Human Cholangiocarcinoma QBC939 Cells Research Articles

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.

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.

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.

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.

Photosan-II loaded hollow silica nanoparticles: Preparation and its effect in killing for QBC939 cells

Nanoparticles have been explored recently as an efficient means to deliver photosensitizers for photodynamic therapy. However, it is largely unknown if polyhematoporphyrin (C34H38N4NaO5, Photosan-II, PS) or other photosensitizers can be efficiently delivered by hollow silica nanoparticles (HSNP). Polyhematoporphyrin (C34H38N4NaO5, Photosan-II, PS) was loaded into hollow silica nanoparticles (HSNP) by one-step wet chemical-based synthetic route. Dynamic light scattering (DLS) and polydispersive index (PDI) were used for measurement of the particles size and size distribution. Transmission electron microscope and scanning electron microscopy were used for the microstructure, morphological and chemical composition analysis. Fourier transform infrared spectrometry spectra and fluorescence emission spectrum were obtained. The photobiological activity of the PS-loaded HSNP was evaluated on human cholangiocarcinoma QBC939 cells. The cellular viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptotic and necrotic cells were measured by flow cytometry. DLS measurements showed that the size of the particles is in the range of 25-90 nm. PDI of the PS-loaded HSNP is 0.121 ± 0.01, indicating that samples have excellent quality with narrow size distribution to monomodal systems. In MTT assay, PS-loaded HSNP and free PS of the same concentration killed about 95.3% ± 2.0% and 55.7% ± 1.9% of QBC939 cells, respectively. The flow cytometry demonstrated that the laser induced cell death with PS-loaded HSNP was much more severe than that of free PS (P<0.05). Photosan-II-loaded hollow silica nanoparticles not only can quickly deliver Photosan-II into cells but also can reach a more high concentration than free Photosan-II. HSNP is a desirable vehicle and the release system that shows promises for photodynamic therapy use, which not only improve the aqueous solubility, stability and transport efficiency of PS, but also increase its photodynamic efficacy compared to free PS.

Over-Expression of Nerve Growth Factor-β in Human Cholangiocarcinoma QBC939 Cells Promote Tumor Progression

AimsIt has been shown that nerve growth factor-β (NGF-β) promoted the initiation and progression of many tumors, and we have previously demonstrated that the expression of NGF-β was associated with tumor stage, nerve infiltration and lymph node metastasis in human hilar cholangiocarcinoma. However, whether NGF-β promotes tumor progression in human cholangiocarcinoma requires further investigation. Therefore, we aimed to determine the effects of NGF-β on the progression of human cholangiocarcinoma.MethodsHuman cholangiocarcinoma QBC939 stable cell lines with over-expressed or silenced NGF-β genes were generated with pEGFP-N1-NGF-β and pGPU6/GFP/Neo-NGF-β-shRNA recombinant plasmids. Cell proliferation assay, colony formation assay, cell cycle analysis, apoptosis assay and tumorigenicity assay were performed to evaluate the role of NGF-β in the progression of human cholangiocarcinoma. In addition, human lymphatic endothelial cells were co-cultured with QBC939 culture supernatants, and the cell proliferation and migration abilities of the lymphatic endothelial cells were evaluated.ResultsForced expression of NGF-β in QBC939 cell lines promoted proliferation, colony formation and tumorigenicity in these cells and inhibited the apoptosis. However, down-regulation of NGF-β inhibited proliferation, colony formation and tumorigenicity, and increased the apoptotic rate of QBC939 cells. In addition, the NGF-β gain-of-function induced a high expression of vascular endothelial growth factor C and enhanced the proliferation and migration of lymphatic endothelial cells, while NGF-β loss-of-function showed opposite effects.ConclusionsWe concluded that NGF-β promoted tumor progression in human cholangiocarcinoma QBC939 cells. Our results provided a new concept to understand the role of NGF-β in cholangiocarcinoma progression, and might provide important information for the development of new targeted therapies in human cholangiocarcinoma.

Mucins 1‐shRNA inhibit the proliferation and HIF‐1alpha protein expression on human cholangiocarcinoma cells

To explore the effects of Mucins (MUC)1-shRNA on the proliferation and hypoxia inducible factor (HIF)-1alpha expression of human cholangiocarcinoma (CCA) QBC939 cells in vitro. MUC1-shRNA was constructed and transfected with Lipofectamine™ 2000 into cultured CCA cells. MUC1 mRNA and protein expression levels were determined by RT-PCR and Western blot, respectively. The cellular proliferation and HIF-1alpha expression of QBC939 cells were evaluated by the MTT assay and Western blot, respectively. After transfection, the expression levels of MUC1 mRNA and protein in the experimental group decreased significantly in QBC939 (P < 0.01). The proliferation of MUC1 shRNA-transfected group was 0.30 ± 0.05, 38.32 ± 1.43%, 15.18 ± 1.32%, and there were remarkable differences when compared with the control groups (P < 0.05). Significant inhibition of HIF-1alpha protein expression in MUC1 shRNA-transfected group was also discovered (P < 0.05). MUC1-shRNA could inhibit proliferation and significantly weaken HIF-1alpha protein expression of QBC939 cells, suggesting its potential as a therapeutic target of CCA.

Preparation, Characterization and Phototoxicity of Photosan-Loaded Hollow Silica Nanospheres for Photodynamic Therapy

In this study, Photosan (PS) was loaded onto hollow silica nanospheres using one-step wet chemical-based synthetic route method. Polydispersive index of the Photosan-loaded hollow Silica Nanospheres (PS-loaded NSs) is 0.121±0.01. The photobiological activity of the PS-loaded NSs was evaluated on human cholangiocarcinoma QBC939 cells. Cells were incubated with free PS or PS-loaded NSs for 2 h and then washed with phosphate-buffered saline. Culture medium was added to the wells containing the cells. Finally, the cells were exposed to red light (630 nm) with a light dose of 10J/cm2. The cellular viability was determined after 24 h of incubation. PS-loaded NSs and free PS eliminated about 96.4%±2.0% and 54.7%±1.9% of QBC939 cells, respectively. The phototoxicity was time dependent up to 2 h and concentration dependent at 1-7.5 mg/l. The cells viability decreased with the increase of the light dose in the range of 5-15 J/cm2. In conclusion, PS-loaded NSs are the release systems that promise photodynamic therapy use.

The Combined Effects of Hematoporphyrin Monomethyl Ether-SDT and Doxorubicin on the Proliferation of QBC939 Cell Lines

It is well established that both hematoporphyrin monomethyl ether-sonodynamic therapy (HMME-SDT) and doxorubicin (DOX) can induce cell apoptosis but each alone has its own limitations. To date, the combined effects of HMME-SDT and DOX on inducing cell apoptosis are little known and the mechanism for the combined effects remains poorly understood. In the present study, we reported the synergistic effects of HMME-SDT and DOX on inhibiting the proliferation of human cholangiocarcinoma QBC939 cells and investigated the mechanism of this synergy. The data from MTT assay, flow cytometer, Hoechst staining and cell arrest analysis showed that the combination of HMME-SDT and DOX exhibited higher inhibiting effects on proliferation of QBC939 cells than the sole application of HMME-SDT or DOX. In addition, the synergistic effects were shown to result from the DNA damage as demonstrated by single cell gel electrophoresis and DNA fragmentation. Furthermore, the expression of p53, Fas, Bax and activated caspase-3 protein was significantly upregulated in cells treated with HMME-SDT and DOX, whereas Bcl-2 protein was downregulated. Taken together, our data suggested that the application of HMME-SDT combined with DOX had better inhibiting effects on QBC939 cells and the effects were caused mainly by DNA damage.

Berberine Inhibits Growth and Induces G1 Arrest and Apoptosis in Human Cholangiocarcinoma QBC939 Cells

The chemotherapeutic approach using non-toxic natural products may be one of the strategies for the management of the cholangiocarcinoma. Here we report that in vitro treatment of human cholangiocarcinoma QBC939 cells with berberine, a naturally occurring isoquinoline alkaloid, decreased cell viability and induced cell death in a dose-dependent manner, which was associated with an increase in G1 arrest. Our western blot analysis showed that berberine-induced G1 cell cycle arrest was mediated through the increased expression of cyclin-dependent kinase inhibitors (Cdki) proteins (Cip1/p21 and Kip1/p27); a simultaneous decrease in Cdk2 and Cdk4 and cyclins D1, and reduced activity of the Cyclins–Cdk complex. In additional studies, treatment of QBC939 cells with different concentrations (10, 40, 80 μM) of berberine for 48 h resulted in a significant dose-dependent increase in apoptosis compared to the non-berberine–treated control, which was associated with an increased expression of pro-apoptotic protein Bax and decreased expression of anti-apoptotic proteins Bcl-2 and Bcl-xL. Together, this study for the first time identified berberine as a chemotherapeutic agent against human cholangiocarcinoma cells QBC939 cells in vitro. Further in vivo studies are required to determine whether berberine could be an effective chemotherapeutic agent for the management of cholangiocarcinoma.

Effect of transfection of WWOX gene on apoptosis of cholangiocarcinoma QBC939 cells and its mechanism

Objective To investigate the effect of transfection of WWOX gene on apoptosis of human cholangiocarcinoma QBC939 cells in vitro andin vivoand its possible mechanism.Methods The recombinant WWOX eukaryotic expression plasmid was introduced into QBC939 cells by liposome-mediated transfection.The mRNA and protein expression levels in QBC939 cells stably transfected with WWOX were detected by using quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting before and after transfection.Cell apoptosis was assessed by flow cytometry (FCM).The alteration of mitochondria membrane potential ( △ Ψm) was assayed by JC-l staining method.The expression change of bcl-2 mRNA and protein was examined by using quantitative RT-PCR and Western blotting.All the experimental subjects were divided into three groups:QBC939 cells were cultured in natural status (nature control group) ; QBC939 cells were transfected with blank plasmid (blank control group),and QBC939 cells were transfected with WWOX/pmCherry-N1 (experimental group).The three groups were subcutaneously inoculated into nude mice and the growth of xenografted tumor was observed.TUNEL was used to evaluate the apoptosis.Results QBC939 cells with stable transfection of WWOX were established.QuantitativeRT-PCR showed that the expression of WWOX mRNA was significantly enhanced and Western blotting demonstrated that WWOX protein expression was markedly increased.FCM analysis showed that the apoptosis rate after transfection was significantly promoted [(1.24 ±0.35)％ vs (1.73 ±0.48)％ vs (21.40 ±2.35)％,P＜0.01].JC-1 staining method indicated that the experimental group was loss of △Ψm [ (4.27 ± 0.64) ％ vs (4.96 ± 0.52 ) ％ vs (28.60 ± 3.94 ) ％,P ＜ 0.01 ].Quantitative RT-PCR and Western blotting showed that the expression of bcl-2 mRNA and protein was markedly decreased (P ＜O.05 ).The growth of implanted tumor of WWOX/pmCherry-N1 cells was significantly slowed down (P ＜0.05 ).Number of apoptotic cells was significantly increased in the experimental group [ (4.1 ± 0.6) ％ vs (4.4±0.5)％ vs (13.6 ± 1.5)％,P＜0.01].Conclusion WWOX gene can promote apoptosis of cholangiocarcinoma cells,which may be associated with downregulation of bcl-2 expression,and activation of the mitochondrial apoptosis pathway. Key words: Cholangiocarcinoma; Gene transfection; Apoptosis

The effect of extracts from Catharanthus roseus on cholangiocarcinoina in vitro

Objective To investigate the potential inhibitory effects of herbal Catharanthus roseus on cholangiocarcinoma in vitro. Methods The whole herbal of Catharanthus roseus was used as the material, and total alkaloid extract and water extract were obtained. The effects of the two extracts on human cholangiocarcinoma QBC939 cells were investigated. The inhibition rate of QBC939 cells was measured by methyl thiazolyl tetrazolium (MTT) method. The morphological change of cells was examined by inverted microscopy. The effect on cell cycles was observed by flow cytometry. Results The two extracts could both inhibit the growth of QBC939 cells, but the total alkaloid extract showed stronger proliferation inhibitory effect comparing to the water extract. IC50 value of total alkaloid and water extract was 0. 16 and 49. 02 mg/L, respectively. Obvious morphological changes were both observed after adding the two extracts. Both the two extracts could change the cell cycle of cholangiocarcinoma cells significantly. The ratio of G0/G1 phase cells was decreased greatly from 53. 10% to 5. 87% and that of G2/M phase cells was increased greatly from 26. 4% to 71. 8% , respectively. Compared to the cells treated with the total alkaloid extract, the ratio of Sub-G1 phase cells treated with the water extract changed greatly. The water extract could induce the apoptosis of the cells. Conclusion The extracts from Catharanthus roseus showed the inhibitory effects on proliferation of cholangiocarcinoma cells. They also could induce the morphological change and cell arrest. Key words: Catharanthus roseus; Cholangiocarcinoma; Proliferation; Apoptosis

FHIT gene transfection inhibits the proliferation and invasiveness of human cholangiocarcinoma QBC939 cells

FHIT gene transfection inhibits the proliferation and invasiveness of human cholangiocarcinoma QBC939 cells

Influence of Connexin43 on the Bystander Effect Induced by Double Suicide Genes System in Vitro and in Vivo

Objective: To observe the influence of connexin 43 (Cx43) on the bystander effect induced by cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-tk) coexpression suicide genes system in human cholangiocarcinoma QBC939 cells and transplantation tumors in nude mice. Methods: In vitro, the CD+tk+ and CD+tk+Cx+ cells were respectively treated with 5-fluorocytosine (5-Fc) and Ganciclovir (GCV). The cytotoxic effect was evaluated by MTT method. In order to investigate the influence of Cx43 on the bystander effect, the size of transplantation tumors of the CD+tk+ and CD+tk+Cx+ cells was measured before and after application of 5-Fc and GCV. Results: CD and tk genes were stably expressed in transfected QBC939 cells. The increased expression of Cx43 was determined by testing for the presence of Cx43 mRNA by RT-PCR and the presence of Cx43 protein by Western Blot in CD+tk+Cx+ cells. The killing effect of 5-Fc and GCV on CD+tk+Cx+ cells was more effective than that on CD+tk+ cells both in vitro and in vivo. Conclusion: Double suicide genes system CD/5-Fc+tk/GCV could induce remarkable killing effect on cholangiocarcinoma cells in vitro and transplantation tumors in vivo. The cotransfection of Cx43 gene could enhance the bystander effect and hence the inhibition of carcinoma cells.

Proliferative activity of bile from congenital choledochal cyst patients.

To explore the potential carcinogenicity of bile from congenital choledochal cyst (CCC) patients and the mechanism of the carcinogenesis in congenital choledochal cyst patients. 20 bile samples from congenital choledochal cyst patients and 10 normal control bile samples were used for this study. The proliferative effect of bile was measured by using Methabenzthiazuron (MTT) assay; Cell cycle and apoptosis were analyzed by using flow cytometry (FCM), and the PGE(2) levels in the supernatant of cultured cholangiocarcinoma cells were quantitated by enzyme-linked immunoabsorbent assay (ELISA). CCC bile could significantly promote the proliferation of human cholangiocarcinoma QBC939 cells compared with normal bile (P=0.001) and negative control group (P=0.002), and the proliferative effect of CCC bile could be abolished by addition of cyclooxygenase-2 specific inhibitor celecoxib (20 microM). The QBC939 cells proliferative index was increased significantly after treated with 1 % bile from CCC patient (P=0.008) for 24 h, the percentage of S phase (29.48+/-3.27)% was increased remarkably (P<0.001) compared with normal bile (11.72+/-2.70) %, and the percentage of G0/G1 phase (54.19+/-9.46) % was decreased remarkably (P=0.042) compared with normal bile (69.16+/-10.88) %, however, bile from CCC patient had no significant influence on apoptosis of QBC939 cells (P=0.719). Bile from congenital choledochal cyst patients can promote the proliferation of human cholangiocarcinoma QBC939 cells via COX-2 and PGE(2) pathway.