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Biochemical Pharmacology (v.81, #8)

Editorial Advisory Board (pp. iii).

Friendly, and not so friendly, roles of Rac1 in islet β-cell function: Lessons learnt from pharmacological and molecular biological approaches by Anjaneyulu Kowluru (pp. 965-975).

A growing body of evidence from multiple laboratories implicated Rac1 in physiological insulin secretion. Emerging evidence is also suggestive of potential involvement of Rac1 in the metabolic dysfunction of the islet β-cell induced by high glucose, palmitate, ceramide or cytokines, specifically at the level of phagocyte-like NADPH oxidase-mediated generation of ROS and associated mitochondrial dysfunction. Potential avenues for future research including development of inhibitors to halt the Rac1–Nox activation and generation of oxidative stress leading to the metabolic dysfunction of the β-cell are also discussed herein.Glucose-stimulated insulin secretion [GSIS] involves a sequence of metabolic events leading to small G-protein [e.g., Rac1]-mediated cytoskeletal remodeling to promote granule mobilization toward the plasma membrane for fusion and release of insulin. Existing evidence supports a positive modulatory role for Rac1 in GSIS. Specific regulatory factors of Rac1 function, including the guanine nucleotide exchange factors [e.g., Tiam1] have also been identified and studied in the islet. Inhibition of Tiam1/Rac1 signaling axis attenuates GSIS suggesting its pivotal role in insulin secretion. In addition to its positive [i.e., friendly] roles in GSIS, Rac1 also plays “non-friendly” role[s] in the islet function. For example, it up-regulates the intracellular reactive oxygen species [ROS] levels via activation of phagocyte-like NADPH oxidase [Nox]. Despite the emerging evidence that a tonic increase in intracellular ROS is necessary for GSIS, experimental evidence also suggests that chronic exposure of β-cells to high glucose, palmitate or cytokines results in the onset of oxidative stress leading to reduction in mitochondrial membrane potential, cytosolic accumulation of cytochrome C and activation of caspase-3 leading to β-cell apoptosis. Pharmacological and molecular biological inhibition of Rac1 activation affords partial protection against Nox-induced oxidative stress and mitochondrial dysfunction induced by elevated glucose, lipids or cytokines. Herein, we overview the existing evidence to suggest positive as well as negative modulatory roles of Rac1 in islet function. Potential avenues for future research including development of inhibitors to halt the Rac1–Nox activation and generation of oxidative stress leading to the metabolic dysfunction of the β-cell are discussed.

Keywords: Abbreviations; CER; ceramide; DPI; diphenyleneiodonium chloride; FTase; farnesyltransferase; FTI; farnesyltransferase inhibitor; GDI; GDP-dissociation inhibitor; GEF; guanine nucleotide exchange factors; GGTase; geranylgeranyltransferase; GGT1; geranylgeranyltransferase inhibitor; GSE; grape seed extract; GSIS; glucose-stimulated insulin secretion; Nox; phagocyte-NADPH-oxidase; OLETF rat; Otsuka Long-Evans Tokushima fatty rat; Rac1; Ras-related C3 botulinum toxin substrate 1; ROS; reactive oxygen species; and Tiam1; T-lymphoma invasion and metastasis1Rac1; Tiam1; NADPH-oxidase; Pancreatic islet; Mitochondrial dysfunction; Insulin secretion

In search of the Holy Grail: Folate-targeted nanoparticles for cancer therapy by Alfonso Garcia-Bennett; Matthias Nees; Bengt Fadeel (pp. 976-984).

Targeted drug therapy or “smart” drug delivery, potentially combined with simultaneous imaging modalities to monitor the delivery of drugs to specific tissues, is arguably the “holy grail” of pharmacology. Therapeutic approaches that exploit nanoparticles to deliver drugs selectively to cancer cells are currently considered one of the most promising avenues in the area of cancer therapeutics and imaging. The potential to deliver active chemotherapeutic drugs in the vicinity or directly within specific tumors via receptor mediated pathways, and to image tumors through the use of nanoparticles has been conceptually and experimentally shown for several classes of nanoparticles. Nanoparticles functionalized with the vitamin folic acid are of particular interest as a variety of malignant tumors are known to overexpress the folate receptor(s). Indeed, several nanoparticle architectures with improved retention time, administration route, biocompatibility, absorption, and clearance are being proposed and are in late stage clinical development. This commentary highlights some of the most important concepts related to nanoparticles and folate-mediated drug delivery and imaging in cancer research.

Keywords: Abbreviations; NPs; nanoparticles; QD; quantum dot; MRI; magnetic resonance imaging; SPIONs; superparamagnetic iron oxide nanoparticles; FOLR; folate receptor; EPR; enhanced permeability and retention; PEG; polyethylene glycolNanoparticle; Folic acid; Targeted delivery; Toxicity

The novel synthetic ether lipid inositol-C2-PAF inhibits phosphorylation of the tyrosine kinases Src and FAK independent of integrin activation in transformed skin cells by Geo Semini; Annette Hildmann; Hans-Ulrich Reissig; Werner Reutter; Kerstin Danker (pp. 985-995).

Ino-C2-PAF at least partially uncouples integrin-mediated attachment from subsequent integrin-dependent signaling steps, which inhibits migration in transformed keratinocyte cell lines.New alkyl-phospholipids that are structurally derived from platelet-activating factor are promising candidates for anticancer treatment. The mechanism of action of derivatives of the platelet-activating factor is distinctly different from that of known DNA- or tubulin-targeting anticancer agents because they are incorporated into cell membranes, where they accumulate and interfere with a wide variety of key enzymes. We recently presented evidence of a novel group of alkyl-phospholipids, glycosidated phospholipids that efficiently inhibit cell proliferation. One member of this group, inositol-C2-PAF (Ino-C2-PAF), displays high efficacy and low cytotoxicity in HaCaT-cells, an immortalized non-tumorigenic skin keratinocyte cell line.Here, we show that Ino-C2-PAF also inhibits the motility of the skin-derived transformed cell lines HaCaT and squamous cell carcinoma (SCC)-25. This decrease in motility is accompanied by an altered F-actin cytoskeleton, increased clustering of integrins, and increased cell–matrix adhesion. Despite enhanced integrin clustering and matrix adhesion, we observed less phosphorylation of the cytoplasmic tyrosine kinases focal adhesion kinase (FAK) and Src, key regulators of cellular motility, at focal adhesion sites. Transient transfection of constitutively active variants of FAK and Src could at least in part bybass this inhibitory effect of Ino-C2-PAF. This fact indicates that Ino-C2-PAF interferes with the fine-tuned balance between adhesion and migration. Ino-C2-PAF at least partially uncouples integrin-mediated attachment from subsequent integrin-dependent signaling steps, which inhibits migration in transformed keratinocyte cell lines.

Keywords: Glycosidated phospholipid; Ino-C2-PAF; Migration; Adhesion; Integrin; SCC-25 cells; HaCaT cells

Opioid-like compound exerts anti-fibrotic activity via decreased hepatic stellate cell activation and inflammation by Stephani A. Day; Ashley M. Lakner; Cathy C. Moore; Mao-Hsiung Yen; Mark G. Clemens; Edwin S. Wu; Laura W. Schrum (pp. 996-1003).

Hepatic fibrosis is characterized by excess type I collagen deposition and exacerbated inflammatory response. Naltrexone, an opioid receptor antagonist used for treating alcohol abuse, attenuates hepatocellular injury in fibrotic animal models, which can be accompanied by deleterious side effects. Additionally, opioid neurotransmission is upregulated in patients with inflammatory liver disease. Several derivatives of Naltrexone, Nalmefene (Nal) and JKB-119, exert immunomodulatory activity; however, unlike Nal, JKB-119 does not show significant opioid receptor antagonism. To delineate the potential hepatoprotective effects of these compounds, we investigated if JKB-119 and Nal could modulate activation of hepatic stellate cells (HSCs), primary effector cells that secrete type I collagen and inflammatory mediators during liver injury. Our results demonstrated that Nal or JKB-119 treatment decreased smooth muscle α-actin, a marker of HSC activation, mRNA and protein expression. Despite decreased collagen mRNA expression, both compounds increased intracellular collagen protein expression; however, inhibition of collagen secretion was observed. To address a possible mechanism for suppressed collagen secretion or retention of intracellular collagen, endoplasmic (ER) protein expression and matrix metalloproteinase (MMP) activity were examined. While no change in ER protein expression (Grp78, PDI, Hsp47) was observed, MMP13 mRNA expression was dramatically increased. In an acute LPS inflammatory injury animal model, JKB-119 treatment decreased liver injury (ALT), plasma TNFα and PMN liver infiltration. Overall, these results suggest that JKB-119 can directly inhibit HSC activation attributed to anti-inflammatory activity and may, therefore, attenuate inflammation associated with HSC activation and liver disease.

Keywords: Hepatic stellate cell; Opioid; Inflammation; Fibrosis; Nalmefene

IL-1β potently stabilizes IL-6 mRNA in human astrocytes by Anneleen Spooren; Pieter Mestdagh; Pieter Rondou; Krzysztof Kolmus; Guy Haegeman; Sarah Gerlo (pp. 1004-1015).

Uncontrolled expression of IL-6 in the central nervous system is associated with neurodegenerative pathology and glioma development. Astrocytes are the predominant source of IL-6 in the central nervous system, and they are characteristically susceptible to synergistic IL-6 expression. Combined β-adrenergic and TNF-receptor triggering induces synergistic IL-6 expression in 1321N1 cells via a transcriptional enhancer mechanism. Here, we have investigated the molecular basis of the very potent “super”-synergistic IL-6 expression that is apparent after combined treatment of astrocytes with a β-adrenergic agonist, isoproterenol, and the inflammatory cytokines TNF-α and IL-1β. We found that IL-1β treatment strengthens the IL-6 synergy by inducing a distinct stabilization of IL-6 mRNA. Surprisingly, the mRNA-stabilizing effect seems to be dependent on protein kinase C (PKC), but not on the prototypical mRNA-stabilizing kinase p38. Moreover, although the mRNA-binding protein HuR basally stabilizes IL-6 mRNA, the mRNA-stabilizing effect of IL-1β is independent of HuR. Our data using pharmacological inhibitors suggest PKC is an important modulator of IL-6 expression in the central nervous system and this might have therapeutic implications.

Keywords: Abbreviations; ARE; AU-rich element; CNS; central nervous system; COX-2; cyclo-oxygenase 2; ELAV; embryonic lethal abnormal vision; HuR; human antigen R; IL-6; interleukin-6; IL-1β; interleukin-1β; iso; isoproterenol; MAPK; mitogen-activated protein kinase; miRNA; microRNA; PKC; protein kinase C; TNF; tumor necrosis factor; 3′UTR; 3′ untranslated region; veh; vehiclemRNA stability; IL-6; Astrocytes; IL-1β; PKC; p38

Arf nucleotide binding site opener [ARNO] promotes sequential activation of Arf6, Cdc42 and Rac1 and insulin secretion in INS 832/13 β-cells and rat islets by Bhavaani Jayaram; Ismail Syed; Chandrashekara N. Kyathanahalli; Christopher J. Rhodes; Anjaneyulu Kowluru (pp. 1016-1027).

A proposed model for the involvement of ARNO signaling axis in GSIS via sequential activation of Arf6, Cdc42 and Rac1 in pancreatic β-cells.Glucose-stimulated insulin secretion [GSIS] involves interplay between small G-proteins and their regulatory factors. Herein, we tested the hypothesis that Arf nucleotide binding site opener [ARNO], a guanine nucleotide-exchange factor [GEF] for the small G-protein Arf6, mediates the functional activation of Arf6, and that ARNO/Arf6 signaling axis, in turn, controls the activation of Cdc42 and Rac1, which have been implicated in GSIS. Molecular biological [i.e., expression of inactive mutants or siRNA] and pharmacological approaches were employed to assess the roles for ARNO/Arf6 signaling pathway in insulin secretion in normal rat islets and INS 832/13 cells. Degrees of activation of Arf6 and Cdc42/Rac1 were quantitated by GST-GGA3 and PAK-1 kinase pull-down assays, respectively. ARNO is expressed in INS 832/13 cells, rat islets and human islets. Expression of inactive mutants of Arf6 [Arf6-T27N] or ARNO [ARNO-E156K] or siRNA-ARNO markedly reduced GSIS in isolated β-cells. SecinH3, a selective inhibitor of ARNO/Arf6 signaling axis, also inhibited GSIS in INS 832/13 cells and rat islets. Stimulatory concentrations of glucose promoted Arf6 activation, which was inhibited by secinH3 or siRNA-ARNO, suggesting that ARNO/Arf6 signaling cascade is necessary for GSIS. SecinH3 or siRNA-ARNO also inhibited glucose-induced activation of Cdc42 and Rac1 suggesting that ARNO/Arf6 might be upstream to Cdc42 and Rac1 activation steps, which are necessary for GSIS. Lastly, co-immunoprecipitation and confocal microscopic studies suggested increased association between Arf6 and ARNO in glucose-stimulated β-cells. These findings provide the first evidence to implicate ARNO in the sequential activation of Arf6, Cdc42 and Rac1 culminating in GSIS.

Keywords: Abbreviations; Arf6; ADP-ribosylation factor 6; ARNO; Arf nucleotide binding site opener; GAP; GTPase activating protein; GDI; GDP-dissociation inhibitor; GEF; guanine nucleotide-exchange factor; GGA3; Golgi-localized c-ear homology domain Arf-binding protein-3; GSIS; glucose-stimulated insulin secretion; PA; phosphatidic acid; PIP; ₂; phosphatidylinositol-4,5-bisphosphate; PLD; phospholipase-DInsulin secretion; Pancreatic islet; ARNO; Arf6; Rac1; SecinH3

Anti-diabetic and anti-adipogenic effects of a novel selective 11β-hydroxysteroid dehydrogenase type 1 inhibitor, 2-(3-benzoyl)-4-hydroxy-1,1-dioxo-2H-1,2-benzothiazine-2-yl-1-phenylethanone (KR-66344) by Ji Seon Park; Sang Dal Rhee; Nam Sook Kang; Won Hoon Jung; Hee Youn Kim; Jun Hyoung Kim; Seung Kyu Kang; Hyae Gyeong Cheon; Jin Hee Ahn; Ki Young Kim (pp. 1028-1035).

The selective inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) have considerable potential for treating type 2 diabetes mellitus and metabolic syndrome. In the present study, we investigated the anti-diabetic and anti-adipogenic effects of 2-(3-benzoyl)-4-hydroxy-1,1-dioxo-2H-1,2-benzothiazine-2-yl-1-phenylethanone (KR-66344), as a 11β-HSD1 inhibitor; we also investigated the underlying molecular mechanisms in the cortisone-induced 3T3-L1 adipogenesis model system and C57BL/6-Lep^ob/ob mice. KR-66344 concentration-dependently inhibited 11β-HSD1 activity in human liver microsome, mouse C2C12 myotube and human SW982 cells. In the C57BL/6-Lep^ob/ob mice study, the administration of KR-66344 (200mg/kg/d, orally for 5 days) improved the glucose intolerance as determined by the oral glucose tolerance test, in which the area under the curve (AUC) of the plasma glucose concentration was significantly reduced by 27% compared with the vehicle treated group. Further, KR-66344 suppressed adipocyte differentiation on cortisone-induced adipogenesis in 3T3-L1 cells is associated with the suppression of the cortisone-induced mRNA levels of FABP4, G3PD, PPARγ2 and Glut4, and 11β-HSD1 expression and activity. Our results additionally demonstrate evidence showing that KR-66344 improved glycemic control and inhibited adipogenesis via 11β-HSD1 enzyme activity. Taken together, these results may provide evidence of the therapeutic potential of KR-66344, as a 11β-HSD1 inhibitor, in obesity and type 2 diabetes patients with metabolic syndrome.

Keywords: Abbreviations; 11β-HSD1; 11β-hydroxysteroid dehydrogenase type 1; G3PD; glycerol-3-phosphate dehydrogenase; PPARγ; peroxisome proliferator-activated receptor γ; FABP4; fatty acid-binding protein 4; GLUT4; glucose transporter 411β-Hydroxysteroid dehydrogenase type 1; Type 2 diabetes; Metabolic syndrome; Adipogenesis; Glucose tolerance

Dose-dependent differential effects of risedronate on gene expression in osteoblasts by J. Wang; P.H. Stern (pp. 1036-1042).

Bisphosphonates have multiple effects on bone. Their actions on osteoclasts lead to inhibition of bone resorption, at least partially through apoptosis. Effects on osteoblasts vary, with modifications in the molecule and concentration both resulting in qualitatively different responses. To understand the mechanism of the differential effects of high and low bisphosphonate concentrations on osteoblast activity, we compared the effects of 10⁻⁸M and 10⁻⁴M risedronate on gene expression in UMR-106 rat osteoblastic cells. Two targeted arrays, an 84-gene signaling array and an 84-gene osteogeneic array were used. Gene expression was measured at 1 and 24h. Although some genes were regulated similarly by low and high concentrations of the drug, there was also differential regulation. At 1h, 11 genes (1 signaling and 10 osteogenesis) were solely regulated by the low concentration, and 7 genes (3 signaling, 4 osteogenesis) were solely regulated by the high concentration. At 24h, 8 genes (3 signaling, 5 osteogenesis) were solely regulated by the low concentration and 30 genes (16 signaling and 14 osteogenesis) were solely regulated by the high concentration. Interestingly, the low, but not the high concentration of risedronate transiently and selectively upregulated several genes associated with cell differentiation. A number of genes related to apoptosis were regulated, and could be involved in effects of bisphosphonates to promote osteoblast apoptosis. Also, observed gene changes associated with decreased angiogenesis and decreased metastasis could, if they occur in other cell types, provide a basis for the effectiveness of bisphosphonates in the prevention of cancer metastases.

Keywords: Bisphosphonate; Risedronate; Osteoblast; Gene; Bone

A comprehensive understanding of thioTEPA metabolism in the mouse using UPLC–ESI-QTOFMS-based metabolomics by Fei Li; Andrew D. Patterson; Constance C. Höfer; Kristopher W. Krausz; Frank J. Gonzalez; Jeffrey R. Idle (pp. 1043-1053).

ThioTEPA, an alkylating agent with anti-tumor activity, has been used as an effective anticancer drug since the 1950s. However, a complete understanding of how its alkylating activity relates to clinical efficacy has not been achieved, the total urinary excretion of thioTEPA and its metabolites is not resolved, and the mechanism of formation of the potentially toxic metabolites S-carboxymethylcysteine (SCMC) and thiodiglycolic acid (TDGA) remains unclear. In this study, the metabolism of thioTEPA in a mouse model was comprehensively investigated using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC–ESI-QTOFMS) based-metabolomics. The nine metabolites identified in mouse urine suggest that thioTEPA underwent ring-opening, N-dechloroethylation, and conjugation reactions in vivo. SCMC and TDGA, two downstream thioTEPA metabolites, were produced from thioTEPA from two novel metabolites 1,2,3-trichloroTEPA (VII) and dechloroethyltrichloroTEPA (VIII). SCMC and TDGA excretion were increased about 4-fold and 2-fold, respectively, in urine following the thioTEPA treatment. The main mouse metabolites of thioTEPA in vivo were TEPA (II), monochloroTEPA (III) and thioTEPA-mercapturate (IV). In addition, five thioTEPA metabolites were detected in serum and all shared similar disposition. Although thioTEPA has a unique chemical structure which is not maintained in the majority of its metabolites, metabolomic analysis of its biotransformation greatly contributed to the investigation of thioTEPA metabolism in vivo, and provides useful information to understand comprehensively the pharmacological activity and potential toxicity of thioTEPA in the clinic.

Keywords: ThioTEPA; Metabolomics; Ultraperformance liquid chromatography; Time-of-flight mass spectrometry; Tandem mass spectrometry

Statin or fibrate chronic treatment modifies the proteomic profile of rat skeletal muscle by Giulia Maria Camerino; Maria Antonietta Pellegrino; Lorenza Brocca; Claudio Digennaro; Diana Conte Camerino; Sabata Pierno; Roberto Bottinelli (pp. 1054-1064).

Molecular mechanisms involved in atorvastatin, fluvastatin and fenofibrate action in skeletal muscle. Statins and fenofibrate impair energy production systems by affecting different oxidative and glycolytic enzymes.Statins and fibrates can cause myopathy. To further understand the causes of the damage we performed a proteome analysis in fast-twitch skeletal muscle of rats chronically treated with different hypolipidemic drugs. The proteomic maps were obtained from extensor digitorum longus (EDL) muscles of rats treated for 2-months with 10mg/kg atorvastatin, 20mg/kg fluvastatin, 60mg/kg fenofibrate and control rats. The proteins differentially expressed were identified by mass spectrometry and further analyzed by immunoblot analysis. We found a significant modification in 40 out of 417 total spots analyzed in atorvastatin treated rats, 15 out of 436 total spots in fluvastatin treated rats and 21 out of 439 total spots in fenofibrate treated rats in comparison to controls. All treatments induced a general tendency to a down-regulation of protein expression; in particular, atorvastatin affected the protein pattern more extensively with respect to the other treatments. Energy production systems, both oxidative and glycolytic enzymes and creatine kinase, were down-regulated following atorvastatin administration, whereas fenofibrate determined mostly alterations in glycolytic enzymes and creatine kinase, oxidative enzymes being relatively spared. Additionally, all treatments resulted in some modifications of proteins involved in cellular defenses against oxidative stress, such as heat shock proteins, and of myofibrillar proteins. These results were confirmed by immunoblot analysis. In conclusions, the proteomic analysis showed that either statin or fibrate administration can modify the expression of proteins essential for skeletal muscle function suggesting potential mechanisms for statin myopathy.

Keywords: Hypolipidemic drugs; Skeletal muscle; Side effects; Myopathy; Proteomic analysis

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Biochemical Pharmacology (v.81, #8)

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Biochemical Pharmacology (v.81, #8)