Lipocalin-2 (LCN2) is a 25-kDa secretory protein with great importance as a sensitive and specific screening and diagnostic biomarker for hepatic inflammation, acute hepatic injury, hepatic cancer, radiation-induced early phase hepatic damage and lipid abnormalities. However, little is known about LCN2's functions in the homeostasis and metabolism of hepatic lipids or in the development of steatosis as well as its actual role in the event of inflammation. The aim of this study was to prospectively evaluate the LCN2 role in the ‘two-hit’ development of Non-alcoholic steatohepatitis (NASH) meaning the steatosis and the injury, observed by the appearance of inflammation. NASH is a condition where fatty liver is accompanied with inflammation. The objective of this study was to determine in wild type (WT) and Lcn2-deficient (Lcn2−/−) mice the possible differences in their biological reactions after a Methionine and Choline deficient diet (MCD), a nutritional model used to induce the pathological condition of NASH. The current study compares initially the intrahepatic lipid accumulation, lipid droplet formation, mitochondrial content, and expression of the Perilipin proteins known to regulate cellular lipid metabolism. Lcn2−/− mice fed with MCD diet accumulated more lipids in the liver than WT controls, and that the basal expression of the lipid droplet coat protein Perilipin 5 (PLIN5) was significantly reduced in these animals. Similarly, the overexpression of LCN2 and PLIN5 were also found in animals that were fed with a high fat diet. Furthermore, the loss of LCN2 and/or PLIN5 in hepatocytes prevented normal intracellular lipid droplet formation both in vitro and in vivo. In in vitro experimentation the restoration of LCN2 in Lcn2−/− primary hepatocytes by either transfection or adenoviral vector infection induced PLIN5 expression and restored proper lipid droplet formation. The other section of this study targeted the role of LCN2 specifically in the inflammation process accompanying hepatic injury after administration of the MCD diet. Therefore, the hepatic LCN2 and inflammatory cytokines’ interleukin- 6 and interleukin-1β (IL-6, IL-1β), tumor necrosis factor α (TNF-α), chemokine (C-C motif) ligand 2 (CCL2), and chemokine (C-C motif) receptor 2 (CCR2) expression was compared between the two genotypes. The expression of the activated forms of the Signal Transducers and Activators of Transcription (STAT1 and STAT3) were tested as well. Additionally, histology inflammation score and immunohistochemistry stainings with leukocytes and neutrophil granulocyte markers were performed. Both genotypes had inflammation sites as a result of the MCD diet, however the WT mice recruited more inflammatory cells than the Lcn2−/− accompanied with a strong upregulation of LCN2. Moreover, the WT mice fed with MCD diet, presented a remarkably stronger expression of phosphorylated forms of STAT1 and STAT3. In conclusion this study shows that LCN2 is a protein of high significance with multiple functions in MCD-induced NASH in mice. The presented findings indicate that first of all, LCN2 is a key modulator of hepatic lipid homeostasis that controls the formation of intracellular lipid droplets by regulating PLIN5 expression while a second significant role in inflammation is that its upregulation serves as a ‘HELP-ME’ signal while inflammation appears. The upregulation of the LCN2-‘HELP-ME’-factor, enhances the recruitment of neutrophil granulocytes and leukocytes towards resolution of the inflammation protecting the liver from progression to further hepatic damage agreeing to previous reports of LCN2’s hepato-protective role in liver injury models. LCN2 may therefore represent a novel therapeutic drug target for the treatment of liver diseases associated with inflammation and elevated fat accumulation.
<p>Lentiviral transduction of miR-371~373 cluster restores miR-371~373 expression in different SCs</p>
Hepatic stellate cells (HSC) are the main effector cells for liver fibrosis. We aimed at optimizing HSC isolation by an additional step of fluorescence-activated cell sorting (FACS) via a UV laser. HSC were isolated from livers of healthy mice and animals subjected to experimental fibrosis. HSC isolation by iohexol- (Nycodenz) based density centrifugation was compared to a method with subsequent FACS-based sorting. We assessed cellular purity, viability, morphology, and functional properties like proliferation, migration, activation marker, and collagen expression. FACS-augmented isolation resulted in a significantly increased purity of stellate cells (>99%) compared to iohexol-based density centrifugation (60-95%), primarily by excluding doublets of HSC and Kupffer cells (KC). Importantly, this method is also applicable to young animals and mice with liver fibrosis. Viability, migratory properties, and HSC transdifferentiation in vitro were preserved upon FACS-based isolation, as assessed using time lapse microscopy. During maturation of HSC in culture, we did not observe HSC cell division using time lapse microscopy. Strikingly, FACS-isolated, differentiated HSC showed very limited molecular and functional responses to LPS stimulation. In conclusion, isolating HSC from mouse liver by additional FACS significantly increases cell purity by removing contaminations from other cell populations especially KC, without affecting HSC viability, migration, or differentiation.
Read more<p>miR-371~373 cluster expression in extracted xenografts and its effect on metastatic outgrowth capacity in SW620 SCs</p>
Read more<div>Abstract<p>The vast majority of colorectal cancer–related deaths can be attributed to metastatic spreading of the disease. Therefore, deciphering molecular mechanisms of metastatic dissemination is a key prerequisite to improve future treatment options. With this aim, we took advantage of different colorectal cancer cell lines and recently established primary cultures enriched in colon cancer stem cells, also known as tumor-initiating cells (TIC), to identify genes and miRNAs with regulatory functions in colorectal cancer progression. We show here that metastasis-derived TICs display increased capacity for self-renewal, TGFβ signaling activity, and reduced expression of the miR-371∼373 cluster compared with nonmetastatic cultures. TGFβ receptor 2 (<i>TGFBR2</i>) and aldehyde dehydrogenase A1 (<i>ALDH1A1</i>) were identified as important target genes of the miR-371∼373 cluster. In addition, TGFBR2 repression, either by direct knockdown or indirectly via overexpression of the entire miR-371∼373 cluster, decreased tumor-initiating potential of TICs. We observed significantly reduced <i>in vitro</i> self-renewal activity as well as lowered tumor initiation and metastatic outgrowth capacity <i>in vivo</i> following stable overexpression of the miR-371∼373 cluster in different colon TIC cultures. Inhibitor of DNA binding 1 (ID1) was affected by both TGFBR2 and miR-371∼373 cluster alterations. Functional sphere and tumor formation as well as metastatic dissemination assays validated the link between miR-371∼373 and ID1. Altogether, our results establish the miR-371∼373/TGFBR2/ID1 signaling axis as a novel regulatory mechanism of TIC self-renewal and metastatic colonization.</p><p><b>Significance:</b> These findings establish the miR-371∼373/TGFBR2/ID1 signaling axis as a novel mechanism regulating self-renewal of tumor-initiating cell and metastatic colonization, potentially opening new concepts for therapeutic targeting of cancer metastasis.</p><p><b>Graphical Abstract:</b> <a href="http://cancerres.aacrjournals.org/content/canres/78/14/3793/F1.large.jpg" target="_blank">http://cancerres.aacrjournals.org/content/canres/78/14/3793/F1.large.jpg</a>. <i>Cancer Res; 78(14); 3793–808. ©2018 AACR</i>.</p></div>
Read moreBackground: Microtubules are attractive targets for anticancer drugs. However, the microtubule-targeting agents (MTAs) currently in clinical use exhibit inevitable drug resistance. Therefore, there is an urgent need to discover novel MTAs for the clinical treatment of cancer. Methods: Bioactive compounds extracted from Lithospermum erythrorhizon were assessed for in vitro anti-proliferative activities against a panel of human cancer cell lines using cell counting kit-8 (CCK-8) assay. Tubulin polymerization inhibition assay, colchicine competitive binding site assay, and immunofluorescence were used to validate the tubulin inhibition effect of acetylshikonin. Flow cytometry, Hoechst staining, and caspase-3 activity evaluation were performed to assess cell cycle arrest and cell apoptosis. 5,5',6,6'-tetrachloro-1,1',3,3'-tetramethylbenzimidazolylcarbocyanine iodide (JC-1) staining and dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining were used to evaluate mitochondrial membrane potential (MMP) and reactive oxygen species (ROS), respectively. Results: Acetylshikonin exhibited potent anti-proliferative activities against a panel of human cancer cell lines (IC50 values: 1.09–7.26 µM) and displayed comparable cytotoxicity against several drug-resistant cell lines. Further mechanism studies revealed that acetylshikonin induced cell cycle arrest of MHCC-97H cells at G2/M phase, and significantly promoted apoptosis marked by a collapse of MMP and abnormal ROS accumulation. Conclusions: In this study, acetylshikonin was identified as MTA against hepatocellular carcinoma and can serve as a promising lead compound for further development of anti-cancer drug, underscoring its potential clinical significance.
Read moreFibrosis is a double-edged sword [...]
Read moreATM received an email from Web of Science informing that it had decided not to index publications from ATM any longer because of two articles published outside the scope of translational medicine (1).
Read more<p>Lentiviral transduction of miR-371~373 cluster restores miR-371~373 expression in different SCs</p>
Read moreBackground: Lipocalin-2 (LCN2), also known as neutrophil gelatinase-associated lipocalin (NGAL), is a small secreted adipokine that belongs to the family of lipocalins that are known for binding and transporting small hydrophobic molecules such as fatty acids, steroids and iron [1]. In the liver, LCN2 plays a protective role in inflammation, infection, injury and other forms of cellular stress. This was proven in the bile duct ligation and the carbon tetrachloride model [2]. Recently, we demonstrated that LCN2 regulates perilipin 5 (PLIN5) expression in primary hepatocytes [3].
Read moreLipocalin-2 (LCN2) performs pleiotropic and tumor context-dependent functions in cancers of diverse etiologies. In prostate cancer (PCa) cells, LCN2 regulates distinct phenotypic features, including cytoskeleton organization and expression of inflammation mediators. Oncolytic virotherapy uses oncolytic viruses (OVs) to kill cancer cells and induce anti-tumor immunity. A main source of specificity of OVs towards tumor cells stems from cancer-induced defects in interferon (IFN)-based cell autonomous immune responses. However, the molecular underpinnings of such defects in PCa cells are only partially understood. Moreover, LCN2 effects on IFN responses of PCa cells and their susceptibility to OVs are unknown. To examine these issues, we queried gene expression databases for genes coexpressed with LCN2, revealing co-expression of IFN-stimulated genes (ISGs) and LCN2. Analysis of human PCa cells revealed correlated expression of LCN2 and subsets of IFNs and ISGs. CRISPR/Cas9-mediated stable knockout of LCN2 in PC3 cells or transient overexpression of LCN2 in LNCaP cells revealed LCN2-mediated regulation of IFNE (and IFNL1) expression, activation of JAK/STAT pathway, and expression of selected ISGs. Accordingly, and dependent on a functional JAK/STAT pathway, LCN2 reduced the susceptibility of PCa cells to infection with the IFN-sensitive OV, EHDV-TAU. In PC3 cells, LCN2 knockout increased phosphorylation of eukaryotic initiation factor 2α (p-eIF2α). Inhibition of PKR-like ER kinase (PERK) in PC3-LCN2-KO cells reduced p-eIF2α while increasing constitutive IFNE expression, phosphorylation of STAT1, and ISG expression; and decreasing EHDV-TAU infection. Together, these data propose that LCN2 regulates PCa susceptibility to OVs through attenuation of PERK activity and increased IFN and ISG expression.
Read more<p>Antibodies, primers, and lentiviral particles (S1); in vivo tumorigenicity for SW480 vs. SW620 (S2); top 20 differentially expressed miRNAs (S3); peritoneal cancer index scores (S4)</p>
Read moreBackground and Aims: Transforming growth factor-β1 (TGF-β1) plays important roles in chronic liver diseases, including metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD involves various biological processes including dysfunctional cholesterol metabolism and contributes to progression to metabolic dysfunction-associated steatohepatitis (MASH) and hepatocellular carcinoma (HCC). However, the reciprocal regulation of TGF-β1 signaling and cholesterol metabolism in MASLD is yet unknown. Methods: Changes in transcription of genes associated with cholesterol metabolism were assessed by RNA-Seq of murine hepatocyte cell line (AML12) and mouse primary hepatocytes (MPH) treated with TGF-β1. Functional assays were performed on AML12 cells (untreated, TGF-β1 treated, or subjected to cholesterol enrichment (CE) or depletion (CD)), and on mice injected with adeno-associated virus 8 (AAV8)-Control/TGF-β1. Results: TGF-β1 inhibited mRNA expression of several cholesterol metabolism regulatory genes, including rate-limiting enzymes of cholesterol biosynthesis in AML12 cells, MPHs, and AAV8-TGF-β1-treated mice. Total cholesterol levels and lipid droplet accumulation in AML12 cells and liver tissue were also reduced upon TGF-β1 treatment. Smad2/3 phosphorylation following 2 h TGF-β1 treatment persisted after CE or CD and was mildly increased following CD, while TGF-β1-mediated AKT phosphorylation (30 min) was inhibited by CE. Furthermore, CE protected AML12 cells from several effects mediated by 72 h incubation with TGF-β1, including EMT, actin polymerization, and apoptosis. CD mimicked the outcome of long term TGF- β1 administration, an effect that was blocked by an inhibitor of the type I TGF-β receptor. Additionally, the supernatant of CE- or CD-treated AML12 cells inhibited or promoted, respectively, the activation of LX-2 hepatic stellate cells. Conclusions: TGF-β1 inhibits cholesterol metabolism while cholesterol attenuates TGF-β1 downstream effects in hepatocytes.
Read moreThe cellular (centralized) communication network (CCN) factor protein family contains six small secreted cysteine-rich proteins sharing high structural similarity. These matricellular proteins have vital biological functions in cell adhesion, migration, cell cycle progression, and control of production and degradation of extracellular matrix. However, in liver the biological functions of CCN proteins become most visible during hepatic injury, disease, and remodeling. In particular, most of the hepatic functions of CCN proteins were derived from CCN2/CTGF, which becomes highly expressed in damaged hepatocytes and acts as a profibrogenic molecule. On the contrary, CCN1/CYR61 seems to have opposite effects, while the biological activity during hepatic fibrosis is somewhat controversially discussed for other CCN family members. In the present study, we analyzed the expression of CCN5/WISP2 in cultures of different types of primary liver cells and in an experimental model of hepatic fibrosis. We found that CCN5 is expressed in hepatic stellate cells, myofibroblasts and portal myofibroblasts, while CCN5 expression is virtually absent in hepatocytes. During hepatic fibrogenesis, CCN5 is significantly upregulated. Overexpression of CCN5 in portal myofibroblasts reduced expression of transforming growth factor-β receptor I (ALK5) and concomitant Smad2 activation, whereas JunB expression is upregulated. Moreover, elevated expression of CCN5 induces endoplasmic reticulum stress, unfolded protein response and apoptosis in portal myofibroblasts. We suggest that upregulated expression of CCN5 might be an intrinsic control mechanism that counteracts overshooting fibrotic responses in profibrogenic liver cells.
Read moreMast cells (MCs) are immune cells of the myeloid lineage distributed in tissues throughout the body. Phenotypically, they are a heterogeneous group characterized by different protease repertoires stored in secretory granules and differential presence of receptors. To adequately address aspects of MC biology either primary MCs isolated from human or mouse tissue or different human MC lines, like HMC-1.1 and -1.2, or rodent MC lines like L138.8A or RBL-2H3 are frequently used. Nevertheless, cellular systems to study MC functions are very limited. We have generated a murine connective tissue-like MC line, termed PMC-306, derived from primary peritoneal MCs (PMCs), which spontaneously transformed. We analyzed PMC-306 cells regarding MC surface receptor expression, effector functions and respective signaling pathways, and found that the cells reacted very similar to primary wildtype (WT) PMCs. In this regard, stimulation with MAS-related G-protein-coupled receptor member B2 (MRGPRB2) ligands induced respective signaling and effector functions. Furthermore, PMC-306 cells revealed significantly accelerated cell cycle progression, which however was still dependent on interleukine 3 (IL-3) and stem cell factor (SCF). Phenotypically, PMC-306 cells adopted an immature connective tissue-like MCs appearance. The observation of cellular transformation was accompanied by the loss of Cdkn2a and Arf expression, which are both described as critical cell cycle regulators. The loss of Cdkn2a and Arf expression could be mimicked in primary bone marrow-derived mast cells (BMMCs) by sustained SCF supplementation strongly arguing for an involvement of KIT activation in the regulation of Cdkn2a / Arf expression. Hence, this new cell line might be a useful tool to study further aspects of PMC function and to address tumorigenic processes associated with MC leukemia.
Read more<p>Investigation of SOX2 and BMP signaling as potential miR-371~373/TGFBR2 targets</p>
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