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Biochemical Pharmacology (v.85, #9)

Editorial Board (pp. iii).

Pro-inflammatory and pathogenic properties of Annexin-A1: The whole is greater than the sum of its parts by Fulvio D’Acquisto; Giuseppa Piras; Lorenza Rattazzi (pp. 1213-1218).

The dual nature of AnxA1.According to Aristotle, “ The whole is greater than the sum of its parts” and yet, although a long time has passed still, we seem to struggle to accept this universal concept. Searching in the literature for the biological function of Annexin-A1, one would find a wealth of information on its homeostatic and protective anti-inflammatory effects. However, very little has been said on its emerging role in a wide variety of pathological conditions ranging from cancer to autoimmunity. In this commentary, we will focus our attention on this novel pro-inflammatory and pathogenic “dark side” of Annexin-A1. We will summarize our current understanding of the signaling pathways regulated by this protein and link it to clinical and experimental evidences. Finally we will discuss assets and limitations of Annexin-A1 therapeutic strategies. Most importantly, we hope that this commentary will provide scientific support to “controversial” findings one might encounter while studying this fascinating protein.

Keywords: Annexin-A1; Formyl peptide agonist; Inflammation; Autoimmunity; Cancer

Molecular aspects of cancer cell resistance to chemotherapy by Magali Rebucci; Carine Michiels (pp. 1219-1226).

Cancer cell resistance to chemotherapy is still a heavy burden that impairs treatment of cancer patients. Both intrinsic and acquired resistance results from the numerous genetic and epigenetic changes occurring in cancer cells. Most of the hallmarks of cancer cells provide general mechanisms to sustain stresses such as the ones induced by chemotherapeutic drugs. Moreover, specific changes in the target bring resistance to specific drugs like modification in nucleotide synthesis enzymes upon anti-metabolite exposure, in microtubule composition upon spindle poison treatment, in topoisomerase activity upon topoisomerase inhibitor incubation or in intracellular signaling pathways when targeting tyrosine kinase receptors.Finally, the stemness properties of a few cancer cells as well as components of the tumor stroma, like fibroblasts and tumor-associated macrophages but also hypoxia, also help tumor to resist to anticancer agents. These processes provide an additional level of complexity to the understanding of the tumor resistance phenomenon.This review aims to describe the different general mechanisms as well as some examples of specific on target modifications inducing cancer cell resistance to chemotherapy at the molecular level. Perspectives to develop more efficient treatment, using genomic signature or more specific biomarkers to characterize putative resistance mechanisms in patients before choosing the more appropriate treatment, will also be discussed.

Keywords: Cancer; Resistance; Anti-cancer agents; Targeted therapies; Hypoxia; Cancer stem cells

Tryptophan hydroxylase-2: An emerging therapeutic target for stress disorders by Guo-Lin Chen; Gregory M. Miller (pp. 1227-1233).

Serotonin (5-HT) has been long recognized to modulate the stress response, and dysfunction of 5-HT has been implicated in numerous stress disorders. Accordingly, the 5-HT system has been targeted for the treatment of stress disorders. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in 5-HT synthesis, and the recent identification of a second, neuron-specific TPH isoform (TPH2) opened up a new area of research. With a decade of extensive investigation, it is now recognized that: (1) TPH2 exhibits a highly flexible gene expression that is modulated by an increasing number of internal and external environmental factors including the biological clock, stressors, endogenous hormones, and antidepressant therapies; and (2) genetically determined TPH2 activity is linked to a growing body of stress-related neuronal correlates and behavioral traits. These findings reveal an active role of TPH2 in the stress response and provide new insights into the long recognized but not yet fully understood 5-HT–stress interaction. As a major modulator of 5-HT neurotransmission and the stress response, TPH2 is of both pathophysiological and pharmacological significance, and is emerging as a new therapeutic target for the treatment of stress disorders. Given that numerous antidepressant therapies influence TPH2 gene expression, TPH2 is already inadvertently targeted for the treatment of stress disorders. With increased understanding of the regulation of TPH2 activity we can now purposely utilize TPH2 as a target to develop new or optimize current therapies, which are expected to greatly improve the prevention and treatment of a wide variety of stress disorders.

Keywords: Tryptophan hydroxylase-2; Serotonin; Stress response; Therapeutic target; Gene expression; Hypothalamus–pituitary–adrenal axis; Sympathetic nervous system

Discovery of a new small-molecule inhibitor of p53–MDM2 interaction using a yeast-based approach by Mariana Leão; Clara Pereira; Alessandra Bisio; Yari Ciribilli; Ana M. Paiva; Neuza Machado; Andreia Palmeira; Miguel X. Fernandes; Emília Sousa; Madalena Pinto; Alberto Inga; Lucília Saraiva (pp. 1234-1245).

Using a yeast-based approach to screen a library of xanthones selected by docking, the pyranoxanthone was identified as p53–MDM2 inhibitor, which activated p53 and downstream signaling in human tumor cells.The virtual screening of a library of xanthone derivatives led us to the identification of potential novel MDM2 ligands. The activity of these compounds as inhibitors of p53–MDM2 interaction was investigated using a yeast phenotypic assay, herein developed for the initial screening. Using this approach, in association with a yeast p53 transactivation assay, the pyranoxanthone (3,4-dihydro-12-hydroxy-2,2-dimethyl-2 H,6 H-pyrano[3,2-b]xanthen-6-one) (1) was identified as a putative small-molecule inhibitor of p53–MDM2 interaction.The activity of the pyranoxanthone1 as inhibitor of p53–MDM2 interaction was further investigated in human tumor cells with wild-type p53 and overexpressed MDM2. Notably, the pyranoxanthone1 mimicked the activity of known p53 activators, leading to p53 stabilization and activation of p53-dependent transcriptional activity. Additionally, it led to increased protein levels of p21 and Bax, and to caspase-7 cleavage. By computational docking studies, it was predicted that, like nutlin-3a, a known small-molecule inhibitor of p53–MDM2 interaction, pyranoxanthone1 binds to the p53-binding site of MDM2.Overall, in this work, a novel small-molecule inhibitor of p53–MDM2 interaction with a xanthone scaffold was identified for the first time. Besides its potential use as molecular probe and possible lead to develop anticancer agents, the pyranoxanthone1 will pave the way for the structure-based design of a new class of p53–MDM2 inhibitors.

Keywords: Xanthone derivatives; Inhibitor of p53–MDM2 interaction; Computational docking; Yeast-based assays; Antitumor activity

Inhibition of gastric tumor growth by a novel Hsp90 inhibitor by Chunwan Lu; Di Liu; Jing Jin; Hemantkumar Deokar; Yi Zhang; John K. Buolamwini; Xiaoming Yu; Chunhong Yan; Xiaoguang Chen (pp. 1246-1256).

Heat shock protein 90 (Hsp90) is a molecular chaperone engaging in multiple cellular signaling by stabilizing oncoproteins (e.g. Akt and c-Raf) in tumor cells. Whereas Hsp90 inhibitors such as 17-AAG exert promising antitumor effects in clinical trials, current efforts focus on developing agents targeting Hsp90 with improved efficacy and lower toxicity. Using a fluorescence polarization assay, we screened over a hundred of synthetic small molecules and identified a resorcinol derivative LD053 that bound the Hsp90 ATP-binding pocket. The binding of LD053 to Hsp90 dissociated the co-chaperone protein cdc37 from Hsp90, resulting in destabilization of Akt and c-Raf and subsequent inhibition of PI3K/Akt and c-Raf/Mek/Erk signaling in BGC823 gastric cancer cells. As a consequence, LD053 decreased cancer cell viability and induced apoptosis evidenced by increased subG0/G1 cell population and increased cleavage of caspase 3 and PARP. Interestingly, normal human cells appeared insensitive to LD053 treatments. Consistent with its in vitro anticancer activities, LD053 significantly inhibited growth of BGC823 xenografts in nude mice without apparent body weight loss. These results thus demonstrate that LD053 is a novel Hsp90 inhibitor and has potential to be used to treat gastric cancer.

Keywords: Abbreviations; 17-AAG; 17-allylamino-17-demethoxyl-geldanamycin; co-IP; co-immunoprecipitation; GA; geldanamycin; Hsp90; heat shock protein 90; IDF; induced fit dockingHsp90; Hsp90 inhibitor; Gastric cancer; Cancer therapy; Molecular chaperone

Parguerenes: Marine red alga bromoditerpenes as inhibitors of P-glycoprotein (ABCB1) in multidrug resistant human cancer cells by Xiao-cong Huang; Yue-Li Sun; Angela A. Salim; Zhe-Sheng Chen; Robert J. Capon (pp. 1257-1268).

Parguerenes I and II, southern Australian marine red alga metabolites, are promising inhibitors of the multi-drug resistant efflux pump P-glycoprotein.High intrinsic or acquired expression of membrane spanning, adenosine triphosphate binding cassette (ABC) transporter proteins, such as P-glycoprotein (P-gp), in cancers represents a major impediment to chemotherapy, with accelerated drug efflux leading to multi-drug resistance (MDR). Although ABC transporter inhibitors offer the prospect of reversing the MDR phenotype, no inhibitors have advanced to the clinic. We employed a range of intracellular fluorescence and radio-ligand accumulation and efflux assays, together with cytotoxicity and MDR reversal assays, as well as flow cytometry, fluorescence microscopy and radioimmunoprecipitation, to discover and evaluate new P-gp inhibitors from a unique library of southern Australian and Antarctic marine natural products. This study successfully characterized two rare bromoditerpenes, parguerenes I and II, sourced from a southern Australian collection of the red alga Laurencia filiformis, as P-gp inhibitors. We determined that the parguerenes were non-cytotoxic, dose-dependent inhibitors of P-gp mediated drug efflux, that modify the extracellular antibody binding epitope of P-gp in a manner that differs markedly from that of the known inhibitors verapamil and cyclosporine A. We confirmed that parguerenes were capable of reversing P-gp mediated vinblastine, doxorubicin and paclitaxel MDR, that inhibitory properties span both P-gp and multidrug resistant protein 1 (MRP1), but do not extend to breast cancer resistance protein (BCRP), and that parguerene II is superior (more potent) to verapamil. Our investigations validate the proposition that marine natural products can deliver new ABC transporter inhibitor scaffolds, with structure characteristics fundamentally different from existing inhibitor classes.

Keywords: Parguerene; P-glycoprotein; Multidrug resistance; ABC transporter; Inhibitor

A high-throughput-compatible, fluorescence anisotropy-based assay for ATP-dependent supercoiled DNA relaxation by human topoisomerase IIα by Adam B. Shapiro (pp. 1269-1277).

A novel, high-throughput-compatible assay for the ATP-dependent supercoiled DNA relaxing activity of human topoisomerase IIα (hTopoIIα) is described. The principle of detection is the preferential binding of the oligodeoxyribonucleotide BODIPY-TMR-5′-TTCTTCTTCT-3′ to relaxed double-stranded plasmid containing the triplex forming sequence (TTC)₉ versus the supercoiled plasmid. Binding of the oligonucleotide to the plasmid increases the fluorescence anisotropy of the BODIPY-TMR label. Optimization of the assay conditions was conducted to maximize the signal and the activity of the topoisomerase. The multiwell assay plate-based fluorescence anisotropy assay gave the same values for the potencies of several previously reported inhibitors of hTopoIIα as a gel electrophoresis-based assay of DNA relaxation.

Keywords: Topoisomerase; DNA relaxation; Fluorescence anisotropy; High-throughput; Inhibitor

Magnolol suppresses hypoxia-induced angiogenesis via inhibition of HIF-1α/VEGF signaling pathway in human bladder cancer cells by Meng-Chuan Chen; Chi-Feng Lee; Wen-Hsin Huang; Tz-Chong Chou (pp. 1278-1287).

Magnolol inhibits the HIF-1α and VEGF expression in hypoxic T24 cells and tumor tissues through suppression of VEGFR2/PI3K/AKT/mTOR/p70S6K1/4E-BP-1 signaling pathway. These effects may account for its anti-angiogenic and anti-cancer activities.The hypoxic environment in tumors is an important factor causing tumor angiogenesis by activating the key transcription factor, hypoxia-inducible factors-1α (HIF-1α). Magnolol isolated from Magnolia officinalis has been reported to exhibit an anticancer activity via elevation of apoptosis. However, whether magnolol inhibits tumor angiogenesis remains unknown. In the present study, we demonstrated that magnolol significantly inhibited angiogenesis in vitro and in vivo evidenced by the attenuation of hypoxia and vascular endothelial growth factor (VEGF)-induced tube formation of human umbilical vascular endothelial cells, vasculature generation in chicken chorioallantoic membrane and Matrigel plug. In hypoxic human bladder cancer cells (T24), treatment with magnolol inhibited hypoxia-stimulated H₂O₂ formation, HIF-1α induction including mRNA, protein expression, and transcriptional activity as well as VEGF secretion. Additionally, the enhanced degradation of HIF-1α protein via enhancing prolyl hydroxylase activity and the decreased newly-synthesized HIF-1α protein in hypoxic T24 cells may involve the reduction of HIF-1α protein accumulation by magnolol. Interestingly, magnolol also acts as a VEGFR2 antagonist, and subsequently attenuates the down-stream AKT/mTOR/p70S6K/4E-BP-1 kinase activation both in hypoxic T24 cells and tumor tissues. As expected, administration of magnolol greatly attenuated tumor growth, angiogenesis and the protein expression of HIF-1α, VEGF, CD31, a marker of endothelial cells, and carbonic anhydrase IX, an endogenous marker for hypoxia, in the T24 xenograft mouse model. Collectively, these findings strongly indicate that the anti-agngiogenic activity of magnolol is, at least in part, mediated by suppressing HIF-1α/VEGF-dependent pathways, and suggest that magnolol may be a potential drug for human bladder cancer therapy.

Keywords: Magnolol; Angiogenesis; Hypoxia-inducible factor 1 alpha; Vascular endothelial growth factor; Human bladder cancer

SIRT1 inhibits NADPH oxidase activation and protects endothelial function in the rat aorta: Implications for vascular aging by María José Zarzuelo; Rocío López-Sepúlveda; Manuel Sánchez; Miguel Romero; Manuel Gómez-Guzmán; Zoltan Ungvary; Francisco Pérez-Vizcaíno; Rosario Jiménez; Juan Duarte (pp. 1288-1296).

Vascular aging is characterized by up-regulation of NADPH oxidase, oxidative stress and endothelial dysfunction. Previous studies demonstrate that the activity of the evolutionarily conserved NAD⁺-dependent deacetylase SIRT1 declines with age and that pharmacological activators of SIRT1 confer significant anti-aging cardiovascular effects. To determine whether dysregulation of SIRT1 promotes NADPH oxidase-dependent production of reactive oxygen species (ROS) and impairs endothelial function we assessed the effects of three structurally different inhibitors of SIRT1 (nicotinamide, sirtinol, EX527) in aorta segments isolated from young Wistar rats. Inhibition of SIRT1 induced endothelial dysfunction, as shown by the significantly reduced relaxation to the endothelium-dependent vasodilators acetylcholine and the calcium ionophore A23187. Endothelial dysfunction induced by SIRT1 inhibition was prevented by treatment of the vessels with the NADPH oxidase inhibitor apocynin or superoxide dismutase. Inhibition of SIRT1 significantly increased vascular superoxide production, enhanced NADPH oxidase activity, and mRNA expression of its subunits p22^phox and NOX4, which were prevented by resveratrol. Peroxisome proliferator-activated receptor-α (PPARα) activation mimicked the effects of resveratrol while PPARα inhibition prevented the effects of this SIRT1 activator. SIRT1 co-precipitated with PPARα and nicotinamide increased the acetylation of the PPARα coactivator PGC-1α, which was suppressed by resveratrol. In conclusion, impaired activity of SIRT1 induces endothelial dysfunction and up-regulates NADPH oxidase-derived ROS production in the vascular wall, mimicking the vascular aging phenotype. Moreover, a new mechanism for controlling endothelial function after SIRT1 activation involves a decreased PGC-1α acetylation and the subsequent PPARα activation, resulting in both decreased NADPH oxidase-driven ROS production and NO inactivation.

Keywords: SIRT1; Endothelial dysfunction; NADPH oxidase; PPARα

Chemical informatics uncovers a new role for moexipril as a novel inhibitor of cAMP phosphodiesterase-4 (PDE4) by Ryan T. Cameron; Ryan G. Coleman; Jon P. Day; Krishna C. Yalla; Miles D. Houslay; David R. Adams; Brian K. Shoichet; George S. Baillie (pp. 1297-1305).

PDE4 is one of eleven known cyclic nucleotide phosphodiesterase families and plays a pivotal role in mediating hydrolytic degradation of the important cyclic nucleotide second messenger, cyclic 3′5′ adenosine monophosphate (cAMP). PDE4 inhibitors are known to have anti-inflammatory properties, but their use in the clinic has been hampered by mechanism-associated side effects that limit maximally tolerated doses. In an attempt to initiate the development of better-tolerated PDE4 inhibitors we have surveyed existing approved drugs for PDE4-inhibitory activity. With this objective, we utilised a high-throughput computational approach that identified moexipril, a well tolerated and safe angiotensin-converting enzyme (ACE) inhibitor, as a PDE4 inhibitor. Experimentally we showed that moexipril and two structurally related analogues acted in the micro molar range to inhibit PDE4 activity. Employing a FRET-based biosensor constructed from the nucleotide binding domain of the type 1 exchange protein activated by cAMP, EPAC1, we demonstrated that moexipril markedly potentiated the ability of forskolin to increase intracellular cAMP levels. Finally, we demonstrated that the PDE4 inhibitory effect of moexipril is functionally able to induce phosphorylation of the small heat shock protein, Hsp20, by cAMP dependent protein kinase A. Our data suggest that moexipril is a bona fide PDE4 inhibitor that may provide the starting point for development of novel PDE4 inhibitors with an improved therapeutic window.

Keywords: Phosphodiesterase inhibitor; Protein kinase A (PKA), PDE4; Catechol ether; Cyclic 3′5′ adenosine monophosphate (cAMP)

Beneficial effect of the non-psychotropic plant cannabinoid cannabigerol on experimental inflammatory bowel disease by Francesca Borrelli; Ines Fasolino; Barbara Romano; Raffaele Capasso; Francesco Maiello; Diana Coppola; Pierangelo Orlando; Giovanni Battista; Ester Pagano; Vincenzo Di Marzo; Angelo A. Izzo (pp. 1306-1316).

Inflammatory bowel disease (IBD) is an incurable disease which affects millions of people in industrialized countries. Anecdotal and scientific evidence suggests that Cannabis use may have a positive impact in IBD patients. Here, we investigated the effect of cannabigerol (CBG), a non-psychotropic Cannabis-derived cannabinoid, in a murine model of colitis. Colitis was induced in mice by intracolonic administration of dinitrobenzene sulphonic acid (DNBS). Inflammation was assessed by evaluating inflammatory markers/parameters (colon weight/colon length ratio and myeloperoxidase activity), by histological analysis and immunohistochemistry; interleukin-1β, interleukin-10 and interferon-γ levels by ELISA, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) by western blot and RT-PCR; CuZn-superoxide dismutase (SOD) activity by a colorimetric assay. Murine macrophages and intestinal epithelial cells were used to evaluate the effect of CBG on nitric oxide production and oxidative stress, respectively. CBG reduced colon weight/colon length ratio, myeloperoxidase activity, and iNOS expression, increased SOD activity and normalized interleukin-1β, interleukin-10 and interferon-γ changes associated to DNBS administration. In macrophages, CBG reduced nitric oxide production and iNOS protein (but not mRNA) expression. Rimonabant (a CB₁ receptor antagonist) did not change the effect of CBG on nitric oxide production, while SR144528 (a CB₂ receptor antagonist) further increased the inhibitory effect of CBG on nitric oxide production. In conclusion, CBG attenuated murine colitis, reduced nitric oxide production in macrophages (effect being modulated by the CB₂ receptor) and reduced ROS formation in intestinal epithelial cells. CBG could be considered for clinical experimentation in IBD patients.

Keywords: Abbreviations; CB; cannabinoid; CBD; cannabidiol; CBG; cannabigerol; CD; Crohn's disease; COX-2; cycloxygenase-2; DNBS; 2,4,6-dinitrobenzene sulphonic acid; H2DCF-DA; 2′,7′-dichlorfluorescein-diacetate; IBD; Inflammatory bowel disease; iNOS; inducible nitric oxide synthase; MPO; myeloperoxidase; ROS; reactive oxygen species; SOD; superoxide dismutase; UC; ulcerative colitisCannabigerol; Phytocannabinoids; Inflammatory bowel disease; Murine colitis; Macrophages; Dinitrobenzene sulphonic acid

The second extracellular loop of GPCRs determines subtype-selectivity and controls efficacy as evidenced by loop exchange study at A₂ adenosine receptors by Benjamin F. Seibt; Anke C. Schiedel; Dominik Thimm; Sonja Hinz; Farag F. Sherbiny; Christa E. Müller (pp. 1317-1329).

The second extracellular loop (EL2) of G protein-coupled receptors (GPCRs), which represent important drug targets, may be involved in ligand recognition and receptor activation. We studied the closely related adenosine receptor (AR) subtypes A_2A and A_2B by exchanging the complete EL2 of the human A_2BAR for the EL2 of the A_2AAR. Furthermore, single amino acid residues (Asp148^45.27, Ser149^45.28, Thr151^45.30, Glu164^45.43, Ser165^45.44, and Val169^45.48) in the EL2 of the A_2BAR were exchanged for alanine. The single mutations did not lead to any major effects, except for the T151A mutant, at which NECA showed considerably increased efficacy. The loop exchange entailed significant effects: The A_2A-selective agonist CGS21680, while being completely inactive at A_2BARs, showed high affinity for the mutant A_2B(EL2-A_2A)AR, and was able to fully activate the receptor. Most strikingly, all agonists investigated (adenosine, NECA, BAY60-6583, CGS21680) showed strongly increased efficacies at the mutant A_2B(EL2-A_2A) as compared to the wt AR. Thus, the EL2 of the A_2BAR appears to have multiple functions: besides its involvement in ligand binding and subtype selectivity it modulates agonist-bound receptor conformations thereby controlling signalling efficacy. This role of the EL2 is likely to extend to other members of the GPCR family, and the EL2 of GPCRs appears to be an attractive target structure for drugs.

Keywords: Mutagenesis; G protein-coupled receptors; Receptor activation; Ligand binding site; Overlap extension mutagenesis; EL2 loop exchange

Betulinic acid alleviates non-alcoholic fatty liver by inhibiting SREBP1 activity via the AMPK–mTOR–SREBP signaling pathway by Hai Yan Quan; Do Yeon Kim; Soo Jung Kim; Hee Kyung Jo; Go Woon Kim; Sung Hyun Chung (pp. 1330-1340).

Proposed mechanism of BA-induced anti-hepatic steatosis by the AMPK–SREBP1 signaling pathway.Non-alcoholic fatty liver disease (NAFLD) is emerging as the most common liver disease in industrialized countries. The discovery of food components that can ameliorate NAFLD is therefore of interest. Betulinic acid (BA) is a triterpenoid with many pharmacological activities, but the effect of BA on fatty liver is as yet unknown. To explore the possible anti-fatty liver effects and their underlying mechanisms, we used insulin-resistant HepG2 cells, primary rat hepatocytes and liver tissue from ICR mice fed a high-fat diet (HFD). Oil Red O staining revealed that BA significantly suppressed excessive triglyceride accumulation in HepG2 cells and in the livers of mice fed a HFD. Ca⁺²-calmodulin dependent protein kinase kinase (CAMKK) and AMP-activated protein kinase (AMPK) were both activated by BA treatment. In contrast, the protein levels of sterol regulatory element-binding protein 1 (SREBP1), mammalian target of rapamycin (mTOR) and S6 kinase (S6K) were all reduced when hepatocytes were treated with BA for up to 24h. We found that BA activates AMPK via phosphorylation, suppresses SREBP1 mRNA expression, nuclear translocation and repressed SREBP1 target gene expression in HepG2 cells and primary hepatocytes, leading to reduced lipogenesis and lipid accumulation. These effects were completely abolished in the presence of STO-609 (a CAMKK inhibitor) or compound C (an AMPK inhibitor), indicating that the BA-induced reduction in hepatic steatosis was mediated via the CAMKK–AMPK–SREBP1 signaling pathway. Taken together, our results suggest that BA effectively ameliorates intracellular lipid accumulation in liver cells and thus is a potential therapeutic agent for the prevention of fatty liver disease.

Keywords: Abbreviations; NAFLD; non-alcoholic fatty liver disease; BA; betulinic acid; HFD; high fat diet; CAMKK; Ca; ⁺²; -calmodulin dependent protein kinase kinase; AMPK; AMP-activated protein kinase; SREBP1; sterol regulatory element-binding protein 1; mTOR; mammalian target of rapamycin; S6K; S6 kinaseBetulinic acid; NAFLD; AMPK; SREBP1; HepG2 cells

Anti-diabetic and anti-lipidemic effects of chlorogenic acid are mediated by ampk activation by Khang Wei Ong; Annie Hsu; Benny Kwong Huat Tan (pp. 1341-1351).

Chlorogenic acid (CGA) has been shown to stimulate glucose uptake in skeletal muscle through the activation of AMPK. However, its effect on other metabolic pathways and likewise its effects after long-term consumption have yet to be understood. We investigated the effects of CGA on glucose tolerance, insulin sensitivity, hepatic gluconeogenesis, lipid metabolism and skeletal muscle glucose uptake in Lepr^db/db mice. Hepatoma HepG2 was used to investigate CGA's effect on hepatic glucose production and fatty acid synthesis. Subsequently, we attempted to evaluate whether these effects of CGA are associated with the activation of AMPK. In Lepr^db/db mice, acute treatment with CGA lowered AUC_glucose in an OGTT. Chronic administration of CGA inhibited hepatic G6Pase expression and activity, attenuated hepatic steatosis, improved lipid profiles and skeletal muscle glucose uptake, which in turn improved fasting glucose level, glucose tolerance, insulin sensitivity and dyslipidemia in Lepr^db/db mice. CGA activated AMPK, leading to subsequent beneficial metabolic outcomes, such as suppression of hepatic glucose production and fatty acid synthesis. Inhibition and knockdown of AMPK abrogated these metabolic alterations. In conclusion, CGA improved glucose and lipid metabolism, via the activation of AMPK.

Keywords: Chlorogenic acid; db/db Mice; HepG2 hepatocytes; Gluconegenesis; Glucose uptake; GLUT 4; AMPK; Fatty acids

Modulation of A-type K⁺ channels by the short-chain cobrotoxin through the protein kinase C-delta isoform decreases membrane excitability in dorsal root ganglion neurons by Qiang Guo; You-Jing Jiang; Hong Jin; Xing-Hong Jiang; Bo Gu; Yi-Ming Zhang; Jian-Gong Wang; Zheng-Hong Qin; Jin Tao (pp. 1352-1362).

A-type K⁺ channels are crucial in controlling neuronal excitability, and their regulation in sensory neurons may alter pain sensation. In this study, we identified the functional role of cobrotoxin, the short-chain α-neurotoxin isolated from Naja atra venom, which acts in the regulation of the transient A-type K⁺ currents ( I_A) and membrane excitability in dorsal root ganglion (DRG) neurons via the activation of the muscarinic M3 receptor (M3R). Our results showed that cobrotoxin increased I_A in a concentration-dependent manner, whereas the sustained delayed rectifier K⁺ currents ( I_DR) were not affected. Cobrotoxin did not affect the activation of I_A markedly, however, it shifted the inactivation curve significantly in the depolarizing direction. The cobrotoxin-induced I_A response was blocked by the M3R-selective antagonists DAU-5884 and 4-DAMP. An siRNA targeting the M3R in small DRG neurons abolished the cobrotoxin-induced I_A increase. In addition, dialysis of the cells with the novel protein kinase C-delta isoform (PKC-δ) inhibitor δv1-1 or an siRNA targeting PKC-δ abolished the cobrotoxin-induced I_A response, whereas inhibition of PKA or classic PKC activity elicited no such effects. Moreover, we observed a significant decrease in the firing rate of the neuronal action potential induced by M3R activation. Pretreatment of the cells with 4-aminopyridine, a selective blocker of I_A, abolished this effect. Taken together, these results suggest that the short-chain cobrotoxin selectively enhances I_A via a novel PKC-δ-dependent pathway. This effect occurred via the activation of M3R and might contribute to its neuronal hypoexcitability in small DRG neurons.

Keywords: A-type K; ⁺; currents; Muscarinic M3 receptor; Cobrotoxin; Dorsal root ganglia

RO4938581, a GABA_Aα5 modulator, displays strong CYP1A2 autoinduction properties in rats by Christoffer Bundgaard; Lassina Badolo; John P. Redrobe (pp. 1363-1369).

Autoinduction in drug metabolism is a known phenomenon observed when a drug induces the enzymes responsible for its own metabolism. The potency, rate and extent of autoinduction following a given treatment paradigm may have therapeutic implications in clinic as well as for in vivo pharmacological assessments in animals. RO4938581, an imidazo-triazolo-benzodiazepine, is a novel GABA_Aα5 negative modulator recently pursued for the treatment of cognitive dysfunctions. As circulating plasma levels of RO4938581 were shown to decrease rapidly after repeated dosing in rats, with CYP1A2 being involved in the metabolism of the compound, we examined the potential role of RO4938581-mediated autoinduction of CYP1A2. Incubation of rat hepatocytes with RO4938581 revealed potent CYP1A2 induction with significant increase in enzymatic activity at concentrations of 0.1nM and RO4938581 was shown to be 700-fold more potent than β-napththoflavone. Ex vivo studies revealed a 7-fold increase in metabolic CYP1A2 activity in liver microsomes prepared from rats administered with 0.1mg/kg of RO4938581 24h before. This induction profile was reflected in vivo in pharmacokinetic studies in rats where an 8-fold reduction in plasma exposure was observed after a second dose. The reduction in plasma exposures due to CYP1A2 autoinduction were confirmed functionally in contextual fear conditioning paradigm in rats, where a positive pharmacological effect observed after acute drug administration disappeared completely after sub-chronic dosing. Together, these findings suggest that RO4938581 possesses potent CYP1A2 autoinductive properties in rats and may serve as a tool for mechanistic metabolism or drug–drug interaction studies encircling this enzyme in rats.

Keywords: Autoinduction; CYP1A2; Contextual fear conditioning; Rat liver; Pharmacokinetics

Carbonyl reduction of triadimefon by human and rodent 11β-hydroxysteroid dehydrogenase 1 by Arne Meyer; Anna Vuorinen; Agnieszka E. Zielinska; Thierry Da Cunha; Petra Strajhar; Gareth G. Lavery; Daniela Schuster; Alex Odermatt (pp. 1370-1378).

11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) catalyzes the conversion of inactive 11-oxo glucocorticoids (endogenous cortisone, 11-dehydrocorticosterone and synthetic prednisone) to their potent 11β-hydroxyl forms (cortisol, corticosterone and prednisolone). Besides, 11β-HSD1 accepts several other substrates. Using rodent liver microsomes and the unspecific inhibitor glycyrrhetinic acid, it has been proposed earlier that 11β-HSD1 catalyzes the reversible conversion of the fungicide triadimefon to triadimenol. In the present study, recombinant human, rat and mouse enzymes together with a highly selective 11β-HSD1 inhibitor were applied to assess the role of 11β-HSD1 in the reduction of triadimefon and to uncover species-specific differences. To further demonstrate the role of 11β-HSD1 in the carbonyl reduction of triadimefon, microsomes from liver-specific 11β-HSD1-deficient mice were employed. Molecular docking was applied to investigate substrate binding. The results revealed important species differences and demonstrated the irreversible 11β-HSD1-dependent reduction of triadimefon. Human liver microsomes showed 4 and 8 times higher activity than rat and mouse liver microsomes. The apparent V_max/ K_m of recombinant human 11β-HSD1 was 5 and 15 times higher than that of mouse and rat 11β-HSD1, respectively, indicating isoform-specific differences and different expression levels for the three species. Experiments using inhibitors and microsomes from 11β-HSD1-deficient mice indicated that 11β-HSD1 is the major if not only enzyme responsible for triadimenol formation. The IC₅₀ values of triadimefon and triadimenol for cortisone reduction suggested that exposure to these xenobiotica unlikely impairs the 11β-HSD1-dependent glucocorticoid activation. However, elevated glucocorticoids during stress or upon pharmacological administration likely inhibit 11β-HSD1-dependent metabolism of triadimefon in humans.

Keywords: Triadimefon; 11β-Hydroxysteroid dehydrogenase; Metabolism; Liver microsomes; Azole fungicide; Molecular docking

Modulation of mitochondrial glutathione status and cellular energetics in primary cultures of proximal tubular cells from remnant kidney of uninephrectomized rats by Bavneet Benipal; Lawrence H. Lash (pp. 1379-1388).

Renal mitochondria from the remnant kidney of uninephrectomized rats undergo hypertrophy, characterized by increased respiration, enhanced formation of reactive oxygen species, but compensatory changes in glutathione status.Compensatory renal hypertrophy following reduction in renal mass leads to a hypermetabolic state and increases in basal mitochondrial oxidative stress and susceptibility to several nephrotoxicants. Previous studies provide conflicting data on whether renal mitochondria after reduction in renal mass undergo proliferation or hypertrophy or both. In the present study, our goal was to determine whether mitochondria of hypertrophied kidney undergo hypertrophy or proliferation after uninephrectomy using the uninephrectomized (NPX) rat model. Renal proximal tubular (PT) cells from NPX rats exhibited increased mitochondrial density, membrane potential and protein but no significant difference in mitochondrial DNA, as compared to PT cells from control rats. Our previous studies showed that overexpression of two mitochondrial anion transporters, the dicarboxylate (DIC, Slc25a10) and oxoglutarate (OGC, Slc25a11) carriers, in NRK-52E cells resulted in increased mitochondrial uptake of glutathione (GSH) and protection from chemically induced apoptosis. In the present study, we overexpressed DIC- and OGC-cDNA plasmids to assess their function in renal PT cells after compensatory renal hypertrophy. PT cells from NPX rats that were first preincubated with GSH were protected from cytotoxicity due to the mitochondrial inhibitor antimycin A by overexpression of either of the two mitochondrial GSH transporters. Our present results provide further evidence that compensatory renal hypertrophy is associated primarily with mitochondrial hypertrophy and hyperpolarization and that manipulation of mitochondrial GSH transporters in PT cells of hypertrophied kidney can alter susceptibility to chemically induced injury under appropriate conditions and may be a suitable therapeutic approach.

Keywords: Abbreviations; AA; antimycin A; BSA; bovine serum albumin; tBH; tert-butyl hydroperoxide; DAPI; 4,6-diamidino-2-phenylindole dihydrochloride; DIC; dicarboxylate carrier; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; GSH; glutathione; JC-1; 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzyimidazolyl-carbocyanine iodide; KHB; Krebs-Henseleit buffer; LDH; lactate dehydrogenase; MVK; methyl vinyl ketone; NPX; uninephrectomized; NRK; normal rat kidney; OGC; 2-oxoglutarate carrier; PT; proximal tubular; RT-PCR; reverse transcription-polymerase chain reactionUninephrectomy; Compensatory renal hypertrophy; Mitochondria; Oxidative stress; Glutathione

Catalytic-site conformational equilibrium in nerve-agent adducts of acetylcholinesterase: Possible implications for the HI-6 antidote substrate specificity by Elisabet Artursson; Per Ola Andersson; Christine Akfur; Anna Linusson; Susanne Börjegren; Fredrik Ekström (pp. 1389-1397).

Nerve agents such as tabun, cyclosarin and Russian VX inhibit the essential enzyme acetylcholinesterase (AChE) by organophosphorylating the catalytic serine residue. Nucleophiles, such as oximes, are used as antidotes as they can reactivate and restore the function of the inhibited enzyme. The oxime HI-6 shows a notably low activity on tabun adducts but can effectively reactivate adducts of cyclosarin and Russian VX. To examine the structural basis for the pronounced substrate specificity of HI-6, we determined the binary crystal structures of Mus musculus AChE ( mAChE) conjugated by cyclosarin and Russian VX and found a conformational mobility of the side chains of Phe338 and His447. The interaction between HI-6 and tabun-adducts of AChE were subsequently investigated using a combination of time resolved fluorescence spectroscopy and X-ray crystallography. Our findings show that HI-6 binds to tabun inhibited Homo sapiens AChE ( hAChE) with an IC₅₀ value of 300μM and suggest that the reactive nucleophilic moiety of HI-6 is excluded from the phosphorus atom of tabun. We propose that a conformational mobility of the side-chains of Phe338 and His447 is a common feature in nerve-agent adducts of AChE. We also suggest that the conformational mobility allow HI-6 to reactivate conjugates of cyclosarin and Russian VX while a reduced mobility in tabun conjugated AChE results in steric hindrance that prevents efficient reactivation.

Keywords: Abbreviations; DFP; diisopropylfluorophosphate; tabun; ethyl N-dimethylphosphoramidocyanidate; soman; pinacolylmethylphosphonofluoridate; sarin; isopropylmethylphosphono- fluoridate; cyclosarin; cyclohexylmethylphosphonofluoridate; VX; O-ethyl S-[2-(diisopropylamino)ethyl) methylphosphonothioate; Russian VX; S-[2-(diethylamino)isobutyl) methylphosphonothioate; obidoxime; 1,10- (oxybis-methylene)bis[4-(hydroxyimino)methyl] pyridinium dichloride; HI 6; 1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2-[(hydroxyimino)- methyl]pyridinium dichloride monohydrate; HLö-7; 1-[[[4-(aminocarbo- nyl)pyridinio]methoxy]methyl]-2,4-bis-[(hydroxyimino)methyl] pyridiniumdimethanesulfonate); K048; 1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide; obidoxime; oxo-[[1-[[4-(oxoazaniumylmethylidene)pyridin-1-yl]methoxymethyl]pyridin-4- ylidene]methyl]azanium; Ortho-7; 1,7-heptylene-bis- N,N0-2-pyridiniumaldoxime dichlorideCrystal structure; Time-resolved fluorescence; Time correlated single photon counting; Fluorescence decay spectroscopy; Acetylcholinesterase; Tabun; Cyclosarin; Russian VX; HI-6; Reactivator; Oxime; Structure activity relationship

Sulforaphane enhances Nrf2 expression in prostate cancer TRAMP C1 cells through epigenetic regulation by Chengyue Zhang; Zheng-Yuan Su; Tin Oo Khor; Limin Shu; Ah-Ng Tony Kong (pp. 1398-1404).

Growing evidence suggests epigenetic alteration is involved during the development and progression of prostate cancer. Previously, we found Nrf2, a key regulator of cellular antioxidant defense systems, was silenced through epigenetic mechanism during tumorigenesis in vivo TRAMP mice and in vitro TRAMP C1 cells. Sulforaphane (SFN) in cruciferous vegetable has been demonstrated to be a potent cancer prevention agent for years. The aim of this study is to investigate the potential of SFN to restore Nrf2 expression in TRAMP C1 cells through epigenetic modifications. Bisulfite genomic sequencing results indicated that SFN treatment led to demethylation of the first 5 CpGs in the promoter region of the Nrf2 gene in TRAMP C1 cells. Using methylation DNA immunoprecipitation (MeDIP) assay, SFN significantly reduced the ratio of anti-mecyt antibody binding to the Nrf2 promoter containing the first 5 CpGs. SFN increased mRNA and protein expressions of Nrf2 and Nrf2 downstream target gene NQO-1. In addition, SFN decreased the protein levels of DNMT1 and DNMT3a. SFN treatment also attenuated the protein expression levels of HDACs 1, 4, 5, and 7 while increased the level of active chromatin marker acetyl-Histone 3 (Ac-H3). SFN treatments also increased chromatin-immunoprecipitated DNA of Nrf2 gene promoter using anti-Ac-H3 antibody. Taken together, our current study shows that SFN regulates Nrf2's CpGs demethylation and reactivation in TRAMP C1 cells, suggesting SFN may exert its chemopreventive effect in part via epigenetic modifications of Nrf2 gene with subsequent induction of its downstream anti-oxidative stress pathway.

Keywords: Epigenetics; DNA methylation; Sulforaphane; Nrf2; Prostate cancer

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Biochemical Pharmacology (v.85, #9)

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Biochemical Pharmacology (v.85, #9)