Masanori Matsushita

Down-regulation of TGF-β receptors in human colorectal cancer : implications for cancer development

Many colorectal cancer cells are resistant to the anti-proliferative effects of transforming growth factor-β (TGF-β). TGF-β also acts as paracrine factor from cancer cells on their mesenchymal cells. The aim of this study was to examine the expression of TGF-β and its receptors in human colorectal cancer tissue and determine any relationship with cancer growth. In situ hybridization and Northern blot hybridization detection of TGF-β1, type I and type II receptor mRNA and immunohistochemical staining of TGF-β1 were performed using 11 human colorectal adenomas, 22 colorectal cancers and ten normal colorectal mucosas as control. TGF-β receptor mRNAs were expressed mainly by normal colorectal epithelial cells and adenoma. However, mRNAs for TGF-β receptors were only faintly, if at all, expressed in eight of 22 human colorectal cancers. In addition, intense signals of TGF-β1 mRNA and the protein were detected in all colorectal cancers. TGF-β receptor mRNAs and TGF-β1 protein were also distributed in fibroblasts and endothelial cells in the interstitium. Moreover, Smad 4 protein was translocated to nucleus in primarily cultured adenoma cells, but not in cancer cells after TGF-β stimulation. The escape of human colon cancer from TGF-β -mediated growth inhibition by down-regulation of TGF-β receptors as well as the effects of TGF-β on stroma formation and angiogenesis indicate a possible role for TGF-β in the progression of colon cancer in an intact host.

Differential regulation of activin A for hepatocyte growth and fibronectin synthesis in rat liver injury

BACKGROUND/AIMS Both hepatocyte growth and production of extracellular matrix such as fibronectin are essential for liver regeneration. Although activin A is reported to inhibit DNA replication in rat hepatocytes, the role of activin A for liver regeneration after acute injury has not been fully assessed. This study investigated the mechanism by which hepatocyte growth is regulated by activin A during liver regeneration and the effects of activin A on extracellular matrix production. METHODS The mRNA for betaA subunit of activin A and activin receptors in hepatocytes and hepatic stellate cells after CCl4 administration were studied by Northern blotting. Binding of 125I-activin A was tested in these cells. Effects of activin A were examined by DNA, collagen and fibronectin synthesis. RESULTS betaA mRNA was expressed in quiescent hepatocytes, and this expression peaked 12 h after CCl4 administration. Activin receptor mRNAs and cross-linked ligand/receptor complexes were expressed in hepatocytes and hepatic stellate cells However, these levels decreased specifically in hepatocytes at 24 h and had normalized by 72 h. The down-regulation of activin receptor was also observed after partial hepatectomy. Antiproliferative response to activin A decreased in hepatocytes at 24 h. Activin A stimulated production of fibronectin by hepatic stellate cells, but the synthesis of collagen was only slightly elevated in hepatic stellate cells following activin stimulation. CONCLUSIONS The down-regulation of activin receptors in hepatocytes may be partly responsible for these cells becoming responsive to mitogenic stimuli. The increase of activin A at the early stage of liver injury has the potential to contribute to the regulation of fibronectin production in hepatic stellate cells.

Neutrophils enhance invasion activity of human cholangiocellular carcinoma and hepatocellular carcinoma cells: An in vitro study

Background and Aim:  Tumor–mesenchymal interactions are involved in the mechanism of tumor invasion in several types of carcinoma. Mutual interactions between carcinoma cells and neutrophils, however, have been poorly understood. In the present study we examined the effect of neutrophils on invasion activities of carcinoma cells in vitro. Role of hepatocyte growth factor (HGF) as a mediator was also evaluated.

Distortion of autocrine transforming growth factor beta signal accelerates malignant potential by enhancing cell growth as well as PAI-1 and VEGF production in human hepatocellular carcinoma cells.

Resistance to growth inhibitory effects of transforming growth factor (TGF)-β is a frequent consequence of malignant transformation. On the other hand, serum concentrations of TGF-β, plasminogen activator inhibitor type 1 (PAI-1), and vascular endothelial growth factor (VEGF) are elevated as tumor progresses. The molecular mechanism of autocrine TGF-β signaling and its effects on PAI-1 and VEGF production in human hepatocellular carcinoma (HCC) is unknown. TGF-β signaling involves TGF-β type I receptor-mediated phosphorylation of serine residues within the conserved SSXS motif at the C-terminus of Smad2 and Smad3. To investigate the involvement of autocrine TGF-β signal in cell growth, PAI-1 and VEGF production of HCC, we made stable transfectants of human HCC line (HuH-7 cells) to express a mutant Smad2(3S-A), in which serine residues of SSXS motif were changed to alanine. The transfectants demonstrated an impaired Smad2 signaling. Along with the resistance to growth inhibition by TGF-β, forced expression of Smad2(3S-A) induced endogenous TGF-β secretion. Moreover, this increased TGF-β enhanced ligand-dependent signaling through the activated Smad3 and Smad4 complex, and transcriptional activities of PAI-1 and VEGF genes. In conclusion, distortion of autocrine TGF-β signals in human HCC accelerates their malignant potential by enhancing cell growth as well as PAI-1 and VEGF production.

Differential expression of transforming growth factor-β and its receptors in hepatocytes and nonparenchymal cells of rat liver after CCl4 administration

BACKGROUND/AIMS Transforming growth factor-beta (TGF-beta) is a family of multifunctional proteins that regulate hepatocyte proliferation, and biosynthesis of the extracellular matrix. In this study we examined whether modulation of TGF-beta receptor expression contributes to the liver diseases. METHODS The mRNA expression of TGF-beta1, TGF-beta type I receptor (TGFbetaRI), TGF-beta type II receptor (TGFbetaRII) and TGF-beta type III receptor (TGFbetaRIII) in rat livers injured by CCl4 administration was studied by Northern blotting. The mRNA expression patterns were confirmed by in situ hybridization. RESULT The peak of TGF-beta1 mRNA expression was observed 48 h after acute intoxication with CCl4 in nonparenchymal cells. However, the levels of TGFbetaRI and TGFbetaRII mRNA expression decreased from 24 h to 48 h and from 12 h to 48 h, respectively, and returned to the normal level by 72 h. TGFbetaRII mRNA expression was depressed more and for longer than that of TGFbetaRI mRNA. Analysis in separated hepatocytes and nonparenchymal cells from the injured livers indicated that the mRNA changes occurred in hepatocytes. Nonparenchymal cells expressed TGFbetaRI and TGFbetaRII mRNAs at constant levels during liver regeneration. TGFbetaRIII mRNA, which also decreased after 12 h, was not apparent in hepatocytes but only in nonparenchymal cells. CONCLUSIONS These observations suggest that: (i) whenever TGF-beta1 is increased in CCl4-treated livers, it may induce liver fibrogenesis via nonparenchymal cells; (ii) the mitoinhibitory effect of TGF-beta1 on hepatocytes is transiently relieved by down-regulation of TGF-beta receptors for 72 h post-damage; and (iii) the resistance to TGF-beta growth inhibition between 24 to 48 h may be predominantly due to down-regulation of the expression of TGFbetaRII.

Ischemia induces metallothionein III expression in neurons of rat brain

Metallothionein III (MT-III) is a brain-specific member of the metallothionein family and binds zinc in vivo. In order to confirm the precise localization of MT-III in normal rat brain and the change of MT-III expression after transient whole brain ischemia, we raised a high affinity phagemid-antibody specific for rat MT-III. Immunohistochemical analysis revealed that MT-III in normal brain is localized abundantly in neuronal cell bodies in CA1-3 regions of hippocampus, dentate gyrus, cerebral cortex, olfactory bulb and Purkinje cells in cerebellum. This expression pattern of MT-III was similar to that of MT-III mRNA observed by in situ hybridization studies. ELISA and Northern blot analysis revealed that MT-III protein as well as mRNA levels were up-regulated in cerebrum soon after ischemic stress. Immunohistochemical analysis also demonstrated intense staining in neurons in injured brain after ischemia, which distributed in the same regions as in normal brain. These results suggest that MT-III plays an important role in protecting neurons from ischemic insult by reducing neurotoxic zinc levels and inhibits uncontrolled growth of neurites after ischemia.

Expression of Transforming Growth Factor-β Receptors in Normal Rat Retina and Experimental Choroidal Neovascularization

PURPOSE Transforming growth factor-beta (TGF-beta) plays an important role in the development of choroidal neovascularization. TGF-beta transduces signals through the mediation of type I and type II receptors. We investigated the expression of TGF-beta receptors in a normal rat retina and a model of experimentally induced choroidal neovascularization. METHODS Choroidal neovascularization was induced by laser photocoagulation in rat eyes. The expression of TGF-beta receptors was determined using immunohistochemical and in situ hybridization methods. RESULTS In normal adult rat retinas, immunoreactivity and mRNA expression of TGF-beta receptor type I (TbetaRI) and TGF-beta receptor type II (TbetaRII) were found in the ganglion cells. During the process of neovascularization, immunoreactivity and mRNA expression of TbetaRI and TbetaRII were widely distributed in laser lesions soon after photocoagulation; thereafter, these receptors were specifically detected in the endothelial cells of choroidal neovascularization. CONCLUSIONS The expression of TGF-beta receptors in normal rat retinas suggests that TGF-beta plays an important role in the homeostasis of normal retina. The upregulation of TGF-beta receptors in choroidal neovascularization strongly suggests that TGF-beta is most likely transduced through specific receptors and plays an important role in the development of choroidal neovascularization.

A Case of Early Gastric Cancer Manifesting as a Submucosal Tumor

Abstract: Gastric cancer manifesting as a submucosal tumor (SMT) is not common. A gastric barium meal and endoscopic studies performed on a 49‐year‐old male with epigastric pain, revealed an elevated lesion with bridging folds and central depression on the posterior wall of the lower body. An endoscopic ultrasonography (EUS) revealed a hypoechoic mass lesion within the submucosal layer. Due to an increase in the size of the tumor and its central depression during the subsequent year and a half, the patient was admitted for closer examination. Endoscopic biopsy material from the deeper layer, obtained by mucosal resection, revealed a poorly differentiated adenocarcinoma. Microscopic examination of the resected stomach showed a poorly differentiated adenocarcinoma within the submucosal layer, with considerable lymphocyte infiltration. Immunohistological examination disclosed marked T cell infiltration adjacent to the cancer cells. We suggest that considerable lymphocyte infiltration, particularly T cells, may have some role in the protective reaction against cancer cells. Our case was diagnosed as being cancer 18 months after the first endoscopic study. The biopsy material taken from the depression at the time of the first examination showed benign findings and a EUS revealed typical SMT. In the case of SMT shown by EUS to be a hypoechoic mass lesion in the submucosal layer, it is recommended that biopsy material be obtained from the deeper layer using methods available such as artificial ulcer formation.