u-0126 and Cholangiocarcinoma

P27 is a putative tumor suppressor when located in the nucleus and AKT is an inhibitor of P27 which promotes growth of cholangiocarcinoma. We hypothesized that AKT-dependent phosphorylation at the P27 nuclear localization sequence T157 leads to nuclear export of P27, and thus loss of its tumor suppressive function. This study investigated whether loss of cell cycle regulation in cholangiocarcinoma due to subcellular localization of P27.. Human cholangiocarcinoma cells were transfected with AKT. P27 was tagged with yellow fluorescence protein. Cell cycle progression was determined by flow cytometry. Migration and invasion of was measured by transwell assay.. Overexpression of wildtype P27 or P27-T157A in Mz-ChA-1 cells resulted in G1 arrest; expression of myr-AKT caused translocation of P27-YFP and endogenous P27 from the nucleus to the cytoplasm, leading to inhibition of P27-dependent G1 arrest; the AKT inhibitor and expression of dnAKT increased P27-YFP accumulation in the nucleus and promoted G1 arrest. In contrast, cells expressing YFP-P27-T157A or P27-YFP accumulated only in the nucleus. Co-expression of myr-AKT failed to induce P27-YFP translocation to the cytoplasm or inhibit G1 arrest. Overexpression of P27-T157A significantly increased migration and invasion.. Cholangiocarcinoma growth is associated with nuclear export of P27 that is due to AKT-mediated phosphorylation of P27 at T157.

Topics: Bile Duct Neoplasms; Butadienes; Cell Line, Tumor; Cell Movement; Cholangiocarcinoma; Chromones; Cyclin-Dependent Kinase Inhibitor p27; Enzyme Inhibitors; G1 Phase Cell Cycle Checkpoints; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Morpholines; Nitriles; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Translocation Systems; Protein Transport; Proto-Oncogene Proteins c-akt; Transfection

miR-21 induces epithelial-mesenchymal transition (EMT) of human cholangiocarcinoma (CCA) cells. However, the mechanism by which this occurs remains unclear. In the present study, high throughput platform was employed to detect the genes that are differential expressed in QBC939 cells transfected with a hsa-miR-21 antagomir or control vectors. The EMT-related Krüppel-like factor 4 (KLF4) gene was downregulated after miR-21 was knocked down. Overexpression of miR-21 upregulated KLF4, Akt, ERK and mesenchymal cell markers (N-cadherin and vimentin), downregulated the expression of epithelial cell marker E-cadherin and reduced cell migration and invasion. Immunohistochemistry showed that KLF4, pAkt and pERK were upregulated in tumor xenografts transfected with miR-21 mimics. Inhibitors of the PI3K-Akt and ERK1/2 pathways, LY294002 and U0126, significantly suppressed the EMT phenotype. The present data demonstrated that overexpression of miR-21, accompanied with KLF4, augmented the EMT via inactivation of Akt and ERK1/2 pathways. In conclusion, we have identified a novel mechanism that may be targeted in an attempt to relieve the malignant biological behavior of CCA cells.

Topics: Antigens, CD; Bile Duct Neoplasms; Butadienes; Cadherins; Cell Line, Tumor; Cell Movement; Cholangiocarcinoma; Chromones; Down-Regulation; Enzyme Inhibitors; Epithelial-Mesenchymal Transition; Humans; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; MAP Kinase Signaling System; MicroRNAs; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; RNA Interference; Vimentin

The Hedgehog (Hh) and pERK1/2 pathways participate in the tumorigenesis of various tissues, but there has been no report on the involvement of these two pathways in cholangiocarcinoma (CCA). The aim of this study was to evaluate the effects of the Hh pathway inhibitor, cyclopamine, and MEK inhibitor, U0126, as a single agent or in combination on CCA cell proliferation and survival.. Seven CCA cell lines were treated with cyclopamine and/or U0126, and cell proliferation was determined by WST-1 assay. The cell cycle was investigated by fluorescence-activated cell sorter analysis. The expression levels of several cell cycle-related genes were determined by western blot analyses.. Cyclopamine decreased cell proliferation and arrested the cell cycle at the G1 phase, while U0126 decreased the proliferation of CCA cells with KRAS mutation stronger than with wild-type KRAS. The combination of both inhibitors had an additive antiproliferative effect, particularly in cells with KRAS mutation, and induced caspase-dependent apoptosis in the CCA cells. The expression levels of cell cycle-related proteins that are targets of the two pathways, such as cyclin D1 and cyclin B1, were strongly decreased in some CCA cell lines after combined inhibitor treatment.. Our results suggest that the Hedgehog and ERK1/2 pathways are important for CCA cell proliferation, and simultaneous inhibition of the two pathways may lead to stronger decreases in cell growth and viability in a subset of CCA cases.

Topics: Apoptosis; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Butadienes; Cell Cycle; Cell Division; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cholangiocarcinoma; Cyclin D1; Drug Synergism; Enzyme Inhibitors; Flow Cytometry; G1 Phase; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Veratrum Alkaloids

To evaluate the expression of C-X-C motif chemokine receptor 4 (CXCR4) and its signaling cascades, which were previously identified as a key factor for cancer cell progression and metastasis, in cholangiocarcinoma cell lines.. The expression of CXCR4 and its signaling cascades were determined in the cholangiocarcinoma cell lines (RMCCA1 and KKU100) by Western blotting. The invasion assays and the detection of actin polymerization were tested in these cholangiocarcinoma cells treated with CXC chemokine ligand -12 (CXCL12).. Expression of CXCR4 was detected in both cholangiocarcinoma cell lines and activation of CXCR4 with CXCL12 triggered the signaling via the extracellular signal-regulated kinase-1/2 (ERK1/2) and phosphoinositide 3-kinase (PI3K) and induction of cholangiocarcinoma cell invasion, and displayed high levels of actin polymerization. Addition of CXCR4 inhibitor (AMD3100) abrogated CXCL12-induced phosphorylation of MEK1/2 and Akt in these cells. Moreover, treatment with MEK1/2 inhibitor (U0126) or PI3K inhibitor (LY294002) also attenuated the effect of CXCL12-induced cholangiocarcinoma cell invasion.. These results indicated that the activation of CXCR4 and its signaling pathways (MEK1/2 and Akt) are essential for CXCL12-induced cholangiocarcinoma cell invasion. This rises Implications on a potential role for the inhibition of CXCR4 or its signal cascades in the treatment of cholangiocarcinoma.

Topics: Benzylamines; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Butadienes; Cell Line, Tumor; Chemokine CXCL12; Chemokines, CXC; Cholangiocarcinoma; Chromones; Cyclams; Cytoskeleton; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Morpholines; Neoplasm Invasiveness; Nitriles; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Receptors, CXCR4; Signal Transduction

Hepatocyte growth factor receptor (c-Met) plays an important role in many functions of cancer cells. We examined the roles of c-Met and its downstream signaling molecules in cholangiocarcinoma cell lines RMCCA1 and HuCCA1.. The expression of c-Met and their signaling cascades were determined in RMCCA1 and HuCCA1 cholangiocarcinoma cell lines by Western blotting. Small interfering RNA (siRNA) specific for c-Met was used to suppress the expression of c-Met. The proliferation, migration and invasion assay were tested in these cholangiocarcinoma cells treated with hepatocyte growth factor (HGF).. Activation of c-Met with HGF triggered the signaling via the ERK cascade mediated by sequential phosphorylation of MEK1/2 and MAPK and induction of cholangiocarcinoma cell invasion. The expression of c-Met in cholangiocarcinoma cells was suppressed by treatment with small interfering RNA (siRNA) specific for c-Met, and resulted in decrease in phosphorylation of MEK1/2. Furthermore, treatment with siRNA specific for c-Met or MEK inhibitor U0126 inhibited cholangiocarcinoma cell invasion induced by HGF.. These results indicated that HGF and c-Met involved in the mechanism of cholangiocarcinoma cell invasion. It implies a potential role for the inhibition of c-Met in the treatment of cholangiocarcinoma.

Topics: Actin Cytoskeleton; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Butadienes; Cell Division; Cell Line, Tumor; Cell Movement; Cholangiocarcinoma; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genetic Therapy; Hepatocyte Growth Factor; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Nitriles; Phosphorylation; Proto-Oncogene Proteins c-met; RNA, Small Interfering