Cholangiocarcinoma RBE Cell Research Articles

Clonorchis sinensis legumain promotes migration and invasion of cholangiocarcinoma cells via regulating tumor-related molecules

BackgroundClonorchis sinensis infection causes serious pathological changes in the bile duct and is highly correlated with cholangiocarcinoma. The excretory–secretory products (ESP) of C. sinensis play a critical role in the oncogenesis and progression of cholangiocarcinoma, while the components and precise mechanism remain unclear. Here, we evaluated the function of C. sinensis legumain (Cslegumain) in promoting the invasion and migration of cholangiocarcinoma cells and the mechanism involved.MethodsThe structural and molecular characteristics of Cslegumain were predicted and analyzed using the online program Phyre2. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical staining were performed to test the transcriptional level of Cslegumain and its localization in the adult. Native Cslegumain was detected by western blotting assay. The effects of Cslegumain on the proliferation, invasion and migration of cholangiocarcinoma cells were checked using CCK-8 assay, Matrigel transwell assay and scratch wound healing assay. Expression levels of tumor-related molecules regulated by Cslegumain were evaluated by qRT-PCR and western blotting assay.ResultsCslegumain showed high similarity with human legumain in the secondary and tertiary structures and displayed higher transcriptional levels in the adult worm than in the metacercariae. Native Cslegumain was detected in a catalytic form and was localized mainly in the intestine of the C. sinensis adult and epithelial cells of the intrahepatic bile duct. After transfection into RBE cells, Cslegumain showed high ability in promoting the invasion and migration but not the proliferation of cholangiocarcinoma RBE cells. Furthermore, the expression levels of some molecules including E-cadherin and N-cadherin were downregulated, while the levels of α-actinin 4, β-catenin and inducible nitric oxide synthase (iNOS) were upregulated.ConclusionsOur findings indicated that Cslegumain showed very similar structures as those of human legumain and could promote the invasion and migration of cholangiocarcinoma cells by regulating some tumor-related molecules.Graphical

Design, synthesis and biological evaluation of novel 4-aminopiperidine derivatives as SMO/ERK dual inhibitors

The hedgehog (Hh) pathway is tightly related with the formation, metastasis and recurrence of various cancers, which makes it a perfect anticancer target. Smoothened (SMO) is one of its key members. Three drugs targeting the Hh pathway have been successfully used in clinic, and they are all known as SMO inhibitors. However, serious drug resistant problem has limited their clinical application. The interaction of oncogenic ERK pathway with the Hh pathway in multiple ways has been proved as one of the main factors that result in drug resistance. Dual inhibition of the Hh and ERK pathways has displayed synergistic suppression to cancer cells overexpressing both pathways. Herein, we designed and synthesized a series of novel 4-aminopiperidine derivatives as SMO/ERK dual inhibitors, and evaluated their biological activities. The results showed that compounds I-13 displayed strong inhibitory activities towards both SMO and ERK, and it also exhibited significant cytotoxicity against human cholangiocarcinoma RBE cells which overexpress both the Hh and ERK pathways. All the results indicate that compound I-13 is a promising anticancer candidate as a SMO/ERK dual inhibitor.

Isocitrate dehydrogenase 1 mutation in cholangiocarcinoma impairs tumor progression by sensitizing cells to ferroptosis.

The present study intends to clarify the hypothesis that isocitrate dehydrogenase 1 (IDH1) mutation in cholangiocarcinoma impairs tumor progression by sensitizing cells to ferroptosis through the in vitro and in vivo experiments. Cholangiocarcinoma RBE cell line was transfected with IDH1 R132C mutation plasmids and treated with erastin to induce ferroptosis, which were then microscopically photographed. Cell viability rate was calculated by trypan blue staining. The lipid ROS level was determined by using flow cytometer. The BALB/c nude mice were injected subcutaneously with IDH1 knockout (KO), WT, or R132C mutation cell line, followed by injecting erastin intraperitoneally. The tumor tissue was surgically separated for the measurement of tumor volume and weight. The results showed that IDH1 mutant RBE cell line are sensitive to erastin-induced ferroptosis, evidenced by the increased number of propidium iodide-positive cells, the decreased cell viability, and increased lipid ROS level. However, current targeted inhibitors of IDH1 mutation (AG120 and IDH305) reversed these effects caused by IDH1 mutation. The in vivo experiment showed that IDH1 mutation in cholangiocarcinoma impairs tumor progression by sensitizing cells to erastin-induced ferroptosis. This study indicated that IDH1 mutation in cholangiocarcinoma impairs tumor progression by sensitizing cells to erastin-induced ferroptosis.

Administration of Iodine-125 Seeds Promotes Apoptosis in Cholangiocarcinoma through the PI3K/Akt Pathway

Purpose. We aimed to examine the effects of 125I seeds on the gene expression of Bcl-2, Bax, and PI3K/Akt pathway components in cholangiocarcinoma cells. Methods. In vitro, human cholangiocarcinoma RBE cells were treated with 125I seeds (0.39 mCi or 0.85 mCi) for 72 h, 120 h, and 168 h. Cell proliferation and apoptosis were assessed. The expression of Bcl-2 and Bax was detected by RT-PCR, and Western blotting was carried out to explore changes in Akt activity. Result. 125I seeds inhibited the proliferation of RBE cells. The apoptosis rate of the RBE cells in the low-activity group was significantly higher than that in the high-activity group at 120 h and 168 h, while no difference was found between the two groups at 72 h. After 120 h of culture, the gene expression of Bcl-2 and Bax decreased in both groups, the ratio of Bcl − 2 / Bax in the low-activity group decreased, and the PI3K/Akt signaling pathway was inhibited in both groups. Conclusion. 125I seeds affect the proliferation and apoptosis of cholangiocarcinoma cells in a dose-dependent manner. The therapeutic effect of low-activity 125I seeds on cancer cells may be better. 125I seed brachytherapy may promote the apoptosis of cholangiocarcinoma cells by inhibiting the PI3K/Akt signaling pathway and regulating the Bcl − 2 / Bax ratio.

MicroRNA-146b-5p suppresses cholangiocarcinoma cells by targeting TRAF6 and modulating p53 translocation

BackgroundIn view of the poor prognosis and high mortality of cholangiocarcinoma, there is a need for new therapeutic strategies. This study aims to reveal the biological function of miR-146b-5p in cholangiocarcinoma cell and its possible mechanism. MethodsThe expression level and prognostic information on miR-146b-5p in cholangiocarcinoma were obtained in TCGA database. The biological function of miR-146b-5p on proliferation and vitality of cholangiocarcinoma cell HUCCT-1 was examined by EdU and MTT assay, and the apoptosis of HUCCT-1 cells transfected with miR-146b-5p mimic, mimic control, inhibitor, inhibitor control was detected by flow cytometry analysis. The western blot was done to evaluate the effect of miR-146b-5p targeting substrate and the expression of p53 in whole-cell protein and mitochondria fractions. ResultsOur finding revealed that miR-146b-5p expression in patients with CHOL was lower than the normal group(p＜0.001). MiR-146b-5p expression was down-regulated in human cholangiocarcinoma HUCCT-1 and RBE cells compared to normal control HIBEC and other cancer cells. The miR-146b-5p mimic could inhibit HUCCT-1 cell proliferation (p＜0.05) and promote HUCCT-1 cell apoptosis significantly (p＜0.05). The results of western blot showed that miR-146b-5p mimic could directly target TRAF6 3′UTR region and up-regulate the expression of p53 in mitochondria and miR-146b-5p inhibitor could down-regulated the level of p53 in mitochondria. ConclusionMiR-146b-5p is a cholangiocarcinoma suppressor by inhibiting cell proliferation and promoting cell apoptosis with targeting TRAF6, possibly via modulating p53 translocation to mitochondria.

Preparation, characterization and primary evaluation of trilayered biliary stent films for anti-cholangiocarcinoma and anti-biofilm formation

Excessive growth of tumor within biliary wall and formation of biofilm on inner surface of stent can cause restenosis or even obstruction after stent implantation. Therefore, it is important and valuable to develop a new biliary stent for anti-cholangiocarcinoma and anti-biofilm formation. Herein, we designed, prepared and primarily evaluated a new trilayered film for biliary stents consisting of one poly (lactic acid) (PLA) layer loaded with anti-tumor paclitaxel (PTX layer), one middle PLA isolation layer (isolation layer) and one PLA layer loaded with antimicrobial ofloxacin (OFLX layer). It is postulated that the PTX layer releases drug towards biliary wall with tumor, the OFLX layer releases drug towards lumen of bile duct and the isolation layer is used to separate from the PTX layer and the OFLX layer and facilitate drug release in unidirectional way. The prepared trilayered films were characterized in terms of morphology, microstructure, crystallinity and biodegradability. It was found that the films could effectively tune drug release by addition of different amounts of drug or PEG, release PTX and OFLX in opposite directions, effectively inhibit the proliferation of human cholangiocarcinoma RBE cells, the adherence of E. coli and S. aureus and the formation of biofilm in vitro. It is potential that the trilayered films can be used to fabricate a new biliary stent with a dual function of anti-cholangiocarcinoma and anti-biofilm formation.

The effect of gemcitabine combined with AMD3100 applying to cholangiocarcinoma RBE cell lines to CXCR4/CXCL12 axis

Purpose To evaluate the effect of AMD3100 treatment to cholangiocarcinoma by analyzing the relationship between them, and provide experimental evidence for whether AMD3100 can become a clinical treatment drug for cholangiocarcinoma. Materials and methods Cholangiocarcinoma RBE cell lines were used in this study. MTT cell proliferation test was used for evaluating the effect of gemcitabine and AMD3100 to cell. CXCR4, N-cadherin, VEGF-C and MMP-9 were detect by RT-PCR and western. Transwell was used for evaluating the invasion effect. Results We demonstrated that as the concentration of gemcitabine increasing from 0.33, 3.33 to 33.33 uM, the cell survival rate was 76.65%, 71.40%, 52.25%, respectively. RT-PCR and Western blot that gemcitabine could affect the expression of CXCR4 protein and the level of mRNA transcription in a dose-dependent manner. N-cadherin VEGF-C, MMP-9 mRNA transcription level showed a significant upward trend in gemcitabine group. In Transwell test, the number of cells in the gemcitabine group was significantly higher than that in the no-medication group (p < .05), the AMD3100 group and the combination group of gemcitabine and AMD3100, the difference between the no-medication group and the AMD3100 monotherapy group was not significant, and the combination group was between them. Conclusions This study showed that gemcitabine significantly inhibited the growth of cholangiocarcinoma RBE cells in a dose-dependent manner, and gemcitabine can affect the expression of CXCR4, N-cadherin, VEGF-C, MMP-9 protein and mRNA. Cell invasion and metastasis-related factors decreased after AMD3100 combined with gemcitabine.

Thymidine phosphorylase promotes angiogenesis and tumour growth in intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer, and thymidine phosphorylase (TP) is a regulator of angiogenesis. To investigate the biological activities of TP in ICC, we established human cholangiocarcinoma RBE cell lines overexpressing TP or silencing TP. Overexpression of TP enhanced viability, suppressed apoptosis and increased tube formation in human umbilical vein endothelial cells, while downregulation of TP reversed these effects. Moreover, an orthotopic xenograft mouse model of ICC was built to further explore TP's function in ICC in vivo. Histological analysis using H&E, TUNEL and Ki67 staining showed that TP promoted tumour growth and inhibited cell apoptosis. Immunostaining for CD31 revealed an elevation in microvessel density in the presence of TP. Besides, upregulation of TP increased the expression of vascular endothelial growth factor, basic fibroblast growth factor, interleukin-8 and tumour necrosis factor alpha. In contrast, TP knockdown inhibited tumour growth, suppressed microvessel formation and decreased the expression of angiogenesis-related proteins. Therefore, we suggest that TP promotes angiogenesis and tumour growth in ICC, which can be a potent therapeutic target for ICC treatment.

Cancer associated fibroblasts promotes cancer metastasis in cholangiocarcinoma RBE cells

Objective Cancer-associated fibroblasts (CAF) have been reported to promote carcinogenesis and tumor progression. However, the role of cancer-associated fibroblasts on the metastasis of cholangiocarcinoma has not been clarified. The aim of this study is to investigate the effect of cancer-associated fibroblasts on progression of cholangiocarcinoma RBE cell in vitro. Methods Cancer-associated fibroblasts were isolated from the tumor tissue of patients. Then, we established primary cultures of CAF and peri-tumor fibroblasts (PTF) from patients with resected cholangiocarcinoma. Flow cytometric analysis, Western blotting and transwell assay were used in our study. Results The results indicated a higher expression of fibroblast secreted protein-1, fibroblasts activating proteinα, α-Smooth muscle actin and Vimentin in CAF compared to that in PTF. Flow cytometric analysis and transwell assay indicated that conditioned medium from CAF cell cultures increased remarkably the proliferative activity of cholangiocarcinoma RBE cell (5.97±1.59)% vs. (1.59±0.35)%, promoted RBE invasion [24 h, (86.76±6.11)% vs. (51.33±4.04)%] and migration [24 h (103.33±5.73)% vs. (87.00±4.85)%] but had no influence on apoptosis of RBE. Conclusion Our study showed that CAF played an important role in reinforcing cholangiocarcinoma metastasis, suggesting as a therapeutic target in cholangiocarcinoma. Key words: Cancer-associated fibroblasts; Proliferation; Invasion; Migration; Cholangiocarcinoma

Induction of cell-cycle arrest and apoptosis in human cholangiocarcinoma cells by pristimerin.

Pristimerin, a triterpenoid isolated from Celastraceae and Hippocrateaceae, is known to induce cytotoxicity in several cancer cell lines. However, whether pristimerin can induce apoptosis in cholangiocarcinoma cells and the underlying mechanism remain unexplored. We assessed the function of human cholangiocarcinoma QBC and RBE cell lines using various experimental methods such as the cell viability assay to elucidate the viability of cells, flow cytometry to detect the death rate of cells, and Western blot analysis to evaluate the expression of cell cycle-related proteins and autophagy-related proteins. Human cholangiocarcinoma QBC cells were transplanted to nude mice to establish an animal model, and the effect of pristimerin on tumor growth in this model was observed. QBC and RBE cell lines treated with pristimerin (0, 5, 10, and 20 μmol/L) demonstrated the induction of apoptosis in a dose-dependent manner. The cell viability assay revealed a reduction in the cell viability with an increase in the pristimerin concentration. Similarly, flow cytometry revealed a gradual increase in the cell death rate with an increase in the pristimerin concentration. In addition, pristimerin significantly lowered the expression of apoptosis-related proteins (Bcl-2, Bcl-xL, and procaspase-3), but increased the Bax expression. Furthermore, pristimerin resulted in the G0/G1 cell-cycle arrest, reducing the expression of cell cycle-related proteins (cyclin E, CDK2, and CDK4), and increased the expression of autophagy-related proteins (LC3) in QBC cell line. Treatment with pristimerin could inhibit tumor growth in the nude mouse model. Overall, this study suggests the potential effect of pristimerin on the cell-cycle arrest and apoptosis in human cholangiocarcinoma cells.

Effects of Transient Receptor Potential Melastain 7 on proliferation and apoptosis of cholangiocarcinoma RBE cells

Effects of Transient Receptor Potential Melastain 7 on proliferation and apoptosis of cholangiocarcinoma RBE cells

Evaluation of NCAM and c-Kit as hepatic progenitor cell markers for intrahepatic cholangiocarcinomas

Intrahepatic cholangiocarcinomas (ICCs) are primary liver malignancies and are the second most common type of malignancy after hepatocellular carcinoma. ICCs are heterogeneous in clinical features, genotype, and biological behavior, suggesting that ICCs can initiate in different cell lineages. We investigated intrahepatic cholangiocarcinoma RBE cell lines for the markers neural cell adhesion molecule (NCAM) and c-Kit, which possess hepatic progenitor cells properties. NCAM + c-Kit + cells were tested for hepatic progenitor cell properties including proliferation ability, colony formation, spheroid formation, and invasiveness in NOD/SCID mice. The Agilent Whole Human Genome Microarray Kit was used to evaluate differences in gene expression related to stem cell signaling pathways between NCAM + c-Kit + and NCAM-c-Kit- subset cells. Microarray results were further confirmed by real-time RT-PCR. NCAM + c-Kit + cells showed hepatic progenitor cell-like traits including the abilities to self-renew and differentiate and tumorigenicity in NOD/SCID mice. Differences were observed in the expression of 421 genes related to stem cell signaling pathways (fc ≥ 2 or fc ≤ 0.5), among which 231 genes were upregulated and 190 genes were downregulated. NCAM + c-Kit + subset cells in RBE may have properties of hepatic progenitor cells. NCAM combined with c-Kit may be a valuable marker for isolating and purifying ICC stem/progenitor cells.

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.

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

Metformin potentiates the anticancer activities of gemcitabine and cisplatin against cholangiocarcinoma cells in vitro and in vivo.

Metformin, an oral biguanide drug used to treat type2 diabetes, has displayed anticancer activities in several types of cancer cells. The combination of gemcitabine and cisplatin is the standard chemotherapy regimen for cholangiocarcinoma, but its benefit is limited. The present study aimed to investigate whether metformin could enhance the activities of gemcitabine and cisplatin against cholangiocarcinoma, and the underlying mechanisms. Metformin inhibited the proliferation of human cholangiocarcinoma RBE and HCCC-9810 cells and induced cell cycle arrest at the G0/G1phase by increasing the activation of AMP-activated protein kinase (AMPK) pathways. Metformin upregulated the expression of p21Waf1 and p27kip1, and downregulated the expression of cyclinD1, a key protein required for cell cycle progression. The combination of gemcitabine and cisplatin inhibited the proliferation and induced the apoptosis of human cholangiocarcinoma cells by inducing the phosphorylation of AMPK, downregulating cyclinD1, and activating caspase-3. Administration of metformin enhanced the efficacy of gemcitabine and cisplatin to suppress the growth of cholangiocarcinoma tumors established in experimental models by inhibiting cell proliferation and inducing cell apoptosis through their effects on AMPK, cyclinD1 and caspase-3. Given that metformin has been used to treat type2 diabetes patients for over half a century due to its superior safety profile, the results presented here indicate that metformin may be a potent agent for enhancing the efficacy of gemcitabine and cisplatin in the treatment of cholangiocarcinoma.

Fascin Overexpression Promotes Cholangiocarcinoma RBE Cell Proliferation, Migration, and Invasion.

Fascin is overexpressed in various tumor tissues and is closely related to tumor metastasis and invasion. However, the role of fascin in cholangiocarcinoma RBE cells has not been clearly reported. This study aimed to establish a cholangiocarcinoma cell line with stable and high expression of fascin to observe the effect of fascin on cell proliferation, migration, and invasion. A fascin overexpression vector, pcDNA3.1-Fascin, was constructed and transfected into the human cholangiocarcinoma RBE cell line. The results of real-time polymerase chain reaction, Western blot, and immunofluorescence indicated that fascin was steadily and highly expressed in RBE cells. The results of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide and colony formation assay indicated that upregulated fascin expression could enhance cholangiocarcinoma cell proliferation. The results of wound healing assay and transwell assay indicated that fascin could promote cholangiocarcinoma cell migration and invasion, and a further study found that the nuclear factor-κB signaling pathway was activated after upregulation of fascin, whereas E-cadherin expression in these cells was significantly decreased. Additionally, E-cadherin expression was significantly increased after inhibiting nuclear factor-κB activity using inhibitor or small interfering RNA, and E-cadherin expression was decreased by fascin overexpression after nuclear factor-κB inhibition, suggesting that nuclear factor-κB signaling pathway was not involved in the regulation of E-cadherin by fascin. In summary, the results of this study demonstrated that fascin effectively promoted cholangiocarcinoma RBE cell proliferation, migration, and invasion. This study provides evidence for fascin as a potential target in the treatment of cholangiocarcinoma.

Effect of norcantharidin on human cholangiocarcinoma cells line RBE: histochemical and electron microscopic study

Objective To observe the effect of norcantharidin on cytohistochemistry and ultrastructure of human cholangiocarcinoma RBE cells, and investigate the action mechanism of norcantharidin inducing apoptosis of RBE cells. Methods The RBE cells were cultured in vitro, followed by treatment with various concentrations of norcantharidin for 24 h.Acridine orange/ethidium bromide (AO/EB)staining was used to determine the inhibition rate and morphological changes. The ultrastructural changes were studied by scanning electron microscopy and transmission electron microscopy. Results AO/EB staining showed that with increasing dose of norcantharidin, apoptosis rate of RBE cells was increased.After treatment with 40 mg/L norcantharidin for 24 h, the apoptotic cells accounted for(83.1±15.6)%.Light microscopy and scanning electron microscopy showed typical apoptotic changes, and the formation of apoptotic bodies. The nucleus crescent- like change also could be observed by transmission electron microscopy. Conclusion Norcantharidin induces the morphological and ultrastructural changes in RBE cells, which have close relationship with apoptosis and invasion. Key words: Cholangiocarcinoma; Norcantharidin; Morphology

Apoptotic Effect of Combination of Epalrestat and Palmitic Acid in Cholangiocarcinoma RBE Cells

Cholangiocarcinoma (CCA) classifies as a very deadly disease due to its detrimental effects, late diagnosis, and limited therapeutic options. AKR1B10, cloned from liver cancer, influences cell survival; the usage of epalrestat (EPS) to inhibit this gene results in the apoptosis of carcinoma cells, and utilizing palmitic acid (PA) partly reduces AKR1B10 silence-induced apoptosis. This investigation observed the combined effect of EPS and PA on the apoptosis of cholangiocarcinoma RBE cell. Our data showed that EPS treatment resulted in RBE cell growth inhibition by a concentration dependent and time independent manner. Strangely, the rescue effect of PA in EPSinduced RBE cell did not occur, and combining PA with EPS caused a synergistic effect in RBE cell apoptosis and growth inhibition in which hindering AKR1B10 activity, the loss of MMP, and the production of ROS might be involved; however, this experiment does not answer the questions of how EPS and PA affect AKR1B10 activity, MMP loss and ROS production, and the correlation between AKR1B10 inhibition and MMP loss and ROS production. This investigation concludes that administering EPS and an optimal concentration of PA in the blood by monitoring the amount of PA in the diet is a potential strategy to treat CCA patients.

Raf kinase inhibitor protein inhibits cholangiocarcinoma cell metastasis by downregulating matrix metalloproteinase 9 and upregulating tissue inhibitor of metalloproteinase 4 expression.

Cholangiocarcinoma cells originate in the biliary epithelium. The cells easily metastasize and cause relapse. The effect of Raf kinase inhibitor protein (RKIP) on the biological behavior of cholangiocarcinoma cells is not yet clear. In the present study, RKIP and cytokeratin 19 expression was detected in the extrahepatic tissues of cholangiocarcinoma patients by immunohistochemistry. RKIP small interfering (si)RNA or an RKIP-overexpressing adenoviral vector were used to infect the human cholangiocarcinoma RBE cell line. RKIP protein or gene expression was analyzed by western blotting or reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively. The cells were assayed for proliferation, apoptosis, invasion and migration. Matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 4 (TIMP-4) mRNA was assayed by RT-qPCR. RKIP expression was reduced in the extrahepatic cholangiocarcinoma tumor compared with the adjacent uninvolved peritumoral tissues. The current study revealed that RKIP expression was positively correlated with cell differentiation, but negatively correlated with lymph node or distant metastasis (P<0.05). RKIP siRNA treatment promoted RBE cell invasion, but RKIP overexpression prevented cell invasion. In the pDC316-siRNA recombinant vector group, the cells migrated more quickly compared with the siRNA-negative control group, and in the RKIP-expressing adenoviral vector group, the cells migrated more slowly compared with the adenoviral negative control group. RKIP inhibited the invasive and metastatic ability of the cholangiocarcinoma cell line, RBE, by downregulating MMP-9 and upregulating TIMP-4 mRNA expression. RKIP is negatively associated with cholangiocarcinoma distant metastasis and prevents cholangiocarcinoma cell metastasis through downregulating MMP-9 expression and upregulating TIMP-4 expression.