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Biochemical Pharmacology (v.77, #8)

Graphical Abstract (pp. e1-e5).

Editorial Advisory Board (pp. iii).

Reactive oxygen species: Destroyers or messengers? by Grzegorz Bartosz (pp. 1303-1315).

Abundant evidence leaves no doubt that reactive oxygen species (ROS) are not only inevitable by-products of oxygen metabolism but also play a role in cellular signaling. ROS are produced by a family of NADPH oxidases for signaling purposes and mediate or augment the effects of insulin, growth factors, cytokines and G-protein-coupled receptors. Disturbances of ROS signaling leading to overproduction of these intermediates inflict oxidative damage of cell components in the course of various diseases. Restoration of proper ROS signaling, especially inhibition of cellular sources of ROS, may thus provide new ways of therapy.

Keywords: Abbreviations; ADCG; anchorage-dependent cell growth; AGE; advanced glycation end-products; AMPK; AMP-activated protein kinase; ASK; apoptotic signal-regulating kinase; Cat; catalase; Duox; dual function oxidase; Cdk; cyclin-dependent kinase; ERK; extracellular signal-regulated kinase; GPx; glutathione peroxidase; Grx; glutaredoxin; GSH; glutathione; HIF; hypoxia-inducible factor; HNE; H; ⁺; /Na; ⁺; exchanger; JNK; c-Jun NH; ₂; -terminal kinase; LPS; lipopolysaccharide; MAPK; mitogen-activated protein kinase; MMP; metalloproteinase; Nox; NADPH oxidase; Nrf; nuclear erythroid 2 p45-related factor; PHD; prolyl hydroxylase; PTEN; phosphatase and tensin homolog deleted on chromosome 10; ROS; reactive oxygen species; SOD; superoxide dismutase; Prx; peroxiredoxin; PTP; protein tyrosine phosphatase; Srx; sulfiredoxin; TNF; tumor necrosis factor; Trx; thioredoxin; TrxR; thioredoxin reductase; TXNIP; thioredoxin-interacting protein; UCP; uncoupling protein; UV; ultraviolet; VEGF; vascular endothelial growth factorNADPH oxidase; Oxidative stress; Protein tyrosine phosphatases; Reactive oxygen species; Signaling

Liver X receptor modulators: Effects on lipid metabolism and potential use in the treatment of atherosclerosis by C. Fiévet; B. Staels (pp. 1316-1327).

Liver X receptors (LXRs) are nuclear receptors that play a crucial role in regulating the expression of genes involved in lipid metabolism. Ligand activation of LXRs improves cholesterol homeostasis via multiple coordinated effects, and this function is likely to explain in part the protective effects of LXR activation on atherosclerosis reported in animal models. However, LXR activation may also induce undesirable side effects, such as lipogenesis and hypertriglyceridemia. This review discusses the potential to develop LXR modulators as therapeutic agents for atherosclerosis.

Keywords: Abbreviations; LXR; Liver X receptor; ABC; ATP-binding cassette transporter; NPC1L1; Niemann-Pick C1 like 1; LDLR; LDL receptor; SREBP; sterol regulatory element-binding protein; SQS; squalene synthase; RCT; reverse cholesterol transport; HDL; high-density lipoproteins; Apo; apolipoprotein; PLTP; phospholipids transfer protein; CETP; cholesteryl ester transfer protein; LPL; lipoprotein lipase; SCD1; stearoyl-coenzyme A desaturase; FAS; fatty acid synthase; ACC; acetyl-coA carboxylase; ChREBP; carbohydrate response element binding protein; Angptl3; angiopoietin-like protein 3; SLXRM; selective LXR modulator; RC; riccardin; AIM; apoptosis inhibitory factorLXRs; Cholesterol homeostasis; Liver steatosis; LXR ligands; Atherosclerosis

Trichostatin A sensitizes human ovarian cancer cells to TRAIL-induced apoptosis by down-regulation of c-FLIP_L via inhibition of EGFR pathway by Soo-Jung Park; Mi-Ju Kim; Hak-Bong Kim; Hee-Young Sohn; Jae-Ho Bae; Chi-Dug Kang; Sun-Hee Kim (pp. 1328-1336).

TRAIL-resistant cancer cells can be sensitized to TRAIL by combination therapy. In this study, we investigated the effect of trichostatin A (TSA), a histone deacetylase inhibitor, to overcome the TRAIL resistance in human ovarian cancer cells. Co-treatment of human ovarian cancer cells with TSA and TRAIL synergistically inhibited cell proliferation and induced apoptosis. The combined treatment of ovarian cancer SKOV3 cells with TSA and TRAIL significantly activated caspase-8 and truncated Bid, resulting in the cytosolic accumulation of cytochrome c as well as the activation of caspase-9 and -3. Moreover, we found that down-regulation of c-FLIP_L might contribute to TSA-mediated sensitization to TRAIL-induced apoptosis in SKOV3 cells, and this result was supported by showing that down- or up-regulation of c-FLIP_L with transfection of siRNA or plasmid sensitized or made SKOV3 cells resistant to TRAIL-induced apoptosis, respectively. TSA or co-treatment with TSA alone and TRAIL also resulted in down-regulation of EGFR1/2 and dephosphorylation of its downstream targets, AKT and ERK. Treatment of SKOV3 cells with PKI-166 (EGFR1/2 inhibitor), LY294002 (AKT inhibitor), and PD98059 (ERK inhibitor) decreased c-FLIP_L expression and co-treatment with TRAIL further reduced the level of c-FLIP_L, respectively, as did TSA. Collectively, our data suggest that TSA-mediated sensitization of ovarian cancer cells to TRAIL is closely correlated with down-regulation of c-FLIP_L via inhibition of EGFR pathway, involving caspase-dependent mitochondrial apoptosis, and combination of TSA and TRAIL may be an effective strategy for treating TRAIL-resistant human ovarian cancer cells.

Keywords: Abbreviations; EGFR; epidermal growth factor receptor; TSA; trichostatin A; HDACI; histone deacetylase inhibitor; PARP; poly(ADP-ribose) polymerase; ERK; extracellular signal-regulated kinase; TRAIL; tumor necrosis factor-related apoptosis-inducing ligand; DR4; death receptor 4; DR5; death receptor 5; c-FLIP; cellular FLICE-inhibitory protein; XIAP; X-linked inhibitor of apoptosis proteinTrichostatin A; TRAIL; Apoptosis; EGFR; c-FLIP; _L

A novel resveratrol derivative, HS1793, overcomes the resistance conferred by Bcl-2 in human leukemic U937 cells by Seung Hun Jeong; Wol Soon Jo; Suhee Song; Hongsuk Suh; So-Young Seol; Sun-Hee Leem; Taeg Kyu Kwon; Young Hyun Yoo (pp. 1337-1347).

A novel resveratrol derivative HS-1793 induces apoptosis and overcomes the resistance conferred by Bcl-2 in human leukemic U937 cells through 14-3-3.The chemopreventive and chemotherapeutic properties associated with resveratrol offer promise for the design of new chemotherapeutic agents. However, resveratrol is not a potent cytotoxic compound when compared with other chemotherapeutic drugs. Thus, several studies were undertaken to obtain synthetic analogues of resveratrol with potent activity. The present study was undertaken to examine whether four resveratrol analogues (HS-1784, -1792, -1791 and -1793) that we had designed and synthesized show antitumor activity. Here, we observed that all of these resveratrol analogues displayed stronger antitumor effects than resveratrol in most cancer cells tested. We further examined whether HS-1793, showing potent antitumor effects in most cancer cells tested, overcomes the resistance conferred by Bcl-2, since overcoming the resistance conferred by Bcl-2 represents an attractive therapeutic strategy against cancer. Our viability assay showed that HS-1793 overcomes the resistance conferred by Bcl-2 in human leukemic U937 cells. Various apoptosis assessment assays demonstrated that HS-1793 overcomes the resistance conferred by Bcl-2 in human leukemic U937 cells by inducing apoptosis. Noticeably, we elucidated the marked downregulation of 14-3-3 protein by HS-1793, indicating that HS-1793 overcomes the resistance conferred by Bcl-2 in U937 cells via 14-3-3. We also observed that HS-1793 exerts its antitumor activity via Bad. However, overall data obtained from methylation specific PCR, RT-PCR and real-time PCR suggest that HS-1793 plays a role in the downregulation of 14-3-3 at a post-transcriptional level. Further understanding exactly how HS-1793 overcomes the resistance conferred by Bcl-2 via 14-3-3 may guide the development of future anticancer agents.

Keywords: Bcl-2; 14-3-3; Resveratrol; Leukemia; Apoptosis

PARP-1 inhibition-induced activation of PI-3-kinase-Akt pathway promotes resistance to taxol by Arpad Szanto; Eva E. Hellebrand; Zita Bognar; Zsuzsanna Tucsek; Aliz Szabo; Ferenc Gallyas Jr.; Balazs Sumegi; Gabor Varbiro (pp. 1348-1357).

PARP-1 inhibition contributes to the activation of PI-3-kinase-Akt pathway which can counteract the effectiveness of paclitaxel administration.PARP inhibitors combined with DNA-damage inducing cytostatic agents can lead to effective tumor therapy. However, inhibition of poly(ADP-ribose) polymerase (PARP-1; EC 2.4.2.30) induces the activation of PI-3-kinase-Akt pathway, which can counteract the effectiveness of this therapy. To understand the role of Akt activation in the combined use of cytostatic agent and PARP inhibition, we used taxol (paclitaxel) as an antineoplastic agent, which targets microtubules and up-regulates mitochondrial ROS production, together with (i) pharmacological inhibition (PJ-34), (ii) siRNA knock-down and (iii) transdominant expression of the DNA binding domain of PARP-1. In all cases, PARP-1 inhibition leads to suppressed poly-ADP-ribosylation of nuclear proteins, prevention of NAD⁺ depletion and significant resistance against taxol induced caspase-3 activation and apoptotic cell death. Paclitaxel induced a moderate increase in Akt activation, which was significantly augmented by PARP inhibition, suggesting that PARP inhibition-induced Akt activation could be responsible for the cytostatic resistance. When activation of the PI-3-kinase-Akt pathway was prevented by LY-294002 or Akt Inhibitor IV, the cytoprotective effect of PARP inhibition was significantly diminished showing that the activation of PI-3-kinase-Akt cascade had significantly contributed to the cytostatic resistance. Our study demonstrates that drug-induced drug resistance can be responsible for the reduced efficacy of antitumor treatment. Although inhibition of PARP-1 can promote cell death in tumor cells by the inhibition of DNA repair, PARP-inhibition promoted activation of the PI-3-kinase-Akt pathway can counteract this facilitating effect, and can cause cytostatic resistance. We suggest augmenting PARP inhibition by the inhibition of the PI-3-kinase-Akt pathway for antitumor therapy.

Keywords: Abbreviations; PARP; poly(ADP-ribose) polymerase; PAR; poly(ADP-ribose); PI-3K; phosphatidylinositol-3-kinase; Akt/PKB; protein kinase B; MTT; ⁺; 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromideAntitumor treatment; Taxol resistance; PARP inhibition; Akt activation; PI-3 kinase

Phenylalanine-induced leucopenia in genetic and dichloroacetic acid generated deficiency of glutathione transferase Zeta by Angelo Theodoratos; Wen Juan Tu; Jean Cappello; Anneke C. Blackburn; Klaus Matthaei; Philip G. Board (pp. 1358-1363).

Glutathione transferase Zeta (GSTZ1-1) is identical to maleylacetoacetate isomerase and catalyses a significant step in the catabolism of phenylalanine and tyrosine. Exposure of GSTZ1-1 deficient mice to high dietary phenylalanine causes a rapid loss of circulating white blood cells (WBCs). The loss was significant ( P<0.05) after 2 days and total WBCs were reduced by 60% after 6 days. The rapid loss of WBCs was attributed to the accumulation of the catabolic intermediates maleylacetoacetate or maleylacetone (MA) in the circulation. Serum from GSTZ1-1 deficient mice treated with phenylalanine was cytotoxic to splenocytes from normal BALB/c mice and direct incubation of normal splenocytes with MA caused a rapid loss of viability. Dichloroacetic acid (DCA) has been used therapeutically to treat lactic acidosis and is potentially of use in cancer chemotherapy. Since DCA can inactivate GSTZ1-1 there is a possibility that long-term treatment of patients with DCA could cause GSTZ1-1 deficiency and susceptibility to oxidative stress and phenylalanine/tyrosine-induced WBC loss. However, although we found that DCA at 200mg/(kgday) causes a severe loss of hepatic GSTZ1-1 activity in BALB/c mice, it did not induce WBC cytotoxicity when combined with high dietary phenylalanine.

Keywords: Glutathione transferase Zeta; Maleylacetoacetate isomerase; Dichloroacetic acid; Phenylalanine-induced leucopenia; Maleylacetone cytotoxicity; GSTZ1-1 deficiency

A new insight of anti-platelet effects of sirtinol in platelets aggregation via cyclic AMP phosphodiesterase by Fu-Chao Liu; Chang-Hui Liao; Yao-Wen Chang; Jiin-Tarng Liou; Yuan-Ji Day (pp. 1364-1373).

The mechanism of sirtinol may include an increase of cAMP level with enhanced VASP-Ser157 phosphorylation via inhibition of cAMP phosphodiesterase activity and subsequent inhibition of intracellular Ca²⁺ mobilization, thromboxane A2 formation, P-selection expression and ATP release during the platelet aggregation.Sirtinol, a cell permeable six-membered lactone ring, is derived from naphthol and potent inhibitor of SIR2 and its naphtholic may have the inhibitory effects on platelets aggregation. In this study, platelet function was examined by collagen/epinephrine (CEPI) and collagen/ADP-induced closure times using the PFA-100 system reveal that CEPI-CT and CADP-CT were prolonged by sirtinol. The platelets aggregation regulated by physiological agonists such as: thrombin, collagen and AA and U46619 were significantly inhibited by sirtinol. Increases cAMP level was observed when sirtinol treated with Prostaglandin E1 in washed platelets. Moreover, sirtinol attenuated intracellular Ca²⁺ release and thromboxane B2 formation stimulated by thrombin, collagen, AA and U46619 in human washed platelets. This study indicated that sirtinol could inhibit the platelet aggregation induced by physiological agonists, AA and U46619. The mechanism of action may include an increase of cAMP level with enhanced VASP-Ser157 phosphorylation via inhibition of cAMP phosphodiesterase activity and subsequent inhibition of intracellular Ca²⁺ mobilization, thromboxane A2 formation, and ATP release during the platelet aggregation.

Keywords: Abbreviations; SIR2; silent information regulator 2; TXB2; thromboxane B2; AA; arachidonic acid; HDAC; histone deacetylase; SIRT1; sirtuin1; FITC; fluorescein isothiocyanate; CEPI-CT; collagen/epinephrine closure time; CADP-CT; collagen/ADP closure time; PGE1; prostaglandin E1; cAMP; cyclic adenosine monophosphate; cGMP; cyclic guanosine monophosphate; ATP; adenosine triphosphate; ADP; adenosine diphosphate; SNP; sodium nitroprusside; IBMX; 3-isobutyl-1-methylxanthine; BSA; bovine serum albumin; EDTA; ethylenediamine tetraacetic acid; DMSO; dimethylsulfoxide; PRP; platelet-rich plasma; PPP; platelet-poor plasma; PKA; protein kinase A; PKG; protein kinase G; PDE; phosphodiesterase; EHNA; erythro-9-(2-hydroxy-3-nonyl)adenine; VASP; vasodilator-stimulated phosphoproteinSirtinol; Platelet aggregation; Prostaglandin E1; Calcium; cAMP; Phosphodiesterase

Identification of transmembrane domain 6 & 7 residues that contribute to the binding pocket of the urotensin II receptor by Brian J. Holleran; Ivana Domazet; Marie-Eve Beaulieu; Li Ping Yan; Gaétan Guillemette; Pierre Lavigne; Emanuel Escher; Richard Leduc (pp. 1374-1382).

Urotensin II (U-II), a cyclic undecapeptide, is the natural ligand of the urotensin II (UT) receptor, a G protein-coupled receptor. In the present study, we used the substituted-cysteine accessibility method to identify specific residues in transmembrane domains (TMDs) six and seven of the rat urotensin II receptor (rUT) that contribute to the formation of the binding pocket of the receptor. Each residue in the R256^(6.32)-Q283^(6.59) fragment of TMD6 and the A295^(7.31)-T321^(7.57) fragment of TMD7 was mutated, individually, to a cysteine. The resulting mutants were expressed in COS-7 cells, which were subsequently treated with the positively charged methanethiosulfonate-ethylammonium (MTSEA) or the negatively charged methanethiosulfonate-ethylsulfonate (MTSES) sulfhydryl-specific alkylating agents. MTSEA treatment resulted in a significant reduction in the binding of TMD6 mutants F268C^(6.44) and W278C^(6.54) and TMD7 mutants L298C^(7.34), T302C^(7.38), and T303C^(7.39) to¹²⁵I-U-II. MTSES treatment resulted in a significant reduction in the binding of two additional mutants, namely L282C^(6.58) in TMD6 and Y300C^(7.36) in TMD7. These results suggest that specific residues orient themselves within the water-accessible binding pocket of the rUT receptor. This approach, which allowed us to identify key determinants in TMD6 and TMD7 that contribute to the UT receptor binding pocket, enabled us to further refine our homology-based model of how U-II interacts with its cognate receptor.

Keywords: Urotensin II; Urotensin II receptor; GPCR; Substituted-cysteine accessibility method; Ligand-binding pocket

Electrophysiologic characterization of a novel hERG channel activator by Zhi Su; James Limberis; Andrew Souers; Philip Kym; Ann Mikhail; Kathryn Houseman; Gilbert Diaz; Xiaoqin Liu; Ruth L. Martin; Bryan F. Cox; Gary A. Gintant (pp. 1383-1390).

Activators of the human ether-a-go-go-related gene (hERG) K⁺ channel have been reported recently to enhance hERG current amplitude (five synthetic small molecules and one naturally occurring substance). Here, we characterize the effects of a novel compound A-935142 ({4-[4-(5-trifluoromethyl-1H-pyrazol-3-yl)-phenyl]-cyclohexyl}-acetic acid) on guinea-pig atrial and canine ventricular action potentials (microelectrode techniques) and hERG channels expressed in HEK-293 cells (whole-cell patch clamp techniques). A-935142 shortened cardiac action potentials and enhanced the amplitude of the hERG current in a concentration- and voltage-dependent manner. The fully activated current–voltage relationship revealed that this compound (60μM) increased both outward and inward K⁺ current as well as the slope conductance of the linear portion of the fully activated I– V relation. A-935142 significantly reduced the time constants ( τ) of hERG channel activation at two example voltages (−10mV: τ=100±17ms vs. 164±24ms, n=6, P<0.01; +30mV: τ=16.7±1.8ms vs. 18.9±1.8ms, n=5, P<0.05) and shifted the voltage-dependence for hERG activation in the hyperpolarizing direction by 9mV. The time course of hERG channel deactivation was slowed at multiple potentials (−120 to −70mV). A-935142 also reduced the rate of inactivation and shifted the voltage-dependence of inactivation in the depolarizing direction by 15mV. Recovery of hERG channel from inactivation was not affected by A-935142. In conclusion, A-935142 enhances hERG current in a complex manner by facilitation of activation, reduction of inactivation, and slowing of deactivation, and abbreviates atrial and ventricular repolarization.

Keywords: hERG activator; K-channel; Action potential; A-935142

Suppression of the TRIF-dependent signaling pathway of Toll-like receptors by luteolin by Jun Kyung Lee; So Young Kim; Yoon Sun Kim; Won-Ha Lee; Daniel H. Hwang; Joo Young Lee (pp. 1391-1400).

Toll-like receptors (TLRs) play important roles in induction of innate immune responses for both host defense against invading pathogens and wound healing after tissue injury. Since dysregulation of TLR-mediated immune responses is closely linked to many chronic diseases, modulation of TLR activation by small molecules may have therapeutic potential against such diseases. Expression of the majority of lipopolysaccharide-induced TLR4 target genes is mediated through a MyD88-independent (TRIF-dependent) signaling pathway. In order to evaluate the therapeutic potential of the flavonoid luteolin we examined its effect on TLR-stimulated signal transduction via the TRIF-dependent pathway. Luteolin suppressed activation of Interferon regulatory factor 3 (IRF3) and NFκB induced by TLR3 and TLR4 agonists resulting in the decreased expression of target genes such as TNF-α, IL-6, IL-12, IP-10, IFNβ, CXCL9, and IL-27 in macrophages. Luteolin attenuated ligand-independent activation of IRF3 or NFκB induced by TLR4, TRIF, or TBK1, while it did not inhibit TLR oligomerization. Luteolin inhibited TBK1-kinase activity and IRF3 dimerization and phosphorylation, leading to the reduction of TBK1-dependent gene expression. Structural analogs of luteolin such as quercetin, chrysin, and eriodictyol also inhibited TBK1-kinase activity and TBK1-target gene expression. These results demonstrate that TBK1 is a novel target of anti-inflammatory flavonoids resulting in the down-regulation of the TRIF-dependent signaling pathway. These results suggest that the beneficial activities of these flavonoids against inflammatory diseases may be attributed to the modulation of TLR-mediated inflammatory responses.

Keywords: Abbreviations; IFNβ; Interferon beta; IKK; IkappaB kinase; IRF3; Interferon regulatory factor 3; MyD88; myeloid differentiation primary response gene 88; TIR; Toll/interleukin-1 receptor; TBK1; TANK-binding kinase 1; TLR; Toll-like receptor; TRIF; TIR-domain-containing adaptor inducing interferon-betaToll-like receptor; Phytochemicals; TRIF; TBK1; Inflammation

Denbinobin inhibits nuclear factor-κB and induces apoptosis via reactive oxygen species generation in human leukemic cells by Gonzalo Sánchez-Duffhues; Marco A. Calzado; Amaya García de Vinuesa; Giovanni Appendino; Bernd L. Fiebich; Ulich Loock; Annette Lefarth-Risse; Karsten Krohn; Eduardo Muñoz (pp. 1401-1409).

Denbinobin, a 1,4-phenanthrenequinone firstly isolated from the stems of Dendrobium moniliforme (Shi-Hu in Chinese medicine), has been reported to exhibit anti-tumoral and anti-inflammatory activities through mechanism(s) not yet fully understood. Because of the critical role of the transcription factor NF-κB and of ROS-induced activation of stress regulated kinases in tumorigenesis, we have investigated the effect of denbinobin on these pathways. We found that denbinobin is a potent inhibitor of TNFα and PMA-induced NF-κB activation, and that it can block the phosphorylation and degradation of IκBα by inhibiting TAK1 activity, an event lying upstream of IKK activation. Moreover, treatment with denbinobin not only elicited apoptotic signalling, including mitochondrial membrane dysfunction, activation of caspases and cleavage of poly(ADP-ribose) polymerase, but also induced intracellular reactive oxygen species (ROS) generation and sustained activation of the mitogen-activated kinases (MAPKs) ERK1+2, p38 and JNK 1+2. The apoptotic effects of denbinobin could be prevented by pre-treatment with the intracellular ROS scavenger N-acetyl-l-cysteine, but not by pharmacological inhibition of MAPKs, suggesting that intracellular ROS generation underlies denbinobin-induced apoptosis, and that this effect takes place in an MAPKs-independent pathway. To define the structural elements critical for these activities, a series of phenanthrenequinones with different substituents in the phenanthrene- and/or in the quinone ring were prepared and assayed for NF-κB inhibition and ROS production. In this way, the major structure–activity relationships and the structural elements critical for the activity of denbinobin could be established.A proposal model for denbinobin-induced apoptosis in leukemic cells

Keywords: Denbinobin; 1,4-Phenanthrenequinones; NF-κB; ROS; MAPKs; Apoptosis

Gene expression profiling of leukemia T-cells resistant to methotrexate and 7-hydroxymethotrexate reveals alterations that preserve intracellular levels of folate and nucleotide biosynthesis by Alan Kambiz Fotoohi; Yehuda G. Assaraf; Ali Moshfegh; Jamileh Hashemi; Gerrit Jansen; Godefridus J. Peters; Catharina Larsson; Freidoun Albertioni (pp. 1410-1417).

In vitro treatment of human T-cell leukemia cells with 7-hydroxymethotrexate, the major metabolite of methotrexate resulted in acquired resistance as a result of the complete loss of folypolyglutamate synthetase (FPGS) activity. This was in contradistinction to the major modality of antifolate resistance of impaired drug transport in leukemia cells exposed to methotrexate. To identify the genes associated with methotrexate and 7-hydroxymethotrexate resistance, we herein explored the patterns of genome-wide expression profiles in these antifolte-resistant leukemia sublines. mRNA levels of the reduced folate carrier, the primary influx transporter of folates and antifolates, were down-regulated more than two-fold in methotrexate-resistant cells. The dramatic loss of FPGS activity in 7-hydroxymethotrexate-resistant cells was associated with alterations in the expression of various genes aimed at preserving reduced folates and/or enhancing purine nucleotide biosynthesis, e.g. methylene tetrahydrofolate reductase, glycinamide ribonucleotide formyltransferase, adenosine deaminase, cystathionine β synthase, as well as the ATP-dependent folate exporters BCRP/ ABCG2 and MRP1/ ABCC1. The observed changes in gene expression were generally not paralleled by acquired DNA copy numbers alterations, suggesting transcriptional regulatory mechanisms. Interestingly, gene expression of DNA/RNA metabolism and transport genes were more profoundly altered in methotrexate-resistant subline, whereas in 7-hydroxymethotrexate-resistant cells, the most profoundly affected groups of genes were those encoding for proteins involved in metabolism and cellular proliferation. Thus, the present investigation provides evidence that 7-hydroxymethotrexate induces gene expression alterations and an antifolate resistance modality that are distinct from its parent drug methotrexate.

Keywords: Abbreviations; MTX; methotrexate; 7-OHMTX; 7-hydroxymethotrexate; IC; ₅₀; the drug concentration inhibiting cell growth by 50%; DHFR; dihydrofolate reductase; TS; thymidylate synthase; RFC; reduced folate carrier; FPGS; folylpolyglutamate synthetase; FPGH; folylpolyglutamate hydrolase; GARFT; glycinamide ribonucleotide formyltransferase; MRP; multidrug resistance-associated proteinMethotrexate; 7-Hydroxymethotrexate; Resistance; Leukemia; Folylpolyglutamate synthetase; Reduced folate carrier; Thymidylate synthase; Microarray; Real-time quantitative RT-PCR

Identification of metabolites of meisoindigo in rat, pig and human liver microsomes by UFLC–MS/MS by Meng Huang; Paul C. Ho (pp. 1418-1428).

In vitro metabolic pathways of meisoindigo were identified in rat, pig and human liver microsomes. Statistical analysis showed both interspecies and gender effects were negligible in metabolic stability profiles of meisoindigo.3-(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)-1,3-dihydro-1-methyl-2H-indol-2-one, abbreviated as meisoindigo, has been a routine therapeutic agent in the clinical treatment of chronic myelogenous leukemia in China since the 1980s. To gain an understanding of the interspecies differences in the metabolism of meisoindigo, the relevant metabolism studies were carried out for the first time in rat, pig and human liver microsomes of different genders by ultra fast liquid chromatography/tandem mass spectrometry (UFLC–MS/MS). The qualitative metabolite identification was accomplished by multiple reaction monitoring (MRM) in combination with Enhanced Product Ion (EPI). The semi-quantitative metabolic stability and metabolite formation were simultaneously measured by MRM. The in vitro metabolic pathways of meisoindigo in three species were proposed as 3,3′ double bond reduction, followed by N-demethylation, and reduction followed by phenyl mono-oxidation. Two novel metabolic pathways involving direct phenyl mono-oxidation without reduction in the three species, and direct N-demethylation without reduction in only pig and human, were also proposed. It may be noted that the two metabolites formed after reduction followed by phenyl mono-oxidation at positions 4, 5, 6 or 7, as well as one metabolite formed from direct phenyl mono-oxidation at either of the two phenyl rings without reduction were found to be uniquely present in human. The in vitro t_1/2 and in vitro CL_int values of meisoindigo were calculated. Statistical analysis showed there were no significant differences in the metabolic stability profiles of meisoindigo among three species, and gender effect on the metabolic stability of meisoindigo was negligible. Formation profiles of the most significant reductive metabolites were obtained in the three species.

Keywords: Abbreviations; CML; chronic myelogenous leukemia; UFLC; ultra fast liquid chromatography; MS; mass spectrometry; MS/MS; tandem mass spectrometry; QTRAP; hybrid triple quadrupole linear ion trap; MRM; multiple reaction monitoring; EPI; Enhanced Product Ion In vitro; metabolism; Meisoindigo; Liver microsomes; Interspecies differences; UFLC–MS/MS

Transport of guanidine compounds by human organic cation transporters, hOCT1 and hOCT2 by Naoko Kimura; Satohiro Masuda; Toshiya Katsura; Ken-ichi Inui (pp. 1429-1436).

Although some guanidine compounds were reported as superior substrates for organic cation transporter (OCT)2 than OCT1, it was unclear whether this guanidino group was an important factor in determining the specificity of hOCT1 and hOCT2. Using HEK293 cells transfected with human (h)OCT1 or hOCT2 cDNA, we assessed the role of hOCT1 and/or hOCT2 in the transport of guanidine compounds such as uremic toxins and therapeutic agents. Guanidine, creatinine and aminoguanidine more markedly inhibited the uptake of [¹⁴C]tetraethylammonium (TEA) by hOCT2 than by hOCT1. [¹⁴C]TEA uptake by hOCT2, but not hOCT1, was trans-stimulated by unlabeled guanidine, methylguanidine, creatinine, aminoguanidine and phenylguanidine. In patients with renal failure, the impairment of hOCT2 might decrease the excretion of guanidine, methylguanidine, and creatinine as uremic toxins. The uptake of aminoguanidine, a candidate for an anti-diabetic agent, was enhanced by hOCT2 with the Michaelis constant ( K_m) of 4.10±0.35mM. Metformin, which was also an anti-diabetic agent, and creatinine more potently inhibited the uptake of [¹⁴C]aminoguanidine by hOCT2 than that by hOCT1. Aminoguanidine had little impact on the uptake of [¹⁴C]metformin by hOCT1, but inhibited that by hOCT2 with the IC₅₀ of 1.49±0.14mM. These results indicated that the specificity of hOCT1 and hOCT2 was not determined simply by guanidino group. Among guanidine compounds, aminoguanidine was identified as a new superior substrate for hOCT2.

Keywords: Guanidine; Aminoguanidine; OCT1; OCT2; Uremic toxins; Kidney

Effects of the anthelmintic drug PF1022A on mammalian tissue and cells by R. Dornetshuber; M.R. Kamyar; P. Rawnduzi; I. Baburin; K. Kouri; E. Pilz; T. Hornbogen; R. Zocher; W. Berger; R. Lemmens-Gruber (pp. 1437-1444).

Nematode infections cause human morbidity and enormous economic loss in livestock. Since resistance against currently available anthelmintics is a worldwide problem, there is a continuous need for new compounds. The cyclooctadepsipeptide PF1022A is a novel anthelmintic that binds to the latrophilin-like transmembrane receptor important for pharyngeal pumping in nematodes. Furthermore, PF1022A binds to GABA receptors, which might contribute to the anthelmintic effect. Like other cyclodepsipeptides, PF1022A acts as an ionophore. However, no correlation between ionophoric activity and anthelmintic properties was found. This is the first study describing the effect of PF1022A on mammalian cells and tissues. While channel-forming activity was observed already at very low concentrations, changes in intracellular ion concentrations and reduction of contractility in isolated guinea pig ileum occurred at multiples of anthelmintically active concentrations. PF1022A did not induce necrotic cell death indicated by complete lack of cellular lactate dehydrogenase release. In contrast, apoptosis induction via the mitochondrial pathway was suggested for long-term drug treatment at high concentrations due to numerous apoptotic morphological changes as well as mitochondrial membrane depolarisation. Short time effects were based on cell cycle blockade in G₀/G₁ phase. Additionally, the cell cycle and apoptosis regulating proteins p53, p21 and bax, but not Bcl-2 were shown to impact on PF1022A-induced cytotoxicity. However, since PF1022A-induced cytotoxicity was found at drug concentrations higher than those used in anthelmintic treatment, it can be suggested that PF1022A intake might not impair human or animal health. Thus, PF1022A seems to be a safe alternative to other anthelmintic drugs.Cytotoxicity of the ionophoric cyclooctadepsipeptide PF1022A is induced by apoptosis via the mitochondrial pathway and cell cycle blockade in G₀/G₁ phase at concentrations higher than those used in anthelmintic treatment.

Keywords: PF1022A; Anthelmintics; Ionophore; Cytotoxicity; Apoptosis

Corrigendum to “Interleukin 10 deficiency exacerbates halothane induced liver injury by increasing interleukin 8 expression and neutrophil infiltration” [Biochem. Pharmacol 77 (2009) 277–284] by Dechun Feng; Ying Wang; Yan Xu; Qingqiong Luo; Bin Lan; Lingyun Xu (pp. 1445-1445).

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Biochemical Pharmacology (v.77, #8)