Induction of apoptotic cell death of cholangiocarcinoma cells by tiliacorinine from Tiliacora triandra: A mechanistic insight.

BACKGROUND: Cholangiocarcinoma (CCA) exhibits poor response to the present chemotherapeutic agents and frequently develops drug resistance. Finding novel anticancer drugs might enhance patient outcomes. Tiliacorinine, a bisbenzylisoquinoline alkaloid from the Thai medicinal plant Tiliacora triandra, effectively induced apoptosis of human CCA cell lines and inhibited tumor growth in mice. Here, we elucidate further the molecular mechanisms underlining the cytotoxicity of tiliacorinine and its implication in overcoming gemcitabine-resistance of CCA cells. METHODS: Cytotoxicity of tiliacorinine against CCA cell lines was assessed using MTT assay. The molecular signaling was determined using Western blot analysis. Molecular docking simulations were applied to predict the binding affinity and orientation of tiliacorinine to the possible binding site(s) of the target proteins. RESULTS: Tiliacorinine induced apoptotic cell death of CCA cells in a dose- and time-dependent manner. Tiliacorinine significantly suppressed the expression of anti-apoptotic proteins, Bcl-xL and XIAP; activated apoptotic machinery proteins, caspase-3, caspase-9, and PARP; and decreased the levels of pAkt and pSTAT3. EGF/EGFR activation model and molecular docking simulations revealed EGFR, Akt, and STAT3 as potent targets of tiliacorinine. Molecular docking simulations indicated a strong binding affinity of tiliacorinine to the ATP-binding pockets of EGFR, PI3K, Akt, JAK2, and SH2 domain of STAT3. Tiliacorinine could synergize with gemcitabine and restore the cytotoxicity of gemcitabine against gemcitabine-resistant CCA cells. CONCLUSION: Tiliacorinine effectively induced apoptosis via binding and blocking the actions of EGFR, Akt, and STAT3. GENERAL SIGNIFICANCE: Tiliacorinine is a novel multi-kinase inhibitor and possibly a potent anti-cancer agent, in cancers with high activation of EGFR.

γ-aminobutyric acid B2 receptor: A potential therapeutic target for cholangiocarcinoma in patients with diabetes mellitus.

BACKGROUND: The association between diabetes mellitus (DM) and the increased risk and progression of cholangiocarcinoma (CCA) has been reported with unclear underlying mechanisms. Previous studies showed that γ-aminobutyric acid (GABA) B2 receptor (GABBR2) was upregulated in CCA cells cultured in high glucose (HG) conditions. Roles of GABA receptors in CCA progression have also been studied, but their association with DM and hyperglycemia in CCA remains unclarified. AIM: To investigate the effects of hyperglycemia on GABBR2 expression and the potential use of GABBR2 as a CCA therapeutic target. METHODS: CCA cells, KKU-055 and KKU-213A, were cultured in Dulbecco Modified Eagle's Medium supplemented with 5.6 mmol/L (normal glucose, NG) or 25 mmol/L (HG) glucose and assigned as NG and HG cells, respectively. GABBR2 expression in NG and HG cells was investigated using real-time quantitative polymerase chain reaction and western blot. Expression and localization of GABBR2 in CCA cells were determined using immunocytofluorescence. GABBR2 expression in tumor tissues from CCA patients with and without DM was studied using immunohistochemistry, and the correlations of GABBR2 with the clinicopathological characteristics of patients were analyzed using univariate analysis. Effects of baclofen, a GABA-B receptor agonist, on CCA cell proliferation and clonogenicity were tested using the MTT and clonogenic assays. Phospho-kinases arrays were used to screen the affected signaling pathways after baclofen treatment, and the candidate signaling molecules were validated using the public transcriptomic data and western blot. RESULTS: GABBR2 expression in CCA cells was induced by HG in a dose- and time-dependent manner. CCA tissues from patients with DM and hyperglycemia also showed a significantly higher GABBR2 expression compared with tumor tissues from those with euglycemia (P < 0.01). High GABBR2 expression was significantly associated with a poorer non-papillary histological subtype but with smaller sizes of CCA tumors (P < 0.05). HG cells of both tested CCA cell lines were more sensitive to baclofen treatment. Baclofen significantly suppressed the proliferation and clonogenicity of CCA cells in both NG and HG conditions (P < 0.05). Phospho-kinase arrays suggested glycogen synthase kinase 3 (GSK3), ß-catenin, and the signal transducer and activator of transcription 3 (STAT3) as candidate signaling molecules under the regulation of GABBR2, which were verified in NG and HG cells of the individual CCA cell lines. Cyclin D1 and c-Myc, the common downstream targets of GSK3/ß-catenin and STAT3 involving cell proliferation, were accordingly downregulated after baclofen treatment. CONCLUSION: GABBR2 is upregulated by HG and holds a promising role as a therapeutic target for CCA regardless of the glucose condition.

High glucose enhances the aggressiveness of lung adenocarcinoma via activating epidermal growth factor receptor/signal transducer and activator of transcription 3 pathways.

Epidemiological studies revealed hyperglycemia as a poor prognostic factor for lung adenocarcinoma with unclear molecular mechanisms. The present study thus aimed to investigate the effects of high glucose on the progression of lung adenocarcinoma and its underlying mechanisms. Lung adenocarcinoma cell lines, A549 and RERF-LC-KJ, were cultured in 5.6 mM glucose (normal glucose; NG) or 25 mM glucose (high glucose; HG) resembling euglycemia and hyperglycemia. Cells were examined for proliferation by the MTT assay, and migration-invasion using Transwell. The expressions of signaling proteins in epidermal growth factor receptor (EGFR) pathways and their downstream targets were investigated using Western blots. The effects of diabetes mellitus (DM) and hyperglycemia on lung adenocarcinoma growth in vivo were studied in streptozotocin-induced diabetic BALB/cAJcl-Nu/Nu mice and their nondiabetic counterparts. High glucose significantly promoted proliferation, migration, and invasion of lung adenocarcinoma cells compared with those in normal glucose (P<.05). Western blot analyses showed the increased ratio of pEGFR/EGFR in cells cultured in high glucose and subsequently activated the signal transducer and activator of transcription 3 (STAT3). Epithelial-mesenchymal (EMT) markers were also altered in lung adenocarcinoma cells in high glucose conditions, corresponding with increased migration and invasion abilities. Erlotinib, an EGFR inhibitor, significantly reversed high glucose-induced aggressive phenotypes confirming high glucose-enhancing lung adenocarcinoma progression via the activation of EGFR. DM and hyperglycemia also promoted the growth of lung adenocarcinoma xenografts in vivo in which erlotinib significantly suppressed the growth of tumors (P<.05) suggesting EGFR inhibitor as an effective therapeutic agent for lung adenocarcinoma with DM.

Diminishing acetyl-CoA carboxylase 1 attenuates CCA migration via AMPK-NF-κB-snail axis.

Cholangiocarcinoma (CCA), a cancer of the biliary tract, is a significant health problem in Thailand. Reprogramming of cellular metabolism and upregulation of lipogenic enzymes have been revealed in CCA, but the mechanism is unclear. The current study highlighted the importance of acetyl-CoA carboxylase 1 (ACC1), a rate-limiting enzyme in de novo lipogenesis, on CCA migration. ACC1 expression in human CCA tissues was determined by immunohistochemistry. The results demonstrated that increased ACC1 was related to the shorter survival of CCA patients. Herein, ACC1-deficient cell lines (ACC1-KD) were generated by the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (cas9) system and were used for the comparative study. The ACC1 levels in ACC1-KD were 80-90 % lower than in parental cells. Suppression of ACC1 significantly reduced intracellular malonyl-CoA and neutral lipid contents. Two-fold growth retardation and 60-80 % reduced CCA cell migration and invasion were observed in ACC1-KD cells. The reduced 20-40 % of intracellular ATP levels, AMPK activation, lowered NF-κB p65 nuclear translocation, and snail expression were emphasized. Migration of ACC1-KD cells was restored by supplementation with palmitic acid and malonyl-CoA. Altogether, the importance of rate-limiting enzyme in de novo fatty acid synthesis, ACC1, and AMPK-NF-κB-snail axis on CCA progression was suggested herein. These might be the novel targets for CCA drug design. (ACC1, AMPK, Cholangiocarcinoma, De novo lipogenesis, NF-κB, Palmitic acid).

High Level of Serum Coiled-coil Domain Containing 25 (CCDC25) as a Diagnostic Marker for Cholangiocarcinoma But Not for Other Cancers.

BACKGROUND/AIM: Recently, we reported that coiled-coil domain containing 25 (CCDC25) protein is elevated in the sera of patients with cholangiocarcinoma (CCA) and is suggested to be a diagnostic biomarker for CCA. This study aimed to examine whether serum CCDC25 level can be a unique biomarker for CCA. Bioinformatic analyses using Human Protein Atlas (HPA) database and Gene Expression Profiling Interactive Analysis 2 (GEPIA2) indicated that CCDC25 protein and mRNA are expressed not only in CCA but also in other cancers, such as colorectal cancer (CRC), breast cancer (BC), and hepatocellular carcinoma (HCC), all of which are the top 5 cancers highly prevalent in Thailand. MATERIALS AND METHODS: Using a quantitative dot blot assay, serum CCDC25 levels were measured for 30 healthy controls (HC), 34 CRC, 42 BC, 43 HCC, and 83 CCA. RESULTS: The serum CCDC25 levels of CCA patients (0.193±0.039 ng/µl) were significantly higher than those of CRC (0.019±0.006 ng/µl), BC (0.036±0.015 ng/µl), HCC (0.035±0.016 ng/µl), and higher than those of HC (0.012±0.003 ng/µl). The serum CCDC25 level can discriminate CCA from the HC, CRC, BC, and HCC with a sensitivity of 100, 99, 94, and 94%, respectively, and specificity of 100, 100, 98, and 95%, respectively. CONCLUSION: CCDC25 is a candidate diagnostic biomarker for CCA.

Lactic acidosis induces metabolic and phenotypic reprogramming in cholangiocarcinoma cells via the upregulation of thrombospondin-1.

The high glycolytic activity of cancer cells leads to lactic acidosis (LA) in the tumor microenvironment. LA is not merely a consequence of metabolic activities but also has functional roles in metabolic reprogramming and cancer progression. Cholangiocarcinoma (CCA) cells exhibit a high dependency on glycolysis for survival and growth, but the specific effects of LA on cellular characteristics remain unknown. Here, we demonstrate that long-term LA (LLA) reprograms the metabolic phenotype of CCA cells from glycolytic to oxidative and enhances their migratory activity. In CCA cell culture, short-term LA (24 h) showed a growth inhibitory effect, while extended LA exposure for more than 2 weeks (LLA) led to enhanced cell motility. Coincidentally, LLA enhanced the respiratory capacity with an increase in mitochondrial mass. Inhibition of mitochondrial function abolished LLA-induced cell motility, suggesting that metabolic remodeling affects the phenotypic outcomes. RNA-sequencing analysis revealed that LLA upregulated genes associated with cell migration and epithelial-mesenchymal transition (EMT), including thrombospondin-1 (THBS1), which encodes a pro-EMT-secreted protein. Inhibition of THBS1 resulted in the suppression of both LLA-induced cell motility and respiratory capacity. Moreover, high THBS1 expression was associated with poor survival in patients with CCA. Collectively, our study suggests that the increased expression of THBS1 by LLA promotes phenotypic alterations, leading to CCA progression.

Anserine/Carnosine-Rich Extract from Thai Native Chicken Suppresses Melanogenesis via Activation of ERK Signaling Pathway.

Skin hyperpigmentation is an aesthetic problem that leads to psychosocial issues. Thus, skin whitening agents from agro- and poultry-industrial co-products are considered high economic value ingredients of interest for sustainable application. Therefore, this study aimed to determine the cosmeceutical potential of anserine/carnosine-rich chicken extract (ACCE) from the Thai native chicken Pradu Hang Dam Mor Kor 55 (PD) meat. The chemical composition was identified and quantified using the HPLC-UV method. Then, the antioxidation potential of the extract was compared to that of L-anserine and L-carnosine, using 1,1-diphenyl-2-picrylhydrazyl assay and shikonin-induced production of reactive oxygen species in CCD-986Sk cell models, and the anti-melanogenesis effect in the MNT-1 melanoma cell line model was investigated. Furthermore, related mechanisms were identified using colorimetric tyrosinase assay and the Western blot technique. The ACCE was composed of L-anserine and L-carnosine as two major constituents. In a dose-dependent manner, ACCE, L-anserine, and L-carnosine manifested significant antioxidation potential and significant reduction of melanin production. Activation of the extracellular signal-regulated kinase (ERK) signaling pathway and inhibition of tyrosinase activity of ACCE were demonstrated as the mechanisms of the anti-melanogenesis effect. In conclusion, ACCE has been revealed as a potential cosmeceutical agent due to its antioxidation and anti-melanogenic activity in association with L-anserine and L-carnosine composition and biomolecular regulating ability. Therefore, further studies and development should be considered to support the utilization of anserine/carnosine-rich chicken extract in the cosmetic industry for economic value creation and sustainability.

Emerging roles of GALNT5 on promoting EGFR activation in cholangiocarcinoma: a mechanistic insight.

Cholangiocarcinoma (CCA) is a lethal cancer in that the incidence is now increasing worldwide. N-acetylgalactosaminyltransferase 5 (GALNT5), an enzyme that initiates the first step of mucin type-O glycosylation, has been reported to promote aggressiveness of CCA cells via the epithelial to the mesenchymal transition (EMT) process, and Akt/Erk activation. In this study, the clinical and biological relevance of GALNT5 and the molecular mechanisms by which GALNT5 modulated EGFR in promoting CCA progression were examined. Using publicly available datasets, upregulation of GALNT5 in patient CCA tissues and its correlation with EGFR expression was noted. High levels of GALNT5 were significantly associated with the short survival of patients, suggesting a prognostic marker of GALNT5 for CCA. GALNT5 modulated EGFR expression as shown in CCA cell lines. Upregulation of GALNT5 significantly increased EGFR mRNA and protein in GALNT5 overexpressing cells, whereas suppression of GALNT5 expression gave the opposite results. The molecular dynamics simulations and MM/PB(GB)SA-based free energy calculations showed that O-glycosylation on the EGFR extracellular domain enhanced the structural stability, compactness, and H-bond formation of the EGF/GalNAc-EGFR complex compared with those of EGF/EGFR. This stabilized the growth factor binding site and fostered stronger interactions between EGF and EGFR. Using the EGF-induced EGFR activation model, GALNT5 was shown to mediate EGFR stability via a decreased rate of EGFR degradation and enhanced EGFR activity by increasing the binding affinity of EGF/EGFR that consequently increasing the activation of EGFR and its downstream effectors Akt and Erk. In summary, GALNT5 was upregulated in CCA tissues and associated with a worse prognosis. The study identified for the first time the impacts of GALNT5 on EGFR activity by increasing: 1) EGFR expression via a transcriptional-dependent mechanism, 2) EGFR stability by reducing EGFR degradation, and 3) EGFR activation through an increased binding affinity of EGF/EGFR which all together fostered the activation of EGFR. These results expanded the understanding of the molecular mechanism of how GALNT5 impacted CCA progression and suggested GALNT5 as a new target for therapeutic intervention against metastatic CCA.

Lactic acidosis promotes aggressive features of cholangiocarcinoma cells via upregulating ALDH1A3 expression through EGFR axis.

AIMS: Lactic acidosis (LA) generated in tumor microenvironment promotes tumor metastasis and drug resistance. This study aimed to demonstrate the impacts and the mechanisms of LA on aldehyde dehydrogenase1A3 (ALDH1A3) in promoting aggressiveness and gemcitabine resistance in cholangiocarcinoma (CCA) cell lines. The clinical relevance and the molecular pathway related to the upregulation of ALDH1A3 in LA cells will be revealed. MAIN METHODS: ALDH1A3 expression and its clinical significances in CCA tissues were analyzed using the GEO databases. Human CCA cell lines, KKU-213A-LA and KKU-213B-LA maintained in the LA medium were studied and compared with its parental cells cultured in normal medium. Aggressive features-proliferation, colony formation, migration, invasion, and gemcitabine response were determined. Expression of ALDH1A3, EGFR and the downstream effectors were analyzed using real-time PCR and Western blotting. KEY FINDINGS: ALDH1A3 was upregulated in patient CCA tissues and correlated with LDHA and shorter survival of CCA patients. mRNA and protein of ALDH1A3 were increased in LA cells. Attenuation of ALDH1A3 expression by siRNA significantly reduced cell proliferation, colony formation, migration, invasion, and gemcitabine resistance of LA cells, and gemcitabine resistant cells. The EGF/EGFR signaling via Erk and STAT3 was pinned to be involved in the induction of ALDH1A3 expression in LA cells. The transcriptomic analysis from TCGA dataset supported the links between LDHA, EGFR and ALDH1A3 in several tumor tissues. SIGNIFICANCE: Lactic acidosis upregulated EGFR and ALDH1A3 expression, leading to the aggressiveness of CCA cells. The EGFR/ALDH1A3 axis could be a novel therapeutic target to eradicate metastatic CCA.

High Glucose Induced Upregulation of Cyclin a Associating with a Short Survival of Patients with Cholangiocarcinoma: A Potential Target for Treatment of Patients with Diabetes Mellitus.

Diabetes mellitus (DM) is associated with an increased risk and progression of cholangiocarcinoma (CCA). High glucose underlying the association between DM and CCA by modulating the intracellular signaling has been demonstrated. However, the effects of DM and hyperglycemia on cell cycle machineries and progression of CCA remain elucidated. CCA cells, KKU-213A and KKU-213B were cultured in normal (NG, 5.6 mM) or high glucose (HG, 25 mM) resembling euglycemia and hyperglycemia. Western blotting was used to determine expressions of cell cycle machineries in CCA cells. The expression of cyclin A in CCA tissues from patients with or without hyperglycemia was determined by immunohistochemistry. Pan-cyclin dependent kinases (CDKs) inhibitor and silencing of cyclin A expression were investigated as a possible modality targeting CCA treatment in patients with DM. High glucose induced expression of cell cycle machinery proteins in both CCA cells. Among these, cyclin A was consistently and significantly upregulated. Nuclear cyclin A was significantly increased in tumor tissues from CCA patients with hyperglycemia and was significantly associated with post-operative survival of shorter than 5 mo. Silencing cyclin A expression sensitized CCA cells to pan-CDKs inhibitor, suggesting the combined treatment as an alternative approach for treatment of CCA patients with DM.

Homophilic Interaction of CD147 Promotes IL-6-Mediated Cholangiocarcinoma Invasion via the NF-κB-Dependent Pathway.

Cholangiocarcinoma (CCA), an aggressive cancer of bile ducts, is a well-known chronic inflammation-related disease. The major impediment in CCA treatment is limited treatment options for advanced disease; hence, an alternative is urgently required. The role of CD147 on cytokine production has been observed in inflammation-related diseases, but not in CCA. Therefore, this study was focused on CD147-promoting proinflammatory cytokine production and functions. Proinflammatory cytokine profiles were compared between CD147 expressing CCA cells and CD147 knockout cells (CD147 KO). Three cytokines, namely interleukin (IL)-6, IL-8, and granulocyte-monocyte colony-stimulating factor (GM-CSF), were dramatically diminished in CD147 KO clones. The involvement of the CD147-related cytokines in CCA invasion was established. CD147-promoted IL-6, IL-8, and GM-CSF secretions were regulated by NF-κB nuclear translocation, Akt activation, and p38 phosphorylation. CD147-fostering IL-6 production was dependent on soluble CD147, CD147 homophilic interaction, and NF-κB function. The overexpression of specific genes in CCA tissues compared to normal counterparts emphasized the clinical importance of these molecules. Altogether, CD147-potentiated proinflammatory cytokine production leading to CCA cell invasion is shown for the first time in the current study. This suggests that modulation of CD147-related inflammation might be a promising choice for advanced CCA treatment.

FOXM1 inhibitor, Siomycin A, synergizes and restores 5-FU cytotoxicity in human cholangiocarcinoma cell lines via targeting thymidylate synthase.

AIMS: 5-Fluorouracil (5-FU), a thymidylate synthase (TS) inhibitor, has been used as the first-line chemotherapeutic drug for cholangiocarcinoma (CCA). The side effects and drug resistance have developed the limits of the clinical application of 5-FU in CCA treatment. Upregulation of Forkhead box M1 (FOXM1) and TS were shown to play a significant role in 5-FU resistance. In this study, the effect of Siomycin A (SioA), a FOXM1 inhibitor, on enhancing 5-FU cytotoxicity and reversing 5-FU resistance in CCA cell lines were demonstrated. MAIN METHODS: Human CCA cell lines, KKU-100 and KKU-213A were used. Cell viability was determined using MTT assay. Expression of FOXM1 and TS proteins were determined using Western blotting. FOXM1 mRNA expression was quantitated using real-time PCR. The combination and dose reduction (DRI) were analyzed according to the Chou and Talalay method. KEY FINDING: Single drug treatment of 5-FU and SioA effectively inhibited CCA cell growth in dose and time dependent fashions. The two CCA cell lines had different responses to 5-FU but exhibited similar sensitivity to SioA. FOXM1 and TS expression were increased in the 5-FU treated cells but were suppressed in the SioA treated cells. A direct binding of SioA, to TS and 5,10-methylene-tetrahydrofolate as an inactive ternary complex was simulated. The combined treatment of 5-FU with SioA showed a synergistic effect with a high DRI and restored 5-FU sensitivity in the 5-FU resistant cells. SIGNIFICANCE: Targeting FOXM1 using SioA in combination with 5-FU might be a strategy to overcome the 5-FU resistance in CCA.

Kallikrein-11, in Association with Coiled-Coil Domain Containing 25, as a Potential Prognostic Marker for Cholangiocarcinoma with Lymph Node Metastasis.

Cholangiocarcinoma (CCA) is a malignancy arising from cholangiocytes. Currently, the treatment and prognosis for CCA are mostly poor. Recently, we have reported that coiled-coil domain containing 25 (CCDC25) protein level in the sera may be a diagnostic marker for CCA. Subsequently, we identified three binding proteins of CCDC25 and found that kallikrein-11 (KLK11) expression was highest among those binding proteins. In this study, we investigated CCDC25 and KLK11 expression in CCA and adjacent normal tissues (n = 18) using immunohistochemistry. The results demonstrated that the expressions of CCDC25 and KLK11 in CCA tissues were both significantly higher than the adjacent tissues (p < 0.001 and p = 0.001, respectively). Then, using GEPIA bioinformatics analysis, KLK11 mRNA was significantly overexpressed in CCA tumor tissues compared with normal tissues (p < 0.05). Moreover, CCDC25 expression was positively correlated with KLK11 expression in CCA with lymph node metastasis (p = 0.028, r = 0.593). An analysis for the interaction of KLK11 with CCDC25 and other proteins, using STRING version 11.0, revealed that CCDC25 and KLK11 correlated with metastasis-related proteins. In addition, Kaplan-Meier survival curve analysis revealed that a high expression of KLK11 was associated with the poor prognosis of CCA. In conclusion, KLK11 is, as a binding protein for CCDC25, possibly involved in the metastatic process of CCA. KLK11 may be used as a prognostic marker for CCA.

High glucose: an emerging association between diabetes mellitus and cancer progression.

The association of cancer and diabetes mellitus (DM) has been studied for decades. Hyperglycemia and the imbalance of hormones are factors that contribute to the molecular link between DM and carcinogenesis and cancer progression. Hyperglycemia alone or in combination with hyperinsulinemia are key factors that promote cancer aggressiveness. Many preclinical studies suggest that high glucose induces abnormal energy metabolism and aggressive cancer via several mechanisms. As evidenced by clinical studies, hyperglycemia is associated with poor clinical outcomes in patients who have comorbid DM. The prognoses of cancer patients with DM are improved when their plasma glucose levels are controlled. This suggests that high glucose level maybe be involved in the molecular mechanism that causes the link between DM and cancer and may also be useful for prognosis of cancer progression. This review comprehensively summarizes the evidence from recent pre-clinical and clinical studies of the impact of hyperglycemia on cancer advancement as well as the underlying molecular mechanism for this impact. Awareness among clinicians of the association between hyperglycemia or DM and cancer progression may improve cancer treatment outcome in patients who have DM.

FOXM1c is the predominant FOXM1 isoform expressed in cholangiocarcinoma that associated with metastatic potential and poor prognosis of patients.

Forkhead box M1 (FOXM1) is a transcriptional factor which plays an important role in oncogenesis. Four FOXM1 isoforms, FOXM1a, FOXM1b, FOXM1c and FOXM1d, are known so far. Different FOXM1 isoforms influence progression of cancer in different cancer types. In this study, the FOXM1c isoform and its impact in cholangiocarcinoma (CCA) was identified. FOXM1c was found to be the predominant isoform in patient-CCA tissues and cell lines. Detection of FOXM1c expression in CCA tissues reflected the worse prognosis of the patients, namely the advanced stage and shorter survival. Suppression of FOXM1 expression using siRNA considerably reduced migration and invasion abilities of CCA cell lines. RNA sequencing analysis revealed claudin-1 as a target of FOXM1. FOXM1 exhibited a negative correlation with claudin-1 expression which was demonstrated in patient CCA tissues and cell lines. FOXM1 may be a potential target for therapeutic treatment of the metastatic CCA.

Bioinformatics analysis identified CDC20 as a potential drug target for cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CCA) is a malignancy that originates from bile duct cells. The incidence and mortality of CCA are very high especially in Southeast Asian countries. Moreover, most CCA patients have a very poor outcome. Presently, there are still no effective treatment regimens for CCA. The resistance to several standard chemotherapy drugs occurs frequently; thus, searching for a novel effective treatment for CCA is urgently needed. METHODS: In this study, comprehensive bioinformatics analyses for identification of novel target genes for CCA therapy based on three microarray gene expression profiles (GSE26566, GSE32225 and GSE76297) from the Gene Expression Omnibus (GEO) database were performed. Based on differentially expressed genes (DEGs), gene ontology and pathway enrichment analyses were performed. Protein-protein interactions (PPI) and hub gene identifications were analyzed using STRING and Cytoscape software. Then, the expression of candidate genes from bioinformatics analysis was measured in CCA cell lines using real time PCR. Finally, the anti-tumor activity of specific inhibitor against candidate genes were investigated in CCA cell lines cultured under 2-dimensional and 3-dimensional cell culture models. RESULTS: The three microarray datasets exhibited an intersection consisting of 226 DEGs (124 up-regulated and 102 down-regulated genes) in CCA. DEGs were significantly enriched in cell cycle, hemostasis and metabolism pathways according to Reactome pathway analysis. In addition, 20 potential hub genes in CCA were identified using the protein-protein interaction (PPI) network and sub-PPI network analysis. Subsequently, CDC20 was identified as a potential novel targeted drug for CCA based on a drug prioritizing program. In addition, the anti-tumor activity of a potential CDC20 inhibitor, namely dinaciclib, was investigated in CCA cell lines. Dinaciclib demonstrated huge anti-tumor activity better than gemcitabine, the standard chemotherapeutic drug for CCA. CONCLUSION: Using integrated bioinformatics analysis, CDC20 was identified as a novel candidate therapeutic target for CCA.

Aberrant GLUT1 Expression Is Associated With Carcinogenesis and Progression of Liver Fluke-associated Cholangiocarcinoma.

BACKGROUND/AIM: Glucose transporter 1 (GLUT1) has been demonstrated to be overexpressed in various cancer tissues and play a significant role on growth, metastasis, and apoptosis in cancer cells. This study aimed to reveal the clinical relevance of glucose transporter 1 (GLUT1) in carcinogenesis and progression on liver fluke-associated cholangiocarcinoma (CCA). MATERIALS AND METHODS: Expression of GLUT1 in CCA tissues from patients, as well as from a liver fluke-induced CCA hamster model, was determined using immunohistochemistry. CCA cell lines were transfected with GLUT1 siRNA and the roles of GLUT1 on cell growth as well as migration and invasion were investigated by using a clonogenic assay and Boyden chamber assays, respectively. RESULTS: GLUT1 was aberrantly expressed in hyperplastic/dysplastic bile ducts and CCA, but not in the normal bile ducts. High GLUT1 expression was significantly associated with non-papillary type, large tumor size, and short survival of patients. GLUT1 was expressed during cholangio-carcinogenesis and gradually increased with progression of histopathologic bile ducts. Silencing of GLUT1 expression significantly suppressed growth, migration, and invasion of CCA cell lines. CONCLUSION: GLUT1 plays important roles in carcinogenesis and progression of liver fluke-associated CCA. Targeting GLUT1 may be a strategy for treatment of metastasis in liver fluke-associated CCA.

High glucose upregulates FOXM1 expression via EGFR/STAT3 dependent activation to promote progression of cholangiocarcinoma.

AIMS: Epidemiological studies indicate diabetes mellitus and hyperglycemia as risk factors of cancers including cholangiocarcinoma (CCA). How high glucose promotes cancer development and progression, however, is still unrevealed. In this study, insight into the molecular pathway of high glucose promoting progression of CCA cells was investigated. MAIN METHODS: Human CCA cell lines, KKU-213A and KKU-213B were cultured in normal glucose (NG; 5.56 mM) or high glucose (HG; 25 mM) and used as NG and HG cells. Forkhead box M1 (FOXM1) expression was transiently suppressed using siFOXM1. Western blotting and image analysis were employed to semi-quantitatively determine the expression levels of the specified proteins. The migration and invasion of CCA cells were revealed using Boyden chamber assays. KEY FINDINGS: All HG cells exhibited higher expression of FOXM1 than the corresponding NG cells in a dose dependent manner. Suppression of FOXM1 expression by siFOXM1 significantly reduced migration and invasion abilities of CCA cells by suppression of Slug and MMP2 expression. Inhibition of STAT3 activation using Stattic, significantly suppressed expression of FOXM1 and Slug and decreased migration and invasion abilities of HG cells. In addition, EGFR expression was significantly higher in HG cells than NG cells and increased dependently with glucose concentration. Inhibition of EGFR activation by cetuximab significantly suppressed STAT3 activation and FOXM1 expression. SIGNIFICANCE: The mechanism of high glucose promoting progression of CCA cells was revealed to be via in part by upregulation of FOXM1 expression under EGF/EGFR and STAT3 dependent activation.

Association of Diabetes Mellitus and Cholangiocarcinoma: Update of Evidence and the Effects of Antidiabetic Medication.

Diabetes mellitus (DM) is a risk factor for cancer in many organs and associated with an increased risk of cholangiocarcinoma (CCA). The molecular linkage between these diseases has been demonstrated in preclinical studies, which have highlighted the role of hyperinsulinemia and hyperglycemia in the carcinogenesis and progression of CCA. Recent studies on the emerging role of antidiabetic medication in the development and progression of CCA showed a subclass of antidiabetic drug with a therapeutic effect on CCA. Although associations between CCA, insulin analogues and sulfonylureas are unclear, incretin-based therapy is likely associated with an increased risk for CCA, and may lead to CCA progression, as demonstrated by in vitro and in vivo experiments. In contrast, biguanides, especially metformin, exert an opposite effect, associated with a reduced risk of CCA and inhibited in vitro and in vivo CCA progression. The association between incretin-based therapy and the risk of CCA needs further clarification, as metformin is being studied in an ongoing clinical trial. Understanding the association between DM and CCA is critical for preventing the development of CCA in patients with DM, and for establishing the appropriateness of antidiabetic medication to treat CCA. Determining how metformin affects CCA can lead to repurposing this safe and well-known drug for improving CCA treatment, regardless of the diabetes status of patients.

High glucose-ROS conditions enhance the progression in cholangiocarcinoma via upregulation of MAN2A2 and CHD8.

Diabetes is a major risk factor in the development and progression of several cancers including cholangiocarcinoma (CCA). However, the molecular mechanism by which hyperglycemia potentiates progression of CCA is not clearly understood. Here, we showed that a high glucose condition significantly increased reactive oxygen species (ROS) production and promoted aggressive phenotypes of CCA cells, including proliferation and migration activities. Mannosidase alpha class 2a member 2 (MAN2A2), was upregulated at both mRNA and protein levels in a high glucose- and ROS-dependent manner. In addition, cell proliferation and migration were significantly reduced by MAN2A2 knockdown. Based on our proteome and in silico analyses, we further found that chromodomain helicase DNA-binding protein 8 (CHD8) was induced by ROS signaling and regulated MAN2A2 expression. Overexpression of CHD8 increased MAN2A2 expression, while CHD8 knockdown dramatically reduced proliferation and migration as well as MAN2A2 expression in CCA cells. Moreover, both MAN2A2 and CHD8 were highly expressed with positive correlation in CCA tumor tissues. Collectively, these data suggested that high glucose conditions promote CCA progression through ROS-mediated upregulation of MAN2A2 and CHD8. Thus, glucose metabolism is a promising therapeutic target to control tumor progression in patients with CCA and diabetes.