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Biochemical Pharmacology (v.84, #2)
The diverse roles and clinical relevance of PARPs in DNA damage repair: Current state of the art by Mike De Vos; Valérie Schreiber; Françoise Dantzer (pp. 137-146).
Poly(ADP-ribose) polymerase (PARP) catalyzed poly(ADP-ribosyl)ation is one of the earliest post-translational modification of proteins detectable at sites of DNA strand interruptions. The considerable recent progress in the science of PARP in the last decade and the discovery of a PARP superfamily (17 members) has introduced this modification as a key mechanism regulating a wide variety of cellular processes including among others transcription, regulation of chromatin dynamics, telomere homeostasis, differentiation and cell death. However, the most extensive studied and probably the best characterized role is in DNA repair where it plays pivotal roles in the processing and resolution of the damaged DNA. Although much of the focus has been on PARP1 in DNA repair, recent advances highlight the emergence of other DNA-dependent PARPs (i.e. PARP2, PARP3 and possibly Tankyrase) in this process. Here we will summarize the recent insights into the molecular functions of these PARPs in different DNA repair pathways in which they emerge as specific actors. Furthermore, the DNA repair functions of PARP1 have stimulated another area of intense research in the field with the development of potent and selective PARP1 inhibitors to promote genome instability and cell death in tumor cells. Their current use in clinical trials have demonstrated potentiation of antitumoral drugs and cytotoxicity in repair deficient tumor cells.
Keywords: Poly(ADP-ribose) polymerase; DNA repair; Chromatin response; Cancer; PARP inhibitors
Vascular endothelin receptor type B: Structure, function and dysregulation in vascular disease by Marc Q. Mazzuca; Raouf A. Khalil (pp. 147-162).
Endothelin-1 (ET-1) is a major regulator of vascular function, acting via both endothelin receptor type A (ETAR) and type B (ETBR). Although the role of ETAR in vascular smooth muscle (VSM) contraction has been studied, little is known about ETBR. ETBR is a G-protein coupled receptor with a molecular mass of ∼50kDa and 442 amino acids arranged in seven transmembrane domains. Alternative splice variants of ETBR and heterodimerization and cross-talk with ETAR may affect the receptor function. ETBR has been identified in numerous blood vessels with substantial effects in the systemic, renal, pulmonary, coronary and cerebral circulation. ETBR in the endothelium mediates the release of relaxing factors such as nitric oxide, prostacyclin and endothelium-derived hyperpolarizing factor, and could also play a role in ET-1 clearance. ETBR in VSM mediates increases in [Ca2+]i, protein kinase C, mitogen-activated protein kinase and other pathways of VSM contraction and cell growth. ET-1/ETAR signaling has been associated with salt-sensitive hypertension (HTN) and pulmonary arterial hypertension (PAH), and ETAR antagonists have shown some benefits in these conditions. In search for other pathogenetic factors and more effective approaches, the role of alterations in endothelial ETBR and VSM ETBR in vascular dysfunction, and the potential benefits of modulators of ETBR in treatment of HTN and PAH are being examined. Combined ETAR/ETBR antagonists could be more efficacious in the management of conditions involving upregulation of ETAR and ETBR in VSM. Combined ETAR antagonist with ETBR agonist may need to be evaluated in conditions associated with decreased endothelial ETBR expression/activity.
Keywords: Abbreviations; AngII; angiotensin II; AT; 1; R; angiotensin type 1 receptor; BP; blood pressure; [Ca; 2+; ]; i; intracellular free Ca; 2+; concentration; CAD; coronary artery disease; CAMK-II; Ca; 2+; /calmodulin-dependent protein kinase II; CHF; chronic heart failure; DAG; diacylglycerol; DOCA; deoxycorticosterone acetate; ECE; endothelin converting enzyme; ECs; endothelial cells; EDHF; endothelium-derived hyperpolarizing factor; EDNRB gene; endothelin receptor type B gene; ET-1; endothelin-1; EDHF; endothelium-derived hyperpolarizing factor; ET; A; R; endothelin receptor type A; ET; B; R; endothelin receptor type B; FBF; forearm blood flow; GPCR; G-protein coupled receptor; GRK; G-protein coupled receptor kinase; HS; high salt; HTN; hypertension; IP; 3; inositol 1,4,5-trisphosphate; KO; knockout; l; -NAME; N; ω; -nitro-; l; -arginine methyl ester; LDL; low-density lipoprotein; MAPK; mitogen-activated protein kinase; MLC; myosin light chain; NO; nitric oxide; PGI; 2; prostacyclin; PAH; pulmonary arterial hypertension; PKC; protein kinase C; PLC-β; phospholipase C-β; S6c; sarafotoxin c; SHR; spontaneously hypertensive rats; TMD; transmembrane domains; VSM; vascular smooth muscleEndothelium; Smooth muscle; Calcium; Blood pressure; Hypertension
The curative efficacy of namitecan (ST1968) in preclinical models of pediatric sarcoma is associated with antiangiogenic effects by Giuliana Cassinelli; Valentina Zuco; Giovanna Petrangolini; Michelandrea De Cesare; Monica Tortoreto; Cinzia Lanzi; Denis Cominetti; Nadia Zaffaroni; Augusto Orlandi; Daniela Passeri; Daniela Meco; Angela Maria Di Francesco; Riccardo Riccardi; Federica Bucci; Claudio Pisano; Franco Zunino (pp. 163-171).
Namitecan (ST1968), a novel hydrophilic camptothecin analog of the 7-oxyiminomethyl series, was selected for clinical development on the basis of its promising preclinical efficacy. Since there is clinical evidence of efficacy of camptothecins against pediatric tumors, this study was performed to explore the antitumor and antiangiogenic activity of the camptothecin derivative in pediatric sarcoma models. With the exception of an undifferentiated rhabdomyosarcoma (A204), namitecan exhibited curative efficacy even at well-tolerated suboptimal doses in a panel of five models. The good therapeutic index of namitecan likely reflected a high and persistent drug accumulation at tumor site. The four responsive tumors were characterized by high topoisomerase I expression. In the RD/TE-671 rhabdomyosarcoma model the drug activity was associated with a marked antiangiogenic effect, which was consistent with the downregulation of proangiogenic factors, including VEGF, bFGF and the multifunctional chemokines CCL-2 and CXCL16. In agreement with this modulation, the combination of low doses of namitecan with other antiangiogenic agents, such as bevacizumab (a humanized anti-VEGF antibody) and sunitinib (a multitarget tyrosine kinase inhibitor effective against receptors implicated in the angiogenesis process), enhanced the antitumor effects. In conclusion, this preclinical study provides evidence of curative efficacy of namitecan at well-tolerated doses against pediatric sarcoma models, likely reflecting a contribution of antiangiogenic effects. Based on the promising therapeutic profile, namitecan is a good candidate for clinical evaluation in pediatric sarcomas.
Keywords: Camptothecin derivative; Antitumor activity; Antiangiogenesis; Pediatric sarcoma
SBF-1, a synthetic steroidal glycoside, inhibits melanoma growth and metastasis through blocking interaction between PDK1 and AKT3 by Wanshuai Li; Ran Song; Xianying Fang; Lu Wang; Wei Chen; Pingping Tang; Biao Yu; Yang Sun; Qiang Xu (pp. 172-181).
SBF-1, a 23-oxa-analogue of OSW-1, inhibits growth and metastasis of melanoma through blocking the interaction between PDK1 and AKT3, and negatively regulating AKT3/Integrin α4/FAK signaling.In the present study, we demonstrate that SBF-1, a synthetic steroidal glycoside, has a strong antitumor activity against melanoma cells in vitro and in vivo. SBF-1 induced cell cycle arrest with a reduced expression of various cell cycle related proteins in B16BL6 melanoma cells without causing apoptosis. SBF-1 dramatically inhibited kinase activity of 3-phosphoinositide dependent protein kinase 1 (PDK1) and thus down-regulated phosphorylation of protein kinase B (AKT). Among three known isoforms of AKT, PDK1 only interacted with AKT3 in B16BL6 melanoma cells, and SBF-1 almost completely blocked this interaction. In addition, adhesion to fibronectin and expression of integrin α4 were significantly reduced in a concentration-dependent manner. Knockdown of AKT3 resulted in the decrease in integrin α4 expression and cell adhesion. Moreover, SBF-1 inhibited the growth of melanoma xenografts and down-regulated the phosphorylation of AKT in vivo. In a mouse model of spontaneous metastasis, SBF-1 at very low doses of 1 and 3μg/kg enormously inhibited melanoma metastasis into draining popliteal lymph nodes. Taken together, this study shows a small molecular compound SBF-1 with a very strong anti-melanoma activity both in vitro and in vivo. Its mechanism underlying such antitumor effect is related to the blockage of the interaction between PDK1 and AKT3.
Keywords: SBF-1; Melanoma; PDK-1; AKT3; Metastasis
5-hydroxy-7-methoxyflavone inhibits N-formyl-l-methionyl-l-leucyl-l-phenylalanine-induced superoxide anion production by specific modulate membrane localization of Tec with a PI3K independent mechanism in human neutrophils by Hsiang-Ruei Liao; Jih-Jung Chen; Yin-Huan Chien; Shinn-Zhi Lin; Shunchih Lin; Ching-Ping Tseng (pp. 182-191).
Respiratory burst mediates crucial bactericidal mechanism in neutrophils. However, undesirable respiratory burst leads to pathological inflammation and tissue damage. This study investigates the effect and the underlying mechanism of 5-hydroxy-7-methoxyflavone (MCL-1), a lignan extracted from the leaves of Muntingia calabura L. (Tiliaceae), on N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP)-induced respiratory burst and cathepsin G release in human neutrophils. Signaling pathways regulated by MCL-1 to oppose fMLP-induced respiratory burst were evaluated by membrane localization of Tec induced by fMLP and by immunoblotting analysis of downstream phosphorylation targets of Tec. Briefly, MCL-1 specific inhibited fMLP-induced superoxide anion production in a concentration-dependent (IC50=0.16±0.01μM) and Tec kinase-dependent manner, however, MCL-1 did not affect fMLP-induced cathepsin G release. Further, MCL-1 suppressed fMLP-induced Tec translocation from the cytosol to the inner leaflet of the plasma membrane, and subsequently activation of phospholipase Cγ2 (PLCγ2). Moreover, MCL-1 attenuated PLCγ2 activity and intracellular calcium concentration notably through extracellular calcium influx. Consequently, fMLP-induced phosphorylation of protein kinase C (PKC) and membrane localization of p47phox were decreased by MCL-1 in a Tec-dependent manner, while the phosphorylation of extracellular signal-regulated kinase (ERK), p38, AKT and Src tyrosine kinase family remained unaffected. In addition, MCL-1 neither inhibited NADPH oxidase activity nor increased cyclicAMP levels. MCL-1 specific opposes fMLP-mediated respiratory burst by inhibition of membrane localization of Tec and subsequently interfered with the activation of PLCγ2, protein kinase C, and p47phox.
Keywords: MCL-1; Neutrophil; fMLP; Tec; Protein kinase C
Incomplete activation of human eosinophils via the histamine H4-receptor: Evidence for ligand-specific receptor conformations by Till M. Reher; Detlef Neumann; Armin Buschauer; Roland Seifert (pp. 192-203).
Eosinophils play a crucial role in the pathogenesis of allergic diseases. Histamine activates eosinophils via the H4-receptor (H4R). However, pharmacological analysis of the H4R in eosinophils is still incomplete, and cell purity is a problem. The H4R antagonist 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine (JNJ7777120) has recently been reported to exhibit paradoxical stimulatory effects in some systems. Therefore, the first aim of our study was to pharmacologically re-examine H xR subtypes on human eosinophils using a highly purified preparation (97±2%). The second aim was to compare the effects of histamine with those induced by well-known activators of eosinophil functions, i.e. eotaxin-1 and formyl peptides. Histamine and the H4R-selective agonist 2-cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-[(2-phenylthio)ethyl]guanidine (UR-PI376) increased intracellular calcium concentration ([Ca2+]i) and activated chemotaxis. JNJ7777120 per se exhibited no stimulatory effects but inhibited stimulation by histamine and UR-PI376. Blockade of the H2R by famotidine enhanced histamine-induced chemotaxis but not rises in [Ca2+]i. Compared to eotaxin and formyl peptides, the effect of histamine on eosinophil chemotaxis was only small. Formyl peptides but not histamine activated reactive oxygen species formation and release of eosinophil peroxidase. In conclusion, histamine is only an incomplete eosinophil activator with the H2R blunting the small chemotactic response to H4R activation. We also noted several differences in potencies of histamine, UR-PI376 and JNJ7777120 in calcium and chemotaxis assays and when compared to results in the literature. This indicates functional selectivity of H4R ligands, thus ligand-specific stabilization of distinct receptor conformations, inducing distinct biological responses.
Keywords: Abbreviations; GPCR; G-protein-coupled receptor; EPO; eosinophil peroxidase; fMLP; N-formyl-; l; -methionyl-; l; -leucyl-; l; -phenylalanine; UR-PI376; 2-cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-[(2-phenylthio)ethyl]guanidine; PMA; phorbol 12-myristate 13-acetate; JNJ7777120; 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine; O; 2; −; superoxide anion; ROS; reactive oxygen species; [Ca; 2+; ]; i; intracellular calcium concentration; CI; confidence interval; H; x; R; histamine H; 1; -, H; 2; -, H; 3; - or H; 4; -receptorEosinophils; Histamine H; 4; -receptor; Chemotaxis; Intracellular calcium concentration; Functional selectivity
Role of chemokine receptor CXCR7 in bladder cancer progression by Mingang Hao; Jianghua Zheng; Kailin Hou; Jinglong Wang; Xiaosong Chen; Xiaojiong Lu; Junjie Bo; Chen Xu; Kunwei Shen; Jianhua Wang (pp. 204-214).
Bladder cancer is one of the most common tumors of the genitourinary tract; however, the molecular events underlying growth and invasion of the tumor remain unclear. Here, role of the CXCR7 receptor in bladder cancer was further explored. CXCR7 protein expression was examined using high-density tissue microarrays. Expression of CXCR7 showed strong epithelial staining that correlated with bladder cancer progression. In vitro and in vivo studies in bladder cancer cell lines suggested that alterations in CXCR7 expression were associated with the activities of proliferation, apoptosis, migration, invasion, angiogenesis and tumor growth. Moreover, CXCR7 expression was able to regulate expression of the proangiogenic factors IL-8 or VEGF, which may involve in the regulation of tumor angiogenesis. Finally, we found that signaling by the CXCR7 in bladder cancer cells activates AKT, ERK and STAT3 pathways. The AKT and ERK pathways may reciprocally regulate, which are responsible for in vitro and in vivo epithelial to mesenchymal transition (EMT) process of bladder cancer. Simultaneously targeting the two pathways by using U0126 and LY294002 inhibitors or using CCX733, a selective CXCR7 antagonist drastically reduced CXCR7-induced EMT process.Taken together, our data show for the first time that CXCR7 plays a role in the development of bladder cancer. Targeting CXCR7 or its downstream-activated AKT and ERK pathways may prove beneficial to prevent metastasis and provide a more effective therapeutic strategy for bladder cancer.
Keywords: CXCR7; Bladder cancer; Chemokine receptor; EMT
Role of human CYP3A4 in the biotransformation of sorafenib to its major oxidized metabolites by Sussan Ghassabian; Tristan Rawling; Fanfan Zhou; Munikumar R. Doddareddy; Bruce N. Tattam; David E. Hibbs; Robert J. Edwards; Pei H. Cui; Michael Murray (pp. 215-223).
The tyrosine kinase inhibitor drug sorafenib is used in the treatment of liver and renal cancers but adverse effects may necessitate dose interruption and under-dosage may lead to therapeutic failure. Sorafenib also undergoes cytochrome P450 (CYP)-dependent biotransformation to the N-oxide and other metabolites. However, although CYPs are major determinants of efficacy and toxicity the roles of these enzymes in the formation of multiple sorafenib metabolites are unclear. In the present study CYP-mediated pathways of sorafenib oxidation in human liver were evaluated. cDNA-expressed CYP3A4 was the major catalyst in the formation of the principal N-oxide and N-hydroxymethyl metabolites of sorafenib, as well as the minor N-desmethyl metabolite. In contrast, CYP3A5 exhibited only ∼5% of the activity of CYP3A4 and eleven other CYPs and three flavin-containing monooxygenases were inactive. In human hepatic microsomes metabolite formation was correlated with CYP3A4-mediated midazolam 1′-hydroxylation, but not with other CYP-specific substrate oxidations. In accord with these findings the CYP3A4 inhibitor ketoconazole selectively inhibited microsomal sorafenib oxidation pathways. From computational modeling studies atoms in the structure of sorafenib that undergo biotransformation were within ∼5.4Å of the CYP3A4 heme. Important hydrogen bonding interactions between sorafenib and amino acids Ser-119 and Glu-374 in the active center of CYP3A4 were identified. These findings indicate that sorafenib is oxidized selectively by human CYP3A4. This information could be adapted in individualized approaches to optimize sorafenib safety and efficacy in cancer patients.
Keywords: Abbreviations; CYP; cytochrome P450; FMO; flavin-containing monooxygenase; HPLC; high-performance liquid chromatography; LC–MS/MS; liquid chromatography–tandem mass spectrometry; TKI; tyrosine kinase inhibitorSorafenib; CYP3A4; Sorafenib biotransformation; Tyrosine kinase inhibitors; Hepatoma
A nano switch mechanism for the redox-responsive sulfotransferase by Chih-Heng Lin; En-Shyh Lin; Tian-Mu Su; Kuo-Sheng Hung; Yuh-Shyong Yang (pp. 224-231).
Redox regulation of sulfotransferase through long distance interaction.Cellular redox signaling is important in diverse physiological and pathological processes. The activity of rat phenol sulfotransferase ( rSULT1A1), which is important for the metabolism of hormone and drug, is subjected to redox regulation. Two cysteines, Cys232 and Cys66, nanometer away from each other and from the enzyme active site were proposed to form disulfide bond to regulate the activity of rSULT1A1. A nano switch, composed of a flexible loop from amino acid residues 59–70, explained how this long distance interaction between two cysteines can be achieved. The enzyme properties were investigated through site-directed muatagnesis, circular dichroism, enzyme kinetics and homologous modeling of the rSULT1A1 structures. We proposed that the formation of disulfide bond between Cys232 and Cys66 induced conformational changes of sulfotransferase, then in turn affected its nucleotide binding and enzyme activity. This discovery was extended to understand the possible redox regulation of other sulfotransferases from different organisms. The redox switch can be created in other redox-insensitive sulfotransferases, such as human phenol sulfotransferase ( hSULT1A1) and human alcohol sulfotransferase ( hSULT2A1), to produce mutant enzymes with redox regulation capacity. This study strongly suggested that redox regulation of drug and hormone metabolism can be significantly varied even though the sequence and structure of SULT1A1 of human and rat have a high degree of homology.
Keywords: Abbreviations; Bis–tris propane; 1,3-bis[tris(hydroxymethyl) methylamino] propane; CD; circular dichroism; DEAE; diethylaminoethyl; GSSG; glutathione (oxidized form); GST; glutathione S-transferase; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SULT; sulfotransferase; TCEP; tris(2-carboxyethyl) phosphineSulfotransferase; Redox regulation; Drug metabolism; Xenobiotics; Glutathione
Age-related inducibility of carboxylesterases by the antiepileptic agent phenobarbital and implications in drug metabolism and lipid accumulation by Da Xiao; Yi-Tzai Chen; Dongfang Yang; Bingfang Yan (pp. 232-239).
Carboxylesterases (CES) constitute a class of hydrolytic enzymes that play critical roles in drug metabolism and lipid mobilization. Previous studies with a large number of human liver samples have suggested that the inducibility of carboxylesterases is inversely related with age. To directly test this possibility, neonatal (10 days of age) and adult mice were treated with the antiepileptic agent phenobarbital. The expression and hydrolytic activity were determined on six major carboxylesterases including ces1d, the ortholog of human CES1. Without exception, all carboxylesterases tested were induced to a greater extent in neonatal than adult mice. The induction was detected at mRNA, protein and catalytic levels. Ces1d was greatly induced and found to rapidly hydrolyze the antiplatelet agent clopidogrel and support the accumulation of neutral lipids. Phenobarbital represents a large number of therapeutic agents that induce drug metabolizing enzymes and transporters in a species-conserved manner. The higher inducibility of carboxylesterases in the developmental age likely represents a general phenomenon cross species including human. Consequently, individuals in the developmental age may experience greater drug–drug interactions. The greater induction of ces1d also provides a molecular explanation to the clinical observation that children on antiepileptic drugs increase plasma lipids.
Keywords: Abbreviations; TCPOBOP; 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene; CES; carboxylesterase; DMEM; Dulbecco's modified eagle medium; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; HPLC; high-performance liquid chromatography; IS; internal standard; PCN; pregnenolone 16α-carbonitrile; RT-qPCR; reverse transcription-quantitative polymerase chain reactionCarboxylesterases; Developmental regulation; Clopidogrel; Aspirin; Liver steatosis