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Biochemical Pharmacology (v.69, #5)


Intracellular generation of reactive oxygen species by mitochondria by Hans Nohl; Lars Gille; Katrin Staniek (pp. 719-723).
Mitochondria have bioenergetic properties that strongly suggest their involvement in the cellular formation of reactive oxygen species (ROS). Apparent confirmation of this process has come from work with isolated mitochondria, which have been shown to produce H2O2 from dismutating superoxide radicals. Two different sites were reported to shuttle single electrons to oxygen out of the normal respiratory sequence. However, the mechanisms for ROS formation at these two sites are controversial. Arguments against mitochondrial ROS formation in the living cell are based on the fact that bioenergetic alterations may result from the mechanical removal of mitochondria from their natural environment. Furthermore, the invasive detection methods that are generally used may be inappropriate because of the possible interaction of the detection system with mitochondrial constituents. The use of non-invasive detection methods has proved that ROS formation does not occur unless changes in the physical state of the membrane are established. The aim of this commentary is to discuss critically the arguments in favor of mitochondria as the main intracellular source of ROS. The pros and cons of working with isolated mitochondria, as well as the detection methodology are carefully analyzed to judge whether or not the above assumption is correct. The conclusion that mitochondria are the main ROS generators in the cell contradicts the fact that ROS release was not observed. However, if electron flow from ubiquinol to the bc1 complex is hindered due to changes in lipid fluidity, single electrons may transfer to dioxygen and produce H2O2 via superoxide radicals.

Keywords: Abbreviations; ROS; reactive oxygen species; O; 2; −; superoxide radical anionMitochondria; Reactive oxygen species; Superoxide radical; Hydrogen peroxide; Phospholipid membrane


Poly(ADP-ribose) polymerase regulates myocardial calcium handling in doxorubicin-induced heart failure by Orsolya Szenczi; Péter Kemecsei; Max F.J. Holthuijsen; Natal A.W. van Riel; Ger J. van der Vusse; Pál Pacher; Csaba Szabó; Márk Kollai; László Ligeti; Tamás Ivanics (pp. 725-732).
Reactive oxygen and nitrogen species are overproduced in the cardiovascular system in response to the exposure to doxorubicin, a cardiotoxic anticancer compound. Oxidant-induced cell injury involves the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) and pharmacological inhibition of PARP has recently been shown to improve myocardial contractility in doxorubicin-induced heart failure models. The current investigation, by utilizing an isolated perfused heart system capable of beat-to-beat intracellular calcium recording, addressed the following questions: (1) is intracellular calcium handling altered in hearts of rats after 6-week doxorubicin treatment, under baseline conditions, and in response to oxidative stress induced by hydrogen peroxide exposure in vitro; and (2) does pharmacological inhibition of PARP with the phenanthridinone-based PARP inhibitor PJ34 affect the changes in myocardial mechanical performance and calcium handling in doxorubicin-treated hearts under normal conditions and in response to oxidative stress. The results showed a marked elevation in intracellular calcium in the doxorubicin-treated hearts which was normalized by pharmacological inhibition of PARP. PARP inhibition also prevented the myocardial contractile disturbances and calcium overload that developed in response to hydrogen peroxide in the doxorubicin-treated hearts. We conclude that PARP activation contributes to the development of the disturbances in cellular calcium handling that develop in the myocardium in response to prolonged doxorubicin exposure.

Keywords: Peroxynitrite; Poly(ADP-ribose) polymerase (PARP); Adriamycin; Heart failure; Superoxide; Calcium


Inhibition of tristetraprolin expression by dexamethasone in activated macrophages by Ulla Jalonen; Aleksi Lahti; Riku Korhonen; Hannu Kankaanranta; Eeva Moilanen (pp. 733-740).
Tristetraprolin (TTP) is a factor that regulates mRNA stability and the expression of certain inflammatory genes. In the present study, we found that TTP expression was increased in macrophages exposed to bacterial lipopolysaccharide (LPS). Dexamethasone and dissociated steroid RU24858 inhibited LPS-induced TTP protein and mRNA expression and the inhibitory effect was reversed by a glucocorticoid receptor antagonist mifepristone. Histone deacetylase inhibitors trichostatin A (TSA) and apicidin reduced the inhibitory effect of dexamethasone and RU24858 on TTP expression, but the glucocorticoids did not alter TTP mRNA half-life. These results suggest that anti-inflammatory steroids reduce TTP expression in activated macrophages by a glucocorticoid response element (GRE)-independent mechanism, possibly through histone deacetylation and transcriptional silencing.

Keywords: Abbreviations; Erk1/2; extracellular signal-regulated kinase 1/2; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; GR; glucocorticoid receptor; GRE; glucocorticoid response element; JNK; c-Jun N-terminal kinase; LPS; lipopolysaccharide; MAPK; mitogen-activated protein kinase; NF-κB; nuclear factor κB; PD 98059; 2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one; PDTC; pyrrolidinedithiocarbamate; SB 202474; 4-ethyl-2-(4-methoxyphenyl)-5-(4-pyridyl)-imidazole; SB 203580; 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)-imidazole; SP 600125; anthra[1,9-; cd; ]pyrazol-6(2; H; )-one; TNF-α; tumor necrosis factor α; TSA; trichostatin A; TTP; tristetraprolinGlucocorticoid; Histone deacetylation; Inflammation; Mitogen-activated protein kinase; mRNA stability; Tristetraprolin


β-Adrenergic receptor modulation of the LPS-mediated depression in CYP1A activity in astrocytes by Dalya Abdulla; Kenneth W. Renton (pp. 741-750).
CYP1A1 and 1A2, two important P450 isoforms in the brain that metabolize many endogenous and exogenous substrates, are downregulated during central nervous system (CNS) inflammation. The stimulation of β-adrenergic receptors has been demonstrated to be anti-inflammatory in many cell types, leading us to hypothesize that stimulation of β-adrenergic receptors could prevent the downregulation in CYP1A1 and 1A2 activity in an in vitro model of CNS inflammation. Isoproterenol, a general β1/β2 receptor agonist, and clenbuterol, a specific β2 receptor agonist, were both able to prevent the LPS-induced downregulation in CYP1A1/2 activity in astrocytes. The involvement of β-adrenergic receptors was confirmed using the general β1/β2 receptor antagonist propranolol, which was able to abrogate the protection conferred by isoproterenol and clenbuterol in astrocytes treated with LPS. The isoproterenol and clenbuterol mediated protective effect on the LPS-induced downregulation in CYP1A activity was a cyclic AMP (cAMP) dependent process, since forskolin was able to mimic the protective effect. Isoproterenol and clenbuterol may also prevent the LPS-induced downregulation in CYP1A activity through changes in TNFα expression. Despite a slight reduction in the LPS-induced nuclear translocation of the p65 subunit of NF-κB, isoproterenol and clenbuterol had no effect on the DNA binding ability of this transcription factor, indicating that the β-adrenergic protective effects on CYP1A activity occurred independent of changes in NF-κB activity. The results presented in this paper reveal that β-adrenergic receptor stimulation can modulate cytochrome P450 activity in an in vitro model of CNS inflammation by a cAMP mediated pathway.

Keywords: Abbreviations; LPS; lipopolysaccharide; CNS; central nervous system; cyt P450; cytochrome P450; DBA; dibenz[a,h]anthracene; NE; norepinephrine; βAR; β-adrenergic receptors; DMEM; Dulbecco's modified Eagle's medium; PMSF; phenylmethylsulfonyl fluoride; DTT; dithiothreitol; TNFα; tumor necrosis factor alpha; EROD; 7-ethoxyresorufin; O; -dealkylase; IL-1β; interleukin-1β; IFNγ; interferon gamma; PDTC; pyrrolidine dithiocarbamate; iNOS; inducible nitric oxide synthase; NOS2; inducible nitric oxide synthaseCytochrome P450; CNS inflammation; Astrocytes; β-Adrenergic receptors; EROD; NF-κB; IκBα; cAMP


Effect of an oversulfated exopolysaccharide on angiogenesis induced by fibroblast growth factor-2 or vascular endothelial growth factor in vitro by Sabine Matou; Sylvia Colliec-Jouault; Isabelle Galy-Fauroux; Jacqueline Ratiskol; Corinne Sinquin; Jean Guezennec; Anne-Marie Fischer; Dominique Helley (pp. 751-759).
The aim of this study was to determine the angiogenic properties of an oversulfated exopolysaccharide (OS-EPS) derived from a polysaccharide secreted by the mesophilic bacterium Alteromonas infernus. We compared the effect of this OS-EPS with that of a non-oversulfated exopolysaccharide (EPS) on human umbilical vein endothelial cell (HUVEC) proliferation, migration and differentiation induced by basic fibroblast growth factor (FGF-2) or vascular endothelial growth factor (VEGF). OS-EPS enhanced HUVEC proliferation by 58% when used alone, and by respectively 30% and 70% in the presence of FGF-2 and VEGF. OS-EPS also increased the density of tubular structures on Matrigel in the presence of FGF-2 or VEGF. Vascular tube formation was related to α6 integrin subunit expression, which was enhanced by 50% in the presence of the growth factors. Indeed, a monoclonal anti-α6 blocking antibody abolished this vascular tube formation. EPS had no effect in any of the experimental conditions, underlying the importance of sulfation in the angiogenic effects of exopolysaccharide. By potentiating the angiogenic activity of FGF-2and/or VEGF, OS-EPS, which possesses low anticoagulant activity and thus a low hemorrhagic risk, could potentially be used to accelerate vascular wound healing or to promote the growth of collateral blood vessels in ischemic tissues.

Keywords: Abbreviations; FGF-2; fibroblast growth factor-2; VEGF; vascular endothelial growth factor; OS-EPS; oversulfated exopolysaccharide; EPS; exopolysaccharide; HUVEC; human umbilical vein endothelial cell; HSPG; heparan sulfate proteoglycans; LMW; low molecular weight; HMW; high molecular weight; FBS; fetal bovine serumAngiogenesis; Polysaccharide; Angiogenic growth factor; α; 6; Integrin subunit; Collagen gel; Matrigel


Regulation of lipid raft proteins by glimepiride- and insulin-induced glycosylphosphatidylinositol-specific phospholipase C in rat adipocytes by Günter Müller; Andrea Schulz; Susanne Wied; Wendelin Frick (pp. 761-780).
The insulin receptor-independent insulin-mimetic signalling provoked by the antidiabetic sulfonylurea drug, glimepiride, is accompanied by the redistribution and concomitant activation of lipid raft-associated signalling components, such as the acylated tyrosine kinase, pp59Lyn, and some glycosylphosphatidylinositol-anchored proteins (GPI-proteins). We now found that impairment of glimepiride-induced lipolytic cleavage of GPI-proteins in rat adipocytes by the novel inhibitor of glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC), GPI-2350, caused almost complete blockade of (i) dissociation from caveolin-1 of pp59Lyn and GPI-proteins, (ii) their redistribution from high cholesterol- (hcDIGs) to low cholesterol-containing (lcDIGs) lipid rafts, (iii) tyrosine phosphorylation of pp59Lyn and insulin receptor substrate-1 protein (IRS-1) and (iv) stimulation of glucose transport as well as (v) inhibition of isoproterenol-induced lipolysis in response to glimepiride. In contrast, blockade of the moderate insulin activation of the GPI-PLC and of lipid raft protein redistribution by GPI-2350 slightly reduced insulin signalling and metabolic action, only. Importantly, in response to both insulin and glimepiride, lipolytically cleaved hydrophilic GPI-proteins remain associated with hcDIGs rather than redistribute to lcDIGs as do their uncleaved amphiphilic versions. In conclusion, GPI-PLC controls the localization within lipid rafts and thereby the activity of certain GPI-anchored and acylated signalling proteins. Its stimulation is required and may even be sufficient for insulin-mimetic cross-talking to IRS-1 in response to glimepiride via redistributed and activated pp59Lyn.

Keywords: Abbreviations; AChE; acetylcholinesterase; aP; alkaline phosphatase; BSA; bovine serum albumin; CBD(P); caveolin-binding domain (peptide); cIP; myo; -inositol-1,2-cyclic phosphate; CRD; cross-reacting determinant; CSD; caveolin-scaffolding domain; DMSO; dimethylsulfoxide; DTT; dithiothreitol; EDTA; ethylene diamine tetraacetic acid; Gce1; GPI-anchored cAMP-binding ectoprotein 1; Glut4; glucose transporter isoform 4; GPI; glycosylphosphatidyl-inositol; GPI-PLC/D; GPI-specific phospholipase C/D; GPI-protein; GPI-anchored plasma membrane protein; HSL; hormone-sensitive lipase; IR(β); insulin receptor (β-subunit); IRS-1/2; insulin receptor substrate-1/2; lc/hcDIGs; low/high cholesterol-containing detergent-insoluble glycolipid-enriched membrane microdomains (lipid rafts); LPL; lipoprotein lipase; m-β-CD; methyl-β-cyclodextrin; MES; morpholinoethane sulfonic acid; NBD-FA; 12-((7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoic acid; NEM; N; -ethylmaleimide; 5′-Nuc; 5′-nucleotidase; p115; 115; kDa PIG receptor protein; PC; phosphatidylcholine; PI; phosphatidylinositol; PIG; phosphoinositolglycans; PI3K; phosphatidylinositol-3′-kinase; PI/PC-PL; PI/PC-specific phospholipase; PL; pancreatic lipase; PLA; 2; /C/D; phospholipase A2/C/D; PM; plasma membranes; SDS-PAGE; sodium dodecylsulfate polyacrylamide gel electrophoresis; TLC; thin layer chromatography; TX-100/114; Triton X-100/114Insulin signalling; Sulfonylurea action; Lipid rafts; Glycosylphosphatidylinositol; Rat adipocytes


Insulin-stimulated endothelial nitric oxide release is calcium independent and mediated via protein kinase B by Nicholas A. Hartell; Helen E. Archer; Clifford J. Bailey (pp. 781-790).
Insulin exerts a vasodilator effect by stimulating endothelial nitric oxide (NO) production. Studies in cultured cells suggest that insulin might activate endothelial nitric oxide synthase (eNOS) by an atypical, calcium-independent mechanism. This study investigates the mechanism of insulin-stimulated endothelial NO production in intact aortic wall. Real time fluorescence imaging with 4,5-diaminofluorescin diacetate (DAF-2 DA) or 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA) and FURA 2-AM was used to simultaneously visualise NO and intracellular calcium concentrations at multiple locations in the endothelium and vascular smooth muscle of isolated rat and mouse aorta after exposure to insulin. Inhibitors of intracellular insulin signalling were used to determine the pathway for insulin-stimulated NO production. Unlike acetylcholine, which stimulated endothelial NO production with a typical increase in free intracellular calcium, insulin (10−8 to 10−6M) stimulated endothelial NO production without elevating intracellular calcium levels. Insulin-stimulated NO production was concentration dependent and detected within 30s of application. Peak increases in NO occurred between 60 and 120s and declined slowly thereafter. Separate measurements of NO production by fluorescence of 2,3-diaminonaphthalene (DAN) noted that selective inhibitors of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB) inhibited insulin-stimulated NO production, whereas these inhibitors alone did not alter NO production or acetylcholine-stimulated NO production. Insulin-stimulated NO production by endothelium is an acute calcium-independent effect mediated via the PI3K-PKB signalling pathway.

Keywords: Abbreviations; DAN; 2,3-diaminonaphthalene; DAF-2 DA; 4,5-diaminofluorescin diacetate; eNOS; endothelial nitric oxide synthase; IGF1; insulin-like growth factor-1; KRB; Krebs Ringer bicarbonate; l; -NAME; N; -nitro-; l; -arginine methyl ester hydrochloride; MAP; mitogen-activated protein; NO; nitric oxide; PI3K; phosphatidylinositol 3-kinase; PKB (Akt); protein kinase B; ROI; region of interest; VSM; vascular smooth muscleInsulin; Endothelium; Nitric oxide; Nitric oxide synthase; Calcium; Phosphatidylinositol 3-kinase; Protein kinase B


Inhibitory effect of 2′-hydroxycinnamaldehyde on nitric oxide production through inhibition of NF-κB activation in RAW 264.7 cells by Seung Ho Lee; Sun Young Lee; Dong Ju Son; Heesoon Lee; Hwan Soo Yoo; Sukgil Song; Ki Wan Oh; Dong Cho Han; Byoung Mog Kwon; Jin Tae Hong (pp. 791-799).
Cinnamomum cassia has been widely used for treating dyspepsia, gastritis, and inflammatory disease. In the present study, several of cinnamaldehyde derivatives were synthesized from various cinnamic acid based on the 2′-hydroxycinnamaldehyde isolated from the bark C. cassia Blume was investigated to compare their NO production and NF-κB activity from Raw 264.7 cell since nitric oxide (NO) and NF-κB have been shown to be implicated factors in the inflammatory disease. The results show that HCA, among the derivatives, most significantly inhibited lipopolysaccharide (LPS)-induced NO production and NF-κB transcriptional activity in a dose-dependent manner with an IC50 value of 8 and 22μM, respectively. We next investigated putative possible mechanisms of inhibitory effect of HCA on NO production. The inhibition of NO by HCA was consistent with the inhibitory effect on LPS-induced inducible nitric oxide synthase (iNOS) expression. Moreover, HCA inhibited LPS-induced p50 and p65 translocation resulting in the inhibition of the DNA binding activity of the NF-κB, a central regulator of iNOS. The present results provided evidence that HCA, among cinnamaledhyde derivatives, has the most inhibitory effect on NO production through inhibition of NF-κB activation, and thus can be used as an anti-inflammatory agent.

Keywords: Abbreviations; NO; nitric oxide; iNOS; inducible nitric oxide synthase; COX-2; cyclooxygenase-2; HCA; 2′-hydroxycinnamaldehyde; LPS; lipopolysacharide; NF-κB; nuclear transcription factor-κB; TNF-α; tumor necrosis factor-α2′-Hydroxycinnamaldehyde; Inducible nitric oxide synthase; Nitric oxide; Nuclear transcription factor-κB; Cyclooxygenase 2; Lipopolysaccharide


Antitumour imidazoacridone C-1311 induces cell death by mitotic catastrophe in human colon carcinoma cells by Magdalena Hyzy; Przemyslaw Bozko; Jerzy Konopa; Andrzej Skladanowski (pp. 801-809).
In this study, we investigated the cell death process induced by imidazoacridone C-1311 (Symadex™) in HT-29 human colon carcinoma cells which have been shown to be preferentially sensitive to this compound in experimental tumour models both in vitro and in nude mice. Compound C-1311 at the EC99 dose delayed progression of cells through the S phase which was followed by G2 arrest. At 48–96h after drug exposure, an increasing fraction of cells rounded up and detached from the substratum which suggested the induction of cell death. This was confirmed by the induction of DNA fragmentation as revealed by pulse field electrophoresis and DNA strand breaks by the TUNEL assay. The dying cells had also mitotic features which were evidenced by various biochemical and morphological criteria such as activation of Cdk1 kinase, presence of the mitotic epitope MPM-2 and condensation of chromatin into mitotic chromosomes in drug-treated cells. These results show that C-1311 does not induce rapid apoptosis in HT-29 cells, instead drug exposure leads to prolonged G2 arrest followed by G2 to M transit and cell death during mitosis in the process of mitotic catastrophe.

Keywords: Abbreviations; PBS; phosphate-buffered saline; DAPI; 4′,6-diamidino-2-phenylindole; BSA; bovine serum albumin; FITC; fluorescein isothiocyanate; SDS; sodium dodecyl sulphateImidazoacridone; C-1311; Cell cycle; G2 arrest; Cell death; Mitotic catastrophe


Molecular modes of action of cantharidin in tumor cells by Thomas Efferth; Rolf Rauh; Stefan Kahl; Maja Tomicic; Herbert Böchzelt; Margaret E. Tome; Margaret M. Briehl; Rudolf Bauer; Bernd Kaina (pp. 811-818).
Cancer chemotherapy is often limited by patient's toxicity and tumor drug resistance indicating that new drug development and modification of existing drugs is critical for improving the therapeutic response. Traditional Chinese medicine is a rich source of potential anticancer agents. In particular, cantharidin (CAN), the active principle ingredient from the blister beetle, Mylabris, has anti-tumor activity, but the cytotoxic mechanism is unknown. In leukemia cells, cantharidin induces apoptosis by a p53-dependent mechanism. Cantharidin causes both DNA single- and double-strand breaks. Colony-forming assays with knockout and transfectant cells lines showed that DNA polymerase β, but not ERCC1, conferred increased cell survival after cantharidin treatment, indicating that base excision repair (BER), rather than nucleotide excision repair (NER), is important for CAN-induced DNA lesions. Oxidative stress-resistant thymic lymphoma-derived WEHI7.2 variants are also more resistant to cantharidin. These data suggest that cantharidin treatment causes oxidative stress that provokes DNA damage and p53-dependent apoptosis.

Keywords: Abbreviations; BER; base excision repair; CAN; cantharidin; NCTD; norcantharidin; NER; nucleotide excision repair; OTM; Olive tail moment; PP1/PP2A; protein phosphatase 1/2AApoptosis; Cancer chemotherapy; DNA repair; Natural compounds; Oxidative stress


Peripheral benzodiazepine receptor (PBR) ligand cytotoxicity unrelated to PBR expression by Gregory Hans; Sabine Wislet-Gendebien; François Lallemend; Pierre Robe; Bernard Rogister; Shibeshih Belachew; Laurent Nguyen; Brigitte Malgrange; Gustave Moonen; Jean-Michel Rigo (pp. 819-830).
Some synthetic ligands of the peripheral-type benzodiazepine receptor (PBR), an 18kDa protein of the outer mitochondrial membrane, are cytotoxic for several tumor cell lines and arise as promising chemotherapeutic candidates. However, conflicting results were reported regarding the actual effect of these drugs on cellular survival ranging from protection to toxicity. Moreover, the concentrations needed to observe such a toxicity were usually high, far above the affinity range for their receptor, hence questioning its specificity. In the present study, we have shown that micromolar concentrations of FGIN-1-27 and Ro 5-4864, two chemically unrelated PBR ligands are toxic for both PBR-expressing SK-N-BE neuroblastoma cells and PBR-deficient Jurkat lymphoma cells. We have thereby demonstrated that the cytotoxicity of these drugs is unrelated to their PBR-binding activity. Moreover, Ro 5-4864-induced cell death differed strikingly between both cell types, being apoptotic in Jurkat cells while necrotic in SK-N-BE cells. Again, this did not seem to be related to PBR expression since Ro 5-4864-induced death of PBR-transfected Jurkat cells remained apoptotic. Taken together, our results show that PBR is unlikely to mediate all the effects of these PBR ligands. They however confirm that some of these ligands are very effective cytotoxic drugs towards various cancer cells, even for reputed chemoresistant tumors such as neuroblastoma, and, surprisingly, also for PBR-lacking tumor cells.

Keywords: Abbreviations; Δ; Ψ; m; mitochondrial membrane potential; PBR; peripheral benzodiazepine receptor; BA; Bongkrekic acid; CsA; cyclosporin A; MTT; 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazam bromide; mPTP; permeability transition pore; VDAC; voltage-dependent anion channel; PARP; poly(ADP-ribose) polymerase-1; ROS; reactive oxygen speciesPeripheral benzodiazepine receptor; Apoptosis; Cancer; Ligands; Mitochondrion; Necrosis


Induction of apoptosis by R-flurbiprofen in human colon carcinoma cells: involvement of p53 by Sabine Grösch; Karin Schilling; Astrid Janssen; Thorsten J. Maier; Ellen Niederberger; Gerd Geisslinger (pp. 831-839).
R-Flurbiprofen, a non cyclooxygenase inhibiting non-steroidal anti-inflammatory drug (NSAID), has been found to inhibit tumor growth in various animal models. In vitro experiments have shown that this effect is based on the induction of a cell cycle block and apoptosis. Cell cycle inhibition has been explained by activation of the c-Jun–N-terminal kinase (JNK) and downregulation of cyclin D1 expression. However, the molecular mechanism leading to apoptosis is unknown. Here, we show that treatment of the human colon carcinoma cell line HCT116 with different concentrations of R-flurbiprofen leads to an accumulation of p53 protein which is accompanied by an increase in phosphorylated p53 at serine 15. Mutation of serine 15 to alanine by site directed mutagenesis and overexpression of the mutated p53 gene in HCT116 cells, revealed that these cells are significantly less sensitive to apoptosis induced by R-flurbiprofen than pcDNA control cells, as measured by PARP-cleavage and flow cytometry. By contrast, no difference was detected between HCT116p53ser15ala cells and HCT116 pcDNA cells with respect to induction of a cell cycle block after R-flurbiprofen treatment. Moreover, in nude mice HCT116p53ser15ala overexpressing xenografts were significantly less sensitive to R-flurbiprofen than HCT116 pcDNA control xenografts. In conclusion, we were able to show that induction of apoptosis in HCT116 cells after R-flurbiprofen treatment is at least partly dependent on the tumor suppressor gene p53 and that mutation of p53 at serine 15 impairs the apoptotic potency of R-flurbiprofen.

Keywords: Abbreviations; COX-2; cyclooxygenase-2; NSAID; non-steroidal anti-inflammatory drugs; FCS; fetal calf serum; PBS; phosphate buffered saline; FACS; fluorescence-activated cell sorter; EDTA; ethylenediaminetetraacetate; PMSF; phenylmethylsulfonylfluoride; DTT; dithiotreitol; SDS–PAGE; sodiumdodecylsulfate–polyacrylamide gel electrophoresis; RT; reverse transcription; PCR; polymerase chain reaction; AUC; area under the curve; ANOVA; univariate analysis of variance; PARP; poly(ADP)-ribose polymeraseG; 1; -phase block; Apoptosis; p53; Colon cancer; Flurbiprofen


Contrasting the effects of nifedipine on subtypes of endogenous and recombinant T-type Ca2+ channels by Alexander Shcheglovitov; Tatiana Zhelay; Yulia Vitko; Vadim Osipenko; Edward Perez-Reyes; Platon Kostyuk; Yaroslav Shuba (pp. 841-854).
There is evidence that nifedipine (Nif) – a dihydropyridine (DHP) Ca2+-channel antagonist mostly known for its L-type-specific action – is capable of blocking low voltage-activated (LVA or T-type) Ca2+ channels as well. However, the discrimination by Nif of either various endogenous T-channel subtypes, evident from functional studies, or cloned Cav3.1, Cav3.2 and Cav3.3 T-channel α1 subunits have not been determined. Here, we investigated the effects of Nif on currents induced by Cav3.1, Cav3.2 and Cav3.3 expression in Xenopus oocytes or HEK-293 cells ( Iα1G, Iα1H and Iα1I, respectively) and two kinetically distinct, “fast� and “slow�, LVA currents in thalamic neurons ( ILVA,f and ILVA,s). At voltages of the maximums of respective currents the drug most potently blocked Iα1H (IC50=5μM, max block 41%) followed by Iα1G (IC50=109μM, 23%) and Iα1I (IC50=243μM, 47%). The mechanism of blockade included interaction with Cav3.1, Cav3.2 and Cav3.3 open and inactivated states. Nif blocked thalamic ILVA,f and ILVA,s with nearly equal potency (IC50=22μM and 28μM, respectively), but with different maximal inhibition (81% and 51%, respectively). We conclude that Cav3.2 is the most sensitive to Nif, and that quantitative characteristics of drug action on T-type Ca2+ channels depend on cellular system they are expressed in. Some common features in the voltage- and state-dependence of Nif action on endogenous and recombinant currents together with previous data on T-channel α1 subunits mRNA expression patterns in the thalamus point to Cav3.1 and Cav3.3 as the major contributors to thalamic ILVA,f and ILVA,s, respectively.

Keywords: Abbreviations; A; max; maximal percentage of inhibition; DMSO; dimethyl sulfoxide; HVA; high voltage-activated; IC; 50; half-inhibitory concentration; I; α1G; current through heterologously expressed α1G (Ca; v; 3.1) subunit of low voltage-activated calcium channel; I; α1H; current through heterologously expressed α1H (Ca; v; 3.2) subunit of low voltage-activated calcium channel; I; α1I; current through heterologously expressed α1I (Ca; v; 3.3) subunit of low voltage-activated calcium channel; I; LVA,f; “fast� low voltage-activated current in thalamic neurons; I; LVA,s; “slow� thalamic low voltage-activated current; I; –; V; current–voltage relationship; k; slope factor; LD; laterodorsal; LVA; low voltage-activated; p; cooperativity coefficient; V; 1/2; potential of half-maximal activation or inactivation; V; m; membrane potentialEndogenous and cloned T-type Ca; 2+; channels; Nifedipine; Thalamic neurons; Xenopus; oocytes


Mechanism of ricin-induced apoptosis in human cervical cancer cells by P.V. Lakshmana Rao; R. Jayaraj; A.S.B. Bhaskar; Om Kumar; R. Bhattacharya; Parag Saxena; P.K. Dash; R. Vijayaraghavan (pp. 855-865).
The mechanism of ricin-induced apoptosis in human cervical cancer cell line HeLa was studied. The present study demonstrated that ricin induces apoptosis of human cervical cancer cells (HeLa) in a time dependent manner with an IC50 for cell viability of 1μg/ml. Ricin treatment resulted in a time dependent increase in LDH leakage, DNA fragmentation, percent apoptotic cells, generation of reactive oxygen species and depletion of intracellular glutathione levels. DNA agarose gel electrophoresis showed typical oligonucleosomal length DNA fragmentation. Additionally, DNA diffusion assay was performed to confirm DNA damage and apoptosis. Ricin activated caspase-3 as evidenced by both proteolytic cleavage of procaspase-3 into 20 and 18kDa subunits, and increased protease activity. Caspase activity was maximum at 4h and led to the cleavage of 116kDa poly(ADP-ribose) polymerase (PARP), resulting in the 85kDa cleavage product. Ricin-induced caspase-3 activation also resulted in cleavage of DNA fragmentation factor-45 (DFF45/ICAD) and DFF40 or caspase-activated DNase in HeLa cells. Activation of caspase-3, cleavage of PARP and DNA fragmentation was blocked by pre-treatment with caspase-3 specific inhibitor Ac-DEVD-CHO (100μM) and broad-spectrum caspase inhibitor Z-VAD-FMK (40μM). Ricin-induced DNA fragmentation was inhibited by pre-treatment with PARP inhibitors 3-aminobenzamide (100μM) and DPQ (10μM). Our results indicate that ricin-induced cell death was mediated by generation of reactive oxygen species and subsequent activation of caspase-3 cascade followed by down stream events leading to apoptotic mode of cell death.

Keywords: Abbreviations; Ac-DEVD-CHO; acetyl-Asp-Glu-Val-Asp aldehyde; Z-VAD-FMK; N; -benzoyloxy-carbonyl-Val-Ala-Asp (; O; -me)-fluoromethyl ketone; GSH; glutathione; PARP; poly(ADP-ribose) polymerase; PMSF; phenylmethylsulfonyl fluoride; DTT; dithiothreitol; OPT; orthopthaldialdehyde; PBS; phosphate buffered saline; DCF-DA; 2,7-dichlorofluorescein diacetate; CHAPS; (3[(3-cholamidopropyl) dimethlylammonio]-1-propanesulfate); DFF/ICAD; DNA fragmentation factor/inhibitor of caspase-activated DNAse; CAD; caspase-activated DNAseRicin; HeLa cells; Apoptosis; Caspase-3; DNA fragmentation factor; Caspase inhibitors


Repression of PXR-mediated induction of hepatic CYP3A gene expression by protein kinase C by Xunshan Ding; Jeff L. Staudinger (pp. 867-873).
Pregnane X receptor (PXR, NR1I2) regulates the inducible expression of the 3A sub-family of cytochrome P450 genes ( CYP3A). CYP3A enzymes are responsible for the oxidative metabolism of a wide array of endobiotic and xenobiotic compounds. Hepatic CYP3A gene expression is rapidly down-regulated during inflammation and sepsis. There are twelve protein kinase C (PKC) isoforms, classified into three subfamilies according to the structure of the N-terminal regulatory domain and their sensitivity to calcium and diacylglycerol. It is now well accepted that cytokine stimulation of hepatocytes increases intracellular PKC activity during inflammation and sepsis. We show here that protein kinase C alpha (PKCα) and phorbol ester-dependent PKC signaling dramatically repressed PXR activity in both, cell-based reporter gene assays and in hepatocytes. Moreover, treatment with the protein phosphatase PP1/PP2A inhibitor okadaic acid (OA) totally abolished PXR activity in reporter gene assays and in cultured hepatocytes. In mammalian two-hybrid assays, treatment with phorbol 12-myristate 13-acetate (PMA) increased the strength of interaction between PXR and the nuclear receptor co-repressor protein (NCoR). Treatment with PMA also abolished the ligand-dependent interaction between PXR and the steroid receptor co-activator 1 protein (SRC1). Our findings suggest that activation of the protein kinase C signaling pathway represses PXR activity through alterations in PXR-protein co-factor complexes, possibly through direct alterations in the phosphorylation status of one or all of these proteins. In addition, our data potentially provide important insights into the molecular mechanism of the repression of hepatic CYP3A gene expression that occurs during the inflammatory response.

Keywords: Abbreviations; PXR; pregnane X receptor; PKC; protein kinase C; PKCα; protein kinase C alpha; CYP; cytochrome P450; PMA; Phorbol 12-myristate 13-acetate; 4-αPMA; 4-α-Phorbol 12-myristate 13-acetate; OA; okadaic acid; PCN; pregnenolone 16α-carbonitrile; XREM; Xenobiotic-responsive enhancer module; PP; protein phosphatase; NCoR; Nuclear receptor co-repressor protein; SRC1; Steroid receptor co-activator 1 protein; TNF-α; tumor necrosis factor-alphaPregnane X receptor; CYP3A; PMA; PCN; PKC; Inflammation
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