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Biochemical Pharmacology (v.74, #12)

Subject Index for Volume 74 (pp. vii-xxxiii).

Editorial Advisory Board (pp. iii).

Author Index for Volume 74 (pp. i-vi).

Harnessing drug resistance: Using ABC transporter proteins to target cancer cells by Heather M. Leitner; Remy Kachadourian; Brian J. Day (pp. 1677-1685).

The ATP-binding cassette (ABC) class of proteins is one of the most functionally diverse transporter families found in biological systems. Although the abundance of ABC proteins varies between species, they are highly conserved in sequence and often demonstrate similar functions across prokaryotic and eukaryotic organisms. Beginning with a brief summary of the events leading to our present day knowledge of ABC transporters, the purpose of this review is to discuss the potential for utilizing ABC transporters as a means for cellular glutathione (GSH) modulation. GSH is one of the most abundant thiol antioxidants in cells. It is involved in cellular division, protein and DNA synthesis, maintenance of cellular redox status and xenobiotic metabolism. Cellular GSH levels are often altered in many disease states including cancer. Over the past two decades there has been considerable emphasis on methods to sensitize cancer cells to chemotherapeutics and radiation therapy by GSH depletion. We contend that ABC transporters, particularly multi-drug resistant proteins (MRPs), may be used as therapeutic targets for applications aimed at modulation of GSH levels. This review will emphasize MRP-mediated modulation of intracellular GSH levels as a potential alternative and adjunctive approach for cancer therapy.

Keywords: Abbreviations; ABC; ATP-binding cassette; BPD; binding protein-dependent; BSO; l; -buthionine sulfoximine; 2′,5′-DHC; 2′,5′-dihydroxychalcone; γ-GCL; γ-glutamylcysteine ligase; GSH; glutathione; MRP; multi-drug resistant protein; NBD; nucleotide-binding domain; PMFD; proton-motive-force-dependent; TMD; trans-membrane domainMultidrug resistance proteins; Glutathione; Oxidative stress; Chemotherapy; Flavonoids

Metallo-β-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily by Carine Bebrone (pp. 1686-1701).

One strategy employed by bacterial strains to resist β-lactam antibiotics is the expression of metallo-β-lactamases requiring Zn²⁺ for activity. In the last few years, many new zinc β-lactamases have been described and several pathogens are now known to synthesize members of this class. Metallo-β-lactamases are especially worrisome due to: (1) their broad activity profiles that encompass most β-lactam antibiotics, including the carbapenems; (2) potential for horizontal transference; and (3) the absence of clinically useful inhibitors. On the basis of the known sequences, three different lineages, identified as subclasses B1, B2, and B3 have been characterized. The three-dimensional structure of at least one metallo-β-lactamase of each subclass has been solved. These very similar 3D structures are characterized by the presence of an αββα-fold.In addition to metallo-β-lactamases which cleave the amide bond of the β-lactam ring, the metallo-β-lactamase superfamily includes enzymes which hydrolyze thiol-ester, phosphodiester and sulfuric ester bonds as well as oxydoreductases. Most of the 6000 members of this superfamily share five conserved motifs, the most characteristic being the His116-X-His118-X-Asp120-His121 signature. They all exhibit an αββα-fold, similar to that found in the structure of zinc β-lactamases. Many members of this superfamily are involved in mRNA maturation and DNA reparation. This fact suggests the hypothesis that metallo-β-lactamases may be the result of divergent evolution starting from an ancestral protein which did not have a β-lactamase activity.

Keywords: Abbreviations; MBL; metallo-β-lactamase; BBL; class B β-lactamase; ISCR; insertion sequences common regionsBacterial resistance; Class B β-lactamases; Zinc binding sites; αββα-fold; Conserved motifs; Metallo-β-lactamase superfamily

Stromal cell-derived factor-1 enhances motility and integrin up-regulation through CXCR4, ERK and NF-κB-dependent pathway in human lung cancer cells by Yi-Chia Huang; Yu-Chun Hsiao; Ying-Ju Chen; Ying-Ying Wei; Tzu-Hsu Lai; Chih-Hsin Tang (pp. 1702-1712).

The chemokine stromal-derived factor-1α (SDF-1α) and its receptor, CXCR4, play a crucial role in adhesion and migration of human cancer cells. Integrins are the major adhesive molecules in mammalian cells. Here we found that SDF-1α increased the migration and cell surface expression of β1 or β3 integrin in human lung cancer cells (A549 cells). CXCR4-neutralizing antibody, CXCR4 specific inhibitor (AMD3100) or small interfering RNA against CXCR4 inhibited the SDF-1α-induced increase in the migration of lung cancer cells. Stimulation of cells with SDF-1α caused an increase in extracellular signal regulated kinase (ERK) phosphorylation in a time-dependent manner. In addition, treatment of A549 cells with ERK inhibitor (PD98059), NF-κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited SDF-1α-induced cells migration and integrins expression. Treatment of A549 cells with SDF-1α induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, IκBα degradation, p65 Ser⁵³⁶ phosphorylation, and κB-luciferase activity. The SDF-1α-mediated increases in IKK α/β phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB-luciferase activity were inhibited by PD98059 and ERK2 mutant. Taken together, these results suggest that SDF-1α acts through CXCR4 to activate ERK, which in turn activates IKKα/β and NF-κB, resulting in the activations of β1 and β3 integrins and contributing the migration of lung cancer cell.

Keywords: Abbreviations; SDF-1; stromal-derived factor-1; ERK; extracellular signal regulated kinase; IKKα/β; IκB kinase α/β; NSCLCs; non-small cell lung cancers; ECM; extracellular matrix; siRNA; small interference RNA; RT-PCR; mRNA analysis by reverse transcriptase-polymerase chain reactionSDF; CXCR4; Lung cancer; Integrin; Migration

Mechanisms underlying the anticancer activities of the angucycline landomycin E by Alla Korynevska; Petra Heffeter; Bohdan Matselyukh; Leonilla Elbling; Michael Micksche; Rostyslav Stoika; Walter Berger (pp. 1713-1726).

Anthracyline antibiotics, produced by Streptomyces sp., still rank among the most efficient anticancer drugs in clinical use. Aim of this study was to gain deeper insight into the anticancer properties of the anthracycline-related angucycline landomycin E (LE). The impact of LE on nuclear morphology was assessed by 4′,6-diamidino-2-phenylindole (DAPI) staining in the human carcinoma cell model KB-3-1. LE treatment led to the appearance of typical morphological signs of programmed cell death like cell shrinkage, chromatin condensation and formation of apoptotic bodies. Apoptotic cell death induced by LE was further characterised by caspase (substrate) cleavage and intense mitochondrial membrane depolarisation (JC-1 and rhodamine 123 staining) already after 1h drug incubation. Moreover, incubation with LE led to reduced intracellular ATP pools suggesting LE-induced apoptotic cell death as a consequence of rapid mitochondrial damage. Furthermore, LE treatment led to profound generation of intracellular oxidative stress, indicated by radical scavenger pre-treatment and dichlorofluorescin diacetate (DCF-DA) staining experiments. Since chemoresistance is a common problem in cancer therapy, we also investigated the influence of ABCB1 (P-glycoprotein, P-gp), ABCC1 (multidrug resistance-related protein, MRP1) and ABCG2 (breast cancer resistance protein, BCRP) overexpression on the anticancer activity of LE. Compared to anthracyclines, cytotoxic activity of LE was only weakly reduced by P-gp and MRP1 overexpression. Moreover, BCRP expression had no influence on LE anticancer activity. In summary, LE exerts anticancer activity via potent induction of apoptosis and has promising anticancer activity even against multidrug resistant (MDR) cells. Taken together, these data suggest further development of LE as a new anticancer drug.

Keywords: Abbreviations; ABC; ATP-binding cassette; ADR; adriamycin (doxorubicin); BCRP; breast cancer resistance protein (ABCG2); BSA; bovine serum albumin; DMSO; dimethyl sulfoxide; DNR; daunomycin; GI; ₅₀; growth inhibition 50%; FACS; fluorescence-activated cell sorting; LE; landomycin E; JC-1; 5,5′,6,6′-tetra-chloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide; MDR; multidrug resistance; MX; mitoxanthrone; MRP; multidrug resistance-related protein (ABCC); NAC; N; -acetyl-; l; -cysteine; PARP; poly(ADP-ribosyl) polymerase; PBS; phosphate-buffered saline; P-gp; P-glycoprotein (ABCB1); PI; propidium iodide; Rh123; rhodamine 123; ROS; reactive oxygen species; TGI; total growth inhibition; Topo; topoisomerase; VP; verapamilLandomycin E; Antibiotic; Adriamycin; Anticancer activity; Apoptosis; Multidrug resistance

Imbalance between CaM kinase II and calcineurin activities impairs caffeine-induced calcium release in hypertrophic cardiomyocytes by Ying-Mei Lu; Norifumi Shioda; Feng Han; Shigeki Moriguchi; Jiro Kasahara; Yasufumi Shirasaki; Zheng-Hong Qin; Kohji Fukunaga (pp. 1727-1737).

Cardiac hypertrophy impairs Ca²⁺ handling in the sarcoplasmic reticulum, thereby impairing cardiac contraction. To identify the mechanisms underlying impaired Ca²⁺ release from the sarcoplasmic reticulum in hypertrophic cardiomyocytes, we assessed Ca²⁺-dependent signaling and the phosphorylation of phospholamban, which regulates Ca²⁺ uptake during myocardial relaxation and is in turn regulated by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and calcineurin. In cultured rat cardiomyocytes, treatment with endothelin-1, angiotensin II, and phenylephrine-induced hypertrophy and increased CaMKII autophosphorylation and calcineurin expression. The calcineurin level reached its maximum at 72h and remained elevated for at least 96h after endothelin-1 or angiotensin II treatment. By contrast, CaMKII autophosphorylation, phospholamban phosphorylation, and caffeine-induced Ca²⁺ mobilization all peaked 48h after these treatments. By 96h after treatment, CaMKII autophosphorylation and phospholamban phosphorylation had returned to baseline, and caffeine-induced Ca²⁺ mobilization was impaired relative to baseline. A similar biphasic change was observed in dystrophin levels in endothelin-1-induced hypertrophic cardiomyocytes, and treatment with the novel CaM antagonists DY-9760e and DY-9836 significantly inhibited the hypertrophy-induced dystrophin breakdown. Taken together, the abnormal Ca²⁺ regulation in cardiomyocytes following hypertrophy is in part mediated by an imbalance in calcineurin and CaMKII activities, which leads to abnormal phospholamban activity.

Keywords: CaMKII; Calcineurin; Hypertrophy; Cardiomyocyte; Phospholamban; DY-9760e

Highly sensitive upregulation of apolipoprotein A-IV by peroxisome proliferator-activated receptor α (PPARα) agonist in human hepatoma cells by Michiaki Nagasawa; Yunike Akasaka; Tomohiro Ide; Tomoko Hara; Naoki Kobayashi; Mari Utsumi; Koji Murakami (pp. 1738-1746).

Peroxisome proliferator-activated receptor α (PPARα) is a key regulator in hepatic lipid metabolism and a potential therapeutic target for dyslipidemia. However, in humans hepatic PPARα-regulated genes remain unclear. To investigate the effect of PPARα agonism on mRNA expressions of lipid metabolism-related genes in human livers, a potent PPARα agonist, KRP-101 (KRP), was used to treat the human hepatoma cell line, HepaRG cells. KRP did not affect AOX or L-PBE, which are involved in peroxisomal β-oxidation. KRP increased L-FABP, CPT1A, VLCAD, and PDK4, which are involved in lipid transport or oxidation. However, the EC₅₀ values (114–2500nM) were >10-fold weaker than the EC₅₀ value (10.9nM) for human PPARα in a transactivation assay. To search for more sensitive genes, we determined the mRNA levels of apolipoproteins, apoA-I, apoA-II, apoA-IV, apoA-V, and apoC-III. KRP had no or little effect on apoA-I, apoC-III, and apoA-II. Interestingly, KRP increased apoA-IV (EC₅₀, 0.99nM) and apoA-V (EC₅₀, 0.29nM) with high sensitivity. We identified apoA-IV as a PPARα-upregulated gene in a study using PPARα siRNA. Moreover, when administered orally to dogs, KRP decreased the serum triglyceride level and increased the serum apoA-IV level in a dose-dependent manner. These findings suggest that apoA-IV, newly identified as a highly sensitive PPARα-regulated gene in human livers, may be one of the mechanisms underlying PPARα agonist-induced triglyceride decrease and HDL elevation.

Keywords: Abbreviations; AOX; acyl-CoA oxidase; apo; apolipoprotein; CHO-K1; Chinese hamster ovary-K1; CPT1A; carnitine palmitoyltransferase 1A; DMEM; Dulbecco's modified Eagle's medium; HDL; high-density lipoprotein; LBD; ligand-binding domain; L-FABP; liver fatty acid binding protein; L-PBE; enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase; LPL; lipoprotein lipase; PDK4; pyruvate dehydrogenase kinase, isozyme 4; PPAR; peroxisome proliferator-activated receptor; PPRE; peroxisome proliferator response element; VLCAD; very long-chain acyl-CoA dehydrogenasePPARα; Apolipoprotein; HDL; Triglyceride; KRP; HepaRG

Antifibrogenic effects of histone deacetylase inhibitors on pancreatic stellate cells by Robin Bülow; Brit Fitzner; Gisela Sparmann; Jörg Emmrich; Stefan Liebe; Robert Jaster (pp. 1747-1757).

Pancreatic stellate cells (PSCs) are essentially involved in pancreatic fibrogenesis and considered as a target for antifibrotic therapies. Here, we have analyzed the effects of three histone deacetylase inhibitors (HDACIs), sodium butyrate, sodium valproate (VPA) and trichostatin A (TSA), on profibrogenic activities of PSC and elucidated molecular targets of HDACI action. Therefore, cultured PSCs were exposed to HDACI. Cell proliferation and viability were assessed by 5-bromo-2′-deoxyuridine (BrdU) incorporation and trypan blue staining assays. Exhibition of the myofibroblastic PSC phenotype was monitored by immunofluorescence analysis of α-smooth muscle actin (α-SMA) expression. [³H]-proline incorporation into acetic acid-soluble proteins was measured to quantify collagen synthesis. Levels of mRNA were determined by quantitative reverse transcriptase real-time PCR. Protein expression, phosphorylation and acetylation were analyzed by immunoblotting, and gel shift assays were performed to study DNA binding of nuclear proteins. HDACI enhanced histone H3 acetylation in a dose-dependent manner. In the same dose range, they strongly inhibited cell proliferation, α-SMA expression and collagen synthesis. A significantly increased rate of cell death was observed in response to TSA at 1μM. While all three HDACI inhibited mRNA expression of endothelin-1, only VPA significantly reduced expression of transforming growth factor-β1. Both mediators exert autocrine profibrogenic effects on PSC. Furthermore, HDACI-treated PSC displayed a diminished DNA binding of AP-1, a key transcription factor in profibrogenic signaling. Together, the results suggest that HDACI exert antifibrogenic effects on PSC. Interruption of AP-1 signaling and autocrine loops enhancing PSC activation might be key mechanisms of HDACI action.

Keywords: Abbreviations; HDACI; histone deacetylase inhibitor; PSC; pancreatic stellate cell; VPA; sodium valproate; TSA; trichostatin A; BrdU; 5-bromo-2′-deoxyuridine; α-SMA; α-smooth muscle actin; ECM; extracellular matrix; HSC; hepatic stellate cell; PDGF; platelet-derived growth factor; TGF; transforming growth factor; ET; endothelin; CTGF; connective tissue growth factor; IFN; interferon; ECL; enhanced chemoluminescence; IMDM; Iscove's modified Dulbecco's medium; HPRT; hypoxanthine-guanine phosphoribosyl transferase; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; EMSA; electrophoretic mobility shift assay; S.E.M.; standard error of the mean; AP-1; activator protein-1; ab; antibodyPancreatic stellate cells; Fibrosis; Histone deacetylase inhibitors; Endothelin-1; Transforming growth factor-β; Activator protein-1

The involvement of AMP-activated protein kinases in the anti-inflammatory effect of nicotine in vivo and in vitro by Pao-Yun Cheng; Yen-Mei Lee; Kwok-Keung Law; Chia-Wei Lin; Mao-Hsiung Yen (pp. 1758-1765).

AMP-activated protein kinase (AMPK) is the downstream component of a kinase cascade that plays a pivotal role in energy homeostasis. AMPK has recently emerged as an attractive and novel target for inflammatory disorders. Thus, the aim of this study was to assess the role of AMPKα in the anti-inflammatory effect of nicotine in carrageenan-induced rat paw edema model and to evaluate the mechanism of nicotine-induced AMPKα phosphorylation in RAW 264.7 cells. The results indicate that nicotine alleviated paw edema and the activation of AMPKα involved in the anti-inflammatory effect of nicotine in vivo. In addition, nicotine was able to activate AMPKα phosphorylation in macrophages and this effect was mediated through nicotinic acetylcholine receptors. Furthermore, nicotine significantly induced the phosphorylation of Akt and the Ca²⁺/calmodulin-dependent protein kinase kinase (CaMKK) protein expression in macrophages. Wortmannin, a specific inhibitor of phosphotidylinositol 3-kinase (PI3K), suppressed nicotine-induced Akt and AMPKα phosphorylation. STO-609, a CaMKK inhibitor, not only inhibited the activation of AMPKα but also suppressed the phosphorylation of Akt induced by nicotine. In conclusion, both of CaMKK and PI3K/Akt pathways are involved in the nicotine-induced AMPKα phosphorylation in macrophages, and the interaction of CaMKK and Akt may exist. AMPKα is a novel and critical component of anti-inflammatory effect of nicotine.

Keywords: Nicotine; AMP-activated protein kinase; iNOS; Carrageenan; Paw edema; CaMKK

Impaired expression and function of breast cancer resistance protein (Bcrp) in brain cortex of streptozocin-induced diabetic rats by Yu-Chun Liu; Hai-Yan Liu; Hui-Wen Yang; Tao Wen; Yang Shang; Xiao-Dong Liu; Lin Xie; Guang-Ji Wang (pp. 1766-1772).

The aim of this study was to investigate whether diabetes mellitus (DM) affected breast cancer resistance protein (Bcrp) function and expression in rat brain. 5-week and 8-week diabetic rats were induced by streptozocin (STZ). Bcrp expression and function in brain cortex were assessed by western blot and measuring the brain-to-plasma concentration ratios of two typical substrates prazosin and cimetidine, respectively. The diabetic rats were treated with three different agents insulin, aminoguanidine (AG) and metformin (MET). It was found that the brain-to-plasma ratios of prazosin and cimetidine in diabetic rats were significantly higher than those of control rats, which were dependent on duration of diabetes. Lower levels of Bcrp were found in brain cortex of diabetic rats, which were in parallel with increase of brain-to-plasma ratios. Insulin treatment may attenuate the impairment of Bcrp expression and function induced by diabetes. Aminoguanidine and metformin treatment did not prevent the impairment of Bcrp function and expression in brain cortex of diabetic rats. All results gave a conclusion that STZ-induced DM may induce the impairment of function and expression of Bcrp in brain cortex, and lower levels of insulin may mainly contribute to Bcrp dysfunction in brain.

Keywords: Abbreviations; Bcrp/ABCG2breast; cancer resistance protein; AG; aminoguanidine; MET; metformin; DM; diabetes mellitus; BBB; blood–brain barrier; STZ; streptozocin; P-gp; P-glycoprotein; MDR; multidrug resistance; MRPs; multidrug resistance-associated proteins; i.p.; intraperitoneally; i.v.; intravenously; AGEs; advanced glycation end products; ANOVA; analysis of variance; S.D.; standard deviation; CYPs; cytochrome P450; BSA; bovine serum albumin; ABCG1; ATP-binding cassette transporter G1Breast cancer resistance protein; Diabetes mellitus; Prazosin; Cimetidine; Insulin; Blood–brain barrier

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Biochemical Pharmacology (v.74, #12)

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Biochemical Pharmacology (v.74, #12)