OBJECTIVETo construct a small interfering RNA (siRNA) vector targeting p63 and observe its effect on the proliferation and invasiveness of human cholangiocarcinoma cells in vitro.

METHODSReal-time PCR was used to examine the expression of p63 in human cholangiocarcinoma QBC939 cells. The recombinant lentivirus shRNA-p63 vector was constructed and transfected into QBC939 cells via Lipofectamine 2000 to establish a cholangiocarcinoma cell line with stable expression of siRNA-p63. The interfering efficiency of the siRNA targeting p63 was assessed using Western blotting. MTT and soft agar colony formation assays were used to evaluate the changes in the cell proliferation, and Boyden test was employed to observe the cell invasiveness after the transfection.

RESULTSQBC939 cells showed a high expression of p63. The recombinant lentivirus shRNA-p63 vector was successfully constructed as verified by sequencing. Transfection with the vector significantly suppressed the proliferation and invasiveness of QBC939 cells.

CONCLUSIONDown-regulation of p63 can inhibit the proliferation and invasiveness of human cholangiocarcinoma QBC939 cells in vitro.

Bile Duct Neoplasms ; genetics ; pathology ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cholangiocarcinoma ; genetics ; pathology ; Humans ; Membrane Proteins ; genetics ; metabolism ; Neoplasm Invasiveness ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo study the effects of anti-oncogene WWOX on cell growth of cholangiocarcinoma.

METHODSThe expression of WWOX protein was detected with immunohistochemical method-SP in 54 patients with cholangiocarcinoma from July 2005 to May 2010 and 12 samples of normal bile duct tissues. The recombinant WWOX eukaryotic expression plasmid was introduced into RBE cells by liposome-mediated transfection and positive cell clones were selected and amplified. The mRNA and protein expressions in RBE cells stably transfected with WWOX were investigated by quantitative RT-PCR and Western Blot before and after transfection. Cell proliferation was tested by MTT, cell apoptosis was assessed by FCM, the alteration of mitochondria membrane potential (ΔΨm) was detected by JC-1 staining method, cell invasion was determined by Transwell chamber assay. The expression change of bcl-2, bax, FasL, caspase-3 mRNA and protein was detected by quantitative RT-PCR and Western Blot.

RESULTSThe expression of WWOX protein was significantly lower in cholangiocarcinoma than that in normal bile duct tissues and loss of WWOX protein expression was found in 40.7% of cholangiocarcinoma specimens (P < 0.05). RBE cells with stable transfection of WWOX were established. Quantitative RT-PCR showed that the expression of WWOX mRNA was significantly enhanced and Western Blot demonstrated that WWOX protein expression was markedly increased. MTT showed that WWOX gene transfection significantly decreased the proliferation of RBE cells (P < 0.05). FCM analysis showed that the apoptosis rate after transfection was significantly promoted [(1.1 ± 0.6)% vs. (1.7 ± 0.5)% vs. (35.2 ± 4.4)%, P < 0.01], JC-1 staining method indicated that the experimental group was loss of ΔΨm [(12.6 ± 1.9)% vs. (13.6 ± 1.8)% vs. (48.7 ± 2.9)%, P < 0.01], transwell chamber assay showed that the number of transfected cells that passed the transwell membrane was significantly less than those of control groups (77 ± 6 vs. 72 ± 8 vs. 48 ± 6, P < 0.01). Quantitative RT-PCR and Western blotting showed that the expression of bcl-2 mRNA and protein was markedly decreased and the expression of bax, caspase-3 were significantly increased. There was no significant change in the expression of FasL.

CONCLUSIONWWOX exerts its antitumor effect against proliferation through inducing cell apoptosis in cholangiocarcinoma.

Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Cholangiocarcinoma ; genetics ; metabolism ; pathology ; Genetic Vectors ; Humans ; Oxidoreductases ; genetics ; metabolism ; Plasmids ; genetics ; Transfection ; Tumor Suppressor Proteins ; genetics ; metabolism ; WW Domain-Containing Oxidoreductase

OBJECTIVETo explore whether hepatitis C virus core protein (HCV C) regulates the expression of NFAT1 to participate in the progression and malignant biological behavior of intrahepatic cholangiocarcinoma cells.

METHODSThe recombinant plasmid pEGFP-N(3)-HCV C and the empty vector pEGFP-N(3) were cotransfected with enhanced green fluorescent protein (EGFP) into RBE cells using liposome. Real-time PCR and Western blotting were used to examine the expression of NFAT1 mRNA and protein in the transfected RBE cells. MTT assay was used to evaluate the changes in the cell proliferation, and the cell cycle changes were analyzed by flow cytometry.

RESULTSHCV C transfection significantly enhanced the expressions of NFAT1 mRNA and protein in RBE cells (P<0.05) and promoted the progression of cell cycle into G(2)/M phase to accelerate the cell proliferation.

CONCLUSIONTransfection with HCV C gene up-regulates NFAT1 expression and promotes the cell cycle progression and proliferation of intrahepatic cholangiocarcinoma cells, suggesting the involvement of HCV C in the progression of intrahepatic cholangiocarcinoma.

Bile Duct Neoplasms ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cholangiocarcinoma ; pathology ; Gene Expression ; Humans ; NFATC Transcription Factors ; genetics ; Plasmids ; Transfection ; Viral Core Proteins ; genetics

OBJECTIVETo compare the immunoprofiles of intrahepatic cholangiocarcinoma and metastatic colorectal adenocarcinoma for mucin glycoproteins (including MUC1, MUC2, MUC5AC and MUC6) and cytokeratins (including CK7, CK19 and CK20), and to assess their diagnostic value.

METHODSOne hundred cases of intrahepatic cholangiocarcinoma and 21 cases of metastatic colorectal adenocarcinoma were enrolled into the study. Immunohistochemical study for MUC1, MUC2, MUC5AC, MUC6, CK7, CK19 and CK20 was carried out in all cases by EnVision method.

RESULTSIn intrahepatic cholangiocarcinoma, the expression rates of MUC1, MUC2, MUC5AC and MUC6 were 61.0%, 2.0%, 22.0% and 8.0% respectively, as compared to 57.1%, 47.6%, 19.0% and 23.8% respectively in metastatic colorectal adenocarcinoma. On the other hand, the expression rates of CK7, CK19 and CK20 in intrahepatic cholangiocarcinoma were 73.0%, 53.0% and 15.0% respectively, in contrast to 14.3%, 90.5% and 85.7% respectively in metastatic colorectal adenocarcinoma. The difference in expressions of MUC2, MUC6, CK7 and CK20 carried statistical significance.

CONCLUSIONSThe immunoprofile for mucin glycoproteins and cytokeratins provides important clues in distinguishing between intrahepatic cholangiocarcinoma and metastatic colorectal adenocarcinoma to liver. The immunophenotype of MUC2-/MUC6-/CK7+/CK20- indicates the diagnosis of intrahepatic cholangiocarcinoma, while MUC2+/MUC6+/CK7-/CK20+ suggests the possibility of metastatic colorectal adenocarcinoma.

Adenocarcinoma ; metabolism ; pathology ; Aged ; Bile Duct Neoplasms ; genetics ; metabolism ; pathology ; Bile Ducts, Intrahepatic ; pathology ; Biomarkers, Tumor ; analysis ; Cholangiocarcinoma ; genetics ; metabolism ; pathology ; Colorectal Neoplasms ; metabolism ; pathology ; Female ; Glycoproteins ; metabolism ; Humans ; Keratins ; metabolism ; Male ; Middle Aged ; Mucins ; metabolism ; Neoplasm Staging ; classification

Bile Duct Neoplasms ; genetics ; pathology ; Bile Ducts, Extrahepatic ; Cholangiocarcinoma ; genetics ; pathology ; Female ; GTP-Binding Protein gamma Subunits ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Middle Aged ; Neoplasm Staging ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo explore the possibility of using melanoma antigen (MAGE)-1 and MAGE-3 gene encoding proteins as an index of potential target for immunotherapy in intrahepatic cholangiocarcinoma (IHCC) patients.

METHODSThe expressions of MAGE-1 and MAGE-3 genes in tumor tissues and tumor adjacent non-IHCC liver tissues were examined by RT-PCR method. The relationship between positive expression rates of MAGE-1 and MAGE-3 genes and clinical data including sex, age, tumor diameters, tumor envelope, tumor nodules number, and hepatitis B virus surface antigen were determined.

RESULTSThe positive expression rates of MAGE-1 (35%) and MAGE-3 genes (45%) were significantly higher in the tumor tissues than in tumor adjacent tissues (0) (P<0.01). The positive expression rates of MAGE-1 and MAGE-3 genes had no relationship with the clinical data (P >0.05), except the morphology of tumor (P <0.05).

CONCLUSIONThe high expression rates of MAGE-1 and MAGE-3 genes in IHCC suggests the MAGE-1 and MAGE-3 gene may be a target for immunotherapy in IHCC patients.

Adult ; Aged ; Antigens, Neoplasm ; genetics ; Bile Duct Neoplasms ; genetics ; Bile Ducts, Intrahepatic ; pathology ; Cholangiocarcinoma ; genetics ; Female ; Humans ; In Vitro Techniques ; Liver Neoplasms ; genetics ; Male ; Melanoma-Specific Antigens ; Middle Aged ; Neoplasm Proteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate and compare the biological characteristics and sensitivity to chemotherapy and radiotherapy of intrahepatic and extrahepatic cholangiocarcinoma cells in vitro.

METHODSThe intrahepatic and extrahepatic cholangiocarcinoma cell lines were established, and cells with steady passage were chosen to study the biological characteristics including morphology, growth dynamics, chromosome, and levels of cancer antigen (CA) 125, CA19-9, alpha-fetoprotein (AFP), and carcino-embryonic antigen (CEA). Meanwhile, MTT assay was used to determine the sensitivity of both kinds of cells to 6 chemotherapeutic drugs, including cisplatin, paclitaxel, harringtonine, 5-fluorouracil, vincristine, and aclacimomycin, and the inhibitory rate of cells under the irradiation of 10 Gy ray was also measured.

RESULTSThe intrahepatic cholangiocarcinoma cells were mostly fusiform in shape, and extrahepatic cholangiocarcinoma cells were mostly round or polygon in shape. Their doubling time was 26. 3 hours and 23. 1 hours, respectively. Their average number of chromosomes was 59 (range, 38-84) and 67 (range, 49-103), respectively. The chromosome karyotypes of most intrahepatic cholangiocarcinoma cells were hyperdiploid and hypotriploid, while hypertriploid was predominant in extrahepatic cholangiocarcinoma cells. The level of CA 125 in supernatant of extrahepatic cholangiocarcinoma cells increased obviously, while levels of other determined tumor markers in both kinds of cells were all within normal range. The intrahepatic cholangiocarcinoma cells were low sensitive to cisplatin and paclitaxel, but not sensitive to the other 4 chemotherapeutic drugs. The extrahepatic cholangiocarcinoma cells were high sensitive to cisplatin, but not sensitive to the other 5 drugs. Both kinds of cells had poor sensitivity to radiotherapy.

CONCLUSIONSIntrahepatic and extrahepatic cholangiocarcinoma cells show differences in shape, doubling time, chromosome karyotype, tumor marker level, and chemosensitivity, whereas they both have poor radiosensitivity. Though they are similar in histopathology, they have different growth characteristics and have discrepancy in treatment and prognosis.

Antineoplastic Agents ; therapeutic use ; Bile Duct Neoplasms ; drug therapy ; genetics ; pathology ; radiotherapy ; Bile Ducts, Intrahepatic ; pathology ; Biomarkers, Tumor ; Cell Line, Tumor ; Cholangiocarcinoma ; drug therapy ; genetics ; pathology ; radiotherapy ; Enzyme-Linked Immunosorbent Assay ; Humans ; Karyotyping ; Radiation Tolerance

BACKGROUND/AIMS: Intrahepatic cholangiocarcinoma is the second most common intrahepatic neoplasm. Carcinogenesis is believed to be a multistage process that occurs as a result of mutations in oncogenes and tumor suppressor genes. Loss of heterozygosity (LOH) is the phenotype of genetic instability which has been used as a tool for detecting genetic phenotype alterations. Large number of the molecular alterations have been described in human cancer. Among them, that of p53 is quite common. The aim of this study was to determine the frequency of LOH at chromosome 17p related with p53. METHODS: Twenty cases who underwent hepatic resection due to intrahepatic cholangiocarcinoma, were included. LOH was analysed with four microsatellite markers by PCR. For the clinicopathologic parameters, tumor size, differentiation, and metastasis were evaluated. RESULTS: Fifteen patients (75%) showed LOH at one of the loci at the least. Five patients were LOH-high and 10 were LOH-low. The highest frequency of LOH was observed at D17S5 by 38.9%. Those of TP53, D17S796 and D17S513 were 29.4%, 21.4% and 35.3%, respectively. In addition, LOH tended to be more frequent when the tumor is mass forming type, poorly differentiated, or has tumor emboli or vascular invasion. CONCLUSIONS: This study showed that LOH was positive in 75% on chromosome 17p in intrahepatic cholangiocarcinoma which was relatively frequent at D17S5.
Aged ; Bile Duct Neoplasms/*genetics/pathology ; *Bile Ducts, Intrahepatic ; Cholangiocarcinoma/*genetics/pathology ; *Chromosomes, Human, Pair 17 ; Female ; Gene Frequency ; Genes, p53 ; Genetic Markers ; Humans ; *Loss of Heterozygosity ; Male ; Middle Aged

OBJECTIVETo investigate the effects of mutant exogenous P27(kip1) gene on chemosensitivity of human cholangiocarcinoma cell line.

METHODSThe recombinant vector was constructed and the mutant P27(kip1) gene was transfected into human cholangiocarcinoma cell line (QBC(939)). RT-PCR and Western blot were used to determine the expression of target genes. The effects of 5-fluorouracil (5-FU), mitomycin C (MMC) and cyclophosphamide (CTX) on the transfected cells were detected by assaying the apoptotic rate and growth inhibition by methabenzthiazuron (MTT) assay and flow cytometry (FCM).

RESULTSThe mutant exogenous P27(kip1) gene was expressed effectively in the cells, and the expression enhanced the apoptosis and growth inhibition of QBC(939) inducted by 5-FU, MMC and CTX. The ratio of growth inhibiting increased significantly from 41.89% (5-FU), 45.59% (MMC), 38.91% (CTX) to 56.15% (5-FU), 55.65% (MMC), 51.69% (CTX), and apoptosis index from 13.76% +/- 3.03% (5-FU), 11.76% +/- 3.99% (MMC), 10.46% +/- 2.10% (CTX) to 41.39% +/- 4.32% (5-FU), 35.94% +/- 2.71% (MMC), 34.46% +/- 2.32% (CTX) (P < 0.05).

CONCLUSIONSThe exogenous P27(kip1) gene transfer can remarkably increase the drug sensibility of the cholangiocarcinoma cells. The strategy targeted to control the cell cycle may be more effective in cancer treatment by combination of P27(kip1) gene therapy.

Adenoviridae ; genetics ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Bile Duct Neoplasms ; pathology ; Bile Ducts, Intrahepatic ; Cell Division ; drug effects ; Cell Line, Tumor ; Cholangiocarcinoma ; pathology ; Cyclin-Dependent Kinase Inhibitor p27 ; genetics ; pharmacology ; Drug Synergism ; Genetic Vectors ; Humans ; Transfection

Fascioliasis is a zoonotic infection caused by Fasciola hepatica or Fasciola gigantica. We report an 87-year-old Korean male patient with postprandial abdominal pain and discomfort due to F. hepatica infection who was diagnosed and managed by endoscopic retrograde cholangiopancreatography (ERCP) with extraction of 2 worms. At his first visit to the hospital, a gallbladder stone was suspected. CT and magnetic retrograde cholangiopancreatography (MRCP) showed an intraductal mass in the common bile duct (CBD) without proximal duct dilatation. Based on radiological findings, the presumed diagnosis was intraductal cholangiocarcinoma. However, in ERCP which was performed for biliary decompression and tissue diagnosis, movable materials were detected in the CBD. Using a basket, 2 living leaf-like parasites were removed. The worms were morphologically compatible with F. hepatica. To rule out the possibility of the worms to be another morphologically close species, in particular F. gigantica, 1 specimen was processed for genetic analysis of its ITS-1 region. The results showed that the present worms were genetically identical (100%) with F. hepatica but different from F. gigantica.
Aged, 80 and over ; Animals ; Base Sequence ; Cholangiocarcinoma/diagnosis ; Cholangiopancreatography, Magnetic Resonance ; Common Bile Duct/*pathology ; DNA, Helminth/*genetics ; DNA, Intergenic/genetics ; Diagnosis, Differential ; Fasciola hepatica/*genetics ; Fascioliasis/*diagnosis/parasitology ; Humans ; Male ; Neglected Diseases/diagnosis/parasitology ; Republic of Korea ; Sequence Analysis, DNA