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Biochemical Pharmacology (v.84, #1)

Editorial Advisory Board (pp. iii).

Searching for molecular targets in sarcoma by Beverly A. Teicher (pp. 1-10).

Sarcoma are about 1% of cancers. Within that 1% are widely varied tumors now divided into types and subtypes. Sarcoma occur in patients of all ages with frequency spread evenly over the human age range. Although the specific cell of origin of many sarcoma remains unclear, sarcoma are all tumors of mesenchymal origin. The mesenchymal stem cell, a pluripotent cell, which gives rise to varied differentiated cells including osteocytes, adipocytes, chondrocytes, muscle cells, fibroblasts, neural cells and stromal cells, is the most likely ultimate cell of origin for sarcoma. When mesenchymal stem cell genetics go awry and malignant transformation occurs sarcoma including osteosarcoma, Ewing's sarcoma, chondrosarcoma, rhabdomyosarcoma, synovial sarcoma fibrosarcoma, liposarcoma and many others can initiate. Our knowledge of sarcoma genetics is increasing rapidly. Two general groups, sarcoma arising from chromosomal translocations and sarcoma with very complex genetics, can be identified. Genes that are frequently mutated in sarcoma include TP53, NF1, PIK3CA, HDAC1, IDH1 and 2, KDR, KIT and MED12. Genes that are frequently amplified in sarcoma include CDK4, YEATS4, HMGA2, MDM2, JUN, DNM3, FLT4, MYCN, MAP3K5, GLI1 and the microRNAs miR-214 and miR-199a2. Genes that are upregulated in sarcoma include MUC4, CD24, FOXL1, ANGPTL2, HIF1α, MDK, cMET, TIMP-2, PRL, PCSK1, IGFR-1, TIE1, KDR, TEK, FLT1 and several microRNAs. While some alterations occur in specific subtypes of sarcoma, others cross several sarcoma types. Discovering and developing new therapeutic approaches for these relentless diseases is critical. The detailed knowledge of sarcoma genetics may allow development of sarcoma subtype-targeted therapeutics.

Keywords: Sarcoma; Gene expression; Ewing's sarcoma; Rhabdomyosarcoma; Osteosarcoma liposarcoma

Searching for molecular targets in sarcoma by Beverly A. Teicher (pp. 1-10).

Keywords: Sarcoma; Gene expression; Ewing's sarcoma; Rhabdomyosarcoma; Osteosarcoma liposarcoma

Complex roles of members of the ADP-ribosyl transferase super family in immune defences: Looking beyond PARP1 by Iain Welsby; David Hutin; Oberdan Leo (pp. 11-20).

ADP ribosylation has been recently recognised as an important posttranslational modification regulating numerous cellular processes. This enzymatic activity is shared by two major families of enzymes, the extracellular ADP-ribosyl-transferases, or ecto-ARTS and the poly-ADP-ribosyltranferases, whose denomination derives from the capacity of its founding member, PARP1, to synthesise large linear or branched polymers of ADP-ribose on target proteins. This latter post-translational modification has recently attracted much interest based on its role in the cellular response to genotoxic and oxidative stress. Accordingly, a series of PARP-specific pharmacological inhibitors have demonstrated cell survival and anti-inflammatory properties in vivo, promoting a renewed interest in the potential immunoregulatory role of this gene family. More recently, the role of ADP-ribosylation in regulating several aspects of intracellular signalling and gene transcription has been uncovered, in particular within cells of the immune system, revealing the potential immunomodulatory role of several members of this family in addition to PARP1. We review herein the experimental evidence illustrating the complex role played by this gene family in regulating multiple aspects of the immune response, including cell survival, cytokine gene transcription and antiviral innate defences. In particular, the unexpected potential anti-inflammatory role of members of this family (including in particular PARP5a, 5b and PARP14) will be briefly discussed, raising some concern on the use of pan-specific PARP inhibitors to treat chronic inflammatory diseases.

Keywords: ADP-ribosyl transferases (ARTs); Poly(ADP-ribose) polymerases (PARPs); Inflammation; Immunomodulation

Complex roles of members of the ADP-ribosyl transferase super family in immune defences: Looking beyond PARP1 by Iain Welsby; David Hutin; Oberdan Leo (pp. 11-20).

Keywords: ADP-ribosyl transferases (ARTs); Poly(ADP-ribose) polymerases (PARPs); Inflammation; Immunomodulation

Discovery of novel A₃ adenosine receptor ligands based on chromone scaffold by Alexandra Gaspar; Joana Reis; Sonja Kachler; Silvia Paoletta; Eugenio Uriarte; Karl-Norbert Klotz; Stefano Moro; Fernanda Borges (pp. 21-29).

Discovery of new chemical entities (NCEs) as potential A₃AR ligands that incorporate a benzo-γ-pyrone substructure.A project focused on the discovery of new chemical entities (NCEs) as AR ligands that incorporate a benzo-γ-pyrone [(4H)-1-benzopyran-4-one] substructure has been developed. Accordingly, two series of novel chromone carboxamides placed at positions C2 (compounds2–13) and C3 (compounds15–26) of the γ-pyrone ring were synthesized using chromone carboxylic acids (compounds1 or14) as starting materials. From this study and on the basis of the obtained structure–activity relationships it was concluded that the chromone carboxamide scaffold represent a novel class of AR ligands. The most remarkable chromones were compounds21 and26 that present a better affinity for A₃AR ( K_i=3680nM and K_i=3750nM, respectively). Receptor-driven molecular modeling studies provide information on the binding/selectivity data of the chromone. The data so far acquired are instrumental for future optimization of chromone carboxamide as a selective A₃AR antagonist.

Keywords: Abbreviations; [; ³; H]CCPA; [; ³; H](2R,3R,4S,5R)-2-[2-chloro-6-(cyclopentylamino)purin-9-yl]-5-(hydroxymethyl)oxolane-3,4-diol; [; ³; H]HEMADO; [; ³; H] 2-(1-Hexynyl)-; N; ⁶; -methyladenosine; [; ³; H]NECA; [; ³; H]adenosine-5′-(; N; -ethylcarboxamide); AR; Adenosine receptor; Arg; arginine; Asn; asparagine; Asp; aspartic acid; BBr; ₃; boron tribromide; BOP; (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; CHO; Chinese hamster ovary cells; Cys; cysteine; DAD; diode-array detector; DIPEA; N; ,; N; -diisopropylethylamine; DMF; dimethylformamide; EI-MS; electron impact mass spectra; Glu; glutamic acid; HPLC; high performance liquid chromatography; Ile; isoleucine; K; _i; inhibition constant; Leu; leucine; MOE; molecular operating environment; MOPAC; molecular orbital PACkage; NCE; new chemical entity; NMR; nuclear magnetic resonance; PLANTS; protein-Ligand ANT System; Phe; phenylalanine; Pro; proline; PyBOP; benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate; R-PIA; N; -R-; N; ⁶; -(1-methyl-2-phenylethyl)adenosine; SAR; structure–affinity relationship; TLC; thin layer chromatography; TMS; tetramethylsilane; Trp; tryptophan; Tyr; tyrosine; UV; ultraviolet; ZM241385; 4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenolDrug discovery; Adenosine receptor ligand; Chromone scaffold

Diminished toxicity of C-1748, 4-methyl-9-hydroxyethylamino-1-nitroacridine, compared with its demethyl analog, C-857, corresponds to its resistance to metabolism in HepG2 cells by Anita Wiśniewska; Magdalena Niemira; Karolina Jagiełło; Agnieszka Potęga; Małgorzata Świst; Colin Henderson; Anna Skwarska; Ewa Augustin; Jerzy Konopa; Zofia Mazerska (pp. 30-42).

The narrow “therapeutic window” of anti-tumour therapy may be the result of drug metabolism leading to the activation or detoxification of antitumour agents. The aim of this work is to examine (i) whether the diminished toxicity of a potent antitumour drug, C-1748, 9-(2′-hydroxyethylamino)-4-methyl-1-nitroacridine, compared with its 4-demethyl analogue, C-857, results from the differences between the metabolic pathways for the two compounds and (ii) the impact of reducing and/or hypoxic conditions on studied metabolism. We investigated the metabolites of C-1748 and C-857 formed in rat and human liver microsomes, with human P450 reductase (POR) and in HepG2 cells under normoxia and hypoxia. The elimination rate of C-1748 from POR knockout mice (HRN) was also evaluated. Three products, 1-amino-9-hydroxyethylaminoacridine, 1-aminoacridinone and a compound with an additional 6-membered ring, were identified for C-1748 and C-857 in all studied metabolic systems. The new metabolite was found in HepG2 cells. We showed that metabolic rate and the reactivity of metabolites of C-1748 were considerably lower than those of C-857, in all investigated metabolic models. Compared with metabolism under normoxia, cellular metabolism under hypoxia led to higher levels of 1-aminoacridine and aza-acridine derivatives of both compounds and of the 6-membered ring metabolite of C-1748. In conclusion, the crucial role of hypoxic conditions and the direct involvement of POR in the metabolism of both compounds were demonstrated. Compared with C-857, the low reactivity of C-1748 and the stability of its metabolites are postulated to contribute significantly to the diminished toxicity of this compound observed in animals.

Keywords: Abbreviations; DTT; dithiothreitol; HLM; human liver microsomes; HRN; Hepatic Reductase Null mouse; POR; NADPH-P450 oxidoreductase; RLM; rat liver microsomes; WT; wild typeAntitumour agents; Capridine β; C-1748; Hypoxia; Metabolism; 1-Nitroacridines

Phosphorylation of deoxycytidine kinase on Ser-74: Impact on kinetic properties and nucleoside analog activation in cancer cells by Rachid Amsailale; Eric Van Den Neste; Angélique Arts; Eliza Starczewska; Françoise Bontemps; Caroline Smal (pp. 43-51).

Deoxycytidine kinase (dCK) (EC 2.7.1.74) is a key enzyme in the activation of several therapeutic nucleoside analogs (NA). Its activity can be increased in vivo by Ser-74 phosphorylation, a property that could be used for enhancing NA activation and clinical efficacy. In line with this, studies with recombinant dCK showed that mimicking Ser-74 phosphorylation by a S74E mutation increases its activity toward pyrimidine analogs. However, purine analogs had not been investigated. Here, we show that the S74E mutation increased the k_cat for cladribine (CdA) by 8- or 3-fold, depending on whether the phosphoryl donor was ATP or UTP, for clofarabine (CAFdA) by about 2-fold with both ATP and UTP, and for fludarabine (F-Ara-A) by 2-fold, but only with UTP. However, the catalytic efficiencies ( k_cat/ K_m) were not, or slightly, increased. The S74E mutation also sensitized dCK to feed-back inhibition by dCTP, regardless of the phosphoryl donor. Importantly, we did not observe an increase of endogenous dCK activity toward purine analogs after in vivo-induced increase of Ser-74 phosphorylation. Accordingly, treatment of CLL cells with aphidicolin, which enhances dCK activity through Ser-74 phosphorylation, did not modify the conversion of CdA or F-Ara-A into their active triphosphate form. Nevertheless, the same treatment enhanced activation of gemcitabine (dFdC) into dFdCTP in CLL as well as in HCT-116 cells and produced synergistic cytotoxicity. We conclude that increasing phosphorylation of dCK on Ser-74 might constitute a valuable strategy to enhance the clinical efficacy of some NA, like dFdC, but not of CdA or F-Ara-A.

Keywords: CLL; dCTP; Deoxycytidine kinase; Nucleoside analogs; Ser-74 phosphorylation

The anticancer thiosemicarbazones Dp44mT and triapine lack inhibitory effects as catalytic inhibitors or poisons of DNA topoisomerase IIα by Jack C. Yalowich; Xing Wu; Rui Zhang; Ragu Kanagasabai; Marisa Hornbaker; Brian B. Hasinoff (pp. 52-58).

The thiosemicarbazones Dp44mT (di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone) and triapine have potent antiproliferative activity and have been evaluated as anticancer agents. While these compounds strongly bind iron and copper, their mechanism(s) of action are incompletely understood. A recent report (Rao et al., Cancer Research 69:948-57, 2009) suggested that Dp44mT may, in part, exert its cytotoxicity through poisoning of DNA topoisomerase IIα. In the present report, a variety of assays were used to determine whether Dp44mT and triapine target topoisomerase IIα. Neither of these compounds inhibited topoisomerase IIα decatenation or induced cleavage of pBR322 DNA in the presence of enzyme. In cells, Dp44mT did not stabilize topoisomerase IIα covalent binding to DNA using an immunoblot band depletion assay, an ICE (immunodetection of complexes of enzyme-to-DNA) assay, and a protein–DNA covalent complex forming assay. Dp44mT did not display cross resistance to etoposide resistant K562 cells containing reduced topoisomerase IIα levels. Synchronized Dp44mT-treated CHO cells did not display a G₂/M cell cycle block expected of a topoisomerase II inhibitor. A COMPARE analysis of Dp44mT using the NCI 60-cell line data indicated that inhibition of cell growth was poorly correlated with DNA topoisomerase IIα mRNA levels. In summary, we found no support for the conclusion that Dp44mT inhibits cell growth through the targeting of topoisomerase IIα. Since clinical trials of triapine are underway, it will be important to better understand the intracellular targeting and mechanisms of action of the thiosemicarbazones to support forward development of these agents and newer analogs.

Keywords: Dp44mT; Triapine; Topoisomerase IIα; Cell cycle analysis; Iron; Thiosemicarbazone

Rottlerin potentiates camptothecin-induced cytotoxicity in human hormone refractory prostate cancers through increased formation and stabilization of topoisomerase I-DNA cleavage complexes in a PKCδ-independent pathway by Jui-Ling Hsu; Yunn-Fang Ho; Tsai-Kun Li; Ching-Shih Chen; Lih-Ching Hsu; Jih-Hwa Guh (pp. 59-67).

Combination therapy, which can optimize killing activity to cancers and minimize drug resistance, is a mainstream therapy against hormone-refractory prostate cancers (HRPCs). Rottlerin, a natural polyphenolic component, synergistically increased PC-3 (a HRPC cell line) apoptosis induced by camptothecin (a topoisomerase I inhibitor). Using siRNA technique to knockdown protein kinase C-δ (PKCδ), the data showed that rottlerin-mediated synergistic effect was PKCδ-independent, although rottlerin has been used as a PKCδ inhibitor. Rottlerin potentiated camptothecin-induced DNA fragmentation at S phase and ATM phosphorylation at Ser¹⁹⁸¹. The effect was correlated to apoptosis ( r²=0.9). To detect upstream signals, the data showed that camptothecin acted on and stabilized topoisomerase I-DNA complex, leading to the formation of camptothecin-trapped cleavage complexes (TOP1cc). The effect was potentiated by rottlerin. To determine DNA repair capability, the time-related γH₂A.X formation was examined after camptothecin removal. Consequently, rottlerin significantly inhibited camptothecin removal-mediated decline of γH₂A.X formation at S phase, indicating the impairment of DNA repair activity in the presence of rottlerin. The combinatory treatment of camptothecin and rottlerin induced conformational change and activation of Bax and formation of truncated Bad, suggesting the contribution of mitochondria stress to apoptosis. In summary, the data suggest that rottlerin-mediated camptothecin sensitization is through the augmented stabilization of TOP1cc, leading to an increase of DNA damage stress and, possibly, an impairment of DNA repair capability. Subsequently, mitochondria-involved apoptosis is triggered through Bax activation and truncated Bad formation. The novel discovery may provide an anticancer approach of combinatory use between rottlerin and camptothecin for the treatment of HRPCs.

Keywords: Camptothecin; Rottlerin; Topoisomerase I-DNA cleavage complex; DNA damage; Hormone-refractory prostate cancer

Binding and activity of the prostacyclin receptor (IP) agonists, treprostinil and iloprost, at human prostanoid receptors: Treprostinil is a potent DP₁ and EP₂ agonist by Brendan J. Whittle; Adam M. Silverstein; David M. Mottola; Lucie H. Clapp (pp. 68-75).

Differential activation of human prostanoid receptors expressed in cells: EC₅₀ values for prostacyclin analogues treprostinil or iloprost in stimulating adenylate cyclase (causing vasodilatation – green) or calcium flux (causing vasoconstriction – red).The prostacyclin analogues, iloprost and treprostinil are extensively used in treating pulmonary hypertension. Their binding profile and corresponding biochemical cellular responses on human prostanoid receptors expressed in cell lines, have now been compared. Iloprost had high binding affinity for EP₁ and IP receptors ( K_i 1.1 and 3.9nM, respectively), low affinity for FP, EP₃ or EP₄ receptors, and very low affinity for EP₂, DP₁ or TP receptors. By contrast, treprostinil had high affinity for the DP₁, EP₂ and IP receptors ( K_i 4.4, 3.6 and 32nM, respectively), low affinity for EP₁ and EP₄ receptors and even lower affinity for EP₃, FP and TP receptors. In functional assays, iloprost had similar high activity in elevating cyclic AMP levels in cells expressing the human IP receptor and stimulating calcium influx in cells expressing EP₁ receptors (EC₅₀ 0.37 and 0.3nM, respectively) with the rank order of activity on the other receptors comparable to the binding assays. As with binding studies, treprostinil elevated cyclic AMP with a similar high potency in cells expressing DP₁, IP and EP₂ receptors (EC₅₀ 0.6, 1.9 and 6.2nM, respectively), but had low activity at the other receptors. Activation of IP, DP₁ and EP₂ receptors, as with treprostinil, can all result in vasodilatation of human pulmonary arteries. However, activation of EP₁ receptors can provoke vasoconstriction, and hence may offset the IP-receptor mediated vasodilator effects of iloprost. Treprostinil may therefore differ from iloprost in its overall beneficial pulmonary vasorelaxant profile and other pharmacological actions, especially in diseases where the IP receptor is down-regulated.

Keywords: Prostacyclin analogues; Radioligand binding; Cyclic AMP; Calcium influx; Prostanoid receptors

The role of the second and third extracellular loops of the adenosine A1 receptor in activation and allosteric modulation by M.C. Peeters; L.E. Wisse; A. Dinaj; B. Vroling; G. Vriend; A.P. IJzerman (pp. 76-87).

The adenosine A₁ receptor is a member of the large membrane protein family that signals through G proteins, the G protein-coupled receptors (GPCRs). GPCRs consist of seven transmembrane domains connected by three intracellular and three extracellular loops. Their N-terminus is extracellular, the C-terminal tail is in the cytoplasm. The transmembrane domains in receptor subfamilies that bind the same endogenous ligand, such as dopamine or adenosine, tend to be highly similar. In contrast, the loop regions can vary greatly, both in sequence and in length, and the role these loops have in the activation mechanism of the receptors remains unclear. Here, we investigated the activating role of the second and third extracellular loop of the human adenosine A₁ receptor. By means of an (Ala)₃ mutagenic scan in which consecutive sets of three amino acids were mutated into alanine residues in EL2 and a classical alanine scan in EL3, we revealed a strong regulatory role for the second extracellular loop (EL2) of the human adenosine A₁ receptor. Besides many residues in the second and the third extracellular loops important for adenosine A₁ receptor activation, we also identified two residues in EL2, a tryptophan and a glutamate, that affect the influence of the allosteric modulator PD81,723. These results, combined with a comparison of the different receptor loop regions, provide insight in the activation mechanism of this typical class A GPCR and further emphasize the unique pharmacological profile the loops can provide to individual receptors, even within subfamilies of GPCRs.

Keywords: G protein-coupled receptor; Adenosine A1 receptor; Receptor activation; S. cerevisiae; Extracellular loops

Eriodictyol prevents early retinal and plasma abnormalities in streptozotocin-induced diabetic rats by Claudio Bucolo; Gian Marco Leggio; Filippo Drago; Salvatore Salomone (pp. 88-92).

Effects of eriodictyol on retinal biomarkers levels in diabetic ratsDiabetic retinopathy is a complex disease that has potential involvement of inflammatory and oxidative stress-related pathways in its pathogenesis. We hypothesized that eriodictyol, one of the most abundant dietary flavonoids, could be effective against diabetic retinopathy, which involves significant oxidative stress and inflammation. The aim of the present study was to investigate the effects of eriodictyol in early retinal and plasma changes of streptozotocin-induced diabetic rats. The effect of eriodictyol treatment (0.1, 1, 10mg/kg daily for 10 days) was evaluated by TNF-α, ICAM-1, VEGF, and eNOS protein levels measurement in the retina, plasma lipid peroxidation, and blood–retinal barrier (BRB) integrity. Increased amounts of cytokines, adhesion molecule, and nitric oxide synthase were observed in retina from diabetic rats. Eriodictyol treatment significantly lowered retinal TNF-α, ICAM-1, VEGF, and eNOS in a dose-dependent manner. Further, treatment with eriodictyol significantly suppressed diabetes-related lipid peroxidation, as well as the BRB breakdown. These data demonstrated that eriodictyol attenuates the degree of retinal inflammation and plasma lipid peroxidation preserving the BRB in early diabetic rats.

Keywords: Diabetes; Retina; Antioxidant; Inflammation; Rat

Eriodictyol prevents early retinal and plasma abnormalities in streptozotocin-induced diabetic rats by Claudio Bucolo; Gian Marco Leggio; Filippo Drago; Salvatore Salomone (pp. 88-92).

Keywords: Diabetes; Retina; Antioxidant; Inflammation; Rat

Estradiol induces cytochrome P450 2B6 expression at high concentrations: Implication in estrogen-mediated gene regulation in pregnancy by Kwi Hye Koh; Steve Jurkovic; Kyunghee Yang; Su-Young Choi; Jin Woo Jung; Kwang Pyo Kim; Wei Zhang; Hyunyoung Jeong (pp. 93-103).

Pregnancy alters the rate and extent of drug metabolism, but little is known about the underlying molecular mechanism. We have found that 17β-estradiol (E2) upregulates expression of the major drug-metabolizing enzyme CYP2B6 in primary human hepatocytes. Results from promoter reporter assays in HepG2 cells revealed that E2 activates constitutive androstane receptor (CAR) and enhances promoter activity of CYP2B6, for which high concentrations of E2 reached during pregnancy were required. E2 triggered nuclear translocation of CAR in primary rat hepatocytes that were transiently transfected with human CAR as well as in primary human hepatocytes, further confirming transactivation of CAR by E2. E2-activated estrogen receptor (ER) also enhanced CYP2B6 promoter activity. The DNA-binding domain of ER was not required for the induction of CYP2B6 promoter activity by E2, suggesting involvement of a non-classical mechanism of ER action. Results from deletion and mutation assays as well as electrophorectic mobility shift and supershift assays revealed that two AP-1 binding sites (−1782/−1776 and −1664/−1658 of CYP2B6) are critical for ER-mediated activation of the CYP2B6 promoter by E2. Concurrent activation of both ER and CAR by E2 enhanced CYP2B6 expression in a synergistic manner. Our data demonstrate that at high concentrations reached during pregnancy, E2 activates both CAR and ER that synergistically induce CYP2B6 expression. These results illustrate pharmacological activity of E2 that would likely become prominent during pregnancy.

Keywords: JEL classification; Pharmacokinetics; Drug metabolismAbbreviations; AP-1; activator protein 1; E2; 17β-estradiol; CYP; cytochrome P450; CAR; constitutive androstane receptor; ER; estrogen receptor; ERE; estrogen responsive element; HH; primary human hepatocyte; PBREM; the phenobarbital-responsive enhancer moduleCytochrome P450 2B6; Estradiol; Nuclear receptors; Pregnancy

Intestinal CYP3A4 and midazolam disposition in vivo associate with VDR polymorphisms and show seasonal variation by Ranjit K. Thirumaran; Jatinder K. Lamba; Richard B. Kim; Brad L. Urquhart; Jamie C. Gregor; Nilesh Chande; Yiping Fan; An Qi; Cheng Cheng; Kenneth E. Thummel; Stephen D. Hall; Erin G. Schuetz (pp. 104-112).

Vitamin D, whose levels vary seasonally with sunlight, is activated to 1α,25-dihydroxyvitamin D₃ that binds the vitamin D receptor (VDR) and transcriptionally regulates intestinal CYP3A4 expression. We genotyped VDR polymorphisms and determined their associations with intestinal CYP3A4 and with midazolam pharmacokinetics, and whether intestinal CYP3A4 levels/activity varied seasonally. The VDR BsmIG>A (rs1544410) polymorphism was significantly associated with CYP3A4 jejunal expression/activity, with CYP3A4 duodenal mRNA, and with midazolam area under the curve (AUC). Intestinal CYP3A4 expression/activity was significantly higher in biopsies with the VDR promoter polymorphisms Cdx2-3731G>A and GATA-1012A>G that increase VDR activation of target genes. Duodenal CYP3A4 mRNA was significantly higher between April and September than between October and March. Midazolam p.o. AUC and oral bioavailability trended higher October through March compared to April through September. These data suggest VDR polymorphisms are predictors of intestinal CYP3A4, and that CYP3A4 intestinal expression varies seasonally – likely related to annual changes in UV sunlight and vitamin D levels.

Keywords: CYP3A4; Vitamin D; VDR; Polymorphism; Seasonal variation

Allopurinol, quercetin and rutin ameliorate renal NLRP3 inflammasome activation and lipid accumulation in fructose-fed rats by Qing-Hua Hu; Xian Zhang; Ying Pan; Yu-Cheng Li; Ling-Dong Kong (pp. 113-125).

The NOD-like receptor 3 (NLRP3) inflammasome-mediated inflammation is recently recognized in the development of renal injury. However, the mechanisms of the inflammasome-mediated inflammation and lipid accumulation in renal injury and the actions of lowing urate agents remain unclear. The present study used fructose to induce hyperuricemia and dyslipidemia, which caused renal NLRP3 inflammasome activation characterized by over-expression of the NLRP3, apoptosis-associated speck-like protein (ASC) and caspase-1, resulting in overproduction of interleukin (IL)-1β and IL-18, as well as IL-6 and tumor necrosis factor α (TNF-α) in rats. The elevated levels of these pro-inflammatory cytokines impaired renal janus-activated kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3)/peroxisome proliferator-activated receptor α (PAPR-α), and insulin receptor (IR)/insulin receptor substrate 1 (IRS1)/protein kinase B (Akt)/extracellular signal-regulated kinase1/2 (ERK1/2) signaling pathways with over-expression of suppressor of cytokine signaling 3 (SOCS3), exacerbating renal lipid accumulation and injury in fructose-fed rats. The restoration of fructose-induced hyperuricemia and dyslipidemia by the treatment of allopurinol, quercetin and rutin blocked the NLRP3 inflammasome activation to improve the signaling impairments and reduce lipid accumulation in the kidney of rats. These results suggest that the activation of renal NLRP3 inflammasome may play an important role in the link among renal inflammation, JAK2/STAT3/PAPR-α and IR/IRS1/Akt/ERK1/2 signaling impairment, and lipid accumulation driven by fructose. The NLRP3 inflammasome may be the target mediating the improvement of urate-lowering agents allopurinol, quercetin and rutin on fructose-induced renal lipid accumulation and injury.

Keywords: NLRP3 inflammasome; Renal inflammation; Lipid accumulation; Urate-lowering agents; Renal injury

Inhibition of thymidine phosphorylase expression by using an HSP90 inhibitor potentiates the cytotoxic effect of cisplatin in non-small-cell lung cancer cells by Shao-Hsing Weng; Sheng-Chieh Tseng; Yu-Ching Huang; Huang-Jen Chen; Yun-Wei Lin (pp. 126-136).

Elevated thymidine phosphorylase (TP) levels, a key enzyme in the pyrimidine nucleoside salvage pathway, are associated with an aggressive disease phenotype and poor prognoses. In this study, we examined the role of TP expression in relation to the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced cytotoxicity in two non-small-cell lung cancer (NSCLC) cell lines, A549 and H1650. Treatment with 17-AAG (0.1–1μM) resulted in a decrease in cellular TP protein and mRNA levels, which was accompanied by a downregulation of phosphorylated MKK1/2-ERK1/2 and AKT protein levels. The 17-AAG treatment disrupted the interaction between HSP90 and TP and triggered TP protein degradation through the ubiquitin-26S proteasome pathway. Specific inhibition of TP expression by siRNA further enhanced the cell death and growth inhibition that had been induced by 17-AAG. An enhancement of ERK1/2 or AKT activation by transfecting the cancer cells with constitutively active MKK1/2 or AKT expression vectors significantly restored the 17-AAG-reduced TP protein levels as well as cell viability. In contrast, a combination of U0126 (MKK1/2 inhibitors) or LY294002 (PI3K inhibitor) further decreased the TP expression and cell viability induced by 17-AAG. Moreover, 17-AAG enhanced the cisplatin-induced cytotoxic effect through downregulation of the cisplatin-induced TP expression and ERK1/2 and AKT activation. Taken together, our results suggest that the down-modulation of TP protein induced by 17-AAG represents a key factor in enhancing the cytotoxic effects of cisplatin in NSCLC cells.

Keywords: HSP90; Thymidine phosphorylase; Cytotoxicity; Non-small cell lung cancer

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Biochemical Pharmacology (v.84, #1)