title: Calpain regulates thymidylate synthase-5-fluoro-dUMP complex levels associated with response to 5-fluorouracil in gastric cancer cells.
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), 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.
authors.affiliation: Division of Gastroenterological Surgery, Chiba Cancer Center, Chiba, Japan. ynabeya@chiba-cc.jp
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Nabeya
authors.forename: Takao
authors.initials: T
authors.lastname: Suzuki
authors.forename: Aki
authors.initials: A
authors.lastname: Furuya
authors.forename: Naoki
authors.initials: N
authors.lastname: Koide
authors.forename: Motohiro
authors.initials: M
authors.lastname: Ohkoshi
authors.forename: Masaki
authors.initials: M
authors.lastname: Takiguchi
authors.forename: Takenori
authors.initials: T
authors.lastname: Ochiai
authors.forename: Hisahiro
authors.initials: H
authors.lastname: Matsubara
authors.forename: Takaki
authors.initials: T
authors.lastname: Hiwasa
mesh_terms: D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D002154:Calpain / Q000032:analysis / Q000502:physiology*; D045744:Cell Line, Tumor; D005468:Fluorodeoxyuridylate / Q000378:metabolism*; D005472:Fluorouracil / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D011480:Protease Inhibitors / Q000494:pharmacology; D013274:Stomach Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D013940:Thymidylate Synthase / Q000378:metabolism*
chemical_list: D000964:Antimetabolites, Antineoplastic; D011480:Protease Inhibitors; D005468:Fluorodeoxyuridylate; D013940:Thymidylate Synthase; D002154:Calpain; D005472:Fluorouracil