Metabolomics and miRNA profiling reveals feature of gallbladder cancer-derived biliary extracellular vesicles.

BACKGROUND: Although there are several studies in the development of various human cancers, the role of exosomes is poorly understood in the progression of gallbladder cancer. This study aims to characterize the metabolic changes occurring in exosomes obtained from patients with gallbladder cancer compared with those from other gallbladder disease groups. METHODS: Biliary exosomes were isolated from healthy donors (n = 3) and from patients with gallbladder cancer (n = 3), gallbladder polyps (n = 4), or cholecystitis (n = 3) using a validated exosome isolation kit. Afterward, we performed miRNA profiling and untargeted metabolomic analysis of the exosomes. The results were validated by integrating the results of the miRNA and metabolomic analyses. RESULTS: The gallbladder cancer group exhibited a significant reduction in the levels of multiple unsaturated phosphatidylethanolamines and phosphatidylcholines compared to the normal group, which resulted in the loss of exosome membrane integrity. Additionally, the gallbladder cancer group demonstrated significant overexpression of miR-181c and palmitic acid, and decreased levels of conjugated deoxycholic acid, all of which are strongly associated with the activation of the PI3K/AKT pathway. CONCLUSIONS: Our findings demonstrate that the contents of exosomes are disease-specific, particularly in gallbladder cancer, and that altered metabolites convey critical information regarding their phenotype. We believe that our metabolomic and miRNA profiling results may provide important insights into the development of gallbladder cancer.

Selective inhibitory effects of suberosin on CYP1A2 in human liver microsomes.

Suberosin is a natural phytoconstituent isolated from Citropsis articulata, especially employed for its anticoagulant properties. Although metabolic studies assessing suberosin have been conducted, it is possible interactions with drugs and food have not yet been investigated. In the present study, we analyzed the selective inhibitory effects of suberosin on cytochrome P450 (CYP) enzymes using a cocktail probe assay. Various concentrations of suberosin (0-50 µM) were incubated with isoform-specific CYP probes in human liver microsomes (HLMs). We found that suberosin significantly inhibited CYP1A2-catalyzed phenacetin O-deethylation, exhibiting IC50 values of 9.39 ± 2.05 and 3.07 ± 0.45 µM with and without preincubation in the presence of ß-NADPH, respectively. Moreover, suberosin showed concentration-dependent, but not time-dependent, CYP1A2 inhibition in HLMs, indicating that suberosin acts as a substrate and reversible CYP1A2 inhibitor. Using a Lineweaver-Burk plot, we found that suberosin competitively inhibited CYP1A2-catalyzed phenacetin O-deethylation. Furthermore, suberosin showed similar inhibitory effects on recombinant human CYP1A1 and 1A2. In conclusion, suberosin may elicit herb-drug interactions by selectively inhibiting CYP1A2 during the concurrent administration of drugs that act as CYP1A2 substrates.

Integrative Profiling of Lysine Acylome in Sepsis-Induced Liver Injury.

Sepsis is one of the life-threatening diseases worldwide. Despite the continuous progress in medicine, the specific mechanism of sepsis remains unclear. A key strategy of pathogens is to use post-translational modification to modulate host factors critical for infection. We employed global immunoprecipitation technology for lysine acetylation (Kac), succinylation (Ksu), and malonylation (Kmal) for the first global lysine acylation (Kacy) analysis in a cecum ligation and puncture (CLP) model in mouse. This was performed to reveal the pathogenic mechanism of integrative Kacy and the changes in modification sites. In total, 2230 sites of 1,235 Kac proteins, 1,887 sites of 433 Ksu proteins, and 499 sites of 276 Kmal proteins were quantified and normalized by their protein levels. We focused on 379 sites in 219 upregulated proteins as the integrative Kacy sites of Kac, Ksu, and Kmal on the basis of sirtuins decreased in the CLP group. KEGG pathway analysis of integrative Kacy in 219 upregulated proteins revealed three central metabolic pathways: glycolysis/gluconeogenesis, pyruvate metabolism, and tricarboxylic acid cycle. These findings reveal the key pathogenic mechanism of integrative PTM alteration in terms of the decreased sirtuins level and provide an important foundation for an in-depth study of the biological function of Kacy in sepsis.

Identification of Histone Lysine Acetoacetylation as a Dynamic Post-Translational Modification Regulated by HBO1.

Ketone bodies have long been known as a group of lipid-derived alternative energy sources during glucose shortages. Nevertheless, the molecular mechanisms underlying their non-metabolic functions remain largely elusive. This study identified acetoacetate as the precursor for lysine acetoacetylation (Kacac), a previously uncharacterized and evolutionarily conserved histone post-translational modification. This protein modification is comprehensively validated using chemical and biochemical approaches, including HPLC co-elution and MS/MS analysis using synthetic peptides, Western blot, and isotopic labeling. Histone Kacac can be dynamically regulated by acetoacetate concentration, possibly via acetoacetyl-CoA. Biochemical studies show that HBO1, traditionally known as an acetyltransferase, can also serve as an acetoacetyltransferase. In addition, 33 Kacac sites are identified on mammalian histones, depicting the landscape of histone Kacac marks across species and organs. In summary, this study thus discovers a physiologically relevant and enzymatically regulated histone mark that sheds light on the non-metabolic functions of ketone bodies.

Novel Time-dependent Multi-omics Integration in Sepsis-associated Liver Dysfunction.

The recently developed technologies that allow the analysis of each single omics have provided an unbiased insight into ongoing disease processes. However, it remains challenging to specify the study design for the subsequent integration strategies that can associate sepsis pathophysiology and clinical outcomes. Here, we conducted a time-dependent multi-omics integration (TDMI) in a sepsis-associated liver dysfunction (SALD) model. We successfully deduced the relation of the toll-like receptor 4 (TLR4) pathway with SALD. Although TLR4 is a critical factor in sepsis progression, it is not specified in single-omics results but only in the TDMI analysis. This result indicates that the TDMI-based approach is more advantageous than single-omics analysis in terms of exploring the underlying pathophysiological mechanism of this disease. Furthermore, this approach can be an ideal paradigm for insightful biological interpretations of multi-omics datasets that will potentially reveal novel insights into basic biology, health, and diseases, thus allowing the identification of promising candidates for therapeutic strategies.

Global Lysine Acetylome Analysis of LPS-Stimulated HepG2 Cells Identified Hyperacetylation of PKM2 as a Metabolic Regulator in Sepsis.

Sepsis-induced liver dysfunction (SILD) is a common event and is strongly associated with mortality. Establishing a causative link between protein post-translational modification and diseases is challenging. We studied the relationship among lysine acetylation (Kac), sirtuin (SIRTs), and the factors involved in SILD, which was induced in LPS-stimulated HepG2 cells. Protein hyperacetylation was observed according to SIRTs reduction after LPS treatment for 24 h. We identified 1449 Kac sites based on comparative acetylome analysis and quantified 1086 Kac sites on 410 proteins for acetylation. Interestingly, the upregulated Kac proteins are enriched in glycolysis/gluconeogenesis pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) category. Among the proteins in the glycolysis pathway, hyperacetylation, a key regulator of lactate level in sepsis, was observed at three pyruvate kinase M2 (PKM2) sites. Hyperacetylation of PKM2 induced an increase in its activity, consequently increasing the lactate concentration. In conclusion, this study is the first to conduct global profiling of Kac, suggesting that the Kac mechanism of PKM2 in glycolysis is associated with sepsis. Moreover, it helps to further understand the systematic information regarding hyperacetylation during the sepsis process.

Extracellular Vesicles Act as Nano-Transporters of Tyrosine Kinase Inhibitors to Revert Iodine Avidity in Thyroid Cancer.

A new approach for using extracellular vesicles (EVs) to deliver tyrosine kinase inhibitors (TKIs) to enhance iodine avidity in radioactive iodine-refractory thyroid cancer is needed. We isolated and characterized primary human adipose-derived stem cells (ADSCs) and isolated their EVs. The EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting. A new TKI was loaded into the EVs by incubation (37 °C; 10 min) or sonication (18 cycles; 4 s per cycle) with 2 s intervals and a 2 min ice bath every six cycles. TKI loading was confirmed and measured by mass spectrometry. EV uptake into radioactive iodine-refractory thyroid cancer cells (SW1736 cells) was confirmed by microscopy. We treated the SW1736 cells with vehicle, TKI, or TKI-loaded EVs (sonication TKI-loaded EVs [EVsTKI(S)]) and examined the expression of iodide-metabolizing proteins and radioiodine uptake in the SW1736 cells. ADSCs cells showed >99% of typical stem cell markers, such as CD90 and CD105. The EVs displayed a round morphology, had an average size of 211.4 ± 3.83 nm, and were positive for CD81 and Alix and negative for cytochrome c. The mass spectrometry results indicate that the sonication method loaded ~4 times more of the TKI than did the incubation method. The EVsTKI(S) were used for further experiments. Higher expression levels of iodide-metabolizing mRNA and proteins in the EVsTKI(S)-treated SW1736 cells than in TKI-treated SW1736 cells were confirmed. EVsTKI(S) treatment enhanced 125I uptake in the recipient SW1736 cells compared with free-TKI treatment. This is the first study that demonstrated successful delivery of a TKI to radioactive iodine-refractory thyroid cancer cells using EVs as the delivery vehicle. This approach can revert radioiodine-resistant thyroid cancer cells back to radioiodine-sensitive thyroid cancer cells.

Discovery and Validation of a Novel Step Catalyzed by OsF3H in the Flavonoid Biosynthesis Pathway.

Kaempferol and quercetin are the essential plant secondary metabolites that confer huge biological functions in the plant defense system. In this study, biosynthetic pathways for kaempferol and quercetin were constructed in Saccharomyces cerevisiae using naringenin as a substrate. OsF3H was cloned into pRS42K yeast episomal plasmid (YEp) vector and the activity of the target gene was analyzed in engineered and empty strains. We confirmed a novel step of kaempferol and quercetin biosynthesis directly from naringenin, catalyzed by the rice flavanone 3-hydroxylase (F3H). The results were confirmed through thin layer chromatography (TLC) followed by western blotting, nuclear magnetic resonance (NMR), and liquid chromatography-mass spectrometry LCMS-MS. TLC showed positive results when comparing both compounds extracted from the engineered strain with the standard reference. Western blotting confirmed the lack of OsF3H activity in empty strains and confirmed high OsF3H expression in engineered strains. NMR spectroscopy confirmed only quercetin, while LCMS-MS results revealed that F3H is responsible for the conversion of naringenin to both kaempferol and quercetin.

Identification of suberosin metabolites in human liver microsomes by high-performance liquid chromatography combined with high-resolution quadrupole-orbitrap mass spectrometer.

Suberosin is a natural prenylated coumarin derivative isolated from Citropsis articulata. It has various pharmacological properties, especially as an anticoagulant, for which it has been used since antiquity. However, its metabolic pathway and metabolites have not yet been studied. Therefore, this study characterizes its metabolic pathway and metabolites in human liver microsomes (HLMs) using high-resolution quadrupole-orbitrap mass spectrometry (HRMS/MS). Eight metabolites (M1-M8) were found, including three monohydroxylated (M1-M3), one hydrated (M4), three dihydroxylated (M5-M7), and one glucuronide conjugate (M8). Furthermore, forms of cytochrome P450 (CYPs) responsible for suberosin metabolism in HLMs were characterized. CYP1A2 was identified as a major enzyme for the production of M1 and M5 metabolites. The M2, M3, and M7 metabolites were predominantly generated by CYP2B6. M8 was the only phase II metabolite, identified as a glucuronide conjugate from either M1 or M2. This glucuronide conjugate may be the only promising metabolite from phase II metabolism. Phase I metabolism, especially hydroxylation, was found to provide a predominant metabolic pathway of suberosin in HLMs. Further studies should be conducted to explore the metabolites, examining their efficacy and their toxicity in an in vivo system.

Assessing Drug Interaction and Pharmacokinetics of Loxoprofen in Mice Treated with CYP3A Modulators.

Loxoprofen (LOX) is a non-selective cyclooxygenase inhibitor that is widely used for the treatment of pain and inflammation caused by chronic and transitory conditions. Its alcoholic metabolites are formed by carbonyl reductase (CR) and they consist of trans-LOX, which is active, and cis-LOX, which is inactive. In addition, LOX can also be converted into an inactive hydroxylated metabolite (OH-LOXs) by cytochrome P450 (CYP). In a previous study, we reported that CYP3A4 is primarily responsible for the formation of OH-LOX in human liver microsomes. Although metabolism by CYP3A4 does not produce active metabolites, it can affect the conversion of LOX into trans-/cis-LOX, since CYP3A4 activity modulates the substrate LOX concentration. Although the pharmacokinetics (PK) and metabolism of LOX have been well defined, its CYP-related interactions have not been fully characterized. Therefore, we investigated the metabolism of LOX after pretreatment with dexamethasone (DEX) and ketoconazole (KTC), which induce and inhibit the activities of CYP3A, respectively. We monitored their effects on the PK parameters of LOX, cis-LOX, and trans-LOX in mice, and demonstrated that their PK parameters significantly changed in the presence of DEX or KTC pretreatment. Specifically, DEX significantly decreased the concentration of the LOX active metabolite formed by CR, which corresponded to an increased concentration of OH-LOX formed by CYP3A4. The opposite result occurred with KTC (a CYP3A inhibitor) pretreatment. Thus, we conclude that concomitant use of LOX with CYP3A modulators may lead to drug-drug interactions and result in minor to severe toxicity even though there is no direct change in the metabolic pathway that forms the LOX active metabolite.

Identification of sulfonyl-loxoprofen as novel phase 2 conjugate in rat.

Loxoprofen is a prodrug that exerts strong analgesic and anti-inflammatory effects through its active trans-alcohol metabolite, which is produced in the liver by carbonyl reductase. Previous metabolic studies have evaluated loxoprofen, but its sulfate and taurine conjugates have not yet been studied. We characterized the metabolomic profile of loxoprofen in rat plasma, urine, and feces using high-resolution mass spectrometry. We identified 17 metabolites of loxoprofen in the three different biological matrices, 13 of which were detected in plasma and feces and 16 in urine. Amongst these metabolites, two novel taurine conjugates (M12 and M13) and two novel acyl glucuronides (M14, M15) were identified for the first time in rats. In addition, we detected three novel sulfate conjugates (M9, M10, and M11) of loxoprofen. Further study of these metabolites of loxoprofen is essential in order to assess their potency and toxicity.

Himalayan honey loaded iron oxide nanoparticles: synthesis, characterization and study of antioxidant and antimicrobial activities.

Background: Himalayan honey, a natural product of wild honey bees found in the Himalayan mountains of Nepal, has been used in medicine for many years. The successful development of nanotechnology and beneficial effects of honey would bring a new opportunity to synthesize hybrid nanomaterials for biomedical applications. Thus, the purpose of this study was to load Himalayan honey onto iron oxide nanoparticles (IO-NPs) and study their antioxidant and antimicrobial activities. Methods: Himalayan honey loaded iron oxide nanoparticles (HHLIO-NPs) were synthesized and X-ray diffraction (XRD) and scanning electron microscope (SEM) analyses were performed for characterization. UV-VIS spectra confirmed the loading of honey onto nanoparticles. The antioxidant activity of these nanoparticles was studied against 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical system. We also tested antimicrobial activity of HHLIO-NPs using well diffusion method towards both Gram-positive and Gram-negative bacterial strains of Staphylococus aureus & Escherichia coli. Results: From XRD analysis, the average particle size was found to be 33-40 nm. The SEM images show needle shape porous structures of HHLIO-NPs compared to free IO-NPs indicating the surfactant-like behaviour of honey. In DPPH radical system, the scavenging activities of Himalayan honey (HH), free IO-NPs and HHLIO-NPs ranged 7.93-35.99%, 11.02-52.02% and 16.10-80.52% respectively, with corresponding IC50 values of 1.36 mg/mL, 1.09 mg/mL and 0.52 mg/mL. The antimicrobial property of all test samples showed a noteworthy inhibition on both bacterial strains. However, the HH and HHLIO-NPs exhibited strong antibacterial activity against E. coli. Conclusion: This work reveals that the biological activity of HH is enhanced significantly after loading into IO-NPs. Thus, the HHLIO-NPs would be a promising alternative for antioxidant and antimicrobial agents.

Characterization of CYPs and UGTs Involved in Human Liver Microsomal Metabolism of Osthenol.

Osthenol is a prenylated coumarin isolated from the root of Angelica koreana and Angelica dahurica, and is an O-demethylated metabolite of osthole in vivo. Its various pharmacological effects have been reported previously. The metabolic pathway of osthenol was partially confirmed in rat osthole studies, and 11 metabolic products were identified in rat urine. However, the metabolic pathway of osthenol in human liver microsomes (HLM) has not been reported. In this study, we elucidated the structure of generated metabolites using a high-resolution quadrupole-orbitrap mass spectrometer (HR-MS/MS) and characterized the major human cytochrome P450 (CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) isozymes involved in osthenol metabolism in human liver microsomes (HLMs). We identified seven metabolites (M1-M7) in HLMs after incubation in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and uridine 5'-diphosphoglucuronic acid (UDPGA). As a result, we demonstrated that osthenol is metabolized to five mono-hydroxyl metabolites (M1-M5) by CYP2D6, 1A2, and 3A4, respectively, a 7-O-glucuronide conjugate (M6) by UGT1A9, and a hydroxyl-glucuronide (M7) from M5 by UGT1A3 in HLMs. We also found that glucuronidation is the dominant metabolic pathway of osthenol in HLMs.

Exploring the Metabolism of Loxoprofen in Liver Microsomes: The Role of Cytochrome P450 and UDP-Glucuronosyltransferase in Its Biotransformation.

Loxoprofen, a propionic acid derivative, non-steroidal anti-inflammatory drug (NSAID) is a prodrug that is reduced to its active metabolite, trans-alcohol form (Trans-OH) by carbonyl reductase enzyme in the liver. Previous studies demonstrated the hydroxylation and glucuronidation of loxoprofen. However, the specific enzymes catalyzing its metabolism have yet to be identified. In the present study, we investigated metabolic enzymes, such as cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT), which are involved in the metabolism of loxoprofen. Eight microsomal metabolites of loxoprofen were identified, including two alcohol metabolites (M1 and M2), two mono-hydroxylated metabolites (M3 and M4), and four glucuronide conjugates (M5, M6, M7, and M8). Based on the results for the formation of metabolites when incubated in dexamethasone-induced microsomes, incubation with ketoconazole, and human recombinant cDNA-expressed cytochrome P450s, we identified CYP3A4 and CYP3A5 as the major CYP isoforms involved in the hydroxylation of loxoprofen (M3 and M4). Moreover, we identified that UGT2B7 is the major UGT isoform catalyzing the glucuronidation of loxoprofen and its alcoholic metabolites. Further experimental studies should be carried out to determine the potency and toxicity of these identified metabolites of loxoprofen, in order to fully understand of mechanism of loxoprofen toxicity.