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Biochemical Pharmacology (v.79, #1)
The Yin and Yang of vitamin D receptor (VDR) signaling in neoplastic progression: Operational networks and tissue-specific growth control by F.C. Campbell; Haibo Xu; M. El-Tanani; P. Crowe; V. Bingham (pp. 1-9).
Substantive evidence implicates vitamin D receptor (VDR) or its natural ligand 1α,25-(OH)2 D3 in modulation of tumor growth. However, both human and animal studies indicate tissue-specificity of effect. Epidemiological studies show both inverse and direct relationships between serum 25(OH)D levels and common solid cancers. VDR ablation affects carcinogen-induced tumorigenesis in a tissue-specific manner in model systems. Better understanding of the tissue-specificity of vitamin D-dependent molecular networks may provide insight into selective growth control by the seco-steroid, 1α,25-(OH)2 D3. This commentary considers complex factors that may influence the cell- or tissue-specificity of 1α,25-(OH)2 D3/VDR growth effects, including local synthesis, metabolism and transport of vitamin D and its metabolites, vitamin D receptor (VDR) expression and ligand-interactions, 1α,25-(OH)2 D3 genomic and non-genomic actions, Ca2+ flux, kinase activation, VDR interactions with activating and inhibitory vitamin D responsive elements (VDREs) within target gene promoters, VDR coregulator recruitment and differential effects on key downstream growth regulatory genes. We highlight some differences of VDR growth control relevant to colonic, esophageal, prostate, pancreatic and other cancers and assess the potential for development of selective prevention or treatment strategies.
Keywords: Abbreviations; 1α,25-(OH); 2; D; 3; one alpha, 25 dihydroxyvitamin D3; APC; adenomatous polyposis coli; CRC; colorectal cancer; DMBA; dimethylbenzanthracene; DR3-type; directly repeated arrangement of the hexameric binding sites with three spacing nucleotides; DRIP; Vitamin D receptor-interacting protein; ERK; extracellular signal-regulated kinase; GSK3β; glycogen synthase kinase beta; HDAC; histone deacetylator co-repressor complex; MAPK; mitogen-activated protein kinase; NCoR; nuclear receptor co-repressor; NHL; non-Hodgkins lymphoma; OPN; osteopontin; RAC3; receptor activated coactivators 3; ROCK; Rho-associated coiled kinase; RXR; retinoid X receptor; SRC-1; steroid receptor coactivators-1; Tcf; T cell factor; TIF2; transcriptional intermediary factor 2; TPA; 12-O-tetradecanoylphorbol-13-acetate; VDRE; vitamin D response element; VDR; vitamin D receptor; WINAC; Williams syndrome transcription factor (WSTF) including nucleosome assembly complex1α,25-(OH); 2; D; 3; Vitamin D receptor; Signaling; Cancer
Differential effects of anti-apoptotic Bcl-2 family members Mcl-1, Bcl-2, and Bcl-xL on Celecoxib-induced apoptosis by Justine Rudner; Simon Johannes Elsaesser; Arndt-Christian Müller; Claus Belka; Verena Jendrossek (pp. 10-20).
The cyclooxygenase-2 inhibitor Celecoxib is a potent inducer of apoptosis in tumor cells. In most cellular systems Celecoxib induces apoptosis via an intrinsic, mitochondrial apoptosis pathway. We recently showed that in Bax-negative Jurkat cells expression of pro-apoptotic Bak is essential for Celecoxib-induced mitochondrial damage and apoptosis induction. Aim of the present study was to identify specific pro- and anti-apoptotic members of the Bcl-2 family involved in the regulation of Bak activation, and subsequent apoptosis upon treatment with Celecoxib in the Jurkat cell model.Our results show that apoptosis in response to Celecoxib required the presence of Noxa and downregulation of the anti-apoptotic protein Mcl-1. Celecoxib-induced Bak activation and subsequent apoptosis could be inhibited by overexpression of Bcl-xL but not by the very similar Bcl-2. In Bcl-xL-overexpressing cells neutralization of both, Mcl-1 and Bcl-xL, was prerequisite for an efficient induction of apoptosis. Our data reveal an important role of the Mcl-1/Noxa axis for Celecoxib-induced apoptosis and suggest that Celecoxib may be of value for treatment of tumors addicted to Mcl-1 and for combined treatment approaches targeting anti-apoptotic Bcl-2 family members.
Keywords: Cancer; Apoptosis; Celecoxib; Mcl-1; Noxa; Bcl-xL; Bcl-2
Cytotoxic activity of gemcitabine in cultured cell lines derived from histologically different types of bladder cancer: Role of thymidine kinase 2 by Swathi Damaraju; Vijaya L. Damaraju; Delores Mowles; Michael B. Sawyer; Sambasivarao Damaraju; Carol E. Cass (pp. 21-29).
World wide incidence of bladder cancer is rising with nearly 13,760 deaths attributed to bladder cancer in 2007 in the USA. Tumor types of the urothelium include transitional cell carcinomas, squamous cell carcinomas, and adenocarcinomas. This study was undertaken to determine gemcitabine's efficacy against bladder cancer cell lines of different origins (HTB2, a papilloma; HTB3, a squamous cell carcinoma; and HTB4, a transitional cell carcinoma). Roles of nucleoside transporters and key enzymes in gemcitabine pharmacology were examined on the premise that cells originating from different types of bladder cancer exhibit different levels and/or types of nucleoside transporters and enzymes and thus may respond differently to gemcitabine. HTB2 cells had the highest transport efficiency and were also most responsive to gemcitabine. HTB3 and HTB4 cells had similar transport efficiencies, but exhibited different sensitivities to gemcitabine (HTB4>HTB3). The highest accumulation of [3H]gemcitabine was in HTB2 cells and the lowest was in HTB3 cells. Sequencing experiments revealed no mutations either in coding exons or intron–exon boundaries of the hENT1 genes of the three cell lines. HTB3 cells exhibited high thymidine kinase 2 (TK2) activity whereas HTB2 and HTB4 cells lacked detectable TK2 activity and pretreatment of HTB3 but not of HTB2 and HTB4 cells with extracellular thymidine resulted in enhanced sensitivity to gemcitabine. Our results highlight the importance of hENT1 and TK2 activities in response to gemcitabine. Elevated TK expression in squamous cell carcinomas warrants further study and offers new insights into rational treatment strategies based on bladder cancer phenotype.
Keywords: Abbreviations; hENT; human equilibrative nucleoside transporters; NBMPR; nitrobenzylmercaptopurine riboside; IC; 50; the concentration of drug that inhibited growth of treated cells by 50% relative to that of untreated cells; TK; thymidine kinase; dCK; deoxycytidine kinase; TK2; thymidine kinase 2Bladder cancer; Nucleoside transporters; Gemcitabine; Thymidine kinase; hENT1
Structure–function analysis of the highly conserved charged residues of the membrane protein FT1, the main folic acid transporter of the protozoan parasite Leishmania by Larbi Dridi; Anass Haimeur; Marc Ouellette (pp. 30-38).
The main plasma membrane folate transporter FT1 of Leishmania belongs to the novel FBT family which is part of the major facilitator superfamily. We have investigated the role of the 10 most conserved charged amino acids of FBTs by site directed mutagenesis. The functions of the mutated proteins were tested for their capacity to transport FA, to sensitize methotrexate resistant cells to methotrexate, for protein production, and for protein localisation. Of the 10 conserved charged amino acids that were mutated to neutral amino acids, all had effects on FT1 transport activities. Only four of the 10 initial mutants (K116L, K133L, R497L, and D529V) retained between 15% and 50% of FT1 activity. The R497 residue was shown to be involved in substrate binding. When the charged conserved residues at position 124, 134, 179, 514, 537 and 565 were changed to neutral amino acids, this led to inactive proteins but the generation of new mutants D124E, R134K, D514E and D537E regained between 20% and 50% of wild-type FT1 activity suggesting that the charge is important for protein function. The mutated protein D179E had, under our standard experimental conditions, no activity, while E565D was completely inactive. The differential activity of the mutated proteins was due either to changes in the apparent Km or Vmax. Mutagenesis experiments have revealed that charged amino acids were essential for FT1 stability or activity and led to a plausible model for the transport of folic acid through FT1.
Keywords: Abbreviations; FA; folic acid; FBT; Folate Biopterin Transporter; MTX; methotrexate; NHS; N; -hydroxysuccinimide; TMS; trans membrane segmentsFolate transporters; Methotrexate; Site directed mutagenesis; Leishmania; Transport studies
Anti-ischemic properties of a new spiro-cyclic benzopyran activator of the cardiac mito-KATP channel by Vincenzo Calderone; Lara Testai; Alma Martelli; Simona Rapposelli; Maria Digiacomo; Aldo Balsamo; Maria C. Breschi (pp. 39-47).
Many activators of KATP channels exhibit cardioprotective effects, mainly mediated by channels expressed on mitochondria (mito-KATP). Previous results showed anti-ischemic effects of the spiro-cyclic derivativeA, on isolated rat hearts. In this work this molecule was more extensively studied and diazoxide was used as reference mito-KATP opener. The studies were performed on an in vivo rat model of myocardial infarct and on heart-derived H9c2 cells exposed to an anoxic environment. The mechanism of action was further investigated on isolated rat heart mitochondria. In the model of myocardial infarct compoundA and diazoxide produced significant cardioprotective effects, antagonised by the selective mito-KATP blocker 5-hydroxydecanoic acid (5-HD). CompoundA, like diazoxide, produced modest and non-significant hypotensive responses, while the hyperglycaemic effects of diazoxide were not observed for the new compound. Protective effects of compoundA and diazoxide were also recorded in H9c2 cells and again were inhibited by 5-HD. CompoundA and diazoxide caused swelling of cardiac mitochondria, in agreement with the profile of mito-KATP openers. Both compounds evoked concentration-dependent Ca2+-release from Ca2+-preloaded mitochondria, prevented mitochondrial Ca2+-uptake and caused mitochondrial membrane depolarisation. These effects were antagonised by ATP, the endogenous KATP inhibitor. In conclusion, compoundA exhibits a promising profile of an anti-ischemic agent, with a mechanism likely to be linked to the activation of mito-KATP channels, and, because of its chemical characteristics such as structural rigidity and chirality due to the spiro-cyclic moiety, represents an interesting template for development of analogues further improved in activity and selectivity.
Keywords: Abbreviations; CsA; cyclosporine A; 5-HD; 5-hydroxydecanoic acid; IPC; ischemic pre-conditioning; I/R; ischemia/reperfusion; LAD; left anterior descending coronary artery; K; ATP; ATP-sensitive potassium channel; MPTP; mitochondrial permeability transition poreMyocardial ischemia/reperfusion; Anti-ischemic drugs; Mitochondrial K; ATP; channel; Benzopyran K; ATP; activators; Spiro-cyclic derivatives
The endocannabinoid 2-arachidonylglycerol is a negative allosteric modulator of the human A3 adenosine receptor by J. Robert Lane; Margot W. Beukers; Thea Mulder-Krieger; Ad P. IJzerman (pp. 48-56).
Studies of endogenous cannabinoid agonists, such as 2-arachidonylglycerol (2-AG), have revealed their potential to exert modulatory actions on other receptor systems in addition to their ability to activate cannabinoid receptors. This study investigated the effect of cannabinoid ligands on the human adenosine A3 (hA3R) receptor. The endocannabinoid 2-AG was able to inhibit agonist ([125I]N6-(4-amino-3-iodobenzyl) adenosine-5′-(N-methyluronamide) – [125I] AB MECA) binding at the hA3R. This inhibition occurred over a narrow range of ligand concentration and was characterized by high Hill coefficients suggesting a non-competitive interaction. Furthermore, in the presence of 2-AG, the rate of [125I] AB MECA dissociation was increased, consistent with an action as a negative allosteric modulator of the hA3R. Moreover, by measuring intracellular cAMP levels, we demonstrate that 2-AG decreases both the potency of an agonist at the hA3R and the basal signalling of this receptor. Since the hA3R has been shown to be expressed in astrocytes and microglia, these findings may be particularly relevant in certain pathological states such as cerebral ischemia where levels of 2-AG and anandamide are raised.
Keywords: G protein coupled receptor (GPCR); Adenosine A; 3; receptor; Endocannabinoid; 2-Arachidonylglycerol; Allosteric modulation
Role of cytochrome P450 2E1 in protein nitration and ubiquitin-mediated degradation during acetaminophen toxicity by Mohamed A. Abdelmegeed; Kwan-Hoon Moon; Chi Chen; Frank J. Gonzalez; Byoung-Joon Song (pp. 57-66).
CYP2E1 is important in protein nitration and ubiquitin-dependent degradation of nitrated proteins during acetaminophen-mediated hepatic necrotic damageIt is well established that following a toxic dose of acetaminophen (APAP), nitrotyrosine protein adducts (3-NT), a hallmark of peroxynitrite production, were colocalized with necrotic hepatic centrilobular regions where cytochrome P450 2E1 (CYP2E1) is highly expressed, suggesting that 3-NT formation may be essential in APAP-mediated toxicity. This study was aimed at investigating the relationship between CYP2E1 and nitration (3-NT formation) followed by ubiquitin-mediated degradation of proteins in wild-type and Cyp2e1-null mice exposed to APAP (200 and 400mg/kg) for 4 and 24h. Markedly increased centrilobular liver necrosis and 3-NT formation were only observed in APAP-exposed wild-type mice in a dose- and time-dependent manner, confirming an important role for CYP2E1 in APAP biotransformation and toxicity. However, the pattern of 3-NT protein adducts, not accompanied by concurrent activation of nitric oxide synthase (NOS), was similar to that of protein ubiquitination. Immunoblot analysis further revealed that immunoprecipitated nitrated proteins were ubiquitinated in APAP-exposed wild-type mice, confirming the fact that nitrated proteins are more susceptible than the native proteins for ubiquitin-dependent degradation, resulting in shorter half-lives. For instance, cytosolic superoxide dismutase (SOD1) levels were clearly decreased and immunoprecipitated SOD1 was nitrated and ubiquitinated, likely leading to its accelerated degradation in APAP-exposed wild-type mice. These data suggest that CYP2E1 appears to play a key role in 3-NT formation, protein degradation, and liver damage, which is independent of NOS, and that decreased levels of many proteins in the wild-type mice (compared with Cyp2e1-null mice) likely contribute to APAP-related toxicity.
Keywords: Liver toxicity; Acetaminophen; CYP2E1; Nitrotyrosine; Ubiquitin; Superoxide dismutase
mTOR inhibitor everolimus ameliorates progressive tubular dysfunction in chronic renal failure rats by Shunsaku Nakagawa; Satohiro Masuda; Kumiko Nishihara; Ken-ichi Inui (pp. 67-76).
The mTOR pathway was activated in the kidney of chronic renal failure rats, and has important roles in the progressive tubular dysfunction.Responsible factors in progressive tubular dysfunction in chronic renal failure have not been fully identified. In the present study, we hypothesized that the mammalian target of rapamycin, mTOR, was a key molecule in the degenerative and progressive tubular damage in chronic renal failure. Everolimus, an mTOR inhibitor, was administered for 14 days in 5/6 nephrectomized (Nx) rats at 2 and 8 weeks after renal ablation. Marked activation of the mTOR pathway was found at glomeruli and proximal tubules in remnant kidneys of Nx rats. The reduced expression levels of the phosphorylated S6 indicated the satisfactory pharmacological effects of treatment with everolimus for 14 days. Everolimus suppressed the accumulation of smooth muscle alpha actin, infiltration of macrophages and expression of kidney injury molecule-1 in the proximal tubules. In addition, everolimus-treatment restored the tubular reabsorption of albumin, and had a restorative effect on the expression levels of membrane transporters in the polarized proximal tubular epithelium, when its administration was started at 8 weeks after Nx. These results indicate that the constitutively activated mTOR pathway in proximal tubules has an important role in the progressive tubular dysfunction, and that mTOR inhibitors have renoprotective effects to improve the proximal tubular functions in end-stage renal disease.
Keywords: Mammalian target of rapamycin; Proximal tubule injury; Albuminuria; Drug transporter; Chronic kidney disease