epidermal-growth-factor and Cholangiocarcinoma

Berberine (BBR), a natural isoquinoline alkaloid, possesses diverse pharmacological properties and anti-cancer effects that have been demonstrated in many in vitro and in vivo studies. In this study, the inhibitory effects and molecular mechanism of low dose BBR on EMT-induced cell migration, and invasion capability of cholangiocarcinoma (CCA) cell lines were demonstrated.. The commercially available BBR chloride powder with purity ≥ 95% was used in this study. Effects of BBR on cell growth of two human CCA cell lines, KKU-213A and KKU-213B were measured using MTT assay. The progressive phenotypes-cell adhesion, migration, and invasion were evaluated using cell adhesion, wound healing, and Boyden chamber assays. Molecular docking analysis was performed to assess the possible binding mode of BBR against EGFR, Erk, STAT3 and Akt. The effects of BBR on the activations of EGF/EGFR and its downstream effectors were demonstrated using Western blotting.. BBR inhibited growth of CCA cells in a dose dependent manner. At sub-cytotoxic dose, BBR significantly inhibited cell adhesion, migration, invasion and decreased expression of vimentin, slug, and VEGFA of both CCA cell lines. Molecular docking suggested the simultaneous inhibitory activity of BBR on EGFR, Erk, STAT3 and Akt. The Western blot analyses revealed that upon the EGF/EGFR activation, BBR considerably attenuated the activations of EGFR, Erk, STAT3 and Akt.. Low dose of BBR suppresses EMT and thus aggressiveness of CCA cells, in part by its multi-kinase inhibitor property on EGFR and its downstream pathways.  BBR might be beneficial for therapy of human CCA.

Topics: Antineoplastic Agents; Berberine; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cholangiocarcinoma; Epidermal Growth Factor; ErbB Receptors; Humans; Molecular Docking Simulation; Proto-Oncogene Proteins c-akt

Aberrant glycosylation is recognized as a cancer hallmark that is associated with cancer development and progression. In this study, the clinical relevance and significance of terminal fucose (TFG), by fucosyltransferase-1 (FUT1) in carcinogenesis and progression of cholangiocarcinoma (CCA) were demonstrated. TFG expression in human and hamster CCA tissues were determined using Ulex europaeus agglutinin-I (UEA-I) histochemistry. Normal bile ducts rarely expressed TFG while 47% of CCA human tissues had high TFG expression and was correlated with shorter survival of patients. In the CCA-hamster model, TFG was elevated in hyperproliferative bile ducts and gradually increased until CCA was developed. This evidence indicates the involvement of TFG in carcinogenesis and progression of CCA. The mechanistic insight was performed in 2 CCA cell lines. Suppression of TFG expression using siFUT1 or neutralizing the surface TFG with UEA-I significantly reduced migration, invasion and adhesion of CCA cells in correlation with the reduction of Akt/Erk signaling and epithelial-mesenchymal transition. A short pulse of EGF could stimulate Akt/Erk signaling via activation of EGF-EGFR cascade, however, decreasing TFG using siFUT1 or UEA-I treatment reduced the EGF-EGFR activation and Akt/Erk signaling. This evidence provides important insight into the relevant role and molecular mechanism of TFG in progression of CCA.

Topics: Adult; Aged; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cholangiocarcinoma; Disease Progression; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Fucose; Fucosyltransferases; Galactoside 2-alpha-L-fucosyltransferase; Humans; Male; MAP Kinase Signaling System; Middle Aged; Proto-Oncogene Proteins c-akt; Signal Transduction; Transcriptome

Seaweeds have a long history of use in Asian countries as functional foods, medicinal herbs, and the treatment of cancer. Polysaccharides from various seaweeds have shown anti-tumor activity. Cholangiocarcinoma (CCA), often with metastatic disease, is highly prevalent in Thailand as a consequence of liver fluke infection. Recently, we extracted sulfated galactans (SG) from Gracilaria fisheri (G. fisheri), a south east Asian seaweed, and found it exhibited anti-proliferation effect on CCA cells.. In the present study, we evaluated the anti-migration activity of SG on CCA cells and its underlined mechanism.. CCA cells were treated with SG alone or drugs targeting to epidermal growth factor (EGF) receptor (EGFR) or pretreated with SG prior to incubation with EGF. Anti-migration activity was determined using a scratch wound-healing assay and zymography. Immunofluorescence staining and western blotting were used to investigate EGFR signaling mediators.. Under basal condition, SG reduced the migration rate of CCA, which was correlated with a decrease in the active-form of matrix metalloproteinases-9. SG decreased expression of phosphorylated focal adhesion kinase (FAK), but increased expression of E-cadherin to promote cells stasis. Moreover, phosphorylation of EGFR and extracellular signal-regulated kinases (ERK), known to stimulate growth of cancer cells, was blocked in a comparable way to EGFR inhibitors Cetuximab and Erlotinib. Pretreatment cells with SG attenuated EGF induced phosphorylation of EGFR, ERK and FAK.. This study reveals that SG from G. fisheri retards migration of CCA cells, and its mechanism of inhibition is mediated, to some extent, by inhibitory effects on MAPK/ERK signal transduction pathway. Our findings suggest that there may be a therapeutic potential of SG in CCA treatment.

Topics: Antigens, CD; Antineoplastic Agents, Phytogenic; Bile Duct Neoplasms; Cadherins; Cell Movement; Cholangiocarcinoma; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Focal Adhesion Kinase 1; Galactans; Gracilaria; Humans; Phosphorylation; Seaweed; Signal Transduction; Tumor Cells, Cultured

To evaluate the expression status of S-phase kinase-associated protein 2 (Skp2)/cyclin-dependent kinases regulatory subunit 1 (Cks1) and p27(kip1), and assess the prognostic significance of Skp2/Cks1 expression with p27(kip1) in patients with extrahepatic cholangiocarcinoma.. Seventy-six patients who underwent curative resection for histologically confirmed extrahepatic cholangiocarcinoma at our institution from December 1994 to March 2008 were enrolled. Immunohistochemical staining for Skp2, Cks1, p27(kip1), and Ki67, along with other relevant molecular biologic experiments, were performed.. By Cox regression analyses, advanced age (> 65 years), advanced AJCC tumor stage, poorly differentiated histology, and higher immunostaining intensity of Skp2 were identified as independent prognostic factors in patients with extrahepatic cholangiocarcinoma. Exogenous epidermal growth factor (EGF, especially 0.1-10 ng/mL) significantly increased the proliferation indices by MTT assay and the mRNA levels of Skp2/Cks1 and p27(kip1) in SNU-1196, SNU-1079, and SNU-245 cells. The protein levels of Skp2/Cks1 (from nuclear lysates) and p27(kip1) (from cytosolic lysate) were also significantly increased in these cells. There were significant reductions in the protein levels of Skp2/Cks1 and p27(kip1) (from nuclear lysate) after the treatment of LY294002. By chromatin immunoprecipitation assay, we found that E2F1 transcription factor directly binds to the promoter site of Skp2.. Higher immunostaining intensity of Skp2/Cks1 was an independent prognostic factor for patients with extrahepatic cholangiocarcinoma. EGF upregulates the mRNA and protein levels of Skp2/Cks1 and p27(kip1) via the PI3K/Akt pathway and direct binding of E2F1 transcription factor with the Skp2 promoter.

Topics: Aged; Bile Duct Neoplasms; Bile Ducts, Extrahepatic; Binding Sites; CDC2-CDC28 Kinases; Cell Line, Tumor; Chi-Square Distribution; Cholangiocarcinoma; Cyclin-Dependent Kinase Inhibitor p27; E2F1 Transcription Factor; Epidermal Growth Factor; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Logistic Models; Male; Middle Aged; Multivariate Analysis; Promoter Regions, Genetic; Proportional Hazards Models; Risk Factors; RNA, Messenger; S-Phase Kinase-Associated Proteins; Signal Transduction; Time Factors; Up-Regulation

Epithelial-mesenchymal transition (EMT) is a cellular process involved in cancer progression. The first step of EMT consists in the disruption of E-cadherin-mediated adherens junctions. Cholangiocarcinoma (CCA), a cancer with a poor prognosis due to local invasion and metastasis, displays EMT features. EGFR, a receptor tyrosine kinase, plays a major role in CCA progression. The aim of the study was to determine if EMT is induced by EGFR in CCA cells.. In vivo, the expression of E-cadherin was analysed in CCA tumours of 100 patients and correlated with pathological features and EGFR expression, and in a xenograft model in mice treated with gefitinib, an inhibitor of EGFR. In vitro, the regulation of EMT by EGFR was investigated in CCA cell lines.. In human CCA, a cytoplasmic localization of E-cadherin occurred in 50% of the tumours was associated with the peripheral type of CCA, tumour size, the presence of satellite nodules and EGFR overexpression. In xenografted tumours, E-cadherin displayed a cytoplasmic pattern whereas the treatment of mice with gefitinib restored the membranous expression of E-cadherin. In vitro, EGF induced scattering of CCA cells that resulted from the disruption of adherens junctions. Internalization and decreased expression of E-cadherin, as well as nuclear translocation of β-catenin, were observed in EGF-treated CCA cells. In these cells, EMT-transcription factors (i.e., Slug and Zeb-1) and mesenchymal markers (i.e., N-cadherin and α-SMA) were induced, favoring cell invasiveness through cytoskeleton remodeling. All these effects were inhibited by gefitinib.. The EGF/EGFR axis triggers EMT in CCA cells highlighting the key role of this pathway in CCA progression.

Topics: Animals; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cadherins; Cell Line, Tumor; Cell Movement; Cholangiocarcinoma; Disease Progression; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Humans; Mice; Neoplasm Invasiveness

MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3'-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376c functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC.

Topics: Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cell Line, Tumor; Cell Movement; Cholangiocarcinoma; DNA Methylation; Down-Regulation; Epidermal Growth Factor; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; GRB2 Adaptor Protein; Humans; Liver Neoplasms; MicroRNAs; Neoplasm Proteins; Proteomics; Reproducibility of Results

Since cholangiocarcinoma has a poor prognosis, several epidermal growth factor receptor (EGFR)-targeted therapies with antibody or small molecule inhibitor treatment have been proposed. However, their effect remains limited. The present study sought to understand the molecular genetic characteristics of cholangiocarcinoma related to EGFR, with emphasis on its degradation and recycling.. We evaluated EGFR expression and colocalization by immunoblotting and immunofluorescence, cell surface EGFR expression by fluorescence-activated cell sorting (FACS), and EGFR ubiquitination and protein binding by immunoprecipitation in the human cholangiocarcinoma RBE and immortalized cholangiocyte MMNK-1 cell lines. Monensin treatment and Rab11a depletion by siRNA were adopted for inhibition of EGFR recycling.. Upon stimulation with EGF, ligand-induced EGFR degradation was impaired and the expression of phospho-tyrosine 1068 and phospho-p44/42 MAPK was sustained in RBE cells as compared with MMNK-1 cells. In RBE cells, the process of EGFR sorting for lysosomal degradation was blocked at the early endosome stage, and non-degradated EGFR was recycled to the cell surface. A disrupted association between EGFR and the E3 ubiquitin ligase c-Cbl, as well as hypo-phosphorylation of EGFR at tyrosine 1045 (Tyr1045), were also observed in RBE cells.. In RBE cells, up-regulation of EGFR Tyr1045 phosphorylation is a potentially useful molecular alteration in EGFR-targeted therapy. The combination of molecular-targeted therapy determined by the characteristics of individual EGFR phosphorylation events and EGFR recycling inhibition show promise in future treatments of cholangiocarcinoma.

Topics: Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cell Line, Tumor; Cholangiocarcinoma; Endosomes; Epidermal Growth Factor; ErbB Receptors; Humans; Phosphorylation; Protein Binding; Protein Transport; Proteolysis; Proto-Oncogene Proteins c-cbl; Signal Transduction; Tyrosine; Ubiquitination

Topics: Animals; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cholangiocarcinoma; Cricetinae; Epidermal Growth Factor; ErbB Receptors; Immunohistochemistry; Transforming Growth Factor alpha

Cholangiocarcinoma is a highly malignant, usually fatal cancer with limited therapeutic options. Receptor tyrosine kinases contribute to the development and progression of this cancer. The relatively selective tyrosine kinase inhibitor imatinib mesylate (STI-571 or Gleevec(R)) has recently been licensed. However, the ability of this drug to inhibit signal transduction and induce apoptosis in human cholangiocarcinoma cells is incompletely studied. Thus, our goal was to examine the ability of STI-571 to induce apoptosis in KMCH-1 cells, a human cholangiocarcinoma cell line.. Apoptosis was assessed morphologically and also biochemically by measuring caspase activity and the mitochondrial membrane potential. STI-571 induced apoptosis and inhibited growth of KMCH-1 cells in a time- and concentration-dependent manner. The induction of apoptosis was accompanied by mitochondrial depolarization followed by a 4.5-fold increase in caspase activation and was abrogated by the pancaspase inhibitor z-VAD(OMe)-fmk. Interestingly, cholangiocarcinoma cells do not express detectable PDGFR, c-Abl or c-Kit, which are protein kinases known to be directly inhibited by STI-571. However, a significant decrease in epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK) phosphorylation was observed following treatment with STI-571. This decrease in EGFR and FAK phosphorylation was associated with a reduction in Akt activity resulting in loss of Mcl-1, a potent anti-apoptotic Bcl-2 family protein.. These results indicate that STI-571 induces caspase-dependent apoptosis in a human cholangiocarcinoma cell line and suggest that STI-571 might warrant further investigation as a possible agent for treatment of human cholangiocarcinoma.

Topics: Analysis of Variance; Apoptosis; Benzamides; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Caspases; Cholangiocarcinoma; Epidermal Growth Factor; Female; Humans; Imatinib Mesylate; Immunoblotting; Male; Mitochondria; Piperazines; Probability; Pyrimidines; Sensitivity and Specificity; Tumor Cells, Cultured