Kenichi Shiiba

Identification of thyroid hormone transporters in humans: different molecules are involved in a tissue-specific manner.

We have recently identified that rat organic anion transporters, polypeptide2 (oatp2) and oatp3, both of which transport thyroid hormones. However, in humans the molecular organization of the organic anion transporters has diverged, and the responsible molecule for thyroid hormone transport has not been clarified, except for human liver-specific transporter (LST-1) identified by us. In this study we isolated and characterized a novel human organic anion transporter, OATP-E from human brain. The isolated complementary DNA encodes a polypeptide of 722 amino acids with 12 transmembrane domains. A rat counterpart, oatp-E, was also identified. Homology analysis and the phylogenetic tree analysis revealed that OATP-E/oatp-E is a subfamily of the organic anion transporter. Human OATP-E transported 3,3′,5-triiodo-l-thyronine (Km, 0.9μ m), thyronine, and rT3 in a Na+-independent manner. Although the clone was isolated from the brain, OATP-E messenger RNA was abundantly expressed in various peripheral tissues. The ...

Introduction of antisense CD44s cDNA down‐regulates expression of overall CD44 isoforms and inhibits tumor growth and metastasis in highly metastatic colon carcinoma cells

We created antisense CD44 transfectants using LS174T, a colon adenocarcinoma cell line and assessed the effects of overall CD44 down‐regulation on colorectal tumor growth and metastasis. The expression of antisense CD44s (the standard form of CD44) cDNA markedly inhibited the overall expression of CD44 variants. In vitro studies showed a significantly reduced ability of the stable antisense transfectants (LS174TAS1 and LS174TAS2) to bind hyaluronate and osteopontin, ligands for CD44. These cells developed tumors more slowly than controls (parental LS174T and mock transfectants) when the cells were subcutaneously injected into SCID mice. However, in vitro proliferation assays demonstrated no significant difference between the antisense transfectants and the controls on a hyaluronate‐coated surface, suggesting the participation of ligands other than hyaluronate in tumor growth in vivo. Intrasplenic injection of parental LS174T cells produced colonies in the liver in 10 of 11 mice, whereas mice injected with the antisense transfectants were completely free of metastasis. In peritoneal dissemination, the weight of nodules and amount of ascites were significantly reduced in LS174TAS1 and AS2 compared with the controls. In vitro adhesion assays between the transfectants or controls and human peritoneal mesothelial cells revealed that the binding of LS174T cells to mesothelial cells was partly mediated by CD44‐hyaluronate interaction. These data suggest that CD44‐hyaluronate interaction plays a crucial role in peritoneal dissemination in colorectal carcinoma. The results of our study demonstrate the possible application of antisense CD44s to the treatment of colorectal carcinoma. Int. J. Cancer 91:67–75, 2001.

Down syndrome candidate region 1,a downstream target of VEGF, participates in endothelial cell migration and angiogenesis.

Vascular endothelial growth factor (VEGF) is a principal stimulator of angiogenesis. However, the downstream targets of VEGF in endothelial cells (ECs) are not entirely clarified. Survey of downstream targets of VEGF in human ECs identified a number of genes, including Down syndrome candidate region 1 (DSCR1). Here, we confirmed the inducible expression of DSCR1 in ECs by Northern and Western blottings. Moreover, VEGF-stimulated induction of DSCR1 was blocked by anti-VEGF receptor-2 monoclonal antibody (mAb), or the specific calcineurin inhibitors cyclosporin A and FK506. The expression of DSCR1 in ECs of neovessels was further shown by immunohistochemical analysis. We therefore examined whether DSCR1 played any roles in angiogenesis. The specific downregulation of DSCR1 expression by antisense oligonucleotide (AS-ODN) inhibited VEGF-stimulated migration of ECs as well as angiogenesis in vivo. AS-ODN inhibited the spreading of ECs on vitronectin, as well as on the immobilized anti-αvβ3 mAb, but not on anti-αvβ5 mAb. Moreover, AS-ODN inhibited tyrosine phosphorylation of focal adhesion kinase when ECs were plated on a vitronectin-coated dish. Immunoprecipitation followed by Western blotting showed the coimmunoprecipitation of DSCR1 and integrin αvβ3. These results suggest that DSCR1 is involved in angiogenesis by regulating adhesion and migration of ECs via the interaction with integrin αvβ3.

Differing expression of MMPs-1 and-9 and urokinase receptor between diffuse-and intestinal-type gastric carcinoma

Gastric cancer is classified into intestinal and diffuse types, which exhibit different biological behavior. Urokinase‐type plasminogen activator (uPA), its receptor (uPAR) and matrix metalloproteinases (MMPs)‐1 and ‐9 are considered to play important roles in cancer invasion and metastasis. We have already suggested a functional duality of these matrix‐degrading enzymes/factors; they may also be involved in the matrix turnover (remodeling) or host immune/inflammatory reactions as far as they are expressed by host cells. We performed a retrospective study on the immuno‐histochemical expression of these enzymes/factors in surgical specimens from patients with gastric cancer, including 26 with the diffuse and 78 with the intestinal type. We also evaluated macrophages since they are major sources of uPAR. The positivity rate for uPA in cancer cells was significantly lower in diffuse‐type than in intestinal‐type. Stromal expression was seen mainly along the invasive margin (tumor–host interface). The degree of stromal expression of uPAR and MMP‐9 and the macrophage number were markedly decreased in diffuse‐type compared with intestinal‐type. Stromal expression of uPAR and macrophage number in intestinal‐type were higher in patients without liver metastasis than in patients with liver metastasis, while uPA expression in cancer cells was more pronounced in patients with liver metastasis. Studies using frozen sections revealed that the expression of MMP‐1, restricted to the stromal area, was more decreased in diffuse‐type (18 patients) than in intestinal‐type (21 patients). Our results show that the in situ expression of matrix‐degrading enzymes/factors in gastric cancer is significantly more diminished in diffuse‐type than in intestinal‐type, suggesting a multifunctional aspect of the matrix‐degradation process in cancer tissue. Int. J. Cancer (Pred. Oncol.) 84:74–79, 1999.

Quantitative evaluation of adhesion of lactobacilli isolated from human intestinal tissues to human colonic mucin using surface plasmon resonance (BIACORE assay)

Aims:  To isolate lactobacilli from the mucus layer of the human intestine and evaluate their adhesion abilities using a BIACORE assay.

Outcomes after Pylorus-preserving Gastrectomy for Early Gastric Cancer: A Prospective Multicenter Trial

The aim of the present study was to compare in a prospective, multicenter trial the results early and late after pylorus-preserving gastrectomy (PPG) versus conventional distal gastrectomy (CDG) with Billroth I anastomosis for early gastric cancer. Eighty-one patients with early gastric cancer were randomized and then underwent either PPG or CDG. Duration of operation, intraoperative blood loss, days until removal of the nasogastric tube, days until start of oral intake, and decrease in body weight were studied as parameters for outcomes early after the surgery. Late results were studied in patients followed for longer than 3 years. Change in body weight, status of oral intake, symptoms suggesting early dumping syndrome, and overall satisfaction were addressed in the questionnaire. The presence of gallstones was examined with ultrasonography. There were no differences in early results between PPG and CDG. The incidence of early dumping syndrome was lower in PPG (8%) than in CDG (33%). Other late results including the incidence of gallstones were not different between the 2 groups. These results indicate that PPG is as safe as CDG and has an advantage in terms of early dumping syndrome.

A New Assay Using Surface Plasmon Resonance (SPR) to Determine Binding of the Lactobacillus acidophilus Group to Human Colonic Mucin

A new binding assay to investigate the mechanism of adhesion of lactic acid bacteria to the human intestine was established by the surface plasmon resonance technique using a biosensor BIACORE1000. Cells of 26 strains of the Lactobacillus acidophilus group as analytes were eluted onto a sensor chip on which were immobilized biotinylated A-trisaccharide polymer probes having human A-type antigen [(GalNAcα1-3(Fucα1-2)Gal)-] or human colonic mucin of blood type A (HCM-A) as ligands. In the first screening, high adhesive affinity to the A-trisaccharide BP-probe was observed in L. acidophilus OLL2769, L. crispatus JCM8778, LA205 and LA206. In the second screening, which used HCM-A, only L. acidophilus OLL2769 and L. crispatus JCM8778 were selected as adhesive strains with specific binding ability to human A-antigen. The results indicated that some strains of the L. acidophilus group could recognize and bind the sugar chain of A-antigen structure on HCM.

Improved Detection Rate of Early Breast Cancer in Mass Screening Combined with Mammography

A cohort study to compare mass screening with and without mammography was conducted in Miyagi Prefecture, Japan in order to establish whether the effectiveness of breast cancer screening would be improved when mammography was combined with physical examination. A trial of mass screening combined with mammography was carried out in 9634 women aged over 50. Lateral imaging of the breast using single‐view film mammography was performed at the first stage of mass screening in addition to physical examination of the breast. Results in the trial were compared with those obtained in 35511 age‐matched subjects without mammography. Thirty breast cancers were found in the trial with a detection rate of 0.31%, which was much higher than that (0.08%) obtained by physical examination without mammography. In 15 of the 30 patients the breast tumor was not palpated at the first screening, but abnormal findings were detected in the mammography. A higher rate (73%) of early breast cancer was obtained in the screening trial with mammography than that (39%) obtained in the screening with physical examination alone. Mass screening combined with mammography is superior to that without mammography for breast cancer screening, especially for the detection of non‐palpable, early breast cancer.

Cell Adhesion Molecule Expression by Vascular Endothelial Cells as an Immune/Inflammatory Reaction in Human Colon Carcinoma

The cell adhesion of inflammatory cells to vascular endothelial cells is an important process in the recruitment of inflammatory cells to the site. In cancer tissue, infiltration of inflammatory cells has been suggested to be a mechanism of host resistance. To clarify this infiltration mechanism, we investigated cell adhesion molecule expression (E‐selectin, P‐selectin, and ICAM‐1) in vascular endothelial cells by immunohistochemistry in colon carcinoma. Venules distributed along the invasive margin expressed E‐ and P‐selectins and ICAM‐1. These phenotypical features are identical to those of endothelial cells observed in active inflammatory lesions, and the vessels can, therefore, be designated as immunologically activated vessels. Nevertheless, the majority of blood vessels within the tumor lacked immunoreactivity for all these adhesion molecules and, therefore, could be designated as immunologically inactive vessels. Granulocytes, lymphocytes and macrophages, bearing the counter‐receptors of these adhesion molecules, were more densely distributed along the invasive margin. In contrast, few inflammatory cells were present within the tumor. In conclusion, the present study has demonstrated the phenotypical heterogeneity of tumor vessels; those for inflammatory cell infiltration to the tumor and those for the nutrient supply to the tumor.

Interleukin 2-activated killer cells: generation in collaboration with interferon and its suppression in cancer patients

SummaryThe generation of lymphokine activated killer (LAK) cells by recombinant IL2 (rIL2) in collaboration with interferonγ (IFNγ) was examined in peripheral blood mononuclear cells (PBMC) from patients with malignant tumors of the digestive organs and breast cancer. LAK cytotoxicity could be induced by rIL2 at 10 units/ml in 10 of 12 patients and 20 of 37 using fresh autologous tumor cells and PK-1, an established solid tumor cell line as a target, respectively. Among 34 patients, in which titers of IFNγ produced were assayed, 12 showed no IFNγ production. All of these 12 patients had no or extremely low LAK activity, suggesting the correlation of LAK generation with the production of IFNγ in response to rIL2. LAK induction by rIL2 in PBMC of cancer patients was almost completely inhibited by addition of anti-IFNγ serum. Depressed LAK generation, which was accompanied by no or low levels of IFNγ production, was partially restored by addition of exogenous recombinant IFNγ. These results indicate that LAK induction by rIL2 in cancer patients involves the production of IFNγ and its interaction with rIL2.The results also suggested the presence of a factor(s) suppressing LAK induction by rIL2 in the serum of cancer patients. Based on these results, the cancer patients could be divided into the following three groups. Group 1, in which the serum suppressor activity was undetectable, had the same level of LAK cytotoxicity in PBMC as healthy controls. Group 2 showed the serum suppressor factor and had the lower level of cytotoxicity in PBMC when cultivated in autologous serum (AS) compared to healthy controls. The depressed LAK induction in AS medium was restored to a normal level in culture with fetal calf serum (FCS) plus rIL2, or by addition of rIFNγ, or high concentrations of rIL2 in AS medium. The last group (group 3), in which the serum suppressor factor was also found, had the lowest level of cytotoxicity compared to healthy controls. The LAK induction in these patients could not be restored to a normal level by culture in FCS medium, addition of exogenous rIFNγ or high concentrations of rIL2, suggesting the possibility that the deficit of LAK generation in this group might involve the dysfunction or the lack of IL2 responder cells, in addition to the presence of a serum suppressor factor(s).