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Methylglyoxal (MG) is a reactive glycation metabolite and potentially induces dicarbonyl stress. The production of MG in cells is increased along with an increase in carbohydrate metabolism. The efficiency of the glyoxalase system, consisting of glyoxalase 1 (GlxI) and glyoxalase 2 (GlxII), is crucial for turning the accumulated MG into nontoxic metabolites. Converting MG-glutathione hemithioacetal to S-d-lactoylglutathione by GlxI is the rate-determining step of the enzyme system. In this study, we found lactic acid accumulated during insulin stimulation in cells, however, cellular MG and S-d-lactoylglutathione also increased due to the massive flux of glycolytic intermediates. The insulin-induced accumulation of MG and S-d-lactoylglutathione were efficiently removed by the treatment of metformin, possibly via affecting the glyoxalase system. With the application of isotopic 13C3-MG, the flux of MG from extracellular and intracellular origins was dissected. While insulin induced an influx of extracellular MG, metformin inhibited the trafficking of MG across the plasma membrane. Therefore, metformin could maintain the extracellular MG by means of reducing the secretion of MG rather than facilitating the scavenging. In addition, metformin may affect the glyoxalase system by controlling the cellular redox state through replenishing reduced glutathione. Overall, alternative biochemical regulation of the glyoxalase system mediated by insulin signaling or molecules like biguanides may control cellular MG homeostasis.

Mu Dan Pi (MDP), also known as Moutan Cortex Radicis, is a traditional Chinese medicine used to treat autoimmune diseases. However, the impact of MDP and its principal active compounds on inflammatory bowel disease (IBD) is uncertain. This study therefore systemically assessed the anti-inflammatory effects of MDP and its known active compounds in IBD. The anti-inflammatory activities of water extract and individual compounds were screened by NF-κB and interferon regulatory factor (IRF) reporter assays in THP-1 cells induced with either Toll-like receptor or retinoic acid inducible gene I/melanoma differentiation-associated gene 5 activators and further verified in bone marrow-derived macrophages. MDP water extract significantly inhibited the activation of NF-κB and IRF reporters, downstream signaling pathways and the production of IL-6 and TNF-α, in a dose-dependent manner. Among 5 known active components identified from MDP (1,2,3,4,6-penta-O-galloyl-β-d-glucose [PGG], gallic acid, methyl gallate, paeoniflorin, and paeonol), PGG was the most efficient at inhibiting both reporters (with an IC50 of 5–10 µM) and downregulating IL-6 and TNF-α. Both MDP powder for clinical use and MDP water extract, but not PGG, reduced colitis and pathological changes in mice. MDP and its water extract show promise as a novel therapy for IBD patients.

Different point stimulations can induce brain activity in specific regions, and however whether these stimulations affect unique neurotransmitter transmission remains unknown. Therefore, we aimed to investigate the effect of point specificity to the brain by resolving the metabolite profiles. Eighteen Sprague–Dawley rats were randomly divided into three groups: (1) the sham group: sham acupuncture at Zusanli (ST36) without electric stimulation; (2) the Zusanli (ST36) group: electroacupuncture (EA) at ST36; and (3) the Neiguan (PC6) group: EA at PC6. Then, the metabolites from rat brain samples were measured by LC–ESI–MS. The results of a partial least squares discriminant analysis revealed the differences among the sham, ST36, and PC6 groups regarding the relative content of metabolites in the cerebral cortex, hippocampus, and hypothalamus. EA at PC6 resulted in downregulation of adenosine, adrenaline, γ-aminobutyric acid, glycine, and glutamate majorly in hippocampus, and then in cerebral cortex. Otherwise, EA at ST6 resulted in upregulation of adrenaline and arginine in hippocampus, and all stimulations showed barely change of identified neurotransmitters in hypothalamus. These differential metabolite and neurotransmitter profiles prove that brain areas can be modulated by point specificity and may provide a maneuver to understand more details of meridian.

Hydrogen sulfide (H2S), previously known as a toxic gas, is now recognized as a gasotransmitter along with nitric oxide and carbon monoxide. However, only few methods are available for quantitative determination of H2S in biological samples. 2-Iodoacetanilide (2-IAN), a thiol-reacting agent, has been used to tag the reduced cysteine residues of proteins for quantitative proteomics and for detection of cysteine oxidation modification. In this article, we proposed a new method for quantitative analyses of H2S and thiol metabolites using the procedure of pre-column 2-IAN derivatization coupled with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI–MS). 13C6-Labeled and label-free 2-IAN efficiently react with H2S and thiol compounds at pH 9.5 and 65 °C. The derivatives exhibit excellent stability at alkaline conditions, high resolution on reverse phase liquid chromatography and great sensitivity for ESI–MS detection. The measurement of H2S, l -cysteine, glutathione, and DL-homocysteine derivatives was validated using 13C6-labeled standard in LC-ESI–MS analyses and exhibited 10 nM–1 μM linear ranges for DL-homocysteine and glutathione and 1 nM–1 μM linear ranges for l -cysteine and H2S. In addition, the sequence of derivatization and extraction of metabolites is important in the quantification of thiol metabolites suggesting the presence of matrix effects. Most importantly, labeling with 2-IAN and 13C6-2-IAN isotopologues could achieve quantitative and matched sample comparative analyses with minimal bias using our extraction and labeling procedures before LC–MS analysis.

Natural phenolic products from herbal medicines and dietary plants constitute the main source of lead compounds for the development of the new drug. 4,4-Dimethylcurcumin (DMCU) is a synthetic curcumin derivative and exhibits anticancer activities against breast, colon, lung, and liver cancers. However, further development of DMCU is limited by unfavorable compound properties such as very low aqueous solubility and moderate stability. To increase its solubility, we installed either or both of the ethylene-carbonate-linked L-valine side chains to DMCU phenolic groups and produced targeted 1-trifluoroacetic acid (1-TFA) and 2-trifluoroacetic acid (2-TFA) derivatives. The terminus L-valine of ethylene-carbonate-linked side chain is known to be a L-type amino acid transporter 1 (LAT1) recognition element and therefore, these two derivatives were expected to readily enter into LAT1-expressing cancer cells. In practice, 1-TFA or 2-TFA were synthesized from DMCU in four steps with 34–48% overall yield. Based on the corresponding LC-MS analysis, water solubility of DMCU, 1-TFA, and 2-TFA at room temperature (25 ± 1 °C) were 0.018, 249.7, and 375.8 mg/mL, respectively, indicating >10,000-fold higher solubility of 1-TFA and 2-TFA than DMCU. Importantly, anti-proliferative assay demonstrated that 2-TFA is a potent anti-cancer agent against LAT1-expressing lung cancer cells NCI-H460, NCI-H358, and A549 cells due to its high intracellular uptake compared to DMCU and 1-TFA. In this study, we logically designed and synthesized the targeted compounds, established the LC-MS analytical methods for evaluations of drug solubility and intracellular uptake levels, and showed improved solubility and anti-cancer activities of 2-TFA. Our results provide a strategical direction for the future development of curcuminoid-like phenolic compounds.

The treatment of cancer cells obtained by blocking cellular metabolism has received a lot of attention recently. Previous studies have demonstrated that Kras mutation-mediated abnormal glucose metabolism would lead to an aberrant cell proliferation in human pancreatic ductal adenocarcinoma (PDAC) cells. Previous literature has suggested that consumption of fish oil is associated with lower risk of pancreatic cancer. In this study, we investigated the anti-cancer effects of docosahexaenoic acid (DHA) in human PDAC cells in vitro and in vivo. Omega-3 polyunsaturated fatty acids (PUFAs) such as DHA and eicosapentaenoic acid (EPA) significantly inhibited the proliferation of human PDAC cells. The actions of DHA were evaluated through an induction of cell cycle arrest at G1 phase and noticed a decreased expression of cyclin A, cyclin E and cyclin B proteins in HPAF-II cells. Moreover, it was found that co-treatment of DHA and gemcitabine (GEM) effectively induced oxidative stress and cell death in HPAF-II cells. Interestingly, DHA leads to an increased oxidative glutathione /reduced glutathione (GSSG/GSH) ratio and induced cell apoptosis in HPAF-II cells. The findings in the study showed that supplementation of GSH or N-Acetyl Cysteine (NAC) could reverse DHA-mediated cell death in HPAF-II cells. Additionally, DHA significantly increased cellular level of cysteine, cellular NADP/NADPH ratio and the expression of cystathionase (CTH) and SLCA11/xCT antiporter proteins in HPAF-II cells. The action of DHA was, in part, associated with the inactivation of STAT3 cascade in HPAF-II cells. Treatment with xCT inhibitors, such as erastin or sulfasalazine (SSZ), inhibited the cell survival ability in DHA-treated HPAF-II cells. DHA also inhibited nucleotide synthesis in HPAF-II cells. It was demonstrated in a mouse-xenograft model that consumption of fish oil significantly inhibited the growth of pancreatic adenocarcinoma and decreased cellular nucleotide level in tumor tissues. Furthermore, fish oil consumption induced an increment of GSSG/GSH ratio, an upregulation of xCT and CTH proteins in tumor tissues. In conclusion, DHA significantly inhibited survival of PDAC cells both in vitro and in vivo through its recently identified novel mode of action, including an increment in the ratio of GSSG/GSH and NADP/NADPH respectively, and promoting reduction in the levels of nucleotide synthesis.

Compound 1 is a curcumin di-O-2,2-bis(hydroxymethyl)propionate that shows significant in vitro and in vivo inhibitory activity against MDA-MB-231 cells with eight to ten-fold higher potency than curcumin. Here, we modified the &alpha, position (C-4 position) of the central 1,3-diketone moiety of 1 with polar or nonpolar functional groups to afford a series of 4,4-disubstituted curcuminoid 2,2-bis(hydroxymethyl)propionate derivatives and evaluated their anticancer activities. A clear structure&ndash, activity relationship of compound 1 derivatives focusing on the functional groups at the C-4 position was established based on their anti-proliferative effects against the MDA-MB-231 and HCT-116 cell lines. Compounds 2&ndash, 6 are 4,4-dimethylated, 4,4-diethylated, 4,4-dibenzylated, 4,4-dipropargylated and 4,4-diallylated compound 1, respectively. Compounds 2m&ndash, 6m, the ester hydrolysis products of compounds 2&ndash, 6, respectively, were synthesized and assessed for anticancer activity. Among all compound 1 derivatives, compound 2 emerged as a potential chemotherapeutic agent for colon cancer due to the promising in vivo anti-proliferative activities of 2 (IC50 = 3.10 &plusmn, 0.29 &mu, M) and its ester hydrolysis product 2m (IC50 = 2.17 &plusmn, 0.16 &mu, M) against HCT-116. The preliminary pharmacokinetic evaluation of 2 implied that 2 and 2m are main contributors to the in vivo efficacy. Compound 2 was further evaluated in an animal study using HCT-116 colon tumor xenograft bearing nude mice. The results revealed a dose-dependent efficacy that led to tumor volume reductions of 27%, 45%, and 60% at 50, 100, and 150 mg/kg doses, respectively. The established structure&ndash, activity relationship and pharmacokinetic outcomes of 2 is the guidance for future development of 4,4-disubstituted curcuminoid 2,2-bis(hydroxymethyl)- propionate derivatives as anticancer drug candidates.

To elucidate the molecular mechanisms underlying the action of bioactive compounds such as metabolites, identification of their binding targets is essential. However, available techniques for enriching metabolite-binding proteins are practically restrained by special equipment requirements and laborious efforts. Here we have developed a novel method, affinity elution in tandem hydrophobic interaction chromatography (AETHIC), which enables enrichment of metabolite-binding proteins from a crude tissue extract. AETHIC constitutes two major steps, protein fractionation and affinity elution. The basic strategy of AETHIC uses a series of HIC matrices encompassing aliphatic chains of different length and thus provides a wide range of hydrophobicity for interactions with most proteins. Thereafter, target proteins are eluted selectively by a given ligand. As our first proof-of-principle, we demonstrated that AETHIC was able to enrich ATP-binding proteins from porcine brain extract. In addition, we have demonstrated that raf kinase inhibitory protein (RKIP) is an ATP-binding protein and ATP attenuates the interaction between RKIP and Raf-1. In parallel, short-term ATP depletion in cultured HEK293 cells augments interaction between RKIP and Raf-1, resulting in decreased activation of the downstream ERK signaling. Therefore, the ATP-binding function renders RKIP's inhibition on Raf-1 modulated by cellular ATP concentrations. These data shed light on how energy levels affect the propagation of cellular signaling. Taken together, the enclosed results advocate the potential of AETHIC in the study of metabolite-protein interactions.

Electrospray ionization (ESI) is the most useful interface for mass spectrometry associated with liquid chromatography (LC). However, analyses of polar metabolites become a challenge because the high polarity impairs the separation of metabolites in reversed-phase liquid chromatography and ionization of metabolites by ESI. In this article, we have used cyanuric chloride to couple the amine-containing molecules with methylamine and found that both resolution on LC chromatogram and ionization by ESI are greatly improved. Derivatives would be obtained in 2-h coupling reactions, and then resolved by LC-ESI-MS in 15 min for each sample. Most amino acids can be quantified with linear range from 1 nM to 1 μM and with an R(2) above 0.979. Although reversed-phase chromatography is suitable for resolving the derivatives, phenyl columns with methanol elution provide optimal separation and signal intensity. Moreover, most structural isomers are well separated following cyanuric chloride and methylamine derivatization. Instead of synthesizing a stable isotope-labeled cyanuric chloride, we can take advantage of using commercially available methyl-d3-amine for a novel stable isotope-coded derivatization method. Each metabolite can be directly quantified by the peak intensity ratio of each derivative isotopologue pair in a single LC-MS analysis. The coupling reactions are relatively easy and accessible to most investigators to generate multiple stable isotope-labeled derivatives of amine-containing compounds for a differential metabolomic analysis.

Glyoxalase 1 (GlxI) is the key enzyme that converts the highly reactive α-oxo-aldehydes into the corresponding α-hydroxy acids using l-glutathione as a cofactor. In our preliminary data, GlxI was identified as a substrate of transglutaminase 2 (TG2), a ubiquitous enzyme with multiple functions. According to the catalytic properties of TG2, protein cross-linking, polyamine conjugation, and/or deamidation are potential post-translational modifications. In this article, we have demonstrated that TG2 catalyzes either polyamine conjugation or deamidation to GlxI depending on the presence of polyamines or not. Deamidation leads to activation of GlxI while polyamine conjugation results in activation of GlxI as well as stabilization of GlxI against denaturation treatment. In cultured HeLa cells, methylglyoxal challenge causes increase in intracellular levels of reactive oxygen species (ROS) and calcium leading to TG2 activation and subsequent transamidation and activation of GlxI. The inhibition of TG2 significantly weakens the cell resistance to the methylglyoxal challenge. Thus, GlxI is a novel substrate of TG2 and is activated by TG2 in vitro and in cellulo. Exposure to methylglyoxal elicits a negative feedback loop entailing ROS, calcium, TG2 and GlxI, thus leading to attenuation of the increase in the methylglyoxal level. The results imply that cancer cells highly express TG2 or GlxI can endure the oxidative stress derived from higher glycolytic flux and may gain extra growth advantage from the aerobic glycolysis., Graphical abstract, Highlights • We have demonstrated novel modifications of glyoxalase I by transglutaminase 2. • The modifications mediated by transglutaminse 2 modulate the glyoxalase I activities. • Methylglyoxal treatment in cells induces increases in the levels of endogenous reactive oxygen species and activation transglutaminase 2 and glyoxalase I. • Cells dispose the accumulated intracellular methylglyoxal by a negative feedback loop consisting of reactive oxygen species, calcium, transglutaminase 2 and glyoxalase I.

Aims: In this study we identified viral gene targets of the important redox regulator thioredoxin (Trx), and explored in depth how Trx interacts with the immediate early gene #1 (IE1) of the white spot syndrome virus (WSSV). Results: In a pull-down assay, we found that recombinant Trx bound to IE1 under oxidizing conditions, and a coimmunoprecipitation assay showed that Trx bound to WSSV IE1 when the transfected cells were subjected to oxidative stress. A pull-down assay with Trx mutants showed that no IE1 binding occurred when cysteine 62 was replaced by serine. Electrophoretic mobility shift assay (EMSA) showed that the DNA binding activity of WSSV IE1 was downregulated under oxidative conditions, and that Penaeus monodon Trx (PmTrx) restored the DNA binding activity of the inactivated, oxidized WSSV IE1. Another EMSA experiment showed that IE1's Cys-X-X-Cys motif and cysteine residue 55 were necessary for DNA binding. Measurement of the ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) in WSSV-infected shrimp showed that oxidative stress was significantly increased at 48 h postinfection. The biological significance of Trx was also demonstrated in a double-strand RNA Trx knockdown experiment where suppression of shrimp Trx led to significant decreases in mortality and viral copy numbers. Innovation and Conclusion: WSSV's pathogenicity is enhanced by the virus' use of host Trx to rescue the DNA binding activity of WSSV IE1 under oxidizing conditions. Antioxid. Redox Signal. 17, 914–926.

Fetuin-A is known for limiting the expansion and formation of hydroxyapatite crystals from calcium phosphate aggregates in circulation by forming a soluble fetuin-mineral complex. This study was aimed to uncover potential proteins involved in the regulation of calcium phosphate precipitation within cells. We found that a novel protein-mineral complex (PMC) can be generated after introduction of calcium chloride and sodium phosphate into the porcine brain protein extract prepared in Tris-HCl buffer. Selectively enriched proteins in the pellet were confirmed by immunoblotting, including heat shock protein 90 (Hsp90), annexin A5, calreticulin, nucleolin, and other proteins. In addition, purified native Hsp90 directly bound both amorphous calcium phosphate and hydroxyapatite and underwent conformational changes and oligomerization in the presence of excess calcium and phosphate. The morphology of the PMC prepared from Hsp90, calcium, and phosphate was distinctly different from that of hydroxyapatite under transmission electron microscope observation. When cultured SiHa cells were treated with a calcium ionophore or damaged by scratch to induce the massive calcium influx, a complex was formed and observed at discrete sites near the plasma membrane as revealed by antibodies against Hsp90, annexin A5, calreticulin, nucleolin, and other proteins. This complex could also be probed in situ with fetuin-A suggesting the existence of calcium phosphate aggregates in this complex. Inhibition of the complex formation by bisphosphonates hindered cell recovery from A23187 assault. Our results show that following membrane damage amorphous calcium phosphate develops at sites near membrane rupture where saturated calcium phosphate concentration is achieved. As a result, Hsp90 and other proteins are recruited, and the cytosolic PMC is formed. Inhibition of the cytosolic PMC formation may in part contribute to the cellular toxicity and in vivo side effects of bisphosphonates, particularly in cells prone to membrane damage under physiological conditions such as gastrointestinal epithelial and oral cavity epithelial cells.