Noboru Ueki

Excessive production of transforming growth-factor β1 can play an important role in the development of tumorigenesis by its action for angiogenesis: validity of neutralizing antibodies to block tumor growth

Angiogenesis is an important part of tumor growth in vivo. We used the transfected Chinese hamster ovary (CHO) cells that overproduced recombinant transforming growth-factor beta 1 (TGF-beta 1) to examine the possible role of this factor in tumor growth and angiogenesis in a nude mouse model. The in-vitro proliferation of TGF-beta 1-transfected CHO cells was unaffected by the treatment of either recombinant TGF-beta 1 or an anti-TGF-beta 1 antibody. The TGF-beta 1-transfected cells grew more rapidly than the parental CHO cells when injected subcutaneously into nude mice. The tumors derived from the TGF-beta 1-transfected cells showed prominent tumor-associated angiogenesis, whereas the parental cells produced tumors without such angiogenesis. In addition, an anti-TGF-beta 1 neutralizing antibody was able to inhibit both growth and angiogenesis in the tumors derived from TGF-beta 1-transfected cells. These findings suggest that the overproduction of TGF-beta 1 by tumor cells can contribute to neovascularization and may help promote tumor development in vivo.

Local production and localization of transforming growth factor-beta in tuberculous pleurisy.

Transforming growth factor‐beta (TGF‐β) is one of the cytokines which play an immunosuppressive role in an inflammatory process. To investigate the local production of TGF‐β, we evaluated the levels of TGF‐β in tuberculous pleural effusions (TBPE) and non‐tuberculous benign pleural effusions (non‐TBPE) by the growth inhibition assay with MvILu mink lung epithelial cells. The mean level of TGF‐β in TBPE (46.1 ± 31.5 pM; mean ± s.d.) was higher than in non‐TBPE (21.7 ± 12.3 pM) (P<0.05). Although the level of interferon‐gamma (IFN‐γ) in TBPE measured by ELISA was significantly higher than in non‐TBPE. there was no significant difference in the levels of tumour necrosis factor‐alpha (TNF‐α) measured by ELISA between these two groups. Moreover, to elucidate localization of TGF‐β in tuberculous pleurisy, immunohistochemical studies of pleura, using the rabbit polyclonal antibody Ab39 against latent TGF‐β1 binding protein (LTBP) were performed. Results revealed that LTBP was localized in immature fibrotic areas where infiltrations of T lymphocytes and macrophages were absent. Importantly, the major sources of LTBP in these areas were thought to be mesothelial cells and fibroblasts. LTBP was not found in granulomas and mature fibrotic areas. Our data suggest that TGF‐β in tuberculous pleurisy may play important roles for regression of granulomatous inflammation and pleural fibrosis for tissue repair.

Induction of Apoptosis in Hepatocellular Carcinoma Cell Lines by Emodin

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 (ΔΨm), 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, ΔΨmdisruption and caspase activation may be involved in the apoptotic pathway induced by emodin.

Stimulation of hyaluronan synthesis by tumor necrosis factor-α is mediated by the p50/p65 NF–κB complex in MRC-5 myofibroblasts

Abstract The lesions of fibrocontractive diseases result from an excessive myofibroproliferative response to numerous forms of inflammatory stimuli, which elicit the net deposition of extracellular matrix (ECM) in the interstitium of the affected tissue. Hyaluronan (HA), reported to be a key player supporting cellular migration and adherence, is a major component of ECM that undergoes dynamic regulation during inflammation. The molecular regulation of HA biosynthesis by inflammatory cytokines on myofibroblasts is not yet completely understood. Here we report the biochemical characteristics of the lung myofibroblast cell line MRC-5, and we demonstrate that the production of HA by this cell line is inducible by the proinflammatory cytokine, tumor necrosis factor-α (TNF-α), at the message level of HA synthase (HAS). In TNF-α-stimulated MRC-5 cells, DNA-binding and competition experiments indicated that the predominant NF–κB binding activity detected with nuclear extract-stimulated cells is mediated by the p50/p65 complex. Using antisense oligonucleotides, we confirmed that the TNF-α-stimulation of HA synthesis by MRC-5 cells is dependent on the activation of the p50/p65 NF–κB complex. These findings indicate that TNF-α production within inflamed tissues may enhance the HA synthesis via the transcriptional induction of HAS on myofibroblasts, thereby providing a provisional matrix for supporting cellular migration and adhesion, and that the p50/p65 NF–κB complex that plays an important role in the regulation of HA production by TNF-α might be an appropriate target for therapeutic compounds to treat tissue fibrosis accompanied by inflammation.

Inhibition of the expression of α-smooth muscle actin in human hepatic stellate cell line, LI90, by a selective cyclooxygenase 2 inhibitor, NS-398

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.

Potentiation of Metastatic Capacity by Transforming Growth Factor-β1 Gene Transfection

This study was designed to assess whether the excessive secretion of transforming growth factor‐β1 (TGF‐β1) by Chinese hamster ovary (CHO) cells transfected with TGF‐β1 gene may be linked to the development of a metastatic phenotype. We observed large numbers of metastatic colonies in the lungs of nude mice inoculated with the transfected CHO cells. The tumors derived from these transfected cells demonstrated marked angiogenesis. We postulate that the overproduction of TGF‐β1 by these tumors may participate in the metastatic progression following establishment of angiogenesis at the primary tumor site.

Transforming growth factor-beta 1 (TGF-β1)- and β2-like activities in malignant pleural effusions caused by malignant mesothelioma or primary lung cancer

We investigated the levels of TGF‐β in malignant pleural effusions (MPE) caused by malignant mesothelioma (MESO) or primary lung cancer. TGF‐β levels in MPE caused by MESO were 283.9 ± 219.2pM (mean plusmn; s.d.) and were three to six times higher than those due to primary lung cancers (P < 0.01 or P < 0.05). We also evaluated TGF‐β1‐ and β2‐like activities in MPE using specific polyclonal antibodies. Although TGF‐β1‐like activity could be detected in all cases, TGF‐β2‐like activities were detected in five of seven in MESO and in a few cases with primary lung cancer. These results demonstrate that the levels of total TGF‐β and TGF‐β2‐like activity may be clinically useful to differentiate MESO from primary lung cancer. Our data also suggest that TGF‐β may help further characterize the clinical features of MESO.

Induction of calponin-h1 by transforming growth factor-β1 in cultured human Ito cells, LI90

We investigated the effect of transforming growth factor-beta1 (TGF-beta1) on the expression of calponin-h1, alpha-smooth muscle actin (alpha-SMA), and extracellular matrix (ECM) components in a cultured human Ito cell line, LI90. The TGF-beta1 treatment stimulated productions of hyaluronic acid and laminin, and significantly decreased the secretion of hepatocyte growth factor in LI90 cells. The functional characteristics of LI90 cells were compatible with those of human-activated Ito cells that are known as pericyte-like mesenchymal liver cells. TGF-beta1 induced a slight growth-inhibition of LI90 cells. TGF-beta1 enhanced the expressions of both alpha-SMA and calponin-h1 at the protein level, while tumor necrosis factor-alpha and interleukin-1alpha did not affect the expressions of these cytoskeletal proteins on LI90 cells. The addition of TGF-beta1 to LI90 cells resulted in a significant increase of calponin-h1 mRNA levels, but not calponin-h2. These data suggest that the expression of calponin-h1 is controlled at the level of mRNA under the coordinate regulation together with alpha-SMA as the process of perpetuation of activated Ito cells promoted by TGF-beta1. The identification of smooth muscle features promoted by TGF-beta1 support the hypothesis that the activation of Ito cells coincides with their contractile behavior, indicating that these cells may be important in vasoregulation during liver injury and fibrosis.

Inhibition of hyaluronan synthesis by vesnarinone in cultured human myofibroblasts

Hyaluronan (HA), which is a major component of the extracellular matrix (ECM), is regulated during myofibroproliferative responses to numerous forms of inflammatory stimuli. It is a key factor involved in cellular migration and adherence. The development of a potent and non-toxic inhibitor of HA synthesis would open up a new avenue for the treatment of fibrocontractive diseases such as pulmonary fibrosis and liver cirrhosis. In this study, the effects of vesnarinone (OPC-8212: 3,4-dihydro-6-[4-(3, 4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone) on the secretion of HA in human myofibroblast cell lines (MRC-5 and LI90 cells, referred to as pulmonary and hepatic myofibroblasts, respectively) were examined. Vesnarinone specifically and dose-dependently inhibited HA secretion by myofibroblasts up-regulated by fetal calf serum (FCS). The treatment of vesnarinone did not modify the phenotype of myofibroblast cells in culture. Vesnarinone also potently inhibited the HA secretion by the two myofibroblast cell lines up-regulated by transforming growth factor-beta1 (TGF-beta1) or tumor necrosis factor-alpha (TNF-alpha). The addition of vesnarinone to myofibroblasts resulted in a significant decrease of HA synthase (HAS) activity, with or without the addition of FCS or either cytokine. These findings suggest that vesnarinone inhibits the secretion of HA in myofibroblasts by specifically suppressing HAS activity, and may therefore prove useful for the treatment of chronic inflammation and tissue fibrosis.

Aspartate aminotransferase-linked immunoglobulin complexes in serum of a patient with primary biliary cirrhosis

A 51-year-old woman who had been treated for primary biliary cirrhosis (PBC) was admitted to our hospital for evaluation of unexplained, isolated, persistently increased aspartate aminotransferase (AST) activity. Results of laboratory tests on admission showed: AST 171 KU, alanine aminotransferase 28 KU, and anti-mitochondrial titer 1/1280. Results of hepatitis B surface antigen (HBs Ag) and hepatitis C virus antibody (HCV Ab; C100-3) assays were negative. Histology of a liver biopsy specimen was compatible with a diagnosis of PBC (stage III of Scheuers classificiation). The molecular size of serum AST was estimated to be more than 500 000 by high-performance size-exclusion liquid chromatography. Electrophoretic analysis showed an abnormal band of AST between supernatant AST (sAST) and mitochondrial AST (mAST), which band was characteristic of AST-immunoglobulin complexes (AST-Ig). Ouchterlony double-diffusion and immunoprecipitation tests identified the immunoglobulin component as IgM. The presence of AST-Ig appeared to be responsible for the elevated serum AST.