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.

胸苷酸合酶（TS）通过直接与5-FU代谢物5-Fluoro-Dump（Fdump）结合，在对5-氟尿嘧啶（5-FU）的反应中起着重要作用。 5-FU给药后TS表达水平的变化与六个人类胃腺癌细胞系中的5-FU反应性并行检查，以阐明5-FU敏感性的可变性来源。 MKN-1，SH-10-TC和MKN-74细胞比MKA-28，Kato III和MKN-45细胞更具耐药性。 Western-Clotting分析表明，这些细胞的5-FU敏感性与基础TS表达水平无关，而是与暴露于5-FU后的TS-FDUMP复合物的快速检测相关。 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.相比之下，导致钙蛋白酶激活的Ono-3403刺激了5-FU敏感细胞中TS-FDUMP复合物的下调。在5-FU敏感的细胞中，内源性钙蛋白酶抑制剂的表达水平高于5-FU的耐药细胞。 ZLLH提高了5-FU耐药细胞的5-FU敏感性，而Ono-3403降低了5-FU敏感细胞的敏感性。此外，siRNA敲低M-钙蛋白会增加5-FU抗性细胞的5-FU敏感性，而敲低calpastatin降低了5-FU敏感细胞的敏感性。这些结果表明，钙蛋白酶可能会通过快速降解TS-FDUMP复合物的快速降解，从而将人类胃癌细胞的化学敏感性降低为5-FU，这一发现被认为具有新颖的治疗意义。