ABSTRACT
Hepatoma exhibits a series of heterogeneous subpopulations in its cell surface markers, tumorigenicity, invasion and metastatic capability. We previously demonstrated that the CD133(-)/EpCAM(-) hepatoma subpopulation was more metastatic than its counterpart; however, the controlling mechanisms are unexplored. The present study aimed to delineate the significance of aberrant hedgehog (Hh) signaling in the mediation of metastases. Fluorescence-activated cell sorting-enriched CD133(-)/EpCAM(-) (double negative, DN), Huh-7 cells underwent a transwell selection for metastatic cells (transwell-selected, TS). The TS cells displayed much greater metastatic activity as evidenced by an increased invasion rate, extremely upregulated expression of matrix metalloproteinase (MMP)-1/2/9 genes compared with DN and double-positive (DP) subpopulations. In contrast to DP cells, TS cells lost E-cadherin and were all vimentin-positive as shown by immunocytochemistry. There was a transitional increase in Gli-1/2 gene expression levels from DP, DN to TS subpopulations, which was consistent with elevated Gli-1/2 or Twist-1 protein levels in the nuclear fraction. Furthermore, truncated Gli-1 (tGli-1), which transactivates molecules involved in metastasis, was detected in the highly invasive Huh-7 cell subpopulation, but not in less metastatic hepatoma cells or normal hepatocytes. The enhanced metastatic features with increased expression of MMPs as well as the presence of twist and snail genes in TS Huh-7 cells were reversed by LDE225, a potent Smoothened antagonist. In conclusion, the highly metastatic capability of a unique TS subpopulation was highly attributed to significant epithelial-mesenchymal transition, enhanced Hh activity and aberrant occurrence of a tGli-1 variant, which appears to be responsible for the highly invasive behavior.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Hedgehog Proteins/metabolism , Liver Neoplasms/metabolism , Nuclear Proteins/metabolism , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Epithelial-Mesenchymal Transition , Hep G2 Cells , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Metalloendopeptidases/genetics , Metalloendopeptidases/metabolism , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolism , Zinc Finger Protein GLI1ABSTRACT
The central role of endoconvertases and HIV-1 protease (HIV-1 PR) in the processing of HIV proproteins makes the design of specific inhibitors important in anti-HIV gene therapy. Accordingly, we tested native alpha(1) antitrypsin (alpha(1)AT) delivered by a recombinant simian virus-40-based vector, SV(AT), as an inhibitor of HIV-1 proprotein maturation. Cell lines and primary human lymphocytes were transduced with SV(AT) without selection and detectable toxicity. Expression of alpha(1)AT was confirmed by Northern blotting, immunoprecipitation and immunostaining. SV(AT)-transduced cells showed no evidence of HIV-1-related cytopathic effects when challenged with high doses of HIV-1(NL4-3). As measured by HIV-1 p24 assay, SV(AT)-transduced cells were protected from HIV-1(NL4-3) at challenge dose of 40 000 TCID(50) (MOI = 0.04). In addition, peripheral blood lymphocytes treated with SV(AT) were protected from HIV doses challenge up to 40 000 TCID(50) (MOI = 0.04). By Western blot analyses, the delivered alpha(1)AT inhibited cellular processing of gp160 to gp120 and decreased HIV-1 virion gp120. SV(AT) inhibited processing of p55(Gag) as well. Furthermore, high levels of uncleaved p55(Gag) protein were detected in HIV virus particles recovered from SV(AT)-transduced cells lines and primary lymphocytes. Thus, delivering alpha(1)AT using SV(AT) to human lymphocytes strongly inhibits replication of HIV-1, most likely by inhibiting the activities both of the cellular serine proteases involved in processing gp160 and of the aspartyl protease, HIV-1 PR, which cleaves p55(Gag). alpha(1)AT delivered by SV(AT) may represent a novel and effective strategy for gene therapy to interfere with HIV replication, by blocking a stage in the virus replicative cycle that has until now been inaccessible to gene therapeutic intervention.
Subject(s)
Gene Products, gag/metabolism , Genetic Therapy/methods , HIV Infections/therapy , Protein Precursors/metabolism , Serine Proteinase Inhibitors/genetics , alpha 1-Antitrypsin/genetics , Animals , COS Cells , Cells, Cultured , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , HIV Envelope Protein gp160/metabolism , HIV Infections/immunology , Humans , Lymphocytes/immunology , Serine Proteinase Inhibitors/analysis , Serine Proteinase Inhibitors/metabolism , Simian virus 40/genetics , Transduction, Genetic/methods , Virus Replication , alpha 1-Antitrypsin/analysis , alpha 1-Antitrypsin/metabolism , gag Gene Products, Human Immunodeficiency Virus , pol Gene Products, Human Immunodeficiency VirusABSTRACT
We have previously demonstrated that liposomes generated from poly(cationic lipid) (PCL) and cholesterol (Chol) have low cytotoxicity, are serum resistant, and display a transfection efficiency in vitro similar to commercially available cationic liposomes. Our in vivo experiments demonstrated that PCL-Chol liposomes bound much less avidly to serum proteins than did liposomes composed of 1,2-bis(dioleoyloxy)-3-(trimethylamonio)propane (DOTAP)-Chol or DOTAP-L-alpha dioleoyl phosphatidylethanolamine (DOPE). Injection of the lipoplexes (PCL-Chol+DNA) through the portal vein after partial hepatectomy (PH) led to much higher reporter gene expression (luciferase) in the liver than did naked DNA injection. Marked green fluorescent protein expression was visualized in almost all hepatocytes in the liver of mice receiving lipoplex injection, even in the absence of PH. Subcutaneous injection of thyroid hormone triiodothyromine (T(3)) significantly promoted hepatocyte regeneration and markedly enhanced PCL-Chol-mediated gene transfer in mouse liver when the lipoplex was administrated through either portal or tail vein. With T(3) pretreatment, PCL-Chol exerted a better gene transfer efficacy in mouse liver than DOTAP-Chol or DOTAP-DOPE. Two injections of lipoplexes through an indwelling catheter in the portal vein extended the transgene expression at a high level when T(3) injection was repeated. In conclusion, our findings demonstrate that the polymerized cationic liposomes are very stable in the blood and are effective agents for in vivo gene delivery, and that thyroid hormone administration offers a non-invasive approach to enhance liposome-mediated liver gene delivery.
Subject(s)
Gene Targeting/methods , Genetic Vectors/administration & dosage , Liver/metabolism , Transfection/methods , Animals , Cholesterol , Genes, Reporter , Green Fluorescent Proteins , Hepatectomy , Injections, Intravenous , Lipids/genetics , Liposomes , Liver/drug effects , Luciferases/analysis , Luciferases/genetics , Luminescent Proteins/analysis , Luminescent Proteins/genetics , Mice , Polyamines , Polyelectrolytes , Portal Vein , Thyroid Hormones/pharmacologyABSTRACT
In vivo gene delivery mediated by cationic lipids is often compromised by aggregation due to complexation with proteins in the blood. To improve the stability of cationic lipid-DNA complexes, the present study aimed to develop a novel approach in which a poly(cationic lipid) (PCL) is utilized to form stable cationic polyplexes for gene transfection. Hydrogenation of the acrylamide analogue of betaAE-DMRI, the polymerizable precursor of PCL, provided a monomeric lipid derivative (MHL) which was used for direct comparison of corresponding lipoplex stability, toxicity, and transfection activity. Various formulations of cationic liposomes, such as MHL, MHL-cholesterol (Chol), PCL, PCL-Chol, DOTAP-Chol, and commercially available lipofectamine were generated and examined in this study. The new poly(cationic lipid) did not display any significant toxicity to rat hepatocytes or Hep G2 cells as indicated by an LDH leakage assay. Furthermore, PCL was significantly less toxic than MHL, DOTAP-Chol or lipofectamine. Suspensions of PCL were resistant to aggregation even after 24 h of exposure to solutions containing 50 and 100% fetal bovine serum (FBS). In contrast, suspensions of lipofectamine extensively aggregated after 24 h of exposure to 50% FBS. To examine the influence of lipid polymerization on gene transfer activity, liposome-mediated transfections of a luciferase vector (pGL3) were performed in Hep G2 and Alexander cell lines. The luciferase activity of the PCL formulations in Hep G2 cells were similar to those of the MHL, DOTAP-Chol and lipofectamine formulations, demonstrating that lipid polymerization does not compromise transfection activity. In comparison to the monomeric precursor MHL and to the industry transfection standards DOTAP and lipofectamine, the novel poly(cationic lipid) exhibited the lowest cytotoxicity, was the most resistant to serum-induced aggregation and had comparable transfection activity when coformulated with cholesterol. This novel polymerization approach for the development of stable and active polyplexes may prove a valuable alternative for in vivo gene delivery.
Subject(s)
Gene Transfer Techniques , Lipids/chemistry , Liposomes/chemistry , Polyamines/chemistry , Transfection/methods , Animals , Cation Exchange Resins/chemistry , Cell Line , Cholesterol/chemistry , Drug Carriers/chemistry , Fatty Acids, Monounsaturated/chemistry , Fluorescent Dyes/chemistry , Hepatocytes/drug effects , Humans , Indicators and Reagents/chemistry , Liposomes/pharmacology , Molecular Structure , Particle Size , Polyelectrolytes , Quaternary Ammonium Compounds/chemistry , Rats , Rats, Sprague-DawleyABSTRACT
Hepatic fibrosis is a wound-healing process that occurs when the liver is injured chronically. Hepatic stellate cells (HSC) are responsible for the excess production of extracellular matrix (ECM) components. The activation of HSC, a key issue in the pathogenesis of hepatic fibrosis, is mediated by various cytokines and reactive oxygen species released from the damaged hepatocytes and activated Kupffer cells. Therefore, inhibition of HSC activation and its related subsequent events, such as increased production of ECM components and enhanced proliferation, are crucial goals for intervention in the hepatic fibrogenesis cascade. This is especially true when the etiology is unknown or there is no established therapy for the cause of the chronic injury. This review explores the rationale for choosing HSC as a target for the pharmacological, molecular, and other novel therapeutics for hepatic fibrosis. One focus of this review is the inhibition of two cytokines, transforming growth factor-beta and platelet-derived growth factor, which are important in hepatic fibrogenesis. A number of new agents, such as Chinese herbal recipes and herbal extracts, silymarin, S-adenosyl-L-methionine, polyenylphosphatidylcholine, and pentoxifylline are also discussed.
Subject(s)
Antioxidants/therapeutic use , Hepatocytes , Liver Cirrhosis/therapy , Cytokines/physiology , Drugs, Chinese Herbal/therapeutic use , Extracellular Matrix/physiology , Growth Substances/physiology , Hepatocytes/pathology , Hepatocytes/physiology , Humans , Liver Cirrhosis/pathology , Silymarin/therapeutic use , Survival Analysis , Tumor Necrosis Factor-alpha/physiologyABSTRACT
The mechanisms by which ethanol inhibits hepatocyte proliferation have been a source of some considerable investigation. Our studies have suggested a possible role for tissue transglutaminase (tTG) in this process. Others have shown that tTG has two distinctly different functions: it catalyzes protein cross-linking, which can lead to apoptosis and enhancement of extracellular matrix stability, and it can function as a G protein (Galpha(h)). Under that circumstance, we speculated that the cross-linking activity would be decreased and that it would function to enhance hepatocyte proliferation in response to adrenergic stimulation. Ethanol treatment inhibited hepatocyte proliferation and led to enhanced tTG cross-linking activity, whereas treatment of hepatocytes with an alpha1 adrenergic agonist, phenylephrine, enhanced hepatocyte proliferation while decreasing tTG cross-linking. However, phenylephrine treatment of several hepatoma cell lines had no effect on cellular proliferation or tTG cross-linking activity, and of note, Northern blot analysis demonstrated that whereas primary hepatocytes had high levels of the alpha1beta adrenergic receptor (alpha1BAR) mRNA, the hepatoma cell lines did not have this mRNA. When the Hep G(2) cell line was stably transduced with an expression vector containing the alpha1BR cDNA, the cell line responded to phenylephrine treatment with enhanced proliferation and with decreased tTG cross-linking activity. Ethanol treatment of the alpha1BAR-transfected cells suppressed the phospholipase C-mediated signaling pathways, as detected in the phenylephrine-induced Ca(2+) response. These results suggest that phenylephrine stimulation of hepatocyte proliferation appears to be occurring through the alpha1BAR, which is known to be coupled with the tTG G protein moiety, Galpha(h), and that tTG appears to play a significant role in either enhancing or inhibiting hepatocyte proliferation, depending on its cellular location and on whether it functions as a cross-linking enzyme or a G protein.
Subject(s)
Ethanol/pharmacology , Liver/metabolism , Liver/pathology , Receptors, Adrenergic, alpha-1/metabolism , Signal Transduction , Transglutaminases/metabolism , Animals , Cell Division/drug effects , Cell Line , Rats , Signal Transduction/drug effectsABSTRACT
BACKGROUND: The course of chronic pancreatitis is often unpredictable and many factors are likely to be involved in the progression of the disease. In physiological condition, pancreatic juice exerts significant antibacterial activity, which is impaired in patients with chronic pancreatitis. AIM: Hypothesizing that Helicobacter pylori could, in these conditions, lead to an ascending infection, we aimed to assess the presence of H. pylori sequences in pancreatic juices of patients with chronic pancreatitis. METHODS: 40 patients (mean age 52+/-3 yr) with alcoholic chronic pancreatitis and H. pylori infection were examined. Pancreatic juices were collected during endoscopic retrograde cholangiopancreatography. Using polymerase chain reaction (PCR) with two primers homologous to a portion of urease-C gene, H. pylori DNA was detected. Gastric biopsies, microscopically positive to H. pylori were used as positive controls. RESULTS: All gastric biopsies produced H. pylori-specific DNA products. Conversely, no H. pylori urease-C gene sequences have been detected in any of the pancreatic juices. CONCLUSION: Our data suggest that the impaired antibacterial activity of pancreatic juices in patients affected by chronic pancreatitis does not have a permissive role for a superimposing H. pylori infection in the pancreas. The possibility that Helicobacter species other than pylori may be involved in a superimposing infection requires further investigation.
Subject(s)
Helicobacter pylori/isolation & purification , Pancreatic Juice/microbiology , Pancreatitis/microbiology , Chronic Disease , Humans , Polymerase Chain Reaction , Sensitivity and Specificity , Urease/geneticsABSTRACT
BACKGROUND & AIMS: The accelerated course of hepatic fibrosis that occurs in some patients after liver transplantation is a major clinical problem. This response may be caused by the antirejection therapeutics, and in an earlier report we showed that FK-506 enhanced the fibrogenic process in in vivo and in vitro models of liver fibrosis. In the present study, the aim was to determine whether a new immunosuppressive agent, rapamycin, enhances or inhibits liver fibrosis. METHODS: Effects of rapamycin were investigated in a carbon tetrachloride model of hepatic fibrosis in rats and on hepatic stellate proliferation in vitro. RESULTS: Rapamycin inhibited extracellular matrix deposition in the rat model of fibrogenesis as determined by histological analysis, collagen content, messenger RNA levels of procollagen and transforming growth factor beta1, and tissue transglutaminase activity. Moreover, rapamycin decreased platelet growth factor-induced proliferation of hepatic stellate cells. CONCLUSIONS: These findings indicate that the new antirejection agent rapamycin inhibits hepatic fibrosis and thus may become a valuable addition to the immunosuppression armamentarium.
Subject(s)
Immunosuppressive Agents/pharmacology , Liver Cirrhosis, Experimental/prevention & control , Liver/cytology , Liver/drug effects , Sirolimus/pharmacology , Animals , Carbon Tetrachloride , Cell Division/drug effects , Cells, Cultured , Collagen/metabolism , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Liver/metabolism , Liver/pathology , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/pathology , Male , Platelet-Derived Growth Factor/pharmacology , Procollagen/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Transforming Growth Factor beta/genetics , Transglutaminases/metabolismABSTRACT
alpha 1-Antitrypsin (alpha 1-AT) deficiency, one of the most common hereditary disorders that mainly affects the lung and liver in Caucasian people, is caused by mutations of alpha 1-AT gene. Decrease of serum alpha 1-AT concentration is directly responsible for lung emphysema, replacement of plasma-derived alpha 1-AT concentrate is administered to patients of alpha 1-AT deficiency with lung emphysema, and lung transplantation is employed for end-stage lung emphysema. Augmentation therapy is not adequate for alpha 1-AT deficiency with liver disease, because liver injury is caused by accumulation of mutated alpha 1-AT protein in hepatocytes. Currently liver transplantation is the only treatment for severe liver cirrhosis. To correct the genetic defects of alpha 1-AT gene, several approaches for gene therapy are under investigation in vitro and in vivo animal models.
Subject(s)
alpha 1-Antitrypsin Deficiency , Genetic Therapy , Humans , Liver Cirrhosis/etiology , Liver Cirrhosis/therapy , Liver Transplantation , Lung Transplantation , Mutation , Pulmonary Emphysema/etiology , Pulmonary Emphysema/therapy , alpha 1-Antitrypsin/administration & dosage , alpha 1-Antitrypsin/genetics , alpha 1-Antitrypsin Deficiency/genetics , alpha 1-Antitrypsin Deficiency/therapyABSTRACT
BACKGROUND/AIMS: Some of the mutant forms of cellular proteins not only lose their function, but also cause diseases by their toxic effects. One of the challenging tasks in the field of gene therapy will be "gene replacement" accomplished by inhibiting mutant gene expression and providing normal function of the same gene, simultaneously. Although lung involvement in alpha1-antrypsin (alpha1-AT) deficiency is caused by the lack of alpha1-AT function, the liver involvement is due to the accumulation of the mutated alpha1-AT protein. Therefore, one possible approach to prevent and treat the disease manifestations of alpha1-AT deficiency is to inhibit the expression of the mutated gene and replace it with normally functioning alpha1-AT protein in the liver. METHODS: For the inhibition of alpha1-AT gene expression, panels of alpha1-AT-specific hammerhead ribozymes designed to target different GUC sites in the alpha1-AT mRNA were evaluated in a human hepatoma cell-line, transduced with retroviral vectors which express ribozymes under the control of a human tRNA promoter. A bi-functional vector was also constructed, which contained a functional alpha1-AT ribozyme and was combined with a modified alpha1-AT gene, whose product was engineered to be resistant to the specific alpha1-AT ribozyme. This construct was transduced into target hepatoma cells. RESULTS: The transduced hepatoma cells showed the effective expression of modified alpha1-AT, under the conditions where the endogenous alpha1-AT gene expression was inhibited. CONCLUSION: This ribozyme-mediated, specific gene replacement is a first step in the gene therapy of alpha1-AT deficiency.
Subject(s)
RNA, Catalytic/metabolism , Transcription, Genetic , Transfection/methods , alpha 1-Antitrypsin/genetics , Base Sequence , Carcinoma, Hepatocellular , Cell-Free System , Genetic Therapy/methods , Genetic Vectors , Humans , Liver Neoplasms , RNA, Catalytic/chemistry , RNA, Messenger/genetics , Retroviridae , Tumor Cells, Cultured , alpha 1-Antitrypsin/biosynthesisABSTRACT
We describe here the development and testing of simian virus 40 (SV40)-derived vectors to deliver foreign genetic material to the liver. Based on current understanding of the biology of wild-type SV40, it should be possible to exploit several important attributes of this virus, including efficient replication and gene expression, almost universal infectivity, and low immunogenicity if large T-antigen is deleted, to deliver DNA to the liver effectively. Our studies in cultured hepatocytes and in vivo, using both reporter constructs and transgenes of therapeutic interest, provide strong experimental support for this prediction. These successes indicate that SV40 may play an important role in gene delivery to the liver.
Subject(s)
Gene Transfer Techniques , Liver/metabolism , Simian virus 40/genetics , Animals , Genetic Vectors , Humans , Liver Diseases/genetics , Liver Diseases/therapyABSTRACT
A successful strategy for the treatment of hepatic damage should be based on the use of nontoxic drugs with a high therapeutic index and safety profile, as well as selective targeting delivery. To achieve these goals, several biological and synthetic vehicles have been formulated with a wide range of chemical and physical properties. These vectors allow the delivery of either traditional drugs or genetic material to the cells. Despite remarkable progress, the design of an ideal carrier is still to be accomplished. Safety issues concerning viral vectors and low efficiency of synthetic delivery systems are the main limitations of present vectors. Once these problems are overcome, the liver-specific targeting systems will provide new opportunities to treat a vast range of hepatic diseases.
Subject(s)
Drug Carriers/therapeutic use , Genetic Vectors/therapeutic use , Liposomes/therapeutic use , Liver Diseases/drug therapy , Drug Carriers/administration & dosage , Genetic Therapy/methods , Humans , Liposomes/administration & dosage , Simian virus 40ABSTRACT
The liver injury caused by hepatotoxins is characterized by varying degrees of hepatocyte degeneration and cell death via either apoptosis or necrosis. Generation of reactive intermediate metabolites from the metabolism of toxins and the occurrence of reactive oxygen species (ROS) during the inflammatory reaction account for a variety of pathophysiologic pathways which lead to cell death. This process can then evoke acute or chronic inflammatory responses if the injury is sustained, and these pathologic alterations eventually progress to cirrhosis. Understanding the function of transcription factors, such as nuclear factor kappa?B (NF-kappa B), in acute liver injury may provide some answers to the molecular mechanisms of toxic insults. Liposomes have been used as vehicles for drug delivery and gene therapy and they have been shown to have substantial potential in the targeting of specific cell types of the liver. Thus, the use of liposomes may improve targeting efficacy in the treatment of a variety of liver diseases.
Subject(s)
Liposomes/therapeutic use , Liver Cirrhosis, Experimental/etiology , Liver Diseases/etiology , Liver Diseases/therapy , NF-kappa B/physiology , Animals , Cations , Liposomes/adverse effects , Liposomes/pharmacokinetics , Mice , Second Messenger Systems , Vitamin E/therapeutic useABSTRACT
The liver can be considered an ideal target to design gene therapy strategies because of the unique properties of hepatic cells. The great amount of work carried out in this field clearly delineates the quality of the new approaches that have been developed in the last few years and demonstrates the feasibility of this strategy in a wide range of multi-factorial and inherited disorders. However, improvement of current vectors to specifically and efficiently deliver the therapeutic gene into the target cell remains a daunting problem. In this review, we will summarize the most important approaches that are currently under investigation, the pathological conditions that can be treated by this strategy and the most important drawbacks that remain to be overcome in order to achieve long-term and high-level gene expression.
Subject(s)
Genetic Therapy , Liver Diseases/therapy , Gene Transfer Techniques , Genetic Vectors , HumansABSTRACT
Alpha 1-antitrypsin (alpha 1AT) deficiency disease is one of the more common hereditary disorders that affects the liver and lung. The liver disease of alpha 1AT deficiency is generally thought to be caused by the accumulation of an abnormal alpha 1AT protein in hepatocytes, whereas the lung disease is thought to be due to a relative lack of the normal protein in the circulation. Therefore, one possible approach to prevent and treat alpha 1AT disease is to both inhibit the expression of the mutated alpha 1AT gene, and to provide a means of synthesizing the normal protein. To do this, we designed specific hammerhead ribozymes that were capable of cleaving the alpha 1AT mRNA at specific sites, and constructed a modified alpha 1AT cDNA not susceptible to ribozyme cleavage. Ribozymes were effective in inhibiting alpha 1AT expression in a human hepatoma cell line using a newly developed simian virus (SV40) vector system. In addition, the hepatoma cell line was stably transduced with a modified alpha 1AT cDNA that was capable of producing wildtype alpha 1AT protein, but was not cleaved by the ribozyme that decreased endogenous alpha 1AT expression. These results suggest that ribozymes can be employed for the specific inhibition for an abnormal alpha 1AT gene product, the first step in designing a gene therapy for the disease. The findings also suggest that the novel SV40-derived vector may represent a fundamental improvement in the gene therapeutic armarmentarium.
Subject(s)
Genetic Therapy/methods , Genetic Vectors , RNA, Catalytic/genetics , Simian virus 40/genetics , alpha 1-Antitrypsin Deficiency/therapy , alpha 1-Antitrypsin/genetics , Blotting, Northern , Carcinoma, Hepatocellular/metabolism , DNA, Complementary/genetics , Gene Expression Regulation , Humans , Mutation , Phenotype , Tumor Cells, CulturedABSTRACT
Liver injury caused by hepatotoxins, such as carbon tetrachloride (CCl4), ethanol, and acetaminophen (APAP), is characterised by varying degrees of hepatocyte degeneration and cell death via either apoptosis or necrosis. The generation of reactive intermediate metabolites from the metabolism of hepatotoxins, and the occurrence of reactive oxygen species (ROS) during the inflammatory reaction account for a variety of pathophysiologic pathways leading to cell death, such as covalent binding, disordered cytosolic calcium homeostasis, glutathione (GSH) depletion, onset of mitochondrial permeability transition (MPT) and associated lipid peroxidation. The metabolism of hepatotoxins by cytochrome P-450 enzyme subtypes is a key step of the intoxication; therefore, enzyme inhibitors are shown to minimise the hepatotoxin-associated liver damage. Understanding the function of transcription factors, such as nuclear factor kappaB (NF-kappaB) in acute liver injury, may provide some answers as to the molecular mechanisms of toxic insults. Moreover, substantial evidence exists that MPT is involved in ROS-associated hepatocellular injury and new findings offer a novel therapeutic approach to attenuate cell damage by blocking the onset of MPT. Thus, oxidant stress and lipid peroxidation are crucial elements leading to hepatotoxin-associated liver injury. In addition to specific treatment for a given hepatotoxin, the general strategy for prevention and treatment of the damage includes reducing the production of reactive metabolites of the hepatotoxins, using anti-oxidative agents, and selectively targeting therapeutics to Kupffer cells or hepatocytes for on-going processes, which play a role in mediating a second phase of the injury.
ABSTRACT
AIMS/BACKGROUND: Potenlini is an injectable compound whose active component is glycyrrhizin, which is extracted from licorice. Previous studies showed that it could reduce liver injury, improve alanine aminotransferase (ALT) levels and act as an antifibrotic agent. However, the mechanism of its action remains unclear. The aim of this study was to determine the molecular mechanism of its action by investigating the effects of potenlini on nuclear factor-kappaB (NF-kappaB) binding activity in an animal model of liver cirrhosis. METHODS: Rats were randomly allocated into a normal control group, a model control group, and a potenlini group. Rats in the latter two groups were treated with CCl4 and ethanol solution in order to induce chronic liver injury. Rats in the potenlini group were given potenlini treatment at the same time. RESULTS: Serum ALT levels were significantly reduced in rats treated with potenlini compared with levels in rats of the model control group, which had dramatically increased ALT levels. Histologically, liver steatosis and fibrosis were severe in the rats of the model group, but were significantly improved in rats of the potenlini group. NF-kappaB binding activity was markedly increased in the liver specimens taken from the rats of the model control group in comparison with the binding of normal livers, but the binding levels were nearly normal in the livers of the potenlini group. CONCLUSIONS: The results suggest that potenlini can inhibit the NF-kappaB binding activity in CCl4 and ethanol-induced chronic liver injury, and that effect may be a possible mechanism by which potenlini protects the liver from hepatotoxin-induced liver injury and cirrhosis.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Glycyrrhizic Acid/pharmacology , Glycyrrhizic Acid/therapeutic use , Liver Cirrhosis, Experimental/drug therapy , NF-kappa B/antagonists & inhibitors , Animals , Carbon Tetrachloride , Ethanol , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/metabolism , Liver Cirrhosis, Experimental/pathology , Male , NF-kappa B/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
The increased deposition of extracellular matrix proteins in the liver is a key factor in the morbidity and mortality of alcoholic liver disease (ALD). This increased fibrosis may be due to a superabundance of profibrogenic factors such as transforming growth factor-beta (TGF-beta). The original peptide is now called TGF-beta 1, and two other isoforms have been recognized in humans (TGF-beta 2 and TGF-beta 3). It was the aim of the present study to determine the expression of the TGF-beta isoforms in different stages of ALD. Thirty patients with ALD had percutaneous liver biopsies performed for diagnostic purposes. They were grouped by clinical findings and by liver histology into four groups: I, steatosis; II, fibrosis; III, hepatitis; and IV, cirrhosis. An unused portion of each biopsy sample was used to evaluate the gene expression of TGF-beta 1, TGF-beta 2, and TGF-beta 3 by reverse transcription polymerase chain reaction (RT-PCR). The expression of all isoforms from patients was significantly greater than their expression in controls. No significant correlation was determined between TGF-beta isoform expression and liver function test results. When the different isoforms were grouped by histology, increased expression with more severe disease was found; however, differences existed among the isoforms. In ALD, all TGF-beta isoforms were increased and their expression was significantly greater in patients with more active and advanced disease. RT-PCR is an effective method for evaluating gene expression in clinical samples which often provide a limited amount of tissue.
Subject(s)
Liver Cirrhosis, Alcoholic/blood , Transforming Growth Factor beta/blood , Adult , Aged , DNA Primers , Female , Gene Expression Regulation, Neoplastic , Humans , Liver Diseases/blood , Male , Middle Aged , Polymerase Chain Reaction/methods , Transcription, Genetic , Transforming Growth Factor beta/geneticsABSTRACT
To improve liposome-directed therapy of liver disease and gene delivery, it would be beneficial to selectively target hepatocytes. For this purpose, conventional liposomes (CL) were labeled with asialofetuin (AF), an asialoglycoprotein. The biodistribution of AF-labeled liposomes (AF-L) in mice and their incorporation into rat hepatocytes, and their potential use in acute liver injury, were investigated. AF-L displayed a quicker plasma clearance than CL, and 25.4%, 2.7%, and 1.2% of the injected dose remained in the plasma versus 47.0%, 26.1%, and 9.5% of CL, respectively at 2, 4, and 20 hours after the injection. Total liver uptake of AF-L (73%+/-3.9%) was markedly higher (P < .005) than CL (16.5%+/-1.8%) 4 hours after the injection. Liposomal radioactivity (cpm/mg) was greatly enhanced in the liver (11-fold) during the first 4 hours after the administration of 14C-AF-L, and was much higher than in 14C-CL-injected mice (1.5-fold). In vitro incubation of isolated rat hepatocytes with 14C-AF-L or intravenous injection of 14C-AF-L in rats resulted in higher hepatocyte-bound radioactivity compared with 14C-CL (P < .01-.005). AF-L-associated 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) fluorescent signals were not only located in Kupffer cells, but also in hepatocytes, in which bile canaliculus networks were imaged. Intravenous administration of vitamin E (VE)-associated CL (VE-CL, 1 mg/mouse) significantly lowered alanine transaminase (ALT) levels in CCl4-treated mice (196+/-79 vs. 2,107+/-235 U/mL; P < .01). The ALT level in CCl4 + VE-AF-L group was decreased to 38+/-16 units/mL, which was significantly lower than the CC14 + VE-CL group (P < .05). In conclusion, labeling liposomes with AF led to a shortened liposome plasma half-life and greatly enhanced uptake of AF-L liposome by the liver. The enhanced uptake resulted from an increased incorporation of hepatocytes with AF-L liposomes. VE-associated AF liposomes further improved the protective effect of VE liposomes on CC14-induced acute liver injury in mice. Preferential hepatocyte incorporation of AF-L liposomes suggests a useful hepatocyte-targeting approach for drug delivery and gene transfection.
Subject(s)
Asialoglycoproteins/metabolism , Liposomes/metabolism , Liver/metabolism , alpha-Fetoproteins/metabolism , Alanine Transaminase/blood , Animals , Carbon Tetrachloride/toxicity , Fetuins , Liver/cytology , Liver/drug effects , Mice , Mice, Inbred ICR , Rats , Rats, Sprague-Dawley , Vitamin E/pharmacologyABSTRACT
One of several postulated roles for tissue transglutaminase (tTG) is the stabilization and assembly of extracellular matrix via peptide cross-linking. We previously determined that tTG activity increased in an animal model of hepatic fibrogenesis and in human liver disease. To further study the role of tTG in liver disease, we initiated investigations into the effect of a proinflammatory mediator, tumor necrosis factor (TNF)-alpha, on tTG activity in cultured liver cells. Treatment of human Hep G2 cells with 1 ng/ml TNF-alpha increased [14C]putrescine cross-linking to cellular proteins. An increase in tTG mRNA content was observed 1 h after addition of TNF-alpha, and levels of tTG mRNA remained elevated after 24 h. Hep G2 cells, transiently transfected with a luciferase reporter containing 1.67 kb of the human tTG promoter, showed an increase in reporter activity after addition of TNF-alpha. Gel shift experiments using nuclear extracts from TNF-alpha-treated cells and oligonucleotides containing the tTG nuclear factor (NF)-kappa B motif revealed increased binding, concordant with mRNA data. Transient transfections with a truncated reporter construct lacking the tTG NF-kappa B sequence showed an attenuated response to TNF-alpha treatment. Similar responses were seen in stably transfected HeLa cells. Primary hepatocytes isolated from a transgenic mouse line containing the mouse tTG promoter driving the beta-galactosidase reporter, show similar time-dependent increases in promoter activity when treated with TNF-alpha. Furthermore, Hep G2 cells are incapable of upmodulating tTG promoter reporter activity in the presence of TNF-alpha when those cells overexpress a transdominant, negative mutant NF-kappa B subunit. Because TNF-alpha expression is upregulated in hepatic inflammation, the data suggest TNF-alpha-mediated increases in tTG expression may play an important role in the process of hepatic fibrogenesis.