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Biochemical Pharmacology (v.75, #4)

Editorial Advisory Board (pp. iii).

Angiotensin-converting enzyme 2 and new insights into the renin–angiotensin system by Daniel W. Lambert; Nigel M. Hooper; Anthony J. Turner (pp. 781-786).

Components of the renin–angiotensin system are well established targets for pharmacological intervention in a variety of disorders. Many such therapies abrogate the effects of the hypertensive and mitogenic peptide, angiotensin II, by antagonising its interaction with its receptor, or by inhibiting its formative enzyme, angiotensin-converting enzyme (ACE). At the turn of the millennium, a homologous enzyme, termed ACE2, was identified which increasingly shares the limelight with its better-known homologue. In common with ACE, ACE2 is a type I transmembrane metallopeptidase; however, unlike ACE, ACE2 functions as a carboxypeptidase, cleaving a single C-terminal residue from a distinct range of substrates. One such substrate is angiotensin II, which is hydrolysed by ACE2 to the vasodilatory peptide angiotensin 1–7. In this commentary we discuss the latest developments in the rapidly progressing study of the physiological and patho-physiological roles of ACE2 allied with an overview of the current understanding of its molecular and cell biology. We also discuss parallel developments in the study of collectrin, a catalytically inactive homologue of ACE2 with critical functions in the pancreas and kidney.

Keywords: Abbreviations; ACE; angiotensin-converting enzyme; ADAM; a disintegrin and metalloproteinase; Ang; angiotensin; ARDS; acute respiratory distress syndrome; AT1; angiotensin receptor, type 1; CoV; coronavirus; HEK; human embryonic kidney; HNF; hepatocyte nuclear factor; mas; receptor for Ang (1-7); MDCK; Madin–Darby Canine Kidney; MODY; maturity-onset diabetes mellitus; RAS; renin–angiotensin system; SARS; severe acute respiratory syndrome; TACE; TNF-α converting enzyme; TNF; tumour necrosis factorACE2; ACE; Angiotensin; ARDS; SARS; Diabetes

Curcumin as “ Curecumin”: From kitchen to clinic by Ajay Goel; Ajaikumar B. Kunnumakkara; Bharat B. Aggarwal (pp. 787-809).

Although turmeric ( Curcuma longa; an Indian spice) has been described in Ayurveda, as a treatment for inflammatory diseases and is referred by different names in different cultures, the active principle called curcumin or diferuloylmethane, a yellow pigment present in turmeric (curry powder) has been shown to exhibit numerous activities. Extensive research over the last half century has revealed several important functions of curcumin. It binds to a variety of proteins and inhibits the activity of various kinases. By modulating the activation of various transcription factors, curcumin regulates the expression of inflammatory enzymes, cytokines, adhesion molecules, and cell survival proteins. Curcumin also downregulates cyclin D1, cyclin E and MDM2; and upregulates p21, p27, and p53. Various preclinical cell culture and animal studies suggest that curcumin has potential as an antiproliferative, anti-invasive, and antiangiogenic agent; as a mediator of chemoresistance and radioresistance; as a chemopreventive agent; and as a therapeutic agent in wound healing, diabetes, Alzheimer disease, Parkinson disease, cardiovascular disease, pulmonary disease, and arthritis. Pilot phase I clinical trials have shown curcumin to be safe even when consumed at a daily dose of 12g for 3 months. Other clinical trials suggest a potential therapeutic role for curcumin in diseases such as familial adenomatous polyposis, inflammatory bowel disease, ulcerative colitis, colon cancer, pancreatic cancer, hypercholesteremia, atherosclerosis, pancreatitis, psoriasis, chronic anterior uveitis and arthritis. Thus, curcumin, a spice once relegated to the kitchen shelf, has moved into the clinic and may prove to be “Curecumin”.

Keywords: Anticancer; Natural products; Curcumin

Induction of γ-globin mRNA, erythroid differentiation and apoptosis in UVA-irradiated human erythroid cells in the presence of furocumarin derivatives by Giampietro Viola; Daniela Vedaldi; Francesco Dall’Acqua; Elena Fortunato; Giuseppe Basso; Nicoletta Bianchi; Cristina Zuccato; Monica Borgatti; Ilaria Lampronti; Roberto Gambari (pp. 810-825).

Psoralens, also known as furocoumarins, are a class of photosensitizers largely used in the therapy of various skin diseases. In this study we have evaluated the combined effects of UVA irradiation and furocoumarins derivatives on (a) erythroid differentiation and apoptosis of human leukemia K562 cells and (b) globin gene expression in cultures of human erythroid progenitors derived from the peripheral blood. To prove the activity of a series of linear and angular furocoumarins derivatives, we employed the human leukemia K562 cell line and the two-phase liquid culture procedure for growing erythroid progenitors. Quantitative real-time reverse transcription polymerase-chain assay (Q-RT-PCR) was employed for quantification of the accumulation of globin mRNAs. The results obtained demonstrate that both linear and angular furocoumarins are strong inducers of erythroid differentiation of K562 cells. From a preliminary screening, we have selected two derivatives, 5-methoxypsoralen (5-MOP) and trimethylangelicin (TMA), for which we have investigated their mechanism of action. The cell cycle analysis showed that these derivatives induce, after irradiation, a cell cycle arrest in the G2/M phase, followed by apoptosis. Mitochondrial depolarisation and caspases activation seem to be involved in the mechanism of cell death. In erythroid precursor cells, psoralens in combination with UVA irradiation, stimulate at very low concentrations a preferential increase of γ-globin mRNA. Altogether, these data suggest that psoralen derivatives warrant further evaluation as potential therapeutic drugs in β-thalassemia and sickle cell anemia.

Keywords: Abbreviations; 5-MOP; 5-methoxypsoralen; 8-MOP; 8-methoxypsoralen; Δ; ψ; _mt; mitochondrial potential; ANG; angelicin; BFUe; erythroid burst forming units; CFUe; erythroid colony forming units; EPO; erythropoietin; HBSS; Hank's balanced salt solution; HE; hydroethidine; ICL; interstrand cross-link; NAO; 10-; N; -nonyl-acridine orange; PI; propidium iodide; PUVA; psoralen plus UVA; ROS; reactive oxygen species; TMA; trimethylangelicinPsoralens; K562 cells; Erythroid precursor; Erythroid differentiation; Apoptosis; Cell cycle

4′-Methoxy-2-styrylchromone a novel microtubule-stabilizing antimitotic agent by Joana Marinho; Madalena Pedro; Diana C.G.A. Pinto; Artur M.S. Silva; José A.S. Cavaleiro; Claudio E. Sunkel; Maria São José Nascimento (pp. 826-835).

4′-Methoxy-2-styrylchromone, a new synthetic chromone was identified as a selective proliferation inhibitor of human tumor (MCF-7 and NCI-H460) cell lines than to non-tumor cells (MRC-5). The antiproliferative activity of this chromone was also extensive to peripheral human lymphocytes. 4′-Methoxy-2-styrylchromone was found to block tumor cells in the G₂/M phase of the cell cycle. The G₂/M arrest of NCI-H460 cells was dose- and time-dependent, reaching a maximum after 12-h treatment while MCF-7 cells reached the maximum value of G₂/M accumulation only after a 24-h treatment. Chromone-treated cells evidenced a high frequency of cells in prometaphase, indicating progression beyond G₂ and arrest early in mitosis. This mitotic arrest was associated with abnormal mitotic spindles characterized by the formation of a monopolar structure, suggesting that the chromone interferes with microtubules. The results of an in vitro tubulin polymerization assay showed that this chromone stabilizes microtubules in a manner similar to paclitaxel.

Keywords: 2-Styrylchromone; Antimitotic drugs; Tubulin; Mitosis; Cell cycle; Cancer chemotherapy

The immunocytokine scFv23/TNF targeting HER-2/neu induces synergistic cytotoxic effects with 5-fluorouracil in TNF-resistant pancreatic cancer cell lines by Mi-Ae Lyu; Razelle Kurzrock; Michael G. Rosenblum (pp. 836-846).

Human pancreatic tumor cells are highly resistant to both tumor necrosis factor (TNF) and to chemotherapeutic agents. HER-2/neu expression has been proposed as a negative prognostic marker in pancreatic intraepithelial neoplasia. Our approach was to utilize HER-2/neu expression on the surface of tumor cells as a therapeutic target employing scFv23/TNF, immunocytokine composed of a single chain Fv antibody (scFv23) targeting the HER-2/neu and the cytokine TNF as the cytotoxic moiety, to deliver TNF directly to TNF-resistant pancreatic tumor cells. Using a panel of human pancreatic cell lines, which overexpress HER-2/neu, we evaluated the in vitro response of cells to TNF, scFv23/TNF, Herceptin, and a combination of scFv23/TNF with various chemotherapeutic agents. We found that all pancreatic cancer cell lines were highly resistant to the cytotoxic effects of TNF and that scFv23/TNF was highly cytotoxic to TNF-resistant HER-2/neu-expressing pancreatic cancer cell lines at levels rivaling that of conventional chemotherapeutic agents. Combination studies demonstrated a synergistic cytotoxic effect of scFv23/TNF with 5-fluorouracil (5-FU) in TNF-resistant pancreatic cancer cell lines. Mechanistic studies demonstrated that the 5-FU plus scFv23/TNF combination specifically resulted in a down-regulation of HER-2/neu, p-Akt and Bcl-2 and up-regulation of TNF-R1. In addition, the combination 5-FU plus scFv23/TNF induced apoptosis and this synergistic effect was dependent on activation of caspase-8 and caspase-3. Delivery of the cytokine TNF to HER-2/neu expressing pancreatic tumor cells, which are inherently resistant to TNF using scFv23/TNF may be an effective therapy for pancreatic cancer especially when utilized in combination with 5-FU.

Keywords: Abbreviations; scFv23/TNF; anti-HER-2/neu single chain Fv antibody fused to TNF; TNF; tumor necrosis factor; HER-2/neu; epidermal growth factor receptor-2; HER-1; epidermal growth factor receptor-1; TNF-R1; TNF receptor-1; TNF-R2; TNF receptor-2; p-Akt; phospho-Akt; 5-FU; 5-fluorouracil; PARP; poly ADP-ribose polymerase; TUNEL; terminal deoxynucleotidyl transferase-mediated nick end labelingHER-2/neu; Pancreatic cancer; scFv23/TNF; 5-Fluorouracil; Combination therapy

WRC-213, anl-methionine-conjugated mitoxantrone derivative, displays anticancer activity with reduced cardiotoxicity and drug resistance: Identification of topoisomerase II inhibition and apoptotic machinery in prostate cancers by Che-Jen Hsiao; Tsia-Kun Li; Ya-Ling Chan; Ling-Wei Hsin; Cho-Hwa Liao; Chien-Hua Lee; Ping-Chiang Lyu; Jih-Hwa Guh (pp. 847-856).

Anthracyclines and anthracenediones are well-known cancer chemotherapeutic agents but their uses are limited with cardiotoxicity and drug resistance. Severall- andd-form amino acids were introduced into the anthraquinone skeleton and numerous derivatives were synthesized for the evaluation of anticancer activity. The screening tests showed that WRC-213, anl-methionine conjugation, was the most effective derivative to inhibit proliferative effect of human androgen-independent prostate cancer PC-3 cells (IC₅₀=50nM). In an extension evaluation, WRC-213 displayed a potent anti-proliferative activity in various cancer cell lines, including non-small cell lung cancer A549, androgen-independent prostate cancer DU145, colorectal cancer HT-29, breast cancer MCF-7 and hepatocellular carcinoma Hep3B and HepG2. It induced cell-cycle arrest at S and G2, but not mitotic phase, in PC-3 cells. The comet assay revealed that induction of DNA damage and inhibition of topoisomerase II were the primary insults. After the checkpoint arrest of the cell-cycle, WRC-213 induced the mitochondria-mediated intrinsic apoptotic pathway, including Mcl-1 cleavage, Bcl-2 down-regulation and activation of caspase-9/caspase-3 cascades. Survivin degradation and caspase-2 activation also contributed to WRC-213-induced apoptosis. Moreover, the assessment of cytotoxicity in H9c2 cardiomyocytes and drug resistance in NCI/ADR-RES cells demonstrated that WRC-213 showed much lower cardiotoxicity and P-glycoprotein-related resistance than those of mitoxantrone, etoposide and doxorubicin. In conclusion, it is suggested that WRC-213 is a potential topoisomerase II inhibitor with reduced cardiotoxicity and drug resistance. It inhibits topoisomerase II activity and induces chromosomal DNA strand breaks, leading to S and G2 arrest of the cell-cycle and activation of mitochondria-mediated apoptotic pathways.

Keywords: Topoisomerase II inhibitor; Cell-cycle arrest; Mitochondria; Cardiotoxicity; Drug resistance

Modulation of the human equilibrative nucleoside transporter1 (hENT1) activity by IL-4 and PMA in B cells from chronic lymphocytic leukemia by Paula Fernández Calotti; Carlos María Galmarini; Cristian Cañones; Romina Gamberale; Daniel Saénz; Julio Sánchez Avalos; Mónica Chianelli; Ruth Rosenstein; Mirta Giordano (pp. 857-865).

Nucleoside transporters (NTs) are essential for the uptake of therapeutic nucleoside analogs, broadly used in cancer treatment. The mechanisms responsible for NT regulation are largely unknown. IL-4 is a pro-survival signal for chronic lymphocytic leukemia (CLL) cells and has been shown to confer resistance to nucleoside analogs. The aim of this study was to investigate whether IL-4 is able to modulate the expression and function of the human equilibrative NT1 (hENT1) in primary cultures of CLL cells and, consequently, to affect cytotoxicity induced by therapeutic nucleosides analogs. We found that treatment with IL-4 (20ng/ml for 24h) increased mRNA hENT1 expression in CLL cells without affecting that of normal B cells. Given that the enhanced mRNA levels of hENT1 in CLL cells did not result in increased transport activity, we examined the possibility that hENT1 induced by IL-4 may require post-translational modifications to become active. We found that the acute stimulation of PKC in IL-4-treated CLL cells by short-term incubation with PMA significantly increased hENT1 transport activity and favoured fludarabine-induced apoptosis. By contrast, and in line with previous reports, IL-4 plus PMA protected CLL cells from a variety of cytotoxic agents. Our findings indicate that the combined treatment with IL-4 and PMA enhances hENT1 activity and specifically sensitizes CLL cells to undergo apoptosis induced by fludarabine.

Keywords: hENT1; IL-4; Chronic lymphocytic leukaemia; PKC; Fludarabine; Nucleoside analogs

On the energy-dependence of Hoechst 33342 transport by the ABC transporter LmrA by Henrietta Venter; Saroj Velamakanni; Lekshmy Balakrishnan; Hendrik W. van Veen (pp. 866-874).

LmrA is an ATP-binding cassette (ABC) multidrug transporter from Lactococcus lactis, and is a structural homologue of the human multidrug resistance P-glycoprotein (ABCB1), the overexpression of which is associated with multidrug resistance in tumours. We recently observed that a truncated version of LmrA lacking the nucleotide-binding domain mediates a proton motive force-dependent ethidium transport reaction by catalyzing proton-ethidium symport. This finding raised the question whether proton motive force-dependent transport can also be observed for other drugs, and whether this reaction is also relevant for full-length LmrA. Furthermore, the observations on LmrA-MD raised the question whether ATP-dependent transport by LmrA in intact cells could be due to the activity of independent ABC transporters that might become upregulated in the lactococcal cells due to the overexpression of LmrA; the recently identified ABC multidrug transporter LmrCD was put forward as a possible candidate. Here, we investigated the energy coupling to the transport of the amphiphilic dye Hoechst 33342 in proteoliposomes containing purified LmrA. For this purpose, LmrA was obtained from lactococcal cells lacking the genomic lmrA and lmrCD genes, in which LmrA was expressed from a plasmid. To separate ATP-dependence from proton motive force-dependence, we also used mutant LmrA proteins, which were affected in their ability to hydrolyse ATP. Our studies in proteoliposomes demonstrate that LmrA can catalyze Hoechst 33342 transport independent of auxiliary proteins, in an ATP-dependent fashion and a transmembrane chemical proton gradient (interior acidic)-dependent fashion.

Keywords: Abbreviations; ABC; ATP binding cassette; ΔpH; transmembrane chemical proton gradient; Δp; proton motive force; MD; membrane domain; NBD; nucleotide-binding domain; TNP-ATP; 2′-(or-3′)-O-(trinitrophenyl)-adenosine 5′-triphosphate; Wt; wildtypeABC transporter; Energy-coupling; Hoechst 33342 transport; LmrA; Proteoliposomes; Nucleotide-binding domain mutants

Differential effects of ABT-510 and a CD36-binding peptide derived from the type 1 repeats of thrombospondin-1 on fatty acid uptake, nitric oxide signaling, and caspase activation in vascular cells by Jeff S. Isenberg; Christine Yu; David D. Roberts (pp. 875-882).

ABT-510 is a potent mimetic of an anti-angiogenic sequence from the second type 1 repeat of thrombospondin-1. ABT-510 and the originald-Ile mimetic from which it was derived, GDGV(dI)TRIR, are similarly active for inhibiting vascular outgrowth in a B16 melanoma explant assay. Because GDGV(dI)TRIR and thrombospondin-1 modulate nitric oxide signaling by inhibiting the fatty translocase activity of CD36, we examined the ability ABT-510 to modulate fatty acid uptake into vascular cells and downstream nitric oxide/cGMP signaling. Remarkably, ABT-510 is less active than GDGV(dI)TRIR for inhibiting myristic acid uptake into both endothelial and vascular smooth muscle cells. Correspondingly, ABT-510 is less potent than GDGV(dI)TRIR for blocking a myristate-stimulated increase in cell adhesion to collagen and nitric oxide-driven accumulation of cGMP. ABT-510 at concentrations sufficient to inhibit CD36 fatty acid translocase activity synergizes with thrombin in aggregating platelets and blunts the activity of NO to delay aggregation, but again less than GDGV(dI)TRIR. In contrast, ABT-510 is more potent than GDGV(dI)TRIR for inducing caspase activation in vascular cells. Thus, we propose that ABT-510 is a drug with at least two mechanisms of action, and its potent anti-tumor activity may be in part independent of CD36 fatty acid translocase inhibition.

Keywords: Abbreviations; DEA/NO; diethylamine NONOate; DETA/NO; diethyltriamine NONOate; eNOS; endothelial nitric oxide synthase; FAF-BSA; fatty acid-free bovine serum albumin; FGF2; fibroblast growth factor-2; HAVSMC; human aortic vascular smooth muscle cells; HUVEC; human umbilical vein endothelial cells; TSP1; thrombospondin-1; VSMC; vascular smooth muscle cellsThrombospondin-1; Nitric oxide; CD36; ABT-510; Cancer; Fatty acid uptake

Proteasomal inhibition stabilizes topoisomerase IIα protein and reverses resistance to the topoisomerase II poison ethonafide (AMP-53, 6-ethoxyazonafide) by Lauren M. Congdon; Alan Pourpak; Aluvia M. Escalante; Robert T. Dorr; Terry H. Landowski (pp. 883-890).

Multiple myeloma (MM) is an incurable malignancy of plasma cells. Although multiple myeloma patients often respond to initial therapy, the majority of patients will relapse with disease that is refractory to further drug treatment. Thus, new therapeutic strategies are needed. One common mechanism of acquired drug resistance involves a reduction in the expression or function of the drug target. We hypothesized that the cytotoxic activity of topoisomerase II (topo II) poisons could be enhanced, and drug resistance overcome, by increasing the expression and activity of the drug target, topo II in myeloma cells. To test this hypothesis, we evaluated the cytotoxicity of the anthracene-containing topo II poison, ethonafide (AMP-53/6-ethoxyazonafide), in combination with the proteasome inhibitor bortezomib (PS-341/Velcade). Combination drug activity studies were done in 8226/S myeloma cells and its drug resistant subclone, 8226/Dox1V. We found that a 24-h treatment of cells with bortezomib maximally increased topo IIα protein expression and activity, and consistently increased the cytotoxicity of ethonafide in the 8226/S and 8226/Dox1V cell lines. This increase in cytotoxicity corresponded to an increase in DNA double-strand breaks, as measured by the neutral comet assay. Therefore, increasing topo IIα expression through inhibition of proteasomal degradation increased DNA double-strand breaks and enhanced the cytotoxicity of the topo II poison ethonafide. These data suggest that bortezomib-mediated stabilization of topo IIα expression may potentiate the cytotoxic activity of topo II poisons and thereby, provide a strategy to circumvent drug resistance.

Keywords: Topoisomerase IIα; Proteasome inhibition; Multiple myeloma; Combination therapy

Therapeutic angiogenesis of mouse hind limb ischemia by novel peptide activating GRP78 receptor on endothelial cells by Britta Hardy; Alexander Battler; Chana Weiss; Orly Kudasi; Annat Raiter (pp. 891-899).

Therapeutic angiogenesis emerged as a non-invasive mean of promoting neovascularization in ischemic tissues. We have searched for new molecules that induce angiogenesis by screening a phage display combinatory peptide library on endothelial cells. One of the selected peptides identified by binding to endothelial cells under hypoxic conditions was further studied. The aim of this study was to assess the therapeutic value of this peptide, RoY, in a mouse hind limb ischemia model and to identify it's receptor on endothelial cells.RoY, a 12 amino-acid synthetic peptide, induced in vitro angiogeneic activity under hypoxic conditions by increasing endothelial cell proliferation, migration and tube formation. In order to assess its therapeutic properties in ischemic tissues, a hind limb ischemia model was induced in C57BL mice by a femoral artery excision. A single local intramuscular injection of RoY peptide to the operated limb, significantly restored blood perfusion and alleviated hind limb ischemia as determined by a laser Doppler imager. Increased capillary density in histological sections corroborated these findings. Protein precipitation and mass spectroscopy studies identified GRP78, a heat shock protein, as the peptide-binding membrane receptor that was increased on endothelial cell membranes under hypoxic conditions. This study demonstrates the efficacy of RoY peptide in alleviation of hind limb ischemia. In addition, it provides evidence that GRP78 is an angiogenic receptor on hypoxic endothelial cells.

Keywords: Hypoxia; Ischemia; Angiogenesis; Peptides; GRP78; Endothelial cells

Reduced expression of thyroid hormone receptors and beta-adrenergic receptors in human failing cardiomyocytes by Pietro Amedeo Modesti; Matilde Marchetta; Tania Gamberi; Gianluca Lucchese; Massimo Maccherini; Mario Chiavarelli; Alessandra Modesti (pp. 900-906).

An altered thyroid hormone profile has been reported in patients with congestive heart failure. However, information regarding the status of thyroid hormone receptors in human failing cardiomyocytes is lacking. Therefore the expression of thyroid hormone and beta-adrenergic receptors was investigated in human ventricular cardiomyocytes isolated from patients with end-stage heart failure (FM, n=12), or from tentative donors (C, n=4). The expression of thyroid (TRalpha1, and TRbeta1) and beta-adrenergic receptors (ARB1 and ARB2) was measured at both the gene, and at the protein level.In FM the reduced mRNA expression of ARB1 ( p<0.05, −37%) and ARB2 ( p<0.05, −42%) was associated with a reduction of the messenger for TRalpha1 ( p<0.05, −85%) and TRalpha2 ( p<0.05, −73%). These findings were confirmed at the protein level for ARB1, ARB2 and TRalpha1.These data reveal that in human heart failure the reduction of beta-adrenergic receptors is associated with reduced expression of both TRalpha1 and TRalpha2 isoforms of thyroid hormone receptors.

Keywords: Heart failure; Cardiomyopathy; 3,5,3′-Triiodo-; l; -thyronine; Myocytes

Reversal of chemoresistance and enhancement of apoptosis by statins through down-regulation of the NF-κB pathway by Kwang Seok Ahn; Gautam Sethi; Bharat B. Aggarwal (pp. 907-913).

We recently found that simvastatin can modulate the nuclear factor-κB (NF-κB) activation pathway, but whether other statins have similar effects to those of simvastatin is unknown. Therefore, we evaluated the effect six different statins on TNF-induced NF-κB activation in human myeloid leukemia cells. We then determined whether the combination of statins and standard chemotherapeutic agents could overcome chemoresistance and augment apoptosis. Of the six statins evaluated, only the natural statins (simvastatin, mevastatin, lovastatin, and pravastatin), not the synthetic statins (fluvastatin and atorvastatin), inhibited TNF-induced NF-κB activation. Simvastatin suppressed the NF-κB activation and potentiated the apoptosis induced by doxorubicin, paclitaxel, and 5-fluorouracil. These results suggest that different statins behave differently from one another and that they may be useful in overcoming chemoresistance.

Keywords: Abbreviations; NF-κB; nuclear factor-κB; TNF; tumor necrosis factor α; EMSA; electrophoretic mobility shift assaySimvastatin; Nuclear factor-kappaB; Chemotherapeutic agents; Apoptosis

Baicalin inhibits macrophage activation by lipopolysaccharide and protects mice from endotoxin shock by Lin-lin Liu; Li-kun Gong; Hui Wang; Ying Xiao; Xiong-fei Wu; Yun-hai Zhang; Xiang Xue; Xin-ming Qi; Jin Ren (pp. 914-922).

Baicalin (BA) exhibits anti-inflammatory effect in vivo and in vitro and is used to treat inflammatory diseases. Here, we report that BA inhibits the activation of macrophage and protects mice from macrophage-mediated endotoxin shock. The experiments in vitro showed BA suppressed the increased generation of nitric oxide (NO) and expression of inducible nitric oxide synthase (iNOS) induced by LPS or Interferon-γ (IFN-γ) without directly affecting iNOS activity in RAW264.7 cells and peritoneal macrophages. Similarly, BA inhibited the production of reactive oxidative species (ROS), whereas augmented the level of intracellular superoxide dismutase (SOD). Moreover, BA inhibited the production of inflammatory mediators including tumor necrosis factor (TNF)-α, endothelin (ET)-1 and thromboxane A₂ (TXA₂) induced by lipopolysaccharide (LPS) in RAW264.7 cells. In animal model, BA protected mice from endotoxin shock induced byd-galactosamine (d-GalN)/LPS possibly through inhibiting the production of cytokine and NO. Collectively, BA inhibited the production of inflammatory mediators by macrophage and may be a potential target for treatment of macrophage-mediated diseases.

Keywords: Baicalin; Macrophage; Lipopolysaccharide; Nitric oxide; Endotoxin shock

Cyclo(dehydrohistidyl-l-tryptophyl) inhibits nitric oxide production by preventing the dimerization of inducible nitric oxide synthase by Mi-Jin Sohn; Gang-Min Hur; Hee-Sun Byun; Won-Gon Kim (pp. 923-930).

Dimerization of inducible NOS has been known to be a potential therapeutic target for iNOS-mediated pathologies. Cyclic dipeptides are among the simplest peptides commonly found as by-products of food processing or metabolites of microorganisms. In this study, we found that cyclo(dehydrohistidyl-l-tryptophyl) (CDHT), a cyclic dipeptide from an unidentified fungal strain Fb956, prevents iNOS dimerization in activated microglial BV-2 cells. CDHT inhibited NO production with an IC₅₀ of 6.5μM in LPS-treated BV-2 cells. Western blot analysis and iNOS activity measurement of fractions from size-exclusion chromatography of cell lysates indicated that CDHT inhibits dimerization of iNOS, while it has no effect on iNOS expression or enzyme activity. The CDHT inhibition of iNOS dimerization was confirmed by partially denaturing SDS-PAGE analysis. In contrast, CDHT did not affect cGMP production in endothelial HUVEC cells, which indicates no inhibition of endothelial NOS activity. These results reveal that CDHT, one of the simplest and cyclic dipeptides, selectively inhibits NO production by inhibiting iNOS dimerization, and could be a useful therapeutic agent for inflammation-mediated diseases.

Keywords: Abbreviations; CDHT; cyclo(dehydrohistidyl-; l; -tryptophyl); NO; nitric oxide; iNOS; inducible nitric oxide synthase; nNOS; neuronal nitric oxide synthase; eNOS; endothelial nitric oxide synthase; LPS; lipopolysaccharide; HUVEC; human umbilical vein endothelial cells; l; -NAME; N; ^G; -nitro-; l; -arginine methyl ester; cGMP; guanosine-3′,5′-cyclic monophosphate; GTP; guanosine triphosphateCyclo(dehydrohistidyl-; l; -tryptophyl); iNOS; Dimerization; Nitric oxide; BV-2 microglia; LPS

Promiscuous coupling and involvement of protein kinase C and extracellular signal-regulated kinase 1/2 in the adenosine A₁ receptor signalling in mammalian spermatozoa by Alba Minelli; Ilaria Bellezza; Giulia Collodel; Bertil B. Fredholm (pp. 931-941).

Mammalian spermatozoa require a maturational event after ejaculation that allows them to acquire the capacity for fertilisation. This process occurs spontaneously during the transit through the female reproductive tract where spermatozoa are in contact with micromolar concentrations of adenosine that might act as a capacitative effector. This study shows that the adenosine A₁ receptor agonist, 2-chloro- N⁶-cyclopentyladenosine, can induce capacitation, i.e., the ability to undergo the acrosome reaction and to become fertile. This receptor, already known to be bound to Gα_i2, is also bound to G_q/11. These G proteins are functional in the signalling pathway elicited by the A₁ receptor and correlate with the multiple intracellular events that follow its activation. The use of protein kinase C isoform inhibitors and MEK inhibitors, resulting in the abolition of the biological response to the selective agonist, indicates the involvement of protein kinase C and MEK in its signalling. In agonist-treated spermatozoa an extracellular calcium influx, involvement of α and γ PKC isoforms and transient phosphorylation of ERK1/2 have been observed. Our results, besides showing that adenosine A₁ receptor prompts mammalian spermatozoa to undergo the acrosome reaction hence supporting a role for adenosine as agent for fertilisation, show that 2-chloro- N⁶-cyclopentyladenosine triggers signalling mechanisms that involve both Gα_i2 and G_q/11, extracellular calcium influx, modulation of classical Ca²⁺-dependent PCK isoforms and up-regulation of the ERK1/2 phosphorylation.

Keywords: Protein G inhibitors; Protein kinase inhibitors; 2-Chloro-; N; ⁶; -cyclopentyladenosine; [Ca; ²⁺; ]; _i; oscillations

Regulation of cyclic AMP response-element binding-protein (CREB) by G_q/11-protein-coupled receptors in human SH-SY5Y neuroblastoma cells by Elizabeth M. Rosethorne; Stefan R. Nahorski; R.A. John Challiss (pp. 942-955).

Human SH-SY5Y neuroblastoma cells have been used to investigate mechanisms involved in CREB phosphorylation after activation of two endogenously expressed G_q/11-protein-coupled receptors, the M₃ muscarinic acetylcholine (mACh) and B₂ bradykinin receptors. Stimulation with either methacholine or bradykinin resulted in maximal increases in CREB phosphorylation within 1min, with either a rapid subsequent decrease (bradykinin) to basal levels, or a sustained response (methacholine). Inhibitor studies were performed to assess the involvement of a number of potential kinases in signalling to CREB phosphorylation. Removal of extracellular Ca²⁺, inhibition of Ca²⁺/calmodulin-dependent protein kinase II and down-regulation of protein kinase C (PKC) resulted in reduced CREB phosphorylation after both M₃ mACh and B₂ bradykinin receptor activation. In contrast, inhibition of MEK1/2 by U0126 resulted in significantly reduced CREB phosphorylation levels after B₂ bradykinin, but not M₃ mACh receptor activation. In addition, we demonstrate that maintained phosphorylation of CREB is necessary for CRE-dependent gene transcription as the M₃ mACh, but not the B₂ bradykinin receptor activates both a recombinant CRE-dependent reporter gene, and the endogenous c-Fos gene. These data highlight the involvement of multiple, overlapping signalling pathways linking these endogenous G_q/11-coupled metabotropic receptors to CREB and emphasize the importance of the duration of signalling pathway activation in converting a CREB phosphorylation event into a significant change in transcriptional activity.

Keywords: Abbreviations; CREB; cyclic AMP response-element binding-protein; ERK; extracellular signal-regulated kinase; GPCR; G-protein-coupled receptor; IBMX; 3-isobutyl-1-methylxanthine; mACh; muscarinic acetylcholine; MAPK; mitogen-activated protein kinase; PDBu; phorbol 12,13-dibutyrate; PKA; cyclic AMP-dependent protein kinase; PKC; protein kinase C; PLC; phospholipase CCyclic AMP response-element binding-protein (CREB); Muscarinic acetylcholine receptor; Bradykinin receptor; SH-SY5Y cells; Intracellular calcium concentration [Ca; ²⁺; ]; _i; Calmodulin; Ca; ²⁺; /calmodulin-dependent protein kinase; Protein kinase C; c-Fos

Effects of memantine on mitochondrial function by Jenna McAllister; Sangeeta Ghosh; Diana Berry; Melissa Park; Sina Sadeghi; Kai Xuan Wang; W. Davis Parker; Russell H. Swerdlow (pp. 956-964).

Because NMDA complex and mitochondrial function are related, we hypothesized memantine would influence mitochondrial function. We addressed this in vitro by studying the effects of chronic and acute memantine exposures on mitochondrial function. For acute exposure experiments, mitochondria were isolated from NT2 cells and assayed for electron transport chain (ETC) enzyme function and peroxide production in buffers containing up to 60uM memantine. For chronic exposure experiments, NT2 cells were maintained for at least two weeks in medium containing up to 60uM memantine, following which we assayed cells or their mitochondria for ETC enzyme activities, cytochrome oxidase protein levels, oxidative stress, calcium levels, and mitochondrial DNA levels. The ability of the NMDA receptor antagonist aminophosphonovaleric acid (APV) to modify memantine's mitochondrial effects was evaluated. Acute and chronic memantine similarly affected complex I (increased at high concentrations) and IV (decreased at high concentrations) V_max activities. APV did not alter the effects of chronic memantine exposure on citrate synthase and complex IV. We detected a lower mitochondrial peroxide production rate with acute exposure, and an increased mitochondrial peroxide production rate with chronic exposure. Micromolar memantine concentrations affect mitochondria, some of these effects are directly mediated, and acute and chronic effects may differ.

Keywords: Alzheimer's disease; Electron transport chain; Memantine; Mitochondria; NMDA; Oxidative stress

Alpha anomer of 5-aza-2′-deoxycytidine down-regulates hTERT mRNA expression in human leukemia HL-60 cells by Miroslav Hájek; Ivan Votruba; Antonín Holý; Marcela Krečmerová; Eva Tloušt’ová (pp. 965-972).

DNA methylation inhibitors are being extensively studied as potential anticancer agents. In the present study, we compared the capability of alpha anomer of 5-aza-2′-deoxycytidine (α-5-azadCyd) to induce down-regulation of hTERT expression in HL-60 cells with other nucleoside analogs that act as DNA methylation inhibitors: β-5-azadCyd (decitabine), ( S)-9-(2,3-dihydroxypropyl)adenine [( S)-DHPA], isobutyl ester of ( R,S)-3-(adenin-9-yl)-2-hydroxypropanoic acid [( R,S)-AHPA- ibu] and prospective DNA methylation inhibitors ( S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine [( S)-HPMPazaC] and 5-fluoro-zebularine (F-PymRf). Exposure to α-5-azadCyd induced the down-regulation of hTERT expression in low micromolar concentrations (0.05–50μM). A more cytotoxic beta anomer caused a transient up-regulation of hTERT and a subsequent reduction in hTERT mRNA levels at concentrations more than 10 times below its GIC₅₀ value. In this respect, ( S)-DHPA and ( R,S)-AHPA- ibu were less efficient, since a similar effect was achieved at concentrations above their GIC₅₀. In contrast, F-PymRf treatment resulted in up to a three-fold induction of hTERT expression within a broad range of concentrations. In all cases, the down-regulation of hTERT expression was concentration-dependent. The correlation was found between c- myc overexpression and transiently elevated hTERT expression after treatment with all tested compounds except for α-5-azadCyd and ( S)-HPMPazaC. Although the primary task of hypomethylating agents in anticancer therapy lies in reactivation of silenced tumour-suppressor genes, the inhibition of hTERT expression might also be a fruitful clinical effect of this approach.

Keywords: Abbreviations; GIC; ₅₀; concentration which causes 50% growth inhibition; hTERT; human telomerase reverse transcriptase; CTCF; CCCTC binding factor; 5-azadCyd; 5-aza-2′-deoxycytidine; 5-azaCyd; 5-azacytidine; HAT; histone acetyltransferase; SAH; S; -adenosyl-; l; -homocysteine; SAM; S; -adenosyl-; l; -methionine; GAPDH; glyceraldehyde-3-phosphate-dehydrogenase; RPII; RNA polymerase II; G6PDH; glucose-6-phosphate dehydrogenase; TBP; TATA box-binding protein; PBGD; porphobilinogen deaminase; PLA; phospholipase A2; Act; β-actinAlpha-5-aza-2′-deoxycytidine; DNA hypomethylation; hTERT; c-; myc

Role of P-glycoprotein in accumulation and cytotoxicity of amrubicin and amrubicinol in MDR1 gene-transfected LLC-PK1 cells and human A549 lung adenocarcinoma cells by Asumi Hira; Hiroshi Watanabe; Yukiko Maeda; Koji Yokoo; Emiko Sanematsu; Junko Fujii; Ji-ichiro Sasaki; Akinobu Hamada; Hideyuki Saito (pp. 973-980).

Amrubicin is a completely synthetic 9-aminoanthracycline agent for the treatment of lung cancer in Japan. The cytotoxicity of C-13 hydroxy metabolite, amrubicinol, is 10 to 100 times greater than that of amrubicin. The transporters responsible for the intracellular pharmacokinetics of amrubicin and amrubicinol remains unclear. This study was aimed to determine whether P-glycoprotein (P-gp) plays functional and preventive role in cellular accumulation and cytotoxicity of amrubicin and its active metabolite amrubicinol by in vitro transport and toxicity experiments. Cytotoxicity and intracellular accumulation of amrubicin and amrubicinol were evaluated by LLC-PK1 cells, MDR1 gene-transfected LLC-PK1 (L-MDR1) cells overexpressing P-gp, and human A549 lung adenocarcinoma cells. L-MDR1 cells showed 6- and 12-fold greater resistance to amrubicin and amrubicinol, respectively, than the parental LLC-PK1 cells. The intracellular accumulation of both drugs in L-MDR1 cells was significantly reduced compared to the LLC-PK1 cells. The basal-to-apical transepithelial transport of both drugs markedly exceeded, whereas the apical-to-basal transport of both drugs was significantly lower in L-MDR1 cells than LLC-PK1 cells. Cyclosporin A (CyA) restored the sensitivity, intracellular accumulation and transport activity for both drugs in L-MDR1 cells. In A549 cells, CyA significantly increased the intracellular accumulation and cytotoxicity of both drugs. These findings indicated that P-gp is responsible for cellular accumulation and cytotoxicity of both amrubicin and amrubicinol, therefore suggesting that the antitumor effect of amrubicin could be affected by the expression level of P-gp in lung cancer cells in chemotherapeutic treatments.

Keywords: Abbreviations; MDR; multidrug resistance; P-gp; P-glycoprotein; CyA; cyclosporine A; MRP; multidrug-resistance-associated proteinAmrubicin; Amrubicinol; P-glycoprotein; Transporter

The pathogenesis of ethanol versus methionine and choline deficient diet-induced liver injury by Maxwell Afari Gyamfi; Ivan Damjanov; Samuel French; Yu-Jui Yvonne Wan (pp. 981-995).

The differences and similarities of the pathogenesis of alcoholic (ASH) and non-alcoholic steatohepatitis (NASH) were examined. Mice (six/group) received one of four Lieber-Decarli liquid diets for 6 weeks: (1) paired-fed control diet; (2) control diet with ethanol (ethanol); (3) paired-fed methionine/choline deficient (MCD) diet; and (4) MCD plus ethanol (combination). Hepatotoxicity, histology, and gene expression changes were examined. Both MCD and ethanol induced macrovesicular steatosis. However, the combination diet produced massive steatosis with minor necrosis and inflammation. MCD and combination diets, but not ethanol, induced serum ALT levels by 1.6- and 10-fold, respectively. MCD diet, but not ethanol, also induced serum alkaline phosphatase levels suggesting bile duct injury. Ethanol increased liver fatty acid binding protein (L-FABP) mRNA and protein levels. In contrast, the combination diet decreased L-FABP mRNA and protein levels and increased hepatic free fatty acid and lipid peroxide levels. Ethanol, but not MCD, reduced hepatic S-adenosylmethionine (SAM) and GSH levels. Hepatic TNFα protein levels were increased in all treatment groups, however, IL-6, a hepatoprotective cytokine which promotes liver regeneration was increased in ethanol-fed mice (2-fold), but decreased in the combination diet-treated mice. In addition, the combination diet reduced phosphorylated STAT3 and Bcl-2 levels. While MCD diet might cause bile duct injury and cholestasis, ethanol preferentially interferes with the SAM-GSH oxidative stress pathway. The exacerbated liver injury induced by the combination diet might be explained by reduced L-FABP, increased free fatty acids, oxidative stress, and decreased IL-6 protein levels. The combination diet is an efficient model of steatohepatitis.

Keywords: Abbreviations; L-FABP; liver fatty acid binding protein; GST; glutathione; S; -transferase; CYP2E1; cytochrome P450 2E1; SAM; S; -adenosylmethionine; SAH; S; -adenosylhomocysteine; MCD; methionine and choline deficient; ROS; reactive oxygen species; PPARα; peroxisome proliferator-activated receptor alpha; TNFα; tumor necrosis factor-alpha; ASH; alcoholic steatohepatitis; NASH; non-alcoholic steatohepatitis; RT-PCR; reverse transcription-polymerase chain reaction; AST; aspartate aminotransferase; ALT; alanine aminotransferase; ALP; alkaline phosphatase; LPO; lipid peroxidation; GSH; reduced glutathione; GPX; glutathione peroxidase; NEFA; nonesterified fatty acid; CDNB; 1-chloro-2,4-dinitrobenzene; TBST; Tris-buffered saline with 0.1% Tween 20; MMLV-RT; Molony murine leukemia virus reverse transcriptase; MAT; methionine adenosyltransferase; AOX; acyl-CoA oxidase; STAT3; signal transducer and activator of transcription factor 3; PC; phosphatidylcholine; VLDL; very low density lipoproteinsAlcoholic steatohepatitis; Non-alcoholic steatohepatitis; Steatosis; Liver fatty acid binding protein; Acyl CoA oxidase; Methionine choline deficient diet; Ethanol

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Biochemical Pharmacology (v.75, #4)