Calpain regulates thymidylate synthase-5-fluoro-dUMP complex levels associated with response to 5-fluorouracil in gastric cancer cells
Calpain regulates thymidylate synthase-5-fluoro-dUMP complex levels associated with response to 5-fluorouracil in gastric cancer cells
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DOI：
10.1111/j.1349-7006.2011.01978.x
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发表时间：
2011-08-01
期刊：
影响因子：
5.7
通讯作者：
Hiwasa, Takaki
中科院分区：
文献类型：
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作者：
Nabeya, Yoshihiro;Suzuki, Takao;Hiwasa, Takaki
胸苷酸合酶 (TS) 通过直接与 5-FU 代谢物 5-氟-dUMP (FdUMP) 结合，在 5-氟尿嘧啶 (5-FU) 反应中发挥重要作用。在六种人胃腺癌细胞系中，同时检查了 5-FU 给药后 TS 表达水平的变化与 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的耐药性比MKN-28、KATO III和MKN-45细胞更强。蛋白质印迹分析显示，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 抗性细胞中，5-FU 暴露后早期非常低水平的 TS-FdUMP 复合物通过用钙蛋白酶抑制剂（例如苄氧羰基-亮氨酰-亮氨酸 (ZLLH)、苄氧羰基-亮氨酰-亮氨酸 (ZLLLH) 和 ALLN）预处理而升高，但其他蛋白酶抑制剂则不会升高。相比之下，引起钙蛋白酶激活的ONO-3403刺激5-FU敏感细胞中TS-FdUMP复合物的下调。 5-FU 敏感细胞中钙蛋白酶抑制剂（一种内源性钙蛋白酶抑制剂）的表达水平高于 5-FU 耐药细胞。 ZLLH 增加了 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，通过siRNA敲低m-钙蛋白酶可增加5-FU抗性细胞中的5-FU敏感性，而敲低钙蛋白酶抑制剂会降低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 TS-FdUMP 复合物的快速降解来降低人胃癌细胞对 5-FU 的化疗敏感性，这一发现被认为具有新的治疗意义。 （癌症科学 2011 年；102：1509-1515）
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), benzyloxycarbonyl-leucyl-leucyl-leucinal (ZLLLH) and ALLN, but not by other protease inhibitors. In contrast, 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. (Cancer Sci 2011; 102: 1509-1515)