The primary mitogen (TCPOBOP)‐induced hepatocyte proliferation is resistant to transforming growth factor‐ β‐1 inhibition

Background: Transforming growth factor (TGF)‐β‐1 is a very efficient inhibitor of hepatocyte proliferation in various in vivo and in vitro experimental systems. However, there are no data on whether it can influence the mitogenic response induced by primary hepatocyte mitogens. Aims: In this study, we compared the proliferative response in the liver between wild‐type and transgenic mice, overexpressing active TGF‐β‐1 in their liver following the treatment by a primary hepatocyte mitogen TCPOBOP (1,4‐bis[2‐(3,5‐dichloropyridyloxy)]benzene). Methods: The proliferative response was characterized by the immunohistochemical examination of pulse and cumulative bromodeoxyuridine labelling and by quantitative real‐time polymerase chain reaction analysis of cell cycle‐related genes. Results: Neither of the applied techniques revealed significant differences between the two groups of mice; furthermore, we observed the upregulation of TGF‐β‐1 expression following the mitogenic treatment. Conclusions: TGF‐β‐1 does not inhibit the primary mitogen‐induced proliferative response of the hepatocytes. This observation may provide an explanation for the divergent consequences of hepatic proliferations induced by partial hepatectomy or primary mitogenic treatment.     

Hypoxia stimulates hepatocyte epithelial to mesenchymal transition by hypoxia‐inducible factor and transforming growth factor‐β‐dependent mechanisms

Background/Aims: During development of liver fibrosis, an important source of myofibroblasts is hepatocytes, which differentiate into myofibroblasts by epithelial to mesenchymal transition (EMT). In epithelial tumours and kidney fibrosis, hypoxia, through activation of hypoxia‐inducible factors (HIFs), is an important stimulus of EMT. Our recent studies demonstrated that HIF‐1α is important for the development of liver fibrosis. Accordingly, the hypothesis was tested that hypoxia stimulates hepatocyte EMT by a HIF‐dependent mechanism. Methods: Primary mouse hepatocytes were exposed to room air or 1% oxygen and EMT evaluated. In addition, bile duct ligations (BDLs) were performed in control and HIF‐1α‐deficient mice and EMT quantified. Results: Exposure of hepatocytes to 1% oxygen increased expression of α‐smooth muscle actin, vimentin, Snail and fibroblast‐specific protein‐1 (FSP‐1). Levels of E‐cadherin and zona occludens‐1 were decreased. Upregulation of FSP‐1 and Snail by hypoxia was completely prevented in HIF‐1β‐deficient hepatocytes and by pretreatment with SB431542, a transforming growth factor‐β (TGF‐β) receptor inhibitor. HIFs promoted TGF‐β‐dependent EMT by stimulating activation of latent TGF‐β1. To determine whether HIF‐1α contributes to EMT in the liver during the development of fibrosis, control and HIF‐1α‐deficient mice were subjected to BDL. FSP‐1 was increased to a greater extent in the livers of control mice when compared with HIF‐1α‐deficient mice. Conclusions: Results from these studies demonstrate that hypoxia stimulates hepatocyte EMT by a HIF and TGF‐β‐dependent mechanism. Furthermore, these studies suggest that HIF‐1α is important for EMT in the liver during the development of fibrosis.     

Transforming Growth Factor‐β:A Promising Target for Anti‐Stenosis Therapy

Transforming growth factor‐β (TGF‐β) is the general name for a family of cytokines which have widespread effects on many aspects of growth and development. The TGF‐β isoforms are produced by most cell types and exert a wide range of effects in a context‐dependent autocrine, paracrine or endocrine fashion via interactions with distinct receptors on the cell surface. TGF‐β is involved in the wound healing process and, thus plays a significant role in the formation of a restenotic lesion after percutaneous transluminal coronary angioplasty (PTCA) or stenting. Perhaps because of its wide‐ranging effects, TGF‐β is usually released from cells in a latent form, and its activation and signaling are complex. Manipulation of the TGF‐β1, TGF‐β2, and TGF‐β3 isoforms by inhibiting their expression, activation, or signaling reduces scarring and fibrosis in animal models. However, to date, few have reached clinical trial. This review summarizes current knowledge on the activation and signaling of TGF‐β, and focuses on the anti‐TGF‐β strategies which may lead to clinical applications in the prevention of restenosis following PTCA or stenting.     

Increased apoptosis in HepG2.2.15 cells with hepatitis B virus expression by synergistic induction of interferon‐γ and tumour necrosis factor‐α

Background: Interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α) were thought to be important immune mediators in host defence against hepatitis B virus (HBV) infection. Aims: To examine the synergistic effect of IFN‐γ and TNF‐α on HBV‐expressing HepG2.2.15 cells and its potential mechanisms. Methods: Cell viability was quantitatively measured by 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide assay. Cell morphology was captured using light microscopy. The typical DNA ladder test was performed using agarose gel electrophoresis. HBsAg and HBeAg titre changes were quantified by the enzyme‐linked immunosorbent assay method. Gene expression was analysed using cDNA macroarrays. Results: Interferon‐γ (1000 U/ml) alone or combined with TNF‐α (5 ng/ml) treatment resulted in apoptosis in HepG2.2.15 cells, but no significant apoptosis in the parent non‐virus expressing HepG2 cells. IFN‐γ‐ and TNF‐α‐mediated apoptosis was reduced by lamivudine treatment in HepG2.2.15 cells. IFN‐γ combined with TNF‐α reduced the titre of hepatitis B surface antigen and hepatitis B e antigen in the HepG2.2.15 cell line. For apoptosis‐related gene changes, IFN regulatory factor 1 (IRF‐1) (12.2‐fold), c‐myc (V00568 4.7‐fold, L00058 2.4‐fold) and caspase 7 (2.3‐fold) genes were upregulated in the combination treatment group. Conclusion: Interferon‐γ and TNF‐α play a role in the cell death of HBV‐expressing HepG2.2.15 cells. Expression of HBV leads to IFN‐γ‐ and TNF‐α‐mediated apoptosis in the cells. Increased IRF‐1, c‐myc and caspase 7 gene expression may be responsible for the synergistic induction of apoptosis by IFN‐γ and TNF‐α.     

Agrocybe aegerita polysaccharide combined with chemotherapy improves tumor necrosis factor‐α and interferon‐γ levels in rat esophageal carcinoma

Natural compounds have generated great interest as alternative treatments of diseases like cancer. Here, we investigated the anti‐tumor mechanism of one such compound, Agrocybe aegerita polysaccharide, by assessing expression of tumor necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ) in rat esophageal carcinoma (EC). EC was induced in healthy Wistar rats by methyl‐n‐amyl nitrosamine. Subsequently, rats were administered cancer treatment daily for 4 weeks, as follows: the normal control group (the only group not treated with methyl‐n‐amyl nitrosamine) and model group received only distilled water; the chemotherapy group received tegafur treatment; and the combination group received tegafur combined with A. aegerita polysaccharide. In normal and combination groups, body weight increased gradually after each week of treatment (P < 0.05), while body weights did not change in model and chemotherapy groups. Using enzyme linked immunosorbent assay, we found serum TNF‐α was lower in the combination group (31.56 ± 7.20 pg/L) than either the model (46.24 ± 8.52 pg/L) or chemotherapy (52.39 ± 9.16 pg/L) group, and, while higher, was more similar to the normal controls (25.08 ± 2.93 pg/L; P < 0.05), a finding that was confirmed by the immunohistochemistry of esophageal samples. In contrast, serum IFN‐γ was higher in the combination group (97.20 ± 10.92 pg/L) than in either the model (76.11 ± 11.92 pg/L) or chemotherapy (76.04 ± 9.85 pg/L) group, but lower than in the normal group (117.56 ± 10.88; P < 0.05), also confirmed by immunohistochemistry. Therefore, Agrocybe aegerita polysaccharide, when combined with chemotherapy, can regulate immune function in EC, potentially by modulating cytokine activity, specifically downregulation of TNF‐α and upregulation of IFN‐γ.     

Peripheral T cell receptors αβ and γδ in patients with Hodgkin's lymphoma

Antigen recognition by T cells is determined by an antigen specific T cell receptor (TCR). Two heterodimeric TCR structures associated with CD3 have been defined: TCR αβ and TCR γδ. TCR αβ and its function are well described but the role of TCR γδ in normal and lymphoproliferative disorders is not well established. In newly diagnosed or relapsed/refractory Hodgkin's disease (HD), a disease associated with defective T cell functions and increased sIL-2R, We determined levels of seven TCR αβ variable regions [βV5(a), βV5(b), βV6(a), βV12(a), αβV(a), αV2(a)] and TCR γδ by using monoclonal antibodies (MCA). TCR γδ levels did not show any difference, but several variable regions of the TCR αβ differed when groups are compared with each other and the control group.     

α‐thalassaemia masked by β gene defects and a new polyadenylation site mutation on the α2‐globin gene

We report three examples of chronic anaemia involving complex combinations of α‐ and β‐globin gene defects. The first case had a potential Hb H disease caused by the classic SEA/RW deletions masked by Hb E [β26(B8)Glu→Lys] in the homozygous state. The second had an unusual Hb H disease caused by compound heterozygosity for two different α2 polyadenylation site mutations masked by a β‐thalassaemia heterozygosity. The third had an intermediate α‐thalassaemia with considerable anaemia caused by an as yet unknown polyadenylation site (AATAAA>AATAAC) mutation in combination with a common RW deletion masked by a common Hb C [β6(A3)Glu→Lys] heterozygosity. Diagnostic methods, genotype/phenotype correlations and the chance of overlooking these combinations during risk assessment in a multiethnic society are discussed.     

New mechanism of transforming growth factor‐β signaling in hepatoma: Dramatic up‐regulation of tumor initiating cells and epidermal growth factor receptor expression

Aim: Transforming growth factor‐β (TGF‐β) has dual activity in tumor cells. We studied the effect of TGF‐β on tumor‐initiating cells (TICs), which are similar in self‐renewal and differentiation features to normal adult stem cells. Methods: We used side population (SP) cells that exclude DNA binding dye Hoechst 33342 to obtain TICs, studied the differences in the kinetics of the SP cell response to TGF‐β treatment between hepatic tumor cell lines, and performed gene analysis. Results: SP cells from all cell lines have higher proliferative ability compared to non‐SP cells and they are drug resistant. TGF‐β treatment increased the percentage of SP cells (%SP) and the survival rate; chemotherapeutic drug resistance developed only in K‐251 SP cells. Gene analysis showed that TGF‐β up‐regulated epidermal growth factor receptor (EGFR) only in K‐251 cells. There were no EGFR mutations in K‐251, which had been reported in lung cancer. Knockdown of Smad4 using the small‐interfering RNA technique in K‐251 cells inhibited EGFR overexpression and significantly decreased the %SP. In contrast, the JNK inhibitor had little effect on EGFR expression or the %SP. Conclusion: TGF‐β treatment of K‐251 cells causes tumor progression and the anti‐cancer drug resistant phenotype by increasing SP.     

Induction of lipocalin‐2 expression in acute and chronic experimental liver injury moderated by pro‐inflammatory cytokines interleukin‐1β through nuclear factor‐κB activation

Background: Lipocalin‐2 (LCN2) belongs to the lipocalin superfamily, sharing a barrel‐shaped tertiary structure with a hydrophobic pocket and an ability to bind lipophilic molecules. LCN2 has recently emerged as an important modulator of cellular homeostasis in several organs, i.e. heart, lung and kidney, but little is known about the expression of LCN2 in acute and chronic liver injury. Aims: In this study, we wanted to analyse the expression and regulation of LCN2 in models of acute and chronic experimental liver injury. Materials and methods: We analysed LCN2 expression in livers of rats subjected to bile duct ligation or repeated doses of carbon tetrachloride and tested the impact of various pro‐inflammatory cytokines in cultured primary liver cells. Results: By using primary cultures of hepatic stellate cells and hepatocytes isolated from normal and injured rat livers, we found a significant LCN2 expression in early hepatic stellate cell cultures, a lower expression in fully transdifferentiated myofibroblasts and no expression in freshly isolated hepatocytes. However, LCN2 expression and secretion in hepatocytes increased dramatically during culturing. In addition, chronic in vivo liver injury resulting from both bile duct ligation and repeated application of carbon tetrachloride resulted in rapid and well‐sustained induction of LCN2 expression. Immunohistochemistry and primary liver cell isolation identified injured hepatocytes as the main source of LCN2 production. LCN2 is strongly induced in both primary hepatocytes and immortalized hepatocellular carcinoma cell line HepG2 by the pro‐inflammatory cytokine interleukin‐1β via nuclear factor‐κB activation, but not by the profibrotic cytokines platelet‐derived growth factor and transforming growth factor‐β. Conclusion: LCN2 expression shows clear correlation to liver damage and resulting inflammatory responses, rather than to the degree of liver fibrosis, which in fact may imply a distinct diagnostic value as an early biomarker of liver inflammation.