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EGFR ligands trafficking into the nucleus in gastric cancer cells

EGFR 配体转运至胃癌细胞的细胞核

基本信息

批准号：
16590614
负责人：
JOH Takashi
金额：
$ 2.3万
依托单位：
Nagoya City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590614/
关键词：
EGF HB-EGF C terminus ADAM trafficking into nucleu HB-EGF C 末端 HB-EGF PLZA c-myc C末端

项目摘要

We have recently shown that Helicobacter pylori as a causal of gastric cancer induces IL-8 production from gastric epithelia, IL-8 subsequently promotes cell migration and proliferation through metalloproteinase-cleavage proHB-EGF. HB-EGF C terminus, trafficking into nucleus, subsequently exerts some effects on regulation of cell growth in fibroblastoma cells. However, little is known about whether this mechanism exists in gastric epithelia. Thus, the presence of HB-EGF C terminus trafficking into nucleus was examined with gastric epithelia and gastric cancer tissues.(method)Gastric cell lines(KATO III, GCIY)were treated with a 6OnM of phorbor ester with or without ADAM inhibitor(KB-R7785). Cell lysates were probed with antibodies against phosphorylation after immunoprecipitation with anti-EGFR antibodies. Cells were transfected with vectors expressing YFP fused to C terminus of proHB-EGF. Cells were treated with a 60nM of phorbor ester with or without KB-R7785, and then were observed under a fluorescent microscopy. Excided stomach samples were immunohistochemically stained with anti-bodies against HB-EGF C-terminus.(result)In KATO III cells, EGFR phosphorylation by phorbor ester peaked within 15 min. That phosphorylation was abolished by KB-R7785. HB-EGF C terminus trafficked into nucleus from membrane with time. In KATO III and GCIY cells, HB-EGF C terminus was stained in the nuclei 60min after stimulation. In carcinomatous components of excided advanced gastric cancer, HB-EGF C terminus was partially stained in the nuclei.(conclusion)ADAM plays an important roles in the EGF signaling pathways, where ADAM cleaves extracellular domain of proHB-EGF and C terminus of proHB-EGF trafficks into the nucleus in gastric cancer cells. Thus, regulation of ADAM may be helpful for control of cell growth, migration and cell cycle, and leads to possible strategy against gastric cancer.

我们最近发现幽门螺杆菌作为胃癌的病因诱导胃上皮细胞产生IL-8，IL-8随后通过金属蛋白酶切割proHB-EGF促进细胞迁移和增殖。HB-EGF C末端进入细胞核，对成纤维细胞瘤细胞的生长调节起一定作用。然而，很少有人知道这一机制是否存在于胃上皮细胞。因此，用胃上皮和胃癌组织检测了HB-EGF C末端进入细胞核的存在。（方法）胃细胞系（KATO III，GCIY）用60 nM的佛波酯（有或没有ADAM抑制剂（KB-R7785））处理。在用抗EGFR抗体免疫沉淀后，用抗磷酸化抗体探测细胞裂解物。用表达与proHB-EGF的C末端融合的YFP的载体转染细胞。在有或没有KB-R7785的情况下，用60 nM佛波酯处理细胞，然后在荧光显微镜下观察。用抗HB-EGF C-末端的抗体对切除的胃样品进行化学染色。（结果）在KATO III细胞中，佛波酯对EGFR的磷酸化作用在15 min内达到峰值，KB-R7785可阻断其磷酸化作用。HB-EGF C末端随时间从细胞膜进入细胞核。在KATO Ⅲ和GCIY细胞中，HB-EGF C端在刺激后60 min染色于细胞核内。在进展期胃癌的癌组织中，HB-EGF C端部分染色于细胞核内。结论：在胃癌细胞中，ADAM在EGF信号通路中起重要作用，其作用是切割proHB-EGF的胞外区，使proHB-EGF的C端进入细胞核。因此，调控ADAM可能有助于控制细胞的生长、迁移和细胞周期，并为胃癌的治疗提供可能的策略。