OBJECTIVES:A cure for cancer is out of reach for most patients due to chemoresistance. Cancer-associated fibroblasts (CAFs) play a vital role in cancer chemoresistance, but detailed understanding of the process particularly in chemoresistant lung cancer is lacking. In this study, we investigated programmed death-ligand 1 (PDL-1) as a potential biomarker for CAF-induced chemoresistance and evaluated its role and the underlying mechanisms of chemoresistance in non-small cell lung cancer (NSCLC).MATERIALS AND METHODS:A systemic search of gene expression profiles of multiple tissues in NSCLC was carried out to determine the expression intensities of traditional fibroblast biomarkers and CAF-secreted protumorigenic cytokines. PDL-1 expression in CAFs was analyzed by ELISA, Western blotting, and flow cytometry. Human cytokine array was used to identify specific cytokines secreted from CAFs. Role of PDL-1 in NSCLC chemoresistance was assessed using CRISPR/Cas9 knockdown and various functional assays including MTT, cell invasion, sphere formation, and cell apoptosis. In vivo experiments were conducted using a co-implantation xenograft mouse model with live cell imaging and immunohistochemistry.RESULTS:We demonstrated that chemotherapy-stimulated CAFs promoted tumorigenic and stem cell-like properties of NSCLC cells, which contribute to their chemoresistance. Subsequently, we revealed that PDL-1 expression is upregulated in chemotherapy-treated CAFs and is associated with poor prognosis. Silencing PDL-1 expression suppressed CAFs' ability to promote stem cell-like properties and invasiveness of lung cancer cells, favoring chemoresistance. Mechanistically, an upregulation of PDL-1 in chemotherapy-treated CAFs led to an increase in hepatocyte growth factor (HGF) secretion, which stimulates cancer progression, cell invasion, and stemness of lung cancer cells, while inhibiting apoptosis.CONCLUSION:Our results show that PDL-1-positive CAFs modulate stem cell-like properties of NSCLC cells by secreting elevated HGF, thereby promoting chemoresistance. Our finding supports PDL-1 in CAFs as a chemotherapy response biomarker and as a drug delivery and therapeutic target for chemoresistant NSCLC.

To design, implement, and assess an integrated virtual escape room that integrates pharmaceutical sciences courses (pharmacokinetics, biopharmaceutics, principles of drug action, medicinal chemistry, and pharmacogenomics) at the end of the semester to improve students' interconnectivity among disciplines.

Spirochaetes are bacteria responsible for several serious diseases, including Lyme disease (Borrelia burgdorferi), syphilis (Treponema pallidum) and leptospirosis (Leptospira interrogans), and contribute to periodontal diseases (Treponema denticola)1. These spirochaetes employ an unusual form of flagella-based motility necessary for pathogenicity; indeed, spirochaete flagella (periplasmic flagella) reside and rotate within the periplasmic space2–11. The universal joint or hook that links the rotary motor to the filament is composed of ∼120–130 FlgE proteins, which in spirochaetes form an unusually stable, high-molecular-weight complex9,12–17. In other bacteria, the hook can be readily dissociated by treatments such as heat18. In contrast, spirochaete hooks are resistant to these treatments, and several lines of evidence indicate that the high-molecular-weight complex is the consequence of covalent crosslinking12,13,17. Here, we show that T. denticola FlgE self-catalyses an interpeptide crosslinking reaction between conserved lysine and cysteine, resulting in the formation of an unusual lysinoalanine adduct that polymerizes the hook subunits. Lysinoalanine crosslinks are not needed for flagellar assembly, but they are required for cell motility and hence infection. The self-catalytic nature of FlgE crosslinking has important implications for protein engineering, and its sensitivity to chemical inhibitors provides a new avenue for the development of antimicrobials targeting spirochaetes. Spirochaete flagella hooks require a lysinoalanine covalent crosslink for motility.

Hydrogen peroxide is a key mediator of oxidative stress known to be important in various cellular processes, including apoptosis. B-cell lymphoma-2 (Bcl-2) is an oxidative stress-responsive protein and a key regulator of apoptosis; however, the underlying mechanisms of oxidative regulation of Bcl-2 are not well understood. The present study investigates the direct effect of H2O2 on Bcl-2 cysteine oxidation as a potential mechanism of apoptosis regulation. Exposure of human lung epithelial cells to H2O2 induces apoptosis concomitant with cysteine oxidation and down-regulation of Bcl-2. Inhibition of Bcl-2 oxidation by antioxidants or by site-directed mutagenesis of Bcl-2 at Cys-158 and Cys-229 abrogates the effects of H2O2 on Bcl-2 and apoptosis. Immunoprecipitation and confocal microscopic studies show that Bcl-2 interacts with mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2 [ERK1/2]) to suppress apoptosis and that this interaction is modulated by cysteine oxidation of Bcl-2. The H2O2-induced Bcl-2 cysteine oxidation interferes with Bcl-2 and ERK1/2 interaction. Mutation of the cysteine residues inhibits the disruption of Bcl-2-ERK complex, as well as the induction of apoptosis by H2O2. Taken together, these results demonstrate the critical role of Bcl-2 cysteine oxidation in the regulation of apoptosis through ERK signaling. This new finding reveals crucial redox regulatory mechanisms that control the antiapoptotic function of Bcl-2.

Objective. Chemoradiation is the mainstay of therapy for advanced cervical cancer, with the most effective treatment regimens involving combinations of radiosensitizing agents. However, administration of radiosensitizing chemotherapeutics concurrently with pelvic radiation is not without side effects. The aim of this study was to examine the utility of localized drug delivery as a means of improving drug targeting of radiosensitizing chemotherapeutics to the cervix while limiting systemic toxicities.Methods. An initial proof-of-concept study was performed in 14 healthy women following local administration of diazepam utilizing a novel cervical delivery device (CeiviPrep (TM)). Uterine vein and peripheral blood samples were collected and diazepam was measured using a GC-MS method. In the follow-up study, gemcitabine was applied to the cervix in 17 women undergoing hysterectomy for various gynecological malignancies. Cervical tissue, uterine vein blood samples, and peripheral plasma were collected, and gemcitabine and its deaminated metabolite 2',2'-difluorodeoxyuridine (dFdU) were measured using HPLC-UV and LC/MS methods.Results. Targeted delivery of diazepam to the cervix was consistent with parent drug detectable in the uterine vein of 13 of 14 women. In the second study, pharmacologically relevant concentrations of gemcitabine (0.01-6.6 nmol/g tissue) were detected in the cervical tissue of 11 of 16 available specimens with dFdU measureable in 15 samples (0.04-8.8 nmol/g tissue). Neither gemcitabine nor its metabolites were detected in the peripheral plasma of any subject.Conclusions. Localized drug delivery to the cervix is possible and may be useful in limiting toxicity associated with intravenous administration of chemotherapeutics for radiosensitization. (C) 2012 Elsevier Inc. All rights reserved.

The Phase 2 drug metabolism of busulfan yields a glutathione conjugate that undergoes a β-elimination reaction. The elimination product is an electrophilic metabolite that is a dehydroalanine-containing tripeptide, γ-glutamyldehydroalanylglycine (EdAG). In the process, glutathione lacks thiol-related redox properties and gains a radical scavenging dehydroalanine group. EdAG scavenged hydroxyl radical generated in the Fenton reaction in a concentration-dependent manner was monitored by electron paramagnetic resonance (EPR) spectroscopy. The apparent rate of hydroxyl radical scavenging was in the same range as published values for known antioxidants, including N-acyl dehydroalanines. A captodatively stabilized carbon-centered radical intermediate was spin trapped in the reaction of EdAG with hydroxyl radical. The proposed structure of a stable product in the Fenton reaction with EdAG was consistent with that of a γ-glutamylserylglycyl dimer. Observation of the hydroxyl trapping properties of EdAG suggests that the busulfan metabolite EdAG may contribute to or mitigate redox-related cytotoxicity associated with the therapeutic use of busulfan, and reaffirms indicators that support a role in free radical biology for dehydroalanine-containing peptides and proteins.

A novel class of N-substituted tetrahydropyridine derivatives was found to have multiple kinetic mechanisms of monoamine oxidase A inhibition. Eleven structurally similar tetrahydropyridine derivatives were synthesized and evaluated as inhibitors of MAO-A and MAO-B. The most potent MAO-A inhibitor in the series, 2,4-dichlorophenoxypropyl analog 12, displayed time-dependent mixed noncompetitive inhibition. The inhibition was reversed by dialysis, indicating reversible enzyme inhibition. Evidence that the slow-binding inhibition of MAO-A with 12 involves a covalent bond was gained from stabilizing a covalent reversible intermediate product by reduction with sodium borohydride. The reduced enzyme complex was not reversible by dialysis. The results are consistent with slowly reversible, mechanism-based inhibition. Two tetrahydropyridine analogs that selectively inhibited MAO-A were characterized by kinetic mechanisms differing from the kinetic mechanism of 12. As reversible inhibitors of MAO-A, tetrahydropyridine analogs are at low risk of having an adverse effect of tyramine-induced hypertension.

Abstract Flavin-dependent histone demethylases play an important role in epigenetic gene regulation. The most studied enzyme, LSD1 (KDM1), catalyzes the oxidative demethylation of methylated Lys4 of histone H3. Known LSD1 inhibitors also inhibit other flavin-dependent enzymes, in particular monoamine oxidase (MAO). Therefore, selective inhibition of LSD1 is a significant drug discovery target. We designed, synthesized and evaluated in vitro a series of eleven N-substituted tetrahydropyridines as inhibitors of flavin-dependent enzymes. Complete chemical structures of the compounds used will be disclosed at the time of presentation. The inhibition of LSD1 was measured in rat glioma C6 cells. Briefly, cells grown in serum-free medium were treated with the inhibitors for 24 h, and the level of Lys4 dimethylated form of histone H3 was measured by immunoblot. MAO A activity was determined using human recombinant enzyme with kynuramine as substrate. The most selective among the inhibitors showed low nanomolar potency toward LSD1 and weak inhibition of MAO A. The observed high potency and substrate selectivity justifies further evaluation of the drug discovery potential of the tetrahydropyridine scaffold. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3255. doi:10.1158/1538-7445.AM2011-3255

We investigated the effects of different carboxylic ester spacers of mono-PEGylated curcumin conjugates on chemical stability, release characteristics, and anticancer activity. Three novel conjugates were synthesized with succinic acid, glutaric acid, and methylcarboxylic acid as the respective spacers between curcumin and monomethoxy polyethylene glycol of molecular weight 2000 (mPEG(2000) ): mPEG(2000) -succinyl-curcumin (PSC), mPEG(2000) -glutaryl-curcumin (PGC), and mPEG(2000) -methylcarboxyl-curcumin (PMC), respectively. Hydrolysis of all conjugates in buffer and human plasma followed pseudo first-order kinetics. In phosphate buffer, the overall degradation rate constant and half-life values indicated an order of stability of PGC > PSC > PMC > curcumin. In human plasma, more than 90% of curcumin was released from the esters after incubation for 0.25, 1.5, and 2 h, respectively. All conjugates exhibited cytotoxicity against four human cancer cell lines: Caco-2 (colon), KB (oral cavity), MCF7 (breast), and NCI-H187 (lung) with half maximal inhibitory concentration (IC(50) ) values in the range of 1-6 µM, similar to that observed for curcumin itself. Our results suggest that mono-PEGylation of curcumin produces prodrugs that are stable in buffer at physiological pH, release curcumin readily in human plasma, and show anticancer activity.

Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess a good leaving group in the β-position. The end products are aminoacrylate and a sulfur-containing fragment. The aminoacrylate tautomerizes and hydrolyzes to pyruvate and ammonia. The mammalian cysteine S-conjugate β-lyases thus far identified are enzymes involved in amino acid metabolism that catalyze β-lyase reactions as non-physiological side reactions. Most are aminotransferases. In some cases the lyase is inactivated by reaction products. The cysteine S-conjugate β-lyases are of much interest to toxicologists because they play an important key role in the bioactivation (toxication) of halogenated alkenes, some of which are produced on an industrial scale and are environmental contaminants. The cysteine S-conjugate β-lyases have been reviewed in this journal previously (Cooper and Pinto in Amino Acids 30:1–15, 2006). Here, we focus on more recent findings regarding: (1) the identification of enzymes associated with high-M r cysteine S-conjugate β-lyases in the cytosolic and mitochondrial fractions of rat liver and kidney; (2) the mechanism of syncatalytic inactivation of rat liver mitochondrial aspartate aminotransferase by the nephrotoxic β-lyase substrate S-(1,1,2,2-tetrafluoroethyl)-l-cysteine (the cysteine S-conjugate of tetrafluoroethylene); (3) toxicant channeling of reactive fragments from the active site of mitochondrial aspartate aminotransferase to susceptible proteins in the mitochondria; (4) the involvement of cysteine S-conjugate β-lyases in the metabolism/bioactivation of drugs and natural products; and (5) the role of cysteine S-conjugate β-lyases in the metabolism of selenocysteine Se-conjugates. This review emphasizes the fact that the cysteine S-conjugate β-lyases are biologically more important than hitherto appreciated.

3,4-Dichloropropionanilide (DCPA), or propanil, a post-emergent herbicide used on rice and wheat crops in the United States, is immunotoxic in vivo and in vitro. Although it has been documented that DCPA exerts differential effects on specific immune cell types and is toxic to the liver, the way in which DCPA modulates intracellular functions leading to these effects is less understood. In this study, Jurkat T cells and hepatocytes from C57Bl/6 mice were exposed to 100 μM DCPA for 1.5 h. Following incubation, subcellular fractions of each cell type were isolated. DCPA, when present, was removed from each cell fraction by liquid–liquid extraction. The extraction product was then analyzed for the presence of DCPA using liquid chromatography–tandem mass spectrometry (LC-MS/MS). The cellular uptake of DCPA was monitored by detection of the molecular ion and product ion of DCPA. The analyses demonstrate that DCPA, a lipophilic compound, localizes primarily in the cytosol of T cells and hepatocytes. These results indicate that DCPA is able to cross the plasma membrane and is accessible to intracellular immunomodulatory effectors.

Isobaric product ions cannot be differentiated by exact mass determinations, although in some cases deuterium labeling can provide useful structural information for identifying isobaric ions. Proposed fragmentation pathways of fentanyl were investigated by electrospray ionization ion trap mass spectrometry coupled with deuterium labeling experiments and spectra of regiospecific deuterium labeled analogs. The major product ion of fentanyl under tandem mass spectrometry (MS/MS) conditions (m/z 188) was accounted for by a neutral loss of N-phenylpropanamide. 1-(2-Phenylethyl)1,2,3,6-tetrahydropyridine (1) was proposed as the structure of the product ion. However, further fragmentation (MS3) of the fentanyl m/z 188 ion gave product ions that were different from the product ion in the MS/MS fragmentation of synthesized 1, suggesting that the m/z 188 product ion from fentanyl includes an isobaric structure different from the structure of 1. MS/MS fragmentation of fentanyl in deuterium oxide moved one of the isobars to 1 Da higher mass, and left the other isobar unchanged in mass. Multistage mass spectral data from deuterium-labeled proposed isobaric structures provided support for two fragmentation pathways. The results illustrate the utility of multistage mass spectrometry and deuterium labeling in structural assignment of isobaric product ions. Copyright (C) 2010 John Wiley & Sons, Ltd.

Fentanyl is a potent opioid analgesic that is increasingly becoming a choice drug of abuse. Fentanyl transdermal patches (FTPs) are easily obtained and consumed by smoking the reservoir gel and/or the whole patch. This allows for an increased bioavailability when inhaled. A method using analytical pyrolysis was developed to identify possible biomarkers associated with smoked fentanyl and FTPs. Pyrolysis was carried out under anaerobic and aerobic conditions using helium and air coupled to a gas chromatograph-mass spectrometer. The presence of a trap enhanced recovery and afforded a positive identification of pyrolytic products. Anaerobic and aerobic pyrolysis of fentanyl and FTPs consistently yielded propionanilide as the major pyrolytic product along with pyridine and previously reported metabolites (norfentanyl and despropionyl fentanyl). Analysis of fentanyl resulted in chlorine-containing compounds, presumably formed from the HCl salt of fentanyl. Analysis of FTPs showed significant polymeric and hydrocarbon compounds and products likely derived from the gel matrix. Fentanyl in the FTPs was in the citrate salt form; therefore, the chlorine-containing pyrolytic products obtained with the neat drug were not observed. Based on this application, it may be possible to identify what salt form of the drug was smoked based on pyrolytic products and to target distinguishing metabolic products for future research.

Diabetic cardiomyopathy is the leading cause of heart failure among diabetic patients, and mitochondrial dysfunction has been implicated as an underlying cause in the pathogenesis. Cardiac mitochondria consist of two spatially, functionally, and morphologically distinct subpopulations, termed subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). SSM are situated beneath the plasma membrane, whereas IFM are embedded between myofibrils. The goal of this study was to determine whether spatially distinct cardiac mitochondrial subpopulations respond differently to a diabetic phenotype. Swiss-Webster mice were subjected to intraperitoneal injections of streptozotocin or citrate saline vehicle. Five weeks after injections, diabetic hearts displayed decreased rates of contraction, relaxation, and left ventricular developed pressures ( P < 0.05 for all three). Both mitochondrial size (forward scatter, P < 0.01) and complexity (side scatter, P < 0.01) were decreased in diabetic IFM but not diabetic SSM. Electron transport chain complex II respiration was decreased in diabetic SSM ( P < 0.05) and diabetic IFM ( P < 0.01), with the decrease being greater in IFM. Furthermore, IFM complex I respiration and complex III activity were decreased with diabetes ( P < 0.01) but were unchanged in SSM. Superoxide production was increased only in diabetic IFM ( P < 0.01). Oxidative damage to proteins and lipids, indexed through nitrotyrosine residues and lipid peroxidation, were higher in diabetic IFM ( P < 0.05 and P < 0.01, respectively). The mitochondria-specific phospholipid cardiolipin was decreased in diabetic IFM ( P < 0.01) but not SSM. These results indicate that diabetes mellitus imposes a greater stress on the IFM subpopulation, which is associated, in part, with increased superoxide generation and oxidative damage, resulting in morphological and functional abnormalities that may contribute to the pathogenesis of diabetic cardiomyopathy.

This case study investigates trans-phenylpropene as a potential marker for smoked methamphetamine. The decedent, a 31-year-old male, was found with paraphernalia that indicated that he may have been smoking abused drugs prior to death. Methamphetamine and cocaine were detected in the residue remaining in the paraphernalia. Markers of thermal degradation of methamphetamine and cocaine were also detected in the paraphernalia. Gas chromatography-mass spectrometry (GC-MS) analysis detected trans-phenylpropene as a marker of smoked methamphetamine and anhydroecgonine methyl ester as a marker of smoked cocaine. Both trans-phenylpropene and anydroecgonine methyl ester were detected in the urine of the decedent, connecting the link between the paraphernalia for smoking and the ingestion of the pyrolysis products of methamphetamine and cocaine. Several other drugs of abuse were identified either in blood and urine or in hexane extracts of the paraphernalia, including phenylacetone, fentanyl, norfentanyl, amphetamine, ecgonine methyl ester, oxycodone, acetaminophen, chlorpheniramine, and caffeine. Using a pyrolysis GC-MS, the characteristic pyrolytic products of cocaine HCl, methamphetamine HCl, and combinations of the two were evaluated and the results showed that combining the drugs in a single run did not alter the pyrolysis pattern. The detection of trans-phenylpropene in both biological specimens and in paraphernalia is the first example of this analyte being applied as evidence of smoked methamphetamine.

The present work documents the first example of an enzyme-catalyzed beta-elimination of a thioether from a sulfonium cysteine S-conjugate. beta-(S-Tetrahydrothiophenium)-L-alanine (THT-A) is the cysteine S-conjugate of busulfan. THT-A slowly undergoes a nonenzymatic beta-elimination reaction at pH 7.4 and 37 degrees C to yield tetrahydrothiophene, pyruvate, and ammonia. This reaction is accelerated by 1) rat liver, kidney, and brain homogenates, 2) isolated rat liver mitochondria, and 3) pyridoxal 5'-phosphate (PLP). A PLP-dependent enzyme in rat liver cytosol that catalyzes a beta-lyase reaction with THT-A was identified as cystathionine gamma-lyase. This unusual drug metabolism pathway represents an alternate route for intermediates in the mercapturate pathway.

Elimination of hydrogen sulfide from glutathione (GSH) converts a well known cellular nucleophile to an electrophilic species, gamma-glutamyldehydroalanylglycine (EdAG). We have found that a sulfonium metabolite formed from GSH and busulfan undergoes a facile beta-elimination reaction to give EdAG, which is an alpha,beta-unsaturated dehydroalanyl analog of GSH. EdAG was identified as a metabolite of busulfan in a human liver cytosol fraction. EdAG condenses with GSH in a Michael addition reaction to produce a lanthionine thioether [(2-amino-5-[[3-[2-[[4-amino-5-hydroxy-5-oxopentanoyl]amino]-3-(carboxymethylamino)-3- oxopropyl]sulfanyl-1-(carboxymethylamino)-1-oxopropan-2-yl]amino]-5-oxopentanoicacid); GSG], which is a nonreducible analog of glutathione disulfide. EdAG was less cytotoxic than busulfan to C6 rat glioma cells. GSH and EdAG were equally effective in displacing a glutathione S-transferase (GST) isozyme (human GSTA1-1) from a GSH-agarose column. The finding of an electrophilic metabolite of GSH suggests that alteration of cellular GSH concentrations, irreversible nonreducible glutathionylation of proteins, and interference with GST function may contribute to the toxicity of busulfan.

Five cases of confirmed multiple-drug overdose were previously screened and quantified by the West Virginia Office of the Chief Medical Examiner; 26 different drugs and metabolites were identified and quantified in blood at > or = 10 ng/mL. In this study, whole blood from those five case samples was analyzed by a direct injection multi-stage mass spectrometric (MSn) method to confirm the identity of 26 analytes at or above 10 ng/mL using 16 different deuterium-labeled internal standards. Samples were spiked with internal standards, precipitated with acetonitrile, and centrifuged. Samples were further diluted with either 0.1% formic acid or 0.1% ammonium hydroxide in methanol prior to injection into an electrospray ionization ion trap mass spectrometer (MS). Ions were monitored as MS-MS or MS3 product ions. In all cases, analysis by MS-MS confirmed the presence of the drugs and metabolites when the internal standards were detected. Detection of characteristic MS3 ions was used for further confirmation of the presence of parent drugs in all but three instances. Total analysis time was less than 1 h. Although only useful for qualitative or confirmatory purposes, this direct injection MSn method provides a simple and rapid confirmation of multiple drugs that have been previously identified and quantified by gas chromatographic-MS or liquid chromatographic-MS analytical methods.