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Biochemical Pharmacology (v.79, #2)

Graphical Abstracts Contents (pp. e1-e6).

Editorial Advisory Board (pp. iii).

Astrocytes in the damaged brain: Molecular and cellular insights into their reactive response and healing potential by Annalisa Buffo; Chiara Rolando; Stefania Ceruti (pp. 77-89).

Long considered merely a trophic and mechanical support to neurons, astrocytes have progressively taken the center stage as their ability to react to acute and chronic neurodegenerative situations became increasingly clear. Reactive astrogliosis starts when trigger molecules produced at the injury site drive astrocytes to leave their quiescent state and become activated. Distinctive morphological and biochemical features characterize this process (cell hypertrophy, upregulation of intermediate filaments, and increased cell proliferation). Moreover, reactive astrocytes migrate towards the injured area to constitute the glial scar, and release factors mediating the tissue inflammatory response and remodeling after lesion. A novel view of astrogliosis derives from the finding that subsets of reactive astrocytes can recapitulate stem cell/progenitor features after damage, fostering the concept of astroglia as a promising target for reparative therapies. But which biochemical/signaling pathways modulate astrogliosis with respect to both the time after injury and the type of damage? Are reactive astrocytes overall beneficial or detrimental for neuroprotection and tissue regeneration? This debate has been animating this research field for several years now, and an integrated view on the results obtained and the possible future perspectives is needed. With this Commentary article we have attempted to answer the above-mentioned questions by reviewing the current knowledge on the molecular mechanisms controlling and sustaining the reaction of astroglia to injury and its stem cell-like properties. Moreover, the cellular/molecular mechanisms supporting the detrimental or beneficial features of astrogliosis have been scrutinized to gain insights on possible pharmacological approaches to enhance astrocyte neuroprotective activities.

Keywords: Abbreviations; Ado; adenosine; AMPA; alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate; AP1; activator protein 1; AQP; acquaporin; BBB; blood–brain barrier; BDNF; brain-derived neurotrophic factor; bFGF; basic fibroblast growth factor; bHLH; basic helix loop helix; BMP; bone morphogenetic protein; CNS; central nervous system; CNTF; ciliary neurotrophic factor; COX-2; cyclooxigenase-2; CREB; cAMP response element binding; CSPGs; chondroitinsulphate proteoglycans; ERK; extracellular signal-regulated kinase; EGF; epidermal growth factor; Eph4A; ephrin 4A; Epo; erythropoietin; ET1; Endothelin 1; ET-R; endothelin receptor; GABA; gamma-aminobutyric acid; GDNF; glial cell-line derived neurotrophic factor; GF; growth factor; GFAP; glial fibrillary acidic protein; GLAST; glutamate/aspartate transporter; GLT1; glutamate transporter 1; GS; glutamine synthase; IFNβ; interferon beta; IFNγ; interferon gamma; IGF1; insulin growth factor 1; IL1β; interleukin 1 beta; IL2; interleukin 2; IL6; interleukin 6; IL10; interleukin 10; JAK; Janus protein tyrosine kinases; Lcn2; Lipocalin 2; MAPK; mitogen-activated protein kinase; MMP; matrix metalloprotease; mTOR; mammalian target of rapamycin; NFAT; nuclear factor of activated T cell; NFkB; nuclear factor kappa B; NGF; nerve growth factor; NT3; neurotrophin 3; p38MAPK; p38 mitogen-activated protein kinase; PTEN; phosphatase and tensin homolog; SOCS; suppressor of cytokine signaling; SOD; superoxide dismutase; STAT; signal transducer and activator of transcription; TGFα; transforming growth factor alpha; TGFβ; transforming growth factor beta; TNFα; tumor necrosis factor alpha; VCAM1; vascular cell adhesion molecule 1; VEGF; vascular endothelial growth factorExtracellular nucleotides; Cytokines; Glial scar; Neural stem cells; Reactive astrogliosis; Brain repair

Cancer cell death induced by phosphine gold(I) compounds targeting thioredoxin reductase by Valentina Gandin; Aristi Potamitou Fernandes; Maria Pia Rigobello; Barbara Dani; Francesca Sorrentino; Francesco Tisato; Mikael Björnstedt; Alberto Bindoli; Alberto Sturaro; Rocco Rella; Cristina Marzano (pp. 90-101).

The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH (nicotinamide adenine dinucleotide phosphate), plays a central role in regulating cellular redox homeostasis and signaling pathways. TrxR, overexpressed in many tumor cells and contributing to drug resistance, has emerged as a new target for anticancer drugs. Gold complexes have been validated as potent TrxR inhibitors in vitro in the nanomolar range. In order to obtain potent and selective TrxR inhibitors, we have synthesized a series of linear, ‘auranofin-like’ gold(I) complexes all containing the [Au(PEt₃)]⁺ synthon and the ligands: Cl⁻, Br⁻, cyanate, thiocyanate, ethylxanthate, diethyldithiocarbamate and thiourea. Phosphine gold(I) complexes efficiently inhibited cytosolic and mitochondrial TrxR at concentrations that did not affect the two related oxidoreductases glutathione reductase (GR) and glutathione peroxidase (GPx). The inhibitory effect of the redox proteins was also observed intracellularly in cancer cells pretreated with gold(I) complexes. Gold(I) compounds were found to induce antiproliferative effects towards several human cancer cells some of which endowed with cisplatin or multidrug resistance. In addition, they were able to activate caspase-3 and induce apoptosis observed as nucleosome formation and sub-G1 cell accumulation. The complexes with thiocyanate and xanthate ligands were particularly effective in inhibiting thioredoxin reductase and inducing apoptosis. Pharmacodynamic studies in human ovarian cancer cells allowed for the correlation of intracellular drug accumulation with TrxR inhibition that leads to the induction of apoptosis via the mitochondrial pathway.

Keywords: Thioredoxin reductase; Phosphine metal complexes; Apoptosis; Drug resistance; Cytotoxicity; Oxidative stress

Testing double mutants of the enzyme nitroreductase for enhanced cell sensitisation to prodrugs: Effects of combining beneficial single mutations by Mansooreh Jaberipour; Simon O. Vass; Christopher P. Guise; Jane I. Grove; Richard J. Knox; Longqin Hu; Eva I. Hyde; Peter F. Searle (pp. 102-111).

Improving prodrug activation gene therapy for cancer by enzyme engineering for better prodrug activation.Prodrug activation gene therapy for cancer involves expressing prodrug-activating enzymes in tumour cells, so they can be selectively killed by systemically administered prodrug. For example, Escherichia coli nfsB nitroreductase (E.C. 1.6.99.7)(NTR), sensitises cells to the prodrug CB1954 (5-[aziridin-1-yl]-2,4-dinitrobenzamide), which it converts to a potent DNA-crosslinking agent. However, low catalytic efficiency with this non-natural substrate appears to limit the efficacy of this enzyme prodrug combination for eliminating the target cancer cells. To improve this, we aim to engineer NTR for improved prodrug activation. Previously, a number of single amino acid substitutions at six positions around the active site of the enzyme were found to increase activity, resulting in up to ∼5-fold enhanced cell sensitisation to CB1954. In this study we have made pairwise combinations among some of the best mutants at each of these 6 sites. A total of 53 double mutants were initially screened in E. coli, then the 7 most promising were inserted into an adenovirus vector and compared in SKOV3 human ovarian carcinoma cells for sensitisation to CB1954 and two alternative prodrugs. The most effective mutants, T41L/N71S and T41L/F70A, were 14–17-fold more potent than WT NTR at sensitising the cancer cells to CB1954. The best mutant for activation of the dinitrobenzamide mustard prodrug SN23862 was T41L/F70A (4.8-fold improvement); and S40A/F124M showed 1.7-fold improvement over WT with the nitrobenzylphosphoramide mustard prodrug LH7. In two tumour xenograft models using SKOV3 or human prostate carcinoma PC3, T41L/N71S NTR demonstrated greater CB1954-dependent anti-tumour activity than WT NTR.

Keywords: Nitroreductase; Prodrugs; CB1954 (5-[(aziridin-1-yl]-2,4-dinitrobenzamide); Virus directed enzyme prodrug therapy (VDEPT); Cancer gene therapy

Conformation and recognition of DNA modified by a new antitumor dinuclear Pt^II complex resistant to decomposition by sulfur nucleophiles by Lenka Zerzankova; Tereza Suchankova; Oldrich Vrana; Nicholas P. Farrell; Viktor Brabec; Jana Kasparkova (pp. 112-121).

A detailed study of the molecular mechanism of action of a new dinuclear platinum complex is reported. DNA adducts of this complex can largely escape repair and yet inhibit very effectively transcription.Reported herein is a detailed biochemical and molecular biophysics study of the molecular mechanism of action of antitumor dinuclear Pt^II complex [{PtCl(DACH)}₂- μ-Y]⁴⁺ [DACH=1,2-diaminocyclohexane, Y=H₂N(CH₂)₆NH₂(CH₂)₂NH₂(CH₂)₆NH₂] (complex1). This new, long-chain bifunctional dinuclear Pt^II complex is resistant to metabolic decomposition by sulfur-containing nucleophiles. The results show that DNA adducts of1 can largely escape repair and yet inhibit very effectively transcription so that they should persist longer than those of conventional cisplatin. Hence, they could trigger a number of downstream cellular effects different from those triggered in cancer cells by DNA adducts of cisplatin. This might lead to the therapeutic effects that could radically improve chemotherapy by platinum complexes. In addition, the findings of the present work make new insights into mechanisms associated with antitumor effects of dinuclear/trinuclear Pt^II complexes possible.

Keywords: Abbreviations; BBR3610; [{; trans; -PtCl(NH; ₃; ); ₂; }; ₂; -; μ; -{; trans; -(H; ₂; N(CH; ₂; ); ₆; NH; ₂; (CH; ₂; ); ₂; NH; ₂; (CH; ₂; ); ₆; NH; ₂; )}](NO; ₃; ); ₄; bp; base pair; CFE; cell-free extract; cisplatin; cis; -diamminedichloridoplatinum(II); complex; 1; [{PtCl(DACH)}; ₂; -; μ; -H; ₂; N(CH; ₂; ); ₆; NH; ₂; (CH; ₂; ); ₂; NH; ₂; (CH; ₂; ); ₆; NH; ₂; ]; ⁴⁺; CT; calf thymus; DACH; 1,2-diaminocyclohexane; dien; diethylenetriamine; DPP; differential pulse polarography; EtBr; ethidium bromide; FAAS; flameless atomic absorption spectrometry; GSH; glutathione; PAA; polyacrylamide; r; _b; the number of molecules of the platinum complex bound per nucleotide residue; r; _i; the molar ratio of free platinum complex to nucleotides at the onset of incubation with DNA; RNA pol II; RNA polymerase IIAntitumor; Dinuclear platinum complex; DNA damage; DNA repair; RNA polymerase II; Glutathione

Aurora kinase inhibitor ZM447439 induces apoptosis via mitochondrial pathways by Minglun Li; Anke Jung; Ute Ganswindt; Patrizia Marini; Anna Friedl; Peter T. Daniel; Kirsten Lauber; Verena Jendrossek; Claus Belka (pp. 122-129).

ZM447439 (ZM) is a potent and selective inhibitor of aurora-A and -B kinase with putative anti-tumoral activity. Inhibitors of aurora kinases were shown to induce apoptosis in vitro and in vivo. To investigate the underlying mechanisms, cell death pathways triggered by ZM was analysed in HCT-116 colorectal cancer cells. Through correlation of polyploidization and apoptosis in different knockout cells, the interrelation of these cellular responses to ZM was investigated. ZM induced apoptosis in a concentration- and time-dependent manner. ZM-induced apoptosis was associated with an upregulation of p53, breakdown of the mitochondrial membrane potential (ΔΨm) and activation of caspase-3. To precisely define key components for ZM-induced apoptosis, knockout cells lacking p53, Bak, Bax or both Bak and Bax were used. Lack of p53 reduced ZM-induced apoptosis and breakdown of ΔΨm, while lack of Bak, Bax or both almost completely inhibited apoptosis and breakdown of ΔΨm. Since no difference in apoptosis induction was detectable between HCT-116 cells lacking Bak, Bax or both, apoptosis induction depended non-redundantly on both Bak and Bax. Phenomenally, ZM induced notable polyploidization in all examined cells, especially in p53−/− cells. A correlation between polyploidization and apoptosis was observed in wild-type, and also in p53−/− cells, albeit with a modest extent of apoptosis. Moreover, in Bak−/−, Bax−/− and Bak/Bax−/− cells apoptosis was totally inhibited in spite of the strongest polyploidization, suggesting apoptosis may be a secondary event following polyploidization in HCT-116 cells. Thus ZM-induced apoptosis depends not only on polyploidization, but also on the intracellular apoptotic signaling.

Keywords: Aurora kinase inhibitor ZM447439; Apoptosis; Mitochondrial pathway; Colorectal tumor cells; Polyploidization

Anti-cancer effects of artesunate in a panel of chemoresistant neuroblastoma cell lines by Martin Michaelis; Malte C. Kleinschmidt; Susanne Barth; Florian Rothweiler; Janina Geiler; Rainer Breitling; Bernd Mayer; Hedwig Deubzer; Olaf Witt; Jörg Kreuter; Hans Wilhelm Doerr; Jaroslav Cinatl; Jindrich Cinatl Jr. (pp. 130-136).

Artemisinin derivatives are well-tolerated anti-malaria drugs that also exert anti-cancer activity. Here, we investigated artemisinin and its derivatives dihydroartemisinin and artesunate in a panel of chemosensitive and chemoresistant human neuroblastoma cells as well as in primary neuroblastoma cultures. Only dihydroartemisinin and artesunate affected neuroblastoma cell viability with artesunate being more active. Artesunate-induced apoptosis and reactive oxygen species in neuroblastoma cells. Of 16 cell lines and two primary cultures, only UKF-NB-3^rCDDP¹⁰⁰⁰ showed low sensitivity to artesunate. Characteristic gene expression signatures based on a previous analysis of artesunate resistance in the NCI60 cell line panel clearly separated UKF-NB-3^rCDDP¹⁰⁰⁰ from the other cell lines.l-Buthionine-S,R-sulfoximine, an inhibitor of GCL (glutamate–cysteine ligase), resensitised in part UKF-NB-3^rCDDP¹⁰⁰⁰ cells to artesunate. This finding together with bioinformatic analysis of expression of genes involved in glutathione metabolism showed that this pathway is involved in artesunate resistance. These data indicate that neuroblastoma represents an artesunate-sensitive cancer entity and that artesunate is also effective in chemoresistant neuroblastoma cells.

Keywords: Neuroblastoma; Artesunate; Artemisinin; Chemoresistance; Cancer; Chemotherapy

Proteasome inhibitors prevent cisplatin-induced mitochondrial release of apoptosis-inducing factor and markedly ameliorate cisplatin nephrotoxicity by Ling Liu; Cheng Yang; Christian Herzog; Rohit Seth; Gur P. Kaushal (pp. 137-146).

Proteasome inhibitors PS-341 and MG-132 are capable of blocking mitochondrial translocation of cisplatin-induced apoptosis-inducing factor (AIF) in renal tubular epithelial cells. Green fluorescence is for AIF and blue staining is for DAPI.We demonstrate the effect of proteasome inhibitors in mitochondrial release of apoptosis-inducing factor (AIF) in cisplatin-exposed renal tubular epithelial cells (LLC-PK₁ cells) and in a model of cisplatin nephrotoxicity. Immunofluorescence and subcellular fractionation studies revealed cisplatin-induced translocation of AIF from the mitochondria to nucleus. Mcl-1, a pro-survival member of the Bcl-2 family, is rapidly eliminated on exposure of renal cells to cisplatin. Proteasome inhibitors PS-341 and MG-132 blocked cisplatin-induced Mcl-1 depletion and markedly prevented mitochondrial release of AIF. PS-341 and MG132 also blocked cisplatin-induced activation of executioner caspases and apoptosis. These studies suggest that proteasome inhibitors prevent cisplatin-induced caspase-dependent and -independent pathways. Overexpression of Mcl-1 was effective in blocking cisplatin-induced cytochrome c and AIF release from the mitochondria. Downregulation of Mcl-1 by small interfering RNA promoted Bax activation and cytochrome c and AIF release, suggesting that cisplatin-induced Mcl-1 depletion and associated Bax activation are involved in the release of AIF. Expression of AIF protein in the mouse was highest in the kidney compared to the heart, brain, intestine, liver, lung, muscle, and spleen. In an in vivo model of cisplatin nephrotoxicity, proteasome inhibitor MG-132 prevented mitochondrial release of AIF and markedly attenuated acute kidney injury as assessed by renal function and histology. These studies provide evidence for the first time that the proteasome inhibitors prevent cisplatin-induced mitochondrial release of AIF, provide cellular protection, and markedly ameliorate cisplatin-induced acute kidney injury. Thus, AIF is an important therapeutic target in cisplatin nephrotoxicity and cisplatin-induced depletion of Mcl-1 is an important pathway involved in AIF release.

Keywords: AIF; Mitochondria; Apoptosis; Mcl-1; Bax; PS-341; MG-132; Acute kidney injury

DNA repair pathways involved in repair of lesions induced by 5-fluorouracil and its active metabolite FdUMP by Renata Matuo; Fabrício Garmus Sousa; Alexandre E. Escargueil; Daniele G. Soares; Ivana Grivicich; Jenifer Saffi; Annette K. Larsen; João Antonio Pêgas Henriques (pp. 147-153).

A systematic study in Saccharomyces cerevisiae reveals important differences in the repair of DNA lesions induced by 5-flourouracil and its major metabolite, FdUMP.5-Fluorouracil (5-FU) is an antitumor antimetabolite that can be converted into fluoronucleotides and FdUMP. Fluoronucleotides are incorporated into DNA and RNA, while FdUMP results in nucleotide pool imbalance. Saccharomyces cerevisiae is unable to convert 5-FU into FdUMP, making yeast a unique model system to study the cellular effects of 5-FU and FdUMP independently. A panel of repair-deficient yeast strains was used to identify the DNA repair pathways needed for repair of lesions generated by 5-FU or FdUMP. This included yeast deficient in base excision repair (BER), nucleotide excision repair (NER), translesion synthesis (TLS), mismatch repair (MMR), post-replication repair (PRR), homologous recombination (HR) and non-homologous end-joining (NHEJ). The results revealed an important role of BER, since BER-mutants (ntg1, ntg2, apn1, apn2) showed pronounced sensitivity to both 5-FU and FdUMP. MMR mutants also showed high sensitivity to both compounds. In contrast, deficiencies in NER, NHEJ and TLS repair had only minor influence on the sensitivity to FU and FdUMP. Interestingly, deficiencies in HR (rad52) and PPR (rad6, rad18) were associated with increased sensitivity to 5-FU, but not to FdUMP. Taken together, our study reveals an important contribution of DNA repair pathways on the sensitivity to 5-FU and its active metabolite FdUMP. Importantly, the repair mechanisms differed for the 2 antimetabolites since lesions induced by 5-FU were repaired by BER, MMR, HR and PRR, while only BER and MMR were required for repair of FdUMP-induced lesions.

Keywords: 5-Fluorouracil; FdUMP; DNA repair; Saccharomyces cerevisiae

Lapatinib and erlotinib are potent reversal agents for MRP7 (ABCC10)-mediated multidrug resistance by Ye-Hong Kuang; Tong Shen; Xiang Chen; Kamlesh Sodani; Elizabeth Hopper-Borge; Amit K. Tiwari; Jeferson W.K.K. Lee; Li-Wu Fu; Zhe-Sheng Chen (pp. 154-161).

In recent years, a number of TKIs (tyrosine kinase inhibitors) targeting epidermal growth factor receptor (EGFR) family have been synthesized and some have been approved for clinical treatment of cancer by the FDA. We recently reported a new pharmacological action of the 4-anilinoquinazoline derived EGFR TKIs, such as lapatinib (Tykerb^®) and erlotinib (Tarceva^®), which significantly affect the drug resistance patterns in cells expressing the multidrug resistance (MDR) phenotype. Previously, we showed that lapatinib and erlotinib could inhibit the drug efflux function of P-glycoprotein (P-gp, ABCB1) and ABCG2 transporters. In this study, we determined if these TKIs have the potential to reverse MDR due to the presence of the multidrug resistance protein 7 (MRP7, ABCC10). Our results showed that lapatinib and erlotinib dose-dependently enhanced the sensitivity of MRP7-transfected HEK293 cells to several established MRP7 substrates, specifically docetaxel, paclitaxel, vinblastine and vinorelbine, whereas there was no or a less effect on the control vector transfected HEK293 cells. [³H]-paclitaxel accumulation and efflux studies demonstrated that lapatinib and erlotinib increased the intracellular accumulation of [³H]-paclitaxel and inhibited the efflux of [³H]-paclitaxel from MRP7-transfected cells but not in the control cell line. Lapatinib is a more potent inhibitor of MRP7 than erlotinib. In addition, the Western blot analysis revealed that both lapatinib and erlotinib did not significantly affect MRP7 expression. We conclude that the EGFR TKIs, lapatinib and erlotinib reverse MRP7-mediated MDR through inhibition of the drug efflux function, suggesting that an EGFR TKI based combinational therapy may be applicable for chemotherapeutic practice clinically.

Keywords: Abbreviations; TKI; tyrosine kinase inhibitor; EGFR; epidermal growth factor receptor; MDR; multidrug resistance; ABC; ATP-binding cassette; P-gp (ABCB1); P-glycoprotein; ABCG2; also called BCRP (breast cancer resistance protein)/MXR (mitoxantrone resistance protein); MRP7 (ABCC10); multidrug resistance protein 7; HER; human epidermal receptorTyrosine kinase inhibitor; EGFR; Lapatinib; Erlotinib; ABC transporters; MRP7/ABCC10; Multidrug resistance

Antroquinonol displays anticancer potential against human hepatocellular carcinoma cells: A crucial role of AMPK and mTOR pathways by Po-Cheng Chiang; Ssu-Chia Lin; Shiow-Lin Pan; Ching-Hua Kuo; I-Lin Tsai; Mao-Tien Kuo; Wu-Che Wen; Peini Chen; Jih-Hwa Guh (pp. 162-171).

5′AMP-activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR) are two serine/threonine protein kinases responsible for cellular energy homeostasis and translational control, respectively. Evidence suggests that these two kniases are potential targets for cancer chemotherapy against hepatocellular carcinoma (HCC). Antroquinonol that is isolated from Antrodia camphorate, a well-known Traditional Chinese Medicine for treatment of liver diseases, displayed effective anticancer activity against both HBV DNA-positive and -negative HCC cell lines. The rank order of potency against HCCs is HepG2>HepG2.2.15>Mahlavu>PLC/PRF/5>SK-Hep1>Hep3B. Antroquinonol completely abolished cell-cycle progression released from double-thymidine-block synchronization and caused a subsequent apoptosis. The data were supported by down-regulation and reduced nuclear translocation of G1-regulator proteins, including cyclin D1, cyclin E, Cdk4 and Cdk2. Further analysis showed that the mRNA expressions of the G1-regulator proteins were not modified by antroquinonol, indicating an inhibition of translational but not transcriptional levels. Antroquinonol induced the assembly of tuberous sclerosis complex (TSC)-1/TSC2, leading to the blockade of cellular protein synthesis through inhibition of protein phosphorylation including mTOR (Ser²⁴⁴⁸), p70^S6K (Thr⁴²¹/Ser⁴²⁴ and Thr³⁸⁹) and 4E-BP1 (Thr³⁷/Thr⁴⁶ and Thr⁷⁰). Furthermore, the AMPK activity was elevated by antroquinonol. Compound C, a selective AMPK inhibitor, significantly reversed antroquinonol-mediated effects suggesting the crucial role of AMPK. Besides, the loss of mitochondrial membrane potential and depletion of mitochondrial content indicated the mitochondrial stress caused by antroquinonol. In summary, the data suggest that antroquinonol displays anticancer activity against HCCs through AMPK activation and inhibition of mTOR translational pathway, leading to G1 arrest of the cell-cycle and subsequent cell apoptosis.

Keywords: Antroquinonol; Hepatocellular carcinoma; AMPK; mTOR; G1 arrest

Cleaved high molecular weight kininogen, a novel factor in the regulation of matrix metalloproteinases in vascular smooth muscle cells by Uwe Vosgerau; Diljara Lauer; Thomas Unger; Elena Kaschina (pp. 172-179).

We previously reported that Brown Norway Katholiek rats, which feature a deficiency of plasma kininogens, develop severe abdominal aortic aneurysm. Increased activity of matrix metalloproteinases (MMPs) in the aortic wall, leading to degradation of extracellular matrix components, is considered to play a crucial role in aneurysm formation. Using an in vitro model of vascular smooth muscle cells (VSMCs), cultured from the rat aorta, we investigated whether the cleaved form of high molecular weight kininogen, designated HKa, affects the expression of MMP-9 and MMP-2 and their tissue inhibitors (TIMPs). Treatment of VSMCs with HKa reduced in a concentration-dependent manner IL-1α-induced release of MMP-9 and MMP-2, associated with decreased MMP enzymatic activity levels in conditioned media, as demonstrated by gelatin zymography and fluorescein-labeled gelatin substrate assay, respectively. Real-time PCR revealed that HKa reduced corresponding MMP-9 mRNA levels. Further investigations showed that this effect did not result from a modified rate of MMP-9 mRNA degradation. TIMP-1 mRNA levels, already increased as a result of cytokine-stimulation, were significantly enhanced by HKa. Furthermore, we found elevated basal mRNA expression levels of MMP-2 and TIMP-2 in VSMCs derived from kininogen-deficient Brown Norway Katholiek rats. These results demonstrate for the first time that HKa affects the regulation of MMPs in VSMCs.

Keywords: Kininogen; Matrix metalloproteinases; Tissue inhibitors of MMPs; Smooth muscle cells; Vascular biology

Adenosine A₃ receptor-mediated cardioprotection against doxorubicin-induced mitochondrial damage by Avishag K. Emanuelov; Asher Shainberg; Yelena Chepurko; Doron Kaplan; Alex Sagie; Eyal Porat; Michael Arad; Edith Hochhauser (pp. 180-187).

Doxorubicin (DOX) causes cardiotoxicity which is accompanied by [Ca²⁺]_i release from the sarcoplasmic reticulum (SR). By activating SERCA2a, the A₃R agonist, Cl-IB-MECA, leads to the restoration of Ca²⁺ homeostasis, thus attenuating DOX cardiotoxicity.Cardiotoxicity associated with doxorubicin (DOX) treatment limits the therapeutic efficiency of this drug against cancer. 2-Chloro-N(6)-(3-iodobenzyl)adenosine-5′-N-methyluronamide (Cl-IB-MECA), a selective agonist of A₃ adenosine receptor (A₃R), reduces DOX toxicity in newborn rat cultured cardiomyocytes. The study's aim was to determine whether the protection demonstrated by Cl-IB-MECA attenuates cardiac depression in vivo. In addition, we wished to examine whether this protective pathway affects the sarcoplasmic reticulum (SR) calcium uptake and release, as well as intramitochondrial Ca²⁺ accumulation induced by DOX.Rats were injected every alternate day (6 times) with (1) saline, (2) 2.5mg/kg i.p. DOX, (3) 33μg/kg i.v. Cl-IB-MECA, (4) DOX+Cl-IB-MECA. Left ventricular functions were assessed by invasive (pressure) and non-invasive (echocardiography) techniques at the end of the injection period and 4 weeks later. Cytosolic and intramitochondrial calcium levels were measured with indo-1 and rhod-2 probes. SR Ca²⁺ content was determined by exposing cultured rat cardiomyocytes to caffeine.Echocardiography data demonstrate left ventricular wall thinning (23%), an increase in the end systolic dimension (170%) and decreased fractional shortening (35±5% vs. 54±5%, p<0.01) in DOX-treated animals, compared to the control group. DOX increased Ca²⁺ levels in the cytosol and in mitochondria by diminishing the SR Ca²⁺ uptake. Pretreatment with Cl-IB-MECA attenuated left ventricular dysfunction, improved SR calcium storage capacity and prevented mitochondrial Ca²⁺ overload.We conclude that the adenosine A₃ receptor agonist is effective in vivo against DOX cardiotoxicity via the restoration of Ca²⁺ homeostasis and prevention of mitochondrial damage that occurs as a result of Ca²⁺ overload.

Keywords: A; ₃; adenosine receptors; Ca; ²⁺; homeostasis; Cl-IB-MECA; Doxorubicin

A novel immunomodulatory mechanism of ribavirin in suppressing natural killer cell function by Henry Ogbomo; Martin Michaelis; Behric Altenbrandt; Hans Wilhelm Doerr; Jindrich Cinatl Jr. (pp. 188-197).

Ribavirin, a broad-spectrum anti-viral drug, exhibits immunomodulatory activities. To study direct effects of ribavirin on natural killer (NK) cell effector functions and signaling, resting NK cells and interleukin (IL)-15-activated NK cells were treated for 5 days with therapeutic ribavirin concentrations ranging from 5μg/ml to 20μg/ml. Both resting and IL-15-activated NK cells that were not treated with ribavirin were used as control. Cytotoxicity assays, flow cytometry, enzyme linked immunosorbent assays, and Western blot experiments were performed to elucidate ribavirin effect on NK cells. Results showed that ribavirin (not toxic at concentrations tested; IC₅₀>80μg/ml) had no influence on lysis of target cells by freshly isolated NK cells. Conversely, ribavirin dose-dependently inhibited lysis of target cells by up to 66% and impaired interferon gamma production when IL-15-activated NK cells were used. IL-15-induced increased expression and hence function of NK cell activating receptors including NKp30, NKp44, NKp46 and NKG2D were selectively down-regulated and impaired. These inhibitory effects were associated with the down-regulation of IL-15 receptor beta and gamma expression. Accordingly, downstream events involved in NK cell signaling via IL-15 receptors including the activation of Janus kinase (Jak)-1, signal transducer and activator of transcription STAT-1, STAT-3, and STAT-5 as well as pathways responsible for NK cell degranulation including extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) were impaired. These results reveal a novel mechanism by which ribavirin exerts its immunomodulatory activities.

Keywords: NK cell activating receptors; NK cell signaling; NK cell degranulation; Perforin and granzyme B release; IL-15 receptors

Beneficial actions of oleanolic acid in an experimental model of multiple sclerosis: A potential therapeutic role by Rubén Martín; Juliana Carvalho-Tavares; Marita Hernández; Mercedes Arnés; Valentina Ruiz-Gutiérrez; María Luisa Nieto (pp. 198-208).

Semiprofilactic and therapeutic administration of the natural triterpene, oleanolic acid, can ameliorate the clinical signs of the experimental autoimmune encephalomyelitis.Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease for which there exist no therapies without undesired side effects. Thus, the establishment of less toxic treatments is an ongoing challenge. Nowadays, research on medicinal plants has been attracting much attention, since screening of its active principles could prove useful in identification of safe and innovative pharmaceutical molecules. In this study we investigated the therapeutic effect of oleanolic acid (OA) a plant-derived triterpene with potent anti-inflammatory and immunomodulatory activities, whose actions on CNS diseases remain far from completely characterized. We focussed on the potential therapeutic effect of oleanolic acid (OA) on an accepted experimental model of MS, the experimental autoimmune encephalomyelitis (EAE). We have found that OA treatment, before or at the early onset of EAE, ameliorates neurological signs of EAE-mice. These beneficial effects of OA seem to be associated with a reduction of blood–brain barrier leakage and lower infiltration of inflammatory cells within the CNS, as well as with its modulatory role in Th1/Th2 polarization: inhibition of proinflammatory cytokines and chemokines, and stimulation of anti-inflammatory ones. Moreover, EAE-animals that were treated with OA had lower levels of anti-MOG antibodies than untreated EAE-mice.Our findings show that the administration of the natural triterpenoid OA reduces and limits the severity and development of EAE. Therefore, OA therapy might be of clinical interest for human MS and other Th1 cell-mediated inflammatory diseases.

Keywords: Encephalomyelitis; Neuroimmunology; Inflammation; Pharmacology; Triterpenes

Involvement of matrix metalloproteinase-3 in CCL5/CCR5 pathway of chondrosarcomas metastasis by Chih-Hsin Tang; Ayaho Yamamoto; Yuh-Tzy Lin; Yi-Chin Fong; Tzu-Wei Tan (pp. 209-217).

CCL5 (previously called RANTES) was originally recognized as a product of activated T cells, and plays a crucial role in the migration and metastasis of human cancer cells. It has been reported that the effect of CCL5 is mediated via CCR receptors. We found that human chondrosarcoma tissues had significant expression of the CCL5 and CCR5, which was higher than that in normal cartilage. We also found CCL5 increased the migration and matrix metalloproteinases-3 (MMP)-3 expression in human chondrosarcoma cells (JJ012 cells). In addition, MMP-3 small interfering RNA and inhibitor inhibited the CCL5-induced cell migration. Activations of phosphatidylinositol 3-kinase (PI3K), Akt and NF-κB pathways after CCL5 treatment was demonstrated, and CCL5-induced expression of MMP-3 and migration activity was inhibited by the specific inhibitor of PI3K, Akt and NF-κB cascades. Taken together, these results indicate that CCL5 and CCR5 interaction enhanced migration of chondrosarcoma cells through the increase of MMP-3 production.

Keywords: CCL5; Migration; CCR5; Chondrosarcoma; Akt

Curcumin potentiates the antitumor effects of gemcitabine in an orthotopic model of human bladder cancer through suppression of proliferative and angiogenic biomarkers by Sheeja T. Tharakan; Teruo Inamoto; Bokyung Sung; Bharat B. Aggarwal; Ashish M. Kamat (pp. 218-228).

Curcumin, a component of turmeric exhibits significant antitumor effects against human bladder cancer and further potentiates the effects of gemcitabine through the modulation of inflammatory transcription factor NF-κB, and gene products linked to survival, proliferation and metastasis of the cancer.Little progress has been made in the last three decades in the treatment of bladder cancer. Novel agents that are nontoxic and can improve the current standard of care of this disease are urgently needed. Curcumin, a component of Curcuma longa (also called turmeric), is one such agent that has been shown to suppress pathways linked to oncogenesis, including cell survival, proliferation, invasion and angiogenesis. We investigated whether curcumin has potential to improve the current therapy for bladder cancer, using an orthotopic mouse model. Curcumin potentiated the apoptotic effects of gemcitabine against human bladder cancer 253JBV cells in culture. Electrophoretic mobility shift assay revealed that curcumin also suppressed the gemcitabine-induced activation of the cell survival transcription factor NF-κB. In an orthotopic mouse model, bioluminescence imaging revealed that while curcumin alone significantly reduced the bladder tumor volume, maximum reduction was observed when curcumin was used in combination with gemcitabine ( P<0.01 versus vehicle; P<0.01 versus gemcitabine alone). Curcumin also significantly decreased the proliferation marker Ki-67 and microvessel density (CD31) ( P<0.01 versus vehicle; P<0.01 versus gemcitabine alone), but maximum reduction occurred when it was combined with gemcitabine ( P<0.01 versus vehicle; P<0.01versus gemcitabine alone). Curcumin abolished the constitutive activation of NF-κB in the tumor tissue; induced apoptosis, and decreased cyclin D1, VEGF, COX-2, c-myc and Bcl-2 expression in the bladder cancer tissue. Overall our results suggest that curcumin alone exhibits significant antitumor effects against human bladder cancer and it further potentiates the effects of gemictabine, possibly through the modulation of NF-κB signaling pathway.

Keywords: Curcumin; Bladder cancer; NF-κB; Gemcitabine

Novel immunomodulatory properties of cirsilineol through selective inhibition of IFN-γ signaling in a murine model of inflammatory bowel disease by Yang Sun; Xing-Xin Wu; Ye Yin; Fang-Yuan Gong; Yan Shen; Tian-Tian Cai; Xiao-Bin Zhou; Xue-Feng Wu; Qiang Xu (pp. 229-238).

Cirsilineol, a natural flavone compound, attenuates TNBS-induced experimental colitis through selective inhibiting IFN-γ/STAT1/T-bet signaling in intestinal CD4⁺ T cells.Regulation of signal transducer and activator of transcription (STAT) 1 signaling is being explored as a new approach to the treatment of inflammatory bowel diseases. However, few chemicals have been reported to inhibit IFN-γ/STAT1 signaling for Crohn's disease therapy. In the present study, we found that cirsilineol, a small natural compound isolated from Artemisia vestita, significantly ameliorated trinitro-benzene sulfonic acid (TNBS)-induced T-cell-mediated experimental colitis in mice, which was closely associated with reduced autoreactive T-cell proliferation and activation. Moreover, the regulatory action of pro-inflammatory and anti-inflammatory cytokine by cirsilineol treatment was found to decrease the activity of effector Th1 cells but increase the activity of regulatory T cells as characterized by down-regulation of IFN-γ and corresponding up-regulation of IL-10 and TGF-β. The therapeutic effect of cirsilineol was attributable to a novel regulatory mechanism with selective inhibiting IFN-γ signaling in colonic lamina propria CD4⁺ T cells, which was mediated through down-regulating STAT1 activation and T-bet expression. Furthermore, cirsilineol was found to down-regulate the activation of JAK2, a critical kinase for IFN-γ/STAT1 signaling, and abrogate the expression of T-bet, resulting in markedly decreased proliferation and activation of T cells in vitro. Importantly, the inhibition of IFN-γ/STAT1 signaling by cirsilineol was reversible in the presence of high level of IFN-γ. These results strongly suggest that cirsilineol might be potentially useful for treating T-cell-mediated human inflammatory bowel diseases.

Keywords: Inflammatory bowel diseases; TNBS-induced colitis; IFN-γ; STAT1; Cirsilineol

Mitochondria and calcium flux as targets of neuroprotection caused by minocycline in cerebellar granule cells by Eva Maria Garcia-Martinez; Sara Sanz-Blasco; Andonis Karachitos; Manuel J. Bandez; Francisco J. Fernandez-Gomez; Sergio Perez-Alvarez; Raquel Maria Melero Fernandez de Mera; Maria J. Jordan; Norberto Aguirre; Maria F. Galindo; Carlos Villalobos; Ana Navarro; Hanna Kmita; Joaquín Jordán (pp. 239-250).

Minocycline, an antibiotic of the tetracycline family, has attracted considerable interest for its theoretical therapeutic applications in neurodegenerative diseases. However, the mechanism of action underlying its effect remains elusive. Here we have studied the effect of minocycline under excitotoxic conditions. Fluorescence and bioluminescence imaging studies in rat cerebellar granular neuron cultures using fura2/AM and mitochondria-targeted aequorin revealed that minocycline, at concentrations higher than those shown to block inflammation and inflammation-induced neuronal death, inhibited NMDA-induced cytosolic and mitochondrial rises in Ca²⁺ concentrations in a reversible manner. Moreover, minocycline added in the course of NMDA stimulation decreased Ca²⁺ intracellular levels, but not when induced by depolarization with a high K⁺ medium. We also found that minocycline, at the same concentrations, partially depolarized mitochondria by about 5–30mV, prevented mitochondrial Ca²⁺ uptake under conditions of environmental stress, and abrogated NMDA-induced reactive oxygen species (ROS) formation. Consistently, minocycline also abrogates the rise in ROS induced by 75μM Ca²⁺ in isolated brain mitochondria. In search for the mechanism of mitochondrial depolarization, we found that minocycline markedly inhibited state 3 respiration of rat brain mitochondria, although distinctly increased oxygen uptake in state 4. Minocycline inhibited NADH–cytochrome c reductase and cytochrome c oxidase activities, whereas the activity of succinate–cytochrome c reductase was not modified, suggesting selective inhibition of complexes I and IV. Finally, minocycline affected activity of voltage-dependent anion channel (VDAC) as determined in the reconstituted system. Taken together, our results indicate that mitochondria are a critical factor in minocycline-mediated neuroprotection.

Keywords: Anti-oxidant; Apoptosis; Voltage-dependent anion channel; Uncoupler; Aequorin; BioluminescenceAbbreviations; DPPH; 2,2-diphenyl-1-picrylhydrazyl; TPTZ; 2,4,6-tripyridyl-; S; -triazine; CM-H; ₂; DCFDA; 5-(and 6-)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate; ANT; adenine nucleotide translocase; ALS; amyotrophic lateral sclerosis; FCCP; carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone; FRAP; ferric reducing ability of plasma; [Ca; ²⁺; ]; _cyt; cytosolic Ca; ²⁺; concentrations; DFCA; fluorescein diacetate; GFP; green fluorescence protein; Het; hydroethidine; [Ca; ²⁺; ]; _m; mitochondrial Ca; ²⁺; concentrations; Δ; Ψ; m; mitochondrial inner membrane potential; MPTP; mitochondrial permeability transition pore; NMDA; N; -methyl-; d; -aspartic acid; ROI; region of interest; ROS; reactive oxygen species; O; ₂; ⁻; superoxide anion radical; TMRM; tetramethylrhodamine methyl ester; VDAC; voltage-dependent anion channel

Metabolism and biological production of resolvins derived from docosapentaenoic acid (DPAn-6) by Bindi Dangi; Marcus Obeng; Julie M. Nauroth; Gloria Chung; Eileen Bailey-Hall; Todd Hallenbeck; Linda M. Arterburn (pp. 251-260).

17S-HDPAn-6 and 10S,17S-HDPAn-6 are resolvins produced from DPAn-6 by the action of lipoxygenase. Analysis of the metabolic stability and identification of metabolites of these compounds could play an important role in the design of better analogs with longer durations of action and hence better efficacy.17S-HDPAn-6 (17S-hydroxydocosa-4 Z,7 Z,10 Z,13 Z,15 E-pentaenoic acid) and 10S,17S-HDPAn-6 (10S,17S-dihydroxydocosa-4 Z,7 Z,11 E,13 Z,15 E-pentaenoic acid) are potent anti-inflammatory resolvins derived from DPAn-6 (docosapentaenoic acid n-6) and are analogous in structure and action to DHA (docosahexaenoic acid)-derived resolvins. These resolvins have proven to be potential drug candidates, albeit with therapeutic profiles that need optimization. The main objectives of this study were to evaluate key features of DPAn-6 derived resolvins that are important for therapeutic efficacy, demonstrate that these DPAn-6 resolvins could be produced naturally, and could therefore have physiological significance. Here we demonstrate biological production, examine pharmacokinetic profiles and identify key routes of metabolic inactivation of DPAn-6 derived resolvins. We compare their metabolic stability to a known resolvin, 17S-HDHA (17S-hydroxydocosa-4 Z,7 Z,10 Z,13 Z,15 E,19Z-hexaenoic acid) and show that order of their stabilities is 10S,17S-HDPAn-6>17S-HDPAn-6>17S-HDHA. We show that both these compounds are not strong inhibitors of cytochrome-P450 enzymes. We evaluate activity of compounds in the delayed-type hypersensitivity model, results of which show that compounds need optimization for enhanced duration and magnitude of action. Analysis of the metabolic stability and identification of metabolites of these compounds could play an important role in the design of better analogs with longer durations of action and hence better efficacy.

Keywords: Abbreviations; 14:0; myristic acid; 15:0; pentadecanoic acid; 16:0; palmitic acid; 17:0; heptadecanoic acid; 18:0; stearic acid; 20:0; arachidic acid; 22:0; behenic acid; 24:0; lignoceric acid; 14:1 n-5; myristoleic acid; 16:1 n-7; palmitoleic acid; 18:1n-7; cis-vaccenic acid; 18:1n-9; oleic acid; 20:1n-9; gondoic acid; 22:1 n-9; erucic acid; 24:1 n-9; nervonic acid; 18:3n-3; α-linolenic acid; 20:5n-3; eicosapentaenoic acid; 22:5n-3; docosapentaenoic acid n-3; 22:6n-3; docosahexaenoic acid; 18:2n-6; linoleic acid; 18:3n-6; α-linolenic acid; 20:2n-6; eicosadienoic acid; 20:3n-6; dihomogamma-linolenic acid; 20:4n-6; arachidonic acid; 22:4n-6; adrenic acid; 22:5n-6; docosapentaenoic acid n-6; CYP; cytochrome P450; FAME; fatty acid methyl ester; LC-PUFA; long-chain polyunsaturated fatty acids; LOD; limit of detection; NPD1; neuroprotectin D1; RVE1; resolvin E1; 15-PGDH; 15-prostaglandin dehydrogenase; PK; pharmacokinetic/s; SRM; selective reaction monitoring; 17S-HDPAn-6; 17S-hydroxydocosa-4; Z,; 7; Z,; 10; Z,; 13; Z,; 15; E; -pentaenoic acid; 10S,17S-HDPAn-6; 10S,17S-dihydroxydocosa-4; Z,; 7; Z,; 11; E,; 13; Z,; 15; E; -pentaenoic acid; 17S-HDHA; 17S-hydroxydocosa-4; Z,; 7; Z,; 10; Z,; 13; Z,; 15; E,19Z; -hexaenoic acid; 10S,17S-HDHA; 10S*17S-dihydroxydocosa-4Z,7Z,11E,13Z,15E, 19Z-hexaenoic acid; LLQ; lower limit of quantitationOxylipin; Anti-inflammatory; Metabolite; Pharmacokinetics

Constitutive androstane receptor transcriptionally activates human CYP1A1 and CYP1A2 genes through a common regulatory element in the 5′-flanking region by Kouichi Yoshinari; Noriaki Yoda; Takayoshi Toriyabe; Yasushi Yamazoe (pp. 261-269).

Phenobarbital has long been known to increase cellular levels of CYP1A1 and CYP1A2 possibly through a pathway(s) independent of aryl hydrocarbon receptor. We have investigated the role of constitutive androstane receptor (CAR), a xenobiotic-responsive nuclear receptor, in the transactivation of human CYP1A1 and CYP1A2. These genes are located in a head-to-head orientation, sharing a 5′-flanking region. Reporter assays were thus performed with dual-reporter constructs, containing the whole or partially deleted human CYP1A promoter between two different reporter genes. In this system, human CAR (hCAR) enhanced the transcription of both genes through common promoter regions from −461 to −554 and from −18089 to −21975 of CYP1A1. With reporter assays using additional deleted and mutated constructs, electrophoresis mobility shift assays and chromatin immunoprecipitation assays, an ER8 motif (everted repeat separated by eight nucleotides), located at around −520 of CYP1A1, was identified as an hCAR-responsive element and a binding motif of hCAR/human retinoid X receptor α heterodimer. hCAR enhanced the transcription of both genes also in the presence of an aryl hydrocarbon receptor ligand. Finally, hCAR activation increased CYP1A1 and CYP1A2 mRNA levels in cultured human hepatocytes. Our results indicate that CAR transactivates human CYP1A1 and CYP1A2 in human hepatocytes through the common cis-element ER8. Interestingly, the ER8 motif is highly conserved in the CYP1A1 proximal promoter sequences of various species, suggesting a fundamental role of CAR in the xenobiotic-induced expression of CYP1A1 and CYP1A2 independent of aryl hydrocarbon receptor.

Keywords: Abbreviations; AhR; aryl hydrocarbon receptor; XRE; xenobiotic-responsive element; CYP; cytochrome P450; CAR; constitutive androstane receptor; RXRα; retinoid X receptor α; h; human; PB; phenobarbital; DMEM; Dulbecco's modified Eagle's medium; FBS; fetal bovine serum; CITCO; 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde; O; -(3,4-dichlorobenzyl)oxime; PCR; polymerase chain reaction; SEAP; secreted alkaline phosphatase; Luc; luciferase; EMSA; electrophoretic mobility shift assay; ChIP; chromatin immunoprecipitation; ANOVA; analysis of varianceCYP induction; Nuclear receptor; HepG2 cell; In vitro reporter assay; Aryl hydrocarbon receptor

Retinoids activate RXR/CAR-mediated pathway and induce CYP3A by Shiyong Chen; Kun Wang; Yu-Jui Yvonne Wan (pp. 270-276).

Retinoids and carotenoids are frequently used as antioxidants to prevent cancer. In this study, a panel of retinoids and carotenoids was examined to determine their effects on activation of RXR/CAR-mediated pathway and regulation of CYP3A gene expression. Transient transfection assays of HepG2 cells revealed that five out of thirteen studied retinoids significantly induced RXRα/CAR-mediated activation of luciferase activity that is driven by the thymidine kinase promoter linked with a PXR binding site in the CYP3A4 gene [tk-(3A4)₃-Luc reporter]. All-trans retinoic acid (RA) and 9-cis RA were more effective than CAR agonist TCBOPOP in induction of the tk-(3A4)₃-Luc reporter. Addition of retinoid and TCBOPOP further enhanced the inducibility and the induction was preferentially mediated by RXRα/CAR and RXRγ/CAR heterodimer. Chromatin immunoprecipitation assay showed that retinoids recruit RXRα and CAR to the proximal ER6 and distal XREM nuclear receptor response elements of the CYP3A4 gene promoter. The experimental data demonstrate that retinoids can effectively regulate CYP3A gene expression through the RXR/CAR-mediated pathway.

Keywords: Abbreviations; ALHD; aldehyde dehydrogenases; CYP; cytochrome P450; RA; retinoic acid; All-trans RP; all-trans retinol palmitate; PXR; pregnane X receptor; CAR; constitutive androstane receptor; RXR; retinoid X receptor; mCAR; mouse constitutive androstane receptor; mRXR; mouse retinoid X receptor; ER; everted repeat; DR; direct repeat; NR; nuclear receptor; NR1, NR2; nuclear receptor site1, 2; XREM; xenobiotic-responsive enhancer module; RT; reverse transcription; Gadph; glyceraldehyde-3-phosphate dehydrogenase; TCPOBOP; 1,4-bis[2-(3,5-dichloropyridyloxy)]benzeneRXR; CAR; Retinoid; CYP3A; Nuclear receptor

Intestinal cell-specific vitamin D receptor (VDR)-mediated transcriptional regulation of CYP3A4 gene by Petr Pavek; Katerina Pospechova; Lucie Svecova; Zdenka Syrova; Lucie Stejskalova; Jana Blazkova; Zdenek Dvorak; Jaroslav Blahos (pp. 277-287).

CYP3A4 is the most important drug-metabolizing enzyme that is involved in biotransformation of more than 50% of drugs. Pregnane X receptor (PXR) dominantly controls CYP3A4 inducibility in the liver, whereas vitamin D receptor (VDR) transactivates CYP3A4 in the intestine by secondary bile acids. Four major functional PXR-binding response elements of CYP3A4 have been discovered and their cooperation was found to be crucial for maximal up-regulation of the gene in hepatocytes. VDR and PXR recognize similar response element motifs and share DR3(XREM) and proximal ER6 (prER6) response elements of the CYP3A4 gene.In this work, we tested whether the recently discovered PXR response elements DR4(eNR3A4) in the XREM module and the distal ER6 element in the CLEM4 module (CLEM4-ER6) bind VDR/RXRα heterodimer, whether the elements are involved in the intestinal transactivation, and whether their cooperation with other elements is essential for maximal intestinal expression of CYP3A4.Employing a series of gene reporter plasmids with various combinations of response element mutations transiently transfected into four intestinal cell lines, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP), we found that the CLEM4-ER6 motif interacts with VDR/RXRα heterodimer and partially cooperates with DR3(XREM) and prER6 in both basal and VDR-mediated inducible CYP3A4 regulation in intestinal cells. In contrast, eNR3A4 is involved only in the basal transactivation in intestinal cells and in the PXR-mediated rifampicin-induced transactivation of CYP3A4 in LS174T intestinal cells.We thus describe a specific ligand-induced VDR-mediated transactivation of the CYP3A4 gene in intestinal cells that differs from PXR-mediated CYP3A4 regulation in hepatocytes.

Keywords: Abbreviations; eNR3A4; essential distal nuclear receptor-binding element for CYP3A4 induction; ER6; everted repeat separated by six nucleotides; prER6; ER6 in the CYP3A4 proximal promoter; DR4; direct repeat separated by four nucleotides; PXR; pregnane X receptor; RE; response element; qRT-PCR; quantitative real-time reverse transcriptase polymerase chain reaction; RXRα; retinoid X receptor α (9-cis retinoic acid receptor-α); VDR; vitamin D receptor; XREM; xenobiotic responsive enhancer module; CLEM4; constitutive liver enhancer module of CYP3A4; 1,25OHvitD; ₃; 1α,25-dihydroxyvitamin D; ₃; LCA; lithocholic acid; CDCA; chenodeoxycholic acid; EMSA; electrophoretic mobility shift assay; ChIP; chromatin immunoprecipitationPharmacokinetics; Metabolism; Cytochrome P450; CYP3A4; Transcriptional regulation; Nuclear receptors

Transcriptional regulation of human carboxylesterase 1A1 by nuclear factor-erythroid 2 related factor 2 (Nrf2) by Taiga Maruichi; Tatsuki Fukami; Miki Nakajima; Tsuyoshi Yokoi (pp. 288-295).

Human CES1A1 is transactivated through binding of Nrf2 to the antioxidant response element at −2025bp.Human carboxylesterase (CES) 1A, which is predominantly expressed in liver and lung, plays an important role in the hydrolysis of endogenous compounds and xenobiotics. CES1A is reported to be induced in human hepatocytes by butylated hydroxyanisole, ticlopidine and diclofenac, and the induction is assumed to be caused by oxidative stress. However, the molecular mechanism remains to be determined. In this study, we sought to investigate whether CES1A is regulated by nuclear factor-erythroid 2 related factor 2 (Nrf2), which is a transcriptional factor activated by oxidative stress, and clarify the molecular mechanism. Real-time reverse transcription-PCR assays revealed that CES1A1 mRNA was significantly induced by tert-butylhydroquinone (tBHQ) and sulforaphane (SFN), which are representative activators of Nrf2 in HepG2, Caco-2 and HeLa cells. The induction was completely suppressed with small interfering RNA for Nrf2. In HepG2 cells, the CES1A protein level and imidapril hydrolase activity, which is specifically catalyzed by CES1A, were also significantly induced by tBHQ and SFN. Luciferase assays revealed that the antioxidant response element (ARE) at −2025 in the CES1A1 gene was responsible for the transactivation by Nrf2. In addition, electrophoretic mobility shift assays and chromatin immunoprecipitation assays revealed that Nrf2 binds to the ARE in the CES1A1 gene. These findings clearly demonstrated that human CES1A1 is induced by Nrf2. This is the first study to demonstrate the molecular mechanism of the inducible regulation of human CES1A1.

Keywords: Carboxylesterase; Gene regulation; Nuclear factor-erythroid 2 related factor 2

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Biochemical Pharmacology (v.79, #2)