Check out our New Publishers' Select for Free Articles

Biochemical Pharmacology (v.70, #3)

Editorial Advisory Board (pp. iii).

Development of 5-lipoxygenase inhibitors—lessons from cellular enzyme regulation by Oliver Werz; Dieter Steinhilber (pp. 327-333).

5-Lipoxygenase (5-LO) catalyzes the first steps in the conversion of arachidonic acid (AA) into leukotrienes (LTs) that are mediators of inflammatory and allergic reactions. Recently, the 5-LO pathway has also been associated with atherosclerosis and osteoporosis. Thus, in addition to the classical applications including asthma and allergic disorders, LT synthesis inhibitors might be of interest for the treatment of cardiovascular diseases and osteoporosis. Recently, it has been shown that cellular 5-LO activity is regulated in a complex manner that can involve different signalling pathways. 5-LO can be activated by an increase in intracellular Ca²⁺ concentration, diacylglycerols, phosphorylation by MAPKAP kinase-2 and ERK. Previous work could demonstrate that cellular 5-LO activity is repressed in a protein kinase A-dependent manner and by glutathione peroxidases. This comment focuses on the impact of these stimulatory and inhibitory pathways on the efficacy of 5-LO inhibitors and suggests additional criteria for the development of this class of compounds.

Keywords: Abbreviations; AA; arachidonic acid; cPLA; ₂; cytosolic phospholipase A; ₂; FLAP; 5-lipoxygenase-activating protein; fMLP; N; -formyl-methionyl-leucyl-phenylalanin; GM-CSF; granulocyte macrophage colony-stimulating factor; GPx; glutathione peroxidase; LPS; lipopolysaccharide; 5-LO; 5-lipoxygenase; LT; leukotriene; MAPK; mitogen-activated protein kinase; MK; MAPKAP kinase (mitogen-activated protein kinase-activated protein kinase); MGST; microsomal glutathione; S; -transferase; PC; phosphatidylcholine; PMNL; polymorphonuclear leukocytes; PKA; protein kinase A; TNFα; tumor necrosis factor alpha

Inhibition of cyclooxygenase with indomethacin phenethylamide reduces atherosclerosis in apoE-null mice by Michael E. Burleigh; Vladimir R. Babaev; Mayur B. Patel; Brenda C. Crews; Rory P. Remmel; Jason D. Morrow; John A. Oates; Lawrence J. Marnett; Sergio Fazio; MacRae F. Linton (pp. 334-342).

Non-selective inhibition of cyclooxygenase (COX) has been reported to reduce atherosclerosis in both rabbit and murine models. In contrast, selective inhibition of COX-2 has been shown to suppress early atherosclerosis in LDL-receptor null mice but not more advanced lesions in apoE deficient (apoE^−/−) mice. We investigated the efficacy of the novel COX inhibitor indomethacin phenethylamide (INDO-PA) on the development of different stages of atherosclerotic lesion formation in female apoE^−/− mice. INDO-PA, which is highly selective for COX-2 in vitro, reduced platelet thromboxane production by 61% in vivo, indicating partial inhibition of COX-1 in vivo. Treatment of female apoE^−/− mice with 5mg/kg INDO-PA significantly reduced early to intermediate aortic atherosclerotic lesion formation (44 and 53%, respectively) in both the aortic sinus and aorta en face compared to controls. Interestingly, there was no difference in the extent of atherosclerosis in the proximal aorta in apoE^−/− mice treated from 11 to 21 weeks of age with INDO-PA, yet there was a striking (76%) reduction in lesion size by en face analysis in these mice. These studies demonstrate the ability of non-selective COX inhibition with INDO-PA to reduce early to intermediate atherosclerotic lesion formation in apoE^−/− mice, supporting a role for anti-inflammatory approaches in the prevention of atherosclerosis.

Keywords: Atherosclerosis; Prostaglandins; Cyclooxygenase; COX inhibition; ApoE; ^−/−; mice; Aorta

Interactions of peroxynitrite with uric acid in the presence of ascorbate and thiols: Implications for uncoupling endothelial nitric oxide synthase by Nermin Kuzkaya; Norbert Weissmann; David G. Harrison; Sergey Dikalov (pp. 343-354).

It has been suggested that uric acid acts as a peroxynitrite scavenger although it may also stimulate lipid peroxidation. To gain insight into how uric acid may act as an antioxidant, we used electron spin resonance to study the reaction of uric acid and plasma antioxidants with ONOO⁻. Peroxynitrite reacted with typical plasma concentrations of urate 16-fold faster than with ascorbate and 3-fold faster than cysteine. Xanthine but not other purine-analogs also reacted with peroxynitrite. The reaction between ONOO⁻ and urate produced a carbon-centered free radical, which was inhibited by either ascorbate or cysteine. Moreover, scavenging of ONOO⁻ by urate was significantly increased in the presence of ascorbate and cysteine. An important effect of ONOO⁻ is oxidation of tetrahydrobiopterin, leading to uncoupling of nitric oxide synthase. The protection of eNOS function by urate, ascorbate and thiols in ONOO⁻-treated bovine aortic endothelial cells (BAECs) was, therefore, investigated by measuring superoxide and NO using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine (CMH) and the NO-spin trap Fe[DETC]₂. Peroxynitrite increased superoxide and decreased NO production by eNOS indicating eNOS uncoupling. Urate partially prevented this effect of ONOO⁻ while treatment of BAECs with the combination of either urate with ascorbate or urate with cysteine completely prevented eNOS uncoupling caused by ONOO⁻. We conclude that the reducing and acidic properties of urate are important in effective scavenging of peroxynitrite and that cysteine and ascorbate markedly augment urate's antioxidant effect by reducing urate-derived radicals.

Keywords: Abbreviations; BAECs; bovine aortic endothelial cells; BH; ₄; tetrahydrobiopterin; CMH; 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine; CM; 3-methoxycarbonyl-proxyl; CP; 3-carboxy-proxyl; CPH; 1-hydroxy-3-carboxy-2,2,5-tetramethyl-pyrrolidine; EMPO; 5-ethoxycarbonyl-5-methyl-1-pyrroline; N; -oxide; SIN-1; 3-morpholinosydnonimine; XXO; xanthine/xanthine oxidaseUrate; Peroxynitrite; eNOS; Ascorbate; Thiols; ESR; Superoxide; Nitric oxide

Effect of thiazolidinediones on equilibrative nucleoside transporter-1 in human aortic smooth muscle cells by George P.H. Leung; Ricky Y.K. Man; Chung-Ming Tse (pp. 355-362).

Thiazolidinediones are a new class of anti-diabetic agents which increase insulin sensitivity by binding to the peroxisome proliferator-activated receptor γ (PPARγ) and stimulating the expression of insulin-responsive genes involved in glucose and lipid metabolism. These drugs also have vasodilatory and anti-proliferative effects on vascular smooth muscle cells. However the mechanisms for these actions are not fully understood. Adenosine is a vasodilator and a substrate of equilibrative nucleoside transporters (ENT). The present study studied the effects of three thiazolidinediones, troglitazone, pioglitazone and ciglitazone, on ENT1 in the human aortic smooth muscle cells (HASMCs). Although incubating HASMCs for 48h with thiazolidinediones had no effect on ENT1 mRNA and protein levels, troglitazone acutely inhibited [³H]adenosine uptake and [³H]NBMPR binding of HASMCs with IC₅₀ values of 2.35±0.35 and 3.99±0.57μM, respectively. The effect of troglitazone on ENT1 was PPARγ-independent and kinetic studies revealed that troglitazone was a competitive inhibitor of ENT1. In contrast, pioglitazone and ciglitazone had minimal effects on [³H]adenosine uptake by HASMCs. Troglitazone differs from pioglitazone and ciglitazone in that its side-chain contains a Vitamin E moiety. The difference in structure of troglitazone did not account for its inhibitory effect on ENT1 because Vitamin E did not inhibit [³H]adenosine uptake by HASMCs. Using the nucleoside transporter deficient PK15NTD cells stably expressing ENT1 and ENT2, it was found that troglitazone inhibited ENT1 but had no effect on ENT2. From these results, it is suggested that troglitazone may enhance the vasodilatory effect of adenosine by inhibiting ENT1. Pharmacologically, troglitazone is a novel inhibitor of ENT1.

Keywords: Abbreviations; ENT; equilibrative nucleoside transporter; HASMCs; human aortic smooth muscle cells; NBMPR; nitrobenzylmercaptopurine riboside; PPARγ; peroxisome proliferator-activated receptor-gammaThiazolidinediones; Troglitazone; Adenosine; Diabetes; Nucleoside transporter; Smooth muscle cells

The licorice root derived isoflavan glabridin increases the function of osteoblastic MC3T3-E1 cells by Eun-Mi Choi (pp. 363-368).

Glabridin, an isoflavan purified from licorice root, exhibits diverse biological activities, including estrogen-like activity. To investigate the bioactivities of glabridin, which act on bone metabolism, the effects of glabridin on the function of mouse osteoblastic cell line (MC3T3-E1) and the production of local factors in osteoblasts were studied. Glabridin (1–10μM) significantly increased the growth of MC3T3-E1 cells and caused a significant elevation of alkaline phosphatase (ALP) activity, collagen content and osteocalcin secretion in the cells ( P<0.05). The effect of glabridin (10μM) in increasing ALP activity and collagen content was completely prevented by the presence of 10⁻⁶M cycloheximide and 10⁻⁶M tamoxifen, suggesting that glabridin's effect results from a newly synthesized protein component and might be partly involved in estrogen action. Then, the effects of glabridin on the TNF-α-induced apoptosis and production of prostaglandin E₂ (PGE₂) and nitric oxide (NO) in osteoblasts were examined. Treatment with glabridin (1–10μM) prevented apoptosis induced by TNF-α (10⁻¹⁰M) in osteoblastic cells. Moreover, glabridin (50μM) decreased the 10⁻¹⁰M TNF-α-induced production of PGE₂ and NO in osteoblasts. Our data indicate that the enhancement of osteoblast function by glabridin may result in the prevention for osteoporosis and inflammatory bone diseases.

Keywords: Glabridin; Osteoblastic MC3T3-E1 cells

The 5-HT3 receptor antagonist tropisetron inhibits T cell activation by targeting the calcineurin pathway by Laureano de la Vega; Eduardo Mu�oz; Marco A. Calzado; Klaus Lieb; Eduardo Candelario-Jalil; Harald Gschaidmeir; Lothar F�rber; Wolfgang Mueller; Thomas Stratz; Bernd L. Fiebich (pp. 369-380).

Tropisetron, an antagonist of serotonin type 3 receptor, has been investigated in chronic inflammatory joint process. Since T cells play a key role in the onset of several inflammatory diseases, we have evaluated the immunosuppressive activity of tropisetron in human T cells, discovering that this compound is a potent inhibitor of early and late events in TCR-mediated T cell activation. Moreover, we found that tropisetron specifically inhibited both IL-2 gene transcription and IL-2 synthesis in stimulated T cells. To further characterize the inhibitory mechanisms of tropisetron at the transcriptional level, we examined the DNA binding and transcriptional activities of NF-κB, NFAT and AP-1 transcription factors in Jurkat T cells. We found that tropisetron inhibited both the binding to DNA and the transcriptional activity of NFAT and AP-1. We also observed that tropisetron is a potent inhibitor of PMA plus ionomycin-induced NF-κB activation but in contrast TNFα-mediated NF-κB activation was not affected by this antagonist. Finally, overexpression of a constitutively active form of calcineurin indicated that this phosphatase may represent one of the main targets for the inhibitory activity of tropisetron. These findings provide new mechanistic insights into the anti-inflammatory activities of tropisetron, which are probably independent of serotonin receptor signalling and highlight their potential to design novel therapeutic strategies to manage inflammatory diseases.

Keywords: Abbreviations; AP-1; activator protein-1; EMSA; electrophoretic mobility shift assay; 5-HT; serotonin; 5-HTR; serotonin receptor; IKK; IκB kinase; IκB; κB inhibitor; JNK; jun kinase; MAPK; mitogen activated kinase; NF-κB; nuclear factor kappa B; NFAT; nuclear factor of activated cells; TNFα; tumor necrosis factor αTropisetron; T cells; IL-2; NF-κB; NFAT; Calcineurin

Amphiphilic pyridinium salts block TNFα/NFκB signaling and constitutive hypersecretion of interleukin-8 (IL-8) from cystic fibrosis lung epithelial cells by Susanna Tchilibon; Jian Zhang; QingFeng Yang; Ofer Eidelman; Haksung Kim; Hung Caohuy; Kenneth A. Jacobson; Bette S. Pollard; Harvey B. Pollard (pp. 381-393).

Cystic fibrosis (CF) is a common, lethal genetic disease, which is due to mutations in the CFTR gene. The CF lung expresses a profoundly proinflammatory phenotype, due to constitutive hypersecretion of IL-8 from epithelial cells lining the airways. In a systematic search for candidate drugs that might be used therapeutically to suppress IL-8 secretion from these cells, we have identified a potent and efficacious series of amphiphilic pyridinium salts. The most potent of these salts is MRS2481, an ( R)-1-phenylpropionic acid ester, with an IC₅₀ of ca. 1μM. We have synthesized 21 analogues of MRS2481, which have proven sufficient to develop a preliminary structure–activity relationship (SAR). For optimal activity, we have found that the ester must be connected to the pyridinium derivative by an eight-carbon chain. An optical isomer of the lead compound, containing an ( S)-1-phenylpropionic acid ester, has been found to be a much less active. The mechanism of action of MRS2481 appears to involve inhibition of signaling of the NFκB and AP-1 transcription factors to the IL-8 promoter. MRS2481 is a potent inhibitor of TNFα-induced phosphorylation and proteosomal destruction of IκBα. Inasmuch as IκBα is the principal inhibitor of the NFκB signaling pathway, preservation of intact IκBα would serve to keep the IL-8 promoter silent. We also find that MRS2481 blocks TNFα-activated phosphorylation of JNK, the c-JUN kinase. The IL-8 promoter is also activated by an AP-1 site, which requires a phospho-c-JUN/c-FOS dimer for activity. We therefore interpret these data to suggest that the mechanism of MRS2481 action is to inhibit both NFκB and AP-1 signaling on the IL-8 promoter. Given the medicinally promising properties of water-solubility, potency in the low μM concentration range, and high efficacy, we anticipate that MRS2481, or a further optimized derivative, may find an important place in the armamentarium of pharmaceutical strategies yet to be arrayed against the inflammatory phenotype of the CF lung.

Keywords: Cystic fibrosis; Inflammation; Lung; Interleukin 8; Epithelium; NFκB; AP-1

HDAC inhibition prevents NF-κB activation by suppressing proteasome activity: Down-regulation of proteasome subunit expression stabilizes IκBα by Robert F. Place; Emily J. Noonan; Charles Giardina (pp. 394-406).

The short chain fatty acid (SCFA) butyrate (BA) and other histone deacetylase (HDAC) inhibitors can rapidly induce cell cycle arrest and differentation of colon cancer cell lines. We found that butyrate and the specific HDAC inhibitor trichostatin A (TSA) can reprogram the NF-κB response in colon cancer cells. Specifically, TNF-α activation is suppressed in butyrate-differentiated cells, whereas IL-1β activation is largely unaffected. To gain insight into the relationship between butyrate-induced differentiation and NF-κB regulation, we determined the impact of butyrate on proteasome activity and subunit expression. Interestingly, butyrate and TSA reduced the cellular proteasome activity in colon cancer cell lines. The drop in proteasome activity results from the reduced expression of the catalytic β-type subunits of the proteasome at both the protein and mRNA level. The selective impact of HDAC inhibitors on TNF-α-induced NF-κB activation appears to relate to the fact that the TNF-α-induced activation of NF-κB is mediated by the proteasome, whereas NF-κB activation by IL-1β is largely proteasome-independent. These findings indicate that cellular differentation status and/or proliferative capacity can significantly impact proteasome activity and selectively alter NF-κB responses in colon cancer cells. This information may be useful for the further development and targeting of HDAC inhibitors as anti-neoplastic and anti-inflammatory agents.

Keywords: Abbreviations; SCFA; short chain fatty acid; HDAC; histone deacetylase; NF-κB; nuclear factor-κB; IκB; inhibitor-κB; IKK; IκB kinase; TNF-α; tumor necrosis factor-α; IL-1β; interleukin-1β; BA; butyrate; TSA; trichostatin A; CT-L; chymotrypsin-like; T-L; trypsyn-like; PGPH; post-glutamyl peptide hydrolyzing; E-MEM; minimal essential media with Earle's salts; EMSA; electrophoretic mobility shift assay; Suc-LLVY-AMC; N; -succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarinNF-κB; IκBα; TNF-α; IL-1β; Butyrate; TSA; HDAC; Proteasome; β5; β1; β2; Caco-2

Orientation of oxazolidinones in the active site of monoamine oxidase by Tadeusz Z.E. Jones; Paul Fleming; Charles J. Eyermann; Michael B. Gravestock; Rona R. Ramsay (pp. 407-416).

Oxazolidinone inhibitors of monoamine oxidase (MAO) and oxazolidinone antibacterials are two distinct classes of drug, often with linear structures and overlapping activities for some derivatives. By synthesizing novel dimerised derivatives with identical substitution of the two C-5 side chains, we have obtained experimental evidence for the orientation of oxazolidinones in the active site of MAO A. Two types of spectral changes, either increasing the absorbance at 510nm or decreasing it at 495nm depending on the group nearest to the flavin cofactor, were seen on ligand binding to MAO A. Side chain derivatives with amine substituents are very poor substrates so that it was possible to examine the spectral change due to binding of a substrate before reduction of the flavin occurred. Binding of these amino derivative substrates to MAO A induced a spectral change characterized by a strong decrease in absorbance at 495nm. These substrates reduced the enzyme fully without any trace of a semiquinone intermediate. Only oxazolidinone inhibitors with a bromo-imidazole substituent increased the yield of semiquinone intermediate obtained during chemical reduction. In accord with the experimental data, results of docking experiments showed that binding of the oxazolidinone ring in the aromatic cage close to the flavin was favored and that the nitrogen of the derivatives that were substrates was within van der Waals distance of N-5 of the flavin.

Keywords: Abbreviation; MAO A; monoamine oxidase AOxazolidinone; Monoamine oxidase; Difference spectra; Flexible docking; Kinetics

Anthocyanidins inhibit cyclooxygenase-2 expression in LPS-evoked macrophages: Structure–activity relationship and molecular mechanisms involved by De-Xing Hou; Takashi Yanagita; Takuhiro Uto; Satoko Masuzaki; Makoto Fujii (pp. 417-425).

The effects of anthocyanidins, the aglycon nucleuses of anthocyanins widely occurring in reddish fruits and vegetables, on the expression of cyclooxygenase-2 (COX-2) were investigated in lipopolysaccharide (LPS)-activated murine macrophage RAW264 cells. Of five anthocyanidins, delphinidin and cyanidin inhibited LPS-induced COX-2 expression, but pelargonidin, peonidin and malvidin did not. The structure–activity relationship suggest that the ortho-dihydroxyphenyl structure of anthocyanidins on the B-ring appears to be related with the inhibitory actions. Delphinidin, the most potent inhibitor, caused a dose-dependent inhibition of COX-2 expression at both mRNA and protein levels. Western blotting analysis indicated that delphinidin inhibited the degradation of IκB-α, nuclear translocation of p65 and CCAAT/enhancer-binding protein (C/EBP)δ and phosphorylation of c-Jun, but not CRE-binding protein (CREB). Moreover, delphinidin suppressed the activations of mitogen-activated protein kinase (MAPK) including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 kinase. MAPK inhibitors (U0126 for MEK1/2, SB203580 for p38 kinase and SP600125 for JNK) specifically blocked LPS-induced COX-2 expression. Thus, our results demonstrated that LPS-induced COX-2 expression by activating MAPK pathways and delphinidin suppressed COX-2 by blocking MAPK-mediated pathways with the attendant activation of nuclear factor-κB (NF-κB), activator protein-1 (AP-1) and C/EBPδ. These findings provide the first molecular basis that anthocyanidins with ortho-dihydroxyphenyl structure may have anti-inflammatory properties through the inhibition of MAPK-mediated COX-2 expression.

Keywords: Anthocyanidins; Delphinidin; Cyclooxygenase-2; Lipopolysaccharide; Transcription factors; Mitogen-activated protein kinaseAbbreviations; AP-1; activator protein-1; C/EBP; CCAAT/enhancer-binding protein; COX-2; cyclooxygenase-2; CREB; CRE-binding protein; ERK; extracellular signal-regulated kinase; JNK; c-Jun; N; -terminal kinase; LPS; lipopolysaccharide; MAPK; mitogen-activated protein kinase; NF-κB; nuclear factor κB

Cytosine arabinoside affects multiple cellular factors and induces drug resistance in human lymphoid cells by Malancha Sarkar; Tieran Han; Vijaya Damaraju; Pat Carpenter; Carol E. Cass; Ram P. Agarwal (pp. 426-432).

Continuous in vitro cultivation of human lymphoid H9 cells in the presence of 0.5μM arabinosyl-cytosine (araC) resulted in cell variant, H9-araC cells, that was >600-fold resistant to the drug and cross resistant to its analogs and other unrelated nucleosides, e.g. dideoxycytidine (5-fold), thiacytidine (2-fold), 2-fluoro-adenine arabinoside (8.3-fold), and 2-chloro-deoxyadenosine (2.1-fold). Compared to the parental cell line, the resistant cells accumulated <1% araCTP, and had reduced deoxycytidine kinase (dCK) activity (31.4%) and equilibrative nucleoside transporter 1 (ENT1) protein. The expression of the dCK gene in araC resistant cells was reduced to 60% of H9 cells, which correlated with lower dCK protein and activity. Whereas, there was no difference in the expression of ENT1 mRNA between the cell lines, ENT1 protein content was much lower in the resistant cells than in H9 cells. The concentrative nucleoside transporter (CNT3) was slightly increased in H9-araC cells, but CNT2, and MDR1 remained unaffected. Although a definitive correlation remains to be established, the amount of Sp1 protein, a transcription factor, that regulates the expressions of dCK, nucleoside transporters and other cellular proteins, was found reduced in H9-araC cells. Like ENT1, the Sp1 mRNA levels remained unaffected in H9-araC whereas protein contents were reduced. These observations are indicative of differences in the production and/or turnover of ENT1 and Sp1 proteins in H9-araC cells. Since nucleoside transporters and dCK play an important role in the activity of potential antiviral and anticancer deoxynucleoside analogs, understanding of their regulation is important. These studies show that the exposure of cells to araC, in vitro, is capable of simultaneously affecting more than one target site to confer resistance. The importance of this observation in the clinical use of araC remains to be determined.

Keywords: Abbreviations; araC; 1-β-; d; arabinofuranosyl-cytosine (cytosine arabinoside); araCTP; 5′-triphosphate of araC; AZT; 3′-azido-2′,3′-dideoxythymidine (zidovudine); CdA; 2-chloro-2′-deoxyadenosine (cladribine); CNT2 and CNT3; concentrative nucleoside transporter 2 and 3; dCK; deoxycytidine kinase (EC 2.7.1.74); ddC; 2′,3′-dideoxycytidine; F-araA; 2-fluoro-arabinosyladenine; FUR; 5-fluoro-uridine; FUdR; 5-fluoro deoxyuridine; ENT1 and ENT2; equilibrative nucleoside transporter 1 and 2; NBMPR; nitrobenzyl-mercaptopurine ribonucleoside; NT; nucleoside transporter; 3TC; thiacytidineTranscription factor; Sp1; Deoxycytidine kinase; Arabinosylcytosine; Nucleoside transport; Cellular resistance; Human lymphoid cells

Dissociated glucocorticoids equipotently inhibit cytokine- and cAMP-induced matrix degrading proteases in rat mesangial cells by Wolfgang Eberhardt; Tobias Kilz; El-Sayed Akool; Roswitha M�ller; Josef Pfeilschifter (pp. 433-445).

Dissociated glucocorticoids are synthetic ligands of the glucocorticoid receptor (GR) and which discriminate between transrepression and transactivation. These compounds were predicted to have large therapeutic benefits when compared to conventional glucocorticoids because of reduced side effects. In this study, we compared the transrepressive properties of different dissociated glucocorticoids on the interleukin-1β (IL-1β)-activated metalloproteinase-9 (MMP-9) and tissue plasminogen activator (tPA) expression in rat mesangial cells (MC). Both proteinases regulate the turnover of extracellular matrix (ECM). We demonstrate that the GR agonist RU 24858, equipotent to dexamethasone (DEX), exhibited strong suppressive effects on the IL-1β-induced MMP-9 and tPA mRNA levels concomitant with an inhibition of corresponding enzyme activities. In contrast, RU 24782 and RU 40066 exhibited weaker inhibitory activities on both proteinases. Mechanistically, the changes in MMP-9 expression level by different RUs were accompanied by an inhibition of cytokine-induced promoter activity indicating that the inhibition occurs on a transcriptional level. In parallel to the reduction in mRNA levels, we observed an attenuation of cytokine-induced DNA binding of nuclear factor kappa B (NF-κB) and reduced contents of the p65 subunit of NF-κB within cell nuclei. Along with these transrepressive activities RU 24858, RU 24782 and RU 40066 displayed similar transactivation potentials as indicated by induction of the glucocorticoid-inducible mouse mammary tumor virus (MMTV) reporter gene and by induced expression level of plasminogen activator inhibitor 1 (PAI-1). Interestingly, the different RUs affected the expression of cAMP-induced tPA and inducible NO synthase with the same potency as the IL-1β-induced protease expression thus indicating that these compounds equipotently modulate cytokine- and cAMP-driven gene expression.

Keywords: Dissociated glucocorticoids; Extracellular matrix; Proteases; Matrix metalloproteinase-9; Tissue plasminogen activator

Effect of repeated systemic administration of selective inhibitors of endocannabinoid inactivation on rat brain endocannabinoid levels by Eva de Lago; Stefania Petrosino; Marta Valenti; Enrico Morera; Silvia Ortega-Gutierrez; Javier Fernandez-Ruiz; Vincenzo Di Marzo (pp. 446-452).

Several selective inhibitors of endocannabinoid inactivation via either the fatty acid amide hydrolase (FAAH) or the putative endocannabinoid transporter have been developed so far. Here, we have studied the effect in rats of a subchronic intraperitoneal treatment with three recently developed selective inhibitors of endocannabinoid uptake (VDM-11, UCM-707 and OMDM-2) or with a selective FAAH inhibitor ( N-arachidonoyl-serotonin, AA-5-HT), on the brain levels of anandamide and 2-arachidonoylglycerol (2-AG) measured by means of isotope dilution LC–MS 1, 5 and 12h after the last treatment. OMDM-2 was the most efficacious compound at enhancing the levels of anandamide at all time points, with a maximal effect (1.9-fold enhancement) after 5h. This compound also enhanced 2-AG levels by ∼1.3-fold, but only 5 and 12h from administration. VDM-11 slightly, albeit significantly, enhanced anandamide levels (1.3-fold) only at 1h from administration and 2-AG levels (1.3-fold) only after 5h. Finally, UCM-707 only affected 2-AG levels (by two-fold) at only 1h from administration. FAAH inhibition by AA-5-HT significantly enhanced the levels of both anandamide (between 1.3- and 1.5-fold, maximal effect after 1h) and 2-AG (between 1.3- and 1.6-fold, maximal effect after 12h) at all time points. Brains from rats treated with AA-5-HT did never exhibit enhanced levels of serotonin, thus pointing to the metabolic stability of this FAAH inhibitor. These data indicate that: (1) the pharmacological effects reported so far for the four compounds under study in animal models of diseases may be due to enhancement of both anandamide and 2-AG levels; (2) 2-AG seems to need a longer time after the last administration in order to be augmented; (3) OMDM-2 and AA-5-HT should be regarded as enhancers of endocannabinoid levels suitable for use in vivo.

Inhibitory effect of conjugated eicosapentaenoic acid on mammalian DNA polymerase and topoisomerase activities and human cancer cell proliferation by Yuko Yonezawa; Takahiko Hada; Keisuke Uryu; Tsuyoshi Tsuzuki; Takahiro Eitsuka; Teruo Miyazawa; Chikako Murakami-Nakai; Hiromi Yoshida; Yoshiyuki Mizushina (pp. 453-460).

Conjugated eicosapentaenoic acid (cEPA) selectively inhibited the activities of mammalian DNA polymerases (pols) and human DNA topoisomerases (topos) [Yonezawa Y, Tsuzuki T, Eitsuka T, Miyazawa T, Hada T, Uryu K, et al. Inhibitory effect of conjugated eicosapentaenoic acid on human DNA topoisomerases I and II. Arch Biochem Biophys 2005;435:197–206]. In this report, we investigated the inhibitory effect of cEPA on a human promyelocytic leukemia cell line, HL-60, to determine which enzymes influence cell proliferation. cEPA inhibited the proliferation of HL-60 cells (LD₅₀=20.0μM), and the inhibitory effect was stronger than that of non-conjugated EPA. cEPA arrested the cells at G1/S-phase, increased cyclin A and E protein levels, and prevented the incorporation of thymidine into the cells, indicating that it blocks the primary step of in vivo DNA replication by inhibiting the activity of replicative pols rather than topos. This compound also induced apoptosis of the cells. These results suggested the therapeutic potential of cEPA as a leading anti-cancer compound that poisons pols.

Keywords: Abbreviations; EPA; eicosapentaenoic acid; cEPA; conjugated eicosapentaenoic acid; DHA; docosahexaenoic acid; PUFA; polyunsaturated fatty acid; Ct; cycle threshold; dTTP; 2′-deoxythymidine 5′-triphosphate; DMSO; dimethylsulfoxide; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; PCR; polymerase chain reaction; pol; DNA polymerase (EC 2.7.7.7); topo; DNA topoisomeraseConjugated eicosapentaenoic acid (cEPA); DNA polymerase; Enzyme inhibitor; DNA replication; Cytotoxicity; Cell proliferation; Apoptosis

The interaction of methylphenidate and benztropine with the dopamine transporter is different than other substrates and ligands by Dalit E. Dar; Cheryl Mayo; George R. Uhl (pp. 461-469).

A substantial body of evidence suggests that the dopamine transporter (DAT) is the principal site for cocaine-induced reward and euphoria. Interactions between the DAT and its substrates and ligands may therefore be of clinical relevance. The pharmacological characteristics of DAT compounds were compared in wild type (WT) and mutant DATs. The DAT mutants chosen for study were those with reduced binding and uptake activities (aspartic acid 79 mutated to alanine, termed D79A), reduced binding but normal uptake (tyrosine 251 mutated to alanine, termed Y251A; tyrosine 273 mutated to alanine, termed, Y273A), and normal binding but reduced uptake (a double mutation: serines 356 and 359 mutated to alanine, termed S356,359A). The WT and mutant DATs were transfected into COS-7 cells, and their pharmacological activities were examined 3 days later. Different patterns of pharmacological activity emerged. GBR 12909, cocaine, and mazindol each showed reduced affinity for the Y251A and the Y273A mutants, but their affinity for the S356,359A mutant was similar to that of the WT DAT. d-Amphetamine, MPP+, and dopamine each showed reduced affinity for the S356,359A mutant. Benztropine and methylphenidate had a different effect. Relative to the WT DAT, they both showed reduced affinity for the S356,359A mutant when displacing radioactive carboxyfluorotropane (CFT) binding, but similar affinity when inhibiting radioactive dopamine uptake. These results indicate that methylphenidate and benztropine may interact with the DAT in a different fashion then other substrates and ligands.

Keywords: Abbreviations; D79A; aspartic acid 79 mutated to alanine; Y251A; tyrosine 251 mutated to alanine; Y273A; tyrosine 273 mutated to alanine; S356,359A; serines 356 and 359 mutated to alanine; DAT; dopamine transporter; WT; wild-type; CFT; carboxyfluorotropane (Win 35,428); TM; transmembrane; DMEM; Dulbecco's modified Eagle's medium; FCS; fetal calf serumDopamine; Cocaine; Transporter; Uptake; Substrates; Ligands

Evaluation of the neuronal apoptotic pathways involved in cytoskeletal disruption-induced apoptosis by Elvira G. Jord�; Ester Verdaguer; Andr�s Jimenez; S. Garcia de Arriba; Clemens Allgaier; Merc� Pall�s; Antoni Camins (pp. 470-480).

The cytoskeleton is critical to neuronal functioning and survival. Cytoskeletal alterations are involved in several neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. We studied the possible pathways involved in colchicine-induced apoptosis in cerebellar granule neurons (CGNs). Although colchicine evoked an increase in caspase-3, caspase-6 and caspase-9 activation, selective caspase inhibitors did not attenuate apoptosis. Inhibitors of other cysteine proteases such as PD150606 (a calpain-specific inhibitor), Z-Phe-Ala fluoromethyl ketone (a cathepsins-inhibitors) and N^α- p-tosyl-l-lysine chloromethyl ketone (serine-proteases inhibitor) also had no effect on cell death/apoptosis induced by colchicine. However, BAPTA-AM 10μM (intracellular calcium chelator) prevented apoptosis mediated by cytoskeletal alteration. These data indicate that calcium modulates colchicine-induced apoptosis in CGNs. PARP-1 inhibitors did not prevent apoptosis mediated by colchicine. Finally, colchicine-induced apoptosis in CGNs was attenuated by kenpaullone, a cdk5 inhibitor. Kenpaullone and indirubin also prevented cdk5/p25 activation mediated by colchicine. These findings indicate that cytoskeletal alteration can compromise cdk5 activation, regulating p25 formation and suggest that cdk5 inhibitors attenuate apoptosis mediated by cytoskeletal alteration. The present data indicate the potential therapeutic value of drugs that prevent the formation of p25 for the treatment of neurodegenerative disorders.

Keywords: Abbreviations; AC-DEVD-CHO; Ac-Asp-Glu-Asp-Val-Aldehyde; AC-LEHD-CHO; Ac-Leu-Glu-His-Asp-aldehyde; AIF; apoptosis inducing factor; BAPTA-AM; 1,2-bis-(; o; -aminophenoxy)-ethane-; N; ,; N; ,-; N; ′,; N; ′-tetraacetic acid tetraacetoxy-methyl ester; CGNs; cerebellar granule neurons; DPQ; 4-dihydro-5[4-(piperindinyl)butoxy]-1(2H)-isoquinoleine; FCS; foetal calf serum; MTT; [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium]; PARP; poly-APD-ribosil polimerase; PI; propidium iodide; PVDF; polyvinylidene fluoride; TLCK; N; α-; p; -tosyl-; l; -lysine chloromethyl ketone; Z-FA-FMK; Z-Phe-Ala fluoromethyl ketone; Z-VEID-FMK; Z-Val-Glu-Ile-Asp-fluoromethylketoneCalcium; Cytoskeleton; Caspases; Calpainsl; Serine-proteases

Depletion of RLIP76 sensitizes lung cancer cells to doxorubicin by Sharad S. Singhal; Sushma Yadav; Jyotsana Singhal; Ewa Zajac; Yogesh C. Awasthi; Sanjay Awasthi (pp. 481-488).

Ral-interacting protein (RLIP76) (RALBP1) is an anti-apoptotic non-ABC glutathione (GSH)-conjugate transporter involved in receptor-ligand endocytosis, as well as in multispecific drug transport and resistance. Partial inhibition of RLIP76 using antibodies in the absence of chemotherapy drug causes apoptosis in multiple small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cell lines and in the presence of doxorubicin (DOX), marked synergy is observed. These findings indicated that RLIP76 should be a good target for cancer cell killing; its down-regulation would promote apoptosis through both drug-dependent and drug-independent effects. To examine the effect of complete and specific RLIP76 depletion on apoptosis, we tested the effects of RLIP76 siRNA in a number of lung cancer cell lines. Growth inhibition and apoptosis was observed in all cases upon RLIP76 depletion. Consistent with these findings, augmenting cellular RLIP76 through transfection or liposomal protein delivery conferred resistance to apoptosis mediated by either DOX or 4-hydroxynonenal (4-HNE). Taken together, our results show that RLIP76 is rational and promising new target for lung cancer therapy.

Keywords: Abbreviations; DNP-; S; G; dinitrophenyl; S; -glutathione; DOX; doxorubicin; GS-E; glutathione-electrophile conjugate; GSH; glutathione; 4HNE; 4-hydroxynonenal; MRP; multi-drug resistance associated protein; NSCLC; non-small cell lung cancer; POB1; partner of RLIP76; RLIP76 (RALBP1); Ral-interacting protein; SCLC; small cell lung cancer; TUNEL; TdT-mediated dUTP nick end labelingTransfection; siRNA; Doxorubicin; Drug-resistance; RLIP76; RALBP1

Sections

Personal tools

Biochemical Pharmacology (v.70, #3)

Sections

Personal tools

Local resources

Biochemical Pharmacology (v.70, #3)