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Raf kinase inhibitor protein inhibits cholangiocarcinoma cell metastasis by downregulating matrix metalloproteinase 9 and upregulating tissue inhibitor of metalloproteinase 4 expression.

ABSTRACT: Cholangiocarcinoma cells originate in the biliary epithelium. The cells easily metastasize and cause relapse. The effect of Raf kinase inhibitor protein (RKIP) on the biological behavior of cholangiocarcinoma cells is not yet clear. In the present study, RKIP and cytokeratin 19 expression was detected in the extrahepatic tissues of cholangiocarcinoma patients by immunohistochemistry. RKIP small interfering (si)RNA or an RKIP-overexpressing adenoviral vector were used to infect the human cholangiocarcinoma RBE cell line. RKIP protein or gene expression was analyzed by western blotting or reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively. The cells were assayed for proliferation, apoptosis, invasion and migration. Matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 4 (TIMP-4) mRNA was assayed by RT-qPCR. RKIP expression was reduced in the extrahepatic cholangiocarcinoma tumor compared with the adjacent uninvolved peritumoral tissues. The current study revealed that RKIP expression was positively correlated with cell differentiation, but negatively correlated with lymph node or distant metastasis (P<0.05). RKIP siRNA treatment promoted RBE cell invasion, but RKIP overexpression prevented cell invasion. In the pDC316-siRNA recombinant vector group, the cells migrated more quickly compared with the siRNA-negative control group, and in the RKIP-expressing adenoviral vector group, the cells migrated more slowly compared with the adenoviral negative control group. RKIP inhibited the invasive and metastatic ability of the cholangiocarcinoma cell line, RBE, by downregulating MMP-9 and upregulating TIMP-4 mRNA expression. RKIP is negatively associated with cholangiocarcinoma distant metastasis and prevents cholangiocarcinoma cell metastasis through downregulating MMP-9 expression and upregulating TIMP-4 expression.

SUBMITTER: Ma J

PROVIDER: S-EPMC4246646 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Raf kinase inhibitor protein inhibits cholangiocarcinoma cell metastasis by downregulating matrix metalloproteinase 9 and upregulating tissue inhibitor of metalloproteinase 4 expression.

Ma Junji J Shi Junli J Zhao Dongqiang D Cheng Lijuan L Wang Wenbin W Li Fangfang F Jiang Xiaoyu X Jiang Huiqing H

Oncology letters 20141024 1

Cholangiocarcinoma cells originate in the biliary epithelium. The cells easily metastasize and cause relapse. The effect of Raf kinase inhibitor protein (RKIP) on the biological behavior of cholangiocarcinoma cells is not yet clear. In the present study, RKIP and cytokeratin 19 expression was detected in the extrahepatic tissues of cholangiocarcinoma patients by immunohistochemistry. RKIP small interfering (si)RNA or an RKIP-overexpressing adenoviral vector were used to infect the human cholangi ...[more]

PMID: 25435928

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Project description:The biological and molecular events that regulate the invasiveness of breast tumour cells need to be further revealed to develop effective therapies that stop breast cancer from expanding and metastasising.Human tissue samples of invasive breast cancer and normal breast, as well as breast cancer cell lines, were evaluated for protein kinase D (PKD) expression, to test if altered expression could serve as a marker for invasive breast cancer. We further utilised specific PKD1-shRNA and a system to inducibly-express PKD1 to analyse the role of PKD1 in the invasive behaviour of breast cancer cell lines in two-dimensional (2D) and three-dimensional (3D) culture. Invasive behaviour in breast cancer cell lines has been linked to matrix metalloproteinases (MMPs), so we also determined if PKD1 regulates the expression and activity of these enzymes.We found that the serine/threonine kinase, PKD1, is highly expressed in ductal epithelial cells of normal human breast tissue, but is reduced in its expression in more than 95% of all analysed samples of human invasive breast tumours. Additionally, PKD1 is not expressed in highly invasive breast cancer cell lines, whereas non-invasive or very low-invasive breast cancer cell lines express PKD1. Our results further implicate that in MDA-MB-231 cells PKD1 expression is blocked by epigenetic silencing via DNA methylation. The re-expression of constitutively-active PKD1 in MDA-MB-231 cells drastically reduced their ability to invade in 2D and 3D cell culture. Moreover, MCF-7 cells acquired the ability to invade in 2D and 3D cell culture when PKD1 expression was knocked-down by shRNA. PKD1 also regulated the expression of breast cancer cell MMPs, MMP-2, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, MMP-14 and MMP-15, providing a potential mechanism for PKD1 mediation of the invasive phenotype.Our results identify decreased expression of the PKD1 as a marker for invasive breast cancer. They further suggest that the loss of PKD1 expression increases the malignant potential of breast cancer cells. This may be due to the function of PKD1 as a negative regulator of MMP expression. Our data suggest re-expression of PKD1 as a potential therapeutic strategy.

Project description:BackgroundThe cleavage product of Raf-1 kinase inhibitor protein (RKIP), hippocampal cholinergic neurostimulating peptide (HCNP) is involved in the promotion of insulin secretion. Studies have shown that liraglutide can inhibit the progression of prediabetes. This study aims to investigate whether the above effects of liraglutide are related to RKIP and HCNP.MethodsInsulin-1 (INS-1) cells were divided into control group (CON), HCNP group, and HCNP + darifenacin group (H-DAR). The three groups were cultured with Roswell Park Memorial Institute (RPMI) 1640, synthetic HCNP (50 pg/mL) and RPMI 1640, and HCNP + RPMI 1640 + darifenacin respectively. Subsequently, twelve 12- to 14-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats were randomly divided into 2 groups: the placebo group (PBO) and the liraglutide treatment group (LIRA). Six Long Evans Tokushima Otsuka (LETO) rats were used as the control group (CON). The LIRA group was given liraglutide 200 µg/kg intraperitoneally twice a day. After 12 weeks, body weight, fasting blood glucose, 2 hours postprandial blood glucose, and insulin resistance index were recorded. Western blot was used to detect expression level of C-RKIP, N-RKIP, and extracellular signal-regulated kinase of phosphorylation (p-ERK). Real-time quantitative polymerase chain reaction (qRT-PCR) to detect pancreatic tissue choline acetyltransferase (ChAT) and M3 cholinergic receptor (M3R) gene expression levels.ResultsAt glucose concentrations of 5.6 and 16.7 mmol/L, the insulin content in the HCNP group was higher than that in the CON and H-DAR groups (all P<0.01). The body weight and fasting serum insulin (FINS) of rats in the PBO group were higher than those in the LIRA group and the CON group (P<0.01). The relative content of C-RKIP protein in the PBO group was higher than that in the LIRA and CON groups (P<0.01). The relative content of N-RKIP protein and p-ERK protein was lower than that in the LIRA and CON group (P<0.05 and P<0.01, respectively). ChAT and M3R gene expression levels in PBO group were lower than those in LIRA and CON group (P<0.01).ConclusionsLiraglutide promotes the production of HCNP, can increase ChAT activity, activate M3R, and further promote the secretion of insulin.

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Raf kinase inhibitor protein inhibits cholangiocarcinoma cell metastasis by downregulating matrix metalloproteinase 9 and upregulating tissue inhibitor of metalloproteinase 4 expression.

Publications

Raf kinase inhibitor protein inhibits cholangiocarcinoma cell metastasis by downregulating matrix metalloproteinase 9 and upregulating tissue inhibitor of metalloproteinase 4 expression.

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