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Mechanism of the regulation of thymidylate synthase levels associated with response to 5-fluorouracil in human gastric cancer cells

人胃癌细胞中胸苷酸合酶水平与5-氟尿嘧啶反应相关的调节机制

基本信息

批准号：
20591562
负责人：
NABEYA Yoshihiro
金额：
$ 2.66万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2008
资助国家：
日本
起止时间：
2008 至 2010
项目状态：
已结题

项目摘要

Thymidylate synthase (TS) plays a major role in the response to 5-fluorouracil (5-FU) by binding directly to the 5-FU metabolite, 5-fluoro-dUMP (FdUMP). The change in the TS expression levels after 5-FU administration was examined in parallel to 5-FU responsiveness in six human gastric adenocarcinoma cell lines to elucidate the source of variability of 5-FU sensitivity. MKN-1, SH-10-TC and MKN-74 cells were more resistant to 5-FU than MKN-28, KATO III and MKN-45 cells. Western-blotting analysis revealed that the 5-FU sensitivity of these cells did not correlate with the basal TS expression levels but did correlate with rapid detection of the TS-FdUMP complex after exposure to 5-FU. In 5-FU-resistant cells, very low levels of the TS-FdUMP complex early after 5-FU exposure were elevated by pretreatment with calpain inhibitors such as benzyloxycarbonyl-leucyl-leucinal (ZLLH) and benzyloxycarbonyl-leucyl-leucyl-leucinal (ZLLLH), but not by other protease inhibitors. In contrastd, ONO-3403, which causes calpain activation, stimulated downregulation of the TS-FdUMP complex in 5-FU-sensitive cells. The expression levels of calpastatin, an endogenous calpain inhibitor, were higher in 5-FU-sensitive cells than in 5-FU-resistant cells. ZLLH increased the 5-FU sensitivity of 5-FU-resistant cells, whereas ONO-3403 decreased the sensitivity of 5-FU-sensitive cells. In addition, knockdown of m-calpain by siRNA increased the 5-FU sensitivity in 5-FU-resistant cells, while knockdown of calpastatin reduced the sensitivity in 5-FU-sensitive cells. These results suggest that calpain might reduce the chemosensitivity of human gastric cancer cells to 5-FU possibly by rapid degradation of the TS-FdUMP complex, a finding that is considered to have novel therapeutic implications.

胸苷酸合成酶（TS）通过直接结合5-氟尿嘧啶（5-FU）代谢产物5-氟-dUMP（FdUMP），在5-FU应答中起主要作用。在6个人胃腺癌细胞系中平行检查5-FU给药后TS表达水平的变化和5-FU反应性，以阐明5-FU敏感性变异的来源。MKN-1、SH-10-TC和MKN-74细胞对5-FU的耐药性高于MKN-28、KATO III和MKN-45细胞。Western-blotting分析显示，这些细胞的5-FU敏感性与基础TS表达水平无关，但与暴露于5-FU后TS-FdUMP复合物的快速检测相关。在5-FU耐药细胞中，5-FU暴露后早期极低水平的TS-FdUMP复合物通过钙蛋白酶抑制剂（如苄氧羰基-亮氨酰-亮氨醛（ZLLH）和苄氧羰基-亮氨酰-亮氨酰-亮氨醛（ZLLLH））预处理而升高，但其他蛋白酶抑制剂未升高。与此相反，ONO-3403，这导致钙蛋白酶激活，刺激下调TS-FdUMP复合物在5-FU敏感细胞。内源性钙蛋白酶抑制剂calpastatin在5-FU敏感细胞中的表达水平高于5-FU耐药细胞。ZLLH增加5-FU耐药细胞的5-FU敏感性，而ONO-3403降低5-FU敏感细胞的敏感性。此外，通过siRNA敲低m-calpain增加了5-FU耐药细胞中的5-FU敏感性，而敲低calpastatin降低了5-FU敏感细胞中的敏感性。这些结果表明，钙蛋白酶可能通过快速降解TS-FdUMP复合物降低人胃癌细胞对5-FU的化学敏感性，这一发现被认为具有新的治疗意义。