Proteomic and redox‐proteomic study on the role of glutathione reductase in human lung cancer cells

Glutathione reductase (GR), a cytosolic protein, plays a vital role in maintaining a correct redox status in cells. However, comprehensive investigations of GR‐modulated cellular responses, including protein level alteration and redox regulation, have yet to be performed. In this study, we cultured a human lung adenocarcinoma line transfected with empty pLKO.1 vector as a control, CL1‐0shControl, and its GR‐knockdown derivative, CL1‐0shΔGR, to evaluate differential protein level alteration and redox regulation of these two cell lines. We identified 34 spots that exhibited marked changes in intensities, and 13 proteins showing significant changes in thiol reactivity, in response to GR depletion. Several proteins involved in redox regulation, calcium signaling, cytoskeleton regulation, and protein folding showed significant changes in expression, whereas proteins involved in redox regulation, protein folding, and glycolysis displayed changes in thiol reactivity. Interestingly, GR knockdown induces peroxiredoxin‐1 overexpression in the air‐exposed tissue and high oxygen consuming tissue such as cornea and liver, but not in the low oxygen consuming tissues such as breast and uterine. In summary, we used a comprehensive lung adenocarcinoma based proteomic approach for identifying GR‐modulated protein expression alteration and redox modification. Based on our research, this is the first comprehensive proteomic and redox‐proteomic analysis used to investigate the role of GR in a mammalian cell model.     

Pharmacokinetics of 2,3,5,4′‐tetrahydroxystilbene‐2‐O‐β‐D‐glucoside in rat using ultra‐performance LC‐quadrupole TOF‐MS

2,3,5,4′‐Tetrahydroxystilbene‐2‐O‐β‐D‐glucoside (THSG) from Polygoni multiflori has been demonstrated to possess a variety of pharmacological activities, including antioxidant, anti‐inflammatory and hepatoprotective activities. Ultra‐performance LC‐quadrupole TOF‐MS with MS Elevated Energy data collection technique and rapid resolution LC with diode array detection and ESI multistage MSⁿ methods were developed for the pharmacokinetics, tissue distribution, metabolism, and excretion studies of THSG in rats following a single intravenous or oral dose. The three metabolites were identified by rapid resolution LC‐MSⁿ. The concentrations of the THSG in rat plasma, bile, urine, feces, or tissue samples were determined by ultra‐performance LC‐MS. The results showed that THSG was rapidly distributed and eliminated from rat plasma. After the intravenous administration, THSG was mainly distributing in the liver, heart, and lung. For the rat, the major distribution tissues after oral administration were heart, kidney, liver, and lung. There was no long‐term storage of THSG in rat tissues. Total recoveries of THSG within 24 h were low (0.1% in bile, 0.007% in urine, and 0.063% in feces) and THSG was excreted mainly in the forms of metabolites, which may resulted from biotransformation in the liver.     

A self‐guided search for good local minima of the sum‐of‐squared‐error in nonlinear least squares regression

Hard modeling of nonlinear chemical or biological systems is highly relevant as a model function together with values for model parameters provides insights in the systems' functionalities. Deriving values for said model parameters via nonlinear regression, however, is challenging as usually one of the numerous local minima of the sum‐of‐squared errors (SSEs) is determined; furthermore, for different starting points, different minima may be found. Thus, nonlinear regression is prone to low accuracy and low reproducibility. Therefore, there is a need for a generally applicable, automated initialization of nonlinear least squares algorithms, which reaches a good, reproducible solution after spending a reasonable computation time probing the SSE‐hypersurface. For this purpose, a three‐step methodology is presented in this study. First, the SSE‐hypersurface is randomly probed in order to estimate probability density functions of initial model parameter that generally lead to an accurate fit solution. Second, these probability density functions then guide a high‐resolution sampling of the SSE‐hypersurface. This second probing focuses on those model parameter ranges that are likely to produce a low SSE. As the probing continues, the most appropriate initial guess is retained and eventually utilized in a subsequent nonlinear regression. It is shown that this “guided random search” derives considerably better regression solutions than linearization of model functions, which has so far been considered the best‐case scenario. Examples from infrared spectroscopy, cell culture monitoring, reaction kinetics, and image analyses demonstrate the broad and successful applicability of this novel method. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous LC‐MS/MS bioanalysis of etoposide and paclitaxel in mouse tissues and plasma after oral administration of self‐microemulsifying drug‐delivery systems

In this study, a liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated to simultaneously determine the anticancer drugs etoposide and paclitaxel in mouse plasma and tissues including liver, kidney, lung, heart, spleen and brain. The analytes were extracted from the matrices of interest by liquid–liquid extraction using methyl tert‐butyl ether–dichloromethane (1:1, v/v). Chromatographic separation was achieved on an Ultimate XB‐C₁₈ column (100 × 2.1 mm, 3 μm) at 40°C and the total run time was 4 min under a gradient elution. Ionization was conducted using electrospray ionization in the positive mode. Stable isotope etoposide‐d3 and docetaxel were used as the internal standards. The lower limit of quantitation (LLOQ) of etoposide was 1 ng/g tissue for all tissues and 0.5 ng/mL for plasma. The LLOQ of paclitaxel was 0.4 ng/g tissue and 0.2 ng/mL for all tissues and plasma, respectively. The coefficients of correlation for all of the analytes in the tissues and plasma were >0.99. Both intra‐ and inter‐day accuracy and precision were satisfactory. This method was successfully applied to measure plasma and tissue drug concentrations in mice treated with etoposide and paclitaxel‐loaded self‐microemulsifying drug‐delivery systems.     

De Sign and Synthesis of 1,2,4‐Oxadiazole Derivatives as Non‐Steroidal 5α‐Reductase Inhibitors

The purpose of this study was to synthesize compounds in which the 1,2,4‐oxadiazole moiety replaced the amide bond of ONO3805 and to evaluate its 5α‐reductase inhibitory activity as a potential benign prostatic hyperplasia therapeutic target. Four 1,2,4‐oxadiazole derivatives, 1,2,8, and 20, were evaluated in vitro against 5α‐reductase of rat liver microsome. The prepared 1 and 2 possessed similar binding affinity (Ki) to that of ONO3805. Therefore, the use of 1,2,4‐oxadiazole ring as surrogate of the amide bond in ONO3805 has a successful result in this study. It leads not only to enhance chemical stability but also to maintain meaningful inhibitory activity. The butyric acid moiety of these inhibitors is considered to play an important role in mimicing the phosphoric acid portion of coenzyme‐NADPH in interacting with the active site of 5α‐reductase.     

Identification of bilobetin metabolites,in vivo and in vitro,based on an efficient ultra‐high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry strategy

Bilobetin, a natural compound extracted from Ginkgo biloba, has various pharmacological activities such as antioxidation, anticancer, antibacterial, antifungal, anti‐inflammatory, antiviral, and promoting osteoblast differentiation. However, few studies have been conducted and there are no reports on its metabolites owing to its low content in nature. In addition, it has been reported to have potential liver and kidney toxicity. Therefore, this study aimed to identify the metabolites of bilobetin in vitro and in vivo. Bilobetin was incubated with liver microsomes to determine metabolites in vitro, and faeces and urine were collected after oral administration to rats to determine metabolites in vivo. After the samples were processed, they were measured using ultra‐high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry. As a result, a total of 21 and 9 metabolites were detected in vivo and in vitro, respectively. Demethylation, demethylation and loss of water, demethylation and hydrogenation, demethylation and glycine conjugation, oxidation, methylation, oxidation and methylation, and hydrogenation were the main metabolic pathways. This study is the first to identify the metabolites of bilobetin and provides a theoretical foundation for the safe use of bilobetin in clinical application and the development of new drugs.     

Pharmacokinetics,protein binding and metabolism of a quinoxaline urea analog as an NF‐κB inhibitor in mice and rats by LC‐MS/MS

13–197 is a novel NF‐κB inhibitor that shows promising in vitro efficacy data against pancreatic cancer. In this study, we characterized the pharmacokinetics, tissue distribution, protein binding and metabolism of 13–197 in mice and rats. A valid, sensitive and selective LC‐MS/MS method was developed. This method was validated for the quantification of 13–197, in the range of 0.1 or 0.2‐500 ng/mL in mouse plasma, liver, kidney, lung, heart, spleen, brain, urine and feces. 13–197 has low bioavailability of 3 and 16% in mice and rats, respectively. It has faster absorption in mice with 12‐fold shorter T_max than in rats. Tissue concentrations were 1.3–69.2‐fold higher in mice than in rats at 72 h after intravenous administration. 13–197 is well distributed to the peripheral tissues and has relatively high tissue–plasma concentration ratios, ranging from 1.8 to 3634, in both mice and rats. It also demonstrated more than 99% binding to plasma proteins in both mice and rats. Finally, <1% of 13–197 is excreted unchanged in urine and feces, and metabolite profiling studies detected more than 20 metabolites in mouse and rat plasma, urine and feces, which indicates that 13–197 is extensively metabolized and primarily eliminated by metabolism rather than by excretion. Copyright © 2013 John Wiley & Sons, Ltd.     

Synthesis of 2‐[4‐(Imidazolin‐2‐Ylideneamino)Benzyl]‐Indan‐1‐Ones as Novel Potent Prostacyclin Antagonists

Prostacyclin is involved in many pathological conditions, such as sensitization of inflammation induced pain and isovolumetic distention. Therefore, antagonism of prostacyclin action may be useful in the alleviation of these conditions. In this study, novel potent prostacyclin antagonists, 2‐[4‐(imidazolin‐2‐ylideneamino)benzyl]‐indan‐1‐ones were synthesized from their respective substituted indanones in three steps. The construction of the amino‐imidazole moiety of these derivatives is achieved by using in situ generation of chloro‐imidazole and reaction with their respective anilines. Thus, these N‐substituted 2‐imidazolines can be prepared safely and efficiently. Moreover, these compounds show potent prostacyclin antagonistic activity by inhibition of prostacyclin agonist induced ERK_1/2 phosphorylation in human erythroleukemia cells. Moreover, we observed an increase in activity with the increase in electro‐donating property of the substitution on the indanone aromatic ring. Prostacyclin antagonists with increased potency may be designed based on these findings. These compounds may also be invaluable tools for the study of the physiological functions of prostacyclin.     

Design of the First‐in‐Class,Highly Potent Irreversible Inhibitor Targeting the Menin‐MLL Protein–Protein Interaction

The structure‐based design of M‐525 as the first‐in‐class, highly potent, irreversible small‐molecule inhibitor of the menin‐MLL interaction is presented. M‐525 targets cellular menin protein at sub‐nanomolar concentrations and achieves low nanomolar potencies in cell growth inhibition and in the suppression of MLL‐regulated gene expression in MLL leukemia cells. M‐525 demonstrates high cellular specificity over non‐MLL leukemia cells and is more than 30 times more potent than its corresponding reversible inhibitors. Mass spectrometric analysis and co‐crystal structure of M‐525 in complex with menin firmly establish its mode of action. A single administration of M‐525 effectively suppresses MLL‐regulated gene expression in tumor tissue. An efficient procedure was developed to synthesize M‐525. This study demonstrates that irreversible inhibition of menin may be a promising therapeutic strategy for MLL leukemia.     

Design of the First‐in‐Class,Highly Potent Irreversible Inhibitor Targeting the Menin‐MLL Protein–Protein Interaction

The structure‐based design of M‐525 as the first‐in‐class, highly potent, irreversible small‐molecule inhibitor of the menin‐MLL interaction is presented. M‐525 targets cellular menin protein at sub‐nanomolar concentrations and achieves low nanomolar potencies in cell growth inhibition and in the suppression of MLL‐regulated gene expression in MLL leukemia cells. M‐525 demonstrates high cellular specificity over non‐MLL leukemia cells and is more than 30 times more potent than its corresponding reversible inhibitors. Mass spectrometric analysis and co‐crystal structure of M‐525 in complex with menin firmly establish its mode of action. A single administration of M‐525 effectively suppresses MLL‐regulated gene expression in tumor tissue. An efficient procedure was developed to synthesize M‐525. This study demonstrates that irreversible inhibition of menin may be a promising therapeutic strategy for MLL leukemia.     

Detection of LacZ‐Positive Cells in Living Tissue with Single‐Cell Resolution

The LacZ gene, which encodes Escherichia coli β‐galactosidase, is widely used as a marker for cells with targeted gene expression or disruption. However, it has been difficult to detect lacZ‐positive cells in living organisms or tissues at single‐cell resolution, limiting the utility of existing lacZ reporters. Herein we present a newly developed fluorogenic β‐galactosidase substrate suitable for labeling live cells in culture, as well as in living tissues. This precisely functionalized fluorescent probe exhibited dramatic activation of fluorescence upon reaction with the enzyme, remained inside cells by anchoring itself to intracellular proteins, and provided single‐cell resolution. Neurons labeled with this probe preserved spontaneous firing, which was enhanced by application of ligands of receptors expressed in the cells, suggesting that this probe would be applicable to investigate functions of targeted cells in living tissues and organisms.     

Silk‐Inspired β‐Peptide Materials Resist Fouling and the Foreign‐Body Response

The functions of implants like medical devices are often compromised by the host's foreign‐body response (FBR). Herein, we report the development of low‐FBR materials inspired by serine‐rich sericin from silk. Poly‐β‐homoserine (β‐HS) materials consist of the hydrophilic unnatural amino acid β‐homoserine. Self‐assembled monolayers (SAMs) of β‐HS resist adsorption by diverse proteins, as well as adhesion by cells, platelets, and diverse microbes. Experiments lasting up to 3 months revealed that, while implantation with control PEG hydrogels induced obvious inflammatory responses, collagen encapsulation, and macrophage accumulation, these responses were minimal with β‐HS hydrogels. Strikingly, the β‐HS hydrogels induce angiogenesis in implant‐adjacent tissues. Molecular dynamics simulations indicated that the low FBR performance of β‐HS results from what we term “dual hydrogen bonding hydration”, wherein both the backbone amide groups and the sidechain hydroxyl groups of β‐HS undergo hydration.     

Blocking of the PD‐1/PD‐L1 Interaction by a D‐Peptide Antagonist for Cancer Immunotherapy

Blockade of the protein–protein interaction between the transmembrane protein programmed cell death protein 1 (PD‐1) and its ligand PD‐L1 has emerged as a promising immunotherapy for treating cancers. Using the technology of mirror‐image phage display, we developed the first hydrolysis‐resistant D ‐peptide antagonists to target the PD‐1/PD‐L1 pathway. The optimized compound ^DPPA‐1 could bind PD‐L1 at an affinity of 0.51 μM in vitro. A blockade assay at the cellular level and tumor‐bearing mice experiments indicated that ^DPPA‐1 could also effectively disrupt the PD‐1/PD‐L1 interaction in vivo. Thus D ‐peptide antagonists may provide novel low‐molecular‐weight drug candidates for cancer immunotherapy.     

Interleukin‐17 Mediated Inflammatory Responses Are Required for Ultraviolet Radiation‐Induced Immune Suppression

Ultraviolet radiation (UVR) induces immunosuppression and is a major factor for development of skin cancer. Numerous efforts have been made to determine mechanisms for UVR‐induced immunosuppression and to develop strategies for prevention and treatment of UVR‐induced cancers. In the current study, we use IL‐17 receptor (IL‐17R) deficient mice to examine whether IL‐17 mediated responses have a role in UVB (290–320)‐induced immunosuppression of contact hypersensitivity responses. Results demonstrate that IL‐17 mediated responses are required for UVB‐induced immunosuppression of contact hypersensitivity responses. The systemic immune suppression and development of regulatory T cells are inhibited in UVB‐treated IL‐17R deficient mice compared to wild‐type animals. The deficiency in IL‐17R inhibits the infiltration and development of a tolerogenic myeloid cell population in UVB‐treated skin, which expresses CD11b and Gr‐1 and produces reactive oxygen species. We speculate that the development of the tolerogenic myeloid cells is dependent on IL‐17‐induced chemokines and inflammatory mediators in UVB‐treated skin. The inhibition of the tolerogenic myeloid cells may be attributed to the suppression of regulatory T cells in UVR‐treated IL‐17R^?/? mice. The findings may be exploited to new strategies for prevention and treatment of UVR‐induced skin diseases and cancers.     

Non‐Viral CRISPR/Cas Gene Editing In Vitro and In Vivo Enabled by Synthetic Nanoparticle Co‐Delivery of Cas9 mRNA and sgRNA

CRISPR/Cas is a revolutionary gene editing technology with wide‐ranging utility. ¹ The safe, non‐viral delivery of CRISPR/Cas components would greatly improve future therapeutic utility. ^1e We report the synthesis and development of zwitterionic amino lipids (ZALs) that are uniquely able to (co)deliver long RNAs including Cas9 mRNA and sgRNAs. ZAL nanoparticle (ZNP) delivery of low sgRNA doses (15 nm ) reduces protein expression by >90 % in cells. In contrast to transient therapies (such as RNAi), we show that ZNP delivery of sgRNA enables permanent DNA editing with an indefinitely sustained 95 % decrease in protein expression. ZNP delivery of mRNA results in high protein expression at low doses in vitro (<600 pM) and in vivo (1 mg kg⁻¹). Intravenous co‐delivery of Cas9 mRNA and sgLoxP induced expression of floxed tdTomato in the liver, kidneys, and lungs of engineered mice. ZNPs provide a chemical guide for rational design of long RNA carriers, and represent a promising step towards improving the safety and utility of gene editing.     

Metabolic alterations in triptolide‐induced acute hepatotoxicity

Triptolide, a major active constitute of Tripterygium wilfordii Hook. F, is prescribed for the treatment of autoimmune diseases in China. One of its most severe adverse effects observed in the clinical use is hepatotoxicity, but the mechanism is still unknown. Therefore, the present study applied an LC/MS‐based metabolomic analysis to characterize the metabolomic changes in serum and liver induced by triptolide in mice. Mice were administered triptolide by gavage to establish the acute liver injury model, and serum biochemical and liver histological analyses were applied to assess the degree of toxicity. Multivariate data analyses were performed to investigate the metabolic alterations. Potential metabolites were identified using variable importance in the projection values and Student's t‐test. A total of 30 metabolites were observed that were significantly changed by triptolide treatment and the abundance of 29 metabolites was correlated with the severity of toxicity. Pathway analysis indicated that the mechanism of triptolide‐induced hepatotoxicity was related to alterations in multiple metabolic pathways, including glutathione metabolism, tricarboxylic acid cycle, purine metabolism, glycerophospholipid metabolism, taurine and hypotaurine metabolism, pantothenate and CoA biosynthesis, pyrimidine metabolism and amino acid metabolism. The current study provides new mechanistic insights into the metabolic alterations that lead to triptolide‐induced hepatotoxicity.     

ROS and p53 in Regulation of UVB‐induced HDM2 Alternative Splicing

Alternative splicing plays an important role in proteasome diversity and gene expression regulation in eukaryotic cells. Hdm2, the human homolog of mdm2 (murine double minute oncogene 2), is known to be an oncogene as its role in suppression of p53. Hdm2 alternative splicing, occurs in both tumor and normal tissues, is believed to be a response of cells for cellular stress, and thus modulate p53 activity. Therefore, understanding the regulation of hdm2 splicing is critical in elucidating the mechanisms of tumor development and progression. In this study, we determined the effect of ultraviolet B light (UVB) on alternative splicing of hdm2. Our data indicated that UVB (50 mJ cm^?2) alone is not a good inducer of alternative splicing of hdm2. The less effectiveness could be due to the induction of ROS and p53 by UVB because removing ROS by L‐NAC (10 mm ) in p53 null cells could lead to alternative splicing of hdm2 upon UVB irradiation.     

Magnetically and Near‐Infrared Light‐Powered Supramolecular Nanotransporters for the Remote Control of Enzymatic Reactions

Cancer is one of the primary causes of death worldwide. A high‐precision analysis of biomolecular behaviors in cancer cells at the single‐cell level and more effective cancer therapies are urgently required. Here, we describe the development of a magnetically‐ and near infrared light‐triggered optical control method, based on nanorobotics, for the analyses of cellular functions. A new type of nanotransporters, composed of magnetic iron nanoparticles, carbon nanohorns, and liposomes, was synthesized for the spatiotemporal control of cellular functions in cells and mice. Our technology will help to create a new state‐of‐the‐art tool for the comprehensive analysis of “real” biological molecular information at the single‐cell level, and it may also help in the development of innovative cancer therapies.     

Inflammation Due to Voriconazole‐induced Photosensitivity Enhanced Skin Phototumorigenesis in Xpa‐knockout Mice

Voriconazole is an antifungal agent and used as a prophylactic measure, especially in immunocompromised patients. However, there have been several reports of its adverse reactions, namely photosensitivity with intense inflammatory rashes and subsequent skin cancer development. To assess the effects of photosensitizing drugs voriconazole and hydrochlorothiazide (HCTZ ) on the enhancement of UV ‐induced inflammatory responses and UV ‐induced tumorigenesis, we utilized Xpa ‐knockout mice, which is DNA repair‐deficient and more susceptible to UV ‐induced inflammation and tumor development than wild‐type mice. Administration of voriconazole prior to broadband UVB exposure significantly upregulated multiple inflammatory cytokines compared with the vehicle‐ or HCTZ ‐administered groups. Voriconazole administration along with chronic UVB exposure produced significantly higher number of skin tumors than HCTZ or vehicle in Xpa ‐knockout mice. Furthermore, the investigation of UVB ‐induced DNA damage using embryonic fibroblasts of Xpa ‐knockout mice revealed a significantly higher 8‐oxo‐7,8‐dihydroguanine level in cells treated with voriconazole N‐oxide, a voriconazole‐metabolite during UV exposure. The data suggest that voriconazole plus UVB ‐induced inflammatory response may be related to voriconazole‐induced skin phototumorigenesis.     

Glutathionylated γG and γA subunits of hemoglobin F: a novel post‐translational modification found in extremely premature infants by LC‐MS and nanoLC‐MS/MS

Oxidative stress plays an important role in the development of various disease processes and is a putative mechanism in the development of bronchopulmonary dysplasia, the most common complication of extreme preterm birth. Glutathione, a major endogenous antioxidant and redox buffer, also mediates cellular functions through protein thiolation. We sought to determine if post‐translational thiol modification of hemoglobin F occurs in neonates by examining erythrocyte samples obtained during the first month of life from premature infants, born at 23 0/7 – 28 6/7 weeks gestational age, who were enrolled at our center in the Prematurity and Respiratory Outcomes Program (PROP). Using liquid chromatography‐mass spectrometry (LC‐MS), we report the novel finding of in vivo and in vitro glutathionylation of γG and γA subunits of Hgb F. Through tandem mass spectrometry (nanoLC‐MS/MS), we confirmed the adduction site as the Cys‐γ94 residue and through high‐resolution mass spectrometry determined that the modification occurs in both γ subunits. We also identified glutathionylation of the β subunit of Hgb A in our patient samples; we did not find modified α subunits of Hgb A or F. In conclusion, we are the first to report that glutathionylation of γG and γA of Hgb F occurs in premature infants. Additional studies of this post‐translational modification are needed to determine its physiologic impact on Hgb F function and if sG‐Hgb is a biomarker for clinical morbidities associated with oxidative stress in premature infants. Copyright © 2014 John Wiley & Sons, Ltd.