ABSTRACT
Cyclooxygenase 2 (COX-2) has been thought to be associated with liver fibrosis whereas it is well known that hepatic stellate cells (HSC) play a central role in the pathogenesis of liver fibrosis. There is little evidence of how COX-2 regulates the activation of human HSC or the mechanism involved. In this study, we investigated the effect of a COX-2 inhibitor, NS-398, on a line of human HSC, LI90. Our findings demonstrated that alpha-smooth muscle actin (alpha-SMA) protein expression was inhibited in a dose-dependent manner by treatment with NS-398. Proliferation cell nuclear antigen (PCNA) expression and cell growth were partially down-regulated. The generation of PGE2, IL-8, IL-6, and hyaluronan in the cultured medium was also inhibited. In conclusion, our findings imply that a selective COX-2 inhibitor might be a potential drug for the chemoprevention and treatment of liver fibrosis by inhibiting the activation of HSC.
Subject(s)
Actins/antagonists & inhibitors , Cyclooxygenase Inhibitors/pharmacology , Isoenzymes/antagonists & inhibitors , Liver/cytology , Muscle, Smooth/metabolism , Nitrobenzenes/pharmacology , Sulfonamides/pharmacology , Blotting, Western , Cell Line , Collagen Type IV/metabolism , Culture Media/pharmacology , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Dinoprostone/biosynthesis , Dose-Response Relationship, Drug , Down-Regulation , Extracellular Matrix/metabolism , Humans , Hyaluronic Acid/biosynthesis , Hyaluronic Acid/pharmacology , Interleukin-6/biosynthesis , Interleukin-8/biosynthesis , Membrane Proteins , Proliferating Cell Nuclear Antigen/biosynthesis , Prostaglandin-Endoperoxide Synthases , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time FactorsABSTRACT
The basic or h1 calponin gene, which encodes an actin-binding protein involved in the regulation of smooth-muscle shortening velocity, is known to be a smooth-muscle differentiation-specific gene. It was found that basic calponin was expressed by cultured mesangial cells and localized along the actin filaments. Among the growth factors involved in the mesangial cell pathophysiology, including platelet-derived growth factor-BB (PDGF-BB), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta1 (TGF-beta1), TNF-alpha potently downregulates basic calponin expression in both the mRNA and protein levels, whereas TGF-beta1 upregulates the calponin expression. PDGF-BB also reduced its mRNA expression. The half-life of basic calponin mRNA was determined to be similar between TNF-alpha-treated and -untreated mesangial cells, whereas cell transfection assays that used a luciferase reporter gene construct containing the functional basic calponin promoter showed that TNF-alpha and PDGF-BB reduced the transcriptional activity. Because stimulation with TNF-alpha and PDGF-BB was associated with mesangial cell proliferation, basic calponin may play a role in the suppression of mesangial cell proliferation. Treatment with anti-glomerular basement membrane antibody in calponin knockout mice induced more severe nephritis than in wild type mice, as judged from an increase in the urinary protein excretion, glomerular cellularity, and number of proliferating cell nuclear antigen-positive cells in glomerulus. These results suggest that basic calponin expression may serve as one of the intrinsic regulators of glomerular nephritis. Elucidation of the molecular mechanisms for regulation of the basic calponin expression in mesangial cells may improve the understanding of the molecular basis and pathogenesis of the glomerular response to injury.
Subject(s)
Anti-Glomerular Basement Membrane Disease/metabolism , Anti-Glomerular Basement Membrane Disease/pathology , Calcium-Binding Proteins/metabolism , Glomerular Mesangium/metabolism , Muscle, Smooth/metabolism , Animals , Becaplermin , Calcium-Binding Proteins/deficiency , Calcium-Binding Proteins/genetics , Cell Division/drug effects , Cells, Cultured , Down-Regulation , Gene Expression/physiology , Glomerular Mesangium/cytology , Glomerular Mesangium/drug effects , Growth Substances/pharmacology , Humans , Mice , Mice, Knockout/genetics , Microfilament Proteins , Platelet-Derived Growth Factor/pharmacology , Proto-Oncogene Proteins c-sis , Tissue Distribution , Transcription, Genetic/physiology , CalponinsABSTRACT
Previous experiments have shown that emodin is highly active in suppressing the proliferation of several tumor cell lines. However, it is not clear that emodin can induce growth inhibition of hepatoma cells. We have found that emodin induces apoptotic responses in the human hepatocellular carcinoma cell lines (HCC) Mahlavu, PLC/PRF/5 and HepG2. The addition of emodin to these three cell lines led to inhibition of growth in a time- and dose-dependent manner. Emodin generated reactive oxygen species (ROS) in these cells which brought about a reduction of the intracellular mitochondrial transmembrane potential (DeltaPsim), followed by the activation of caspase-9 and caspase-3, leading to DNA fragmentation and apoptosis. Our findings demonstrate that ROS and the resulting oxidative stress play a pivotal role in apoptosis. Preincubation of hepatoma cell lines with the hydrogen peroxide-scavenging enzyme, catalase (CAT) and cyclosporin A (CsA), partially inhibited apoptosis. These results demonstrate that enhancement of generation of ROS, DeltaPsim disruption and caspase activation may be involved in the apoptotic pathway induced by emodin.
