Human leukocyte elastase counteracts matrix metalloproteinase-7 induced apoptosis resistance of tumor cells.

Matrix metalloproteinase-7 (MMP-7/Matrilysin) is a component of the tumor microenvironment associated with malignant progression. Its expression in tumors protects tumor cells from CD95-mediated apoptosis and the cytotoxic activity of tumor specific CD8(+) T cells. In the present study, we show that human leukocyte elastase (HLE) secreted by polymorphonuclear leukocytes cleaves MMP-7 resulting in loss of enzymatic activity. The anti-apoptotic effect of MMP-7 is reduced in the presence of HLE for CD95-, doxorubicin- and CTL-mediated apoptosis. Our data indicates that HLE may be a natural inactivator of MMP-7 which can counteract MMP-7-induced apoptosis resistance.

Tissue factor pathway inhibitor primes monocytes for antiphospholipid antibody-induced thrombosis

Antiphospholipid antibodies (aPLs) with complex lipid and/or protein reactivities cause complement-dependent thrombosis and pregnancy complications. Although cross-reactivities with coagulation regulatory proteins contribute to the risk for developing thrombosis in patients with antiphospholipid syndrome, the majority of pathogenic aPLs retain reactivity with membrane lipid components and rapidly induce reactive oxygen species-dependent proinflammatory signaling and tissue factor (TF) procoagulant activation. Here, we show that lipid-reactive aPLs activate a common species-conserved TF signaling pathway. aPLs dissociate an inhibited TF coagulation initiation complex on the cell surface of m…

Melatonin induces transcriptional regulation of Bim by FoxO3a in HepG2 cells

Background: Melatonin induces apoptosis in many different cancer cell lines, including hepatocellular carcinoma cells. However, the responsible pathways have not been clearly elucidated. A member of the forkhead transcription factors' family, FoxO3a, has been implicated in the expression of the proapoptotic protein Bim (a Bcl-2-interacting mediator of cell death). In this study, we used human HepG2 liver cancer cells as an in vitro model to investigate whether melatonin treatment induces Bim through regulation by the transcription factor FoxO3a. Methods: Cytotoxicity of melatonin was compared in HepG2 hepatoblastoma cells and primary human hepatocytes. Proapoptotic Bim expression was analys…

Placenta-derived CD95 ligand causes liver damage in hemolysis, elevated liver enzymes, and low platelet count syndrome.

Background & Aims: The HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome is a life-threatening complication during pregnancy. The associated liver disease may be severe, and maternal hepatic complications may progress to the point that transplantation becomes necessary. CD95 (APO-1, Fas)-mediated apoptosis of liver cells is one of the major pathogenic mechanisms during liver disease. The interaction of CD95 with its ligand, CD95L(FasL), induces apoptosis and thus the source of the death-inducing ligand is critical for understanding the pathomechanism of liver damage involving the CD95-system. Methods: Sera from HELLP patients were analyzed and used in cell culture experiment…

Assessing the Histone Deacetylase Activity of SIRT6 in Primary Murine Hepatocytes via Proximity Ligation Assay

Generation of primary cell culture of hepatocytes by mouse liver perfusion (MLP) combines the advantages of in vivo and in vitro models. It provides hepatocytes that grow under physiological conditions in mice, with the genotype of the whole organism or a specific gene knockout. In contrast to immortalized cell cultures, primary murine hepatocytes (pmHep) are non-cancerous cells with a limited lifespan but still amenable to classical in vitro methods such as treatment with drugs, small molecule inhibitors, and agonistic/antagonistic antibodies of surface receptors as well as transfection. One technique, which has gained popularity recently, is the analysis of protein-protein interactions by…

Amine functionalized ZrO2 nanoparticles as biocompatible and luminescent probes for ligand specific cellular imaging

Surface functionalized ZrO2 nanoparticles show strong photoluminescence and are a versatile tool for cellular targeting due to their chemical functionality. They are highly photostable, biocompatible and amenable to coupling with bioligands (e.g. secondary goat anti-rabbit antibody (GAR) and tri-phenyl phosphine (TPP)) via carbodiimide chemistry. Antibody (GAR) functionalized ZrO2 nanoparticles were used to image the nuclear protein Sirt6, whereas triphenyl phosphonium ion (TPP) functionalized ZrO2 nanoparticles specifically targeted the mitochondria. The versatility and easiness of the ZrO2 surface modification opens up new possibilities for designing non-toxic water dispersible and photos…

Multi-photon imaging of amine-functionalized silica nanoparticles.

A convenient and simple strategy for preparing water soluble, photoluminescent functionalized silica nanoparticles (M-dots) in the absence of fluorophores or metal doping is demonstrated. These M-dots can be used for bioimaging using one and two-photon microscopy. Because of their high photostability, low toxicity and high biocompatibility compared with Lumidot™ CdSe/ZnS quantum dots, functionalized silica particles are superior alternatives for current bioimaging platforms. Moreover, the presence of a free amine group at the surface of the M-dots allows biomolecule conjugation (e.g. with antibodies, proteins) in a single step for converting these photoluminescent SiO(2) nanoparticles into …

Monophosphoryl lipid A coating of hydroxyethyl starch nanocapsules drastically increases uptake and maturation by dendritic cells while minimizing the adjuvant dosage.

Enhancing delivery of antigens to dendritic cells (DCs) is essential for the induction of vigorous antigen-specific cellular immune responses. Aim of the present study was to evaluate the properties of hydroxyethyl starch nanocapsules (HES-NCs) functionalized with anti-CD40, anti-DEC205, interferon-γ (IFNγ) and/or monophosphoryl lipid A (MPLA) with respect to the overall uptake, the released cytokine profile, and the influence on phenotypic maturation of human monocyte-derived DCs using flow cytometry, confocal microscopy and enzyme-linked immunosorbent assays. NC uptake by DCs was significantly enhanced by functionalizing NCs with anti-CD40 or MPLA. With respect to the cytokine profile and…

Reduced expression of Hugl-1, the human homologue of Drosophila tumour suppressor gene lgl, contributes to progression of colorectal cancer.

The human gene, human giant larvae (Hugl-1/Llg1/Lgl1) has significant homology to the Drosophila tumour suppressor gene lethal(2)giant larvae (lgl). The lgl gene codes for a cortical cytoskeleton protein, Lgl, that binds Myosin II and is involved in maintaining cell polarity and epithelial integrity. The human protein, Hugl-1 contains several conserved functional domains found in Lgl, suggesting that these proteins may have closely related functions. Whether loss of Hugl expression plays a role in human tumorigenesis has so far not been extensively investigated. Thus, we evaluated tumour tissues from 94 patients undergoing surgery for colorectal cancer (CRC) for loss of Hugl-1 transcription…

Dinitrosyl-iron triggers apoptosis in Jurkat cells despite overexpression of Bcl-2

Cells expressing the cytokine-inducible NO synthase are known to trigger apoptosis in neighboring cells. Paramagnetic dinitrosyl nonheme iron complexes (DNIC) were found in tumor tissue about 40 years ago; however, the role of these NO(+)-bearing species is not completely understood. In the human Jurkat leukemia cell line, the application of the model complex DNIC-thiosulfate (50-200 microM) induced apoptosis (defined by phosphatidylserine externalization) in a concentration- and time-dependent manner. In Jurkat cells, the pan-caspase inhibitor, zVADfmk (50 microM), and/or stable transfection of antiapoptotic protein, Bcl-2, was unable to afford protection against DNIC-induced apoptosis. Th…

Activation of silent mating type information regulation 2 homolog 1 by human chorionic gonadotropin exerts a therapeutic effect on hepatic injury and inflammation

Incidence and prevalence of inflammatory liver diseases has increased over the last years, but therapeutic options are limited. Pregnancy induces a state of immune tolerance, which can result in spontaneous improvement of clinical symptoms of certain autoimmune diseases including autoimmune hepatitis (AIH). We investigated the immune-suppressive mechanisms of the human pregnancy hormone, chorionic gonadotropin (hCG), in the liver. hCG signaling activates silent mating type information regulation 2 homolog 1 (SIRT1), which deacetylates forkhead box o3 (FOXO3a), leading to repression of proapoptotic gene expression, because the immunosuppressive consequence attributed to the absence of caspas…

Inhibition of histone deacetylation rescues phenotype in a mouse model of Birk-Barel intellectual disability syndrome

Mutations in the actively expressed, maternal allele of the imprinted KCNK9 gene cause Birk-Barel intellectual disability syndrome (BBIDS). Using a BBIDS mouse model, we identify here a partial rescue of the BBIDS-like behavioral and neuronal phenotypes mediated via residual expression from the paternal Kcnk9 (Kcnk9pat) allele. We further demonstrate that the second-generation HDAC inhibitor CI-994 induces enhanced expression from the paternally silenced Kcnk9 allele and leads to a full rescue of the behavioral phenotype suggesting CI-994 as a promising molecule for BBIDS therapy. Thus, these findings suggest a potential approach to improve cognitive dysfunction in a mouse model of an impri…

SIRT1 prevents genotoxic stress-induced p53 activation in acute myeloid leukemia

SIRT1 is an important regulator of cellular stress response and genomic integrity. Its role in tumorigenesis is controversial. Whereas sirtuin 1 (SIRT1) can act as a tumor suppressor in some solid tumors, increased expression has been demonstrated in many cancers, including hematologic malignancies. In chronic myeloid leukemia, SIRT1 promoted leukemia development, and targeting SIRT1 sensitized chronic myeloid leukemia progenitors to tyrosine kinase inhibitor treatment. In this study, we investigated the role of SIRT1 in acute myeloid leukemia (AML). We show that SIRT1 protein, but not RNA levels, is overexpressed in AML samples harboring activating mutations in signaling pathways. In FMS-l…

Dominant negative MORT1/FADD rescues mice from CD95 and TNF-induced liver failure

Derangement of the apoptotic program is considered an important cause of liver disease. It became clear that receptor-mediated apoptosis is of specific interest in this context, and CD95 and CD120a, both members of the tumor necrosis factor (TNF) receptor superfamily, are the most prominent cell death receptors involved. The death signal is induced upon ligand binding by recruitment of caspases via the adapter molecule MORT1/FADD to the receptor and their subsequent activation. To investigate the role of MORT1/FADD in hepatocyte apoptosis, we generated transgenic mice expressing liver-specific dominant negative mutant. Mice looked grossly normal; breeding and liver development were not diff…

Application of patient derived liver cancer cell lines for phenotypic characterization and therapeutic target identification

Cleavage of CD95 by matrix metalloproteinase-7 induces apoptosis resistance in tumour cells

The ability of tumour cells to resist apoptosis-inducing signals by cytotoxic T cells may decide the success or failure of tumour elimination. An important effector of apoptosis is the CD95/CD95 ligand system (APO-1/Fas) that mediates perforin-independent cytotoxic T-cell killing of tumour cells. We propose a new strategy by which tumour cells can resist CD95-induced apoptosis. We identified matrix metalloproteinase-7, MMP-7 (Martilysin), as the first physiologically relevant protease that can specifically cleave CD95. MMP-7 is of unique importance because it is produced by the tumour cells themselves at early stages of tumour development. Microsequencing of the positions in CD95 cleaved by…

Expression of Hugl-1 is strongly reduced in malignant melanoma.

The human gene Hugl-1 (Llgl/Lgl1) has significant homology to the Drosophila tumor suppressor gene lethal(2)giant larvae (lgl). The lgl gene codes for a cortical cytoskeleton protein, Lgl, that is involved in maintaining cell polarity and epithelial integrity. We speculate that Hugl-1 might play a role in epithelial-mesenchymal transition (EMT) and that loss of Hugl-1 expression plays a role in the development or progression of malignant melanoma. Thus, we evaluated melanoma cell lines and tissue samples of malignant melanoma for loss of Hugl-1 transcription. We found that Hugl-1 was downregulated or lost in all cell lines and in most of the tumor samples analysed, and that these losses wer…

Ginkgo biloba induces different gene expression signatures of oncogenic pathways in malignant and non-malignant cells in the liver

Role of SIRT1 and FOXO factors in eNOS transcriptional activation by resveratrol.

Many of the cardiovascular protective effects of resveratrol are attributable to an enhanced production of nitric oxide (NO) by the endothelial NO synthase (eNOS). Resveratrol has been shown to enhance eNOS gene expression as well as eNOS enzymatic activity. The aim of the present study was to analyze the molecular mechanisms of eNOS transcriptional activation by resveratrol. Treatment of human EA.hy 926 endothelial cells with resveratrol led to a concentration-dependent upregulation of eNOS expression. In luciferase reporter gene assay, resveratrol enhanced the activity of human eNOS promoter fragments (3500, 1600, 633 and 263bp in length, respectively), indicating that the proximal promot…

Molybdenum Trioxide Nanoparticles with Intrinsic Sulfite Oxidase Activity

Sulfite oxidase is a mitochondria-located molybdenum-containing enzyme catalyzing the oxidation of sulfite to sulfate in the amino acid and lipid metabolism. Therefore, it plays a major role in detoxification processes, where defects in the enzyme cause a severe infant disease leading to early death with no efficient or cost-effective therapy in sight. Here we report that molybdenum trioxide (MoO3) nanoparticles display an intrinsic biomimetic sulfite oxidase activity under physiological conditions, and, functionalized with a customized bifunctional ligand containing dopamine as anchor group and triphenylphosphonium ion as targeting agent, they selectively target the mitochondria while bein…

Polymeric hepatitis C virus non-structural protein 5A nanocapsules induce intrahepatic antigen-specific immune responses

Targeting antigen combined with adjuvants to hepatic antigen-presenting cells (APCs) is essential for the induction of intrahepatic T cellular immunity controlling and resolving viral infections of the liver. Intravenous injection of antigen-loaded nanoparticles is a promising approach for the delivery of antigens to liver APCs. Accordingly, polymeric nanocapsules (NCs) synthesized exclusively of hepatitis C virus non-structural protein 5A (NS5A) and the adjuvant monophosphoryl lipid A (MPLA) adsorbed to the nanocapsule surface were developed. Aim of the present study was the evaluation of the in vitro and in vivo behavior of MPLA-functionalized NS5A-NCs regarding the interaction with liver…

Ginkgo biloba induces different gene expression signatures and oncogenic pathways in malignant and non-malignant cells of the liver

Ginkgo biloba (EGb761) is a widely used botanical drug. Several reports indicate that EGb761 confers preventive as well as anti-tumorigenic properties in a variety of tumors, including hepatocellular carcinoma (HCC). We here evaluate functional effects and molecular alterations induced by EGb761 in hepatoma cells and non-malignant hepatocytes. Hepatoma cell lines, primary human HCC cells and immortalized human hepatocytes (IH) were exposed to various concentrations (0-1000 μg/ml) of EGb761. Apoptosis and proliferation were evaluated after 72h of EGb761 exposure. Response to oxidative stress, tumorigenic properties and molecular changes were further investigated. While anti-oxidant effects w…

Identification of a classic nuclear localization signal at the N terminus that regulates the subcellular localization of Rbfox2 isoforms during differentiation of NMuMG and P19 cells.

Nuclear localization of the alternative splicing factor Rbfox2 is achieved by a C-terminal nuclear localization signal (NLS) which can be excluded from some Rbfox2 isoforms by alternative splicing. While this predicts nuclear and cytoplasmic localization, Rbfox2 is exclusively nuclear in some cell types. Here, we identify a second NLS in the N terminus of Rbfox2 isoform 1A that is not included in Rbfox2 isoform 1F. Rbfox2 1A isoforms lacking the C-terminal NLS are nuclear, whereas equivalent 1F isoforms are cytoplasmic. A shift in Rbfox2 expression toward cytoplasmic 1F isoforms occurs during epithelial to mesenchymal transition (EMT) and could be important in regulating the activity and fu…

Glucagon-like peptide-1 receptor signalling reduces microvascular thrombosis, nitro-oxidative stress and platelet activation in endotoxaemic mice

Background and purpose Excessive inflammation in sepsis causes microvascular thrombosis and thrombocytopenia associated with organ dysfunction and high mortality. The present studies aimed to investigate whether inhibition of dipeptidyl peptidase-4 (DPP-4) and supplementation with glucagon-like peptide-1 (GLP-1) receptor agonists improved endotoxaemia-associated microvascular thrombosis via immunomodulatory effects. Experimental approach Endotoxaemia was induced in C57BL/6J mice by a single injection of LPS (17.5 mg kg-1 for survival and 10 mg kg-1 for all other studies). For survival studies, treatment was started 6 h after LPS injection. For all other studies, drugs were injected 48 h bef…

A systems biology perspective on cholangiocellular carcinoma development: focus on MAPK-signaling and the extracellular environment.

Background/Aims Multiple genes have been implicated in cholangiocellular carcinoma (CCC) development. However, the overall neoplastic risk is likely associated with a much lower number of critical physiological pathways. Methods To investigate this hypothesis, we extracted all published genetic associations for the development of CCC from PubMed (genetic association studies, but also studies associating genes and CCC in general, i.e. functional studies in cell lines, genetic studies in humans, knockout mice etc.) and integrated CCC microarray data. Results We demonstrated the MAPK pathway was consistently enriched in CCC. Comparing our data to genetic associations in HCC often successfully …

Histone deacetylase inhibition by valproic acid down-regulates c-FLIP/CASH and sensitizes hepatoma cells towards CD95-and TRAIL receptor-mediated apoptosis and chemotherapy

Hepatocellular carcinoma (HCC) is highly resistant to chemotherapy, leading to a poor prognosis of advanced disease. Inhibitors of histone deacetylase (HDACi) induce re-differentiation in tumor cells and thereby re-establish sensitivity towards apoptotic stimuli. HDACi are entering the clinical stage of tumor treatment, and several substances are currently being tested in clinical trials to prove their efficacy in the treatment of leukemias and solid tumors. In this study, we investigated the impact of the HDACi valproic acid (VA) on TRAIL- and CD95-mediated apoptosis in hepatoma cells, as well as its sensitizing effect on a chemotherapeutic agent. Treatment of HepG2 cells with VA increased…

Cloning and characterization of the promoter of Hugl-2, the human homologue of Drosophila lethal giant larvae (lgl) polarity gene.

The human lgl gene, Hugl-2 (llgl2, Lgl2), codes for a cytoskeletal protein involved in regulating cell polarity. Here, we report the identification and functional characterization of the promoter region ( approximately 1.2kb) of the Hugl-2 gene. Luciferase expression assays show a high basal Hugl-2 promoter activity in different cell lines and primary human hepatocytes. Truncations of the promoter identified a GC-rich region important for this activity. Alignment of human and mouse genomic sequences demonstrate that this is an evolutionary conserved region fcontaining putative binding sites for several transcription factors including Elk-1 and Sp-1. Mithramycin A reduces Hugl-2 expression i…

Nitric Oxide Promotes Resistance to Tumor Suppression by CTLs

Abstract Many human tumors express inducible NO synthetase (NOS2), but the roles of NO in tumor development are not fully elucidated. An important step during tumor development is the acquisition of apoptosis resistance. We investigated the dose-dependent effects of endogenously produced NO on apoptosis using ecdysone-inducible NOS2 cell lines. Our results show that short-term NOS2 expression enhances CD95-mediated apoptosis and T cell cytotoxicity dose dependently. Furthermore, we could show that during chronic exposure to NO, besides the primary cytotoxic NO effect, there is selection of cell clones resistant to NO that show cross-resistance to CD95-induced apoptosis and the killing by CT…

TGF-β Suppresses Tumor Progression in Colon Cancer by Inhibition of IL-6 trans-Signaling

Alterations of TGF-beta signaling have been described in colorectal cancer, although the molecular consequences are largely unknown. By using transgenic mice overexpressing TGF-beta or a dominant-negative TGF-betaRII, we demonstrate that TGF-beta signaling in tumor infiltrating T lymphocytes controls the growth of dysplastic epithelial cells in experimental colorectal cancer, as determined by histology and a novel system for high-resolution chromoendoscopy. At the molecular level, TGF-beta signaling in T cells regulated STAT-3 activation in tumor cells via IL-6. IL-6 signaling required tumor cell-derived soluble IL-6R rather than membrane bound IL-6R and suppression of such TGF-beta-depende…

Cooperation of Human Tumor-Reactive CD4+ and CD8+ T Cells after Redirection of Their Specificity by a High-Affinity p53A2.1-Specific TCR

Abstract Efficient immune attack of malignant disease requires the concerted action of both CD8 + CTL and CD4 + Th cells. We used human leukocyte antigen (HLA)-A*0201 (A2.1) transgenic mice, in which the mouse CD8 molecule cannot efficiently interact with the α3 domain of A2.1, to generate a high-affinity, CD8-independent T cell receptor (TCR) specific for a commonly expressed, tumor-associated cytotoxic T lymphocyte (CTL) epitope derived from the human p53 tumor suppressor protein. Retroviral expression of this CD8-independent, p53-specific TCR into human T cells imparted the CD8 + T lymphocytes with broad tumor-specific CTL activity and turned CD4 + T cells into potent tumor-reactive, p53…

Damage-associated molecular pattern activated Toll-like receptor 4 signalling modulates blood pressure in L-NAME-induced hypertension

Aims Recent publications have shed new light on the role of the adaptive and innate immune system in the pathogenesis of hypertension. However, there are limited data whether receptors of the innate immune system may influence blood pressure. Toll-like receptor 4 (TLR4), a pattern recognition receptor, is a key component of the innate immune system, which is activated by exogenous and endogenous ligands. Hypertension is associated with end-organ damage and thus might lead to the release of damage-associated molecular patterns (DAMPs), which are endogenous activators of TLR4 receptors. The present study aimed to elucidate whether TLR4 signalling is able to modulate vascular contractility in …

CAF-like state in primary skin fibroblasts with constitutional BRCA1 epimutation sheds new light on tumor suppressor deficiency-related changes in healthy tissue

Constitutive epimutations of tumor suppressor genes are increasingly considered as cancer predisposing factors equally to sequence mutations. In light of the emerging role of the microenvironment for cancer predisposition, initiation, and progression, we aimed to characterize the consequences of a BRCA1 epimutation in cells of mesenchymal origin. We performed a comprehensive molecular and cellular comparison of primary dermal fibroblasts taken from a monozygous twin pair discordant for recurrent cancers and BRCA1 epimutation, whose exceptional clinical case we previously reported in this journal. Comparative transcriptome analysis identified differential expression of extracellular matrix-r…

mTOR Driven Gene Transcription Is Required for Cholesterol Production in Neurons of the Developing Cerebral Cortex

AbstractDysregulated mammalian target of rapamycin (mTOR) activity is associated with various neurodevelopmental disorders ranging from idiopathic autism spectrum disorders to syndromes caused by single gene defects. This suggests that maintaining mTOR activity levels in a physiological range is essential for brain development and functioning. Upon activation, mTOR regulates a variety of cellular processes such as cell growth, autophagy and metabolism. On a molecular level, however, the consequences of mTOR activation in the brain are not well understood.Low levels of cholesterol are associated with a wide variety of neurodevelopmental disorders. We here describe numerous genes of the stero…

Lipid presentation by the protein C receptor links coagulation with autoimmunity.

A lipid-protein autoimmunity target Several autoimmune diseases, including systemic lupus erythematosus and primary antiphospholipid syndrome, are characterized by the presence of antiphospholipid antibodies (aPLs). These molecules can activate the complement and coagulation cascades, which contributes to pathologies such as thrombosis, stroke, and pregnancy complications. Müller-Calleja et al. found that endothelial protein C receptor (EPCR) in complex with lysobisphosphatidic acid (LBPA) is the cell-surface target for aPL and mediates its internalization (see the Perspective by Kaplan). aPL binding to EPCR-LBPA resulted in the activation of tissue factor–mediated coagulation and interfero…

Superior antitumor in vitro responses of allogeneic matched sibling compared with autologous patient CD8+ T cells.

AbstractAllogeneic cell therapy as a means to break immunotolerance to solid tumors is increasingly used for cancer treatment. To investigate cellular alloimmune responses in a human tumor model, primary cultures were established from renal cell carcinoma (RCC) tissues of 56 patients. In three patients with stable RCC line and human leukocyte antigen (HLA)-identical sibling donor available, allogeneic and autologous RCC reactivities were compared using mixed lymphocyte/tumor cell cultures (MLTC). Responding lymphocytes were exclusively CD8+ T cells, whereas CD4+ T cells or natural killer cells were never observed. Sibling MLTC populations showed higher proliferative and cytolytic antitumor …

Liver-specific Ldb1 deletion results in enhanced liver cancer development.

Background & Aims LIM-domain-binding (Ldb) proteins have been demonstrated to be essential not only to key embryonic developmental processes but also to carcinogenesis. We have previously demonstrated Ldb1 to be of high biological and developmental relevance, as a targeted deletion of the Ldb1 gene in mice results in an embryonic lethal and pleiotropic phenotype. Methods We have now established a liver-specific Ldb1 knock out to investigate the role of Ldb1 in carcinogenesis, in particular in hepatocellular carcinoma (HCC) development, in vivo . Results These mice demonstrated a significantly enhanced growth of liver cancer by means of tumor size and number, advocating for an essential role…

Glycine-functionalized copper(ii) hydroxide nanoparticles with high intrinsic superoxide dismutase activity

Superoxide dismutases (SOD) are a group of enzymes that catalyze the dismutation of superoxide (O2−) radicals into molecular oxygen (O2) and H2O2 as a first line of defense against oxidative stress. Here, we show that glycine-functionalized copper(II) hydroxide nanoparticles (Gly-Cu(OH)2 NPs) are functional SOD mimics, whereas bulk Cu(OH)2 is insoluble in water and catalytically inactive. In contrast, Gly-Cu(OH)2 NPs form water-dispersible mesocrystals with a SOD-like activity that is larger than that of their natural CuZn enzyme counterpart. Based on this finding, we devised an application where Gly-Cu(OH)2 NPs were incorporated into cigarette filters. Cigarette smoke contains high concent…

CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes

Azathioprine and its metabolite 6-mercaptopurine (6-MP) are immunosuppressive drugs that are used in organ transplantation and autoimmune and chronic inflammatory diseases such as Crohn disease. However, their molecular mechanism of action is unknown. In the present study, we have identified a unique and unexpected role for azathioprine and its metabolites in the control of T cell apoptosis by modulation of Rac1 activation upon CD28 costimulation. We found that azathioprine and its metabolites induced apoptosis of T cells from patients with Crohn disease and control patients. Apoptosis induction required costimulation with CD28 and was mediated by specific block- ade of Rac1 activation thro…

Getting the Fat out of Met and Fas

Lack of Fas antagonism by Met in human fatty liver disease. Zou C, Ma J, Wang X, Guo L, Zhu Z, Stoops J, Eaker AE, Johnson CJ, Strom S, Michalopoulos GK, DeFrances MC, Zarnegar R. Hepatocytes in fatty livers are hypersensitive to apoptosis and undergo escalated apoptotic activity via death receptor-mediated pathways, particularly that of Fas–FasL, causing hepatic injury that can eventually proceed to cirrhosis and end-stage liver disease. Here we report that the hepatocyte growth factor receptor, Met, plays an important part in preventing Fas-mediated apoptosis of hepatocytes by sequestering Fas. We also show that Fas antagonism by Met is abrogated in human fatty liver disease (FLD). Throug…

Increased stability of the TM helix oligomer abrogates the apoptotic activity of the human Fas receptor

Human death receptors control apoptotic events during cell differentiation, cell homeostasis and the elimination of damaged or infected cells. Receptor activation involves ligand-induced structural reorganizations of preformed receptor trimers. Here we show that the death receptor transmembrane domains only have a weak intrinsic tendency to homo-oligomerize within a membrane, and thus these domains potentially do not significantly contribute to receptor trimerization. However, mutation of Pro183 in the human CD95/Fas receptor transmembrane helix results in a dramatically increased interaction propensity, as shown by genetic assays. The increased interaction of the transmembrane domain is co…

Application of Patient-Derived Liver Cancer Cells in Personalized Treatment Approach: Phenotypic Characterization and Therapeutic Target Identification

Application of patient-derived liver cancer cells for phenotypic characterization and therapeutic target identification.

Primary liver cancer (PLC) ranks among the most lethal solid cancers worldwide due to lack of effective biomarkers for early detection and limited treatment options in advanced stages. Development of primary culture models that closely recapitulate phenotypic and molecular diversities of PLC is urgently needed to improve the patient outcome. Long-term cultures of 7 primary liver cancer cell lines of hepatocellular and cholangiocellular origin were established using defined culture conditions. Morphological and histological characteristics of obtained cell lines and xenograft tumors were analyzed and compared to original tumors. Time course analyses of transcriptomic and genomic changes were…

EGF and HGF levels are increased during active HBV infection and enhance survival signaling through extracellular matrix interactions in primary human hepatocytes

The hepatitis B virus (HBV) is a major causative agent of chronic liver disease and subsequent liver cirrhosis worldwide. The reduced sensitivity of virus-infected liver cells to apoptosis may play a role in the failure to remove virus-infected cells and eventually promote viral chronicity. The purpose of our study was to investigate whether survival factors induced during compensatory liver regeneration may protect hepatocytes against apoptosis. We evaluated the serum levels of hepatocyte growth factor (HGF) and epidermal growth factor (EGF) in HBV-infected patients and found significant increases in HGF and EGF in patients with active virus infection. In primary human hepatocytes we show …

Sirtuin-6-dependent genetic and epigenetic alterations are associated with poor clinical outcome in hepatocellular carcinoma patients

Sirtuin 6 (SIRT6) is a member of the sirtuin family of NAD+–dependent deacetylases. Genetic deletion of Sirt6 in mice results in a severe degenerative phenotype with impaired liver function and premature death. The role of SIRT6 in development and progression of hepatocellular carcinoma is currently unknown. We first investigated SIRT6 expression in 153 primary human liver cancers and in normal and cirrhotic livers using microarray analysis. SIRT6 was significantly down-regulated in both cirrhotic livers and cancer. A Sirt6 knockout (KO) gene expression signature was generated from primary hepatoctyes isolated from 3-week-old Sirt6-deficient animals. Sirt6-deficient hepatocytes showed up-re…

Regulation of the hDlg/hScrib/Hugl-1 tumour suppressor complex.

The proper function of the Scribble tumour suppressor complex is dependent upon the correct localisation of its components. Previously we observed dynamic relocalisation of the hDlg component under conditions of osmotic stress. We now show that the other two components of the complex, hScrib and Hugl-1 display similar patterns of expression. We demonstrate, by shRNA ablation of hScrib expression, that hDlg and Hugl-1 are in part dependent upon hScrib for their correct localization. However under conditions of osmotic stress this apparent dependency no longer exists: hDlg and Hugl-1 localise to cell membranes independently of hScrib. We also demonstrate an interaction between the three compo…

Targeting positive regulatory domain I-binding factor 1 and X box-binding protein 1 transcription factors by multiple myeloma-reactive CTL.

Abstract Growing evidence indicates that multiple myeloma (MM) and other malignancies are susceptible to CTL-based immune interventions. We studied whether transcription factors inherently involved in the terminal differentiation of mature B lymphocytes into malignant and nonmalignant plasma cells provide MM-associated CTL epitopes. HLA-A*0201 (A2.1) transgenic mice were used to identify A2.1-presented peptide Ag derived from the plasma cell-associated transcriptional regulators, positive regulatory domain I-binding factor 1 (PRDI-BF1) and X box-binding protein 1 (XBP-1). A2.1-restricted CTL specific for PRDI-BF1 and XBP-1 epitopes efficiently killed a variety of MM targets. PRDI-BF1- and X…

The human Lgl polarity gene, Hugl-2, induces MET and suppresses Snail tumorigenesis

Lethal giant larvae proteins have key roles in regulating polarity in a variety of cell types and function as tumour suppressors. A transcriptional programme initiated by aberrant Snail expression transforms epithelial cells to potentially aggressive cancer cells. Although progress in defining the molecular determinants of this programme has been made, we have little knowledge as to how the Snail-induced phenotype can be suppressed. In our studies we identified the human lethal giant larvae homologue 2, Hugl-2, (Llgl2/Lgl2) polarity gene as downregulated by Snail. Snail binds E-boxes in the Hugl-2 promoter and represses Hugl-2 expression, whereas removal of the E-boxes releases Hugl-2 from …