Cholangiocarcinoma QBC939 Cells Research Articles

Effect of FXR agonist GW4064 in the treatment of hilar cholangiocarcinoma in rats

The study objective was to observe the treatment effect of the farnesoid X receptor (FXR) agonist GW4064 in a rat model of hilar cholangiocarcinoma to explore a new therapeutic target for gene therapy for hilar cholangiocarcinoma. Eighty male Wistar rats were randomly divided into four groups (treatment group, model group, control group and sham operation group, 20 rats in each group). The four groups were fed a standard diet. The treatment group and the model group were injected with a suspension of cholangiocarcinoma QBC939 cells into the hilar bile duct with a microsyringe, the control group was injected with normal saline, and the sham operation group was not injected with anything. A modified tail suspension test (TST) was used to evaluate the vitality of the rats. At 4 weeks, one rat in the treatment group and model group was euthanized, and the changes in the hilar bile duct were recorded. The procedure was repeated at 6 weeks. After 6 weeks, hilar cholangiocarcinoma occurred in the treatment group and model group. Then, the treatment group was injected with GW4064 intraperitoneally at a dose of 50 mg/kg/day. One week after injection, the rats in the four groups were euthanized. Pathological examination confirmed that tumours had formed, and hilar bile duct tissues were taken from the four groups. FXR, Bsep, Ntcp and NF-κB expression in the hilar bile duct was detected by real-time polymerase chain reaction (RT–PCR) and immunohistochemistry. After three weeks, the rats in the treatment group and model group ate less, and their weight was significantly reduced. Six weeks later, hilar cholangiocarcinoma was detected in the treatment group and model group. After treatment with GW4064, the ratios of FXR/GAPDH mRNA, Bsep/GAPDH mRNA, Ntcp/GAPDH mRNA and NF-κBp65/GAPDH mRNA were significantly different among the four groups. Under a light microscope, FXR protein reacted with anti-FXR antibody, Bsep protein reacted with anti-Bsep antibody, Ntcp protein reacted with anti-Ntcp antibody and NF-κBp65 protein reacted with anti-NF-κBp65 antibody, and they showed granular expression. Every pathological section included 4,800 cells, and there were different numbers of positive cells in each group. FXR expression in the hilar cholangiocarcinoma of rats was significantly lower than that in normal hilar bile duct tissues. GW4064 increased the expression of FXR in tumour tissues. These findings suggest that FXR may be a new therapeutic target and that GW4064 may be helpful in the treatment of hilar cholangiocarcinoma.

Effects of 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione on autophagy and the PI3K/AKT/mTOR signaling pathway in human cholangiocarcinoma QBC939 cells.

2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD) has been reported to have good antitumor effects. The aim of this study was to investigate whether DMDD induces apoptosis and autophagy in human cholangiocarcinoma (CCA) QBC939 cells and determine its effect on the PI3K/AKT/mTOR signaling pathway. QBC939 cells were cultured in vitro and changes in cell viability were detected by the Cell Counting Kit (CCK8) assay after treatment with different concentrations of DMDD for 24, 48, and 72 h. The cells were divided into control and DMDD-treated groups (treated concentrations were 10, 15, and 20 µM/L), and the cell cycle, apoptosis, and autophagic vesicles were assessed. The expression levels of PI3K, AKT, mTOR, microtubule-associated protein 1 light chain 3 beta (LC3-II)/I, Beclin-1, and P62 were detected by Western blot. A xenograft mouse model was constructed to detect the effect of DMDD on CCA. The experimental results showed that DMDD was able to inhibit proliferation, migration, and invasion and induce cell cycle arrest and autophagy of QBC939 cells. In addition, DMDD decreased the protein expression of PI3K, AKT, and mTOR and increased the expression of LC3-II/I, Beclin-1, and P62. In mice, DMDD was able to inhibit the growth of tumors. DMDD inhibits CCA cell viability and induces cell cycle arrest and autophagy by a mechanism that may be related to the downregulation of the PI3K/AKT/mTOR signaling pathway.

MiR-29b restrains cholangiocarcinoma progression by relieving DNMT3B-mediated repression of CDKN2B expression.

Numerous studies have reported the important role of microRNAs (miRNAs) in human cancers. Although abnormal miR-29b expression has been linked to tumorigenesis in several cancers, its role in cholangiocarcinoma remains largely unknown. We found that miR-29b expression is frequently downregulated in human cholangiocarcinoma QBC939 cells and in clinical tumor samples. In cholangiocarcinoma patients, low miR-29b expression predicts poor overall survival. Overexpression of miR-29b in QBC939 cells inhibited proliferation, induced G1 phase cycle arrest, and promoted apoptosis. Methylation-specific PCR (MSP) analysis revealed a decreased methylation imprint at the promoter of the cell cycle inhibitor gene CDKN2B in cells overexpressing miR-29b. After identifying the DNA methyltransferase DNMT3B as a putative miR-29b target, luciferase reporter assays confirmed a suppressive effect of miR-29b on DNMT3B expression. Accordingly, we detected an inverse correlation between miR-29b and DNMT3B expression in clinical cholangiocarcinoma specimens. In QBC939 cells, DNMT3B overexpression promoted proliferation and inhibited apoptosis. DNMT3B silencing, in turn, led to increased CDKN2B expression. We also observed significant growth arrest in subcutaneous tumors formed in nude mice by QBC939 cells overexpressing miR-29b. These findings suggest miR-29b functions as a tumor suppressor in cholangiocarcinoma by relieving DNMT3B-mediated repression of CDKN2B expression.

Bsep expression\xa0in hilar cholangiocarcinoma of rat model

Develop a rat model of hilar cholangiocarcinoma for detecting bile salt export pump (Bsep) expression in hilar cholangiocarcinoma tissues, in order to provide a new therapeutic target for the gene therapy of hilar cholangiocarcinoma. Sixty male Wistar rats (body weight, 190 ± 8 g) were randomly divided into three groups (the experimental group, the control group and the sham operation group, n = 20 each) as follows: The three groups were fed a standard diet, the experimental group was injected by cholangiocarcinoma QBC939 cell suspension along the hilar bile duct into the bile duct bifurcation with microsyringe, the control group was injected by normal saline, the sham operation group did not inject anything. Every day assess the rats’ mental state, diet, and motion by using Basso–Beattie–Bresnahan and combined behavioral score. At 4 weeks, one rat of the experimental group was sacrificed after it was administered anesthesia, and we recorded changes in hilar bile duct size, texture, and form. This procedure was repeated at 6 weeks. After 6 weeks, hilar cholangiocarcinoma developed only in the experimental group, thereby establishing an experimental model for studying QBC939-induced hilar cholangiocarcinoma. Tumor formation was confirmed by pathological examination, and hilar bile duct tissues were harvested from both the groups. A real-time polymerase chain reaction assay and an immunohistochemical assay were used to analyze the expression of Bsep in hilar bile duct tissues of each group. From the second week, the rats in experimental group began to eat less, and their body mass decreased compared with control group and sham operation group. After 6 weeks, we detected hilar cholangiocarcinoma in the hilar bile duct tissues of 18 rats (90%) in the experimental group. In the experimental group with hilar cholangiocarcinoma, we found that the levels of total cholesterol, total bilirubin, and direct bilirubin were higher compared with those in the control group and sham operation group. Simultaneously, muddy stones emerged from the bile ducts of rats in the experimental group. The Bsep/Gapdh mRNA ratio in hilar cholangiocarcinoma, control group and sham operation group differed markedly. Light microscopy revealed a granular pattern of Bsep protein expression which reacted with the anti-Bsep antibody. Each section was randomly divided into six regions, with 80 cells were observed in every region. Sections with > 10% positive cells were designated positive, Sections with < 10% positive cells were designated negative. Each group included 4800 cells. In the experimental group, 1200 cells (25%) were positive, in the control group, 3648 cells (76%) were positive and in the sham operation group 3598 cells (75%) were positive, and this difference was statistically significant. Bsep expression significantly decreased in hilar cholangiocarcinoma of rats than those in control group and sham operation group, suggesting that drugs targeting Bsep are a new strategy for hilar cholangiocarcinoma.

Anticancer Effect of Radix Astragali on Cholangiocarcinoma In Vitro and Its Mechanism via Network Pharmacology.

BackgroundThis study used network pharmacology method and cell model to assess the effects of Radix Astragali (RA) on cholangiocarcinoma (CCA) and to predict core targets and molecular mechanisms.Material/MethodsWe performed an in vitro study to assess the effect of RA on CCA using CCK8 assay, the Live-Cell Analysis System, and trypan blue staining. The components and targets of RA were analyzed using the Traditional Chinese Medicine Systems Pharmacology database, and genes associated with CCA were retrieved from the GeneCards and OMIM platforms. Protein–protein interactions were analyzed with the STRING platform. The components–targets–disease network was built by Cytoscape. The TIMER database revealed the expression of core targets with diverse immune infiltration levels. GO and KEGG analyses were performed to identify molecular-biology processes and signaling pathways. The predictions were verified by Western blotting.ResultsConcentration-dependent antitumor activity was confirmed in the cholangiocarcinoma QBC939 cell line treated with RA. RA contained 16 active compounds, with quercetin and kaempferol as the core compounds. The most important biotargets for RA in CCA were caspase 3, MAPK8, MYC, EGFR, and PARP. The TIMER database revealed that the expression of caspase3 and MYC was related with diverse immune infiltration levels of CCA. The results of Western blotting showed RA significantly influenced the expression of the 5 targets that network pharmacology predicted.ConclusionsRA is an active medicinal material that can be developed into a safe and effective multi-targeted anticancer treatment for CCA.

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.

Inhibiting the proliferation and metastasis of hilar cholangiocarcinoma cells by blocking the expression of vascular endothelial growth factor with small interfering RNA.

The aim of the present study was to investigate whether the proliferation and metastasis of hilar cholangiocarcinoma cells can be suppressed and whether apoptosis can be induced by small interfering RNA (siRNA) repression of vascular endothelial growth factor (VEGF). siRNA sequences targeting the VEGF gene were designed and the human hilar cholangiocarcinoma QBC939, HCCC-9810 and RBE cell lines were transfected with VEGF-siRNA plasmids for 48 h. Reverse transcription-quantitative polymerase chain reaction and western blotting measured the levels of VEGF-A, VEGF-C and matrix metalloproteinase 2 (MMP2) mRNA expression and protein content. The cell invasion potential was evaluated using the Transwell invasion and migration assay and the MTT assay was employed to detect the proliferation of hilar cholangiocarcinoma cells. Flow cytometry was used to quantify cell apoptosis and necrosis. Following the transfection of VEGF-siRNA, a significant reduction of mRNA and protein levels of VEGF-A, VEGF-C and MMP2 was observed in the hilar cholangiocarcinoma cells. The invasion, migration and proliferation of tumor cells were also notably decreased. The rate of tumor cell apoptosis was increased in the VEGF-siRNA group (15.42%) compared with the non-siRNA control (2.22%) and the negative control (2.71%) groups. It was concluded that blocking the expression of VEGF via VEGF-siRNA effectively inhibited the invasion, migration and proliferation, and induced apoptosis in hilar cholangiocarcinoma cells. These observations suggested that targeting VEGF with RNAi may be an effective therapeutic strategy for treating hilar cholangiocarcinoma.

The effect of docosahexaenoic acid combined with cyclooxygenase-2 selective inhibitor NS-398 on the apoptosis of bile duct cancer cells QBC939

Objective To investigate the effect of docosahexaenoic acid (DHA) combined with cyclooxygenase-2 (COX-2) selective inhibitor NS-398 on the apoptosis of cholangiocarcinoma QBC939 cells and the mechanism. Methods In vitro, cholangiocarcinoma QBC939 cells were treated with 0, 15, 30, 45, 60 and 75 μg/ml DHA with 0, 25, 50, 100, 150 and 200 μmol/L NS-398, respectively. The absorbances of the QBC939 cells were measured by CCK8 and its growth inhibition ratios were analyzed. Flow cytometry was applied to detect cell apoptosis. The level of β-catenin and COX-2 mRNA and protein were measured by real-time PCR, immunocytochemistry and enzyme-linked immunoadsordent assay, respectively. Results DHA combined with NS-398 could significantly suppress the growth of QBC939 cells (P<0.05). When the concentration of DHA went up to 45 μg/ml and NS-398 was 100 μmol/L, the relative growth inhibition rate of QBC939 cells was 90.0%. If the concentrations were increased, the result showed no significant differences. Furthermore, flow cytometry analysis indicated that DHA combined with NS-398 could induce QBC939 cells apoptosis at the early stage, and the apoptosis rate was significantly different between the experimental and control groups (P<0.01). Real-time PCR showed low β-catenin and COX-2 expression in QBC939 cells disposed by DHA combined with NS-398, and their expression were significantly different between the experimental and control groups (P<0.01). Immunocytochemistry and ELISA demonstrated that DHA combined with NS-398 could decrease β-catenin and COX-2 protein expression in QBC939 cells. Conclusion DHA combined with NS-398 induced apoptosis and inhibited proliferation of cholangiocarcinoma cells QBC939 in vitro through targeting β-catenin and COX-2. Key words: Docosahexaenoic acid (DHA); NS-398; Cholangiocarcinoma; Apoptosis; β-catenin; Cyclooxygenase-2 (COX-2)

Effect of microRNA-148b and microRNA-152 on proliferation and apoptosis of cholangiocarcinoma cells

Objective To investigate the microRNA (miRNA, miR)-148b and miR-152 expression and their biological functions in cholangiocarcinoma cells. Methods The expression levels of miR-148b and miR-152 in cholangiocarcinoma simples and QBC939 cells were detected using real-time quantitative polymerase chain reaction (Real-time PCR). After transfection with miR-148b/miR-152 mimics, cell proliferation, cell cycle and apoptosis were assessed using methyl thiazol tetrazolium (MTT) method, clone formation test and flow cytometry, respectively. Results The expression levels of miR-148b and miR-152 were significantly down-regulated [(2.75±0.16) folds and (3.05±0.18) folds] in cholangiocarcinoma samples as compared with normal samples (P=0.013, 0.019, respectively), and [(2.52±0.03) folds and (3.96±0.02) folds] in QBC939 cells as compared with H-69 cells (P=0.018, 0.012, respectively). Overexpression of miR-148b or miR-152 could significantly inhibit cell proliferation [(38.49±0.31)% and (52.69±1.01)%, respectively], and suppress cell clone formation (P=0.027, 0.017, respectively), block cell cycle in the S phase and induce cell apoptosis. Conclusion MiR-148b and miR-152 were down-regulated in cholangiocarcinoma cells, and overexpression of miR-148b and miR-152 could inhibit cell proliferation and induce cell apoptosis. Key words: MicroRNA-148b; MicroRNA-152; Cholangiocarcinoma; Proliferation; Apoptosis

PF-2341066 combined with celecoxib promotes apoptosis and inhibits proliferation in human cholangiocarcinoma QBC939 cells.

Intrahepatic cholangiocarcinoma (ICC) is a malignant tumor with high incidence and an average age of onset of 50-70 years old. However, at present there is no effective treatment for this disease. The aim of the present study was to investigate the effects of a c-Met inhibitor, PF-2341066 and a cyclooxygenase-2 (COX-2) inhibitor, celecoxib, on c-Met and COX-2 expression, proliferation and apoptosis. The results demonstrated that c-Met and COX-2 are highly expressed in hepatobiliary calculus with cholangiocarcinoma. PF-2341066 was able to downregulate the expression of c-Met and COX-2 in a dose-dependent manner at the mRNA and protein levels in human cholangiocarcinoma QBC939 cells. Furthermore, combined treatment with PF-2341066 with celecoxib downregulated the mRNA expression of both genes, inhibited cell proliferation and promoted cell apoptosis. It was also demonstrated that PF-2341066 and celecoxib treatment was able to restrict the expression of vascular endothelial growth factor (VEGF). The results of the present study suggest that PF-2341066 and celecoxib may inhibit the development of cholangiocarcinoma by downregulating the expression of c-Met and COX-2 to inhibit cell proliferation, promote apoptosis and prevent VEGF-mediated tumor angiogenesis. Co-treatment with PF-2341066 and celecoxib may be a potential therapeutic strategy for hepatobiliary calculus with cholangiocarcinoma.

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.

Autophagy inhibitor chloroquine increases sensitivity to cisplatin in QBC939 cholangiocarcinoma cells by mitochondrial ROS

The tumor cells have some metabolic characteristics of the original tissues, and the metabolism of the tumor cells is closely related to autophagy. However, the mechanism of autophagy and metabolism in chemotherapeutic drug resistance is still poorly understood. In this study, we investigated the role and mechanism of autophagy and glucose metabolism in chemotherapeutic drug resistance by using cholangiocarcinoma QBC939 cells with primary cisplatin resistance and hepatocellular carcinoma HepG2 cells. We found that QBC939 cells with cisplatin resistance had a higher capacity for glucose uptake, consumption, and lactic acid generation, and higher activity of the pentose phosphate pathway compared with HepG2 cells, and the activity of PPP was further increased after cisplatin treatment in QBC939 cells. It is suggested that there are some differences in the metabolism of glucose in hepatocellular carcinoma and cholangiocarcinoma cells, and the activation of PPP pathway may be related to the drug resistance. Through the detection of autophagy substrates p62 and LC3, found that QBC939 cells have a higher flow of autophagy, autophagy inhibitor chloroquine can significantly increase the sensitivity of cisplatin in cholangiocarcinoma cells compared with hepatocellular carcinoma HepG2 cells. The mechanism may be related to the inhibition of QBC939 cells with higher activity of the PPP, the key enzyme G6PDH, which reduces the antioxidant capacity of cells and increases intracellular ROS, especially mitochondrial ROS. Therefore, we hypothesized that autophagy and the oxidative stress resistance mediated by glucose metabolism may be one of the causes of cisplatin resistance in cholangiocarcinoma cells. It is suggested that according to the metabolism characteristics of tumor cells, inhibition of autophagy lysosome pathway with chloroquine may be a new route for therapeutic agents against cholangiocarcinoma.

Natural compound oblongifolin C inhibits autophagic flux, and induces apoptosis and mitochondrial dysfunction in human cholangiocarcinoma QBC939 cells.

The compounds, which are obtained from natural plants or microbes may offer potential as one of the strategies for the management of cholangiocarcinoma. Oblongifolin C (OC), a natural small molecule compound extracted and purified from Garcinia yunnanensis Hu, can activate the mitochondrial apoptotic pathway in human cervical cancer cells. However, the direct effects of OC on cholangiocarcinoma cells are not well defined. The effect of OC on cell apoptosis and its underlying mechanisms were investigated in cultured QBC939 cells by the methyl thiazol tetrazolium assay, mitochondrial membrane potential, ATP content and western blot analysis. The present study reported that the in vitro treatment of human cholangiocarcinoma QBC939 cells with different concentrations (5, 10, 20 and 40 μM) of OC decreased cell viability and induced apoptosis in a dose-dependent manner. The results of the present study also showed that OC-induced QBC939 cell apoptosis was mediated through the inhibition of autophagy and mitochondrial dysfunction (MtD). Additionally, inhibiting autophagy increased OC-induced apoptosis and MtD, whereas exposure to the autophagy inducer, rapmycin, attenuated these changes. Together, the results of the present study are the first, to the best of our knowledge, to identify OC as a chemotherapeutic agent against human cholangiocarcinoma QBC939 cells in vitro via the regulation of autophagy and MtD.

The mechanism of γ-aminobutyric acid inhibitory on the growth of cholangiocarcinoma cell line

Objective To explore the molecular mechanism of cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signaling pathway in γ-aminobutyric acid inhibitory on the growth of cholangiocarcinoma QBC939 cell line. Methods QBC939 cells were cultured in different groups and treated withγ-aminobutyric acid (GABA), GABA+ 8 Br (cAMP agonists), GABA+ H89 (PKA antagonist) for 48 hours. (4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to determine the proliferation of QBC939 cells. Annexin V-fluoresceine isothiocyanate (FITC)/propidium iodide (PI) binding assay was used to detect apoptosis in the QBC939 cells. Enzymelinkedimmunosorbentassay (ELISA) assay was detected to the expression of cAMP and PKA. Western blotting was applied to check the expression of PKAI and PKAII and extracellular regulated protein kinases (ERK) proteins in different groups of QBC939 cells. Animal models of cholangiocarcinoma bearing nude mice were established by subcutaneous injection of QBC939 cells and randomized into 2 groups: control and GABA-treated groups. The effect of GABA was evaluated after 5 weeks, including tumor volume. The expression of PKA Ⅰ and PKA Ⅱ and ERK was detected by Western blotting in xenograft tumors. Results MTT and flow cytometry (FCM) assays all showed that the effect of GABA inhibitory on the proliferation and induced apoptosis of QBC939 cells could be antagonized by H89, but not 8 Br. GABA significantly increased the content of cAMP and PKA, the content of cAMP was enhanced by 8 Br, the content of PKA was antagonized by H89. Western blotting analysis showed that GABA significantly down-regulated the expression of PAK I protein (0.087 8±0.003 0 vs. 0.152 1±0.003 0, t=29.687, P<0.05), up-regulated the expression of PKAⅡ, and also decreased the expression of ERK protein protein (0.368 3±0.007 0 vs. 0.468 7±0.010 0, t=13.647, P<0.05). Xenograft tumor volume [ (0.500±0.020 vs. 0.320±0.030) cm3,t=15.354, P<0.05]. The expression of PKAI and ERK were significantly decreased, and PKA II was increased in GABA-treated group as compared with control group. Conclusion GABA may inhibit the growth of cholangiocarcinoma cells QBC939 through cAMP/PKA, down-regulated PAK I, up-regulated PKA II, and decreased the expression of the ERK perhaps is one of its anti-tumor mechanisms. Key words: Bile duct neoplasms; Gamma-aminobutyric acid; Cyclic adenosine monophosphate; Protein kinase A; Extracellular regulated protein kinases

Influence of Photodynamic Therapy on Apoptosis and Invasion of Human Cholangiocarcinoma QBC939 Cell Line.

To investigate the effect of photodynamic therapy (PDT) mediated by hematoporphyrin derivative (HPD) on apoptosis and invasion of cholangiocarcinoma QBC939 cell lines. In vitro cultured cholangiocarcinoma QBC939 cell line was exposed to 2, 4, 6, 8, 10, 12, and 14 μg/ml HPD with 5, 10, and 15 J/cm2 light intensity, respectively. The optical density at 450 nm of the QBC939 cells was measured by CCK8 assay and its growth inhibition ratio was calculated. Flow cytometry and transwell migration assay were applied to detect cell apoptosis and invasion respectively. RT-PCR and immunocytochemistry analyses were used to detect expressions of vascular endothelial growth factor-C (VEGF-C), cyclooxygenase-2 (COX-2), and proliferating cell nuclear antigen (PCNA). Enzyme-linked immunosorbent assay (ELISA) was carried out to examine the secretion of VEGF-C and COX-2 in QBC939 cells. Exposure to HPD-PDT can significantly suppress the growth of QBC939 cells (all P<0.05). HPD-PDT can promote apoptosis of QBC939 cells at the early stage. When the concentration of HPD was 2 μg/ml and light irradiation was 5 J/cm2, HPD-PDT had no obvious inhibitory effect on QBC939 cell growth, but can obviously inhibit cell invasion, and significant difference was observed between the HPD-PDT and control groups (P<0.01). The HPD-PDT can reduce the mRNA and protein expressions of VEGF-C, COX-2, and PCNA, and decrease the secretion of VEGF-C and COX-2 in QBC939 cells. PDT could promote apoptosis and inhibit growth and invasion of cholangiocarcinoma cells QBC939 in vitro.

Upregulation of the MCL-1S protein variant following dihydroartemisinin treatment induces apoptosis in cholangiocarcinoma cells.

The aim of the present study was to determine whether dihydroartemisinin (DHA) induces apoptosis in the human cholangiocarcinoma QBC939 cell line through the regulation of myeloid cell leukemia-1 (MCL-1) expression. The inhibitory rates of DHA on QBC939 cell proliferation and the effects of DHA on the cell death rates at various DHA concentrations and following various treatment times were examined. The rate of apoptosis and cell cycle changes following DHA treatment were examined and the changes in the expression of MCL-1 mRNAs and MCL-1 proteins following DHA treatment were also examined. The MTT assay and trypan blue staining demonstrated that DHA significantly inhibited the proliferation (P<0.05) and promoted the death of QBC939 cells (P<0.05). The DNA ladder assay and flow cytometry (FCM) analysis demonstrated that the rate of apoptosis in the experimental group was significantly increased following DHA treatment (P<0.01). FCM analysis also demonstrated that DHA treatment led to a reduction in the percentage of QBC939 cells in the G0/G1 and G2/M phases, and the majority of the DNA-treated cells were arrested in the S phase of the cell cycle (P<0.01). Western blot analysis demonstrated that DHA treatment significantly upregulated the expression of the pro-apoptotic MCL-1S protein. In contrast, no significant difference in the expression of the anti-apoptotic MCL-1L protein was observed following DHA treatment. DHA affected the expression of the apoptosis-associated protein MCL-1 through multiple mechanisms. DHA treatment increased the ratio of MCL-1S/MCL-1L protein, thus inducing apoptosis in cholangiocarcinoma cells.

Expression and clinical significance of PIWIL2 in hilar cholangiocarcinoma tissues and cell lines.

The objective of this study was to explore the relationship between PIWI-like protein 2 (PIWIL2) and clinicopathological charac-teristics and prognosis after radical resection. To accomplish this, we analyzed PIWIL2 expression in hilar cholangiocarcinoma tissues and cell lines. PIWIL2 expression was detected by immunohistochemistry in 41 hilar cholangiocarcinoma samples and 10 control tissues. Western blotting and immunocytofluorescence were used to investigate PIWIL2 expression in the cholangiocarcinoma cell line QBC939 and the bile duct epithelial cell line HIBEpic. Univariate and multivariate surviv-al analyses were performed using the Kaplan-Meier method for hilar cholangiocarcinoma patients who underwent radical resection. PIWIL2 expression was significantly higher in the hilar cholangiocarcinoma tissues and QBC939 cells than in control tissues and HIBEpic cells, respectively (P < 0.05). Poorly and moderately differentiated cholan-giocarcinoma tissues had significantly higher PIWIL2 expression than well-differentiated tissues (P < 0.05). Univariate analysis demonstrated that high PIWIL2 expression was associated with shorter survival time after radical resection (P < 0.05). Multivariate analysis showed that PI-WIL2 expression was an independent prognostic factor after radical re-section of hilar cholangiocarcinoma (P < 0.05). PIWIL2 expression was also associated with tumor-node-metastasis stage and differentiation. PIWIL2 was an independent prognostic factor after radical resection of hilar cholangiocarcinoma.

Cell-SELEX based selection and optimization of DNA aptamers for specific recognition of human cholangiocarcinoma QBC-939 cells.

Cholangiocarcinoma (CCA) is a very aggressive biliary tract malignancy with no efficient early diagnosis and therapeutics available, so there is a call for effective molecular probes. Herein, we performed cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX) to obtain aptamers for the specific recognition of human cholangiocarcinoma QBC-939 cells. By coordinating sequence homology analysis and secondary structure analysis, we successfully obtained two aptamers with dissociation constants (Kd) in the low nanomolar range. A 23 nt truncated sequence was identified after further analysis on the secondary structure. More importantly, because hepatocellular carcinoma SMMC-7721 cells were employed as the control in the counter selection, the obtained aptamers demonstrated excellent specificity to the target cells, and no binding to several other hepatocellular carcinoma cell lines was observed. Moreover, the aptamers were initially found to recognize membrane proteins, giving them great potential in the field of biomarker discovery. These newly generated aptamers may play a key role in the early diagnosis and clinical treatment of CCA.

Expression and clinical significance of PIWIL2 in hilar cholangiocarcinoma tissues and in cell lines

Objective To explore the relationship between PIWIL2 and clinicopathological characteristics and prognosis after radical resection by detecting the expression of PIWIL2 in hilar cholangiocarcinoma tissues and in cell lines.Methods The expression of PIWIL2 was detected by immunohistochemistry in 41 hilar cholangiocarcinomas and 10 control tissues,Western-blot and immunocytofluorescent were used to investigate the PIWIL2 expression in cholangiocarcinoma cell lines QBC939 and bile duct epithelial cell lines HIBEpic.Univariate and multivariate analysis of survival was performed using the Kaplan-Meier curve in hilar cholangiocarcinoma patients who bad underwent radical resection.Result The expression of PIWIL2 in hilar cholangiocarcinoma tissues and QBC939 cells were significantly higher than the control tissues and HIBEpic cells (P ＜ 0.05).The expression of PIWIL2 in poor and moderate differentiated cholangiocarcinoma was significantly higher than the well differentiated group (P ＜ 0.05).Univariate analysis showed that high expression of PIWIL2 was associated with short-survival time after radical resection (P ＜ 0.05).Multivariate analysis showed that the expression of PIWIL2 was an independent prognostic factor after radical resection of hilar cholangiocarcinoma (P ＜ 0.05).Conclusions The expression of PIWIL2 was associated with TNM stage and differentiation; PIWIL2 was an independent factor for prognosis after radical resection of hilar cholangiocarcinoma. Key words: PIWI protein, PIWIL2 ; Hilar cholangiocarcinoma; Prognosis ; Risk factors

Fragile histidine triad (FHIT) suppresses proliferation and promotes apoptosis in cholangiocarcinoma cells by blocking PI3K-Akt pathway.

Fragile histidine triad (FHIT) is a tumor suppressor protein that regulates cancer cell proliferation and apoptosis. However, its exact mechanism of action is poorly understood. Phosphatidylinositol 3-OH kinase (PI3K)-Akt-survivin is an important signaling pathway that was regulated by FHIT in lung cancer cells. To determine whether FHIT can regulate this pathway in cholangiocarcinoma QBC939 cells, we constructed an FHIT expression plasmid and used it to transfect QBC939 cells. Protein and mRNA expression were measured by western blotting and qRT-PCR, respectively. The viability and apoptosis of QBC939 cells were then assessed using MTT assays and flow cytometry. Our results revealed that the expression of survivin and Bcl-2 was downregulated, and caspase 3 was upregulated, in cells overexpressing FHIT. In addition, FHIT suppressed the phosphorylation of Akt. The changes in cell proliferation and apoptosis were obvious in cells overexpressing FHIT which parallels that of treatment with LY294002, a potent inhibitor of phosphoinositide 3-kinases. Treatment with LY294002 further decreased the expression of survivin and Bcl-2 and increased caspase-3 levels. These results suggest that FHIT can block the PI3K-Akt-survivin pathway by suppressing the phosphorylation of Akt and the expression of survivin and Bcl-2 and upregulating caspase 3.