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Biochemical Pharmacology (v.83, #6)

Editorial Board (pp. iii).

Role of the endogenous elastase inhibitor, elafin, in cardiovascular injury by Shirjel R. Alam; David E. Newby; Peter A. Henriksen (pp. 695-704).

Pre-clinical studies indicate the therapeutic potential of the human neutrophil elastase inhibitor “elafin” in inflammatory cardiovascular pathology.Neutrophils and neutrophil-derived elastases play a major role in the regulation of vascular injury and inflammation, such as ischemia–reperfusion injury. Elafin is an endogenous inhibitor of neutrophil-derived elastases with numerous anti-inflammatory functions that include modulation of inflammatory cytokine release as well as innate and adaptive immunity. It is produced by epithelial tissues including the skin and respiratory system that have adapted to respond to the microbial and chemical insults that lead to inflammation. The production of peptides like elafin with multi-faceted anti-inflammatory activity is an important part of this adaptation. Although not directly expressed within the cardiovascular system itself, pre-clinical studies have suggested therapeutic benefit of elafin in cardiovascular disease.The aim of this review is to highlight the role of neutrophil-derived elastases in vascular inflammation and injury. We will discuss the beneficial effects of elafin inhibition of neutrophil elastase and its extended anti-inflammatory activity in pre-clinical models of inflammatory vascular injury.

Keywords: Abbreviations; HNE; human neutrophil elastase; PAR; protease activated receptor; MMP; matrix metalloproteinase; TIMP; tissue inhibitors of metalloproteases; PAI-1; plasminogen activator inhibitor type-1; TFPI; tissue factor pathway inhibitor; α1-PI; α1-antitrypsin; SLPI; secretory leucocyte protease inhibitor; WAP; whey acidic protein; LPS; lipopolysaccharide; EMPIRE; elafin myocardial protection from ischemia reperfusionCardiovascular; Elafin; Elastase; Proteinase-3; Neutrophil

Cyclic AMP response element-binding protein (CREB) phosphorylation: A mechanistic marker in the development of memory enhancing Alzheimer's disease therapeutics by R. Scott Bitner (pp. 705-714).

CREB-mediated transcription can be initiated by membrane receptor stimulation and subsequent activation of intracellular pathways to the cell nucleus, and has been described as a molecular switch required for learning and memory. While CREB dimers are thought to be constitutively bound to response elements on DNA under basal conditions, it is CREB phosphorylation that is believed to be responsible for transcriptional activation leading to gene products such as BDNF that play a key role in synaptic plasticity and cognitive function. Conversely, preclinical and clinical findings now suggest that impaired CREB phosphorylation may be a pathological component in neurodegenerative disorders, in particular Alzheimer's disease (AD). In this regard, pharmacological-induced CREB phosphorylation in brain regions associated with cognition, i.e. cortex and hippocampus may represent a mechanistic basis for the development of novel AD therapeutics. The purpose of this commentary is to describe an experimental strategy to biochemically characterize the pharmacological induction of CREB phosphorylation as a mechanistic marker across different pharmacological classes of compounds for the potential treatment of AD that include: α7 nicotinic agonists, H3 antagonists and 11β HSD1 inhibitors.

Keywords: CREB phosphorylation; Alzheimer's disease; α7 nAChR agonist; H3 antagonist; HSD1 inhibitor

Hydrogen sulfide-releasing NSAIDs inhibit the growth of human cancer cells: A general property and evidence of a tissue type-independent effect by Mitali Chattopadhyay; Ravinder Kodela; Niharika Nath; Yosef M. Dastagirzada; Carlos A. Velázquez-Martínez; Daniel Boring; Khosrow Kashfi (pp. 715-722).

H₂S-releasing-NSAIDs are more potent in inhibiting the growth of colon, breast, pancreas, lung, prostate, and leukemia cancer cell lines, than their parent NSAIDs.Hydrogen sulfide-releasing non-steroidal anti-inflammatory drugs (HS-NSAIDs) are an emerging novel class of compounds with significant anti-inflammatory properties. They consist of a traditional NSAID to which an H₂S-releasing moiety is covalently attached. We examined the effects of four different HS-NSAIDs on the growth properties of eleven different human cancer cell lines of six different tissue origins. Human colon, breast, pancreatic, prostate, lung, and leukemia cancer cell lines were treated with HS-aspirin, -sulindac, -iburofen, -naproxen, and their traditional counterparts. HS-NSAIDs inhibited the growth of all cancer cell lines studied, with potencies of 28- to >3000-fold greater than that of their traditional counterparts. HS-aspirin (HS-ASA) was consistently the most potent. HS-NSAIDs inhibited cell proliferation, induced apoptosis, and caused G₀/G₁ cell cycle block. Metabolism of HS-ASA by colon cells showed that the acetyl group of ASA was hydrolyzed rapidly, followed by hydrolysis of the ester bond linking the salicylate anion to the H₂S releasing moiety, producing salicylic acid and ADT-OH from which H₂S is released. In reconstitution studies, ASA and ADT-OH were individually less active than the intact HS-ASA towards cell growth inhibition. Additionally, the combination of these two components representing a fairly close approximation to the intact HS-ASA, was 95-fold less active than the intact HS-ASA for growth inhibition. Taken together, these results demonstrate that HS-NSAIDs have potential anti-growth activity against a wide variety of human cancer cells.

Keywords: Hydrogen sulfide; NSAIDs; Cancer prevention; COX-independent

Hydrogen sulfide-releasing aspirin suppresses NF-κB signaling in estrogen receptor negative breast cancer cells in vitro and in vivo by Mitali Chattopadhyay; Ravinder Kodela; Niharika Nath; Arpine Barsegian; Daniel Boring; Khosrow Kashfi (pp. 723-732).

H₂S-releasing aspirin reduces tumor mass, inhibits cell proliferation, induces apoptosis and decreases NF-κB levels in estrogen receptor negative MDA-MB-231 human tumor xenografts in mice.Hormone-dependent estrogen receptor positive (ER+) breast cancers generally respond well to anti-estrogen therapy. Unfortunately, hormone-independent estrogen receptor negative (ER−) breast cancers are aggressive, respond poorly to current treatments and have a poor prognosis. New approaches and targets are needed for the prevention and treatment of ER− breast cancer. The NF-κB signaling pathway is strongly implicated in ER− tumor genesis, constituting a possible target for treatment. Hydrogen sulfide-releasing aspirin (HS-ASA), a novel and safer derivative of aspirin, has shown promise as an anti-cancer agent. We examined the growth inhibitory effect of HS-ASA via alterations in cell proliferation, cell cycle phase transitions, and apoptosis, using MDA-MB-231 cells as a model of triple negative breast cancer. Tumor xenografts in mice, representing human ER− breast cancer, were evaluated for reduction in tumor size, followed by immunohistochemical analysis for proliferation, apoptosis and expression of NF-κB. HS-ASA suppressed the growth of MDA-MB-231 cells by induction of G₀/G₁ arrest and apoptosis, down-regulation of NF-κB, reduction of thioredoxin reductase activity, and increased levels reactive oxygen species. Tumor xenografts in mice, were significantly reduced in volume and mass by HS-ASA treatment. The decrease in tumor mass was associated with inhibition of cell proliferation, induction of apoptosis and decrease in NF-κB levels in vivo. HS-ASA has anti-cancer potential against ER− breast cancer and merits further study.

Keywords: Hydrogen sulfide; Estrogen receptor negative breast cancer; NF-κB; Thioredoxin reductase; Chemoprevention

Hydrogen sulfide-releasing aspirin modulates xenobiotic metabolizing enzymes in vitro and in vivo by Mitali Chattopadhyay; Ravinder Kodela; Niharika Nath; Cherease R. Street; Carlos A. Velázquez-Martínez; Daniel Boring; Khosrow Kashfi (pp. 733-740).

The balance between phase-I carcinogen-activating and phase-II detoxifying xenobiotic metabolizing enzymes is critical to determining an individual's risk for cancer. We evaluated the effect of Hydrogen sulfide-releasing aspirin (HS-ASA) on xenobiotic metabolizing enzymes in HT-29 human colon and Hepa 1c1c7 mouse liver adenocarcinoma cells and in Wistar rats. HS-ASA inhibited the growth of HT-29 and Hepa 1c1c7 cells, with an IC₅₀ of 3.2±0.3μM and 4.2±0.4μM, respectively. The IC₅₀ for ASA in both cell lines was greater than 5000μM at 24h. In these cell lines, HS-ASA caused a dose-dependent increase in activity and expression of the phase-II enzymes glutathione S-transferase (GST) and NAD(P)H:quinoneoxireductase (NQO1). It also caused an increase in UDP-glucuronosyltransferase (UGT) expression. The levels of CYP 1A1 a phase-I enzyme was increased by HS-ASA in both cell lines. Pretreatment of cells with NaF, an esterase inhibitor, abrogated the HS-ASA-mediated increases in NQO1 enzyme activity. HS-ASA increased the protein levels of the transcription factor Nrf2, which is a regulator of the phase-II enzymes. In vivo, HS-ASA at 100mg/kg/day had no effect on rat's weights; it induced a 3.4-fold and 1.4-fold increase in hepatic GST and NQO1 enzyme activities, respectively. GST and NQO1 protein levels were also increased. In contrast to that in cultured cells, CYP 1A1 protein levels were not altered in vivo. Therefore, HS-ASA induces phase-II enzymes, at least in part, through the action of H₂S and by modulating Nrf2; these effects may be part of its mechanism of action against carcinogenesis.

Keywords: Xenobiotic metabolizing enzymes; GST; NOQ1; UGT; CYP 1A1; Hydrogen sulfide; NSAIDs

Expression and regulation of RAD51 mediate cellular responses to chemotherapeutics by Zhengguan Yang; Alan S. Waldman; Michael D. Wyatt (pp. 741-746).

There is evidence that RAD51 expression associates with resistance to commonly used chemotherapeutics. Our previous work demonstrated that inhibitors of thymidylate synthase (TS) induced RAD51-dependent homologous recombination (HR), and depleting the RAD51 recombinase sensitized cells to TS inhibitors. In this study, the consequences of RAD51 over-expression were studied. Over-expression of wild-type RAD51 (∼6-fold above endogenous RAD51) conferred resistance to TS inhibitors. In contrast, over-expression of a mutant RAD51 (T309A) that is incapable of being phosphorylated rendered cells more chemosensitive. Moreover, over-expression of the T309A mutant acted in a dominant negative manner over endogenous RAD51 by causing the reduced localization of RAD51 foci following treatment with TS inhibitors. To measure the effect of mutant RAD51 on the cellular response to other DNA damaging chemotherapeutics, the topoisomerase poison etoposide was utilized. Cells over-expressing wild-type RAD51 showed reduced DNA strand breaks, while cells over-expressing the mutant RAD51 showed more than twice as many strand breaks, suggesting that the mutant RAD51 was actively inhibiting strand break resolution. To directly demonstrate an effect on HR, wild-type RAD51 and T309A mutant RAD51 were transiently expressed in HeLa cells that contained an HR reporter construct. HR events provoked by DNA breaks induced by the I-SceI endonuclease increased in cells expressing wild-type RAD51 and decreased in cells expressing the T309A mutant. Collectively, the data suggest that interference with the activation of RAD51-mediated HR represents a potentially useful anticancer target for combination therapies.

Keywords: Abbreviations; HR; homologous recombination; DSBs; DNA double strand breaks; TS; thymidylate synthase; RTX; raltitrexed (Tomudex™); 5-FU; 5-fluorouracil; FdUrd; fluorodeoxyuridine; UCN-01; 7-Hydroxy-staurosporine; GFP; green fluorescent proteinThymidylate synthase; Homologous recombination; Replication protein A; RAD51; Etoposide

Role of autophagy in chemoresistance: Regulation of the ATM-mediated DNA-damage signaling pathway through activation of DNA–PKcs and PARP-1 by Jung-Hoon Yoon; Sang-Gun Ahn; Byung-Hoon Lee; Sung-Hoo Jung; Seon-Hee Oh (pp. 747-757).

DNA-damage signaling pathway after genotoxic stress. Capsaicin-induced autophagy regulates the DNA-repair signaling pathway, thereby prolonging cancer cell survival.Capsaicin treatment was previously reported to reduce the sensitivity of breast cancer cells, but not normal MCF10A cells, to apoptosis. The present study shows that autophagy is involved in cellular resistance to genotoxic stress, through DNA repair. Capsaicin treatment of MCF-7 cells induced S-phase arrest and autophagy through the AMPKα–mTOR signaling pathway and the accumulation of p53 in the nucleus and cytosol, including a change in mitochondrial membrane potential. Capsaicin treatment also activated δ-H2AX, ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase catalytic subunit (DNA–PKcs), and poly(ADP-ribose) polymerase (PARP)-1. Genetic or pharmacological disruption of autophagy attenuated capsaicin-induced phospho-ATM and phospho-DNA–PKcs and enhanced apoptotic cell death. ATM inhibitors, including Ku55933 and caffeine, and the genetic or pharmacological inhibition of p53 prevented capsaicin-induced DNA–PKcs phosphorylation and stimulated PARP-1 cleavage, but had no effect on microtubule-associated protein light chain 3 (LC3)-II levels. Ly294002, a DNA–PKcs inhibitor, boosted the capsaicin-induced cleavage of PARP-1. In M059K cells, but not M059J cells, capsaicin induced ATM and DNA–PKcs phosphorylation, p53 accumulation, and the stimulation of LC3II production, all of which were attenuated by knockdown of the autophagy-related gene atg5. Ku55933 attenuated capsaicin-induced phospho-DNA–PKcs, but not LC3II, in M059K cells. In human breast tumors, but not in normal tissues, AMPKα, ATM, DNA–PKcs, and PARP-1 were activated and LC3II was induced. The induction of autophagy by genotoxic stress likely contributes to the sustained survival of breast cancer cells through DNA repair regulated by ATM–mediated activation of DNA–PKcs and PARP-1.

Keywords: Abbreviations; ATM; ataxia telangiectasia mutated; DNA–PKcs; DNA-dependent protein kinase catalytic subunit; PARP-1; poly(ADP-ribose) polymerase; PAR; anti-poly(ADP-ribose); 3-MA; 3-methyladenine; 3-AB; 3-aminobenzamide; AMPKα; AMP-activated protein kinase-α; LC3; microtubule-associated protein 1 light chain 3; mTOR; mammalian target of rapamycin; siRNA; small interfering RNA; MTT; 3-(4,5-dimetylthiazole-2-yl)-2,5-diphenyltetrazolium bromideAutophagy; ATM; DNA–PKcs; PARP-1; DNA repair

Knockdown of HURP inhibits the proliferation of hepacellular carcinoma cells via downregulation of gankyrin and accumulation of p53 by Tzu-Ching Kuo; Pu-Yuan Chang; Shiu-Feng Huang; Chen-Kung Chou; Chuck C.-K. Chao (pp. 758-768).

We determined earlier that the hepatoma upregulated protein (HURP) is overexpressed in hepatocellular carcinoma (HCC), but the role of this protein during cancer development and progression remains unknown. Here, we observed that the overexpression of HURP in HEK293 cells promoted the ubiquitination of p53 and its degradation by the proteasome. In contrast, HURP knockdown using short-hairpin RNA reversed these effects. Knockdown of HURP promoted the accumulation of p53 in SK-Hep-1 cells (p53+/−), and these cells showed reduced proliferation, while the p53-mutant Mahlavu cells were not affected. HURP knockdown did not affect the proliferation of H1299 lung carcinoma cells and Hep3B HCC cells which lack p53. Knockdown of HURP also sensitized SK-Hep-1 cells to cisplatin. On the other hand, the expression of exogenous p53 in H1299 and Hep3B cells was decreased following overexpression of HURP, and these cells showed decreased sensitivity to cisplatin-induced apoptosis. Importantly, overexpression of HURP promoted the proliferation of HEK293 cells in an anchorage-independent manner, and inoculation of SK-Hep-1 cancer cells that expressed short-hairpin RNA to knockdown HURP resulted in smaller tumors in nude mice. Gankyrin, a positive regulator of the E3 ubiquitin ligase MDM2, was found to be upregulated following HURP expression, and gankyrin knockdown decreased the HURP-mediated downregulation of p53. Notably, we detected a positive correlation between elevated HURP and gankyrin protein levels in HCC patients ( r²=0.778; N=9). Taken together, these results indicate that HURP represents an oncogene that may play a role in HCC progression and chemoresistance.

Keywords: Hepatocellular carcinoma; HURP; Gankyrin; p53; Oncogenesis; Chemoresistance

Dovitinib sensitizes hepatocellular carcinoma cells to TRAIL and tigatuzumab, a novel anti-DR5 antibody, through SHP-1-dependent inhibition of STAT3 by Kuen-Feng Chen; Hui-Ling Chen; Chun-Yu Liu; Wei-Tien Tai; Kimihisa Ichikawa; Pei-Jer Chen; Ann-Lii Cheng (pp. 769-777).

Hepatocellular carcinoma (HCC) often displays resistance to recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Dovitinib, a multiple tyrosine kinase inhibitor, and tigatuzumab, a novel humanized anti-human death receptor 5 (DR5) agonistic antibody, are both under clinical investigations in HCC. Here, we report that dovitinib sensitizes resistant HCC cells to TRAIL- and tigatuzumab-induced apoptosis through inhibition of signal transducers and activators of transcription 3 (STAT3). Our data indicate that HCC cells showed significant resistance to TRAIL- and tigatuzumab-induced apoptosis. The combination of dovitinib and tigatuzumab restored the sensitivity of HCC cells to TRAIL- and tigatuzumab-induced apoptosis. Dovitinib down-regulated phospho-STAT3 (Tyr705) (p-STAT3) and subsequently reduced the protein levels of STAT3-regulated proteins, Mcl-1, survivin and cylcin D1, in TRAIL-treated HCC cells. Knockdown of STAT3 by RNA-interference overcame apoptotic resistance to TRAIL in HCC cells, and ectopic expression of STAT3 in HCC cells abolished the sensitizing effect of dovitinib on TRAIL-induced apoptosis. Importantly, silencing SHP-1 by RNA-interference reduced the effects of dovitinib and TRAIL on p-STAT3 and apoptosis, whereas co-treatment of TRAIL and dovitinib increased the activity of SHP-1. Moreover, in vivo the combination of tigatuzumab and dovitinib inhibited Huh-7 xenograft tumor growth. In conclusion, dovitinib sensitizes resistant HCC cells to TRAIL- and tigatuzumab-induced apoptosis through a novel machinery: SHP-1 dependent STAT3 inhibition.

Keywords: Abbreviations; HCC; hepatocellular carcinoma; TRAIL; tumor necrosis factor-related apoptosis-inducing ligand; STAT3; signal transducers and activators of transcription 3; FADD; fas associated protein with death domain; c-FLIP; cellular FLICE-inhibitory protein; PARP; polypolymerase; DMEM; Dulbecco's modified Eagle's medium; FBS; fetal bovine serumDovitinib; TRAIL; Tigatuzumab; STAT3; HCC

Orally bioavailable allosteric CCR8 antagonists inhibit dendritic cell, T cell and eosinophil migration by Stephen Connolly; Marco Skrinjar; Alexander Rosendahl (pp. 778-787).

The chemokine receptor CCR8 is associated with asthma. Herein, we describe that both mature and immature dendritic cells (DC) express CCR8, whereas only mature DC migrate towards CCL1. Moreover, transient LPS challenge significantly down-regulates CCR8 expression hence attenuating CCL1 chemotaxis.To inhibit CCR8 pathophysiology, we recently developed a novel series of small molecule CCR8 antagonists containing a diazaspiroundecane scaffold, which had micromolar potency. However, these first generation antagonists had high lipophilicity that endowed the compounds with poor physicochemical properties, and were thus not suitable for further development. By introducing polar bicyclic groups on the N-benzyl substituent and building in further polar interactions on the amide group we now show second generation diazospiroundecane antagonists with significantly improved overall properties. Potency is substantially improved from micromolar to nanomolar potency in CCR8 binding and inhibition of chemotaxis in human primary T cells, DC and in an eosinophil cell line. In addition to high potency, the most attractive antagonist, AZ084 showed excellent selectivity, high metabolic stability in vitro and an attractive in vivo PK profile with a long half-life in rat.Interestingly, in ligand saturation experiments and in wash-off experiments, CCL1 was shown to have two binding sites to CCR8 with K_d at 1.2/68pM respectively, and on-off rates of 0.004 and 0.0035/0.02pMmin, respectively. The lead antagonist, AZ084, appears to act as an allosteric inhibitor with a K_i at 0.9nM.Taken together, we herein report a novel oral allosteric CCR8 antagonist with predicted low once-daily dosing capable of potent inhibition of both human T cell and DC functions.

Keywords: Abbreviations; DC; dendritic cell; LPS; lipopolysaccharide; PK; pharmacokinetics; GPCR; G-protein-coupled receptor; hERG; human ether-à-go-go related gene; FEV1; forced expiratory volume 1; KO; knock-out; wt; wild-type; CYP; cytochrome P450; MEC; minimal effective concentration; DtM; dose-to-man; SPA; scintillation proximity assay; BSA; bovine serum albumin; HLM; human liver microsomesChemokine; Dendritic cell; Asthma; Receptor inhibitor; Mode of binding

A selective reversible azapeptide inhibitor of human neutrophil proteinase 3 derived from a high affinity FRET substrate by Christophe Epinette; Cécile Croix; Lucie Jaquillard; Sylvain Marchand-Adam; Christine Kellenberger; Gilles Lalmanach; Martine Cadene; Marie-Claude Viaud-Massuard; Francis Gauthier; Brice Korkmaz (pp. 788-796).

The biological functions of human neutrophil proteinase 3 (PR3) remain unclear because of its close structural resemblance to neutrophil elastase and its apparent functional redundancy with the latter. Thus, all natural inhibitors of PR3 preferentially target neutrophil elastase. We have designed a selective PR3 inhibitor based on the sequence of one of its specific, sensitive FRET substrates. This azapeptide, azapro-3, inhibits free PR3 in solution, PR3 bound to neutrophil membranes, and the PR3 found in crude lung secretions from patients with chronic inflammatory pulmonary diseases. But it does not inhibit significantly neutrophil elastase or cathepsin G. Unlike most of azapeptides, this inhibitor does not form a stable acyl–enzyme complex; it is a reversible competitive inhibitor with a K_i comparable to the K_m of the parent substrate. Low concentrations (60μM) of azapro-3 totally inhibited the PR3 secreted by triggered human neutrophils (200,000cells/100μL) and the PR3 in neutrophil homogenates and in lung secretions of patients with lung inflammation for hours. Azapro-3 also resisted proteolysis by all proteases contained in these samples for at least 2h.

Keywords: Abbreviations; PR3; proteinase 3; HNE; human neutrophil elastase; CG; cathepsin G; NSPs; neutrophil serine proteases; α1-PI; alpha-1-antitrypsin; Abz; ortho; -aminobenzoic acid; (nor)V/(nor)Val; norValine; ANCAs; anti-neutrophil cytoplasmic antibodies; FRET; fluorescence resonance energy transfer; EDDnp; N-(2,4-dinitrophenyl)ethylenediamine; PBS; phosphate-buffered saline; HPLC; high-performance liquid chromatography; MALDI-TOF; matrix assisted laser desorption ionization; BALF; broncho alveolar lavage fluidProteinase 3 (myeloblastin); Azapeptide; Inhibitor; Drug development; Lung diseases

Curcumin as anti-endometriotic agent: Implication of MMP-3 and intrinsic apoptotic pathway by Sayantan Jana; Sumit Paul; Snehasikta Swarnakar (pp. 797-804).

The disease of reproductive women, endometriosis represents implantation of functional endometrial glands outside uterine cavity. This invasive disorder is associated with dysregulation of matrix metalloproteases (MMP)s and extracellular matrix (ECM) remodeling. In this study, we investigated the role of MMP-3 on apoptosis during endometriosis. We also checked whether curcumin has potency to regress endometriosis by modulating MMP-3 and apoptotic pathway. Mouse model of endometriosis was designed by intraperitoneal inoculation of endometrial tissues to syngeneic female BALB/c. At 15th day, stable endometriotic developments were observed with increased MMP-3 expression. TUNEL positive cells were also found with endometriotic progression, which might resulted from destruction of local immune cells. We speculate that increased MMP-3 activity might be involved in the Fas mediated apoptosis. Curcumin treatment regressed endometriosis by inhibiting NFκB translocation and MMP-3 expression. It also accelerated apoptosis in endometriomas predominantly via cytochrome-c mediated mitochondrial pathway. Involvement of mitochondria in apoptosis was further confirmed by atomic force microscopy (AFM). These results were also supported by our therapeutic study, where curcumin induced apoptosis both by p53 dependent and independent manner, while celecoxib followed only p53 independent pathway. Altogether, our study establishes the novel role of curcumin as a potent anti-endometriotic compound.

Keywords: Abbreviations; PBS; phosphate-buffered saline; SDS; sodium dodecyl sulfate; Cur; curcumin; FasL; fas ligand; Cyt-c; cytochrome-c; MMP; matrix metalloproteinase; ECM; extracellular matrix; NFκB; nuclear factor-κB; JNK; c-jun N-terminal kinaseEndometriosis; Matrix metalloproteinase; Curcumin; Apoptosis

Lamotrigine is a substrate for OCT1 in brain endothelial cells by David Dickens; Andrew Owen; Ana Alfirevic; Athina Giannoudis; Andrea Davies; Babette Weksler; Ignacio A. Romero; Pierre-Olivier Couraud; Munir Pirmohamed (pp. 805-814).

The mechanisms that underpin the passage of lamotrigine at the blood–brain barrier to its site of action in the brain is poorly understood. Lamotrigine has been postulated to be delivered to its site of action in the brain favourably despite its physicochemical properties. The aim of this study was to investigate the transport of lamotrigine in an in-vitro model of the BBB. In this study, lamotrigine was found to have a distribution coefficient of 0 at pH 7.4 indicating that it was not highly lipophilic. Human brain endothelial cells (hCMEC/D3) were used to probe the interaction of lamotrigine with drug transporters. The uptake of lamotrigine into hCMEC/D3 cells was found to be an active process ( K_m=62±14μM; V_max=385±30pmol/min/million cells). Furthermore, use of a panel of transporter inhibitors indicated that this active uptake was mediated by organic cation transporter 1 (OCT1). OCT1 mRNA and protein were shown to be expressed in hCMEC/D3 cells. KCL22 cells overexpressing OCT1 were then used to validate these findings. Lamotrigine was confirmed to be a substrate and inhibitor in OCT1-transfected KCL22 cells. A putative pharmacokinetic drug–drug interaction (DDI) between quetiapine and lamotrigine was recently reported in patients and we show here that quetiapine is a potent inhibitor of the OCT1-mediated transport of lamotrigine. This is the first time that a specific influx transporter has been shown to transport lamotrigine. The clinical implications of these findings with respect to the efficacy of lamotrigine and its potential for DDI require further investigation.

Keywords: Abbreviations; BBB; blood–brain barrier; CSF; cerebrospinal fluid; FBS; fetal bovine serum; HBSS; hanks balanced salt solution; OCT; Organic cation transporters; TEA; tetraethylammonium chloride; OCT1; organic cation transporter 1; AED; anti-epileptic drugSLC22A1; OCT1; Lamotrigine; Blood–brain barrier; hCMEC/D3; Quetiapine; Drug–drug interaction

Pyrroloquinoline quinone modulates the kinetic parameters of the mammalian selenoprotein thioredoxin reductase 1 and is an inhibitor of glutathione reductase by Jianqiang Xu; Elias S.J. Arnér (pp. 815-820).

Pyrroloquinoline quinone (PQQ) is a redox active cofactor for bacterial quinoproteins. Dietary PQQ also has prominent physiological effects in mammals although no mammalian quinoprotein has yet been conclusively identified. Here we found that PQQ has substantial effects on the redox active mammalian selenoprotein thioredoxin reductase 1 (TrxR1). PQQ efficiently inhibited the activity of TrxR1 with its main native substrate thioredoxin and acted as a low efficiency substrate in a Sec-dependent TrxR1-catalyzed reduction. Interestingly, PQQ also stimulated redox cycling of TrxR1 with another quinone substrate, juglone, as much as 13-fold ( k_cat/ K_m increased from 105min⁻¹μM⁻¹ to 1331min⁻¹μM⁻¹ for juglone in the presence of 50μM PQQ, mainly through a lowered apparent K_m for juglone). Glutathione reductase was also inhibited by PQQ but in contrast to the effects of PQQ on TrxR1, its quinone reduction was not further stimulated. These results reveal that glutathione reductase and the mammalian selenoprotein TrxR1 are direct PQQ protein targets, although not being genuine quinoproteins. These findings may help explain several of the effects of PQQ seen in mammals.

Keywords: Pyrroloquinoline quinone; Juglone; Redox cycling; Thioredoxin reductase; Selenoprotein; Glutathione reductase

Correspondence regarding “Long term treatment with ACE inhibitor enalapril decreases body weight gain and increases life span in rats” by Abdoreza Davoodi-Semiromi; Rhonda Cooper-DeHoff (pp. 821-821).

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Biochemical Pharmacology (v.83, #6)

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Biochemical Pharmacology (v.83, #6)