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| 1.
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Watanabe, Toshifumi ; Tajima, Hidehiro ; Hayashi, Hironori ; Nakagawara, Hisatoshi ; Ohnishi, Ichiro ; Takamura, Hiroyuki ; Ninomiya, Itasu ; Kitagawa, Hirohisa ; Fushida, Sachio ; Tani, Takashi ; Fujimura, Takashi ; Ohta, Tetsuo ; Wakayama, Tomohiko ; Iseki, Shoichi ; Harada, Shinichi ; 田島, 秀浩 ; 林, 泰寛 ; 中川原, 寿俊 ; 高村, 博之 ; 二宮, 致 ; 北川, 裕久 ; 伏田, 幸夫 ; 谷, 卓 ; 藤村, 隆 ; 太田, 哲生 ; 若山, 友彦 ; 井関, 尚一 ; 原田, 真市
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金沢大学医薬保健研究域医学系<br />Histone acetylation and deacetylation have been thought to be related to gene expression, and there
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are many reports indicating that histone deacetylase inhibitors (HDACis) exert antifibrogenic effects in several organs. In injured livers, hepatic stellate cells (HSCs) are activated in response to profibrogenic mediators and produce large amounts of extracellular matrix. In particular, transforming growth factor-β1 (TGF-β1) is considered as a key factor in accelerating hepatic fibrosis because it is released from activated HSCs and further stimulates them. The present study aimed to clarify whether sodium valproate (VPA) has suppressive effects on cultured human HSCs (LI90). We showed that treatment with VPA had no significantly suppressive effect on cell proliferation at a concentration of 1 mM, which corresponded approximately to the serum concentration obtained by the administration of a clinical dose. However, VPA prevented the morphological changes characteristic for activation and inhibited the expression of collagen type 1 α1 (COL1A1) and TGF-β1 in activated LI90 cells at the mRNA and protein levels. Our results support the hypothesis that VPA exerts antifibrogenic activity with little cytotoxicity at 1 mM, and HDACis are expected to be used in clinical practice for the treatment of fibrotic diseases.<br />Embargo Period 6 months
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| 2.
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Nakanuma, Shinichi ; Tajima, Hidehiro ; Okamoto, Koichi ; Hayashi, Hironori ; Nakagawara, Hisatoshi ; Onishi, Ichiro ; Takamura, Hiroyuki ; Kitagawa, Hirohisa ; Fushida, Sachio ; Tani, Takashi ; Fujimura, Takashi ; Kayahara, Masato ; Ohta, Tetsuo ; Wakayama, Tomohiko ; Iseki, Shoichi ; Harada, Shinichi ; 中村, 信一 ; 田島, 秀浩 ; 岡本, 浩一 ; 林, 泰寛 ; 中川原, 寿俊 ; 高村, 博之 ; 北川, 裕久 ; 伏田, 幸夫 ; 谷, 卓 ; 藤村, 隆 ; 萱原, 正都 ; 太田, 哲生 ; 若山, 友彦 ; 井関, 尚一 ; 原田, 真市
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金沢大学医薬保健研究域医学系<br />In primary malignant liver tumors, trypsinogen-immunoreactivity was present in 70% of intrahepatic c
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holangiocarcinoma (ICC) specimens, but absent in hepato-cellular carcinoma (HCC) specimens. We suggest the secretion of trypsinogen to be a key difference in biological behavior between ICC and HCC cells. The purpose of this study was to investigate the secretion of tumor-derived trypsin and the expression of its specific receptor, protease-activated receptor-2 (PAR-2), in ICC using cell lines and surgical specimens. The expression of trypsinogen-1 mRNA was observed in three of four ICC cell lines, but none of three HCC cell lines. Western blot analysis detected trypsinogen-1 in serum-free conditioned medium from one of the ICC cell lines positive for the mRNA. Gelatin zymography revealed a gelatinolytic activity for trypsin, the activated form of trypsinogen, in the same conditioned medium. PAR-2 mRNA and protein were observed in ICC cell lines. The proliferative activity of ICC cells was increased by concentrations of trypsin as low as 10 nM, and peaked at 100 nM. The effect of trypsin was suppressed by a serine protease inhibitor, gabexate mesilate. PAR-2 expression was detected in 64% of ICC surgical specimens immunohistochemically. In addition, stroma fibroblasts expressed PAR-2 in 52% of ICC specimens. These results suggest that trypsinogen-1 contributes to the growth of ICC cells and also tumor-associated fibroblasts.<br />Embargo Period 6 months
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| 3.
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Okamoto, Koichi ; Tajima, Hidehiro ; Nakanuma, Shinichi ; Sakai, Seisho ; Makino, Isamu ; Kinoshita, Jun ; Hayashi, Hironori ; Nakamura, Keishi ; Oyama, Katsunobu ; Nakagawara, Hisatoshi ; Fujita, Hideto ; Takamura, Hiroyuki ; Ninomiya, Itasu ; Kitagawa, Hirohisa ; Fushida, Sachio ; Fujimura, Takashi ; Harada, Shinichi ; Wakayama, Tomohiko ; Iseki, Shoichi ; Ohta, Tetsuo ; 岡本, 浩一 ; 田島, 秀浩 ; 中村, 信一 ; 牧野, 勇 ; 木下, 淳 ; 林, 泰寛 ; 中村, 慶史 ; 尾山, 勝信 ; 中川原, 寿俊 ; 藤田, 秀人 ; 高村, 博之 ; 二宮, 致 ; 北川, 裕久 ; 伏田, 幸夫 ; 藤村, 隆 ; 原田, 真市 ; 若山, 友彦 ; 井関, 尚一 ; 太田, 哲生
概要:
金沢大学医薬保健研究域医学系<br />We previously reported that hepatic stellate cells (HSCs) activated by angiotensin II (AngII) facili
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tate stromal fibrosis and tumor progression in intrahepatic cholangiocarcinoma (ICC). AngII has been known as a growth factor which can promote epithelial-to-mesenchymal transition (EMT) in renal epithelial cells, alveolar epithelial cells and peritoneal mesothelial cells. However, in the past, the relationship between AngII and stromal cell-derived factor-1 (SDF-1) in the microenvironment around cancer and the role of AngII on EMT of cancer cells has not been reported in detail. SDF-1 and its specific receptor, CXCR4, are now receiving attention as a mechanism of cell progression and metastasis. In this study, we examined whether activated HSCs promote tumor fibrogenesis, tumor progression and distant metastasis by mediating EMT via the AngII/AngII type 1 receptor (AT-1) and the SDF-1/CXCR4 axis. Two human ICC cell lines and a human HSC line, LI-90, express CXCR4. Significantly higher concentration of SDF-1αwas released into the supernatant of LI-90 cells to which AngII had been added. SDF-1α increased the proliferative activity of HSCs and enhanced the activation of HSCs as a growth factor. Furthermore, addition of SDF-1α and AngII enhanced the increase of the migratory capability and vimentin expression, reduced E-cadherin expression, and translocated the expression of β-catenin into the nucleus and cytoplasm in ICC cells. Co-culture with HSCs also enhanced the migratory capability of ICC cells. These findings suggest that SDF-1α, released from activated HSCs and AngII, play important roles in cancer progression, tumor fibrogenesis, and migration in autocrine and paracrine fashion by mediating EMT. Our mechanistic findings may provide pivotal insights into the molecular mechanism of the AngII and SDF-1α-initiated signaling pathway that regulates fibrogenesis in cancerous stroma, tumor progression and metastasis of tumor cells expressing AT-1 and CXCR4.<br />Embargo Period 6 months
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| 4.
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Ohe, Kazuyo ; Watanabe, Takuo ; Harada, Shinichi ; Munesue, Seiichi ; Yamamoto, Yasuhiko ; Yonekura, Hideto ; Yamamoto, Hiroshi
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金沢大学医薬保健研究域医学系<br />Receptor for advanced glycation endproducts (RAGE) is a cell-surface receptor. The binding of ligand
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s to membrane-bound RAGE (mRAGE) evokes cellular responses involved in various pathological processes. Previously, we identified a novel soluble form, endogenous secretory RAGE (esRAGE) generated by alternative 5′ splice site selection in intron 9 that leads to extension of exon 9 (exon 9B). Because esRAGE works as an antagonistic decoy receptor, the elucidation of regulatory mechanism of the alternative splicing is important to understand RAGE-related pathological processes. Here, we identified G-rich cis-elements within exon 9B for regulation of the alternative splicing using a RAGE minigene. Mutagenesis of the G-rich cis-elements caused a drastic increase in the esRAGE/mRAGE ratio in the minigene-transfected cells and in loss of binding of the RNA motif to heterogenous nuclear ribonucleoprotein (hnRNP) H. On the other hand, the artificial introduction of a G-stretch in exon 9B caused a drastic decrease in the esRAGE/mRAGE ratio accompanied by the binding of hnRNP H to the RNA motif. Thus, the G-stretches within exon 9B regulate RAGE alternative splicing via interaction with hnRNP H. The findings should provide a molecular basis for the development of medicines for RAGE-related disorders that could modulate esRAGE/mRAGE ratio. © The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.
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| 5.
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Ooi, Akishi ; Inokuchi, Masafumi ; Harada, Shinichi ; Inazawa, Johji ; Tajiri, Ryousuke ; Sawada-Kitamura, Seiko ; Ikeda, Hiroko ; Kawashima, Hiroko ; Dobashi, Yoh
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Oestrogen receptor-alpha (ERα), encoded by the ESR1 gene located on 6q25, is a nuclear transcription factor. Since it wa
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s reported in 2007 that more than 20% of breast cancers show ESR1 gene amplification, there has been considerable controversy about its frequency and clinical significance. We set out to assess the frequency and levels of ESR1 amplification in breast cancers. In a total of 106 breast needle biopsy specimens examined by immunohistochemistry, 78 tumours contained more than 10% ERα-positive cancer cells. In fluorescence in situ hybridization (FISH) analysis with an ESR1-specific probe, variously extended ESR1 signals were found in ERα-expressing cells. Some of these were indistinguishable from large clustered signals generally accepted to mean high-level gene amplification in homogeneously staining regions (HSRs), and could be considered to represent gene amplification. However, with RNase treatment, the 'HSR-like' signals changed to small compact signals, and are thus thought to represent concentrated RNA. FISH using two differently labelled probes corresponding to the non-overlapping 5'- and 3'-end portions of the ESR1 gene on touch smears showed a preserved spatial relationship of the 3' to 5' sequence of ESR1, therefore strongly suggesting that the RNA consisted of primary transcripts. Using touch smears obtained from 51 fresh tumours, precise enumeration of ESR1 signals with a correction by the number of centromere 6 on FISH after RNase A treatment revealed that three tumours (5.9%) had tumour cells with one to three additional copies of ESR1 as predominant subpopulations. This infrequent and low level of gene amplification of ESR1 was also detected as a 'gain' of the gene by analysis with multiplex ligation-dependent probe amplification (MLPA). The consistent results from immunohistochemistry, FISH, and MLPA in the present study settle the long-standing debate concerning gene amplification of ESR1 in breast carcinoma. © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd
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| 6.
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Tsukada, Tomoya ; Fushida, Sachio ; Harada, Shinichi ; Terai, Shiroh ; Yagi, Yasumichi ; Kinoshita, Jun
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Background: Adiponectin is inversely related to BMI, positively correlates with insulin sensitivity, and has anti-athero
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genic effects. In recent years, adiponectin has been well studied in the field of oncology. Adiponectin has been shown to have antiproliferative effects on gastric cancer, and adiponectin expression is inversely correlated with clinical staging of the disease. However, no studies have reported the correlation between serum adiponectin and receptor expression with disease progression. Methods. In this study, we evaluated expression levels of 2 adiponectin receptors - AdipoR1 and AdipoR2 - and attempted to correlate their expression with prognosis in gastric cancer patients. AdipoR1 and AdipoR2 expression in gastric cancer cell lines (MKN45, TMK-1, NUGC3, and NUGC4) was evaluated by western blotting analysis, and the antiproliferative potential of adiponectin was examined in vitro. Serum adiponectin levels were evaluated in 100 gastric cancer patients, and the expression of AdipoR1 and AdipoR2 was assessed by immunohistochemical staining. Results: MKN45 and NUGC3 expressed higher levels of AdipoR1 compared to NUGC4, even though there was no significance in AdipoR2 expression. The antiproliferative effect of adiponectin was confirmed in MKN45 and NUGC3 at 10 g/ml. No significant associations were observed between serum adiponectin levels and clinicopathological characteristics, but lymphatic metastasis and peritoneal dissemination were significantly higher in the negative AdipoR1 immunostaining group (24/32, p = 0.013 and 9/32, p = 0.042, respectively) compared to the positive AdipoR1 group (lymphatic metastasis, 33/68; peritoneal dissemination, 8/68). On the other hand, AdipoR2 expression was only associated with histopathological type (p = 0.001). In survival analysis, the AdipoR1 positive staining group had significantly longer survival rates than the negative staining group (p = 0.01). However, multivariate analysis indicated that AdipoR1 was not an independent prognostic factor on patient's survival on gastric cancer. Conclusions: In gastric cancer, adiponectin has the possibility to be involved in cell growth suppression via AdipoR1. The presence of AdipoR1 could be a novel anticancer therapeutic target in gastric cancer. © 2011Tsukada et al; licensee BioMed Central Ltd.
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| 7.
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Yagi, Yasumichi ; Fushida, Sachio ; Harada, Shinichi ; Kinoshita, Jun ; Makino, Isamu ; Oyama, Katsunobu
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Background. Management of peritoneal dissemination is the most critical problem in gastric cancer. This study was perfor
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med to investigate the inhibitory effects of valproic acid (VPA) on a highly peritoneal-seeding cell line of human scirrhous gastric cancer, OCUM-2MD3, and to explore the mechanism and the potential of VPA. Methods. The effects of VPA on the growth of OCUM-2MD3 cells were assessed by MTT assay. In addition, paclitaxel (PTX) was combined with VPA to evaluate their synergistic effects. HDAC1 and HDAC2 expression were evaluated by western blotting in OCUM-2MD3 cells and other gastric cancer cell lines (TMK-1, MKN-28). The acetylation status of histone H3 and α-tubulin after exposure to VPA were analyzed by western blotting. The activities of cell cycle regulatory proteins and apoptosis-modulating proteins were also examined by western blotting. The effects of VPA in vivo were evaluated in a xenograft model, and apoptotic activity was assessed by TUNEL assay. Results. OCUM-2MD3 cells showed high levels of HDAC1 and HDAC2 expression compared with TMK-1 and MKN-28. The concentration of VPA required for significant inhibition of cell viability (P < 0.05) was 5 mM at 24 h and 0.5 mM at 48 h and 72 h. The inhibition of VPA with PTX showed dose-dependent and combinatorial effects. VPA increased acetyl-histone H3, acetyl α-tubulin, and p21WAF1 levels accompanied by upregulation of p27, caspase 3, and caspase 9, and downregulation of bcl-2, cyclin D1, and survivin. In the xenograft model experiment, the mean tumor volume of the VPA-treated group was significantly reduced by 36.4%, compared with that of the control group at 4 weeks after treatment (P < 0.01). The apoptotic index was significantly higher in the VPA-treated group (42.3% ± 3.5%) than in the control group (7.7% ± 2.5%) (P < 0.001). Conclusions. VPA induced dynamic modulation of histone H3 and α-tubulin acetylation in relation with the anticancer effect and the enhancement of PTX in the OCUM-2MD3 cell line. Therefore, VPA in combination with PTX is expected to be a promising therapy for peritoneal dissemination of scirrhous gastric cancer. © 2010 Yagi et al; licensee BioMed Central Ltd.
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| 8.
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Hirata, Miki ; Tajima, Hidehiro ; Miyashita, Tomoharu ; Miyata, Takashi ; Nakanuma, Shinichi ; Makino, Isamu ; Hayashi, Hironori ; Oyama, Katsunobu ; Takamura, Hiroyuki ; Ninomiya, Itasu ; Fushida, Sachio ; Nakata, Hiroki ; Iseki, Shoichi ; Harada, Shinichi ; Wakayama, Tomohiko ; Ohta, Tetsuo
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Oxaliplatin-based chemotherapy plays an important role in the treatment of colorectal liver metastases. Oxaliplatin, however, causes sinusoidal obstruction syndrome (SOS), which is characterized by portal hypertension, splenomegaly,
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thrombocytopenia, and liver dysfunction. SOS is diagnosed histopathologically by disruption of the sinusoidal endothelium, collagen deposition, fbrosis especially around zone 3, dilatation of the sinusoidal space and congestion. This study assessed the characteristics of a rat model of SOS. SOS was induced in rats by administration of monocrotaline (MCT). Blood chemistries and macroscopic and microscopic fndings were compared in rats administered MCT and vehicle (control group). Levels of expression in the liver of CD41, P-selectin, rat endothelial cell antigen-1, CD34, and cleaved caspase-3 were analyzed immunohistochemically. Moreover, livers of these rats were analyzed by electron microscopy. Macroscopically, MCT-treated rats showed accumulation of bloody ascites and blue liver and were diagnosed with SOS histologically. Serum concentrations of aspartate aminotransferase (P=0.003), alanine aminotransferase (P=0.008), total-bilirubin (P=0.012), direct-bilirubin (P=0.007), indirect-bilirubin (P=0.003), lactate dehydrogenase (P<0.001) and hyaluronic acid (P=0.016) were signifcantly higher, and platelet counts signifcantly lower (P=0.004), in MCT-treated than in control rats. The livers of MCT-treated rats were immunohistochemically positive for CD41 and P-selectin, suggesting platelet aggregates; for rat endothelial cell antigen-1 and CD34, suggesting sinusoidal endothelial disorder; and for cleaved caspase-3, suggesting hepatocyte apoptosis. Electron microscopic fndings revealed platelet aggregation in the space of Disse in the MCT group. Extravasated platelet aggregation in Disse's space may be involved in the development of SOS.
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| 9.
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Okamoto, Koichi ; Tajima, Hidehiro ; Ohta, Tetsuo ; Nakanuma, Shinichi ; Hayashi, Hironori ; Nakagawara, Hisatoshi ; Onishi, Ichiro ; Takamura, Hiroyuki ; Ninomiya, Itasu ; Kitagawa, Hirohisa ; Fushida, Sachio ; Tani, Takashi ; Fujimura, Takashi ; Kayahara, Masato ; Harada, Shinichi ; Wakayama, Tomohiko ; Iseki, Shoichi
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富山県立中央病院<br />金沢大学医薬保健研究域医学系<br />金沢大学附属病院胃腸外科<br />Intrahepatic cholangiocarcinoma (ICC) is characterized as a highly f
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atal tumor with poor prognosis because of its strong progression, early invasion, widespread metastasis and rich cancerous stroma. Although it is widely accepted that fibroblasts facilitate stromal fibrosis and tumor progression, the mechanisms of the interaction between cancer cells and activated fibroblasts have not been fully elucidated thus far. In this study, we demonstrate the presence of angiotensin II (AngII) in ICC tissues and explore the interaction between hepatic stellate cells (HSCs) and ICC cells as one of the sources of stromal fibrosis and tumor progression through the interaction of the AngII/AngII type 1 receptor (AT-1) axis. The concentrations of AngII in ICC tissues were significantly higher than those of HCC and normal liver. Two human ICC cell lines (HuCCT-1, CCKS-1) and a human HSC cell line (LI-90) expressed AT-1 mRNA and protein. The proliferative activity of ICC cells and HSCs to which AngII was added dose-dependently increased and AT-1 antagonist inhibited the proliferative effects. HSCs to which AngII was added showed a higher expression of α-smooth muscle actin (α-SMA, a marker of activated HSCs and myofibroblasts), glial fibrillary acidic protein (GFAP, a specific marker of HSCs) and collagen type I than control cells. AT-1 antagonist also inhibited the activation and transformation of HSCs stimulated by AngII. These findings suggested that locally formed AngII in ICC tissues plays a role in the proliferation and activation of ICC cells and HSCs expressing AT-1 as a growth factor in autocrine and paracrine fashions. Our mechanistic findings provide the first insight into an autocrine and paracrine AngII-initiated signaling pathway that regulates ICC proliferation and fibrosis.
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| 10.
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原田, 真市 ; Harada, Shinichi
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金沢大学医薬保健研究域医学系<br />LED光源の中でも、特に青色LEDに着目し線虫の寿命に与える影響について検討した。青色LEDにより線虫の寿命は有意に短くなった。さらに青色LED曝露による線虫の活性酸素種を測定したところ僅かではあるが
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酵素活性の上昇が認められた。遺伝子発現解析からは、触感覚神経で発現するmec-5遺伝子や神経系カルシウムセンサーとして機能すると考えられるncs-1遺伝子の発現が有意に増減していた。また線虫の行動解析からはコントロールに比べ青色LED照射により行動距離が短くなった。以上のことから線虫では青色LED曝露により酸化ストレスが上昇し、行動が抑制されることが明らかとなった。<br />To invesigate the biological disadvantage of blue LED light, we used the free-living nematode Caenorhabditis elegans, as a model. By exposing blue LED light at 465nm, the lifespan of C. elegans was decreased, and these reactive oxygen species (ROS) activity as a marker of stress response were slightly increased. Using Real-Time RT-PCR analysis to measure gene expression levels by exposure of LED light, we found that mec-5 gene which express in sensory neuron was highly expressed and neural calcium sensor (ncs-1) gene was significantly decreased in worms with blue LED light as compared to dark exposure as a control. Furthermore, we found to cause a marked deterioration in the worm's behavior with blue LED light. It is concluded that exposure of blue LED light may cause oxidative stress, and result in abnormal locomotion and survival in the nematode C. elegans. However, further investigation is required to clarify the toxicological effect of the LEDs light.<br />研究課題/領域番号:26430068, 研究期間(年度):2014-04-01 - 2017-03-31
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