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The role of NK4, a competitive antagonist for hepatocyte growth factor (HGF) and the Met receptor, in regulating the response of cholangiocarcinoma (CCA) cells to 5-Fluorouracil (5-FU)was investigated.We established cholangiocarcinoma cell line HuCC-T1, to produce abundant NK4 (Hu-NK4).5-FU metabolism, cell proliferation, cell cycle, apoptosis and intracellular signaling were examined.There was no difference in thymidylate synthase (TS), thymidine phosphorylase (TP)[1] and dihydro-pyrimidine dehydrogenase (DPD) mRNA level between mock-transfected control Hu-Em cells and Hu-NK4 cells, suggesting that NK4 expression does not change 5-FU metabolism.Moreover, Cell cycle analysis showed that 5-FU treatment caused a decrease in the proportion of G2 cells while NK4 gene expression had little effect on it.5-FU-induced apoptosis was significantly increased in Hu-NK4 compared to Hu-Em cell lines.Further investigation revealed that NK4 gene expression enhanced 5-FU-induced caspase-3 and caspase-9 activation.Furthermore, apoptosis of cells were associated with modulation of expression of Bcl-2 family members.In addition, western bloting analysis revealed that HGF induced phosphorylation of Met was mainly inhibited by NK4, not significantly influenced by the addition of 5-FU.Collectively, these results suggest that following 5-FU treatment in CCA cell lines, NK4 is involved in apoptosis induction through the intrinsic mitochondrial pathway.This indicates that NK4 may be an important mediator of 5-FU-induced cell death.Moreover,down-regulation of NK4 in response to 5-FU may represent an intrinsic mechanism of resistance to this anti-cancer drug.