Anticancer properties and metabolomic profiling of Shorea roxburghii extracts toward gastrointestinal cancer cell lines.

BACKGROUND: Gastrointestinal cancer (GIC) ranks as the highest cause of cancer-related deaths globally. GIC patients are often diagnosed at advanced stages, limiting effective treatment options. Chemotherapy, the common GIC recommendation, has significant disadvantages such as toxicity and adverse effects. Natural products contain substances with diverse pharmacological characteristics that promise for use in cancer therapeutics. In this study, the flower of renowned Asian medicinal plant, Shorea roxburghii was collected and extracted to investigate its phytochemical contents, antioxidant, and anticancer properties on GIC cells. METHODS: The phytochemical contents of Shorea roxburghii extract were assessed using suitable methods. Phenolic content was determined through the Folin-Ciocalteu method, while flavonoids were quantified using the aluminum chloride (AlCl3) method. Antioxidant activity was evaluated using the FRAP and DPPH assays. Cytotoxicity was assessed in GIC cell lines via the MTT assay. Additionally, intracellular ROS levels and apoptosis were examined through flow cytometry techniques. The correlation between GIC cell viability and phytochemicals, 1H-NMR analysis was conducted. RESULTS: Among the four different solvent extracts, ethyl acetate extract had the highest phenolic and flavonoid contents. Water extract exhibited the strongest reducing power and DPPH scavenging activity following by ethyl acetate. Interestingly, ethyl acetate extract demonstrated the highest inhibitory activity against three GIC cell lines (KKU-213B, HepG2, AGS) with IC50 values of 91.60 µg/ml, 39.38 µg/ml, and 35.59 µg/ml, while showing less toxicity to normal fibroblast cells. Ethyl acetate extract induced reactive oxygen species and apoptosis in GIC cell lines by downregulating anti-apoptotic protein Bcl-2. Metabolic profiling-based screening revealed a positive association between reduced GIC cell viability and phytochemicals like cinnamic acid and its derivatives, ferulic acid and coumaric acid. CONCLUSIONS: This study highlights the potential of natural compounds in Shorea roxburghii in the development of more effective and safer anticancer agents as options for GIC as well as shedding light on new avenues for cancer treatment.

Iron-induced kidney cell damage: insights into molecular mechanisms and potential diagnostic significance of urinary FTL.

Background: Iron overload can lead to organ and cell injuries. Although the mechanisms of iron-induced cell damage have been extensively studied using various cells, little is known about these processes in kidney cells. Methods: In this study, we first examined the correlation between serum iron levels and kidney function. Subsequently, we investigated the molecular impact of excess iron on kidney cell lines, HEK293T and HK-2. The presence of the upregulated protein was further validated in urine. Results: The results revealed that excess iron caused significant cell death accompanied by morphological changes. Transcriptomic analysis revealed an up-regulation of the ferroptosis pathway during iron treatment. This was confirmed by up-regulation of ferroptosis markers, ferritin light chain (FTL), and prostaglandin-endoperoxide synthase 2 (PTGS2), and down-regulation of acyl-CoA synthetase long-chain family member 4 (ACSL4) and glutathione peroxidase 4 (GPX4) using real-time PCR and Western blotting. In addition, excess iron treatment enhanced protein and lipid oxidation. Supportively, an inverse correlation between urinary FTL protein level and kidney function was observed. Conclusion: These findings suggest that excess iron disrupts cellular homeostasis and affects key proteins involved in kidney cell death. Our study demonstrated that high iron levels caused kidney cell damage. Additionally, urinary FTL might be a useful biomarker to detect kidney damage caused by iron toxicity. Our study also provided insights into the molecular mechanisms of iron-induced kidney injury, discussing several potential targets for future interventions.

Cholangiocarcinoma: Recent Advances in Molecular Pathobiology and Therapeutic Approaches.

Cholangiocarcinomas (CCA) pose a complex challenge in oncology due to diverse etiologies, necessitating tailored therapeutic approaches. This review discusses the risk factors, molecular pathology, and current therapeutic options for CCA and explores the emerging strategies encompassing targeted therapies, immunotherapy, novel compounds from natural sources, and modulation of gut microbiota. CCA are driven by an intricate landscape of genetic mutations, epigenetic dysregulation, and post-transcriptional modification, which differs based on geography (e.g., for liver fluke versus non-liver fluke-driven CCA) and exposure to environmental carcinogens (e.g., exposure to aristolochic acid). Liquid biopsy, including circulating cell-free DNA, is a potential diagnostic tool for CCA, which warrants further investigations. Currently, surgical resection is the primary curative treatment for CCA despite the technical challenges. Adjuvant chemotherapy, including cisplatin and gemcitabine, is standard for advanced, unresectable, or recurrent CCA. Second-line therapy options, such as FOLFOX (oxaliplatin and 5-FU), and the significance of radiation therapy in adjuvant, neoadjuvant, and palliative settings are also discussed. This review underscores the need for personalized therapies and demonstrates the shift towards precision medicine in CCA treatment. The development of targeted therapies, including FDA-approved drugs inhibiting FGFR2 gene fusions and IDH1 mutations, is of major research focus. Investigations into immune checkpoint inhibitors have also revealed potential clinical benefits, although improvements in survival remain elusive, especially across patient demographics. Novel compounds from natural sources exhibit anti-CCA activity, while microbiota dysbiosis emerges as a potential contributor to CCA progression, necessitating further exploration of their direct impact and mechanisms through in-depth research and clinical studies. In the future, extensive translational research efforts are imperative to bridge existing gaps and optimize therapeutic strategies to improve therapeutic outcomes for this complex malignancy.

Integrative multiomics enhancer activity profiling identifies therapeutic vulnerabilities in cholangiocarcinoma of different etiologies.

OBJECTIVES: Cholangiocarcinoma (CCA) is a heterogeneous malignancy with high mortality and dismal prognosis, and an urgent clinical need for new therapies. Knowledge of the CCA epigenome is largely limited to aberrant DNA methylation. Dysregulation of enhancer activities has been identified to affect carcinogenesis and leveraged for new therapies but is uninvestigated in CCA. Our aim is to identify potential therapeutic targets in different subtypes of CCA through enhancer profiling. DESIGN: Integrative multiomics enhancer activity profiling of diverse CCA was performed. A panel of diverse CCA cell lines, patient-derived and cell line-derived xenografts were used to study identified enriched pathways and vulnerabilities. NanoString, multiplex immunohistochemistry staining and single-cell spatial transcriptomics were used to explore the immunogenicity of diverse CCA. RESULTS: We identified three distinct groups, associated with different etiologies and unique pathways. Drug inhibitors of identified pathways reduced tumour growth in in vitro and in vivo models. The first group (ESTRO), with mostly fluke-positive CCAs, displayed activation in estrogen signalling and were sensitive to MTOR inhibitors. Another group (OXPHO), with mostly BAP1 and IDH-mutant CCAs, displayed activated oxidative phosphorylation pathways, and were sensitive to oxidative phosphorylation inhibitors. Immune-related pathways were activated in the final group (IMMUN), made up of an immunogenic CCA subtype and CCA with aristolochic acid (AA) mutational signatures. Intratumour differences in AA mutation load were correlated to intratumour variation of different immune cell populations. CONCLUSION: Our study elucidates the mechanisms underlying enhancer dysregulation and deepens understanding of different tumourigenesis processes in distinct CCA subtypes, with potential significant therapeutics and clinical benefits.

Overexpression of microRNA-205-5p promotes cholangiocarcinoma growth by reducing expression of homeodomain-interacting protein kinase 3.

The microRNA miR-205-5p has diverse effects in different malignancies, including cholangiocarcinoma (CCA), but its effects on CCA progression is unclear. Here we investigated the role and function of miR-205-5p in CCA. Three CCA cell lines and human serum samples were found to have much higher expression levels of miR-205-5p than seen in typical cholangiocyte cell lines and healthy controls. Inhibition of miR-205-5p suppressed CCA cell motility, invasion and proliferation of KKU-213B whereby overexpression of miR-205-5p promoted cell proliferation and motility of KKU-100 cells. Bioinformatics tools (miRDB, TargetScan, miRWalk, and GEPIA) all predicted various miR-205-5p targets. Experiments using miR-205-5p inhibitor and mimic indicated that homeodomain-interacting protein kinase 3 (HIPK3) was a potential direct target of miR-205-5p. Overexpression of HIPK3 using HIPK3 plasmid cloning DNA suppressed migration and proliferation of KKU-100 cells. Notably, HIPK3 expression was lower in human CCA tissues than in normal adjacent tissues. High HIPK3 expression was significantly associated with longer survival time of CCA patients. Multivariate regression analysis indicated tissue HIPK3 levels as an independent prognostic factor for CCA patients. These findings indicate that overexpression of miR-205-5p promotes CCA cells proliferation and migration partly via HIPK3-dependent way. Therefore, targeting miR-205-5p may be a potential treatment approach for CCA.

The Hallmarks of Liver Fluke Related Cholangiocarcinoma: Insight into Drug Target Possibility.

Cholangiocarcinoma (CCA) is a malignant tumor of the biliary tree that is classified into three groups based on its anatomic location: intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA). Perihilar CCA is the most common type and accounts for 50-60% of CCA cases. It is followed by distal CCA and then intrahepatic CCA that account for 20-30% and 10-20% of cases, respectively. This chapter discusses the hallmarks of liver fluke related CCA and explores insights into drug target possibilities.

Alteration of STK11 Expression Associated With Cholangiocarcinoma Progression.

BACKGROUND/AIM: Serine/threonine kinase 11 (STK11), a tumor suppressor, controls 5' AMP-activated protein kinase (AMPK) signaling in a variety of cellular functions. Mutated STK11 has been identified as a novel driver gene that promotes cancer progression. The purpose of this study was to investigate the alteration of STK11 and its correlation with clinicopathological data in cholangiocarcinoma (CCA). MATERIALS AND METHODS: Gene mutation and expression analyses were performed using cBioportal and Gene Expression Profiling Interactive Analysis version 2 (GEPIA2). qRT-PCR was performed to measure STK11 mRNA levels and immunohistochemistry was performed to investigate STK11 protein expression in CCA tissues. RESULTS: The results from publicly available cancer datasets showed that 2.7% of CCA cases had STK11 mutations. Most of STK11 gene mutations are of the truncating type and result in low STK11 mRNA and protein expression. We detected a correlation between STK11 mutation status and the tendency for shorter patient survival. The results of qRT-PCR revealed that STK11 mRNA levels were statistically significantly lower in CCA patients with mutated STK11 compared to those with wild-type STK11 (p-value=0.013). Immunohistochemical staining showed high STK11 expression in 43.8% and low expression in 56.2% of CCA tissues examined. Low STK11 protein expression resulted in poor prognosis compared with high STK11 expression, especially in CCA papillary carcinoma. Univariate and multivariate analysis revealed that high STK11 expression was associated with a decreased hazard ratio of patient survival rates (HR=0.696, p-value=0.06 and HR=0.666, p-value=0.04, respectively). CONCLUSION: Alteration of STK11 mutational or mRNA/protein status might be used as a potential predictive biomarker for the prognosis of the clinical outcomes in CCA patients.

FGF10/FGFR2 Signaling: Therapeutically Targetable Vulnerability in Ligand-responsive Cholangiocarcinoma Cells.

BACKGROUND/AIM: Increasing evidence has revealed FGFR2 as an attractive therapeutic target for cancer including cholangiocarcinoma (CCA). The present study investigated the oncogenic mechanisms by which FGF10 ligand activates FGFR2 in CCA cells and determined whether FGFR inhibitors could suppress FGF10-mediated migration of CCA cells. MATERIALS AND METHODS: Effects of FGF10 on the proliferation, migration, and invasion of KKU-M213A cells were assessed using clonogenic and transwell assays. Protein expression levels of FGFR2 and pro-angiogenic factors were determined via immunoblotting and antibody array analysis. FGFR2 knockdown using a small interfering RNA was used to validate the role of FGF10 in promoting cell migration via FGFR2. The effects of infigratinib (FGFR inhibitor) on cell viability, were determined in KKU-100, KKU-M213A, KKU-452 cells. Moreover, the efficacy of the FGFR inhibitor in suppressing migration via FGF10/FGFR2 stimulation was assessed in KKU-M213A cells. RESULTS: FGF10 significantly increased the expression of phospho-FGFR/FGFR2 and promoted the proliferation, migration, and invasion of KKU-M213A cells. FGF10 increased the expression levels of p-Akt, p-mTOR, VEGF, Slug, and pro-angiogenic proteins related to metastasis. Cell migration mediated by FGF10 was markedly decreased in FGFR2-knockdown cells. Moreover, FGF10/FGFR2 promoted the migration of cells, which was suppressed by the FGFR inhibitor. CONCLUSION: FGF10/FGFR2 activates the Akt/mTOR and VEGF/Slug pathways, which are associated with the stimulation of migration and invasion in CCA. Moreover, the FGF10/FGFR2 signaling was inhibited by an FGFR inhibitor resulting suppression of cell migration, which warrants further studies on their clinical utility for CCA treatment.

Serum α2,6-sialylated glycoform of serotransferrin as a glycobiomarker for diagnosis and prediction of clinical severity in cholangiocarcinoma.

BACKGROUND: Glycoprotein sialylation changes are associated with severe development of various cancers. We previously discovered the sialylation of serotransferrin (TF) in cholangiocarcinoma (CCA) using glycoproteomics approach. However, a simple and reliable method for validating sialylation of a specific glycobiomarker is urgently needed. METHODS: We identified the altered glycosylation in CCA tissues by glycoproteomics approach using mass spectrometry. An enzyme-linked lectin assay (ELLA) was developed for determining the serum levels of sialylated TF in CCA, hepatocellular carcinoma (HCC) and healthy controls in training and validation cohorts. RESULTS: The nine highly sialylated glycoforms of TF were markedly abundant in CCA tumor tissues than in control. Serum SNA-TF and MAL1-TF were significantly higher in CCA patients. Under receiver operating characteristic curve, serum SNA-TF concentrations significantly differentiated CCA from healthy control. Higher SNA-TF were significantly correlated with severe tumor stages and lymph node metastasis. The combined SNA-TF, MAL1-TF, and CA19-9 as a novel glycobiomarkers panel demonstrated the highest specificity (96.2%) for distinguishing CCA from HCC patients. In CCA patients with low CA19-9 levels, SNA-TF in combination with CA19-9 achieved in 97% diagnostic accuracy. CONCLUSIONS: Sialylated serotransferrin glycoforms could be used as a novel glycobiomarker for diagnosis and prediction of clinical severity in CCA patients.

Prediction of Angiopoietin-like Protein 4-related Signaling Pathways in Cholangiocarcinoma Cells.

BACKGROUND/AIM: Angiopoietin-like protein 4 (ANGPTL4) is a multifunctional signaling protein implicated in carbohydrate metabolism, inflammation, cancer growth and progression, anoikis resistance, angiogenesis, and metastasis. However, signaling pathways of ANGPTL4 in cholangiocarcinoma (CCA) remain unknown. The aim of this study was to explore ANGPTL4-related signaling proteins and pathways associated with CCA biology. MATERIALS AND METHODS: ANGPTL4 of CCA cells was silenced by small interfering RNA (siRNA) with scramble control and ANGPTL4-related signaling proteins were investigated using mass spectrometry, bioinformatics tools and molecular docking. RESULTS: Among the 321 differentially expressed proteins, 151 were down-regulated. Among them, bioinformatic analyses revealed that ANGPTL4 interacts with DNA-dependent protein kinase catalytic subunit (PRKDC) and 60S ribosomal protein L21 (RPL21) via AKT serine/threonine kinase 1 (AKT1), mechanistic target of rapamycin kinase (MTOR) and ribosomal protein L5 (RPL5). Online database analysis showed that mRNA and protein expression levels of ANGPTL4-related signaling proteins were significantly higher in CCA than in normal tissues. Moreover, a high mRNA expression level was associated with high tumor grade (p<0.0001) and lymph node metastasis (p<0.0001). CONCLUSION: The signaling pathway of ANGPTL4 in CCA progression might be regulated by PRKDC and RPL21. Furthermore, high expression of ANGPTL4-related signaling proteins has potential to be used in clinical prognosis.

Serum Angiopoietin-Like Protein 4: A Potential Prognostic Biomarker for Prediction of Vascular Invasion and Lymph Node Metastasis in Cholangiocarcinoma Patients.

Cholangiocarcinoma (CCA) is a tumor arising from cholangiocytes lining the bile ducts. Vascular invasion and lymph node metastasis are important prognostic factors for disease staging as well as clinical therapeutic decisions for CCA patients. In the present study, we applied CCA sera proteomic analysis to identify a potential biomarker for prognosis of CCA patients. Then, using bioinformatics tools, we identified angiopoietin-like protein 4 (ANGPTL4) which expressed highest signal intensity among candidate proteins in proteomic analysis of CCA sera. Expression of ANGPTL4 in CCA tissues was determined using immunohistochemistry. The results showed that ANGPTL4 was stained at higher level in CCA cells when compared with normal cholangiocytes. The high expression of ANGPTL4 was associated with lymph node metastasis and advanced tumor stage (p = 0.013 and p = 0.031, respectively). Furthermore, serum ANGPTL4 levels in CCA and healthy control (HC) were analyzed using a dot blot assay. And it was found that ANGPTL4 level was significantly higher in CCA than HC group (p < 0.0001). ROC curve analysis revealed that serum ANGPTL4 level was effectively distinguished CCA from healthy patients (cutoff = 0.2697 arbitrary unit (AU), 80.0% sensitivity, 72.7% specificity, AUC = 0.825, p < 0.0001). Serum ANGPTL4 level was associated with vascular invasion and lymph node metastasis (p = 0.0004 and p = 0.006), so that it differentiated CCA with vascular invasion from CCA without vascular invasion (cutoff = 0.5526 AU, 64.9% sensitivity, 92.9% specificity, AUC = 0.751, p = 0.006) and it corresponded to CCA with/without lymph node metastasis (cutoff = 0.5399 AU, 71.4% sensitivity, 70.8% specificity, AUC = 0.691, p = 0.01) by ROC analysis. Serum ANGPTL4 levels showed superior predictive efficiency compared with CA 19-9 and CEA for vascular invasion and lymph node metastasis. In addition, serum ANGPTL4 level was an independent predictive indicator by multivariate regression analysis. In conclusion, serum ANGPTL4 could be a novel prognostic biomarker for prediction of vascular invasion and lymph node metastasis of CCA patients.

Inhibition of FGFR2 enhances chemosensitivity to gemcitabine in cholangiocarcinoma through the AKT/mTOR and EMT signaling pathways.

AIM: To investigate the oncogenic role of FGFR2 in carcinogenesis in cholangiocarcinoma (CCA) cells. In addition, the feasibility of using FGFR inhibitors in combination with standard chemotherapy was also explored for the chemosensitizing effect in CCA cells. MAIN METHODS: Five CCA cell lines were used to screen FGFR2 expression by Western immunoblotting. Two CCA cell lines, KKU-100 and KKU-213A, were knocked down of the FGFR2 gene using siRNA. Cell viability was assessed by the MTS cell proliferation assay. Reproductive cell death was assessed by clonogenic assay. The effects on cell migration and invasion were analyzed by the Transwell chamber method. Cell cycle analysis was performed by flow cytometry. Cell angiogenesis was assessed by HUVEC tube formation and human angiogenesis antibody array analysis. Proteins associated with proliferative and metastatic properties were evaluated by Western blotting. KEY FINDINGS: Knockdown of FGFR2 suppressed cell growth and colony formation in CCA cells in association with G2/M cell cycle arrest and downregulation of STAT3, cyclin A and cyclin B1. Silencing FGFR2 enhanced the suppressive effect of gemcitabine (Gem) on cell migration and invasion. The combination of infigratinib, an FGFR inhibitor, and Gem, interrupted cell growth, migration, and invasion via downregulation of FGFR/AKT/mTOR pathways and the EMT-associated proteins vimentin and slug. Moreover, the combination also suppressed tube formation together with decreased expression of the proangiogenic factor VEGF. SIGNIFICANCE: Inhibition of FGFRs by infigratinib enhanced the antitumor effect of Gem in CCA cells through downregulation of the FGFR/AKT/mTOR, FGFR/STAT3 and EMT signaling pathways.

Therapeutic targeting of ARID1A and PI3K/AKT pathway alterations in cholangiocarcinoma.

BACKGROUND: Genetic alterations in ARID1A were detected at a high frequency in cholangiocarcinoma (CCA). Growing evidence indicates that the loss of ARID1A expression leads to activation of the PI3K/AKT pathway and increasing sensitivity of ARID1A-deficient cells for treatment with the PI3K/AKT inhibitor. Therefore, we investigated the association between genetic alterations of ARID1A and the PI3K/AKT pathway and evaluated the effect of AKT inhibition on ARID1A-deficient CCA cells. METHODS: Alterations of ARID1A, PI3K/AKT pathway-related genes, clinicopathological data and overall survival of 795 CCA patients were retrieved from cBio Cancer Genomics Portal (cBioPortal) databases. The association between genetic alterations and clinical data were analyzed. The effect of the AKT inhibitor (MK-2206) on ARID1A-deficient CCA cell lines and stable ARID1A-knockdown cell lines was investigated. Cell viability, apoptosis, and expression of AKT signaling were analyzed using an MTT assay, flow cytometry, and Western blots, respectively. RESULTS: The analysis of a total of 795 CCA samples revealed that ARID1A alterations significantly co-occurred with mutations of EPHA2 (p < 0.001), PIK3CA (p = 0.047), and LAMA1 (p = 0.024). Among the EPHA2 mutant CCA tumors, 82% of EPHA2 mutant tumors co-occurred with ARID1A truncating mutations. CCA tumors with ARID1A and EPHA2 mutations correlated with better survival compared to tumors with ARID1A mutations alone. We detected that 30% of patients with PIK3CA driver missense mutations harbored ARID1A-truncated mutations and 60% of LAMA1-mutated CCA co-occurred with truncating mutations of ARID1A. Interestingly, ARID1A-deficient CCA cell lines and ARID1A-knockdown CCA cells led to increased sensitivity to treatment with MK-2206 compared to the control. Treatment with MK-2206 induced apoptosis in ARID1A-knockdown KKU-213A and HUCCT1 cell lines and decreased the expression of pAKTS473 and mTOR. CONCLUSION: These findings suggest a dependency of ARID1A-deficient CCA tumors with the activation of the PI3K/AKT-pathway, and that they may be more vulnerable to selective AKT pathway inhibitors which can be used therapeutically.

Concentration of Urine Samples Improves Sensitivity in Detection of Strongyloides-Specific IgG Antibody in Urine for Diagnosis of Strongyloidiasis.

Detection of IgG in urine is an efficient method comparable to that in serum for diagnosis of strongyloidiasis, but the effects of daily variation in urine dilution on diagnostic accuracy are not clearly known. This study evaluated the effects of urine concentration on the detection of parasite-specific IgG by urine enzyme-linked immunosorbent assay (ELISA), particularly in individuals with borderline results or false-negative diagnosis. Optimal concentration conditions were established by comparing Strongyloides-specific IgG antibody levels between unconcentrated and concentrated urine in participants with different infection intensities, namely, healthy control (HC), low-negative (LN), high-negative (HN), and low-positive (LP) groups. The optimal condition was selected and validated in a field trial study. The final urine concentration protocol required centrifugation at 4,000 × g at 4°C for 10 mins using the Amicon concentrator tube. This protocol was validated in groups of participants with various diagnoses according to urine ELISA and fecal examination (n = 148). The concentrated-urine ELISA increased the proportion of positive results in the LN group by 68.2% and by 100% in the HN group. Significantly elevated IgG antibody levels were seen in the LP group. In the group that was false negative by urine ELISA but positive by fecal examination (n = 28), concentrated-urine ELISA yielded 100% positive results. Overall, the frequency estimates of Strongyloides stercoralis were 23.6% by fecal culture, 27% by standard urine ELISA, and 90.5% by concentrated-urine ELISA. The concentration of urine samples prior to analysis by ELISA improved the sensitivity for diagnosis and is potentially useful in the diagnosis of strongyloidiasis in immunocompromised individuals or in low-prevalence areas.

Arctigenin inhibits cholangiocarcinoma progression by regulating cell migration and cell viability via the N-cadherin and apoptosis pathway.

Northeast Thailand has the highest incidence of cholangiocarcinoma (CCA) in the world. The lack of promising diagnostic markers and appropriate therapeutic drugs is the main problem for metastatic stage CCA patients who have a poor prognosis. N-cadherin, a cell adhesion molecule, is usually upregulated in cancers and has been proposed as an important mediator in epithelial-mesenchymal transition (EMT), one of the metastasis processes. Additionally, it has been shown that arctigenin, a seed isolated compound from Arctium lappa, can inhibit cancer cell progression via suppression of N-cadherin pathway. In this study, we investigated the protein expression of N-cadherin and its correlation with clinicopathological data of CCA patients, as well as the impact of arctigenin on KKU-213A and KKU-100 CCA cell lines and its underlying mechanisms. Immunohistochemistry results demonstrated that high expression of N-cadherin was significantly associated with severe CCA stage (p = 0.027), and shorter survival time (p = 0.002) of CCA patients. The mean overall survival times between low and high expression of N-cadherin were 31.6 and 14.8 months, respectively. Wound healing assays showed that arctigenin significantly inhibited CCA cell migration by downregulating N-cadherin whereas upregulating E-cadherin expression. Immunocytochemical staining revealed that arctigenin suppressed the expression of N-cadherin in both CCA cell lines. Furthermore, flow cytometry and western blot analysis revealed that arctigenin significantly reduced CCA cell viability and induced apoptosis via the Bax/Bcl-2/caspase-3 pathway. This research supports the use of N-cadherin as a prognostic marker for CCA and arctigenin as a potential alternative therapy for improving CCA treatment outcomes.

Diagnostic and Prognostic Value of Circulating Cell-Free DNA for Cholangiocarcinoma.

The analysis of cfDNA has been applied as a liquid biopsy in several malignancies. However, its value in the diagnosis and prognosis of cholangiocarcinoma (CCA) have not been well defined. We aimed to investigate the diagnostic and prognostic values of cfDNA level and tumor-specific mutation in circulating DNA (ctDNA) in CCA. The plasma cfDNA levels from 62 CCA patients, 33 benign biliary disease (BBD) patients and 30 normal controls were quantified by fluorescent assay. Targeted probe-based sequencing of 60 genes was applied for mutation profiling in 10 ctDNA samples and their corresponding treatment-naïve tissues. cfDNA levels in CCA were significantly higher than those in BBD and normal controls. We found that cfDNA levels at 0.2175 and 0.3388 ng/µL significantly discriminated CCA from healthy controls and BBD with 88.7 and 82.3% sensitivity and 96.7 and 57.6% specificity, respectively. cfDNA levels showed superior diagnostic efficacy in detecting CCA compared to CEA and CA19-9. ARID1A (30%), PBRM1 (30%), MTOR (30%), and FGFR3 (30%) mutations were the most common. Using nine frequently mutated genes in the ctDNA samples, the diagnostic accuracy of cfDNA sequencing was 90.8%, with 96.7% average sensitivity and 72.4% specificity. This study supports the use of cfDNA as a diagnosis and prognostic biomarker for CCA.

Promoter hypermethylation of early B cell factor 1 (EBF1) is associated with cholangiocarcinoma progression.

DNA hypermethylation in a promoter region causes gene silencing via epigenetic changes. We have previously reported that early B cell factor 1 (EBF1) was down-regulated in cholangiocarcinoma (CCA) tissues and related to tumor progression. Thus, we hypothesized that the DNA hypermethylation of EBF1 promoter would suppress EBF1 expression in CCA and induce its progression. In this study, the DNA methylation status of EBF1 and mRNA expression levels were analyzed in CCA and normal bile duct (NBD) tissues using a publicly available database of genome-wide association data. The results showed that the DNA methylation of EBF1 promoter region was significantly increased in CCA tissues compared with those of NBD. The degree of methylation was negatively correlated with EBF1 mRNA expression levels. Using methylation-specific PCR technique, the DNA methylation rates of EBF1 promoter region were investigated in CCA tissues (n=72). CCA patients with high methylation rates of EBF1 promoter region in the tumor tissues (54/72) had a poor prognosis. Higher methylation rates of EBF1 promoter region have shown in all CCA cell lines than that of an immortal cholangiocyte cell line (MMNK1). Upon treatment with the DNA methyltransferase inhibitor 5-Aza-dC, increased EBF1 expression levels and reduced DNA methylation rates were observed in CCA cells. Moreover, restoration of EBF1 expression in CCA cells led to inhibition of cell growth, migration and invasion. In addition, RNA sequencing analysis suggested that EBF1 is involved in suppression of numerous pathways in cancer. Taken together, DNA hypermethylation in the EBF1 promoter region suppresses EBF1 expression and induces CCA progression with aggressive clinical outcomes.

Establishment of a Potential Serum Biomarker Panel for the Diagnosis and Prognosis of Cholangiocarcinoma Using Decision Tree Algorithms.

Potential biomarkers which include S100 calcium binding protein A9 (S100A9), mucin 5AC (MUC5AC), transforming growth factor ß1 (TGF-ß1), and angiopoietin-2 have previously been shown to be effective for cholangiocarcinoma (CCA) diagnosis. This study attempted to measure the sera levels of these biomarkers compared with carbohydrate antigen 19-9 (CA19-9). A total of 40 serum cases of CCA, gastrointestinal cancers (non-CCA), and healthy subjects were examined by using an enzyme-linked immunosorbent assay. The panel of biomarkers was evaluated for their accuracy in diagnosing CCA and subsequently used as inputs to construct the decision tree (DT) model as a basis for binary classification. The findings showed that serum levels of S100A9, MUC5AC, and TGF-ß1 were dramatically enhanced in CCA patients. In addition, 95% sensitivity and 90% specificity for CCA differentiation from healthy cases, and 70% sensitivity and 83% specificity for CCA versus non-CCA cases was obtained by a panel incorporating all five candidate biomarkers. In CCA patients with low CA19-9 levels, S100A9 might well be a complementary marker for improved diagnostic accuracy. The high levels of TGF-ß1 and angiopoietin-2 were both associated with severe tumor stages and metastasis, indicating that they could be used as a reliable prognostic biomarkers panel for CCA patients. Furthermore, the outcome of the CCA burden from the Classification and Regression Tree (CART) algorithm using serial CA19-9 and S100A9 showed high diagnostic efficiency. In conclusion, results have shown the efficacy of CCA diagnosis and prognosis of the novel CCA-biomarkers panel examined herein, which may prove be useful in clinical settings.

Vitamin C supplementation reduces expression of circulating miR-451a in subjects with poorly controlled type 2 diabetes mellitus and high oxidative stress.

BACKGROUND: Vitamin C is an essential element required for normal metabolic function. We investigated the effect of vitamin C supplementation on circulating miRNA (miR) expression in subjects with poorly controlled type 2 diabetes mellitus (T2DM). Changes in miR expression were also correlated with clinical measures of disease. METHODS: Pre- and post-vitamin C supplementation samples from five participants who had increased vitamin C levels, improved oxidative status and polymorphonuclear (PMN) function after receiving 1,000 mg of vitamin C daily for six weeks were screened for miRNA expression using the NanoString miRNA assay. Differences in miRNA expression identified from the miRNA screen were validated by qRT-PCR. RESULTS: Four miRNAs showed significantly different expression post-vitamin C supplementation relative to baseline, including the down-regulation of miR-451a (-1.72 fold change (FC), p = 0.036) and up-regulation of miR-1253 (0.62 FC, p = 0.027), miR-1290 (0.53 FC, p = 0.036) and miR-644a (0.5 FC, p = 0.042). The validation study showed only miR-451a expression was significantly different from baseline with vitamin C supplementation. MiR-451a expression was negatively correlated with vitamin C levels (r =  - 0.497, p = 0.049) but positively correlated with levels of malondialdehyde (MDA) (r = 0.584, p = 0.017), cholesterol (r = 0.564, p = 0.022) and low-density lipoproteins (LDL) (r = 0.522, p = 0.037). Bioinformatics analysis of the putative target genes of miR-451a indicated gene functions related to signaling pathways involved in cellular processes, such as the mammalian target of rapamycin (mTOR) signaling pathway. CONCLUSIONS: Vitamin C supplementation altered circulating miR-451a expression. The results from this pilot study suggest that miRNAs could be used as biomarkers to indicate oxidative status in subjects with T2DM and with poor glycemic control and could lead to a novel molecular strategy to reduce oxidative stress in T2DM.

ARID1A alterations and their clinical significance in cholangiocarcinoma.

BACKGROUND: ARID1A is a member of the SWI/SNF chromatin remodeling complex. It functions as a tumor suppressor and several therapeutic targets in ARID1A-mutated cancers are currently under development, including EZH2. A synthetic lethal relationship between ARID1A and EZH2 has been revealed in several tumor entities. Although genomic alterations of ARID1A have been described in various cancers, no study has examined correlations between ARID1A gene mutation and protein expression with clinicopathologic parameters and prognosis, particularly in liver fluke-related cholangiocarcinoma (Ov-CCA). Here, we investigated the clinical significance of ARID1A mutations and protein expression in CCA tissues and determined whether there is a correlation with EZH2 protein expression. METHODS: We evaluated ARID1A and EZH2 immunoreactivity using immunohistochemistry in 98 Ov-CCA with a wide range of clinicopathological features. Somatic mutations of ARID1A were analyzed using the ICGC sequencing data in 489 of Ov and non Ov-CCA and assessed prognostic values. RESULTS: While detecting a loss or reduction of ARID1A expression in 54 cases (55%) in Ov-CCA, ARID1A expression was associated with ARID1A mutations (p < 0.001, adjusted p-value < 0.001). We observed that 12 of 13 tumors (92%) with loss of ARID1A expression had truncating mutations. There were nine of 13 tumors (69%) with loss of ARID1A expression and 25 of 41 tumors (61%) with low ARID1A expression exhibited distant metastasis (p = 0.028, adjusted p-value = 0.168). ARID1A was predominantly mutated in Ov-CCA compared to non Ov-CCA (24% and 14% in Ov-CCA and non Ov-CCA, respectively, p = 0.027). There were 36 of 72 (50%) and 52 of 79 (66%) tumors with ARID1A mutation showed tumor stage IV and T3/T4, respectively. The significant mutual exclusivity and co-occurrence between ARID1A and TP53/KRAS mutations were not found in ICGC cohort. In addition, high EZH2 expression, a potential synthetic lethal target in ARID1A-mutated tumors, was detected in 49 of 98 Ov-CCA (50%). Importantly, neither ARID1A expression nor ARID1A mutations correlated with EZH2 expression in this cohort. CONCLUSION: We found that ARID1A inactivation, by somatic mutation or by loss of expression, frequently occurs in Ov-CCA. Reduction of ARID1A expression and/or somatic mutation was shown to be associated with CCA progression. These findings suggest that ARID1A may serve as a prognostic biomarker, and thus may be a promising therapeutic target for CCA.