Check out our New Publishers' Select for Free Articles

Biochemical Pharmacology (v.79, #11)

Graphical Abstracts Contents (pp. e1-e5).

Editorial Advisory Board (pp. iii).

Ryanodine receptor calcium channels and their partners as drug targets by John J. Mackrill (pp. 1535-1543).

Ryanodine receptors (RyRs) are high conductance intracellular cation channels that release calcium ions from stores such as the endoplasmic reticulum and sarcoplasmic reticulum. Although RyRs are expressed in many cell types, their roles have only been extensively characterised in tissues in which they are abundant: RyR1 is essential for excitation–contraction coupling in skeletal muscle; whereas RyR2 is required for the analogous signal transduction pathway in heart. Defects in RyR1 cause malignant hyperthermia and a spectrum of myopathies in skeletal muscle; whereas RyR2 dysregulation can result in fatal cardiac arrhythmias and is involved in heart failure. Altered RyR gating has been implicated in a range of other diseases, including epilepsy, neurodegeneration, pain and cancer. RyRs interact with a range of toxic substances, providing insights into their functional and structural properties. Consequently, these channel complexes represent potential therapeutic targets for treatment of numerous diseases. Furthermore, strategies for combating multicellular parasites and agricultural pests could exploit pharmacological differences between their RyRs and those of vertebrates. However, available pharmacological tools for manipulation of RyR gating are generally unsuitable for clinical, veterinary or agricultural use, owing to their lack of selectivity, inappropriate solubility in the aqueous or lipid environment, or generation of side-effects. The expression, subcellular distribution and gating of RyRs is modified by a wide variety of cellular proteins, some of which are expressed in a developmentally or tissue-restricted manner. This commentary examines the possibility of manipulating the expression and function of such proteins in order develop new drugs acting on RyR channel complexes.

Keywords: Abbreviations; FKBP12, FKBP12.6; 12/12.6; kDa FK506-binding proteins; AKAP; a-kinase anchoring protein; AVRD2; arrhythmogenic right ventricular dysplasia type 2; CASQ; calsequestrin; CREB; cAMP response element binding protein; CREM; cAMP response element modulator; CPVT1; catecholaminergic polymorphic ventricular tachycardia type 1; CCD; central core disease; ER; endoplasmic reticulum; EC-; excitation–contraction; HF; heart failure; InsP; ₃; Rs; inositol 1,4,5-trisphosphate receptors; MHS; malignant hyperthermia susceptibility; MTMR14; myotubularin-related protein 14; RyR; ryanodine receptor; SR; sarcoplasmic reticulum; Sp1; specificity protein-1Ryanodine receptor; Calcium channel; Interacting protein; Drug target; Disease

Emerging immunotherapies targeting CD30 in Hodgkin's lymphoma by Hans-Peter Gerber (pp. 1544-1552).

Complex nature of HL.The immunotherapy of Hodgkin's lymphoma (HL) has been particularly challenging because of the unique features of tumor intrinsic and host mediated factors, interfering with the antitumor activities of therapeutic antibodies. Despite a wide array of compounds tested successfully in preclinical studies, immunotherapy in HL patients resulted in only limited success when compared to the significant improvements in patient survival provided by chemotherapeutic agents. Antibody–drug conjugates (ADCs) may surmount the restrictions posed by the unique pathobiology of HL tumors as they combine the selective tumor targeting of monoclonal antibodies with the potent anti-neoplastic activities of cytotoxic drugs. In early clinical trials, this class of compounds induced robust antitumor effects in patients with relapsed or refractory lymphoproliferative diseases, in the absence of overt toxicities, while naked antibodies failed to induce therapeutic benefit. Here we review some of the unique features of HL tumor biology and the key advantages of ADC-based lymphoma therapies, which may ultimately account for the improved therapeutic benefit provided by ADCs compared to first generation immunotherapeutics tested in HL patients.

Keywords: Cancer; Tumor necrosis factor (TNF); Hodgkin's lymphoma (HL); Anaplastic large cell lymphomas (ALCL); Hematologic malignancies; Lymphoproliferative diseases; Reed–Sternberg cells; Antibody–drug conjugates; Immunotoxins; Immune evasion

Reductive activation of the prodrug 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) selectively occurs in oxygen-deficient cells and overcomes O⁶-alkylguanine-DNA alkyltransferase mediated KS119 tumor cell resistance by Raymond P. Baumann; Philip G. Penketh; Kimiko Ishiguro; Krishnamurthy Shyam; Yong L. Zhu; Alan C. Sartorelli (pp. 1553-1561).

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) is a prodrug of the 1,2-bis(sulfonyl)hydrazine class of antineoplastic agents designed to exploit the oxygen-deficient regions of cancerous tissue. Thus, under reductive conditions in hypoxic cells this agent decomposes to produce the reactive intermediate 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE), which in turn generates products that alkylate the O⁶-position of guanine in DNA. Comparison of the cytotoxicity of KS119 in cultured cells lacking O⁶-alkylguanine-DNA alkyltransferase (AGT) to an agent such as Onrigin™, which through base catalyzed activation produces the same critical DNA G-C cross-link lesions by the generation of 90CE, indicates that KS119 is substantially more potent than Onrigin™ under conditions of oxygen deficiency, despite being incompletely activated. In cell lines expressing relatively large amounts of AGT, the design of the prodrug KS119, which requires intracellular activation by reductase enzymes to produce a cytotoxic effect, results in an ability to overcome resistance derived from the expression of AGT. This appears to derive from the ability of a small portion of the chloroethylating species produced by the activation of KS119 to slip through the cellular protection afforded by AGT to generate the few DNA G-C cross-links that are required for tumor cell lethality. The findings also demonstrate that activation of KS119 under oxygen-deficient conditions is ubiquitous, occurring in all of the cell lines tested thus far, suggesting that the enzymes required for reductive activation of this agent are widely distributed in many different tumor types.

Keywords: Abbreviations; AGT; O; ⁶; -alkylguanine-DNA alkyltransferase; 90CE; 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine; KS119; 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine; Onrigin™; 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylaminocarbonyl)hydrazine; O; ⁶; -BG; O; ⁶; -benzylguanineOxygen-deficient cells; O; ⁶; -Alkylguanine-DNA alkyltransferase; 1,2-Bis(sulfonyl)hydrazines; KS119; Onrigin™

Synchronised phosphorylation of BNIP3, Bcl-2 and Bcl-xL in response to microtubule-active drugs is JNK-independent and requires a mitotic kinase by Howard R. Mellor; Kasper M. Rouschop; Simon M. Wigfield; Bradly G. Wouters; Adrian L. Harris (pp. 1562-1572).

BNIP3 is a hypoxia-inducible BH3-only member of the Bcl-2 family of proteins that regulate apoptosis and autophagy. However the role of BNIP3 in the hypoxia response has proved difficult to define and remains controversial. In this study we show that in cancer cells, knockdown or forced expression of BNIP3 fails to modulate cell survival under hypoxic or normoxic conditions. However, we demonstrate that BNIP3 is regulated post-translationally, existing as multiple monomeric and dimeric phosphorylated forms. Upon treatment with microtubule inhibitors, but not other classes of chemotherapeutics, BNIP3 becomes hyperphosphorylated. We demonstrate that the phosphorylation of BNIP3 occurs in synchrony with phosphorylation of its binding partners Bcl-2 and Bcl-xL. Microtubule inhibitor-induced phosphorylation of these proteins occurs independently of the AKT/mTor and JNK kinase pathways and requires Mps1 mitotic checkpoint kinase activity. Inhibition of mitotic arrest in the presence of paclitaxel blocks the phosphorylation of BNIP3, Bcl-2 and Bcl-xL, demonstrating that these proteins are phosphorylated by a mitochondrially active mitotic kinase. We show that phosphorylation increases the stability of BNIP3 and that BNIP3 predominantly interacts with the phosphorylated form of Bcl-2. This study provides new insight into the post-translational functional control of these Bcl-2 family members.

Keywords: Abbreviations; BAD; Bcl-2-associated death promoter; Bcl-2; B-cell CLL/lymphoma 2; Bcl-xL; B-cell leukemia XL; Bcl-w; B-cell leukemia w; BID; Bcl-2 interacting domain; BIK; BCL2-interacting killer (apoptosis-inducing); BNIP3; BCL2/adenovirus E1B 19; kDa protein-interacting protein 3; BNIP3L; BCL2/adenovirus E1B 19; kDa protein-interacting protein 3 like; DMEM; Dulbecco's Modified Eagle Medium; DTT; dithiothreitol; ECL; enhanced chemoilluminescence; FBS; foetal bovine serum; HIF-1; hypoxia-inducible factor-1; HRK; harakiri; HRP; horse radish peroxidase; Mcl-1; myeloid cell leukemia-1; NOXA; Phorbol-12-myristate-13-acetate-induced protein 1; NP40; nonyl phenoxylpolyethoxylethanol; PUMA; p53-upregulated modulator of apoptosis; RIPA; radioimmunoprecipitation assay; RPMI; Roswell Park Memorial Institute MediumBNIP3; Bcl-2; Paclitaxel; Phosphorylation; Mitosis; Hypoxia

Inhibition of inducible nitric oxide synthase by bis(helenalinyl)glutarate in RAW264.7 macrophages by V. Badireenath Konkimalla; Martina Blunder; Rudolf Bauer; Thomas Efferth (pp. 1573-1580).

Screening a phytochemical library for novel nitric oxide (NO) inhibitors, we identified bis(helalinyl)glutarate (BHG) as candidate compound. Indeed, BHG inhibited NO production and expression of inducible NO synthase. The glucocorticoid receptor and interleukin-1 and interleukin-10 signaling pathways were found as possible modes of action of BHG by means of mRNA microarray hybridization.Nitric oxide (NO) plays a role in various physiological and pathophysiological conditions such as immunoregulatory and inflammatory processes. Hence, NO and its generating enzyme, inducible nitric oxide synthase (iNOS) may not only be of diagnostic and prognostic value, but may also serve as targets for novel therapeutic options. In the present investigation, we have screened a phytochemical library by correlating the IC₅₀ values for 531 natural products of 60 cell lines with the microarray-based mRNA expression of 95 genes known to be involved in NO metabolism and signaling with the aim to identify candidate compounds as inhibitors for NO metabolism and signaling. We identified bis(helenalinyl)glutarate (BHG) as putative candidate compound. Indeed, BHG inhibited NO production (IC₅₀ value: 0.90±0.04μM) and down-regulated iNOS protein expression (IC₅₀ value: 1.12±0.16μM) of RAW264.7 mouse macrophages in the presence of lipopolysaccharide and interferon-γ. Performing XTT cytotoxicity assays, we found that BHG inhibited cell growth in a dose-dependent manner with an IC₅₀ value of 5.6μM. To gain insight into molecular pathways involved in NO inhibition and cytotoxicity, we performed microarray experiments which were exemplarily validated by real-time RT-PCR. A total of 227 genes (67 up- and 160 down-regulated) were obtained, which exhibited significant differences in mRNA regulation between BHG-treated and untreated RAW264.7 macrophages. Sixteen of 227 genes are known to be involved in NO-signaling. Pathway analyses revealed that further five and four down-regulated genes belong to the glucocorticoid receptor and interleukin-1 and interleukin-10 pathways, respectively. An interference of these two pathways and NO is known for inflammation and auto-immune diseases. The therapeutic potential of this compound has to be explored in the future.

Keywords: Cytotoxicity; Glucocorticoid receptor signaling pathway; Griess; assay; Interleukin-10 signaling pathway; Microarrays; Nitric oxide

A simple yeast-based system for analyzing inhibitor resistance in the human cancer drug targets Hsp90α/β by Stefan H. Millson; Chrisostomos Prodromou; Peter W. Piper (pp. 1581-1588).

Heat shock protein 90 (Hsp90), a highly conserved molecular chaperone, is one of the most promising targets for cancer drug development. Whether any resistance to these Hsp90 inhibitor drugs could arise by Hsp90 mutation is still unknown. Yeast is readily engineered so that its essential Hsp90 function is provided by either isoform of the human cytosolic Hsp90, Hsp90α or Hsp90β. However, its high intrinsic resistance to most drugs poses a major obstacle to the use of such Hsp90α- or Hsp90β-expressing yeast cells as a model system to analyse whether drug resistance might arise by Hsp90 mutation. In order to overcome this problem, we have generated a strain that is both hypersensitive to Hsp90 inhibitors as it lacks multiple drug resistance genes, and in which different heterologous and mutant Hsp90s can be expressed by plasmid exchange. It is not rendered appreciably stress sensitive when made to express Hsp90α or Hsp90β as its sole form of Hsp90. Should there be any development of resistance to the Hsp90 drugs now in cancer clinic trials, this system can provide a rapid initial test of whether any single nucleotide polymorphism appearing within the coding regions of Hsp90α or Hsp90β could be a contributory factor in this resistance. We have used this strain to demonstrate that significant levels of resistance to the Hsp90 inhibitors radicicol and 17-allylamino-demethoxygeldanamycin (17-AAG) are generated as a result of the same single point mutation within the native Hsp90 of yeast (A107N), the human Hsp90α (A121N) and the human Hsp90β (A116N).

Keywords: Abbreviations; RAD; radicicol; GdA; geldanamycin; 17-AAG; 17-allylamino-demethoxygeldanamycin; DMSO; dimethylsulphoxide; 5-FOA; 5-fluoroorotic acidHsp90; Molecular chaperone; Cancer; Drug target; Drug resistance; Yeast

Bax deficiency mediated drug resistance can be reversed by endoplasmic reticulum stress induced death signaling by Bhavya Balan Chandrika; Sathish Kumar Maney; Swathi U. Lekshmi; Jeena Joseph; Mahendra Seervi; Praveen K.S.; Santhoshkumar T.R. (pp. 1589-1599).

Tumors often acquire drug resistance due to functional loss of pro apoptotic gene Bax, a critical and essential component of cell death rendering them insensitive to most anti-tumor agents. Compounds that can induce Bax independent apoptotic cell death are expected to overcome such drug resistance. We have employed a live cell based screening platform to identify potential compounds that can induce programmed cell death in Bax deficiency. Release of cytochrome C from mitochondria into the cytosol is a decisive initial event required for the caspase dependent cell death. We have engineered both wild type and Bax knock out colon cancer cells stably expressing cytochrome C with EGFP fusion protein to identify compounds that can trigger cytochrome C release in both cells with equal efficiency. In the fluorescent translocation assay, most of the drugs tested failed to induce cytochrome C release in Bax deficient cells validating the sensitivity of the assay. This study identified five lead compounds such as thapsigargin, tunicamycine, MG132, kaempferol and camptothecin that could induce cytochrome C release in both wild type and Bax deficient cells with equal potency. All the positive hits induced ER stress signaling as evidenced by up-regulation of Grp78. Studies with a Bak deficient cells indicate that Bak deficiency confers protection to cells from ER stress through autophagy. Further studies revealed that ER stress inducing agents are capable of triggering classical mitochondrial apoptotic cell death through the conformational activation of Bak, substantiating the potential of this pathway in designing drugs against Bax deficiency mediated drug resistance.

Keywords: Abbreviations; BaxKO; Bax knock out; cyt.C-EGFP; Cytochrome C-enhanced green fluorescent protein; ER; Endoplasmic reticulumDrug resistance; ER stress; Bax mutation; Apoptosis; Autophagy

Flavonoids inhibit hypoxia-induced vascular endothelial growth factor expression by a HIF-1 independent mechanism by Elena Ansó; Alicia Zuazo; Marta Irigoyen; María C. Urdaci; Ana Rouzaut; Juan J. Martínez-Irujo (pp. 1600-1609).

Flavonoids are a group of polyphenolic dietary compounds that have been proposed to possess chemopreventive properties against lung cancer. In this work we analyzed the effect of a group of 20 structurally related flavonoids, including flavones, flavonols and isoflavones, on the production of vascular endothelial growth factor (VEGF) induced by hypoxia in NCI-H157 cells. VEGF is the main regulator of physiological and pathological angiogenesis and is highly stimulated by hypoxia-inducible factor 1 (HIF-1). We found that apigenin, luteolin, fisetin and quercetin inhibited hypoxia-induced VEGF expression in the low micromolar range. Structure–activity relationships demonstrated that flavone derivatives were the most active compounds and that hydroxylation of the A ring at the positions 5 and 7 and of the B ring at the 4′ position were important for this activity. Interestingly, only a group of VEGF inhibitors, including apigenin, flavone and 4′,7-dihydroxiflavone, reduced the expression of HIF-1α under these conditions, whereas others, such as fisetin, luteolin, galangin or quercetin, induced HIF-1α expression while reducing those of VEGF. When cells were exposed to hypoxia in the presence of these flavonoids, HIF-1α translocated to the nucleus and interacted with p300/CBP, but this complex was transcriptionally inactive. Taken together these findings indicate that flavonoids impair VEGF transcription by an alternative mechanism that did not depend on nuclear HIF levels. We also found that flavonoids suppressed hypoxia-induced STAT3 tyrosine phosphorylation and that this activity correlated with their potency as VEGF inhibitors, suggesting that inhibition of STAT3 function may play a role in this process.

Keywords: Flavonoids; Hypoxia; HIF; VEGF; Angiogenesis; STAT3

The effect of stereochemistry on the thermodynamic characteristics of the binding of fenoterol stereoisomers to the β₂-adrenoceptor by Krzysztof Jozwiak; Lawrence Toll; Lucita Jimenez; Anthony Yiu-Ho Woo; Rui-Ping Xiao; Irving W. Wainer (pp. 1610-1615).

The binding thermodynamics of the stereoisomers of fenoterol, (R,R′)-, (S,S′)-, (R,S′)-, and (S,R′)-fenoterol, to the β₂-adrenergic receptor (β₂-AR) have been determined. The experiments utilized membranes obtained from HEK cells stably transfected with cDNA encoding human β₂-AR. Competitive displacement studies using [³H]CGP-12177 as the marker ligand were conducted at 4, 15, 25, 30 and 37°C, the binding affinities calculated and the standard enthalpic (Δ H°) and standard entropic (Δ S°) contribution to the standard free energy change (Δ G°) associated with the binding process determined through the construction of van’t Hoff plots. The results indicate that the binding of (S,S′)- and (S,R′)-fenoterol were predominately enthalpy-driven processes while the binding of (R,R′)- and (R,S′)-fenoterol were entropy-driven. All of the fenoterol stereoisomers are full agonists of the β₂-AR, and, therefore, the results of this study are inconsistent with the previously described “thermodynamic agonist–antagonist discrimination”, in which the binding of an agonist to the β-AR is entropy-driven and the binding of an antagonist is enthalpy-driven. In addition, the data demonstrate that the chirality of the carbon atom containing the β-hydroxyl group of the fenoterol molecule (the β-OH carbon) is a key factor in the determination of whether the binding process will be enthalpy-driven or entropy-driven. When the configuration at the β-OH carbon is S the binding process is enthalpy-driven while the R configuration produces an entropy-driven process.

Keywords: Enantiomers; Enantiospecific binding; Thermodynamic agonist–antagonist discrimination; Binding mechanisms; Binding thermodynamics; Chirality

Antithrombotic activity of F 16618, a new PAR1 antagonist evaluated in extracorporeal arterio-venous shunt in the rat by Robert Létienne; Anne Leparq-Panissié; Yannick Calmettes; Florence Nadal-Wollbold; Michel Perez; Bruno Le Grand (pp. 1616-1621).

A new PAR1 antagonist, F 16618 exerted a potent antithrombotic activity by intravenous and oral routes, without affecting bleeding time, this activity was potentiated when combined with aspirin or clopidogrel.The purpose of the present work was the evaluation of the antithrombotic activity of a new PAR1 antagonist, F 16618 in arterio-venous shunt in the rat. Arterial thrombosis was induced by insertion of a silk thread (thrombogenic substrate) into an extracorporeal shunt. F 16618 was administered either by intravenous route (0.63–2.5mg/kg) or by oral route (20–80mg/kg). Oral activity of F 16618 was compared to that of aspirin (20–80mg/kg) and clopidogrel (0.63–10mg/kg). Finally, F 16618 was associated to aspirin and/or clopidogrel to test for possible antithrombotic activity and its effects on bleeding time. SFLLR-induced human platelet aggregation was evaluated in the presence of F 16618, demonstrating the anti-aggregant activity of this compound. F 16618 (1.25mg/kg) significantly delayed the time leading to occlusion by 52±17%, without affecting bleeding time and in absence of hemodynamic effects. F 16618 given orally dose-dependently increased the time to occlusion. The maximal effect was observed at 40mg/kg (984±95s versus 644±17s in vehicle group). Aspirin and clopidogrel also dose-dependently lengthened time to occlusion, but this effect was associated with an increase of bleeding time. F 16618 (20mg/kg) orally associated with either aspirin (40mg/kg) or with clopidogrel (1.25mg/kg) potentiated the antithrombotic effects of both compounds without further increasing of bleeding time. In conclusion, F 16618 exerted a potent antithrombotic activity by intravenous and oral routes, without affecting bleeding time. Furthermore, the antithrombotic activity was potentiated when combined with aspirin or clopidogrel.

Keywords: PAR1 antagonist; Thrombosis model; Rat

Protective effect of rebamipide against celecoxib-induced gastric mucosal cell apoptosis by Tomoaki Ishihara; Ken-Ichiro Tanaka; Saki Tashiro; Kosuke Yoshida; Tohru Mizushima (pp. 1622-1633).

A major clinical problem encountered with the use of non-steroidal anti-inflammatory drugs (NSAIDs) is gastrointestinal complications. We have previously suggested that both decreases in prostaglandin E₂ (PGE₂) levels and mucosal apoptosis are involved in the development of NSAID-produced gastric lesions and that this apoptosis is mediated by an increase in the intracellular Ca²⁺ concentration and the resulting endoplasmic reticulum (ER) stress response and mitochondrial dysfunction. Celecoxib and rebamipide are being used clinically as a safer NSAID and an anti-ulcer drug, respectively. In this study, we have examined the effect of rebamipide on celecoxib-induced production of gastric lesions. In mice pre-administered with a low dose of indomethacin, orally administered rebamipide suppressed celecoxib-induced mucosal apoptosis and lesion production but did not decrease in PGE₂ levels in the stomach. Rebamipide also suppressed celecoxib-induced increases in intracellular Ca²⁺ concentration, the ER stress response, mitochondrial dysfunction and apoptosis in vitro. We also found that rebamipide suppresses the increases in intracellular Ca²⁺ concentration induced by an activator of voltage-dependent L-type Ca²⁺ channels and that another blocker of this channel suppresses celecoxib-induced increases in intracellular Ca²⁺ concentration. These results suggest that celecoxib activates voltage-dependent L-type Ca²⁺ channels and that rebamipide blocks this activation, resulting in suppression of celecoxib-induced apoptosis. We believe that this novel activity of rebamipide may play an important role in the protection of gastric mucosa against the formation of celecoxib-induced lesions.

Keywords: Celecoxib; Rebamipide; Ulcer; Apoptosis; Voltage-dependent L-type Ca; ²⁺; channel

Role of modulation of vascular endothelial growth factor and tumor necrosis factor-alpha in gastric ulcer healing in diabetic rats by Azza M. Baraka; Aida Guemei; Hala Abdel Gawad (pp. 1634-1639).

The aim of the present study was to assess the effect of drugs that increase gastric vascular endothelial growth factor (VEGF) and suppress gastric tumor necrosis factor-alpha (TNF-α) in gastric ulcer healing in streptozotocin-induced diabetic rats. Sixty male albino rats were made diabetic by intraperitoneal (i.p.) streptozotocin injection and ten rats were injected i.p. by a single dose of saline. Six weeks following streptozotocin or saline injection, gastric ulcers were induced by serosal application of acetic acid. Three days after acetic acid application, rats were divided into: group I (non-diabetic control), group II (streptozotocin-injected), groups III–VII (streptozotocin-injected rats treated with insulin, insulin and pentoxifylline, insulin and simvastatin, pentoxifylline as well as simvastatin, respectively, for 7 days following acetic acid application. The use of insulin, combinations of insulin and pentoxifylline or simvastatin resulted in a significant decrease in gastric ulcer area, significant increase in epithelial regeneration assessed histologically, significant increase in gastric VEGF concentration, and gastric von Willebrand factor (vWF) as well as significant decrease in gastric TNF-α. A significant difference in gastric ulcer area as well as in gastric TNF-α, VEGF and vWF levels could be observed between rats that received combinations of insulin and pentoxifylline or simvastatin compared to rats that received either drug alone. Our results suggest the feasibility of a novel treatment strategy, namely pentoxifylline and simvastatin, for patients in whom impairment of ulcer healing constitutes a secondary complication of diabetes mellitus.

Keywords: Vascular endothelial growth factor; Tumor necrosis factor; Simvastatin; Pentoxifylline; Diabetes mellitus; Peptic ulcer; Streptozotocin

Thymoquinone poly (lactide-co-glycolide) nanoparticles exhibit enhanced anti-proliferative, anti-inflammatory, and chemosensitization potential by Jayaraj Ravindran; Hareesh B. Nair; Bokyung Sung; Sahdeo Prasad; Rajeshwar R. Tekmal; Bharat B. Aggarwal (pp. 1640-1647).

Our results demonstrate that encapsulation of thymoquinone, TQ, derived from the medicinal spice Nigella sativa (also called black cumin), into nanoparticles enhances its anti-proliferative, anti-inflammatory, and chemosensitizing effects.Thymoquinone (TQ), derived from the medicinal spice Nigella sativa (also called black cumin), has been shown to exhibit anti-inflammatory and anti-cancer activities. In this report we employed polymer-based nanoparticle approach to improve upon its effectiveness and bioavailability. TQ was encapsulated with 97.5% efficiency in biodegradable nanoparticulate formulation based on poly (lactide-co-glycolide) (PLGA) and the stabilizer polyethylene glycol (PEG)-5000. Dynamic laser light scattering and transmission electron microscopy confirmed particle diameter between 150 and 200nm. Electrophoretic gel shift mobility assay showed that TQ nanoparticles (NP) were more active than TQ in inhibiting NF-κB activation and in suppressing the expression of cyclin D1, matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF), those are markers of cell proliferation, metastasis and angiogenesis, respectively. TQ-NP were also more potent than TQ in suppressing proliferation of colon cancer, breast cancer, prostate cancer, and multiple myeloma cells. Esterase staining for plasma membrane integrity revealed that TQ-NP were more potent than TQ in sensitizing leukemic cells to TNF- and paclitaxel-induced apoptosis. Overall our results demonstrate that encapsulation of TQ into nanoparticles enhances its anti-proliferative, anti-inflammatory, and chemosensitizing effects.

Keywords: Nanoparticles; Thymoquinone; Apoptosis; Inflammation; NF-κB

Lipoteichoic acid enhances IL-6 production in human synovial fibroblasts via TLR2 receptor, PKCδ and c-Src dependent pathways by Chih-Hsin Tang; Chin-Jung Hsu; Wei-Hung Yang; Yi-Chin Fong (pp. 1648-1657).

Patients with rheumatoid arthritis (RA) are at increased risk of developing infections and appear to be particularly susceptible to septic arthritis. Lipoteichoic acid (LTA), a cell wall component of Gram-positive bacteria is an amphiphilic, negatively charged glycolipid. However, the effects of LTA on human synovial fibroblasts are largely unknown. We investigated the signaling pathway involved in IL-6 production stimulated by LTA in rheumatoid arthritis synovial fibroblasts (RASF). LTA caused concentration- and time-dependent increases in IL-6 production. LTA-mediated IL-6 production was attenuated by Toll-like receptor 2 (TLR2) monoclonal antibody or siRNA. Pretreatment with PKCδ inhibitor (rottlerin), c-Src inhibitor (PP2), AP-1 inhibitor (tanshinone IIA) and NF-κB inhibitor (PDTC and TPCK) also inhibited the potentiating action of LTA. However, focal adhesion kinase (FAK) mutant and siRNA did not affect LTA-mediated IL-6 production. Stimulation of cells with LTA increased the PKCδ and c-Src phosphorylation and kinase activity. LTA increased the accumulation of p-c-Jun and p-p65 in the nucleus, as well as AP-1 and NF-κB luciferase activity. LTA-mediated increase of AP-1 and NF-κB luciferase activity was inhibited by rottlerin and PP2 or TLR2 and PKCδ siRNA or c-Src mutant. Our results suggest that LTA-increased IL-6 production in human synovial fibroblasts via the TLR2 receptor, PKCδ, c-Src, AP-1 and NF-κB signaling pathways.

Keywords: Abbreviations; LTA; lipoteichoic acid; PGN; peptidoglycan; siRNA; small interference RNA; LPS; lipopolysaccharide; IL; interleukin; TNF; tumor necrosis factor; RA; rheumatoid arthritis; RASF; rheumatoid arthritis synovial fibroblasts; TLR; Toll-like receptor; ODN; oligonucleotide; qPCR; quantitative real-time PCRIL-6; LTA; RA; PKC; c-Src

Bisdemethylcurcumin and structurally related hispolon analogues of curcumin exhibit enhanced prooxidant, anti-proliferative and anti-inflammatory activities in vitro by Jayaraj Ravindran; Gottumukkala V. Subbaraju; Modukuri V. Ramani; Bokyung Sung; Bharat B. Aggarwal (pp. 1658-1666).

While turmeric ( Curcuma longa) is the source of curcumin, the mushroom Phellinus linteus called “meshimakobu” in Japanese, “song gen” in Chinese, and “Sang-Hwang” in Korean is the best source of hispolon.Curcumin, a component of turmeric ( Curcuma longa), exhibits anti-inflammatory and anti-proliferative activities through the generation of reactive oxygen species (ROS). Curcumin (diferuloylmethane) contains two hydroxyl, two methoxy and two phenyl groups but how these groups contribute to its activity is poorly understood. We synthesized analogues that varied in inclusion of these groups and compared their activity. We found that bisdemethylcurcumin (BDC) was more potent than curcumin as an anti-inflammatory agent as indicated by suppression of TNF-induced NF-κB activation, more potent as an anti-proliferative agent, and more potent in inducing ROS. Hispolon, which lacks one aromatic unit in relation to curcumin, also exhibited enhanced anti-inflammatory and anti-proliferative activities. When synthetic curcumin (Cur-S) was compared with bisdemethylcurcumin (BDC), hispolon, hispolon methyl ether (HME), dehydroxy hispolon (DH), hydroxy hispolon (HH), methoxy hispolon methyl ether (MHME), and methoxy hispolon (MH), we found that following order of anti-inflammatory activity: BDC=Hispolon>HME>HH>Cur-S>MHME>MH>DH; for anti-proliferative: Hispolon>BDC>MHME>Cur-S>MH>HME=HH>DH; and for prooxidant: BDC>Cur-S=MHME>HH>MH+HME>DH (254-1414 mean fluorescence intensity). Thus, dehydroxy hispolon was least potent for all three activities. Overall the results indicate that the substitution of a hydroxyl group for a methoxy group at the meta positions of the phenyl rings in curcumin significantly enhanced the anti-inflammatory activity, and the removal of phenyl ring at the 7^th position of the heptadiene back bone and addition of hydroxyl group significantly increased the anti-proliferative activity of curcumin.

Keywords: Curcumin; Hispolon; NF-κB; TNF; Anti-proliferation

Activation of the A₃ adenosine receptor inhibits fMLP-induced Rac activation in mouse bone marrow neutrophils by Dharini van der Hoeven; Elizabeth T. Gizewski; John A. Auchampach (pp. 1667-1673).

Adenosine is released from injured or hypoxic tissues where it exerts numerous anti-inflammatory effects including suppression of neutrophil functions. Although most previous work has implicated the A_2AAR, we have recently shown that selective activation of the abundantly expressed A₃AR inhibits neutrophil superoxide production and chemotaxis providing a potential mechanistic explanation for the efficacy of A₃AR agonists in experimental animal models of inflammation. In this study, we hypothesized that the A₃AR suppresses neutrophil functions by inhibiting the monomeric GTPase Rac, a central regulator of chemokine-directed neutrophil migration and superoxide production. We found that pre-treating neutrophils with the highly selective A₃AR agonist CP-532,903 reduced fMLP-induced Rac activation using an ELISA-based assay that detects all three Rac isoforms. CP-532,903 also inhibited fMLP-induced F-actin formation, a downstream effector function of Rac relevant to neutrophil migration, but not activation of ERK1/2 or p38. Pre-treating neutrophils with CP-532,903 did not stimulate cAMP production or alter fMLP-induced calcium transients, implicating that A₃AR stimulation does not inhibit Rac activation or neutrophil activities by suppressing Ca²⁺ signaling, elevating the intracellular concentration of cAMP, or by cross-desensitizing fMLP receptors. Our results suggest that activation of the A₃AR signals to suppress neutrophil functions by interfering with the monomeric GTPase Rac, thus contributing to the ant-inflammatory actions of adenosine.

Keywords: Abbreviations; ADA; adenosine deaminase; AR; adenosine receptor; C5a; complement component 5a; CGS 21680; 2-[p-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamidoadenosine; CP-532,903; (2S,3S,4R,5R)-3-amino-5-[6-(2,5-dichlorobenzylamino)purin-9-yl]-4-hydroxytetrahydrofuran-2-carboxylic acid methylamide; fMLP; formylated-methionine-leucine-phenylalanine; GEF; guanine nucleotide exchange factor; KO; knockout; WT; wild-typeAdenosine; Adenosine receptors; Neutrophils; Inflammation; Rac; Cell signaling

Nobiletin improves hyperglycemia and insulin resistance in obese diabetic ob/ ob mice by Young-Sil Lee; Byung-Yoon Cha; Kiyoto Saito; Hiroshi Yamakawa; Sun-Sil Choi; Kohji Yamaguchi; Takayuki Yonezawa; Toshiaki Teruya; Kazuo Nagai; Je-Tae Woo (pp. 1674-1683).

Nobiletin is a polymethoxylated flavone found in certain citrus fruits that exhibits various pharmacological effects including anti-inflammatory, antitumor and neuroprotective properties. The present study investigated the effects of nobiletin on insulin sensitivity in obese diabetic ob/ ob mice, and the possible mechanisms involved. The ob/ ob mice were treated with nobiletin (200mg/kg) for 5 weeks. Nobiletin significantly improved the plasma glucose levels, homeostasis model assessment index, glucose tolerance in an oral glucose tolerance test and plasma adiponectin levels. In white adipose tissue (WAT), nobiletin significantly decreased the mRNA expression levels of inflammatory adipokines such as interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1 and increased the mRNA expression levels of adiponectin, peroxisome proliferator-activated receptor (PPAR)-γ and its target genes. At the same time, nobiletin increased the glucose transporter (Glut) 4 expression levels in the whole plasma membrane, and Glut1 and phospho-Akt expression in the whole cell lysates in WAT and muscle. Nobiletin also increased Glut4 protein expression level in the whole cell lysates of the muscle. Taken together, the present results suggest that nobiletin improved the hyperglycemia and insulin resistance in obese diabetic ob/ ob mice by regulating expression of Glut1 and Glut4 in WAT and muscle, and expression of adipokines in WAT.

Keywords: Nobiletin; Homeostasis model assessment index; Glucose tolerance; Adipokine; Glucose transporter

Bupropion metabolism by human placenta by Xiaoming Wang; Doaa R. Abdelrahman; Olga L. Zharikova; Svetlana L. Patrikeeva; Gary D.V. Hankins; Mahmoud S. Ahmed; Tatiana N. Nanovskaya (pp. 1684-1690).

Human placental 11β-hydroxysteroid dehydrogenases were identified as the major carbonyl-reducing enzymes responsible for reduction of bupropion to threo- and erythrohydrobupropion in microsomal fractions.Smoking during pregnancy is the largest modifiable risk factor for pregnancy-related morbidity and mortality. The success of bupropion for smoking cessation warrants its investigation for the treatment of pregnant patients. Nevertheless, the use of bupropion for the treatment of pregnant smokers requires additional data on its bio-disposition during pregnancy. Therefore, the aim of this investigation was to determine the metabolism of bupropion in placentas obtained from nonsmoking and smoking women, identify metabolites formed and the enzymes catalyzing their formation, as well as the kinetics of the reaction. Data obtained revealed that human placentas metabolized bupropion to hydroxybupropion, erythro- and threohydrobupropion. The rates for formation of erythro- and threohydrobupropion exceeded that for hydroxybupropion by several folds, were dependent on the concentration of bupropion and exhibited saturation kinetics with an apparent K_m value of 40μM. Human placental 11β-hydroxysteroid dehydrogenases were identified as the major carbonyl-reducing enzymes responsible for the reduction of bupropion to threo- and erythrohydrobupropion in microsomal fractions. On the other hand, CYP2B6 was responsible for the formation of OH-bupropion. These data suggest that both placental microsomal carbonyl-reducing and oxidizing enzymes are involved in the metabolism of bupropion.

Keywords: Abbreviations; V; _max; maximum velocity; CL; _in; intrinsic clearance (; V; _max; /; K; _m; ); K; _m; substrate concentration at 50% of; V; _max; CYP450; cytochrome P450, ADHs, alcohol dehydrogenases; AKRs; aldo-keto reductases; CRs; carbonyl reductases; 11β-HSD; 11β-hydroxysteroid dehydrogenases; 18β-GA; 18β-glycyrrhetinic acidBupropion; Metabolism; Human placenta

Characterization of major phytocannabinoids, cannabidiol and cannabinol, as isoform-selective and potent inhibitors of human CYP1 enzymes by Satoshi Yamaori; Mika Kushihara; Ikuo Yamamoto; Kazuhito Watanabe (pp. 1691-1698).

Inhibitory effects of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), cannabidiol (CBD), and cannabinol (CBN), the three major constituents in marijuana, on catalytic activities of human cytochrome P450 (CYP) 1 enzymes were investigated. These cannabinoids inhibited 7-ethoxyresorufin O-deethylase activity of recombinant CYP1A1, CYP1A2, and CYP1B1 in a competitive manner. CBD most potently inhibited the CYP1A1 activity; the apparent K_i value (0.155μM) was at least one-seventeenth of the values for other CYP1 isoforms. On the other hand, CBN more effectively decreased the activity of CYP1A2 and CYP1B1 ( K_i=0.0790 and 0.148μM, respectively) compared with CYP1A1 ( K_i=0.541μM). Δ⁹-THC less potently inhibited the CYP1 activity than CBD and CBN, and showed low selectivity against the CYP1 inhibition ( K_i=2.47–7.54μM). The preincubation of CBD resulted in a time- and concentration-dependent decrease in catalytic activity of all the recombinant CYP1 enzymes and human liver microsomes. Similarly, the preincubation of Δ⁹-THC or CBN caused a time- and concentration-dependent inhibition of recombinant CYP1A1. The inactivation of CYP1A1 by CBD indicated the highest k_inact/ K_I value (540l/mmol/min) among the CYP1 enzyme sources tested. The inactivation of recombinant CYP1A1 and human liver microsomes by CBD required NADPH, was not influenced by dialysis and by glutathione, N-acetylcysteine, and superoxide dismutase as trapping agents. These results indicated that CBD and CBN showed CYP1 isoform-selective direct inhibition and that CBD was characterized as a potent mechanism-based inhibitor of human CYP1 enzymes, especially CYP1A1.

Keywords: Abbreviations; BSA; bovine serum albumin; CBD; cannabidiol; CBN; cannabinol; CYP; cytochrome P450; EROD; 7-ethoxyresorufin; O; -deethylase; GSH; glutathione; HLMs; human liver microsomes; NAC; N; -acetylcysteine; SOD; superoxide dismutase; Δ; ⁹; -THC; Δ; ⁹; -tetrahydrocannabinolCannabidiol; Cannabinol; CYP1; Inhibition; Mechanism-based inactivation

Sections

Personal tools

Biochemical Pharmacology (v.79, #11)

Sections

Personal tools

Local resources

Biochemical Pharmacology (v.79, #11)