FAME4-associating YEATS2 knockdown impairs dopaminergic synaptic integrity and leads to seizure-like behaviours in Drosophila melanogaster.

Familial adult myoclonus epilepsy (FAME) is a neurological disorder caused by a TTTTA/TTTCA intronic repeat expansion. FAME4 is one of the six types of FAME that results from the repeat expansion in the first intron of the gene YEATS2. Although the RNA toxicity is believed to be the primary mechanism underlying FAME, the role of genes where repeat expansions reside is still unclear, particularly in the case of YEATS2 in neurons. This study used Drosophila to explore the effects of reducing YEATS2 expression. Two pan-neuronally driven dsDNA were used for knockdown of Drosophila YEATS2 (dYEATS2), and the resulting molecular and behavioural outcomes were evaluated. Drosophila with reduced dYEATS2 expression exhibited decreased tolerance to acute stress, disturbed locomotion, abnormal social behaviour, and decreased motivated activity. Additionally, reducing dYEATS2 expression negatively affected tyrosine hydroxylase (TH) gene expression, resulting in decreased dopamine biosynthesis. Remarkably, seizure-like behaviours induced by knocking down dYEATS2 were rescued by the administration of L-DOPA. This study reveals a novel role of YEATS2 in neurons in regulating acute stress responses, locomotion, and complex behaviours, and suggests that haploinsufficiency of YEATS2 may play a role in FAME4.

1H NMR fecal metabolic phenotyping of periductal fibrosis- and cholangiocarcinoma-specific metabotypes defining perturbation in gut microbial-host co-metabolism.

Background: The liver fluke Opisthorchis viverrini (OV), which subsequently inhabits the biliary system and results in periductal fibrosis (PDF), is one of the primarily causes of cholangiocarcinoma (CCA), a bile duct cancer with an exceptionally high incidence in the northeast of Thailand and other Greater Mekong Subregion (GMS) countries. Insights in fecal metabolic changes associated with PDF and CCA are required for further molecular research related to gut health and potential diagnostic biological marker development. Methods: In this study, nuclear magnetic resonance (NMR) metabolomics was applied for fecal metabolic phenotyping from 55 fecal water samples across different study groups including normal bile duct, PDF and CCA groups. Results: By using NMR spectroscopy-based metabolomics, fecal metabolic profiles of patients with CCA or PDF and of individuals with normal bile duct have been established with a total of 40 identified metabolites. Further multivariate statistical analysis and hierarchical clustering heat map have demonstrated the PDF- and CCA-specific metabotypes through various altered metabolite groups including amino acids, alcohols, amines, anaerobic glycolytic metabolites, fatty acids, microbial metabolites, sugar, TCA cycle intermediates, tryptophan catabolism substrates, and pyrimidine metabolites. Compared to the normal bile duct group, PDF individuals showed the significantly elevated relative concentrations of fecal ethanol, glycine, tyrosine, and N-acetylglucosamine whereas CCA patients exhibited the remarkable fecal metabolic changes that can be evident through the increased relative concentrations of fecal uracil, succinate, and 5-aminopentanoate. The prominent fecal metabolic alterations between CCA and PDF were displayed by the reduction of relative concentration of methanol observed in CCA. The metabolic alterations associated with PDF and CCA progression have been proposed with the involvement of various metabolic pathways including TCA cycle, ethanol biogenesis, hexamine pathway, methanol biogenesis, pyrimidine metabolism, and lysine metabolism. Among them, ethanol, methanol, and lysine metabolism strongly reflect the association of gut-microbial host metabolic crosstalk in PDF and/or CCA patients. Conclusion: The PDF- and CCA-associated metabotypes have been investigated displaying their distinct fecal metabolic patterns compared to that of normal bile duct group. Our study also demonstrated that the perturbation in co-metabolism of host and gut bacteria has been involved from the early step since OV infection to CCA tumorigenesis.

The impact of hypoxia and oxidative stress on proteo-metabolomic alterations of 3D cholangiocarcinoma models.

The three-dimensional multicellular spheroid (3D MCS) model has been employed in cholangiocarcinoma research as it generates 3D architecture and includes more physiological relevance with the multicellular arrangement. However, it is also essential to explain the molecular signature in this microenvironment and its structural complexity. The results indicated that poorly differentiated CCA cell lines were unable to form 3D MCS due to the lack of cell adhesion molecules with more mesenchymal marker expression. The well-differentiated CCA and cholangiocyte cell lines were able to develop 3D MCSs with round shapes, smooth perimeter, and cell adhesion molecules that led to the hypoxic and oxidative microenvironment detected. For MMNK-1, KKU-213C, and KKU-213A MCSs, the proteo-metabolomic analysis showed proteins and metabolic products altered compared to 2D cultures, including cell-cell adhesion molecules, energy metabolism-related enzymes and metabolites, and oxidative-related metabolites. Therefore, the 3D MCSs provide different physiological states with different phenotypic signatures compared to 2D cultures. Considering the 3D model mimics more physiological relevance, it might lead to an alternate biochemical pathway, targeting to improve drug sensitivity for CCA treatment.

1H NMR metabolic profiling of Staphylococcus pseudintermedius isolated from canine uroliths.

Staphylococcus pseudintermedius is a urease-producing bacteria which is a major cause of magnesium ammonium phosphate (MAP) urolithiasis in canine. A positive urolith culture is an important risk factor for MAP urolithiasis in canine. The mechanism underlying the metabolic changes of S. pseudintermedius after crystallization in artificial urine (AU) needs more defined baseline metabolic information. Therefore, we extensively investigated the metabolic changes of S. pseudintermedius extensively after crystallization in AU. A high urease activity and positive biofilm formation strain, entitled the S. pseudintermedius (SPMAP09) strain, was isolated from canine MAP uroliths, and analyzed using nuclear magnetic resonance (NMR) spectroscopy-based metabolomics. The molecular mechanism-specific metabolic phenotypes were clearly observed after crystallization in AU at day 3. The crystals induced by SPMAP09 were also confirmed and the major chemical composition identified as struvite. Interestingly, our findings demonstrated that a total of 11 identified metabolites were significantly changed. The levels of formate, homocarnosine, tyrosine, cis-aconitate, glycolate, ethyl malonate, valine and acetate level were significantly higher, accompanied with decreased levels of inosine, glucose, and threonine at day 3 compared with the initial time-point (day 0). In addition, our results exhibited that the glyoxylate and dicarboxylate metabolism was significantly related to the SPMAP09 strain at day 3 in AU. Thus, metabolic changes of the SPMAP09 strain after crystallization in AU potentially helps to explain the preliminary molecular mechanism for the crystals induced by S. pseudintermedius.

Interleukin-6-derived cancer-associated fibroblasts activate STAT3 pathway contributing to gemcitabine resistance in cholangiocarcinoma.

Cancer-associated fibroblasts (CAFs) are the dominant component of the tumor microenvironment (TME) that can be beneficial to the generation and progression of cancer cells leading to chemotherapeutic failure via several mechanisms. Nevertheless, the roles of CAFs on anti-cancer drug response need more empirical evidence in cholangiocarcinoma (CCA). Herein, we examined the oncogenic roles of CAFs on gemcitabine resistance in CCA cells mediated via IL-6/STAT3 activation. Our findings showed that CCA-derived CAFs promote cell viability and enhance gemcitabine resistance in CCA cells through the activation of IL-6/STAT3 signaling. High expression of IL-6R was correlated with a poor overall survival rate and gemcitabine resistance in CCA, indicating that IL-6R can be a prognostic or predictive biomarker for the chemotherapeutic response of CCA patients. Blockade of IL-6R on CCA cells by tocilizumab, an IL-6R humanized antihuman monoclonal antibody, contributed to inhibition of the CAF-CCA interaction leading to enhancement of gemcitabine sensitivity in CCA cells. The results of this study should be helpful for modifying therapeutic regimens aimed at targeting CAF interacting with cancer cells resulting in the suppression of the tumor progression but enhancement of drug sensitivity.

Chemotherapeutic resistant cholangiocarcinoma displayed distinct intratumoral microbial composition and metabolic profiles.

Background: Cholangiocarcinoma (CCA) is a malignancy of the cholangiocytes. One of the major issues regarding treatment for CCA patients is the development of chemotherapeutic resistance. Recently, the association of intratumoral bacteria with chemotherapeutic response has been reported in many cancer types. Method: In the present study, we aimed to investigate the association between the intratumoral microbiome and its function on gemcitabine and cisplatin response in CCA tissues using 16S rRNA sequencing and 1H NMR spectroscopic analysis. Result: The results of 16S rRNA sequencing demonstrated that Gammaproteobacteria were significantly higher in both gemcitabine- and cisplatin-resistance groups compared to sensitive groups. In addition, intratumoral microbial diversity and abundance were significantly different compared between gemcitabine-resistant and sensitive groups. Furthermore, the metabolic phenotype of the low dose gemcitabine-resistant group significantly differed from that of low dose gemcitabine-sensitive group. Increased levels of acetylcholine, adenine, carnitine and inosine were observed in the low dose gemcitabine-resistant group, while the levels of acetylcholine, alpha-D-glucose and carnitine increased in the low dose cisplatin-resistant group. We further performed the intergrative microbiome-metabolome analysis and revealed a correlation between the intratumoral bacterial and metabolic profiles which reflect the chemotherapeutics resistance pattern in CCA patients. Conclusion: Our results demonstrated insights into the disruption of the microbiome and metabolome in the progression of chemotherapeutic resistance. The altered microbiome-metabolome fingerprints could be used as predictive markers for drug responses potentially resulting in the development of an appropriate chemotherapeutic drug treatment plan for individual CCA patients.

Metabolic Phenotyping Predicts Gemcitabine and Cisplatin Chemosensitivity in Patients With Cholangiocarcinoma.

Gemcitabine and cisplatin serve as appropriate treatments for patients with cholangiocarcinoma (CCA). Our previous study using histoculture drug response assay (HDRA), demonstrated individual response patterns to gemcitabine and cisplatin. The current study aimed to identify predictive biomarkers for gemcitabine and cisplatin sensitivity in tissues and sera from patients with CCA using metabolomics. Metabolic signatures of patients with CCA were correlated with their HDRA response patterns. The tissue metabolic signatures of patients with CCA revealed the inversion of the TCA cycle that is evident with increased levels of citrate and amino acid backbones as TCA cycle intermediates, and glucose which corresponds to cancer stem cell (CSC) properties. The protein expression levels of CSC markers were examined on tissues and showed the significantly inverse association with the responses of patients to cisplatin. Moreover, the elevation of ethanol level was observed in gemcitabine- and cisplatin-sensitive group. In serum, a lower level of glucose but a higher level of methylguanidine was observed in the gemcitabine-responders as non-invasive predictive biomarker for gemcitabine sensitivity. Collectively, our findings indicate that these metabolites may serve as the predictive biomarkers in clinical practice which not only predict the chemotherapy response in patients with CCA but also minimize the adverse effect from chemotherapy.

Bacterial challenge-associated metabolic phenotypes in Hermetia illucens defining nutritional and functional benefits.

Black soldier fly (BSF, Hermetia illucens) is popular for its applications in animal feed, waste management and antimicrobial peptide source. The major advantages of BSF larva include their robust immune system and high nutritional content that can be further developed into more potential agricultural and medical applications. Several strategies are now being developed to exploit their fullest capabilities and one of these is the immunity modulation using bacterial challenges. The mechanism underlying metabolic responses of BSF to different bacteria has, however, remained unclear. In the current study, entometabolomics was employed to investigate the metabolic phenoconversion in response to either Escherichia coli, Staphylococcus aureus, or combined challenges in BSF larva. We have, thus far, characterised 37 metabolites in BSF larva challenged with different bacteria with the major biochemical groups consisting of amino acids, organic acids, and sugars. The distinct defense mechanism-specific metabolic phenotypes were clearly observed. The combined challenge contributed to the most significant metabolic phenoconversion in BSF larva with the dominant metabolic phenotypes induced by S. aureus. Our study suggested that the accumulation of energy-related metabolites provided by amino acid catabolism is the principal metabolic pathway regulating the defense mechanism. Therefore, combined challenge is strongly recommended for raising BSF immunity as it remarkably triggered amino acid metabolisms including arginine and proline metabolism and alanine, aspartate and glutamate metabolism along with purine metabolism and pyruvate metabolism that potentially result in the production of various nutritional and functional metabolites.

Therapeutic challenges at the preclinical level for targeted drug development for Opisthorchis viverrini-associated cholangiocarcinoma.

INTRODUCTION: Cholangiocarcinoma (CCA) is a malignant tumor of bile duct epithelium with the highest incidence found in Thailand. Some patients are considered suitable for adjuvant therapy and surgical resection is currently the curative treatment for CCA patients. Tumor recurrence is still a hurdle after treatment; hence, finding novel therapeutic strategies to combat CCA is necessary for improving outcome for patients. AREAS COVERED: We discuss targeted therapies and other novel treatment approaches which include protein kinase inhibitors, natural products, amino acid transporter-based inhibitors, immunotherapy, and drug repurposing. We also examine the challenges of tumor heterogeneity, cancer stem cells (CSCs), the tumor microenvironment, exosomes, multiomics studies, and the potential of precision medicine. EXPERT OPINION: Because CCA is difficult to diagnose at the early stage, the traditional treatment approaches are not effective for many patients and most tumors recur. Consequently, researchers are exploring multi-aspect molecular carcinogenesis to uncover molecular targets for further development of novel targeted drugs.

High Levels of Serum IgG for Opisthorchis viverrini and CD44 Expression Predict Worse Prognosis for Cholangiocarcinoma Patients after Curative Resection.

BACKGROUND: Opisthorchis viverrini (OV)-associated cholangiocarcinoma (CCA) has a high immune response with chronic inflammation and oxidative stress. CD44 and Nestin, two cancer stem cell (CSC) markers, play major roles in cancer cell survival. Effects of immune response and expression CSC markers on survival of patients with CCA remain unclear. OBJECTIVE: To investigate the effects of level of OV IgG together with CSC marker expression and also the combination of these markers on survival of CCA patients after curative resection. METHODS: All serum specimens from CCA patients who underwent curative surgery from 2005 to 2015 were examined for IgG for OV antigen by ELISA. Tissue specimens were studied for CD44 and Nestin expression. Survival analysis by Cox proportional hazard model was used for estimating hazard ratio (HR) with a 95% confidence interval (CI). RESULTS: In this study, 122 (69.3%) of 176 were positive for OV IgG, and 35 (19.9%) were considered to have high-positive OV IgG. CD44s positive expression was found in 54 (40%), CD44v6 high expression in 96 (69.6%), CD44v8-10 high expression in 87 (63.5%) and Nestin high expression in 21 (16.1%). Multivariate survival analysis found that high-positive OV IgG and late stage tumor were independent prognostic factors with the adjusted HR of 2.24 (95% CI 1.27-3.93) and 2.78 (95% CI 1.46-5.29), respectively. Subgroup analysis in early and late stage CCA showed that a combined positive OV IgG and CD44s expression with the high expression of CD44v8-10 had the significantly poorest prognosis with HR of 3.75 (95% CI 1.61-8.72) and HR of 1.76 (95% CI 1.02-3.03), respectively. CONCLUSION: A high level of OV IgG as well as a high level of CSC markers resulted in an aggressive CCA. OV IgG level together with CSC markers can be used as the prognostic markers for CCA patients' survival. The study of the CD44 pathway is promising for adjuvant treatment.

Sulfasalazine modifies metabolic profiles and enhances cisplatin chemosensitivity on cholangiocarcinoma cells in in vitro and in vivo models.

BACKGROUND: Sulfasalazine (SSZ) is widely known as an xCT inhibitor suppressing CD44v9-expressed cancer stem-like cells (CSCs) being related to redox regulation. Cholangiocarcinoma (CCA) has a high recurrence rate and no effective chemotherapy. A recent report revealed high levels of CD44v9-positive cells in CCA patients. Therefore, a combination of drugs could prove a suitable strategy for CCA treatment via individual metabolic profiling. METHODS: We examined the effect of xCT-targeted CD44v9-CSCs using sulfasalazine combined with cisplatin (CIS) or gemcitabine in CCA in vitro and in vivo models and did NMR-based metabolomics analysis of xenograft mice tumor tissues. RESULTS: Our findings suggest that combined SSZ and CIS leads to a higher inhibition of cell proliferation and induction of cell death than CIS alone in both in vitro and in vivo models. Xenograft mice showed that the CD44v9-CSC marker and CK-19-CCA proliferative marker were reduced in the combination treatment. Interestingly, different metabolic signatures and significant metabolites were observed in the drug-treated group compared with the control group that revealed the cancer suppression mechanisms. CONCLUSIONS: SSZ could improve CCA therapy by sensitization to CIS through killing CD44v9-positive cells and modifying the metabolic pathways, in particular tryptophan degradation (i.e., kynurenine pathway, serotonin pathway) and nucleic acid metabolism.

1H NMR metabolic phenotyping of Dipterocarpus alatus as a novel tool for age and growth determination.

Dipterocarpus alatus belongs to Family Dipterocarpaceae that can be commonly found in Southeast Asian countries. It is a perennial plant with oval-shaped leaves and oleoresin-rich wood. It has been considered as a multipurpose plant since all parts can be practically utilized. One of the major problems for utilizing Dipterocarpus alatus is the difficulty knowing the exact age as this kind of plant is ready for multipurpose use after 20 years of age. At present, the most commonly used method for determining age of Dipterocarpus alatus is the annual ring estimation. However, this conventional method is unable to provide the high precision and accuracy of age determination due to its limitation including blurry annual rings caused by enriched oleoresin in the wood. The current study aimed to investigate the differences of 1H -NMR spectroscopy-based metabolic profiles from bark and leaf of Dipterocarpus alatus at different ages including 2, 7, 15 and 25 years. Our findings demonstrated that there is a total of 56 metabolites shared between bark and leaf. It is noticeable that bark at different ages exhibited the strongest variation and sugar or sugar derivatives that were found in higher concentrations in bark compared with those in leaf. We found that decreasing levels of certain metabolites including tagatose, 1'kestose and 2'-fucosyllactose exhibited the promising patterns. In conclusion, panel metabolites involved in the sucrose biosynthesis can precisely determine the age and growth of Dipterocarpus alatus.

In vitro and in vivo Anti-Tumor Effects of Pan-HER Inhibitor Varlitinib on Cholangiocarcinoma Cell Lines.

BACKGROUND: Cholangiocarcinoma (CCA) is a slowly progressing but highly aggressive malignancy. Targeting the HER protein family represents a potential therapeutic strategy for CCA treatment. The pan-HER inhibitor varlitinib is being developed for the treatment of breast cancer, gastric cancer, and biliary tract cancer, which includes CCA. This study aims to evaluate the anti-tumor effect of varlitinib on CCA using both in vitro and in vivo models. MATERIALS AND METHODS: HER family expression profiles and the cytotoxic activity of varlitinib were determined in CCA cell lines (KKU-214, KKU-213, KKU-156 and KKU-100) and cholangiocyte (MMNK-1). Anti-proliferation and apoptosis induction were examined in KKU-214 and KKU-100 cell lines. A combination of varlitinib with PI3K inhibitor, BKM-120, was explored for efficacy in the KKU-100 cell line. In addition, the anti-tumor activity of varlitinib on CCA and the key metabolites were evaluated in tumor tissues from CCA xenograft model. RESULTS: Elevated expressions of EGFR and HER2 were observed in KKU-214 and KKU-100 cells and varlitinib can suppress CCA cell growth in the micromolar range. Varlitinib inhibits cell proliferation and enhances cell death via the suppression of Akt and Erk1/2 activity in the KKU-214 cell line. While KKU-100 cells showed a poor response to varlitinib, a combination of varlitinib with BKM-120 improved anti-tumor activity. Varlitinib can significantly suppress tumor growth in the CCA xenograft model after oral administration for 15 days without noticeable toxicity, and aspartate can be the key metabolite to correlate with varlitinib response. CONCLUSION: Our study indicates that varlitinib is a promising therapeutic agent for CCA treatment via the inhibition of EGFR/HER2. The anti-tumor effect of varlitinib on CCA also showed synergism in combination with PI3K inhibition. Aspartate metabolite level was correlated with varlitinib response. Combination of varlitinib with targeted drug or cytotoxic drug was recommended.

In vitro and molecular chemosensitivity in human cholangiocarcinoma tissues.

Adjuvant chemotherapy is required for cholangiocarcinoma (CCA) patients after surgical treatment. Gemcitabine and gemcitabine plus cisplatin are considered the appropriate regimen; however, the response spectrum to chemotherapy differs between patients. Thus, the present study aims to evaluate the response pattern of individual CCA patients by using an in vitro method, histoculture drug response assay (HDRA), to predict the chemosensitivity of individual patients in a prospective study. Moreover, we also investigate the expression of gemcitabine and cisplatin sensitivity factors in CCA tissues in the same cases. Based on the dose response curve, 1000 and 1500 µg/ml of gemcitabine were used as the testing concentrations. For cisplatin, concentrations of 20 and 25 µg/ml were selected for testing and for the combination regimen, 1000 µg/ml of gemcitabine and 20 µg/ml of cisplatin were chosen. The median %IR of each drug was measured as the cut-off to categorize the response pattern into response and non-response groups. In addition, we compared the effectiveness of the chemotherapy regimens between gemcitabine alone and gemcitabine plus cisplatin. The %IR of the combination of gemcitabine and cisplatin was significantly higher than gemcitabine alone. The relationship between the expression level of gemcitabine and cisplatin sensitive factors and the individual response pattern as well as clinicopathological data of CCA patients were analyzed. The results indicated that a low expression of the gemcitabine sensitive factor hENT-1 was significantly associated with the non-response group in vitro (p = 0.002). Moreover, the low expression of hENT-1 was also significantly associated with advanced stages CCA in the patients (p = 0.025). A low expression of MT and ERCC1 was significantly correlated with the response group in the in vitro experiments (p = 0.015 and p = 0.037 for MT and ERCC1, respectively). Therefore, HDRA may serve as an aid to selecting chemotherapy, and the expression of hNET-1, MT and ERCC1 may serve as biomarkers for predicting chemotherapy success.

Inhibition of endothelial nitric oxide synthase in cholangiocarcinoma cell lines - a new strategy for therapy.

The isoform of nitric oxide synthase (NOS) found in endothelial cells (eNOS) plays a crucial role in vasodilation. We recently reported the activation of eNOS in cholangiocarcinoma (CCA) tissues and cell lines. Moreover, we also reported that the abundance of eNOS and phosphorylated eNOS (p-eNOS), as well as its upstream regulator proteins, is significantly associated with the metastatic status of CCA patients. However, the function of eNOS in CCA progression has not been addressed. Therefore, the present study aimed to investigate the function of eNOS involved in the migration and invasion ability of CCA cell lines. The results reveal that eNOS activation significantly increases migration and invasion ability of CCA cells via the up-regulation of phosphorylated vasodilator-stimulated protein (p-VASP). A combination treatment with recombinant human vascular endothelial growth factor C and eNOS inhibitor (Nω-nitro-l-arginine methyl ester hydrochloride) resulted in the down-regulation of p-VASP, as well as a decreased migration and invasion ability of the CCA cell line. Thus, this work suggests that eNOS can serve as an attractive target to inhibit the progression of CCA.

Upregulation of endothelial nitric oxide synthase (eNOS) and its upstream regulators in Opisthorchis viverrini associated cholangiocarcinoma and its clinical significance.

Endothelial nitric oxide synthase (eNOS) is an isoform of the enzyme nitric oxide synthase (NOS) which is constitutively expressed in endothelial cells and plays important roles in vasodilation. We previously reported the importance of eNOS activation in cholangiocarcinoma (CCA) tissues and cell lines. The present study aims to investigate the relative abundance of eNOS and phosphorylated eNOS (P-eNOS) and their upstream regulators VEGFR3, VEGFC, EphA3 and ephrin-A1, in the Opisthorchis viverrini (Ov)/N-nitrosodimethylamine (NDMA)-induced hamster CCA model and in human CCA by semiquantitative immunohistochemical analysis of the relevant tissues. Results from the hamster model suggested an increase in eNOS and P-eNOS and upstream regulators during CCA genesis. In human CCA, high immunohistochemical staining intensity of all investigated proteins was associated with the presence of metastasis. A pairwise analysis of the staining data for eNOS and its upstream regulators showed that a concurrent increase in eNOS/VEGFR3, eNOS/ephrin-A1, eNOS/VEGFC and eNOS/EphA3 was significantly associated with metastasis. An increase in eNOS/VEGFR3, eNOS/ephrin-A1 was also associated with non-papillary type CCA. Additionally, an increase in eNOS and P-eNOS was significantly correlated with a high micro-vessel level (P=0.04). Our results indicate that the development of CCA involves upregulation of eNOS and P-eNOS and their regulators. This may drive angiogenesis and metastasis in CCA.