Human Cholangiocarcinoma Cell Research Articles

VX-680 induces p53-mediated apoptosis in human cholangiocarcinoma cells.

VX-680 is one selective small-molecule inhibitor of the Aurora kinases. It has been shown to disrupt motosis and induce apoptosis in a wide variety of tumor cell lines. However, its effect on human cholangiocarcinoma (CCA) cells remains uncharacterized. In the current study, we observed the effects of VX-680 on the human CCA (QBC939 and HCCC-9810) cell line. In cell culture, VX-680 inhibited proliferation and induced apoptosis of tumor cell growth in a dose-dependent and time-dependent manner, and exerted the most effective cytotoxicity against HCCC-9810 cells. The proliferation inhibition rate increased from 5.39 to 51.74%, whereas the apoptosis rate increased from 9.59 to 50.02% when HCCC-9810 cells were cultured with 5 µmol/l VX-680 for 48 h. Immunoblot analysis showed that the expression of phospho-p53(Ser-15) was upregulated after 48 h treatment of the cancer cells with VX-680. This activation in p53 was associated with a decrease in Bcl-2 and an increase in Bax, which led to the expression of its downstream effectors (caspase-9 and caspase-3). We further found that pifithrin-α, a p53 inhibitor, attenuated the anticancer effects of VX-680 and downregulated the expression of apotosis-related proteins (Bax and caspase-9). These results suggest that VX-680 could mediate cell death by acting on a P53/Bax/ caspase-3-dependent pathway in human CCA cells.

Effects of Irisin on proliferation, apoptosis, migration and invasion of human cholangiocarcinoma cell line Hucct-1

Objective To investigate the effect of Irisin on proliferation, apoptosis, migration and invasion of human cholangiocarcinoma cell line Hucct-1. Methods After treatment with Irisin, CCK-8 assay and flow cytometry assay were conducted to investigate the effect of Irisin on proliferation and apoptosis of cholangiocarcinoma cells. Scratch test and transwell invasion assay were used to studythe effect of Irisin on the migration and invasion ability of cholangiocarcinoma cells. Western blot was utilized to detect the expression of E-cadherin, N-cadherin and Vimentin in cholangiocarcinoma cells. Results CCK-8 assay showed that Irisin inhibited cholangiocarcinoma cell proliferationin a dose-dependent manner. Flow cytometry assay showed that the apoptosis rate of Irisin group [(14.8±0.9)%] was higher than that in the control group [(5.4±0.6)%], (P<0.05). The scratch test showed that the rate of cell scratch healing in Irisin group [(15.0±1.0)%] was significantly lower than that in the control group [(28.0±2.0)%] (P<0.05). Transwell invasion test showed that the number of cells in Irisin group was (96.0±7.0), which was significantly lower than that in control group (155.0±9.0) (P<0.05). Western blot showed that the expression of E-cadherin increased and N-cadherin and Vimentin decreased after Irisin treatment. Conclusion Irisin inhibits proliferation, migration and invasion and promote apoptosis of cholangiocarcinoma cells. Key words: Intrahepatic cholangiocarcinoma; Irisin; Proliferation; Apoptosis; Migration; Invasion

Enhanced chemosensitivity of cholangiocarcinoma cell line QBC939 to gemcitabine induced by curcumin

Objective To investigate the effect of curcumin in enhancing chemosensitivity of cholangiocarcinoma cell line QBC939 to gemcitabine and its mechanism. Methods QBC939 cells were divided into control group, 5 μmol/L curcumin group, 0.625 μg/ml gemcitabine group and 5 μmol/L curcumin combined 0.625 μg/ml gemcitabine group. Methyl thiazol tetrazolium (MTT) assay was used to detect the proliferation inhibition effect, flow cytometry was used to detected the change of cell cycle and apoptosis rate. Expressions of nuclear factor-κB (NF-κB), Cyclin B1 and inhibitor of apoptosis proteins (IAP)-1 in different groups were detected by Western blotting. Results Compared with control group, curcumin group and gemcitabine group, the inhibitory rates of cells in curcumin combined gemcitabine group at 12, 24, 48 and 72 h were (10.00±0.02)%, (23.10±0.03)%, (33.23±0.01)%, (48.30±0.05)% respectively, which were significantly higher than those of control group and single medicaments treatment group (P=0.001, 0.000, 0.000, 0.000); besides, curcumin combined with gemcitabine blocked (28.75±3.23)% human cholangiocarcinoma cell line QBC939 in G2/M phase, which was significantly higher than those of curcumin group and gemcitabine group [(14.49±2.20)%, (11.82±2.10)%; P=0.000], the apoptosis rate in combined group was (13.64±3.40)%, significantly higher than those of control group, curcumin group and gemcitabine group [(4.53±0.56)%, (6.47±0.98)%, (9.87±1.12)%; P=0.000]; moreover, the relative expression levels of NF-κB and its downstream moleculor Cyclin B1 and IAP-1 proteins in curcumin combined with gemcitabine group were 0.300±0.001, 0.407±0.036, 0.475±0.032 respectively, significantly lower than those of control group, curcumin group and gemcitabine group (P=0.000, 0.001, 0.003). Conclusion Curcumin can enhance the sensitivity of human cholangiocarcinoma cell line QBC939 to gemcitabine through the inactivation of NF-κB signaling pathway. Key words: Cholangiocarcinoma cell; Curcumin; Gemcitabine; Cholangiocarcinoma cell line QBC939; Nuclear factor-κB

Establishment of Cas9 stably expressed human hepatocellular carcinoma and cholangiocarcinoma cell lines

Objective: To facilitate using the CRISPR/Cas9 gene editing system in human liver and gallbladder cancer cells, we established Cas9 stably expressed human liver and gallbladder cancer cell lines, and validated the gene editing activity of Cas9. Methods: Human liver cancer cell lines (Huh7, PLC/PRF/5, HepG2, Hep3b, SK-HEP-1 and Li-7), human cholangiocarcinoma cells (RBE) and human gallbladder cancer cells (GBC-SD) were infected with 3 Cas9-expressing lentivirus vectors (pLv-EF1α-Cas9-Flag-Neo, pLv-EF1α-Cas9-Flag-Puro, Cas9m1.1), respectively, and Cas9 stably expressed colonies were screened and selected. We extracted the genomic DNA and protein, validated the stable expression of Cas9 by using genomic polymerase chain reaction (PCR) and western blot. Three of cell lines were further infected with Lv-EF1α-mCherry. Then mCherry positive cells were sorted by flow cytometry and infected with designed guide RNA (gRNA) vectors which targeted mCherry gene. Subsequently the gene editing activity of Cas9 was detected by genomic PCR, fluorescence microscopic observation and flow cytometry analysis. Results: One hundred Cas9-expressing human liver and gallbladder cancer cell lines were selected. Among them, 35 cell lines expressed Cas9-Neo, 25 expressed Cas9-puro, and 40 expressed mutant Cas9 (mCas9). We also established 3 cell lines with stable expression of mCherry (Huh7-mCas9-M, PLC/PRF/5-Cas9-M and SK-HEP-1-Cas9-M). The results of genomic PCR and sequencing showed that by lentiviral infection with 2 types of designed gRNA, the long fragment deletion of mCherry gene was found in these 3 cell lines. Moreover, mCherry(-)EGFP(+) cells infected with 2 types of gRNA were observed by fluorescence microscope. The results of flow cytometry showed that mCherry(-)EGFP(+) cells accounted from 0.3% to 93.6%. Conclusion: We successfully establish 100 human liver and gallbladder cancer cell lines with stable expression of Cas9 protein and validate their activities of gene editing.

High promoter activity of cytokeratin-19 gene in cholangiocarcinoma.

Conditionally replicating adenoviral vectors constructed with tumor-specific promoters (TSPs) offers a viable tool for the treatment of cholangiocarcinoma. The aim of the present study was to investigate cholangiocarcinoma-specific TSPs that remain active in adenoviral constructs in gene therapy. The mRNA expressions of cyclooxygenase-2, cytokeratin-19 (CK19), mucin-1, midkine and telomerase reverse transcriptase were determined in human cholangiocarcinoma cell lines, primary human hepatocytes and cholangiocytes using reverse transcription-quantitative polymerase chain reaction. The candidate promoters constructed in adenoviral vectors were analyzed for their activities in cholangiocarcinoma cell lines, primary human hepatocytes and cholangiocytes using dual-luciferase reporter assays. The mRNA expression of CK19 was markedly higher in the QBC939 cell line, indicating specificity to cholangiocarcinoma. Moreover, the promoter activity of CK19 in the adenoviral vector in infected cholangiocarcinoma cells was found to be significantly stronger compared with that in infected primary human hepatocytes and cholangiocytes. CK19 may be implicated in the pathogenesis of cholangiocarcinoma, as demonstrated by the stronger activity of its promoter, as well as the higher expression of mRNA in tumor cells. Therefore, the use of the promoter sequence of the CK19 gene may represent a potential tool in cholangiocarcinoma-specific adenoviral gene therapy.

Antitumor effects of candidone extracted from &lt;i&gt;Derris indica&lt;/i&gt; (Lamk) Bennet in cholangiocarcinoma cells

Purpose : To investigate the antitumor effect of candidone extracted from Derris indica , against human cholangiocarcinoma (CCA) cells. Methods : Candidone was purified from the hexane extract of Derris indica fruit. CCA cell lines, KKUM156 and KKU-M213, were treated with candidone. Sulforhodamine B (SRB) assay and acridine orange/ethidium bromide (AO/EB) staining were used to investigate the effects of candidone on cell proliferation and induction of apoptosis, respectively. The effect on cell migration was assessed by wound healing assay. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was employed to assess the effects of candidone on the expression of genes that regulate proliferation and apoptosis. Results : Candidone exerted strong anticancer effects on CCA cells. The agent suppressed CCA cell proliferation and induced apoptotic cell death. RT-qPCR assay revealed that candidone significantly increased the expression of anti-proliferative and pro-apoptotic genes, including p21 and Bax, and decreased the expression of anti-apoptotic genes, including Bcl-2 and survivin. Moreover, candidone inhibited the migration of CCA cells induced by IGF-1. Conclusion : Candidone exhibits potent antitumor effect on CCA cells. These findings suggest that candidone is potentially suitable for the management of CCA and, therefore, warrants further investigation. Keywords : Candidone, Derris indica, Cholangiocarcinoma, Cytotoxicity, Apoptosis

Anti-cancer activity of asiatic acid against human cholangiocarcinoma cells through inhibition of proliferation and induction of apoptosis

Plant-derived anti-cancer agents have been of considerable interest due to their promising effectiveness with low side effects. Asiatic acid, the main constituent of the medicinal plant Centella asiatica (L.) Urban, has a wide range of biological properties such as antioxidant, anti-inflammatory and anti-cancer activities. Cholangiocarcinoma (CCA), which is a malignant tumor of bile duct epithelium, is one of the leading cancers in Southeast Asia, notably the northeast of Thailand where the liver fluke, Opisthorchis viverrini predominates. Many in vitro and in vivo studies have provided evidence supporting that oxidative stress induced by chronic inflammation is involved in CCA genesis with aggressive clinical outcomes. This study was performed to evaluate the cytotoxic effects of asiatic acid on two human CCA cell lines (KKU-156 and KKU-213). Cell viability was determined by a sulforhodamine B (SRB) assay. Morphological changes of the cells were observed by microscopy. Cell apoptosis was detected by flow cytometry using annexin V and propidium iodide (PI) staining. Messenger RNA (mRNA) expression levels of BAX, BCL2 and Survivin/BIRC5 were analyzed by real-time polymerase chain reaction (PCR). It was found that asiatic acid efficiently suppressed CCA cellular viability via induction of apoptosis. In addition, the occurrence of asiatic acid-induced apoptosis was confirmed by microscopic observation of apoptotic vesicles, down-regulation of anti-apoptotic genes (BCL2 and Survivin/BIRC5) and increased early and late apoptotic cells. Our results showed the chemotherapeutic activities of asiatic acid, suggesting the anti-cancer properties of this compound should be clinically assessed and its supplementation may lead to an improvement of survival of CCA patients.

The effect of microRNA-224 on the anti-apoptosis and proliferation promoting of cholangiocarcinoma cells in vitro

Objective To explore the regulatory mechanism of microRNA-224 (miR-224) in the apoptosis and proliferation of human cholangiocarcinoma (CC) cells and examine whether the pathway of miR-224-HOXD10-PAK4 exist in cc cells or not. Methods The expression of miR-224 in CC and adjacent normal tissue were measured by RT-PCR. QBC939 cells were chosen and divided into two groups: cell groups transducted with negative control viruses (NC group) and cell groups transducted with target gene miRNA-down virus (DOMN group). The rate of apoptosis and the number of colonies formed in QBC939 cell lines of DOWN group and NC group were counted. The expression of miR-224, API5, PAK4 and HOXD10 in QBC939 cell lines of DOWN group and NC group were measured by RT-PCR and western blotting. Results (1) MiR-224 expression was higher in CC tissues than that in normal bile duct tissues (P<0.05). (2) In QBC939 cells lines, miR-224 expression was lower in DOMN group than that in NC group. The clone formation of QBC939 cells in DOWN group was significantly lower than that of NC group. The apoptosis rate of QBC939 cells of DOWN group was significantly higher than that of NC group. (3)The expression of API5 and HOXD10 in QBC939 cells of DOWN group was significantly higher than that of NC group in the RT-PCR and western blotting experiments. (4) The expression of PAK4 in QBC939 cells of DOWN group was significantly lower than that of NC group in the RT-PCR and western blotting. Conclusions MiR-224 has anti-apoptotic effects and can promote the proliferation of QBC939 cells and may serve as an important regulator of QBC939 cell proliferation in vitro. There are miR-224-HOXD10-PAK4 signaling pathways in cholangiocarcinoma. Key words: Cholangiocarcinoma; microRNA-224 (miR-224); Cell apoptosis; Proliferation; Signal transduction, API5P1, PAK4, HOXD10

Gene expression profile of TLR7 signaling pathway in the liver of rhesus rotavirus-induced murine biliary atresia

PurposeTo identify genes potentially involved in the pathogenesis of bile duct obstruction in biliary atresia (BA). MethodsWe used rhesus rotavirus (RRV) Balb/c mouse BA model to study BA. Liver and serum samples were harvested from BA and normal control (NC) groups at 1, 3, 5, 7, 10 and 14 days postinoculation. Serum total bilirubin (STB) and conjugated bilirubin (CB) were measured. Livers of each group at day 7 were used for a genome-wide expression analysis. Expression of TLR7 signaling pathway in liver was measured by immunohistochemical staining and western blotting, including expression of TLR7, activation of phosphorylated IRF7 and secretion of IFN-β, IL-1α and IL-6. Cell viability and survival rate after RRV infection were measured by using TLR7 knockdown human cholangiocarcinoma cell RBE. ResultsSTB was significantly elevated from day 5 postinoculation and CB was from day 7 postinoculation, while CK19 (the biomarker of biliary epithelial cells) expression by western blotting was decreased. By microarray analysis of liver tissues at day 7 postinoculation, TLR7 signaling pathway was up-regulated in BA mice. Based on the results of microarray analysis, the protein expression of TLR7 in the liver tissues of BA groups were found to be up-regulated from day 5 comparing to respective NC groups, although it was increased as pups aged in NC groups. And the level of p-IRF7 and secretion of cytokines were also statistically significant in BA groups. In vitro, TLR7 knockdown cell line showed less cellular proliferation and more susceptible to RRV infection. ConclusionBy in vivo study, TLR7 signal pathway was up-regulated in BA group; by additional in vitro study, intact TLR7 signal pathway might have some protective abilities in BA pathogenesis.

Deoxypodophyllotoxin induces cell cycle arrest and apoptosis in human cholangiocarcinoma cells.

Deoxypodophyllotoxin (DPT), a naturally occurring flavolignan, has a broad range of biological effects, including anti-inflammatory, anti-viral and anticancer properties. The present study investigated the anti-proliferative effect of DPT on human cholangiocarcinoma QBC939 and RBE cell lines and its underlying mechanisms of inducing cytotoxicity. MTT assays demonstrated that DPT inhibited the viability of the QBC939 and RBE cells in a dose and time-dependent manner. In addition, DPT treatment resulted in G2/M phase cell cycle arrest associated with the downregulation of Cyclin B and cyclin dependent kinase 1 and caused an increase in apoptosis that was confirmed by characteristic morphological changes. Apoptosis was accompanied by increasing B-cell lymphoma-2 (Bcl-2)/Bcl-2 associated X protein ratios and activated expression of caspase-3, -8 and -9. These findings suggested that DPT may be a novel anticancer agent against human cholangiocarcinoma.

Transglutaminase 2 Mediates the Cytotoxicity of Resveratrol in a Human Cholangiocarcinoma and Gallbladder Cancer Cell Lines

ABSTRACTResveratrol is a polyphenolic compound extracted from plants and is also a constituent of red wine. Our aim was to evaluate if the cytotoxic effect of resveratrol (RES) on cholangiocarcinoma (CC) and gallbladder cancer (GBC) cell lines could be abolished by TG2 inhibition. Human CC and GBC cell lines (SK-ChA-1 and MZ-ChA-1), grown in a three-dimensional cell culture system (MCTS, multicellular tumor spheroids), were treated for 72 h with RES (32, 64 µM) alone or combined with different TG2 inhibitors (Cystamine, B003, T101). We investigated: cells viability; cell morphology with light microscopy (LM) and transmission electron microscopy (TEM); immunoreactivity with immunohistochemistry; Q-Banding karyotype analysis; TG2 activity; Western blotting. RES treatment induced a significant inhibition of cell growth, ranging from 24% to 76% in both cell lines. The inhibitors successfully reduced TG2 activity without any variation of protein quantity as demonstrated by immunohistochemistry and Western blot. TG2 inhibition resulted in cell growth normalization. In addition, morphologic analysis by light and transmission electron microscopy confirmed the cytotoxic effect of RES and its reduction consequent to TG2 inhibition. Our data demonstrated a connection between the cytotoxic effect of RES in SK-ChA-1 and MZ-ChA-1 and TG2 activity.

SAT-161 - Obeticholic acid, a FXR agonist, inhibits the cancerogenic potential of primary human cholangiocarcinoma (CCA) cells cultures

SAT-161 - Obeticholic acid, a FXR agonist, inhibits the cancerogenic potential of primary human cholangiocarcinoma (CCA) cells cultures

Curcumol Exerts Anticancer Effect in Cholangiocarcinoma Cells via Down-Regulating CDKL3.

Curcumol is the major component extracted from root of Rhizoma Curcumae. Recent studies have shown that curcumol exerts therapeutic effects against multiple conditions, particularly cancers. However, the therapeutic role and mechanism of curcumol against cholangiocarcinoma cells are still unclear. In our current research, we tested the effect of curcumol in cholangiocarcinoma cells, and using two-dimensional electrophoresis, proteomics and bioinformatics, we identified cyclin-dependent kinase like 3 (CDKL3) as a potential target for curcumol. We have demonstrated that curcumol can evidently suppress growth and migration of cholangiocarcinoma cells. Furthermore, curcumol could significantly block the cell cycle progression of the cholangiocarcinoma cells. These effects could be largely attributed to the inhibition of CDKL3 by curcumol. Further studies have recapitulated the oncogenic role of CDKL3 in that knockdown of CDKL3 by lentiviral mediated transfection of shRNA against CDKL3 also led to a significant inhibition on cell proliferation, migration, invasion, and cell cycle progression. Given the high level of CDKL3 expression in human cholangiocarcinoma tissues and cell lines, we speculated that CDKL3 may constitute a potential biological target for curcumol in cholangiocarcinoma.

Downregulation of microRNA-193-3p inhibits the progression of intrahepatic cholangiocarcinoma cells by upregulating TGFBR3.

The aim of this study was to investigate the function of microRNA (miR)-193-3p in human intrahepatic cholangiocarcinoma (ICC) tissues and cells. To evaluate whether miR-193-3p was aberrantly upregulated, we used reverse transcription-quantitative polymerase chain reaction to detect the level of miR-193-3p in ICC tissues and ICC-9810 cells. The effects of miR-193-3p downregulation on ICC cell proliferation, migration and invasion were also measured by MTT, wound-healing and Transwell assays. Additionally, transforming growth factor-β receptor type 3 (TGFBR3) was investigated as a direct target of miR-193-3p by dual-luciferase reporter assays and western blot analyses. The results demonstrated that miR-193-3p was aberrantly upregulated in ICC tissues and cell lines. Furthermore, TGFBR3 was confirmed to be a target of miR-193-3p in ICC and was notably upregulated by miR-193-3p knockdown in the ICC-9810 cells. The knockdown of miR-193-3p also exerted direct inhibitory effects on the proliferation, migration and invasion of the ICC-9810 cells. Therefore, we present evidence that miR-193-3p plays a key role in promoting human ICC by regulating TGFBR3.

Fibroblast growth factor receptor inhibition induces loss of matrix MCL1 and necrosis in cholangiocarcinoma

Myeloid cell leukemia 1 (MCL1), a prosurvival member of the BCL2 protein family, has a pivotal role in human cholangiocarcinoma (CCA) cell survival. We previously reported that fibroblast growth factor receptor (FGFR) signalling mediates MCL1-dependent survival of CCA cells in vitro and in vivo. However, the mode and mechanisms of cell death in this model were not delineated. Human CCA cell lines were treated with the pan-FGFR inhibitor LY2874455 and the mode of cell death examined by several complementary assays. Mitochondrial oxidative metabolism was examined using a XF24 extracellular flux analyser. The efficiency of FGFR inhibition in patient-derived xenografts (PDX) was also assessed. CCA cells expressed two species of MCL1, a full-length form localised to the outer mitochondrial membrane, and an N terminus-truncated species compartmentalised within the mitochondrial matrix. The pan-FGFR inhibitor LY2874455 induced non-apoptotic cell death in the CCA cell lines associated with cellular depletion of both MCL1 species. The cell death was accompanied by failure of mitochondrial oxidative metabolism and was most consistent with necrosis. Enforced expression of N terminus-truncated MCL1 targeted to the mitochondrial matrix, but not full-length MCL1 targeted to the outer mitochondrial membrane, rescued cell death and mitochondrial function. LY2874455 treatment of PDX-bearing mice was associated with tumour cell loss of MCL1 and cell necrosis. FGFR inhibition induces loss of matrix MCL1, resulting in cell necrosis. These observations support a heretofore unidentified, alternative MCL1 survival function, namely prevention of cell necrosis, and have implications for treatment of human CCA. Herein, we report that therapeutic inhibition of a cell receptor expressed by bile duct cancer cells resulted in the loss of a critical survival protein termed MCL1. Cellular depletion of MCL1 resulted in the death of the cancer cells by a process characterised by cell rupture. Cell death by this process can stimulate the immune system and has implications for combination therapy using receptor inhibition with immunotherapy.

Solamargine derived from Solanum nigrum induces apoptosis of human cholangiocarcinoma QBC939 cells.

Solamargine, an active ingredient of Solanum nigrum, has been previously revealed to inhibit the proliferation of cancer cells. However, the effect of solamargine on human cholangiocarcinoma cells and the underlying molecular mechanism remain unknown. In the present study, the molecular mechanism underlying the anti-cancer effect of solamargine was assessed in human cholangiocarcinoma QBC939 cells. The results of the present study revealed that solamargine inhibited the viability of QBC939 cells in a dose-dependent manner. Furthermore, solamargine significantly induced the apoptosis of QBC939 cells and altered the mitochondrial membrane potential of cells. Quantitative polymerase chain reaction analysis revealed that solamargine decreased the mRNA level of B-cell lymphoma-2 (Bcl-2), Bcl-extra-large and X-linked inhibitor of apoptosis protein but increased the mRNA level of Bcl-2-associated X protein (Bax). In addition, western blot analysis demonstrated that solamargine inhibited the protein expression of Bcl-2 and poly ADP ribose polymerase (PARP), and promoted the protein expression of Bax, cleaved PARP, caspase 3, cleaved caspase 3 and caspase 7. Therefore, the results of the present study revealed that solamargine may induce apoptosis via the mitochondrial pathway and alter the level of apoptosis-associated proteins in human cholangiocarcinoma QBC939 cells. This in vitro study demonstrated that solamargine may be an effective chemotherapeutic agent against cholangiocarcinoma in clinical practice.

Mir-637 inhibits the proliferation of cholangiocarcinoma cell QBC939 through interfering CTSB expression.

To investigate the role of mir-637 on the proliferation, migration and apoptosis in human cholangiocarcinoma (CCA) cell line QBC939, and the impact of mir-637 on Cathepsin B (CTSB) expression. Expression of mir-637 and CTSB in CCA tissues and adjacent non-tumor tissues were measured by Real-time PCR. CTSB expression in human CCA cell lines was detected with or without mir-637 exogenous overexpression through fluorescent quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) method. The efficiency of lentivirus-mir-637 vector infection, and the effect of mir-637 on the proliferation, migration ability, apoptosis and cell cycle of CCA cell were detected, respectively. The possibility of mir-637 targeting CTSB was also tested by bioinformatics method, correlation analysis and molecular biology method. Decreased mir-637 and increased CTSB expression were observed in CCA tissue compared with adjacent non-tumor tissues. CTSB expression in mir-637 exogenously overexpressed QBC939 cells decreased compared with negative control. Mir-637 overexpression caused significant decrease of proliferation and migration ability of QBC939 cell. In addition, mir-637 overexpression induced that the cell cycle was blocked in G0/G1 phase, and cell apoptosis rate increased significantly. mir-637 and CTSB play an important role in the proliferation and migration of CCA cells. Mir-637 could inhibit CTSB expression significantly, which in-turn down-regulates the proliferation, migration and invasion ability of QBC939 cell, and promotes apoptosis in QBC939 cell line.

Effect of AdipoR agonist in cholangiocarcinoma.

323 Background: Adiponectin is the key adipokine, which plays an important role in health and disease such as obesity, diabetes, and cancer. Adiponectin is reduced in different tumor types, especially in obesity-related cancer, and recent studies showed that Adiponectin had a potential anti-cancer effect. Obesity is a risk factor for various tumor diseases including cholangiocarcinoma (CC), the second most common primary hepatic cancer. The aim of this study is to investigate for the first time the anti-cancer effect of AdipoR agonist in CC cell lines and a CC engineered mouse model. Methods: Human CC cell lines (TFK-1 and SZ-1) and CC engineered mice (Alb-Cre/KRASG12D/p53L/L) were used to investigate the anti-cancer effects of an AdipoR agonist (2-(4-Benzoylphenoxy)-N-[1-(phenylmethyl)-4-piperidinyl]-acetamide). Cell proliferation, migration, invasion, colony formation, apoptosis assay were applied to evaluate the effect of AdipoR agonist in vitro. In addition, important cancer signalling pathways and targets were analysed by Western Blot. Mice (n = 12) were treated with AdipoR or verhicle and tumor burden and survival were monitored. Results: AdipoR agonist suppressed proliferation, migration, invasion, colony formation and apoptosis in CC cells. AdipoR agonist regulated the expression of different proteins such as EMT markers, pAMPK, pSTAT3, and PARP, which have pivotal functions in cholangiocarcinogenesis. AdipoR agonist also prolonged survival time in a CC engineered mouse model. Conclusions: Our data revealed that AdipoR agonist inhibited successfully cell proliferation, migration, invasion, colony formation and apoptosis in vitro, and prolonged mice survival in vivo through regulation of crucial signaling pathways in cholangiocarcinogenesis. These results suggested that AdipoR agonist might become a new potential candidate for CC treatment in the future.

Silencing of Kangai 1 C-terminal interacting tetraspanin suppresses progression of cholangiocarcinoma

Cholangiocarcinoma (CC) is the second most common primary hepatic malignancy. CC treatment options are very limited especially for patients with distant metastasis. Kangai 1 C-terminal interacting tetraspanin (KITENIN) is highly expressed in numerous cancers, but the role of KITENIN in CC remains unknown. Here, we have investigated for the first time the function of KITENIN in human CC cell lines (TFK-1, SZ-1), tissues and a CC mouse model (Alb-Cre/LSL-KRASG12D/p53L/L). KITENIN was expressed in 92.2% of human CC tissues, in murine CC samples and also in human CC cell lines. Knockdown of KITENIN by small interfering RNA (siRNA) effectively reduced proliferation, migration, invasion and colony formation in both intra- and extra-hepatic CC cells. The expression of epithelial-mesenchymal transition (EMT) markers like N-cadherin, Vimentin, Snail and Slug were suppressed in KITENIN knockdown CC cells. Our results indicate that KITENIN is crucial for cholangiocarcinogenesis and it might become a potential therapeutic target for human CC treatment.

Effect of puerarin on the apoptosis of human cholangiocarcinoma RBE cells

Objective To study the effects of puerarin on the proliferation and apoptosis of human cholangiocarcinoma (RBE) cells and the expression of related genes. Methods RBE cells cultured were treated with puerarin at different concentrations (0, 100, 250, 500, 1 000, 1 500 μg/ml). And then methyl thiazol tetrazolium (MTT) assay was used to measure cell viability. Flow cytometry (FCM) was used to detect the apoptosis of RBE cells treated with puerarin at (500, 1 000 μg/ml) concentrations. The expression levels of B cell lymphoma/leukemia-2 (bcl-2) and bcl-2 associated X protein (bax) genes were detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). Western blotting was used to detect the expression of apoptosis-related proteins. Results MTT assay revealed that puerarin (500, 1 000, 1 500 μg/ml) inhibited the proliferation of RBE cells in a time- and dose-dependent manner, with inhibition rate at 12, 24 and 48 h being (8.14±1.85)%, (12.57±3.10)% and (18.26±2.74)% (P=0.000, 0.005, 0.003), (22.38±2.77)%, (29.14±3.85)% and (37.43±2.11)% (P=0.005, 0.007, 0.002), and (35.47±3.75)%, (40.81±2.52)% and (52.29±3.98)% (P=0.007, 0.000, 0.002) respectively. After RBE cells were treated with puerarin at (500, 1 000 μg/ml) for 24 h, the apoptosis rate [(14.21±2.53)% and (22.87±2.74)%] was increased greatly as compared with the control group [(3.246±2.25)%] (P=0.013, 0.008). The expression of bax was up-regulated (2.37±0.17 and 3.78±0.41) times as compared with the control group (0.98±0.04) (P=0.005, 0.003), and the expression of bcl-2 was down-regulated (1.57±0.14 and 2.86±0.55) times as compared with the control group (0.99±0.03) (P=0.002 and 0.008). The expression levels of Cleaved-Caspase-8, Cleaved-Caspase-9 and bax protein were increased (P=0.015, 0.003 and 0.008) and those of bcl-2 protein were decreased (P=0.021). Conclusion Puerarin can inhibit the proliferation of RBE cells and promote the apoptosis. Key words: Puerarin; Cholangiocarcinoma; Proliferation; Apoptosis