Overexpression of Insulin Receptor Substrate 1 (IRS1) Relates to Poor Prognosis and Promotes Proliferation, Stemness, Migration, and Oxidative Stress Resistance in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is one of the oxidative stress-driven carcinogenesis through chronic inflammation. Insulin receptor substrate 1 (IRS1), an adaptor protein of insulin signaling pathways, is associated with the progression of many inflammation-related cancers. This study hypothesized that oxidative stress regulates IRS1 expression and that up-regulation of IRS1 induces CCA progression. The localizations of IRS1 and an oxidative stress marker (8-oxodG) were detected in CCA tissues using immunohistochemistry (IHC). The presence of IRS1 in CCA tissues was confirmed using immortal cholangiocyte cells (MMNK1), a long-term oxidative-stress-induced cell line (ox-MMNK1-L), and five CCA cell lines as cell culture models. IRS1 was overexpressed in tumor cells and this was associated with a shorter patient survival time and an increase in 8-oxodG. IRS1 expression was higher in ox-MMNK1-L cells than in MMNK1 cells. Knockdown of IRS1 by siRNA in two CCA cell lines led to inhibition of proliferation, cell cycle progression, migration, invasion, stemness, and oxidative stress resistance properties. Moreover, a transcriptomics study demonstrated that suppressing IRS1 in the KKU-213B CCA cell line reduced the expression levels of several genes and pathways involved in the cellular functions. The findings indicate that IRS1 is a key molecule in the connection between oxidative stress and CCA progression. Therefore, IRS1 and its related genes can be used as prognostic markers and therapeutic targets for CCA therapy.

Opposing Roles of FoxA1 and FoxA3 in Intrahepatic Cholangiocarcinoma Progression.

Cholangiocarcinoma (CCA), a malignancy of biliary epithelium, is related to liver stem cell deregulation. FoxAs are a group of transcription factors that play critical roles in liver stem cell differentiation. In this study, the expression levels of FoxAs (i.e., FoxA1, FoxA2 and FoxA3) were detected in intrahepatic CCA tissues and the functions of FoxAs were studied in CCA cell lines. FoxA1 and FoxA2 were mainly localized in the nuclei of normal bile duct (NBD) cells and some of the cancer cells. Low expression of FoxA1 in CCA tissues (72%) was significantly correlated with poor prognosis. FoxA3 expression of CCA cells was localized in the nucleus and cytoplasm, whereas it was slightly detected in NBDs. High expression of FoxA3 in cancer tissues (61%) was significantly related to high metastasis status. These findings suggest the opposing roles of FoxA1 and FoxA3 in CCA. Moreover, the FoxA1-over-expressing CCA cell line exhibited a significant reduction in proliferative and invasive activities compared to control cells. Knockdown of FoxA3 in CCA cells resulted in a significant decrease in proliferative and invasive activities compared with control cells. Taken together, in CCA, FoxA1 is down-regulated and has tumor suppressive roles, whereas FoxA3 is up-regulated and has oncogenic roles.

Syzygium gratum Extract Alleviates Vascular Alterations in Hypertensive Rats.

Background and Objectives: Syzygium gratum (SG) is a local vegetable and widely consumed in Thailand. Previously, a strong antioxidative effect of SG extract has been reported. The effects of SG extract on hypertension have remained unknown. The effect of SG aqueous extract on blood pressure and vascular changes were examined in L-NAME-induced hypertensive rats (LHR), and its potential active constituents were also explored. Materials and Methods: Male Sprague Dawley rats were allocated to control, L-NAME (40 mg/kg/day), L-NAME + SG (100, 300, 500 mg/kg/day), or captopril (5 mg/kg/day) groups. The components of SG extract were analyzed. Results: The analysis of aqueous SG extract was carried out using HPLC-Mass spectroscopy, and phenolic compounds could be identified as predominant components which might be responsible for its antihypertensive effects observed in the LHR model (p < 0.05). Additionally, SG extract also improved vascular responses to acetylcholine and decreased vascular remodeling in LHR (p < 0.05). Enhancements of eNOS expression and plasma nitric oxide metabolite levels, and attenuation of angiotensin converting enzyme (ACE) activity and plasma angiotensin II levels were observed in the LHR group treated with SG. Moreover, SG exhibited strong antioxidant activities by reducing vascular superoxide generation and systemic malondialdehyde in LHRs. Captopril suppressed high blood pressure and alleviated vascular changes and ACE activity in LHRs, similar to those of the SG extract (p < 0.05). Conclusion: Our results suggest that the SG extract exhibited antihypertensive effects, which is relevant to alleviation of vascular dysfunction and vascular remodeling of LHRs. These effects might be mediated by phenolic compounds to inhibit ACE activity and scavenge reactive oxygen species in LHR.

Insight into the Molecular Mechanisms of AuNP-Based Aptasensor for Colorimetric Detection: A Molecular Dynamics Approach.

Colorimetric aptasensor based on assembly of salt-induced gold nanoparticles (AuNPs) is a promising biosensor. However, the molecular mechanism of the aptasensor is far from being fully understood. Herein, molecular dynamics (MD) simulation was used to investigate molecular interactions in the detection of ochratoxin A (OTA) including the following: (i) the molecular recognition of the anti-OTA aptamer, (ii) OTA-aptamer interactions in monovalent (Na+) and divalent (Mg2+) electrolytes, (iii) the binding mode of citrate on the AuNP surface, (iv) interactions of the aptamer with citrate-capped AuNPs, and (v) a detailed mechanism of the aptasensor. Our MD simulations revealed a specific binding of the OTA-aptamer complex, compared with OTB and warfarin. Compared with Na+, Mg2+ ions exerted a more effective attractive force between OTA and anti-OTA aptamer. Three different binding modes of citrate on AuNP surfaces were found. The kinetics of the adsorption of unfolded aptamers onto the citrate-capped AuNP was also elucidated. Most importantly, our MD simulation revealed an insightful analysis of the molecular mechanisms in the AuNP-based aptasensor and paved the way for the design of a novel colorimetric aptasensor for other target molecules, which is not limited to OTA detection.

Anti-cancer activity of asiatic acid against human cholangiocarcinoma cells through inhibition of proliferation and induction of apoptosis.

Plant-derived anti-cancer agents have been of considerable interest due to their promising effectiveness with low side effects. Asiatic acid, the main constituent of the medicinal plant Centella asiatica (L.) Urban, has a wide range of biological properties such as antioxidant, anti-inflammatory and anti-cancer activities. Cholangiocarcinoma (CCA), which is a malignant tumor of bile duct epithelium, is one of the leading cancers in Southeast Asia, notably the northeast of Thailand where the liver fluke, Opisthorchis viverrini predominates. Many in vitro and in vivo studies have provided evidence supporting that oxidative stress induced by chronic inflammation is involved in CCA genesis with aggressive clinical outcomes. This study was performed to evaluate the cytotoxic effects of asiatic acid on two human CCA cell lines (KKU-156 and KKU-213). Cell viability was determined by a sulforhodamine B (SRB) assay. Morphological changes of the cells were observed by microscopy. Cell apoptosis was detected by flow cytometry using annexin V and propidium iodide (PI) staining. Messenger RNA (mRNA) expression levels of BAX, BCL2 and Survivin/BIRC5 were analyzed by real-time polymerase chain reaction (PCR). It was found that asiatic acid efficiently suppressed CCA cellular viability via induction of apoptosis. In addition, the occurrence of asiatic acid-induced apoptosis was confirmed by microscopic observation of apoptotic vesicles, down-regulation of anti-apoptotic genes (BCL2 and Survivin/BIRC5) and increased early and late apoptotic cells. Our results showed the chemotherapeutic activities of asiatic acid, suggesting the anti-cancer properties of this compound should be clinically assessed and its supplementation may lead to an improvement of survival of CCA patients.

Dissecting Multivalent Lectin-Carbohydrate Recognition Using Polyvalent Multifunctional Glycan-Quantum Dots.

Multivalent protein-carbohydrate interactions initiate the first contacts between virus/bacteria and target cells, which ultimately lead to infection. Understanding the structures and binding modes involved is vital to the design of specific, potent multivalent inhibitors. However, the lack of structural information on such flexible, complex, and multimeric cell surface membrane proteins has often hampered such endeavors. Herein, we report that quantum dots (QDs) displayed with a dense array of mono-/disaccharides are powerful probes for multivalent protein-glycan interactions. Using a pair of closely related tetrameric lectins, DC-SIGN and DC-SIGNR, which bind to the HIV and Ebola virus glycoproteins (EBOV-GP) to augment viral entry and infect target cells, we show that such QDs efficiently dissect the different DC-SIGN/R-glycan binding modes (tetra-/di-/monovalent) through a combination of multimodal readouts: Förster resonance energy transfer (FRET), hydrodynamic size measurement, and transmission electron microscopy imaging. We also report a new QD-FRET method for quantifying QD-DC-SIGN/R binding affinity, revealing that DC-SIGN binds to the QD >100-fold tighter than does DC-SIGNR. This result is consistent with DC-SIGN's higher trans-infection efficiency of some HIV strains over DC-SIGNR. Finally, we show that the QDs potently inhibit DC-SIGN-mediated enhancement of EBOV-GP-driven transduction of target cells with IC50 values down to 0.7 nM, matching well to their DC-SIGN binding constant (apparent Kd = 0.6 nM) measured by FRET. These results suggest that the glycan-QDs are powerful multifunctional probes for dissecting multivalent protein-ligand recognition and predicting glyconanoparticle inhibition of virus infection at the cellular level.

Molecular Recognition-Mediated Transformation of Single-Chain Polymer Nanoparticles into Crosslinked Polymer Films.

We describe single-chain polymer nanoparticles (SCNPs) possessing intramolecular dynamic covalent crosslinks that can transform into polymer films through a molecular recognition-mediated crosslinking process. The SCNPs utilise molecular recognition with surface-immobilised proteins to concentrate upon a substrate, bringing the SCNPs into close spatial proximity with one another and allowing their dynamic covalent crosslinkers to undergo intra- to interpolymer chain crosslinking leading to the formation of polymeric film. SCNPs must possess both the capacity for specific molecular recognition and a dynamic nature to their intramolecular crosslinkers to form polymer films, and an investigation of the initial phase of film formation indicates it proceeds from features which form upon the surface then grow predominantly in the xy directions. This approach to polymer film formation presents a potential method to "wrap" surfaces displaying molecular recognition motifs-which could potentially include viral, cellular and bacterial surfaces or artificial surfaces displaying multivalent recognition motifs-within a layer of polymer film.

Compact, Polyvalent Mannose Quantum Dots as Sensitive, Ratiometric FRET Probes for Multivalent Protein-Ligand Interactions.

A highly efficient cap-exchange approach for preparing compact, dense polyvalent mannose-capped quantum dots (QDs) has been developed. The resulting QDs have been successfully used to probe multivalent interactions of HIV/Ebola receptors DC-SIGN and DC-SIGNR (collectively termed as DC-SIGN/R) using a sensitive, ratiometric Förster resonance energy transfer (FRET) assay. The QD probes specifically bind DC-SIGN, but not its closely related receptor DC-SIGNR, which is further confirmed by its specific blocking of DC-SIGN engagement with the Ebola virus glycoprotein. Tuning the QD surface mannose valency reveals that DC-SIGN binds more efficiently to densely packed mannosides. A FRET-based thermodynamic study reveals that the binding is enthalpy-driven. This work establishes QD FRET as a rapid, sensitive technique for probing structure and thermodynamics of multivalent protein-ligand interactions.

Templating carbohydrate-functionalised polymer-scaffolded dynamic combinatorial libraries with lectins.

A conceptually new approach to the design of macromolecular receptors for lectins is outlined. Carbohydrate-functionalised Polymer-Scaffolded Dynamic Combinatorial Libraries (PS-DCLs) have been prepared in aqueous solution by the reversible conjugation of carbohydrates possessing acylhydrazide functionalities in their aglycone on to an aldehyde-functionalised polymer scaffold. PS-DCLs have been shown to undergo compositional change in response to the addition of lectin templates, with polymer scaffolds preferentially incorporating carbohydrate units which recognise the lectin added. This compositional change has been shown to generate polymers of significantly enhanced affinity for the lectin added, with enhancements in free energy of binding in the range of 5.2-8.8 kJ mol(-1) observed. Experiments indicate that these enhancements are not only as a consequence of increased display of the preferred carbohydrate upon the polymer scaffold, but that templation also reorganises key residues into strategic positions in order to interact more strongly with the target.

Corosolic Acid Induced Apoptosis via Upregulation of Bax/Bcl-2 Ratio and Caspase-3 Activation in Cholangiocarcinoma Cells.

Cholangiocarcinoma (CCA), an aggressive malignancy arising from the biliary epithelium, exhibits a high incidence in Thailand. CCA usually lacks specific symptoms and is typically diagnosed in its advanced stages, presenting significant treatment challenges. Current CCA therapeutic options, including surgery, chemotherapy, and radiation, have limited success rates and often cause side effects. Nature-derived compounds hold promise for reducing undesirable adverse effects and are an excellent source of anticancer drugs. Corosolic acid (CA), a triterpenoid found in Lagerstroemia speciosa L. leaves, exhibits anticancer properties; however, the effectiveness of CA against CCA and its molecular mechanisms remained unexplored. Herein, the anti-CCA and apoptosis-inducing effects of CA were investigated using various techniques, i.e., the MTT assay, flow cytometry with FITC-labeled Annexin V (Annexin V-FITC) and propidium iodide double staining, JC-1 staining, western blot analysis, caspase-3 activity assay, and molecular dynamics (MD) simulations. CA inhibited the proliferation of KKU-213A and KKU-213B CCA cells and triggered apoptosis through alterations in mitochondrial membrane potential (ΔΨm), and increases in the Bax/Bcl-2 expression ratio, cytochrome c release, and caspase-3 activity. As indicated by MD simulations, CA has the potential to bind to Bcl-2 through hydrogen bonds between amino acid residues R146 and N143. These findings underscore the potential of CA as a promising candidate for treatment of CCA.

Sustainable methods for the carboxymethylation and methylation of ursolic acid with dimethyl carbonate under mild and acidic conditions.

Ursolic acid is a triterpene plant extract that exhibits significant potential as an anti-cancer, anti-tumour, and anti-inflammatory agent. Its direct use in the pharmaceutical industry is hampered by poor uptake of ursolic acid in the human body coupled with rapid metabolism causing a decrease in bioactivity. Modification of ursolic acid can overcome such issues, however, use of toxic reagents, unsustainable synthetic routes and poor reaction metrics have limited its potential. Herein, we demonstrate the first reported carboxymethylation and/or methylation of ursolic acid with dimethyl carbonate (DMC) as a green solvent and sustainable reagent under acidic conditions. The reaction of DMC with ursolic acid, in the presence of PTSA, ZnCl2, or H2SO4-SiO2 yielded the carboxymethylation product 3ß-[[methoxy]carbonyl]oxyurs-12-en-28-oic acid, the methylation product 3ß-methoxyurs-12-en-28-oic acid and the dehydration product urs-2,12-dien-28-oic acid. PTSA demonstrated high conversion and selectivity towards the previously unreported carboxymethylation of ursolic acid, while the application of formic acid in the system led to formylation of ursolic acid (3ß-formylurs-12-en-28-oic acid) in quantitative yields via esterification, with DMC acting solely as a solvent. Meanwhile, the methylation product of ursolic acid, 3ß-methoxyurs-12-en-28-oic acid, was successfully synthesised with FeCl3, demonstrating exceptional conversion and selectivity, >99% and 99%, respectively. Confirmed with the use of qualitative and quantitative green metrics, this result represents a significant improvement in conversion, selectivity, safety, and sustainability over previously reported methods of ursolic acid modification. It was demonstrated that these methods could be applied to other triterpenoids, including corosolic acid. The study also explored the potential pharmaceutical applications of ursolic acid, corosolic acid, and their derivatives, particularly in anti-inflammatory, anti-cancer, and anti-tumour treatments, using molecular ADMET and docking methods. The methods developed in this work have led to the synthesis of novel molecules, thus creating opportunities for the future investigation of biological activity and the modification of a wide range of triterpenoids applying acidic DMC systems to deliver novel active pharmaceutical intermediates.

Dual-Responsive Carbon Quantum Dots for the Simultaneous Detection of Cytosine and 5-Methylcytosine Interpreted by a Machine Learning-Assisted Smartphone.

DNA methylation is an epigenetic alteration that results in 5-methylcytosine (5-mC) through the addition of a methyl group to the fifth carbon of a cytosine (C) residue. The methylation level, the ratio of 5-mC to C, in urine might be related to the whole-body epigenetic status and the occurrence of common cancers. To date, never before have any nanomaterials been developed to simultaneously determine C and 5-mC in urine samples. Herein, a dual-responsive fluorescent sensor for the urinary detection of C and 5-mC has been developed. This assay relied on changes in the optical properties of nitrogen-doped carbon quantum dots (CQDs) prepared by microwave-assisted pyrolysis. In the presence of C, the blue-shifted fluorescence intensity of the CQDs increased. However, fluorescence quenching was observed upon the addition of 5-mC. This was primarily due to photoinduced electron transfer as confirmed by the density functional theory calculation. In urine samples, our sensitive fluorescent sensor had detection limits for C and 5-mC of 43.4 and 74.4 µM, respectively, and achieved satisfactory recoveries ranging from 103.5 to 115.8%. The simultaneous detection of C and 5-mC leads to effective methylation level detection, achieving recoveries in the range of 104.6-109.5%. Besides, a machine learning-enabled smartphone was also developed, which can be effectively applied to the determination of methylation levels (0-100%). These results demonstrate a simple but very effective approach for detecting the methylation level in urine, which could have significant implications for predicting the clinical prognosis.

Rapid detection of cancer DNA in human blood using cysteamine-capped AuNPs and a machine learning-enabled smartphone.

DNA methylation occurs when a methyl group is added to a cytosine (C) residue's fifth carbon atom, forming 5-methylcytosine (5-mC). Cancer genomes have a distinct methylation landscape (Methylscape), which could be used as a universal cancer biomarker. This study developed a simple, low-cost, and straightforward Methylscape sensing platform using cysteamine-decorated gold nanoparticles (Cyst/AuNPs), in which the sensing principle is based on methylation-dependent DNA solvation. Normal and cancer DNAs have distinct methylation profiles; thus, they can be distinguished by observing the dispersion of Cyst/AuNPs adsorbed on these DNA aggregates in MgCl2 solution. After optimising the MgCl2, Cyst/AuNPs, DNA concentration, and incubation time, the optimised conditions were used for leukemia screening, by comparing the relative absorbance (ΔA 650/525). Following the DNA extraction from actual blood samples, this sensor demonstrated effective leukemia screening in 15 minutes with high sensitivity, achieving 95.3% accuracy based on the measurement by an optical spectrophotometer. To further develop for practical realisation, a smartphone assisted by machine learning was used to screen cancer patients, achieving 90.0% accuracy in leukemia screening. This sensing platform can be applied not only for leukemia screening but also for other cancers associated with epigenetic modification.

Aptamer-based colorimetric detection of the DNA damage marker 8-oxo-dG using cysteamine-stabilised gold nanoparticles.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) is a crucial biomarker for oxidative DNA damage and carcinogenesis. Current strategies for 8-oxo-dG detection often require sophisticated instruments and qualified personnel. In this study, cysteamine-stabilised gold nanoparticles (cyst-AuNPs) were synthesised and used for colorimetric detection of 8-oxo-dG in urine. Sensing of 8-oxo-dG is based on the anti-aggregation of cyst-AuNPs, mediated by the specific recognition of 8-oxo-dG and its aptamer. In the absence of 8-oxo-dG, the aptamer was adsorbed onto the surface of cyst-AuNPs, resulting in aggregation and the development of a purple colour solution. Upon addition of the target molecule 8-oxo-dG, the aptamer specifically bound to it and could not induce the aggregation of cyst-AuNPs, leading to the dispersion of cyst-AuNPs in the solution. Simple visual examination could be used to monitor the purple-to-red colour change that started at 12 nM, a threshold concentration for visual analysis. The absorbance at 525 nm increased in direct relation to the number of the target molecule 8-oxo-dG. This aptamer/cyst-AuNPs system showed excellent sensing ability for the 8-oxo-dG concentration in the range of 15-100 nM, with a detection limit as low as 10.3 nM and a detection time of 30 min. Interference experiments showed that the developed colorimetric strategy had a good sensitivity. This simple and rapid colorimetric method has successfully been applied to inspect 8-oxo-dG concentration in real urine samples and provided recoveries between 93.6 and 94.1%, with a limit of quantification (LOQ) of 34.3 nM, which was comparable with an enzyme-linked immunosorbent-based detection of 8-oxo-dG. This new, easy-to-use, and rapid method could be used as an alternative and initiative strategy for the development of an on-site analysis of 8-oxo-dG in urine.

Computational Insights into Molecular Adsorption Characteristics of Methylated DNA on Graphene Oxide for Multicancer Early Detection.

DNA methylation is an epigenetic modification involving the transfer of a methyl group to cytosine residues of a DNA molecule. Altered DNA methylation of certain genes is associated with several diseases including cancer. Nanomaterials, such as graphene oxide (GO), offer great potential as sensing elements for methylated DNA (mDNA) detection due to their distinct properties. Understanding molecular interactions between mDNA and GO can make provision for developing a universal cancer screening test. Molecular dynamics (MD) simulation and density functional theory (DFT) calculation have been employed for investigating their detailed macro- and microscale interactions. Based upon the MD simulation, different adsorption levels of methylated and unmethylated DNAs on GO were represented by a contacting surface area (CSA), which depends on surrounding conditions (in water or a MgCl2 solution). In water, the CSAs of the methylated and unmethylated single-stranded DNA (ssDNA) were ≈13 and ≈5 nm2, respectively, representing more preferable adsorption on GO for the methylated ssDNA. In the presence of divalent ions (Mg2+), the CSAs of both methylated and unmethylated DNA molecules were ≈8 nm2, suggesting that there was no significant difference in adsorption in a saline solution. To reveal the electrical property of GO covered by either methylated or unmethylated DNA, its electronic structure was investigated by the DFT calculation. The energy gaps of pristine graphene (pG) and GO adsorbed by 5-methylcytosine (5mC) were 1.6 and 12.9 meV, respectively, while cytosine adsorption resulted in lower energy gaps (1.2 meV for pG and 9.5 meV for GO). When comparing methylated DNA-covered GO with that covered with unmethylated DNA, remarkable differences in electrical conductivity, which were caused by the electronic structure of GO, were observed. These findings will provide a new route for an efficient detection method of DNA methylation, which can further be used to develop a universal cancer test.

Adaptor protein XB130 regulates the aggressiveness of cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a group of heterogenous malignancies arising from bile duct epithelium with distinct pathological features. Adaptor proteins have implicated in cell proliferation, migration, and invasion of different cancer cells. The objective of this study was to assess whether the adaptor protein XB130 (AFAP1L2) is a critical biological determinant of CCA outcome. XB130 expression levels were investigated in four CCA cell lines compared to an immortalized cholangiocyte cell line by Western blotting. Small interfering (si) RNA-mediated XB130 gene silencing was conducted to evaluate the effects of reduced XB130 expression on cell proliferation, migration, and invasion by MTT, transwell migration and cell invasion assay. The immunohistochemical quantification of XB130 levels were performed in surgically resected formalin-fixed, paraffin-embedded specimens obtained from 151 CCA patients. The relationship between XB130 expression and the clinicopathological parameters of CCA patients were analyzed. Our results showed that XB130 was highly expressed in KKU-213A cell line. Knockdown of XB130 using siRNA significantly decreased the proliferation, migration, and invasion properties of KKU-213A cells through the inhibition of PI3K/Akt pathway, suggesting that XB130 plays an important role in CCA progression. Moreover, elevated XB130 expression levels were positive relationship with lymphovascular space invasion (LVSI), intrahepatic type of CCA, high TNM staging (stage III, IV), high T classification (T3, T4), and lymph node metastasis. We provide the first evidence that the overexpression of XB130 is associated with tumorigenic properties of CCA cells, leading to CCA progression with aggressive clinical outcomes.

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.

Synchrotron FTIR microspectroscopy revealed apoptosis-induced biomolecular changes of cholangiocarcinoma cells treated with ursolic acid.

BACKGROUND: Ursolic acid (UA) is a natural triterpenoid which possesses anti-cancer activity. However, little is known regarding the activity and molecular mechanism of UA in cholangiocarcinoma (CCA). Thus, we investigated the effects of UA on growth inhibition and apoptosis induction through biomolecular changes in KKU-213 and KKU-055 CCA cell lines. METHODS: The anti-proliferative effect of UA against CCA cells was evaluated using SRB assay. Changes in biomolecules were assessed by SR-FTIR microspectroscopy combined with PCA and conventional methods (i.e., Annexin V-FITC/PI staining for lipid alteration and apoptosis induction; Western blot analysis and caspase-3/7 activity assay for apoptotic protein detection). RESULTS: UA suppressed the proliferation of CCA cells in a dose- and time-dependent manner. SR-FTIR data revealed a significant alteration in lipids attributable to changes in apoptotic cell membranes, confirmed by Annexin V-FITC/PI staining. SR-FTIR data showed that UA promoted changes in the protein secondary structure. Elevated expression of Bax and decreased expression of Bcl-2 and survivin/BIRC5 along with augmented caspase-3/7 activity supported alterations in apoptosis-related proteins. CONCLUSIONS: SR-FTIR microspectroscopy was successfully used as a label-free technique to monitor apoptosis-induced biomolecular changes in UA-treated CCA cells. UA exerted the cytotoxic and apoptotic activities in CCA cells through alterations in membrane lipids and apoptotic proteins. UA could be a potential anti-CCA candidate and a chemical starting point for the discovery of novel anti-cancer agents. SIGNIFICANCE: Our present study showed the first evidence that UA exhibited the anti-proliferative and pro-apoptotic activities toward CCA cells through changes in biomolecules, notably lipids and proteins.

Development of Low-Cost AuNP-Based Aptasensors with Truncated Aptamer for Highly Sensitive Detection of 8-Oxo-dG in Urine.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), an oxidized form of guanosine residues, is a critical biomarker for various cancers. Herein, a sensitive citrate-capped gold nanoparticle-based aptasensor device has been developed for the detection of 8-oxo-dG in urine. We previously designed a 38-nt anti-8-oxo-dG-aptamer by a computer simulation and the experimental validation has been performed in the present work. The analytical performance of the 38-nt aptamer from the in silico design was compared with the parent 66-nt aptamer. This assay is based on the principle of salt-induced aggregation of citrate-capped gold nanoparticles. Based on this sensing mechanism, the difference between the absorbance in the presence and absence of 8-oxo-dG at λ = 525 nm (ΔA525) increased linearly as a function of 8-oxo-dG concentrations in the ranges of 10-100 and 15-100 nM for 38-nt and 66-nt aptasensors, respectively. This method can provide detection limits of 6.4 nM for 8-oxo-dG in the 38-nt aptasensor and 13.2 nM in the 66-nt aptasensor. Similar to the 66-nt aptamer, the shortened aptamer, 38-nt long, can provide high sensitivity and selectivity with rapid detection time. In addition, using the 38-nt aptamer as a recognition component in the developed portable low-cost device showed high sensitivity in the detection range of 15-100 nM with a detection limit of 12.9 nM, which is much lower than the threshold value (280 nM) for normal human urine. This easy-to-use device could effectively and economically be utilized for monitoring 8-oxo-dG in real urine samples and potentially serve as a prototype for a commercial device.