NK4 regulates 5-fluorouracil sensitivity in cholangiocarcinoma cells by modulating the intrinsic apoptosis pathway

Ge,Xianxiu; Wang,Youli; Li,Quanpeng; Yu,Hong; Ji,Guozhong; Miao,Lin

doi:10.3892/or.2013.2427

NK4 regulates 5-fluorouracil sensitivity in cholangiocarcinoma cells by modulating the intrinsic apoptosis pathway

Authors:
- Xianxiu Ge
- Youli Wang
- Quanpeng Li
- Hong Yu
- Guozhong Ji
- Lin Miao
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Affiliations: Institute of Digestive Endoscopy and Medical Center for Digestive Diseases, Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210011, P.R. China, The Clinical Laboratory of Nanjing Hospital Affiliated to Nanjing Medical University, Nanjing, Jiangsu, P.R. China
Published online on: April 25, 2013 https://doi.org/10.3892/or.2013.2427
Pages: 448-454

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Abstract

The aim of the present study was to investigate the role of NK4, an antagonist for hepatocyte growth factor (HGF) and the Met receptor, in regulating the response of cholangiocarcinoma (CCA) cells to 5-fluorouracil (5-FU). We established the CCA cell line, HuCC-T1, to produce abundant NK4 (Hu-NK4). Cell proliferation, cell cycle distribution, apoptosis, 5-FU metabolism and intracellular signaling were examined. There were no significant differences in the mRNA levels of thymidylate synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase between the mock-transfected control Hu-Em cells and Hu-NK4 cells, suggesting that NK4 expression does not alter 5-FU metabolism. Moreover, cell cycle analysis showed that 5-FU treatment caused a decrease in the proportion of cells in the G2/M phase while NK4 gene expression had little effect on the cell cycle distribution. However, 5-FU-induced apoptosis was significantly increased in the Hu-NK4 cells when compared to that in the Hu-Em cells. Further investigation revealed that NK4 gene expression enhanced 5-FU-induced caspase-3 and caspase-9 activation, and that the apoptosis of cells was associated with modulation of expression of the Bcl-2 family members. Furthermore, western blot analysis revealed that both NK4 and 5-FU were inhibitors for HGF-induced phosphorylation of Met, but they may be independent factors. Collectively, these results suggest that following 5-FU treatment in CCA cell lines, NK4 was 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, downregulation of NK4 in response to 5-FU may represent an intrinsic mechanism of resistance to this anticancer drug.

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1	Marsh Rde W, Alonzo M, Bajaj S, et al: Comprehensive review of the diagnosis and treatment of biliary tract cancer 2012. Part I: diagnosis-clinical staging and pathology. J Surg Oncol. 106:332–338. 2012.PubMed/NCBI
2	Blechacz B, Komuta M, Roskams T and Gores GJ: Clinical diagnosis and staging of cholangiocarcinoma. Nat Rev Gastroenterol Hepatol. 8:512–522. 2011. View Article : Google Scholar : PubMed/NCBI
3	Romiti A, D’Antonio C, Zullo A, et al: Chemotherapy for the biliary tract cancers: moving toward improved survival time. J Gastrointest Cancer. 43:396–404. 2012. View Article : Google Scholar : PubMed/NCBI
4	Nakamura T, Nishizawa T, Hagiya M, et al: Molecular cloning and expression of human hepatocyte growth factor. Nature. 342:440–443. 1989. View Article : Google Scholar : PubMed/NCBI
5	Date K, Matsumoto K, Shimura H, Tanaka M and Nakamura T: HGF/NK4 is a specific antagonist for pleiotrophic actions of hepatocyte growth factor. FEBS Lett. 420:1–6. 1997. View Article : Google Scholar : PubMed/NCBI
6	Comoglio PM, Giordano S and Trusolino L: Drug development of MET inhibitors: targeting oncogene addiction and expedience. Nat Rev Drug Discov. 7:504–516. 2008. View Article : Google Scholar : PubMed/NCBI
7	Sattler M and Salgia R: The MET axis as a therapeutic target. Update Cancer Ther. 3:109–118. 2009. View Article : Google Scholar : PubMed/NCBI
8	Engelman JA, Zejnullahu K, Mitsudomi T, et al: MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science. 316:1039–1043. 2007. View Article : Google Scholar : PubMed/NCBI
9	Ohuchida K, Mizumoto K, Murakami M, et al: Radiation to stromal fibroblasts increases invasiveness of pancreatic cancer cells through tumor-stromal interactions. Cancer Res. 64:3215–3222. 2004. View Article : Google Scholar
10	Yano S, Wang W, Li Q, et al: Hepatocyte growth factor induces gefitinib resistance of lung adenocarcinoma with epidermal growth factor receptor-activating mutations. Cancer Res. 68:9479–9487. 2008. View Article : Google Scholar : PubMed/NCBI
11	Onimaru M, Ohuchida K, Egami T, et al: Gemcitabine synergistically enhances the effect of adenovirus gene therapy through activation of the CMV promoter in pancreatic cancer cells. Cancer Gene Ther. 17:541–549. 2010. View Article : Google Scholar : PubMed/NCBI
12	Taiyoh H, Kubota T, Fujiwara H, et al: NK4 gene expression enhances 5-fluorouracil-induced apoptosis of murine colon cancer cells. Anticancer Res. 31:2217–2224. 2011.PubMed/NCBI
13	Crea F, Nobili S, Paolicchi E, et al: Epigenetics and chemoresistance in colorectal cancer: an opportunity for treatment tailoring and novel therapeutic strategies. Drug Resist Updat. 14:280–296. 2011. View Article : Google Scholar : PubMed/NCBI
14	Banerjee D, Mayer-Kuckuk P, Capiaux G, Budak-Alpdogan T, Gorlick R and Bertino JR: Novel aspects of resistance to drugs targeted to dihydrofolate reductase and thymidylate synthase. Biochim Biophys Acta. 1587:164–173. 2002. View Article : Google Scholar : PubMed/NCBI
15	Longley DB, Allen WL and Johnston PG: Drug resistance, predictive markers and pharmacogenomics in colorectal cancer. Biochim Biophys Acta. 1766:184–196. 2006.
16	Leichman CG, Lenz HJ, Leichman L, et al: Quantitation of intratumoral thymidylate synthase expression predicts for disseminated colorectal cancer response and resistance to protracted-infusion fluorouracil and weekly leucovorin. J Clin Oncol. 15:3223–3229. 1997.
17	Metzger R, Danenberg K, Leichman CG, et al: High basal level gene expression of thymidine phosphorylase (platelet-derived endothelial cell growth factor) in colorectal tumors is associated with nonresponse to 5-fluorouracil. Clin Cancer Res. 4:2371–2376. 1998.
18	Nishi M, Shimada M, Utsunomiya T, et al: Role of dihydropyrimidine dehydrogenase and thymidylate synthase expression in immunohistochemistry of intrahepatic cholangiocarcinoma. Hepatol Res. 41:64–70. 2011. View Article : Google Scholar : PubMed/NCBI
19	Morine Y, Shimada M, Utsunomiya T, et al: Role of thymidylate synthase and dihydropyrimidine dehydrogenase mRNA in intrahepatic cholangiocarcinoma. Surg Today. 42:135–140. 2012. View Article : Google Scholar : PubMed/NCBI
20	Wilson TR, Johnston PG and Longley DB: Anti-apoptotic mechanisms of drug resistance in cancer. Curr Cancer Drug Targets. 9:307–319. 2009. View Article : Google Scholar : PubMed/NCBI
21	Huang L, Wong YP, Cai YJ, Lung I, Leung CS and Burd A: Low-dose 5-fluorouracil induces cell cycle G2 arrest and apoptosis in keloid fibroblasts. Br J Dermatol. 163:1181–1185. 2010. View Article : Google Scholar : PubMed/NCBI
22	Okamoto S, Sakai M, Uchida J and Saito H: 5-Fluorouracil induces apoptotic cell death with G2 phase arrest in human breast cancer grafted in nude mice. Anticancer Res. 16:2699–2704. 1996.PubMed/NCBI
23	Naus PJ, Henson R, Bleeker G, Wehbe H, Meng F and Patel T: Tannic acid synergizes the cytotoxicity of chemotherapeutic drugs in human cholangiocarcinoma by modulating drug efflux pathways. J Hepatol. 46:222–229. 2007. View Article : Google Scholar : PubMed/NCBI
24	Ikeguchi M, Hirooka Y, Makino M and Kaibara N: Dihydropyrimidine dehydrogenase activity of cancerous and non-cancerous tissues in liver and large intestine. Oncol Rep. 8:621–625. 2001.PubMed/NCBI
25	Salonga D, Danenberg KD, Johnson M, et al: Colorectal tumors responding to 5-fluorouracil have low gene expression levels of dihydropyrimidine dehydrogenase, thymidylate synthase, and thymidine phosphorylase. Clin Cancer Res. 6:1322–1327. 2000.
26	Namwat N, Amimanan P, Loilome W, et al: Characterization of 5-fluorouracil-resistant cholangiocarcinoma cell lines. Chemotherapy. 54:343–351. 2008. View Article : Google Scholar : PubMed/NCBI
27	Danial NN: BCL-2 family proteins: critical checkpoints of apoptotic cell death. Clin Cancer Res. 13:7254–7263. 2007. View Article : Google Scholar : PubMed/NCBI
28	Zhang L, Yu J, Park BH, Kinzler KW and Vogelstein B: Role of BAX in the apoptotic response to anticancer agents. Science. 290:989–992. 2000. View Article : Google Scholar : PubMed/NCBI
29	Heiser D, Labi V, Erlacher M and Villunger A: The Bcl-2 protein family and its role in the development of neoplastic disease. Exp Gerontol. 39:1125–1135. 2004. View Article : Google Scholar : PubMed/NCBI
30	Oltvai ZN, Milliman CL and Korsmeyer SJ: Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 74:609–619. 1993. View Article : Google Scholar : PubMed/NCBI
31	Yin XM, Oltvai ZN and Korsmeyer SJ: BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature. 369:321–323. 1994. View Article : Google Scholar : PubMed/NCBI
32	Zou H, Li Y, Liu X and Wang X: An APAF-1. cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem. 274:11549–11556. 1999. View Article : Google Scholar : PubMed/NCBI
33	Nachmias B, Ashhab Y and Ben-Yehuda D: The inhibitor of apoptosis protein family (IAPs): an emerging therapeutic target in cancer. Semin Cancer Biol. 14:231–243. 2004. View Article : Google Scholar : PubMed/NCBI