2010 Fiscal Year Final Research Report
Mechanism of the regulation of thymidylate synthase levels associated with response to 5-fluorouracil in human gastric cancer cells
| Project/Area Number |
20591562
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Digestive surgery
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| Research Institution | Chiba University |
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Principal Investigator |
NABEYA Yoshihiro Chiba University, 医療局・消化器外科, 主任医長 (40322028)
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| Co-Investigator(Kenkyū-buntansha) |
HIWASA Takaki 千葉大学, 大学院・医学研究院遺伝子生化学, 准教授 (30260251)
MATSUBARA Hisahiro 千葉大学, 大学院・医学研究院・先端応用外科学, 教授 (20282486)
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| Project Period (FY) |
2008 – 2010
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| Keywords | 胃癌 / 化学療法 / 5-FU / チミジル酸合成酵素 / TS-FdUMP複合体 / カルパイン / カルパスタチン / ノックダウン |
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| Research Abstract |
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.
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Research Products
(6 results)
