Human Cholangiocarcinoma Cell Research Articles

Inhibitory activities of plumbagin on cell migration and invasion and inducing activity on cholangiocarcinoma cell apoptosis

Objective: To investigate the cytotoxic, apoptotic and inhibitory activities on cell migration and invasion of plumbagin in the human cholangiocarcinoma (CCA) cell line (CL-6) in comparison with human embryonic fibroblast cell line (OUMS). Methods: Cytotoxicity activity was evaluated using MTT assay. Inhibitory effect on cell migration and invasion were investigated using label-free real-time cell analysis and QCM ECMatrix cell invasion chamber, respectively. Apoptotic activity was evaluated using flow cytometry and CellEvent™ Caspase 3/7 assay. Results: Based on results of the cytotoxicity test in CL-6 cells, 50% inhibitory concentration (IC50, Mean±SD) values of plumbagin and the standard drug 5-fluorouracil were (24.00±3.33) and (1 036.00±137.77) μmol/L, respectively. The corresponding values for OUMS cells were (57.00±5.23) and (2 147.00±209.98) μmol/L, respectively. The selectivity index was 2.28. The inhibitory activities of plumbagin on cell migration and invasion were potent and concentration-dependent with IC50 of 25.0 μmol/L and complete inhibition at 25.0 μmol/L. Flow cytometry analysis showed that plumbagin at 12.5 μmol/L (half IC50) induced CL-6 cell apoptosis (43.24% of control) through stimulation of caspase 3/7 activities. Complete cell apoptosis was observed at 12.5 μmol/L. Conclusions: The cytotoxic activity and inhibition of migration and invasion including apoptosis induction in the human CCA cell line (CL-6) suggest that plumbagin could be a promising candidate for CCA chemotherapeutics. However, its relatively low selective cytotoxic effect on CCA cells is a major concern.

Peanut testa extracts possessing histone deacetylase inhibitory activity induce apoptosis in cholangiocarcinoma cells

Previous studies demonstrated that peanut testa extracts (KK4 and ICG15042) containing natural histone deacetylase (HDAC) inhibitors inhibited the growth of several human cancer cell lines via apoptosis induction. The aims of this study were to investigate the anti-proliferative effects and the mechanism(s) responsible for apoptosis induction mediated by these peanut testa extracts in human cholangiocarcinoma cell lines (KKU-M214 and KKU-100). The anti-proliferative effects were assessed by MTT assay. Apoptotic cell death and cell cycle arrest were analyzed by flow cytometry. The caspase activities were studied using colorimetric caspase activity assay and western blot analysis. Our results revealed that KK4 and ICG15042 extracts inhibited cell proliferation of both KKU-M214 and KKU-100 cells in a dose- and time-dependent manner, with IC50 values of 38.28 ± 0.29 (KK4), 43.91 ± 1.94 (ICG15042) μg/mL for KKU-M214 and 78.40 ± 1.74 (KK4), 82.77 ± 0.94 (ICG15042) μg/mL for KKU-100 at 72 h. Apoptosis induction by these peanut testa extracts were observed in both KKU-M214 and KKU-100 cells in a concentration-dependent manner. Moreover, the percentage of cells in the sub-G1 phase was significantly increased in both KKU-M214 and KKU-100 cells. Cell cycle arrest was not observed in other cell cycle phases. Activation of caspases 8 and 3 were apparent integral parts of apoptosis induction in both cells. Both peanut testa extracts also caused down-regulation of p53, p21, Bcl-2 and pERK1/2 protein expression in these cells. These results suggest that peanut testa extracts may be potential anti-cancer agents for cholangiocarcinoma chemoprevention or chemotherapy.

Loading 5-Fluorouracil into calcined Mg/Al layered double hydroxide on AZ31 via memory effect

The clinic application of magnesium is mainly impeded by its poor corrosion resistance and implants with localized drug delivery attract tremendous interest. Here, Mg/Al layered double hydroxide (Mg-Al LDH) film was in situ prepared on AZ31 alloy by a hydrothermal process, and further calcined to form metastable mixed oxides, also named Mg/Al layered double oxide (LDO). Finally, immersing in 5-Fluorouracil (5-FU) solution, Mg/Al LDO would revert to Mg/Al LDH via a memory effect, and be loaded with 5-FU (#LDO-5FU). Comparing to AZ31 alloy, the corrosion resistance of the as-prepared #LDO-5FU sample was enhanced. The amount of released 5-FU from #LDO-5FU sample was ∼993 ng/cm2. The proliferation of human cholangiocarcinoma cell (RBE) was effectively inhibited on the surface of #LDO-5FU sample owing to the release of 5-FU. This work broadens the application of calcined LDH and provides a new strategy in the field of surface modification of magnesium alloy.

Activation of Fas/FasL pathway and the role of c-FLIP in primary culture of human cholangiocarcinoma cells

Intrahepatic cholangiocarcinoma (iCCA) represents a heterogeneous group of malignancies emerging from the biliary tree, often in the context of chronic bile ducts inflammation. The immunological features of iCCA cells and their capability to control the lymphocytes response have not yet been investigated. The aims of the present study were to evaluate the interaction between iCCA cells and human peripheral blood mononuclear cells (PBMCs) and the role of Fas/FasL in modulating T-cells and NK-cells response after direct co-culture. iCCA cells express high levels of Fas and FasL that increase after co-culture with PBMCs inducing apoptosis in CD4+, CD8+ T-cells and in CD56+ NK-cells. In vitro, c-FLIP is expressed in iCCA cells and the co-culture with PBMCs induces an increase of c-FLIP in both iCCA cells and biliary tree stem cells. This c-FLIP increase does not trigger the caspase cascade, thus hindering apoptotis of iCCA cells which, instead, underwent proliferation. The increased expression of Fas, FasL and c-FLIP is confirmed in situ, in human CCA and in primary sclerosing cholangitis. In conclusion our data indicated that iCCA cells have immune-modulatory properties by which they induce apoptosis of T and NK cells, via Fas/FasL pathway, and escape inflammatory response by up-regulating c-FLIP system.

Antiproliferative and apoptotic effects of xanthohumol in cholangiocarcinoma

Cholangiocarcinoma remains the second most prevalent hepatic neoplasm in the United States with a 5-year survival rate of less than 10%. Currently, no systemic therapy has demonstrated efficacy. Therefore, an urgent need for the identification of molecularly targeted compound(s) remains. The Notch signaling pathway has been shown to be dysregulated in cholangiocarcinoma, exhibiting hyperactivity while also possibly mediating chemotherapeutic resistance. We analyzed the effects of xanthohumol, a prenylated chalcone, on cholangiocarcinoma proliferation utilizing human cholangiocarcinoma cell lines CCLP1, SG-231 and CC-SW-1 while gaining insight into the associated mechanism. Xanthohumol potently reduced cellular proliferation, colony formation, and cell confluency in all three cell lines. Xanthohumol induced cell cycle arrest as well as apoptosis through the reduction of cell cycle regulatory proteins as well as an increase in pro-apoptotic markers (cleaved poly ADP ribose polymerase, cleaved caspase-3) and a decrease in anti-apoptotic markers (X-linked inhibitor of apoptosis and survivin). At the molecular level, xanthohumol reduced Notch1 and AKT expression in a step-wise and time-dependent fashion, with Notch1 reductions preceding AKT. Additionally, xanthohumol reduced cholangiocarcinoma growth in both CCLP-1 and SG-231 derived mice xenografts. In summary, we show that xanthohumol significantly reduced cholangiocarcinoma growth through the Notch1/AKT signaling axis. Furthermore, known pharmacokinetics and bioavailability of XN supports continued development of treatment for cholangiocarcinoma.

TGF-β signaling is an effective target to impair survival and induce apoptosis of human cholangiocarcinoma cells: A study on human primary cell cultures.

Cholangiocarcinoma (CCA) and its subtypes (mucin- and mixed-CCA) arise from the neoplastic transformation of cholangiocytes, the epithelial cells lining the biliary tree. CCA has a high mortality rate owing to its aggressiveness, late diagnosis and high resistance to radiotherapy and chemotherapeutics. We have demonstrated that CCA is enriched for cancer stem cells which express epithelial to mesenchymal transition (EMT) traits, with these features being associated with aggressiveness and drug resistance. TGF-β signaling is upregulated in CCA and involved in EMT. We have recently established primary cell cultures from human mucin- and mixed-intrahepatic CCA. In human CCA primary cultures with different levels of EMT trait expression, we evaluated the anticancer effects of: (i) CX-4945, a casein kinase-2 (CK2) inhibitor that blocks TGF-β1-induced EMT; and (ii) LY2157299, a TGF-β receptor I kinase inhibitor. We tested primary cell lines expressing EMT trait markers (vimentin, N-cadherin and nuclear catenin) but negative for epithelial markers, and cell lines expressing epithelial markers (CK19-positive) in association with EMT traits. Cell viability was evaluated by MTS assays, apoptosis by Annexin V FITC and cell migration by wound-healing assay. Results: at a dose of 10 μM, CX4945 significantly decreased cell viability of primary human cell cultures from both mucin and mixed CCA, whereas in CK19-positive cell cultures, the effect of CX4945 on cell viability required higher concentrations (>30μM). At the same concentrations, CX4945 also induced apoptosis (3- fold increase vs controls) which correlated with the expression level of CK2 in the different CCA cell lines (mucin- and mixed-CCA). Indeed, no apoptotic effects were observed in CK19-positive cells expressing lower CK2 levels. The effects of CX4945 on viability and apoptosis were associated with an increased number of γ-H2ax (biomarker for DNA double-strand breaks) foci, suggesting the active role of CK2 as a repair mechanism in CCAs. LY2157299 failed to influence cell proliferation or apoptosis but significantly inhibited cell migration. At a 50 μM concentration, in fact, LY2157299 significantly impaired (at 24, 48 and 120 hrs) the wound-healing of primary cell cultures from both mucin-and mixed-CCA. In conclusion, we demonstrated that CX4945 and LY2157299 exert relevant but distinct anticancer effects against human CCA cells, with CX4945 acting on cell viability and apoptosis, and LY2157299 impairing cell migration. These results suggest that targeting the TGF-β signaling with a combination of CX-4945 and LY2157299 could have potential benefits in the treatment of human CCA.

Apigenin inhibits growth and induces apoptosis in human cholangiocarcinoma cells.

A promising nutraceutical, apigenin, was recently revealed to exhibit biological activity in inhibiting several types of cancer. The effects of apigenin on the growth inhibition and apoptosis of the cholangiocarcinoma HuCCA-1 cell line were investigated. Protein alterations subsequent to apigenin treatment were studied using a proteomic approach. The values of 20, 50 and 90% inhibition of cell growth (IC20, IC50 and IC90) were determined by MTT cell viability assay. Apoptotic cell death was detected using two different methods, a flow cytometric analysis (Muse Cell Analyzer) and DNA fragmentation assay. A number of conditions including attached and detached cells were selected to perform two-dimensional gel electrophoresis (2-DE) to study the alterations in the expression levels of treated and untreated proteins and identified by liquid chromatography (LC)/tandem mass spectrometry (MS/MS). The IC20, IC50 and IC90 values of apigenin after 48 h treatment in HuCCA-1 cells were 25, 75 and 200 µM, respectively, indicating the cytotoxicity of this compound. Apigenin induced cell death in HuCCA-1 cells via apoptosis as detected by flow cytometric analysis and exhibited, as confirmed with DNA fragmentation, characteristics of apoptotic cells. A total of 67 proteins with altered expression were identified from the 2-DE analysis and LC/MS/MS. The cleavage of proteins involved in cytoskeletal, cytokeratin 8, 18 and 19, and high expression of S100-A6 and S100-A11 suggested that apoptosis was induced by apigenin via the caspase-dependent pathway. Notably, two proteins, heterogeneous nuclear ribonucleoprotein H and A2/B1, disappeared completely subsequent to treatment, suggesting the role of apigenin in inducing cell death. The present study indicated that apigenin demonstrates an induction of growth inhibition and apoptosis in cholangiocarcinoma cells and the apoptosis pathway was confirmed by proteomic analysis.

Abstract 2095: E7090, a novel and selective FGFR inhibitor, for the treatment of cholangiocarcinoma cells harboring FGFR2-fusion genes

Abstract Genetic abnormalities (gene fusion, mutation and amplification) of fibroblast growth factor receptor (FGFR) family members are known to cause constitutive activation of their signaling pathways, which play an important role in proliferation, survival and migration of cancer cells, as well as tumor angiogenesis. Several recent studies have identified that FGFR2 gene fusions were found in about 10% of intrahepatic cholangiocarcinoma and acted as oncogenes. E7090, an orally available FGFR1, 2 and 3 inhibitor, whose chemical-structure and basic kinase inhibitory profile have been disclosed at AACR2015, is currently under a first-in-human study (NCT02275910). The antitumor activity of E7090 against NIH3T3 and human cholangiocarcinoma cells harboring FGFR2-fusion genes was investigated. NIH3T3 cells stably infected of five FGFR2-fusion genes (FGFR2-AHCYL1, FGFR2-BICC1 type1, FGFR2-BICC1 type2, FGFR2-TXLNA, or FGFR2-KCTD1), all of which were discovered in human intrahepatic cholangiocarcinoma patients, were successfully established and exhibited anchorage-independent cell growth in soft agar assay. E7090 revealed an inhibitory activity on soft agar colony formation of these cells with IC50 values of 0.9 to 17.3 nmol/L in a dose dependent manner. E7090 also reduced activation of downstream pathways of FGFR signaling (MAPK and STAT3) in these cells. Subcutaneous transplantation of FGFR2-BICC1 type2, FGFR2-TXLNA, or FGFR2-KCTD1 expressing cells into immune-deficient mice produced tumor growth, and then oral administration of E7090 at 12.5, 25 and 50 mg/kg showed significant tumor growth inhibition. Finally, antitumor activity of E7090 was examined in a patient derived xenograft (PDX) model of human cholangiocarcinoma harboring FGFR2-BICC1 fusion gene. E7090 also showed dose-dependent antitumor activity and more than 50% of tumor regression was achieved at a dose of 50 mg/kg. In conclusion, E7090 showed potent antitumor activity against FGFR2-fusion genes found in intrahepatic cholangiocarcinoma both in vitro and in vivo models, suggesting that E7090 may provide therapeutic opportunities for cholangiocarcinoma harboring FGFR2-fusion genes. Citation Format: Saori W. Miyano, Yasuhito Arai, Junji Matsui, Tatsuhiro Shibata. E7090, a novel and selective FGFR inhibitor, for the treatment of cholangiocarcinoma cells harboring FGFR2-fusion genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2095. doi:10.1158/1538-7445.AM2017-2095

Effect of monoamine oxidase A on anti-cancer immunity of CD8+ T cells in cholangiocarcinoma

Objective To investigate the effect of monoamine oxidase A (MAOA) in cholangiocarcinoma on anti-tumor immunity of CD8+ T cells. Methods We established stably inducible MAOA expression human cholangiocarcinoma cell lines by transfecting Tet-on 3G system. CD8+ T cells were isolated from peripheral blood donated by healthy donors. After coculture with above celllines for 48 h, CD8+ T cells were analyzed by flow cytometry to detect the expression of granzyme B, perforin, programmed death 1 (PD-1), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), as well as apoptosis rate. Enzyme linked immunosorbent assay (ELISA) was applied to assess the excretion of IFN-γ from CD8+ T cells. Lactate dehydrogenase (LDH) test was performed to evaluate the cytotoxicity against the mixed cultured tumor cells by CD8+ T cells. Results CD8+ T cells in mock transfected groups expressed lower level of granzyme B (44.2% vs. 51.5%, P=0.000), perforin (51.8% vs. 74.3%, P=0.000), expressed higher level of PD-1 and CTLA-4 (81.70% vs. 21.30%, P=0.000; 16.00% vs. 8.49%, P=0.000 respectively), secreted fewer IFN-γ (7 562.0±927.5) pg/ml vs. (10 107.0±1 162.0) pg/ml, P=0.229) and had a higher apoptosis rate (26.43% vs. 13.35%, P=0.000), and the cytotoxicity CD8+ T cells was also suppressed [(12.70±1.49)% vs. (22.88±2.93)%, P=0.036]. Conclusion Down-regulation of MAOA in cholangiocarcinoma would impede anti-tumour immunity of CD8+ T cells. Key words: Monoamine oxidase A; Cholangiocarcinoma; CD8+ T cells; Tumor immunity

The effect and mechanism of chaetocin on the intrahepatic cholangiocarcinoma cells

Objective To investigate the effect of chaetocin on the intrahepatic cholangiocarcinoma CCLP-1 and explore the potential mechanism. Methods The concentration of chaetocin were divided into 4 groups: Group 1: 0 nmol/L, Group 2: 50 nmol/L, Group 3: 100 nmol/L and Group 4: 200 nmol/L, and all were co-cultured with CCLP-1 cell, respectively. Cell viability was detected by Cell Counting Kit-8 assay; Giemsa staining was used to observe the cell morphological and flow cytometry technique were used to detect cell apoptosis; The intracellular reactive oxygen species (ROS) level was detected using fluorescent probe under fluorescence microscope; Western blotting were used to measure the expression of Apoptosis signal-regulating kinase 1 (ASK-1) and c-Jun N-terminal kinase (JNKs). Results The proliferation of CCLP-1 cell was inhibited by chaetocin in dose dependent (F=102.369, P=0.000) or in time dependent (F=141.377, P=0.000). Furthermore, both have interaction (F=15.418, P=0.000). Giemsa staining showed chaetocin brought about CCLP-1 cell to present morphological characteristics of apoptosis, and flow cytometry indicated title rate of apoptosis of CCLP-1 cell were (6.42±3.10)%, (10.82±2.84)%, (13.67±1.71)% and (17.91±6.16)%, and apoptosis was significantly increased (F=4.788, P=0.034). The intracellular ROS level of treatment group (24.126±9.587) was much higher than that of control group (5.114±1.937) and had significantly difference (P=0.006). Western blotting showed the expressions of p-ASK-1 were 0.255±0.150, 0.680±0.039, 1.356±0.125 and 1.916±0.367. The expressions of JNKs were 0.493±0.233, 1.542±0.573, 1.639±0.342 and 2.480±1.001.Both proteins expressed increasingly as dose of chaetocin increased. (ASK-1: F=36.946, P=0.000, JNKs: F=5.291, P=0.027). Conclusion Chaetocin could inhibit CCLP-1 cell and induce it apoptosis via promote ROS mediate ASK-1/JNK signal. Key words: Chaetocin; Human intrahepatic cholangiocarcinoma cell; Apoptosis; Oxygen species

Chaetocin induces cell cycle arrest and apoptosis by regulating the ROS-mediated ASK-1/JNK signaling pathways.

The present study demonstrated that chaetocin, a natural small-molecule product produced by Chaetomium fungal species and a potential anticancer agent, inhibited the viability and invasive ability of the human intrahepatic cholangio-carcinoma cell line CCLP-1 invivo and invitro as revealed by CCK-8 and Transwell invasion assays and mouse xenograft tumor experiments. As determined using flow cytometry and intracellular ROS assays, chaetocin was found to induce cell cycle arrest and oxidative stress, leading to CCLP-1 cell apoptosis. Cell apoptosis can be initiated via different apoptotic signaling pathways under oxidative stress. As determined by western blot analysis, expression levels of the apoptosis signal-regulating kinase1 (ASK-1) signalosome and its downstream c-Jun N-terminal kinase (JNK) signaling pathway were increased under oxidative stress stimulation. These findings indicate that chaetocin arrests the cell cycle and induces apoptosis by regulating the reactive oxygen species-mediated ASK-1/JNK signaling pathways.

C‑Myc promotes cholangiocarcinoma cells to overcome contact inhibition via the mTOR pathway.

The loss of contact inhibition is a hallmark of a wide range of human cancer cells. Yet, the precise mechanism behind this process is not fully understood. c‑Myc plays a pivotal role in carcinogenesis, but its involvement in regulating contact inhibition has not been explored to date. Here, we report that c‑Myc plays an important role in abrogating contact inhibition in human cholangiocarcinoma (CCA) cells. Our data show that the protein level of c‑Myc obviously decreased in contact-inhibited normal biliary epithelial cells. However, CCA cells sustain high protein levels of c‑Myc and keep strong proliferation ability in confluent conditions. Importantly, the suppression of c‑Myc by inhibitor or siRNA induced G0/G1 phase cell cycle arrest in confluent CCA cells. We demonstrate that the inhibition of c‑Myc suppressed the activity of mammalian target of rapamycin (mTOR) in confluent CCA cells, and mTOR inhibition induced G0/G1 phase cell cycle arrest in confluent CCA cells. In confluent CCA cells, the activity of Merlin is downregulated, and Yes-associated protein (YAP) sustains high levels of activity. Furthermore, YAP inhibition not only induced G0/G1 phase cell cycle arrest, but also decreased c‑Myc expression in confluent CCA cells. These results indicate that Merlin/YAP/c‑Myc/mTOR signaling axis promotes human CCA cell proliferation by overriding contact inhibition. We propose that overriding c‑Myc‑mediated contact inhibition is implicated in the development of CCA.

Nicotine Promotes Cholangiocarcinoma Growth in Xenograft Mice

Nicotine, the main addictive substance in tobacco, is known to play a role in the development and/or progression of a number of malignant tumors. However, nicotine's involvement in the pathogenesis of cholangiocarcinoma is controversial. Therefore, we studied the effects of nicotine on the growth of cholangiocarcinoma cells invitro and the progression of cholangiocarcinoma in a mouse xenograft model. The predominant subunit responsible for nicotine-mediated proliferation in normal and cancer cells, the α7 nicotinic acetylcholine receptor (α7-nAChR), was more highly expressed in human cholangiocarcinoma cell lines compared with normal human cholangiocytes. Nicotine also stimulated the proliferation of cholangiocarcinoma cell lines and promoted α7-nAChR-dependent activation of proliferation and phosphorylation of extracellular-regulated kinase in Mz-ChA-1 cells. In addition, nicotine and PNU282987 (α7-nAChR agonist) accelerated the growth of the cholangiocarcinoma tumors in our xenograft mouse model and increased fibrosis, proliferation of the tumor cells, and phosphorylation of extracellular-regulated kinase activation. Finally, α7-nAChR was expressed at significantly higher levels in human cholangiocarcinoma compared with normal human control liver samples. Taken together, results of this study suggest that nicotine acts through α7-nAChR and plays a novel role in the pathogenesis of cholangiocarcinoma. Furthermore, nicotine may act as a mitogen in cholestatic liver disease processes, thereby facilitating malignant transformation.

Reduced EBP50 expression levels are correlated with unfavorable clinicopathological features of extrahepatic bile duct carcinoma and promote the proliferation and migration of QBC939 cells.

The present study aimed to clarify the association between ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) expression level and the tumor phenotype and clinicopathological features of extrahepatic bile duct carcinoma. Tissue samples from patients with extrahepatic bile duct carcinoma (54 cases) and patients with normal bile duct epithelia from gallbladder of cholecystitis (20 cases) were collected, and immunohistochemical staining was used to detect the expression levels of EBP50 in these tissues. In addition, small interfering (si)RNA-EBP50 was used to knock down the expression of EBP50 in the QBC939 human cholangiocarcinoma (CC) cell line. The effect of EBP50 expression on QBC939 cell proliferation and migration was analyzed using the Cell Counting kit-8 and wound healing assays, respectively. EBP50 expression was significantly downregulated in CC tissue samples (P<0.01), with low EBP50 expression levels positively correlated with a high pathological stage and a poor differentiation degree (P<0.01 and P<0.001, respectively). EBP50 expression in QBC939 cells was knocked down by ≤80% using siRNA-EBP50, and EBP50 knockdown significantly promoted QBC939 cell proliferation, as compared with the vector control cells (P=0.04). EBP50 knockdown also significantly enhanced the wound healing ability of QBC939 cells (P=0.02). These results demonstrated that EBP50 expression levels are significantly correlated with a malignant phenotype in patients with CC, and decreased expression levels of EBP50 may promote CC cell proliferation and migration. These findings provide insight into novel potential diagnostic and therapeutic approaches for patients with CC.

MiR-21 and KLF4 jointly augment epithelial‑mesenchymal transition via the Akt/ERK1/2 pathway.

miR-21 induces epithelial-mesenchymal transition (EMT) of human cholangiocarcinoma (CCA) cells. However, the mechanism by which this occurs remains unclear. In the present study, high throughput platform was employed to detect the genes that are differential expressed in QBC939 cells transfected with a hsa-miR-21 antagomir or control vectors. The EMT-related Krüppel-like factor 4 (KLF4) gene was down-regulated after miR-21 was knocked down. Overexpression of miR-21 upregulated KLF4, Akt, ERK and mesenchymal cell markers (N-cadherin and vimentin), downregulated the expression of epithelial cell marker E-cadherin and reduced cell migration and invasion. Immunohistochemistry showed that KLF4, pAkt and pERK were upregulated in tumor xenografts transfected with miR-21 mimics. Inhibitors of the PI3K-Akt and ERK1/2 pathways, LY294002 and U0126, significantly suppressed the EMT phenotype. The present data demonstrated that overexpression of miR-21, accompanied with KLF4, augmented the EMT via inactivation of Akt and ERK1/2 pathways. In conclusion, we have identified a novel mechanism that may be targeted in an attempt to relieve the malignant biological behavior of CCA cells.

FRI-117 - Insulin/IGF-1 receptors mediate acquired resistance to anti-EGFR therapy in human cholangiocarcinoma cells

FRI-117 - Insulin/IGF-1 receptors mediate acquired resistance to anti-EGFR therapy in human cholangiocarcinoma cells

Inhibitory Effect of Aspirin on Cholangiocarcinoma Cells

Aspirin and other non-steroidal anti-inflammatory drugs reduce the risk of cancer due to their anti-proliferative and apoptotic effects, which are the important mechanisms for their anti-tumor activity. Here, the effect of aspirin on human cholangiocarcinoma cells (KKU-214) and the underlying mechanisms of its action were explored. Cell proliferation was measured by sulforhodamine B (SRB) assay, while cell cycle distribution and apoptosis were determined by flow cytometry. Western blotting was used to explore protein expression underlying molecular mechanisms of anti-cancer treatment of aspirin. Aspirin reduced cell proliferation in a dose- and time-dependent manner, and altered the cell cycle phase distribution of KKU-214 cells by increasing the proportion of cells in the G0/G1 phase and reducing the proportion in the S and G2/M phases. Consistent with its effect on the cell cycle, aspirin also reduced the expression of cyclin D1 and cyclin-dependent kinase 4 (Cdk-4), which are important for G0/G1 cell cycle progression. Treatment with aspirin led to increased induction of apoptosis in a dose-dependent manner. Further analysis of the mechanism underlying the effect of this drug showed that aspirin induced the expression of the tumor-suppressor protein p53 while inhibiting the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2). Correspondingly, the activation of caspase-9 and -3 was also increased. These findings suggest that aspirin causes cell cycle arrest and apoptosis, both of which could contribute to its anti-proliferative effect.

Cordycepin inhibits cell growth and induces apoptosis in human cholangiocarcinoma.

The purpose of this study was to explore the role of cordycepin in human cholangiocarcinoma (CCA) cell growth and apoptosis. In the present study, colony formation assay, cell-counting kit-8 (CCK-8) assay and tumor xenograft experiment were performed to evaluate the effect of cordycepin on human CCA cell growth in vitro and in vivo; flow cytometric analysis was performed to evaluate the effect of cordycepin on cell apoptosis; quantitative real-time reverse transcription PCR (qRT-PCR) and western blot assays were performed to evaluate the expression levels of Caspase-3, Bcl-2 and Bax. The results showed that cordycepin inhibited cell growth in QBC939 and RBE cells in vitro and it could also inhibit QBC939 cells growth in vivo. Furthermore, the flow cytometric analysis, qRT-PCR and western blot assays showed that cordycepin could trigger QBC939 and RBE cells apoptosis by regulating the expression levels of Caspase-3, Bcl-2 and Bax. And we proposed that cordycepin could inhibit human CCA cell growth in vitro and in vivo, while, this function is related to the induction of cell apoptosis.

In Vitro and In Vivo Inhibitory Effects of α-Mangostin on Cholangiocarcinoma Cells and Allografts

We investigated the anti-cholangiocarcinoma effect of α-mangostin from Garcinia mangostana pericarp extract (GM) in a human cholangiocarcinoma (CCA) cell line and a hamster CCA allograft model. In vitro, human CCA cells were treated with GM at various concentrations and for different time periods; then cell-cycle arrest and apoptosis were evaluated using flow cytometry, and metastatic potential with wound healing assays. In vivo, hamster allografts were treated with GM, gemcitabine (positive control) and a placebo (negative control) for 1 month; tumor weight and volume were then determined. Histopathological features and immunostaining (CK19 and PCNA) characteristics were examined by microscopy. The present study found that α-mangostin could: inhibit CCA cell proliferation by inducing apoptosis through the mitochondrial pathway; induce G1 cell-cycle arrest; and inhibit metastasis. Moreover, α-mangostin could inhibit CCA growth, i.e. reduce tumor mass (weight and size) and alter CCA pathology, as evidenced by reduced positive staining for CK19 and PCNA. The present study thus suggested that α-mangostin is a promising anti-CCA compound whose ready availability in tropical countries might indicate use for prevention and treatment of CCA.

Dysregulated Expression of MITF in Subsets of Hepatocellular Carcinoma and Cholangiocarcinoma.

Cholangiocarcinoma represents the second most common primary liver tumor after hepatocellular carcinoma. Mahanine, a carbazole alkaloid derived from Murraya koenigii (Linn.) Spreng, has been used as folk medicine in Thailand, where the liver fluke-associated cholangiocarcinoma is common. The expression of microphthalmia-associated transcription factor (MITF) is maintained at immunohistochemically undetectable levels in hepatocytes and cholangiocytes. To explore the regulation of MITF expression in the liver, we immunohistochemically analyzed the MITF expression using hepatocellular carcinoma and cholangiocarcinoma specimens of the human liver cancer tissue array. MITF immunoreactivity was detected in subsets of hepatocellular carcinoma (6 out of 38 specimens; 16%) and cholangiocarcinoma (2/7 specimens; 29%). Moreover, immunoreactivity for glioma-associated oncogene 1 (GLI1), a transcription factor of the Hedgehog signaling pathway, was detected in 55% of hepatocellular carcinoma (21/38 specimens) and 86% of cholangiocarcinoma (6/7 specimens). Importantly, MITF was detectable only in the GLI1-positive hepatocellular carcinoma and cholangiocarcinoma, and MITF immunoreactivity is associated with poor prognosis in patients with hepatocellular carcinoma. Subsequently, the effect of mahanine was analyzed in HepG2 human hepatocellular carcinoma and HuCCT1 and KKU-100 human cholangiocarcinoma cells. Mahanine (25 µM) showed the potent cytotoxicity in these hepatic cancer cell lines, which was associated with increased expression levels of MITF, as judged by Western blot analysis. MITF is over-expressed in subsets of hepatocellular carcinoma and cholangiocarcinoma, and detectable MITF immunoreactivity is associated with poor prognosis in patients with hepatocellular carcinoma. MITF expression levels may be determined in hepatic cancer cells by the balance between the Hedgehog signaling and the cellular stress.