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

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.

Thymineless death in F10-treated AML cells occurs via lipid raft depletion and Fas/FasL co-localization in the plasma membrane with activation of the extrinsic apoptotic pathway

“…FdU and 5-FU) [23] as well as folate-based TS inhibitors [24] demonstrated thymineless cell death was consequent to DNA damage. While both the intrinsic [25] and extrinsic [13] apoptotic pathways have been implicated previously in 5-FU-induced apoptosis, our studies indicate the extrinsic pathway initiates apoptosis in F10-treated AML cells. Our studies show the Fas death pathway is activated in AML cells in response to F10-treatment (Figures 2–5) which provides new insights into how this agent may be used clinically.…”

Cholangiocarcinoma: current knowledge and future perspectives consensus statement from the European Network for the Study of Cholangiocarcinoma (ENS-CCA)

“…NK4 is a fragment of hepatocyte growth factor (HGF) that blocks its binding to HGF-receptor (HGFR), which enhances 5-fluorouracil-induced activation of caspases 3 and 9 (REF. 306). Downregulation of NK4 in response to 5-fluorouracil treatment constitutes an intrinsic mechanism of CCA resistance to this drug 306 .…”

“…306). Downregulation of NK4 in response to 5-fluorouracil treatment constitutes an intrinsic mechanism of CCA resistance to this drug 306 . In addition, CCAs are frequently resistant to TRAIL-mediated apoptosis 307,308 .…”

SP/NK‐1R promotes gallbladder cancer cell proliferation and migration

“…As described by L. Miao et al 25 Total RNA was extracted from cells using a RNA Rapid Extraction Kit (BioTeke Corporation, Peking, China) and reversely transcribed using a reverse transcription kit (Thermo Fisher Scientific, MA, USA) according to the manufacturer's instructions. The subsequent Real-time PCR was performed using the SYBR ® Green Master Mix (Bio-Rad Laboratories, CA, USA).…”

“…As described by L. Miao et al 25 Total proteins were isolated from the cell lines. Protein lysates were run on SDS PAGE gels and then moved to PVDF membrane.…”