Human Bile Duct Carcinoma Cell Research Articles

Autophagy impairment in human bile duct carcinoma cells.

Bile duct epithelial cells, named cholangiocytes, may undergo a neoplastic transformation leading to cholangiocarcinoma. The role autophagy plays in cancer is still debated and few information are available in cholangiocarcinoma. We report in vitro data, at least in part validated in vivo,i ndicating that autophagy is impaired in intrahepatic cholangiocarcinoma cells, as compared to healthy cholangiocytes, evaluated through LC3II and p62 Western blot analyses. Autophagy impairment was found to be associated with low expression of TFEB protein and high expression of three proteins i.e., c-FLIP, caspase-10 and cleaved BCLAF-1, as compared to healthy cholangiocytes. We highlight biological effects of autophagy impairment in cholangiocarcinoma showing that autophagy induction, via rapamycin, as well as caspase inhibition, via Q-VD-OPh, are able to reduce proliferation marker PCNA level, colony size and protein content of cultured cholangiocarcinoma cells. The increased protein expression of p62, c-FLIP, caspase-10 observed in vitro in cholangiocarcinoma cells was paralleled by significant increase at gene expression levels in vivo; in fact, significant increase of transcript levels of p62, c-FLIP and caspase-10 was observed in 34 biopsies from human cholangiocarcinoma patients compared to 9 biopsies from 9 healthy controls, as reported in the GEPIA2 public database. The significant increase of p62 level in cholangiocarcinoma was found as a relatively uncommon finding in solid cancers, since it was also found in only 7 cancer types out of 31 cancer types investigated, including melanoma and hepatocarcinoma. In conclusion, we present data suggesting a molecular machinery controlling autophagy in cholangiocytes and autophagy impairment in cholangiocarcinoma.

Photodynamic efficiency of a chlorophyll-a derivative in vitro and in vivo

This paper reports the antitumor activity of a chlorophyll-a derivative, 2-[1-hydroxyethyl]-2-devinylpyropheophorbide-a (HEPa). Photophysical characteristics of HEPa were measured. And its cytotoxicity, intracellular localization, biodistribution, efficiency of photodynamic therapy (PDT), histological analysis were investigated using human bile duct carcinoma cells (QBC-939) and QBC-939 tumor bearing BABL/c nude mice as animal model. The results showed that HEPa was localized mainly within the cytoplasmic region and partially in lysosome. Biodistribution of HEPa in QBC-939 tumor bearing BABL/c nude mice showed its fast rate of clearance and high tumor selectivity. In vitro, HEPa had low dark toxicity and high photoxicity against QBC-939 cells. The inhibition rate of QBC-939 tumor could increase up to 92.3%, and H&E staining confirmed that HEPa could cause serious damage to the tumor with light dose of 100J/cm(2), implying that HEPa has potential to be a new antitumor candidate for photodynamic therapy (PDT).

Experimental study of emodin inducing apoptosis of bile duct carcinoma cell QBC939

Objective To investigate the effect and mechanism of emodin inducing the apoptosis of human bile duct carcinoma cell QBC939 in vitro. Methods The inhibition of cell proliferation was detected by MTT. The morphological changes of apoptotic cells were observed under fluorescence microscopy.The apoptosis rates and intracellular reactive oxygen species levels were detected by flow cytometry.The intracellular relative activities of caspase-9 and caspase-3 were tested through colorimetric method.Results Emodin inhibited cell proliferation of human bile duct carcinoma cell QBC939 in time-dose dependent fashion.Apoptotic cells displayed bright,nuclear presented divided leaves,debris and a set of edges under fluorescence microscopy.Treated with 30 μmol/L and 50 μmol/L emodin,the apoptosis rates of 24 hours were respectively 38.9％ ± 9.07％ and 67.09％ ± 4.08％ (P ＜ 0.05).The intracellular reactive oxygen species levels after 30 minutes treatment were 1.65 ± 0.08 and 2.28 ± 0.04folds of the control group (P ＜ 0.05). Emodin could activate caspase-9 and caspase-3,leading to elevations of their activities (P ＜ 0.05).Conclusion Emodin inhibites cell proliferation of human bile duct carcinoma cell QBC939 through inducing apoptosis.The mechanism is associated with the elevated levels of reactive oxygen species and the activation of caspase-9 cells and caspase-3. Key words: Emodin; Apoptosis; Reactive oxygen species; Caspases

Lysophosphatidylcholine induces apoptosis in human bile duct carcinoma cell HuCCT-1

Lysophosphatidylcholine induces apoptosis in human bile duct carcinoma cell HuCCT-1

Effect of adenovirus-mediated apoptin gene on apoptosis of human bile duct carcinoma cells

目的 探讨凋亡素基因转染对人胆管癌细胞凋亡的影响,为胆管癌的治疗探索新方法.方法 采用含凋亡素基因的重组腺病毒感染胆管癌细胞株QBC939,在确定其感染复数后通过光镜、荧光观察其凋亡情况,同时通过TUNEL染色统计凋亡率.结果 胆管癌细胞株QBC939的重组腺病毒的感染复数为65,在感染后第3天凋亡素基因能引发胆管癌细胞株QBC939的凋亡,并且随着时间的推移其作用越明显,在第7天凋亡率达到61.9%.结论 凋亡素能明显引发人胆管癌细胞的凋亡,在胆管癌的治疗上具有较大潜力。

The effect of hepatitis B virus X gene transfection on expression of human telomerase reverse transcriptase mRNA in human bile duct carcinoma cell lines

To study the effect of Hepatitis B virus X (HBx) gene transfection on expression of human telomerase reverse transcriptase (hTERT) mRNA in human bile duct carcinoma cell lines QBC939 and to elucidate the significance of cis-activation of hTERT mRNA by HBx gene on the carcinogenesis of bile duct. QBC939 were cultured in vitro and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein (EGFP) coding sequence using liposome-mediated gene transduction technique. Thirty six hours after transfection, EGFP expression, the indicator of successful transfection in cells, was determined. Flow cytometry was applied to determine the transfection efficiency. Cells were harvested and total RNA was extracted with TRI(ZOL) Reagent. The expression of hTERT mRNA in QBC939 was assayed by Reverse Transcription Polymerase Chain Reaction. The expression of HBx protein in QBC939 was detected by immunocytochemistry staining and western blotting. The transfection efficiency was 29.6% for both HBx expression vector and vector control group. The expression of hTERT mRNA was significantly increased when transfected with HBx expression vector than that transfected with OPTI-MEM medium and vector only. The expression of HBx protein could only be found in the cells when transfected with HBx expression vector by immunocytochemistry staining and western blotting. HBx gene transfection may up-regulate the transcriptional expression of hTERT mRNA in bile duct carcinoma cells. The cis-activation of hTERT gene by HBx gene is primary mechanism for carcinogenesis of biliary epithelia after HBV infection.

Effect of down regulation of laminin receptor on urokinase-type plasminogen activator expression in human bile duct carcinoma cells

To investigate the effect of antisense oligonucleotide (AS-OD) of laminin receptor (LNR) on urokinase-type plasminogen activator (u-PA) expression in human bile duct carcinoma cells. Bile duct carcinoma cells of QBC939 line were cultured. Compounds of lipofectAMINE and AS-OD phosphorothioate of LNR of the concentrations of 3, 6, and 12 micromol/L respectively were prepared and then transfected into the QBC939 cells. Sense oligodeoxynucleotide (S-OD) of LNR and blank vector were used as controls. After culture of 48 hours, flow cytometry was used to detect the protein expression of LNR and the u-PA mRNA expression was detected by reverse-transcriptase polymerase chain reaction (RT-PCR). Cell invasion was examined by culture plate with Transwell microporous film before and after transfection. Flow cytometry showed down-regulation of LNR protein expression in the cells transfected with AS-OD dose-dependently. The LNR protein expression of the cells transfected with AS-OD of the concentration of 6 micromol/L was down-regulated by over 25% in comparison with those of the blank vector group and S-OD group (both P < 0.05). RT-PCR showed that the u-PA mRNA expression of the AS-OD 6 micromol/L group was down-regulated by 30% in comparison with the 2 control groups. The number of migrating cells in the AS-OD group was 78 +/- 6, significantly lower than those in the S-OD group (105 +/- 11), and blank vector group (101 +/- 7), both P < 0.05. The expression of laminin receptor in the bile duct carcinoma cells is related with the expression of u-PA gene expression. Inhibition of LNR expression decreases the u-PA gene expression, suggesting that LNR plays an important role in the invasion and metastasis of bile duct carcinoma.

SEA-scFv as a Bifunctional Antibody: Construction of a Bacterial Expression System and Its Functional Analysis

A SEA-antibody single chain Fv (SEA-scFv) fusion protein was produced by bacterial expression system in this study. SEA-scFv has both staphylococcal enterotoxin A (SEA) effects and antibody activity directed at the epithelial mucin core protein MUC1, a cancer associated antigen. It was expressed mostly in the cytoplasm as an insoluble form. The gene product was solubilized by guanidine hydrochloride, refolded by conventional dilution method, and purified using metal-chelating chromatography. The resulting SEA-scFv fusion protein preparation was found to react with MUC1 and MHC class II antigens and had the ability to enhance cytotoxicity of lymphokine activated killer cells with a T cell phenotype against a human bile duct carcinoma cell line, TFK-1, expressing MUC1. This genetically engineered SEA-scFv fusion protein promises to be an important reagent for cancer immunotherapy.

Transcriptional Regulation of the N-Acetylglucosaminyltransferase V Gene in Human Bile Duct Carcinoma Cells (HuCC-T1) Is Mediated by Ets-1

N-Acetylglucosaminyltransferase V (GnT-V) catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to alpha-6-D-mannoside to produce the beta1-6 linked branching of N-glycan oligosaccharides, which controls the polylactosamine content. The expression of N-acetylglucosaminyltransferase V, which contains 17 exons and spans 155 kilobase pairs, is expressed in a tissue- and cell type-specific manner and is regulated at the level of transcription by multiple promoters (Saito, H., Gu, J., Nishikawa, A., Ihara, Y., Fujii, J., Kohgo, Y., and Taniguchi, N. (1995) Eur. J. Biochem. 233, 18-26). To elucidate the mechanism by which the GnT-V gene is expressed in a cell- and tissue-specific manner, cell-restricted expression was analyzed using the 5'-upstream regions of the human GnT-V gene spanning base pairs -2760 to +23 in a human bile duct carcinoma cell line, HuCC-T1. We characterized two cis-acting elements that are potentially important in HuCC-T1 cell-specific expression. The two elements each contain an Ets-1 binding site, 5'-GGA-3'. Specific binding of Ets-1 to the respective elements was demonstrated by competition analysis as well as by antibody supershift experiments. Cotransfection of an Ets-1 expression plasmid along with a GnT-V promoter-luciferase reporter plasmid revealed the participation of Ets-1 in the regulation of the GnT-V gene transcription. These data indicated that the transcriptional regulation of the GnT-V gene was mediated by transcription factor Ets-1.

Chromosomal breakpoints in cholangiocarcinoma cell lines

Little is known about the genetics and biology of cholangiocarcinoma (intrahepatic bile duct carcinoma). Only three human bile duct carcinoma cell lines have been described in the literature. We present the first detailed cytogenetic analysis of two cell lines; a new cell line designated PCI:SG231, established in our laboratory, and RPMI-7451, a previously established cell line. Both lines had highly aneuploid karyotypes with complex rearrangements including marker chromosomes. PCI:SG231, harvested after 50 days in culture, had a modal and median chromosome number of 65, and many cells contained double-minute chromosomes. RPMI-7451 had a modal and median chromosome number of 67. C-banding confirmed the presence of dicentric chromosomes in PCI:SG231. Q-banding confirmed the absence of the Y chromosome in PCI:SG231 and the presence of a der(1)t(Y;1)(q11;p11) chromosome in RPMI-7451. Numerical abnormalities common to both lines included trisomies 2, 5, 11, and 20. Chromosomes 1, 5, 7, and 12 were most commonly involved in structural abnormalities in both lines. Consistent chromosomal breakpoints included 7q22 and 12p11-12. PCI:SG231 was tumorigenic in immunosuppressed nude mice and was histologically similar to the original tumor. Additional cholangiocarcinoma cell lines are being developed to continue the study of the genetics and cell biology of this disorder.

Human bile duct carcinoma cell line producing abundant mucin in vitro.

A human bile duct carcinoma cell line, designated OZ, was established from ascitic effusion of a patient who suffered from obstructive jaundice due to the clogging of the common bile duct with mucinous substances secreted by the cancer cells. OZ was found to be capable of producing mucin in vitro and pools of mucin were macroscopically identified on the monolayer of the cells. On the electron micrographs, cell coat type mucin and abundant intracytoplasmic desmosomes were observed. The OZ cells secreted carcinoembryonic antigen in culture and had high enzymatic activity of gamma-glutamyl transpeptidase. The tumor heterotransplanted into nude mice also showed mucin production.