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Biochemical Pharmacology (v.79, #9)

Editorial Advisory Board (pp. iii).

Graphical Abstracts Contents (pp. e1-e5).

Thiopurines: Factors influencing toxicity and response by Alan Kambiz Fotoohi; Sally A. Coulthard; Freidoun Albertioni (pp. 1211-1220).

When inside the cell 6-TG is converted directly by hypoxanthine-guanine phosphoribosyl transferase (HGPRT) through addition of ribose-5-phosphate to 6-thioguanosine-5′-monophosphate (TGMP), 6-MP is converted first to 6-thioinosine-5′-monophosphate (TIMP) by HGPRT then to 6-thioxanthine-5′-monophosphate (TXMP) by inosine monophosphate dehydrogenase (IMPDH) and finally to TGMP by guanosine monophosphate synthetase (GMPS). Both 6-MP and 6-TG and their respective monophosphates (TIMP and TGMP) are extensively inactivated inside the cell by thiopurine-S-methyltransferase (TPMT). Methylthioinosine monophosphate (meTIMP) is a strong inhibitor of DNPS. The remaining TGMP is converted to 6-thioguanosine-5′-diphosphate (TGDP), reduced to deoxy-6-thioguanosine-5′-diphosphate (dTGDP) by ribonucleotide reductase (RR) and phosphorylated by nucleoside diphosphate kinase (NDPK) to dTGTP, a DNA polymerase substrate.Thiopurines are the backbone of current anti-leukemia regimens and have also been effective immunosuppressive agents for the past half a century. Extensive research on their mechanism of action has been undertaken, yet many issues remain to be addressed to resolve unexplained cases of thiopurine toxicity or treatment failure. The aim of this review is to summarize current knowledge of the mechanism of thiopurine action in experimental models and put into context with clinical observations. Clear understanding of their metabolism will contribute to maximizing efficacy and minimizing toxicity by individually tailoring therapy according to the expression profile of relevant factors involved in thiopurine activation pathway.

Keywords: Abbreviations; 6-MP; 6-mercaptopurine; 6-TG; 6-thioguanine; ALL; acute lymphocytic leukemia; AML; acute myeloid leukemia; AZA; azathioprine; TGN; thioguanine nucleotide; meMP; methylmercaptopurine; HGPRT; hypoxanthine-guanine phosphoribosyl transferase; DNPS; de novo; purine synthesis; TIMP; 6-thioinosine-5′-monophosphate; IMPDH; inosine monophosphate dehydrogenase; GMPS; guanosine monophosphate synthetase; meTIMP; methylthioinosine monophosphate; TGTP; 6-thioguanosine-5′-triphosphate; dTGTP; deoxy-6-thioguanosine-5′-triphosphate; MMR; mismatch repair; meMP; 6-methylmercaptopurine; meTG; 6-methylthioguanine; XO; xanthine oxidase; TU; thiouric acid; AO; aldehyde oxidase; PRPP; phosphoribosyl pyrophosphate; ITPase; inosine triphosphate pyrophosphatase; MRP; multidrug resistance-associated proteins; MDR1; multidrug resistance-1; SAM; S-adenosylmethionine; medTGTP; methyl deoxythioguanosine-5′-triphosphate; TXMP; thioxanthosine monophosphate; TGMP; 6-thioguanosine-5′-monophosphate; NDPK; nucleoside diphosphate kinase; ENT; equilibrative nucleoside transporters; CNT; sodium-dependent transporters; NBTI; nitrobenzylthioinosine; meMPR; methylmercaptopurine riboside; DNMT; DNA methyltransferaseAcute lymphoblastic leukemia; 6-Mercaptopurine; 6-Thioguanine; Azathioprine; Hypoxanthine-guanine phosphoribosyl transferase; Thiopurine methyltransferase; Inosine 5′-monophosphate dehydrogenase; Transport; Pharmacogenetics

Endoplasmic reticulum calcium release potentiates the ER stress and cell death caused by an oxidative stress in MCF-7 cells by Nicolas Dejeans; Nicolas Tajeddine; Raphaël Beck; Julien Verrax; Henryk Taper; Philippe Gailly; Pedro Buc Calderon (pp. 1221-1230).

Increase in cytosolic calcium concentration ([Ca²⁺]_c), release of endoplasmic reticulum (ER) calcium ([Ca²⁺]_er) and ER stress have been proposed to be involved in oxidative toxicity. Nevertheless, their relative involvements in the processes leading to cell death are not well defined. In this study, we investigated whether oxidative stress generated during ascorbate-driven menadione redox cycling (Asc/Men) could trigger these three events, and, if so, whether they contributed to Asc/Men cytoxicity in MCF-7 cells. Using microspectrofluorimetry, we demonstrated that Asc/Men-generated oxidative stress was associated with a slow and moderate increase in [Ca²⁺]_c, largely preceding permeation of propidium iodide, and thus cell death. Asc/Men treatment was shown to partially deplete ER calcium stores after 90min (decrease by 45% compared to control). This event was associated with ER stress activation, as shown by analysis of eIF2 phosphorylation and expression of the molecular chaperone GRP94. Thapsigargin (TG) was then used to study the effect of complete [Ca²⁺]_er emptying during the oxidative stress generated by Asc/Men. Surprisingly, the combination of TG and Asc/Men increased ER stress to a level considerably higher than that observed for either treatment alone, suggesting that [Ca²⁺]_er release alone is not sufficient to explain ER stress activation during oxidative stress. Finally, TG-mediated [Ca²⁺]_er release largely potentiated ER stress, DNA fragmentation and cell death caused by Asc/Men, supporting a role of ER stress in the process of Asc/Men cytotoxicity. Taken together, our results highlight the involvement of ER stress and [Ca²⁺]_er decrease in the process of oxidative stress-induced cell death in MCF-7 cells.

Keywords: Oxidative stress; Breast cancer; Calcium homeostasis; Endoplasmic reticulum stress; MCF-7 cells; Ascorbate-driven menadione redox cycling

Antitumor 1-nitroacridine derivative C-1748, induces apoptosis, necrosis or senescence in human colon carcinoma HCT8 and HT29 cells by Ewa Augustin; Anna Moś-Rompa; Dorota Nowak-Ziatyk; Jerzy Konopa (pp. 1231-1241).

C-1748 is a DNA-binding agent with potent antitumor activity, especially towards prostate and colon carcinoma xenografts in mice. Here, we elucidated the nature of cellular response of human colon carcinoma HCT8 and HT29 cells to C-1748 treatment, at biologically relevant concentrations (EC₉₀ and their multiplicity). Cell cycle analysis showed gradual increase in HCT8 cells with sub-G1 DNA content (25% after 72h) considered as apoptotic. Hypodiploid cell population increased up to 60% upon treatment with 4× EC₉₀ concentration of the drug. Compared with HCT8 cells, the fraction of sub-G1 HT29 cells did not exceed 14%, even following 4-fold dose escalation. Morphological changes and biochemical markers such as: phosphatydylserine externalization, apoptotic DNA breaks, mitochondrial dysfunction and caspase activation confirmed the presence of considerable amount of apoptotic HCT8 cells but only a low amount of apoptotic HT29 cells. Next, we demonstrated that HCT8 cells surviving after exposure to C-1748 were in the state of senescence, based on altered cell morphology and expression of a pH 6-dependent β-galactosidase. On the contrary, no β-galactosidase staining was observed in HT29 cells after C-1748 treatment. Moreover, prolonged drug incubation (up to 168h) resulted in massive detachment of cells from culture plates, which together with Annexin V/PI results, indicated that necrosis was the main response of HT29 cells to C-1748 treatment. We also determined the ability of C-1748 to induce reactive oxygen species (ROS) in colon cancer cells and demonstrated, that generation of ROS was not essential for C-1748-induced apoptosis and cytotoxic activity of this drug.

Keywords: C-1748; Apoptosis; Necrosis; Senescence; Reactive oxygen species (ROS)

Inhibition of the JAK-STAT3 pathway by andrographolide enhances chemosensitivity of cancer cells to doxorubicin by Jing Zhou; Choon-Nam Ong; Gang-Min Hur; Han-Ming Shen (pp. 1242-1250).

Andro serves as a promising anticancer agent via its potent inhibitory effect on JAK-STAT3 pathway. Inhibition of STAT3 activity by Andro enhanced chemosensitivity of tumor cells to doxorubicin.Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess potent anti-inflammatory and anticancer properties. In this study, we sought to examine the effect of Andro on signal transducer and activator of transcription 3 (STAT3) pathway and evaluate whether suppression of STAT3 activity by Andro could sensitize cancer cells to a chemotherapeutic drug doxorubicin. First, we demonstrated that Andro is able to significantly suppress both constitutively activated and IL-6-induced STAT3 phosphorylation and subsequent nuclear translocation in cancer cells. Such inhibition is found to be achieved through suppression of Janus-activated kinase (JAK)1/2 and interaction between STAT3 and gp130. For understanding the biological significance of the inhibitory effect of Andro on STAT3, we next investigated the effect of Andro on doxorubicin-induced apoptosis in human cancer cells. In our study the constitutive activation level of STAT3 was found to be correlated to the resistance of cancer cells to doxorubicin-induced apoptosis. Both the short-term MTT assay and the long-term colony formation assay showed that Andro dramatically promoted doxorubicin-induced cell death in cancer cells, indicating that Andro enhances the sensitivity of cancer cells to doxorubicin mainly via STAT3 suppression. These observations thus reveal a novel anticancer function of Andro and suggest a potential therapeutic strategy of using Andro in combination with chemotherapeutic agents for treatment of cancer.

Keywords: Abbreviations; Andro; andrographolide; DAPI; 4,6-diamidino-2-phenylindole; DMEM; Dulbecco's modified Eagle's medium; DMSO; dimethyl sulfoxide; DN; dominant negative; FBS; fetal bovine serum; IFN; interferon; IL; interleukin; JAK; Janus kinase; MTT; 3(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide N-acetylcysteine; NF-; κ; B; nuclear transcription factor-kappaB; PARP; poly(ADP-ribose) polymerase; PBS; phosphate buffered saline; SDS; sodium dodecyl sulfate; STAT; signal transducers and activators of transcription; TRAIL; TNF-related apoptosis-inducing ligand; XIAP; X-linked inhibitor of apoptosisAndro; STAT3; Apoptosis; Doxorubicin

Induction of apoptosis by thymoquinone in lymphoblastic leukemia Jurkat cells is mediated by a p73-dependent pathway which targets the epigenetic integrator UHRF1 by Mahmoud Alhosin; Abdurazzag Abusnina; Mayada Achour; Tanveer Sharif; Christian Muller; Jean Peluso; Thierry Chataigneau; Claire Lugnier; Valérie B. Schini-Kerth; Christian Bronner; Guy Fuhrmann (pp. 1251-1260).

The salvage anti-tumoral pathway which implicates the p53-related p73 gene is not yet fully characterized. We therefore attempted to identify the up- and down-stream events involved in the activation of the p73-dependent pro-apoptotic pathway, by focusing on the anti-apoptotic and epigenetic integrator UHRF1 which is essential for cell cycle progression. For this purpose, we analyzed the effects of a known anti-neoplastic drug, thymoquinone (TQ), on the p53-deficient acute lymphoblastic leukemia (ALL) Jurkat cell line. Our results showed that TQ inhibits the proliferation of Jurkat cells and induces G1 cell cycle arrest in a dose-dependent manner. Moreover, TQ treatment triggers programmed cell death, production of reactive oxygen species (ROS) and alteration of the mitochondrial membrane potential (ΔΨm). TQ-induced apoptosis, confirmed by the presence of hypodiploid G0/G1 cells, is associated with a rapid and sharp re-expression of p73 and dose-dependent changes of the levels of caspase-3 cleaved subunits. These modifications are accompanied by a dramatic down-regulation of UHRF1 and two of its main partners, namely DNMT1 and HDAC1, which are all involved in the epigenetic code regulation. Knockdown of p73 expression restores UHRF1 expression, reactivates cell cycle progression and inhibits TQ-induced apoptosis. Altogether our results showed that TQ mediates its growth inhibitory effects on ALL p53-mutated cells via the activation of a p73-dependent mitochondrial and cell cycle checkpoint signaling pathway which subsequently targets UHRF1.

Keywords: Apoptosis; Caspase; Thymoquinone; Tumor suppressor protein p73; UHRF1

Chamaecypanone C, a novel skeleton microtubule inhibitor, with anticancer activity by trigger caspase 8-Fas/FasL dependent apoptotic pathway in human cancer cells by Cheng-Chih Hsieh; Yueh-Hsiung Kuo; Ching-Chuan Kuo; Li-Tzong Chen; Chun-Hei Antonio Cheung; Tsu-Yi Chao; Chi-Hung Lin; Wen-Yu Pan; Chi-Yen Chang; Shih-Chang Chien; Tung-Wei Chen; Chia-Chi Lung; Jang-Yang Chang (pp. 1261-1271).

Microtubule is a popular target for anticancer drugs. Chamaecypanone C, is a natural occurring novel skeleton compound isolated from the heartwood of Chamaecyparis obtusa var. formosana. The present study demonstrates that chamaecypanone C induced mitotic arrest through binding to the colchicine-binding site of tubulin, thus preventing tubulin polymerization. In addition, cytotoxic activity of chamaecypanone C in a variety of human tumor cell lines has been ascertained, with IC₅₀ values in nanomolar ranges. Flow cytometric analysis revealed that chamaecypanone C treated human KB cancer cells were arrested in G₂–M phases in a time-dependent manner before cell death occurred. Additional studies indicated that the effect of Chamaecypanone C on cell cycle arrest was associated with an increase in cyclin B1 levels and a mobility shift of Cdc2/Cdc25C. The changes in Cdc2 and Cdc25C coincided with the appearance of phosphoepitopes recognized by a marker of mitosis, MPM-2. Interestingly, this compound induced apoptotic cell death through caspase 8-Fas/FasL dependent pathway, instead of mitochondria/caspase 9-dependent pathway. Notably, several KB-derived multidrug resistant cancer cell lines overexpressing P-gp170/MDR and MRP were sensitive to Chamaecypanone C. Taken together, these findings indicated that Chamaecypanone C is a promising anticancer compound that has potential for management of various malignancies, particularly for patients with drug resistance.

Keywords: Abbreviations; CA-4; combretastatin A-4; CCD; charge-coupled device; DIC; differential interference contrast; DMSO; dimethyl sulfoxide; FITC; fluorescent isothiocyanate; GFP; green fluorescent protein; HRP; horseradish peroxidase; MAP; Microtubule-associated protein; MDR; multidrug resistance; MRP; multidrug resistance-associated proteins; PI; propidium iodide; SPA; scintillation proximity assay; VP16; etoposideChamaecypanone C; Microtubule inhibitor; Anticancer; Drug resistance; Fas/FasL

Identification of known drugs that act as inhibitors of NF-κB signaling and their mechanism of action by Susanne C. Miller; Ruili Huang; Srilatha Sakamuru; Sunita J. Shukla; Matias S. Attene-Ramos; Paul Shinn; Danielle Van Leer; William Leister; Christopher P. Austin; Menghang Xia (pp. 1272-1280).

Nuclear factor-kappa B (NF-κB) is a transcription factor that plays a critical role across many cellular processes including embryonic and neuronal development, cell proliferation, apoptosis, and immune responses to infection and inflammation. Dysregulation of NF-κB signaling is associated with inflammatory diseases and certain cancers. Constitutive activation of NF-κB signaling has been found in some types of tumors including breast, colon, prostate, skin and lymphoid, hence therapeutic blockade of NF-κB signaling in cancer cells provides an attractive strategy for the development of anticancer drugs. To identify small molecule inhibitors of NF-κB signaling, we screened approximately 2800 clinically approved drugs and bioactive compounds from the NIH Chemical Genomics Center Pharmaceutical Collection (NPC) in a NF-κB mediated β-lactamase reporter gene assay. Each compound was tested at fifteen different concentrations in a quantitative high throughput screening format. We identified nineteen drugs that inhibited NF-κB signaling, with potencies as low as 20nM. Many of these drugs, including emetine, fluorosalan, sunitinib malate, bithionol, narasin, tribromsalan, and lestaurtinib, inhibited NF-κB signaling via inhibition of IκBα phosphorylation. Others, such as ectinascidin 743, chromomycin A3 and bortezomib utilized other mechanisms. Furthermore, many of these drugs induced caspase 3/7 activity and had an inhibitory effect on cervical cancer cell growth. Our results indicate that many currently approved pharmaceuticals have previously unappreciated effects on NF-κB signaling, which may contribute to anticancer therapeutic effects. Comprehensive profiling of approved drugs provides insight into their molecular mechanisms, thus providing a basis for drug repurposing.

Keywords: Caspase 3/7; Cervical cancer; IκBα phosphorylation; NCGC Pharmaceutical Collection; NF-κB signaling

The alkylating prodrug J1 can be activated by aminopeptidase N, leading to a possible target directed release of melphalan by Malin Wickström; Kristina Viktorsson; Lovisa Lundholm; Reidun Aesoy; Helen Nygren; Linda Sooman; Mårten Fryknäs; Lotte Katrine Vogel; Rolf Lewensohn; Rolf Larsson; Joachim Gullbo (pp. 1281-1290).

J1, a new alkylating prodrug, can be activated by aminopeptidases to release melphalan (A). In experiments with transfected cells overexpressing aminopeptidase N (full length and soluble form), the cytotoxicity was increased (B).The alkylating prodrug of melphalan, J1 (melphalanyl-l- p-fluorophenylalanyl ethyl ester) is currently in early clinical trials. Preclinical studies have shown that J1-mediated cytotoxicity is dependent on hydrolytic activity of tumor cells. In this report we have analyzed potential peptidases and esterases of importance for release of free melphalan from J1. Exposure of tumor cell lines to J1 resulted in a significant increased level of free intracellular melphalan, at least tenfold at C_max, compared to exposure to melphalan at the same molar concentration. This efficient intracellular delivery could be inhibited in both magnitude and in time by bestatin, a broad spectrum inhibitor of the aminopeptidases, including the metalloproteinase aminopeptidase N (APN, EC 3.4.11.2.), and ebelactone A, an esterase inhibitor. These effects resulted, as expected, in decreased cytotoxic effects of J1. A specific role of APN in hydrolyzing J1 releasing free melphalan was demonstrated in vitro with pure APN enzyme. By using plasmid-based overexpression of APN or down regulation of endogenous APN with siRNA in different tumor cell lines we here confirm the involvement of APN in J1-mediated cytotoxic and apoptotic signaling. In conclusion, this study demonstrates a role of APN in the activation of the melphalan prodrug J1 and subsequently, its cytotoxicity. Given that APN is shown to be overexpressed in several solid tumors our data suggest that J1 may be activated in a tumor selective manner.

Keywords: J1; Aminopeptidase N; Esterases; Prodrug; Cancer therapeutics; Alkylating agents

Inhibition of Plasmodium falciparum pH regulation by small molecule indole derivatives results in rapid parasite death by Donelly A. van Schalkwyk; Xie W.A. Chan; Paola Misiano; Stefania Gagliardi; Carlo Farina; Kevin J. Saliba (pp. 1291-1299).

The V-type H⁺-ATPase is critical during the intraerythrocytic stage of the human malaria parasite Plasmodium falciparum. It is responsible for maintaining a near-neutral cytosolic pH (pH 7.3), an acidic digestive vacuole (pH 4.5–5.5) and the generation of an inside-negative plasma membrane potential (∼−95mV). Inhibition of this pump is therefore likely to result in profound physiological disturbances within the parasite and parasite death, as illustrated previously by the antiplasmodial activity of the potent and specific inhibitors of the V-type H⁺-ATPase, bafilomycin A₁ and concanamycin A. In this study we examined the antiplasmodial activity of a series of compounds previously designed, on the basis of the active structural constituents of bafilomycin A₁, to inhibit the osteoclast V-type H⁺-ATPase. The compounds were tested against up to 4 strains of P. falciparum with varying chloroquine sensitivities. Of the 30 novel compounds tested, 9 had sub-micromolar antiplasmodial IC₅₀ values, with the most active compound having an IC₅₀ of 160±20nM. The activity of a number of these compounds was investigated in more detail. We show that these inhibitors acidify the parasite cytosol within seconds and that some inhibitors irreversibly kill the parasite within 0.5–4h. The antiplasmodial activity of the V-type H⁺-ATPase inhibitors was strongly correlated with their ability to acidify the parasite cytosol (correlation coefficient 0.98). In combination studies, we show that the inhibitors act indifferently when combined with current antimalarials. Our data support the disruption of parasite pH regulation through inhibition of its V-type H⁺-ATPase as an antimalarial approach.

Keywords: V-type H; ⁺; -ATPase; Antimalarial; pH regulation; Proton pump inhibitors; Plasmodium

Rosiglitazone increases cell surface GLUT4 levels in 3T3-L1 adipocytes through an enhancement of endosomal recycling by Laurène Martinez; Marion Berenguer; M. Christine Bruce; Yannick Le Marchand-Brustel; Roland Govers (pp. 1300-1309).

Insulin induces a translocation of the glucose transporter GLUT4 from intracellular storage compartments towards the cell surface in adipocytes and skeletal muscle cells, allowing the cells to take up glucose. In type 2 diabetes-associated insulin resistance, the efficiency of this process is reduced. The thiazolidinediones, widely prescribed as anti-diabetic therapy, are generally regarded as insulin-sensitizers. The aim of this study was to evaluate the effect of the thiazolidinedione rosiglitazone (BRL 49653) on GLUT4 in adipocytes. When applied during differentiation, rosiglitazone dose dependently augmented GLUT4 expression along with the formation of lipid droplets. Intriguingly, its presence during differentiation led to increases in both cell surface GLUT4 levels and insulin sensitivity of GLUT4 translocation in mature adipocytes. Treatment of fully differentiated adipocytes with rosiglitazone also led to increases in GLUT4 at the plasma membrane. Rosiglitazone similarly affected cell surface levels of the endosomal transferrin receptor, but did not alter the GLUT4 internalization rate. The augmentation in cell surface GLUT4 levels was maintained in adipocytes that were rendered insulin-resistant in vitro by a 24h insulin treatment and moreover in these cells rosiglitazone also fully restored insulin-induced GLUT4 translocation. We conclude that in adipocytes, rosiglitazone increases cell surface GLUT4 levels by increasing its endosomal recycling and restores insulin-induced GLUT4 translocation in insulin resistance. These results implicate novel modes of action on GLUT4 that are all likely to contribute to the insulin-sensitizing effect of rosiglitazone in type 2 diabetes.

Keywords: GLUT4; Thiazolidinedione; Rosiglitazone; Diabetes type 2; Insulin resistance; Adipocyte

Proton pump inhibitor Lansoprazole is a nuclear liver X receptor agonist by Andrea A. Cronican; Nicholas F. Fitz; Tam Pham; Allison Fogg; Brionna Kifer; Radosveta Koldamova; Iliya Lefterov (pp. 1310-1316).

The liver X receptors (LXRα and LXRβ) are transcription factors that control the expression of genes primarily involved in cholesterol metabolism. In the brain, in addition to normal neuronal function, cholesterol metabolism is important for APP proteolytic cleavage, secretase activities, Aβ aggregation and clearance. Particularly significant in this respect is LXR mediated transcriptional control of APOE, which is the only proven risk factor for late onset Alzheimer’s disease. Using a transactivation reporter assay for screening pharmacologically active compounds and off patent drugs we identified the proton pump inhibitor Lansoprazole as an LXR agonist. In secondary screens and counter-screening assays, it was confirmed that Lansoprazole directly activates LXR, increases the expression of LXR target genes in brain-derived human cell lines, and increases Abca1 and Apo-E protein levels in primary astrocytes derived from wild type but not LXRα/β double knockout mice. Other PPIs activate LXR as well, but the efficiency of activation depends on their structural similarities to Lansoprazole. The identification of a widely used drug with LXR agonist-like activity opens the possibility for systematic preclinical testing in at least two diseases—Alzheimer’s disease and atherosclerosis.

Keywords: Abbreviations; LXR; liver X receptor; PPI; proton pump inhibitor; ABCA1; ATP binding cassette transporter A1; CompL; Lansoprazole; A; β; amyloid; β; RXR; retinoid X receptor; IFN; interferon; NSAID; non-steroidal antiinflammatory drugLiver X receptors; Proton pump inhibitors; Gene expression; LXR knockout mice; Primary astrocytes; ABCA1; APOE

Activation of distinct P2Y receptor subtypes stimulates insulin secretion in MIN6 mouse pancreatic β cells by Ramachandran Balasubramanian; Inigo Ruiz de Azua; Jürgen Wess; Kenneth A. Jacobson (pp. 1317-1326).

Activation of P2Y₁ and P2Y₆ receptors as targets for therapeutic intervention for diabetes.Extracellular nucleotides and their receptor antagonists have therapeutic potential in disorders such as inflammation, brain disorders, and cardiovascular diseases. Pancreatic β cells express several purinergic receptors, and reported nucleotide effects on insulin secretion are contradictory. We studied the effect of P2Y receptors on insulin secretion and cell death in MIN6, mouse pancreatic β cells. Expression of P2Y₁ and P2Y₆ receptors was revealed by total mRNA analysis using RT-PCR. MIN6 cells were stimulated in the presence of 16.7mM glucose with or without P2Y₁ and P2Y₆ agonists, 2-MeSADP and Up₃U, respectively. Both the agonists increased insulin secretion with EC₅₀ values of 44.6±7.0nM and 30.7±12.7nM respectively. The insulin secretion by P2Y₁ and P2Y₆ agonists was blocked by their selective antagonists MRS2179 and MRS2578, respectively. Binding of the selective P2Y₁ receptor antagonist radioligand [¹²⁵I]MRS2500 in MIN6 cell membranes was saturable ( K_D 4.74±0.47nM), and known P2Y₁ ligands competed with high affinities. Inflammation and glucose toxicity lead to pancreatic β cell death in diabetes. Flow cytometric analysis revealed that Up₃U but not 2-MeSADP protected MIN6 cells against TNF-α induced apoptosis. Overall, the results demonstrate that selective stimulation of P2Y₁ and P2Y₆ receptors increases insulin secretion that accompanies intracellular calcium release, suggesting potential application of P2Y receptor ligands in the treatment of diabetes.

Keywords: Abbreviations; DMEM; Dulbecco's modified Eagle's medium; FBS; fetal bovine serum; KRBB; Kreb's ringer bicarbonate buffer; MRS2179; N; ⁶; -methyl-2′-deoxyadenosine-3′,5′,-bisphosphate; MRS2211; pyridoxal-5′-phosphate-6-azo-(2-chloro-5-nitrophenyl)-2,4-disulfonic acid; MRS2279; 2-chloro-; N; ⁶; -methyl-(; N; )-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate; MRS2365; (; N; )-methanocarba-2′-deoxy-2-methylthio-adenosine-5′-diphosphate; MRS2500; 2-iodo-; N; ⁶; -methyl-(; N; )-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate; 2-MeSADP; 2-methylthioadenosine 5′-diphosphate; Tris; tris(hydroxymethyl) aminomethane; Up; ₃; U; P; ¹; ,P; ⁴; -di(uridine 5′-)triphosphate; HO342; Hoechst 33342; PI; propidium iodide; TNF-α; Tumor necrosis factor-alphaNucleotides; G protein-coupled receptors; Phospholipase C; Radioligand binding

St. John's Wort reduces neuropathic pain through a hypericin-mediated inhibition of the protein kinase C γ and ɛ activity by Nicoletta Galeotti; Elisa Vivoli; Anna Rita Bilia; Franco Francesco Vincieri; Carla Ghelardini (pp. 1327-1336).

SJW relieves neuropathic pain in different rat models through a decrease of PKC γ and ɛ isoform expression and phosphorylation.Current pharmacological treatments for neuropathic pain have limited efficacy and severe side-effect limitations. St. John's Wort (SJW) is a medicinal plant, mainly used as antidepressant, with a favourable side-effect profile. We here demonstrate the ability of SJW to relieve neuropathic pain in rat models. The antihyperalgesic profile and mechanism of action of SJW and its main components were studied in two rat models of neuropathic pain: the chronic constriction injury and the repeated administration of oxaliplatin. SJW, acutely administered at low doses (30–60mgkg⁻¹ p.o.), reversed mechanical hyperalgesia with a prolonged effect, being effective up to 180min after injection. Further examinations of the SJW main components revealed that hyperforin and hypericin were responsible for the antihyperalgesic properties whereas flavonoids were ineffective. The effect of SJW on the PKC expression and activation was investigated in the periaqueductal grey (PAG) area by immunoblotting experiments. Mechanistic studies showed a robust over-expression and hyperphosphorylation of the PKCγ (227.0±15.0% of control) and PKCɛ (213.9±17.0) isoforms in the rat PAG area. A single oral administration of SJW produced a significant decrease of the PKCγ (131.8±10.0) and PKCɛ (105.2±12.0) phosphorylation in the PAG area due to the presence of hypericin. Furthermore, SJW showed a dual mechanism of action since hyperforin antinociception involves an opioid-dependent pathway. Rats undergoing treatment with SJW and purified components did not show any behavioural side effects or signs of altered locomotor activity. Our results indicate SJW as a prolonged antihyperalgesic treatment through inhibition of PKC isoforms and their phosphorylation.

Keywords: Abbreviations; CCI; chronic constriction injury; CHL; chloroformic fraction; i.c.v.; intracerebroventricular; i.p.; intraperitoneal; MET; methanolic fraction; OXA; oxaliplatin repeated administration; p.o.; per os; PAG; periaqueductal grey area; PKC; protein kinase C; PMA; phorbol-12-myristate-13-acetate; SJW; St. John's Wort; s.c.; subcutaneous Hypericum perforatum; St. John's Wort; Neuropathic pain; Hypericin; Hyperforin; PKC

GABA_A receptor subtype selectivity underlying anxiolytic effect of 6-hydroxyflavone by Lihuan Ren; Feng Wang; Zhiwen Xu; Wing Man Chan; Cunyou Zhao; Hong Xue (pp. 1337-1344).

6-Hydroxyflavone (6HF), a naturally occurring flavonoid, herein brought about anxiolysis without the sedative, cognitive impairment, myorelaxant, motor incoordination, or anticonvulsant activity.6-Hydroxyflavone (6HF), a naturally occurring flavonoid, was previously reported to bind to type A γ-aminobutyric acid (GABA_A) receptors benzodiazepine (BZ) site with moderate binding affinity. In the present study, we showed that 6HF partially potentiated GABA-induced currents in native GABA_A receptors expressed in cortical neurons via BZ site, as the enhancement was blocked by the antagonist flumazenil. Furthermore, in patch clamp studies, 6HF displayed significant preference for α₂- and α₃-containing subtypes, which were thought to mediate anxiolytic effect, compared to α₁- and α₅-containing subtypes expressed in HEK 293T cells. In mice, 6HF exhibited anxiolytic-like effect in the elevated plus-maze test, unaccompanied at anxiolytic doses by the sedative, cognitive impairing, myorelaxant, motor incoordination and anticonvulsant effects commonly associated with classical BZs when tested in the hole-board, step-through passive avoidance, horizontal wire, rotarod, and pentylenetetrazol (PTZ)-induced seizure tests, respectively. The findings therefore identified 6HF as a promising drug candidate for the treatment of anxiety-like disorders.

Keywords: GABA; _A; receptor; Benzodiazepine; Flavonoids; 6-Hydroxyflavone; Subtype selectivity

Isoform distinct time-, dose-, and castration-dependent alterations in flavin-containing monooxygenase expression in mouse liver after 2,3,7,8-tetrachlorodibenzo- p-dioxin treatment by Rachel M. Novick; Chad M. Vezina; Adnan A. Elfarra (pp. 1345-1351).

Flavin-containing monooxygenase (FMO) expression in male mouse liver is altered after 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) exposure or castration. Because TCDD is slowly eliminated from the body, we examined hepatic Fmo mRNA alterations for up to 32 days following 10 or 64μg/kg TCDD exposure by oral gavage in male C57BL/6J mice. Fmo2 mRNA was significantly induced at 1, 4, and 8 days whereas Fmo3 mRNA was also induced at 32 days relative to controls. Fmo3 mRNA levels exhibited a dose-dependent increase at 4, 8, and 32 days after exposure; Fmo1, Fmo4, and Fmo5 mRNA did not exhibit clear trends. Because castration alone also increased Fmo2, Fmo3, and Fmo4 mRNA we examined the combined effects of castration and TCDD treatment on FMO expression. A greater than additive effect was observed with Fmo2 and Fmo3 mRNA expression. Fmo2 mRNA exhibited a 3–5-fold increase after castration or 10μg/kg TCDD exposure by oral gavage, whereas an approximately 20-fold increase was observed between the sham-castrated control and castrated TCDD-treated mice. Similarly, treatment with 10μg/kg TCDD alone increased Fmo3 mRNA 130- and 180-fold in the sham-castrated and castrated mice compared to their controls respectively, whereas, Fmo3 mRNA increased approximately 1900-fold between the sham control and castrated TCDD-treated mice. An increase in hepatic Fmo3 protein in TCDD-treated mice was observed by immunoblotting and assaying methionine S-oxidase activity. Collectively, these results provide evidence for isoform distinct time-, dose-, and castration-dependent effects of TCDD on FMO expression and suggest cross-talk between TCDD and testosterone signal transduction pathways.

Keywords: Abbreviations; FMO; flavin-containing monooxygenase; TCDD; 2,3,7,8-tetrachlorodibenzo-; p; -dioxin; AHR; aryl-hydrocarbon receptorFlavin-containing monooxygenase; Aryl-hydrocarbon receptor; 2,3,7,8,-Tetrachlorodibenzo-p-dioxin; Testosterone; Castration

AMPK-mediated GSK3β inhibition by isoliquiritigenin contributes to protecting mitochondria against iron-catalyzed oxidative stress by Song Hwa Choi; Young Woo Kim; Sang Geon Kim (pp. 1352-1362).

Isoliquiritigenin (ILQ), a flavonoid compound originated from Glycyrrhiza species, is known to activate SIRT1. Arachidonic acid (AA) in combination with iron (a catalyst of auto-oxidation) leads cells to produce excess reactive species with a change in mitochondrial permeability transition. In view of the importance of oxidative stress in cell death and inflammation, this study investigated the potential of ILQ to protect cells against the mitochondrial impairment induced by AA+iron and the underlying basis for this cytoprotection. Treatment with ILQ inhibited apoptosis induced by AA+iron, as evidenced by alterations in the levels of the proteins associated with cell viability: ILQ prevented a decrease in Bcl-x_L, and cleavage of poly(ADP-ribose)polymerase and procaspase-3. Moreover, ILQ inhibited the ability of AA+iron to elicit mitochondrial dysfunction. In addition, superoxide generation in mitochondria was attenuated by ILQ treatment. Consistently, ILQ prevented cellular H₂O₂ production increased by AA+iron, thereby enabling cells to restore GSH content. ILQ treatment enhanced inhibitory phosphorylation of glycogen synthase kinase-3β (GSK3β), and prevented a decrease in the GSK3β phosphorylation elicited by AA+iron, which contributed to protecting cells and mitochondria. GSK3β phosphorylation by ILQ was preceded by AMP-activated protein kinase (AMPK) activation, which was also responsible for mitochondrial protection, as shown by reversal of its effect in the experiments using a dominant negative mutant of AMPK and compound C. Moreover, the AMPK activation led to GSK3β phosphorylation. These results demonstrate that ILQ has the ability to protect cells from AA+iron-induced H₂O₂ production and mitochondrial dysfunction, which is mediated with GSK3β phosphorylation downstream of AMPK.

Keywords: Abbreviations; AA; arachidonic acid; ACC; acetyl-CoA carboxylase; AICAR; 5-aminoimidazole-4-carboxamide-1-β-; d; -ribofuranoside; AMPK; AMP-activated protein kinase; CaMKK; calcium/calmodulin-dependent kinase kinase; DCFH-DA; 2′,7′-dichlorofluorescein diacetate; DPPH; 2,2-diphenyl-1-picrylhydrazyl; FACS; fluorescence-activated cell sorter; GSK3β; glycogen synthase kinase-3β; ILQ; isoliquiritigenin; Keap1; Kelch-like ECH-associated protein 1; LQ; liquiritigenin; MMP; mitochondrial membrane potential; mPTP; mitochondrial permeability transition pore; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; Nrf2; NF-E2-related factor-2; NTA; nitrilotriacetic acid; Rh123; rhodamine 123; ROS; reactive oxygen species; SOD; superoxide dismutase; trolox; (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid; VDAC; voltage-activated anion channelIsoliquiritigenin; Mitochondrial dysfunction; Cytoprotection; GSK3β; AMPK

Susceptibility to acetaminophen (APAP) toxicity unexpectedly is decreased during acute viral hepatitis in mice by Yonas Getachew; Laura James; William M. Lee; Dwain L. Thiele; Bonnie C. Miller (pp. 1363-1371).

Acetaminophen (APAP) hepatotoxicity results from cytochrome P450 metabolism of APAP to the toxic metabolite, n-acetyl-benzoquinone imine (NAPQI), which reacts with cysteinyl residues to form APAP adducts and initiates cell injury. As APAP is commonly used during viral illnesses there has been concern that APAP injury may be additive to that of viral hepatitis, leading physicians to advise against its use in such patients; this has not been investigated experimentally. We infected C57BL/6 male mice with replication-deficient adenovirus to produce moderately severe acute viral hepatitis and observed that APAP doses that were hepatotoxic or lethal in control mice produced neither death nor additional increase in serum ALT when administered to infected mice at the peak of virus-induced liver injury. Moreover, the concentration of hepatic APAP-protein adducts formed in these mice was only 10% that in control mice. Protection from APAP hepatotoxicity also was observed earlier in the course of infection, prior to the peak virus-induced ALT rise. Hepatic glutathione limits APAP-protein adduct formation but glutathione levels were similar in control and infected mice. Cyp1a2 (E.C. 1.14.14.1) and Cyp2e1 (E.C. 1.14.13.n7) mRNA expression decreased by 3 days post-infection and hepatic Cyp2e1 protein levels were reduced almost 90% at 7 days, when adduct formation was maximally inhibited. In vitro, hepatocytes from virally infected mice also were resistant to APAP-induced injury but sensitive to NAPQI. Rather than potentiating APAP-induced liver injury, acute viral hepatitis in this model resulted in selective down-regulation of APAP metabolizing P450s in liver and decreased the risk of APAP hepatotoxicity.

Keywords: Abbreviations; ALT; alanine aminotrasferase; APAP; acetaminophen, paracetamol; CYP; designation of specific cytochrome P450 isomers in humans, rats and most species other than mouse; Cyp; cytochrome isomer designation in mice; LDH; lactate dehydrogenase; NAPQI; n-acetyl-benzoquinone imine; P450; cytochrome P450; o.p.u.; optical particle unitAcetaminophen; Liver; DILI (drug induced liver injury); Cytochrome P450; Adenovirus

Corrigendum to “Comparative pharmacology and computational modelling yield insights into allosteric modulation of human α7 nicotinic acetylcholine receptors” [Biochem. Pharmacol. 78 (2009) 836–843] by David B. Sattelle; Steven D. Buckingham; Miki Akamatsu; Kazuhiko Matsuda; Ilse S. Pienaar; Andrew K. Jones; Benedict M. Sattelle; Andrew Almond; Charles D. Blundell (pp. 1372-1372).

Acknowledgement (pp. 1373-1378).

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Biochemical Pharmacology (v.79, #9)