Hirotaka Kuga

Interleukin 1β Enhances Invasive Ability of Gastric Carcinoma through Nuclear Factor-κB Activation

Purpose: We examined the role of interleukin (IL)-1β in activation of nuclear factor κB (NF-κB) and the biological function of activated NF-κB in gastric carcinoma cells. Experimental Design: Human gastric carcinoma cell line GCTM-1 was used to examine NF-κB activation by immunostaining and electrophoretic mobility shift assay. Matrix metalloproteinase (MMP)-9 expression, which plays an important role in tumor invasion, was assessed by semiquantitative reverse transcription-PCR, Western blotting, and immunostaining. The invasive ability of GCTM-1 cells was measured by Matrigel invasion assay. In vivo expression of IL-1β and MMP-9 and activation of NF-κB in 10 surgically resected gastric carcinoma specimens were examined immunohistochemically. Results: IL-1β enhanced NF-κB activation, MMP-9 expression, and the invasive ability of GCTM-1. A NF-κB inhibitor, pyrrolidine dithiocarbamate, suppressed both MMP-9 expression and invasiveness of IL-1β-treated GCTM-1 cells. IL-1β did not increase the invasive ability of GCTM-1 cells transfected with MMP-9 antisense oligonucleotide. Concomitant expression of IL-1β and nuclear NF-κB was observed in 3 of 10 gastric carcinoma specimens. Cells producing IL-1β were tumor-infiltrating macrophages in two specimens and gastric carcinoma cells in one specimen. Conclusions: One of the molecules that may play a role in NF-κB activation in some gastric carcinomas is IL-1β. The present results suggest that IL-1β increases the invasive ability of carcinoma cells through activation of NF-κB and the resulting MMP-9 expression.

Interferon-gamma suppresses transforming growth factor-beta-induced invasion of gastric carcinoma cells through cross-talk of Smad pathway in a three-dimensional culture model.

We reconstituted a three-dimensional gastric carcinoma model similar to invasive gastric carcinoma tissue. This model consists of a human gastric carcinoma cell line, GCTM-1, a human fibroblast cell line, TIG-1-20, and transforming growth factor-β (TGF-β)-containing type I collagen gel. Using this model, we were able to observe the growth of the two cell types, especially carcinoma cell invasive growth, in real time for more than 30 days. TGF-β and TIG-1-20 were essential for GCTM-1 invasive growth and proliferation, respectively. TGF-β induced the enhanced expression of matrix metalloproteinase 9 (MMP9) and urokinase-type plasminogen activator (uPA) in GCTM-1 at both the protein and enzymatic activity levels. The TGF-β-induced invasion of GCTM-1 was inhibited by MMP9- or uPA-antisense (AS) oligonucleotide transfection to GCTM-1. When exogenous interferon-γ (IFN-γ) was added to this model, TGF-β-dependent GCTM-1 invasion was significantly inhibited, concomitant with the decreased expression of MMP9 and uPA. The intracellular signal transduction of Smad was examined to analyse the mechanism of the inhibitory effect of IFN-γ. TGF-β accelerated the phosphorylation of Smad2/3 and nuclear translocation of the Smad2/3–Smad4 complex in GCTM-1, but these TGF-β-induced effects were significantly inhibited by IFN-γ-induced Smad7 expression. When GCTM-1 was cotransfected with AS oligonucleotide of Smad2 and Smad3, the TGF-β-induced invasion of GCTM-1 disappeared. In addition, the inhibitory effect of IFN-γ on TGF-β-dependent GCTM-1 invasion vanished by the AS oligonucleotide of Smad7 transfection. These results indicate that IFN-γ inhibits TGF-β-dependent GCTM-1 invasion through cross-talk in the Smad pathway. IFN-γ may be a new therapeutic tool for TGF-β-expressed invasive carcinomas.

Unproductive cleavage and the inactivation of protease‐activated receptor‐1 by trypsin in vascular endothelial cells

Using fura‐2 fluorometry of [Ca2+]i in response to thrombin, trypsin and protease‐activated receptor activating peptides (PAR‐APs), we determined whether trypsin cleaves protease‐activated receptor 1 (PAR1) and activates it in the endothelial cells of the porcine aortic valves and human umbilical vein. Once stimulated with thrombin, the subsequent application of trypsin induced a [Ca2+]i elevation similar to that obtained without the preceding stimulation with thrombin in the valvular endothelial cells. However, the preceding stimulation with trypsin abolished the subsequent response to thrombin, but not to bradykinin or substance P. The response to PAR1‐AP (SFLLRNP) was significantly (P<0.05) reduced by the preceding stimulation with thrombin and PAR1‐AP in the valvular endothelial cells, while, importantly, it remained unaffected by the preceding stimulation with either trypsin or PAR2‐AP (SLIGRL). The response to PAR2‐AP was reduced by the preceding stimulation with trypsin and PAP2‐AP. PAR1‐AP attenuated the subsequent responses not only to thrombin and PAR1‐AP but also to trypsin and PAR2‐AP, while PAR2‐AP specifically attenuated the subsequent responses to trypsin and PAR2‐AP. In human umbilical vein endothelial cells, a higher affinity PAR1‐AP (haPAR1‐AP) (Ala‐pF‐Arg‐Cha‐HArg‐Tyr‐NH2) specifically attenuated the responses to thrombin but not trypsin. On the other hand, the response to haPAR1‐AP was significantly (P<0.05) attenuated by the preceding stimulation with thrombin but not trypsin. In conclusion, trypsin cleaved PAR1 but did not activate it in the endothelial cells. Moreover, the trypsin‐cleaved PAR1 was no longer responsive to thrombin.

Construction of a Transplantable Tissue-Engineered Artificial Peritoneum

Background: Peritoneal defects lead to serious postoperative problems. Thus the development of physiological material to cover peritoneal defects is very desirable. Aim: The aim of this study was to develop a transplantable artificial peritoneum. Method: The artificial peritoneum consisted of collagen gel, fibroblasts, and mesothelial cells, and histological features were analyzed. The artificial peritoneum at the site of a peritoneal defect in the rat was transplanted to the abdominal wall. Results: Histological examination revealed that the artificial peritoneum consisted of a flat mesothelial monolayer upon a stromal matrix. All transplanted artificial peritoneums adapted well to the host and prevented severe adhesion. Conclusion: Our artificial peritoneum may be a useful transplantable bioengineered material for repair of surgical peritoneal defects.

A large quantity of CD3-/CD19-/CD16- lymphocytes in malignant pleural effusion from a patient with recurrent cholangio cell carcinoma.

Tumor infiltrating lymphocytes (TILs) are candidates for adoptive cellular immunotherapy. Here we report on a patient whose TILs presented unusual lymphocyte antigens. Pleural effusions were collected from a 47-year-old man with recurrent cholangio cell carcinoma and malignant effusion. Effusion- associated lymphocytes (EALs) were separated by Ficoll-Hypaque gradient, and the EAL phenotype was determined by flow cytometry. The percentage of positive cells was determined for each lymphocyte-related differentiation antigen. The percentages of CD3+, CD19+, and CD16+ lymphocyte subpopulations among EALs were 20%, 7%, and 3%, respectively. Nearly 70% of EALs were CD3− /CD19− /CD56− /CD16−cells. The phenotypes of peripheral blood lymphocytes (PBLs) collected simultaneously from the patients peripheral blood were CD3+ (52%), CD19+ (20%), and CD16+ (20%).When EALs were cultured in medium without pleural effusion, T cell-related antigens, but not B cell- or natural killer (NK) cell-related antigens, were newly expressed on EALs, and this expression reached a plateau after 48 h in culture. The proportions of CD3+, CD19+, and CD16+ cells were 69%, 7%, and 3%, respectively. However, when EALs were cultured in medium with pleural effusion, increased expression of T cell-related antigens was not observed; the proportions of CD3+, CD19+, and CD16+ cells were 16%, 6%, and 1%, respectively. Neither total cell numbers nor cellular viability of EALs changed significantly after in-vitro culture, suggesting that significant proliferation or death of EALs did not occur during the culture period. Co-culture of the patients PBLs with autologous pleural effusion for 96 h did not alter the expression of lymphocyte-related antigens on the PBLs. These results indicate that expression of T cell-related antigens, but not B cell- or NK cell-related antigens, on EALs was blocked temporarily by the malignant pleural effusion. This is the first report concerning the existence of a large quantity of unclassified lymphocytes in which the T cell-related antigens were reversibly masked in the malignant pleural effusion.