Researchers Database
TANIDA SatoshiGraduate School of Medical Sciences Core Laboratory | |||
Last Updated :2024/03/19
Research Grants & Projects
- 日本学術振興会：科学研究費助成事業Date (from‐to) : 2019/04 -2022/03Author : 谷田 諭史; 前川 大志1.胃癌細胞株におけるPD-L1蛋白発現量の検討。11種類の胃癌細胞株においてPD-L1蛋白量を比較し、PD-L1発現量の変化を検討した。NUGC3細胞がPD-L1をconstitutiveに多く発現しており、CUL3欠失により、PD-L1発現が増加していることを確認し、以後NUGC3を使用した。2.CUL3-ANKFY1結合の確認。 NUGC3細胞をlysisした後、抗ANKFY1抗体で免疫沈降した後、抗CUL3抗体でブロットした。CUL3-ANKFY1の結合を確認し、ANKFY1 siRNAオリゴにてノックダウンするとその結合が消失することを確認した。3.PD-L1の細胞内膜輸送を制御するCUL3-ANKFY1の基質探索。 CUL3-ANKFY1の基質の同定は、本申請課題の最重要課題である。各種培養消化器がん細胞株において、アスコルビン酸ペルオキシダーゼ変異体を用いた生細胞でのin vivoビオチン標識法を行い。CUL3-ANKFY1の基質の一つは、Rab5だということを同定した。4.CUL3-ANKFY1の基質欠失時のPD-L1の細胞内膜輸送変化の検討。 NUGC3細胞にANKFY1 siRNAオリゴまたは、コントロールオリゴをトランスフェクション施し、ANKFY1欠失後、蛍光標識したPD-L1抗体処置後、細胞膜から細胞内への輸送系をmicroscopeにて観察した。また、細胞膜、細胞質、細胞核のフラクションでのPD-L1の局在発現も確認した。ANKFY1欠失により、ウェスタン解析および免疫染色により、PD-L１は、コントロールに比較して細胞膜に多く局在することが明らかになった。5.Rab5欠失時のPD-L1の細胞内膜輸送阻害効果の検討。 Rab5の欠失後ウェスタン解析および免疫染色により、PD-L１は、局在は、細胞質に留まり細胞膜へ移動が阻害されることが明らかになった。
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 2016/04 -2019/03Author : Tanida Satoshi; HIGASHIYAMA shigekiAn analyzed system for membrane trafficking of intracellular proteins was established based on confocal images in NUGC3 gastric cancer cells which are fixed after transfection of siRNA of 175 BTBPs and stained for integrin β1.CUL3 was critical for the cell surface level of integrin β1. After depletion of 175 BTBPs by siRNA, a family of adaptor proteins for CUL3, we discovered that ANKFY1, an early endosomal BTBP, wasalso critical for localization of surface integrin β1.CUL3 interacted with ANKFY1 and was required for the early endosomal localization of ANKFY1. These data suggest that CUL3/ANKFY1 regulates endosomal membrane traffic of integrin β1.Next, we examine membrane trafficking of PD-L1 during depletion of CUL3 and ANKFY1 by siRNA.
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 2014/04 -2018/03Author : Mizoshita TsutomuThe analyses in long-term three-dimensional method for primary mouse glandular stomach culture have shown that the gastric mesenchymal myofibroblast (GMF) is important for the differentiation and proliferation of gastric epithelial cell. Growth arrest specific 1（GAS1）expression is higher in GMF, and Homeobox C8（HoxC8）、Notch 1、SRY-box containing gene 10（Sox10） are higher in intestinal mesenchymal myofibroblast, suggesting that these factors play the key roles in the gastric and intestinal differentiation by epithelial-mesenchymal interactions. We consider that the ectopic MUC5AC expression in the intestinal epithelial cells may be a predictive factor of recurrence or remission in the cases of Crohn's disease and intestinal Behçet's disease.
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 2013/04 -2016/03Author : Tanida Satoshi; JOH Takashi; HIGASHIYAMA ShigekiInhibition of Annexin A2 may be a new therapeutic strategy for the prevention of TNF-α shedding during inflammatory bowel disease inflammation.We try to propose the potential of Annexin A2 as a new molecular target for the treatment of IBD by using Annexin A2 knockout mouse. Annexin A2 knockout mouse cannnot be established becase chimera mouse's genital organ was immature. Annexin A2 is likely to be associated with mouse's genital organ formation.
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 2012/04 -2015/03Author : JOH Takashi; TANIDA Satoshi; HIGASHIYAMA ShigekiColon epithelial cells, many expressed in monocyte cells I was paying attention to the ARP 36-2 seen. When IL-1β stimulates colon epithelial cell line, TNFα concentration in the culture solution in over time increased but TPA, LPS, increase was observed. On the other hand, when the monocytic cell line TPA stimulation, TNFα concentration in the culture solution is increased, IL-1β, the stimulation with TPA, increase was observed. Further, when the deletion of the ADAM17 by KB-R7785, siRNA, colon epithelial cells, IL-1β in the monocyte cell, the concentration increase due to TPA stimulation during TNFαshedding, was significantly inhibited. Furthermore, even if lacking the ARP36-2 by siRNA, similarly concentration rise due to IL-1β, TPA stimulus during TNFαshedding has been significantly suppressed. ARP36, it seemed to be made in inflammatory bowel disease novel therapeutic target to control the TNFαshedding.
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 2011 -2013Author : MIZOSHITA Tsutomu; TANIDA Satoshi; JOH TakashiWe established a long-term three-dimensional primary culture of mouse glandular stomach epithelial cells, being cultivated for more than 2 months and maintaining the gastric phenotype without intestinal one. We consider that the shift from gastric to intestinal phenotype of stem cell in the stomach mucosa may be clarified by applying the above-mentioned three-dimensional primary culture to the glandular stomach of Helicobacter pylori-infected Mongolian gerbil model in which the intestinal metaplasia often occurs.
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 2010 -2012Author : TANIDA Satoshi; JOH Takashi; KATAOKA Hiromi; HIGASHIYAMA ShigekiCurrent treatment target toward advanced colorectal cancers is mainly focused on the epidermal growth factor receptor (EGFR) signaling, but its additive effects with chemotherapy are still limited. A disintegrin and metalloproteinase (ADAM) cleaves the proheparin-binding epidermal growth factor like growth factor (proHB-EGF). And soluble HB-EGF activates EGFR. In parallel, the carboxy-terminal fragment of proHB-EGF (HB-EGF-CTF) translocate into the inner nuclear membrane, subsequently exerts on the regulation of cell proliferation by binding nuclear promyelocytic leukemia zinc finger (PLZF) protein, a transcriptional repressor, thereby causing its nuclear export. We hypothesized that the inhibition of HB-EGF-CTF nuclear translocation may be a new strategy in preventing cell proliferation.Methods:12-O-tetradecanoylphorbor-13-acetate (TPA) was treated to activate ADAM. Nine-thousand chemical compounds were screened for their efficacies in blocking the binding of HB-EGF-CTF to promyelocytic leukemia zinc finger (PLZF) with Alphascreen system. The obtained candidates were then used to block the binding of HB-EGF-CTF to PLZF in colon cancer cells, HT29 and HCT116. Cell proliferation was investigated with a growth curve assay. The intracellular localization, and association between HB-EGF-CTF and PLZF, was assessed with immunofluorescent staining, and immunoprecipitation and Western blotting, respectively. The effects of obtained candidates on EGFR phosphorylation and on nuclear translocation of HB-EGF-CTF and export of PLZF during the angiotensin II type1 receptor (AT1R) knockdown were also investigated. Results: Telmisartan and candesartan were found to be potential candidates. Telmisartan inhibited TPA-induced cell proliferation stronger than candesartan. Telmisartan, but not candesartan blocked the nuclear translocation of HB-EGF-CTF, and binding of HB-EGF-CTF to PLZF, during TPA stimulation. Both telmisartan and candesartan did not inhibit TPA-induced EGFR phosphorylation, and telmisartan, but not candesartan, inhibited TPA-induced nuclear translocation of HB-EGF-CTF after knockdown of AT1R. Conclusions: The inhibition of HB-EGF-CTF nuclear translocation with telmisartan may be a novel strategy in preventing cell proliferation.
- Japan Society for the Promotion of Science：Grants-in-Aid for Scientific ResearchDate (from‐to) : 2009 -2011Author : JOH Takashi; KATAOKA Hiromi; TANIDA Satoshi; HIGASHIYAMA ShigekiBCL6 is a transcriptional repressor that has important functions in lymphocyte differentiation and lymphomagenesis, but there have been no reports of BCL6 expression in gastric cancers. In the present study, we investigated the BCL6 function in gastric cancers. Treatment with TPA resulted in BCL6 degradation and cyclin D2 upregulation. This phenomenon was inhibited by the suppression of the nuclear translocation of HB-EGF-CTF(C-terminal fragment of pro-HB-EGF). The HB-EGF-CTF nuclear translocation leads to the interaction of BCL6 with HB-EGF-CTF and the nuclear export of BCL6, and after that BCL6 degradation was mediated by ubiquitin/proteasome pathway. Real-time RT. PCR and siRNA targeting BCL6 revealed that BCL6 suppresses cyclin D2 expression. Our data indicate that BCL6 interacts with nuclear-translocated HB-EGF-CTF and that the nuclear export and degradation of BCL6 induces cyclin D2 upregulation. We performed immunohistochemical analyses of BCL6, HB-EGF and cyclin D2 in human gastric cancers. The inverse correlation between BCL6 and cyclin D2 was also found in HB-EGF-positive human gastric cancers. BCL6 degradation caused by the HB-EGF-CTF also might induce cyclin D2 expression in human gastric cancers. Inhibition of HB-EGF-CTF nuclear translocation and maintenance of BCL6 function are important for the regulation of gastric cancer progression.
- 炎症性サイトカイン・上皮増殖因子（EGFファミリー）を介した発癌メカニズムの解明
- 炎症から発がんのメカニズム
- 炎症性腸疾患治療の開発