GLRX3, a novel cancer stem cell-related secretory biomarker of pancreatic ductal adenocarcinoma.

BACKGROUND: Cancer stem cells (CSCs) are implicated in carcinogenesis, cancer progression, and recurrence. Several biomarkers have been described for pancreatic ductal adenocarcinoma (PDAC) CSCs; however, their function and mechanism remain unclear. METHOD: In this study, secretome analysis was performed in pancreatic CSC-enriched spheres and control adherent cells for biomarker discovery. Glutaredoxin3 (GLRX3), a novel candidate upregulated in spheres, was evaluated for its function and clinical implication. RESULTS: PDAC CSC populations, cell lines, patient tissues, and blood samples demonstrated GLRX3 overexpression. In contrast, GLRX3 silencing decreased the in vitro proliferation, migration, clonogenicity, and sphere formation of cells. GLRX3 knockdown also reduced tumor formation and growth in vivo. GLRX3 was found to regulate Met/PI3K/AKT signaling and stemness-related molecules. ELISA results indicated GLRX3 overexpression in the serum of patients with PDAC compared to that in healthy controls. The sensitivity and specificity of GLRX3 for PDAC diagnosis were 80.0 and 100%, respectively. When GLRX3 and CA19-9 were combined, sensitivity was significantly increased to 98.3% compared to that with GLRX3 or CA19-9 alone. High GLRX3 expression was also associated with poor disease-free survival in patients receiving curative surgery. CONCLUSION: Overall, these results indicate GLRX3 as a novel diagnostic marker and therapeutic target for PDAC targeting CSCs.

Identification of Circulating Serum miRNAs as Novel Biomarkers in Pancreatic Cancer Using a Penalized Algorithm.

Pancreatic cancer (PC) is difficult to detect in the early stages; thus, identifying specific and sensitive biomarkers for PC diagnosis is crucial, especially in the case of early-stage tumors. Circulating microRNAs are promising non-invasive biomarkers. Therefore, we aimed to identify non-invasive miRNA biomarkers and build a model for PC diagnosis. For the training model, blood serum samples from 63 PC patients and 63 control subjects were used. We selected 39 miRNA markers using a smoothly clipped absolute deviation-based penalized support vector machine and built a PC diagnosis model. From the double cross-validation, the average test AUC was 0.98. We validated the diagnosis model using independent samples from 25 PC patients and 81 patients with intrahepatic cholangiocarcinoma (ICC) and compared the results with those obtained from the diagnosis using carbohydrate antigen 19-9. For the markers miR-155-5p, miR-4284, miR-346, miR-7145-5p, miR-5100, miR-661, miR-22-3p, miR-4486, let-7b-5p, and miR-4703-5p, we conducted quantitative reverse transcription PCR using samples from 17 independent PC patients, 8 ICC patients, and 8 healthy individuals. Differential expression was observed in samples from PC patients. The diagnosis model based on the identified markers showed high sensitivity and specificity for PC detection and is potentially useful for early PC diagnosis.

Neuroprotective and Anti-Inflammatory Effects of Low-Moderate Dose Ionizing Radiation in Models of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia. The neuropathological features of AD include amyloid-ß (Aß) deposition and hyperphosphorylated tau accumulation. Although several clinical trials have been conducted to identify a cure for AD, no effective drug or treatment has been identified thus far. Recently, the potential use of non-pharmacological interventions to prevent or treat AD has gained attention. Low-dose ionizing radiation (LDIR) is a non-pharmacological intervention which is currently being evaluated in clinical trials for AD patients. However, the mechanisms underlying the therapeutic effects of LDIR therapy have not yet been established. In this study, we examined the effect of LDIR on Aß accumulation and Aß-mediated pathology. To investigate the short-term effects of low-moderate dose ionizing radiation (LMDIR), a total of 9 Gy (1.8 Gy per fraction for five times) were radiated to 4-month-old 5XFAD mice, an Aß-overexpressing transgenic mouse model of AD, and then sacrificed at 4 days after last exposure to LMDIR. Comparing sham-exposed and LMDIR-exposed 5XFAD mice indicated that short-term exposure to LMDIR did not affect Aß accumulation in the brain, but significantly ameliorated synaptic degeneration, neuronal loss, and neuroinflammation in the hippocampal formation and cerebral cortex. In addition, a direct neuroprotective effect was confirmed in SH-SY5Y neuronal cells treated with Aß1-42 (2 µM) after single irradiation (1 Gy). In BV-2 microglial cells exposed to Aß and/or LMDIR, LMDIR therapy significantly inhibited the production of pro-inflammatory molecules and activation of the nuclear factor-kappa B (NF-κB) pathway. These results indicate that LMDIR directly ameliorated neurodegeneration and neuroinflammation in vivo and in vitro. Collectively, our findings suggest that the therapeutic benefits of LMDIR in AD may be mediated by its neuroprotective and anti-inflammatory effects.

Novel Gastric Cancer Stem Cell-Related Marker LINGO2 Is Associated with Cancer Cell Phenotype and Patient Outcome.

The expression of leucine-rich repeat and immunoglobulin-like domain-containing nogo receptor-interacting protein 2 (LINGO2) has been reported in Parkinson's disease; however, its role in other diseases is unknown. Gastric cancer is the second leading cause of cancer death. Cancer stem cells (CSC) are a subpopulation of cancer cells that contribute to the initiation and invasion of cancer. We identified LINGO2 as a CSC-associated protein in gastric cancers both in vitro and in patient-derived tissues. We studied the effect of LINGO2 on cell motility, stemness, tumorigenicity, and angiogenic capacity using cells sorted based on LINGO2 expression and LINGO2-silenced cells. Tissue microarray analysis showed that LINGO2 expression was significantly elevated in advanced gastric cancers. The overall survival of patients expressing high LINGO2 was significantly shorter than that of patients with low LINGO2. Cells expressing high LINGO2 showed elevated cell motility, angiogenic capacity, and tumorigenicity, while LINGO2 silencing reversed these properties. Silencing LINGO2 reduced kinase B (AKT)/extracellular signal-regulated kinase (ERK)/ERK kinase (MEK) phosphorylation and decreased epithelial-mesenchymal transition (EMT)-associated markers-N-Cadherin and Vimentin and stemness-associated markers- POU class 5 homeobox 1 (OCT4) and Indian hedgehog (IHH), and markedly decreased the CD44⁺ population. These indicate the involvement of LINGO2 in gastric cancer initiation and progression by altering cell motility, stemness, and tumorigenicity, suggesting LINGO2 as a putative target for gastric cancer treatment.

Neurogenic Effects of Ghrelin on the Hippocampus.

Mammalian neurogenesis continues throughout adulthood in the subventricular zone of the lateral ventricle and in the subgranular zone of the dentate gyrus in the hippocampus. It is well known that hippocampal neurogenesis is essential in mediating hippocampus-dependent learning and memory. Ghrelin, a peptide hormone mainly synthesized in the stomach, has been shown to play a major role in the regulation of energy metabolism. A plethora of evidence indicates that ghrelin can also exert important effects on neurogenesis in the hippocampus of the adult brain. The aim of this review is to discuss the current role of ghrelin on the in vivo and in vitro regulation of neurogenesis in the adult hippocampus. We will also discuss the possible role of ghrelin in dietary restriction-induced hippocampal neurogenesis and the link between ghrelin-induced hippocampal neurogenesis and cognitive functions.

Mdivi-1 Protects Adult Rat Hippocampal Neural Stem Cells against Palmitate-Induced Oxidative Stress and Apoptosis.

Palmitate concentrations in type 2 diabetic patients are higher than in healthy subjects. The prolonged elevation of plasma palmitate levels induces oxidative stress and mitochondrial dysfunction in neuronal cells. In this study, we examined the role of mdivi-1, a selective inhibitor of mitochondrial fission protein dynamin-regulated protein 1 (Drp1), on the survival of cultured hippocampal neural stem cells (NSCs) exposed to high palmitate. Treatment of hippocampal NSCs with mdivi-1 attenuated palmitate-induced increase in cell death and apoptosis. Palmitate exposure significantly increased Drp1 protein levels, which were prevented by pretreatment of cells with mdivi-1. We found that cytosolic Drp1 was translocated to the mitochondria when cells were exposed to palmitate. In contrast, palmitate-induced translocation of Drp1 was inhibited by mdivi-1 treatment. We also investigated mdivi-1 regulation of apoptosis at the mitochondrial level. Mdivi-1 rescued cells from palmitate-induced lipotoxicity by suppressing intracellular and mitochondrial reactive oxygen species production and stabilizing mitochondrial transmembrane potential. Mdivi-1-treated cells showed an increased Bcl-2/Bax ratio, prevention of cytochrome c release, and inhibition of caspase-3 activation. Our data suggest that mdivi-1 protects hippocampal NSCs against lipotoxicity-associated oxidative stress by preserving mitochondrial integrity and inhibiting mitochondrial apoptotic cascades.

Embigin is overexpressed in pancreatic ductal adenocarcinoma and regulates cell motility through epithelial to mesenchymal transition via the TGF-ß pathway.

Embigin is a member of the immunoglobulin superfamily and encodes a transmembrane glycoprotein. There have been reports of Embigin involvement in neuromuscular junction formation and plasticity; however, the molecular functions of Embigin in other organs are unknown. Our aim was to investigate the possible role of Embigin in pancreatic cancer. In pancreatic ductal adenocarcinoma tissues, Embigin expression was higher than that in normal pancreatic tissues. Immunohistochemical analysis revealed expression of Embigin in pancreatic cancer cells, as well as expression of monocarboxylate transporter 2 (MCT2) in cancer tissues. To gain further insight, we transfected BxPC-3 and HPAC pancreatic cancer cells with siRNA or shRNA targeting Embigin and observed reductions in cell proliferation, migration, invasion, wound healing, and reduced levels of matrix metalloproteinases-2 and -9. Silencing of Embigin increased intracellular L-lactate concentration by 1.5-fold and decreased MCT2 levels at the plasma membrane. Furthermore, Embigin silencing led to a reduced expression of PI3K, GSK3-ß, and Snail/Slug. Upon treating BxPC-3 cells with transforming growth factor-ß (TGF-ß), we observed elevated expression of Snail/Slug, Embigin, and Vimentin; meanwhile, when treating cells with SB-216763, a GSK3-ß inhibitor, we noted decreases in GSK3-ß, Snail/Slug, and Embigin expression, suggesting that the TGF-ß signaling cascade, comprising PI3K, GSK3-ß, Snail/Slug, and Embigin signals, mediates epithelial to mesenchymal transition (EMT) in pancreatic cancer cells. These findings indicate the involvement of Embigin in EMT in pancreatic cancer progression and suggest Embigin as a putative target for the detection and/or treatment of pancreatic cancer.

Ghrelin is required for dietary restriction-induced enhancement of hippocampal neurogenesis: lessons from ghrelin knockout mice.

Neurogenesis occurs in the adult hippocampus and is enhanced by dietary restriction (DR), and neurogenesis enhancement is paralleled by circulating ghrelin level enhancement. We have previously reported that ghrelin modulates adult neurogenesis in the hippocampus. In order to investigate the possible role of ghrelin in DR-induced hippocampal neurogenesis in adult mice, ghrelin knockout (GKO) mice and wild-type (WT) mice were maintained for 3 months on DR or ad libitum (AL) diets. Protein levels of ghrelin in the stomach and the hippocampus were increased by DR in WT mice. One day after BrdU administration, the number of BrdU-labeled cells in the hippocampal dentate gyrus was decreased in GKO mice maintained on the AL diet. DR failed to alter the proliferation of progenitor cells in both WT and GKO mice. Four weeks after BrdU injection, the number of surviving cells in the dentate gyrus was decreased in AL-fed GKO mice. DR increased survival of newborn cells in WT mice, but not in GKO mice. Levels of brain-derived neurotrophic factor protein in the hippocampus were similar between WT and GKO mice, and were increased by DR both in WT and GKO mice. These results suggest that elevated levels of ghrelin during DR may have an important role in the enhancement of neurogenesis induced by DR.

PUM1-TRAF3 fusion protein activates non-canonical NF-κB signaling via rescued NIK in biliary tract cancer.

Discovery and verification of diagnostic or therapeutic biomarkers for biliary tract cancer (BTC) is challenging owing to the low prevalence of the disease. Here, we identified and investigated the clinical impact of a fusion gene, Pumilio1-tumor necrosis factor receptor-associated factor 3 (PUM1-TRAF3), caused by 1;14 chromosomal translocation in BTC. PUM1-TRAF3 was initially identified in the RNA-sequencing of five BTC surgical tissues and confirmed by fluorescence in situ hybridization. Expression of the fusion gene was validated in an expanded cohort (5/55, 9.1%). Establishment and molecular assessment of PUM1-TRAF3 expressing BTC cells revealed that PUM1-TRAF3 activates non-canonical NF-κB signaling via NF-κB-inducing kinase (NIK). Abnormal TRAF3 activity, driven by competitive binding of PUM1-TRAF3 and TRAF3 to NIK, led to NIK rescue followed by P52/RelB nuclear translocation, all of which were reverted by an NIK inhibitor. The elevated expression of NIK and activated NF-κB signaling was observed in the PUM1-TRAF3-expressing regions of patient tissues. Expression of the PUM1-TRAF3 fusion was significantly correlated with strong NIK expression, which is associated with a poorer prognosis for patients with BTC. Overall, our study identifies a new fusion gene, PUM1-TRAF3, that activates NIK and non-canonical NF-κB signaling, which may be beneficial for developing precise treatment strategies for BTC.

Integrative analysis of multiple genomic data from intrahepatic cholangiocarcinoma organoids enables tumor subtyping.

As genomic analysis technology has advanced, it has become possible to sub-classify intrahepatic cholangiocarcinoma (ICC) at the histological or molecular level. Here, we verify the recently suggested two subgroups of ICC in the organoids model, compare the characteristics between types. ICC patients are subclassified into small-duct (SD) and large-duct (LD) subtype according to histological characteristics. ICC organoids are established, and unsupervised principal component analysis clustering separates each type of ICC. Differential gene expression reveals enrichment on KRAS, TGFß and ERBB2 signaling pathways in LD-type compared with SD-type (P < 0.05). Gene set enrichment analysis demonstrates that the cholangiocarcinoma class 2 signature, defined by Andersen et al., is enriched in the LD-type (enrichment Score = 2.19, P < 0.001). A protein-protein interaction network analysis identifies ZNF217 as a significant hub protein (odds ratio = 4.96, P = 0.0105). We perform prospective modeling of histological subtype using patient-derived organoids. Moreover, gene expression profiling of ICC organoids enables identification of type-specific targetable pathways.

Sorption of anthracene (C14H10) and 9-anthroic acid (C15H10O2) onto biochar-amended soils as affected by field aging treatments.

There is a lack of information on how aging affects the sorption of ionizable organic compounds on biochar-amended soils. To that end, this study investigates the sorption of two hydrophobic solutes (anthracene (ANT) and 9-anthroic acid (9-ANCA)) onto biochar-amended (5%, w/w) soils as a function of aging period (0, 6, and 12 months), electrolyte (5 mM CaCl2 and 10 mM KCl), and aqueous pH. The isotherm plot of both solutes was fairly linear (r2 > 0.998) and the linear sorption coefficient (Kd, L kg-1) was obtained from this. In CaCl2 solution at pH 5, the log Kd of ANT tended to decrease (from 3.90 to 3.72) with an increasing aging period, which was attributed to clogged pore surface, whereas the differences in 9-ANCA sorption (from 2.56 to 2.51) were not significant (α = 0.05). The increased ANT sorption at acidic pH (<4) could be attributed to π-π interaction. Aqueous Ca2+ ions played an important role in 9-ANCA sorption by forming a Ca-bridge between anionic solute and negatively charged adsorbent surface, thus accounting for up to 35% of its sorption at alkaline pH (>8). The spectroscopic data and isoelectric point measurement results indicated that the number of oxygen-containing functional groups and the content of elemental oxygen were both higher in aged samples, resulting in a more polar (negatively charged) surface. The formation of surface polar groups and the associated deformation altered the adsorbent nature of the tested biochar, thereby fortifying the hydrophilic retention propensity for ionizable organic solutes.

Physicochemical characteristics of Ethiopian Coffea arabica cv. Heirloom coffee extracts with various roasting conditions.

This study aimed to investigate the physicochemical characteristics of Ethiopian Coffea arabica cv. Heirloom coffee extracts with various roasting conditions. Green coffee beans were roasted at four different conditions (Light-medium, Medium, Moderately dark, and Very dark) and used to extract espresso and drip coffee. Moisture content in coffee beans was decreased as the roasting degree increased. The contents of crude fat and ash were lower in the Light-medium roasted coffee beans than in green coffee beans but increased as the roasting degree increased. The values of lightness (L*), redness (a*), yellowness (b*), and browning index of coffee extracts were decreased as the roasting degree increased. Total dissolved solids in espresso coffee were increased with increasing roasting degree but decreased in drip coffee. In both the extracts, the contents of reducing sugar, titratable acidity, organic acids, and chlorogenic acid were decreased, but that of caffeine was increased with the roasting degree increased. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-020-00865-w.

Identification of potential biomarkers for diagnosis of pancreatic and biliary tract cancers by sequencing of serum microRNAs.

BACKGROUND: Pancreatic and biliary tract cancer (PC and BTC, respectively) are difficult to diagnose because of their clinical characteristics; however, recent studies suggest that serum microRNAs (miRNAs) might be the key to developing more efficient diagnostic methods for these cancers. METHODS: We analysed the genome-wide expression of serum miRNAs in PC and BTC patients to identify novel biomarker candidates using high-throughput sequencing and experimentally validated miRNAs on clinical samples. RESULTS: Statistical and classification analysis of the serum miRNA-expression profiles of 55 patient samples showed distinguishable patterns between cancer patients and healthy controls; however, we were unable to distinguish the two cancers. We found that three of the highest performing miRNAs were capable of distinguishing cancer patients from controls, with an accuracy of 92.7%. Additionally, dysregulation of these three cancer-specific miRNAs was demonstrated in an independent sample group by quantitative reverse transcription polymerase chain reaction. CONCLUSIONS: These results suggested three candidate serum miRNAs (mir-744-5p, mir-409-3p, and mir-128-3p) as potential biomarkers for PC and BTC diagnosis.

IGF-1 protects SH-SY5Y cells against MPP+-induced apoptosis via PI3K/PDK-1/Akt pathway.

Insulin-like growth factor (IGF)-1 is a well-known anti-apoptotic pro-survival factor and phosphatidylinositol-3-kinase (PI3K)/Akt pathway is linked to cell survival induced by IGF-1. It is also reported that Akt signaling is modulated by 3-phosphoinositide-dependent kinase-1 (PDK1). In the current study, we investigated whether the anti-apoptotic effect of IGF-1 in SH-SY5Y cells exposed to 1-methyl-4-phenylpyridinium (MPP+) is associated with the activity of PI3K/PDK1/Akt pathway. Treatment of cells with IGF-1 inhibited MPP+-induced apoptotic cell death. IGF-1-induced activation of Akt and the protective effect of IGF-1 on MPP+-induced apoptosis were abolished by chemical inhibition of PDK1 (GSK2334470) or PI3K (LY294002). The phosphorylated levels of Akt and PDK1 were significantly suppressed after MPP+ exposure, while IGF-1 treatment completely restored MPP+-induced reductions in phosphorylation. IGF-1 protected cells from MPP+ insult by suppressing intracellular reactive oxygen species (ROS) production and malondialdehyde levels and increasing superoxide dismutase activity. Mitochondrial ROS levels were also increased during MPP+ exposure, which were attenuated by IGF-1 treatment. In addition, IGF-1-treated cells showed increased activities of succinate dehydrogenase and citrate synthase, stabilization of mitochondrial transmembrane potential, increased ratio of Bcl-2 to Bax, prevention of cytochrome c release and inhibition of caspase-3 activation with PARP cleavage. Furthermore, the protective effects of IGF-1 on oxidative stress and mitochondrial dysfunction were attenuated when cells were preincubated with GSK2334470 or LY294002. Our data suggest that IGF-1 protects SH-SY5Y cells against MPP+-associated oxidative stress by preserving mitochondrial integrity and inhibiting mitochondrial apoptotic cascades via the activation of PI3K/PDK1/Akt pathway.

CG200745, an HDAC inhibitor, induces anti-tumour effects in cholangiocarcinoma cell lines via miRNAs targeting the Hippo pathway.

Cholangiocarcinoma is a devastating malignancy with fatal complications that exhibits low response and resistance to chemotherapy. Here, we evaluated the anticancer effects of CG200745, a novel histone deacetylase inhibitor, either alone or in combination with standard chemotherapy drugs in cholangiocarcinoma cells. CG200745 dose-dependently reduced the viability of cholangiocarcinoma cells in vitro and decreased tumour volume and weight in a xenograft model. Administering CG200745 along with other chemotherapeutic agents including gemcitabine, 5-fluorouracil (5-FU), cisplatin, oxaliplatin, or gemcitabine plus cisplatin further decreased cholangiocarcinoma cell viability, with a combination index < 1 that indicated synergistic action. CG200745 also enhanced the sensitivity of gemcitabine-resistant cells to gemcitabine and 5-FU, thereby decreasing cell viability and inducing apoptosis. This was accompanied by downregulation of YAP, TEAD4, TGF-ß2, SMAD3, NOTCH3, HES5, Axl, and Gas6 and upregulation of the miRNAs miR-22-3p, miR-22-5p, miR-194-5p, miR-194-3p, miR-194-5p, miR-210-3p, and miR-509-3p. The Ingenuity Pathway Analysis revealed that CG200745 mainly targets the Hippo signalling pathway by inducing miR-509-3p expression. Thus, CG200745 inhibits cholangiocarcinoma growth in vitro and in vivo, and acts synergistically when administered in combination with standard chemotherapeutic agents, enabling dose reduction. CG200745 is therefore expected to improve the outcome of cholangiocarcinoma patients who exhibit resistance to conventional therapies.

Ghrelin gene products rescue cultured adult rat hippocampal neural stem cells from high glucose insult.

Adult hippocampal neurogenesis is decreased in type 2 diabetes, and this impairment appears to be important in cognitive dysfunction. Previous studies suggest that ghrelin gene products (acylated ghrelin (AG), unacylated ghrelin (UAG) and obestatin (OB)) promote neurogenesis. Therefore, we hypothesize that ghrelin gene products may reduce the harmful effects of high glucose (HG) on hippocampal neural stem cells (NSCs). The aim of this study was to investigate the role of these peptides on the survival of cultured hippocampal NSCs exposed to HG insult. Treatment of hippocampal NSCs with AG, UAG or OB inhibited HG-induced cell death and apoptosis. Exposure of cells to the growth hormone secretagogue receptor 1a antagonist abolished the protective effects of AG against HG toxicity, whereas those of UAG or OB were preserved. All three peptides attenuated HG-induced decrease in BrdU-labeled and phosphohistone-H3-labeled cells. We also investigated the effects of ghrelin gene products on the regulation of apoptosis at the mitochondrial level. AG, UAG or OB rescued hippocampal NSCs from HG insult by inhibiting intracellular and mitochondrial reactive oxygen species generation and stabilizing mitochondrial transmembrane potential. In addition, cells treated with ghrelin gene products showed an increased Bcl-2 and decreased Bax levels, thereby increasing the Bcl-2/Bax ratio, inhibiting cytochrome c release and preventing caspase-3 activation. Finally, AG-, UAG- or OB-mediated protection was dependent on the activities of adenosine monophosphate-activated protein kinase/uncoupling protein 2 pathway. Our data indicate that ghrelin gene products may act as survival factors that preserve mitochondrial function and inhibit oxidative stress-induced apoptosis.