FGF21 therapy normalizes blood glucose in animal models of type 2 diabetes, the induction of hepatic FGF21 by metformin might play an important role in metformin’s antidiabetic effect.

  15. The use of pig hepatocytes for biotransformation and toxicity studies

    NARCIS (Netherlands)

    Hoogenboom, L.A.P.

    1991-01-01

    The three main objectives of this study were, (1) to investigate the possibility to isolate viable hepatocytes from liver samples of pigs, (2) to study their use for biotransformation and toxicity studies, and (3) to demonstrate the value of this model, in particular in the field of residue

  16. 3D Cultivation Techniques for Primary Human Hepatocytes

    Directory of Open Access Journals (Sweden)

    Anastasia Bachmann

    2015-02-01

    Full Text Available One of the main challenges in drug development is the prediction of in vivo toxicity based on in vitro data. The standard cultivation system for primary human hepatocytes is based on monolayer cultures, even if it is known that these conditions result in a loss of hepatocyte morphology and of liver-specific functions, such as drug-metabolizing enzymes and transporters. As it has been demonstrated that hepatocytes embedded between two sheets of collagen maintain their function, various hydrogels and scaffolds for the 3D cultivation of hepatocytes have been developed. To further improve or maintain hepatic functions, 3D cultivation has been combined with perfusion. In this manuscript, we discuss the benefits and drawbacks of different 3D microfluidic devices. For most systems that are currently available, the main issues are the requirement of large cell numbers, the low throughput, and expensive equipment, which render these devices unattractive for research and the drug-developing industry. A higher acceptance of these devices could be achieved by their simplification and their compatibility with high-throughput, as both aspects are of major importance for a user-friendly device.

  17. Hepatocytes: a key cell type for innate immunity.

    Science.gov (United States)

    Zhou, Zhou; Xu, Ming-Jiang; Gao, Bin

    2016-05-01

    Hepatocytes, the major parenchymal cells in the liver, play pivotal roles in metabolism, detoxification, and protein synthesis. Hepatocytes also activate innate immunity against invading microorganisms by secreting innate immunity proteins. These proteins include bactericidal proteins that directly kill bacteria, opsonins that assist in the phagocytosis of foreign bacteria, iron-sequestering proteins that block iron uptake by bacteria, several soluble factors that regulate lipopolysaccharide signaling, and the coagulation factor fibrinogen that activates innate immunity. In this review, we summarize the wide variety of innate immunity proteins produced by hepatocytes and discuss liver-enriched transcription factors (e.g. hepatocyte nuclear factors and CCAAT/enhancer-binding proteins), pro-inflammatory mediators (e.g. interleukin (IL)-6, IL-22, IL-1β and tumor necrosis factor-α), and downstream signaling pathways (e.g. signal transducer and activator of transcription factor 3 and nuclear factor-κB) that regulate the expression of these innate immunity proteins. We also briefly discuss the dysregulation of these innate immunity proteins in chronic liver disease, which may contribute to an increased susceptibility to bacterial infection in patients with cirrhosis.

  18. Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes

    Directory of Open Access Journals (Sweden)

    Yue Wang

    2016-11-01

    Full Text Available The toxicity and carcinogenicity of aniline in humans and animals have been well documented. However, the molecular mechanism involved in aniline-induced liver toxicity and carcinogenesis remains unclear. In our research, primary cultured hepatocytes were exposed to aniline (0, 1.25, 2.50, 5.0 and 10.0 μg/mL for 24 h in the presence or absence of N-acetyl-l-cysteine (NAC. Levels of reactive oxygen species (ROS, malondialdehyde (MDA, and glutathione (GSH, activities of superoxide dismutase (SOD and catalase (CAT, mitochondrial membrane potential, DNA damage, cell viability, and apoptosis were detected. Levels of ROS and MDA were significantly increased and levels of GSH and CAT, activity of SOD, and mitochondrial membrane potential in hepatocytes were significantly decreased by aniline compared with the negative control group. The tail moment and DNA content of the tail in exposed groups were significantly higher than those in the negative control group. Cell viability was reduced and apoptotic death was induced by aniline in a concentration-dependent manner. The phenomena of ROS generation, oxidative damage, loss of mitochondrial membrane potential, DNA damage and apoptosis could be prevented if ROS inhibitor NAC was added. ROS generation is involved in the loss of mitochondrial membrane potential and DNA injury, which may play a role in aniline-induced apoptosis in hepatocytes. Our study provides insight into the mechanism of aniline-induced toxicity and apoptosis of hepatocytes.

  19. Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes.

    Science.gov (United States)

    Wang, Yue; Gao, Hong; Na, Xiao-Lin; Dong, Shu-Ying; Dong, Hong-Wei; Yu, Jia; Jia, Li; Wu, Yong-Hui

    2016-11-30

    The toxicity and carcinogenicity of aniline in humans and animals have been well documented. However, the molecular mechanism involved in aniline-induced liver toxicity and carcinogenesis remains unclear. In our research, primary cultured hepatocytes were exposed to aniline (0, 1.25, 2.50, 5.0 and 10.0 μg/mL) for 24 h in the presence or absence of N-acetyl-l-cysteine (NAC). Levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH), activities of superoxide dismutase (SOD) and catalase (CAT), mitochondrial membrane potential, DNA damage, cell viability, and apoptosis were detected. Levels of ROS and MDA were significantly increased and levels of GSH and CAT, activity of SOD, and mitochondrial membrane potential in hepatocytes were significantly decreased by aniline compared with the negative control group. The tail moment and DNA content of the tail in exposed groups were significantly higher than those in the negative control group. Cell viability was reduced and apoptotic death was induced by aniline in a concentration-dependent manner. The phenomena of ROS generation, oxidative damage, loss of mitochondrial membrane potential, DNA damage and apoptosis could be prevented if ROS inhibitor NAC was added. ROS generation is involved in the loss of mitochondrial membrane potential and DNA injury, which may play a role in aniline-induced apoptosis in hepatocytes. Our study provides insight into the mechanism of aniline-induced toxicity and apoptosis of hepatocytes.

  20. Hepatocyte growth factor activator inhibitor-2 prevents shedding of matriptase

    DEFF Research Database (Denmark)

    Larsen, Brian R; Steffensen, Simon D R; Nielsen, Nis Valentin Ladefoged

    2013-01-01

    Hepatocyte growth factor activator inhibitor-2 (HAI-2) is an inhibitor of many proteases in vitro, including the membrane-bound serine protease, matriptase. Studies of knock-out mice have shown that HAI-2 is essential for placental development only in mice expressing matriptase, suggesting that HAI...

  1. Biotransformation of dimetridazole by primary cultures of pig hepatocytes

    NARCIS (Netherlands)

    Hoogenboom, L.A.P.; Polman, Th.H.G.; Rhijn, van J.A.; Heskamp, H.H.; Foster, B.C.; Kuiper, H.A.

    1997-01-01

    Monolayer cultures of pig hepatocytes were used to investigate the role of the liver in the biotransformation of the veterinary drug dimetridazole (1,2-dimethyl-5-nitroimidazole). 14C-Labeled dimetridazole (DMZ) was primarily hydroxylated to 1-methyl-2-hydroxymethyl-5-nitroimidazole (up to 90%) and

  2. Gene expression and functional analyses of primary rat hepatocytes on nanofiber matrices.

    Science.gov (United States)

    Brophy, Colleen M; Luebke-Wheeler, Jennifer L; Amiot, Bruce P; Remmel, Rory P; Rinaldo, Piero; Nyberg, Scott L

    2010-01-01

    Long term culture of primary hepatocytes is valuable for diagnostic and therapeutic applications. However, standard monolayer culture of primary hepatocytes on tissue culture plastic (TCP) - either uncoated or coated with a biological material such as collagen or laminin - is problematic. Thus, novel support matrices are under development to better maintain gene expression and differentiated function of primary hepatocytes in vitro. In this study, a fabricated nanofiber matrix was compared to control conditions of uncoated and laminin-coated TCP. Gene expression and biochemical analyses were performed to compare functional abilities of the hepatocytes in the different conditions. Hepatocytes cultured on nanofibers maintained higher cytochrome P450 1A activity (0.49 +/- 0.08 ng resorufin/ml/min) compared to hepatocytes on laminin (0.11 +/- 0.05 ng resorufin/ml/min). In addition, albumin production of hepatocytes on nanofibers was greater than twice the production of hepatocytes on laminin (day 14, 34.4 +/- 1.8 vs. 15.9 +/- 4.5 microg albumin/ml/day). Hepatocytes demonstrated the ability to generate urea from ammonia in all conditions; however, hepatocytes performed ureagenesis more effectively on nanofibers than on laminin (0.55 +/- 0.25 microM vs. 0.36 +/- 0.24 microM urea, day 14). Gene expression of hepatocytes cultured on nanofiber and laminin conditions were similar on a per cell basis determined by analysis using a custom microarray of 250 genes expressed in hepatocytes. Similar cell attachment data between conditions and similar numbers of cells expressing the hepatocyte marker hepatocyte nuclear factor 4alphaindicates that hepatocytes grown on nanofibers only marginally display improved hepatic functions compared to laminin control conditions. Copyright 2009 S. Karger AG, Basel.

  3. Toluene metabolism in isolated rat hepatocytes: effects of in vivo pretreatment with acetone and phenobarbital.

    Science.gov (United States)

    Smith-Kielland, A; Ripel, A

    1993-01-01

    Hepatocytes isolated from control, acetone- and phenobarbital-pretreated rats were used to study the metabolic conversion of toluene to benzyl alcohol, benzaldehyde, benzoic acid and hippuric acid at low (toluene concentrations. The baseline formation rates of toluene metabolites (benzyl alcohol, benzoic acid and hippuric acid) were 2.9 +/- 1.7 and 10.0 +/- 2.3 nmol/mg cell protein/60 min at low and high toluene concentrations, respectively. In vivo pretreatment of rats with acetone and phenobarbital increased the formation of metabolites: at low toluene concentrations 3- and 5-fold, respectively; at high toluene concentrations no significant increase (acetone) and 8-fold increase (phenobarbital). Apparent inhibition by ethanol, 7 and 60 mM, was most prominent at low toluene concentrations: 63% and 69%, respectively, in control cells; 84% and 91% in acetone-pretreated cells, and 32% (not significant) and 51% in phenobarbital-pretreated cells. Ethanol also caused accumulation of benzyl alcohol. The apparent inhibition by isoniazid was similar to that of ethanol at low toluene concentrations. Control and acetone-pretreated cells were apparently resistant towards metyrapone; the decrease was 49% and 64% in phenobarbital-pretreated cells at low and high toluene concentrations, respectively. In these cells, the decrease in presence of combined ethanol and metyrapone was 95% (low toluene concentrations). 4-Methyl-pyrazole decreased metabolite formation extensively in all groups. Benzaldehyde was only found in the presence of an aldehyde dehydrogenase inhibitor. Increased ratio benzoic/hippuric acid was observed at high toluene concentrations. These results demonstrate that toluene oxidation may be studied by product formation in isolated hepatocytes. However, the influence of various enzymes in the overall metabolism could not be ascertained due to lack of inhibitor specificity.

  4. Toluene metabolism in isolated rat hepatocytes: effects of in vivo pretreatment with acetone and phenobarbital

    Energy Technology Data Exchange (ETDEWEB)

    Smith-Kielland, A.; Ripel, A. (National Inst. of Forensic Toxicology, Oslo (Norway))

    1993-02-01

    Hepatocytes isolated from control, acetone- and phenobarbital-pretreated rats were used to study the metabolic conversion of toluene to benzyl alcohol, benzaldehyde, benzoic acid and hippuric acid at low (<100 [mu]M) and high (100-500 [mu]M) toluene concentrations. The baseline formation rates of toluene metabolites (benzyl alcohol, benzoic acid and hippuric acid) were 2.9[+-]1.7 and 10.0[+-]2.3 nmol/mg cell protein/60 min at low and high toluene concentrations, respectively. In vivo pretreatment of rats with acetone and phenobarbital increased the formation of metabolites: at low toluene concentrations 3- and 5-fold, respectively; at high toluene concentrations no significant increase (acetone) and 8-fold increase (phenobarbital). Apparent inhibition by ethanol, 7 and 60 mM, was most prominent at low toluene concentrations: 63% and 69%, respectively, in control cells; 84% and 91% in acetone-pretreated cells, and 32% (not significant) and 51% in phenobarbital-pretreated cells. Ethanol also caused accumulation of benzyl alcohol. The apparent inhibition by isoniazid was similar to that of ethanol at low toluene concentrations. Control and acetone-pretreated cells were apparently resistant towards metyrapone; the decrease was 49% and 64% in phenobarbital-pretreated cells at low and high toluene concentrations, respectively. In these cells, the decrease in presence of combined ethanol and metyrapone was 95% (low toluene concentrations). 4-Methylpyrazole decreased metabolite formation extensively in all groups. Benzaldehyde was only found in the presence of an aldehyde dehydrogenase inhibitor. Increased ratio benzoic/hippuric acid was observed at high toluene concentrations. These results demonstrate that toluene oxidation may be studied by product formation in isolated hepatocytes. However, the influence of various enzymes in the overall metabolism could not be ascertained due to lack of inhibitor specificity. (orig.).

  5. Genomics and proteomics analysis of cultured primary rat hepatocytes.

    Science.gov (United States)

    Beigel, Juergen; Fella, Kerstin; Kramer, Peter-Juergen; Kroeger, Michaela; Hewitt, Philip

    2008-02-01

    The use of animal models in pharmaceutical research is a costly and sometimes misleading method of generating toxicity data and hence predicting human safety. Therefore, in vitro test systems, such as primary rat hepatocytes, and the developing genomics and proteomics technologies, are playing an increasingly important role in toxicological research. Gene and protein expression analysis were investigated in a time series (up to 5 days) of primary rat hepatocytes cultured on collagen coated dishes. Especially after 24h, a significant down-regulation of many important Phase I and Phase II enzymes (e.g., cytochrome P450's, glutathione-S-transferases, sulfotransferases) involved in xenobiotic metabolism, and antioxidative enzymes (e.g., catalase, superoxide dismutase, glutathione peroxidase) was observed. Acute-phase-response enzymes were frequently up-regulated (e.g., LPS binding protein, alpha-2-macro-globulin, ferritin, serine proteinase inhibitor B, haptoglobin), which is likely to be a result of cellular stress caused by the cell isolation procedure (perfusion) itself. A parallel observation was the increased expression of several structural genes (e.g., beta-actin, alpha-tubulin, vimentin), possibly caused by other proliferating cell types in the culture, such as fibroblasts or alternatively by hepatocyte dedifferentiation. In conclusion, the careful interpretation of data derived from this in vitro system indicates that primary hepatocytes can be successfully used for short-term toxicity studies up to 24h. However, culturing conditions need to be further optimized to reduce the massive changes of gene and protein expression of long-term cultured hepatocytes to allow practical applications as a long-term toxicity test system.

  6. [Proliferation and cell death of hepatocytes in regenerating rat fetal liver].

    Science.gov (United States)

    El'chaninov, A V; Bol'shakova, G B

    2012-01-01

    Proliferation and death of hepatocytes in regenerating liver of 17-day white rat fetuses were investigated. During 2 days after liver resection (20%), animals were sacrificed every 3 h. In experimental groups, the index of Ki67-positive hepatocytes increased sharply in 15 h after liver resection. In all experimental and control groups, the ratio of the metaphase, the longest phase of mitosis, and index to mitotic index remained unchanged, indicating identical duration of hepatocytes mitoses in regenerating liver. In the regenerating and intact liver hepatocytes labeled with antibodies to caspase 3 were not detected. Thus, resection of 20% rat fetal liver did not contribute to increased apoptosis of hepatocytes.

  7. Antiviral activity and host gene induction by tamarin and marmoset interferon-alpha and interferon-gamma in the GBV-B primary hepatocyte culture model.

    Science.gov (United States)

    Chavez, Deborah; Guerra, Bernadette; Lanford, Robert E

    2009-08-01

    GBV-B induces hepatitis in tamarins and marmosets and is a surrogate model for HCV infections. Here, we cloned and characterized the antiviral activity of tamarin and marmoset interferon (IFN)alpha and IFN gamma. Potent antiviral activity was observed for tamarin and marmoset IFN alpha in primary hepatocyte cultures infected with GBV-B. The antiviral activity was greater in cultures exposed to IFN alpha prior to GBV-B infection, suggesting that either GBV-B was capable of inhibition of the antiviral activity of exogenous IFN alpha or that the preexisting endogenous IFN response to the virus reduced efficacy to exogenous IFN alpha. IFN gamma also exhibited antiviral activity in GBV-B infected hepatocytes. The transcriptional response to IFN alpha in marmoset hepatocytes was characterized using human genome microarrays. Since the GBV-B hepatocyte culture model possesses a functional innate immune response, it will provide opportunities to explore the nature of the antiviral response to a virus closely related to HCV.

  8. In vitro toxicity of perfluorooctane sulfonate on rat liver hepatocytes: probability of distructive binding to CYP 2E1 and involvement of cellular proteolysis.

    Science.gov (United States)

    Khansari, Mehdi Rajabnia; Yousefsani, Bahareh Sadat; Kobarfard, Farzad; Faizi, Mehrdad; Pourahmad, Jalal

    2017-10-01

    Perfluorooctanesulfonate (PFOS), an anthropogenic fluorosurfactant, is one of the most common global pollutants. PFOS is used in various consumer products to provide soil, oil, and water resistance to materials used in clothing, upholstery, and food packaging. PFOS is persistent, bioaccumulative, and toxic to mammalian species. In this study, the cellular mechanisms involved in PFOS hepatotoxicity were evaluated. For this purpose, we determined oxidative stress markers including cell lysis, ROS generation, lipid peroxidation, glutathione depletion, mitochondrial membrane potential decrease, lysosomal membrane leakiness, and cellular proteolysis. Our results demonstrated that PFOS liver cytotoxicity was associated with reactive oxygen species (ROS) formation and lipid peroxidation in isolated rat hepatocytes. Incubation of hepatocytes with PFOS caused rapid depletion of hepatocyte glutathione (GSH), an important marker of cellular oxidative stress. Most of the PFOS-induced GSH depletion could be attributed to the expulsion of glutathione disulfide (GSSG). PFOS hepatotoxicity was inhibited by antioxidants and ROS scavengers, mitochondrial permeability transition (MPT) pore sealing agents, and endocytosis inhibitors. Our results suggest that PFOS hepatotoxicity might be the result of oxidative stress-induced lysosomal membrane leakiness and cellular proteolysis in rat hepatocytes.

  9. Activation of AMPK by Buddleja officinalis Maxim. Flower Extract Contributes to Protecting Hepatocytes from Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Ji Yun Jung

    2017-01-01

    Full Text Available The Buddleja officinalis Maxim. flower is used in traditional Chinese and Korean medicine to treat inflammation, vascular diseases, headache, and stroke, as well as enhance liver function. This research investigated the effects of B. officinalis Maxim. flower extract (BFE on hepatotoxicity. The cytoprotective effects and mechanism of BFE against severe mitochondrial dysfunction and H2O2 production in hepatotoxicity induced by coadministration of arachidonic acid (AA and iron were observed in the HepG2 cell line. In addition, we performed blood biochemical, histopathological, and histomorphometric analyses of mice with carbon tetrachloride- (CCl4- induced acute liver damage. BFE inhibited the AA + iron-mediated hepatotoxicity of HepG2 cells. Moreover, it inhibited mitochondrial dysfunction, H2O2 production, and glutathione depletion mediated by AA + iron in the same cells. Meanwhile, the cytoprotective effects of BFE against oxidative stress were associated with the activation of AMP-activated protein kinase (AMPK. In particular, based on the histopathological observations, BFE (30 and 100 mg/kg showed clear hepatoprotective effects against CCl4-induced acute hepatic damage. Furthermore, it inhibited 4-hydroxynonenal and nitrotyrosine immunoreactivity in hepatocytes. These results provide evidence that BFE has beneficial hepatoprotective effects against hepatic damage via the activation of AMPK pathway. Accordingly, BFE may have therapeutic potential for diverse liver disorders.

  10. Activation of AMPK by Buddleja officinalis Maxim. Flower Extract Contributes to Protecting Hepatocytes from Oxidative Stress

    Science.gov (United States)

    Jung, Ji Yun; Jegal, Kyung Hwan; Kim, Jae Kwang; Park, Chung A.; Jung, Dae Hwa; Ku, Sae Kwang

    2017-01-01

    The Buddleja officinalis Maxim. flower is used in traditional Chinese and Korean medicine to treat inflammation, vascular diseases, headache, and stroke, as well as enhance liver function. This research investigated the effects of B. officinalis Maxim. flower extract (BFE) on hepatotoxicity. The cytoprotective effects and mechanism of BFE against severe mitochondrial dysfunction and H2O2 production in hepatotoxicity induced by coadministration of arachidonic acid (AA) and iron were observed in the HepG2 cell line. In addition, we performed blood biochemical, histopathological, and histomorphometric analyses of mice with carbon tetrachloride- (CCl4-) induced acute liver damage. BFE inhibited the AA + iron-mediated hepatotoxicity of HepG2 cells. Moreover, it inhibited mitochondrial dysfunction, H2O2 production, and glutathione depletion mediated by AA + iron in the same cells. Meanwhile, the cytoprotective effects of BFE against oxidative stress were associated with the activation of AMP-activated protein kinase (AMPK). In particular, based on the histopathological observations, BFE (30 and 100 mg/kg) showed clear hepatoprotective effects against CCl4-induced acute hepatic damage. Furthermore, it inhibited 4-hydroxynonenal and nitrotyrosine immunoreactivity in hepatocytes. These results provide evidence that BFE has beneficial hepatoprotective effects against hepatic damage via the activation of AMPK pathway. Accordingly, BFE may have therapeutic potential for diverse liver disorders. PMID:28473864

  11. [Current status and future perspectives of hepatocyte transplantation].

    Science.gov (United States)

    Pareja, Eugenia; Cortés, Miriam; Gómez-Lechón, M José; Maupoey, Javier; San Juan, Fernando; López, Rafael; Mir, Jose

    2014-02-01

    The imbalance between the number of potential beneficiaries and available organs, originates the search for new therapeutic alternatives, such as Hepatocyte transplantation (HT).Even though this is a treatment option for these patients, the lack of unanimity of criteria regarding indications and technique, different cryopreservation protocols, as well as the different methodology to assess the response to this therapy, highlights the need of a Consensus Conference to standardize criteria and consider future strategies to improve the technique and optimize the results.Our aim is to review and update the current state of hepatocyte transplantation, emphasizing the future research attempting to solve the problems and improve the results of this treatment. Copyright © 2013 AEC. Published by Elsevier Espana. All rights reserved.

  12. Insulin induces calcium signals in the nucleus of rat hepatocytes.

    Science.gov (United States)

    Rodrigues, Michele A; Gomes, Dawidson A; Andrade, Viviane A; Leite, M Fatima; Nathanson, Michael H

    2008-11-01

    Insulin is an hepatic mitogen that promotes liver regeneration. Actions of insulin are mediated by the insulin receptor, which is a receptor tyrosine kinase. It is currently thought that signaling via the insulin receptor occurs at the plasma membrane, where it binds to insulin. Here we report that insulin induces calcium oscillations in isolated rat hepatocytes, and that these calcium signals depend upon activation of phospholipase C and the inositol 1,4,5-trisphosphate receptor, but not upon extracellular calcium. Furthermore, insulin-induced calcium signals occur in the nucleus, and are temporally associated with selective depletion of nuclear phosphatidylinositol bisphosphate and translocation of the insulin receptor to the nucleus. These findings suggest that the insulin receptor translocates to the nucleus to initiate nuclear, inositol 1,4,5-trisphosphate-mediated calcium signals in rat hepatocytes. This novel signaling mechanism may be responsible for insulin's effects on liver growth and regeneration.

  13. DIFFERENTIAL-EFFECTS OF EICOSAPENTAENOIC ACID ON GLYCEROLIPID AND APOLIPOPROTEIN-B METABOLISM IN PRIMARY HUMAN HEPATOCYTES COMPARED TO HEPG2 CELLS AND PRIMARY RAT HEPATOCYTES

    NARCIS (Netherlands)

    LIN, YG; SMIT, MJ; HAVINGA, R; VERKADE, HJ; VONK, RJ; KUIPERS, F

    1995-01-01

    We compared the effects of eicosapentaenoic acid (EPA) and oleic acid (OA) on glycerolipid and apolipoprotein B (apoB) metabolism in primary human hepatocytes, HepG2 cells and primary rat hepatocytes. Cells were incubated for 1 to 5 h with 0.25 mM bovine serum albumin in the absence (control) or

  14. A role for lipid rafts in the protection afforded by docosahexaenoic acid against ethanol toxicity in primary rat hepatocytes.

    Science.gov (United States)

    Aliche-Djoudi, Fatiha; Podechard, Normand; Collin, Aurore; Chevanne, Martine; Provost, Emilie; Poul, Martine; Le Hégarat, Ludovic; Catheline, Daniel; Legrand, Philippe; Dimanche-Boitrel, Marie-Thérèse; Lagadic-Gossmann, Dominique; Sergent, Odile

    2013-10-01

    Previously, we demonstrated that eicosapentaenoic acid enhanced ethanol-induced oxidative stress and cell death in primary rat hepatocytes via an increase in membrane fluidity and lipid raft clustering. In this context, another n-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA), was tested with a special emphasis on physical and chemical alteration of lipid rafts. Pretreatment of hepatocytes with DHA reduced significantly ethanol-induced oxidative stress and cell death. DHA protection could be related to an alteration of lipid rafts. Indeed, rafts exhibited a marked increase in membrane fluidity and packing defects leading to the exclusion of a raft protein marker, flotillin. Furthermore, DHA strongly inhibited disulfide bridge formation, even in control cells, thus suggesting a disruption of protein-protein interactions inside lipid rafts. This particular spatial organization of lipid rafts due to DHA subsequently prevented the ethanol-induced lipid raft clustering. Such a prevention was then responsible for the inhibition of phospholipase C-γ translocation into rafts, and consequently of both lysosome accumulation and elevation in cellular low-molecular-weight iron content, a prooxidant factor. In total, the present study suggests that DHA supplementation could represent a new preventive approach for patients with alcoholic liver disease based upon modulation of the membrane structures. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Glutathione deficiency induced by cystine and/or methionine deprivation does not affect thyroid hormone deiodination in cultured rat hepatocytes and monkey hepatocarcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Sato, K.; Robbins, J.

    1981-09-01

    To elucidate the recently advanced hypothesis that glutathione (L-gamma-glutamyl-L-cysteinyl glycine (GSH)) regulates deiodinating enzyme activities, accounting for the decreased conversion of T4 to T3 in the liver of fetal and starved animals, we investigated thyroid hormone metabolism in GSH-depleted neoplastic and normal hepatocytes. In monkey hepatocarcinoma cells, intracellular total GSH decreased below 10% of the control value (approximately 25 micrograms/mg protein) when cells were grown for 44 h in medium deficient in cystine and methionine or in cystine alone. The latter finding indicated that transsulfuration from methionine to cysteine was defective in these neoplastic cells. In primary cultured adult rat hepatocytes, on the other hand, the transsulfuration pathway was intact, and total GSH decreased below 10% of control (approximately 20 micrograms/mg protein) only in cells grown in cystine- and methionine-deficient medium. In both cell types, the oxidized GSH fraction remained constant (2-5% of total). Incubation with 125I-labeled T4 and T3, followed by chromatography, was used to evaluate 5-deiodination in hepatocarcinoma cells and both 5- and 5'-deiodination in normal hepatocytes. Deiodination was not decreased by GSH deficiency in either case, but was actually increased in hepatocarcinoma cells. This resulted from an increase in the Vmax of 5-deiodinase related to growth arrest. Diamide at 2 mM reversibly inhibited both 5'- and 5'-deiodination in rat hepatocytes, accompanied by decreased total GSH as well as increased GSH disulfide (27% of total). The data suggest that GSH is so abundant in the liver that hepatocytes can tolerate a greater than 90% decrease in intracellular concentration without any change in thyroid hormone deiodination and indicate that altered thyroid hormone metabolism in the fetus and in starvation cannot be accounted for by a decreased hepatic GSH concentration.

  16. Ectopic adenine nucleotide translocase activity controls extracellular ADP levels and regulates the F1-ATPase-mediated HDL endocytosis pathway on hepatocytes.

    Science.gov (United States)

    Cardouat, G; Duparc, T; Fried, S; Perret, B; Najib, S; Martinez, L O

    2017-09-01

    Ecto-F 1 -ATPase is a complex related to mitochondrial ATP synthase which has been identified as a plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein of high-density lipoprotein (HDL), and has been shown to contribute to HDL endocytosis in several cell types. On hepatocytes, apoA-I binding to ecto-F 1 -ATPase stimulates extracellular ATP hydrolysis into ADP, which subsequently activates a P2Y 13 -mediated HDL endocytosis pathway. Interestingly, other mitochondrial proteins have been found to be expressed at the plasma membrane of several cell types. Among these, adenine nucleotide translocase (ANT) is an ADP/ATP carrier but its role in controlling extracellular ADP levels and F 1 -ATPase-mediated HDL endocytosis has never been investigated. Here we confirmed the presence of ANT at the plasma membrane of human hepatocytes. We then showed that ecto-ANT activity increases or reduces extracellular ADP level, depending on the extracellular ADP/ATP ratio. Interestingly, ecto-ANT co-localized with ecto-F 1 -ATPase at the hepatocyte plasma membrane and pharmacological inhibition of ecto-ANT activity increased extracellular ADP level when ecto-F 1 -ATPase was activated by apoA-I. This increase in the bioavailability of extracellular ADP accordingly translated into an increase of HDL endocytosis on human hepatocytes. This study thus uncovered a new location and function of ANT for which activity at the cell surface of hepatocytes modulates the concentration of extracellular ADP and regulates HDL endocytosis. Copyright © 2017. Published by Elsevier B.V.

  17. Alginate Encapsulation of Human Hepatocytes and Assessment of Microbeads.

    Science.gov (United States)

    Mitry, Ragai R; Jitraruch, Suttiruk; Iansante, Valeria; Dhawan, Anil

    2017-01-01

    Alginate encapsulation of cells is an attractive technique in which alginate becomes polymerized entrapping the cells. The structure of formed microbeads/microcapsules is semipermeable as it allows oxygen and nutrients to go in, and waste products and other materials produced by the cells to go out. Here, we describe basic protocols for alginate encapsulation of human hepatocytes and methods for assessing the microbeads produced.

  18. Toxic compounds of Curcuma aeruginosa causes necrosis of mice hepatocytes

    Directory of Open Access Journals (Sweden)

    Eka Pramyrtha Hestianah

    2014-08-01

    Full Text Available BACKGROUND People have been using Curcuma aeruginosa rhizome as a traditional herbal medicine as appetite stimulant, without realizing its side effects. Herbal plants contain tens to hundreds of compounds, some of which are toxic. The aim of this research was to determine which toxic compound of Curcuma aeruginosa rhizome has an impact on apoptosis and PARP-1 expression of hepatocytes in male mice. METHODS Eighty eight male Balb C mice were divided into 10 groups treated respectively with Curcuma aeruginosa rhizome cloroform extract, methanol extract, essential oil, infusion, and press juice, at dosages of 0.004g/kgBW and 0.06g/kgBW, and 1 control group. The treatment was given orally once a day for 10 days and on the 11th day, the research animals were sacrificed, and their liver taken for histopathologic slide preparation with Apopteq Detection Kit, and immunofluorescence. Compounds in Curcuma aeruginosa rhizome were analyzed with gas chromatography/mass spectrometry. The data obtained were analyzed by one-way ANOVA, and Partial Least Squares to determine which compounds had an impact on murine hepatocytes. RESULTS The result of one way ANOVA showed that the chloroform groups at dosages of 0.004g/kgBW and 0.06g/kgBW showed the highest apoptosis of mice’s hepatocytes (p<0.05. There were significant differences in PARP-1 expression between control and treatment groups. The highest PARP-1 expression was in the essential oil group at a dosage of 0.06g/kg BW (p<0.05. CONCLUSION Curcuma aeruginosa rhizome given to mice orally causes necrosis of mice’s hepatocytes.

  19. Toxic compounds of Curcuma aeruginosa causes necrosis of mice hepatocytes

    Directory of Open Access Journals (Sweden)

    Eka Pramyrtha Hestianah

    2015-12-01

    Full Text Available BACKGROUND People have been using Curcuma aeruginosa rhizome as a traditional herbal medicine as appetite stimulant, without realizing its side effects. Herbal plants contain tens to hundreds of compounds, some of which are toxic. The aim of this research was to determine which toxic compound of Curcuma aeruginosa rhizome has an impact on apoptosis and PARP-1 expression of hepatocytes in male mice. METHODS Eighty eight male Balb C mice were divided into 10 groups treated respectively with Curcuma aeruginosa rhizome cloroform extract, methanol extract, essential oil, infusion, and press juice, at dosages of 0.004g/kgBW and 0.06g/kgBW, and 1 control group. The treatment was given orally once a day for 10 days and on the 11th day, the research animals were sacrificed, and their liver taken for histopathologic slide preparation with Apopteq Detection Kit, and immunofluorescence. Compounds in Curcuma aeruginosa rhizome were analyzed with gas chromatography/mass spectrometry. The data obtained were analyzed by one-way ANOVA, and Partial Least Squares to determine which compounds had an impact on murine hepatocytes. RESULTS The result of one way ANOVA showed that the chloroform groups at dosages of 0.004g/kgBW and 0.06g/kgBW showed the highest apoptosis of mice’s hepatocytes (p<0.05. There were significant differences in PARP-1 expression between control and treatment groups. The highest PARP-1 expression was in the essential oil group at a dosage of 0.06g/kg BW (p<0.05. CONCLUSION Curcuma aeruginosa rhizome given to mice orally causes necrosis of mice’s hepatocytes.

  20. Hepatocyte-specific ABCA1 transfer increases HDL cholesterol but impairs HDL function and accelerates atherosclerosis.

    Science.gov (United States)

    Feng, Yingmei; Lievens, Joke; Jacobs, Frank; Hoekstra, Menno; Van Craeyveld, Eline; Gordts, Stephanie C; Snoeys, Jan; De Geest, Bart

    2010-11-01

    The ATP-binding cassette transporter A1 (ABCA1) lipidates apolipoprotein (apo) A-I. The hypothesis that hepatocyte-specific ABCA1 overexpression results in high-density lipoprotein (HDL) dysfunction was evaluated by comparing the effects of murine ABCA1 (AdABCA1) and human apo A-I (AdA-I) transfer on lipoprotein profile, HDL function, and progression of atherosclerosis. Gene transfer in male and female C57BL/6 apo E(-/-) mice was performed at the age of 3 months with E1E3E4-deleted adenoviral vectors containing hepatocyte-specific expression cassettes. Atherosclerosis was quantified at baseline and 56 days later in AdABCA1, AdA-I, and control mice. HDL cholesterol after AdA-I transfer was 1.7-fold (P < 0.001) and 1.8-fold (P < 0.001) higher in male and female mice, respectively, and potently inhibited atherosclerosis progression compared with respective controls. Notwithstanding a 1.4-fold (P < 0.01) and a 1.7-fold (P < 0.01) increase of HDL cholesterol in male and female mice, respectively, after AdABCA1 transfer, the intima was 2.2-fold (P < 0.001) larger in male and 1.3-fold (P = NS) larger in female mice compared with respective controls. HDL isolated from control and AdA-I mice but not from AdABCA1 mice enhanced endothelial progenitor cell (EPC) migration in vitro and reduced endothelial cell death in vitro after serum and growth factor withdrawal. Scavenger receptor class B type I (SR-BI) protein level in the liver was significantly lower in AdABCA1 mice than in control and AdA-I mice. Hepatocyte-specific ABCA1 transfer decreases SR-BI protein level in the liver and abrogates beneficial effects of HDL on EPCs and endothelial cells. Decreased HDL function may underlie accelerated atherosclerosis in AdABCA1 apo E(-/-)mice.

  1. Direct lineage conversion of terminally differentiated hepatocytes to functional neurons.

    Science.gov (United States)

    Marro, Samuele; Pang, Zhiping P; Yang, Nan; Tsai, Miao-Chih; Qu, Kun; Chang, Howard Y; Südhof, Thomas C; Wernig, Marius

    2011-10-04

    Several recent studies have showed that mouse and human fibroblasts can be directly reprogrammed into induced neuronal (iN) cells, bypassing a pluripotent intermediate state. However, fibroblasts represent heterogeneous mesenchymal progenitor cells that potentially contain neural crest lineages, and the cell of origin remained undefined. This raises the fundamental question of whether lineage reprogramming is possible between cell types derived from different germ layers. Here, we demonstrate that terminally differentiated hepatocytes can be directly converted into functional iN cells. Importantly, single-cell and genome-wide expression analyses showed that fibroblast- and hepatocyte-derived iN cells not only induced a neuronal transcriptional program, but also silenced their donor transcriptome. The remaining donor signature decreased over time and could not support functional hepatocyte properties. Thus, the reprogramming factors lead to a binary lineage switch decision rather than an induction of hybrid phenotypes, but iN cells retain a small but detectable epigenetic memory of their donor cells. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Hepatocyte and Sertoli Cell Aquaporins, Recent Advances and Research Trends

    Directory of Open Access Journals (Sweden)

    Raquel L. Bernardino

    2016-07-01

    Full Text Available Aquaporins (AQPs are proteinaceous channels widespread in nature where they allow facilitated permeation of water and uncharged through cellular membranes. AQPs play a number of important roles in both health and disease. This review focuses on the most recent advances and research trends regarding the expression and modulation, as well as physiological and pathophysiological functions of AQPs in hepatocytes and Sertoli cells (SCs. Besides their involvement in bile formation, hepatocyte AQPs are involved in maintaining energy balance acting in hepatic gluconeogenesis and lipid metabolism, and in critical processes such as ammonia detoxification and mitochondrial output of hydrogen peroxide. Roles are played in clinical disorders including fatty liver disease, diabetes, obesity, cholestasis, hepatic cirrhosis and hepatocarcinoma. In the seminiferous tubules, particularly in SCs, AQPs are also widely expressed and seem to be implicated in the various stages of spermatogenesis. Like in hepatocytes, AQPs may be involved in maintaining energy homeostasis in these cells and have a major role in the metabolic cooperation established in the testicular tissue. Altogether, this information represents the mainstay of current and future investigation in an expanding field.

  3. Evidence for a channel for the electrogenic transport of chloride ion in the rat hepatocyte

    Energy Technology Data Exchange (ETDEWEB)

    Bear, C.E.; Petrunka, C.N.; Strasberg, S.M.

    1985-05-01

    Chloride is the major inorganic anion in bile but its mechanism of passage from blood to bile is uncertain. Specific membrane channels account for most net inorganic anion flux in other cell types such as the proximal tubular cell and red blood cell; disulfonic stilbenes inhibit anion movement through these channels. Therefore, we have sought the presence of similar channels in the hepatocyte. Net inorganic anion flux or conductance was initiated in isolated rat hepatocytes by valinomycin in the presence of an outward potassium gradient. Potassium concentration in the extracellular medium increased from 2.75 +/- 0.02 in control cell suspensions to 3.15 +/- 0.04 in valinomycin-treated cell suspensions. Membrane potential difference (Em) (mV), determined as the distribution of (/sup 14/C)tetraphenyl phosphonium ion was -28 mV in control cells and -42 mV in valinomycin-treated cells. Intracellular chloride concentration (/sup 36/Cl-) (mEq per liter of cell water) decreased significantly from 38.6 in control cells to 32.0 in valinomycin-treated cells. The observed intracellular concentrations (/sup 36/Cl-) in both control and valinomycin-treated cell suspensions closely approximates values predicted on the basis of the Nernst equation: 41 and 29 (mEq per liter of cell water), respectively, suggesting that the chloride ion is passively distributed on the basis of the membrane potential difference. Furthermore, net rate-limited cell water loss of approximately 15% of control values was associated with the above valinomycin-stimulated changes in ion distribution, as assessed using three methods of cell water volume determination.

  4. 5'-methylthioadenosine modulates the inflammatory response to endotoxin in mice and in rat hepatocytes.

    Science.gov (United States)

    Hevia, Henar; Varela-Rey, Marta; Corrales, Fernando J; Berasain, Carmen; Martínez-Chantar, María L; Latasa, M Ujue; Lu, Shelly C; Mato, José M; García-Trevijano, Elena R; Avila, Matías A

    2004-04-01

    5'-methylthioadenosine (MTA) is a nucleoside generated from S-adenosylmethionine (AdoMet) during polyamine synthesis. Recent evidence indicates that AdoMet modulates in vivo the production of inflammatory mediators. We have evaluated the anti-inflammatory properties of MTA in bacterial lipopolysaccharide (LPS) challenged mice, murine macrophage RAW 264.7 cells, and isolated rat hepatocytes treated with pro-inflammatory cytokines. MTA administration completely prevented LPS-induced lethality. The life-sparing effect of MTA was accompanied by the suppression of circulating tumor necrosis factor-alpha (TNF-alpha), inducible NO synthase (iNOS) expression, and by the stimulation of IL-10 synthesis. These responses to MTA were also observed in LPS-treated RAW 264.7 cells. MTA prevented the transcriptional activation of iNOS by pro-inflammatory cytokines in isolated hepatocytes, and the induction of cyclooxygenase 2 (COX2) in RAW 264.7 cells. MTA inhibited the activation of p38 mitogen-activated protein kinase (MAPK), c-jun phosphorylation, inhibitor kappa B alpha (IkappaBalpha) degradation, and nuclear factor kappaB (NFkappaB) activation, all of which are signaling pathways related to the generation of inflammatory mediators. These effects were independent of the metabolic conversion of MTA into AdoMet and the potential interaction of MTA with the cAMP signaling pathway, central to the anti-inflammatory actions of its structural analog adenosine. In conclusion, these observations demonstrate novel immunomodulatory properties for MTA that may be of value in the management of inflammatory diseases.

  5. Efavirenz and 8-hydroxyefavirenz induce cell death via a JNK- and BimEL-dependent mechanism in primary human hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bumpus, Namandje N., E-mail: nbumpus1@jhmi.edu

    2011-12-15

    Chronic use of efavirenz (EFV) has been linked to incidences of hepatotoxicity in patients receiving EFV to treat HIV-1. While recent studies have demonstrated that EFV stimulates hepatic cell death a role for the metabolites of efavirenz in this process has yet to be examined. In the present study, incubation of primary human hepatocytes with synthetic 8-hydroxyEFV (8-OHEFV), which is the primary metabolite of EFV, resulted in cell death, caspase-3 activation and reactive oxygen species formation. The metabolite exerted these effects at earlier time points and using lower concentrations than were required for the parent compound. In addition, pharmacological inhibition of cytochrome P450-dependent metabolism of EFV using 1-aminobenzotriazole markedly decreased reactive oxygen species formation and cell death. Treatment of primary human hepatocytes with EFV and 8-OHEFV also stimulated phosphorylation of c-Jun N-terminal kinase (JNK) as well as phosphorylation of the JNK substrate c-Jun. Further, the mRNA and protein expression of an isoform of Bim (Bcl-2 interacting mediator of cell death) denoted as BimEL, which is proapoptotic and has been shown to be modulated by JNK, was increased. Inhibition of JNK using SP600125 prevented the EFV- and 8-OHEFV-mediated cell death. Silencing of Bim using siRNA transfected into hepatocytes also prevented cell death resulting from 8-OHEFV-treatment. These data suggest that the oxidative metabolite 8-OHEFV is a more potent inducer of hepatic cell death than the parent compound EFV. Further, activation of the JNK signaling pathway and BimEL mRNA expression appear to be required for EFV- and 8-OHEFV-mediated hepatocyte death. -- Highlights: Black-Right-Pointing-Pointer 8-Hydroxyefavirenz is a more potent stimulator of cell death than efavirenz. Black-Right-Pointing-Pointer Efavirenz and 8-hydroxyefavirenz increase JNK activity and BimEL mRNA expression. Black-Right-Pointing-Pointer JNK and Bim are required for efavirenz- and 8

  6. Alginate-based microcapsules with galactosylated chitosan internal for primary hepatocyte applications.

    Science.gov (United States)

    Lou, Ruyun; Xie, Hongguo; Zheng, Huizhen; Ren, Ying; Gao, Meng; Guo, Xin; Song, Yizhe; Yu, Weiting; Liu, Xiudong; Ma, Xiaojun

    2016-12-01

    Alginate-galactosylated chitosan/polylysine (AGCP) microcapsules with excellent stability and high permeability were developed and employed in primary hepatocyte applications. The galactosylated chitosan (GC), synthesized via the covalent coupling of lactobionic acid (LA) with low molecular weight and water-soluble chitosan (CS), was ingeniously introduced into the core of alginate microcapsules by regulating the pH of gelling bath. The internal GC of the microcapsules simultaneously provided a large number of binding sites for the hepatocytes and further promoted the hepatocyte-matrix interactions via the recognition of asialoglycoprotein receptors (ASGPRs) on the hepatocyte surface, and afforded the AGCP microcapsules an excellent stability via the electrostatic interactions with alginate. As a consequence, primary hepatocytes in AGCP microcapsules demonstrated enhanced viability, urea synthesis, albumin secretion, and P-450 enzyme activity, showing great prospects for hepatocyte applications in microcapsule system. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Freshly isolated hepatocyte transplantation in acetaminophen-induced hepatotoxicity model in rats

    Directory of Open Access Journals (Sweden)

    Daniela Rodrigues

    2012-12-01

    Full Text Available CONTEXT: Hepatocyte transplantation is an attractive therapeutic modality for liver disease as an alternative for orthotopic liver transplantation. OBJECTIVE: The aim of the current study was to investigate the feasibility of freshly isolated rat hepatocyte transplantation in acetaminophen-induced hepatotoxicity model. METHODS: Hepatocytes were isolated from male Wistar rats and transplanted 24 hours after acetaminophen administration in female recipients. Female rats received either 1x10(7 hepatocytes or phosphate buffered saline through the portal vein or into the spleen and were sacrificed after 48 hours. RESULTS: Alanine aminotransferase levels measured within the experiment did not differ between groups at any time point. Molecular analysis and histology showed presence of hepatocytes in liver of transplanted animals injected either through portal vein or spleen. CONCLUSION: These data demonstrate the feasibility and efficacy of hepatocyte transplantation in the liver or spleen in a mild acetaminophen-induced hepatotoxicity model.

  8. Fermented wheat powder induces the antioxidant and detoxifying system in primary rat hepatocytes.

    Directory of Open Access Journals (Sweden)

    Margherita La Marca

    Full Text Available Many plants exhibit antioxidant properties which may be useful in the prevention of oxidative stress reactions, such as those mediated by the formation of free radical species in different pathological situations. In recent years a number of studies have shown that whole grain products in particular have strong antioxidant activity. Primary cultures of rat hepatocytes were used to investigate whether and how a fermented powder of wheat (Lisosan G is able to modulate antioxidant and detoxifying enzymes, and whether or not it can activate Nrf2 transcription factor or inhibit NF-kB activation. All of the antioxidant and detoxifying enzymes studied were significantly up-regulated by 0.7 mg/ml Lisosan G treatment. In particular,quinone oxidoreductase and heme oxygenase-1 were induced, although to different degrees, at the transcriptional, protein and/or activity levels by the treatment. As for the Nrf2 transcription factor, a partial translocation of its protein from the cytosol to the nucleus after 1 h of Lisosan G treatment was revealed by immunoblotting. Lisosan G was also observed to decrease H2O2-induced toxicity Taken together, these results show that this powder of wheat is an effective inducer of ARE/Nrf2-regulated antioxidant and detoxifying genes and has the potential to inhibit the translocation of NF-kB into the nucleus.

  9. The Slit/Robo system suppresses hepatocyte growth factor-dependent invasion and morphogenesis.

    Science.gov (United States)

    Stella, Maria Cristina; Trusolino, Livio; Comoglio, Paolo M

    2009-01-01

    The Slit protein acts through the Roundabout receptor as a paracrine chemorepellent in axon guidance and as an inhibitor in leukocyte chemotaxis, but its role in epithelial cell motility and morphogenesis remains largely unexplored. We report that nontransformed epithelial cells and cancerous cells empower the Slit-2/Robo1 signaling system to limit outward migration in response to motogenic attractants and to remain positionally confined within their primitive location. Short hairpin RNA-mediated depletion of SLIT-2 or ectopic expression of a soluble decoy Robo enhance hepatocyte growth factor (HGF)-induced migration, matrix invasion, and tubulogenesis, concomitantly with the up-regulation of Cdc-42 and the down-modulation of Rac-1 activities. Accordingly, autocrine overexpression or exogenous administration of Slit-2 prevent HGF-triggered motile responses, reduce Cdc-42 activation, and stimulate Rac-1. This antimigratory activity of Slit-2 derives from the inhibition of actin-based protrusive forces and from an increased adhesive strength of cadherin-mediated intercellular contacts. These results disclose a novel function for Slit and Robo in the inhibition of growth factor-mediated epithelial cell motility and morphogenesis, invoking a critical role for both molecules as natural antagonists of neoplastic invasive growth.

  10. Antioxidant effect of a fermented powder of Lady Joy bean in primary rat hepatocytes.

    Science.gov (United States)

    La Marca, Margherita; Pucci, Laura; Bollini, Roberto; Russo, Rossella; Sparvoli, Francesca; Gabriele, Morena; Longo, Vincenzo

    2015-03-01

    The role and beneficial effects of plant and food extracts against various diseases induced by oxidative stress have received much attention in recent years. Legumes are rich in bioactive compounds, and some studies suggest a correlation between their consumption and a reduced incidence of diseases. Primary cultures of rat hepatocytes were used to investigate whether and how an extract obtained from a fermented powder of bean named Lady Joy (Phaseolus vulgaris L.) is able to regulate antioxidant and detoxifying enzymes through the NRF2 pathway, inhibit NF-kB activation, and reduce H2O2-induced endoplasmic reticulum (ER) stress. All of the antioxidant and detoxifying enzymes studied were significantly up-regulated by Lady Joy treatment. Western blot showed that Nrf2 was activated by Lady Joy treatment. Also, cells treated with this fermented bean were partially protected against NF-kB activation resulting from H2O2 stress. As a link between oxidative stress and ER dysfunction is hypothesized, we verified whether Lady Joy was able to protect cells from H2O2-induced ER stress, by studying the response of the proteins CHOP, BiP and caspase 12. The results of this study show that Lady Joy can induce the Nrf2 pathway, inhibit NF-kB, and protect ER from stress induced by H2O2.

  11. Fermented wheat powder induces the antioxidant and detoxifying system in primary rat hepatocytes.

    Science.gov (United States)

    La Marca, Margherita; Beffy, Pascale; Pugliese, Annalisa; Longo, Vincenzo

    2013-01-01

    Many plants exhibit antioxidant properties which may be useful in the prevention of oxidative stress reactions, such as those mediated by the formation of free radical species in different pathological situations. In recent years a number of studies have shown that whole grain products in particular have strong antioxidant activity. Primary cultures of rat hepatocytes were used to investigate whether and how a fermented powder of wheat (Lisosan G) is able to modulate antioxidant and detoxifying enzymes, and whether or not it can activate Nrf2 transcription factor or inhibit NF-kB activation. All of the antioxidant and detoxifying enzymes studied were significantly up-regulated by 0.7 mg/ml Lisosan G treatment. In particular, quinone oxidoreductase and heme oxygenase-1 were induced, although to different degrees, at the transcriptional, protein and/or activity levels by the treatment. As for the Nrf2 transcription factor, a partial translocation of its protein from the cytosol to the nucleus after 1 h of Lisosan G treatment was revealed by immunoblotting. Lisosan G was also observed to decrease H2O2-induced toxicity Taken together, these results show that this powder of wheat is an effective inducer of ARE/Nrf2-regulated antioxidant and detoxifying genes and has the potential to inhibit the translocation of NF-kB into the nucleus.

  12. Allicin Modulates the Antioxidation and Detoxification Capabilities of Primary Rat Hepatocytes

    OpenAIRE

    Wu, Chih-Chung; Chu, Yung-Lin; Sheen, Lee-Yan

    2012-01-01

    The effect of allicin, an active ingredient of garlic, on lactate dehydrogenase (LDH) leakage, lipid peroxidation, glutathione (GSH) content, and GSH-related enzyme activity was investigated in primary hepatocytes. In this study, allicin was synthesized in our laboratory as an experimental material, and primary hepatocytes isolated from Sprague-Dawley rats were used as an experimental model. According to the results, hepatocytes treated with 10 μM allicin did not differ from the control on LD...

  13. Cholesterol Enhances the Toxic Effect of Ethanol and Acetaldehyde in Primary Mouse Hepatocytes

    OpenAIRE

    L?pez-Islas, Anayelly; Chagoya-Hazas, Victoria; P?rez-Aguilar, Benjamin; Palestino-Dom?nguez, Mayrel; Souza, Ver?nica; Miranda, Roxana U.; Bucio, Leticia; G?mez-Quiroz, Luis Enrique; Guti?rrez-Ruiz, Mar?a-Concepci?n

    2015-01-01

    Obesity and alcohol consumption are risk factors for hepatic steatosis, and both commonly coexist. Our objective was to evaluate the effect of ethanol and acetaldehyde on primary hepatocytes obtained from mice fed for two days with a high cholesterol (HC) diet. HC hepatocytes increased lipid and cholesterol content. HC diet sensitized hepatocytes to the toxic effect of ethanol and acetaldehyde. Cyp2E1 content increased with HC diet, as well as in those treated with ethanol or acetaldehyde,...

  14. Transcriptional Up-Regulation of APE1/Ref-1 in Hepatic Tumor: Role in Hepatocytes Resistance to Oxidative Stress and Apoptosis.

    Science.gov (United States)

    Di Maso, Vittorio; Mediavilla, María Gabriela; Vascotto, Carlo; Lupo, Francesco; Baccarani, Umberto; Avellini, Claudio; Tell, Gianluca; Tiribelli, Claudio; Crocè, Lory Saveria

    2015-01-01

    Human Hepatocellular Carcinoma (HCC) is the fifth most frequent neoplasm worldwide and the most serious complication of long-standing chronic liver diseases (CLD). Its development is associated with chronic inflammation and sustained oxidative stress. Deregulation of apurinic apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1), a master regulator of cellular response to oxidative stress, has been associated with poor prognosis in several cancers including HCC. In the present study we investigated the APE1/Ref-1 mRNA levels in cirrhotic and HCC tissues obtained during HCC resection. The possible protective role of APE1/Ref-1 against oxidative stress and apoptosis was evaluated in vitro in immortalized human hepatocytes (IHH) over-expressing APE1/Ref-1. APE1/Ref-1 was up-regulated in HCC, regulation occurring at the transcriptional level. APE1/Ref-1 mRNA content increased with the progression of liver disease with the transcriptional up-regulation present in cirrhosis significantly increased in HCC. The up-regulation was higher in the less differentiated cancers. In vitro, over-expression of APE1/Ref-1 in normal hepatocytes conferred cell protection against oxidative stress and it was associated with BAX inhibition and escape from apoptosis. APE1/Ref-1 is up-regulated in HCC and this over-expression correlates with cancer aggressiveness. The up-regulation occurs at the transcriptional level and it is present in the earliest phases of hepatocarcinogenesis. The APE-1/Ref-1 over-expression is associated with hepatocyte survival and inhibits BAX activation and apoptosis. These data suggest a possible role of APE1/Ref-1 over-expression both in hepatocyte survival and HCC development calling attention to this molecule as a promising marker for HCC diagnosis and treatment.

  15. Transcriptional Up-Regulation of APE1/Ref-1 in Hepatic Tumor: Role in Hepatocytes Resistance to Oxidative Stress and Apoptosis.

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    Vittorio Di Maso

    Full Text Available Human Hepatocellular Carcinoma (HCC is the fifth most frequent neoplasm worldwide and the most serious complication of long-standing chronic liver diseases (CLD. Its development is associated with chronic inflammation and sustained oxidative stress. Deregulation of apurinic apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1, a master regulator of cellular response to oxidative stress, has been associated with poor prognosis in several cancers including HCC.In the present study we investigated the APE1/Ref-1 mRNA levels in cirrhotic and HCC tissues obtained during HCC resection. The possible protective role of APE1/Ref-1 against oxidative stress and apoptosis was evaluated in vitro in immortalized human hepatocytes (IHH over-expressing APE1/Ref-1.APE1/Ref-1 was up-regulated in HCC, regulation occurring at the transcriptional level. APE1/Ref-1 mRNA content increased with the progression of liver disease with the transcriptional up-regulation present in cirrhosis significantly increased in HCC. The up-regulation was higher in the less differentiated cancers. In vitro, over-expression of APE1/Ref-1 in normal hepatocytes conferred cell protection against oxidative stress and it was associated with BAX inhibition and escape from apoptosis.APE1/Ref-1 is up-regulated in HCC and this over-expression correlates with cancer aggressiveness. The up-regulation occurs at the transcriptional level and it is present in the earliest phases of hepatocarcinogenesis. The APE-1/Ref-1 over-expression is associated with hepatocyte survival and inhibits BAX activation and apoptosis. These data suggest a possible role of APE1/Ref-1 over-expression both in hepatocyte survival and HCC development calling attention to this molecule as a promising marker for HCC diagnosis and treatment.

  16. Three-dimensional (3D) printing of mouse primary hepatocytes to generate 3D hepatic structure.

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    Kim, Yohan; Kang, Kyojin; Jeong, Jaemin; Paik, Seung Sam; Kim, Ji Sook; Park, Su A; Kim, Wan Doo; Park, Jisun; Choi, Dongho

    2017-02-01

    The major problem in producing artificial livers is that primary hepatocytes cannot be cultured for many days. Recently, 3-dimensional (3D) printing technology draws attention and this technology regarded as a useful tool for current cell biology. By using the 3D bio-printing, these problems can be resolved. To generate 3D bio-printed structures (25 mm × 25 mm), cells-alginate constructs were fabricated by 3D bio-printing system. Mouse primary hepatocytes were isolated from the livers of 6-8 weeks old mice by a 2-step collagenase method. Samples of 4 × 107 hepatocytes with 80%-90% viability were printed with 3% alginate solution, and cultured with well-defined culture medium for primary hepatocytes. To confirm functional ability of hepatocytes cultured on 3D alginate scaffold, we conducted quantitative real-time polymerase chain reaction and immunofluorescence with hepatic marker genes. Isolated primary hepatocytes were printed with alginate. The 3D printed hepatocytes remained alive for 14 days. Gene expression levels of Albumin, HNF-4α and Foxa3 were gradually increased in the 3D structures. Immunofluorescence analysis showed that the primary hepatocytes produced hepatic-specific proteins over the same period of time. Our research indicates that 3D bio-printing technique can be used for long-term culture of primary hepatocytes. It can therefore be used for drug screening and as a potential method of producing artificial livers.

  17. Cholesterol Enhances the Toxic Effect of Ethanol and Acetaldehyde in Primary Mouse Hepatocytes

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    Anayelly López-Islas

    2016-01-01

    Full Text Available Obesity and alcohol consumption are risk factors for hepatic steatosis, and both commonly coexist. Our objective was to evaluate the effect of ethanol and acetaldehyde on primary hepatocytes obtained from mice fed for two days with a high cholesterol (HC diet. HC hepatocytes increased lipid and cholesterol content. HC diet sensitized hepatocytes to the toxic effect of ethanol and acetaldehyde. Cyp2E1 content increased with HC diet, as well as in those treated with ethanol or acetaldehyde, while the activity of this enzyme determined in microsomes increased in the HC and in all ethanol treated hepatocytes, HC and CW. Oxidized proteins were increased in the HC cultures treated or not with the toxins. Transmission electron microscopy showed endoplasmic reticulum (ER stress and megamitochondria in hepatocytes treated with ethanol as in HC and the ethanol HC treated hepatocytes. ER stress determined by PERK content was increased in ethanol treated hepatocytes from HC mice and CW. Nuclear translocation of ATF6 was observed in HC hepatocytes treated with ethanol, results that indicate that lipids overload and ethanol treatment favor ER stress. Oxidative stress, ER stress, and mitochondrial damage underlie potential mechanisms for increased damage in steatotic hepatocyte treated with ethanol.

  18. Cholesterol Enhances the Toxic Effect of Ethanol and Acetaldehyde in Primary Mouse Hepatocytes.

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    López-Islas, Anayelly; Chagoya-Hazas, Victoria; Pérez-Aguilar, Benjamin; Palestino-Domínguez, Mayrel; Souza, Verónica; Miranda, Roxana U; Bucio, Leticia; Gómez-Quiroz, Luis Enrique; Gutiérrez-Ruiz, María-Concepción

    2016-01-01

    Obesity and alcohol consumption are risk factors for hepatic steatosis, and both commonly coexist. Our objective was to evaluate the effect of ethanol and acetaldehyde on primary hepatocytes obtained from mice fed for two days with a high cholesterol (HC) diet. HC hepatocytes increased lipid and cholesterol content. HC diet sensitized hepatocytes to the toxic effect of ethanol and acetaldehyde. Cyp2E1 content increased with HC diet, as well as in those treated with ethanol or acetaldehyde, while the activity of this enzyme determined in microsomes increased in the HC and in all ethanol treated hepatocytes, HC and CW. Oxidized proteins were increased in the HC cultures treated or not with the toxins. Transmission electron microscopy showed endoplasmic reticulum (ER) stress and megamitochondria in hepatocytes treated with ethanol as in HC and the ethanol HC treated hepatocytes. ER stress determined by PERK content was increased in ethanol treated hepatocytes from HC mice and CW. Nuclear translocation of ATF6 was observed in HC hepatocytes treated with ethanol, results that indicate that lipids overload and ethanol treatment favor ER stress. Oxidative stress, ER stress, and mitochondrial damage underlie potential mechanisms for increased damage in steatotic hepatocyte treated with ethanol.

  19. Magnetic cell labeling of primary and stem cell-derived pig hepatocytes for MRI-based cell tracking of hepatocyte transplantation.

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    Dwayne R Roach

    Full Text Available Pig hepatocytes are an important investigational tool for optimizing hepatocyte transplantation schemes in both allogeneic and xenogeneic transplant scenarios. MRI can be used to serially monitor the transplanted cells, but only if the hepatocytes can be labeled with a magnetic particle. In this work, we describe culture conditions for magnetic cell labeling of cells from two different pig hepatocyte cell sources; primary pig hepatocytes (ppHEP and stem cell-derived hepatocytes (PICM-19FF. The magnetic particle is a micron-sized iron oxide particle (MPIO that has been extensively studied for magnetic cell labeling for MRI-based cell tracking. ppHEP could endocytose MPIO with labeling percentages as high as 70%, achieving iron content as high as ~55 pg/cell, with >75% viability. PICM-19FF had labeling >97%, achieving iron content ~38 pg/cell, with viability >99%. Extensive morphological and functional assays indicated that magnetic cell labeling was benign to the cells. The results encourage the use of MRI-based cell tracking for the development and clinical use of hepatocyte transplantation methodologies. Further, these results generally highlight the importance of functional cell assays in the evaluation of contrast agent biocompatibility.

  20. Magnetic cell labeling of primary and stem cell-derived pig hepatocytes for MRI-based cell tracking of hepatocyte transplantation.

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    Roach, Dwayne R; Garrett, Wesley M; Welch, Glenn; Caperna, Thomas J; Talbot, Neil C; Shapiro, Erik M

    2015-01-01

    Pig hepatocytes are an important investigational tool for optimizing hepatocyte transplantation schemes in both allogeneic and xenogeneic transplant scenarios. MRI can be used to serially monitor the transplanted cells, but only if the hepatocytes can be labeled with a magnetic particle. In this work, we describe culture conditions for magnetic cell labeling of cells from two different pig hepatocyte cell sources; primary pig hepatocytes (ppHEP) and stem cell-derived hepatocytes (PICM-19FF). The magnetic particle is a micron-sized iron oxide particle (MPIO) that has been extensively studied for magnetic cell labeling for MRI-based cell tracking. ppHEP could endocytose MPIO with labeling percentages as high as 70%, achieving iron content as high as ~55 pg/cell, with >75% viability. PICM-19FF had labeling >97%, achieving iron content ~38 pg/cell, with viability >99%. Extensive morphological and functional assays indicated that magnetic cell labeling was benign to the cells. The results encourage the use of MRI-based cell tracking for the development and clinical use of hepatocyte transplantation methodologies. Further, these results generally highlight the importance of functional cell assays in the evaluation of contrast agent biocompatibility.

  1. Effect of Non-Esterified Fatty Acids on Fatty Acid Metabolism-Related Genes in Calf Hepatocytes Cultured in Vitro

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    Peng Li

    2013-11-01

    Full Text Available Background: NEFA plays numerous roles in the metabolism of glucose, lipids, and proteins. A number of experimental studies have shown that NEFA may have an important role in fatty acid metabolism in the liver, especially in dairy cows that experience negative energy balance (NEB during early lactation. Methods: In this study, using fluorescent quantitative RT-PCR, ELISA, and primary hepatocytes cultured in vitro, we examined the effect of NEFA (0, 0.2, 0.4, 0.8, 1.6, and 3.2 mmol/L on fatty acid metabolism by monitoring the mRNA and protein expression of the following key enzymes: long chain acyl-CoA synthetase (ACSL, carnitine palmitoyltransferase IA (CPT IA, long chain acyl-CoA dehydrogenase (ACADL, and acetyl-CoA carboxylase (ACC. Results: The mRNA and protein expression levels of ACSL and ACADL markedly increased as the concentration of NEFA in the media was increased. The mRNA and protein expression levels of CPT IA were enhanced significantly when the NEFA concentrations increased from 0 to 1.6 mmol/L and decreased significantly when the NEFA concentrations increased from 1.6 to 3.2 mmol/L. The mRNA and protein expression of ACC decreased gradually with increasing concentrations of NEFA. Conclusion: These findings indicate that increased NEFA significantly promote the activation and β-oxidation of fatty acids, but very high NEFA concentrations may inhibit the translocation of fatty acids into mitochondria of hepatocytes. This may explain the development of ketosis or liver lipidosis in dairy cows. CPT IA might be the key control enzyme of the fatty acid oxidation process in hepatocytes.

  2. Inhibitory effects of metformin on insulin and glucagon action in rat hepatocytes involve post-receptor alterations.

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    Alengrin, F; Grossi, G; Canivet, B; Dolais-Kitabgi, J

    1987-01-01

    The effect of the hypoglycaemic biguanide, metformin, on insulin binding and insulin action was investigated in rat hepatocyte monolayers. The binding of insulin was not modified in cultured cells exposed for 24 or 48 h to metformin at concentrations ranging from 1 mumol/l to 1 mmol/l, and no effect could be detected on insulin-induced down regulation. Metformin did not alter insulin stimulation of amino acid transport but the stimulatory effect of insulin on glycogen synthesis was reduced by 20%, 30% and 63% for metformin at 0.01, 0.1 and 1 mmol/l, respectively. Both responsiveness and sensitivity were altered by the biguanide. Metformin also inhibited basal glycogen synthesis and cellular glycogen contents were markedly decreased after exposure of cells to metformin (0.01-1 mmol/l). We also investigated the effect of metformin on glucagon action and metformin (0.1-1 mmol/l) was found to decrease the stimulatory effect of glucagon on amino acid uptake and on gluconeogenesis from alanine. These inhibitory effects of the biguanide were still observed when glucagon was replaced by dibutyryl cAMP. These in vitro studies demonstrate that: 1) metformin has no direct effect on insulin binding in hepatocytes, indicating that alteration of insulin stimulation of glycogen synthesis is due to modifications at the post receptor level. 2) metformin alters the action of glucagon in hepatocytes at a post AMP cyclase step. They also suggest that one of the mechanism of action of metformin may be to antagonize the effect of glucagon rather than to potentiate the action of insulin.

  3. EPA, DHA, and Lipoic Acid Differentially Modulate the n-3 Fatty Acid Biosynthetic Pathway in Atlantic Salmon Hepatocytes.

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    Bou, Marta; Østbye, Tone-Kari; Berge, Gerd M; Ruyter, Bente

    2017-03-01

    The aim of the present study was to investigate how EPA, DHA, and lipoic acid (LA) influence the different metabolic steps in the n-3 fatty acid (FA) biosynthetic pathway in hepatocytes from Atlantic salmon fed four dietary levels (0, 0.5, 1.0 and 2.0%) of EPA, DHA or a 1:1 mixture of these FA. The hepatocytes were incubated with [1-14C] 18:3n-3 in the presence or absence of LA (0.2 mM). Increased endogenous levels of EPA and/or DHA and LA exposure both led to similar responses in cells with reduced desaturation and elongation of [1-14C] 18:3n-3 to 18:4n-3, 20:4n-3, and EPA, in agreement with reduced expression of the Δ6 desaturase gene involved in the first step of conversion. DHA production, on the other hand, was maintained even in groups with high endogenous levels of DHA, possibly due to a more complex regulation of this last step in the n-3 metabolic pathway. Inhibition of the Δ6 desaturase pathway led to increased direct elongation to 20:3n-3 by both DHA and LA. Possibly the route by 20:3n-3 and then Δ8 desaturation to 20:4n-3, bypassing the first Δ6 desaturase step, can partly explain the maintained or even increased levels of DHA production. LA increased DHA production in the phospholipid fraction of hepatocytes isolated from fish fed 0 and 0.5% EPA and/or DHA, indicating that LA has the potential to further increase the production of this health-beneficial FA in fish fed diets with low levels of EPA and/or DHA.

  4. Phenobarbital Regulates Nuclear Expression of HNF-4α in Mouse and Rat Hepatocytes Independent of CAR and PXR

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    Bell, Aaron W.; Michalopoulos, George K.

    2007-01-01

    Phenobarbital is a lipophilic molecule used as a sedative and antiepileptic drug that elicits a multitude of effects in the liver, including gross liver enlargement, hepatocyte hypertrophy, and induced expression of drug-metabolizing enzymes and other liver-specific genes. The constitutive androstane receptor (CAR; NR1I3) and to a lesser extent the pregnane X receptor (PXR; NR1I2) are responsible for mediating induction of many phenobarbital-responsive genes. However, CAR-mediated transcriptional control of some genes is critically dependent on hepatocyte nuclear factor 4 alpha (HNF-4α; NR2A1), which itself regulates multiple liver-specific genes involved in hepatic growth, metabolism, and differentiation. We studied the effects of phenobarbital on HNF-4α expression in hepatocytes and provide evidence that HNF-4α nuclear expression is regulated in response to phenobarbital. Real-time polymerase chain reaction analyses revealed that HNF-4α mRNA is modestly up-regulated by phenobarbital. In addition, nuclear expression of HNF-4α protein is significantly elevated 3 hours after the administration of phenobarbital in wild-type, CAR−/−, and CAR−/−/PXR−/− mice. In vitro analysis revealed that phenobarbital-induced HNF-4α expression is both time- and dose dependent. In addition, the phosphatase inhibitor okadaic acid and the Ca2−/calmodulin-dependent protein kinase II inhibitor KN62 block nuclear induction of HNF-4α by phenobarbital. Furthermore, HNF-4α nuclear expression is enhanced by inhibition of cyclic AMP– dependent protein kinase A. In conclusion, induced nuclear expression of HNF-4α and CAR is an integral part of the phenobarbital response, aimed at coordinated regulation of genes involved in drug metabolism and detoxification as well as maintenance of liver function. PMID:16799975

  5. Effects of ethanol on antioxidant capacity in isolated rat hepatocytes

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    Yang, Sien-Sing; Huang, Chi-Chang; Chen, Jiun-Rong; Chiu, Che-Lin; Shieh, Ming-Jer; Lin, Su-Jiun; Yang, Suh-Ching

    2005-01-01

    AIM: To investigate dose-response and time-course of the effects of ethanol on the cell viability and antioxidant capacity in isolated rat hepatocytes. METHODS: Hepatocytes were isolated from male adult Wistar rats and seeded into 100-mm dishes. Hepatocytes were treated with ethanol at concentrations between 0 (C), 10 (E10), 50 (E50), and 100 (E100) mmol/L (dose response) for 12, 24, and 36 h (time course). Then, lactate dehydrogenase (LDH) leakage, malondialdehyde (MDA) concentration, glutathione (GSH) level, and activities of glutathione peroxidase (GPX), glutathione reductase (GRD), superoxide dismutase (SOD), and catalase (CAT) were measured. RESULTS: Our data revealed that LDH leakage was significantly increased by about 30% in group E100 over those in groups C and E10 at 24 and 36 h, The MDA concentration in groups C, E10 and E50 were significantly lower than that in group E100 at 36 h. Furthermore, the concentration of MDA in group E100 at 36 h was significantly higher by 4.5- and 1.7-fold, respectively, than that at 12 and 24 h. On the other hand, the GSH level in group E100 at 24 and 36 h was significantly decreased, by 32% and 28%, respectively, compared to that at 12 h. The activities of GRD and CAT in group E100 at 36 h were significantly less than those in groups C and E10. However, The GPX and SOD activities showed no significant change in each group. CONCLUSION: These results suggest that long-time incubation with higher concentration of ethanol (100 mmol/L) decreased the cell viability by means of reducing GRD and CAT activities and increasing lipid peroxidation. PMID:16437627

  6. Human hepatocyte functions in a crossed hollow fiber membrane bioreactor.

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    De Bartolo, Loredana; Salerno, Simona; Curcio, Efrem; Piscioneri, Antonella; Rende, Maria; Morelli, Sabrina; Tasselli, Franco; Bader, Augustinus; Drioli, Enrico

    2009-05-01

    An important challenge in liver tissue engineering is the development of bioartificial systems that are able to favour the liver reconstruction and to modulate liver cell behaviour. A crossed hollow fiber membrane bioreactor was developed to support the long-term maintenance and differentiation of human hepatocytes. The bioreactor consists of two types of hollow fiber (HF) membranes with different molecular weight cut-off (MWCO) and physico-chemical properties cross-assembled in alternating manner: modified polyetheretherketone (PEEK-WC) and polyethersulfone (PES), used for the medium inflow and outflow, respectively. The combination of these two fiber set produces an extracapillary network for the adhesion of cells and a high mass exchange through the cross-flow of culture medium. The transport of liver specific products such as albumin and urea together with the transport of drug such as diazepam was modelled and compared with the experimental metabolic data. The theoretical metabolite concentration differed 7.5% for albumin and 5% for urea with respect to experimental data. The optimised perfusion conditions of the bioreactor allowed the maintenance of liver functions in terms of urea synthesis, albumin secretion and diazepam biotransformation up to 18 days of culture. In particular the good performance of the bioreactor was confirmed by the high rate of urea synthesis (28.7 microg/h 10(6) cells) and diazepam biotransformation. In the bioreactor human hepatocytes expressed at high levels the individual cytochrome P450 isoenzymes involved in the diazepam metabolism. The results demonstrated that crossed HF membrane bioreactor is able to support the maintenance of primary human hepatocytes preserving their liver specific functions for all investigated period. This device may be a potential tool in the liver tissue engineering for drug metabolism/toxicity testing and study of disease pathogenesis alternatively to animal experimentation.

  7. The activation by glucose of liver membrane nitric oxide synthase in the synthesis and translocation of glucose transporter-4 in the production of insulin in the mice hepatocytes.

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    Suman Bhattacharya

    Full Text Available INTRODUCTION: Glucose has been reported to have an essential role in the synthesis and secretion of insulin in hepatocytes. As the efflux of glucose is facilitated from the liver cells into the circulation, the mechanism of transportation of glucose into the hepatocytes for the synthesis of insulin was investigated. METHODS: Grated liver suspension (GLS was prepared by grating intact liver from adult mice by using a grater. Nitric oxide (NO was measured by methemoglobin method. Glucose transporter-4 (Glut-4 was measured by immunoblot technique using Glut-4 antibody. RESULTS: Incubation of GLS with different amounts of glucose resulted in the uptake of glucose by the suspension with increased NO synthesis due to the stimulation of a glucose activated nitric oxide synthase that was present in the liver membrane. The inhibition of glucose induced NO synthesis resulted in the inhibition of glucose uptake. Glucose at 0.02M that maximally increased NO synthesis in the hepatocytes led to the translocation and increased synthesis of Glut-4 by 3.3 fold over the control that was inhibited by the inhibition of NO synthesis. The glucose induced NO synthesis was also found to result in the synthesis of insulin, in the presence of glucose due to the expression of both proinsulin genes I and II in the liver cells. CONCLUSION: It was concluded that glucose itself facilitated its own transportation in the liver cells both via Glut-4 and by the synthesis of NO which had an essential role for insulin synthesis in the presence of glucose in these cells.

  8. Allicin Modulates the Antioxidation and Detoxification Capabilities of Primary Rat Hepatocytes

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    Chih-Chung Wu

    2012-10-01

    Full Text Available The effect of allicin, an active ingredient of garlic, on lactate dehydrogenase (LDH leakage, lipid peroxidation, glutathione (GSH content, and GSH-related enzyme activity was investigated in primary hepatocytes. In this study, allicin was synthesized in our laboratory as an experimental material, and primary hepatocytes isolated from Sprague-Dawley rats were used as an experimental model. According to the results, hepatocytes treated with 10 μM allicin did not differ from the control on LDH leakage during various incubation times. When the hepatocytes were treated with 10 μM allicin, their levels of thiobarbituric acid reactive-substances (TBARS did not differ significantly from that of the control within the 8-h incubation. However, the TBARS values of hepatocytes treated with 30 and 50 μM allicin were higher compared to the control after incubation for 4 h and 8 h, respectively. The hepatocyte intracellular GSH content was significantly higher than that of the control after 30 μM allicin treatment, but treatment with 50 μM allicin caused a significant GSH depletion after incubation for 4 h or longer. In addition, when hepatocytes were treated for 24 h with 10 or 30 μM allicin, glutathione peroxidase (GPx activity was significantly increased compared to that of the control, whereas 50 μM allicin treatment for 24 h or longer significantly decreased the GPx activity. Glutathione reductase (GRd activity was significantly increased when the hepatocytes were treated with 10 μM allicin for 24 h, but GRd activity significantly decreased when the hepatocytes were treated with 50 μM allicin. However, hepatocytes treated for 24 h with 10 or 30 μM allicin showed significantly increased glutathione S-transferase (GST activity compared to the control. These results suggest that 10 μM allicin potentially enhances the antioxidation and detoxification capabilities of primary rat hepatocytes.

  9. Treatment of nonalbumin rats by transplantation of immortalized hepatocytes using artificial human chromosome.

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    Ito, M; Ikeno, M; Nagata, H; Yamamoto, T; Hiroguchi, A; Fox, I J; Miyakawa, S

    2009-01-01

    The shortage of organ donors has impeded the development of human hepatocyte transplantation. Immortalized hepatocytes, however, could provide an unlimited supply of transplantable cells. To determine whether immortalized hepatocytes could provide global metabolic support in end-stage liver disease, rat hepatocyte clones were developed by transduction with the gene encoding the simian virus 40 T antigen (SVLT) using the new technique of human artificial mini chromosome (HAC). Immortalized rat hepatocyte clones were developed by transduction with the gene encoding the SV40 using HAC. Many clones were obtained using this technique. From comparison of the properties of all these clones using the normal hepatocytes and reverse transcription-polymerase chain reaction (RT-PCR), the characteristics of the cell clones (at least partially characterized, and assayed for albumin, glucose-6-phosphate and dipeptidyl peptidase-4, gamma-glutamyltranspeptidase, SVLT and beta-actin expression by RT-PCR) showed no differences other than the immortalization. We compared the albumin bands of the first-day (0-day) and 30-day cells by RT-PCR, showing conditions to be stable for at least 1 month. Three experimental animal model groups were used for albumin analysis: nonalbumin rats with 2/3 hepatectomy only (R-NARs; n = 4); R-NARs with intrasplenic transplantation of 3 x 10(7) primary hepatocytes (pHTx; n = 4); and R-NARs with intrasplenic transplantation of 3 x 10(7) immortalized hepatocytes (iHTx; n = 4). All HTx groups produced albumin, but the immortalized hepatocyte group did not generate significantly elevated albumin levels compared with primary hepatocytes. The results presented herein have demonstrated an initial step toward the development of immortalized hepatocytes for transplantable cells or artificial organs using HAC technology.

  10. Characterization of a transporting system in rat hepatocytes. Studies with competitive and non-competitive inhibitors of phalloidin transport.

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    Münter, K; Mayer, D; Faulstich, H

    1986-08-07

    Primary cultures of rat hepatocytes were used for assaying several drugs not previously known for inhibiting the transport of phalloidin. In order to have 50% inhibition (IC50) of the entrance of a tritiated phallotoxin derivative ([3H]demethylphalloin, 1 microM) from the medium into the cells the following concentrations (microM) of the various inhibitors were determined: cyclolinopeptide (0.5), Nocloprost (5.0), Nileprost (7.0), beta-estradiol (42), Verapamil (70). For comparison, the corresponding IC50 values of some known antagonists of phalloidin toxicity were determined by the same method. Moreover, we studied several natural and synthetic phallotoxins and alpha-amanitin for their ability to displace [3H]demethylphalloin from the transporting system. Lineweaver-Burk plots made it obvious that two groups of inhibitors exist. Competitive inhibitors are, for example, antamanide, beta-estradiol, silybin, Nileprost, taurocholate, and the cyclic somatostatin analog cyclo[Phe-Thr-Lys-Trp-Phe-D-Pro], whereas Verapamil and monensin inhibit phallotoxin uptake in a non-competitive way. Considering the very different chemical features of the competitive inhibitors, we tentatively conclude that the phallotoxin transport system selects compounds not on the basis of their chemical features, but rather their physical properties. The physical properties of a typical substrate are low molecular mass, lipophilic nature, and, possibly the presence of rigid ring structures. Negative charges accelerate the transport of a substrate, while positive charges have the opposite effect. The phalloidin-transporting system may represent part of a hepatic equipment which clears portal blood from, for example, bile acids, lipophilic hormones, or xenobiotics. By chance, the transporting system incorporates phallotoxins into the hepatocytes leading to the death of these cells.

  11. Effects of eugenol on hepatic glucose production and AMPK signaling pathway in hepatocytes and C57BL/6J mice.

    Science.gov (United States)

    Jeong, Kyong Ju; Kim, Do Yeon; Quan, Hai-Yan; Jo, Hee Kyung; Kim, Go Woon; Chung, Sung Hyun

    2014-03-01

    Eugenol is a phenylpropanoid with many pharmacological activities, but its anti-hyperglycemic activity is not yet fully explored. For in vitro study, HepG2 cells and primary rat hepatocytes were used, and glucose production was induced by adding 100 nM of glucagon in the presence of gluconeogenic substrates. In animal study, hyperglycemia was induced by high fat diet (HFD) in male C57BL/6J mice, and eugenol was orally administered at 20 or 40 mg per kg (E20, E40) for 15 weeks. Eugenol significantly inhibited glucagon-induced glucose production and phosphorylated AMPK in the HepG2 and primary rat hepatocytes, and these effects were reversed in the presence of compound C (an AMPK inhibitor) or STO-609 (a CAMKK inhibitor). In addition, the protein and gene expression levels of CREB, CRTC2·CREB complex, PGC-1α, PEPCK and G6Pase were all significantly suppressed. Moreover, inhibition of AMPK by over-expression of dominant negative AMPK prevented eugenol from suppressions of gluconeogenic gene expression and hepatic glucose production. In animal study, plasma glucose and insulin levels of the E40 group were decreased by 31% and 63%, respectively, when compared to those of HFD control. In pyruvate tolerance tests, pyruvate-induced glucose excursions were decreased, indicating that the anti-hyperglycemic activity of eugenol is primarily due to the suppression of hepatic gluconeogenesis. In summary, eugenol effectively ameliorates hyperglycemia through inhibition of hepatic gluconeogenesis via modulating CAMKK-AMPK-CREB signaling pathway. Eugenol or eugenol-containing medicinal plants could represent a promising therapeutic agent to prevent type 2 diabetes. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Regulation of tissue inhibitor of metalloproteinases-1 gene expression by cytokines and dexamethasone in rat hepatocyte primary cultures.

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    Roeb, E; Graeve, L; Hoffmann, R; Decker, K; Edwards, D R; Heinrich, P C

    1993-12-01

    The steady-state levels of extracellular matrix proteins are regulated by the rates of their synthesis and degradation. Metalloproteinases and their specific inhibitors, tissue inhibitor of metalloproteinases-1 and -2 are believed to play a crucial role in extracellular matrix protein degradation. Here we show that the tissue inhibitor of metalloproteinases-1 is expressed in rat hepatocytes in primary culture and regulated by inflammatory cytokines. Rat hepatocytes constitutively express mRNA of tissue inhibitors of metalloproteinases-1 at a low level. Incubation with conditioned medium from lipopolysaccharide-stimulated human monocytes led to a dramatic induction of mRNA of tissue inhibitors of metalloproteinases-1. The inflammatory cytokines interleukin-1 beta, interleukin-6, interleukin-11, leukemia inhibitory factor and ciliary neurotrophic factor were also capable of stimulating expression of mRNA of tissue inhibitors of metalloproteinases-1. Among these cytokines interleukin-6 was the most potent stimulator. The combination of interleukin-1 beta, interleukin-6 and interleukin-11 synergistically up-regulated mRNA of tissue inhibitors of metalloproteinases-1. The synthetic glucocorticoid dexamethasone dose dependently inhibited constitutive and interleukin-6-induced expression of tissue inhibitors of metalloproteinases-1. A possible involvement of tissue inhibitor of metalloproteinases-1 in the pathogenesis of liver fibrosis and cirrhosis is discussed.

  13. Pharmacokinetics and effects on serum cholinesterase activities of organophosphorus pesticides acephate and chlorpyrifos in chimeric mice transplanted with human hepatocytes.

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    Suemizu, Hiroshi; Sota, Shigeto; Kuronuma, Miyuki; Shimizu, Makiko; Yamazaki, Hiroshi

    2014-11-01

    Organophosphorus pesticides acephate and chlorpyrifos in foods have potential to impact human health. The aim of the current study was to investigate the pharmacokinetics of acephate and chlorpyrifos orally administered at lowest-observed-adverse-effect-level doses in chimeric mice transplanted with human hepatocytes. Absorbed acephate and its metabolite methamidophos were detected in serum from wild type mice and chimeric mice orally administered 150mg/kg. Approximately 70% inhibition of cholinesterase was evident in plasma of chimeric mice with humanized liver (which have higher serum cholinesterase activities than wild type mice) 1day after oral administrations of acephate. Adjusted animal biomonitoring equivalents from chimeric mice studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Estimated plasma concentrations of acephate and chlorpyrifos in humans were consistent with reported concentrations. Acephate cleared similarly in humans and chimeric mice but accidental/incidental overdose levels of chlorpyrifos cleared (dependent on liver metabolism) more slowly from plasma in humans than it did in mice. The data presented here illustrate how chimeric mice transplanted with human hepatocytes in combination with a simple PBPK model can assist evaluations of toxicological potential of organophosphorus pesticides. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Reprogramming factors involved in hybrids and cybrids of human embryonic stem cells fused with hepatocytes.

    Science.gov (United States)

    Guo, Jitong; Tecirlioglu, R Tayfur; Nguyen, Linh; Koh, Karen; Jenkin, Graham; Trounson, Alan

    2010-10-01

    Embryonic stem cells (ESCs) have the potential to reprogram somatic cells into ESC-like cells through cell fusion. In the present study, the potential of human (h)ESC cytoplasts and karyoplasts to reprogram human hepatocytes was evaluated. Green fluorescent protein (GFP) transfected hESCs (ENVY cells) were fused with SNARF-1 (CellTracker)-labeled human hepatocytes using polyethylene glycol (PEG) and fluorescence-activated cell sorting (FACS) to produce hESC-hepatocyte hybrids. Immunocytochemical analysis of ESC markers showed that the hybrids expressed OCT4, TRA-1-60, TRA-1-81, SSEA-4, and GCTM-2. However, SSEA-1, which is typically low or absent on hESCs, was detected on hESC–hepatocyte hybrids. Moreover, reverse transcriptase polymerase chain reaction (RT-PCR) showed that alpha-fetoprotein, which is highly expressed in hepatocytes, was erased in the hybrids. These results indicated that hESCs have the potential to reprogram hepatocyte phenotype to a relatively undifferentiated state, but such hybrid cells are not identical to hESCs. Although hESC–hepatocyte hybrids were aneuploid, they were able to differentiate into embryoid bodies and some types of somatic cells. Furthermore, cybrids of enucleated hESCs and hepatocytes were produced by cell fusion, but the cybrids were unable to self-renew in the same way as hESCs. Presumably, the reprogramming factors are associated with the karyoplast and not the cytoplast of hESCs.

  15. Partial hepatectomy induces delayed hepatocyte proliferation and normal liver regeneration in ovariectomized mice

    Directory of Open Access Journals (Sweden)

    Umeda M

    2015-07-01

    Full Text Available Makoto Umeda,1 Masaki Hiramoto,1,2 Takeshi Imai1 1Department of Aging Intervention, National Center for Geriatrics and Gerontology, Obu, Aichi, Japan; 2Department of Biochemistry, Tokyo Medical University, Tokyo, Japan Abstract: Estrogens play central roles in sexual development, reproduction, and hepatocyte proliferation. The ovaries are one of the main organs for estradiol (E2 production. Ovariectomies (OVXs were performed on the female mice, and hepatocyte proliferation was analyzed. The ovariectomized mice exhibited delayed hepatocyte proliferation after partial hepatectomy (PH and also exhibited delayed and reduced E2 induction. Both E2 administration and PH induced the gene expression of estrogen receptor α (ERα. The transcripts of ERα were detected specifically in periportal hepatocytes after E2 administration and PH. Moreover, the E2 concentrations and hepatocyte proliferation rates were highest in the proestrus period of the estrous cycle. Taken together, these findings indicate that E2 accelerated ERα expression in periportal hepatocytes and hepatocyte proliferation in the female mice.Keywords: estrogen, ER, estrous cycle, hepatocyte proliferation, liver regeneration

  16. Measurement of acetyl-CoA carboxylase activity in isolated hepatocytes

    NARCIS (Netherlands)

    Bijleveld, C.; Geelen, M.J.H.

    1987-01-01

    An assay is described for acetyl-CoA carboxylase activity in isolated hepatocytes. The assay is based on two principles: (a) The hepatocytes are made permeable by digitonin. 64 μg of digitonin per mg of cellular protein were most effective in exposing enzyme activity without a significant effect on

  17. Eight paths of ERK1/2 signalling pathway regulating hepatocyte ...

    Indian Academy of Sciences (India)

    2011-12-05

    Dec 5, 2011 ... Abstract. Although it is known that hormones, growth factors and integrin promote hepatocyte proliferation in liver regeneration. (LR) through ERK1/2 signalling pathway, reports about regulating processes of its intracellular paths in hepatocytes of LR are limited. This study aims at exploring which paths of ...

  18. High throughput micro-well generation of hepatocyte micro-aggregates for tissue engineering.

    Science.gov (United States)

    Gevaert, Elien; Dollé, Laurent; Billiet, Thomas; Dubruel, Peter; van Grunsven, Leo; van Apeldoorn, Aart; Cornelissen, Ria

    2014-01-01

    The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the high throughput generation of hepatocyte aggregates, uniform in size. In our study we observed that aggregation of hepatocytes had a beneficial effect on the expression of certain hepatocyte specific markers. Moreover we observed that the beneficial effect was dependent on the aggregate dimensions, indicating that aggregate parameters should be carefully considered. In a second part of the study, the selected aggregates were immobilized by encapsulation in methacrylamide-modified gelatin. Phenotype evaluations revealed that a stable hepatocyte phenotype could be maintained during 21 days when encapsulated in the hydrogel. In conclusion we have demonstrated the beneficial use of micro-well chips for hepatocyte aggregation and the size-dependent effects on hepatocyte phenotype. We also pointed out that methacrylamide-modified gelatin is suitable for the encapsulation of these aggregates.

  19. High Throughput Micro-Well Generation of Hepatocyte Micro-Aggregates for Tissue Engineering

    NARCIS (Netherlands)

    Gevaert, Elien; Dollé, Laurent; Billiet, Thomas; Dubruel, Peter; van Grunsven, Leo; van Apeldoorn, Aart A.; Cornelissen, Ria

    2014-01-01

    The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the

  20. High throughput micro-well generation of hepatocyte micro-aggregates for tissue engineering.

    Directory of Open Access Journals (Sweden)

    Elien Gevaert

    Full Text Available The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the high throughput generation of hepatocyte aggregates, uniform in size. In our study we observed that aggregation of hepatocytes had a beneficial effect on the expression of certain hepatocyte specific markers. Moreover we observed that the beneficial effect was dependent on the aggregate dimensions, indicating that aggregate parameters should be carefully considered. In a second part of the study, the selected aggregates were immobilized by encapsulation in methacrylamide-modified gelatin. Phenotype evaluations revealed that a stable hepatocyte phenotype could be maintained during 21 days when encapsulated in the hydrogel. In conclusion we have demonstrated the beneficial use of micro-well chips for hepatocyte aggregation and the size-dependent effects on hepatocyte phenotype. We also pointed out that methacrylamide-modified gelatin is suitable for the encapsulation of these aggregates.

  1. Human Cytotoxic T Lymphocyte-Mediated Acute Liver Failure and Rescue by Immunoglobulin in Human Hepatocyte Transplant TK-NOG Mice.

    Science.gov (United States)

    Uchida, Takuro; Hiraga, Nobuhiko; Imamura, Michio; Tsuge, Masataka; Abe, Hiromi; Hayes, C Nelson; Aikata, Hiroshi; Ishida, Yuji; Tateno, Chise; Yoshizato, Katsutoshi; Ohdan, Hideki; Murakami, Kazunari; Chayama, Kazuaki

    2015-10-01

    Hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) are critical in eliminating infection. We developed an animal model in which HBV-infected human hepatocytes are targeted by HBV-specific CTLs. After HBV inoculation in human hepatocyte-transplanted herpes simplex virus type-1 thymidine kinase-NOG mice, human peripheral blood mononuclear cells (PBMCs) were administered, and albumin, HBV DNA, alanine aminotransferase (ALT), and cytokine levels were analyzed. Histopathological and flow-cytometric analysis of infiltrating human immune cells were performed, and the efficacy of CTL-associated antigen-4 immunoglobulin (CTLA4Ig) against liver damage was evaluated. PBMC treatment resulted in massive hepatocyte damage with elevation of ALT, granzyme A, and gamma interferon and decrease in albumin and HBV DNA. The number of liver-infiltrating human lymphocytes and CD8-positive cells was significantly higher in HBV-infected mice. HBV-specific CTLs were detected by core and polymerase peptide-major histocompatibility complex-tetramer, and the population of regulatory T cells was significantly decreased in HBV-infected mice. Serum hepatitis B surface (HBs) antigen became negative, and HBs antibody appeared. CTLA4Ig treatment strongly inhibited infiltration of mononuclear cells. CTLA4Ig treatment will be used to treat patients who develop severe acute hepatitis B to prevent liver transplantation or lethality. This animal model is useful for virological and immunological analysis of HBV infection and to develop new therapies for severe acute hepatitis B. Without liver transplantation, some HBV-infected patients will die from severe liver damage due to acute overreaction of the immune system. No effective treatment exists, due in part to the lack of a suitable animal model. An animal model is necessary to investigate the mechanism of hepatitis and to develop therapeutic strategies to prevent acute liver failure in HBV infection. We developed an animal model in which

  2. Study of Valproic Acid-Enhanced Hepatocyte Steatosis

    Directory of Open Access Journals (Sweden)

    Renin Chang

    2016-01-01

    Full Text Available Valproic acid (VPA is one of the most widely used antiepilepsy drugs. However, several side effects, including weight gain and fatty liver, have been reported in patients following VPA treatment. In this study, we explored the molecular mechanisms of VPA-induced hepatic steatosis using FL83B cell line-based in vitro model. Using fluorescent lipid staining technique, we found that VPA enhanced oleic acid- (OLA- induced lipid accumulation in a dose-dependent manner in hepatocytes; this may be due to upregulated lipid uptake, triacylglycerol (TAG synthesis, and lipid droplet formation. Real-time PCR results showed that, following VPA treatment, the expression levels of genes encoding cluster of differentiation 36 (Cd36, low-density lipoprotein receptor-related protein 1 (Lrp1, diacylglycerol acyltransferase 2 (Dgat2, and perilipin 2 (Plin2 were increased, that of carnitine palmitoyltransferase I a (Cpt1a was not affected, and those of acetyl-Co A carboxylase α (Acca and fatty acid synthase (Fasn were decreased. Furthermore, using immunofluorescence staining and flow cytometry analyses, we found that VPA also induced peroxisome proliferator-activated receptor γ (PPARγ nuclear translocation and increased levels of cell-surface CD36. Based on these results, we propose that VPA may enhance OLA-induced hepatocyte steatosis through the upregulation of PPARγ- and CD36-dependent lipid uptake, TAG synthesis, and lipid droplet formation.

  3. Hepatocytes Polyploidization and Cell Cycle Control in Liver Physiopathology

    Directory of Open Access Journals (Sweden)

    Géraldine Gentric

    2012-01-01

    Full Text Available Most cells in mammalian tissues usually contain a diploid complement of chromosomes. However, numerous studies have demonstrated a major role of “diploid-polyploid conversion” during physiopathological processes in several tissues. In the liver parenchyma, progressive polyploidization of hepatocytes takes place during postnatal growth. Indeed, at the suckling-weaning transition, cytokinesis failure events induce the genesis of binucleated tetraploid liver cells. Insulin signalling, through regulation of the PI3K/Akt signalling pathway, is essential in the establishment of liver tetraploidization by controlling cytoskeletal organisation and consequently mitosis progression. Liver cell polyploidy is generally considered to indicate terminal differentiation and senescence, and both lead to a progressive loss of cell pluripotency associated to a markedly decreased replication capacity. Although adult liver is a quiescent organ, it retains a capacity to proliferate and to modulate its ploidy in response to various stimuli or aggression (partial hepatectomy, metabolic overload (i.e., high copper and iron hepatic levels, oxidative stress, toxic insult, and chronic hepatitis etc.. Here we review the mechanisms and functional consequences of hepatocytes polyploidization during normal and pathological liver growth.

  4. Ultrastructural hepatocytic alterations induced by silver nanoparticle toxicity.

    Science.gov (United States)

    Almansour, Mansour; Sajti, Laszlo; Melhim, Walid; Jarrar, Bashir M

    2016-01-01

    Silver nanoparticles (SNPs) are widely used in nanomedicine and consuming products with potential risk to human health. While considerable work was carried out on the molecular, biochemical, and physiological alterations induced by these particles, little is known of the ultrastructural pathological alterations that might be induced by nanosilver materials. The aim of the present work is to investigate the hepatocyte ultrastructural alterations that might be induced by SNP exposure. Male rats were subjected to a daily single dose (2 mg/kg) of SNPs (15-35 nm diameter) for 21 days. Liver biopsies from all rats under study were processed for transmission electron microscopy examination. The following hepatic ultrastructural alterations were demonstrated: mitochondria swelling and crystolysis, endoplasmic reticulum disruption, cytoplasmic vacuolization, lipid droplets accumulation, glycogen depletion, karyopyknosis, apoptosis, sinusoidal dilatation, Kupffer cells activation, and myelin figures formation. The current findings may indicate that SNPs can induce hepatocyte organelles alteration, leading to cellular damage that may affect the function of the liver. These findings might indicate that SNPs potentially trigger heptocyte ultrastructural alterations that may affect the function of the liver with potential risk on human health in relation to numerous applications of these particles. More work is needed to elucidate probable ultrastructural alterations in the vital organs that might result from nanosilver toxicity.

  5. Rapid and sensitive measure of gluconeogenesis in isolated bovine hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Azain, M.J.; Kasser, T.R.; Atwell, C.A.; Baile, C.A.

    1986-03-05

    Available methods for determining glucose synthesis from radiolabelled precursors using ion exchange column chromatography limit the number of samples that can be processed. To facilitate this process, a rapid method for determining glucose synthesis from 3-carbon precursors was developed using suspensions of anion and cation exchange resins. Hepatocytes were prepared from calf liver by collagenase perfusion of the caudate lobe. Isolated cells were incubated with /sup 14/C-labelled lactate or propionate in the presence or absence of glucagen and/or palmitate. Glucose synthesis was determined by vortexing an aliquot of cell suspension with a 50% slurry of anion exchange resin (acetate form), followed by cation exchange resin. After centrifugation /sup 14/C-glucose was recovered in the supernatant and measured by scintillation counting. Using this method, more than 95% of unused labelled precursor was bound to the ion exchange resin and essentially 100% of /sup 14/C-glucose tracer was recovered in the supernatant. In hepatocyte suspensions, radioactivity recovered in the supernatants was confirmed to be glucose by pre-incubating aliquots of media with glucose oxidase prior to addition of ion exchange resins. The present system allows determination of hepatic gluconeogenesis, is sensitive to substrate and hormonal manipulations and has the capacity for processing several hundred samples per day.

  6. Effectiveness of Intracavernous Delivery of Recombinant Human Hepatocyte Growth Factor on Erectile Function in the Streptozotocin-Induced Diabetic Mouse.

    Science.gov (United States)

    Das, Nando Dulal; Yin, Guo Nan; Choi, Min Ji; Song, Kang-Moon; Park, Jin-Mi; Limanjaya, Anita; Ghatak, Kalyan; Minh, Nguyen Nhat; Ock, Jiyeon; Park, Soo-Hwan; Kim, Ho Min; Ryu, Ji-Kan; Suh, Jun-Kyu

    2016-11-01

    Diabetic erectile dysfunction is a disease mostly of vascular origin and men with diabetic erectile dysfunction respond poorly to oral phosphodiesterase-5 inhibitors. Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an essential role in the regulation of cell proliferation, survival, and angiogenesis. To determine the effectiveness of recombinant human (rh)-HGF in restoring erectile function in diabetic mice. Four groups of mice were used: control non-diabetic mice and streptozotocin-induced diabetic mice receiving two successive intracavernous injections of phosphate buffered saline (days -3 and 0), a single intracavernous injection of rh-HGF (day 0), or two successive intracavernous injections of rh-HGF (days -3 and 0). We also examined the effect of rh-HGF in primary cultured mouse cavernous endothelial cells and in major pelvic ganglion culture in vitro, which was incubated under a normal-glucose (5 mmol/L) or a high-glucose (30 mmol/L) condition. Two weeks after treatment, we measured erectile function by electrical stimulation of the cavernous nerve and the penis was harvested for histologic studies. Repeated intracavernous injections of rh-HGF protein induced significant restoration of erectile function in diabetic mice (89-100% of control values), whereas a single intracavernous injection of rh-HGF protein elicited modest improvement. Rh-HGF significantly induced phosphorylation of its receptor c-Met, increased the content of endothelial cells and smooth muscle cells, and decreased the generation of reactive oxygen species (superoxide anion and peroxynitrite) and extravasation of oxidized low-density lipoprotein in diabetic mice. Under the high-glucose condition, rh-HGF protein also promoted tube formation in mouse cavernous endothelial cells and enhanced neurite sprouting in major pelvic ganglion culture in vitro. The dual angiogenic and neurotrophic effects of HGF could open a new avenue through which diabetic erectile dysfunction

  7. A feedback loop between the liver-enriched transcription factor network and mir-122 controls hepatocyte differentiation.

    OpenAIRE

    Laudadio, Ilaria; Manfroid, Isabelle; Achouri, Younes; Schmidt, Dominic; Wilson, Michael D; Cordi, Sabine; Thorrez, Lieven; Knoops, Laurent; Jacquemin, Patrick; Schuit, Frans; Pierreux, Christophe E; Odom, Duncan T; Peers, Bernard; Lemaigre, Frederic P

    2012-01-01

    BACKGROUND & AIMS: Hepatocyte differentiation is controlled by liver-enriched transcription factors (LETFs). We investigated whether LETFs control microRNA expression during development and whether this control is required for hepatocyte differentiation. METHODS: Using in vivo DNA binding assays, we identified miR-122 as a direct target of the LETF hepatocyte nuclear factor (HNF) 6. The role and mechanisms of the HNF6-miR-122 gene cascade in hepatocyte differentiation were studied in vivo and...

  8. Measurement of Blood Coagulation Factor Synthesis in Cultures of Human Hepatocytes.

    Science.gov (United States)

    Heinz, Stefan; Braspenning, Joris

    2015-01-01

    An important function of the liver is the synthesis and secretion of blood coagulation factors. Within the liver, hepatocytes are involved in the synthesis of most blood coagulation factors, such as fibrinogen, prothrombin, factor V, VII, IX, X, XI, XII, as well as protein C and S, and antithrombin, whereas liver sinusoidal endothelial cells produce factor VIII and von Willebrand factor. Here, we describe methods for the detection and quantification of most blood coagulation factors in hepatocytes in vitro. Hepatocyte cultures indeed provide a valuable tool to study blood coagulation factors. In addition, the generation and expansion of hepatocytes or hepatocyte-like cells may be used in future for cell-based therapies of liver diseases, including blood coagulation factor deficiencies.

  9. In vivo N-acetyl cysteine reduce hepatocyte death by induced acetaminophen

    Science.gov (United States)

    Lin, Chih-Ju; Li, Feng-Chieh; Wang, Sheng-Shun; Lee, Hsuan-Shu; Dong, Chen-Yuan

    2011-07-01

    Acetaminophen (APAP) is the famous drug in global, and taking overdose Acetaminophen will intake hepatic cell injure. Desptie substantial progress in our understanding of the mechanism of hepatocellular injury during the last 40 years, many aspects of the pathophysiology are still unknown or controversial.1 In this study, mice are injected APAP overdose to damage hepatocyte. APAP deplete glutathione and ATP of cell, N-Acetyl Cysteine (NAC) plays an important role to protect hepatocytes be injury. N-Acetyl Cysteine provides mitochondrial to produce glutathione to release drug effect hepatocyte. By 6-carboxyfluorescein diacetate (6-CFDA) metabolism in vivo, glutathione keep depleting to observe the hepatocyte morphology in time. Without NAC, cell necrosis increase to plasma membrane damage to release 6-CFDA, that's rupture. After 6-CFDA injection, fluorescence will be retained in hepatocyte. For cell retain with NAC and without NAC are almost the same. With NAC, the number of cell rupture decreases about 75%.</